**2. Luteinizing hormone-induced signaling in StAR gene expression and testosterone biosynthesis in Leydig cell aging**

StAR gene expression and testosterone biosynthesis in testicular Leydig cells are mainly regulated by luteinizing hormone (LH) secreted from pituitary gland. LH stimulation of Leydig cells induces two messengers: cyclic AMP (cAMP) and arachidonic acid (AA). These two messengers transduce signals to the nucleus through two separated pathways to coregulate StAR gene expression. Both pathways are required with neither one alone being sufficient for LH-induced StAR gene expression and testosterone production (Wang & Stocco, 1999). In addition, these two pathways transduce both positive and negative signals to regulate StAR gene expression. The negative signals increase in Leydig cell aging, resulting in decreases in StAR gene expression and testosterone biosynthesis.

#### **2.1 LH-induced positive signaling through cAMP-protein kinase A pathway**

It is documented that LH-induced signaling through cAMP-protein kinase A (PKA) pathway phosphorylates the transcription factors regulating StAR gene expression (Reinhart et al., 1999b). PKA-phosphorylation is important for the activities of several groups of transcription factors, including cAMP-responsive element binding (CREB) protein, steroidogenic factor-1 (SF-1) and GATA-4, which bind on StAR promoter to induce StAR gene transcription. The cAMP-responsive element (CRE)-like sites were found in StAR promoter DNA sequences. The results from electrophoretic mobility shift assays (EMSA) demonstrated the binding of CREB to StAR promoter (Manna et al., 2002; Manna et al., 2003). PKA-phosphorylation is critical for the activity of CREB (Meyer & Habener, 1993; Montminy et al., 1986), because the phosphorylation of CREB is needed for the recruitment

the mitochondrial cholesterol transfer is defective in aged Leydig cells (Culty et al., 2002; Leers-Sucheta et al., 1999; Luo et al., 2001). Therefore, understanding the mechanism for the decline in StAR gene expression becomes an important consideration in the research on the

Further studies observed that cyclooxygenase-2 (COX2) in Leydig cells increased in male aging, and the increase in COX2 depressed StAR gene expression and testosterone production. Inhibition of COX2 activity in aged Leydig cells reversed the declines in StAR protein and testosterone production. In addition, feeding aged rats with a COX2 inhibitor reversed the decreased StAR protein and blood testosterone concentrations (Wang et al., 2005). These observations suggest a novel mechanism involving COX2 in the age-related decline in testosterone biosynthesis. The results from the studies indicate that it is possible to delay the decline using COX2 inhibitors. However, aging is a long process and long-term application of pharmacological levels of COX2 inhibitors is limited by their potential side effects. In search for safe and practical approaches, a group of natural flavonoids in food and food supplements has been identified being able to block the COX2-dependent signaling. These flavonoids acted at the different steps of the COX2 signaling pathway and significantly enhanced StAR gene expression and testosterone biosynthesis in Leydig cells. This chapter will describe the mechanism involving COX2 in the declines in StAR gene expression and testosterone biosynthesis in Leydig cell aging. The effects of the flavonoids on this mechanism will be reviewed. The potential application of the natural flavonoids in delaying the declines in StAR gene expression and testosterone biosynthesis will be

**2. Luteinizing hormone-induced signaling in StAR gene expression and** 

resulting in decreases in StAR gene expression and testosterone biosynthesis.

**2.1 LH-induced positive signaling through cAMP-protein kinase A pathway**

StAR gene expression and testosterone biosynthesis in testicular Leydig cells are mainly regulated by luteinizing hormone (LH) secreted from pituitary gland. LH stimulation of Leydig cells induces two messengers: cyclic AMP (cAMP) and arachidonic acid (AA). These two messengers transduce signals to the nucleus through two separated pathways to coregulate StAR gene expression. Both pathways are required with neither one alone being sufficient for LH-induced StAR gene expression and testosterone production (Wang & Stocco, 1999). In addition, these two pathways transduce both positive and negative signals to regulate StAR gene expression. The negative signals increase in Leydig cell aging,

It is documented that LH-induced signaling through cAMP-protein kinase A (PKA) pathway phosphorylates the transcription factors regulating StAR gene expression (Reinhart et al., 1999b). PKA-phosphorylation is important for the activities of several groups of transcription factors, including cAMP-responsive element binding (CREB) protein, steroidogenic factor-1 (SF-1) and GATA-4, which bind on StAR promoter to induce StAR gene transcription. The cAMP-responsive element (CRE)-like sites were found in StAR promoter DNA sequences. The results from electrophoretic mobility shift assays (EMSA) demonstrated the binding of CREB to StAR promoter (Manna et al., 2002; Manna et al., 2003). PKA-phosphorylation is critical for the activity of CREB (Meyer & Habener, 1993; Montminy et al., 1986), because the phosphorylation of CREB is needed for the recruitment

**testosterone biosynthesis in Leydig cell aging** 

age-related decline in testosterone biosynthesis.

discussed.

of its cofactor CBP (CREB binding protein) that is involved in the transcriptional activity of CREB (Chrivia et al., 1993; Mayr & Montminy, 2001; Mayr et al., 2001). SF-1 binding sites were also located on StAR promoter (Sandhoff et al., 1998). Binding of SF-1 to these binding sites is crucial for StAR gene transcription (Caron et al., 1997a; Caron et al., 1997b; Sandhoff et al., 1998; Sugawara et al., 1997). PKA-phosphorylation of SF-1 protein was detected at serine and threonine residues (Zhang & Mellon, 1996), which was further confirmed by the study with R2C rat Leydig cells (Carlone & Richards, 1997). It appears that PKAphosphorylation is needed for the maximal activity of SF-1 in the regulation of StAR gene transcription (Lopez et al., 2001; Sugawara et al., 1996). GATA-4 is another transcription factor binding on StAR promoter to regulate StAR gene expression. A GATA-4 binding site was found at -61 to -66 in StAR promoter sequences (Silverman et al., 1999; Silverman et al., 2006). Stimulation of MA-10 mouse Leydig cells with cAMP dramatically increased phosphorylated GATA-4 protein (Tremblay et al., 2002), resulting in the increase in StAR promoter activity. The essential role of PKA-phosphorylation in the activity of GATA-4 was further confirmed by over-expression of PKA catalytic subunit. While expression of PKA catalytic subunit significantly increased GATA-4-supported StAR promoter activity, the increased promoter activity was reversed by co-expression of the protein kinase inhibitor (Tremblay et al., 2002).

The signaling through cAMP-PKA pathway is able to increase the activities of a group of transcription factors that bind on StAR promoter and regulate StAR gene transcription, including C/EBPβ (Christenson et al., 1999), Sp-1 (Sugawara et al., 2000), activator protein-1 (AP-1) (Manna et al., 2004), and sterol regulatory element binding protein (SREBP) (Shea-Eaton et al., 2001), by inducing the interaction and cooperation among them. The cAMP or PKA-induced interactions among these transcription factors generated synergistic effects that increased the cAMP-induced StAR gene transcription and steroid hormone production in Leydig cells (Reinhart et al., 1999a; Silverman et al., 1999; Silverman et al., 2006; Sugawara et al., 2000; Tremblay et al., 2002).

In addition, it was observed that cAMP stimulation of Leydig cells reduced DAX-1 (dosagesensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1) protein. DAX-1 is a strong transcriptional repressor that binds to a hairpin structure of StAR promoter between -20 to -61 and depresses StAR gene expression (Zazopoulos et al., 1997). The reduction of DAX-1 by cAMP stimulation results in dramatic increases in StAR gene expression and steroid hormone production in Leydig cells (Jo & Stocco, 2004). Although it is still not clear how the signaling through cAMP-PKA pathway reduces DAX-1 protein, the observations indicate that this signaling pathway generates positive signals to increase StAR gene transcription by reduction of the transcriptional repressor.

#### **2.2 LH-induced positive signaling through AA pathway**

In addition to the cAMP-PKA signaling pathway, LH induces positive signaling through AA pathway. Stimulation of Leydig cells with LH induced AA release in one minute (Cooke et al., 1991). Previous studies have described three mechanisms for the AA release by LH or hCG: 1) Binding of the trophic hormones to their receptors activates G protein. G protein directly activates phospholipase A2 (PLA2) that catalyzes AA release from phospholipids (Axelrod et al., 1988; Ronco et al., 2002); 2) G protein increases cAMP formation that induces AA release, possibly through the activation of PLA2 by PKA-phosphorylation (Piomelli & Greengard, 1991; Wang et al., 2002); 3) AA is released by the co-regulation of acyl-CoA synthetase 4 (ACS-4) and acyl-CoA thioesterase (Acot2). Trophic hormone stimulation

Natural Flavonoids in StAR Gene Expression

expression and testosterone biosynthesis in Leydig cells.

**2.3 LH-induced negative signaling in StAR gene expression** 

depress StAR gene transcription in Leydig cells (Wang et al., 2003b).

al., 2007b).

and Testosterone Biosynthesis in Leydig Cell Aging 183

metabolites act on StAR promoter is not clear, the studies showed that LH- or cAMPreleased AA is converted to positive signals, 5-HPETE, 5-HETE, 5,6-EET, 8,9-EET and 11,12- EET through lipoxygenase and epoxygenase pathways, which enhance StAR gene

Whereas LH-released AA is metabolized to the positive signals by lipoxygenase and epoxygenase, it is converted to negative signals by cyclooxygenase (COX). It was found that inhibition of COX activity using an inhibitor significantly increased cAMP-induced StAR protein and steroid hormone production in MA-10 mouse Leydig cells. Similar increases were observed in StAR promoter activity and StAR mRNA levels. Although inhibition of COX activity significantly increased the cAMP-induced StAR gene expression, in the absence of cAMP the inhibitor alone did not increase StAR protein expression and steroid hormone production. The studies suggest that the COX inhibitor itself is not able to induce StAR gene expression, but rather reduce the negative signals, resulting in significant increases in the cAMP-induced StAR gene expression and steroidogenesis. There are two isoforms of COX enzymes, COX1 and COX2, in Leydig cells. To determine which isoform is responsible for the inhibitory effect on StAR gene expression, Leydig cells were treated with the selective COX1 inhibitor SC560 or COX2 inhibitor NS398, respectively. Whereas inhibition of COX1 activity did not change StAR protein level, inhibition of COX2 activity dramatically enhanced cAMPinduced StAR protein expression and steroid hormone production. Further studies observed that while COX2 inhibitor significantly increased StAR protein, it did not affect PKA activity. In addition, over-expression of COX2 reduced StAR promoter activity. The observations indicate that COX2, not COX1, converts the LH- or cAMP-released AA to negative signals that

The signaling through cAMP-PKA pathway also generates negative signals to inhibit StAR gene expression and steroidogenesis in Leydig cells by increasing COX2 expression. The mechanism responsible for the cAMP-increased COX2 may involve the signaling through cAMP-PKA pathway in the activation of several transcription factors that regulate COX2 promoter activity. One is CREB that binds on the CRE site at -53 to -59 of human COX2 promoter sequences (Schroer et al., 2002). CREB activation by PKA increased its binding to the CRE site and induced COX2 gene transcription. While forskolin or cAMP activated PKA and increased COX2 promoter activity, inhibition of PKA activity reversed the increase in the promoter activity. Another transcription factor NF-κB plays an essential role in regulating COX2 gene expression (Arun et al., 2009). This transcription factor was activated by PKA-phosphorylation of NF-κB p65 sub-unit. Inhibition of its phosphorylation using H89 reduced COX2 gene transcription. The involvement of cAMP-PKA signaling pathway in the LH-induced COX2 expression was supported by the studies with Leydig cells. An earlier study reported that hCG stimulation of Leydig cells increased COX2 expression (Frungieri et al., 2006). This observation was corroborated by a latter study that reported a LH-increased COX2 protein in rat Leydig cells. The increase in COX2 was almost abolished by inhibition of PKA activity with its inhibitor H89, indicating that cAMP-PKAphosphorylation is required for the LH-induced COX2 expression in Leydig cells (Chen et

As LH stimulates testosterone biosynthesis, the increase in testosterone may feed back to induce COX2 expression in Leydig cells. A recent study reported that testosterone induced COX2 expression and prostaglandin F2a production in hamster Leydig cells (Matzkin et al.,

induces ACS-4, and activates Acot2 by cAMP-PKA-phosphorylation. Co-action of these two enzymes catalyzes AA release from arachidonyl CoA (Cano et al., 2006; Cornejo Maciel et al., 2005; Finkielstein et al., 1998; Maloberti et al., 2005; Maloberti et al., 2007; Paz et al., 1994). The LH-induced AA release is required for testosterone biosynthesis. It was observed that incubation of Leydig cells with AA significantly increased testosterone production (Didolkar & Sundaram, 1987, 1989). When the AA release was blocked using PLA2 inhibitors, the LH-induced testosterone production was significantly reduced although the inhibitors did not affect the cAMP formation (Abayasekara et al., 1990). Inhibiting AA metabolism in rat Leydig cells produce similar results (Cooke et al., 1984; Dix et al., 1984; Mele et al., 1997; Sullivan & Cooke, 1985). In addition, blocking AA release by knockdown of the gene expressions of ACS-4 and Acot2 significantly reduced steroidogenesis (Maloberti et al., 2005). The studies indicated that LH induces positive signaling through AA pathway to regulate testosterone biosynthesis in Leydig cells.

To understand how the released AA plays such an important role in LH-induced steroidogenesis in Leydig cells, its effect on mitochondrial cholesterol transfer was investigated. It was found that blocking AA release failed to affect steroidogenesis when Leydig cells were incubated with 22(R)hydroxycholesterol (Abayasekara et al., 1990; Dix et al., 1984; Mele et al., 1997), a water soluble substrate that is able to diffuse to the mitochondrial inner membrane. The studies suggest that AA enhance testosterone biosynthesis by increasing the mitochondrial cholesterol transfer. The observations were corroborated by the increases in StAR protein and steroidogenesis in the Leydig cells incubated with AA. In addition, blocking AA release using PLA2 inhibitors dramatically reduced LH- or cAMP-induced StAR promoter activity, StAR mRNA, StAR protein and steroidogenesis in Leydig cells. Importantly, the reduced StAR gene expression and steroidogenesis by PLA2 inhibitor were reversed by addition of AA in the cell cultures (Wang et al., 2000; Wang et al., 1999). It is clear that AA increases testosterone biosynthesis by enhancing StAR gene expression. Moreover, the results from EMSA show that AA enhanced the binding of nuclear protein(s) to the StAR promoter DNA sequences between - 67 and -96 (Wang et al., 2003a). The observation indicates that AA acts on StAR promoter and regulates StAR gene transcription.

AA is metabolized mainly through three metabolic pathways, the lipoxygenase, epoxygenase and cyclooxygenase pathways, generating various metabolites. To determine which pathways are involved in the LH-induced StAR gene expression and steroidogenesis, MA-10 Leydig cells pre-loaded with 3H-AA were used to study the effects of cAMP on AA metabolism. The results from HPLC analysis of the cell extract showed that cAMP stimulation significantly increased two groups of AA metabolites: the lipoxygenasegenerated metabolites, 5-hydroxyeicosatetraenoic acid (5-HETE) and 5 hydroperoxyeicosatetraenoic acid (5-HPETE) (Wang et al., 2003a); and the epoxygenasegenerated metabolites, 5,6-epoxyeicosatrienoic acid (5,6-EET), 8,9-epoxyeicosatrienoic acid (8,9-EET), and 11,12-epoxyeicosatrienoic acid (11,12-EET) (Wang et al., 2006). The roles of these AA metabolites in StAR gene expression were investigated with Leydig cells. It was observed that addition of each of these metabolites into the cultures of Leydig cells significantly increased cAMP-induced StAR promoter activity, StAR mRNA, StAR protein and steroidogenesis. In addition, when activity of lipoxygenase or epoxygenase was inhibited, the cAMP-induced StAR gene expression and steroidogenesis were significantly reduced (Wang et al., 2000). The observations indicated that each of the metabolites enhanced StAR gene expression at the level of transcription. Although how these AA

induces ACS-4, and activates Acot2 by cAMP-PKA-phosphorylation. Co-action of these two enzymes catalyzes AA release from arachidonyl CoA (Cano et al., 2006; Cornejo Maciel et al., 2005; Finkielstein et al., 1998; Maloberti et al., 2005; Maloberti et al., 2007; Paz et al., 1994). The LH-induced AA release is required for testosterone biosynthesis. It was observed that incubation of Leydig cells with AA significantly increased testosterone production (Didolkar & Sundaram, 1987, 1989). When the AA release was blocked using PLA2 inhibitors, the LH-induced testosterone production was significantly reduced although the inhibitors did not affect the cAMP formation (Abayasekara et al., 1990). Inhibiting AA metabolism in rat Leydig cells produce similar results (Cooke et al., 1984; Dix et al., 1984; Mele et al., 1997; Sullivan & Cooke, 1985). In addition, blocking AA release by knockdown of the gene expressions of ACS-4 and Acot2 significantly reduced steroidogenesis (Maloberti et al., 2005). The studies indicated that LH induces positive signaling through AA pathway

To understand how the released AA plays such an important role in LH-induced steroidogenesis in Leydig cells, its effect on mitochondrial cholesterol transfer was investigated. It was found that blocking AA release failed to affect steroidogenesis when Leydig cells were incubated with 22(R)hydroxycholesterol (Abayasekara et al., 1990; Dix et al., 1984; Mele et al., 1997), a water soluble substrate that is able to diffuse to the mitochondrial inner membrane. The studies suggest that AA enhance testosterone biosynthesis by increasing the mitochondrial cholesterol transfer. The observations were corroborated by the increases in StAR protein and steroidogenesis in the Leydig cells incubated with AA. In addition, blocking AA release using PLA2 inhibitors dramatically reduced LH- or cAMP-induced StAR promoter activity, StAR mRNA, StAR protein and steroidogenesis in Leydig cells. Importantly, the reduced StAR gene expression and steroidogenesis by PLA2 inhibitor were reversed by addition of AA in the cell cultures (Wang et al., 2000; Wang et al., 1999). It is clear that AA increases testosterone biosynthesis by enhancing StAR gene expression. Moreover, the results from EMSA show that AA enhanced the binding of nuclear protein(s) to the StAR promoter DNA sequences between - 67 and -96 (Wang et al., 2003a). The observation indicates that AA acts on StAR promoter

AA is metabolized mainly through three metabolic pathways, the lipoxygenase, epoxygenase and cyclooxygenase pathways, generating various metabolites. To determine which pathways are involved in the LH-induced StAR gene expression and steroidogenesis, MA-10 Leydig cells pre-loaded with 3H-AA were used to study the effects of cAMP on AA metabolism. The results from HPLC analysis of the cell extract showed that cAMP stimulation significantly increased two groups of AA metabolites: the lipoxygenasegenerated metabolites, 5-hydroxyeicosatetraenoic acid (5-HETE) and 5 hydroperoxyeicosatetraenoic acid (5-HPETE) (Wang et al., 2003a); and the epoxygenasegenerated metabolites, 5,6-epoxyeicosatrienoic acid (5,6-EET), 8,9-epoxyeicosatrienoic acid (8,9-EET), and 11,12-epoxyeicosatrienoic acid (11,12-EET) (Wang et al., 2006). The roles of these AA metabolites in StAR gene expression were investigated with Leydig cells. It was observed that addition of each of these metabolites into the cultures of Leydig cells significantly increased cAMP-induced StAR promoter activity, StAR mRNA, StAR protein and steroidogenesis. In addition, when activity of lipoxygenase or epoxygenase was inhibited, the cAMP-induced StAR gene expression and steroidogenesis were significantly reduced (Wang et al., 2000). The observations indicated that each of the metabolites enhanced StAR gene expression at the level of transcription. Although how these AA

to regulate testosterone biosynthesis in Leydig cells.

and regulates StAR gene transcription.

metabolites act on StAR promoter is not clear, the studies showed that LH- or cAMPreleased AA is converted to positive signals, 5-HPETE, 5-HETE, 5,6-EET, 8,9-EET and 11,12- EET through lipoxygenase and epoxygenase pathways, which enhance StAR gene expression and testosterone biosynthesis in Leydig cells.

## **2.3 LH-induced negative signaling in StAR gene expression**

Whereas LH-released AA is metabolized to the positive signals by lipoxygenase and epoxygenase, it is converted to negative signals by cyclooxygenase (COX). It was found that inhibition of COX activity using an inhibitor significantly increased cAMP-induced StAR protein and steroid hormone production in MA-10 mouse Leydig cells. Similar increases were observed in StAR promoter activity and StAR mRNA levels. Although inhibition of COX activity significantly increased the cAMP-induced StAR gene expression, in the absence of cAMP the inhibitor alone did not increase StAR protein expression and steroid hormone production. The studies suggest that the COX inhibitor itself is not able to induce StAR gene expression, but rather reduce the negative signals, resulting in significant increases in the cAMP-induced StAR gene expression and steroidogenesis. There are two isoforms of COX enzymes, COX1 and COX2, in Leydig cells. To determine which isoform is responsible for the inhibitory effect on StAR gene expression, Leydig cells were treated with the selective COX1 inhibitor SC560 or COX2 inhibitor NS398, respectively. Whereas inhibition of COX1 activity did not change StAR protein level, inhibition of COX2 activity dramatically enhanced cAMPinduced StAR protein expression and steroid hormone production. Further studies observed that while COX2 inhibitor significantly increased StAR protein, it did not affect PKA activity. In addition, over-expression of COX2 reduced StAR promoter activity. The observations indicate that COX2, not COX1, converts the LH- or cAMP-released AA to negative signals that depress StAR gene transcription in Leydig cells (Wang et al., 2003b).

The signaling through cAMP-PKA pathway also generates negative signals to inhibit StAR gene expression and steroidogenesis in Leydig cells by increasing COX2 expression. The mechanism responsible for the cAMP-increased COX2 may involve the signaling through cAMP-PKA pathway in the activation of several transcription factors that regulate COX2 promoter activity. One is CREB that binds on the CRE site at -53 to -59 of human COX2 promoter sequences (Schroer et al., 2002). CREB activation by PKA increased its binding to the CRE site and induced COX2 gene transcription. While forskolin or cAMP activated PKA and increased COX2 promoter activity, inhibition of PKA activity reversed the increase in the promoter activity. Another transcription factor NF-κB plays an essential role in regulating COX2 gene expression (Arun et al., 2009). This transcription factor was activated by PKA-phosphorylation of NF-κB p65 sub-unit. Inhibition of its phosphorylation using H89 reduced COX2 gene transcription. The involvement of cAMP-PKA signaling pathway in the LH-induced COX2 expression was supported by the studies with Leydig cells. An earlier study reported that hCG stimulation of Leydig cells increased COX2 expression (Frungieri et al., 2006). This observation was corroborated by a latter study that reported a LH-increased COX2 protein in rat Leydig cells. The increase in COX2 was almost abolished by inhibition of PKA activity with its inhibitor H89, indicating that cAMP-PKAphosphorylation is required for the LH-induced COX2 expression in Leydig cells (Chen et al., 2007b).

As LH stimulates testosterone biosynthesis, the increase in testosterone may feed back to induce COX2 expression in Leydig cells. A recent study reported that testosterone induced COX2 expression and prostaglandin F2a production in hamster Leydig cells (Matzkin et al.,

Natural Flavonoids in StAR Gene Expression

and various inflammatory factors (Chung et al., 2006).

mechanism in the age-related declines in testosterone biosynthesis.

have significantly improved the understanding of the mechanism.

**3.1 AA distribution between COX2 and lipoxygenase metabolic pathways** 

**testosterone biosynthesis in Leydig cell aging** 

and Testosterone Biosynthesis in Leydig Cell Aging 185

(TGF)-β and tumor necrosis factor (TNF) (Chung et al., 2001; Morley & Baumgartner, 2004). IL-1β was reported to be inhibitory in hCG-, cAMP-, and forskolin-induced testosterone production (Calkins et al., 1990). While it is able to reduce activities of steroidogenic enzymes (Hales, 1992; Lin et al., 1991), IL-1β induced COX2 expression (Chen et al., 2007b; Walch & Morris, 2002) and reduced StAR protein expression (Ogilvie et al., 1999). TGF-β in testis increases with age (Jung et al., 2004). The increase in TGF-β may significantly reduce trophic hormone-induced cAMP formation and testosterone production in Leydig cells (Avallet et al., 1987). TNF-α reduced hCG-stimulated cAMP formation in a concentration-dependent manner in MA-10 mouse Leydig cells (Budnik et al., 1999), which was associated with a reduction in StAR protein. In addition, it was reported that the transcription factor NF-kB increases in the aging process (Chung et al., 2009; Chung et al., 2006). This increase would contribute to the increase in COX2, because NF-kB plays an essential role in COX2 gene transcription. Another group of the biological factors is reactive oxidative species (ROS). An age-related increase in ROS was previously described (Chen et al., 2001b). While ROS may inhibit testosterone through different mechanisms, it is able to activate NF-kB, resulting in the increases in COX2

The age-related increases in the biological factors, such as IL-1β, NF-kB and ROS, suggest an increase in COX2 expression in Leydig cell aging, because these factors induce COX2 as mentioned above. This was supported by the increase in COX2 mRNA level detected in aged Leydig cells of rats (Syntin et al., 2001). It is known that COX2 produces an inhibitory effect on StAR gene expression and steroidogenesis (Wang et al., 2003b). These observations suggest a possibility of involving an age-related increase of COX2 in the declines in StAR gene expression and testosterone production. This possibility was demonstrated by the studies with aged rats (Wang et al., 2005). It was found that from 3 to 30 months of age, the levels of COX2 protein in aged rat Leydig cells increased by 346% over that of young Leydig cells. The increase in COX2 was associated with the decreases in StAR protein, testosterone biosynthesis, and blood testosterone concentration in aged rats. Inhibition of COX2 activity with the selective COX2 inhibitor NS398 reversed the decreases in StAR protein and testosterone production in aged Leydig cells. In addition, while over-expression of COX2 reduced cAMP-stimulated StAR protein and steroidogenesis, co-incubation with NS398 reversed the reduced StAR protein and steroid hormone. These findings reveal a novel

**3. Mechanism for COX2-dependent inhibition of StAR gene expression and** 

The studies described above indicated an involvement of COX2 in the age-related declines in StAR gene expression and testosterone biosynthesis. However, the mechanism for the COX2-dependent inhibition of StAR gene expression in Leydig cells needs to be elucidated. While further investigations are needed for the elucidation, the studies in the recent years

It is known that the LH-released AA is converted to positive signals through lipoxygenase and epoxygenase pathways (Wang et al., 2006; Wang et al., 2003a), and also negative signals through COX2 pathway (Wang et al., 2003b). This suggests that StAR gene expression and testosterone biosynthesis in Leydig cell aging are regulated by the activities of these AA

2009). These effects of testosterone were abolished by the anti-androgen bicalutamide. A testosterone-stimulated phosphorylation of the mitogen-activated protein kinase (MAPK) was also observed in the study. It is known that the activity of MAPK is important in COX2 gene expression (Chun & Surh, 2004), because MAPK is able to activate the transcription factors that regulate COX2 gene transcription.

The studies described above indicate that when LH stimulates positive signals through cAMP-PKA pathway and AA pathway, it also induces negative signals through these two pathways. Therefore, the total effect of LH on StAR gene expression and testosterone biosynthesis depends on the ratio and levels of the positive and negative signals. The alterations of the signals in Leydig cell aging affect StAR gene expression and testosterone biosynthesis.

#### **2.4 The age-related alterations of the positive and negative signals in StAR gene expression**

Previous studies have reported the multiple alterations or defects during male aging that are involved in the decline in testosterone biosynthesis (Wang & Stocco, 2005). Many of these alterations or defects in the aging process increase the negative signals and reduce the positive signals in StAR gene expression. These changes reduce the sensitivity of Leydig cells to trophic hormone stimulation and result in the declines in StAR gene expression and testosterone biosynthesis.

It was observed that the age-related alteration in hypothalamic-pituitary-adrenal axis attenuates the sensitivity of the feedback control of adrenal steroidogenesis, resulting in the increases in basal and stress-induced glucocorticoid levels (Hardy & Cooper, 2010). The inhibitory effect of glucocorticoid on testosterone biosynthesis was reported by previous studies with rat Leydig cells. This steroid hormone was shown being able to inhibit PLA2 activity and block LH-induced AA release (Abayasekara et al., 1990). Further studies observed that blocking AA release using dexamethasone dramatically reduced StAR protein and steroidogenesis in MA-10 mouse Leydig cells. Similar results were obtained in the analyses of StAR promoter activity and StAR mRNA levels. The reduced StAR gene expression and steroidogenesis were reversed by addition of AA to the cell cultures (Wang et al., 2000). The studies suggest that the age-related defect in hypothalamic-pituitaryadrenal axis result in reduction in the LH-induced positive signals through AA pathway.

The positive signaling through cAMP-PKA pathway is also reduced in Leydig cell aging. The previous studies with aged Leydig cells reported a defect in this signaling pathway that caused the decreases in LH-induced cAMP formation and PKA activity (Lin et al., 1980; Luo et al., 2005). The decrease in LH-induced cAMP formation may significantly contribute to the reduced sensitivity of aged Leydig cells to LH stimulation. The mechanism for this defect in aged Leydig cells has not been completely understood. It was observed that the number of the LH-binding sites and their binding affinity decreased in aged Leydig cells, but how the reduced LH-binding capacity affected the cAMP formation remains to be elucidated (Chen et al., 2002). In addition, defect of G protein was investigated. It is possible that inefficiency of G protein or defect in coupling among the signal cascades affects the LHinduced cAMP formation (Chen et al., 2004).

In the aging process, the levels of many biological factors in blood or testis are altered. These alterations may reduce the positive signaling through cAMP-PKA pathway and enhance the negative signaling through COX2 pathway. One group of the biological factors that increase in aging is the inflammatory cytokines, such as Interlukin-1 (IL-1), Transforming growth factor

2009). These effects of testosterone were abolished by the anti-androgen bicalutamide. A testosterone-stimulated phosphorylation of the mitogen-activated protein kinase (MAPK) was also observed in the study. It is known that the activity of MAPK is important in COX2 gene expression (Chun & Surh, 2004), because MAPK is able to activate the transcription

The studies described above indicate that when LH stimulates positive signals through cAMP-PKA pathway and AA pathway, it also induces negative signals through these two pathways. Therefore, the total effect of LH on StAR gene expression and testosterone biosynthesis depends on the ratio and levels of the positive and negative signals. The alterations of the signals in Leydig cell aging affect StAR gene expression and testosterone

**2.4 The age-related alterations of the positive and negative signals in StAR gene** 

Previous studies have reported the multiple alterations or defects during male aging that are involved in the decline in testosterone biosynthesis (Wang & Stocco, 2005). Many of these alterations or defects in the aging process increase the negative signals and reduce the positive signals in StAR gene expression. These changes reduce the sensitivity of Leydig cells to trophic hormone stimulation and result in the declines in StAR gene expression and

It was observed that the age-related alteration in hypothalamic-pituitary-adrenal axis attenuates the sensitivity of the feedback control of adrenal steroidogenesis, resulting in the increases in basal and stress-induced glucocorticoid levels (Hardy & Cooper, 2010). The inhibitory effect of glucocorticoid on testosterone biosynthesis was reported by previous studies with rat Leydig cells. This steroid hormone was shown being able to inhibit PLA2 activity and block LH-induced AA release (Abayasekara et al., 1990). Further studies observed that blocking AA release using dexamethasone dramatically reduced StAR protein and steroidogenesis in MA-10 mouse Leydig cells. Similar results were obtained in the analyses of StAR promoter activity and StAR mRNA levels. The reduced StAR gene expression and steroidogenesis were reversed by addition of AA to the cell cultures (Wang et al., 2000). The studies suggest that the age-related defect in hypothalamic-pituitaryadrenal axis result in reduction in the LH-induced positive signals through AA pathway. The positive signaling through cAMP-PKA pathway is also reduced in Leydig cell aging. The previous studies with aged Leydig cells reported a defect in this signaling pathway that caused the decreases in LH-induced cAMP formation and PKA activity (Lin et al., 1980; Luo et al., 2005). The decrease in LH-induced cAMP formation may significantly contribute to the reduced sensitivity of aged Leydig cells to LH stimulation. The mechanism for this defect in aged Leydig cells has not been completely understood. It was observed that the number of the LH-binding sites and their binding affinity decreased in aged Leydig cells, but how the reduced LH-binding capacity affected the cAMP formation remains to be elucidated (Chen et al., 2002). In addition, defect of G protein was investigated. It is possible that inefficiency of G protein or defect in coupling among the signal cascades affects the LH-

In the aging process, the levels of many biological factors in blood or testis are altered. These alterations may reduce the positive signaling through cAMP-PKA pathway and enhance the negative signaling through COX2 pathway. One group of the biological factors that increase in aging is the inflammatory cytokines, such as Interlukin-1 (IL-1), Transforming growth factor

factors that regulate COX2 gene transcription.

induced cAMP formation (Chen et al., 2004).

biosynthesis.

**expression** 

testosterone biosynthesis.

(TGF)-β and tumor necrosis factor (TNF) (Chung et al., 2001; Morley & Baumgartner, 2004). IL-1β was reported to be inhibitory in hCG-, cAMP-, and forskolin-induced testosterone production (Calkins et al., 1990). While it is able to reduce activities of steroidogenic enzymes (Hales, 1992; Lin et al., 1991), IL-1β induced COX2 expression (Chen et al., 2007b; Walch & Morris, 2002) and reduced StAR protein expression (Ogilvie et al., 1999). TGF-β in testis increases with age (Jung et al., 2004). The increase in TGF-β may significantly reduce trophic hormone-induced cAMP formation and testosterone production in Leydig cells (Avallet et al., 1987). TNF-α reduced hCG-stimulated cAMP formation in a concentration-dependent manner in MA-10 mouse Leydig cells (Budnik et al., 1999), which was associated with a reduction in StAR protein. In addition, it was reported that the transcription factor NF-kB increases in the aging process (Chung et al., 2009; Chung et al., 2006). This increase would contribute to the increase in COX2, because NF-kB plays an essential role in COX2 gene transcription. Another group of the biological factors is reactive oxidative species (ROS). An age-related increase in ROS was previously described (Chen et al., 2001b). While ROS may inhibit testosterone through different mechanisms, it is able to activate NF-kB, resulting in the increases in COX2 and various inflammatory factors (Chung et al., 2006).

The age-related increases in the biological factors, such as IL-1β, NF-kB and ROS, suggest an increase in COX2 expression in Leydig cell aging, because these factors induce COX2 as mentioned above. This was supported by the increase in COX2 mRNA level detected in aged Leydig cells of rats (Syntin et al., 2001). It is known that COX2 produces an inhibitory effect on StAR gene expression and steroidogenesis (Wang et al., 2003b). These observations suggest a possibility of involving an age-related increase of COX2 in the declines in StAR gene expression and testosterone production. This possibility was demonstrated by the studies with aged rats (Wang et al., 2005). It was found that from 3 to 30 months of age, the levels of COX2 protein in aged rat Leydig cells increased by 346% over that of young Leydig cells. The increase in COX2 was associated with the decreases in StAR protein, testosterone biosynthesis, and blood testosterone concentration in aged rats. Inhibition of COX2 activity with the selective COX2 inhibitor NS398 reversed the decreases in StAR protein and testosterone production in aged Leydig cells. In addition, while over-expression of COX2 reduced cAMP-stimulated StAR protein and steroidogenesis, co-incubation with NS398 reversed the reduced StAR protein and steroid hormone. These findings reveal a novel mechanism in the age-related declines in testosterone biosynthesis.
