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PubMed1993

Regulation of 3 beta-hydroxysteroid dehydrogenase and 17 beta-hydroxysteroid dehydrogenase messenger ribonucleic acid levels by cyclic adenosine 3',5'-monophosphate and phorbol myristate acetate in human choriocarcinoma cells.

3 beta-Hydroxysteroid dehydrogenase (3 beta HSD) in human placenta converts 3 beta-hydroxy-5-ene steroids producing progesterone, whereas 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) mediates the interconversion of estrone and estradiol. We first showed that the expression of type I 17 beta HSD (17 beta HSD-I) gene was undetectable in human JEG-3 cells. We then studied the effects of cAMP- and protein kinase-C-dependent pathways on the expression of 3 beta HSD-I and 17 beta HSD-II genes using an analog of cAMP [8-(4-chlorophenylthio)cAMP (8CPTcAMP)] and a protein kinase-C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), in JEG-3 cells. Novel inhibitors of protein kinase-A (PKA) and PKC were also used. The 3 beta HSD cDNA probe hybridized to a single 1.7-kilobase (kb) 3 beta HSD mRNA species corresponding to the transcript of the 3 beta HSD-I gene. The 17 beta HSD cDNA probe hybridized to two 17 beta HSD transcripts of 1.3 and 2.2 kb. The 1.3-kb 17 beta HSD mRNA species was regulated, whereas the 2.2-kb species was constitutively expressed in JEG-3 cells. When JEG-3 cells were exposed to 8CPTcAMP or PMA, 3 beta HSD-I and 17 beta HSD-II gene transcriptions were increased in a dose- and time-dependent manner. Moreover, the combined effects of PMA and 8CPTcAMP on 3 beta HSD-I mRNA levels was additive and synergistic on 17 beta HSD-II mRNA levels. The mechanism by which cAMP activated accumulation of 3 beta HSD-I and 17 beta HSD-II mRNAs involved an activation of the cyclase. The effects of a cAMP-dependent kinase inhibitor and a diacylglycerol-dependent kinase inhibitor in JEG-3 cells indicated that cAMP acts on 3 beta HSD-I mRNA via a PKA-dependent mechanism, but on 17 beta HSD-II mRNA via another nonclassical cAMP-dependent mechanism. Finally, the effect of activation of both signaling pathways on expression of the 17 beta HSD-II gene as well as the effect of PMA on the 3 beta HSD-I gene did not require protein synthesis. These data provide strong evidence for the regulation of the 3 beta HSD-I and 17 beta HSD-II genes by cAMP and PKC and, thus, indicate an important endocrine and/or paracrine regulation of steroid hormone production in human placenta.

17-Hydroxysteroid Dehydrogenases↗
PubMed2001

Immunoelectron microscopic localization of 3beta-hydroxysteroid dehydrogenase and type 5 17beta-hydroxysteroid dehydrogenase in the human prostate and mammary gland.

The subcellular distribution of steroidogenic enzymes has so far been studied mostly in classical endocrine glands and in the placenta. In the peripheral intracrine organs which synthesize sex steroids there is no indication about the organelles which contain the enzymes involved in steroid biosynthesis. We have thus investigated the subcellular localization of two enzymes involved in the production of sex steroids, namely 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and type 5 17beta-hydroxysteroid dehydrogenase (17beta-HSD). Using specific antibodies to these enzymes, we conducted immunoelectron microscopic studies in two peripheral tissues, namely the human prostate and mammary gland. In the prostate, immunolabelling for both 3beta-HSD and type 5 17beta-HSD was detected in the basal cells of the tube-alveoli as well as in fibroblasts and endothelial cells lining the blood vessels. In all the labelled cell types, the gold particles were distributed throughout the cytoplasm. No obvious association with any specific organelle could be observed, although some concentration of gold particles was occasionally found over bundles of microfilaments. In mammary gland sections immunolabelled for 3beta-HSD or type 5 17beta-HSD localization, labelling was observed in the cytoplasm of the secretory epithelial cells in both the acini and terminal ducts. Immunolabelling was also found in the endothelial cells as well as in fibroblasts in stroma and blood vessels. The gold particles were not detected over any organelles, except with the occasional accumulation of gold particles over microfilaments. The present data on the localization of two steroidogenic enzymes leading to the synthesis of testosterone indicate that these enzymes are located not only in epithelial cells but also in stromal and endothelial cells in both tissues studied. The absence of any association of the enzymes with membrane-bound organelles appears as a common finding in the reactive cell types of two peripheral tissues.

3-Hydroxysteroid Dehydrogenases↗
PubMed1996

Luteolytic effect of LH: inhibition of 3 beta-hydroxysteroid dehydrogenase and stimulation of 20 alpha-hydroxysteroid dehydrogenase luteal activities in late pregnant rats.

The mechanisms associated with the onset of luteolysis in the pregnant rat are not well known. The effect of a specific rat LH antiserum (AS-rLH) and of ovine LH (oLH) on luteal steroidogenesis on day 19 of pregnancy was examined. Rat LH antiserum administered intrabursally at 1000-1100 h on day 19 of pregnancy prevented the physiological decrease in 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity, the increase in 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity and the fall in serum progesterone (P4) level observed at 1800 h on day 21 of pregnancy. To see if oLH has a direct effect on luteal steroidogenesis, the gonadotrophin was injected into the periovarian bursa. The intrabursa treatment with 1 microgram oLH on day 19 of pregnancy at 0800-0900 h did not modify corpus luteal function 36 h after treatment, but treatment with 4 micrograms oLH per ovary induced a significant progressive decrease in luteal 3 beta-HSD activity starting 12 h after treatment, while a significant increase in 20 alpha-HSD activity, concomitant with a decrease in serum P4 level, occurred 48 h after treatment. Luteal P4 content decreased with respect to control groups 36 and 48 h after intrabursal treatment with 4 micrograms oLH. The intrabursal administration of 8 micrograms oLH induced an increase in 20 alpha-HSD activity and a decrease in 3 beta-HSD activity 36 h after treatment. Administration of 4 micrograms oLH per ovary on day 8 of pregnancy induced a significant increase in serum P4 levels without modifying 3 beta-HSD activity. In rats treated with oLH on day 19 of pregnancy the decrease in 3 beta-HSD activity occurred 36 h before the significant increase in 20 alpha-HSD activity and serum P4 level. In conclusion, the luteal enzymatic activity changes and the significant decrease in the intraluteal P4 concentration induced by the intrabursal administration of oLH and the clear effect of AS-rLH preventing the physiological luteal changes preceding parturition provide good evidence of an intraovarian action of LH during the normal progression of luteolysis in late pregnant rats.

20-Hydroxysteroid Dehydrogenases↗
PubMed1997

Regulation of cytochrome P450 cholesterol side-chain cleavage, 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase type 1 and estradiol-17 beta-hydroxysteroid dehydrogenase mRNA levels by calcium in human choriocarcinoma JEG-3 cells.

In human placenta the cytochrome P450 side-chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase type 1 (3 beta-HSD-1) convert cholesterol and pregnenolone producing progesterone, whereas 17 beta-hydroxysteroid dehydrogenase type 1 (17 beta-HSD-1) mediates the interconversion of estrone and estradiol. We have examined the effects of calcium on phorbol ester- and cAMP-induced P450scc, 3 beta-HSD-1 and 17 beta-HSD-1 mRNAs in human JEG-3 cells. A23187 increased in a dose-dependent fashion in the 1.3 kb 17 beta-HSD-1 mRNA whereas a weaker increase followed by a gradual depletion effect of A23187 was observed on 3 beta-HSD-1 mRNA. No significant effect of A23187 on P450scc mRNA was observed. Using 0.50 microM of A23187 the induction of 3 beta-HSD-1 and 17 beta-HSD-1 mRNAs was maximum within about 6 h whereas P450scc mRNA levels stayed unaffected throughout the time-course period. The action of A23187 was synergistic on cAMP-stimulated 17 beta-HSD-1 mRNA levels, while in a dose-dependent manner A23187 progressively depleted 3 beta-HSD-1 and P450scc mRNA abundance probably by activation of a calcium-/calmodulin-dependent phosphodiesterase. On the phorbol 12-myristate, 13-acetate (PMA)-stimulated 3 beta-HSD-1, 17beta-HSD-1 and P450scc mRNA levels only the lowest concentration of A23187 potentialized the PMA effect on the 17 beta-HSD-1 mRNA levels. Using thapsigargin (TG), a cell-permeable sesquiterpene lactone that releases calcium by inhibiting sarco/endoplasmic reticular calcium-ATPase, our data indicated the presence in JEG-3 cells of TG-sensitive and TG-insensitive calcium-ATPases regulating 3 beta-HSD-1 and 17 beta-HSD-1 mRNA levels. These results emphasized the complexity of calcium contribution with the protein kinase A and C pathways in the regulation of P450scc, 3 beta-HSD-1 and 17 beta-HSD-1 mRNA levels. In addition, the different sensitivity of these genes to calcium suggest they could be activated by different subclasses of PKCs.

17-Hydroxysteroid Dehydrogenases↗
PubMed1982

Formation of ursodeoxycholic acid from chenodeoxycholic acid by a 7 beta-hydroxysteroid dehydrogenase-elaborating Eubacterium aerofaciens strain cocultured with 7 alpha-hydroxysteroid dehydrogenase-elaborating organisms.

A gram-positive, anaerobic, chain-forming, rod-shaped anaerobe (isolate G20-7) was isolated from normal human feces. This organism was identified by cellular morphology as well as fermentative and biochemical data as Eubacterium aerofaciens. When isolate G20-7 was grown in the presence of Bacteroides fragilis or Escherichia coli (or another 7 alpha-hydroxysteroid dehydrogenase producer) and chenodeoxycholic acid, ursodeoxycholic acid produced. Time course curves revealed that 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid produced by B. fragilis or E. coli or introduced into the medium as a pure substance was reduced by G20-7 specifically to ursodeoxycholic acid. The addition of glycine- and taurine-conjugated primary bile acids (chenodeoxycholic and cholic acids) and other bile acids to binary cultures of B. fragilis and G20-7 revealed that (i) both conjugates were hydrolyzed to give free bile acids, (ii) ursocholic acid (3 alpha, 7 beta, 12 alpha-trihydroxy-5 beta-cholanoic acid) was produced when conjugated (or free) cholic acid was the substrate, and (iii) the epimerization reaction was at least partially reversible. Corroborating these observations, an NADP-dependent 7 beta-hydroxysteroid dehydrogenase (reacting specifically with 7 beta-OH-groups) was demonstrated in cell-free preparations of isolate G20-7; production of the enzyme was optimal at between 12 and 18 h of growth. This enzyme, when measured in the oxidative direction, was active with ursodeoxycholic acid, ursocholic acid, and the taurine conjugate of ursodeoxycholic acid (but not with chenodeoxycholic, deoxycholic, or cholic acids) and displayed an optimal pH range of 9.8 to 10.2

Bacteroides fragilis↗
PubMed1996

Molecular cloning of human type 3 3 alpha-hydroxysteroid dehydrogenase that differs from 20 alpha-hydroxysteroid dehydrogenase by seven amino acids.

We have isolated, by screening a lambda gt11 human prostatic cDNA library, a cDNA clone that shows after transfection into transformed human embryonal kidney (293) cells high 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) activity that catalyzes efficiently the transformation of dihydrotestosterone to 5 alpha-androstane-3 alpha, 17 beta-diol. Chronologically, we name this enzyme type 3 3 alpha-HSD (3 alpha-HSD3). Surprisingly, human 3 alpha-HSD3 shares much higher amino acids sequence identity with human 20 alpha-HSD (97.8%) than with human type 1 and type 2 3 alpha-HSD (81.1 and 85.7% identity, respectively). DNA analysis predicts a protein of 323 amino acids with a molecular mass of 36,844. Alignment of the amino acid sequence of 3 alpha-HSD3 with other related 3 alpha- and 20 alpha-HSDs indicates that 3 alpha-HSD3 shares 68.1, 78.3, and 67.4% identity with rat 3 alpha-HSD and rabbit and rat 20 alpha-HSD, respectively. 3 alpha-HSD3 belongs to the aldo-keto reductase family and like almost all the members of this family preferred NADPH as cofactor.

20-Hydroxysteroid Dehydrogenases↗
PubMed1999

Conversion of mammalian 3alpha-hydroxysteroid dehydrogenase to 20alpha-hydroxysteroid dehydrogenase using loop chimeras: changing specificity from androgens to progestins.

Hydroxysteroid dehydrogenases (HSDs) regulate the occupancy and activation of steroid hormone receptors by converting potent steroid hormones into their cognate inactive metabolites. 3alpha-HSD catalyzes the inactivation of androgens in the prostate by converting 5alpha-dihydrotestosterone to 3alpha-androstanediol, where excess 5alpha-dihydrotestosterone is implicated in prostate disease. By contrast, 20alpha-HSD catalyzes the inactivation of progestins in the ovary and placenta by converting progesterone to 20alpha-hydroxyprogesterone, where progesterone is essential for maintaining pregnancy. Mammalian 3alpha-HSDs and 20alpha-HSDs belong to the aldo-keto reductase superfamily and share 67% amino acid sequence identity yet show positional and stereospecificity for the formation of secondary alcohols on opposite ends of steroid hormone substrates. The crystal structure of 3alpha-HSD indicates that the mature steroid binding pocket consists of 10 residues located on five loops, including loop A and the mobile loops B and C. 3alpha-HSD was converted to 20alpha-HSD by replacing these loops with those found in 20alpha-HSD. However, when pocket residues in 3alpha-HSD were mutated to those found in 20alpha-HSD altered specificity was not achieved. Replacement of loop A created a 17beta-HSD activity that was absent in either 3alpha- or 20alpha-HSD. Once loops A and C were replaced, the chimera had both 3alpha- and 20alpha-HSD activity. When loops A, B, and C were substituted, 3alpha-HSD was converted to a stereospecific 20alpha-HSD with a resultant shift in k(cat)/K(m) for the desired reaction of 2 x 10(11). This study represents an example where sex hormone specificity can be changed at the enzyme level.

20-Hydroxysteroid Dehydrogenases↗
PubMed2003

Activity and expression of progesterone metabolizing 5alpha-reductase, 20alpha-hydroxysteroid oxidoreductase and 3alpha(beta)-hydroxysteroid oxidoreductases in tumorigenic (MCF-7, MDA-MB-231, T-47D) and nontumorigenic (MCF-10A) human breast cancer cells.

BACKGROUND: Recent observations indicate that human tumorous breast tissue metabolizes progesterone differently than nontumorous breast tissue. Specifically, 5alpha-reduced metabolites (5alpha-pregnanes, shown to stimulate cell proliferation and detachment) are produced at a significantly higher rate in tumorous tissue, indicating increased 5alpha-reductase (5alphaR) activity. Conversely, the activities of 3alpha-hydroxysteroid oxidoreductase (3alpha-HSO) and 20alpha-HSO enzymes appeared to be higher in normal tissues. The elevated conversion to 5alpha-pregnanes occurred regardless of estrogen (ER) or progesterone (PR) receptor levels. To gain insight into these differences, the activities and expression of these progesterone converting enzymes were investigated in a nontumorigenic cell line, MCF-10A (ER- and PR-negative), and the three tumorigenic cell lines, MDA-MB-231 (ER- and PR-negative), MCF-7 and T-47D (ER- and PR-positive). METHODS: For the enzyme activity studies, either whole cells were incubated with [14C]progesterone for 2, 4, 8, and 24 hours, or the microsomal/cytosolic fraction was incubated for 15-60 minutes with [3H]progesterone, and the metabolites were identified and quantified. Semi-quantitative RT-PCR was employed to determine the relative levels of expression of 5alphaR type1 (SRD5A1), 5alphaR type 2 (SRD5A2), 20alpha-HSO (AKR1C1), 3alpha-HSO type 2 (AKR1C3), 3alpha-HSO type 3 (AKR1C2) and 3beta-HSO (HSD3B1/HSD3B2) in the four cell lines using 18S rRNA as an internal control. RESULTS: The relative 5alpha-reductase activity, when considered as a ratio of 5alpha-pregnanes/4-pregnenes, was 4.21 (+/- 0.49) for MCF-7 cells, 6.24 (+/- 1.14) for MDA-MB-231 cells, 4.62 (+/- 0.43) for T-47D cells and 0.65 (+/- 0.07) for MCF-10A cells, constituting approximately 6.5-fold, 9.6-fold and 7.1 fold higher conversion to 5alpha-pregnanes in the tumorigenic cells, respectively, than in the nontumorigenic MCF-10A cells. Conversely, the 20alpha-HSO and 3alpha-HSO activities were significantly higher (p < 0.001) in MCF-10A cells than in the other three cell types. In the MCF-10A cells, 20alpha-HSO activity was 8-14-fold higher and the 3alpha-HSO activity was 2.5-5.4-fold higher than in the other three cell types. The values of 5alphaR:20alpha-HSO ratios were 16.9-32.6-fold greater and the 5alphaR:3alpha-HSO ratios were 5.2-10.5-fold greater in MCF-7, MDA-MB-231 and T-47D cells than in MCF-10A cells. RT-PCR showed significantly higher expression of 5alphaR1 (p < 0.001), and lower expression of 20alpha-HSO (p < 0.001), 3alpha-HSO2 (p < 0.001), 3alpha-HSO3 (p < 0.001) in MCF-7, MDA-MB-231 and T-47D cells than in MCF-10A cells. CONCLUSION: The findings provide the first evidence that the 5alphaR activity (leading to the conversion of progesterone to the cancer promoting 5alpha-pregnanes) is significantly higher in the tumorigenic MCF-7, MDA-MB-231 and T-47D breast cell lines than in the nontumorigenic MCF-10A cell line. The higher 5alphaR activity coincides with significantly greater expression of 5alphaR1. On the other hand, the activities of 20alpha-HSO and 3alpha-HSO are higher in the MCF-10A cells than in MCF-7, MDA-MB-231 and T-47D cells; these differences in activity correlate with significantly higher expression of 20alpha-HSO, 3alpha-HSO2 and 3alpha-HSO3 in MCF-10A cells. Changes in progesterone metabolizing enzyme expression (resulting in enzyme activity changes) may be responsible for stimulating breast cancer by increased production of tumor-promoting 5alpha-pregnanes and decreased production of anti-cancer 20alpha--and 3alpha-4-pregnenes.

20-alpha-Hydroxysteroid Dehydrogenase↗
PubMed2000

Androgen formation and metabolism in the pulmonary epithelial cell line A549: expression of 17beta-hydroxysteroid dehydrogenase type 5 and 3alpha-hydroxysteroid dehydrogenase type 3.

Surfactant synthesis within developing fetal lung type II cells is affected by testosterone and 5alpha-dihydrotestosterone (5alpha-DHT). The pulmonary epithelial cell line A549, isolated from a human lung carcinoma, like normal lung type II cell, produces disaturated phosphatidylcholines and has been widely used for studying the regulation of surfactant production. Androgen receptor has been detected in A549 cells; however, the capacity of these cells for androgen synthesis and metabolism has not been investigated at molecular level. This study was undertaken to identify the steroidogenic enzymes involved in the formation and metabolism of androgens from adrenal C19 steroid precursors in A549 cells. When cultured in the presence of normal FCS, A549 intact cells converted DHEA to androstenediol, androstenedione principally to testosterone, and 5alpha-DHT to 5alpha-androstane 3alpha,17beta-diol. High levels of 17beta-hydroxysteroid dehydrogenase (HSD) and 3alpha-HSD activities were detected in both cytosol and microsomes isolated from homogenates. Analysis of A549 RNA indicated the presence of 17beta-HSD type 4 and type 5, and of 3alpha-HSD type 3 messenger RNAs. Very low levels of 3beta-HSD type 1 and 5alpha-reductase type 1 messenger RNAs and activities were detected. With regard to active androgen formation, there was little or no capacity for the conversion of DHEA to 5alpha-DHT. In contrast, androstenedione was rapidly transformed to testosterone. The pattern of steroid metabolism was not affected by the use of charcoal-stripped FCS or by the synthetic glucocorticoid dexamethasone. Together, our findings show that A549 cells express a pattern of steroid metabolism in which 17beta-HSD type 5 and 3alpha-HSD type 3 are the predominant enzymes. The level of androgens is regulated at the level of catalysis in intact cells such that the intracellular level of testosterone is stabilized, whereas 5alpha-DHT is rapidly inactivated by reduction to 3alpha,17beta-diol. This pattern of androgen metabolism has implications for the relative importance of testosterone and 5alpha-DHT in normal lung development and surfactant production.

17-Hydroxysteroid Dehydrogenases↗
PubMed1984

[Changes in ovarian 3 beta-hydroxysteroid dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase activities in pregnant rats].

Changes in ovarian 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 20 alpha-HSD activities were studied in pregnant rats. The activity of 3 beta-HSD was determined by measuring the rate of conversion of pregnenolone to progesterone. The activity of 20 alpha-HSD was determined by measuring the rate of conversion of 20 alpha-hydroxy-pregn-4-en-3-one to progesterone. 3 beta-HSD activity in corpora lutea (CL) was low between days 8 and 12, increased rapidly on day 15, and then gradually decreased until day 23. 20 alpha-HSD activity in CL markedly decreased from days 8 to 18, and then increased until day 23. The activities of 3 beta-HSD and 20 alpha-HSD in non-luteal ovarian compartment (NLO) showed no significant changes between days 8 and 23. These results indicated the important role of these enzymes in the secretion of progesterone from CL. It was also suggested that these enzyme activities in NLO might be controlled by a different mechanism.

20-Hydroxysteroid Dehydrogenases↗
PubMed1993

Expression cloning and characterization of human 17 beta-hydroxysteroid dehydrogenase type 2, a microsomal enzyme possessing 20 alpha-hydroxysteroid dehydrogenase activity.

17 beta-Hydroxysteroid dehydrogenase (17 beta-HSD) is an enzyme crucial to the regulation of intracellular levels of biologically active steroid hormones in a variety of tissues. Here, we report the isolation, structure, and characterization of a cDNA encoding the human 17 beta-HSD type 2. A 1.4-kilobase cDNA was identified, and DNA sequence analysis indicated that 17 beta-HSD type 2 was a protein of 387 amino acids with a predicted molecular weight of 42,782. The protein contained an amino-terminal type II signal-anchor motif and a carboxyl-terminal endoplasmic reticulum retention motif, which suggested that 17 beta-HSD type 2 was associated with the membranes of the endoplasmic reticulum. 17 beta-HSD type 2 was capable of catalyzing the interconversion of testosterone and androstenedione as well as estradiol and estrone. The enzyme also demonstrated 20 alpha-HSD activity toward 20 alpha-dihydroprogesterone. The amount of 17 beta-HSD type 2 mRNA in placenta was found to be high. The data suggest that the 17 beta-HSD type 2 cDNA encodes the microsomal 17 beta-HSD of human placenta, described by several laboratories.

17-Hydroxysteroid Dehydrogenases↗
PubMed1994

Comparative aspects of 11 beta-hydroxysteroid dehydrogenase. Testicular 11 beta-hydroxysteroid dehydrogenase: development of a model for the mediation of Leydig cell function by corticosteroids.

It has been shown that stress or disease-induced increases in plasma corticosterone result in diminished testosterone secretion from the testes. This article reviews investigations from our laboratories that explore the role of 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) in this process. It is proposed that the level of 11 beta-OHSD in Leydig cells dictates the level of intracellular glucocorticoid available to the glucocorticoid receptor and thus the potency of corticosteroid as an inhibitor of testosterone secretion. Stressed and unstressed rats were housed under simulated natural conditions in a Visible Burrow System. Stressed animals showed elevated plasma corticosteroid, lowered plasma testosterone, and diminished testicular 11 beta-OHSD, Immunocytochemical analysis showed that only Leydig cells of the rat testis contain 11 beta-OHSD and glucocorticoid receptors. Half-maximal inhibition of testosterone by Leydig cells required 1.5 nM dexamethasone or 0.4 microM corticosterone. Glycyrrhetinic acid, an inhibitor of 11 beta-OHSD, increased the potency of corticosterone, but did not affect dexamethasone based inhibition. The glucocorticoid receptor blocker, RU 486, prevented inhibition by both corticosterone and dexamethasone. Other classes of steroid were not inhibitors of testosterone biosynthesis. Thus, 11 beta-OHSD oxidizes corticosterone to the inactive metabolite 11-dehydrocorticosterone, relieving steroid-dependent inhibition of Leydig cell function. Lowered enzyme activity increases glucocorticoid dependent inhibition of testosterone production. We conclude that the evidence supports a role of 11 beta-OHSD in testosterone secretion by the testes.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1992

3 alpha-hydroxysteroid dehydrogenase activity catalyzed by purified pig adrenal 20 alpha-hydroxysteroid dehydrogenase.

In earlier studies, two distinct molecules, 20 alpha-HSD-I and 20 alpha-HSD-II, responsible for 20 alpha-HSD activity of pig adrenal cytosol were purified to homogeneity and characterized [S. Nakajin et al., J. Steroid Biochem. 33 (1989) 1181-1189]. We report here that the purified 20 alpha-HSD-I, which mainly catalyzes the reduction of 17 alpha-hydroxyprogesterone to 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one, catalyzes 3 alpha-hydroxysteroid oxidoreductase activity for 5 alpha (or 5 beta)-androstanes (C19), 5 alpha (or 5 beta)-pregnanes (C21) in the presence of NADPH as the preferred cofactor. The purified enzyme has a preference for the 5 alpha (or 5 beta)-androstane substrates rather than 5 alpha (or 5 beta)-pregnane substrates, and the 5 beta-isomers rather than 5 alpha-isomers, respectively. Kinetic constants in the reduction for 5 alpha-androstanedione (Km; 3.3 microM, Vmax; 69.7 nmol/min/mg) and 5 beta-androstanedione (Km; 7.7 microM, Vmax; 135.7 nmol/min/mg) were demonstrated for comparison with those for 17 alpha-hydroxyprogesterone (Km; 26.2 microM, Vmax; 1.3 nmol/min/mg) which is a substrate for 20 alpha-HSD activity. Regarding oxidation, the apparent Km and Vmax values for 3 alpha-hydroxy-5 alpha-androstan-17-one were 1.7 microM and 43.2 nmol/min/mg, and 1.2 microM and 32.1 nmol/min/mg for 3 alpha-hydroxy-5 beta-androstan-17-one, respectively. 20 alpha-HSD activity in the reduction of 17 alpha-hydroxyprogesterone catalyzed by the purified enzyme was inhibited competitively by addition of 5 alpha-DHT with a Ki value of 2.0 microM. Furthermore, 17 alpha-hydroxyprogesterone inhibited competitively 3 alpha-HSD activity with a Ki value of 150 microM.

20-Hydroxysteroid Dehydrogenases↗
PubMed2003

Oxidative 3alpha-hydroxysteroid dehydrogenase activity of human type 10 17beta-hydroxysteroid dehydrogenase.

In vitro enzyme assays have demonstrated that human type 10 17beta-hydroxysteroid dehydrogenase (17beta-HSD10) catalyzes the oxidation of 5alpha-androstane-3alpha,17beta-diol (adiol), an almost inactive androgen, to dihydrotestosterone (DHT) rather than androsterone or androstanedione. To further investigate the role of this steroid-metabolizing enzyme in intact cells, we produced stable transfectants expressing 17beta-HSD10 or its catalytically inactive Y168F mutant in human embryonic kidney (HEK) 293 cells. It was found that DHT levels in HEK 293 cells expressing 17beta-HSD10, but not its catalytically inactive mutant, will dramatically increase if adiol is added to culture media. Moreover, certain malignant prostatic epithelial cells have more 17beta-HSD10 than normal controls, and can generate DHT, the most potent androgen, from adiol. This event might promote prostate cancer growth. Analysis of the 17beta-HSD10 sequence shows that this enzyme does not have any ER retention signal or transmembrane segments and has not originated by divergence from a retinol dehydrogenase. The data suggest that the unique mitochondrial location of this HSD [Eur. J. Biochem. 268 (2001) 4899] does not prevent it from oxidizing the 3alpha-hydroxyl group of a C19 sterol in living cells. The experimental results lead to the conclusion that mitochondrial 17beta-HSD10 plays a significant part in a non-classical androgen synthesis pathway along with microsomal retinol dehydrogenases.

17-Hydroxysteroid Dehydrogenases↗
PubMed2001

17beta-Hydroxysteroid dehydrogenase type 9 and other short-chain dehydrogenases/reductases that catalyze retinoid, 17beta- and 3alpha-hydroxysteroid metabolism.

Subgroups of related short-chain dehydrogenase/reductase (SDR) family members serve as retinoid/androgen/estrogen metabolizing enzymes. These include retinol dehydrogenases (RoDHs) 1-3, cis-retinol/androgen dehydrogenase 1 and 2 (CRAD), retSDRs1-4, 9/11-cis-retinol dehydrogenase, and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) types 6 and 9. Interaction with cellular retinol-binding protein (CRBP), the major physiological form of retinol, led to the identification and cDNA cloning of RoDH1. Probes for RoDH1 contributed to cDNA cloning many of the others. Some of these SDRs show specificity with all-trans-retinol (RoDH, retSDR, 17beta-HSD6 and 9) and others with 9 and/or 11-cis-retinol (CRAD, 9/11-cis-retinol dehydrogenase). Many have 3alpha-HSD activities with 3alpha-androstandiol as the most efficiently used substrate, followed by androsterone. In addition to 3alpha-HSD activity, CRAD2 shows relatively weak 17beta-HSD activity with testosterone. Rat 17beta-HSD6 and mouse 17beta-HSD9, which are not interspecies homologs, have efficient 17beta-HSD activities. 17beta-HSD6 has approximately 50% greater 17beta-HSD activity with estradiol than with 3alpha-androstandiol. With 3alpha-androstandiol, 17beta-HSD9 operates equally efficiently as a 17beta-HSD or a 3alpha-HSD. The multi-substrate nature of these SDRs allows for retinoid/steroid interactions. The ability of some these SDRs to access retinol bound with CRBP provides specificity in retinoid metabolism and allows retinoic acid biosynthesis and retinol esterification to continue, as CRBP protects retinol from the general cellular milieu.

17-Hydroxysteroid Dehydrogenases↗
PubMed1999

Localization of type 5 17beta-hydroxysteroid dehydrogenase, 3beta-hydroxysteroid dehydrogenase, and androgen receptor in the human prostate by in situ hybridization and immunocytochemistry.

An important source of androgens in the human prostate are those synthesized locally from the inactive adrenal precursor dehydroepiandrosterone (DHEA) and its sulfated derivative DHEA-S. Three beta-HSD (hydroxysteroid dehydrogenase) converts DHEA into androstenedione (4-dione), whereas type 5 17beta-HSD catalyzes the reduction of 4-dione into testosterone in the human prostate and other peripheral intracrine tissues. In the present study, we have used two complementary approaches, namely in situ hybridization and immunocytochemistry, to identify the cells that contain the type 5 17beta-HSD messenger RNA and enzyme in human benign prostatic hyperplasia (BPH). Localization of 3beta-HSD and of the androgen receptor (AR) was also investigated by immunostaining in the same tissue. To find out whether there are any differences between BPH and normal prostate tissue, the localization of type 5 17beta-HSD was reexamined by immunocytochemistry in the normal human prostate samples and also in normal prostate epithelial cell line (PrEC). The in situ hybridization results obtained with a tritiated uridine triphosphate (3H-UTP)-labeled type 5 17beta-HSD riboprobe are in agreement with the immunostaining data obtained with a specific antibody to the enzyme. The immunostaining results obtained from normal prostate tissue and BPH were found to be similar. Thus, in the glandular epithelium, basal cells highly express the messenger RNA and the enzyme, whereas luminal cells show a much lower and variable level of expression. In the stroma and walls of blood vessels, fibroblasts and the endothelial cells lining the blood vessels show positive staining. Similar results are observed when the cellular distribution of 3beta-HSD is investigated. AR immunoreactivity, however, shows a different distribution because, in the epithelium, most of the nuclei of basal cells are negative, whereas the majority of nuclei of the luminal cells show positive staining. A strong reaction for AR is also found in most stromal cell nuclei and in the nuclei of most endothelial cells, as well as in some other cells of the walls of blood vessels. In conclusion, human type 5 17beta-HSD, as well as 3beta-HSD, are highly expressed, not only in the basal epithelial cells and stromal fibroblasts but also in the endothelial cells and fibroblasts of the blood vessels. AR, on the other hand, is highly expressed in the luminal cells. The present data suggest that DHEA is transformed in the basal cells of the glandular epithelium into 4-dione by 3beta-HSD and then into testosterone by type 5 17beta-HSD, whereas dihydrotestosterone is synthesized in the luminal cells after diffusion of testosterone from the underlying layer of basal cells. The potential role of androgen formation and action in blood vessels is unknown and opens new avenues of investigation for a better understanding of the multiple roles of androgens.

17-Hydroxysteroid Dehydrogenases↗
PubMed2004

Evolutionary analysis of 11beta-hydroxysteroid dehydrogenase-type 1, -type 2, -type 3 and 17beta-hydroxysteroid dehydrogenase-type 2 in fish.

Steroid dehydrogenases regulate the access of active steroids to their receptors. In particular, 11beta-hydroxysteroid dehydrogenase-type 1 (11beta-HSD1) and 11beta-HSD2 regulate the levels of glucocorticoids, such as cortisol, and 17beta-HSD1 and 17beta-HSD2 regulate the levels of androgens and estrogens. Human 11beta-HSD1 and 11beta-HSD2 are distant homologs, with less than 25% amino acid sequence identity, as are human 17beta-HSD1 and 17beta-HSD2. In contrast, human 11beta-HSD2 and 17beta-HSD2 are close homologs, with about 43% sequence identity. Until recently, deciphering early events in the evolution of 11beta-HSD2 and 17beta-HSD2 was difficult because only mammalian sequences were available. The completely sequenced Takifugu, Tetraodon and medaka genomes and the almost completed zebrafish genome provide an opportunity to investigate the evolution of 11beta-HSD2, 17beta-HSD2, and 11beta-HSD1. Unexpectedly, a search of the Takifugu, Tetraodon and medaka genomes only found an ortholog to 11beta-HSD2 and none to 17beta-HSD2, while the zebrafish genome contains orthologs of both enzymes. This suggests that 17beta-HSD2 was lost in teleosts after the divergence of zebrafish and medaka. Also unexpectedly, searches with 11beta-HSD1 only identified several fish 11beta-HSD3s, as well as an ortholog in Ciona, indicating that 11beta-HSD3 is the ancestor of 11beta-HSD1.

Animals↗
PubMed1997

Structure-function relationships of 3 beta-hydroxysteroid dehydrogenase: contribution made by the molecular genetics of 3 beta-hydroxysteroid dehydrogenase deficiency.

The transformation of delta 5-3 beta-hydroxysteroids into the corresponding delta 4-3-keto-steroids is an essential step for the biosynthesis of all classes of active steroids: progesterone, mineralocorticoids, glucocorticoids, androgens, and estrogens. These steroid hormones play a crucial role in the differentiation, development, growth, and physiological function of most human tissues. The structures of several cDNAs encoding 3 beta-HSD isoenzymes have been characterized in human and several other vertebrate species: human types I and II; macaque; bovine; rat types I, II, III, and IV; mouse types I, II, III, IV, V and VI; hamster types I, II, and III; and rainbow trout. Their transient expression reveals that 3 beta-HSD and delta 5-delta 4-isomerase activities reside within a single protein. Distinct approaches have been used for a better understanding of the structure-function relationships of these 3 beta-HSD enzymes: i) affinity radiolabeling studies of the human type I 3 beta-HSD; ii) identification and the functional consequences of the human type-II 3 beta-HSD mutations detected in patients with 3 beta-HSD deficiency. Taken together, all of these data were examined to determine whether the relationship between the genotype and the phenotype of these patients were consistent with in vitro mutagenesis studies. 3 beta-HSD deficiency, transmitted in an autosomic recessive disorder, is characterized by varying degrees of salt wasting; in genetic males, fetal testicular 3 beta-HSD deficiency causes an undervirilized male genitalia (male pseudohermaphroditism); females exhibit either normal sexual differentiation or mild virilization. All mutations were detected in the type II 3 beta-HSD gene, which is expressed almost exclusively in the adrenals and gonads. No mutation was detected in the type I 3 beta-HSD gene, which is expressed in peripheral tissues. The finding of a normal type I 3 beta-HSD gene explains the elevated delta 5-steroids and mild virilization of affected girls at birth. To date, 24 mutations have been identified in 25 distinct families with 3 beta-HSD deficiencies. All nonsense and frameshift mutations introducing a premature termination codon were associated with the classical salt-losing form. The locations of these nonsense mutations suggest that at least the first 318 amino acids out of 371 are required for 3 beta-HSD activity. The consequences of the missense mutations on some domains of the 3 beta-enzyme, such as membrane-spanning domains, cofactor-binding site, and steroid-binding site, were reviewed. The future crystallization of the overexpressed normal and mutant-type II-3 beta-HSD enzymes should contribute to a better understanding of the structure-function relationships of this enzyme, especially for missense mutations located outside the putative functional regions.

3-Hydroxysteroid Dehydrogenases↗