11β-HSD Type Research Articles

Steroidogenic gene expression during sex determination in the frog Rana rugosa

Rana rugosa is unique among frog species in that it has two distinct types of sex chromosomes in two separate forms (XX/XY and ZZ/ZW). Treatment with sex steroids can reverse its gender from female to male or male to female. This phenomenon makes it a novel model for studying gonadal differentiation. The physiological role of sex steroids in sex differentiation in amphibians is yet unclear, however. To address this issue, we cloned the cDNAs of 17βHSD types 8 ( 17βHSD8) and 12 ( 17βHSD12), 5α-reductase type 1 ( 5αRed1), and the steroidogenic acute regulatory protein known as StAR in the steroidogenic pathway. Then, we measured the mRNA levels of these genes during sex differentiation by real-time RT-PCR. The levels of CYP11A1, 3βHSD, CYP17 and CYP19 mRNA were also measured by real-time RT-PCR. As a result, we detected transcripts of all such genes except for that of 17βHSD8 in the indifferent gonad before the onset of sex determination. The expression of CYP17 occurred in indifferent gonads in both sexes; and its transcript levels were much higher in the male gonads. By contrast, the levels for CYP19 were much higher in the female gonads. CYP11A1, 3βHSD, 17βHSD12, 5αRed1 and StAR showed no sexually dimorphic expression during gonadal sex differentiation. Taken together, the results suggest that CYP17 has a major influence on testis development and that CYP19 plays a similar role in ovary development. However, the factors that up-regulate their expression remain to be identified.

Obesity-Related Modification of Ovarian Gene Expression and Glucocorticoid Metabolism in Aging Lethal Yellow Mice.

Mice possessing the lethal yellow (LY) agouti mutation (C57BL/6J Ay/a) exhibit adult-onset obesity, altered metabolic regulation, and early reproductive senescence. We have previously reported that aging (180-day-old) LY mice exhibit altered ovarian expression of genes involved in sterol synthesis and metabolism relative to age-matched lean non-mutant black mice (B; C57BL/6J a/a). The present study was designed to test the hypothesis that the altered expression of ovarian genes is related to acquired obesity rather than to a direct consequence of the agouti mutation. Differential gene expression in young (90-day-old) LY and B mice, which have similar body weights, were compared with aging mice in which the LY animals are obese. To synchronize ovarian state and exclude gonadotropin-mediated effects, 90-day-old diestrous LY (n = 3) and B (n = 3) mice were suppressed with GnRH antagonist (Antide, Sigma, St. Louis, MO) for five days, then injected with 5 IU eCG (Sigma). Mice were killed 36 h after eCG, and ovaries were immediately removed, carefully trimmed of adhering tissue, and placed in RNALater (Ambion, Austin, TX ) for subsequent RNA extraction. cRNA derived from RNA extracts were run individually on Codelink Mouse Whole Genome Bioarrays (GE Healthcare Life Sciences). Relative gene expression was compared between B and LY mice by t-test using GeneSpring software. Unnamed genes, EST, genes whose expression was below sensitivity limits, and those with less than a 2-fold difference in expression between groups were excluded from further analysis. After these exclusions, 45 genes were differentially expressed in young B and LY mice, compared to 110 differentially expressed genes in aging mice. Both 90- and 180-day-old LY mice exhibited elevated ovarian expression relative to B mice of the agouti gene (330x,350x) as expected. In contrast, numerous genes whose expression was elevated in aging LY mice relative to B mice, e.g. leptin, HMG-CoA reductase, CYP51, and steroidogenic acute regulatory protein (StAR), were not differentially expressed in young LY and B mice. Notably, aging LY mice had a 2-fold greater expression of 11β-HSD type 1 and a 2-fold lesser expression of 11β-HSD type 2 relative to B mice. There was no difference in expression of these genes in young B and LY mice. Consistent with altered 11β-HSD gene expression, ovarian concentrations of corticosterone (C) were elevated in aging LY mice relative to B mice (1.7 ng/mg vs. 0.8 ng/mg, respectively), but C levels were similar in young B and LY mice (0.8 ng/mg vs. 0.9 ng/mg). Results suggest that differential ovarian gene expression and glucocorticoid metabolism observed in aging LY mice is directly related to acquired obesity. (Supported by NIH INBRE 2P20RR016479, NIH R15 HD044438, and Sanford Research/USD)

In situ estrogen metabolism in proliferative endometria from untreated women with polycystic ovarian syndrome with and without endometrial hyperplasia

The aim of the present investigation was to study whether the endocrinological status of women bearing polycystic ovarian syndrome (PCOS) affects the endometrial in situ steroid metabolism. For this purpose, we evaluated the mRNA levels (RT-PCR), and the activity of steroid metabolic enzymes: P450 aromatase, steroid sulfatase (STS), estrogen sulfotransferase (EST) and 17β-hydroxysteroid dehydrogenase (17β-HSD) in 23 samples of normal endometria (CE), 18 PCOS endometria without treatment (PCOSE), 10 specimens from PCOS women with endometrial hyperplasia (HPCOSE), and 7 endometria from patients with endometrial hyperplasia not associated to PCOS (EH). The data showed lower levels of STS mRNA for PCOSE and HPCOSE ( p < 0.05, p < 0.01, respectively) and of EST for HPCOSE and EH compared to control ( p < 0.05). However, higher levels for EST mRNA were obtained in PCOSE ( p < 0.05) versus CE. The mRNA and protein levels for P450 aromatase were undetectable in all analyzed endometria. The relationship between the activities of STS and EST was lower in PCOSE and HPCOSE ( p < 0.05) versus CE. The ratio betweeen the mRNA from 17β-HSD type 1/type 2 was higher in PCOSE ( p < 0.05), whereas, a diminution in the 17β-HSD type 2 activity was observed in PCOSE ( p < 0.05). These results indicate that the activity of enzymes related to the steroid metabolism in analyzed PCOSE differ from those found in the CE. Consequently, PCOSE may present an in situ deregulation of the steroid metabolism.

17β-Hydroxysteroid dehydrogenase type 13 is a liver-specific lipid droplet-associated protein

17β-Hydroxysteroid dehydrogenase (17βHSD) type 13 is identified as a new lipid droplet-associated protein. 17βHSD type 13 has an N-terminal sequence similar to that of 17βHSD type 11, and both sequences function as an endoplasmic reticulum and lipid droplet-targeting signal. Localization of native 17βHSD type 13 on the lipid droplets was confirmed by subcellular fractionation and Western blotting. In contrast to 17βHSD type 11, however, expression of 17βHSD type 13 is largely restricted to the liver and is not enhanced by peroxisome proliferator-activated receptor α and its ligand. Instead the expression level of 17βHSD type 13 in the receptor-null mice was increased several-fold. 17βHSD type 13 may have a distinct physiological role as a lipid droplet-associated protein in the liver.

Novel inhibitors of 17β-hydroxysteroid dehydrogenase type 1: Templates for design

The 17β-hydroxysteroid dehydrogenases (17β-HSDs) catalyze the interconversion between the oxidized and reduced forms of androgens and estrogens at the 17 position. The 17β-HSD type 1 enzyme (17β-HSD1) catalyzes the reduction of estrone (E1) to estradiol and is expressed in malignant breast cells. Inhibitors of this enzyme thus have potential as treatments for hormone dependent breast cancer. Syntheses and biological evaluation of novel non-steroidal inhibitors designed to mimic the E1 template are reported using information from potent steroidal inhibitors. Of the templates investigated biphenyl ethanone was promising and led to inhibitors with IC 50 values in the low micromolar range.

Osteoblasts Directly Control Lineage Commitment of Mesenchymal Progenitor Cells through Wnt Signaling

Lineage commitment of mesenchymal progenitor cells is still poorly understood. Here we demonstrate that Wnt signaling by osteoblasts is essential for mesenchymal progenitor cells to differentiate away from a default adipogenic into an osteoblastic lineage. Dominant adipogenesis and reduced osteoblastogenesis were observed in calvarial cell cultures from transgenic mice characterized by osteoblast-targeted disruption of glucocorticoid signaling. This phenotypic shift in mesenchymal progenitor cell commitment was associated with reciprocal regulation of early adipogenic and osteoblastogenic transcription factors and with a reduction in Wnt7b and Wnt10b mRNA and beta-catenin protein levels in transgenic versus non-transgenic cultures. Transwell co-culture of transgenic mesenchymal progenitor cells with wild type osteoblasts restored commitment to the osteoblast lineage. This effect was blocked by adding sFRP1, a Wnt inhibitor, to the co-culture. Treatment of transgenic cultures with Wnt3a resulted in stimulation of osteoblastogenesis and suppression of adipogenesis. Our findings suggest a novel cellular mechanism in bone cell biology in which osteoblasts exert direct control over the lineage commitment of their mesenchymal progenitor through Wnt signaling. This glucocorticoid-dependent forward control function indicates a central role for osteoblasts in the regulation of early osteoblastogenesis.

Differential gene expression in ACTH -secreting and non-functioning pituitary tumors

Differential expression of several genes between ACTH-secreting pituitary tumors causing Cushing' disease (CD), silent corticotroph adenoma (SCA), and non-functioning pituitary tumors (NFT) was investigated. We used tissue specimens from 35 pituitary tumors (12 CD, 8 SCA, and 15 NFT). Steady-state mRNA levels of the genes related to proopiomelanocortin (POMC) transcription, synthesis, processing, and secretion, such as neurogenic differentiation 1 (NeuroD1), T-box 19 (Tpit), corticotropin releasing hormone receptor (CRHR), vasopressin receptor 1b (V1bR), prohormone convertase (PC) 1/3 and PC2, 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 1 and type 2, glucocorticoid receptor alpha (GRalpha), annexin A1, histone deacetylase 2 (HDAC2), and BRM/SWI2-related gene 1, were determined by real-time RT-PCR. POMC and Tpit mRNA levels were greater in CD and SCA than those in NFT. NeuroD1 mRNA levels were less in CD than those in NFT, but almost comparable between SCA and NFT. PC1/3 mRNA levels were greater in CD, but less in SCA than those in NFT. PC2 mRNA levels in CD and SCA were less than those in NFT. CRHR, V1bR, and 11beta-HSD2 mRNA levels in CD were greater than those in SCA and NFT. HDAC2 mRNA levels in CD and SCA were lower than those in NFT. In conclusion, our study demonstrated that the genes related to transcription, synthesis, processing, and secretion of POMC are differentially regulated in ACTH-secreting pituitary tumors causing CD and SCA compared with those in NFT. This may partly explain the development of clinically active and inactive CD.

Crystallogenesis of Steroid-Converting Enzymes and Their Complexes: Enzyme–Ligand Interaction Studies and Inhibitor Design Facilitated by Complex Structures

Crystal growth of steroid enzymes in complex with hydrophobic ligands is hindered by the low solubility of both partners. However, their interaction can promote highly soluble complexes, leading to well diffracting crystals and high-quality structures. The moderate amphiphilic property of polyethylene glycol increases steroid solubility and facilitates complex formation, but altered orders of soaking can lead to a variety of complex formations and different structures. Understanding the latter mechanism is important for inhibitor design. Thus, different enzyme–ligand complex structures have provided detailed pictures of steroid alternative binding and multispecificity of the enzymes, as, for example, in the case of human 17β-hydroxysteroid dehydrogenases (17β-HSD) type 1 due to C19 steroid pseudosymmetry or in human 17β-HSD type 5 due to the spatial binding site. These complex structures also provide insights into the dynamics of the enzyme binding and catalytic process. Hybrid inhibitors constituted by the steroid and adenine cores of the native substrate and cofactor can properly occupy their original binding sites in 17β-HSD1, leading to an inhibition constant at the nanomolar level. Altered complex crystallization and even a modified order of soaking may result in different complex structures, shedding light on detailed protein–ligand interactions as well as on the enzyme reaction mechanism. These results highlight the important contribution and high significance of the crystal growth mechanism to protein structure determination and function study, as well as to drug design for medicinal applications.

Estrogen metabolizing enzymes in endometrium and endometriosis

Estradiol (E(2)) is an important promoter of the growth of both eutopic and ectopic endometrium. The findings with regard to the expression and activity of steroidogenic enzymes in endometrium of controls, in endometrium of endometriosis patients and in endometriotic lesions are not consistent. In this study, we have looked at the mRNA expression and protein levels of a range of steroidogenic enzymes [aromatase, 17beta-hydroxysteroid dehydrogenases (17beta-HSD) type 1, 2 and 4, estrogen sulfotransferase (EST) and steroid sulfatase (STS)] in eutopic and ectopic endometrium of patients (n = 14) with deep-infiltrative endometriosis as well as in disease-free endometrium (n = 48) using real-time PCR and immunocytochemistry. In addition, we evaluated their menstrual cycle-related expression patterns, and investigated their steroid responsiveness in explant cultures. Aromatase and 17beta-HSD type 1 mRNA levels were extremely low in normal human endometrium, while mRNAs for types 2 and 4 17beta-HSD, EST and STS were readily detectable. Only 17beta-HSD type 2 and EST genes showed sensitivity to progesterone in normal endometrium. Types 1 and 2 17beta-HSD and STS protein was detected in normal endometrium using new polyclonal antibodies. In endometriosis lesions, the balance is tilted in favor of enzymes producing E(2). This is due to a suppression of types 2 and 4 17beta-HSD, and an increased expression of aromatase and type 1 17beta-HSD in ectopic endometrium.

Role of Luteal Glucocorticoid Metabolism during Maternal Recognition of Pregnancy in Women

The human corpus luteum (hCL) is an active, transient, and dynamic endocrine gland. It will experience extensive tissue and vascular remodeling followed by 1) demise of the whole gland without any apparent scarring or 2) maintenance of structural and functional integrity dependent on conceptus-derived human chorionic gonadotropin (hCG). Because cortisol has well-characterized roles in tissue remodeling and repair, we hypothesized that it may have a role in controlling luteal dissolution during luteolysis and would be locally produced toward the end of the luteal cycle. Glucocorticoid-metabolizing enzymes [11beta-hydroxysteroid dehydrogenase (11betaHSD) types 1 and 2] and the glucocorticoid receptor (GR) were assessed in hCL and cultures of luteinized granulosa cells (LGC) using immunofluorescence and quantitative RT-PCR. Furthermore, the effect of cortisol on steroidogenic cell survival and fibroblast-like cell activity was explored in vitro. The hCL expressed 11betaHSD isoenzymes in LGC and nuclear GR in several cell types. hCG up-regulated the expression and activity of 11betaHSD type 1 (P < 0.05) and down-regulated type 2 enzyme (P < 0.05) in vitro and tended to do the same in vivo. Cortisol increased the survival of LGC treated with RU486 (P < 0.05) and suppressed the activity of a proteolytic enzyme associated with luteolysis in fibroblast-like cells (P < 0.05). Our results suggest that, rather than during luteolysis, it is luteal rescue with hCG that is associated with increased local cortisol generation by 11betaHSD type 1. Locally generated cortisol may therefore act on the hCL through GR to have a luteotropic role in the regulation of luteal tissue remodeling during maternal recognition of pregnancy.

Leydig cells from neonatal pig testis abundantly express 11β-hydroxysteroid dehydrogenase (11β-HSD) type 2 and effectively inactivate cortisol to cortisone

11β-Hydroxysteroid dehydrogenase (11β-HSD) isozymes, designated types 1 and 2, catalyze the interconversion of physiologically active glucocorticoids and inactive 11-keto forms. The presence of types 1 and 2 was determined in neonatal pig testis and Leydig cells purified from testes by reverse transcription polymerase chain reaction, Western blotting, and immunohistochemical staining. Type 2 mRNA was expressed at a high level in neonatal pig testis. In particular, in the entire testis, a higher level of type 2 was expressed compared to type 1. Furthermore, these expression levels in the testis were compared with the expression levels of the respective isozymes in pig liver and kidney, which are known to have high levels. Next, the direction of glucocorticoid metabolism in intact Leydig cells was examined, and only oxidation from cortisol to cortisone was detected. Virtually no reduction of cortisone to cortisol was detected. Using a microsomal enzyme preparation from Leydig cells, type 2 exhibited potent oxidation activity, and the activity was higher than the oxidation activity catalyzed by the type 1 isozyme. In kinetic analysis, the K m and V max for type 1 were 1.36 μM and 0.91 nmol/(h mg), respectively, and 0.38 μM and 1.25 nmol/(h mg), respectively, for type 2. The results of the present study using neonatal pig testis suggest that not only 11β-HSD type 1 but also type 2, which is abundantly expressed, plays important roles in cortisol inactivation in pig Leydig cells, and furthermore, that excess cortisol will cause glucocorticoid-mediated suppression of testosterone production in even neonatal pig Leydig cells.

Hydroxysteroid Dehydrogenase (17β -HSD3, 17β-HSD5, and 3α-HSD3) Inhibitors:Extragonadal Regulation of Intracellular Sex Steroid Hormone Levels

Sex hormone signaling regulates the growth, differentiation and development of many tissues. The intracellular concentrations of sex hormones are regulated by several enzymes, including the 17β-hydroxysteroid dehydrogenases (17β-HSDs) and 3α- hydroxysteroid dehydrogenases (3α-HSDs). Most notably, these enzymes are involved in the oxidation and reduction of ketone and β- hydroxyl groups at the C17 position of androgens and estrogens. Fourteen mammalian 17β-HSDs have been identified to date; and are grouped into oxidative enzymes (17β-HSD types 2, 4, 6, 8, 9, 10, 11 and 14) that catalyze the NAD+-dependent inactivation of sex hormones and reductive enzymes (17β-HSD types 1, 3, 5 and 7) that catalyze the formation of more potent steroid receptor ligands. The proliferative effects of androgens and estrogens in target tissues and over-expression of 17β-HSDs in cancer have led to intense drug discovery efforts to identify and develop 17β-HSD inhibitors that can be used for the treatment of breast, prostate and endometrial cancers, neurological disorders, endometriosis, acne, hirsutism and other hormone dependent and independent diseases. Potent and selective inhibitors of intracellular androgen biosynthesis have been reported and, recent proof-of-concept data suggests that these agents have utility in the treatment of androgen-dependent diseases. This review summarizes recent patents and scientific literature regarding steroidal and nonsteroidal 17β-HSD3, 17β-HSD5, and 3α-HSD3 inhibitors and their promise for treatment of androgen-dependent diseases.

Estradiol inhibits the estrone sulfatase activity in normal and cancerous human breast tissues

It is well accepted that estradiol (E 2) plays an important role in the genesis and evolution of breast cancer. Quantitative evaluation indicates that in human breast tumor, estrone sulfate (E 1S) ‘via sulfatase’ is a much more likely precursor for E 2 than is androstenedione ‘via aromatase’. In previous studies, it was demonstrated that in isolated MCF-7 and T-47D breast cancer cell lines, estradiol can block estrone sulfatase activity. In the present study, the effect of E 2 was explored using total normal and cancerous breast tissues. This study was carried out with post-menopausal patients with breast cancer. None of the patients had a history of endocrine, metabolic or hepatic diseases or had received treatment in the previous 2 months. Each patient received local anaesthetic (lidocaine 1%) and two regions of the mammary tissue were selected: (A) the tumoral tissue and (B) the distant zone (glandular tissue) which was considered as normal. Samples were placed in liquid nitrogen and stored at –80 °C until enzyme activity analysis. Breast cancer histotypes were ductal and post-menopausal stages were T2. Homogenates of tumoral or normal breast tissues (45–75 mg) were incubated in 20 mM Tris–HCl, pH 7.2 with physiological concentrations of [ 3H]-E 1S (5 × 10 −9 M) alone or in the presence of E 2 (5 × 10 −5 to 5 × 10 −7 M) during 30 min or 3 h. E 1S, E 1 and E 2 were characterized by thin layer chromatography and quantified using the corresponding standard. The sulfatase activity is significantly more intense with the breast cancer tissue than normal tissue, since the concentration of E 1 was 3.20 ± 0.15 and 0.42 ± 0.07 pmol/mg protein, respectively after 30 min incubation. The values were 27.8 ± 1.8 and 3.5 ± 0.21 pmol/mg protein, respectively after 3 h incubation. Estradiol at the concentration of 5 × 10 −7 M inhibits this conversion by 33% and 31% in cancerous and normal breast tissues, respectively and by 53% and 88% at the concentration of 5 × 10 −5 M after 30 min incubation. The values were 24% and 18% for 5 × 10 −7 M and 49% and 42% for 5 × 10 −5 M, respectively after 3 h incubation. It was observed that [ 3H]-E 1S is only converted to [ 3H]-E 1 and not to [ 3H]-E 2 in normal or cancerous breast tissues, which suggests a low or no 17β-hydroxysteroid dehydrogenase (17β-HSD) Type 1 reductive activity in these experimental conditions. In conclusion, estradiol is a strong anti-sulfatase agent in cancerous and normal breast tissues. This data can open attractive perspectives in clinical trials using this hormone.

A sensitive HPLC method for the assessment of metabolic conversion of estrogens

Disorders of estrogen-responsive tissues are frequently associated with aberrations in steroid metabolism due to altered expression of synthesizing and metabolizing enzymes. For instance, overexposure to unopposed 17β-estradiol has been associated with the pathogenesis of endometrial proliferative disorders, such as endometriosis. Investigations into the metabolic conversion in tissues and cells have been rather limited. This is mostly due to fact that such studies have to make use of radioactive steroid hormones and expensive equipment to obtain sufficient sensitivity. We adapted a sensitive non-radioactive HPLC method to study estrogen metabolism in more detail. This HPLC method is based on the solid phase extraction of estrogens and the derivatization of the steroids with 2-(4-carboxy-phenyl)-5,6-dimethylbenzimidazole. The technique is sensitive, robust and is useful for the detection of aromatase, 17β-HSD types 1 and 2 and sulfatase activities in lysates of placenta and endometrium.

Alteration of the cortisol–cortisone shuttle in leprosy type 1 reactions in leprosy patients in Hyderabad, India

Regulation of inflammation in leprosy may be influenced by local concentrations of active cortisol and inactive cortisone, whose concentrations are regulated by enzymes in the cortisol–cortisone shuttle. We investigated the cortisol–cortisone shuttle enzymes in the skin of leprosy patients with type 1 reactions (T1R), which are characterised by skin and nerve inflammation. Gene expression of the shuttle enzymes were quantified in skin biopsies from 15 leprosy patients with new T1R before and during prednisolone treatment and compared with levels in skin biopsies from 10 borderline leprosy patients without reactions. Gene expression of 11β-hydroxysteroid dehydrogenase (11β-HSD) type 2, which converts cortisol to cortisone, is down-regulated in skin from T1R lesions. However expression levels of 11β-HSD type 1, which converts cortisone to cortisol, were similar in skin with and without reactions and did not change during anti-leprosy drug treatment. Prednisolone treatment of patients with reactions is associated with an upregulation of 11β-HSD2 expression in skin. The down regulation of 11β-HSD2 at the beginning of a reaction may be caused by pro-inflammatory cytokines in the leprosy reactional lesion and may be a local attempt to down-regulate inflammation. However in leprosy reactions this local response is insufficient and exogenous steroids are required to control inflammation.

Expression analysis of the genes involved in estradiol and progesterone action in human ovarian endometriosis

Endometriosis is defined as the presence of endometrial glands and stroma within extrauterine sites, and it is well known that endometriosis is an estrogen-dependent disease. The defective formation and metabolism of steroid hormones is responsible for the promotion and development of endometriosis. In the present study we examined the mRNA levels of six enzymes that are involved in the metabolism of estrogen and progesterone – aromatase, 17β-hydroxysteroid dehydrogenase (17β-HSD) types 1, 2 and 7, sulfatase and sulfotransferase – and of the steroid receptors – estrogen receptors α and β (ERα, ERβ) and progesterone receptors A and B (PRAB) – implicated in human ovarian endometriosis. We analyzed 16 samples of ovarian endometriosis and 9 of normal endometrium. The real-time polymerase chain reaction analyses revealed that six of the nine genes investigated are differentially regulated. Aromatase, 17β-HSD types 1 and 7, sulfatase and ERβ were statistically significantly upregulated, while ERα was significantly downregulated, in the endometriosis group compared with the control group. There were no significant differences in 17β-HSD type 2, sulfotransferase and PRAB gene expression. Our results indicate that, in addition to the previously reported upregulation of aromatase, upregulation of 17β-HSD types 1 and 7 and sulfatase can also increase the local estradiol concentration. This could thus be responsible for the estrogen-dependent growth of endometriotic tissue. Surprisingly ERα was downregulated.

A ação das isoenzimas 11B-hidroxiesteróide desidrogenase tipo 1 e tipo 2 na patogênese da Síndrome de Cushing

The action of glucocorticoids is modulated by isoenzymes 11β-hidroxiesteroide desidrogenases (11β-HSD) type 1 and 2. The knowledge concerning these isoenzymes contribute to the understanding of the regulatory mechanisms involved in several disease processes of the Cushing’s syndrome, such as obesity, osteoporosis and hypertension. With the aim at describing the action of isoenzymes 11β-HSD type 1 and 2 in the Cushing’s syndrome, a literature review was done from 1990 - 2006 using the Medline data base, searching for the following key-words: Cushing’s syndrome, glucocorticoids, 11β-hydroxysteroid dehydrogenase, hypertension, osteoporosis and obesity. Review studies, meta-analysis and original articles were selected and chosen on the basis of methodological aspects and relevance. The exact mechanism by which cortisol increases blood pressure is not completely understood, but it involves, among others factors, changes in the sodium homeostasis. The conversion of cortisone to cortisol through expression of 11β-HSD1 induces the differentiation of preadipoctyes to mature adipoctyes and such patients develop an increase in visceral fat. The prevalence of osteoporosis in adult patients with Cushing’s syndrome is approximately 50% and glucocorticoids play a strong effect on the bone and calcium metabolism. The isoenzymes 11β-HSD1 and 11β-HSD2 have an important function in these several pathophysiology processes; however the isoenzymes action in the pathophysiology of the Cushing’s syndrome need to be more investigated

Expression of aromatase and 17β-hydroxysteroid dehydrogenase types 1, 7 and 12 in breast cancer: An immunocytochemical study

It is known that there is a local biosynthesis of estradiol (E2) in breast carcinoma. The steroidogenic enzymes involved in E2 formation are aromatase which transforms testosterone into E2 and androstenedione into estrone (E1) and reductive 17β-hydroxysteroid dehydrogenases (17β-HSDs) which convert E1 into E2. Using immunocytochemistry, we have studied the expression of aromatase and the three reductive 17β-HSDs 17β-HSD types 1, 7 and 12 in 41 specimens of female human breast carcinoma and adjacent non-malignant tissues. These results were correlated with the estrogen receptor α (ERα) and β (ERβ), progesterone receptor, androgen receptor, CDC47 and c-erb B-2 expressions and with the tumor stages. Aromatase was found in 58%, 17β-HSD type 7 in 47% and 17β-HSD type 12 in 83% of the breast cancer specimens. The 17β-HSD type 1 could be detected in only one tumor. A significant correlation was observed between the aromatase, 17β-HSD type 7 and 17β-HSD type 12 expression, as well as between each of the two enzymes 17β-types 7 and 12 and the ERβ expression. The expression of 17β-HSD type 12 was significantly higher in breast carcinoma specimens than in normal tissue. There was also a significant association of CDC 47 expression with ERβ, AR and 17β-HSD type 12. The results indicate that aromatase, 17β-HSD type 7 and 17β-HSD type 12, but not 17β-HSD type 1, are commonly expressed in human breast cancer. Moreover, the high expression of both 17β-HSD type 12 and ERβ in breast carcinoma cells may play a role in the development and/or progression of breast cancer.

Rational proteomics V: Structure-based mutagenesis has revealed key residues responsible for substrate recognition and catalysis by the dehydrogenase and isomerase activities in human 3β-hydroxysteroid dehydrogenase/isomerase type 1

Mammalian 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) is a member of the short chain dehydrogenase/reductase. It is a key steroidogenic enzyme that catalyzes the first step of the multienzyme pathway conversion of circulating dehydroepiandrosterone and pregnenolone to active steroid hormones. A three dimensional model of a ternary complex of human 3β-HSD type 1 (3β-HSD_1) with an NAD cofactor and androstenedione product has been developed based upon X-ray structures of the ternary complex of E. coli UDP-galactose 4-epimerase (UDPGE) with an NAD cofactor and substrate (PDB_AC: 1NAH) and the ternary complex of human type 1 17β-hydroxysteroid dehydrogenase (17β-HSD_1) with an NADP cofactor and androstenedione (PDB_AC: 1QYX). The dimeric structure of the enzyme was built from two monomer models of 3β-HSD_1 by respective 3D superposition with A and B subunits of the dimeric structure of Streptococcus suis DTDP- d-glucose 4,6-dehydratase (PDB_AC: 1KEP). The 3D model structure of 3β-HSD_1 has been successfully used for the rational design of mutagenic experiments to further elucidate the key substrate binding residues in the active site as well as the basis for dual function of the 3β-HSD_1 enzyme. The structure based mutant enzymes, Asn100Ser, Asn100Ala, Glu126Leu, His232Ala, Ser322Ala and Asn323Leu, have been constructed and functionally characterized. The mutagenic experiments have confirmed the predicted roles of the His232 and Asn323 residues in recognition of the 17-keto group of the substrate and identified Asn100 and Glu126 residues as key residues that participate for the dehydrogenase and isomerization reactions, respectively.

MDM2: A Novel Mineralocorticoid-Responsive Gene Involved in Aldosterone-Induced Human Vascular Structural Remodeling

Aldosterone has been demonstrated to play an important role in the pathogenesis of various cardiovascular diseases. Vascular structural remodeling, including vascular smooth muscle cell (VSMC) proliferation, has been also reported in small resistance arteries of patients with primary aldosteronism. Therefore, in this study, we examined whether genes involved in the regulation of the cell cycle were induced by aldosterone alone in cultured human VSMCs and in human small resistance arteries. Results of these studies eventually demonstrated that MDM2, one of the genes involved in anti-apoptosis and cell growth, was markedly increased in mineralocorticoid receptor (MR)-positive VSMCs by aldosterone in all microarray, reverse transcriptase-polymerase chain reaction, immunoblotting, and immunofluorescence analyses. In addition, an analysis using small interfering RNA demonstrated that this gene product was involved in cell proliferation of VSMCs induced by aldosterone. Eplerenone, a specific MR antagonist, inhibited this gene induction by aldosterone in VSMCs. MDM2 protein was also more abundant in VSMCs of small resistance arteries in patients with primary aldosteronism compared with a control population. MDM2 is therefore considered one of the mineralocorticoid-responsive genes that regulates cell proliferation of VSMCs induced by MR-mediated aldosterone stimulation, possibly playing an important role in aldosterone-induced vascular structural remodeling.