Crystal structure of human estrogenic 17 beta-hydroxysteroid dehydrogenase complexed with 17 beta-estradiol.
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Binding Sites↗
Biomedical subjects
F Labrie
Publications and source records attributed to F Labrie.
Isolation and characterization of a stereospecific 3beta-hydroxysteriod sulfotransferase (pregnenolone sulfotransferase) cDNA.
In contrast to humans, who possess a hydroxysteroid sulfotransferase (HSST), namely, DHEA sulfotransferase (DHEA-ST), that displays broad substrate specificities, HSSTs of the guinea pig show a high substrate stereoselectivity, as shown by the recent cloning of a chiral-specific 3alpha-hydroxysteroid sulfotransferase. Herein, we report the cloning and expression of the substrate and chiral-specific pregnenolone sulfotransferase (PREG-ST). Transfection of the pCMV expression vector containing PREG-ST cDNA in transformed human embryonal kidney (293) cells showed that the expressed enzyme selectively catalyzes the 3beta-hydroxysteroid substrate. It converts pregnenolone to pregnenolone sulfate most efficiently, whereas dehydroepiandrosterone and epiandrosterone were transformed at a much lower rate, and androsterone, a 3alpha-hydroxysteroid, was not significantly metabolized (30-fold lower). Thus, the enzyme was identified as pregnenolone sulfotransferase. DNA analysis predicts a protein of 287 amino acids with a calculated molecular mass of 34,199 daltons. Alignment of the amino acid sequence with other sulfotransferases indicated that guinea pig pregnenolone sulfotransferase shares 75 and 80% homology with human DHEA sulfotransferase and rat hydroxysteroid dehydrogenase, respectively. RNA blot analysis using guinea pig liver, intestine, adrenal, kidney, epididymis, testis, and lung showed a single RNA species at 1.3 kb is expressed in liver, intestine, and kidney. Guinea pig 3beta-hydroxysteroid sulfotransferase is thus different from that in humans, who possess two mRNA species of 1.3 and 1.8 kb.
Amino Acid Sequence↗
Comparison of BRCA1 polymorphisms, rare sequence variants and/or missense mutations in unaffected and breast/ovarian cancer populations.
Inherited mutations in the BRCA1 gene are known to confer a predisposition to breast and ovarian cancer. We have first characterized 19 sequence variants in the BRCA1 gene during mutation screening by direct sequencing using DNA samples from breast/ovarian cancer patients or obligate carriers. The frequencies of these sequence variants were then compared with those found in control populations of women. Among the 10 sequence variants showing an estimated frequency of the less common allele above 0.05, Q/R356, L/P871, E/G1038, K/R1183 and S/G1613 result in a change of amino acids, 2201C/T, 2430T/C and 4427C/T are silent mutations and the two others, 4209-141C/A and 5272 + 66A/G, are intronic polymorphisms. These frequent polymorphisms, with the exception of Q/R356, were in complete or significant pairwise linkage disequilibrium as evaluated in our control populations. With one exception (L/P871), none of these variants had statistically significant (P < 0.05) differences in allele frequency between breast/ovarian cancer patients or obligate carriers and our control populations. Four rare sequence variants designated 710C-->T, D693N, R841W and S1040N were found in both unaffected and breast/ovarian cancer populations, while the missense mutations M1008I, E1219D, R1347G, T1561I and M1628V were detected only once in our patient population. When a functional test is available, it will be important to determine the consequence on the BRCA1 activity of these rare sequence variants and missense mutations.
BRCA1 Protein↗
Effect of neoadjuvant endocrine therapy (combined androgen blockade) on normal prostate and prostatic carcinoma. A randomized study.
The morphologic changes induced by neoadjuvant combination endocrine therapy were evaluated in prostatectomy specimens from patients diagnosed with localized prostate cancer. These patients participated in a prospective, randomized clinical trial investigating the effect of 3 months of combination therapy with flutamide and an LHRH agonist prior to radical prostatectomy versus radical prostatectomy alone. Ninety-six radical prostatectomy specimens processed according to the same protocol were evaluated without knowledge of prior treatment. Forty-seven patients were randomly assigned to the neoadjuvant combination therapy group and 49 to the control arm. Compared with the control group, several changes were strongly and significantly associated with exposure to neoadjuvant combination therapy. The nonmalignant prostatic tissue showed strong prominence and hyperplasia of the basal cell layer, accompanied by epithelial cell vacuolization and markedly reduced occurrence of prostatic intraepithelial neoplasia (p < 0.001) after combination therapy. Prostate cancer tissue, on the other hand, showed smaller nucleoli (p < 0.001), cell vacuolization (p < 0.001), rare intraluminal crystalloids (p < 0.001), higher Gleason grade (p < 0.001), lower prevalence of capsular penetration (p < 0.001), and less frequent invasion of the perineural spaces (p < 0.001) and surgical margins (p = 0.002). Tumor volume, was also reduced by more than 40% in the treated group (p = 0.007). The present findings show that preoperative endocrine combination therapy induces highly characteristic changes in both nonmalignant and cancerous prostatic tissue. Furthermore, following endocrine treatment, the surgical margins are less likely to be involved by cancer and capsular penetration is reduced.
Adult↗
Localization and regulation of expression of the FAR-17A gene in the hamster flank organs.
A quantitative in situ hybridization study was carried out to determine the precise localization and androgen regulation of the flank organ regulated (FAR-17A) mRNA expression in the different cellular components of the hamster flank organs. Although FAR-17A mRNA was highly expressed in the epithelial cells of the sebaceous glands, it was also found in the outer root sheath of the hair follicles and in melanocytes. The changes in FAR-17A mRNA levels, in the size of the flank organ and sebaceous gland areas as well as in the weight of the seminal vesicles and prostate, were compared following castration and after 5alpha-dihydrotestosterone treatment. FAR-17A mRNA levels were already significantly decreased 1 d after castration, in parallel with a concomitant decrease in the number of labeled cells with the FAR-17A probe. A maximal decrease was found 7 d after castration. The other parameters were significantly reduced later. After 7 d of treatment with dihydrotestosterone, all values returned to those found in intact animals. Similar stimulatory effects on these parameters were observed after treatment with the adrenal sex steroid precursor dehydroepiandrosterone. These data show that all of the components of the flank organs (sebaceous glands, hair follicles, and melanocytes) express the flank organ regulated (17A) type gene (FAR-17A) gene and that its expression is stimulated by treatment with either dihydrotestosterone or dehydroepiandrosterone. Moreover, FAR-17A mRNA levels respond to androgen stimulation more rapidly than the standard morphologic parameters, revealing that the FAR-17A gene could be a more sensitive and cell specific marker to study the mechanisms of androgen action in the skin.
Androgens↗
Genetic linkage mapping of the human steroid 5 alpha-reductase type 2 gene (SRD5A2) close to D2S352 on chromosome region 2p23-->p22.
Two steroid 5 alpha-reductase isoenzymes catalyze the conversion of testosterone into dihydrotestosterone, the more bioactive androgen, which is essential for male phenotypic sexual differentiation and for androgen-mediated growth of such tissues and organs as the prostate. Inherited mutations in SRD5A2 cause male pseudohermaphroditism. The SRD5A1 and SRD5A2 genes encoding the steroid 5 alpha-reductase type 1 and type 2 isoenzymes have been previously assigned by in situ hybridization to 5p15 and 2p23, respectively. To map the SRD5A2 gene by linkage analysis, a novel RsaI RFLP detected in exon I and a TA repeat polymorphism found in exon V were genotyped in eight CEPH reference families. A two-point linkage analysis was performed between these polymorphisms and the chromosome 2 microsatellite markers of Généthon and NIH/CEPH. The closest linkage was observed with D2S352 (Zmax = 24.06; thetamax = 0.001) in the region 2p23-->p22. To further define the localization of SRD5A2, a framework map, including nine Généthon markers flanking the polymorphic SRD5A2 locus, was built by multipoint linkage analysis. This led to a high-resolution genetic map of the region flanking the polymorphic SRD5A2 gene, including the nine Généthon markers and three NIH/CEPH markers, yielded the following order: tel-D2S48-D2S149-D2S320-D2S171-D2S165- [D2S352/SRD5A2]-D2S367-[D2S19/D2S177]-[ D2S391/CALM]-D2S378-cen.
Base Sequence↗
Adrenal steroid precursors exert potent androgenic action in the hamster sebaceous glands of flank organs and ears.
To assess the effect of the androgen precursors dehydroepiandrosterone (DHEA) and androstenedione (4-ene-dione) on androgen-sensitive parameters in the skin of the hamster, these two steroids were released from SILASTIC implants inserted sc into castrated male hamsters. The pigmented area of the flank organs, the size of the underlying sebaceous glands, [3H]thymidine incorporation into these sebaceous glands, and the size of the ear sebaceous glands were measured. The decrease in flank organ size caused by orchidectomy was partially reversed by DHEA and completely reversed by 4-ene-dione, testosterone (T), or dihydrotestosterone (DHT) implants. Similarly, the size of the sebaceous glands of the flank organs was reduced 88.1% by castration, whereas DHEA, 4-ene-dione, T, and DHT implants reversed the effect of orchidectomy. Orchidectomy also decreased the sizes of the sebaceous glands of the ears; DHEA, 4-ene-dione, T, and DHT implants increased their sizes to 50.6%, 81.9%, 91.6%, and 105.8% of the values found in intact hamsters, respectively. Parallel results were observed on the labeling of flank organ sebaceous glands with [3H]thymidine as well as on prostate weight. Serum concentrations of T and DHT became undetectable in castrated hamsters and were increased to intact or slightly elevated values in animals receiving implants of DHEA, 4-ene-dione, or T. The present results show that DHEA and 4-ene-dione are potent stimulators of androgen-sensitive parameters in the sebaceous glands of both the flank organs and ears in the hamster and illustrate the importance of extragonadal or peripheral intracrine formation of active steroids. It is suggested that the castrated hamster supplemented with adrenal precursor steroids is a good model that can closely mimic the human situation, where adrenal steroids play an important role in androgen formation and action in peripheral tissues.
Androgens↗
Mutation R96W in cytochrome P450c17 gene causes combined 17 alpha-hydroxylase/17-20-lyase deficiency in two French Canadian patients.
Congenital adrenal hyperplasia (CAH) is the most frequent cause of adrenal insufficiency and ambiguous genitalia in newborn children. In contrast to CAH caused by 21 alpha-hydroxylase and 11 beta-hydroxylase deficiencies, which impairs steroid formation in the adrenal exclusively, 17 alpha-hydroxylase/17,20-lyase deficiency impairs steroid biosynthesis in the adrenals and gonads. The sequence of CYP17 gene was determined by direct sequencing of asymmetric PCR products in two French-Canadian 46,XY pseudohermaphrodite siblings suffering from combined 17 alpha-hydroxylase/17,20-lyase deficiency. The two patients are homozygous for the novel missense mutation R96W caused by a C to T transition converting codon Arg96 (CGG) into a Trp (TGG) in exon 1. The both parents are heterozygous for this missense mutation. We assessed the effect of the R96W mutation on 17 alpha-hydroxylase/17,20-lyase activity by analysis of mutant enzyme, generated by site-directed mutagenesis, expressed in COS-1 cells. The presence of R96W substitution almost completely abolished the activity of the mutant protein. The present findings provide a molecular explanation for the signs and symptoms of combined 17 alpha-hydroxylase/17,20-lyase deficiency in these two patients and provide useful information on the structure-activity relationships of the P450c17, enzyme.
Adolescent↗
The dense LDL phenotype. Association with plasma lipoprotein levels, visceral obesity, and hyperinsulinemia in men.
OBJECTIVE: To investigate the potential relationship between the cluster of metabolic abnormalities found in visceral obesity and the small dense LDL phenotype. RESEARCH DESIGN AND METHODS: We have estimated LDL peak particle size by nondenaturing 2-16% gradient gel electrophoresis in a sample of 79 men. Glucose tolerance and fasting plasma insulin and lipoprotein levels were also measured. RESULTS: The LDL particle score, calculated from migration, distances and relative band intensities and reflecting the proportion of small dense LDL particles, was positively correlated with plasma triglyceride (TG) (r = 0.60, P < 0.0001) and negatively correlated with HDL cholesterol (r = -0.56, P < 0.0001) levels. Although the LDL particle score was not associated with variations in plasma LDL cholesterol or LDL apolipoprotein (apo) B concentrations, it was significantly correlated with the LDL apo B-to-LDL cholesterol ratio (r = 0.60, P < 0.0001). Fasting plasma insulin and visceral adipose tissue (AT) areas measured by computed tomography were weakly but significantly correlated with the LDL particle score (r = 0.23 and 0.29, respectively, P < 0.05). LDL peak particle size showed similar but inverse correlations with anthropometric and metabolic variables. Subjects classified as having small dense LDL particles (by comparing subjects in the highest tertile versus those in the lowest tertile of the LDL particle score distribution) were characterized by increased plasma TG, reduced HDL cholesterol, higher fasting insulin levels, and elevated visceral AT accumulation. However, multiple regression analyses revealed that visceral AT accumulation was not an independent predictor of the dense LDL phenotype after inclusion of TG and HDL cholesterol levels and lipoprotein ratios in the model. CONCLUSIONS: It thus appears that the high TG-low HDL cholesterol dyslipidemia frequently found in visceral obesity and in a hyperinsulinemic state is a strong correlate of the small dense LDL phenotype. Although associated with the dense LDL phenotype, visceral obesity and hyperinsulinemia were not independent predictors of an increased proportion of small dense LDL particles after controlling for TG and HDL cholesterol levels.
Adipose Tissue↗
Studies on the three-dimensional structure of estrogenic 17 beta-hydroxysteroid dehydrogenase.
The structure-function relationship of the estrogenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD1), a pivotal enzyme in the synthesis of active sex hormones, has been studied via protein chemistry and crystallography. A highly active and homogeneous 17 beta-HSD1 was prepared with a rapid purification from human placenta. We then characterized the native and expressed enzyme, and concluded, for the first time, that 17 beta-HSD1 is formed by two identical subunits. The enzyme was also overproduced in insect cells with a baculovirus expression system. The highly active 17 beta-HSD1 preparation was successfully crystallized in the presence of NADP-, polyethylene glycol, beta-octylglucoside and glycerol, resulting in the first diffraction quality crystals of any steroid-converting enzyme from a human source. The three-dimensional structure of 17 beta-HSD1 was determined at 2.2 A resolution, showing that the overall structure of the enzyme is similar to the other enzymes in the short-chain dehydrogenase family, with a conserved Tyr-X-X-X-Lys sequence and a serine residue in the active site. It is distinguished from the other known structures reported for short-chain dehydrogenases by the insertion of two helix-turn-helix motifs that appear to govern membrane association and substrate specificity [corrected]. More recently, the complex of 17 beta-HSD1 with estradiol has been successfully crystallized and its structure determined. The latter demonstrates detailed information of the interactions between the substrate and residues Ser142, Tyr155, His221 and Glu282 of the enzyme. These interactions and the complementarity of the substrate with the hydrophobic binding pocket make critical contributions to the enzyme specificity. The above results provide a strong basis for the design of potent inhibitors of this pivotal steroid dehydrogenase.
Estradiol Dehydrogenases↗
Metabolic effects of 12-month percutaneous dehydroepiandrosterone replacement therapy in postmenopausal women.
We have evaluated the effect of dehydroepiandrosterone (DHEA) replacement therapy in 60- to 70-year-old women (n = 15) who received a single daily percutaneous application of a 10% DHEA cream for 12 months. While anthropometric measurements showed no change in body weight, we observed a 9.8% decrease in subcutaneous skinfold thickness at 12 months (P < 0.05). This was confirmed by measurements of midthigh fat and muscle areas by computed tomography where a 3.8% decrease (P < 0.05) in femoral fat and a 3.5% increase (P < 0.05) in femoral muscular areas were observed at 12 months. There was no significant change in abdominal fat measurements but the waist-to-hip ratio was only 0.83 at the onset of treatment. These changes in body fat and muscular mass were associated with a 11% decrease (P < 0.05) in fasting plasma glucose and a 17% decrease (P < 0.05) in fasting insulin levels. Treatment with DHEA had no adverse effect on the lipid or lipoprotein profile. In fact, an overall trend towards a decrease in total cholesterol and its lipoprotein fractions was observed. Plasma triglycerides were not affected. Plasma high-density lipoprotein (HDL) cholesterol decreased by 8% but the ratio HDL/cholesterol was unchanged by DHEA treatment because of a parallel decrease in total cholesterol. The index of sebum secretion showed a 73% increase (P < 0.05) during the 12 months of DHEA therapy followed by a return to pretreatment values 3 months after cessation of therapy. At the same time, sex hormone-binding globulin levels decreased (P < 0.05) during treatment and returned to pretreatment values 3 months after the end of therapy. Serum gonadotropins were not changed by DHEA treatment. Although not significant, we observed a tendency towards an elevation in serum GH levels. Values of serum IGF-I remained unchanged while plasma IGF-binding protein-3 levels significantly decreased (P < 0.05) during treatment and returned to pretreatment values after cessation of DHEA therapy. The present data clearly indicate the beneficial effects of DHEA therapy in postmenopausal women through its transformation into androgens and/or estrogens in specific intracrine tissues without any significant side effects.
Adipose Tissue↗
High bioavailability of dehydroepiandrosterone administered percutaneously in the rat.
Dehydroepiandrosterone (DHEA) administered percutaneously by twice daily application for 7 days to the dorsal skin of the rat stimulates an increase in ventral prostate weight with approximately one third the potency of the compound given by subcutaneous injection. The doses required to achieve a 50% reversal of the inhibitory effect of orchiectomy are approximately 3 and 1 mg respectively. By the oral route, on the other hand. DHEA has only 10-15% of the activity of the compound given percutaneously. Taking the bioavailability obtained by the subcutaneous route as 100%, it is estimated that the potencies of DHEA by the percutaneous and oral routes are approximately 33 and 3% respectively. Similar ratios of activity were obtained when dorsal prostate and seminal vesicle weight were used as parameters of androgenic activity. When examined on an estrogen-sensitive parameter, namely uterine weight in ovariectomized rats, the stimulatory effect of DHEA was much less potent than its androgenic activity measured in the male animal, a 50% reversal of the inhibitory effect of ovariectomy on uterine weight being observed at the 3 and 30 mg doses of DHEA administered by the subcutaneous and percutaneous routes respectively. When measured on uterine weight, percutaneous DHEA thus shows a 10% potency compared with the subcutaneous route. The sulfate of DHEA (DHEA-S), on the other hand, was approximately 50% as potent as DHEA at increasing ventral prostate weight after subcutaneous or percutaneous administration. When the effect was measured on dorsal prostate and seminal vesicle weight, percutaneous DHEA-S had 10-25% of the activity of DHEA. DHEA decreased serum LH levels in ovariectomized animals, an effect which was completely reversed by treatment with the antiandrogen flutamide. On the other hand, flutamide had no significant effect on the increase in uterine weight caused by DHEA, thus suggesting a predominant estrogenic effect of DHEA at the level of the uterus and an estrogenic effect on the feedback control of LH secretion. The present data show a relatively high bioavailability of percutaneous DHEA as measured by its androgenic and/or estrogenic biological activity in well-characterized peripheral target intracrime tissues in the rat.
Administration, Cutaneous↗
Obesity and metabolic complications: contribution of dehydroepiandrosterone and other steroid hormones.
Obesity is a heterogeneous condition and not every obese patient is at increased risk of cardiovascular diseases (CVD). It is now well established that the regional distribution of body fat is a critical correlate of the metabolic complications of obesity. Studies that have assessed adipose tissue distribution by imaging techniques such as computed tomography have demonstrated the importance of the intra-abdominal (visceral) fat depot as a marker of a cluster of metabolic abnormalities which include glucose intolerance, insulin resistance, hyper-insulinemia, hypertriglyceridemia, elevated number of apo B-carrying lipoproteins as well as hypoalphalipoproteinemia. Although the association between visceral obesity and metabolic complications can hardly be questioned, it has been suggested that it may not necessarily represent a causal relationship. For instance, concomitant alterations in sex steroid levels have been found in both men and women with abdominal (visceral) obesity which have also been reported to be significantly correlated with the insulin resistant-dyslipidemic state found in abdominal obese subjects. In women, abdominal obesity is associated with increased free testosterone concentrations and reduced sex hormone binding globulin (SHBG) levels, whereas in men this condition is associated with reduced testosterone and adrenal C12 steroid (dehydroepiandrosterone, androstenedione, androstene-3 beta, 17 beta-diol) levels as well as decreased SHBG concentrations. These altered steroid and SHBG; levels have been reported to be independent correlates of the metabolic complications of visceral obesity although they cannot solely account for the increased CVD risk found in these patients. In this regard, intervention studies are clearly warranted to better quantity the respective contribution of excess visceral adipose tissue and of the concomitant alterations in sex steroid levels as modulators of metabolic disturbances increasing CVD risk in obesity.
Adipose Tissue↗
Molecular biology and genetics of the 3 beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase gene family.
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3-Hydroxysteroid Dehydrogenases↗
DHEA and peripheral androgen and estrogen formation: intracinology.
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11-beta-Hydroxysteroid Dehydrogenases↗
Immunocytochemical localization of type I 17 beta-hydroxysteroid dehydrogenase in the rat brain.
Type I 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) is mainly involved in the reductive transformation of estrone to estradiol. Such a conversion is known to occur in mammalian brain. In order to determine the brain areas and the nerve cell types containing this enzyme, we have proceeded to its immunocytochemical localization in the adult rat brain. Immunoblot analysis showed that the antibodies used could specifically bind to one brain protein band corresponding to purified 17 beta-HSD. Immunolabelled cells were found in high concentration in the hypothalamus, thalamus, hippocampus, cerebral cortex, caudate putamen and pineal gland. At the light microscopic level, 17 beta-HSD immunoreactive material appeared to be present only in glial and ependymal cells, including tanycytes. Double staining procedures showed that the 17 beta-HSD nerve cells also contained glial fibrillary acidic protein (GFAP), a specific marker for glial cells. Immunoelectron microscopic studies demonstrated that immunoreactive material was diffusely distributed throughout the cytoplasm of glial and ependymal cells, thus confirming the association of 17 beta-HSD immunoreactivity with nonneuronal cells. These data suggest that glial cells play an important role in the conversion of a weak estrogen, estrone, to a more potent estrogen, estradiol.
17-Hydroxysteroid Dehydrogenases↗
Anti-androgens in treatment of prostate cancer.
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Androgen Antagonists↗
Interleukin-6 inhibits the potent stimulatory action of androgens, glucocorticoids and interleukin-1 alpha on apolipoprotein D and GCDFP-15 expression in human breast cancer cells.
Our study was designed to investigate the potential interaction between steroid hormones and interleukin-6 (IL-6) in the regulation of apolipoprotein D (apo-D) and gross cystic disease fluid protein 15 (GCDFP-15) expression in ZR-75-1 human breast cancer cells. We first observed that exposure to IL-6 for 6-14 days decreased basal apo-D and GCDFP-15 secretion by 50% and 23%, respectively. In the same experiment, such treatment with IL-6 decreased cell proliferation by approximately 40% after 6 and 14 days of incubation. Exposure to IL-6 markedly decreased dihydrotestosterone (DHT)-induced apo-D and GCDFP-15 release, with a half-maximal effect measured at 13 U/ml. A similar inhibitory action of IL-6 was observed on the glucocorticoid dexamethasone (DEX)-induced apo-D and GC-DFP-15 secretion. The sensitivity of the apo-D and GCDFP-15 response to the stimulatory action of DHT or DEX was, however, not changed by concomitant exposure to IL-6. The inhibitory effect of IL-6 on the secretion of these two biochemical markers was additive to that of 17 beta-estradiol. In addition, IL-6 blocked the stimulatory effect of interleukin-1 alpha (IL-1 alpha) on apo-D and GCDFP-15 secretion. Our results show that IL-6 is a potent inhibitory of basal as well as androgen-, glucocorticoid- and IL-1 alpha-induced apo-D and GCDFP-15 secretion in ZR-75-1 human breast cancer cells, while cell proliferation is inhibited by this cytokine.
Androgens↗