Molecular cloning, modeling, and localization of rat type 10 17beta-hydroxysteroid dehydrogenase

Rat and mouse complementary DNAs of type 10 17beta-hydroxysteroid dehydrogenase were cloned and sequenced. The mouse cDNA clone's sequence corrected the previously published nucleotide and amino acid sequence of mouse endoplasmic reticulum-associated beta-amyloid-binding protein. A subunit of the rat enzyme consists of 261 amino acid residues with a calculated molecular mass of 27250 Da. Compared with its human counterpart, rat 17betaHSD type 10 shows 88% identity. Mouse 17betaHSD type 10 is composed of 261 amino acid residues with a calculated molecular mass of 27274 Da. There is 95% identity between the two rodent enzymes. A stereostructure model of rat 17betaHSD type 10 was constructed based on its amino acid sequence. Similar to human type 10 17betaHSD, the rodent enzymes also displayed relatively higher 3alphaHSD activity than 17betaHSD activity. Intracellular localization of rat 17betaHSD type 10 has been determined by subcellular fractionation and confocal microscopy studies. The results unequivocally establish that this is a nuclear gene-encoded mitochondrial enzyme, and that this 17betaHSD is not associated with the endoplasmic reticulum. The unique location distinguishes type 10 from other types of 17beta-hydroxysteroid dehydrogenases.     

Androgenic 17 beta-hydroxysteroid dehydrogenase activity of expressed rat type I 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase.

Transient expression in nonsteroidogenic mammalian cells of the rat wild type I and type II 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) cDNAs shows that the encoded proteins, in addition to being able to catalyze the oxidation and isomerization of delta 5-3 beta-hydroxysteroid precursors into the corresponding delta 4-3-ketosteroids, interconvert 5 alpha-dihydrotestosterone (DHT) and 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol). When homogenate from cells transfected with a plasmid vector containing type I 3 beta-HSD is incubated in the presence of DHT using NAD+ as cofactor, a somewhat unexpected metabolite is formed, namely 5 alpha-androstanedione (A-dione), thus indicating an intrinsic androgenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity of this 3 beta-HSD isoform. Although the relative Vmax of 17 beta-HSD activity is 14.9-fold lower than that of 3 beta-HSD activity, the Km value for the 17 beta-HSD activity of type I 3 beta-HSD is 7.97 microM, a value which is in the same range as the conversion of DHT into 3 beta-diol which shows a Km value of 4.02 microM. Interestingly, this 17 beta-HSD activity is highly predominant in unbroken cells in culture, thus supporting the physiological relevance of this "secondary" activity. Such 17 beta-HSD activity is inhibited by the classical substrates of 3 beta-HSD, namely pregnenolone (PREG), dehydroepiandrosterone (DHEA), delta 5-androstene-3 beta,17 beta-diol (delta 5-diol), 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol) and DHT, with IC50 values of 2.7, 1.0, 3.2, 6.2, and 6.3 microM, respectively. Although dual enzymatic activities have been previously reported for purified preparations of other steroidogenic enzymes, the present data demonstrate the multifunctional enzymatic activities associated with a recombinant oxidoreductase enzyme. In addition to its well known 3 beta-HSD activity, this enzyme possesses the ability to catalyze DHT into A-dione thus potentially controlling the level of the active androgen DHT in classical steroidogenic as well as peripheral intracrine tissues.     

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.     

The regulation and inhibition of 17beta-hydroxysteroid dehydrogenase in breast cancer

17Beta-hydroxysteroid dehydrogenase Type 1 (17beta-HSD1) has a pivotal role in regulating the synthesis of oestradiol (E2) within breast tumours. In whole body studies in postmenopausal women with breast cancer the conversion of oestrone (E1) to E2 (4.4+/-1.1%) was much lower than the inactivation of E2 to E1 (17.3+/-5.0%). In contrast, an examination of in vivo oestrogen metabolism within breast tumours revealed that whereas little metabolism of E2 occurred, E1 was converted to E2 to a much greater extent in malignant (48+/-14%) than in normal (19+/-6%) breast tissue. Findings from these studies originally suggested that oestrogen metabolism within breast tumours may differ from the mainly oxidative direction found in most other body tissues and that the activity of 17beta-HSD1 might be regulated by tumour-derived factors. Several growth factors (e.g. IGF-I, IGF-II) and cytokines (e.g. IL-6, TNFalpha) have now been identified which can markedly stimulate the activity of 17beta-HSD1 and such a mechanism may account for the high concentrations of E2 found in most breast tumours. Cells of the immune system, which can infiltrate breast tumours, are thought to be a major source of the growth factors and cytokines which can modulate 17beta-HSD1 activity. Given the central role that 17beta-HSD1 has in regulating breast tumour E2 concentrations the development of potent inhibitors of this enzyme has recently attracted considerable attention. Our initial studies in this area explored the use of derivatives of E1 as inhibitors, with 2-ethyl- and 2-methoxy E1 being found to inhibit 17beta-HSD1 activity in T-47D breast cancer cells by 96+/-2 and 91+/-1% respectively at 10 microM, but with a lack of specificity. Using the E1 scaffold a number of potent, selective 17beta-HSD1 inhibitors have now been identified including E1- and 2-ethyl-E1 containing a side chain with a m-pyridylmethylamidomethyl functionality extending from the 16beta position of the steroid nucleus. At 10 microM these compounds both inhibited 17beta-HSD1 activity by >90%, however some inhibition of 17beta-HSD2 activity was exhibited by the E1 derivative (25%) but not the 2-ethyl analogue. It is now apparent that 17beta-HSD1 activity contributes to the high E2 concentrations found in most breast tumours. The identification of potent, selective novel 17beta-HSD1 inhibitors will allow their efficacy to be tested in in vitro and in vivo studies.     

Clinical, endocrine, and molecular findings in 17beta-hydroxysteroid dehydrogenase type 3 deficiency

The 17beta-hydroxysteroid dehydrogenases (17betaHSD) gene family comprises different enzymes involved in the biosynthesis of active steroid hormones. The 17betaHSD type 3 (17betaHSD3) isoenzyme catalyzes the reductive conversion of the inactive C19-steroid, Delta4-androstenedione (Delta4- A), into the biologically active androgen, testosterone (T), in the Leydig cells of the testis. It is encoded by the 17beta-hydroxysteroid dehydrogenase type 3 (HSD17B3) gene, which maps to chromosome 9q22. Mutations in the HSD17B3 gene are associated with a rare form of 46,XY disorder of sex development referred to as 17betaHSD3 deficiency (or as 17-ketosteroid reductase deficiency), due to impaired testicular conversion of Delta4-A into T. 46,XY patients with 17betaHSD3 deficiency are usually classified as female at birth, raised as such, but develop secondary male features at puberty. Diagnosis, and consequently early treatment, is difficult because clinical signs from birth until puberty may be mild or absent. Biochemical diagnosis of 17betaHSD3 deficiency requires measurement of serum T/Delta4-A ratio after hCG stimulation test in pre-pubertal subjects, while baseline values seem to be informative in early infancy and adolescence. However, low basal T/Delta4-A ratio is not specific for 17betaHSD3 deficiency, being sometimes also found in patients with other defects in T synthesis or with Leydig cells hypoplasia. Mutational analysis of the 17HSDB3 gene is useful in confirming the clinical diagnosis of 17betaHSD3 deficiency. This review describes clinical findings, diagnosis, and molecular basis of this rare disease.     

Phytoestrogens inhibit human 17beta-hydroxysteroid dehydrogenase type 5

The 17beta-hydroxysteroid dehydrogenase type 5 (17beta-HSD 5) is involved in estrogen and androgen metabolism. In our study we tested the influence of environmental hormones, such as phytoestrogens (flavonoids, coumarins, coumestans), on reductive and oxidative 17beta-HSD activity of the human 17beta-hydroxysteroid dehydrogenase type 5 (17beta-HSD 5). These dietary substances were shown to be potent inhibitors of aromatase, different 17beta-HSDs and seem to play an important role in delay of development of hormone dependent cancers. Our studies show that reductive and oxidative activity of the enzyme are inhibited by many dietary compounds, especially zearalenone, coumestrol, quercetin and biochanin A. Among the group of flavones inhibitor potency is growing with increasing number of hydroxylations. We suggest that these substances are bound to the hydrophilic cofactor-binding pocket of the enzyme. An interesting inhibition pattern is observed for 18beta-glycyrrhetinic acid, which has no influence on the oxidative but only on the reductive reaction. This indicates that this substrate binds to pH- and cofactor-depending sites at the active center of the enzyme.     

The design of novel 17beta-hydroxysteroid dehydrogenase type 3 inhibitors

17beta-Hydroxysteroid dehydrogenase type 3 (17beta-HSD3) is expressed at high levels in the testes and seminal vesicles but has also been shown to be present in prostate tissue, suggesting its potential involvement in both gonadal and non-gonadal testosterone biosynthesis. The role of 17beta-HSD3 in testosterone biosynthesis makes this enzyme an attractive molecular target for small molecule inhibitors for the treatment of prostate cancer. Here we report the design of selective inhibitors of 17beta-HSD3 as potential anti-cancer agents. Due to 17beta-HSD3 being a membrane-bound protein a crystal structure is not yet available. A homology model of 17beta-HSD3 has been built to aid structure-based drug design. This model has been used with docking studies to identify a series of lead compounds that may give an insight as to how inhibitors interact with the active site. Compound 1 was identified as a potent selective inhibitor of 17beta-HSD3 with an IC(50)=700nM resulting in the discovery of a novel lead series for further optimisation. Using our homology model as a tool for inhibitor design compound 5 was discovered as a novel potent and selective inhibitor of 17beta-HSD3 with an IC(50) approximately 200nM.     

New inhibitors of 17beta-hydroxysteroid dehydrogenase type 1

The estradiol-synthesizing enzyme 17beta-hydroxysteroid dehydrogenase type 1 (17betaHSD1) is mainly responsible for the conversion of estrone (E1) to the potent estrogen estradiol (E2). It is a key player to control tissue levels of E2 and is therefore an attractive target in estradiol-dependent diseases like breast cancer or endometriosis. We selected a unique non-steroidal pyrimidinone core to start a lead optimization program. We optimized this core by modulation of R1-R6. Its binding mode at the substrate-binding site of 17betaHSD1 is complex and difficult to predict. Nevertheless, some basic structure-activity relationships could be identified. In vitro, the most active pyrimidinone derivative showed effective inhibition of recombinant human 17betaHSD1 at nanomolar concentrations. In intact cells overexpressing the human enzyme, IC50 values in the lower micromolar range were determined. Furthermore, the pyrimidinone proved its use in vivo by significantly reducing 17betaHSD1-dependent tumor growth in a new nude mouse model.     

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.     

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.     

Novel, potent inhibitors of 17beta-hydroxysteroid dehydrogenase type 1

Many breast tumours are hormone-responsive and rely on estrogens for their sustained growth and development. The enzyme 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) is primarily responsible for the conversion of estrone (E1) into the most potent of the human estrogens 17beta-estradiol (E2). Here we report the syntheses, inhibitory activities and docking studies for a novel series of pyrazole amides which have been discovered with the aim of probing the structure activity relationships (SAR) for such a template and of using this template to mimic the potent inhibitor 1 (Fig. 1). Amides containing an aromatic pyridyl moiety have been found to give the best inhibition, indicating that the pyridyl group interacts beneficially in the active site. This work has shown that extension from this position on the pyrazole template is well tolerated and the optimization of such systems is under investigation.     

17 beta-hydroxysteroid dehydrogenase type XI localizes to human steroidogenic cells

We searched expressed sequence tag databases with conserved domains of the short-chain alcohol dehydrogenase superfamily and identified another isoform of 17 beta-hydroxysteroid dehydrogenase, 17 beta HSDXI. This enzyme converts 5 alpha-androstane-3 alpha, 17 beta-diol to androsterone. The substrate has been implicated in supporting gestation and modulating gamma-aminobutyric acid receptor activity. 17 beta HSDXI is colinear with human retinal short-chain dehydrogenase/reductase retSDR2, a protein with no known biological activity (accession no. AAF06939). Of the proteins with known function, 17 beta HSDXI is most closely related to the retinol-metabolizing enzyme retSDR1, with which it has 30% identity. There is a polymorphic stretch of 15 adenosines in the 5' untranslated region of the cDNA sequence and a silent polymorphism at C719T. A 17 beta HSDXI construct with a stretch of 20 adenosines was found to produce significantly more enzyme activity than constructs containing 15 or less adenosines (43% vs. 26%, P < 0.005). The C719T polymorphism is present in 15% of genomic DNA samples. Northern blot analysis showed high levels of 17 beta HSDXI expression in the pancreas, kidney, liver, lung, adrenal, ovary, and heart. Immunohistochemical staining for 17 beta HSDXI is strong in steroidogenic cells such as syncytiotrophoblasts, sebaceous gland, Leydig cells, and granulosa cells of the dominant follicle and corpus luteum. In the adrenal 17 beta HSDXI, staining colocalized with the distribution of 17 alpha-hydroxylase but was stronger in the mid to outer cortex. 17 beta HSDXI was also found in the fetus and increased after birth. Liver parenchymal cells and epithelium of the endometrium and small intestine also stained. Regulation studies in mouse Y1 cells showed that cAMP down-regulates 17 beta HSDXI enzymatic activity (40% vs. 32%, P < 0.05) and reduces gene expression to undetectable levels. All-trans-retinoic acid did not affect 17 beta HSDXI expression or activity, but addition of the retinoid together with cAMP significantly decreased activity over cAMP alone (32% vs. 23%, P < 0.05). Cloning and sequencing of the 17 beta HSDXI promoter identified the potential nuclear receptor steroidogenic factor-1 half-site TCCAAGGCCGG, and a cluster of three other potential steroidogenic factor-1 half-sites were found in the distal part of intron 1. Collectively, these results suggest a role for 17 beta HSDXI in androgen metabolism during steroidogenesis and a possible role in nonsteroidogenic tissues including paracrine modulation of 5 alpha-androstane-3 alpha, 17 beta-diol levels. 17 beta HSDXI could act by metabolizing compounds that stimulate steroid synthesis and/or by generating metabolites that inhibit it.     

Interspecies comparison of gene structure and computational analysis of gene regulation of 17beta-hydroxysteroid dehydrogenase type 1

17Beta-hydroxysteroid dehydrogenase type 1 (HSD17B1) is a key enzyme of 17beta-estradiol biosynthesis, and in rodents is additionally involved in testosterone biosynthesis. The human HSD17B1 gene, located on chromosome 17q12-21, is duplicated in tandem, with the 3'-copy being the functional gene. Here we show by sequencing the gene from a diverse set of related species that this duplication is of very recent evolutionary origin, having occurred in the common ancestor of Hominoidae (apes and humans) while being absent in the closely related Old World monkeys (Macaca) and the outgroup species Tupaia belangeri and Mus musculus. By computational analysis of the conserved regulatory elements in the 5'-untranslated (5'-UTR) and putative promoter region of the HSD17B1 gene and, where present, pseudogene, across our broad sample of species we can show significant differences that might point to the origin of the divergent substrate specificity of human and rodent HSD17B1 and highlight potential functionally relevant differences in regulatory patterns in different evolutionary lineages.     

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.     

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.