Showing papers on "Hydroxysteroid dehydrogenase published in 1999"
TL;DR: Two complementary approaches are used, namely in situ hybridization and immunocytochemistry, to identify the cells that contain the type 5 17β-HSD messenger RNA and enzyme in human benign prostatic hyperplasia (BPH).
Abstract: 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 β-HSD (hydroxysteroid dehydrogenase) converts DHEA into androstenedione (4-dione), whereas type 5 17β-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 17β-HSD messenger RNA and enzyme in human benign prostatic hyperplasia (BPH). Localization of 3β-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 17β-HSD was reexamined by immunocytochemistry in the normal human prostate samples and also in normal prostate epithelial cell line ...
142 citations
TL;DR: Because the catalytic functions of human brain short chain l-3-hydroxyacyl-CoA dehydrogenase could weaken the protective effects of estrogen and generate aldehydes in neurons, it is proposed that a high concentration of this enzyme in brain is a potential risk factor for Alzheimer’s disease.
136 citations
TL;DR: Gen deletion experiments in the mouse indicate that this enzyme is important both for the maintenance of normal serum glucocorticoid levels, and in the activation of key hepatic gluconeogenic enzymes, as well as as a mechanism providing ligand for the putative 11-dehydrocorticosterone receptor.
123 citations
TL;DR: The novel human cDNA encodes a 37 kDa protein that shows 78 and 74% amino acid identity with rat and mouse 17β‐HSD 7, respectively, which are responsible for estradiol production in the corpus luteum during pregnancy, but are also present in placenta and several steroid target tissues (breast, testis and prostate) as revealed by RT‐PCR.
118 citations
TL;DR: It is concluded that in 3 alpha-HSD, the time dependence of the change in Trp 86 fluorescence is due to cofactor anchoring, and thus,trp 86 is a distal reporter of this event, and the loop movement that accompanies cofactor binding is spectrally silent.
Abstract: Fluorescence stopped-flow studies were conducted with recombinant rat liver 3α-HSD, an aldo-keto reductase (AKR) that plays critical roles in steroid hormone inactivation, to characterize the bindi...
66 citations
TL;DR: These studies demonstrate transcriptional regulation by PRL of the gene encoding the final enzymatic step in progesterone biosynthesis: 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD).
Abstract: Altered PRL levels are associated with infertility in women. Molecular targets at which PRL elicits these effects have yet to be determined. These studies demonstrate transcriptional regulation by PRL of the gene encoding the final enzymatic step in progesterone biosynthesis: 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3beta-HSD). A 9/9 match with the consensus Stat5 response element was identified at -110 to -118 in the human Type II 3beta-HSD promoter. 3beta-HSD chloramphenicol acetyltransferase (CAT) reporter constructs containing either an intact or mutated Stat5 element were tested for PRL activation. Expression vectors for Stat5 and the PRL receptor were cotransfected with a -300 --> +45 3beta-HSD CAT reporter construct into HeLa cells, which resulted in a 21-fold increase in reporter activity in the presence of PRL. Promoter activity showed an increased response with a stepwise elevation of transfected Stat5 expression or by treatment with increasing concentrations of PRL (max, 250 ng/ml). This effect was dramatically reduced when the putative Stat5 response element was removed by 5'-deletion of the promoter or by the introduction of a 3-bp mutation into critical nucleotides in the element. Furthermore, 32P-labeled promoter fragments containing the Stat5 element were shifted in electrophoretic mobility shift assay experiments using nuclear extracts from cells treated with PRL, and this complex was supershifted with antibodies to Stat5. These results demonstrate that PRL has the ability to regulate expression of a key human enzyme gene (type II 3beta-HSD) in the progesterone biosynthetic pathway, which is essential for maintaining pregnancy.
58 citations
TL;DR: The isolation of two cDNAs which encode proteins with high homology to carbonyl reductase-like 20beta- HSD (CR/20beta-HSD) from rainbow trout (Oncorhynchus mykiss) ovarian follicles clearly demonstrate the presence of two distinct CR/20 BetaHSD transcripts in the trout ovary.
57 citations
TL;DR: The time-course of glucocorticoid regulation of 11beta-HSD-1 in the liver, hippocampus and kidney of adult male rats in vivo is re-examined, showing a pattern of regulation more consistent with the evidence that 11 beta-HSd-1 is an 11Beta-reductase in vivo.
56 citations
TL;DR: This study represents an example where sex hormone specificity can be changed at the enzyme level with a resultant shift in k(cat)/K(m) for the desired reaction of 2 x 10(11).
Abstract: Hydroxysteroid dehydrogenases (HSDs) regulate the occupancy and activation of steroid hormone receptors by converting potent steroid hormones into their cognate inactive metabolites. 3α-HSD catalyzes the inactivation of androgens in the prostate by converting 5α-dihydrotestosterone to 3α-androstanediol, where excess 5α-dihydrotestosterone is implicated in prostate disease. By contrast, 20α-HSD catalyzes the inactivation of progestins in the ovary and placenta by converting progesterone to 20α-hydroxyprogesterone, where progesterone is essential for maintaining pregnancy. Mammalian 3α-HSDs and 20α-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 3α-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. 3α-HSD was converted to 20α-HSD by replacing these loops with those found in 20α-HSD. However, when pocket residues in 3α-HSD were mutated to those found in 20α-HSD altered specificity was not achieved. Replacement of loop A created a 17β-HSD activity that was absent in either 3α- or 20α-HSD. Once loops A and C were replaced, the chimera had both 3α- and 20α-HSD activity. When loops A, B, and C were substituted, 3α-HSD was converted to a stereospecific 20α-HSD with a resultant shift in kcat/Km for the desired reaction of 2 × 1011. This study represents an example where sex hormone specificity can be changed at the enzyme level.
51 citations
TL;DR: Seven women who are sisters of men with 17HSD3 deficiency and who are either homozygotes or compound heterozytotes for the same mutations as their affected brothers are ascertained, confirming the concept that women with such mutations are asymptomatic.
Abstract: In genetic males, mutation of the 17β-hydroxysteroid dehydrogenase 3 (17HSD3)gene that is normally expressed in the testes impairs testosterone formation and causes development of male pseudohermaphroditism We have ascertained seven women who are sisters of men with 17HSD3 deficiency and who are either homozygotes or compound heterozytotes for the same mutations as their affected brothers Our findings confirm the concept that women with such mutations are asymptomatic
49 citations
TL;DR: Substrate specificity and cofactor requirement patterns as well as pH optima and kinetic properties suggest the occurrence of 17β‐HSD type 3 and type 4 in the human temporal lobe.
Abstract: Sex steroids exert important effects on the central nervous system (CNS). Although the formation of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) metabolites in the CNS was discovered almost 30 years ago, conclusive studies concerning 17beta-HSD activity in the human brain are still lacking. Therefore, we investigated 17beta-HSD in vitro activity in human temporal lobe biopsies of 13 women and 13 men using radioactively labelled androstenedione, testosterone, oestrone and 17beta-oestradiol and compared it to that in human placenta, liver, testis and prostate. We could demonstrate androgenic and oestrogenic 17beta-HSD activities in all tissues under investigation. The reduction of androstenedione and oestrone in brain was NADPH dependent with a broad pH optimum between 6.5 and 9.0, whereas the oxidation of testosterone and 17beta-oestradiol was NAD dependent with a pH optimum of >/=9.0. Using optimum cofactors sex differences of brain 17beta-HSD activities were not observed. Conversion of androstenedione, testosterone, oestrone and 17beta-oestradiol was significantly higher in the subcortical white matter than in the cerebral cortex. We could demonstrate a significant formation of testosterone in the brain tissue of all patients under investigation. Substrate specificity and cofactor requirement patterns as well as pH optima and kinetic properties suggest the occurrence of 17beta-HSD type 3 and type 4 in the human temporal lobe.
TL;DR: It was found that a para-substituted benzylthio group at the 7alpha-position enhances the inhibitory potency of spironolactone derivatives on type II 17beta-HSD, and offer an interesting tool to study the regulation of this enzyme in several biological systems.
TL;DR: The cloned 17βHSD expressed in mammalian cell lines specifically catalyzed the reductive reaction in androgen formation with androstenedione as the preferred substrate, and this reaction was significantly reduced in the absence of glucose.
Abstract: 17β-Hydroxysteroid dehydrogenase (17βHSD), the enzyme that catalyzes the final step of testosterone biosynthesis in the testis, was cloned from a rat Leydig cell complementary DNA library to gain insights into the functional requirements, activation mechanisms, and molecular regulation The 17βHSD complementary DNA encoded 306 amino acids (molecular mass of 337 kDa) and displayed 75% and 85% amino acid sequence homology to the human and mouse 17βHSD type III enzymes, respectively Northern analysis revealed a single 14-kb messenger RNA (mRNA) species in rat Leydig cells, whereas ovarian mRNA was detected only by RT-PCR amplification The cloned 17βHSD expressed in mammalian cell lines specifically catalyzed the reductive reaction in androgen formation with androstenedione as the preferred substrate This reaction was significantly reduced in the absence of glucose Expression of the endogenous 17βHSD gene in rat Leydig cells was inhibited by a single dose of hCG in vivo, with maximum reduction of steady
TL;DR: While some studies demonstrate impaired 11 beta-HSD activity in broader populations of patients with hypertension, further studies are required to clarify the role of 11beta- HSD2 in 'essential' hypertension.
TL;DR: The consequences of one frequent mutation, G16 S, which results in inactivation of the enzyme due to loss of interaction with NAD+.
TL;DR: The findings indicate that genes encoding soluble proteins should be examined as candidates for actual steroid dehydrogenase activity in Schizosaccharomyces pombe, and more than one steroid dehydration homologue is likely to occur.
Abstract: The fungal biotransformation of steroids is of applied interest due to the economic importance of such stereo- and regiospecific reactions and also in the context of ergosterol pathway engineering to produce vitamin D and steroidal products. In Schizosaccharomyces pombe no steroid hydroxylation as is found in filamentous fungi was observed, but a cytosolic NAD(H)/NADP(H)-dependent hydroxysteroid dehydrogenase activity was identified. Progesterone was reduced at the Δ4 double bond (in vivo only) as well as at the C-3 and C-20 keto groups. Testosterone and 4-androstene-3,17-dione were interconverted and 5α-pregnane-3,20-dione and 5β-pregnane-3,20-dione were reduced to 3-hydroxy products. The reactions were sometimes reversible and showed regio- and stereo specificity. In S. pombe more than one steroid dehydrogenase homologue is likely to occur, as has been observed in Saccharomyces cerevisiae. Our findings indicate that genes encoding soluble proteins should be examined as candidates for actual steroid dehydrogenase activity. Copyright © 1999 John Wiley & Sons, Ltd.
TL;DR: The cloning and characterization of the mouse type 5 17beta-HSD belonging to the aldo-keto reductase superfamily is reported, in contrast with types 1, 2, 3, 4, 6, and 7 17 beta-HSDs which belong to the short-chain alcohol dehydrogenase family.
TL;DR: Together, 20α-HSD mRNA was localized in cells that surround fetuses during the mid and late stages of gestation and was not detected in endometrial cells during pseudopregnancy as well as early pregnancy.
Abstract: 20α-Hydroxysteroid dehydrogenase (20α-HSD) converts progesterone to a biologically inactive steroid, 20α-dihydroprogesterone. To examine a potential role of 20α-HSD in tissues other than corpus luteum (CL) during pregnancy, mouse 20α-HSD cDNA was cloned and was used for the examination of its mRNA localization in the conceptus and uterus at different stages of gestation. Using probes based on the rat sequence, mouse 20α-HSD cDNA clone was isolated from a cDNA library prepared from ovaries of day 12 pseudopregnant mice. The mouse 20α-HSD cDNA was 1193 bp in length and encoded 323 amino acids with an estimated molecular weight of 37 kD. The mouse 20α-HSD was homologous to the rat 20α-HSD in nucleotide and amino acid sequences (both in 93% homology), and to other members of aldo-keto reductase superfamily at the cofactor (NADP(H)) binding site. By in situ hybridization, 20α-HSD mRNA was localized in endometrial epithelial cells on days 10, 15 and 18 of pregnancy, maternal placental endothelial cells on day 10, and fetal epidermal cells on day 15. During pseudopregnancy as well as early pregnancy, however, 20α-HSD mRNA was not detected in endometrial cells. Together, 20α-HSD mRNA was localized in cells that surround fetuses during the mid and late stages of gestation.
TL;DR: A convenient overexpression system that permits the purification of milligram quantities of homogeneous recombinant 20alpha-HSD with wild-type enzyme activity is reported, suggesting that selective non-steroidal AKR inhibitors could be developed for 20alpha -HSD that might be useful in maintaining pregnancy and that specific inhibitors might be developed from either N-phenylanthranilates or biphenols.
Abstract: In rat ovary, 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), a member of the aldo-keto reductase (AKR) superfamily, converts progesterone into the inactive progestin 20alpha-hydroxyprogesterone and has been implicated in the termination of pregnancy. Here we report a convenient overexpression system that permits the purification of milligram quantities of homogeneous recombinant 20alpha-HSD with wild-type enzyme activity. The availability of this enzyme has permitted detailed kinetic, inhibition and fluorescence analyses. The enzyme exhibited narrow steroid specificity, catalysing reactions only at C-20; it reduced progesterone and 17alpha-hydroxyprogesterone and oxidized 20alpha-hydroxypregnanes. It also turned over common AKR substrates, such as 9, 10-phenanthrenequinone and 4-nitrobenzaldehyde. The intrinsic fluorescence spectrum of 20alpha-HSD was characterized and was quenched on the binding of NADP(H), yielding a KNADPd of 0.36 microM and a KNADPHd of 0.64 microM. NADP(H) binding generated an energy transfer band that could not be quenched by steroids. Inhibition studies conducted with non-steroidal and steroidal anti-inflammatory drugs and synthetic oestrogens indicated that even though rat ovarian 20alpha-HSD and rat liver 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) share more than 67% amino acid identity, their inhibition profiles are markedly different. Unlike 3alpha-HSD, most of these compounds did not inhibit 20alpha-HSD. Only meclofenamic acid and hexoestrol were potent competitive inhibitors for 20alpha-HSD, yielding K(i) values of 18.9 and 14.3 microM respectively. These studies suggest that selective non-steroidal AKR inhibitors could be developed for 20alpha-HSD that might be useful in maintaining pregnancy and that specific inhibitors might be developed from either N-phenylanthranilates or biphenols.
TL;DR: The inhibition provoked by Medrogestone on the reductive 17beta-HSD activity involved in the local biosynthesis of the biologically active estrogen estradiol, may constitute a new therapeutic approach for the treatment of breast cancer.
TL;DR: The pc17bHSD is the cDNA clone for the ovary-specific molecular species of 17beta-HSD in chickens, and five sequence regions common to the short-chain alcohol dehydrogenase superfamily are well conserved.
TL;DR: All 4 17BetaHSD isoforms are variably expressed in human anterior pituitary adenomas, which also show 17betaHSD enzyme activity, suggesting that 17 betaHSD may play an important role in regulating the local cellular levels of estradiol.
Abstract: 17Beta-hydroxysteroid dehydrogenase (17betaHSD) isoforms reversibly catalyze the final step in the formation of estradiol (E2) from estrone (E1) and the formation of testosterone from androstenedione. We have investigated 17betaHSD type 1, 2, 3, and 4 gene expression and 17betaHSD estrogenic activity in human anterior pituitary adenomas. 17BetaHSD messenger ribonucleic acid (mRNA) expression was studied by RT-PCR in 42 pituitary tumors and 3 normal pituitaries, 17betaHSD activity was studied in 11 tumors and 17betaHSD type 1 was immunolocalized in vitro in 6 tumors. 17BetaHSD type 1 gene expression was detected in 34 of 42 adenomas in all tumor subtypes; 17betaHSD type 2 mRNA was detected in 18 of 42 adenomas, but not in prolactinomas; 17betaHSD type 3 mRNA was detected in 12 of 42 adenomas, but not in corticotropinomas; 17betaHSD type 4 was expressed in 20 of 42 adenomas by all adenoma subtypes. Reversible 17betaHSD activity was found in 9 of 11 adenomas, and 17betaHSD type 1 immunopositivity was cytoplasmically distributed in all 6 adenomas in vitro. All 4 17betaHSD isoforms are variably expressed in human anterior pituitary adenomas, which also show 17betaHSD enzyme activity, suggesting that 17betaHSD may play an important role in regulating the local cellular levels of estradiol.
TL;DR: The preparative-scale regio- and stereo-specific oxidation of hydroxy groups and reduction of keto functions at C(3) of several C 21 bile acids, catalyzed by a new 3α-hydroxysteroid dehydrogenase (3α-HSDH), is reported.
Journal Article•
TL;DR: It was found that T3 acts on luteal cell steroidogenesis via the activation of 3beta-hydrosysteroid dehydrogenase in these cells.
Abstract: Objective. To study the mechanism of thyroid hormone action on the activity of 3β-hydroxysteroid dehydrogenase in the porcine corpus luteum. Methods. Pig ovaries were obtained from slaughterhouse animals. Luteal cells were isolated from mid-developing (5-7 days after ovulation) corpora lutea and incubated for 24 h with or without triiodothyronine. Trilostane, an inhibitor of 3β-HSD that blocks the conversion of pregnenolone to progesterone, was added to the medium in doses of 0, 0.1, 1, 10, and 100 μmol. Each treatment was performed in triplicate and each culture system was set up in triplicate. Progesterone concentrations in culture media were determined by radioimmunoassays. Results. Trilostane in a dose of 100 μM significantly decreased the basal progesterone secretion from luteal cells by 26 % (P<0.05). However, such secretion was increased by triiodothyronine (T 3 ) in a dose of 10 -9 M. In addition, in T 3 -treated cells dose dependent inhibitory effect of trilostane on progesterone secretion was observed. Control cultures grown in control medium revealed a relatively weak 3s-HSD activity which, however, markedly increased after the addition of T 3 to the culture medium. Trilostane remarkably decreased 3s-HSD activity in T 3 -stimulated cells. Conclusion. It was found that T 3 acts on luteal cell steroidogenesis via the activation of 3βhydroxysteroid dehydrogenase in these cells.
TL;DR: The reticularis increased in size in the 8-13-year-olds and adults, but 3bHSD activity was markedly reduced, which allows amplified production of adrenal androgens.
Abstract: 3bHSD and 21-hydroxylase (CYP21) activities were assessed by immunohistochemical staining of adrenal tissue fractions from 30 children aged 4 months-4 years, 5-7 years, 8-13 years, as well as eight adults, who died ofvarious nonadrenal causes. No differences in 3bHSD activities of the zona fasciculata and glomerulosa were found, but 3bHSD activity of the zona reticularis varied strikingly by age. The zona reticularis in children less than 7 years old was thin but had a high 3bHSD activity. The reticularis increased in size in the 8-13-year-olds and adults, but 3bHSD activity was markedly reduced. This allows amplified production of adrenal androgens.
01 Jan 1999
TL;DR: The purified enzyme was characterized with respect to kinetic parameters, substrate specificity and localization, and yielded three fragments with high sequence homology to members of the superfamily of short-chain dehydrogenases/reductases.
Abstract: Delta(5)-3beta-Etaydroxysteroid dehydrogenase (Delta(5)-3beta-HSD; EC 1.1.1.145), an enzyme converting pregn-5-ene-3beta-ol-20-one (pregnenolone) to pregn-5-ene-3,20-dione (isoprogesterone), was isolated from the soluble fraction of suspension-cultured cells of Digitalis lanata L. strain VIII. Starting with acetone dry powder the enzyme was purified in three steps using column chromatography on Fractogel-TSK DEAE, hydroxyapatite and Sephacryl G-200. Fractions with highest Delta(5)-3beta-HSD activity were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After in-situ digestion the resulting bands were sequenced N-terminally. The 29-kDa band yielded three fragments with high sequence homology to members of the superfamily of short-chain dehydrogenases/reductases. High similarity was found to microbial hydroxysteroid dehydrogenases. The band may therefore represent the Delta(5)-3beta-HSD. The purified enzyme was characterized with respect to kinetic parameters, substrate specificity and localization. The function of the enzyme in steroid metabolism is discussed.
TL;DR: Identity of naloxone reductase and 3alpha-hydroxysteroid dehydrogenase in rat liver was demonstrated by comparing the elution profiles of the two enzyme activities during purification, the ratios of theTwo enzyme activities at each purification steps, and thermal stability and susceptibility to inhibitors for the two enzymes activities.
Abstract: 1. Rat liver cytosol produced exclusively 6β-naloxol from naloxone in the presence of either NADPH or NADH at pH 7.4. The amount of 6β-naloxol formed with NADPH was about four times that with NADH. The enzyme responsible for this reaction, termed naloxone reductase, was purified to a homogeneous protein by various chromatographic techniques. 2. The purified enzyme is a monomeric protein with a molecular weight of 34000 and an isoelectric point of 5.9, and it has a dual co-factor specificity for NADPH and NADH. The enzyme catalysed the reduction of various carbonyl compounds as well as naloxone analogues, and the dehydrogenation of 3α-hydroxysteroids and alicyclic alcohols. Indomethacin, quercetin and sulphhydryl reagents potently inhibited the enzyme, but pyrazole and barbital had no effect on the enzyme activity. 3. Identity of naloxone reductase and 3alpha-hydroxysteroid dehydrogenase in rat liver was demonstrated by comparing the elution profiles of the two enzyme activities during purification, the ra...
TL;DR: Medroxyprogesterone Acetate Inhibits Pituitary-Independent Steroidogenesis by Inhibiting 3 β-Hydroxysteroid Dehydrogenase Type II (3 β-HSDII)
Abstract: Medroxyprogesterone Acetate (MPA) Inhibits Pituitary-Independent Steroidogenesis by Inhibiting 3 β-Hydroxysteroid Dehydrogenase Type II (3 β-HSDII)