Prostaglandin dehydrogenase activity of purified rat liver 3 alpha-hydroxysteroid dehydrogenase

Homogeneous 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) from rat liver cytosol displays 9, 11, and 15-hydroxyprostaglandin dehydrogenase activity. Using [14C]-PGF2 alpha as substrate the products of this reaction were separated by TLC and identified by autoradiography as PGE2 and PGB2. The purified enzyme catalyzes this reaction at a rate 200 times faster than cytosol. This corresponds to the rate enhancement observed when the enzyme is purified from cytosol using androsterone (a 3 alpha-hydroxysteroid) as substrate and suggests that it may represent a major 9-hydroxyprostaglandin dehydrogenase in this tissue. Although the 3 alpha-HSD has many properties in common with the 9-hydroxyprostaglandin dehydrogenase of rat kidney, rat kidney contains no protein that is immunodetectable with polyclonal antibody raised against the purified 3 alpha-HSD.     

20 beta-hydroxysteroid dehydrogenase of neonatal pig testis: 3 alpha/beta-hydroxysteroid dehydrogenase activities catalyzed by highly purified enzyme

Pig testicular 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) has also 3 alpha- and 3 beta-HSD (3 alpha/beta-HSD) activities. The purified 20 beta-HSD preparation from neonatal pig testes could catalyze the conversion of 5 alpha-dihydrotestosterone (5 alpha-DHT) in the presence of beta-NADPH to 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol at the ratio of 4:3, and the specific 3 alpha/beta-HSD activity of 20 beta-HSD for 5 alpha-DHT was about 10 or 15 times larger than the 20 beta-HSD activities for 17 alpha-hydroxypregn-4-ene-3,20-dione (17 alpha-hydroxyprogesterone) or progesterone, respectively. The result indicates that the testicular 20 beta-HSD has high 3 alpha(axial, 3R)- and 3 beta(equatorial, 3S)-HSD activity. The testicular 20 beta-HSD could catalyze the reversible conversion of various 5 alpha- or 5 beta-dihydrosteroids which have a 3-carbonyl or 3-hydroxyl group with beta-NADP(H) as the preferred cofactor. The enzyme transferred the 4-proS hydrogen of NADPH to the 5 alpha-DHT for both 3 alpha- and 3 beta-hydroxylation and it was the same as the 20 beta-hydroxylation of 17 alpha-hydroxyprogesterone. Although the 3 alpha/beta-HSD activity has been known to be present in 3 alpha,20 beta-HSD of Streptomyces hydrogenans, the enzymological properties for 3 alpha/beta-HSD activity catalyzed by testicular 20 beta-HSD were different from the properties for 3 alpha/beta-HSD activity catalyzed by prokaryotic 3 alpha, 20 beta-HSD with respect to the specificity of the catalytic reaction and the cofactor requirement.     

3(20)alpha-hydroxysteroid dehydrogenase activity of monkey liver indanol dehydrogenase

Homogeneous indanol dehydrogenase from monkey liver catalyzed the reversible conversion of 3 alpha- or 20 alpha-hydroxy groups of several bile acids and 5 beta-pregnanes to the corresponding 3- or 20-ketosteroids. The kcat values for the steroids determined at pH 7.4 were low, but the kcat/Km values for the 3-ketosteroids were comparable to or exceeded those for 1-indanol and xenobiotic carbonyl substrates. The enzyme transferred the 4-pro-R-hydrogen atom of NADPH to the 3 beta- or 20 beta-face of the ketosteroid substrate. Competitive inhibition of the hydroxysteroid dehydrogenase activity of the enzyme by medroxyprogesterone acetate, hexestrol, and 1,10-phenanthroline suggests that both 1-indanol and hydroxysteroid are oxidized at the same active site on the enzyme. The specific inhibitor of the enzyme, 1,10-phenanthroline, suppressed the 3 alpha-hydroxysteroid dehydrogenase activity in the crude extract of monkey liver by 50%. The results strongly suggest that indanol dehydrogenase acts as a 3(20)alpha-hydroxysteroid dehydrogenase in the metabolism of certain steroid hormones and bile acids.     

Microsomal 20 alpha-hydroxysteroid dehydrogenase activity for progesterone in human placenta

Placental 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity was studied in order to evaluate the mechanism of continuation of pregnancy and initiation of labor. The placentas obtained at various gestational weeks were homogenized and fractionated into "nuclear", "mitochondrial", "microsomal" and "supernatant" fractions. Each fraction was incubated with 14C-progesterone and a hydrogen donor. Enzymatic activity was measured by the conversion of progesterone to 20 alpha-dihydroprogesterone. The highest activity of 20 alpha-HSD for progesterone was found to be localized in "microsomal" fraction. The Km constant of 20 alpha-HSD was 4.5 X 10(-6)M for progesterone in "microsomal" fraction. It was found that placental microsomal 20 alpha-HSD required NADPH as well as NADH. 20 alpha-HSD activity for progesterone increased as gestational weeks advanced. The addition of DHA-sulfate and DHA inhibited 20 alpha-HSD activity for progesterone significantly, suggesting that the steroid produced by the feto-placental unit may be involved in the metabolism of progesterone in human placenta.     

Camphoroquinone reduction: another reaction catalyzed by rat liver cytosol 3 alpha-hydroxysteroid dehydrogenase

A new purification scheme is described for the female rat liver cytosolic enzyme dually catalyzing the oxidation of androsterone (3 alpha-hydroxysteroid:NAD(P)+ oxidoreductase, EC 1.1.1.50) and acenaphthenol (trans-1,2-dihydrobenzene-1,2-diol:NADP+ oxidoreductase, EC 1.3.1.20). This purification procedure yielded the most highly purified preparation of this enzyme thus far published as adjudged from its androsterone oxidation activity. In addition, we have demonstrated that this purified enzyme also catalyzes the reduction of camphoroquinone, a natural monoterpene, non-aromatic quinone. The nature of the products of the camphoroquinone reduction has been partially elucidated and agrees with previously published results (Robertson, J.S. and Solomon, E. (1971) Biochem. J. 121, 503-509). Kinetic studies of the metabolism of androsterone, camphoroquinone and acenaphthenol by the enzyme have been performed, yielding respective Km and Vmax values. The results of these studies allow a clarification of the mechanism of action of this enzyme, particularly with respect to its dihydrodiol dehydrogenase activity.     

Characteristics and inhibition by flavonoids of 20alpha-hydroxysteroid dehydrogenase activity in mouse tissues

Progesterone was stereoselectively reduced to a metabolite 20alpha-hydroxy-4-pregnen-3-one in the cytosolic fraction from the liver of male mice, indicating that the reduction of progesterone is catalyzed by 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD). The cytosolic 20alpha-HSD activity was observed not only in the liver, but also in the kidney and lung. In liver cytosol, both NADPH and NADH were effective as cofactors for 20alpha-HSD activity, although NADPH was better than NADH for the enzyme activity. On the other hand, 20alpha-HSD activity in kidney cytosol required only NADPH as a cofactor. No significant sex-related difference of 20alpha-HSD activity was observed in liver and kidney cytosols. Flavonoids have been reported to inhibit the biosynthesis and metabolism of steroids. However, little is known about inhibitory effects of flavonoids on 20alpha-HSD activity. Thus, the effects of 16 flavonoids on 20alpha-HSD activity were examined, using liver cytosol of male mice. Among flavonoids tested, fisetin, apigenin, naringenin, luteolin, quercetin and kaempferol exhibited high inhibitory potencies for the 20alpha-HSD activity. We propose the possibility that these flavonoids augment progesterone signaling by inhibiting potently 20alpha-HSD activity in non-reproductive tissues.     

20alpha-hydroxysteroid dehydrogenase: a T lymphocyte-associated enzyme.

20alpha-Hydroxysteroid dehydrogenase (20alpha-SDH), an enzyme which reduces progesterone to 20alpha-dihydroprogesterone, was found to be associated with T lymphocytes. 20alphaSDH activity was present in spleen cells bearing theta antigen, spleen cells nonadherent to nylon wool (T lymphocyte-enriched population), and in thymocytes. Almost no enzymatic activity was found in bone marrow cells from normal mice and in spleen cells from neonatally thymectomized or athymic nude mice. T cell mitogens (PHA and Con A), but not the B cell mitogen LPS, induced high levels of enzymatic activity 48 hr after addition to spleen cell cultures. The level of 20alphaSDH activity in lymphocytes was age dependent. At the age of 4 weeks 20alphaSDH activity in thymocytes, spleen cells, and lymph node lymphocytes was 3 to 5 times higher than at 8 and 16 weeks. Progesterone (5.0 X 10(-7) M) was found to inhibit thymocyte proliferation after exposure to mitogens, but not 20alpha-dihydroprogesterone (10(-6) M). 20alpha SDH may protect the embryonic thymocytes against high concentrations of progesterone.     

The 20 alpha-hydroxysteroid dehydrogenase of Streptomyces hydrogenans

In addition to the well-known 3 alpha,20 beta-hydroxysteroid dehydrogenase ('cortisone reductase'), Streptomyces hydrogenans produces a relatively stable, NAD-dependent 20 alpha-hydroxysteroid dehydrogenase of molecular mass approximately 48 kDa. This enzyme catalyzes the transfer of hydrogen from the 4-pro-S position of NADH.     

Stereospecificity of hydrogen transfer by bovine testicular 20 alpha-hydroxysteroid dehydrogenase

The stereospecificity of hydrogen transfer between steroid (17-hydroxyprogesterone) and both natural cofactors by bovine testicular 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) has been determined. Cofactors used in these studies, [4-pro-S-3H]NADH ([4B-3H]NADH) and [4-pro-S-3H]NADPH ([4B-3H]NADPH) were generated with human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) utilizing [17 alpha-3H]estradiol-17 beta and NAD+ or NADP+, respectively. The resulting [4B-3H]NADH and [4B-3H]NADPH were purified by ion-exchange chromatography and separately incubated with molar excess of 17-hydroxyprogesterone as substrate in the presence of 20 alpha-HSD. Following incubation, steroid reactant and product were extracted, separated by HPLC and quantitated as to mass and content of tritium. The oxidized and reduced cofactors were separated by ion-exchange chromatography and quantitated as to mass and tritium content. In all incubations, equimolar amounts of 17,20 alpha-dihydroxy-4-pregnen-3-one and oxidized cofactor were obtained. Further, all recovered radioactivity remained with cofactor and none was found in the steroid product. In additional experiments, both reduced cofactors were separately incubated with glutamate dehydrogenase, an enzyme known to transfer from the B-side of the nicotinamide ring. Here radioactivity was present only in the unreacted cofactor fractions and in the product, glutamic acid. The results indicate that bovine testicular 20 alpha-HSD catalyzes transfer of the 4A-hydrogen from the dihydronicotinamide moiety of the reduced cofactor. Finally, this work described modifications that represent considerable improvement in the purification and assay of bovine 20 alpha-HSD as originally described.     

Characterization of the oxidative 3alpha-hydroxysteroid dehydrogenase activity of human recombinant 11-cis-retinol dehydrogenase.

11-cis-Retinol dehydrogenase catalyzes the oxidation of cis-retinols, a rate-limiting step in the biosynthesis of 9-cis-retinoic acid. It is also active toward 3alpha-hydroxysteroids, and thus might be involved in steroid metabolism. To better understand the role of this enzyme, we produced stable transfectants expressing 11-cis-retinol dehydrogenase in human embryonic kidney 293 cells. In vitro enzymatic assays have demonstrated that, with an appropriate exogenous cofactor, the enzyme catalyzes the interconversion of 5alpha-androstane-3alpha,17beta-diol and dihydrotestosterone and that of androsterone and androstanedione. However, using intact transfected cells, we found that the enzyme catalyzes reactions only in the oxidative direction. Thus, it is possible that 5alpha-androstane-3alpha,17beta-diol (an inactive androgen) can be converted into dihydrotestosterone, the most potent androgen, by the action of 11-cis-retinol dehydrogenase. This reaction could constitute a non-classical pathway of production of active androgens in the peripheral tissues. We also showed that all-trans-, 9-cis- and 13-cis-retinol inhibit the oxidative 3alpha-hydroxysteroid steroid activity of 11-cis-retinol dehydrogenase with similar K(i) values. Since all-trans-retinol is a precursor of cis-retinols, its inhibitory effect on the activity suggests that it could play an important role in modulating the formation of 9-cis-retinoic acid. In addition, we examined the effect of several known enzyme modulators, namely carbenoxolone, phenylarsine oxide and phosphatidylcholine, on 11-cis-retinol dehydrogenase activity. Taken together, our results suggest that, in humans, this enzyme might play a role in the biosynthesis of both 9-cis-retinoic acid and dihydrotestosterone.     

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.     

Active-site directed inactivation of rat ovarian 20 alpha-hydroxysteroid dehydrogenase.

Rat ovarian 20 alpha-hydroxysteroid dehydrogenase plays a pivotal role in leuteolysis and parturition by catalysing the reduction of progesterone to give the progestationally inactive steroid 20 alpha-hydroxyprogesterone. Putative mechanism based inhibitors of this enzyme were synthesized as potential progestational maintaining agents, including the epimeric allylic alcohol pair 3 beta-hydroxy-alpha-vinyl-5 alpha-androstane-17 beta-methanol and the related vinyl ketone 1-(3 beta-hydroxy-5 alpha-androstan-17 beta-yl)-2-propen-1-one. The vinyl ketone inactivates rat ovarian 20 alpha-hydroxysteroid dehydrogenase, semi-purified by poly(L-lysine)-agarose column chromatography, in a rapid time-dependent manner. Analysis of the pseudo-first-order inactivation plots gave a Ki of 2.0 microM for the inhibitor and a t1/2 for the enzyme of 20 s at saturation. These data indicate that the vinyl ketone is a potent and efficient inactivator of the ovarian dehydrogenase. Neither dialysis in the presence or absence of a competing nucleophile nor gel filtration reserves the inactivation, suggesting that a stable covalent bond is formed between the enzyme and steroid ligand. Both substrates (20 alpha-hydroxyprogesterone and NADP+) protect the enzyme from inactivation; moreover, initial velocity measurements in the presence of saturating concentrations of both substrates indicate that the vinyl ketone can behave as a competitive inhibitor, yielding a Ki value identical with that obtained in the inactivation experiments. Our results imply that the vinyl ketone is an active-site directed alkylating agent. By contrast the allylic alcohol pair 3 beta-hydroxy-alpha-vinyl-5 alpha-androstane-17 beta-methanol are neither substrates nor inhibitors of the ovarian enzyme and appear to be excluded from the catalytic site. The rapid inactivation observed with the vinyl ketone suggests that this compound may be useful as a progestational maintaining agent.     

In vitro secretion of progestins by rat luteal cells and their 20 alpha-hydroxysteroid dehydrogenase activity

Functionally active or regressing corpora lutea were harvested from pseudopregnant (psp) rats between days 5-8 of psp or day 15 of psp, respectively. They were enzymatically dispersed and cultured for 24 h to assess progestins in the medium and 20 alpha-hydroxysteroid dehydrogenase [20 alpha-HSD, catalyzing the conversion of progesterone to 20 alpha-dihydroprogesterone (20 alpha-OH-P)] activity in the cell. Though the active luteal cells retained low 20 alpha-HSD activity, they secreted 6-7 times more 20 alpha-OH-P than progesterone as the regressing luteal cells did. There was no significant difference between the total amounts of progestins in the 2 groups. When increasing doses of pregnenolone were added to the media, progesterone secretion from the active luteal cells was promoted and the progesterone to 20 alpha-OH-P ratio became comparable to the circulating progestins ratio during the mid-luteal phase. In contrast, from the regressing luteal cells only 20 alpha-OH-P secretion was promoted. These results indicate that an insufficient precursor supply results in the catabolism of a large part of synthesized progesterone before its release from luteal cells and suggest the presence of a high affinity but low capacity 20 alpha-HSD in active corpora lutea.     

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.     

Several distinct enzymes catalyze 20alpha-hydroxysteroid dehydrogenase activity in mouse liver and kidney

The effects of flavonoids and quinones on NADPH- and NADH-dependent 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) activities were examined in cytosolic fractions from the liver and kidney of mice. Judging from the data for the inhibition of NADPH- and NADH-dependent 20alpha-HSD activities by flavonoids and quinones, enzyme catalyzing renal NADPH-dependent 20alpha-HSD activity was found to be distinct from enzyme(s) catalyzing hepatic NADPH- and NADH-dependent 20alpha-HSD activities. Sulfobromophthalein (SBP) had little ability to inhibit hepatic NADPH-dependent 20alpha-HSD activity and bromophenol blue (BPB) exhibited a weak activation against the enzyme activity, whereas SBP and BPB were potent and moderate inhibitors, respectively, of hepatic NADH-dependent 20alpha-HSD activity. Thus, enzyme catalyzing hepatic NADPH-dependent 20alpha-HSD activity appeared to be distinct from enzyme catalyzing hepatic NADH-dependent 20alpha-HSD activity. The data for the pH profiles of hepatic NADPH- and NADH-dependent 20alpha-HSD activities also led us to the conclusion. Based on these results, we propose the possibility that several distinct enzymes catalyze NADPH- and NADH-dependent 20alpha-HSD activities in cytosolic fractions from the liver and kidney of mice.     

20 alpha-hydroxysteroid dehydrogenase expression in a murine virus-induced myeloproliferative syndrome.

The myeloproliferative sarcoma virus (MPSV) infection in DBA/2 mice leads to important quantitative and qualitative changes in their hemopoiesis. These findings suggest a disturbance in the production and action of a certain hemopoietic factor similar to IL3. Here, we show that the level of the 20 alpha-hydroxysteroid dehydrogenase (20 alpha-SDH) expression, which can be induced by IL3, is dramatically increased in spleen and thymus of MPSV-infected mice. Our results suggest that quantification of 20 alpha-SDH activity can be used to indicate abnormal production of a growth factor similar to IL3 in hemopoietic system diseases.     

Alterations of 20 alpha-hydroxysteroid dehydrogenase activity in cultured rat granulosa cells by follicle-stimulating hormone and testosterone

The effect of follicle-stimulating hormone (FSH) and testosterone (T) on rat granulosa cell progestin metabolism was investigated by incubation of the cells for 24 h with FSH and/or T and subsequent reincubation with an appropriate rabiolabeled steroid for 3 h. Exposure to varying concentrations of FSH (8-1000 ng/ml) and T (4-500 nM) decreased overall 4-[14C] progesterone utilization and accumulation of 20 alpha-reduced metabolites of progesterone in a dose-related manner. The accumulation of 5 alpha-reduced metabolites was not markedly changed by FSH and T treatments. Treatments with FSH and/or T decreased utilization of all progestins studied: progesterone by 30-50%, 20 alpha-hydroxy-4-pregnen-3-one by 23-31%, 3 alpha-hydroxy-5 alpha-pregnan-20-one by 41-64%, and 5 alpha-pregnane-3 alpha,20 alpha-diol by 26-34%. The greatest effects were observed following FSH + T treatments. Decreased utilization of substrates was associated with the decrease of 20 alpha-hydroxy-steroid dehydrogenase activity; the conversion of progesterone to 20 alpha-hydroxy-4-pregnen-3-one was decreased by 44-62%, the conversion of 20 alpha-hydroxy-4-pregnen-3-one to progesterone was decreased by 41-61%, the conversion of 3 alpha-hydroxy-5 alpha-pregnan-20-one to 5 alpha-pregnane-3 alpha,20 alpha-diol was decreased by 42-69%, and the conversion of 5 alpha-pregnane-3 alpha,20 alpha-diol to 3 alpha-hydroxy-5 alpha-pregnan-20-one was decreased by 53-60%. The incubation of granulosa cells with cyanoketone (10(-6)M), an inhibitor of delta 5,3 beta-hydroxysteroid dehydrogenase, virtually eliminated de novo progesterone production but did not alter the inhibitory effect of FSH and T on radiolabeled progesterone utilization and accumulation of 20 alpha-reduced metabolites, indicating that the observed effects are not influenced by endogenous production of progesterone. It was concluded from these studies that both FSH and testosterone inhibit the 20 alpha-hydroxysteroid dehydrogenase activity and consequently decrease progesterone catabolism by granulosa cells.