**WW Domain-Containing Oxidoreductase is a Potential Receptor for Sex Steroid Hormones**

Won-Pei Su1, Shu-Hui Chen2, Szu-Jung Chen1, Pei-Yi Chou1, Chun-Cheng Huang1 and Nan-Shan Chang1,3,4 *1Institute of Molecular Medicine, National Cheng Kung University, Tainan, Taiwan, 2Department of Chemistry, National Cheng Kung University, Tainan, Taiwan, 3Department of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY, 4Department of Neurochemistry, NYS Institute of Basic Research in Developmental Disabilities, Staten Island, NY, 1,2ROC 3,4USA* 

#### **1. Introduction**

332 Sex Hormones

Yadav BR, King WA, & Betteridge KJ (1993). Relationships between the completion of first

Yong LC, Kuller LH, Rutan G, & Bunker C (1993). Longitudinal Study of Blood Pressure:

Zechner U, Wilda M, Kehrer-Sawatzki H, Vogel W, Fundele R, & Hameister H (2001). A

bovine embryos generated in vitro. *Mol Reprod Dev* 36, 434-439.

shaping human evolution? *Trends Genet* 17, 697-701.

138, 973-983.

cleavage and the chromosomal complement, sex, and developmental rates of

Changes and Determinants from Adolescence to Middle Age. The Dormont High School Follow-up Study, 1957-1963 to 1989-1990. *American Journal of Epidemiology*

high density of X-linked genes for general cognitive ability: a run-away process

#### **1.1 Biosynthesis and metabolism of estrogens**

Estrogen is a steroid hormone that comprises a group of compounds, including estrone (E1), estradiol (E2) and estriol (E3). E2 is an ovarian hormone necessary for the development of secondary sexual characteristics and function of the reproductive system in females. It also plays important roles in non-reproductive organs by multiple pathways. Estrogens are produced primarily by developing follicles in the ovaries, the corpus luteum, and the placenta. Some estrogens are also produced in smaller amounts by other tissues such as the liver, adrenal glands, and the breasts. E2 is converted from testosterone and E1 from rostenedione; both conversions are regulated by a dehydrogenase enzyme, aromatase. Estrogens are eliminated from the body by metabolic conversion to hormonally inactive and water-soluble metabolites that are excreted in the urine and/or feces. The metabolic disposition of estrogens includes oxidative metabolism (Martucci et al., 1993) and conjugative metabolism by glucuronidation (Zhu, et al., 1996), sulfonation (Hernandez et al., 1992) and/or *O*-methylation (Ball & Knuppen, 1980). Hydroxylation at the C-2 and C-4 position of E2 (17-Estradiol) yields the catecholestrogens (CEs), 2-hydroxyestrone (2- OHE1) and 2-hydroxyestradiol (2-OHE2), 4-hydroxyestrone (4-OHE1) and 4 hydroxyestradiol (4-OHE2) while hydroxylation at the C-16 position yields 16 hydroxyestrone (16 -OHE1), which is subsequently converted to estriol (E3) (Ball & Knuppen, 1980; Zhu & Conney, 1998). The hydroxylated products exert very different biological properties: the 16 -hydroxy and 4-hydroxy metabolites are active estrogens,

WW Domain-Containing Oxidoreductase is a Potential Receptor for Sex Steroid Hormones 335

Based on a review of data scattered in the literature, we suggest that some of the effects exerted by active estrogen may be mediated by specific intracellular receptors or effectors, which are different from the classical estrogen receptor. It is most likely that additional isoforms of the classical ERs or putative receptors with the ligand binding domain are potential candidates of E2 receptors. Moreover, active estrogen metabolites such as catechol estrogens are not merely to simplify the secretion of estrogen, but may have their own biological roles (Zhu & Conney, 1998). Receptors of estrogen metabolites are distinct and different from classical ERs (Markides & Liehr, 2005). A locally formed estrogen metabolite may exert a biological effect important for the action of the parent hormone. Cytochrome P450 family are the major enzymes catalyzing nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH\*)-dependent oxidative metabolism of estrogens to multiple hydroxylated metabolites. The estrogen biosynthesis enzyme, aromatase, whose function is to aromatize androgens in order to produce estrogens, is a member of the cytochrome P450 superfamily. Since estrogen and estrogen metabolites are substrates of specific reductases or oxidases, we suspect that cellular proteins, which possess an oxidoreductase domain, are candidates of novel estrogen receptors. These novel receptors may possess important and unique biological functions that are not directly associated with the classical estrogen action.

Biologically active sex steroid hormones are metabolically converted in normal and cancerous tissues and organs. Estrogen provides a proliferative effect in majority of ERpositive breast cancer cells. Enzymes responsible for metabolizing steroid hormones are aromatase, estrone sulfatases, and 17β-hydroxysteroid dehydrogenases (17β-HSDs) (Jansson, 2009; Aka et al., 2009). These enzymes are present in breast cancer tissues (Miki et al., 2009). There are reductive and oxidative 17β-HSDs. The reductive 17β-HSDs are responsible for manufacturing active androgens and estrogens by catalyzing the formation of the hydroxy group at position 17β of the steroid backbone. The oxidative 17β-HSD transforms the hydroxy group into keto and inactivates the steroids. The type 3 17β-HSD (17β-HSD3) is reductive, structurally similar to 17β-HSD12, and present in the testis. 17β-HSD3 recognizes androgen by catalyzing the transformation of 4-androstenedione into testosterone (Geissler et al., 1994). 17β-HSD12 catalyzes the transformation of both androgens and estrogens (Blanchard & Luu-The, 2007; Liu et al., 2007). *Caenorhabditis elegans* LET-767 is known to metabolize androgens and estrogens, and the gene appears to share a common ancestor with human types 17β-HSD3 and HSD12 (Desnoyers et al., 2007). High levels of expression of 17β-HSD1 have been shown to be associated with poor prognosis in breast cancer and late relapse among patients with ER-positive breast tumors (Sasaki et al., 2010; Jansson et al., 2009). In contrast, significant downregulation of 17β-HSD2 is also correlated with decreased survival in ER-positive breast cancer (Sasaki et al., 2010; Jansson et al., 2009). Similarly, significantly reduced expression of 17β-HSD14 mRNA in breast cancer is also associated with decreased survival (Jansson et al., 2009). Overall, there are 14 different types of 17β-HSDs (Marchais-Oberwinkler et al., 2011). These oxidoreductases are central to the estrogen and androgen steroid metabolism by catalyzing final steps of the steroid biosynthesis. Indeed, 17β-HSDs act like receptor molecules. While these proteins are involved in many diseases such as breast cancer, prostate cancer, endometriosis, osteoporosis, and brain

**1.4 Non-classical estrogen receptors** 

**2. Oxidoreductases and sex steroid hormones** 

cancer, 17β-HSDs are of considerable interest in therapeutic targeting.

**2.1 17β-hydroxysteroid dehydrogenases** 

whereas the 2-hydroxy metabolites are not as active (Fishman & Martucci, 1980; Swaneck & Fishman, 1988). However, the binding and redox cycling activities of CEs can be blocked via *O*-methylation by catechol-O-methyltransferase (COMT), which converts 2-OHE1/E2 and 4- OHE1/E2 to their methoxy derivatives 2-MeOHE1, 2-MeOHE2, 4-MeOHE1, and 4- MeOHE2, respectively (Albin et al., 1993; Cheng et al., 1998; Falany & Falany, 1996). Although liver is the major organ of the estrogen metabolism, some estrogen hydroxylation enzymes are selectively expressed in other tissues. Our recent data indicated that trace amounts (<0.9 fg/cell) of estrogens are produced in the endogenous breast cancer cells (MCF-7) (Huang et al., 2011). Moreover, E2 treatment substantially induced E1 and estrogen metabolites in MCF-7 cells, indicating the expression of estrogen metabolizing enzymes in breast cancer cells as well.

#### **1.2 Estrogen receptors**

E2 is most known to act by binding to and activating two estrogen receptors (ERs), ER and ER (Mosselman et al., 1996), which belongs to the super-family of nuclear receptors *(*McDonnel & Norris, 2002)*.* Like many nuclear receptors, ERs are consisted of hypervariable *N*-termini that contribute to the transaction function; namely, a highly conserved DNA binding domain responsible for DNA binding and dimerization and *C*-terminal domain, which is involved in ligand binding, nuclear localization, and ligand-dependent transaction function. It is well established that E2 can activate ER and promote cancer formation in experimental animals, which is associated with cell proliferation. In contrast, the activated ER suppresses cell proliferation and colon cancer xenograft growth, probably as a consequence of ER-mediated inhibition of cell-cycle pathways (Hartman et al., 2009). E2 action involves ligand-mediated activation of ER and ER, which binds directly with estrogen response element (ERE) in the promoters of target genes and recruits various coactivators to mediate transcriptional regulation. There is a general consensus that hormonally active compounds may directly or indirectly activate transcription factors through ER binding and promote gene transcription and cell proliferation, in particular in cells responding to the hormones by growth. Many anti-cancer drugs for estrogendependent breast tumor have been developed based on their antagonistic effect on E2 binding so as to affect protein expression.

#### **1.3 Non-classical estrogen actions**

E2, however, could also induce estrogenic effects in ER-negative systems through signaling pathways more commonly associated with growth factor activation of cell surface receptors such as G-protein-coupled receptor (GPCR) GPR30 to transactivate epidermal growth factor receptor (EGFR) and activate the MAPK cascade *via* the release of surface-associated heparin binding epidermal growth factor (Filardo et al., 2002). E2 may also trigger the transcription of non-estrogen responsive genes through kinase activation. It has been demonstrated that this GPR30-dependent estrogen induction of MAPK is transient and under the control of a cAMP-dependent negative feedback loop. Whereas, our phosphoproteomics data (Wu et al., 2011) suggested that the growth factor-mediated pathways also occur in ER-dependent cells. Furthermore, accumulating evidence reveals that many unexpected non-classical responses such as estrogen-derived reactive oxidative stress (ROS) may also be induced (Yeh et al., 2005; Miro et al., 2011). The interaction between estrogen-derived ROS and proliferation machinery has not been elucidated yet.

#### **1.4 Non-classical estrogen receptors**

334 Sex Hormones

whereas the 2-hydroxy metabolites are not as active (Fishman & Martucci, 1980; Swaneck & Fishman, 1988). However, the binding and redox cycling activities of CEs can be blocked via *O*-methylation by catechol-O-methyltransferase (COMT), which converts 2-OHE1/E2 and 4- OHE1/E2 to their methoxy derivatives 2-MeOHE1, 2-MeOHE2, 4-MeOHE1, and 4- MeOHE2, respectively (Albin et al., 1993; Cheng et al., 1998; Falany & Falany, 1996). Although liver is the major organ of the estrogen metabolism, some estrogen hydroxylation enzymes are selectively expressed in other tissues. Our recent data indicated that trace amounts (<0.9 fg/cell) of estrogens are produced in the endogenous breast cancer cells (MCF-7) (Huang et al., 2011). Moreover, E2 treatment substantially induced E1 and estrogen metabolites in MCF-7 cells, indicating the expression of estrogen metabolizing enzymes in

E2 is most known to act by binding to and activating two estrogen receptors (ERs), ER and ER (Mosselman et al., 1996), which belongs to the super-family of nuclear receptors *(*McDonnel & Norris, 2002)*.* Like many nuclear receptors, ERs are consisted of hypervariable *N*-termini that contribute to the transaction function; namely, a highly conserved DNA binding domain responsible for DNA binding and dimerization and *C*-terminal domain, which is involved in ligand binding, nuclear localization, and ligand-dependent transaction function. It is well established that E2 can activate ER and promote cancer formation in experimental animals, which is associated with cell proliferation. In contrast, the activated ER suppresses cell proliferation and colon cancer xenograft growth, probably as a consequence of ER-mediated inhibition of cell-cycle pathways (Hartman et al., 2009). E2 action involves ligand-mediated activation of ER and ER, which binds directly with estrogen response element (ERE) in the promoters of target genes and recruits various coactivators to mediate transcriptional regulation. There is a general consensus that hormonally active compounds may directly or indirectly activate transcription factors through ER binding and promote gene transcription and cell proliferation, in particular in cells responding to the hormones by growth. Many anti-cancer drugs for estrogendependent breast tumor have been developed based on their antagonistic effect on E2

E2, however, could also induce estrogenic effects in ER-negative systems through signaling pathways more commonly associated with growth factor activation of cell surface receptors such as G-protein-coupled receptor (GPCR) GPR30 to transactivate epidermal growth factor receptor (EGFR) and activate the MAPK cascade *via* the release of surface-associated heparin binding epidermal growth factor (Filardo et al., 2002). E2 may also trigger the transcription of non-estrogen responsive genes through kinase activation. It has been demonstrated that this GPR30-dependent estrogen induction of MAPK is transient and under the control of a cAMP-dependent negative feedback loop. Whereas, our phosphoproteomics data (Wu et al., 2011) suggested that the growth factor-mediated pathways also occur in ER-dependent cells. Furthermore, accumulating evidence reveals that many unexpected non-classical responses such as estrogen-derived reactive oxidative stress (ROS) may also be induced (Yeh et al., 2005; Miro et al., 2011). The interaction between estrogen-derived ROS and proliferation

breast cancer cells as well.

**1.2 Estrogen receptors** 

binding so as to affect protein expression.

**1.3 Non-classical estrogen actions** 

machinery has not been elucidated yet.

Based on a review of data scattered in the literature, we suggest that some of the effects exerted by active estrogen may be mediated by specific intracellular receptors or effectors, which are different from the classical estrogen receptor. It is most likely that additional isoforms of the classical ERs or putative receptors with the ligand binding domain are potential candidates of E2 receptors. Moreover, active estrogen metabolites such as catechol estrogens are not merely to simplify the secretion of estrogen, but may have their own biological roles (Zhu & Conney, 1998). Receptors of estrogen metabolites are distinct and different from classical ERs (Markides & Liehr, 2005). A locally formed estrogen metabolite may exert a biological effect important for the action of the parent hormone. Cytochrome P450 family are the major enzymes catalyzing nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH\*)-dependent oxidative metabolism of estrogens to multiple hydroxylated metabolites. The estrogen biosynthesis enzyme, aromatase, whose function is to aromatize androgens in order to produce estrogens, is a member of the cytochrome P450 superfamily. Since estrogen and estrogen metabolites are substrates of specific reductases or oxidases, we suspect that cellular proteins, which possess an oxidoreductase domain, are candidates of novel estrogen receptors. These novel receptors may possess important and unique biological functions that are not directly associated with the classical estrogen action.

#### **2. Oxidoreductases and sex steroid hormones**

#### **2.1 17β-hydroxysteroid dehydrogenases**

Biologically active sex steroid hormones are metabolically converted in normal and cancerous tissues and organs. Estrogen provides a proliferative effect in majority of ERpositive breast cancer cells. Enzymes responsible for metabolizing steroid hormones are aromatase, estrone sulfatases, and 17β-hydroxysteroid dehydrogenases (17β-HSDs) (Jansson, 2009; Aka et al., 2009). These enzymes are present in breast cancer tissues (Miki et al., 2009). There are reductive and oxidative 17β-HSDs. The reductive 17β-HSDs are responsible for manufacturing active androgens and estrogens by catalyzing the formation of the hydroxy group at position 17β of the steroid backbone. The oxidative 17β-HSD transforms the hydroxy group into keto and inactivates the steroids. The type 3 17β-HSD (17β-HSD3) is reductive, structurally similar to 17β-HSD12, and present in the testis. 17β-HSD3 recognizes androgen by catalyzing the transformation of 4-androstenedione into testosterone (Geissler et al., 1994). 17β-HSD12 catalyzes the transformation of both androgens and estrogens (Blanchard & Luu-The, 2007; Liu et al., 2007). *Caenorhabditis elegans* LET-767 is known to metabolize androgens and estrogens, and the gene appears to share a common ancestor with human types 17β-HSD3 and HSD12 (Desnoyers et al., 2007). High levels of expression of 17β-HSD1 have been shown to be associated with poor prognosis in breast cancer and late relapse among patients with ER-positive breast tumors (Sasaki et al., 2010; Jansson et al., 2009). In contrast, significant downregulation of 17β-HSD2 is also correlated with decreased survival in ER-positive breast cancer (Sasaki et al., 2010; Jansson et al., 2009). Similarly, significantly reduced expression of 17β-HSD14 mRNA in breast cancer is also associated with decreased survival (Jansson et al., 2009). Overall, there are 14 different types of 17β-HSDs (Marchais-Oberwinkler et al., 2011). These oxidoreductases are central to the estrogen and androgen steroid metabolism by catalyzing final steps of the steroid biosynthesis. Indeed, 17β-HSDs act like receptor molecules. While these proteins are involved in many diseases such as breast cancer, prostate cancer, endometriosis, osteoporosis, and brain cancer, 17β-HSDs are of considerable interest in therapeutic targeting.

WW Domain-Containing Oxidoreductase is a Potential Receptor for Sex Steroid Hormones 337

Mare et al., 2009; Chang et al., 2010; Chang et al., 2001). It is documented that there is a relative high percentage of loss of heterozygosity (LOH) from 30 to 50% in human *WWOX* gene in many types of cancer cells (Chang et al., 2007; Smith et al., 2007; Del Mare et al.,

The WWOX/WOX1 protein is composed of a nuclear localization sequence (NLS), two *N*terminal WW domains (containing conserved tryptophan residues), a *C*-terminal short-chain alcohol dehydrogenase/reductase (SDR) domain, and probably a functional *C*-terminal tail named D3 (Hong et al., 2007; Hsu et al., 2008; Lin et al., 2011) (Figure 1). The putative tertiary structures of the first WW domain and the C-terminal SDR domain are shown. The solution structure of the second WW domain has been documented (Wang et al., 2007). The *N*-terminal conserved first WW domain, which has been categorized as a group I WW domain, binds many proteins containing a PPXY motif(s), where P is proline, Y is tyrosine and X is any amino acid (Chang et al., 2007; Smith et al., 2007; Del Mare et al., 2009; Chang et al., 2010). Among these WWOX/WOX1-binding protein targets are p73, activator protein 2 (AP-2, ErbB4, Ezrin, small integral membrane protein of the lysosome/late endosome (SIMPLE), c-Jun, and runt-related transcription factor 2 (RUNX2) (Chang et al., 2007; Del Mare et al., 2009; Chang et al., 2010). While most of the observations were from ectopic expression to enhance the binding, physiological consequences of the binding interactions

Fig. 1. WWOX and simulated tertiary structures. The predicted amino acid sequence of WW domain-containing oxidoreductase, designated WWOX, FOR, or WOX1, possesses two *N*terminal WW domains, a nuclear localization signal sequence (NLS), and a *C*-terminal shortchain alcohol dehydrogenase/reductase (SDR) domain, where Tyr33 and Tyr287 are the hosphorylation sites, and NSYK is the binding motif for sex steroid hormones. Simulated structures of the first WW domain and the SDR domain are shown (1= 1st tryptophan; 2= Try33 phosphorylation site; 3= 2nd tryptophan; see yellow). Also, NSYK residues are marked

2009; Chang et al., 2010).

are largely unknown.

in yellow.

#### **2.2 Estrogen metabolites and biological effects**

Despite the wealthy knowledge of estrogen/ER in signaling, metabolism and diseases (Tam et al., 2011; Okoh et al., 2011; Nilsen, 2008; Mueck & Seeger, 2007; Straub, 2007), the signal pathways underlying the biological effects of estrogen metabolites are largely unknown. Estrogen metabolites could provide growth signal for cancer cells, and yet they may become toxic to normal cells (Obi et al., 2011; Sepkovic & Bradlow, 2009; Chen at al., 2008). The metabolites may invoke inflammatory lung diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease in women (Tam et al., 2011). Estrogen metabolite 16α-hydroxyestrone exerts estrogenic activity through covalent ER binding, whereas 2 hydroxyestrone would have anti-estrogenic capabilities (Obi et al., 2011). The ratios of these metabolites appear to be critical in controlling breast cancer cell growth. 2-Hydroxyestradiol and 4-hydroxyestradiol are implicated in tumorigenesis via increasing cell proliferation and the formation of reactive oxygen species for possibly generating deoxyribonucleic acid mutations (Joubert et al., 2009). The E2 metabolite 2-methoxyestradiol exerts apoptosis in many cancer cell types (Verenich & Gerk, 2010).

#### **2.3 Short chain alcohol dehydrogenase/reductase (SDR)**

Long-term exposure to estrogen and metabolites influences the development of breast cancer in women. The underlying mechanisms appear to be mainly involved in 1) estradiol/ER signaling for stimulation of cell proliferation, and 2) formation of genotoxic metabolites of estradiol for binding to DNA and causing depurination and mutations (Santen et al., 2009). We suspect that naturally occurring dehydrogenases/reductases (including 17β-HSDs), which possess binding sites for sex steroid hormones, may act as receptors and play an alternative role in breast cancer progression. For example, short-chain dehydrogenases/reductases (SDRs) are composed of a large family of NAD(P)(H) dependent oxidoreductases, sharing sequence motifs with similar functions (Kavanagh et al., 2008; Jörnvall et al., 2010). SDR enzymes play critical roles in metabolism for lipid, amino acid, carbohydrate, cofactor, and hormones, as well as in redox sensor mechanisms (Kavanagh et al., 2008). The SDR enzymes are normally 250–300 amino acid residues in length, which possesses a catalytic tetrad of Asn-Ser-Tyr-Lys (N-S-Y-K), and provides a platform for enzymatic activities encompassing several EC classes, including oxidoreductases, epimerases and lyases (Kavanagh et al., 2008).

#### **3. WW domain-containing Oxidoreductase**

#### **3.1 Tumor suppressor WWOX/FOR/WOX1 – a protein possessing WW and SDR domains**

WW domain-containing oxidoreductase, designated WWOX, FOR, or WOX1, is a protein possessing both WW domains and an SDR domain. The human and mouse *WWOX*/*Wwox* gene was first isolated independently by 3 laboratories in year 2000 (Smith et al., 2007; Del Mare et al., 2009; Chang et al., 2007, 2010; reviews). Human *WWOX* gene possesses approximately 1 million bases with 9 exons and codes for a 46-kDa protein containing 414 amino acids. Due to frequent genetic alterations, *WWOX* gene is generally considered as a tumor suppressor. The reason for the genetic alterations is probably associated with its localization on a common fragile site *FRA16D* on chromosome ch16q23.3-24.1. The *WWOX* gene encodes the WWOX/WOX1 protein. Substantial evidence reveals that this protein possesses a tumor suppressor function (Chang et al., 2007; Smith et al., 2007; Del

Despite the wealthy knowledge of estrogen/ER in signaling, metabolism and diseases (Tam et al., 2011; Okoh et al., 2011; Nilsen, 2008; Mueck & Seeger, 2007; Straub, 2007), the signal pathways underlying the biological effects of estrogen metabolites are largely unknown. Estrogen metabolites could provide growth signal for cancer cells, and yet they may become toxic to normal cells (Obi et al., 2011; Sepkovic & Bradlow, 2009; Chen at al., 2008). The metabolites may invoke inflammatory lung diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease in women (Tam et al., 2011). Estrogen metabolite 16α-hydroxyestrone exerts estrogenic activity through covalent ER binding, whereas 2 hydroxyestrone would have anti-estrogenic capabilities (Obi et al., 2011). The ratios of these metabolites appear to be critical in controlling breast cancer cell growth. 2-Hydroxyestradiol and 4-hydroxyestradiol are implicated in tumorigenesis via increasing cell proliferation and the formation of reactive oxygen species for possibly generating deoxyribonucleic acid mutations (Joubert et al., 2009). The E2 metabolite 2-methoxyestradiol exerts apoptosis in

Long-term exposure to estrogen and metabolites influences the development of breast cancer in women. The underlying mechanisms appear to be mainly involved in 1) estradiol/ER signaling for stimulation of cell proliferation, and 2) formation of genotoxic metabolites of estradiol for binding to DNA and causing depurination and mutations (Santen et al., 2009). We suspect that naturally occurring dehydrogenases/reductases (including 17β-HSDs), which possess binding sites for sex steroid hormones, may act as receptors and play an alternative role in breast cancer progression. For example, short-chain dehydrogenases/reductases (SDRs) are composed of a large family of NAD(P)(H) dependent oxidoreductases, sharing sequence motifs with similar functions (Kavanagh et al., 2008; Jörnvall et al., 2010). SDR enzymes play critical roles in metabolism for lipid, amino acid, carbohydrate, cofactor, and hormones, as well as in redox sensor mechanisms (Kavanagh et al., 2008). The SDR enzymes are normally 250–300 amino acid residues in length, which possesses a catalytic tetrad of Asn-Ser-Tyr-Lys (N-S-Y-K), and provides a platform for enzymatic activities encompassing several EC classes, including

**2.2 Estrogen metabolites and biological effects** 

many cancer cell types (Verenich & Gerk, 2010).

**2.3 Short chain alcohol dehydrogenase/reductase (SDR)** 

oxidoreductases, epimerases and lyases (Kavanagh et al., 2008).

**3.1 Tumor suppressor WWOX/FOR/WOX1 – a protein possessing WW and SDR** 

WW domain-containing oxidoreductase, designated WWOX, FOR, or WOX1, is a protein possessing both WW domains and an SDR domain. The human and mouse *WWOX*/*Wwox* gene was first isolated independently by 3 laboratories in year 2000 (Smith et al., 2007; Del Mare et al., 2009; Chang et al., 2007, 2010; reviews). Human *WWOX* gene possesses approximately 1 million bases with 9 exons and codes for a 46-kDa protein containing 414 amino acids. Due to frequent genetic alterations, *WWOX* gene is generally considered as a tumor suppressor. The reason for the genetic alterations is probably associated with its localization on a common fragile site *FRA16D* on chromosome ch16q23.3-24.1. The *WWOX* gene encodes the WWOX/WOX1 protein. Substantial evidence reveals that this protein possesses a tumor suppressor function (Chang et al., 2007; Smith et al., 2007; Del

**3. WW domain-containing Oxidoreductase** 

**domains** 

Mare et al., 2009; Chang et al., 2010; Chang et al., 2001). It is documented that there is a relative high percentage of loss of heterozygosity (LOH) from 30 to 50% in human *WWOX* gene in many types of cancer cells (Chang et al., 2007; Smith et al., 2007; Del Mare et al., 2009; Chang et al., 2010).

The WWOX/WOX1 protein is composed of a nuclear localization sequence (NLS), two *N*terminal WW domains (containing conserved tryptophan residues), a *C*-terminal short-chain alcohol dehydrogenase/reductase (SDR) domain, and probably a functional *C*-terminal tail named D3 (Hong et al., 2007; Hsu et al., 2008; Lin et al., 2011) (Figure 1). The putative tertiary structures of the first WW domain and the C-terminal SDR domain are shown. The solution structure of the second WW domain has been documented (Wang et al., 2007).

The *N*-terminal conserved first WW domain, which has been categorized as a group I WW domain, binds many proteins containing a PPXY motif(s), where P is proline, Y is tyrosine and X is any amino acid (Chang et al., 2007; Smith et al., 2007; Del Mare et al., 2009; Chang et al., 2010). Among these WWOX/WOX1-binding protein targets are p73, activator protein 2 (AP-2, ErbB4, Ezrin, small integral membrane protein of the lysosome/late endosome (SIMPLE), c-Jun, and runt-related transcription factor 2 (RUNX2) (Chang et al., 2007; Del Mare et al., 2009; Chang et al., 2010). While most of the observations were from ectopic expression to enhance the binding, physiological consequences of the binding interactions are largely unknown.

Fig. 1. WWOX and simulated tertiary structures. The predicted amino acid sequence of WW domain-containing oxidoreductase, designated WWOX, FOR, or WOX1, possesses two *N*terminal WW domains, a nuclear localization signal sequence (NLS), and a *C*-terminal shortchain alcohol dehydrogenase/reductase (SDR) domain, where Tyr33 and Tyr287 are the hosphorylation sites, and NSYK is the binding motif for sex steroid hormones. Simulated structures of the first WW domain and the SDR domain are shown (1= 1st tryptophan; 2= Try33 phosphorylation site; 3= 2nd tryptophan; see yellow). Also, NSYK residues are marked in yellow.

WW Domain-Containing Oxidoreductase is a Potential Receptor for Sex Steroid Hormones 339

Tyr33 phosphorylation and accumulation in the mitochondria and nuclei (Chen et al., 2005; Lo et al., 2008; Li et al., 2009). Tyr287 in WWOX/WOX1 can undergo phosphorylation by activated tyrosine kinase 1 (Ack1) for polyubiquitination and protein degradation in

Cumulative reports have shown deletion or epigenetic alteration of human *WWOX* gene induces loss of protein expression in malignant cancer (Del Mare et al., 2009; Chang et al., 2007, 2010). For example, as demonstrated in most recent reports, both tumor suppressor genes *FHIT* and *WWOX* are deleted in primary effusion lymphoma (PEL) cell lines (Roy et al., 2011). Loss of WWOX occurs during the progression and development of gastric cancer (Maeda et al., 2010). *Helicobacter pylori / H. pylori* infection induces methylation of *WWOX* gene in human gastric cancer, suggestive of the role of epigenetic modification by H. pylori in causing cancer (Yan et al., 2011). Interestingly, polymorphism Pro-282-Ala in *WWOX* gene may have a risk factor for differentiated thyroid carcinoma (Cancemi et al., 2011). Also, hypermethylation of *WWOX* gene promoter region and mutations in the gene, encoding the SDR domain, appears to contribute to lung carcinogenesis (Baykara et al., 2010). Overall, it is not surprising to observe complete loss of *WWOX* gene and protein in invasive or metastatic

In most cases, cancer specimens from patients cannot represent the very early stages of cancer development. In this regard, our knowledge concerning how and when *WWOX* gene is altered is still lacking. We have examined the time-related *Wwox* gene alteration in hairless mice during the initiation and progression of cutaneous squamous cell carcinoma (SCC) (Lai et al., 2005). During the acute phase of UVB exposure in hairless mice, WOX1 protein was significantly upregulated and became activated in epidermal cells in 24 hours. After the inflammatory phase, the mice developed cutaneous SCC in 3 months, with significant reduction of WOX1 protein and its Tyr33 phosphorylation, but without downregulation of *Wwox* mRNA. In normal human and mouse skin, keratinocyte differentiation involves upregulation of human WWOX/WOX1, isoform WOX2, and Tyr33 phosphorylation prior to cornification and death (Lai et al., 2005). However, there are significant reductions in WOX1 and WOX2 proteins and their Tyr33 phosphorylation in non-metastatic and metastatic cutaneous SCC, but without down-regulation of *WWOX* mRNA. These observations suggest an additional mechanism for the inactivation of *WWOX*

By immunohistochemistry, it was reported that WWOX protein levels are not decreased but rather elevated in gastric and breast carcinoma (Watanabe et al., 2003), challenging the notion of WWOX as a classical tumor suppressor. Nonetheless, the stages of cancer cells are unknown. We have examined the hyperplasia stage of prostate cancer development and shown the increased expression levels of WWOX/WOX1 protein and isoforms (Chang et al.,

Normal cells of the epithelial origin express WWOX/WOX1. These cells include skin keratinocytes and sebaceous gland cells, lung epithelial cells, epithelial cells of the digestive system, Leydig cells, follicular cells, prostate epithelial cells, and mammary gland cells.

mRNA and a translational blockade of *WWOX* mRNA to protein.

**3.4 WWOX/WOX1 localization and signaling** 

prostate cancer cells (Mahajan et al., 2005).

cancer cells.

2005b)

**3.3 Alteration of human** *WWOX* **gene in cancer** 

When WWOX/WOX1 becomes activated by stress stimuli such as UV light and tumor necrosis factor, Tyr33 is phosphorylated in the first WW domain (Chang, 2002; Chang et al., 2003a, 2003b, 2005a, 2007; Lai et al., 2005; Lo et al., 2008). Tyrosine kinase Src is known to phosphorylate Tyr33 in WWOX/WOX1 (Aqeilan et al., 2004a). The activated WWOX/WOX1 interacts with a large spectrum of proteins without possessing a PPXY motif(s), including proteins in the stress signaling and apoptotic response, as well as transcription factors (Chang et al., 2007, 2010; Del Mare et al., 2009). These proteins are p53 (Lo et al., 2008; Chang et al., 2001, 2003a, 2005a, 2005b; Lai et al., 2005), JNK1 (Lo et al., 2008; Chang et al., 2003a), MDM2 (Chang et al., 2005a), Zfra (Hong et al., 2007; Hsu et al., 2008), and Hyal-2 (Hsu et al., 2009).

The *C*-terminal SDR domain in WOX1/WWOX has been shown to bind Tau, a microtubulebinding protein involved in neurodegeneration (Sze et al., 2004). Functional consequence of this binding is also unknown. It is postulated that WOX1/WWOX binds Tau to prevent hyperphosphorylation by enzymes such as ERK, Cdk5, GSK-3 and JNK, thereby preventing tau aggregation as found in the hippocampi of patients with Alzheimer's disease (Sze et al., 2004). WOX1/WWOX physically interacts with MEK1 in T leukemia cells, and PMA (phorbol myristate acetate) modulates the binding interactions (Lin et al., 2011). PMAinduced dissociation of the WOX1/MEK1 interactions leads to apoptosis of Jurkat T cells, suggesting there is a critical switch in cell death for T cell leukemia upon the dissociation of WOX1/MEK1 (Lin et al., 2011). MEK1 has been shown to bind to both the WW and SDR domain of WOX1 with differential affinities. How this differential binding strength affects cell growth and death and correlates with biological activities is unknown and remains to be established.

#### **3.2 WWOX/WOX1 activation and its role in multiple signaling networks**  *in vitro* **and** *in vivo*

WWOX/WOX1 interacts with many proteins in the stress signaling, growth, gene transcription, and apoptosis regulations, suggesting it is involved in multiple signal networks. For example, WWOX/WOX1 controls the activation of transcription factors, including p53 (Lo et al., 2008; Chang et al., 2001, 2003a, 2003b, 2005a, 2005b; Lai et al., 2005), p73 (Aqeilan et al., 2004a), AP2 (Aqeilan et al., 2004b), c-Jun (Gaudio et al., 2006; Li et al., 2009), and CREB (Li et al., 2009). By immunoelectron microscopy, FRET (Förster resonance energy transfer) and co-immunoprecipitation, we have revealed the complex formation of the Tyr33-phosphorylated or activated WOX1 with p-CREB and p-c-Jun *in vivo* (Li et al., 2009). Interestingly, WOX1 blocks the prosurvival CREB-, CRE-, and AP-1-mediated promoter activation *in vitro*. In contrast, WOX1 enhances promoter activation regulated by c-Jun, Elk-1 and NF-B (Li et al., 2009).

Tyr33-phosphorylated WOX1 is central to the stability and function of tumor suppressor p53. The activated WOX1 binds and stabilizes p53 with Ser46 phosphorylation, which is necessary for the apoptotic function of p53 (Chang et al., 2005a).

Numerous factors are known to induce Tyr33 phosphorylation in WWOX/WOX1, including sex steroid hormones (Chang et al., 2005b), transforming growth factor beta (Hsu et al., 2009), complement C1q (Hong et al., 2009), UV light, and anisomycin (Chang et al., 2001, 2003a, 2005a). Stress stimuli induce relocation of WWOX/WOX1 to the mitochondria and nuclei both *in vitro* and *in vivo*. When neurons are subjected to injury by axotomy, neurotoxin and long-term exposure to constant light in rats, WOX1 becomes activated via Tyr33 phosphorylation and accumulation in the mitochondria and nuclei (Chen et al., 2005; Lo et al., 2008; Li et al., 2009). Tyr287 in WWOX/WOX1 can undergo phosphorylation by activated tyrosine kinase 1 (Ack1) for polyubiquitination and protein degradation in prostate cancer cells (Mahajan et al., 2005).

#### **3.3 Alteration of human** *WWOX* **gene in cancer**

338 Sex Hormones

When WWOX/WOX1 becomes activated by stress stimuli such as UV light and tumor necrosis factor, Tyr33 is phosphorylated in the first WW domain (Chang, 2002; Chang et al., 2003a, 2003b, 2005a, 2007; Lai et al., 2005; Lo et al., 2008). Tyrosine kinase Src is known to phosphorylate Tyr33 in WWOX/WOX1 (Aqeilan et al., 2004a). The activated WWOX/WOX1 interacts with a large spectrum of proteins without possessing a PPXY motif(s), including proteins in the stress signaling and apoptotic response, as well as transcription factors (Chang et al., 2007, 2010; Del Mare et al., 2009). These proteins are p53 (Lo et al., 2008; Chang et al., 2001, 2003a, 2005a, 2005b; Lai et al., 2005), JNK1 (Lo et al., 2008; Chang et al., 2003a), MDM2 (Chang et al., 2005a), Zfra (Hong et al., 2007; Hsu et al., 2008),

The *C*-terminal SDR domain in WOX1/WWOX has been shown to bind Tau, a microtubulebinding protein involved in neurodegeneration (Sze et al., 2004). Functional consequence of this binding is also unknown. It is postulated that WOX1/WWOX binds Tau to prevent hyperphosphorylation by enzymes such as ERK, Cdk5, GSK-3 and JNK, thereby preventing tau aggregation as found in the hippocampi of patients with Alzheimer's disease (Sze et al., 2004). WOX1/WWOX physically interacts with MEK1 in T leukemia cells, and PMA (phorbol myristate acetate) modulates the binding interactions (Lin et al., 2011). PMAinduced dissociation of the WOX1/MEK1 interactions leads to apoptosis of Jurkat T cells, suggesting there is a critical switch in cell death for T cell leukemia upon the dissociation of WOX1/MEK1 (Lin et al., 2011). MEK1 has been shown to bind to both the WW and SDR domain of WOX1 with differential affinities. How this differential binding strength affects cell growth and death and correlates with biological activities is unknown and remains to be

**3.2 WWOX/WOX1 activation and its role in multiple signaling networks** 

necessary for the apoptotic function of p53 (Chang et al., 2005a).

WWOX/WOX1 interacts with many proteins in the stress signaling, growth, gene transcription, and apoptosis regulations, suggesting it is involved in multiple signal networks. For example, WWOX/WOX1 controls the activation of transcription factors, including p53 (Lo et al., 2008; Chang et al., 2001, 2003a, 2003b, 2005a, 2005b; Lai et al., 2005), p73 (Aqeilan et al., 2004a), AP2 (Aqeilan et al., 2004b), c-Jun (Gaudio et al., 2006; Li et al., 2009), and CREB (Li et al., 2009). By immunoelectron microscopy, FRET (Förster resonance energy transfer) and co-immunoprecipitation, we have revealed the complex formation of the Tyr33-phosphorylated or activated WOX1 with p-CREB and p-c-Jun *in vivo* (Li et al., 2009). Interestingly, WOX1 blocks the prosurvival CREB-, CRE-, and AP-1-mediated promoter activation *in vitro*. In contrast, WOX1 enhances promoter activation regulated by

Tyr33-phosphorylated WOX1 is central to the stability and function of tumor suppressor p53. The activated WOX1 binds and stabilizes p53 with Ser46 phosphorylation, which is

Numerous factors are known to induce Tyr33 phosphorylation in WWOX/WOX1, including sex steroid hormones (Chang et al., 2005b), transforming growth factor beta (Hsu et al., 2009), complement C1q (Hong et al., 2009), UV light, and anisomycin (Chang et al., 2001, 2003a, 2005a). Stress stimuli induce relocation of WWOX/WOX1 to the mitochondria and nuclei both *in vitro* and *in vivo*. When neurons are subjected to injury by axotomy, neurotoxin and long-term exposure to constant light in rats, WOX1 becomes activated via

and Hyal-2 (Hsu et al., 2009).

established.

*in vitro* **and** *in vivo*

c-Jun, Elk-1 and NF-B (Li et al., 2009).

Cumulative reports have shown deletion or epigenetic alteration of human *WWOX* gene induces loss of protein expression in malignant cancer (Del Mare et al., 2009; Chang et al., 2007, 2010). For example, as demonstrated in most recent reports, both tumor suppressor genes *FHIT* and *WWOX* are deleted in primary effusion lymphoma (PEL) cell lines (Roy et al., 2011). Loss of WWOX occurs during the progression and development of gastric cancer (Maeda et al., 2010). *Helicobacter pylori / H. pylori* infection induces methylation of *WWOX* gene in human gastric cancer, suggestive of the role of epigenetic modification by H. pylori in causing cancer (Yan et al., 2011). Interestingly, polymorphism Pro-282-Ala in *WWOX* gene may have a risk factor for differentiated thyroid carcinoma (Cancemi et al., 2011). Also, hypermethylation of *WWOX* gene promoter region and mutations in the gene, encoding the SDR domain, appears to contribute to lung carcinogenesis (Baykara et al., 2010). Overall, it is not surprising to observe complete loss of *WWOX* gene and protein in invasive or metastatic cancer cells.

In most cases, cancer specimens from patients cannot represent the very early stages of cancer development. In this regard, our knowledge concerning how and when *WWOX* gene is altered is still lacking. We have examined the time-related *Wwox* gene alteration in hairless mice during the initiation and progression of cutaneous squamous cell carcinoma (SCC) (Lai et al., 2005). During the acute phase of UVB exposure in hairless mice, WOX1 protein was significantly upregulated and became activated in epidermal cells in 24 hours. After the inflammatory phase, the mice developed cutaneous SCC in 3 months, with significant reduction of WOX1 protein and its Tyr33 phosphorylation, but without downregulation of *Wwox* mRNA. In normal human and mouse skin, keratinocyte differentiation involves upregulation of human WWOX/WOX1, isoform WOX2, and Tyr33 phosphorylation prior to cornification and death (Lai et al., 2005). However, there are significant reductions in WOX1 and WOX2 proteins and their Tyr33 phosphorylation in non-metastatic and metastatic cutaneous SCC, but without down-regulation of *WWOX* mRNA. These observations suggest an additional mechanism for the inactivation of *WWOX* mRNA and a translational blockade of *WWOX* mRNA to protein.

By immunohistochemistry, it was reported that WWOX protein levels are not decreased but rather elevated in gastric and breast carcinoma (Watanabe et al., 2003), challenging the notion of WWOX as a classical tumor suppressor. Nonetheless, the stages of cancer cells are unknown. We have examined the hyperplasia stage of prostate cancer development and shown the increased expression levels of WWOX/WOX1 protein and isoforms (Chang et al., 2005b)

#### **3.4 WWOX/WOX1 localization and signaling**

Normal cells of the epithelial origin express WWOX/WOX1. These cells include skin keratinocytes and sebaceous gland cells, lung epithelial cells, epithelial cells of the digestive system, Leydig cells, follicular cells, prostate epithelial cells, and mammary gland cells.

WW Domain-Containing Oxidoreductase is a Potential Receptor for Sex Steroid Hormones 341

Purified serum C1q is able to rapidly induce the activation of WWOX/WOX1 (Hong et al., 2009). Complement C1q induces apoptosis of cancer cells overexpressing WWOX/WOX1, and the induced cell death is independent of the complement classical activation pathway. When WWOX/WOX1 is deficient in cells, C1q fails to cause apoptosis, indicating the presence of a novel pathway of programmed cell death. As determined by time-lapse surface plasmon-enhanced two-photon total internal reflection fluorescence (TIRF) microscopy (He et al., 2009, 2010), C1q induces the formation of clusters of microvilli and destabilizes the adherence in WOX1-overexpressing prostate DU145 cancer cells, without causing exposure of phosphatidylserine (PS) on the outer leaflet of the plasma membrane (Hong et al., 2009). Ultimately, these cells undergo shrinkage, membrane blebbing, and death (Hong et al., 2009). The observations suggest a critical role of WWOX/WOX1 in cell adherence and microvillus formation. Indeed, benign prostatic hyperplasia and prostate cancer have a significantly reduced expression of tissue C1q, compared to age-matched normal prostate tissues (Hong et al., 2009), suggesting that they can grow favorably as long

Low serum HDL-cholesterol (HDL-C) is known to be one of the risk factors for coronary artery disease. Three recent studies demonstrated that *WWOX* gene is associated with the alterations of plasma HDL levels (Lee et al., 2008; Sáez et al., 2010; Leduc et al., 2011). By genotyping of single nucleotide polymorphisms (SNPs), Lee et al. identified one SNP, rs2548861, in the intron 8 of *WWOX* gene with region-wide significance for low HDL-C in dyslipidemic families of Mexican and European descent and in low-HDL-C cases and controls of European descent. They concluded that there is a significant association between HDL-C and a *WWOX* variant with an allele-specific cis-regulatory function. Similar approaches, coupled with mouse genome mapping, were also used to indicate the association of *WWOX* gene with HDL cholesterol and triglyceride levels (Sáez et al., 2010;

Genetic knockout models have revealed the functional properties of WWOX. In a *Drosophila*  model, Wwox is shown to play a key role in aerobic metabolism probably via functional interactions with CG6439/isocitrate dehydrogenase (*Idh*) and Cu-Zn superoxide dismutase (*Sod*) (O'Keefe et al., 2011). Varied Wwox expression also causes altered levels of endogenous reactive oxygen species. A direct interaction between Wwox and the functional

Targeted ablation of mouse *Wwox* gene at exons 2-4 appears to increase the incidence of spontaneous formation of tumors in heterozygous mice (Aqeilan et al., 2007). Importantly, the effect of *Wwox* gene knockout has a significant effect on bone metabolism defects (Aqeilan et al., 2008). The whole body *Wwox* gene-ablated mice can only survive for approximately one month. The molecular mechanism of this regard is not known. In

**3.4.3 Complement protein C1q as an activator of WWOX/WOX1** 

as WWOX/WOX1 is also downregulated.

**3.5 A role of WWOX/WOX1 in metabolism** 

Leduc et al., 2011).

**3.5.1 WWOX/WOX1 is associated with plasma HDL levels** 

**3.5.2 WWOX/WOX1 plays a role in aerobic metabolism** 

interactors has not been demonstrated.

**3.5.3** *Wwox* **gene knockout mice models** 

Many of these cells are responsive to stimulation by sex steroid hormones. During terminal differentiation of kerationcytes, WWOX/WOX1 expression is increased steadily prior to cornification. Whether this also reflects an increased oxidoreductase activity of WWOX/WOX1 in the keratinocytes is unknown. WWOX/WOX1 is accumulated in the nuclei during the terminal differentiation of keratinocytes (Lai et al., 2005). Substantial evidence shows that accumulation of WWOX/WOX1 in the nuclei may induce death of cancer cells in culture (Chang et al., 2007, 2010). Also, during axotomy, WWOX/WOX1, along with CREB, NF-B and many transcription factors, relocates to the nuclei, and this appears to contribute to the eventual death of neurons (Li et al., 2009). Similar observations for the accumulation of WWOX/WOX1 in the nuclei have been shown in animal models using neurotoxin MPP+ and long-term constant light exposure to cause neuronal death (Lo et al., 2008; Chen at al., 2005).

#### **3.4.1 WWOX/Ezrin interactions**

WWOX/WOX1 is known to be associated in part with the cell membrane/cytoskeleton area, and thereby serves as a sensor of environmental cues (Chang et al., 2010). WWOX/WOX1 receives and integrates signals from cell surface by undergoing Tyr33-phosphorylation and relocation to the nuclei *in vitro* and *in vivo* (Chang et al., 2010; review). Nuclear WWOX may either enhance or inhibit the promoter activities regulated by SMAD, NF-B, c-JUN, CREB and other transcription factors (Gaudio et al., 2006; Li et al., 2009; Chang et al., 2010). By immunoelectron microscopy, WWOX/WOX1 can exist alone at the membrane/cytosleleton (Hsu et al., 2009), or it can be in binding with Ezrin (Jin el al., 2006), Hyal-2 (Hsu et al., 2009), or other cytoskeletal proteins (Cheng et al., unpublished). PKA-mediated phosphorylation of ezrin is central to the apical localization of WWOX protein in parietal cells, and that disruption of ezrin-WWOX interaction reduces the apical localization of WWOX (Jin et al., 2006). Ezrin directly binds to the first WW domain of WWOX via its *C*-terminal tyrosinecontaining polyproline sequence (470)PPPPPPVY(477) (Jin et al., 2006).

#### **3.4.2 TGF-/Hyal-2/WWOX/Smad4 signal pathway**

We have recently demonstrated that transforming growth factor beta (TGF- induces relocation of WWOX/WOX1 to the nuclei in response to TGF- in many types of cells, except in certain breast cancer cells (Hsu et al., 2009). Under physiological conditions, TGF binds membrane TRII as a cognate receptor for recruiting TRI, followed by phosphorylating Smad2 and 3, recruiting Smad4, and the Smad2/3/4 complex binding to responsive elements in the nucleus. In TRII-deficient HCT116 cells, we showed that membrane hyaluronidase Hyal-2 acts as a cognate receptor for TGF-1 (Hsu et al., 2009). TGF-1 binds to a surface-exposed segment in the catalytic domain of Hyal-2 in the microvilli, followed by rapidly recruiting WWOX. The WWOX/Hyal-2 complex appears to recruit Smad4 for enhancing SMAD-responsive promoter activation. Hyaluronan is also a ligand for Hyal-2, suggesting that both hyaluronan and TGF-1 may compete for the binding with membrane Hyal-2. Thus, we propose an alternative scenario that hyaluronan enhances the binding of TGF-1 with Hyal-2 without transmitting the signal. Presumably, TGF-1 is trapped on the cell surface by hyaluronan and Hyal-2. Upon hyaluronan degradation, the signal event may start. Two reports showed that hyaluronan blocks TGF signaling by inducing trafficking of TGF-β receptors to lipid raft-associated pools, which facilitates increased receptor turnover (Ito et al., 2004; Webber et al., 2009).

#### **3.4.3 Complement protein C1q as an activator of WWOX/WOX1**

Purified serum C1q is able to rapidly induce the activation of WWOX/WOX1 (Hong et al., 2009). Complement C1q induces apoptosis of cancer cells overexpressing WWOX/WOX1, and the induced cell death is independent of the complement classical activation pathway. When WWOX/WOX1 is deficient in cells, C1q fails to cause apoptosis, indicating the presence of a novel pathway of programmed cell death. As determined by time-lapse surface plasmon-enhanced two-photon total internal reflection fluorescence (TIRF) microscopy (He et al., 2009, 2010), C1q induces the formation of clusters of microvilli and destabilizes the adherence in WOX1-overexpressing prostate DU145 cancer cells, without causing exposure of phosphatidylserine (PS) on the outer leaflet of the plasma membrane (Hong et al., 2009). Ultimately, these cells undergo shrinkage, membrane blebbing, and death (Hong et al., 2009). The observations suggest a critical role of WWOX/WOX1 in cell adherence and microvillus formation. Indeed, benign prostatic hyperplasia and prostate cancer have a significantly reduced expression of tissue C1q, compared to age-matched normal prostate tissues (Hong et al., 2009), suggesting that they can grow favorably as long as WWOX/WOX1 is also downregulated.

#### **3.5 A role of WWOX/WOX1 in metabolism**

340 Sex Hormones

Many of these cells are responsive to stimulation by sex steroid hormones. During terminal differentiation of kerationcytes, WWOX/WOX1 expression is increased steadily prior to cornification. Whether this also reflects an increased oxidoreductase activity of WWOX/WOX1 in the keratinocytes is unknown. WWOX/WOX1 is accumulated in the nuclei during the terminal differentiation of keratinocytes (Lai et al., 2005). Substantial evidence shows that accumulation of WWOX/WOX1 in the nuclei may induce death of cancer cells in culture (Chang et al., 2007, 2010). Also, during axotomy, WWOX/WOX1, along with CREB, NF-B and many transcription factors, relocates to the nuclei, and this appears to contribute to the eventual death of neurons (Li et al., 2009). Similar observations for the accumulation of WWOX/WOX1 in the nuclei have been shown in animal models using neurotoxin MPP+ and long-term constant light exposure to cause neuronal death (Lo

WWOX/WOX1 is known to be associated in part with the cell membrane/cytoskeleton area, and thereby serves as a sensor of environmental cues (Chang et al., 2010). WWOX/WOX1 receives and integrates signals from cell surface by undergoing Tyr33-phosphorylation and relocation to the nuclei *in vitro* and *in vivo* (Chang et al., 2010; review). Nuclear WWOX may either enhance or inhibit the promoter activities regulated by SMAD, NF-B, c-JUN, CREB and other transcription factors (Gaudio et al., 2006; Li et al., 2009; Chang et al., 2010). By immunoelectron microscopy, WWOX/WOX1 can exist alone at the membrane/cytosleleton (Hsu et al., 2009), or it can be in binding with Ezrin (Jin el al., 2006), Hyal-2 (Hsu et al., 2009), or other cytoskeletal proteins (Cheng et al., unpublished). PKA-mediated phosphorylation of ezrin is central to the apical localization of WWOX protein in parietal cells, and that disruption of ezrin-WWOX interaction reduces the apical localization of WWOX (Jin et al., 2006). Ezrin directly binds to the first WW domain of WWOX via its *C*-terminal tyrosine-

We have recently demonstrated that transforming growth factor beta (TGF- induces relocation of WWOX/WOX1 to the nuclei in response to TGF- in many types of cells, except in certain breast cancer cells (Hsu et al., 2009). Under physiological conditions, TGF binds membrane TRII as a cognate receptor for recruiting TRI, followed by phosphorylating Smad2 and 3, recruiting Smad4, and the Smad2/3/4 complex binding to responsive elements in the nucleus. In TRII-deficient HCT116 cells, we showed that membrane hyaluronidase Hyal-2 acts as a cognate receptor for TGF-1 (Hsu et al., 2009). TGF-1 binds to a surface-exposed segment in the catalytic domain of Hyal-2 in the microvilli, followed by rapidly recruiting WWOX. The WWOX/Hyal-2 complex appears to recruit Smad4 for enhancing SMAD-responsive promoter activation. Hyaluronan is also a ligand for Hyal-2, suggesting that both hyaluronan and TGF-1 may compete for the binding with membrane Hyal-2. Thus, we propose an alternative scenario that hyaluronan enhances the binding of TGF-1 with Hyal-2 without transmitting the signal. Presumably, TGF-1 is trapped on the cell surface by hyaluronan and Hyal-2. Upon hyaluronan degradation, the signal event may start. Two reports showed that hyaluronan blocks TGF signaling by inducing trafficking of TGF-β receptors to lipid raft-associated pools, which

containing polyproline sequence (470)PPPPPPVY(477) (Jin et al., 2006).

facilitates increased receptor turnover (Ito et al., 2004; Webber et al., 2009).

**3.4.2 TGF-/Hyal-2/WWOX/Smad4 signal pathway**

et al., 2008; Chen at al., 2005).

**3.4.1 WWOX/Ezrin interactions** 

#### **3.5.1 WWOX/WOX1 is associated with plasma HDL levels**

Low serum HDL-cholesterol (HDL-C) is known to be one of the risk factors for coronary artery disease. Three recent studies demonstrated that *WWOX* gene is associated with the alterations of plasma HDL levels (Lee et al., 2008; Sáez et al., 2010; Leduc et al., 2011). By genotyping of single nucleotide polymorphisms (SNPs), Lee et al. identified one SNP, rs2548861, in the intron 8 of *WWOX* gene with region-wide significance for low HDL-C in dyslipidemic families of Mexican and European descent and in low-HDL-C cases and controls of European descent. They concluded that there is a significant association between HDL-C and a *WWOX* variant with an allele-specific cis-regulatory function. Similar approaches, coupled with mouse genome mapping, were also used to indicate the association of *WWOX* gene with HDL cholesterol and triglyceride levels (Sáez et al., 2010; Leduc et al., 2011).

#### **3.5.2 WWOX/WOX1 plays a role in aerobic metabolism**

Genetic knockout models have revealed the functional properties of WWOX. In a *Drosophila*  model, Wwox is shown to play a key role in aerobic metabolism probably via functional interactions with CG6439/isocitrate dehydrogenase (*Idh*) and Cu-Zn superoxide dismutase (*Sod*) (O'Keefe et al., 2011). Varied Wwox expression also causes altered levels of endogenous reactive oxygen species. A direct interaction between Wwox and the functional interactors has not been demonstrated.

#### **3.5.3** *Wwox* **gene knockout mice models**

Targeted ablation of mouse *Wwox* gene at exons 2-4 appears to increase the incidence of spontaneous formation of tumors in heterozygous mice (Aqeilan et al., 2007). Importantly, the effect of *Wwox* gene knockout has a significant effect on bone metabolism defects (Aqeilan et al., 2008). The whole body *Wwox* gene-ablated mice can only survive for approximately one month. The molecular mechanism of this regard is not known. In

WW Domain-Containing Oxidoreductase is a Potential Receptor for Sex Steroid Hormones 343

(Amplified in Breast Cancer-1) is responsible for E2-mediated apoptosis in breast MCF-7: cells (Hu et al., 2011). Computational analysis revealed that AIB1 integrates signals from Gprotein-coupled receptors, PI3 kinase, Wnt and Notch signal pathways, which affect cell growth and death. Interestingly, it has been hypothesized that ER conformation affects E2-

Fig. 2. 17-estradiol (E2) stimulates phosphorylation of WWOX/WOX1 at Tyr33, and cotranalocation of WOX1 with E2 to the nucleus in COS7 fibroblasts. Stimulation of COS7

phosphorylation (p-WOX1) and nuclear translocation, along with E2. Both p-WOX1 and E2 were stained with specific antibodies. WOX1 undergoes activation in ER-positive MCF-7 cells, whereas E2 is retained in the cytoplasm. Both WOX1 and E2 are retained in the cytoplasm without undergoing nuclear translocation in AR-negative DU145 cells.

fibroblasts with E2 (40 nM) for 1 hr resulted in activation of WOX1 via Tyr33

induced cell death (Maximov et al., 2011).

addition, the knockout mice are also defective in the reproductive system (Ludes-Meyers et al., 2009). Inactivation of *Wwox* gene induces mammary tumorigenesis, and the tumors tend to have loss of estrogen receptor-α (ER) and progesterone receptor (Abdeen et al., 2011).

#### **4. WWOX/WOX1 is a candidate hormone receptor**

How breast cancer cells develop estrogen-independent growth is not known. Hormoneindependent breast cancer cells are normally ER-negative and highly invasive. Prognosis for patients is poor. WWOX/WOX1 possesses an NSYK motif for hormone binding. Depending upon cell lines, estrogen or androgen may induce WWOX/WOX1 phosphorylation at Tyr33 (Chang et al., 2005b). Activated WWOX/WOX1 relocates to the nucleus to induce apoptosis in certain cells. Conceivably, loss of WWOX/WOX1 in invasive breast cancer allows them to grow independently of hormones. TFAP2C plays a critical role in gene regulation in hormone responsive breast cancer. *WWOX* gene is one of the transcriptional targets of TFAP2C (Woodfield et al., 2010), suggesting a role of WWOX in the hormonal response.

#### **4.1 17β-estradiol (E2) induces WWOX/WOX1 activation**

The NSYK motif for binding with estrogen and androgen in WWOX/WOX1 is predicted to be N232, S281, Y293, and K297 (Chang et al., 2003b; review). We have investigated whether androgen and estrogen activate WWOX/WOX1 (Chang et al., 2005b). In COS7 fibroblasts, E2 induces Tyr33 phosphorylation in WWOX/WOX1, and both E2 and WWOX/WOX1 cotranslocate to the nuclei (Chang et al., 2005b) (Figure 2). E2 at M levels induces apoptosis of COS7 cells. It appears that when a sufficient amount of WWOX/WOX1 is accumulated in the nucleus, apoptosis occurs. However, it is not clear whether E2 binds to the NSYK motif. Indeed, E2 stimulates the formation of p53 and WOX1 complex, which is found in the nucleus (Chang et al., 2005b) (Figure 3). In contrast, JNK1 blocks the relocation of p53/WOX1 to the nucleus (Chang et al., 2005b). JNK1 binds and blocks WOX1 and p53 activation *in vivo* (Chang et al., 2003), and that dominant-negative JNK1 spontaneously induces WOX1 nuclear translocation. Whether there is a direct binding interaction between E2 and p53 or JNK1 is unknown.

E2 could not induce accumulation of WWOX/WOX1 in the nuclei of ER-positive breast MCF-7. ER-negative breast MDA-MB-231 and MDA-MB-435S are metastatic and have very low levels of WWOX/WOX1. Reconstitution of WWOX/WOX1 in these cells is expected to restore their sensitivity to estrogen. Interestingly, E2 and androsterone induce WWOX/WOX1 activation in androgen receptor (AR)-negative prostate DU145 cells, indicating that ER and AR are probably not involved in the E2-induced WWOX/WOX1 activation. Taken together, WWOX/WOX1 is a potential receptor for sex steroid hormones (Figure 4). Whether this protein metabolizes estrogen or androgen remains to be determined. Also, whether WWOX/WOX1 possesses an enzymatic activity in oxidation/reduction is still elusive.

#### **4.2 Estrogen-induced apoptosis**

Majority of ER-positive breast cancer cells depend upon estrogen for growth. It appears that these cells may become sensitive to estrogen-mediated apoptosis upon long-term deprivation of estrogen, followed by re-introducing estrogen. Whether WWOX/WOX1 is involved in the conferred sensitivity is not known. A recent study showed that AIB1

addition, the knockout mice are also defective in the reproductive system (Ludes-Meyers et al., 2009). Inactivation of *Wwox* gene induces mammary tumorigenesis, and the tumors tend to have loss of estrogen receptor-α (ER) and progesterone receptor (Abdeen et al., 2011).

How breast cancer cells develop estrogen-independent growth is not known. Hormoneindependent breast cancer cells are normally ER-negative and highly invasive. Prognosis for patients is poor. WWOX/WOX1 possesses an NSYK motif for hormone binding. Depending upon cell lines, estrogen or androgen may induce WWOX/WOX1 phosphorylation at Tyr33 (Chang et al., 2005b). Activated WWOX/WOX1 relocates to the nucleus to induce apoptosis in certain cells. Conceivably, loss of WWOX/WOX1 in invasive breast cancer allows them to grow independently of hormones. TFAP2C plays a critical role in gene regulation in hormone responsive breast cancer. *WWOX* gene is one of the transcriptional targets of TFAP2C (Woodfield et al., 2010), suggesting a role of WWOX in the hormonal response.

The NSYK motif for binding with estrogen and androgen in WWOX/WOX1 is predicted to be N232, S281, Y293, and K297 (Chang et al., 2003b; review). We have investigated whether androgen and estrogen activate WWOX/WOX1 (Chang et al., 2005b). In COS7 fibroblasts, E2 induces Tyr33 phosphorylation in WWOX/WOX1, and both E2 and WWOX/WOX1 cotranslocate to the nuclei (Chang et al., 2005b) (Figure 2). E2 at M levels induces apoptosis of COS7 cells. It appears that when a sufficient amount of WWOX/WOX1 is accumulated in the nucleus, apoptosis occurs. However, it is not clear whether E2 binds to the NSYK motif. Indeed, E2 stimulates the formation of p53 and WOX1 complex, which is found in the nucleus (Chang et al., 2005b) (Figure 3). In contrast, JNK1 blocks the relocation of p53/WOX1 to the nucleus (Chang et al., 2005b). JNK1 binds and blocks WOX1 and p53 activation *in vivo* (Chang et al., 2003), and that dominant-negative JNK1 spontaneously induces WOX1 nuclear translocation. Whether there is a direct binding interaction between

E2 could not induce accumulation of WWOX/WOX1 in the nuclei of ER-positive breast MCF-7. ER-negative breast MDA-MB-231 and MDA-MB-435S are metastatic and have very low levels of WWOX/WOX1. Reconstitution of WWOX/WOX1 in these cells is expected to restore their sensitivity to estrogen. Interestingly, E2 and androsterone induce WWOX/WOX1 activation in androgen receptor (AR)-negative prostate DU145 cells, indicating that ER and AR are probably not involved in the E2-induced WWOX/WOX1 activation. Taken together, WWOX/WOX1 is a potential receptor for sex steroid hormones (Figure 4). Whether this protein metabolizes estrogen or androgen remains to be determined. Also, whether WWOX/WOX1 possesses an enzymatic activity in

Majority of ER-positive breast cancer cells depend upon estrogen for growth. It appears that these cells may become sensitive to estrogen-mediated apoptosis upon long-term deprivation of estrogen, followed by re-introducing estrogen. Whether WWOX/WOX1 is involved in the conferred sensitivity is not known. A recent study showed that AIB1

**4. WWOX/WOX1 is a candidate hormone receptor** 

**4.1 17β-estradiol (E2) induces WWOX/WOX1 activation** 

E2 and p53 or JNK1 is unknown.

oxidation/reduction is still elusive.

**4.2 Estrogen-induced apoptosis** 

(Amplified in Breast Cancer-1) is responsible for E2-mediated apoptosis in breast MCF-7: cells (Hu et al., 2011). Computational analysis revealed that AIB1 integrates signals from Gprotein-coupled receptors, PI3 kinase, Wnt and Notch signal pathways, which affect cell growth and death. Interestingly, it has been hypothesized that ER conformation affects E2 induced cell death (Maximov et al., 2011).

Fig. 2. 17-estradiol (E2) stimulates phosphorylation of WWOX/WOX1 at Tyr33, and cotranalocation of WOX1 with E2 to the nucleus in COS7 fibroblasts. Stimulation of COS7 fibroblasts with E2 (40 nM) for 1 hr resulted in activation of WOX1 via Tyr33 phosphorylation (p-WOX1) and nuclear translocation, along with E2. Both p-WOX1 and E2 were stained with specific antibodies. WOX1 undergoes activation in ER-positive MCF-7 cells, whereas E2 is retained in the cytoplasm. Both WOX1 and E2 are retained in the cytoplasm without undergoing nuclear translocation in AR-negative DU145 cells.

WW Domain-Containing Oxidoreductase is a Potential Receptor for Sex Steroid Hormones 345

Fig. 4. Schematic illustration of E2/WWOX signaling. (**A**) In ER-positive cells, E2 binds ER and other proteins, and the complex translocates the nucleus to control gene transcription by binding to estrogen responsive elements (EREs) in chromosomal DNA. Alternatively, E2 may co-translocate with WOX1 to the nuclei. (**B**) In ER-negative, metastatic breast cancer cells, the wild type WWOX or WOX1 is deficient, whereas isoforms WOX2 and WOX8 may be present. These proteins provide the NSYK motif for binding with estrogen or androgen

Research was supported, in part, by the Department of Defense, USA (W81XWH-08-1-0682), the National Science Council, Taiwan, ROC (NSC96-2320-B-006-014, 98-2628-B-006-041-MY3, and 98-2628-B-006-045-MY3), the National Health Research Institute, Taiwan, ROC (NHRI-EX99-9705BI), and the National Cheng Kung University Landmark Projects (C0167 & R026)

Abdeen S.K.; Salah Z.; Maly B.; Smith Y.; Tufail R.; Abu-Odeh M.; Zanesi N.; Croce C.M.;

tumorigenesis. *Oncogene*, (Apr 2011), ISSN

Nawaz Z. & Aqeilan R.I. (2011). Wwox inactivation enhances mammary

for relocating to the nucleus.

**5. Acknowledgement** 

(to NS Chang).

**6. References** 

Fig. 3. E2 induces co-translocation of p53 and WWOX/WOX1 to the nuclei of COS7. *In vitro* experiments support the likely scenario that E2 induces the complex formation of Tyr33 phosphorylated WOX1 and Ser15-phosphorylated p53, and the complex relocates to the nuclei (Chang et al., 2005b). JNK1 is also associated with the p53/WOX1 in the cytosol, but fails to undergo nuclear relocation. JNK1 blocks the nuclear accumulation of p53/WOX1.

#### **4.3 Hormone-independence in breast cancer and perspectives**

Development of hormone-independence in breast cancer patients involves a complicated event that underlies a network structure rather than individual molecular components. It is critical to probe the "disease systems" from a gene regulatory network to a cell, a tissue, or even an entire organism. Areas of this regard in terms of development independence in breast cancer are largely unknown. Invasive breast cancer cells exhibit a high frequency of loss of heterozygosity of *WWOX* gene. Wild type WWOX/WOX1 is responsive to estrogeninduced activation, via Tyr33 phosphorylation and nuclear translocation, for controlling cell growth. Thus, loss of *WWOX* gene in invasive breast cancer cells is likely to result in hormone resistance.

Fig. 4. Schematic illustration of E2/WWOX signaling. (**A**) In ER-positive cells, E2 binds ER and other proteins, and the complex translocates the nucleus to control gene transcription by binding to estrogen responsive elements (EREs) in chromosomal DNA. Alternatively, E2 may co-translocate with WOX1 to the nuclei. (**B**) In ER-negative, metastatic breast cancer cells, the wild type WWOX or WOX1 is deficient, whereas isoforms WOX2 and WOX8 may be present. These proteins provide the NSYK motif for binding with estrogen or androgen for relocating to the nucleus.

#### **5. Acknowledgement**

344 Sex Hormones

Fig. 3. E2 induces co-translocation of p53 and WWOX/WOX1 to the nuclei of COS7. *In vitro* experiments support the likely scenario that E2 induces the complex formation of Tyr33 phosphorylated WOX1 and Ser15-phosphorylated p53, and the complex relocates to the nuclei (Chang et al., 2005b). JNK1 is also associated with the p53/WOX1 in the cytosol, but fails to undergo nuclear relocation. JNK1 blocks the nuclear accumulation of p53/WOX1.

Development of hormone-independence in breast cancer patients involves a complicated event that underlies a network structure rather than individual molecular components. It is critical to probe the "disease systems" from a gene regulatory network to a cell, a tissue, or even an entire organism. Areas of this regard in terms of development independence in breast cancer are largely unknown. Invasive breast cancer cells exhibit a high frequency of loss of heterozygosity of *WWOX* gene. Wild type WWOX/WOX1 is responsive to estrogeninduced activation, via Tyr33 phosphorylation and nuclear translocation, for controlling cell growth. Thus, loss of *WWOX* gene in invasive breast cancer cells is likely to result in

**4.3 Hormone-independence in breast cancer and perspectives** 

hormone resistance.

Research was supported, in part, by the Department of Defense, USA (W81XWH-08-1-0682), the National Science Council, Taiwan, ROC (NSC96-2320-B-006-014, 98-2628-B-006-041-MY3, and 98-2628-B-006-045-MY3), the National Health Research Institute, Taiwan, ROC (NHRI-EX99-9705BI), and the National Cheng Kung University Landmark Projects (C0167 & R026) (to NS Chang).

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**17** 

*Italy* 

**Sex Steroids in Insects and the** 

**A New Perspective** 

*1DIVAPRA - University of Turin* 

Ilaria Negri1 and Marco Pellecchia2

*2Koiné - Consulenze Ambientali S.n.c. Parma* 

**Role of the Endosymbiont** *Wolbachia***:** 

Sex steroids play a pivotal role in sex differentiation and sex reversal in several species of vertebrates, both with genotypic and environmental sex determination systems (Nakamura, 2010; Norris & Carr, 2006). Steroidal sex hormones can be found naturally in both sexes of vertebrates, although the proportions of hormones may differ between males and females. Feminization of males or masculinization of females can be induced by altering the levels of 'female' and 'male' hormones, respectively. Estrogens for example have a feminizing effect on gonadal differentiation in many species of fish, amphibians, reptiles, and birds (Guiguen et al., 2010; Nakamura, 2009, 2010). In humans, androgen receptor defect disorder may lead to gonadal feminization and, in its complete form, the syndrome causes sex reversal of

Vertebrate-like sex steroids occur in several groups of invertebrates including nematodes, arthropods, echinoderms, but full information on the precise action and function of sex steroids is still missing (Janer & Porte, 2007). Some intriguing data have been provided in mollusks, where an involvement of steroids in gender determination and sexual differentiation of the brain, and even in a "superfeminization syndrome", has been

In insects the existence of sex hormones is under debate. Indeed sex differentiation is generally thought to be a strictly genetic process, in which each cell decides its own sexual fate autonomously, based on its sex chromosome constitution. Therefore, differentiation of primary and secondary sexual characteristics should be exclusively under the control of the genotype of each single cell (Schütt & Nöthiger, 2000; Steinmann-Zwicky et al., 1989). This hypothesis was born studying insect gynandromorphs, i.e. aberrant specimens with an intermediate feature between female and male (according to the Greek roots gyne = female, aner = male, morphe = form; Fig. 1). In the fruit fly *Drosophila melanogaster*, gynandromorphs may arise when one embryonic nucleus loses an X chromosome and the insects possess a mixture of XX (i.e. female) and X0 (i.e. male) tissues. According to Gilbert (2000), because there are no sex hormones in insects to modulate such events, each cell makes its own sexual

genotypical (XY) males and a female phenotype (Oakes et al., 2008).

demonstrated (Oehlmann et al., 2006; Wang & Croll, 2004).

**1. Introduction** 

"decision".


### **Sex Steroids in Insects and the Role of the Endosymbiont** *Wolbachia***: A New Perspective**

Ilaria Negri1 and Marco Pellecchia2 *1DIVAPRA - University of Turin 2Koiné - Consulenze Ambientali S.n.c. Parma Italy* 

#### **1. Introduction**

352 Sex Hormones

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female hormones on lung function in chronic lung diseases. *B.M.C. Womens Health*,

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Aburatani H. (2003) An opposing view on WWOX protein function as a tumor

TGFbeta1-dependent myofibroblast differentiation by hyaluronan. *Am J Pathol*,

primary gene targets of TFAP2C in hormone responsive breast carcinoma cells.

using stable isotope dimethyl labeling coupled with IMAC-HILIC-nanoLC-MS/MS for estrogen-induced transcriptional regulation. *J. Proteome Res,* Vol.10, No.3, (Mar

methylation of WWOX gene in human gastric cancer. Biochem Biophys Res

methylation of WWOX gene in human gastric cancer. Biochem Biophys Res

in prostate and prostate cancer, in: Basic Mechanisms and Therapeutic Approaches.

the glucuronidation of estradiol and estrone by UDPglucuronosyltransferase in liver microsomes from male and female rats. *Biochem. Pharmacol.*, Vol.51, No.9, Sex steroids play a pivotal role in sex differentiation and sex reversal in several species of vertebrates, both with genotypic and environmental sex determination systems (Nakamura, 2010; Norris & Carr, 2006). Steroidal sex hormones can be found naturally in both sexes of vertebrates, although the proportions of hormones may differ between males and females. Feminization of males or masculinization of females can be induced by altering the levels of 'female' and 'male' hormones, respectively. Estrogens for example have a feminizing effect on gonadal differentiation in many species of fish, amphibians, reptiles, and birds (Guiguen et al., 2010; Nakamura, 2009, 2010). In humans, androgen receptor defect disorder may lead to gonadal feminization and, in its complete form, the syndrome causes sex reversal of genotypical (XY) males and a female phenotype (Oakes et al., 2008).

Vertebrate-like sex steroids occur in several groups of invertebrates including nematodes, arthropods, echinoderms, but full information on the precise action and function of sex steroids is still missing (Janer & Porte, 2007). Some intriguing data have been provided in mollusks, where an involvement of steroids in gender determination and sexual differentiation of the brain, and even in a "superfeminization syndrome", has been demonstrated (Oehlmann et al., 2006; Wang & Croll, 2004).

In insects the existence of sex hormones is under debate. Indeed sex differentiation is generally thought to be a strictly genetic process, in which each cell decides its own sexual fate autonomously, based on its sex chromosome constitution. Therefore, differentiation of primary and secondary sexual characteristics should be exclusively under the control of the genotype of each single cell (Schütt & Nöthiger, 2000; Steinmann-Zwicky et al., 1989). This hypothesis was born studying insect gynandromorphs, i.e. aberrant specimens with an intermediate feature between female and male (according to the Greek roots gyne = female, aner = male, morphe = form; Fig. 1). In the fruit fly *Drosophila melanogaster*, gynandromorphs may arise when one embryonic nucleus loses an X chromosome and the insects possess a mixture of XX (i.e. female) and X0 (i.e. male) tissues. According to Gilbert (2000), because there are no sex hormones in insects to modulate such events, each cell makes its own sexual "decision".

Sex Steroids in Insects and the Role of the Endosymbiont *Wolbachia*: A New Perspective 355

Insect moulting is induced by the steroid hormone 20-hydroxyecdysone (20E). Ecdysone pulses in the insects' hemolymph trigger moulting, and the presence or absence of juvenile hormone determines whether moults will lead to another larval stage or, through metamorphosis, to a pupa and an adult form (Gilbert at al., 2002). The 20E precursor is secreted by the prothoracic glands after their stimulation by the brain prothoracicotropic hormone (PTTH) whose release is governed by both intrinsic factors, like the body size, and

Dietary cholesterol is then converted to 20E thanks to many hydroxylation reactions catalysed by cytochrome P450 enzymes of microsomal and/or mitochondrial origin, the final step being characterised by the action of a P450 monooxygenase that hydroxylates the

Cytochrome P450s are encoded by the Halloween genes family, first characterised in *D. melanogaster* and then described in lots of insect species (Christiaens et al., 2010; Rewitz et al.,

Once 20E is biosynthesized, it binds the heterodimeric nuclear receptor EcR/USP composed of EcR (Ecdysone Receptor) and USP (Ultraspiracle, homologous to the vertebrate retinoid-X receptor), which shares many commonalities with the human thyroid hormone receptor. Then, the EcR/USP complex activates the transcriptional processes underlying the cellular and morphogenetic moulting cascade events (Gilbert et al., 2002). In *D. melanogaster*, pulses of 20E throughout fly development have proved to regulate cell proliferation, differentiation, and programmed cell death in a highly controlled manner. During metamorphosis, for example, ecdysone is a primary regulator of apoptosis in larval tissues such as salivary glands, midgut and neural tissues which are destroyed or remodelled into an "adult" form (Mottier et al., 2004; Tsuzuki et al., 2001). The activation and execution of ecdysis (i.e. shedding of the old cuticle during embryonic and larval development) are controlled by a series of peptide hormones produced by Inka cells and neuropeptides within the central nervous system, whose expression is again under ecdysteroid control (Zitnan et

In adults the role played by ecdysteroids is much less explored: for example, it has been demonstrated that they control several important aspects of reproduction, including ovarian development and oogenesis (Carney & Bender, 2001; Raikhel et al., 2005; Riddifort, 1993; Swevers & Iatrou, 2003). In many insect species 20E is also directly involved in the regulation of vitellogenin biosynthesis by the female fat body, a metabolic tissue functionally analogous to the vertebrate liver, and it can also induce vitellogenin synthesis in males (Bownes et al., 1983; Bownes et al., 1996; Huybrechts & De Loof, 1977; Zhu et al., 2007). The 20E has also been shown to affect sexual behaviour, having a role in courtship

initiation by males, and promoting male–male sexual attraction (Ganter et al., 2007).

De Loof (2006, 2008) suggests that ecdysteroids already served as sex hormones long before they acquired a function in moulting. In particular, 20E secreted by the follicle cells of the insect ovary could be the physiological equivalent of vertebrate estrogens, while E - the precursor of the active moulting hormone 20E - should act as a distinct hormone, being the physiological equivalent of the vertebrate testosterone (De Loof & Huybrechts, 1998; De Loof, 2006). Indeed, by using *Drosophila* larval organ culture Beckstead and colleagues (2007) demonstrate that E can regulate a set of genes that are distinct from those controlled by 20E, thus confirming that it may exert different biological (=hormonal) functions from 20E.

extrinsic factors, like photoperiod and temperature (Gilbert at al., 2002).

ecdysone s.s. (E) at carbon 20.

2007).

al., 2007).

Fig. 1. Gynandromorphs, i.e. aberrant specimens made up of both female and male cells, are common in insects. On the left: *Polyommatus bellargus* (Lepidoptera, Lycaenidae) mosaic gynandromorph in which male (blue) and female (brown) features are mixed (Courtesy of R. Villa, Bologna, Italy). On the right: *D. melanogaster* bilateral gynandromorph in which one side is female and the other male (Modified from Griffiths et al., (2000). *An introduction to genetic analysis*, 7th edition, New York: W. H. Freeman).

However recent data demonstrate that in insects as in vertebrates, non-autonomous (= hormonal) sex determination controls sex dimorphism (DeFalco et al., 2008). In the germ line of the *D. melanogaster* embryo there is evidence for both autonomous and nonautonomous regulation of sexual identity, but non-autonomous signals from the soma are dominant, and germ cells establish their sexual identity as they contact the somatic gonad (Casper & Van Doren, 2009; DeFalco et al., 2008). In fact, XX (i.e. female) and XY or X0 (i.e. male) germ cells are not irrevocably committed to female or male identity, respectively (Waterbury et al., 2000).

According to these results, the presence of signals coordinating the development of a gender-specific phenotype (i.e. sex hormones) is conceivable. In his fine review, De Loof (2006) suggests that the loss of an X chromosome in *Drosophila* embryonic cells possibly makes the mutant cells react differently to a given hormonal environment and/or signals from their neighbours than XX cells.

Finally, it is noteworthy to note that, in addition to gynandromorphs, intersexes specimens do exist in insects. As previously discussed, gynandromorphism is the simultaneous presence within the same organism of genotypically and phenotypically male and female tissues rather than of masculinized or feminized tissues, as is the case with intersexes. Indeed intersexes are characterised by phenotypically male and female regions, but genetically homogeneous pattern (Laugé, 1985; White, 1973). In 1934, for example, Whiting and colleagues described individuals of the hymenopteran *Habrobracon juglandis* which were found to be genetically male but with feminized genitalia.

How can the existence of intersexes be explained, if each cell makes its own sexual decision?

#### **2. Ecdysteroids: A role as sex hormones in insects?**

De Loof (2006) proposes that ecdysteroids are the best candidates for a role as sex steroids in insects since, for example, they are involved in the appearance of sex dimorphic structures; are produced by the gonads; and induce different gender-specific physiological effects. Indeed the role of ecdysteroids is not restricted to moulting but they have a much wider effect on the insect biology, both at the larval and adult stages.

Fig. 1. Gynandromorphs, i.e. aberrant specimens made up of both female and male cells, are common in insects. On the left: *Polyommatus bellargus* (Lepidoptera, Lycaenidae) mosaic gynandromorph in which male (blue) and female (brown) features are mixed (Courtesy of R. Villa, Bologna, Italy). On the right: *D. melanogaster* bilateral gynandromorph in which one side is female and the other male (Modified from Griffiths et al., (2000). *An introduction to* 

However recent data demonstrate that in insects as in vertebrates, non-autonomous (= hormonal) sex determination controls sex dimorphism (DeFalco et al., 2008). In the germ line of the *D. melanogaster* embryo there is evidence for both autonomous and nonautonomous regulation of sexual identity, but non-autonomous signals from the soma are dominant, and germ cells establish their sexual identity as they contact the somatic gonad (Casper & Van Doren, 2009; DeFalco et al., 2008). In fact, XX (i.e. female) and XY or X0 (i.e. male) germ cells are not irrevocably committed to female or male identity, respectively

According to these results, the presence of signals coordinating the development of a gender-specific phenotype (i.e. sex hormones) is conceivable. In his fine review, De Loof (2006) suggests that the loss of an X chromosome in *Drosophila* embryonic cells possibly makes the mutant cells react differently to a given hormonal environment and/or signals

Finally, it is noteworthy to note that, in addition to gynandromorphs, intersexes specimens do exist in insects. As previously discussed, gynandromorphism is the simultaneous presence within the same organism of genotypically and phenotypically male and female tissues rather than of masculinized or feminized tissues, as is the case with intersexes. Indeed intersexes are characterised by phenotypically male and female regions, but genetically homogeneous pattern (Laugé, 1985; White, 1973). In 1934, for example, Whiting and colleagues described individuals of the hymenopteran *Habrobracon juglandis* which were

How can the existence of intersexes be explained, if each cell makes its own sexual decision?

De Loof (2006) proposes that ecdysteroids are the best candidates for a role as sex steroids in insects since, for example, they are involved in the appearance of sex dimorphic structures; are produced by the gonads; and induce different gender-specific physiological effects. Indeed the role of ecdysteroids is not restricted to moulting but they have a much wider

*genetic analysis*, 7th edition, New York: W. H. Freeman).

found to be genetically male but with feminized genitalia.

**2. Ecdysteroids: A role as sex hormones in insects?** 

effect on the insect biology, both at the larval and adult stages.

(Waterbury et al., 2000).

from their neighbours than XX cells.

Insect moulting is induced by the steroid hormone 20-hydroxyecdysone (20E). Ecdysone pulses in the insects' hemolymph trigger moulting, and the presence or absence of juvenile hormone determines whether moults will lead to another larval stage or, through metamorphosis, to a pupa and an adult form (Gilbert at al., 2002). The 20E precursor is secreted by the prothoracic glands after their stimulation by the brain prothoracicotropic hormone (PTTH) whose release is governed by both intrinsic factors, like the body size, and extrinsic factors, like photoperiod and temperature (Gilbert at al., 2002).

Dietary cholesterol is then converted to 20E thanks to many hydroxylation reactions catalysed by cytochrome P450 enzymes of microsomal and/or mitochondrial origin, the final step being characterised by the action of a P450 monooxygenase that hydroxylates the ecdysone s.s. (E) at carbon 20.

Cytochrome P450s are encoded by the Halloween genes family, first characterised in *D. melanogaster* and then described in lots of insect species (Christiaens et al., 2010; Rewitz et al., 2007).

Once 20E is biosynthesized, it binds the heterodimeric nuclear receptor EcR/USP composed of EcR (Ecdysone Receptor) and USP (Ultraspiracle, homologous to the vertebrate retinoid-X receptor), which shares many commonalities with the human thyroid hormone receptor. Then, the EcR/USP complex activates the transcriptional processes underlying the cellular and morphogenetic moulting cascade events (Gilbert et al., 2002). In *D. melanogaster*, pulses of 20E throughout fly development have proved to regulate cell proliferation, differentiation, and programmed cell death in a highly controlled manner. During metamorphosis, for example, ecdysone is a primary regulator of apoptosis in larval tissues such as salivary glands, midgut and neural tissues which are destroyed or remodelled into an "adult" form (Mottier et al., 2004; Tsuzuki et al., 2001). The activation and execution of ecdysis (i.e. shedding of the old cuticle during embryonic and larval development) are controlled by a series of peptide hormones produced by Inka cells and neuropeptides within the central nervous system, whose expression is again under ecdysteroid control (Zitnan et al., 2007).

In adults the role played by ecdysteroids is much less explored: for example, it has been demonstrated that they control several important aspects of reproduction, including ovarian development and oogenesis (Carney & Bender, 2001; Raikhel et al., 2005; Riddifort, 1993; Swevers & Iatrou, 2003). In many insect species 20E is also directly involved in the regulation of vitellogenin biosynthesis by the female fat body, a metabolic tissue functionally analogous to the vertebrate liver, and it can also induce vitellogenin synthesis in males (Bownes et al., 1983; Bownes et al., 1996; Huybrechts & De Loof, 1977; Zhu et al., 2007). The 20E has also been shown to affect sexual behaviour, having a role in courtship initiation by males, and promoting male–male sexual attraction (Ganter et al., 2007).

De Loof (2006, 2008) suggests that ecdysteroids already served as sex hormones long before they acquired a function in moulting. In particular, 20E secreted by the follicle cells of the insect ovary could be the physiological equivalent of vertebrate estrogens, while E - the precursor of the active moulting hormone 20E - should act as a distinct hormone, being the physiological equivalent of the vertebrate testosterone (De Loof & Huybrechts, 1998; De Loof, 2006). Indeed, by using *Drosophila* larval organ culture Beckstead and colleagues (2007) demonstrate that E can regulate a set of genes that are distinct from those controlled by 20E, thus confirming that it may exert different biological (=hormonal) functions from 20E.

Sex Steroids in Insects and the Role of the Endosymbiont *Wolbachia*: A New Perspective 357

Such phenotypic variability is thought to be linked to high genome plasticity of insect-borne *Wolbachia*, since all the sequenced genomes of the symbiont contain high number of repetitive sequences, including IS (insertion sequences) elements and prophage-like

According to us, except for cytoplasmic incompatibility that is a secondary effect of the infection, the phenotypic effects observed in arthropods might not be so different, but

Indeed, male killing could be just an unsuccessful "attempt" at feminization by *Wolbachia*. Male-killing is known in several insect species, where males die during embryogenesis or development. Insight into the mechanism of male killing comes from the moths *Ostrinia scapulalis* and *O. furnacalis*, where *Wolbachia* kills genetic males during the larval development. Intriguingly, a partial *Wolbachia* curing leads to the appearance of lepidopteran intersexes having exclusively male genotype (Kageyama & Traut, 2003). Accordingly, a partial feminization of genetic males does occur, while a complete feminization is incompatible with the survival of the male genotype (Kageyama & Traut,

Regarding the parthenogenesis induction by *Wolbachia*, this phenomenon has been demonstrated in several haplodiploid species of mites, hymenopterans and thrips, where males naturally develop (parthenogenetically) from unfertilized haploid eggs and females from fertilized diploid eggs (Arakaki et al., 2001; Stouthamer et al., 1990; Weeks & Breeuwer, 2001). In *Wolbachia*-infected species, unfertilized eggs are subjected to a "diploidy" restoration, giving origin to (infected) females. Recently, Giorgini and colleagues (2009) observed that in the (haplodiploid) wasp *Encarsia hispida*, the symbiont *Cardinium* (which belongs to the only bacterial group known to cause similar reproductive manipulations of *Wolbachia*) doesn't induce, as expected, thelytokous parthenogenesis but feminization. In fact antibiotic treatment results in uninfected diploid male offspring, thus demonstrating that diploidy restoration is a necessary condition, but not sufficient, to elicit female development. Therefore, *Cardinium* is responsible for the feminization of the hymenopteran

Since in studies concerning the parthenogenesis induction by *Wolbachia* no cytogenetic analyses have been performed on males produced by cured females, the hypothesis that the symbiont actually induces feminization rather than parthenogenesis may be conceivable. As will be discussed later, feminization deals with sex determination and differentiation much more directly than the other *Wolbachia*-induced phenotypes, thus offering the opportunity to shed light on processes governing arthropod development and reproduction,

On the whole, the data available in the literature suggest that the phenotypic effects induced by *Wolbachia* may be linked to differences in host physiology, and in particular to endocrinerelated processes governing development and reproduction which in insects display high

Interestingly, *Wolbachia* bacteria are known to localize in many hosts' steroidogenic tissues. In different insect species, the endosymbiont has been observed in the cytoplasm of the follicular cells (Gonella et al. 2011; Sacchi et al., 2010) (Fig. 2). In *Drosophila*, *Wolbachia* microinjected into the abdominal cavity has shown a tropism towards somatic stem cells that differentiate in follicular cells (Frydman et al., 2006). In insects the follicular epithelium is one of the major niches deputed to the synthesis of ecdysteroids (Swevers et al., 2005).

and on the involvement of the endosymbiont in such processes.

sequences (Iturbe-Ormaetxe & O'Neill, 2007; Wu et al., 2004).

2003; Sakamoto et al., 2007).

genetic males.

variability.

strictly interconnected, and possibly all ascribable to feminization.

#### **3. Ecdysteroids and** *Wolbachia***: Different roles and different manipulations**

*Wolbachia* are members of the order Rickettsiales (α-Proteobacteria), a diverse group of symbionts with parasitic, mutualistic or commensal lifestyle. The genus *Wolbachia* is known to infect exclusively invertebrates, namely nematodes and arthropods, being widely spread in insects where it is estimated to occur in up to 66% of the species (Hilgenboecker et al., 2008; Werren et al., 2008). *Wolbachia* bacteria, and specifically the species *W. pipientis*, are transmitted through the germ line from the mother to the offspring and, occasionally, between individuals of phylogenetically distant species (Stouthamer et al., 1999). The transovarial inheritance of *Wolbachia* in insects seems to be mediated by bacteryocite-like cells (cells specialized for harbouring endosymbionts) in the ovary of the infected mother, which degenerate thus ensuring transmission of bacteria to germ line cells and then to the progeny (Sacchi et al., 2010).

Phylogenetic studies based on 16S ribosomal sequences reveal that *Wolbachia* bacteria are divided into eight different supergroups: two are commonly found in Nematoda (mainly in filarial but also in non filarial species), whereas the other six supergroups are found primarily in Arthropoda, including insects, mites, spiders, scorpions and isopod crustaceans (Werren et al., 2008).

A unique feature shared by Arthropoda and Nematoda is the ability to replace the exoskeleton, a process known as ecdysis. This shared characteristic is thought to reflect a common ancestry, giving rise to the clade Ecdysozoa (Ewer, 2005a). Although the exoskeleton composition varies among ecdysozoans, the process of moulting itself is similar within the clade: the epidermis undergoes cell division producing a larger surface and separates from the exoskeleton. Then the epidermis secretes a new exoskeleton that remains soft until the residues of the old cuticle are shed at ecdysis. The new cuticle then expands and hardens (Ewer, 2005a, 2005b).

As previously discussed, arthropod moulting is induced by the steroid hormone 20E and a role for ecdysteroids in nematode ecdysis has also been observed. In filarial nematodes, moulting seems to be regulated by ecdysteroid-like hormones: in *Dirofilaria immitis*, for example, moulting from the third to the fourth larval stage can be induced in vitro by the 20E of insects (Wabrick et al., 1993), and orthologs of insects nuclear receptors involved in ecdysone response have been found (Crossgrove et al., 2008; Ghedin et al., 2007; Tzertzinis et al., 2010). In *Caenorhabditis elegans* these nuclear receptors are also involved in the regulation of sex determination and reproductive development (Höss & Weltje, 2007; Motola et al., 2006) and, interestingly, ecdysone has also a role in the fertility and microfilaria release in filarial worms (Barker et al., 1991).

In nematodes, *Wolbachia* is an obligate symbiont, as worms depend on bacteria for survival. Antibiotic curing of *Wolbachia* "infection" inhibits nematode fertility and development, suggesting a specific role for the symbiont in host oogenesis, embryogenesis and moulting (Arumugam et al., 2008; Casiraghi et al., 2002; Frank et al., 2010).

In arthropods the bacterium is able to manipulate the host reproduction in order to increase the number of infected females. The effects of the *Wolbachia* infection include cytoplasmic incompatibility, that is an aberrant or considerably reduced offspring production if uninfected females mate with infected males, or if the parents are infected with different *Wolbachia* strains; thelytokous parthenogenesis, in which infected virgin females produce daughters; feminization, in which infected genetic males develop as females; and malekilling, in which infected males die (Stouthamer et al., 1999; Werren et al., 2008).

Phylogenetic studies based on 16S ribosomal sequences reveal that *Wolbachia* bacteria are divided into eight different supergroups: two are commonly found in Nematoda (mainly in filarial but also in non filarial species), whereas the other six supergroups are found primarily in Arthropoda, including insects, mites, spiders, scorpions and isopod crustaceans

A unique feature shared by Arthropoda and Nematoda is the ability to replace the exoskeleton, a process known as ecdysis. This shared characteristic is thought to reflect a common ancestry, giving rise to the clade Ecdysozoa (Ewer, 2005a). Although the exoskeleton composition varies among ecdysozoans, the process of moulting itself is similar within the clade: the epidermis undergoes cell division producing a larger surface and separates from the exoskeleton. Then the epidermis secretes a new exoskeleton that remains soft until the residues of the old cuticle are shed at ecdysis. The new cuticle then expands

As previously discussed, arthropod moulting is induced by the steroid hormone 20E and a role for ecdysteroids in nematode ecdysis has also been observed. In filarial nematodes, moulting seems to be regulated by ecdysteroid-like hormones: in *Dirofilaria immitis*, for example, moulting from the third to the fourth larval stage can be induced in vitro by the 20E of insects (Wabrick et al., 1993), and orthologs of insects nuclear receptors involved in ecdysone response have been found (Crossgrove et al., 2008; Ghedin et al., 2007; Tzertzinis et al., 2010). In *Caenorhabditis elegans* these nuclear receptors are also involved in the regulation of sex determination and reproductive development (Höss & Weltje, 2007; Motola et al., 2006) and, interestingly, ecdysone has also a role in the fertility and

In nematodes, *Wolbachia* is an obligate symbiont, as worms depend on bacteria for survival. Antibiotic curing of *Wolbachia* "infection" inhibits nematode fertility and development, suggesting a specific role for the symbiont in host oogenesis, embryogenesis and moulting

In arthropods the bacterium is able to manipulate the host reproduction in order to increase the number of infected females. The effects of the *Wolbachia* infection include cytoplasmic incompatibility, that is an aberrant or considerably reduced offspring production if uninfected females mate with infected males, or if the parents are infected with different *Wolbachia* strains; thelytokous parthenogenesis, in which infected virgin females produce daughters; feminization, in which infected genetic males develop as females; and male-

killing, in which infected males die (Stouthamer et al., 1999; Werren et al., 2008).

**3. Ecdysteroids and** *Wolbachia***: Different roles and different manipulations**  *Wolbachia* are members of the order Rickettsiales (α-Proteobacteria), a diverse group of symbionts with parasitic, mutualistic or commensal lifestyle. The genus *Wolbachia* is known to infect exclusively invertebrates, namely nematodes and arthropods, being widely spread in insects where it is estimated to occur in up to 66% of the species (Hilgenboecker et al., 2008; Werren et al., 2008). *Wolbachia* bacteria, and specifically the species *W. pipientis*, are transmitted through the germ line from the mother to the offspring and, occasionally, between individuals of phylogenetically distant species (Stouthamer et al., 1999). The transovarial inheritance of *Wolbachia* in insects seems to be mediated by bacteryocite-like cells (cells specialized for harbouring endosymbionts) in the ovary of the infected mother, which degenerate thus ensuring transmission of bacteria to germ line cells and then to the progeny

(Sacchi et al., 2010).

(Werren et al., 2008).

and hardens (Ewer, 2005a, 2005b).

microfilaria release in filarial worms (Barker et al., 1991).

(Arumugam et al., 2008; Casiraghi et al., 2002; Frank et al., 2010).

Such phenotypic variability is thought to be linked to high genome plasticity of insect-borne *Wolbachia*, since all the sequenced genomes of the symbiont contain high number of repetitive sequences, including IS (insertion sequences) elements and prophage-like sequences (Iturbe-Ormaetxe & O'Neill, 2007; Wu et al., 2004).

According to us, except for cytoplasmic incompatibility that is a secondary effect of the infection, the phenotypic effects observed in arthropods might not be so different, but strictly interconnected, and possibly all ascribable to feminization.

Indeed, male killing could be just an unsuccessful "attempt" at feminization by *Wolbachia*. Male-killing is known in several insect species, where males die during embryogenesis or development. Insight into the mechanism of male killing comes from the moths *Ostrinia scapulalis* and *O. furnacalis*, where *Wolbachia* kills genetic males during the larval development. Intriguingly, a partial *Wolbachia* curing leads to the appearance of lepidopteran intersexes having exclusively male genotype (Kageyama & Traut, 2003). Accordingly, a partial feminization of genetic males does occur, while a complete feminization is incompatible with the survival of the male genotype (Kageyama & Traut, 2003; Sakamoto et al., 2007).

Regarding the parthenogenesis induction by *Wolbachia*, this phenomenon has been demonstrated in several haplodiploid species of mites, hymenopterans and thrips, where males naturally develop (parthenogenetically) from unfertilized haploid eggs and females from fertilized diploid eggs (Arakaki et al., 2001; Stouthamer et al., 1990; Weeks & Breeuwer, 2001). In *Wolbachia*-infected species, unfertilized eggs are subjected to a "diploidy" restoration, giving origin to (infected) females. Recently, Giorgini and colleagues (2009) observed that in the (haplodiploid) wasp *Encarsia hispida*, the symbiont *Cardinium* (which belongs to the only bacterial group known to cause similar reproductive manipulations of *Wolbachia*) doesn't induce, as expected, thelytokous parthenogenesis but feminization. In fact antibiotic treatment results in uninfected diploid male offspring, thus demonstrating that diploidy restoration is a necessary condition, but not sufficient, to elicit female development. Therefore, *Cardinium* is responsible for the feminization of the hymenopteran genetic males.

Since in studies concerning the parthenogenesis induction by *Wolbachia* no cytogenetic analyses have been performed on males produced by cured females, the hypothesis that the symbiont actually induces feminization rather than parthenogenesis may be conceivable.

As will be discussed later, feminization deals with sex determination and differentiation much more directly than the other *Wolbachia*-induced phenotypes, thus offering the opportunity to shed light on processes governing arthropod development and reproduction, and on the involvement of the endosymbiont in such processes.

On the whole, the data available in the literature suggest that the phenotypic effects induced by *Wolbachia* may be linked to differences in host physiology, and in particular to endocrinerelated processes governing development and reproduction which in insects display high variability.

Interestingly, *Wolbachia* bacteria are known to localize in many hosts' steroidogenic tissues. In different insect species, the endosymbiont has been observed in the cytoplasm of the follicular cells (Gonella et al. 2011; Sacchi et al., 2010) (Fig. 2). In *Drosophila*, *Wolbachia* microinjected into the abdominal cavity has shown a tropism towards somatic stem cells that differentiate in follicular cells (Frydman et al., 2006). In insects the follicular epithelium is one of the major niches deputed to the synthesis of ecdysteroids (Swevers et al., 2005).

Sex Steroids in Insects and the Role of the Endosymbiont *Wolbachia*: A New Perspective 359

In arthropods, feminization induced by *Wolbachia* was first described in isopod crustaceans (Bouchon et al., 2008; Martin et al., 1973; Rigaud et al., 1999) and, later, the phenomenon was studied in the lepidopteran species *Eurema hecabe*, *Ostrinia scapulalis* and *O. furnacalis*, and the hemipteran species *Zyginidia pullula* (Hiroki et al., 2002; Kageyama & Traut, 2003; Negri

In Crustacea, which are phylogenetically close to insects, sex differentiation and development of secondary sexual characteristics are driven by an androgenic hormone (AH), secreted by the androgenic gland (AG), whose action inhibits female differentiation (Legrand et al., 1987; Sagi & Khalaila, 2001). In fact, Crustacea are by default female and the expression of male secondary characteristics is only possible by the production of AH. Indeed, the ablation of the AG results in the degeneration from male to the female form, whereas injection with purified extracts of the AH or implantation of AGs into females results in the development of external male sexual characteristics or the complete sex reversal (Charniaux-Cotton, 1954; Sagi et al., 1997; Suzuki & Yamasaki, 1998). Therefore, it has been suggested that in Crustacea sex reversal is actually due to masculinisation of

The feminization effect induced by *Wolbachia* in isopods is thought to be linked to interactions between the bacterium and the AG differentiation process or, more probably, the AH receptors (Bouchon et al., 2008; Rigaud & Juchault, 1998). Indeed, in *Armadillidium vulgare* genetic males, AH mRNA can be detected at the beginning of male gonad differentiation, and AH may thus have an early and local action by inducing male differentiation of embryonic gonads (Negri et al., 2010). *Wolbachia* could then induce feminization (or de-masculinisation?) by targeting AH receptors, thereby inhibiting AG differentiation (Juchault & Legrand, 1985). If *Wolbachia* bacteria are experimentally inoculated in adult males, the AG become hypetrophic, but the host soon develops female genital apertures, probably because the AH receptors are no longer

In insect species, *Wolbachia* is able to feminize genetical males and, in all these cases, the existence of intersexes linked to *Wolbachia* effects has been described: in the presence of signals coordinating the development of a gender specific phenotype, intersexes might arise from a conflict between male and female sex hormones and/or receptors (Hiroki et al., 2002;

In *E. hecabe*, feminizing *Wolbachia* acts continuously throughout the larval development to produce the female phenotype (Narita et al., 2007). As a consequence, if the bacteria act on sex differentiation rather than sex determination, sex hormone (i.e. ecdysteroid) pathways should be involved. Some clues are provided by studies on infected *E. hecabe*, where an incomplete *Wolbachia* suppression during host development, i.e. when host sex differentiation is not yet completed, leads to larval/pupal moulting defects (Narita et al., 2007). In particular, some individuals show morphological abnormalities (i.e. curled, folded or asymmetric wings), while a certain number of insects do not pupate: dissection of dead pupae reveals that many of them have actually completed adult morphogenesis but failed to escape from the pupal case (Narita et al., 2007). Interestingly, similar moulting defects may be obtained in knockdown insects using RNA interference techniques on ecdysone receptors. For example, some treated nymphs of the german cockroach *Blattella germanica* do not moult into adults, maintaining both nymphal and adult structures of ectodermal origin duplicated, whereas those nymphs that moulted into adults show characteristic

**4.** *Wolbachia* **and the feminization of the arthropod host** 

et al., 2006; Sakamoto et al., 2007).

females or de-masculinisation of males (Ford, 2008).

functional due to the infection (Martin et al., 1973; Martin et al., 1999).

Kageyama & Traut, 2003; Negri et al., 2006; Sakamoto et al., 2007).

Upper left and right: A *Zyginidia pullula* (Hemiptera, Cicadellidae) oocyte surrounded by a single layer of follicle cells (gallocyanin–chrome alum reaction on leafhopper ovary sections). Lower left: TEM micrograph of a *Wolbachia*-infected *Z. pullula* follicle cell filled with bacteria. Lower right: Immunohistochemical reactions showing strong positivity (brown) to anti-wsp (*Wolbachia* surface protein) antibody in the leafhopper's follicular epithelium.

Fig. 2. *Wolbachia* localization in the follicular epithelium of the gonad's host.

Moreover, the endosymbiont is frequently associated to host's fat bodies, the other major niche for steroid synthesis (Kamoda et al., 2000; Thummel & Chory, 2002) (Fig. 3).

Therefore, it is conceivable that *Wolbachia* may interfere with insect reproduction and development by modulating host hormonal pathways, as it has already been shown for isopod crustaceans and will be explained in the following section.

Fig. 3. In-situ hybridization with a specific probe for *Wolbachia* 16S rRNA on *Zyginidia pullula*  fat body shows positive staining (red), indicating that the tissue is filled with bacteria.

#### **4.** *Wolbachia* **and the feminization of the arthropod host**

358 Sex Hormones

Upper left and right: A *Zyginidia pullula* (Hemiptera, Cicadellidae) oocyte surrounded by a single layer of follicle cells (gallocyanin–chrome alum reaction on leafhopper ovary sections). Lower left: TEM micrograph of a *Wolbachia*-infected *Z. pullula* follicle cell filled with bacteria. Lower right: Immunohistochemical reactions showing strong positivity (brown) to anti-wsp (*Wolbachia* surface protein)

Moreover, the endosymbiont is frequently associated to host's fat bodies, the other major

Therefore, it is conceivable that *Wolbachia* may interfere with insect reproduction and development by modulating host hormonal pathways, as it has already been shown for

Fig. 3. In-situ hybridization with a specific probe for *Wolbachia* 16S rRNA on *Zyginidia pullula*  fat body shows positive staining (red), indicating that the tissue is filled with bacteria.

Fig. 2. *Wolbachia* localization in the follicular epithelium of the gonad's host.

isopod crustaceans and will be explained in the following section.

niche for steroid synthesis (Kamoda et al., 2000; Thummel & Chory, 2002) (Fig. 3).

antibody in the leafhopper's follicular epithelium.

In arthropods, feminization induced by *Wolbachia* was first described in isopod crustaceans (Bouchon et al., 2008; Martin et al., 1973; Rigaud et al., 1999) and, later, the phenomenon was studied in the lepidopteran species *Eurema hecabe*, *Ostrinia scapulalis* and *O. furnacalis*, and the hemipteran species *Zyginidia pullula* (Hiroki et al., 2002; Kageyama & Traut, 2003; Negri et al., 2006; Sakamoto et al., 2007).

In Crustacea, which are phylogenetically close to insects, sex differentiation and development of secondary sexual characteristics are driven by an androgenic hormone (AH), secreted by the androgenic gland (AG), whose action inhibits female differentiation (Legrand et al., 1987; Sagi & Khalaila, 2001). In fact, Crustacea are by default female and the expression of male secondary characteristics is only possible by the production of AH. Indeed, the ablation of the AG results in the degeneration from male to the female form, whereas injection with purified extracts of the AH or implantation of AGs into females results in the development of external male sexual characteristics or the complete sex reversal (Charniaux-Cotton, 1954; Sagi et al., 1997; Suzuki & Yamasaki, 1998). Therefore, it has been suggested that in Crustacea sex reversal is actually due to masculinisation of females or de-masculinisation of males (Ford, 2008).

The feminization effect induced by *Wolbachia* in isopods is thought to be linked to interactions between the bacterium and the AG differentiation process or, more probably, the AH receptors (Bouchon et al., 2008; Rigaud & Juchault, 1998). Indeed, in *Armadillidium vulgare* genetic males, AH mRNA can be detected at the beginning of male gonad differentiation, and AH may thus have an early and local action by inducing male differentiation of embryonic gonads (Negri et al., 2010). *Wolbachia* could then induce feminization (or de-masculinisation?) by targeting AH receptors, thereby inhibiting AG differentiation (Juchault & Legrand, 1985). If *Wolbachia* bacteria are experimentally inoculated in adult males, the AG become hypetrophic, but the host soon develops female genital apertures, probably because the AH receptors are no longer functional due to the infection (Martin et al., 1973; Martin et al., 1999).

In insect species, *Wolbachia* is able to feminize genetical males and, in all these cases, the existence of intersexes linked to *Wolbachia* effects has been described: in the presence of signals coordinating the development of a gender specific phenotype, intersexes might arise from a conflict between male and female sex hormones and/or receptors (Hiroki et al., 2002; Kageyama & Traut, 2003; Negri et al., 2006; Sakamoto et al., 2007).

In *E. hecabe*, feminizing *Wolbachia* acts continuously throughout the larval development to produce the female phenotype (Narita et al., 2007). As a consequence, if the bacteria act on sex differentiation rather than sex determination, sex hormone (i.e. ecdysteroid) pathways should be involved. Some clues are provided by studies on infected *E. hecabe*, where an incomplete *Wolbachia* suppression during host development, i.e. when host sex differentiation is not yet completed, leads to larval/pupal moulting defects (Narita et al., 2007). In particular, some individuals show morphological abnormalities (i.e. curled, folded or asymmetric wings), while a certain number of insects do not pupate: dissection of dead pupae reveals that many of them have actually completed adult morphogenesis but failed to escape from the pupal case (Narita et al., 2007). Interestingly, similar moulting defects may be obtained in knockdown insects using RNA interference techniques on ecdysone receptors. For example, some treated nymphs of the german cockroach *Blattella germanica* do not moult into adults, maintaining both nymphal and adult structures of ectodermal origin duplicated, whereas those nymphs that moulted into adults show characteristic

Sex Steroids in Insects and the Role of the Endosymbiont *Wolbachia*: A New Perspective 361

*Wolbachia*-cured mothers, both female- and male-specific isoforms are present, suggesting that the symbiont may interfere either with the sex-specific splicing of the gene *dsx* itself or (more probably) with another upstream process involved in sex determination/differentiation

Male- and female-specific isoforms of DSX share a zinc finger DNA-binding domain (designated as the DM motif), which is widely conserved in the Animal Kingdom, from corals to nematodes, from arthropods to vertebrates, and characterize the *dmrt* family of genes (Erdman & Burtis, 1993; Murphy et al., 2010; Matsuda et al., 2002; Raymond et al.,

Despite the attention that *dmrt* factors have received, to date it has not been well elucidated how *dmrts* mediate their activities, and putative downstream targets have yet to be

In some vertebrates, such as fish, it has been demonstrated that sex steroid hormones affect *dmrt1* expression (Herpin & Schartl, 2011), thus it would be of capital interest to verify changes in *O. scapulalis dsx* expression following steroid treatments, and if feminizing *Wolbachia* may

New insights into the mechanisms underlying the bacterium-host interaction have been provided by studies on the leafhopper *Z. pullula*. In this hemipteran species, *Wolbachia*infected genetic males develop into intersexes with a female phenotype, which retain

Fig. 4. *Zyginidia pullula* males feminized by *Wolbachia* maintain typical male structures (the socalled upper pygofer appendages) localized in the last abdominal segments. These forked chitinous structures are completely absent in normal females. In feminized males they appear well developed (upper left), or not completely developed but reduced to a stump (lower right). Leafhopper feminized males are vital and even active reproductively. In laboratory rearing, couplings are often observed (Fig. 5), meaning that these individuals have a feminine 'sex appeal', and progeny is occasionally obtained (Negri et al., 2006). In addition to feminized males with ovaries ("intersex females"), some rare intersexes bear male gonads ("intersex males") (Negri et al., 2009a) (Fig. 5). Interestingly, "intersex males" possess a *Wolbachia* density approximately four orders of magnitude lower than "intersex females" (Negri et al., 2009a).

play a role in modulating *dsx* expression by interaction with hormonal pathways.

secondary male features in the ano-genital zone (Negri et al., 2006) (Fig. 4).

1998; Smith et al., 2009; Yoshimoto et al., 2008; Zhu et al., 2000).

(Sugimoto et al., 2010).

characterized.

deformations in the wing extension (Cruz et al., 2006). Also *Drosophila* EcR mutants are characterised by pupal lethality: specimens rarely eclose and the pharate adults dissected from the pupal case show abnormalities (Davies et al., 2005).

Moreover, since in some lepidopteran species the ecdysteroid titer has been proven to regulate sex specific wing development (Lobbia et al., 2003), sexually intermediate traits in wing morphology observed in *E. hecabe* specimens subjected to a partial *Wolbachia* curing could also be attributed to the ecdysteroid action.

In the other lepidopteran species *Ostrinia scapulalis* and *O. furnacalis*, *Wolbachia* has the ability to feminize genetic males, but – as discussed above - a complete feminization is fatal, and genetical males die (Kageyama and Traut, 2003; Sakamoto et al., 2007). In these species male-killing occurs during the larval development while the role played by ecdysteroids is crucial. In other insects, the sex-specific killing action by *Wolbachia* occurs during embryogenesis (Dyer& Jaenike, 2004; Fialho & Stevens, 2000; Jiggins et al., 2001; Zeh et al., 2005). Embryogenesis takes place in a steroid hormone-enriched environment where steroid hormones act for the coordination of morphogenetic movements (De Loof, 2006; Kozlova & Thummel, 2003; Gaziova et al., 2004). Thus, if male-killing *Wolbachia* interacts with the host hormonal pathway involving ecdysteroids, this could interfere with the processes required for a normal development of males.

Unfortunately, little information about sex-specific action of ecdysteroids during insect embryogenesis and development is available, and it mainly concerns the effects of endocrine disrupting chemicals. For example, in the housefly *Musca domestica* and in the midge *Chironomus riparius* the sex ratio is affected by the ecdysteroid agonist bisphenol A (Izumi et al., 2008; Lee & Choi, 2007). Another ecdysteroid agonist, tebufenozide, exerts similar effects on *C. riparius* and the moth *Platynota idaeusalis* (Biddinger et al., 2006; Hahn et al., 2001). Female-biased sex ratios are also obtained after a treatment performed on the midge larvae with the ecdysteroid antagonist ethynil estradiol (Hahn et al., 2001; Lee & Choi, 2007).

According to some authors, the observed sex-specific effect could be explained by considering insect steroids as sex hormones. In particular, larval or embryo males die because they are subjected to an unsuitable, i.e. female, hormonal environment (Hahn et al., 2001).

Recent studies on the moth *Ostrinia scapulalis* are providing new data on the molecular bases of the *Wolbachia*-host interaction. Sugimoto and colleagues (2010) analysed the expression of the *doublesex* gene (*dsx*) in *Ostrinia* intersexes (= partially feminized males) generated from antibiotic treated mothers. *Doublesex* is the highly conserved gene at the bottom of the sex determination cascades in insects, and it is known to regulate the somatic sexual differentiation through the sex specific proteins DSXf (female) and DSXm (male). (Burtis & Baker, 1989). In particular, *dsx* resides at the junction of a complex network of regulatory interactions that include homeotic genes, ligand-based signal transduction cascades, and other transcriptional regulators for the differentiation of sexually dimorphic structures (Burtis, 2002; Rideout et al., 2010). In *Drosophila* males, feminization may be induced by modifying dsx expression. The ectopical expression of DSXf with the complete removal of endogenous DSXm may cause external complete feminization (Waterbury et al., 1999); even the XY (male) germ line may be feminized by ectopical expression of DSXf (Waterbury et al., 2000).

As expected, in somatic tissues of *O. scapulalis* males and females, the sex-specific isoform of DSX was found; while in the gonads the opposite sex was also weakly expressed, maybe because reproductive organs comprise also undifferentiated germ cells where both DSXf and DSXm could be expressed (Sugimoto et al., 2010). In intersex individuals originated from

deformations in the wing extension (Cruz et al., 2006). Also *Drosophila* EcR mutants are characterised by pupal lethality: specimens rarely eclose and the pharate adults dissected

Moreover, since in some lepidopteran species the ecdysteroid titer has been proven to regulate sex specific wing development (Lobbia et al., 2003), sexually intermediate traits in wing morphology observed in *E. hecabe* specimens subjected to a partial *Wolbachia* curing

In the other lepidopteran species *Ostrinia scapulalis* and *O. furnacalis*, *Wolbachia* has the ability to feminize genetic males, but – as discussed above - a complete feminization is fatal, and genetical males die (Kageyama and Traut, 2003; Sakamoto et al., 2007). In these species male-killing occurs during the larval development while the role played by ecdysteroids is crucial. In other insects, the sex-specific killing action by *Wolbachia* occurs during embryogenesis (Dyer& Jaenike, 2004; Fialho & Stevens, 2000; Jiggins et al., 2001; Zeh et al., 2005). Embryogenesis takes place in a steroid hormone-enriched environment where steroid hormones act for the coordination of morphogenetic movements (De Loof, 2006; Kozlova & Thummel, 2003; Gaziova et al., 2004). Thus, if male-killing *Wolbachia* interacts with the host hormonal pathway involving ecdysteroids, this could interfere with the processes required

Unfortunately, little information about sex-specific action of ecdysteroids during insect embryogenesis and development is available, and it mainly concerns the effects of endocrine disrupting chemicals. For example, in the housefly *Musca domestica* and in the midge *Chironomus riparius* the sex ratio is affected by the ecdysteroid agonist bisphenol A (Izumi et al., 2008; Lee & Choi, 2007). Another ecdysteroid agonist, tebufenozide, exerts similar effects on *C. riparius* and the moth *Platynota idaeusalis* (Biddinger et al., 2006; Hahn et al., 2001). Female-biased sex ratios are also obtained after a treatment performed on the midge larvae with the ecdysteroid antagonist ethynil estradiol (Hahn et al., 2001; Lee & Choi, 2007). According to some authors, the observed sex-specific effect could be explained by considering insect steroids as sex hormones. In particular, larval or embryo males die because they are

Recent studies on the moth *Ostrinia scapulalis* are providing new data on the molecular bases of the *Wolbachia*-host interaction. Sugimoto and colleagues (2010) analysed the expression of the *doublesex* gene (*dsx*) in *Ostrinia* intersexes (= partially feminized males) generated from antibiotic treated mothers. *Doublesex* is the highly conserved gene at the bottom of the sex determination cascades in insects, and it is known to regulate the somatic sexual differentiation through the sex specific proteins DSXf (female) and DSXm (male). (Burtis & Baker, 1989). In particular, *dsx* resides at the junction of a complex network of regulatory interactions that include homeotic genes, ligand-based signal transduction cascades, and other transcriptional regulators for the differentiation of sexually dimorphic structures (Burtis, 2002; Rideout et al., 2010). In *Drosophila* males, feminization may be induced by modifying dsx expression. The ectopical expression of DSXf with the complete removal of endogenous DSXm may cause external complete feminization (Waterbury et al., 1999); even the XY (male) germ line may be feminized by ectopical expression of DSXf (Waterbury et al.,

As expected, in somatic tissues of *O. scapulalis* males and females, the sex-specific isoform of DSX was found; while in the gonads the opposite sex was also weakly expressed, maybe because reproductive organs comprise also undifferentiated germ cells where both DSXf and DSXm could be expressed (Sugimoto et al., 2010). In intersex individuals originated from

subjected to an unsuitable, i.e. female, hormonal environment (Hahn et al., 2001).

from the pupal case show abnormalities (Davies et al., 2005).

could also be attributed to the ecdysteroid action.

for a normal development of males.

2000).

*Wolbachia*-cured mothers, both female- and male-specific isoforms are present, suggesting that the symbiont may interfere either with the sex-specific splicing of the gene *dsx* itself or (more probably) with another upstream process involved in sex determination/differentiation (Sugimoto et al., 2010).

Male- and female-specific isoforms of DSX share a zinc finger DNA-binding domain (designated as the DM motif), which is widely conserved in the Animal Kingdom, from corals to nematodes, from arthropods to vertebrates, and characterize the *dmrt* family of genes (Erdman & Burtis, 1993; Murphy et al., 2010; Matsuda et al., 2002; Raymond et al., 1998; Smith et al., 2009; Yoshimoto et al., 2008; Zhu et al., 2000).

Despite the attention that *dmrt* factors have received, to date it has not been well elucidated how *dmrts* mediate their activities, and putative downstream targets have yet to be characterized.

In some vertebrates, such as fish, it has been demonstrated that sex steroid hormones affect *dmrt1* expression (Herpin & Schartl, 2011), thus it would be of capital interest to verify changes in *O. scapulalis dsx* expression following steroid treatments, and if feminizing *Wolbachia* may play a role in modulating *dsx* expression by interaction with hormonal pathways.

New insights into the mechanisms underlying the bacterium-host interaction have been provided by studies on the leafhopper *Z. pullula*. In this hemipteran species, *Wolbachia*infected genetic males develop into intersexes with a female phenotype, which retain secondary male features in the ano-genital zone (Negri et al., 2006) (Fig. 4).

Fig. 4. *Zyginidia pullula* males feminized by *Wolbachia* maintain typical male structures (the socalled upper pygofer appendages) localized in the last abdominal segments. These forked chitinous structures are completely absent in normal females. In feminized males they appear well developed (upper left), or not completely developed but reduced to a stump (lower right).

Leafhopper feminized males are vital and even active reproductively. In laboratory rearing, couplings are often observed (Fig. 5), meaning that these individuals have a feminine 'sex appeal', and progeny is occasionally obtained (Negri et al., 2006). In addition to feminized males with ovaries ("intersex females"), some rare intersexes bear male gonads ("intersex males") (Negri et al., 2009a) (Fig. 5). Interestingly, "intersex males" possess a *Wolbachia* density approximately four orders of magnitude lower than "intersex females" (Negri et al., 2009a).

Sex Steroids in Insects and the Role of the Endosymbiont *Wolbachia*: A New Perspective 363

suggests that *Wolbachia* is not only able to induce a feminization of genetic males, but may also cause the inheritance of female imprinting in gonads of feminized males. This is particularly intriguing since in gonads the parental imprinting is generally erased and reestablished on the basis of the parent sex, and clearly indicates that feminized males act as true females establishing a female genomic imprinting in their genome. On the whole data demonstrate that Wolbachia may be considered an 'environmental' factor that promotes heritable epigenetic changes in the host gene expression: the epigenetic effects of Wolbachia symbiosis are manifested as a 'maternal effect', in which infection of the mother alters the

**5. Possible interplay between steroid signalling and epigenetic pathways** 

In humans the male-determining gene *Sry* on the male-specific Y chromosome is known to promote sexual development by inducing the bipotential gonads of the embryo to form testes. Then, the differentiated gonad produces the male sex steroid (i.e. testosterone) which activates gene transcription via androgen and estrogen receptors, thus driving the masculinisation processes of the whole body (Anway et al., 2005; Chang et al., 2006). In particular, sex hormone synthesis induces not only the sexual differentiation of the reproductive system, but also the sexual differentiation of the brain. This is known to occur in a carefully defined critical period, where a brief hormone exposure permanently organizes the brain sex differences (Dohier, 1998; Gabory et al., 2009; McCarthy et al., 2009). Indeed, gonadal hormones defeminize and masculinize the male brain, while a lack of gonadal steroids allows for feminization in the female. In rodents, for example, treatments with steroids during the critical period leads to a defeminized and masculinized neural phenotype, while blocking aromatization of testosterone to estradiol or antagonizing estrogen receptor binding inhibits a correct brain organization in males (Barraclough, 1961;

The mechanisms exerted by sex hormones are strictly linked to the epigenetic machinery. For example, gonadal hormones are able to induce sex differences in DNA methylation, methyl-binding proteins and chromatin modifications necessary for a correct sexual

The role for steroids in modulating epigenetic changes is attracting the growing interest of many researchers. In particular, the field of endocrine disruption is shedding new light on the discipline of basic reproductive neuro-endocrinology, through studies on how early life exposures to endocrine-disrupting chemicals may alter gene expression via epigenetic mechanisms, including DNA methylation and histone acetylation/methylation. Importantly, these effects may be transmitted to future generations if the germ line is

Recent evidence shows for example that androgen and estrogen receptors interact with histone modifying enzymes (Tsai et al., 2009). Measuring levels of acetylation and methylation of histones in neonatal mouse brains, Tsai and colleagues (2009) found that H3 histone modification is sexually dimorphic in some areas of the neonatal brain, and prenatal

In another study, tamoxifen - a selective estrogen receptor modulator - has been shown to interfere with imprinting at the specific locus Insulin-like growth factor 2/H19 in rat spermatozoa (Pathak et al., 2010). Since imprint at this locus is acquired during

**5.1 Role of sex steroids in mammal sex differentiation** 

differentiation of the brain (Nugent & McCarthy, 2011).

affected via trans-generational, epigenetic actions.

testosterone interacts with H3 acetylation to reverse this dimorphism.

offspring phenotype.

Baum, 1979; Vreeburg et al., 1977).

Fig. 5. On the left: a spermatheca of an intersex female of *Zyginidia pullula* full of sperms after after mating (haematoxylin/eosin stain on leafhopper gonad sections). On the right: testis of an intersex male showing different stages of spermatogenesis (gallocyanin–chrome alum reaction on leafhopper gonad sections).

Recent data demonstrate that *Wolbachia* infection is able to modulate the leafhopper's genomic imprinting through cytosine methylation of the host DNA (Negri et al., 2009a, 2009b).

Genomic imprinting is a phenomenon whereby a gene, or a region of a chromosome, is reversibly modified so that it retains a sort of "memory" of its own genetic history. The term imprinting, originally coined referring to a complex behaviour of the X chromosome in the dipteran insect *Sciara coprophila* (Crouse, 1960), indicates a situation in which the activity of the imprinted genes or chromosomes is determined by the sex of the parent that transmits them, and the altered expression is limited to the somatic tissue of the progeny, whereas the germ line is not permanently altered (Surani, 1998). Epigenetic changes are based on molecular mechanisms including methylation of cytosines, remodelling of chromatin structure through histone chemical modifications and RNA interference. These molecular processes can activate, reduce or completely disable the activity of genes.

Methylation of cytosine residues in the DNA is currently one of the most studied epigenetic mechanisms (Bender, 2004). This robust but reversible marking of genomic DNA is catalyzed by a conserved family of enzymes called DNA methyltransferases (DNMTs), which have been extensively studied in mammals, plants and fungi (Goll & Bestor, 2005).

Until now, the genomic imprinting has been found in vertebrates (Martin & McGowan 1995; Sharman 1971; Surani 1998) and invertebrates, including lots of insect species (Rewieved in Lyko & Maleszka, 2011). In particular, in the hymenopteran wasp *Nasonia vitripennis* and in the coccid *Planococcus citri* imprinting is related to sex determination (Beukeboom et al. 2007; Field et al. 2004); in *P. citri* it has been clearly assessed that DNA methylation is deeply involved in the establishment of the differential sex-specific genomic imprinting.

At a molecular level, in the hemipteran *Z. pullula* the occurrence of sex specific differences in the methylation pattern was observed (Negri et al., 2009a). Surprisingly, Random Amplification of Polymorphic DNA (RAPD) PCRs showed that *Wolbachia*-infected "intersex females" possess the same imprinting pattern of uninfected females (Negri et al., 2009a, 2009b). These data demonstrate that the infection disrupts the male imprinting thus influencing the expression of genes involved in sex differentiation and development. In addition, the alteration occurs only if the bacterium exceeds a density threshold, as "intersex males" maintain a male genome—methylation pattern (Negri et al., 2009a). Methylationsensitive RAPD analyses were also carried out on gonads (testes and ovaries), confirming the occurrence of a sex-specific methylation of the genome, and strengthening the results obtained with somatic tissues in *Wolbachia-*infected specimens (Negri et al., 2009b). This suggests that *Wolbachia* is not only able to induce a feminization of genetic males, but may also cause the inheritance of female imprinting in gonads of feminized males. This is particularly intriguing since in gonads the parental imprinting is generally erased and reestablished on the basis of the parent sex, and clearly indicates that feminized males act as true females establishing a female genomic imprinting in their genome. On the whole data demonstrate that Wolbachia may be considered an 'environmental' factor that promotes heritable epigenetic changes in the host gene expression: the epigenetic effects of Wolbachia symbiosis are manifested as a 'maternal effect', in which infection of the mother alters the offspring phenotype.

#### **5. Possible interplay between steroid signalling and epigenetic pathways**

#### **5.1 Role of sex steroids in mammal sex differentiation**

362 Sex Hormones

Fig. 5. On the left: a spermatheca of an intersex female of *Zyginidia pullula* full of sperms after after mating (haematoxylin/eosin stain on leafhopper gonad sections). On the right: testis of an intersex male showing different stages of spermatogenesis (gallocyanin–chrome

Recent data demonstrate that *Wolbachia* infection is able to modulate the leafhopper's genomic imprinting through cytosine methylation of the host DNA (Negri et al., 2009a,

Genomic imprinting is a phenomenon whereby a gene, or a region of a chromosome, is reversibly modified so that it retains a sort of "memory" of its own genetic history. The term imprinting, originally coined referring to a complex behaviour of the X chromosome in the dipteran insect *Sciara coprophila* (Crouse, 1960), indicates a situation in which the activity of the imprinted genes or chromosomes is determined by the sex of the parent that transmits them, and the altered expression is limited to the somatic tissue of the progeny, whereas the germ line is not permanently altered (Surani, 1998). Epigenetic changes are based on molecular mechanisms including methylation of cytosines, remodelling of chromatin structure through histone chemical modifications and RNA interference. These molecular

Methylation of cytosine residues in the DNA is currently one of the most studied epigenetic mechanisms (Bender, 2004). This robust but reversible marking of genomic DNA is catalyzed by a conserved family of enzymes called DNA methyltransferases (DNMTs), which have been extensively studied in mammals, plants and fungi (Goll & Bestor, 2005). Until now, the genomic imprinting has been found in vertebrates (Martin & McGowan 1995; Sharman 1971; Surani 1998) and invertebrates, including lots of insect species (Rewieved in Lyko & Maleszka, 2011). In particular, in the hymenopteran wasp *Nasonia vitripennis* and in the coccid *Planococcus citri* imprinting is related to sex determination (Beukeboom et al. 2007; Field et al. 2004); in *P. citri* it has been clearly assessed that DNA methylation is deeply

At a molecular level, in the hemipteran *Z. pullula* the occurrence of sex specific differences in the methylation pattern was observed (Negri et al., 2009a). Surprisingly, Random Amplification of Polymorphic DNA (RAPD) PCRs showed that *Wolbachia*-infected "intersex females" possess the same imprinting pattern of uninfected females (Negri et al., 2009a, 2009b). These data demonstrate that the infection disrupts the male imprinting thus influencing the expression of genes involved in sex differentiation and development. In addition, the alteration occurs only if the bacterium exceeds a density threshold, as "intersex males" maintain a male genome—methylation pattern (Negri et al., 2009a). Methylationsensitive RAPD analyses were also carried out on gonads (testes and ovaries), confirming the occurrence of a sex-specific methylation of the genome, and strengthening the results obtained with somatic tissues in *Wolbachia-*infected specimens (Negri et al., 2009b). This

processes can activate, reduce or completely disable the activity of genes.

involved in the establishment of the differential sex-specific genomic imprinting.

alum reaction on leafhopper gonad sections).

2009b).

In humans the male-determining gene *Sry* on the male-specific Y chromosome is known to promote sexual development by inducing the bipotential gonads of the embryo to form testes. Then, the differentiated gonad produces the male sex steroid (i.e. testosterone) which activates gene transcription via androgen and estrogen receptors, thus driving the masculinisation processes of the whole body (Anway et al., 2005; Chang et al., 2006). In particular, sex hormone synthesis induces not only the sexual differentiation of the reproductive system, but also the sexual differentiation of the brain. This is known to occur in a carefully defined critical period, where a brief hormone exposure permanently organizes the brain sex differences (Dohier, 1998; Gabory et al., 2009; McCarthy et al., 2009). Indeed, gonadal hormones defeminize and masculinize the male brain, while a lack of gonadal steroids allows for feminization in the female. In rodents, for example, treatments with steroids during the critical period leads to a defeminized and masculinized neural phenotype, while blocking aromatization of testosterone to estradiol or antagonizing estrogen receptor binding inhibits a correct brain organization in males (Barraclough, 1961; Baum, 1979; Vreeburg et al., 1977).

The mechanisms exerted by sex hormones are strictly linked to the epigenetic machinery. For example, gonadal hormones are able to induce sex differences in DNA methylation, methyl-binding proteins and chromatin modifications necessary for a correct sexual differentiation of the brain (Nugent & McCarthy, 2011).

The role for steroids in modulating epigenetic changes is attracting the growing interest of many researchers. In particular, the field of endocrine disruption is shedding new light on the discipline of basic reproductive neuro-endocrinology, through studies on how early life exposures to endocrine-disrupting chemicals may alter gene expression via epigenetic mechanisms, including DNA methylation and histone acetylation/methylation. Importantly, these effects may be transmitted to future generations if the germ line is affected via trans-generational, epigenetic actions.

Recent evidence shows for example that androgen and estrogen receptors interact with histone modifying enzymes (Tsai et al., 2009). Measuring levels of acetylation and methylation of histones in neonatal mouse brains, Tsai and colleagues (2009) found that H3 histone modification is sexually dimorphic in some areas of the neonatal brain, and prenatal testosterone interacts with H3 acetylation to reverse this dimorphism.

In another study, tamoxifen - a selective estrogen receptor modulator - has been shown to interfere with imprinting at the specific locus Insulin-like growth factor 2/H19 in rat spermatozoa (Pathak et al., 2010). Since imprint at this locus is acquired during

Sex Steroids in Insects and the Role of the Endosymbiont *Wolbachia*: A New Perspective 365

methylation. In *Wolbachia*-infected insects, alterations of the methylation patterns may be due to hypothetical *Wolbachia* products (directly binding the nuclear-receptor or functioning as/or interfering with nuclear receptor co-regulators) that could inhibit EcR binding to DNA or DNA-methyltransferases (and/or histone modifying enzymes)

Accordingly, studies on *Wolbachia*-host interactions should give great attention for example to selective nuclear receptor modulators; substances with an antagonist action on the ecdysone nuclear receptor; or co-regulators of nuclear receptors, in view of their emerging role in integrating transcriptional co-regulation with epigenetic regulation (Rosenfeld et al., 2006; Kato et al., 2011). This could eventually clarify the nature of this fascinating microbial symbiosis and the extraordinary effects on the host sexual development and reproduction.

An interaction between *Wolbachia* and host hormonal signalling pathways involving ecdysteroids may suggest the mechanistic way the bacterium uses for manipulating the host sexual behaviour and reproduction. Thus, the various phenotypic effects induced by the symbiont may be due to differences in the host physiology, considering that endocrinerelated processes governing host development and reproduction display an enormous

Recent data demonstrate a role of the symbiont in inducing epigenetic trans-generational changes in the host: by establishing intimate relationships with germ-line cells, epigenetic effects of *Wolbachia* symbiosis are manifested as a 'maternal effect', in which infection of the mother modulates the offspring phenotype. Indeed the *Wolbachia* infection is known to disrupt male imprinting, corresponding to changes in the genomic methylation pattern and

These observations raise a key question: what is the molecular basis of such an interaction? Some fascinating clues are provided by the recent demonstrations of interplay between

The mechanisms exerted by hormones are strictly linked to the epigenetic machinery, where steroids promote sex differences in DNA methylation, methyl-binding proteins and chromatin modifications, even if some epigenetic sex differences can also be directly attributed to the sex chromosomes. According to recent studies, selective nuclear receptor modulators and co-regulators of nuclear receptors are key factors in inducing epigenetic changes via DNA methylation and histone chemical modifications. These complex interactions influence the transcriptional output of many gene networks: the disruption of their normal function or expression by environmental factors can contribute to a vast

Hence, we propose a new perspective supporting a role of the symbiont *Wolbachia* as an "environmental factor" experienced by a mother that promotes heritable epigenetic changes by interaction with hormonal signalling pathways. Although further efforts are needed to fully clarify the genetic and molecular bases of such an interaction, new work hypotheses have been now offered for the study of the mechanisms (yet largely unknown) used by symbionts to dialogue with their hosts. Likewise, the *Wolbachia*-host interaction could become an emerging model system for the study of hormone signalling orchestration by nuclear receptors, and for shedding light on the role of nuclear receptor coregulators in

recruitment, respectively.

**6. Conclusion** 

variability.

in the host sexual phenotype towards females.

hormone signalling and epigenetic pathways.

spectrum of physiological abnormalities and disorders.

integrating transcriptional coregulation with epigenetic regulation.

spermatogenesis in the male germ line, a role for estrogen signalling in the methylation dynamics of the testis is hypothesized. In particular it has been hypothesized that tamoxifen could exert an epigenetic action by directly affecting DNA methylation in the male germ cells. The observed reduction in sperm DNA methylation suggests imprinting error in the male germ-line mediated by defective estrogen signalling (Pathak et al., 2009; Pathak et al., 2010). Hence decipher interaction between estrogen signalling and DNA methylation pathways is of primary importance.

#### **5.2 The** *Wolbachia***-host interaction: A new perspective**

The model proposed in Fig. 6 tries to explain a possible *Wolbachia*/host interaction involving host hormonal signalling and epigenetic regulation. In view of the absence of genes codifying for typical eukaryotic DNA methyltransferases in the sequenced genomes of *Wolbachia* strains isolated from *D. melanogaster* and the nematode *B. Malayi* (Foster et al., 2005; Wu et al., 2004), we cannot exclude that the bacterium encodes for some proteins interfering with ecdysteroids signalling pathway thus modulating the expression of the host DNMTs and/or histone modifying enzymes.

Hormone signalling orchestration is done by nuclear receptors, and over the past decade it has become increasingly clear that the recruitment of co-regulatory proteins to nuclear receptors is required for hormone-mediated transcriptional and biological activities. Many nuclear receptor co-regulators are key epigenetic regulators and utilize enzymatic activities to epigenetically modify the DNA and chromatin, through DNA methylation and histone acetylation/methylation (Hsia et al., 2010 Mahajan & Samuels, 2000; Rosenfeld et al., 2006).

20E = 20-hydroxyecdysone; EcR = Ecdysone receptor; USP = Ultraspiracle; NRc = Nuclear Receptor coregulator; Dmt = DNA-methyltransferase; dmr = Differentially metylated regions; Wp = *Wolbachia* product; WNRc = *Wolbachia* Nuclear Receptor co-regulator.

Filled lollypops and open lollypops indicate methylated and unmethylated CpGs, respectively.

Fig. 6. Model illustrating the possible interplay between ecdysone signaling and epigenetic regulation. For simplicity, among epigenetic mechanisms, only DNA methylation is considered.

In particular, as proposed in Fig. 6, once 20E is biosynthesized, it binds the nuclear receptor EcR which heterodimerizes with USP. Then, the EcR/USP complex binds DNA constitutively and complexes with nuclear receptors co-regulators, thus catalyzing DNA methyltransferases (and/or histone modifying enzymes) which results in a proper DNA methylation. In *Wolbachia*-infected insects, alterations of the methylation patterns may be due to hypothetical *Wolbachia* products (directly binding the nuclear-receptor or functioning as/or interfering with nuclear receptor co-regulators) that could inhibit EcR binding to DNA or DNA-methyltransferases (and/or histone modifying enzymes) recruitment, respectively.

Accordingly, studies on *Wolbachia*-host interactions should give great attention for example to selective nuclear receptor modulators; substances with an antagonist action on the ecdysone nuclear receptor; or co-regulators of nuclear receptors, in view of their emerging role in integrating transcriptional co-regulation with epigenetic regulation (Rosenfeld et al., 2006; Kato et al., 2011). This could eventually clarify the nature of this fascinating microbial symbiosis and the extraordinary effects on the host sexual development and reproduction.

### **6. Conclusion**

364 Sex Hormones

spermatogenesis in the male germ line, a role for estrogen signalling in the methylation dynamics of the testis is hypothesized. In particular it has been hypothesized that tamoxifen could exert an epigenetic action by directly affecting DNA methylation in the male germ cells. The observed reduction in sperm DNA methylation suggests imprinting error in the male germ-line mediated by defective estrogen signalling (Pathak et al., 2009; Pathak et al., 2010). Hence decipher interaction between estrogen signalling and DNA methylation

The model proposed in Fig. 6 tries to explain a possible *Wolbachia*/host interaction involving host hormonal signalling and epigenetic regulation. In view of the absence of genes codifying for typical eukaryotic DNA methyltransferases in the sequenced genomes of *Wolbachia* strains isolated from *D. melanogaster* and the nematode *B. Malayi* (Foster et al., 2005; Wu et al., 2004), we cannot exclude that the bacterium encodes for some proteins interfering with ecdysteroids signalling pathway thus modulating the expression of the host

Hormone signalling orchestration is done by nuclear receptors, and over the past decade it has become increasingly clear that the recruitment of co-regulatory proteins to nuclear receptors is required for hormone-mediated transcriptional and biological activities. Many nuclear receptor co-regulators are key epigenetic regulators and utilize enzymatic activities to epigenetically modify the DNA and chromatin, through DNA methylation and histone acetylation/methylation (Hsia et al., 2010 Mahajan & Samuels, 2000; Rosenfeld et al., 2006).

20E = 20-hydroxyecdysone; EcR = Ecdysone receptor; USP = Ultraspiracle; NRc = Nuclear Receptor coregulator; Dmt = DNA-methyltransferase; dmr = Differentially metylated regions; Wp = *Wolbachia*

Fig. 6. Model illustrating the possible interplay between ecdysone signaling and epigenetic regulation. For simplicity, among epigenetic mechanisms, only DNA methylation is

In particular, as proposed in Fig. 6, once 20E is biosynthesized, it binds the nuclear receptor EcR which heterodimerizes with USP. Then, the EcR/USP complex binds DNA constitutively and complexes with nuclear receptors co-regulators, thus catalyzing DNA methyltransferases (and/or histone modifying enzymes) which results in a proper DNA

Filled lollypops and open lollypops indicate methylated and unmethylated CpGs, respectively.

pathways is of primary importance.

DNMTs and/or histone modifying enzymes.

product; WNRc = *Wolbachia* Nuclear Receptor co-regulator.

considered.

**5.2 The** *Wolbachia***-host interaction: A new perspective** 

An interaction between *Wolbachia* and host hormonal signalling pathways involving ecdysteroids may suggest the mechanistic way the bacterium uses for manipulating the host sexual behaviour and reproduction. Thus, the various phenotypic effects induced by the symbiont may be due to differences in the host physiology, considering that endocrinerelated processes governing host development and reproduction display an enormous variability.

Recent data demonstrate a role of the symbiont in inducing epigenetic trans-generational changes in the host: by establishing intimate relationships with germ-line cells, epigenetic effects of *Wolbachia* symbiosis are manifested as a 'maternal effect', in which infection of the mother modulates the offspring phenotype. Indeed the *Wolbachia* infection is known to disrupt male imprinting, corresponding to changes in the genomic methylation pattern and in the host sexual phenotype towards females.

These observations raise a key question: what is the molecular basis of such an interaction? Some fascinating clues are provided by the recent demonstrations of interplay between hormone signalling and epigenetic pathways.

The mechanisms exerted by hormones are strictly linked to the epigenetic machinery, where steroids promote sex differences in DNA methylation, methyl-binding proteins and chromatin modifications, even if some epigenetic sex differences can also be directly attributed to the sex chromosomes. According to recent studies, selective nuclear receptor modulators and co-regulators of nuclear receptors are key factors in inducing epigenetic changes via DNA methylation and histone chemical modifications. These complex interactions influence the transcriptional output of many gene networks: the disruption of their normal function or expression by environmental factors can contribute to a vast spectrum of physiological abnormalities and disorders.

Hence, we propose a new perspective supporting a role of the symbiont *Wolbachia* as an "environmental factor" experienced by a mother that promotes heritable epigenetic changes by interaction with hormonal signalling pathways. Although further efforts are needed to fully clarify the genetic and molecular bases of such an interaction, new work hypotheses have been now offered for the study of the mechanisms (yet largely unknown) used by symbionts to dialogue with their hosts. Likewise, the *Wolbachia*-host interaction could become an emerging model system for the study of hormone signalling orchestration by nuclear receptors, and for shedding light on the role of nuclear receptor coregulators in integrating transcriptional coregulation with epigenetic regulation.

Sex Steroids in Insects and the Role of the Endosymbiont *Wolbachia*: A New Perspective 367

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**18** 

*Sweden* 

**Tissue-Specific Regulation of Sex** 

Maria Norlin and Kjell Wikvall *Department of Pharmaceutical Biosciences,* 

*Uppsala University, Uppsala,* 

**Hormone Biosynthesis and Metabolism:** 

**Maintenance of Cellular Steroid Levels** 

**Novel Aspects on Hormonal Signalling and** 

In this chapter, we intend to review our recent research in the context of contemporary research in the field of sex hormone biosynthesis and metabolism. Our findings have revealed novel aspects on the regulation of sex hormone metabolism and the metabolic control of cellular levels and effects of estrogens, androgens and neurosteroids. We will discuss our results in relation to current knowledge of metabolism and actions of estrogens

The sex hormones, estrogens and androgens, are present in almost all tissues and affect such diverse processes as bone formation, sexual function, brain development, cardiovascular and immune systems and growth of various organs (Arnal et al., 2007; Cheskis et al., 2007; Folkerd et al., 2010; Li & Al-Azzawi, 2009). One of the tissues, where hormonal control of growth is essential, is the prostate, where androgens as well as estrogens play a role (Prins & Korach, 2008; Weihua et al., 2002). Although the physiological levels are different, both

The biosynthesis and metabolism of estrogens and androgens involve many different enzymes expressed in multiple organs (Miller et al., 2008; Norlin, 2008; Simard et al., 2005; Vihko et al., 2006). Large amounts of steroids, including sex hormone precursors are enzymatically formed in the adrenals, using cholesterol as starting material, and secreted to the circulation (Fig. 1). The formed sex hormone precursors may then be taken up by gonads and other organs for further metabolism to different androgenic and estrogenic compounds. The most important precursors for sex hormones are androstenedione and dehydroepiandrosterone (DHEA) and its sulphate, DHEA-S (Fig. 1). This large-scale production of precursors, available for transport to other tissues, is essential for reproductive functions and sexual development, including the formation of genitalia

and androgens. First, an introduction to this research field will be given.

**2.1 Biosynthesis and metabolism of sex hormones: An introduction** 

**2. Sex hormones: Biosynthesis, metabolism and actions** 

androgens and estrogens are needed in both sexes.

**1. Introduction** 


### **Tissue-Specific Regulation of Sex Hormone Biosynthesis and Metabolism: Novel Aspects on Hormonal Signalling and Maintenance of Cellular Steroid Levels**

Maria Norlin and Kjell Wikvall *Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden* 

#### **1. Introduction**

374 Sex Hormones

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In this chapter, we intend to review our recent research in the context of contemporary research in the field of sex hormone biosynthesis and metabolism. Our findings have revealed novel aspects on the regulation of sex hormone metabolism and the metabolic control of cellular levels and effects of estrogens, androgens and neurosteroids. We will discuss our results in relation to current knowledge of metabolism and actions of estrogens and androgens. First, an introduction to this research field will be given.

#### **2. Sex hormones: Biosynthesis, metabolism and actions**

#### **2.1 Biosynthesis and metabolism of sex hormones: An introduction**

The sex hormones, estrogens and androgens, are present in almost all tissues and affect such diverse processes as bone formation, sexual function, brain development, cardiovascular and immune systems and growth of various organs (Arnal et al., 2007; Cheskis et al., 2007; Folkerd et al., 2010; Li & Al-Azzawi, 2009). One of the tissues, where hormonal control of growth is essential, is the prostate, where androgens as well as estrogens play a role (Prins & Korach, 2008; Weihua et al., 2002). Although the physiological levels are different, both androgens and estrogens are needed in both sexes.

The biosynthesis and metabolism of estrogens and androgens involve many different enzymes expressed in multiple organs (Miller et al., 2008; Norlin, 2008; Simard et al., 2005; Vihko et al., 2006). Large amounts of steroids, including sex hormone precursors are enzymatically formed in the adrenals, using cholesterol as starting material, and secreted to the circulation (Fig. 1). The formed sex hormone precursors may then be taken up by gonads and other organs for further metabolism to different androgenic and estrogenic compounds. The most important precursors for sex hormones are androstenedione and dehydroepiandrosterone (DHEA) and its sulphate, DHEA-S (Fig. 1). This large-scale production of precursors, available for transport to other tissues, is essential for reproductive functions and sexual development, including the formation of genitalia

Tissue-Specific Regulation of Sex Hormone Biosynthesis and Metabolism:

development and testicular function.

reductases

Novel Aspects on Hormonal Signalling and Maintenance of Cellular Steroid Levels 377

Several androgens are formed in humans (Bauman et al., 2006; Hudak et al., 2006; Weihua et al., 2002). A well-known androgen, testosterone, is formed in large amounts in several tissues, including the Leydig cells of the testes (Fig. 2). Testosterone levels are strongly influenced by the levels of its precursors, androstenedione and DHEA, in the blood circulation. Target tissues can convert testosterone into dihydrotestosterone (DHT), the most potent androgen, 10-fold more potent than testosterone (Fig. 2). Maintenance of adequate cellular levels of DHT is essential for a number of physiological processes, including sexual

Fig. 3. Formation of some estrogens. HSD, hydroxysteroid dehydrogenases; AKR, aldo-keto

The physiologically most potent estrogen is estradiol (17-estradiol). The levels of estradiol are dependent on the enzymatic activity of aromatase (CYP19A1) which forms estradiol from testosterone (Bulun et al., 2009; Simpson et al., 2002) (Fig. 3). In addition, there are several other endogenous estrogens that affect various organs and cells (Norlin et al., 2008; Pettersson et al., 2008; Weihua et al., 2002; Zhu et al., 2005). Individual estrogens may be of different importance in different tissues. For instance, 5-androstane-3,17-diol (3-Adiol) has been described as particularly important for estrogenic function in the prostate (Weihua et al., 2002). An

The total body levels of sex hormones are regulated by signalling from the pituitary and by mechanisms for excretion by the kidneys or in the bile (Bourdeau & Stratakis, 2002; Hum et al., 1999; Waxman & Holloway, 2009). In addition, cell-specific factors and mechanisms are important for regulation of the local sex hormone synthesis and elimination (Jellinck et al., 2007; Norlin, 2008; Penning et al., 2000; Reddy, 2004). Estrogens and androgens are removed from the body via metabolism into inactive metabolites that are excreted in the urine and/or feces. Local metabolism of importance for tissue hormone levels vary for different steroids

overview of enzymes in the formation of some estrogenic steroids is shown in Fig. 3.

Fig. 1. A simplified overview on the biosynthesis of steroid hormones in the adrenal cortex.

(Rainey et al., 2004; Sultan et al., 2001). A sex hormone precursor may undergo different metabolic transformations in different cells, depending on which enzymes that are expressed in a certain tissue and how these enzymes are regulated. In addition to the uptake of precursors transported from the adrenals, many tissues have the ability to carry out all the steps in estrogen and androgen synthesis. Thus, cell-specific needs for these hormones may be controlled locally in each tissue (Hudak et al., 2006; Penning et al., 2000; Tsuchiya et al., 2005). Local metabolism, dependent on various tissue-specific enzymes, is essential to achieve hormonal effects and to eliminate excess hormone from the cells.

Fig. 2. Formation of androgens. HSD, hydroxysteroid dehydrogenases.

Fig. 1. A simplified overview on the biosynthesis of steroid hormones in the adrenal cortex. (Rainey et al., 2004; Sultan et al., 2001). A sex hormone precursor may undergo different metabolic transformations in different cells, depending on which enzymes that are expressed in a certain tissue and how these enzymes are regulated. In addition to the uptake of precursors transported from the adrenals, many tissues have the ability to carry out all the steps in estrogen and androgen synthesis. Thus, cell-specific needs for these hormones may be controlled locally in each tissue (Hudak et al., 2006; Penning et al., 2000; Tsuchiya et al., 2005). Local metabolism, dependent on various tissue-specific enzymes, is essential to

achieve hormonal effects and to eliminate excess hormone from the cells.

Fig. 2. Formation of androgens. HSD, hydroxysteroid dehydrogenases.

Several androgens are formed in humans (Bauman et al., 2006; Hudak et al., 2006; Weihua et al., 2002). A well-known androgen, testosterone, is formed in large amounts in several tissues, including the Leydig cells of the testes (Fig. 2). Testosterone levels are strongly influenced by the levels of its precursors, androstenedione and DHEA, in the blood circulation. Target tissues can convert testosterone into dihydrotestosterone (DHT), the most potent androgen, 10-fold more potent than testosterone (Fig. 2). Maintenance of adequate cellular levels of DHT is essential for a number of physiological processes, including sexual development and testicular function.

Fig. 3. Formation of some estrogens. HSD, hydroxysteroid dehydrogenases; AKR, aldo-keto reductases

The physiologically most potent estrogen is estradiol (17-estradiol). The levels of estradiol are dependent on the enzymatic activity of aromatase (CYP19A1) which forms estradiol from testosterone (Bulun et al., 2009; Simpson et al., 2002) (Fig. 3). In addition, there are several other endogenous estrogens that affect various organs and cells (Norlin et al., 2008; Pettersson et al., 2008; Weihua et al., 2002; Zhu et al., 2005). Individual estrogens may be of different importance in different tissues. For instance, 5-androstane-3,17-diol (3-Adiol) has been described as particularly important for estrogenic function in the prostate (Weihua et al., 2002). An overview of enzymes in the formation of some estrogenic steroids is shown in Fig. 3.

The total body levels of sex hormones are regulated by signalling from the pituitary and by mechanisms for excretion by the kidneys or in the bile (Bourdeau & Stratakis, 2002; Hum et al., 1999; Waxman & Holloway, 2009). In addition, cell-specific factors and mechanisms are important for regulation of the local sex hormone synthesis and elimination (Jellinck et al., 2007; Norlin, 2008; Penning et al., 2000; Reddy, 2004). Estrogens and androgens are removed from the body via metabolism into inactive metabolites that are excreted in the urine and/or feces. Local metabolism of importance for tissue hormone levels vary for different steroids

Tissue-Specific Regulation of Sex Hormone Biosynthesis and Metabolism:

others, resulting in different tissue-specific responses.

**related compounds** 

2010; Schaeffer et al., 2006).

**and effects of sex hormones** 

Novel Aspects on Hormonal Signalling and Maintenance of Cellular Steroid Levels 379

Many of the cellular effects of estrogens and androgens are mediated via the classical sex hormone receptors, the androgen receptor, AR, and the two estrogen receptors, ER and ER (Brinkmann et al., 1999; Cheskis et al., 2007; Li & Al-Azzawi, 2009; Prins & Korach, 2008). The actions of estrogens and androgens are often mediated via hormone-responsive sequences in target gene promoters, the "androgen response elements" (ARE) and "estrogen response elements" (ERE). Sex hormones are also able to act via mechanisms independent of the AR and ER (Bryant et al., 2006; Cheskis et al., 2007; Lorenzo & Saatcioglu 2008). Such mechanisms may involve signal transduction pathways, for instance MAPK (mitogen-activated protein kinase) or PI3K (phosphoinositide 3-kinase)/Akt signal pathways. Crosstalk between proteins of these signal pathways and estrogen receptors also have been reported for some hormonal targets. Additional pathways for hormonal action are believed to be mediated by different types of hormone receptors located at the cell membrane. Sex hormone-related compounds used in therapy include antagonists of ER and AR and selective hormone receptor modulators (SERMs and SARMs, respectively) (Bhasin & Jasuja, 2009; Cheskis et al., 2007; Jordan, 2007). Selective hormone receptor modulators function as agonists in some tissues and antagonists in

Despite the essential roles of sex hormones in a large number of physiological processes, excess amounts of these compounds can have a negative impact and even contribute to disease. Adverse effects of estrogens include e. g. intrahepatic cholestasis, which may occur in some women using oral contraceptives or during pregnancy, where this condition can result in premature delivery or fetal death (Yamamoto et al., 2006). Many breast tumours are dependent on estrogen for growth and are reported to exhibit increased estradiol biosynthesis. For this reason, inhibitors of the estradiol-forming aromatase (CYP19A1) are routinely used in treatment of breast cancer (Chen, 1998). Furthermore, although the growth-inducing effect of androgens are required for formation and normal function of the prostate, overproduction of androgens can contribute substantially to unwanted growth of this tissue e. g. in malignancy. Treatment to suppress androgen action and/or formation have therefore proven very useful in prostate cancer therapy as well as in treatment of benign prostate hyperplasia (Bauman et al., 2006; Hudak et al., 2006). In general, transformation into malignancy may elicit changes in both sex hormone concentrations and effects of sex hormones on growth (Bauman et al., 2006; Vihko et al., 2006). Marked changes in androgen and estrogen metabolism have been reported for many malignant cells. However, abnormalities in hormone synthesis and metabolism are not only found in malignancy. Although the functions of sex hormones formed in the CNS have been much less studied than those of the reproductive organs, disturbed synthesis and/or metabolism of several brain steroids have been described in neurodegenerative disease (Cossec et al.,

**2.3 Cell- and tissue-specific steroid metabolism – role(s) for controlling cellular levels** 

Steroid hormone metabolism is not the same everywhere in the body. Due to the different physiological demands that will arise in various tissues and during different conditions, the level of a certain hormone at a given time need to be carefully controlled. Also, not all cells need the same types of hormones. Often, a steroid hormone may potentially undergo several different metabolic pathways. However, one pathway may be particularly active in a

**2.2 Physiological and pharmacological actions of sex hormones and sex hormone-**

and different tissues. Mechanisms believed to be of importance involve e. g. hydroxylation by cytochrome P450 (CYP) enzymes and conjugation with sulfate or glucuronic acid by the UDP-glucuronosyl transferases (UGT) and sulfotransferases (SULT) (Tang et al., 2006; Turgeon et al., 2001 Zhu et al., 2005) (Figs. 4 and 5).

Fig. 4. Metabolic pathways that may affect the levels of estradiol and estrone. Please note that this figure is intended as an overview of potentially important pathways and does not necessarily describe the situation in any particular cell. AKR, aldo-keto reductases; CYP, cytochrome P450; HSD, hydroxysteroid dehydrogenases; SULT, sulfotransferases; UGT, UDP-glucuronosyl transferases.

Fig. 5. Metabolic pathways that may affect the cellular levels of testosterone and its metabolites. Please note that this figure is intended as an overview of potentially important pathways and does not necessarily describe the situation in any particular cell. For abbreviations, see legend of Fig. 4.

and different tissues. Mechanisms believed to be of importance involve e. g. hydroxylation by cytochrome P450 (CYP) enzymes and conjugation with sulfate or glucuronic acid by the UDP-glucuronosyl transferases (UGT) and sulfotransferases (SULT) (Tang et al., 2006;

Fig. 4. Metabolic pathways that may affect the levels of estradiol and estrone. Please note that this figure is intended as an overview of potentially important pathways and does not necessarily describe the situation in any particular cell. AKR, aldo-keto reductases; CYP, cytochrome P450; HSD, hydroxysteroid dehydrogenases; SULT, sulfotransferases; UGT,

Fig. 5. Metabolic pathways that may affect the cellular levels of testosterone and its

pathways and does not necessarily describe the situation in any particular cell. For

metabolites. Please note that this figure is intended as an overview of potentially important

Turgeon et al., 2001 Zhu et al., 2005) (Figs. 4 and 5).

UDP-glucuronosyl transferases.

abbreviations, see legend of Fig. 4.

#### **2.2 Physiological and pharmacological actions of sex hormones and sex hormonerelated compounds**

Many of the cellular effects of estrogens and androgens are mediated via the classical sex hormone receptors, the androgen receptor, AR, and the two estrogen receptors, ER and ER (Brinkmann et al., 1999; Cheskis et al., 2007; Li & Al-Azzawi, 2009; Prins & Korach, 2008). The actions of estrogens and androgens are often mediated via hormone-responsive sequences in target gene promoters, the "androgen response elements" (ARE) and "estrogen response elements" (ERE). Sex hormones are also able to act via mechanisms independent of the AR and ER (Bryant et al., 2006; Cheskis et al., 2007; Lorenzo & Saatcioglu 2008). Such mechanisms may involve signal transduction pathways, for instance MAPK (mitogen-activated protein kinase) or PI3K (phosphoinositide 3-kinase)/Akt signal pathways. Crosstalk between proteins of these signal pathways and estrogen receptors also have been reported for some hormonal targets. Additional pathways for hormonal action are believed to be mediated by different types of hormone receptors located at the cell membrane. Sex hormone-related compounds used in therapy include antagonists of ER and AR and selective hormone receptor modulators (SERMs and SARMs, respectively) (Bhasin & Jasuja, 2009; Cheskis et al., 2007; Jordan, 2007). Selective hormone receptor modulators function as agonists in some tissues and antagonists in others, resulting in different tissue-specific responses.

Despite the essential roles of sex hormones in a large number of physiological processes, excess amounts of these compounds can have a negative impact and even contribute to disease. Adverse effects of estrogens include e. g. intrahepatic cholestasis, which may occur in some women using oral contraceptives or during pregnancy, where this condition can result in premature delivery or fetal death (Yamamoto et al., 2006). Many breast tumours are dependent on estrogen for growth and are reported to exhibit increased estradiol biosynthesis. For this reason, inhibitors of the estradiol-forming aromatase (CYP19A1) are routinely used in treatment of breast cancer (Chen, 1998). Furthermore, although the growth-inducing effect of androgens are required for formation and normal function of the prostate, overproduction of androgens can contribute substantially to unwanted growth of this tissue e. g. in malignancy. Treatment to suppress androgen action and/or formation have therefore proven very useful in prostate cancer therapy as well as in treatment of benign prostate hyperplasia (Bauman et al., 2006; Hudak et al., 2006). In general, transformation into malignancy may elicit changes in both sex hormone concentrations and effects of sex hormones on growth (Bauman et al., 2006; Vihko et al., 2006). Marked changes in androgen and estrogen metabolism have been reported for many malignant cells. However, abnormalities in hormone synthesis and metabolism are not only found in malignancy. Although the functions of sex hormones formed in the CNS have been much less studied than those of the reproductive organs, disturbed synthesis and/or metabolism of several brain steroids have been described in neurodegenerative disease (Cossec et al., 2010; Schaeffer et al., 2006).

#### **2.3 Cell- and tissue-specific steroid metabolism – role(s) for controlling cellular levels and effects of sex hormones**

Steroid hormone metabolism is not the same everywhere in the body. Due to the different physiological demands that will arise in various tissues and during different conditions, the level of a certain hormone at a given time need to be carefully controlled. Also, not all cells need the same types of hormones. Often, a steroid hormone may potentially undergo several different metabolic pathways. However, one pathway may be particularly active in a

Tissue-Specific Regulation of Sex Hormone Biosynthesis and Metabolism:

as well (Lou et al., 2004, 2010; Tuohimaa et al., 2005).

Fig. 6. Bioactivation of vitamin D3.

Verstuyf et al., 2010).

Novel Aspects on Hormonal Signalling and Maintenance of Cellular Steroid Levels 381

transcription factors (Armas & Heaney, 2011; Atkins et al., 2007; Norman, 2006, 2008;

The active vitamin D3 hormone, 1,25-dihydroxy-vitamin D3 (calcitriol), is formed through metabolic bioactivation by cytochrome P450 (CYP450) enzymes (Jones et al., 1998; Prosser & Jones, 2004; Wikvall, 2001). The activation of vitamin D requires two sequential hydroxylations (Fig. 6). The first step is a 25-hydroxylation of vitamin D3 producing 25 hydroxyvitamin D3 or calcidiol. A number of cytochrome P450-enzymes are capable of performing the 25-hydroxylation and in most organisms studied at least two 25 hydroxylases have been found – the mitochondrial CYP27A1 and the microsomal CYP2R1 (Cheng et al., 2003, 2004; Dahlbäck & Wikvall, 1988; Gascon-Barre et al., 2001). The second bioactivation step is a 1-hydroxylation of calcidiol producing 1,25-dihydroxyvitamin D3 or calcitriol. The 1-hydroxylation is carried out by CYP27B1 (Fu et al., 1997). Production of the circulating 1,25-dihydroxyvitamin D3 is initiated by hepatic 25-hydroxylation followed by renal 1-hydroxylation. The circulating vitamin D hormone has mainly endocrine function e.g. in regulation of calcium homeostasis and maintenance of bone health. The normal serum levels of 25-hydroxyvitamin D3 (20-250 nmol/L) are thousand times higher than the levels of 1,25-dihydroxyvitamin D3 (20-250 pmol/L). 1,25-Dihydroxyvitamin D3 is the most potent form of vitamin D3 but 25-hydroxyvitamin D3 can exert biological effects

Both calcidiol and calcitriol undergo further metabolism by the catabolizing enzyme CYP24A1 (Fig. 7). This enzyme 24-hydroxylates vitamin D metabolites and the 24 hydroxylation is a key point at which degradation of vitamin D begins. 24-Hydroxylated vitamin D metabolites are less biologically active and are then further metabolized by the multicatalytic CYP24A1 via side chain hydroxylation and oxidation to less active substances. Finally, after side chain cleavage, calcitroic acid is formed. Calcitroic acid is then excreted

The expression of both the activating (25- and 1-hydroxylases) and the catabolizing (24 hydroxylase) enzymes by cells of certain tissues, indicates that the multifunctional hormone 1,25-dihydroxyvitamin D3 can be produced locally in some tissues, including cells in the skin, breast, colon, prostate, lung, and various cells of the immune system. The intracellular

into the bile (Makin et al., 1989; Reddy and Tserng, 1989; Zimmerman et al., 2001).

certain tissue but absent in another tissue where a different pathway dominates. Metabolism of a steroid could also lead to a host of metabolites in the same tissue, via different enzymatic steps, all of which can be differentially affected by endogenous or exogenous regulators. Consequently, since the effect(s) of a hormone are dependent on its concentration, the metabolic pathways in a cell or tissue can have a considerable impact on which cellular actions that takes place.

An example of a steroid metabolized into many different compounds is dehydroepiandrosterone (DHEA) (Norlin, 2008; Rainey, 2004). This steroid is reported to have a number of effects of its own but, as described in the Introductory section, it is also essential as a precursor for a number of other hormones, with their own individual effects. The activity of the enzymes and genes involved in formation of these different products can all be differentially regulated.

Our knowledge on the cellular metabolic events that involve steroid hormones is far from sufficient. A proper understanding of these events would substantially increase the possibilities to intervene in physiological processes involving these hormones, such as cellular growth, dysfunctions of the reproductive and immune systems and a number of processes important for brain function. Even though there is a clear link between the serum levels of a hormone and its physiological effect(s), the levels of hormones present in a tissue often differs substantially from the levels measured in serum. Unfortunately, the steroid levels in tissue material are much more problematical to assay than the serum levels. Other complicating factors are the complex interplay of enzymes and regulatory molecules leading to formation of a certain compound and the existence of different cell-types with specific properties within the same tissue.

#### **3. Sex hormones and vitamin D**

In recent years, vitamin D has attracted increasing attention for its ability to regulate numerous genes in several physiological processes. In the following sections, a brief overview of the vitamin D hormone and discussion of data regarding the effects on sex hormone biosynthesis and metabolism will be given.

#### **3.1 Vitamin D is activated to a multifunctional hormone with effects on gene expression**

The prohormone vitamin D3 (cholecalciferol) is synthesized in the skin on exposure to ultraviolet light and is also acquired from the diet (Holick, 1987). Vitamin D is needed for regulation of calcium levels in the body and vitamin D deficiency leads to skeletal diseases such as rickets in children and osteomalacia/osteoporosis in adults (Brown et al., 1999; DeLuca, 2004; Dusso et al., 2005; Jones et al., 1998). The role of vitamin D in human health and disease has received increased recognition in recent years. Although it was previously considered to be limited to regulation of calcium levels, recent data from epidemiological studies and basic sciences have expanded our understanding of its pivotal role in many biological processes. Vitamin D is important not only for endocrine functions, such as calcium homeostasis, but also for autocrine and/or paracrine functions, such as regulation of immune system, brain and fetal development, insulin secretion, apoptosis, cell proliferation and differentiation as well as involvement in cancer and the cardiovascular system. Most of these actions are mediated by transcriptional regulation of target genes through the vitamin D receptor (VDR), a member of the nuclear receptor superfamily of

certain tissue but absent in another tissue where a different pathway dominates. Metabolism of a steroid could also lead to a host of metabolites in the same tissue, via different enzymatic steps, all of which can be differentially affected by endogenous or exogenous regulators. Consequently, since the effect(s) of a hormone are dependent on its concentration, the metabolic pathways in a cell or tissue can have a considerable impact on

An example of a steroid metabolized into many different compounds is dehydroepiandrosterone (DHEA) (Norlin, 2008; Rainey, 2004). This steroid is reported to have a number of effects of its own but, as described in the Introductory section, it is also essential as a precursor for a number of other hormones, with their own individual effects. The activity of the enzymes and genes involved in formation of these different products can

Our knowledge on the cellular metabolic events that involve steroid hormones is far from sufficient. A proper understanding of these events would substantially increase the possibilities to intervene in physiological processes involving these hormones, such as cellular growth, dysfunctions of the reproductive and immune systems and a number of processes important for brain function. Even though there is a clear link between the serum levels of a hormone and its physiological effect(s), the levels of hormones present in a tissue often differs substantially from the levels measured in serum. Unfortunately, the steroid levels in tissue material are much more problematical to assay than the serum levels. Other complicating factors are the complex interplay of enzymes and regulatory molecules leading to formation of a certain compound and the existence of different cell-types with specific

In recent years, vitamin D has attracted increasing attention for its ability to regulate numerous genes in several physiological processes. In the following sections, a brief overview of the vitamin D hormone and discussion of data regarding the effects on sex

The prohormone vitamin D3 (cholecalciferol) is synthesized in the skin on exposure to ultraviolet light and is also acquired from the diet (Holick, 1987). Vitamin D is needed for regulation of calcium levels in the body and vitamin D deficiency leads to skeletal diseases such as rickets in children and osteomalacia/osteoporosis in adults (Brown et al., 1999; DeLuca, 2004; Dusso et al., 2005; Jones et al., 1998). The role of vitamin D in human health and disease has received increased recognition in recent years. Although it was previously considered to be limited to regulation of calcium levels, recent data from epidemiological studies and basic sciences have expanded our understanding of its pivotal role in many biological processes. Vitamin D is important not only for endocrine functions, such as calcium homeostasis, but also for autocrine and/or paracrine functions, such as regulation of immune system, brain and fetal development, insulin secretion, apoptosis, cell proliferation and differentiation as well as involvement in cancer and the cardiovascular system. Most of these actions are mediated by transcriptional regulation of target genes through the vitamin D receptor (VDR), a member of the nuclear receptor superfamily of

**3.1 Vitamin D is activated to a multifunctional hormone with effects on gene** 

which cellular actions that takes place.

all be differentially regulated.

properties within the same tissue.

**expression** 

**3. Sex hormones and vitamin D** 

hormone biosynthesis and metabolism will be given.

transcription factors (Armas & Heaney, 2011; Atkins et al., 2007; Norman, 2006, 2008; Verstuyf et al., 2010).

The active vitamin D3 hormone, 1,25-dihydroxy-vitamin D3 (calcitriol), is formed through metabolic bioactivation by cytochrome P450 (CYP450) enzymes (Jones et al., 1998; Prosser & Jones, 2004; Wikvall, 2001). The activation of vitamin D requires two sequential hydroxylations (Fig. 6). The first step is a 25-hydroxylation of vitamin D3 producing 25 hydroxyvitamin D3 or calcidiol. A number of cytochrome P450-enzymes are capable of performing the 25-hydroxylation and in most organisms studied at least two 25 hydroxylases have been found – the mitochondrial CYP27A1 and the microsomal CYP2R1 (Cheng et al., 2003, 2004; Dahlbäck & Wikvall, 1988; Gascon-Barre et al., 2001). The second bioactivation step is a 1-hydroxylation of calcidiol producing 1,25-dihydroxyvitamin D3 or calcitriol. The 1-hydroxylation is carried out by CYP27B1 (Fu et al., 1997). Production of the circulating 1,25-dihydroxyvitamin D3 is initiated by hepatic 25-hydroxylation followed by renal 1-hydroxylation. The circulating vitamin D hormone has mainly endocrine function e.g. in regulation of calcium homeostasis and maintenance of bone health. The normal serum levels of 25-hydroxyvitamin D3 (20-250 nmol/L) are thousand times higher than the levels of 1,25-dihydroxyvitamin D3 (20-250 pmol/L). 1,25-Dihydroxyvitamin D3 is the most potent form of vitamin D3 but 25-hydroxyvitamin D3 can exert biological effects as well (Lou et al., 2004, 2010; Tuohimaa et al., 2005).

Fig. 6. Bioactivation of vitamin D3.

Both calcidiol and calcitriol undergo further metabolism by the catabolizing enzyme CYP24A1 (Fig. 7). This enzyme 24-hydroxylates vitamin D metabolites and the 24 hydroxylation is a key point at which degradation of vitamin D begins. 24-Hydroxylated vitamin D metabolites are less biologically active and are then further metabolized by the multicatalytic CYP24A1 via side chain hydroxylation and oxidation to less active substances. Finally, after side chain cleavage, calcitroic acid is formed. Calcitroic acid is then excreted into the bile (Makin et al., 1989; Reddy and Tserng, 1989; Zimmerman et al., 2001).

The expression of both the activating (25- and 1-hydroxylases) and the catabolizing (24 hydroxylase) enzymes by cells of certain tissues, indicates that the multifunctional hormone 1,25-dihydroxyvitamin D3 can be produced locally in some tissues, including cells in the skin, breast, colon, prostate, lung, and various cells of the immune system. The intracellular

Tissue-Specific Regulation of Sex Hormone Biosynthesis and Metabolism:

Novel Aspects on Hormonal Signalling and Maintenance of Cellular Steroid Levels 383

Accumulated data have revealed that sex hormone biosynthesis and metabolism may be regulated by vitamin D (Barrera et al., 2007; Krishnan et al., 2010; Lou et al., 2005; Lundqvist et al., 2011; Tanaka et al., 1996). Important reactions in the metabolism of androgens and estrogens are catalyzed by 5-reductase and aromatase (CYP19A1) (Fig. 8). These two key enzymes determine the balance between androgen production and estrogen production. In addition to 5-reductase and aromatase, the 17-hydroxysteroid dehydrogenases are enzymes which regulate intracellular concentrations of active sex steroid hormones. Calcitriol has been found to up-regulate some types of 17-hydroxysteroid dehydrogenase in human prostate

Estrogens are produced from androgenic precursors in a reaction catalyzed by aromatase (CYP19A1) (Figs. 3 and 8). Calcitriol increases aromatase activity in placental cells (Barrera et al., 2007), prostate cells (Lou et al., 2005) and osteoblasts (Tanaka et al., 1996) and vitamin D receptor null mutant mice have a decreased aromatase activity in the ovary, testis and epididymis (Kinuta et al., 2000). Interestingly, it was recently reported that 1,25 dihydroxyvitamin D3 regulates the expression of aromatase in a tissue-selective manner. Thus, calcitriol significantly decreased aromatase expression in human breast cancer cells and adipocytes but caused increased aromatase expression in human osteosarcoma cells and ovarian cancer cells (Krishnan et al., 2010). Calcitriol exerts cell line-specific effects on both estrogen and androgen metabolism, including the production of estrogens and androgens (Lundqvist et al., 2011). In breast cancer MCF-7 cells, aromatase gene expression and estradiol production were decreased, while production of androgens was markedly increased. In human adrenocortical NCI-H295R cells, 1,25-dihydroxyvitamin D3 stimulated aromatase expression and decreased dihydrotestosterone production. In prostate cancer LNCaP cells, aromatase expression increased after the same treatment, as did production of testosterone and dihydrotestosterone (Table 1). These findings are of interest for the research fields of breast cancer and prostate cancer. Vitamin D seems to be involved in the control also of prostate cancer cell growth (Flanagan et al., 2010; Tuohimaa et al., 2005). Analysis of effects of 1,25-dihydroxyvitamin D3 on aromatase promoter activities revealed differences between NCI-H295R cells and MCF-7 cells, where promoter I.3 and promoter I.4 were stimulated and promoter II were down regulated in NCI-H295R cells (Lundqvist et al., 2011) while all three promoters are down regulated in breast cancer MCF-7 cells (Krishnan

The findings that 1,25-dihydroxyvitamin D3 specifically down regulates aromatase gene expression and activity and decreases production of estradiol in breast cancer cells are interesting in the context of 1,25-dihydroxyvitamin D3 as an anti cancer agent (Krishnan et

Fig. 8. Some important enzyme reactions in androgen and estrogen metabolism*.* 

cancer LNCaP and PC3 cells but not in stromal cells (Wang & Tuohimaa, 2007).

et al., 2010).

al., 2010, Lundqvist et al., 2011)

Fig. 7. Metabolism of 1,25-dihydroxyvitamin D3 (calcitriol) by CYP24A1 into less active metabolites. 25-Hydroxyvitamin D3 is also catabolized by CYP24A1 in a similar way.

1,25-dihydroxyvitamin D3 is mainly used in an autocrine manner as a cofactor in the expression of many genes. This autocrine 1,25-dihydroxyvitamin D3 binds to VDR and modifies gene transcription. For example, genes involved in cell proliferation, differentiation and apoptosis are believed to be regulated by the internal 1,25-dihydroxyvitamin D3 of the cell. The activation, effects on gene expression and inactivation of 1,25-dihydroxyvitamin D3, is contained within the host cell. This active form of vitamin D is the major player in the internal autocrine action, but also 25-hydroxyvitamin D3 can be formed within the cell and regulate gene expression (Lou et al., 2004; Tuohimaa et al., 2005; Verstuyf et al., 2010).

Recent data have revealed that vitamin D deficiency in the general population and even among young and healthy people is much more common than previously believed. Vitamin D deficiency not only causes rickets among children but also precipitates and exacerbates osteoporosis among adults and causes the painful bone disease osteomalacia. Interestingly, vitamin D deficiency is associated with increased risks of cardiovascular disease, multiple sclerosis, rheumatoid arthritis, type 1 diabetes mellitus and deadly cancers, such as prostate, breast and colon cancers (Adorini, 2002; Armas & Heaney, 2011; Giovannucci, 2007; Pérez-López, 2008; Schwartz, 2005; Zittermann, 2003).

The vitamin D receptor is widely expressed and it has been suggested that the active vitamin D3 hormone may affect the expression of up to 200 genes in humans (Jones et al., 1998; Norman, 2008; Ramagopalan et al., 2010). It is probable that vitamin D3 may have other roles yet undiscovered.

#### **3.2 Vitamin D3 exerts tissue-specific effects on estrogen and androgen metabolism**

In recent reports from our laboratory, data are presented revealing vitamin D-mediated effects on the production of steroid hormones and expression of crucial steroidogenic enzymes in various cell lines (Lundqvist et al., 2010, 2011). As an example, the active vitamin D hormone 1,25-dihydroxyvitamin D3 decreases the production of dehydroepiandrosterone (DHEA) and DHEA-sulphate in adrenocortical NCI-H295R cells. DHEA is a precursor for both estrogen and androgen production. mRNA levels and enzyme activities for key enzymes in the steroidogenesis, such as CYP17A1 and CYP21A2 (cf. Fig. 1), were found to be altered by 1,25-dihydroxyvitamin D3 treatment (Lundqvist et al., 2010).

Fig. 7. Metabolism of 1,25-dihydroxyvitamin D3 (calcitriol) by CYP24A1 into less active metabolites. 25-Hydroxyvitamin D3 is also catabolized by CYP24A1 in a similar way.

López, 2008; Schwartz, 2005; Zittermann, 2003).

roles yet undiscovered.

1,25-dihydroxyvitamin D3 is mainly used in an autocrine manner as a cofactor in the expression of many genes. This autocrine 1,25-dihydroxyvitamin D3 binds to VDR and modifies gene transcription. For example, genes involved in cell proliferation, differentiation and apoptosis are believed to be regulated by the internal 1,25-dihydroxyvitamin D3 of the cell. The activation, effects on gene expression and inactivation of 1,25-dihydroxyvitamin D3, is contained within the host cell. This active form of vitamin D is the major player in the internal autocrine action, but also 25-hydroxyvitamin D3 can be formed within the cell and regulate gene expression (Lou et al., 2004; Tuohimaa et al., 2005; Verstuyf et al., 2010). Recent data have revealed that vitamin D deficiency in the general population and even among young and healthy people is much more common than previously believed. Vitamin D deficiency not only causes rickets among children but also precipitates and exacerbates osteoporosis among adults and causes the painful bone disease osteomalacia. Interestingly, vitamin D deficiency is associated with increased risks of cardiovascular disease, multiple sclerosis, rheumatoid arthritis, type 1 diabetes mellitus and deadly cancers, such as prostate, breast and colon cancers (Adorini, 2002; Armas & Heaney, 2011; Giovannucci, 2007; Pérez-

The vitamin D receptor is widely expressed and it has been suggested that the active vitamin D3 hormone may affect the expression of up to 200 genes in humans (Jones et al., 1998; Norman, 2008; Ramagopalan et al., 2010). It is probable that vitamin D3 may have other

**3.2 Vitamin D3 exerts tissue-specific effects on estrogen and androgen metabolism**  In recent reports from our laboratory, data are presented revealing vitamin D-mediated effects on the production of steroid hormones and expression of crucial steroidogenic enzymes in various cell lines (Lundqvist et al., 2010, 2011). As an example, the active vitamin D hormone 1,25-dihydroxyvitamin D3 decreases the production of dehydroepiandrosterone (DHEA) and DHEA-sulphate in adrenocortical NCI-H295R cells. DHEA is a precursor for both estrogen and androgen production. mRNA levels and enzyme activities for key enzymes in the steroidogenesis, such as CYP17A1 and CYP21A2 (cf. Fig. 1), were found to be altered by 1,25-dihydroxyvitamin D3 treatment (Lundqvist et al., 2010).

Fig. 8. Some important enzyme reactions in androgen and estrogen metabolism*.* 

Accumulated data have revealed that sex hormone biosynthesis and metabolism may be regulated by vitamin D (Barrera et al., 2007; Krishnan et al., 2010; Lou et al., 2005; Lundqvist et al., 2011; Tanaka et al., 1996). Important reactions in the metabolism of androgens and estrogens are catalyzed by 5-reductase and aromatase (CYP19A1) (Fig. 8). These two key enzymes determine the balance between androgen production and estrogen production. In addition to 5-reductase and aromatase, the 17-hydroxysteroid dehydrogenases are enzymes which regulate intracellular concentrations of active sex steroid hormones. Calcitriol has been found to up-regulate some types of 17-hydroxysteroid dehydrogenase in human prostate cancer LNCaP and PC3 cells but not in stromal cells (Wang & Tuohimaa, 2007).

Estrogens are produced from androgenic precursors in a reaction catalyzed by aromatase (CYP19A1) (Figs. 3 and 8). Calcitriol increases aromatase activity in placental cells (Barrera et al., 2007), prostate cells (Lou et al., 2005) and osteoblasts (Tanaka et al., 1996) and vitamin D receptor null mutant mice have a decreased aromatase activity in the ovary, testis and epididymis (Kinuta et al., 2000). Interestingly, it was recently reported that 1,25 dihydroxyvitamin D3 regulates the expression of aromatase in a tissue-selective manner. Thus, calcitriol significantly decreased aromatase expression in human breast cancer cells and adipocytes but caused increased aromatase expression in human osteosarcoma cells and ovarian cancer cells (Krishnan et al., 2010). Calcitriol exerts cell line-specific effects on both estrogen and androgen metabolism, including the production of estrogens and androgens (Lundqvist et al., 2011). In breast cancer MCF-7 cells, aromatase gene expression and estradiol production were decreased, while production of androgens was markedly increased. In human adrenocortical NCI-H295R cells, 1,25-dihydroxyvitamin D3 stimulated aromatase expression and decreased dihydrotestosterone production. In prostate cancer LNCaP cells, aromatase expression increased after the same treatment, as did production of testosterone and dihydrotestosterone (Table 1). These findings are of interest for the research fields of breast cancer and prostate cancer. Vitamin D seems to be involved in the control also of prostate cancer cell growth (Flanagan et al., 2010; Tuohimaa et al., 2005). Analysis of effects of 1,25-dihydroxyvitamin D3 on aromatase promoter activities revealed differences between NCI-H295R cells and MCF-7 cells, where promoter I.3 and promoter I.4 were stimulated and promoter II were down regulated in NCI-H295R cells (Lundqvist et al., 2011) while all three promoters are down regulated in breast cancer MCF-7 cells (Krishnan et al., 2010).

The findings that 1,25-dihydroxyvitamin D3 specifically down regulates aromatase gene expression and activity and decreases production of estradiol in breast cancer cells are interesting in the context of 1,25-dihydroxyvitamin D3 as an anti cancer agent (Krishnan et al., 2010, Lundqvist et al., 2011)

Tissue-Specific Regulation of Sex Hormone Biosynthesis and Metabolism:

dependent and anti-estrogen resistant breast cancer.

Novel Aspects on Hormonal Signalling and Maintenance of Cellular Steroid Levels 385

Masuda & Jones, 2006). Estrogens and androgens play a role in the pathogenesis of prostate cancer and a large group of all breast cancers involves estrogen-dependent mechanisms, i.e. they rely on estrogens to proliferate (Mathiasen et al., 2002; Sasano et al., 2009). The recent findings indicating regulation of intracellular levels of androgens and estrogens by vitamin

Aromatase (CYP19A1), the enzyme catalyzing the conversion of testosterone to estradiol, is critical for the progression of estrogen receptor-positive breast cancer in postmenopausal women. The aromatase expression is higher in breast cancer tissue than in normal breast tissue and the local estrogen levels in breast cancer tissue are higher than the circulating levels (Chen, 1998; Miller et al., 1990). Regulation of estradiol production and estrogenic signalling are key strategies in breast cancer treatment. Aromatase inhibitors and antiestrogens have therefore become important drugs in breast cancer treatment. Due to its effects on aromatase gene expression and enzyme activity, 1,25-dihydroxyvitamin D3 has been proposed as an interesting substance in breast cancer treatment and prevention. The vitamin D-mediated inhibition of aromatase seems to be tissue specific for breast cells, indicating that it is a potential drug target in treatment aiming to prevent estradiol production in breast but not in other tissues. This would reduce the risk of adverse effects due to effects on peripheral estrogen metabolism (e.g. osteoporosis). The vitamin D-induced tissue-selective regulation of aromatase expression and activity is an interesting strategy to affect estrogen levels in breast without effects on the peripheral estrogen metabolism. High-dose vitamin D treatment will lead to adverse effects e.g. hypercalcemia. Therefore, synthetic vitamin D analogs with less pronounced hypercalcemic effect are potential drugs in treatment aiming to prevent estradiol production in breast but not in other tissues, a strategy that would lead to less adverse effects than the existing treatments. However, the mechanisms for these effects of vitamin D need to be clarified before vitamin D or analogs can be used in breast cancer treatment. It has been reported that the vitamin D analog EB1089 decreases the proliferation of breast cancer cells, especially anti-estrogen resistant breast cancer cells (Christensen et al., 2004; Larsen et al., 2001). EB1089 has undergone clinical trials phase I and II and was found to be a well tolerated substance (Dalhoff et al., 2003). However, it has not yet been tested in clinical trials against breast cancer. The new findings on vitamin D as a tissue-selective modulator of aromatase reinforce the interest for EB1089 as a potential drug in treatment of breast cancer, which may inhibit both estrogen-

Human adrenocortical NCI-H295R cells are widely used as a model for human adrenal cortex. It has been proposed that this adrenocortical carcinoma cell line could be suitable in a screening assay to study the effects of different chemicals on estradiol and testosterone production (Gracia et al., 2006; Hecker et al., 2006, 2007; Higley et al, 2010). We have examined whether adrenocortical NCI-H295R cells react in the same way as prostate cells and breast cells when they are treated with 1,25-dihydroxyvitamin D3. Interestingly, both estrogen and androgen metabolism were affected in a cell line-specific way (Table 1). The largest differences were observed between NCI-H295R cells and MCF-7 cells, where aromatase gene expression, estradiol production, aromatase promoter activity, testosterone production and dihydrotestosterone production were affected in opposite ways in the two cell lines. The discrepancies between NCI-H29R cells and LNCaP cells were smaller, but still noteworthy. Production of both testosterone and dihydrotestosterone was affected differentially in the two cell lines, as was the gene expression of 5-reductase. Our data

D open new possibilities in prevention and treatment of prostate and breast cancer.


Table 1. Effects of calcitriol on estrogen and androgen metabolism. Please note that this table only summarizes the data obtained by our group (Lundqvist et al., 2011). Arrows indicate up- and down-regulation; -, unaltered.

Interestingly, 1,25-dihydroxyvitamin D3 increases androgen production in breast cancer MCF-7 cells (Lundqvist et al., 2011). The production of testosterone was increased by 60% and the production of dihydrotestosterone was increased 4-fold. The markedly increased production of dihydrotestosterone in MCF-7 cells after 1,25-dihydroxyvitamin D3 treatment appears not to be the result of increased 5-reductase expression. An explanation for this effect could be that the decreased aromatase activity increases the concentration of testosterone, which is the precursor for dihydrotestosterone. The increased androgen production in breast cancer cells following vitamin D treatment needs to be studied further to elucidate its potential physiological roles.

The data showing that 1,25-dihydroxyvitamin D3 exerts tissue-specific effects on sex hormone production and metabolism provide important knowledge for further research in the fields of prostate and breast cancer. Prostate and breast are key tissues for estrogenic and androgenic pathways. Vitamin D deficiency is associated with increased risks of prostate and breast cancers (Holick, 2006; Thorne & Campbell, 2008; Bouillon et al., 2006). It is wellknown that the active form of vitamin D, 1,25-dihydroxyvitamin D3, and analogs via binding to the vitamin D receptor exert anti-proliferative and pro-differentiative effects and have therefore been proposed to be of potential use as anti cancer agents (Deeb et al., 2007;

Table 1. Effects of calcitriol on estrogen and androgen metabolism. Please note that this table only summarizes the data obtained by our group (Lundqvist et al., 2011). Arrows indicate

Interestingly, 1,25-dihydroxyvitamin D3 increases androgen production in breast cancer MCF-7 cells (Lundqvist et al., 2011). The production of testosterone was increased by 60% and the production of dihydrotestosterone was increased 4-fold. The markedly increased production of dihydrotestosterone in MCF-7 cells after 1,25-dihydroxyvitamin D3 treatment appears not to be the result of increased 5-reductase expression. An explanation for this effect could be that the decreased aromatase activity increases the concentration of testosterone, which is the precursor for dihydrotestosterone. The increased androgen production in breast cancer cells following vitamin D treatment needs to be studied further

The data showing that 1,25-dihydroxyvitamin D3 exerts tissue-specific effects on sex hormone production and metabolism provide important knowledge for further research in the fields of prostate and breast cancer. Prostate and breast are key tissues for estrogenic and androgenic pathways. Vitamin D deficiency is associated with increased risks of prostate and breast cancers (Holick, 2006; Thorne & Campbell, 2008; Bouillon et al., 2006). It is wellknown that the active form of vitamin D, 1,25-dihydroxyvitamin D3, and analogs via binding to the vitamin D receptor exert anti-proliferative and pro-differentiative effects and have therefore been proposed to be of potential use as anti cancer agents (Deeb et al., 2007;

up- and down-regulation; -, unaltered.

to elucidate its potential physiological roles.

Masuda & Jones, 2006). Estrogens and androgens play a role in the pathogenesis of prostate cancer and a large group of all breast cancers involves estrogen-dependent mechanisms, i.e. they rely on estrogens to proliferate (Mathiasen et al., 2002; Sasano et al., 2009). The recent findings indicating regulation of intracellular levels of androgens and estrogens by vitamin D open new possibilities in prevention and treatment of prostate and breast cancer.

Aromatase (CYP19A1), the enzyme catalyzing the conversion of testosterone to estradiol, is critical for the progression of estrogen receptor-positive breast cancer in postmenopausal women. The aromatase expression is higher in breast cancer tissue than in normal breast tissue and the local estrogen levels in breast cancer tissue are higher than the circulating levels (Chen, 1998; Miller et al., 1990). Regulation of estradiol production and estrogenic signalling are key strategies in breast cancer treatment. Aromatase inhibitors and antiestrogens have therefore become important drugs in breast cancer treatment. Due to its effects on aromatase gene expression and enzyme activity, 1,25-dihydroxyvitamin D3 has been proposed as an interesting substance in breast cancer treatment and prevention. The vitamin D-mediated inhibition of aromatase seems to be tissue specific for breast cells, indicating that it is a potential drug target in treatment aiming to prevent estradiol production in breast but not in other tissues. This would reduce the risk of adverse effects due to effects on peripheral estrogen metabolism (e.g. osteoporosis). The vitamin D-induced tissue-selective regulation of aromatase expression and activity is an interesting strategy to affect estrogen levels in breast without effects on the peripheral estrogen metabolism.

High-dose vitamin D treatment will lead to adverse effects e.g. hypercalcemia. Therefore, synthetic vitamin D analogs with less pronounced hypercalcemic effect are potential drugs in treatment aiming to prevent estradiol production in breast but not in other tissues, a strategy that would lead to less adverse effects than the existing treatments. However, the mechanisms for these effects of vitamin D need to be clarified before vitamin D or analogs can be used in breast cancer treatment. It has been reported that the vitamin D analog EB1089 decreases the proliferation of breast cancer cells, especially anti-estrogen resistant breast cancer cells (Christensen et al., 2004; Larsen et al., 2001). EB1089 has undergone clinical trials phase I and II and was found to be a well tolerated substance (Dalhoff et al., 2003). However, it has not yet been tested in clinical trials against breast cancer. The new findings on vitamin D as a tissue-selective modulator of aromatase reinforce the interest for EB1089 as a potential drug in treatment of breast cancer, which may inhibit both estrogendependent and anti-estrogen resistant breast cancer.

Human adrenocortical NCI-H295R cells are widely used as a model for human adrenal cortex. It has been proposed that this adrenocortical carcinoma cell line could be suitable in a screening assay to study the effects of different chemicals on estradiol and testosterone production (Gracia et al., 2006; Hecker et al., 2006, 2007; Higley et al, 2010). We have examined whether adrenocortical NCI-H295R cells react in the same way as prostate cells and breast cells when they are treated with 1,25-dihydroxyvitamin D3. Interestingly, both estrogen and androgen metabolism were affected in a cell line-specific way (Table 1). The largest differences were observed between NCI-H295R cells and MCF-7 cells, where aromatase gene expression, estradiol production, aromatase promoter activity, testosterone production and dihydrotestosterone production were affected in opposite ways in the two cell lines. The discrepancies between NCI-H29R cells and LNCaP cells were smaller, but still noteworthy. Production of both testosterone and dihydrotestosterone was affected differentially in the two cell lines, as was the gene expression of 5-reductase. Our data

Tissue-Specific Regulation of Sex Hormone Biosynthesis and Metabolism:

Lundqvist J., unpublished results).

et al., 2006; Norlin; 2008; Sharifi, 2010; Vihko et al., 2006).

Novel Aspects on Hormonal Signalling and Maintenance of Cellular Steroid Levels 387

examined the role of CYP7B1-mediated catalysis for activation of the ER (Pettersson et al., 2008, 2010). Our studies, using ER-dependent luciferase reporter systems and ER-target genes, indicate significant stimulation of ER-response by the CYP7B1 substrates 5 androstene-3,17-diol (Aene-diol) and 5-androstane-3,17-diol (3-Adiol), for both ER and . In contrast, the CYP7B1-formed metabolites from these steroids have little or no estrogenic effects, indicating that CYP7B1-mediated metabolism abolishes the ERstimulating effect of these compounds (Pettersson et al., 2010). In the course of our studies we have also found that DHEA induces both ER-dependent and AR-dependent responses in some cell types whereas 7-hydroxy-DHEA has no or diminished effect (Norlin M. &

Our findings seem to indicate that actions by CYP7B1 might be a way to decrease estrogenic response, at least in some tissues. One of these may be the prostate, where signalling via ER has been reported to play a role in growth suppression. Gustafsson and collaborators proposed a pathway for hormonal control of proliferation where the role of CYP7B1 would be to counteract anti-proliferative action of ER by metabolizing its ligand 3-Adiol (Weihua et al., 2002). This concept is supported by findings indicating that prostates of CYP7B1-/ mice are hypoproliferative. The roles of estrogens and estrogen receptors in prostate growth are however not well understood (Morani et al., 2008; Prins & Korach, 2008). Interestingly, Olsson et al. (2007) reported high expression of CYP7B1 protein in human high-grade prostatic intraepithelial neoplasia and adenocarcinomas. Enzymatic events of potential importance for intraprostatic hormone levels are of course not limited to actions by CYP7B1. Other enzymes of relevance include e. g. the conjugating enzymes, particularly some of the UDP-glucuronosyltransferases (UGT) expressed in prostate (Barbier & Bélanger, 2008). Also, the enzymes known to form important hormones in this tissue such as CYP17A1, 5 reductase and the 3- and 17-hydroxysteroid dehydrogenases have attracted interest as potential or existing targets for therapy aimed at controlling cellular hormone levels (Hudak

Considering its role in metabolism of ER ligands, CYP7B1 has been linked to estrogenic action by several studies and investigators (Petterson et al., 2010; Sugiyama et al., 2009; Weihua et al., 2002). However, the actions of CYP7B1 in sex hormone metabolism do not seem to be limited to estrogenic signalling. For instance, some of the steroids that are substrates for this enzyme may affect both estrogen and androgen receptors. This includes DHEA and Aenediol which are reported to be able to trigger both ER and AR signalling. It seems likely that the actual effects of these steroids in vivo may strongly depend on their local concentration. In addition, the presence or absence of other hormone ligands with higher affinitity for the receptor as well as the effects of tissue-specific comodulators should play important roles. These different possibilities open for enzymatic control of cell-specific actions, depending on e. g. comodulator expression, substrate availability, and substrate

The most potent androgenic hormone in the human body is dihydrotestosterone (DHT). Maintenance of normal DHT levels is essential for a number of physiological processes. On the other hand, excess levels of androgens, which strongly stimulate growth, may have a negative impact in disease. We recently identified a previously unknown androgenic substrate for CYP7B1 which is a metabolite of DHT (Pettersson et al., 2009) (Fig. 10). This steroid, 5-androstane-3,17-diol (3-Adiol), can itself induce androgenic responses, but since the effects of 3-Adiol are weaker that those of DHT, conversion into 3-Adiol is

competition (Pettersson et al., 2008; Shapiro et al., 2011; Sugiyama et al., 2009).

show that NCI-H295R cells respond in a different way than cells derived from important target tissues in estrogen and androgen production and metabolism (Lundqvist et al., 2011). These differences between NCI-H295R cells and cells derived from key endocrine target tissues need to be addressed and clarified if NCI-H295R cells should be used as a model for effects of different chemicals on estrogen and androgen metabolism.

#### **4. Actions of CYP7B1 - potential role(s) for the levels and effects of estrogens, androgens and neurosteroids**

In recent years we have carried out several studies on the effects and regulation of catalytic reactions mediated by CYP7B1, a widely expressed enzyme with a number of steroid substrates including DHEA. CYP7B1-mediated catalysis leads to formation of 6- or 7 hydroxymetabolites, mainly 7-hydroxyderivatives (Pettersson et al, 2008; Rose et al., 1997; Tang et al., 2006; Stiles et al., 2009; Wu et al., 1999) (Fig. 9). This enzyme has been associated with several physiological processes, including brain function, immune system, cholesterol homeostasis and cellular viability and growth. Scientific publications in various areas have linked altered CYP7B1 levels and/or function to neurodegenerative processes, arthritis, and prostate cancer (Dulos et al., 2004; Olsson et al., 2007; Tsaousidou et al., 2008; Yau et al., 2003). However, the manner in which CYP7B1 affects these processes are in many cases unclear.

Fig. 9. CYP7B1-mediated catalytic reactions.

Substrates for CYP7B1 are neurosteroids, cholesterol derivatives and sex hormones, including some of the ligands for the estrogens receptors (ER). In recent studies we

show that NCI-H295R cells respond in a different way than cells derived from important target tissues in estrogen and androgen production and metabolism (Lundqvist et al., 2011). These differences between NCI-H295R cells and cells derived from key endocrine target tissues need to be addressed and clarified if NCI-H295R cells should be used as a model for

In recent years we have carried out several studies on the effects and regulation of catalytic reactions mediated by CYP7B1, a widely expressed enzyme with a number of steroid substrates including DHEA. CYP7B1-mediated catalysis leads to formation of 6- or 7 hydroxymetabolites, mainly 7-hydroxyderivatives (Pettersson et al, 2008; Rose et al., 1997; Tang et al., 2006; Stiles et al., 2009; Wu et al., 1999) (Fig. 9). This enzyme has been associated with several physiological processes, including brain function, immune system, cholesterol homeostasis and cellular viability and growth. Scientific publications in various areas have linked altered CYP7B1 levels and/or function to neurodegenerative processes, arthritis, and prostate cancer (Dulos et al., 2004; Olsson et al., 2007; Tsaousidou et al., 2008; Yau et al., 2003). However, the manner in which CYP7B1 affects these processes are in many cases

Substrates for CYP7B1 are neurosteroids, cholesterol derivatives and sex hormones, including some of the ligands for the estrogens receptors (ER). In recent studies we

effects of different chemicals on estrogen and androgen metabolism.

**estrogens, androgens and neurosteroids** 

Fig. 9. CYP7B1-mediated catalytic reactions.

unclear.

**4. Actions of CYP7B1 - potential role(s) for the levels and effects of** 

examined the role of CYP7B1-mediated catalysis for activation of the ER (Pettersson et al., 2008, 2010). Our studies, using ER-dependent luciferase reporter systems and ER-target genes, indicate significant stimulation of ER-response by the CYP7B1 substrates 5 androstene-3,17-diol (Aene-diol) and 5-androstane-3,17-diol (3-Adiol), for both ER and . In contrast, the CYP7B1-formed metabolites from these steroids have little or no estrogenic effects, indicating that CYP7B1-mediated metabolism abolishes the ERstimulating effect of these compounds (Pettersson et al., 2010). In the course of our studies we have also found that DHEA induces both ER-dependent and AR-dependent responses in some cell types whereas 7-hydroxy-DHEA has no or diminished effect (Norlin M. & Lundqvist J., unpublished results).

Our findings seem to indicate that actions by CYP7B1 might be a way to decrease estrogenic response, at least in some tissues. One of these may be the prostate, where signalling via ER has been reported to play a role in growth suppression. Gustafsson and collaborators proposed a pathway for hormonal control of proliferation where the role of CYP7B1 would be to counteract anti-proliferative action of ER by metabolizing its ligand 3-Adiol (Weihua et al., 2002). This concept is supported by findings indicating that prostates of CYP7B1-/ mice are hypoproliferative. The roles of estrogens and estrogen receptors in prostate growth are however not well understood (Morani et al., 2008; Prins & Korach, 2008). Interestingly, Olsson et al. (2007) reported high expression of CYP7B1 protein in human high-grade prostatic intraepithelial neoplasia and adenocarcinomas. Enzymatic events of potential importance for intraprostatic hormone levels are of course not limited to actions by CYP7B1. Other enzymes of relevance include e. g. the conjugating enzymes, particularly some of the UDP-glucuronosyltransferases (UGT) expressed in prostate (Barbier & Bélanger, 2008). Also, the enzymes known to form important hormones in this tissue such as CYP17A1, 5 reductase and the 3- and 17-hydroxysteroid dehydrogenases have attracted interest as potential or existing targets for therapy aimed at controlling cellular hormone levels (Hudak et al., 2006; Norlin; 2008; Sharifi, 2010; Vihko et al., 2006).

Considering its role in metabolism of ER ligands, CYP7B1 has been linked to estrogenic action by several studies and investigators (Petterson et al., 2010; Sugiyama et al., 2009; Weihua et al., 2002). However, the actions of CYP7B1 in sex hormone metabolism do not seem to be limited to estrogenic signalling. For instance, some of the steroids that are substrates for this enzyme may affect both estrogen and androgen receptors. This includes DHEA and Aenediol which are reported to be able to trigger both ER and AR signalling. It seems likely that the actual effects of these steroids in vivo may strongly depend on their local concentration. In addition, the presence or absence of other hormone ligands with higher affinitity for the receptor as well as the effects of tissue-specific comodulators should play important roles. These different possibilities open for enzymatic control of cell-specific actions, depending on e. g. comodulator expression, substrate availability, and substrate competition (Pettersson et al., 2008; Shapiro et al., 2011; Sugiyama et al., 2009).

The most potent androgenic hormone in the human body is dihydrotestosterone (DHT). Maintenance of normal DHT levels is essential for a number of physiological processes. On the other hand, excess levels of androgens, which strongly stimulate growth, may have a negative impact in disease. We recently identified a previously unknown androgenic substrate for CYP7B1 which is a metabolite of DHT (Pettersson et al., 2009) (Fig. 10). This steroid, 5-androstane-3,17-diol (3-Adiol), can itself induce androgenic responses, but since the effects of 3-Adiol are weaker that those of DHT, conversion into 3-Adiol is

Tissue-Specific Regulation of Sex Hormone Biosynthesis and Metabolism:

**5.1 CYP7B1 is regulated by sex hormones** 

\* Involvement of the PI3K/Akt pathway (Tang et al., 2008)

2006, 2008; Tang & Norlin, 2006; Fex Svenningsen et al., 2011)

Novel Aspects on Hormonal Signalling and Maintenance of Cellular Steroid Levels 389

endogenous and synthetic estrogens strongly decreases CYP7B1-mediated DHEA hydroxylation in primary cultures of rat astrocytes (Fex Svenningsen et al., 2011). Since CYP7B1-mediated metabolism appears to be the main pathway for DHEA metabolism in these cells, we believe that estrogenic effects on this enzyme may lead to increase of the levels of DHEA via suppression of its metabolism. This could be one of the potential mechanisms whereby estrogens affect CNS, and may play a role for estrogen-dependent

protection of CNS cells against injury (Brann et al., 2007; Melcangi & Panzica 2006).

**5. Role of sex hormones in regulation of steroid-metabolizing enzymes** 

Our studies, carried out in several different cell types, indicate regulatory effects of both estrogen and androgens on CYP7B1 expression (Fex Svenningsen et al., 2011; Tang et al., 2006; Tang & Norlin, 2006) (Fig. 11). As described in the previous section, our results in primary rat CNS cultures showed decreased CYP7B1-mediated catalytic activity following estrogen treatment (Fex Svenningsen et al., 2011). In the studies on astrocytes we also found suppressive effects of estrogen on CYP7B1 mRNA levels. These findings, indicating suppression of the CYP7B1 gene, are different compared to some of our previous data concerning estrogen-dependent regulation of CYP7B1 in human kidney- and liver-derived cell lines (Tang et al., 2006, 2008). In kidney-derived HEK293 cells and liver-derived HepG2

Fig. 11. Simplified overview of our findings on the effects of estrogens on the regulation of CYP7B1 in different cell types. For more information see text and references (Tang et al.,

considered a means to reduce DHT-mediated effects on cell growth and other processes. 3- Adiol can easily be converted back to DHT and is believed to serve as a source for this hormone (Auchus, 2004; Penning et al., 2000). 3-Adiol is also believed to be of importance in the CNS, where it can modulate the action of gamma-amino butyric acid A (GABAA) receptors and is reported to have anticonvulsant and analgesic properties (Reddy 2004; Frye, 2007).

Fig. 10. CYP7B1-mediated metabolism of 5-androstane-3,17-diol (3-Adiol)

Local formation and functions in the CNS are features shared by other, previously characterized, substrates for CYP7B1, including DHEA, pregnenolone, Aenediol and 27 hydroxycholesterol, a cholesterol derivative believed to serve as a regulator of cholesterol homeostasis (Norlin & Wikvall 2007, Pikuleva; 2006). An important role for CYP7B1 in brain physiology is indicated by the recent studies revealing that disturbed function of this enzyme is linked to a human motor-neuron degenerative disease. Tsaousidou et al. (2008) first showed that hereditary spastic paraplegia (HSP) is associated with mutations in the CYP7B1 gene. Mutations in the coding region of this gene, which affects the functionality of the enzyme, is believed to be a frequent cause of this disease. The etiology and molecular mechanisms that underlies hereditary spastic paraplegia are however not known. It has been proposed that the mutated forms of CYP7B1 in patients suffering from HSP might lead to an abnormal brain cholesterol homeostasis due to an increase in 27-hydroxycholesterol levels (Tsaousidou et al., 2008). Despite the role of this steroid for maintenance of cholesterol homeostasis, 27-hydroxycholesterol negatively affects viability of some cells (Dasari et al., 2010). This steroid can also modulate estrogen receptor signalling in some tissues and has been described as an endogenous SERM (Du Sell et al. 2008; Umetani et al., 2008) However, it is also possible that the pathogenic basis for hereditary spastic paraplegia is connected to abnormal levels of other CYP7B1 substrates present in the brain, including DHEA. Although there is still much to be learned in this field, brain steroids are believed to influence several aspects of CNS function (Charalampopoulos et al., 2006; Maninger et al., 2009; Melcangi & Panzica, 2006). Various neurosteroids are considered to be involved in e. g. neuronal development, regulation of inflammatory responses, effects on cellular viability and modulation of the actions of various neurotransmitter receptors.

The concept of widely varying cell- and tissue-specific steroid metabolism is illustrated by the differences in metabolism of DHEA in different CNS cell types. Thus, whereas rat microglia, a cell type important for brain immune function, is reported to exclusively convert this steroid to Aenediol, the major route for DHEA metabolism in rat astrocytes seems to be 7-hydroxylation, carried out by CYP7B1 (Fex Svenningsen et al., 2011; Jellinck et al., 2001; Jellinck et al, 2007). In contrast, the pathways using DHEA as an obligatory precursor for formation of testosterone and estradiol (see Introductory section), are much more dominant in cells outside the brain and are indeed essential for the development and functions of many endocrine organs. Very recently, we found that treatment with endogenous and synthetic estrogens strongly decreases CYP7B1-mediated DHEA hydroxylation in primary cultures of rat astrocytes (Fex Svenningsen et al., 2011). Since CYP7B1-mediated metabolism appears to be the main pathway for DHEA metabolism in these cells, we believe that estrogenic effects on this enzyme may lead to increase of the levels of DHEA via suppression of its metabolism. This could be one of the potential mechanisms whereby estrogens affect CNS, and may play a role for estrogen-dependent protection of CNS cells against injury (Brann et al., 2007; Melcangi & Panzica 2006).

### **5. Role of sex hormones in regulation of steroid-metabolizing enzymes**

#### **5.1 CYP7B1 is regulated by sex hormones**

388 Sex Hormones

considered a means to reduce DHT-mediated effects on cell growth and other processes. 3- Adiol can easily be converted back to DHT and is believed to serve as a source for this hormone (Auchus, 2004; Penning et al., 2000). 3-Adiol is also believed to be of importance in the CNS, where it can modulate the action of gamma-amino butyric acid A (GABAA) receptors and is reported to have anticonvulsant and analgesic properties (Reddy 2004; Frye, 2007).

Fig. 10. CYP7B1-mediated metabolism of 5-androstane-3,17-diol (3-Adiol)

modulation of the actions of various neurotransmitter receptors.

Local formation and functions in the CNS are features shared by other, previously characterized, substrates for CYP7B1, including DHEA, pregnenolone, Aenediol and 27 hydroxycholesterol, a cholesterol derivative believed to serve as a regulator of cholesterol homeostasis (Norlin & Wikvall 2007, Pikuleva; 2006). An important role for CYP7B1 in brain physiology is indicated by the recent studies revealing that disturbed function of this enzyme is linked to a human motor-neuron degenerative disease. Tsaousidou et al. (2008) first showed that hereditary spastic paraplegia (HSP) is associated with mutations in the CYP7B1 gene. Mutations in the coding region of this gene, which affects the functionality of the enzyme, is believed to be a frequent cause of this disease. The etiology and molecular mechanisms that underlies hereditary spastic paraplegia are however not known. It has been proposed that the mutated forms of CYP7B1 in patients suffering from HSP might lead to an abnormal brain cholesterol homeostasis due to an increase in 27-hydroxycholesterol levels (Tsaousidou et al., 2008). Despite the role of this steroid for maintenance of cholesterol homeostasis, 27-hydroxycholesterol negatively affects viability of some cells (Dasari et al., 2010). This steroid can also modulate estrogen receptor signalling in some tissues and has been described as an endogenous SERM (Du Sell et al. 2008; Umetani et al., 2008) However, it is also possible that the pathogenic basis for hereditary spastic paraplegia is connected to abnormal levels of other CYP7B1 substrates present in the brain, including DHEA. Although there is still much to be learned in this field, brain steroids are believed to influence several aspects of CNS function (Charalampopoulos et al., 2006; Maninger et al., 2009; Melcangi & Panzica, 2006). Various neurosteroids are considered to be involved in e. g. neuronal development, regulation of inflammatory responses, effects on cellular viability and

The concept of widely varying cell- and tissue-specific steroid metabolism is illustrated by the differences in metabolism of DHEA in different CNS cell types. Thus, whereas rat microglia, a cell type important for brain immune function, is reported to exclusively convert this steroid to Aenediol, the major route for DHEA metabolism in rat astrocytes seems to be 7-hydroxylation, carried out by CYP7B1 (Fex Svenningsen et al., 2011; Jellinck et al., 2001; Jellinck et al, 2007). In contrast, the pathways using DHEA as an obligatory precursor for formation of testosterone and estradiol (see Introductory section), are much more dominant in cells outside the brain and are indeed essential for the development and functions of many endocrine organs. Very recently, we found that treatment with Our studies, carried out in several different cell types, indicate regulatory effects of both estrogen and androgens on CYP7B1 expression (Fex Svenningsen et al., 2011; Tang et al., 2006; Tang & Norlin, 2006) (Fig. 11). As described in the previous section, our results in primary rat CNS cultures showed decreased CYP7B1-mediated catalytic activity following estrogen treatment (Fex Svenningsen et al., 2011). In the studies on astrocytes we also found suppressive effects of estrogen on CYP7B1 mRNA levels. These findings, indicating suppression of the CYP7B1 gene, are different compared to some of our previous data concerning estrogen-dependent regulation of CYP7B1 in human kidney- and liver-derived cell lines (Tang et al., 2006, 2008). In kidney-derived HEK293 cells and liver-derived HepG2

\* Involvement of the PI3K/Akt pathway (Tang et al., 2008)

Fig. 11. Simplified overview of our findings on the effects of estrogens on the regulation of CYP7B1 in different cell types. For more information see text and references (Tang et al., 2006, 2008; Tang & Norlin, 2006; Fex Svenningsen et al., 2011)

Tissue-Specific Regulation of Sex Hormone Biosynthesis and Metabolism:

Fig. 13. Formation and metabolism of 27-hydroxycholesterol.

for regulation of the human CYP27A1 gene are of great interest.

different effects observed with different cell types and receptor subtypes.

**5.2 CYP27A1 is regulated by sex hormones** 

below.

Novel Aspects on Hormonal Signalling and Maintenance of Cellular Steroid Levels 391

above, studies on this enzyme in the CNS point towards a potential role connected to neuroprotective events (Bean et al., 2001; Fex Svenningsen et al., 2011; Tsaousidou et al., 2008). Although CYP7B1 expression is abundant in tissues of humans and animals most immortalized cell lines lose their expression of CYP7B1. The reason for this is not known. The estrogen-mediated regulation of CYP7B1 is of interest also because this enzyme catabolizes 27-hydroxycholesterol, recently identified as the first endogenous selective estrogen receptor modulator, SERM (Fig. 13). 27-Hydroxycholesterol is produced from cholesterol by the sterol 27-hydroxylase CYP27A1. Interestingly, CYP27A1 is also regulated by estrogens and androgens. The regulation of CYP27A1 by sex hormones is discussed

The sterol 27-hydroxylase CYP27A1 is an enzyme with several important roles (Norlin & Wikvall, 2007). CYP27A1 regulates cholesterol homeostasis including bile acid biosynthesis, cholesterol transport and cholesterol elimination. CYP27A1 is also a vitamin D 25 hydroxylase, catalyzing the first step in the bioactivation of vitamin D into the multifunctional hormone 1,25-dihydroxyvitamin D (Fig. 6). CYP27A1 is essential for the production of 27-hydroxycholesterol (Fig. 13), an oxysterol which has recently been identified as an endogenous selective estrogen receptor modulator, SERM (DuSell et al., 2008, 2010; Umetani et al., 2007, 2011). Considering these important functions, mechanisms

In studies carried out by our laboratory, we found that the cellular mRNA levels, enzyme activity and promoter activity of CYP27A1 are regulated by estrogens and androgens (Norlin et al., 2011; Tang et al., 2007) (Figs. 14 and 15). The responses to sex hormones are different in various cell lines and cells from different tissues. The hormonal action on the CYP27A1 promoter appears to be complex. In addition to cell-dependent effects, there are also differences between receptor subtypes and different promoter deletion constructs. For instance, whereas ER suppressed the full-length promoter in HepG2 cells, deletion of a 3.4 kb long part of the promoter resulted in the opposite response. On the contrary, the response of different promoter constructs to ER was similar. The data available indicate that the CYP27A1 promoter contains sequences able to mediate both stimulation and suppression by ER (Tang et al., 2007). ER-mediated regulation of transcription is often associated with binding of ER homodimers to estrogen response elements (ERE) in target promotors. However, regulation by ER can also involve interaction with sequences containing Sp1 and activator protein (AP-1) sites (Safe, 2001; Schultz et al., 2005). Interestingly, it has been reported that ER-mediated regulation involving AP-1 sites may lead to opposite effects depending on ER subtype (Paech et al., 1997). As mentioned above, the CYP27A1 promoter contains several putative binding sites for ER, AP-1 and Sp1. It seems possible that cell-specific interactions with coactivators may be the reason for the

cells, we have found that estradiol up-regulates CYP7B1 gene expression in the presence of estrogen receptors. Without overexpression of ER however, the kidney HEK293 cells, which have very low endogenous ER expression, react similarly as the astrocytes to estradiol treatment, suggesting that there might be both ER-dependent and ER-independent pathways for estrogen-mediated regulation of CYP7B1. Other possibilities are tissue- and/or speciesspecific differences in regulatory mechanisms. Species differences in enzymes may include enzyme localization. Data obtained by us and others show higher CYP7B1 levels in rat astrocytes than in rat neurons, whereas in humans CYP7B1 expression has been reported to be predominantly located in neurons (Fex Svenningsen et al., 2011; Trap et al., 2005; Zhang et al., 1997). However, in similarity with our results in human renal and hepatic cells, estrogen receptor-mediated upregulation of CYP7B1 has been shown also in mouse kidney and liver, indicating a similar effect in rodents as in humans and supporting an in vivo role for this regulatory mechanism (Jelinsky et al., 2003; Yamamoto et al., 2006).

Thus, from the results obtained by us and other investigators it seems that CYP7B1 not only affects hormonal actions but is itself regulated by hormones. Our studies have shown effects on CYP7B1 transcription and/or activity by both estrogens and androgens, although we observed differences depending on cell type (Fex Svenningsen et al., 2011; Tang et al., 2006, 2008; Tang & Norlin 2006). This may reflect different functions of CYP7B1 in different tissues or cells. In cell types where formation of estradiol is quantitatively important, the observed ER-mediated induction by estradiol on the CYP7B1-mediated pathway may be a means to divert DHEA from estradiol production, by increasing the amount of DHEA metabolized to 7-hydroxyDHEA (Tang et al., 2006) (Fig. 12). In this way, estradiolmediated regulation of CYP7B1 may decrease the levels of DHEA available for synthesis of estradiol in some tissues, functioning as a feed-back mechanism to balance the amount of estradiol formed. This could be of particular importance during fetal development as placental estrogen formation is dependent on C19-steroid precursors such as DHEA which is secreted in large amounts by the fetal adrenal cortex. Although there are few data on the role(s) of CYP7B1 in fetal development, studies on tissues from both humans and rodents show markedly higher CYP7B1 mRNA levels in extrahepatic fetal tissues compared with the corresponding adult ones (Bean et al., 2001; Tang et al., 2006).

$$\begin{array}{ccc} \text{7a-OH-} \overset{\text{CYP7BI}}{\underset{\text{DHA}}{\rightleftharpoons}} \text{DHA} & & \\ \text{DHA} & \downarrow & \\ & \downarrow & \\ \text{Testosterone} & \xrightarrow{\text{CYP19A1}} \text{Estradiol} \\ 5a\text{-}Reductase & \\ & \text{DHT} & \\ \end{array}$$

#### Fig. 12. Some of the alternate pathways for DHEA metabolism

Our data on estrogen-mediated upregulation of the CYP7B1 gene promoter in liver-derived human HepG2 cells showed involvement of the Akt/PI3K (phosphoinositide 3-kinase) cascade in the ER-mediated effects on CYP7B1 (Tang et al., 2008). The link between CYP7B1 and this signalling pathway, which is known to be of importance for cellular survival, suggests a possible connection between CYP7B1 action and viability. Also, as outlined above, studies on this enzyme in the CNS point towards a potential role connected to neuroprotective events (Bean et al., 2001; Fex Svenningsen et al., 2011; Tsaousidou et al., 2008). Although CYP7B1 expression is abundant in tissues of humans and animals most immortalized cell lines lose their expression of CYP7B1. The reason for this is not known.

The estrogen-mediated regulation of CYP7B1 is of interest also because this enzyme catabolizes 27-hydroxycholesterol, recently identified as the first endogenous selective estrogen receptor modulator, SERM (Fig. 13). 27-Hydroxycholesterol is produced from cholesterol by the sterol 27-hydroxylase CYP27A1. Interestingly, CYP27A1 is also regulated by estrogens and androgens. The regulation of CYP27A1 by sex hormones is discussed below.

Fig. 13. Formation and metabolism of 27-hydroxycholesterol.

#### **5.2 CYP27A1 is regulated by sex hormones**

390 Sex Hormones

cells, we have found that estradiol up-regulates CYP7B1 gene expression in the presence of estrogen receptors. Without overexpression of ER however, the kidney HEK293 cells, which have very low endogenous ER expression, react similarly as the astrocytes to estradiol treatment, suggesting that there might be both ER-dependent and ER-independent pathways for estrogen-mediated regulation of CYP7B1. Other possibilities are tissue- and/or speciesspecific differences in regulatory mechanisms. Species differences in enzymes may include enzyme localization. Data obtained by us and others show higher CYP7B1 levels in rat astrocytes than in rat neurons, whereas in humans CYP7B1 expression has been reported to be predominantly located in neurons (Fex Svenningsen et al., 2011; Trap et al., 2005; Zhang et al., 1997). However, in similarity with our results in human renal and hepatic cells, estrogen receptor-mediated upregulation of CYP7B1 has been shown also in mouse kidney and liver, indicating a similar effect in rodents as in humans and supporting an in vivo role for this

Thus, from the results obtained by us and other investigators it seems that CYP7B1 not only affects hormonal actions but is itself regulated by hormones. Our studies have shown effects on CYP7B1 transcription and/or activity by both estrogens and androgens, although we observed differences depending on cell type (Fex Svenningsen et al., 2011; Tang et al., 2006, 2008; Tang & Norlin 2006). This may reflect different functions of CYP7B1 in different tissues or cells. In cell types where formation of estradiol is quantitatively important, the observed ER-mediated induction by estradiol on the CYP7B1-mediated pathway may be a means to divert DHEA from estradiol production, by increasing the amount of DHEA metabolized to 7-hydroxyDHEA (Tang et al., 2006) (Fig. 12). In this way, estradiolmediated regulation of CYP7B1 may decrease the levels of DHEA available for synthesis of estradiol in some tissues, functioning as a feed-back mechanism to balance the amount of estradiol formed. This could be of particular importance during fetal development as placental estrogen formation is dependent on C19-steroid precursors such as DHEA which is secreted in large amounts by the fetal adrenal cortex. Although there are few data on the role(s) of CYP7B1 in fetal development, studies on tissues from both humans and rodents show markedly higher CYP7B1 mRNA levels in extrahepatic fetal tissues compared with the

regulatory mechanism (Jelinsky et al., 2003; Yamamoto et al., 2006).

corresponding adult ones (Bean et al., 2001; Tang et al., 2006).

Fig. 12. Some of the alternate pathways for DHEA metabolism

Our data on estrogen-mediated upregulation of the CYP7B1 gene promoter in liver-derived human HepG2 cells showed involvement of the Akt/PI3K (phosphoinositide 3-kinase) cascade in the ER-mediated effects on CYP7B1 (Tang et al., 2008). The link between CYP7B1 and this signalling pathway, which is known to be of importance for cellular survival, suggests a possible connection between CYP7B1 action and viability. Also, as outlined The sterol 27-hydroxylase CYP27A1 is an enzyme with several important roles (Norlin & Wikvall, 2007). CYP27A1 regulates cholesterol homeostasis including bile acid biosynthesis, cholesterol transport and cholesterol elimination. CYP27A1 is also a vitamin D 25 hydroxylase, catalyzing the first step in the bioactivation of vitamin D into the multifunctional hormone 1,25-dihydroxyvitamin D (Fig. 6). CYP27A1 is essential for the production of 27-hydroxycholesterol (Fig. 13), an oxysterol which has recently been identified as an endogenous selective estrogen receptor modulator, SERM (DuSell et al., 2008, 2010; Umetani et al., 2007, 2011). Considering these important functions, mechanisms for regulation of the human CYP27A1 gene are of great interest.

In studies carried out by our laboratory, we found that the cellular mRNA levels, enzyme activity and promoter activity of CYP27A1 are regulated by estrogens and androgens (Norlin et al., 2011; Tang et al., 2007) (Figs. 14 and 15). The responses to sex hormones are different in various cell lines and cells from different tissues. The hormonal action on the CYP27A1 promoter appears to be complex. In addition to cell-dependent effects, there are also differences between receptor subtypes and different promoter deletion constructs. For instance, whereas ER suppressed the full-length promoter in HepG2 cells, deletion of a 3.4 kb long part of the promoter resulted in the opposite response. On the contrary, the response of different promoter constructs to ER was similar. The data available indicate that the CYP27A1 promoter contains sequences able to mediate both stimulation and suppression by ER (Tang et al., 2007). ER-mediated regulation of transcription is often associated with binding of ER homodimers to estrogen response elements (ERE) in target promotors. However, regulation by ER can also involve interaction with sequences containing Sp1 and activator protein (AP-1) sites (Safe, 2001; Schultz et al., 2005). Interestingly, it has been reported that ER-mediated regulation involving AP-1 sites may lead to opposite effects depending on ER subtype (Paech et al., 1997). As mentioned above, the CYP27A1 promoter contains several putative binding sites for ER, AP-1 and Sp1. It seems possible that cell-specific interactions with coactivators may be the reason for the different effects observed with different cell types and receptor subtypes.

Tissue-Specific Regulation of Sex Hormone Biosynthesis and Metabolism:

\* Involvement of the JNK/c-jun kinase signalling pathway (Norlin et al., 2011).

**5.3 Effects of sex hormones on vitamin D metabolism** 

Fig. 15. Simplified overview of our findings on the effects of androgens on the gene

regulation of CYP27A1 in different cell types. For more information see text and references

Activated vitamin D metabolites, that can be formed by CYP27A1, are known to have beneficial effects on cell growth in extrahepatic tissues, such as in prostate cells and prostate cancer cells. As discussed above, novel data indicate that estrogens and androgens might regulate the intracellular levels of active hydroxyvitamin D3 metabolites in prostate cells via regulation of CYP27A1 gene expression (Norlin et al., 2011; Tang et al., 2007). Another observation indicating that androgens can influence intracellular levels of active vitamin D metabolites was reported some years ago (Lou and Tuohimaa, 2006). These authors demonstrated that dihydrotestosterone (DHT) significantly suppressed the expression of the catabolizing enzyme CYP24A1 in androgen-sensitive prostate cancer LNCaP cells. Their data demonstrated that DHT enhances the antiproliferative activity of vitamin D3 hormones by inhibiting their inactivating enzyme at physiological concentration of androgen. They suggested that the combined use of androgen and vitamin D3 metabolites could be a

Recent research using cell models has revealed novel and tissue-specific actions of sex hormones and vitamin D3 in the regulation of enzymes in steroid metabolism. The active vitamin D hormone, calcitriol, has been found to affect genes in androgen and estrogen metabolism. Sex hormones regulate genes in neurosteroid metabolism and cholesterol

remain to be established.

(Norlin et al., 2011; Tang et al., 2007).

promising treatment for prostate cancer.

**6. Concluding remarks** 

Novel Aspects on Hormonal Signalling and Maintenance of Cellular Steroid Levels 393

activity. The results also show that effects of sex hormones on CYP27A1 regulation are different in non-cancerous prostate RWPE-1 compared with prostate cancer LNCaP cells. Whether this difference in regulatory effects is due to different properties of different prostate cell lines or to altered properties of the CYP27A1 regulation in prostate cancer

Fig. 14. Simplified overview of our findings on the effects of estrogens on the gene regulation of CYP27A1 in different cell types. For more information see text and references (Norlin et al., 2011; Tang et al., 2007).

Our studies on the mechanisms for the hormonal regulation of CYP27A1 have indicated the involvement of the JNK (c-jun N-terminal kinase)/c-jun pathway in androgen-mediated regulation of this enzyme (Norlin et al., 2011). It has been reported that the androgen receptor (AR) is phosphorylated by JNK and that stress kinase signaling regulates AR phosphorylation, transcription, and localization. Crosstalk between the JNK protein kinase and AR has been reported in several studies (Gioeli et al., 2006; Lazarevic et al, 2008; Lorenzo & Saatcioglu, 2008;). The link to JNK signaling is interesting since inflammatory processes, which can induce the JNK/c-jun pathway, may upregulate CYP27A1 to clear cholesterol from peripheral tissues.

The findings that estrogens downregulate and androgens upregulate CYP27A1 expression in liver-derived HepG2 cells are of interest for several reasons (Tang et al., 2007). The results are consistent with reports on an increased risk for cardiovascular disease in postmenopausal women treated with estrogen plus progestin. Also, increased testosterone levels in men have been associated with a favorable lipid profile (Alexandersen & Christiansen, 2004; Steinberg, 2006; Stoll & Bendszus, 2006; Tchernof et al., 1997; Zmuda et al., 1997)*.* The difference in prevalence of atherosclerotic coronary disease between men and women can not be explained by effects of estrogens and androgens solely on CYP27A1 expression. However, the effects by sex hormones on the expression of CYP27A1 may have impact on several processes in cholesterol homeostasis. The findings that CYP27A1 is regulated by sex hormones imply that endogenous sex hormones as well as pharmacological compounds with estrogenic and androgenic effects may have an impact on several processes related to CYP27A1-mediated metabolism, such as cellular survival and growth, CNS function and cholesterol homeostasis. Because estrogens are used in oral contraceptives, in hormone therapy of postmenopausal women and in cancer treatment, the question arises how CYP27A1 is influenced by estrogens in different tissues. This question is of particular interest considering that anti-atherogenic properties have been ascribed to CYP27A1. The possibility of a tissue-specific regulation by sex hormones is supported by results with prostate cancer cells where estrogen increases the CYP27A1 transcriptional

Fig. 14. Simplified overview of our findings on the effects of estrogens on the gene regulation of CYP27A1 in different cell types. For more information see text and references

Our studies on the mechanisms for the hormonal regulation of CYP27A1 have indicated the involvement of the JNK (c-jun N-terminal kinase)/c-jun pathway in androgen-mediated regulation of this enzyme (Norlin et al., 2011). It has been reported that the androgen receptor (AR) is phosphorylated by JNK and that stress kinase signaling regulates AR phosphorylation, transcription, and localization. Crosstalk between the JNK protein kinase and AR has been reported in several studies (Gioeli et al., 2006; Lazarevic et al, 2008; Lorenzo & Saatcioglu, 2008;). The link to JNK signaling is interesting since inflammatory processes, which can induce the JNK/c-jun pathway, may upregulate CYP27A1 to clear

The findings that estrogens downregulate and androgens upregulate CYP27A1 expression in liver-derived HepG2 cells are of interest for several reasons (Tang et al., 2007). The results are consistent with reports on an increased risk for cardiovascular disease in postmenopausal women treated with estrogen plus progestin. Also, increased testosterone levels in men have been associated with a favorable lipid profile (Alexandersen & Christiansen, 2004; Steinberg, 2006; Stoll & Bendszus, 2006; Tchernof et al., 1997; Zmuda et al., 1997)*.* The difference in prevalence of atherosclerotic coronary disease between men and women can not be explained by effects of estrogens and androgens solely on CYP27A1 expression. However, the effects by sex hormones on the expression of CYP27A1 may have impact on several processes in cholesterol homeostasis. The findings that CYP27A1 is regulated by sex hormones imply that endogenous sex hormones as well as pharmacological compounds with estrogenic and androgenic effects may have an impact on several processes related to CYP27A1-mediated metabolism, such as cellular survival and growth, CNS function and cholesterol homeostasis. Because estrogens are used in oral contraceptives, in hormone therapy of postmenopausal women and in cancer treatment, the question arises how CYP27A1 is influenced by estrogens in different tissues. This question is of particular interest considering that anti-atherogenic properties have been ascribed to CYP27A1. The possibility of a tissue-specific regulation by sex hormones is supported by results with prostate cancer cells where estrogen increases the CYP27A1 transcriptional

(Norlin et al., 2011; Tang et al., 2007).

cholesterol from peripheral tissues.

activity. The results also show that effects of sex hormones on CYP27A1 regulation are different in non-cancerous prostate RWPE-1 compared with prostate cancer LNCaP cells. Whether this difference in regulatory effects is due to different properties of different prostate cell lines or to altered properties of the CYP27A1 regulation in prostate cancer remain to be established.

\* Involvement of the JNK/c-jun kinase signalling pathway (Norlin et al., 2011).

Fig. 15. Simplified overview of our findings on the effects of androgens on the gene regulation of CYP27A1 in different cell types. For more information see text and references (Norlin et al., 2011; Tang et al., 2007).

#### **5.3 Effects of sex hormones on vitamin D metabolism**

Activated vitamin D metabolites, that can be formed by CYP27A1, are known to have beneficial effects on cell growth in extrahepatic tissues, such as in prostate cells and prostate cancer cells. As discussed above, novel data indicate that estrogens and androgens might regulate the intracellular levels of active hydroxyvitamin D3 metabolites in prostate cells via regulation of CYP27A1 gene expression (Norlin et al., 2011; Tang et al., 2007). Another observation indicating that androgens can influence intracellular levels of active vitamin D metabolites was reported some years ago (Lou and Tuohimaa, 2006). These authors demonstrated that dihydrotestosterone (DHT) significantly suppressed the expression of the catabolizing enzyme CYP24A1 in androgen-sensitive prostate cancer LNCaP cells. Their data demonstrated that DHT enhances the antiproliferative activity of vitamin D3 hormones by inhibiting their inactivating enzyme at physiological concentration of androgen. They suggested that the combined use of androgen and vitamin D3 metabolites could be a promising treatment for prostate cancer.

#### **6. Concluding remarks**

Recent research using cell models has revealed novel and tissue-specific actions of sex hormones and vitamin D3 in the regulation of enzymes in steroid metabolism. The active vitamin D hormone, calcitriol, has been found to affect genes in androgen and estrogen metabolism. Sex hormones regulate genes in neurosteroid metabolism and cholesterol

Tissue-Specific Regulation of Sex Hormone Biosynthesis and Metabolism:

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#### **7. Acknowledgments**

The research in the authors` laboratory was supported by grants from the Swedish Research Council Medicine and the Åke Wiberg foundation (Sweden). We are grateful to Dr. Johan Lundqvist for assistance with some of the illustrations.

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**1. Introduction** 

**19** 

*Thailand* 

**Solid State and Thermal Behavior** 

**of 17-Estradiol in Ammonioethyl** 

17-estradiol is the most potent form of naturally occurring estrogen secreted during the reproductive years (Andersson 2000; Paoletti 2001). It is an essential steroid hormone that regulates numerous endocrine functions through binding to two estrogen receptor (ER) isoforms, i.e., ER and ER. The concentration of these receptor subtypes and the corresponding cofactors vary with tissue types, leading to tissue specific regulation of estrogen response and toxicity. However, the content of ER in tissues has been assumed to be time-invariant (Plowchalk 2002). This indicates that multiple or prolonged exposure to E2

Estradiol (E2) is carried in the plasma in two forms, bound to plasma binding proteins (95-98 %) and unbound form or free estradiol (2-5 %) (Dunn 1983; Pardridge 1986; Plowchalk 2002). Both unbound and bound forms of E2 manifest pharmacological actions. Bound form acts as a reservoir for E2 in blood circulation (Plowchalk 2002). After menopause the primary circulating estrogen is estrone, which is less biologically active than E2 (Kuhl 2005; Margolis 2010). Thus, E2 is usually administered to control early menopausal symptoms such as hot flashes and night sweats. For long-term administration E2 can prevent cardiovascular diseases and osteoporosis (Andersson 2000; Paoletti 2001). After administered E2 reaches to blood circulation, it rapidly undergoes chemical conversions to estrone, driven in part by the body's need to maintain homeostasis. Other estrogens in conjugated forms either equine- or synthetic-derived also exist as a balance between the ingested form and estrone at steady state in the body. Margolis (Margolis 2010) indicated that the conversion of E2 to estrone was reversible reaction. Therefore, estrone plays as a hormonally inert reservoir, capably converting to E2. These conversions make the administered estrogen

However, hormone therapy is not without risks. The FDA and several professional organizations currently recommend prescribing the lowest effective dose for the shortest duration of time in accordance with treatment goals for an individual woman (Mueck 2003; Utian 2008; Margolis 2010). Nowaday there are a wide variety of estrogen products, including oral tablets, transdermal patches, and topical sprays, gels, and lotions, as well as vaginal creams, tablets, and rings. Selection of an appropriate product is often based on patient's preference. Oral estrogen dosage form is preferred due to its convenient route of

of ER in various tissues should not alter physiological and toxic responses.

indistinguishable from naturally occurring E2 in the body.

**Methacrylate Ester Copolymer** 

*Faculty of Pharmacy, Srinakharinwirot University* 

Chutima Wiranidchapong

