Localization of 17beta-hydroxysteroid dehydrogenase type 1 mRNA in mouse tissues

The enzyme 17beta-hydroxysteroid dehydrogenase (17beta-HSD) type 1 catalyzes the conversion of estrone (E1) into 17beta estradiol (E2). To gain information about the cellular localization of 17beta-HSD mRNA type 1 expression, we performed in situ hybridization using a 35S-labeled cRNA probe in several tissues of adult mice of both sexes. In the ovary, high expression was found in granulosa cells of growing follicles. No specific labeling could be observed in corpora lutea or interstitial cells. In the pituitary gland of animals of both sexes, 17beta-HSD type 1 mRNA was expressed in the intermediate lobe melanotrophs while no specific signal could be detected in the anterior or posterior lobes of the pituitary. In the prostate, 17beta-HSD type 1 mRNA was exclusively found in the epithelial cells. In both male and female mouse dorsal skin, a specific hybridization signal was seen in the sebaceous glands while the epidermis, stroma, hair follicles and sweat glands were unlabeled. In the testis, a hybridization signal was detected in germ cells of the seminiferous tubules, Leydig cells being unlabeled. The present data indicate that E2 can be formed through the action of 17beta-HSD type 1 in specific cells of the gonads and peripheral tissues. In the testes and peripheral tissues, the action of E2 is probably limited to the cells involved in its formation in an intracrine fashion.     

Promiscuous 3beta-hydroxysteroid dehydrogenases: testosterone 17beta-hydroxysteroid dehydrogenase activities of mouse type I and VI 3beta-hydroxysteroid dehydrogenases

3Beta-hydroxysteroid dehydrogenase (3beta-HSD) activity is essential for the synthesis of all classes of steroid hormones, converting various delta5-3beta-hydroxysteroids into hormonally active delta4-3-ketosteroids in NAD+ -dependent reactions. Certain 3beta-HSD isoforms have been reported to exhibit additional dehydrogenase character (e.g., 17-hydroxysteroid dehydrogenase/reductase). We have investigated whether mouse type I (adrenal/gonadal) and type VI 3beta-HSDs (uterine/embryonic) display significant 17beta-HSD-like activity. Nonsteroidogenic HEK 293T cells were transiently transfected with pCMV-based expression vectors containing mouse type I and type VI 3beta-HSDs. Transfected cells expressing either mouse type I or type VI 3beta-HSD converted testosterone to androstenedione, albeit at rates one-tenth of those of pregnenolone to progesterone in similarly transfected 293T cells. Our findings demonstrate that the mouse 3beta-HSD I and VI isoforms can inactivate testosterone within an intact cell milieu. These findings are important not only in establishment of structure-function relationships, but also whenever murine systems are used for developmental/reproductive paradigms associated with human disorders.     

Localization of 11beta-hydroxysteroid dehydrogenase types 1 and 2 in the male reproductive tract

The action of glucocorticoids in target tissues is dependent on the local expression of glucocorticoid receptors and two 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzymes, 11beta-HSD1 and 11beta-HSD2, which interconvert active and inactive glucocorticoids. This study examined expression of the 11beta-HSD enzymes in the male reproductive tract of the adult rat. 11beta-HSD1 was immunolocalized to the apical region of principal epithelial cells of the caput epididymis, with the less numerous clear cells devoid of signal. Epididymal 11beta-HSD1 expression was confirmed by Western blot analysis, with immunoreactive species identified at 34 kDa (the expected size for 11beta-HSD1) and at approximately 48 kDa. 11beta-HSD bioactivity was readily detectable in the epididymis, with 11-oxoreductase activity clearly the favored reaction (as observed in liver), consistent with 11beta-HSD1 expression. The epithelium of the vas deferens, seminal vesicle, and penile urethra were also immunopositive for 11beta-HSD1, as were smooth muscle cells of the vas deferens and penile blood vessels. 11beta-HSD2 was also immunolocalized to the epididymal epithelium, but its distribution was complementary to that of 11beta-HSD1 (i.e. clear cells showing intense 11beta-HSD2 staining but principal cells devoid of signal). 11beta-HSD2 was also present in the corpora cavernosa of the penis but not in other tissues. In conclusion, the differential expression of 11beta-HSD1 and 11beta-HSD2 throughout the male reproductive tract suggests that these enzymes locally modulate glucocorticoid and mineralocorticoid actions, particularly in the epididymis and penile vasculature.     

Mechanisms of androgen production in male pseudohermaphroditism due to 17 beta-hydroxysteroid dehydrogenase deficiency.

17 beta-Hydroxysteroid dehydrogenase (17 beta HSD) deficiency is a rare cause of male pseudohermaphroditism, but is a frequent disorder among a highly inbred Arab population in the Gaza strip. Affected individuals are born and reared as females until puberty, when marked virilization occurs, leading in many cases to the spontaneous adoption of a male gender role. To investigate the mechanisms and site(s) of androgen production, we determined the gonadal and extragonadal steroid patterns in two postpubertal male pseudohermaphroditism patients, who were castrated and reared as females. Before castration, both patients had very high plasma levels of androstenedione (delta 4-A), normal or moderately low levels of testosterone (T), and significantly elevated delta 4-A/T ratios (P less than 0.01). Dihydrotestosterone (DHT) levels were normal or high, while the DHT/T ratios were lower than normal (P less than 0.01), suggesting enhanced 5 alpha-reductase activity. These abnormalities were much more severe in spermatic venous blood. 17 beta HSD deficiency was also found in the delta 5-pathway, by high dehydroepiandrosterone (DHEA) levels and very high dehydroxyepiandrosterone/delta 5-androstenediol (DHEA/delta 5-diol) ratios, and in peripheral tissue metabolites, by very high androsterone glucuronide/3 alpha-androstanediol glucuronide ratios (P less than 0.01). The estrogen pathway was also impaired (P less than 0.01), even though both estrone and estradiol levels were elevated. Gonadectomy significantly reduced all androgens and estrogens (P less than 0.01), but when compared to 42 castrated controls, both patients had lower delta 4-A and higher T levels. The delta 4-A/T ratio was lower than that in controls, indicating normal to enhanced extragonadal 17 beta HSD activity. A similar pattern was observed in the delta 5- and estrogen pathways. DHT levels were within normal limits, and 3 alpha-diol was moderately decreased. These data suggest that testicular 17 beta HSD activity is under a different genetic control from that in extragonadal tissues. Affected males lack the testicular enzyme, but their extragonadal 17 beta HSD activity is normal or enhanced. Together with enhanced 5 alpha-reductase activity, this represents a highly efficient compensatory mechanism for androgen and estrogen production after puberty.     

Expression of the 17beta-hydroxysteroid dehydrogenase type 5 mRNA in the human brain

An enzyme-mediated metabolism of androgens and estrogens including 17beta-HSD activity in the brain of vertebrates was discovered approximately 30 years ago. Mainly 5alpha-reductase and aromatase have been studied in detail. Recently we could demonstrate reductive and oxidative 17beta-HSD activity as well as considerable mRNA expression of the 17beta-HSD types 3 and 4 in the human brain. In the present study, we report on 17beta-HSD type 5 mRNA expression in brain tissue of women and men. Data analysis did not reveal sex specific differences, but we determined a significantly higher mRNA concentration in the subcortical white matter (SC) than in the cerebral cortex (CX). Investigation of reductive 17beta-HSD in vitro activity with 2 microM androstenedione as the substrate revealed no sex specific differences. Testosterone formation was significantly higher in SC than in CX. Moreover, enzyme activity was significantly higher in brain tissue of adults compared to that of children.     

Localization of type 5 17beta-hydroxysteroid dehydrogenase, 3beta-hydroxysteroid dehydrogenase, and androgen receptor in the human prostate by in situ hybridization and immunocytochemistry

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 beta-HSD (hydroxysteroid dehydrogenase) converts DHEA into androstenedione (4-dione), whereas type 5 17beta-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 17beta-HSD messenger RNA and enzyme in human benign prostatic hyperplasia (BPH). Localization of 3beta-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 17beta-HSD was reexamined by immunocytochemistry in the normal human prostate samples and also in normal prostate epithelial cell line (PrEC). The in situ hybridization results obtained with a tritiated uridine triphosphate (3H-UTP)-labeled type 5 17beta-HSD riboprobe are in agreement with the immunostaining data obtained with a specific antibody to the enzyme. The immunostaining results obtained from normal prostate tissue and BPH were found to be similar. Thus, in the glandular epithelium, basal cells highly express the messenger RNA and the enzyme, whereas luminal cells show a much lower and variable level of expression. In the stroma and walls of blood vessels, fibroblasts and the endothelial cells lining the blood vessels show positive staining. Similar results are observed when the cellular distribution of 3beta-HSD is investigated. AR immunoreactivity, however, shows a different distribution because, in the epithelium, most of the nuclei of basal cells are negative, whereas the majority of nuclei of the luminal cells show positive staining. A strong reaction for AR is also found in most stromal cell nuclei and in the nuclei of most endothelial cells, as well as in some other cells of the walls of blood vessels. In conclusion, human type 5 17beta-HSD, as well as 3beta-HSD, are highly expressed, not only in the basal epithelial cells and stromal fibroblasts but also in the endothelial cells and fibroblasts of the blood vessels. AR, on the other hand, is highly expressed in the luminal cells. The present data suggest that DHEA is transformed in the basal cells of the glandular epithelium into 4-dione by 3beta-HSD and then into testosterone by type 5 17beta-HSD, whereas dihydrotestosterone is synthesized in the luminal cells after diffusion of testosterone from the underlying layer of basal cells. The potential role of androgen formation and action in blood vessels is unknown and opens new avenues of investigation for a better understanding of the multiple roles of androgens.     

Extragonadal 17 beta-hydroxysteroid dehydrogenase activity in rainbow trout.

Blood cells of male and female rainbow trout showed 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) activity in vitro, reducing 11 beta-hydroxyandrostenedione and 11-ketoandrostenedione (OA) to 11 beta-hydroxytestosterone and 11-ketotestosterone (OT), respectively. Enzyme activity did not vary with gonadal development in either sex. The conversion of tritiated precursors was partly inhibited in the presence of steroid-free serum or radioinert steroid, but inhibition was less strong when radioinert androgens were added to steroid-free serum or when the serum contained endogenous steroids. Treatment of male trout with salmon gonadotropin in vivo and/or incubation with a pituitary extract of mature salmon in vitro did not affect OA conversion when blood cells were incubated in the absence of serum, whereas it was slightly but significantly higher when they were incubated in the presence of serum and pituitary extract. In addition to blood cells and steroidogenic tissues, spleen, intestine, brain, liver, excretory kidney, and skin tissue also produced an OA metabolite isopolar to OT in vitro, so that 17 beta HSD appears to be present in a variety of trout issues. With respect to the biological significance of extragonadal steroid metabolism in vivo, the ligand binding characteristics of circulating steroid binding proteins may be of primary relevance in regulating substrate availability.     

Coenzyme specificity in fungal 17beta-hydroxysteroid dehydrogenase

The 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus is an NADP(H)-dependent member of the short-chain dehydrogenase/reductase superfamily (SDR) that belongs to the cP1 classical subfamily. Here, we have created several mutants by site-directed mutagenesis, and through these we have studied the amino acid residues that are responsible for coenzyme binding and specificity. The Thr202Val and Thr202Ile mutants were inactive, thus confirming the importance of Thr202 for the appropriate orientation of the coenzyme that enables the hydride transfer. The Ala50Arg and Asn51Arg mutants had increased rates of NADPH dissociation, and thus an enhanced substrate oxidation with NADP+, while the Asn51Arg mutant also showed an increased rate of NADP+ dissociation, and thus an enhanced substrate reduction with NADPH. Addition of a negatively-charged amino acid residue at the first position after the second beta-strand (Tyr49Asp) affected the coenzyme specificity and turned the enzyme into an NAD+-dependent oxidase resembling the cD1d subfamily members.     

Multiple functions of type 10 17beta-hydroxysteroid dehydrogenase.

Human 17beta-hydroxysteroid dehydrogenase type 10 (17beta-HSD10) is a mitochondrial enzyme encoded by the SCHAD gene, which escapes chromosome X inactivation. 17Beta-HSD10/SCHAD mutations cause a spectrum of clinical conditions, from mild mental retardation to progressive infantile neurodegeneration. 17Beta-HSD10/SCHAD is essential for the metabolism of isoleucine and branched-chain fatty acids. It can inactivate 17beta-estradiol and steroid modulators of GABA(A) receptors, and convert 5alpha-androstanediol into 5alpha-dihydrotestosterone (DHT). Certain malignant prostatic epithelial cells contain high levels of 17beta-HSD10, generating 5alpha-DHT in the absence of testosterone. 17Beta-HSD10 has an affinity for amyloid-beta peptide, and might be linked to the mitochondrial dysfunction seen in Alzheimer's disease. This versatile enzyme might provide a new drug target for neuronal excitability control and for intervention in Alzheimer's disease and certain cancers.     

Inhibitors of type II 17beta-hydroxysteroid dehydrogenase

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) are involved in the last step of the biosynthesis of sex steroids from cholesterol. This family of steroidogenic enzymes constitutes an interesting target in the control of the concentration of estrogens and androgens. Among the isoforms of 17beta-HSD, type II preferentially catalyzes the oxidation of estradiol (E(2)), testosterone (T), dihydrotestosterone (DHT), and 20alpha-dihydroprogesterone (20alpha-DHP). Based on structure-activity relationship studies, we have developed steroidal spirolactones as inhibitors of type II 17beta-HSD using different steroid nuclei: a C18-steroid (lactones 1 and 10), an antiestrogenic nucleus (lactone 2), and a C19-steroid (lactone 28). We know these inhibitors are selective for type II 17beta-HSD as no or only weak inhibition was observed for types I and III. They also have no proliferative (androgenic) activity on androgen sensitive (AR(+)) Shionogi cells whereas their proliferative (estrogenic) activity on estrogen sensitive (ER(+)) ZR-75-1 cells depends on the nature of the steroid nucleus. Lactones 1 and 10 are weak estrogens, while lactones 2 and 28 do not exert estrogenic activity, in fact lactone 2 is an antiestrogen. Lactones 1, 2, 10 and 28 were also tested in an identical assay with a series of enzyme substrates, C19-steroid diols, and known inhibitors, for the oxidation of testosterone and estradiol into androstenedione and estrone, respectively. From this comparative study, the best inhibitors of type II 17beta-HSD (oxidase activity) were identified, but none of them were clearly more potent than the hydroxylated (reduced) forms of enzyme substrates, E2, T, and DHT. Such inhibitors remain, however, useful tools to, (1) further elucidate the role of type II 17beta-HSD, and (2) regulate the level of active estrogens, androgens and progesterone.     

Ontogeny of 11 beta-hydroxysteroid dehydrogenase in rat brain and kidney.

Close regulation of circulating corticosteroid levels during the early postnatal period is crucial for normal development and maturation of the central nervous system. In the first weeks of life cerebral glucocorticoid receptor concentrations are low and the hypothalamic-pituitary-adrenal axis is relatively unresponsive to stress, which might, in part, protect the developing brain from elevated corticosteroid levels. However, central mineralocorticoid receptors are at near adult levels and free glucocorticoid concentrations may approximate adult values as corticosteroid binding globulin is absent. Thus other mechanisms controlling cerebral exposure to corticosteroids may be of importance. 11 beta-Hydroxysteroid dehydrogenase (11 beta-OHSD) determines the access of corticosterone to peripheral mineralocorticoid and glucocorticoid receptors in adults in vivo by metabolizing corticosterone to inactive 11-dehydrocorticosterone. The enzyme has recently been demonstrated in brain subregions and may modulate local corticosteroid-receptor interactions. We therefore examined 11 beta-OHSD bioactivity and messenger RNA (mRNA) expression in the brain, compared with kidney, during the neonatal period. 11 beta-OHSD bioactivity (expressed as the percentage conversion of corticosterone to 11-dehydrocorticosterone) was moderately high in hippocampus and parietal cortex at birth (46 +/- 4% and 48 +/- 5%, respectively), fell significantly to a nadir (32 +/- 1% and 30 +/- 1%, respectively) at postnatal day 10 and then gradually rose to adult values (52 +/- 3% and 58 +/- 3%). By contrast, 11 beta-OHSD activity in cerebellum was high at birth (60 +/- 3%), rose significantly to a peak at postnatal day 10 (74 +/- 3%), and then fell to adult values by postnatal day 15 (64 +/- 3%). Renal 11 beta-OHSD activity was moderately high (69 +/- 3%) at birth and reached adult values (80 +/- 2%) by postnatal day 5. Northern blots showed high and similar expression of a single species of 11 beta-OHSD mRNA from birth to adulthood in the hippocampus. Only low expression of 11 beta-OHSD (two or three separate species) was found in the kidney during the first 2 weeks of life, whereas, in adults high expression of 11 beta-OHSD mRNA was detected in kidney (four species). Using in situ hybridization high 11 beta-OHSD mRNA expression was localized to the neuronal layers of the postnatal hippocampus, neocortex, and cerebellum, and low but detectable expression was found in the neonatal renal cortex. Thus, 11 beta-OHSD is highly expressed in rat brain subregions in the early postnatal period with specific developmental patterns of activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cinnamates and cinnamamides inhibit fungal 17beta-hydroxysteroid dehydrogenase

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) have important roles in the regulation of steroid hormone actions through their catalysis of the oxidation or reduction of estrogens and androgens at position 17. Dysfunctions of the human 17beta-HSDs have been associated with reproduction disorders, neuronal diseases and the development of hormone-dependent forms of cancers. Therefore, these enzymes represent interesting targets for the development of new drugs. Here we present a series of new cinnamic acid esters and amides that inhibit the oxidative and reductive reaction catalyzed by 17beta-HSD from the fungus Cochliobolus lunatus, a model enzyme of the short-chain dehydrogenase/reductase superfamily. We found that esters of unsubstituted cinnamic acid were better inhibitors than esters of 3,4,5-trimethoxycinnamic acid. Cinnamates were also more potent inhibitors than structurally related cinnamamides. The compounds presented in this paper are potential leads for the development of inhibitors of human 17beta-HSD isoforms, which may prove to have different therapeutic applications.     

Increased 17 beta-hydroxysteroid dehydrogenase activity in a masculinizing adrenal adenoma in a patient with isolated testosterone overproduction

The patient studied had noted the onset of virilization shortly after menopause. Urinary 17-ketosteroid levels were normal, as were fractionated 17-ketosteroid levels by gas liquid chromatography, but for 3 yr, serum testosterone levels had been greater than 490 ng/dl. The ovaries were found to be normal by laparoscopy. Abdominal exploration revealed a 1-cm adenoma in the right adrenal. A part of the adenoma excised from our patient was homogenized and incubated with 5 microCi [14C]androstenedione. Five percent of the 14C was converted by the tumor homogenate to a metabolite with the same mobility as testosterone on LH-20 chromatography. After thin layer chromatography, the radiolabeled material together with 3H-labeled authentic testosterone were crystallized to a constant specific activity. The net rate of testosterone synthesis by the tumor was 26 pmol/mg wet tissue wt.h vs. 0.56 pmol/mg.h by a control adrenal homogenate. Thus, the tumor demonstrated a 50-fold increase in 17 beta-hydroxysteroid dehydrogenase activity compared to normal adrenal tissue. This is the first report to identify altered activity of a specific enzyme system in this syndrome of isolated adrenal testosterone overproduction.     

Expression and regulation of 17beta-hydroxysteroid dehydrogenase 7 in the rabbit

A recent study from our laboratory demonstrated a strong upregulation of activin expression during cutaneous wound healing. To further analyze the role of activin A in skin morphogenesis and wound repair, we generated transgenic mice that overexpress activin A under the control of the keratin 14 promoter. The latter targets expression of transgenes to the basal, proliferating layer of the epidermis. Hetero- as well as homozygous transgenic animals were viable and fertile. However, they were smaller than non-transgenic littermates and they had smaller ears and shorter tails. Histological analysis of their skin revealed dermal hyperthickening, mainly due to the replacement of fatty tissue by connective tissue, and an increase in suprabasal, partially differentiated epidermal layers. After cutaneous injury, a strong enhancement of granulation tissue formation was observed. Furthermore, the extent of re-epithelialization was increased in some of the wounds. These data demonstrate that activin A is a potent stimulator of the wound healing process. Using an in vivo model of local brain injury, we found that activin A also plays a significant role in the early cellular response to neuronal damage. Expression of activin mRNA and protein is markedly upregulated within a few hours of injury. If applied exogenously, recombinant activin A is capable of rescuing neurons from acute cell death. Studying the interaction between bFGF, a well-established neuroprotective agent, which is currently being tested in stroke patients, and activin A, we arrived at the unexpected conclusion that it is the strong induction of activin A by bFGF which endows the latter with its beneficial actions in patients. These findings suggest that the development of substances directly targeting activin expression or receptor binding should offer new possibilities in the acute treatment of stroke and brain trauma.     

Immunoelectron microscopic localization of 3beta-hydroxysteroid dehydrogenase and type 5 17beta-hydroxysteroid dehydrogenase in the human prostate and mammary gland

The subcellular distribution of steroidogenic enzymes has so far been studied mostly in classical endocrine glands and in the placenta. In the peripheral intracrine organs which synthesize sex steroids there is no indication about the organelles which contain the enzymes involved in steroid biosynthesis. We have thus investigated the subcellular localization of two enzymes involved in the production of sex steroids, namely 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and type 5 17beta-hydroxysteroid dehydrogenase (17beta-HSD). Using specific antibodies to these enzymes, we conducted immunoelectron microscopic studies in two peripheral tissues, namely the human prostate and mammary gland. In the prostate, immunolabelling for both 3beta-HSD and type 5 17beta-HSD was detected in the basal cells of the tube-alveoli as well as in fibroblasts and endothelial cells lining the blood vessels. In all the labelled cell types, the gold particles were distributed throughout the cytoplasm. No obvious association with any specific organelle could be observed, although some concentration of gold particles was occasionally found over bundles of microfilaments. In mammary gland sections immunolabelled for 3beta-HSD or type 5 17beta-HSD localization, labelling was observed in the cytoplasm of the secretory epithelial cells in both the acini and terminal ducts. Immunolabelling was also found in the endothelial cells as well as in fibroblasts in stroma and blood vessels. The gold particles were not detected over any organelles, except with the occasional accumulation of gold particles over microfilaments. The present data on the localization of two steroidogenic enzymes leading to the synthesis of testosterone indicate that these enzymes are located not only in epithelial cells but also in stromal and endothelial cells in both tissues studied. The absence of any association of the enzymes with membrane-bound organelles appears as a common finding in the reactive cell types of two peripheral tissues.     

Studies on 17beta-hydroxysteroid dehydrogenase in human endometrium and endometrial carcinoma.

Kinetic parameters, substrate specificity and stability of a cytoplasmic 17beta-hydroxysteroid dehydrogenase of human secretory endometrium were studied. Using oestradiol as substrate, oestrone formation was found to be linear with time and the concentration of protein. The optimum temperature was 40 degrees C and the optimum pH 9.5. For the reduction of oestrone the optimal pH was 6. With NADP the maximal velocity was about 1/3 of that with NAD (0.23 nmoles/mg protein/10 min). The Km for oestradiol was 3.3 times 10- minus 6 M. Testosterone and androstenedione also served as substrates but they were interconverted more slowly than oestradiol and oestrone. Sulphhydryl groups were shown to be essential for catalysis. The enzyme is cold sensitive but cold inactivation can be reduced by NAD, NADP, oestradiol or glycerol.