**3. 3β-HSD gene family – function, tissues distribution, regulation and clinical importance**

The 3β-HSD was described in 1951 and later characterized as bifunctional dimeric enzyme required for the biosynthesis of all classes of steroid hormones (glucocorticoids, mineralocorticoids, progestagens, androgens, and estrogens). Therefore the 3β-HSD controls the critical steroidogenic reactions in the adrenal cortex, gonads, placenta, and peripheral target tissues [31]. The 3β-HSD isoforms catalyze the conversion of the ∆5-3βhydoxysteroids - pregnenolone, 17α-hydroxypregnenolone, and DHEA, to the ∆4-3 ketosteroids - progesterone, 17α-hydroxyprogesterone, and androstenedione, respectively. Two sequential reactions are involved in the conversion of the ∆5-3β-hydroxysteroid to a ∆4-3 ketosteroid. The first reaction is the dehydrogenation of the 3β-hydroxysteroid, requiring the coenzyme NAD+, yielding the ∆5-3-keto intermediate, and reduced NADH. The reduced NADH, activates the isomerization of the ∆5-3- keto steroid to yield the ∆4-3 ketosteroid (Figure 2.). Stopped-flow spectroscopy studies show that NADH activates the isomerase activity by inducing a time-dependant conformational change in the enzyme [15, 32]. Using histochemical and imunohistochemical techniques 3β-HSD activity was detected to the smooth endoplasmic reticulum and mitochondrial cristae and later in the microsomal fraction suggesting that 3β-HSD is a membrane-associated enzyme [16]. Submitochondrial fractionation studies showed that 3β-HSD is in a functional steroidogenic complex with P450scc located in the inner mitochondrial membrane [33, 34], that provides the enzyme with immediate substrate metabolized from cholesterol. However, 3β-HSD activity could be preferentially distributed to the mitochondria under certain physiological conditions [35, 36].

Hydrohysteroid Dehydrogenases – Biological Role and Clinical Importance – Review 121

[45, 46, 47]. Concerning to an enzyme having dual activity, such secondary activity could be explained by binding of the steroid in the inverted substrate orientation, in this case C-17

To date, six distinct cDNAs encoding murine members of the 3β-HSD family have been cloned and all of them are highly homologous and encode a protein of 372 amino acids. Functionally, the different forms fall into two distinct classes of enzymes - 3β-HSD types I, II and III function as dehydrogenase/isomerases, and are essential for the biosynthesis of active steroid hormones whereas 3β-HSD type IV and type V (analogous to rat type III) function as 3-KSRs and they are involved in the inactivation of active steroid hormones [48, 49]. In the adult mouse 3β HSD I is expressed in gonads and adrenal gland, whereas 3β-HSD II and III are expressed in liver and kidney. The type V isoenzyme is expressed only in the liver of the male mouse and the expression starts in late puberty. The type VI isoenzyme is the earliest isoform expressed during the first half of pregnancy in cells of embryonic origin and in uterine tissue suggesting that this isoenzyme may be involved in the local production of progesterone, required for the successful implantation and/or maintenance of pregnancy [50]. In the adult mouse, 3β-HSD type VI appears to be the only isoenzyme expressed in skin. The aminoacid sequences among the different isoforms and between mouse and human isoforms show a high degree of identity. Mouse 3β-HSD I has 84% identity to mouse VI, and 71% identity to human

**Tissue distribution:** As 3β-HSD gene family is widely expressed within the steroidogenic organs (adrenal, ovary and testis) as well as in peripheral tissues, the distribution and local

*Adrenal*: The onset of 3β-HSD expression in the fetal primate adrenal cortex correlates with the ability of the definitive zone to synthesize aldosterone and also allows cortisol production by transitional zone cells. Although 3β-HSD is not expressed to a high degree in the fetal cortex, P450c17 is expressed, thereby directing the steroidogenic pathway toward ∆5-hydroxysteroid (*i*.*e*., DHEA) production. There is zone-specific steroid secretion pattern dependent on the relative expression levels of 3β-HSD, P450c17 and P450 21α-hydroxylase (P450c21) that serve as molecular markers of the adrenocortical developmental state [51, 52]. After birth, the coexpression of 3β-HSD and P450c21 leads to aldosterone production, whereas the coexpression of 3β-HSD and P450c17 results in production of cortisol. The expression of P450c17 along with low levels of 3β-HSD expression leads to synthesis of DHEA. The differential expression of the enzymes required for zonal-specific steroid production in the adrenal is under the control of multiple factors as Adrenocorticotropic hormone (ACTH), Epidermal Growth Factor (EGF), Fibroblast Growth Factors (FGFs), Insulin-like Growth Factors (IGFs), thyroid hormone (T3), Transforming Growth Factor-β (TGFβ) [31, 53, 54]. Therefore, there appears to be a complex interplay of factors controlling adrenal development, and combinations of these factors could be involved in the regulation

*Ovary:* Ontological studies for 3β-HSD have shown that fetal human ovaries are steroidogenically quiescent except for a window late in gestation [55], so most of the

regulation will be described separately for each organ.

of 3β-HSD and other steroidogenic enzymes *in vivo*.

rather than C-3 possition. [47].

II [31, 50].

Isoforms: Structural studies of 3β-HSD family characterized several isoforms, products of distint genes. The number of isozymes varies in different species. The isoenzymes differ in tissue distribution, catalytic activity (whether they function predominantly as dehydrogenases or reductases), in substrate and cofactor specificity, and in subcellular distribution [6]. So far, two isoforms were reported in human (h) 3β-HSD, six in mouse, four in rat and three in hamster. Multiple 3β-HSD isoenzymes have been cloned from several other species, further illustrating that the 3β-HSD gene family is conserved in vertebrate species The human type I 3β-HSD gene (*HSD3B1*) encodes an enzyme of 372 amino acids predominantly expressed in the placenta and peripheral tissues (skin, mammary gland, prostate, and several other normal and tumor tissues) [37, 38]. In comparison, the type II gene (*HSD3B2*), which encodes a protein of 371 amino acids, shares 93.5% identity with the type I and it is almost exclusively expressed in the adrenals ovaries and testes. It is most homologous to the type I gene expressed in mice, rats and other species [39, 40]. The structure of h*HSD3B1* and h*HSD3B2* genes consists of four exons which are included within a DNA fragment of 7.8 kb and genes are assigned to chromosome 1p13.1 [41].

The rat type I and II 3β-HSD proteins are expressed in the adrenals, gonads, kidney, placenta, adipose tissue, and uterus and share 93.8% identity. The type III protein shares 80% identity with the type I and II proteins but, in contrast to other types, it is a specific 3 ketosteroid reductase (KSR) [42, 43]. The type III gene is exclusively expressed in male liver, and there is marked sexual dimorphic expression, which results in pituitary hormoneinduced gene repression in the female rat liver [44]. The rat type IV protein shares 90.9%, 87.9%, and 78.8% identity with types I, II, and III proteins, respectively. Furthermore, types I and IV possess a 17β-HSD activity specific to 5α-androstane-17β-ol steroids, thus suggesting a key role in controlling the bioavailibility of the active androgen dihydrotestosterone DHT [45, 46, 47]. Concerning to an enzyme having dual activity, such secondary activity could be explained by binding of the steroid in the inverted substrate orientation, in this case C-17 rather than C-3 possition. [47].

120 Dehydrogenases

36].

the critical steroidogenic reactions in the adrenal cortex, gonads, placenta, and peripheral target tissues [31]. The 3β-HSD isoforms catalyze the conversion of the ∆5-3βhydoxysteroids - pregnenolone, 17α-hydroxypregnenolone, and DHEA, to the ∆4-3 ketosteroids - progesterone, 17α-hydroxyprogesterone, and androstenedione, respectively. Two sequential reactions are involved in the conversion of the ∆5-3β-hydroxysteroid to a ∆4-3 ketosteroid. The first reaction is the dehydrogenation of the 3β-hydroxysteroid, requiring the coenzyme NAD+, yielding the ∆5-3-keto intermediate, and reduced NADH. The reduced NADH, activates the isomerization of the ∆5-3- keto steroid to yield the ∆4-3 ketosteroid (Figure 2.). Stopped-flow spectroscopy studies show that NADH activates the isomerase activity by inducing a time-dependant conformational change in the enzyme [15, 32]. Using histochemical and imunohistochemical techniques 3β-HSD activity was detected to the smooth endoplasmic reticulum and mitochondrial cristae and later in the microsomal fraction suggesting that 3β-HSD is a membrane-associated enzyme [16]. Submitochondrial fractionation studies showed that 3β-HSD is in a functional steroidogenic complex with P450scc located in the inner mitochondrial membrane [33, 34], that provides the enzyme with immediate substrate metabolized from cholesterol. However, 3β-HSD activity could be preferentially distributed to the mitochondria under certain physiological conditions [35,

Isoforms: Structural studies of 3β-HSD family characterized several isoforms, products of distint genes. The number of isozymes varies in different species. The isoenzymes differ in tissue distribution, catalytic activity (whether they function predominantly as dehydrogenases or reductases), in substrate and cofactor specificity, and in subcellular distribution [6]. So far, two isoforms were reported in human (h) 3β-HSD, six in mouse, four in rat and three in hamster. Multiple 3β-HSD isoenzymes have been cloned from several other species, further illustrating that the 3β-HSD gene family is conserved in vertebrate species The human type I 3β-HSD gene (*HSD3B1*) encodes an enzyme of 372 amino acids predominantly expressed in the placenta and peripheral tissues (skin, mammary gland, prostate, and several other normal and tumor tissues) [37, 38]. In comparison, the type II gene (*HSD3B2*), which encodes a protein of 371 amino acids, shares 93.5% identity with the type I and it is almost exclusively expressed in the adrenals ovaries and testes. It is most homologous to the type I gene expressed in mice, rats and other species [39, 40]. The structure of h*HSD3B1* and h*HSD3B2* genes consists of four exons which are included within

a DNA fragment of 7.8 kb and genes are assigned to chromosome 1p13.1 [41].

The rat type I and II 3β-HSD proteins are expressed in the adrenals, gonads, kidney, placenta, adipose tissue, and uterus and share 93.8% identity. The type III protein shares 80% identity with the type I and II proteins but, in contrast to other types, it is a specific 3 ketosteroid reductase (KSR) [42, 43]. The type III gene is exclusively expressed in male liver, and there is marked sexual dimorphic expression, which results in pituitary hormoneinduced gene repression in the female rat liver [44]. The rat type IV protein shares 90.9%, 87.9%, and 78.8% identity with types I, II, and III proteins, respectively. Furthermore, types I and IV possess a 17β-HSD activity specific to 5α-androstane-17β-ol steroids, thus suggesting a key role in controlling the bioavailibility of the active androgen dihydrotestosterone DHT To date, six distinct cDNAs encoding murine members of the 3β-HSD family have been cloned and all of them are highly homologous and encode a protein of 372 amino acids. Functionally, the different forms fall into two distinct classes of enzymes - 3β-HSD types I, II and III function as dehydrogenase/isomerases, and are essential for the biosynthesis of active steroid hormones whereas 3β-HSD type IV and type V (analogous to rat type III) function as 3-KSRs and they are involved in the inactivation of active steroid hormones [48, 49]. In the adult mouse 3β HSD I is expressed in gonads and adrenal gland, whereas 3β-HSD II and III are expressed in liver and kidney. The type V isoenzyme is expressed only in the liver of the male mouse and the expression starts in late puberty. The type VI isoenzyme is the earliest isoform expressed during the first half of pregnancy in cells of embryonic origin and in uterine tissue suggesting that this isoenzyme may be involved in the local production of progesterone, required for the successful implantation and/or maintenance of pregnancy [50]. In the adult mouse, 3β-HSD type VI appears to be the only isoenzyme expressed in skin. The aminoacid sequences among the different isoforms and between mouse and human isoforms show a high degree of identity. Mouse 3β-HSD I has 84% identity to mouse VI, and 71% identity to human II [31, 50].

**Tissue distribution:** As 3β-HSD gene family is widely expressed within the steroidogenic organs (adrenal, ovary and testis) as well as in peripheral tissues, the distribution and local regulation will be described separately for each organ.

*Adrenal*: The onset of 3β-HSD expression in the fetal primate adrenal cortex correlates with the ability of the definitive zone to synthesize aldosterone and also allows cortisol production by transitional zone cells. Although 3β-HSD is not expressed to a high degree in the fetal cortex, P450c17 is expressed, thereby directing the steroidogenic pathway toward ∆5-hydroxysteroid (*i*.*e*., DHEA) production. There is zone-specific steroid secretion pattern dependent on the relative expression levels of 3β-HSD, P450c17 and P450 21α-hydroxylase (P450c21) that serve as molecular markers of the adrenocortical developmental state [51, 52]. After birth, the coexpression of 3β-HSD and P450c21 leads to aldosterone production, whereas the coexpression of 3β-HSD and P450c17 results in production of cortisol. The expression of P450c17 along with low levels of 3β-HSD expression leads to synthesis of DHEA. The differential expression of the enzymes required for zonal-specific steroid production in the adrenal is under the control of multiple factors as Adrenocorticotropic hormone (ACTH), Epidermal Growth Factor (EGF), Fibroblast Growth Factors (FGFs), Insulin-like Growth Factors (IGFs), thyroid hormone (T3), Transforming Growth Factor-β (TGFβ) [31, 53, 54]. Therefore, there appears to be a complex interplay of factors controlling adrenal development, and combinations of these factors could be involved in the regulation of 3β-HSD and other steroidogenic enzymes *in vivo*.

*Ovary:* Ontological studies for 3β-HSD have shown that fetal human ovaries are steroidogenically quiescent except for a window late in gestation [55], so most of the

estrogens seen by the primate fetus are of placental origin [56]. 3β-HSD is not expressed in mouse and rat ovary until first week after birth.This is in contrast to testicular expression because androgen production by the male embryo is critical for male sexual development [57]. PCOS is an ovarian disorder associated with hyperthecosis of the ovary and elevated serum LH, insulin, and androgen levels. Several studies provide evidence of aberrant 3β-HSD regulation in polycystic thecal cells although the mechanisms are unclear [58].

Hydrohysteroid Dehydrogenases – Biological Role and Clinical Importance – Review 123

LC expressed P450scc and P450c17, as well. Therefore an antibody against 3β-HSD is highly applicable as a marker for visualization both, fetal and adult LC. The expression of 3β-HSD protein overlapped with expression of other steroidogenic enzymes, P450scc and P450c17, clearly demonstrated on Figure 3 and that was confirmed by other authors [70]. Development of triple co-localization immunohistochemical technique allows distinguishing of presumptive progenitors cells form adult or fetal LC that is very helpful to study kinetic and differentiation pattern of LCs (Figure 4) [71]. Application of IHC for 3β-HSD is widely used by many authors in quantification studies of LC under normal and experimental/pathological conditions, especially those of hormonal manipulations [72]. 3β-HSD immunohistochemistry is also useful tool for validation of EDS (ethane dimethanesulfonate) model for selective ablation of adult LC and thus testosterone withdrawal. The major regulator of postnatal testicular expression of 3β-HSD in rodents and human is the LH, acting via LH receptor located in LCs. That is in contrast to the fetal testis where an independent mechanism is suggested [73]. Steroids and growth factors (EGF, TGFβ, FGFs, Activin A) are also suggested to control the expression of 3β-

*Peripheral tissues;* Expression of 3β-HSD in peripheral tissues such breast, prostate, placenta, liver, blain and skin will be briefly described in relation to clinical importance. Sex steroids are well recognized to play a predominant role in the regulation of cell growth and differentiation of normal mammary gland as well as in hormone-sensitive breast carcinomas. Estrogens stimulate cell growth of hormonesensitive breast cancer cells, whereas androgens exert an antiproliferative action in breast cancer cells [74]. Stage II/III infiltrating ductal primary breast tumors demonstrated 3β-HSD activity [75], and 3β-HSD protein was seen in 36% of breast carcinoma samples tested [76, 77]. The 3β-HSD expressed in human placenta is the peripheral isoform, type I 3β-HSD, and it is under differential regulatory control than the adrenal/gonadal isoform, type II 3β-HSD [78, 79]. In the prostate epithelium 3β-HSD expression was colocalized with 17β-HSD type V in normal conditions. 3β-HSD was found in human hyperplastic prostates suggesting the capacity of the human prostate for local androgen production, that increase the hypertrophic potential of the organ [80, 81]. Hepatic 3β-HSD expression is presumed to be important in the metabolism and inactivation of steroids. 3β-HSD activity in human liver microsomes was shown to be three times higher for the reduction of DHT to 3∆-DIOL than the reverse reaction [82]. The circulating levels of steroids might affect regulation of 3β-HSD activity in the liver, principally through altering Growth Hotmone (GH) and PRL levels, and thereby resulting in feedback on steroid degradation [83]. In skin 3β-HSD was confined to keretinocytes, co-expressed with 17β-HSD. Aberrant expression of these enzymes results in increased scalp DHT levels and possibly acceleration of the balding process in genetically predisposed men and women [84, 85]. 3β-HSD expression was reported in the central nervous system (CNS) and peripheral neurons demonstrating the importance of steroid hormones for growth maturation and differentiation of nerve tissue. For instance, 3β-HSD together with P450scc are expressed in the hippocampus, dentate gyrus, cerebellum, olfactory bulb, and Purkinje cells of the rat brain

with highest levels in cerebellum [86] as well as in cultured neuronal cells [87]

HSD [31].

Preantral/antral *follicular* expression studies show 3β-HSD mRNA and protein expression in the human ovary initially in the theca and then in the granulosa layer as folliculogenesis continues [59]. In nonprimate species, 3β-HSD has been shown to have different expression patterns. In the rat, preantral, antral, and preovulatory rat follicles showed 3β-HSD expression in the theca, but no expression was seen in the granulosa layer [60]. In contrast to rodents, pigs, and primates, 3β-HSD expression in the cow was seen in all the stages of the preovulatory follicle in both theca and granulosa layers [61]. Pituitary hormones are the primary means of the regulation of the steroidogenesis in the ovary.The gonadotropins, FSH and LH cause an increase in 3β-HSD expression concomitantly with other steroidogenic enzymes. The role of prolactin (PRL) on primate 3β-HSD is unclear, although PRL was shown to be inhibitory. Interestingly in postmenopausal women 30% of circulating ∆4- DIONE is of ovarian origin [62]. These studies suggest that ovarian steroid production in postmenopausal women continues, but the decline in pituitary control dramatically changes the steroid profile. After ovulation, *Corpus Luteum (CL)* is developed to secrete large amount of progesterone that is controlled in part by the amount of 3β-HSD. The enzyme is considered as a marker for progesterone production of the CL [63]. In primates, LH/hCG action through LH receptor provides a primary mean of luteotropic support [64, 65]. In addition, FSH increased 3β-HSD protein and mRNA levels in human granulosa-lutal cells, and this effect could be enhanced by insulin [66]. Although the direct control of 3β-HSD by PRL in humans has yet to be demonstrated, PRL has been shown to up-regulate 3β-HSD transcriptional activity *in vitro* [67]. During regression of CL (luteolysis) the expression of 3β-HSD dramatically decreased and there is evidence that LH is mainly involved in induction of luteolysis [68].

*Testis:* Testis is the major place for production of androgens, mainly testosterone although local conversion/reduction of testosterone to dihydrotestosterone (DHT) by 5α-Reductase (5α-Red) occurred in the following part of reproductive system (epididymis and prostate). Within the testis, the Leydig cells (LC) are primary place for steroidogenesis as they are only cell type in the male that expressed all of the enzymes essential for the conversion of cholesterol to testosterone [16]. During development two distinct population of LCs arise sequentially, namely fetal and adult LC population, being differentially regulated [20]. Immunohistochemical studies have revealed that human Leydig cells express 3β-HSD as early as 18 wk of gestation. During gestation in human, 3β-HSD expression is an indicator of testicular androgen production. Adult Leydig cells arise postnatally and encompass three developmental stages: progenitor, immature and adult Leydig cells [69]. Rat testes of postnatal day 15 showed 3β-HSD localization to the smooth ER in precursor Leydig cells and that points the beginning of differentiation of adult LC population. At this time point

LC expressed P450scc and P450c17, as well. Therefore an antibody against 3β-HSD is highly applicable as a marker for visualization both, fetal and adult LC. The expression of 3β-HSD protein overlapped with expression of other steroidogenic enzymes, P450scc and P450c17, clearly demonstrated on Figure 3 and that was confirmed by other authors [70]. Development of triple co-localization immunohistochemical technique allows distinguishing of presumptive progenitors cells form adult or fetal LC that is very helpful to study kinetic and differentiation pattern of LCs (Figure 4) [71]. Application of IHC for 3β-HSD is widely used by many authors in quantification studies of LC under normal and experimental/pathological conditions, especially those of hormonal manipulations [72]. 3β-HSD immunohistochemistry is also useful tool for validation of EDS (ethane dimethanesulfonate) model for selective ablation of adult LC and thus testosterone withdrawal. The major regulator of postnatal testicular expression of 3β-HSD in rodents and human is the LH, acting via LH receptor located in LCs. That is in contrast to the fetal testis where an independent mechanism is suggested [73]. Steroids and growth factors (EGF, TGFβ, FGFs, Activin A) are also suggested to control the expression of 3β-HSD [31].

122 Dehydrogenases

induction of luteolysis [68].

estrogens seen by the primate fetus are of placental origin [56]. 3β-HSD is not expressed in mouse and rat ovary until first week after birth.This is in contrast to testicular expression because androgen production by the male embryo is critical for male sexual development [57]. PCOS is an ovarian disorder associated with hyperthecosis of the ovary and elevated serum LH, insulin, and androgen levels. Several studies provide evidence of aberrant 3β-

Preantral/antral *follicular* expression studies show 3β-HSD mRNA and protein expression in the human ovary initially in the theca and then in the granulosa layer as folliculogenesis continues [59]. In nonprimate species, 3β-HSD has been shown to have different expression patterns. In the rat, preantral, antral, and preovulatory rat follicles showed 3β-HSD expression in the theca, but no expression was seen in the granulosa layer [60]. In contrast to rodents, pigs, and primates, 3β-HSD expression in the cow was seen in all the stages of the preovulatory follicle in both theca and granulosa layers [61]. Pituitary hormones are the primary means of the regulation of the steroidogenesis in the ovary.The gonadotropins, FSH and LH cause an increase in 3β-HSD expression concomitantly with other steroidogenic enzymes. The role of prolactin (PRL) on primate 3β-HSD is unclear, although PRL was shown to be inhibitory. Interestingly in postmenopausal women 30% of circulating ∆4- DIONE is of ovarian origin [62]. These studies suggest that ovarian steroid production in postmenopausal women continues, but the decline in pituitary control dramatically changes the steroid profile. After ovulation, *Corpus Luteum (CL)* is developed to secrete large amount of progesterone that is controlled in part by the amount of 3β-HSD. The enzyme is considered as a marker for progesterone production of the CL [63]. In primates, LH/hCG action through LH receptor provides a primary mean of luteotropic support [64, 65]. In addition, FSH increased 3β-HSD protein and mRNA levels in human granulosa-lutal cells, and this effect could be enhanced by insulin [66]. Although the direct control of 3β-HSD by PRL in humans has yet to be demonstrated, PRL has been shown to up-regulate 3β-HSD transcriptional activity *in vitro* [67]. During regression of CL (luteolysis) the expression of 3β-HSD dramatically decreased and there is evidence that LH is mainly involved in

*Testis:* Testis is the major place for production of androgens, mainly testosterone although local conversion/reduction of testosterone to dihydrotestosterone (DHT) by 5α-Reductase (5α-Red) occurred in the following part of reproductive system (epididymis and prostate). Within the testis, the Leydig cells (LC) are primary place for steroidogenesis as they are only cell type in the male that expressed all of the enzymes essential for the conversion of cholesterol to testosterone [16]. During development two distinct population of LCs arise sequentially, namely fetal and adult LC population, being differentially regulated [20]. Immunohistochemical studies have revealed that human Leydig cells express 3β-HSD as early as 18 wk of gestation. During gestation in human, 3β-HSD expression is an indicator of testicular androgen production. Adult Leydig cells arise postnatally and encompass three developmental stages: progenitor, immature and adult Leydig cells [69]. Rat testes of postnatal day 15 showed 3β-HSD localization to the smooth ER in precursor Leydig cells and that points the beginning of differentiation of adult LC population. At this time point

HSD regulation in polycystic thecal cells although the mechanisms are unclear [58].

*Peripheral tissues;* Expression of 3β-HSD in peripheral tissues such breast, prostate, placenta, liver, blain and skin will be briefly described in relation to clinical importance. Sex steroids are well recognized to play a predominant role in the regulation of cell growth and differentiation of normal mammary gland as well as in hormone-sensitive breast carcinomas. Estrogens stimulate cell growth of hormonesensitive breast cancer cells, whereas androgens exert an antiproliferative action in breast cancer cells [74]. Stage II/III infiltrating ductal primary breast tumors demonstrated 3β-HSD activity [75], and 3β-HSD protein was seen in 36% of breast carcinoma samples tested [76, 77]. The 3β-HSD expressed in human placenta is the peripheral isoform, type I 3β-HSD, and it is under differential regulatory control than the adrenal/gonadal isoform, type II 3β-HSD [78, 79]. In the prostate epithelium 3β-HSD expression was colocalized with 17β-HSD type V in normal conditions. 3β-HSD was found in human hyperplastic prostates suggesting the capacity of the human prostate for local androgen production, that increase the hypertrophic potential of the organ [80, 81]. Hepatic 3β-HSD expression is presumed to be important in the metabolism and inactivation of steroids. 3β-HSD activity in human liver microsomes was shown to be three times higher for the reduction of DHT to 3∆-DIOL than the reverse reaction [82]. The circulating levels of steroids might affect regulation of 3β-HSD activity in the liver, principally through altering Growth Hotmone (GH) and PRL levels, and thereby resulting in feedback on steroid degradation [83]. In skin 3β-HSD was confined to keretinocytes, co-expressed with 17β-HSD. Aberrant expression of these enzymes results in increased scalp DHT levels and possibly acceleration of the balding process in genetically predisposed men and women [84, 85]. 3β-HSD expression was reported in the central nervous system (CNS) and peripheral neurons demonstrating the importance of steroid hormones for growth maturation and differentiation of nerve tissue. For instance, 3β-HSD together with P450scc are expressed in the hippocampus, dentate gyrus, cerebellum, olfactory bulb, and Purkinje cells of the rat brain with highest levels in cerebellum [86] as well as in cultured neuronal cells [87]

Hydrohysteroid Dehydrogenases – Biological Role and Clinical Importance – Review 125

**Figure 4.** Triple immunostaining for 3β-HSD (blue), α-smooth muscle actin (red) and COUP TFII (brown) in fetal (embryonal day 21.5) and postnatal (pubertal-d25 and adult-d75) rat testes. Fetal and adult LCs (arrows) are clrearly distinguishable from presumptive ptogenitors cells (arrowheads) x400.

gene are not clear yet. [31].

**Regulation:** The regulation of 3β-HSD gene family is quite complex process involving multiple signal transduction pathways that are activated by growth factors, steroids and cytokines and they are differentially dependent on ontogeny and tissue distribution. Initial studies investigating the transcriptional regulation of the human *HSD3B2* gene are primarily focused on the trophic hormones, including ACTH in the adrenal cortex, LH/human chorionic gonadotropin (hCG) in theca cells and corpus luteum, as well as LH in testicular Leydig cells. cAMP is well known intracellular mediator of trophic hormone stimulation of 3β-HSD expression but mechanisms by which cAMP stimulate transcription of the *HSD3B2* 

Gonadal expression of human 3β-HSD II and mouse 3β-HSD I is dependent on SF-1 as described for the gonadal-specific expression of the P450 steroidogenic enzymes [88]. Studies on mouse *Hsd3b1* promoter identified three potential SF-1 consensus binding sites [89]. The regulation of *HSD3B2* human gene expression involved the transcription factors of Stat family (signal transducers and activators of transcription) [90]. Interestingly, the Stat5 knockout mice displays luteal failure [91]. DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on X chromosome gene-1) was originally isolated by positional cloning from patients with DAX-mutation exhibiting adrenal congenita hypoplasia associated with hypogonadotropic hypogonadism. The studies examining the effects of DAX-1 overexpression on adrenal cell showed suppression of steroidogenesis associated with inhibition of the expression of StAR, P450scc, and 3β-HSD [92]. The exact mechanisms by which DAX-1 overexpression affects 3β-HSD expression remain unclear. Interestingly, transcription factors belonging to the GATA family are emerging as novel regulators of steroidogenesis. In fetal and adult adrenals and gonads several target genes for GATA protein were identified such as StAR, *CYP11A, CYP17A, CYP19A, HSD17B1,* human *HSD3B1* and *HSD3B2* [93]. Moreover, deregulation of GATA expression and/or activity might be relevant to pathological processes associated with aberrant *HSD3B2*

**Figure 3.** Immunoexpression of steroideogenic ezymes (3β-HSD, cytochrome P450scc and cytochrome P450c17 in the Leydig cells (DAB-brown) of postnatal mouse testis after birth to sexual maturity (d2 neonatal, d12-prepubertal, d20-pubertal, d50-adult) x400.

**Figure 3.** Immunoexpression of steroideogenic ezymes (3β-HSD, cytochrome P450scc and cytochrome P450c17 in the Leydig cells (DAB-brown) of postnatal mouse testis after birth to sexual maturity (d2-

neonatal, d12-prepubertal, d20-pubertal, d50-adult) x400.

**Figure 4.** Triple immunostaining for 3β-HSD (blue), α-smooth muscle actin (red) and COUP TFII (brown) in fetal (embryonal day 21.5) and postnatal (pubertal-d25 and adult-d75) rat testes. Fetal and adult LCs (arrows) are clrearly distinguishable from presumptive ptogenitors cells (arrowheads) x400.

**Regulation:** The regulation of 3β-HSD gene family is quite complex process involving multiple signal transduction pathways that are activated by growth factors, steroids and cytokines and they are differentially dependent on ontogeny and tissue distribution. Initial studies investigating the transcriptional regulation of the human *HSD3B2* gene are primarily focused on the trophic hormones, including ACTH in the adrenal cortex, LH/human chorionic gonadotropin (hCG) in theca cells and corpus luteum, as well as LH in testicular Leydig cells. cAMP is well known intracellular mediator of trophic hormone stimulation of 3β-HSD expression but mechanisms by which cAMP stimulate transcription of the *HSD3B2*  gene are not clear yet. [31].

Gonadal expression of human 3β-HSD II and mouse 3β-HSD I is dependent on SF-1 as described for the gonadal-specific expression of the P450 steroidogenic enzymes [88]. Studies on mouse *Hsd3b1* promoter identified three potential SF-1 consensus binding sites [89]. The regulation of *HSD3B2* human gene expression involved the transcription factors of Stat family (signal transducers and activators of transcription) [90]. Interestingly, the Stat5 knockout mice displays luteal failure [91]. DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on X chromosome gene-1) was originally isolated by positional cloning from patients with DAX-mutation exhibiting adrenal congenita hypoplasia associated with hypogonadotropic hypogonadism. The studies examining the effects of DAX-1 overexpression on adrenal cell showed suppression of steroidogenesis associated with inhibition of the expression of StAR, P450scc, and 3β-HSD [92]. The exact mechanisms by which DAX-1 overexpression affects 3β-HSD expression remain unclear. Interestingly, transcription factors belonging to the GATA family are emerging as novel regulators of steroidogenesis. In fetal and adult adrenals and gonads several target genes for GATA protein were identified such as StAR, *CYP11A, CYP17A, CYP19A, HSD17B1,* human *HSD3B1* and *HSD3B2* [93]. Moreover, deregulation of GATA expression and/or activity might be relevant to pathological processes associated with aberrant *HSD3B2*

expression such as adrenal insufficiency, male pseudohermaphroditism and polycystic ovary syndrome (PCOS) [31]. Immune cell populations in the ovary undergo changes during the reproductive cycle and cytokines from these immune cells (Interleukin-4, IL-4) have been shown to affect steroidogenesis, mediated by Stat [94]. Some growth factors like members of the TGFβ family and nerves growth factor have been shown to regulate *HSD3B2* gene expression [95-97]. There is growing evidence in the literature that steroid hormones modulate type II 3β-HSD expression. For example, glucocorticoids stimulate the expression of 3β-HSD in adrenal cells [98], whereas androgens inhibit 3β-HSD expression in the adrenal cortical cells and in testicular Leydig cells [99, 100]. There are number of questions concerning the mechanisms of steroids and the action of their receptors. In relation to structure-function aspects the question is what is the influence of known steroid agonists and antagonists on the efficacy of activation? What is the effect of other nonsteroid factors, which are known to activate other intracellular signaling pathways on steroidregulated transcription?

Hydrohysteroid Dehydrogenases – Biological Role and Clinical Importance – Review 127

considered to be the best biological parameter for the diagnosis of 3β- HSD deficiency. The best criteria for the correct diagnosis of this disorder now appears to be a plasma level of 17OH-PREG but 17OH Progesterone (17OH-PROG) also should be measured for correct diagnosis of 3β-HSD deficiency. It is well recognized that plasma levels of 17OH-PROG and ∆4-DIONE and other ∆4-steroids are frequently elevated in 3β-HSD-deficient patients. Such observations are consistent with a functional type I 3β-HSD enzyme that is expressed in peripheral tissues. Moreover, the peripheral type I 3β-HSD activity could explain why certain patients were initially misdiagnosed as suffering from 21-hydroxylase deficiency, in view of elevated levels of 17OH-PROG and mild virilization seen in girls at birth. Therefore, measurement of the levels of 17OH-PREG should be performed when an elevated level of 17OH-PROG has been observed in a female neonate without ambiguity of external genitalia

**4. 11-hydroxysteroid dehydrogenase – biological role in the regulation** 

The glucocorticosteroids exert diverse actions throughout the body and many of them have important implications in the reproduction and metabolite syndrome. It was recognized that within potential target cells, the actions of glucocorticoids are modulated by 11 hydroxysteroid dehydrogenases (11β-HSD) which catalyse the reversible inactivation of cortisol and corticosterone to their inert 11-ketosteroid metabolites, cortisone and 11 dehydrocorticosterone, respectively [101]. The actions of physiological glucocorticoids (cortisol and corticosterone) are modulated by isoforms of the enzyme 11β-HSD (Figure 5, [108]). To date, two isoforms of 11β-HSD have been identified: 1) 11β-HSD1 acts predominantly as an NADP(H)-dependent reductase that converts inactive circulating 11 ketosteroids, into active glucocorticoids generating active cortisol or corticosterone; 2) 11β-HSD2 is a high affinity NAD+-dependent enzyme that catalyses the inactivation of glucocorticoids [102-107]. Although the biochemistry of 11β-HSD is well established, the physiological significance of glucocorticoid metabolism by these enzymes is still not fully

**Figure 5.** 11-hydroxysteroid dehydrogenase (11-HSD) (Adopted by Seckl et al., 2004 [108])

or if the patient is a male pseudohermaphrodite [31].

**of glucocorticoid metabolisms and cortisol levels** 
