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PubMed1995

Properties and regulation of 17 beta-hydroxysteroid oxidoreductase of OVCAR-3, CAOV-3, and A431 cells: effects of epidermal growth factor, estradiol, and progesterone.

Although there is a growing body of evidence that 17 beta-hydroxysteroid oxidoreductase plays a role in the regulation of steroid levels in epithelial tumors of the endometrium and breast, our knowledge of its role in other gynecologic tumors is limited. In this investigation, the 17 beta-hydroxysteroid oxidoreductase activity of cell lines derived from two ovarian tumors (OVCAR-3, CAOV-3) and an epidermoid tumor of the vulva (A431) was assayed under conditions which differentiate between 17 beta-hydroxysteroid oxidoreductase type 1, a cytosolic isoform highly specific for estradiol, and type 2, a membrane bound isoform reactive with both estradiol and testosterone. On the basis of estradiol/testosterone activity ratios, all three cell lines appear to have type 2-like activity, with the specific activity of A431 markedly greater than that of the other cell lines. Estradiol, progesterone, or EGF, alone or in combination, were without effect on the enzymatic activity of OVCAR-3 cells. EGF decreased the activity of CAOV-3 cells slightly. In contrast, EGF stimulated A431 17 beta-hydroxysteroid oxidoreductase activity 7-8-fold over a 5-day exposure. Estradiol or progesterone, singly or in combination, also did not effect the enzymatic activity of A431 cells. However, progesterone inhibited the increase in activity seen in the presence of EGF. With EGF, estradiol, and progesterone together, the increase in enzymatic activity was comparable to that with EGF alone. The effects of estradiol and progesterone appear to result from steroid actions following binding of EGF to low-affinity receptors on A431 cells.

Epidermal Growth Factor↗

PubMed1998

Human hydroxysteroid sulfotransferase SULT2B1: two enzymes encoded by a single chromosome 19 gene.

We have cloned and characterized cDNAs that encode two human hydroxysteroid sulfotransferase (SULT) enzymes, SULT2B1a and SULT2B1b, as well as the single gene that encodes both of these enzymes. The two cDNAs differed at their 5'-termini and had 1050- and 1095-bp open reading frames that encoded 350 and 365 amino acids, respectively. The amino acid sequences encoded by these cDNAs included "signature sequences" that are conserved in all known cytosolic SULTs. Both cDNAs appeared, on the basis of amino acid sequence analysis, to be members of the hydroxysteroid SULT "family, " SULT2, but they were only 48% identical in amino acid sequence with the single known member of that family in humans, SULT2A1 (also referred to as DHEA ST). Northern blot analysis demonstrated the presence of SULT2B1 mRNA species approximately 1.4 kb in length in human placenta, prostate, and trachea and-faintly-in small intestine and lung. Expression of the two human SULT2B1 cDNAs in COS-1 cells showed that both of the encoded proteins catalyzed sulfation of the prototypic hydroxysteroid SULT substrate, dehydroepiandrosterone, but both failed to catalyze the sulfate conjugation of 4-nitrophenol or 17beta-estradiol, prototypic substrates for the phenol and estrogen SULT subfamilies. Both of these cDNAs were encoded by a single gene, SULT2B1. The locations of most exon-intron splice junctions in SULT2B1 were identical to those of the only other known human hydroxysteroid SULT gene SULT2A1 (previously STD). The divergence in 5'-terminal sequences of the two SULT2B1 cDNAs resulted from alternative transcription initiation prior to different 5' exons, combined with alternative splicing. SULT2B1 mapped to human chromosome band 19q13.3, approximately 500 kb telomeric to the location of SULT2A1.

Alternative Splicing↗

PubMed1996

Differential expression and immunohistochemical localisation of the phenol and hydroxysteroid sulphotransferase enzyme families in the developing lung.

Reversible sulphation, catalysed by sulphotransferases and sulphatases, of biologically active compounds such as androgens and oestrogens is a sensitive mechanism for regulating their bioavailability, and we have previously hypothesised that this process plays a significant role in the regulation of human fetal lung development. Sulphation is also a major detoxification reaction, contributing significantly to the body's chemical defence mechanism. We have used qualitative and semiquantitative immunological studies to determine the temporal expression and localisation of phenol and hydroxysteroid sulphotransferases during human lung development. Our results show that in the early fetal lung, phenol sulphotransferase expression is at its highest, and is most widely distributed throughout the developing respiratory epithelium. With later development, expression levels decrease and become predominantly restricted to the more proximal airways. In contrast, hydroxysteroid sulphotransferase is present only at very low levels in the early-gestation lung but expression increases rapidly through gestation to reach an apparent peak by 1 year postnatal age. The proximal-to-distal gradients of phenol and hydroxysteroid sulphotransferase expression were similar in mature respiratory epithelium, with immunoreactivity in ciliated cells, non-ciliated secretory cells and basal cells, but with no apparent expression in mucus-secreting cells. These studies provide supporting evidence for the hypothesis that hydroxysteroid sulphotransferase, an androgen-inactivating enzyme, contributes to the role of androgens in retarding the maturation of human lung in utero.

Arylsulfotransferase↗

PubMed1999

Testosterone sulfotransferase: evidence in the guinea pig that this reaction is carried out by 3 alpha-hydroxysteroid sulfotransferase.

During the course of isolating, characterizing, and cloning estrogen and 3-hydroxysteroid sulfotransferases from the guinea pig adrenal gland, it was noted that cytosolic preparations from this tissue would also sulfonate testosterone. Therefore, we set out to isolate and clone the enzyme that performs this reaction. Testosterone sulfotransferase (TST) was isolated from the guinea pig adrenal by using the standard procedures of ion exchange, affinity, and high-performance liquid chromatography. When purified, TST was examined by liquid-phase nondenaturing isoelectric focusing, it was found that the TST activity profile completely overlapped with the activity profile of the 3alpha-hydroxysteroid sulfotransferase (3alphaHST) isoform, but not the 3beta-hydroxysteroid sulfotransferase (3betaHST) isoform. This finding was further investigated by overexpressing the cDNAs for 3alphaHST and 3betaHST in Escherichia coli and examining the expressed proteins for TST activity. This experiment confirmed that 3alphaHST does indeed function as a TST. In addition, 3alphaHST was also found to sulfonate estradiol but not estrone, a finding that further suggested that 3alphaHST may function as a general 17beta-hydroxysteroid sulfotransferase.

Adrenal Glands↗

PubMed1985

Strain differences in purified rat hepatic 3 alpha-hydroxysteroid UDP-glucuronosyltransferase.

Qualitative and quantitative differences of purified hepatic 3 alpha-hydroxysteroid UDP-glucuronosyltransferase were investigated in Wistar and Sprague-Dawley rats. Individual differences in the glucuronidation rate of androsterone and chenodeoxycholic acid were observed in hepatic microsomal fractions from Wistar but not Sprague-Dawley rats. No individual variation was observed in the glucuronidation of testosterone, p-nitrophenol or oestrone. The 3 alpha-hydroxysteroid UDP-glucuronosyltransferases from livers of Wistar and Sprague-Dawley rats were isolated and highly purified by using Chromatofocusing and affinity chromatography. The amount of 3 alpha-hydroxysteroid UDP-glucuronosyltransferase in the liver of Wistar rats exhibiting low rates for androsterone glucuronidation is about 10% or less than that found in hepatic microsomal fractions obtained from Wistar rats having high rates for androsterone glucuronidation. The apparent Km for androsterone with purified 3 alpha-hydroxysteroid UDP-glucuronosyltransferase from Wistar rats with high glucuronidation activity (6 microM) was not different from that observed for the enzyme purified from Sprague-Dawley animals, whereas that for the enzyme purified from Wistar rats with low glucuronidation activity was substantially higher (120 microM). Despite the differences in apparent Km values for androsterone, the apparent Km for UDP-glucuronic acid (0.3 mM) was not different in the different populations of rats.

Androsterone↗

PubMed2000

Molecular characterization of a first human 3(alpha-->beta)-hydroxysteroid epimerase.

In this report, we describe the isolation and characterization of a cDNA encoding an enzyme that exhibits catalytic characteristics of a 3(alpha-->beta)-hydroxysteroid epimerase (3(alpha-->beta)-HSE). The enzyme overexpressed in human 293 embryonic kidney cells transforms androsterone into epi-androsterone in two steps: the oxidation of androsterone to 5 alpha-androstane-3,17-dione, followed by the reduction of the latter to epi-androsterone. The reverse reaction, 3(beta-->alpha)-hydroxysteroid epimeration, is approximately 10-fold weaker. These results are confirmed by V(max)/K(m) determination, which shows that the enzyme catalyzes the oxidation of androsterone to 5 alpha-androstane-3,17-dione and the reduction of 5 alpha-androstane-3,17-dione to epi-androsterone more efficiently than the reverse reactions. The selective catalysis of the reaction following the 3(alpha-->beta) direction is also observed in intact transfected cells in culture, which better reflect physiological conditions. In vitro assays reveal that the recombinant enzyme prefers NAD(+) and NADH as cofactors and could recognize both C-19 and C-21 3 alpha-hydroxysteroids as substrates. DNA sequence analysis predicts a protein of 317 amino acids. Tissue distribution analysis using RT-PCR reveals that the mRNA of the enzyme is expressed in various tissues, including liver, brain, prostate, adrenal, and uterus, with the most abundant expression in the liver. Because active hydroxysteroids generally exert their effect in a stereo-specific manner, 3(alpha-->beta)-HSE could thus potentially play an important role in regulating the biological activities of various steroids.

Amino Acid Sequence↗

PubMed1975

Enzymatic preparation of 20 beta-hydroxysteroids in a two-phase system.

The behavior of 20 beta-hydroxysteroid dehydrogenase in a two-phase system consisting of an emulsion of water with an immiscible organic solvent was investigated. The effect of several organic solvents on the stability, activity, and kinetic profile of the enzyme was considered. The most suitable system for carrying out the enzymatic reaction proved to be water-butyl acetate. The production of high quantitied of 20 beta-hydroxysteroids in 100% yield using catalytic amount of cofactor was achieved by coupling the 20 beta-hydroxysteroid dehydrogenase-and the alcohol dehydrogenase-catalyzed reactions.

20-Hydroxysteroid Dehydrogenases↗

PubMed1995

Structures stabilizing the dimer interface on human 11 beta-hydroxysteroid dehydrogenase types 1 and 2 and human 15-hydroxyprostaglandin dehydrogenase and their homologs.

Human 11 beta-hydroxysteroid dehydrogenase-types 1 and 2 and human 15-hydroxyprostaglandin dehydrogenase belong to a large family of oxidoreductases that includes human dihydropteridine reductase and Streptomyces hydrogenans 20 beta-hydroxysteroid dehydrogenase, for which 3D structures are available. Almost all of these enzymes are either dimers or tetramers. The dimer interface of rat dihydropteridine reductase consists of alpha-helices E and F from each monomer arranged in a four alpha-helix bundle [Varughese et al. (1992) Proc. Natl. Acad. Sci. USA 89, 6080-6084]. Alpha-helix F contains tyrosine-146 and lysine-150, residues that are highly conserved in this protein superfamily and have been proposed to be at the catalytic site. We have examined the dimer interface between alpha-helix F in human and rat dihydropteridine reductase and Streptomyces hydrogenans 20 beta-hydroxysteroid dehydrogenase as well as modeled 3D structures of steroid and prostaglandin dehydrogenases and homologs for stabilizing interactions. We find a site in the middle of alpha-helix F that stabilizes the dimer. This anchor is adjacent to conserved lysine on alpha-helix F. Our analysis suggests that sequence variation in the anchor may be important in substrate specificity.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1984

5 Alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase activities in isolated canine prostatic epithelial cells.

Kinetic parameters of two enzymes, 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase, have been calculated for freshly isolated canine prostatic epithelial cells separated into secretory and non-secretory cells on the basis of their density in Percoll gradients. For 5 alpha-reductase, a Km value of 3 X 10(-6) M was obtained in both epithelial cell types, and similar Vmax values of 1.7 X 10(-12) and 1.9 X 10(-12) moles of dihydrotestosterone formed/min/10(6) cells (P greater than 0.50) were calculated in secretory and non-secretory cells, respectively. The Km of 3 alpha-hydroxysteroid dehydrogenase (dihydrotestosterone----3 alpha-androstanediol) varied between 2.2 and 2.8 X 10(-6) M (P greater than 0.50) with respective Vmax's of 9 and 24 X 10(-12) moles of 3 alpha-androstanediol formed/min/10(6) cells (P less than 0.005) in secretory and non-secretory cells. For the reverse reaction, that is the transformation of 3 alpha-androstanediol to dihydrotestosterone, the Km's obtained were 0.4 and 0.5 X 10(-6) M (P greater than 0.50) with Vmax's of 14 and 19 X 10(-12) moles of dihydrotestosterone formed/min/10(6) cells (P less than 0.50) in secretory and non-secretory cells, respectively. Vmax values for 3 alpha-hydroxysteroid dehydrogenase are 10-fold higher than the ones for 5 alpha-reductase. Moreover, Km values for the reaction 3 alpha-androstanediol----DHT are 5-fold lower than those calculated for the reverse reaction and for 5 alpha-reductase. This could explain why the major metabolic pathway in the canine prostate gland is the conversion of 3 alpha-androstanediol to dihydrotestosterone.

3-Hydroxysteroid Dehydrogenases↗

PubMed1986

3 beta-hydroxysteroid isomerase dehydrogenase in guinea-pig kidney: possible involvement in 11-deoxycorticosterone formation in situ.

3 beta-Hydroxysteroid isomerase dehydrogenase, capable of acting on C21- and C19-3 beta-hydroxy-5-ene-steroids has been found in guinea-pig kidney at equivalent levels to those in guinea pig testes. Of the 3 beta-hydroxy-5-ene-steroids present in guinea pig serum, 21-hydroxypregnenolone occurs in highest concentration (17 nM) followed by pregnenolone (10 nM), whereas 17 alpha-hydroxy-pregnenolone and dehydroepiandrosterone occur in very low concentrations (less than 0.5 nM). Furthermore, the concentration of 21-hydroxypregnenolone relative to 11-deoxycorticosterone (the mineralocorticoid of the guinea pig), is 10:1 (Nishikawa and Strott, Steroids 41 (1983) 105-120). The apparent Km value for 21-hydroxypregnenolone, for the reaction yielding 11-deoxycorticosterone as catalysed by guinea pig kidney microsomes, was 85 nM and the Vmax 33 pmol/min per mg protein. Pregnenolone was a competitive inhibitor (apparent Ki = 5 microM) in the above reaction. A sex difference in the level of the enzyme in the kidney was found (activity in the female was one-third of that in the male) which may indicate that the enzyme is under partial androgen control. 3 beta-Hydroxysteroid isomerase dehydrogenase activity was also detected in guinea pig liver and again it was lower in the female. Whilst the exact role of 3 beta-hydroxysteroid isomerase dehydrogenase in guinea-pig kidney remains uncertain, the data suggest that it may utilise blood-borne 21-hydroxypregnenolone, the later then playing the role of a prohormone.

17-Hydroxysteroid Dehydrogenases↗

PubMed1987

Cofactor requirements of steroid-17-20-desmolase and 20 alpha-hydroxysteroid dehydrogenase activities in cell extracts of Clostridium scindens.

Two neutral steroid-transforming activities were demonstrated in cell extracts of Clostridium scindens. Steroid-17-20-desmolase and 20 alpha-hydroxysteroid dehydrogenase were found to be inducible in cells cultured in the presence of cortisol. Both activities required manganese ions and NAD+ or NADH for activity. Cortisol, cortisone and 11-desoxycortisol were substrates as well as inducers of steroid-17-20-desmolase and 20 alpha-hydroxysteroid dehydrogenase activities. 17 alpha-Hydroxyprogesterone was an effective inducer but did not serve as a substrate for either enzyme activity. C. scindens is the first bacterial species of the normal human intestinal flora reported to elaborate inducible steroid-17-20-desmolase and 20 alpha-hydroxysteroid dehydrogenase activities. The results of cofactor, substrate specificity and induction studies suggest that these two activities may reside in the same enzyme complex.

20-Hydroxysteroid Dehydrogenases↗

PubMed1990

17 Beta-hydroxysteroid oxidoreductase activity: age-dependent profile in rat liver and kinetic properties of the hepatic microsomal enzyme in relation to cytochrome P450-dependent steroid hydroxylation.

The functional relationship between the microsomal cytochrome P450 and 17 beta-hydroxysteroid oxidoreductase (HSOR) enzymes involved in steroid metabolism was investigated in rat liver. In male and female rat hepatic microsomes the NADPH-dependent conversion of androstenedione (AD) to testosterone (T) was approx. 4-fold greater at 6 weeks of age than in 1 week old animals. In hepatic microsomes from 15 week old rats the activity of the HSOR pathway was greater in males than in females (1.51 compared to 0.80 nmol T formed/min/mg protein). However, oestradiol administration to intact adult male rats did not decrease HSOR activity. Thus, androgen is not essential for maintenance of HSOR enzymes. Instead, it is likely that irreversible androgen imprinting of the HSOR enzyme occurs during the prepubertal period. The in vitro characteristics of HSOR activity were also assessed. The Km for NADH-dependent reduction of AD to T was 9.2 microM and the Vmax was 3.0 nmol/min/mg protein but the NAD-mediated formation of AD from T did not follow Michaelis-Menton kinetics. pH markedly influenced HSOR-mediated AD/T interconversion with 17-ketosteroid reduction facilitated at low pH, and 17 beta-hydroxysteroid dehydrogenation about 2-fold more efficient at pH 8.0 than at pH 5.5. Product steroid activation of HSOR activity was noted. 17 beta-Hydroxysteroids, including T and oestradiol, activated the rate of conversion of AD to T and 17-ketosteroids such as oestrone and AD activated the NAD-dependent dehydrogenation of T. Activation was not observed at low steroid substrate concentrations so that it was not possible to analyse this phenomenon by a conventional kinetic approach.

17-Hydroxysteroid Dehydrogenases↗

PubMed1983

The distribution of 5 alpha-reductase and 3 alpha(beta)-hydroxysteroid dehydrogenase activities in the hyperplastic human prostate gland.

This study examines the distribution of 5 alpha-reductase and 3 alpha(beta)-hydroxysteroid dehydrogenase activities throughout the intact hyperplastic prostate gland and relate these measurements to the fibromuscular/epithelial composition and to the gross glandular morphology. The relative capacities of the stroma and epithelium to metabolize testosterone and dihydrotestosterone were also examined. The results indicate that under optimum reaction conditions an uneven distribution of 5 alpha-reductase and 3 alpha(beta)-hydroxysteroid dehydrogenase could be measured across the prostate. These regional variations reflect true differences in metabolic activity and were independent of any morphological changes: caution is therefore advised when interpreting hormonal metabolic data obtained from single sampling of the gland. Our investigations also suggest that the capacity to metabolize testosterone was evenly distributed between stroma and epithelium and that both tissue components are primary sites for 5 alpha-reductase activity. The reductive 3 alpha(beta)-hydroxysteroid dehydrogenase was also found in both tissue types but the mean stromal activity was marginally higher than the levels measured in the epithelium.

3-Hydroxysteroid Dehydrogenases↗

PubMedDate not supplied

Rat liver 3 alpha-hydroxysteroid dehydrogenase.

3 alpha-HSD appears to be a multifunctional enzyme. In addition to its traditional role of catalyzing early steps in androgen metabolism, it will also oxidoreduce prostaglandins and detoxify trans-dihydrodiols (proximate carcinogens). Since these novel reactions have been quantified using homogeneous enzyme it is necessary to interpret the role of the enzyme in these processes in vivo with some caution. However, it is rare that such observations on a purified hydroxysteroid dehydrogenase have led to such important questions. Is the 3 alpha-HSD the only steroid dehydrogenase that transforms prostaglandins and trans-dihydrodiols? Are hydroxysteroid dehydrogenases and prostaglandin dehydrogenases the same enzymes in certain tissues? Does 3 alpha-HSD protect against chemical carcinogenesis in vivo? The inhibition of the purified dehydrogenase by therapeutically relevant concentrations of anti-inflammatory drugs also deserves comment. Is this hydroxysteroid dehydrogenase really an in vivo target for anti-inflammatory drug action? Could these drugs exert some of their pharmacological effect either by preventing glucocorticoid metabolism in some tissues or by preventing the transformation of PGF2 alpha (non-inflammatory prostanoid) to PGE2 (a pro-inflammatory prostanoid)? Could these drugs, by inhibiting trans-dihydrodiol oxidation, potentiate the initiation of chemical carcinogenesis? These and other important questions can be answered only by developing specific inhibitors for the dehydrogenase to decipher its function in vivo.

3-Hydroxysteroid Dehydrogenases↗

PubMed1994

Compartmentalization of type I 17 beta-hydroxysteroid oxidoreductase in the human ovary.

The steroid-metabolizing enzyme, type I 17 beta-hydroxysteroid oxidoreductase (17 beta-HSOR) also called 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) plays a key role in ovarian synthesis of 17 beta-estradiol. This is the only enzyme in the steroid-metabolizing pathway which has not been localized in the human ovary by immunohistochemistry. In this study, using antibody directed against human placental cytosolic 17 beta-HSOR (type I), a single protein band with a relative molecular mass of approximately 34 kDa was demonstrated by Western analysis in both human luteinized granulosa cells and placental tissue. In placental tissue, immunoreactive type I 17 beta-HSOR was demonstrated within the syncytiotrophoblast using immunohistochemistry. In human ovary, immunoreactive type I 17 beta-HSOR was localized exclusively in granulosa cells of developing follicles, ranging from primary follicles with a single layer of cuboidal-shaped granulosa cells, preantral follicles with multiple layers of granulosa cells, and large antral follicles. No immunoreactivity was detected in spindle-shaped granulosa cells of primordial follicles, theca interna, theca externa or surrounding stroma. In the corpus luteum, type I 17 beta-HSOR immunoreactivity was localized solely in granulosa-lutein cells. For comparison, immunoreactive 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) was examined in the same tissues. Both theca interna and granulosa cells of preantral and antral follicles exhibited 3 beta-HSD staining. Primary follicles did not exhibit detectable 3 beta-HSD in either granulosa or theca cells.(ABSTRACT TRUNCATED AT 250 WORDS)

17-Hydroxysteroid Dehydrogenases↗

PubMed1996

Expression of multiple forms of 3 beta-hydroxysteroid dehydrogenase in the mouse liver during fetal and postnatal development.

The enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) is essential for the biosynthesis of all steroid hormones. To date this laboratory has isolated and characterized five distinct 3 beta HSD cDNAs in the mouse (3 beta HSD I-V). The different isoforms fall into two functionally distinct groups. 3 beta HSD I and III function as dehydrogenase/isomerases and 3 beta HSD IV and V function as 3-ketosteroid reductases. Previously it was shown that the liver of the adult mouse expresses 3 beta HSD II, III and V, with 3 beta HSD III being the major isoform. This study examines the expression of the different forms of 3 beta HSD mRNAs and proteins in the livers of male and female mice during fetal and postnatal development. 3 beta HSD I, which in the adult mouse is expressed only in the gonads and adrenal glands, is the major isoform expressed in both male and female livers during fetal development until the first postnatal (pn) day after which time 3 beta HSD III becomes the major isoform. Expression of 3 beta HSD I mRNA and protein completely ceases after day 20 pn. The expression of 3 beta HSD V is first detected at day 40 pn and is observed only in the male. Very low expression of 3 beta HSD II mRNA is detected throughout development. Previous characterization of enzymatic activity of the expressed proteins showed that 3 beta HSD I exhibits lower Km values for the delta 5-3 beta-hydroxysteroids than 3 beta HSD III, indicating that 3 beta HSD I functions as a more efficient 3 beta-hydroxysteroid dehydrogenase/isomerase than 3 beta HSD III. The results of this study suggest that the liver may play an important role in the biosynthesis of steroid hormones during murine fetal development.

3-Hydroxysteroid Dehydrogenases↗

PubMed1993

Molecular cloning of multiple cDNAs encoding human enzymes structurally related to 3 alpha-hydroxysteroid dehydrogenase.

Rat liver 3 alpha-hydroxysteroid dehydrogenase cDNA was previously cloned by us. In this study, we used the rat cDNA as the probe to screen a human liver lambda gt11 cDNA library. A total of four different cDNAs were identified and sequenced. The sequence of one of the cDNAs is identical to that of the human chlordecone reductase cDNA except that our clone contains a much longer 5'-coding sequence than previously reported. The other three cDNAs display high degrees of sequence homology to those of both rat 3 alpha-hydroxysteroid dehydrogenase and human chlordecone reductase. Because 3 alpha-hydroxysteroid dehydrogenase and human chlordecone reductase belong to the aldo-keto reductase superfamily, we named these human clones HAKRa to HAKRd. Northern blot analysis showed that the liver expresses the highest levels of all four clones. Expression of all four clones was also detected in the brain, kidney, lung, and testis, whereas the placenta expressed only the messenger RNA for HAKRb. Genomic blot analysis using HAKRb as the probe detected multiple DNA fragments hybridized to the probe and a high degree of restriction fragment length polymorphism, suggesting the complexity of this supergene family.

3-Hydroxysteroid Dehydrogenases↗

PubMed2003

Role of human type 3 3alpha-hydroxysteroid dehydrogenase (AKR1C2) in androgen metabolism of prostate cancer cells.

Four human aldo-keto reductases (AKRs) that belong to the AKR1C subfamily function in vitro as 3-keto-, 17-keto- and 20-ketosteroid reductases or as 3alpha-, 17beta- and 20alpha- hydroxysteroid oxidases to varying degrees. By acting as ketosteroid reductases or hydroxysteroid oxidases these AKRs can either convert potent sex hormones (androgens, estrogens and progestins) into their inactive metabolites or they can form potent hormones by catalyzing the reverse reaction. In this manner they may regulate occupancy and trans-activation of steroid hormone receptors. Tissue distribution studies previously indicated that AKR1C2 (type 3 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD)) and AKR1C3 (type 2 3alpha-HSD) are highly expressed in human prostate. An assessment of the directionality of these AKR1C isozymes in a cellular environment would help identify which isozymes are responsible for 5alpha-dihydrotestosterone (5alpha-DHT) formation or its elimination in the prostate. An imbalance in 5alpha-DHT levels has been implicated in development of prostate carcinoma and benign prostatic hyperplasia. We focused our attention on AKR1C2 since this is the isoform that will oxidize 3alpha-androstanediol (3alpha-diol) to 5alpha-DHT in vitro, suggesting it could elevate 5alpha-DHT levels. To determine whether AKR1C2 preferentially functions as a reductase or an oxidase in a cellular context, we transiently transfected AKR1C2 (pcDNA3-AKR1C2) into COS-1 cells and stably transfected pcDNA3-AKR1C2 and pLNCX-AKR1C2 constructs into PC-3 and LNCaP cells, respectively. COS-1 is a monkey kidney cell line, while PC-3 and LNCaP cells are androgen receptor (-) and (+) prostate adenocarcinoma cell lines, respectively. In transient COS-1-AKR1C2 and in stable PC3-AKR1C2 transfectants, AKR1C2 functioned as a 3-ketosteroid reductase inactivating 5alpha-DHT. In androgen dependent human prostate cancer cells LNCaP, it was not possible to ascertain the preferred direction of AKR1C2 by stable transfection due to the high rate of 5alpha-DHT and 3alpha-diol glucuronidation. Based on these findings AKR1C2 may diminish 5alpha-DHT and prevent this ligand from activating the androgen receptor in situ.

Androgens↗