**2. Steroid hormones and role of hydroxysteroid dehydrogenases in steroidogenesis: steroidogenic pathways and general regulatory mechanisms**

Steroid hormones are produced by the gonads, adrenal gland and placenta and they play vital role in physiological and reproductive processes. Structurally, steroids have a basic or common nucleus called the cyclopentanoperhydrophenanthrene, consisting of three, sixmembered fully hydrogenated (perhydro) phenanthrene rings designated A, B and C, and one five-membered cyclopentane ring designated D (Fig 1, right top). In 1967, the International Union of Pure and Applied Chemistry (IUPAC) established rules for the number of carbons in a steroid and thus its biological action can be predicted. For instance, 21-carbon steroids have progestogenic or corticoid activity, 19-carbon steroids have androgenic activity and 18-carbon steroids have estrogenic activity. Cholesterol is a 27 carbon steroid that gives rise pregnenolone (21-carbon) after cleavage of its side chain. Pregnenolone is subsequently converted to progesterone, which in turn give rise androgens or corticoids. Androgens are subjected to aromatization of ring A thus giving rise estrogens [11]. The pathways of steroidogenesis differ between species, but the pathways of human steroidogenesis are shown in the Figure 1. [12]. Cholesterol is the precursor of the steroid hormones, providing backbone of the steroid molecule. The enzymes involved in the synthesis of steroid hormones can be divided into two major classes of proteins: the cytochrome P450 heme-containing proteins (CYP) and the hydroxysteroid dehydrogenases (HSD) [13, 14]. These enzymes are primarily expressed in the gonads, adrenal and placenta. Interestingly, some of these enzyme activities have been demonstrated in non-endocrine tissues, where they may be involved in important paracrine and autocrine actions. This is particularly the case in the human fetus where steroid precursors circulates at high levels and could be metabolized within tissues to produce active steroid hormones. The first class of steroidogenic enzymes, CYP proteins called hydroxylases catalyze reaction of xydroxylation (introduction of hydroxyl group –OH into organic compound) and cleavage of the steroid substrate utilizing molecular oxygen and nicotinamide adenine dinucleotide phosphate (NADPH, reduced) as the source of reductive potential. Several enzymes are included: cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc, *CYP11A1)*, cytochrome P450 17α-hydroxylase (P450c17, 17α-hydroxylase, 17-20 lyase, *CYP17A1*), P450 aromatase (aromatase, *CYP19A1*), 21α-hydroxylase (*CYP21A*), 11β-hydroxylase (*CYP11B1)* and aldosterone synthase (*CYP11B2*). The second class of steroidogenic enzymes, HSD enzymes called alcohol oxydoreductases catalyze the dehydrogenation of hydroxysteroids. Acting as oxydoreductases, HSD enzymes require nicotinamide adenine dinucleotide (NAD, oxidized) and/or NADPH as electron acceptor/donor. HSD enzymes include: 3βhydroxysteroid dehydrogenase (3β-HSD),11β-hydroxysteroid dehydrogenase (11β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD). While each P450 enzyme is the product of a single gene, the HSD enzymes have several isoforms that are products of distinct genes [15]. There are four types, classified by the number of the carbon acted upon.

116 Dehydrogenases

deceases.

**mechanisms** 

regulation of intratissue hormone concentrations by expression of steroidogenic enzymes. The modulation of local sex steroid production using pharmaceutical compounds is also a valuable treatment option for developing of novel therapies against hormonal diseases [7]. In the view of successful practice of inhibiting of non-HSD enzymes (aromatase and 5αreductase) [8, 9], recent attempt are made for development of HSD inhibitors as therapeutic strategy. Several of HSD enzymes are also considered as promising drug targets and

In this review, we summarise the data from the literatute and our own data on the main HSDs (11β-HSD, 3β-HSD 17β-HSD) focusing our attention on the localization/tissue distribution and regulation of the enzyme isoforms and their role in normal and pathological processes as revealed by experimental models and clinical observations. The review would provide better understanding on multifunctionality of HSDs and their relevance to the clinic and that would be helpful for scientists and clinicians, working in a new challenging area of development of HSD-inhibitors as new drugs for hormone-related

**2. Steroid hormones and role of hydroxysteroid dehydrogenases in steroidogenesis: steroidogenic pathways and general regulatory** 

Steroid hormones are produced by the gonads, adrenal gland and placenta and they play vital role in physiological and reproductive processes. Structurally, steroids have a basic or common nucleus called the cyclopentanoperhydrophenanthrene, consisting of three, sixmembered fully hydrogenated (perhydro) phenanthrene rings designated A, B and C, and one five-membered cyclopentane ring designated D (Fig 1, right top). In 1967, the International Union of Pure and Applied Chemistry (IUPAC) established rules for the number of carbons in a steroid and thus its biological action can be predicted. For instance, 21-carbon steroids have progestogenic or corticoid activity, 19-carbon steroids have androgenic activity and 18-carbon steroids have estrogenic activity. Cholesterol is a 27 carbon steroid that gives rise pregnenolone (21-carbon) after cleavage of its side chain. Pregnenolone is subsequently converted to progesterone, which in turn give rise androgens or corticoids. Androgens are subjected to aromatization of ring A thus giving rise estrogens [11]. The pathways of steroidogenesis differ between species, but the pathways of human steroidogenesis are shown in the Figure 1. [12]. Cholesterol is the precursor of the steroid hormones, providing backbone of the steroid molecule. The enzymes involved in the synthesis of steroid hormones can be divided into two major classes of proteins: the cytochrome P450 heme-containing proteins (CYP) and the hydroxysteroid dehydrogenases (HSD) [13, 14]. These enzymes are primarily expressed in the gonads, adrenal and placenta. Interestingly, some of these enzyme activities have been demonstrated in non-endocrine tissues, where they may be involved in important paracrine and autocrine actions. This is particularly the case in the human fetus where steroid precursors circulates at high levels and could be metabolized within tissues to produce active steroid hormones. The first class of steroidogenic enzymes, CYP proteins called hydroxylases catalyze reaction of

inhibitors, for example most of the isoforms of 17β-HSD enzyme [10].

In all species, the first and rate-limiting step in steroidogenesis, in particular androgen biosynthesis, is conversion of the C27 cholesterol to the C21 steroid, pregnenolone (Figure 1). This reaction is catalyzed by cytochrome P450scc enzyme located in the inner mitochondrial membrane. Pregnenolone diffuses across the mitochondrial membrane and it is further metabolized by enzymes associated with the smooth endoplasmic reticulum. These enzymes are: 1) cytochrome P450c17, which catalyzes the conversion of the C21 steroids pregnenolone or progesterone to the C19 steroids dehydroepiandrosterone or androstenedione, respectively; 2) 3β-HSD (∆5-∆4 isomerase), which catalyzes the conversion of the ∆5 hydroxysteroids - pregnenolone or dehydroepiandrosterone to the ∆4 ketosteroids - progesterone or androstenedione, respectively; 3) 17β-HSD (17-ketosteroid reductase), which catalyzes the final step in the biosynthesis of testosterone [16].

Corticosteroids (mineralocorticoids and glucocorticoids, C-21 cabons) derive from progestagens (progesterone and 17α-OH progesterone) after hydroxylation of carbon-21 by the enzyme 21α-hydroxylase. So, aldosterone and corticosterone share the first part of their biosynthetic pathway. The last part is mediated either by aldosterone synthase (for aldosterone) or by 11β-hydroxylase (for corticosterone). These enzymes are nearly identical (they share 11β-hydroxylation and 18-hydroxylation functions). Aldosterone synthase is also able to perform 18-oxidation. 11β-hydroxysteroid dehydrogenase (11β-HSD) catalyzes the conversion of active cortisol to inert 11 keto-products (cortisone), or vice versa, thus regulating the access of glucocorticoids to the steroid receptors.

The steroidogenic pathays/steroid output are controlled by complex regulatory mechanisms that involved wide range of factors like pituitary trophic hormones, growth factors, cytokines and steroids. The major factors, expressed since early fetal life, are steroidogenic acute regulatory protein (StAR) and Steroidogenic Factor-1 (SF-1). StAR actively transports cholesterol from the outer to the inner mitochondrial membrane and allows *CYP11A* (located in the inner membrane) access to cholesterol [17]. Cell specific expression of StAR and P450 enzymes are regulated by Steroidogenic Factor-1 (SF-1), which binds to promoter region of StAR gene and of all CYP genes, activating their expression [18, 19]. The most compelling evidence for the essential requirement for StAR in steroidogenesis is provided by StARspecific knockout mice and human mutations that caused the potentially lethal condition known as congenital lipoid adrenal hyperplasia. It is not surprising that 46XY individuals with mutated SF1 have XY sex reversal, indicative of disrupted fetal testosterone biosynthesis and masculinization. In mice with Leydig cell-specific knockout of SF-1 gene there is lack of *CYP11A* and StAR expression resulting in adrenal and gonadal agenesis [20-23]. The activity of P450scc enzyme is regulated by mitochondrial environment [24] and the vital role of this enzyme is demonstrated by homozygous mutation of *CYP11A* gene that is lethal due to inability of placenta to produce progesterone [25]. Consequently, 46XY genetic males with partial inactivation of *CYP11A* exhibit major deficiencies in masculinization [26, 27].

Hydrohysteroid Dehydrogenases – Biological Role and Clinical Importance – Review 119

**Figure 2.** Steroid biosynthetic pathways as adapted to Payne, 2007 [16]

enzyme [6, 14].

**clinical importance** 

The ∆4 pathway (pregnenolone, progesterone, androstenedione, testosterone) was the first indentified route in rat testis and subsequently shown to be preferred one. In the human and higher primates, as well as in pig and rabbit the ∆5 pathway predominates in the adult and fetal testis because human P450c17 enzyme readily converts 17α-hydroxypregnenolone to dehydroepiandrosterone (DHEA), but has little emzyme activity when 17αhydroxyprogesterone is the substrate. In the rat, P450c17 readily cleaves both the ∆4 and ∆5 C21 steroids, but in contrast to the human, it has a preference for the ∆4 pathway. In the mouse the ∆4 pathway dominates before puberty but in adult animals the ∆5 pathway may also contribute to overall testosterone production. Therefore, differences in preferred pathways between species are likely to depend upon relative substrate affinity of P450c17

The clinical importance of P450c17 enzyme is demonstrated by numerous reports on *CYP17A* gene mutations [28, 29, 30]. Both male and female patients are hypertensive because overproduction of mineralocorticoids as well as impaired production of cortisol. Affected females exhibit abnormal sexual development resulting in primary amenorrhea. Male

patients are phenotypic females due to the deficiency of testosterone production.

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

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 combined enzymatic actions of 3β-HSD and P450c17 catalyze the overall conversion of pregnenolone to androstenedione, the precursor of testosterone. This conversion can occur via one of two main pathways, either via ∆4 or ∆5 pathway and the preferred route is both species- and age-dependent. [14] (Figure 2.).

**Figure 1.** Pathways of human steroidogenesis [12].

**Figure 2.** Steroid biosynthetic pathways as adapted to Payne, 2007 [16]

evidence for the essential requirement for StAR in steroidogenesis is provided by StARspecific knockout mice and human mutations that caused the potentially lethal condition known as congenital lipoid adrenal hyperplasia. It is not surprising that 46XY individuals with mutated SF1 have XY sex reversal, indicative of disrupted fetal testosterone biosynthesis and masculinization. In mice with Leydig cell-specific knockout of SF-1 gene there is lack of *CYP11A* and StAR expression resulting in adrenal and gonadal agenesis [20-23]. The activity of P450scc enzyme is regulated by mitochondrial environment [24] and the vital role of this enzyme is demonstrated by homozygous mutation of *CYP11A* gene that is lethal due to inability of placenta to produce progesterone [25]. Consequently, 46XY genetic males with

partial inactivation of *CYP11A* exhibit major deficiencies in masculinization [26, 27].

species- and age-dependent. [14] (Figure 2.).

**Figure 1.** Pathways of human steroidogenesis [12].

The combined enzymatic actions of 3β-HSD and P450c17 catalyze the overall conversion of pregnenolone to androstenedione, the precursor of testosterone. This conversion can occur via one of two main pathways, either via ∆4 or ∆5 pathway and the preferred route is both

> The ∆4 pathway (pregnenolone, progesterone, androstenedione, testosterone) was the first indentified route in rat testis and subsequently shown to be preferred one. In the human and higher primates, as well as in pig and rabbit the ∆5 pathway predominates in the adult and fetal testis because human P450c17 enzyme readily converts 17α-hydroxypregnenolone to dehydroepiandrosterone (DHEA), but has little emzyme activity when 17αhydroxyprogesterone is the substrate. In the rat, P450c17 readily cleaves both the ∆4 and ∆5 C21 steroids, but in contrast to the human, it has a preference for the ∆4 pathway. In the mouse the ∆4 pathway dominates before puberty but in adult animals the ∆5 pathway may also contribute to overall testosterone production. Therefore, differences in preferred pathways between species are likely to depend upon relative substrate affinity of P450c17 enzyme [6, 14].

> The clinical importance of P450c17 enzyme is demonstrated by numerous reports on *CYP17A* gene mutations [28, 29, 30]. Both male and female patients are hypertensive because overproduction of mineralocorticoids as well as impaired production of cortisol. Affected females exhibit abnormal sexual development resulting in primary amenorrhea. Male patients are phenotypic females due to the deficiency of testosterone production.
