Intracrinology: role of the family of 17 beta-hydroxysteroid dehydrogenases in human physiology and disease

In women and men, an important proportion of estrogens and androgens are synthesized locally at their site of action in peripheral target tissues. This new field of endocrinology has been called intracrinology. In postmenopausal women, 100% of active sex steroids are synthesized in peripheral target tissues from inactive steroid precursors while, in adult men, approximately 50% of androgens are made locally in intracrine target tissues. The last and key step in the formation of all estrogens and androgens is catalyzed by members of the family of 17beta-hydroxysteroid dehydrogenases (17 beta-HSDs) while different 17 beta-HSDs inactivate these steroids in the same cell where synthesis takes place. To date, seven human 17 beta-HSDs have been cloned, sequenced and characterized. The 17 beta-HSDs provide each cell with the means of precisely controlling the intracellular concentration of each sex steroid according to local needs.     

Evolution of 17beta-hydroxysteroid dehydrogenases and their role in androgen, estrogen and retinoid action

17beta-Hydroxysteroid dehydrogenases (17beta-HSDs) regulate androgen and estrogen concentrations in mammals. By 1995, four distinct enzymes with 17beta-HSD activity had been identified--17beta-HSD-types 1 and 3, which, in vivo, are NADPH-dependent reductases; and 17beta-HSD-types 2 and 4, which, in vivo, are NAD(+)-dependent oxidases. Since then, six additional enzymes with 17beta-HSD activity have been isolated from mammals. With the exception of 17beta-HSD-type 5, which belongs to the aldoketo-reductase (AKR) family, these 17beta-HSDs belong to the short chain dehydrogenase/reductase (SDR) family. Several 17beta-HSDs appear to be examples of convergent evolution. That is, 17beta-HSD activity arose several times from different ancestors. Some 17beta-HSDs share a common ancestor with retinoid oxido-reductases and have retinol dehydrogenase activity. 17beta-HSD-types 2, 6 and 9 appear to have diverged from ancestral retinoid dehydrogenases early in the evolution of deuterostomes during the Cambrian, about 540 million years ago. This coincided with the origin of nuclear receptors for androgens and estrogens suggesting that expression of 17beta-HSDs had an important role in the early evolution of the physiological response to androgens and estrogens.     

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.     

Design and validation of specific inhibitors of 17beta-hydroxysteroid dehydrogenases for therapeutic application in breast and prostate cancer, and in endometriosis

17beta-Hydroxysteroid dehydrogenases (17beta-HSDs) are enzymes that are responsible for reduction or oxidation of hormones, fatty acids and bile acids in vivo, regulating the amount of the active form that is available to bind to its cognate receptor. All require NAD(P)(H) for activity. Fifteen 17beta-HSDs have been identified to date, and with one exception, 17beta-HSD type 5 (17beta-HSD5), an aldo-keto reductase, they are all short-chain dehydrogenases/reductases, although overall homology between the enzymes is low. Although named as 17beta-HSDs, reflecting the major redox activity at the 17beta-position of the steroid, the activities of these 15 enzymes vary, with several of the 17beta-HSDs able to reduce and/or oxidise multiple substrates at various positions. These activities are involved in the progression of a number of diseases, including those related to steroid metabolism. Despite the success of inhibitors of steroidogenic enzymes in the clinic, such as those of aromatase and steroid sulphatase, the development of inhibitors of 17beta-HSDs is at a relatively early stage, as at present none have yet reached clinical trials. However, many groups are now working on inhibitors specific for several of these enzymes for the treatment of steroid-dependent diseases, including breast and prostate cancer, and endometriosis, with demonstrable efficacy in in vivo disease models. In this review, the recent advances in the validation of these enzymes as targets for the treatment of these diseases, with emphasis on 17beta-HSD1, 3 and 5, the development of specific inhibitors, the models used for their evaluation, and their progress towards the clinic will be discussed.     

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.     

Cloning, expression and characterization of three types of 17beta-hydroxysteroid dehydrogenases from the Nile tilapia, Oreochromis niloticus

In order to elucidate the roles of 17beta-HSDs in fish gonadal steroidogenesis, three types of 17beta-HSDs (17beta-HSD1, 17beta-HSD8 and putative 17beta-HSD12) were cloned and characterized from the Nile tilapia, Oreochromis niloticus. The cloned cDNAs of 17beta-HSD type 1, 8 and 12 were 1504, 1006 and 1930 bp long, with open reading frames encoding proteins of 289, 256 and 314 aminoacids, respectively. Tissue distribution pattern analyzed by RT-PCR and Northern blot showed that 17beta-HSD1 was dominantly expressed in the ovary, while the putative 17beta-HSD12, one of the two duplicates found in fish, is a male specific enzyme and expressed exclusively in testis (detected by RT-PCR only). On the other hand, 17beta-HSD8 was expressed in the brain, gill, heart, liver, intestine, gonad, kidney and muscle of both male and female. Enzymatic assays of the three types of 17beta-HSDs were performed using recombinant proteins expressed in E. coli or HEK 293 cells. Tilapia 17beta-HSD1 expressed in E. coli had the preference for NADP(H) as cofactor and could catalyze the inter-conversion between estrone and estradiol efficiently as well as the inter-conversion between androstenedione and testosterone, but less efficiently. Tilapia 17beta-HSD8 recombinant protein expressed in HEK 293 cells could catalyze the conversion of testosterone to androstenedione, as well as the inter-conversion between estrone and estradiol. However, the putative 17beta-HSD12 expressed in E. coli or in HEK 293 cells showed no conversion to any of the four substrates tested in this study. Based on enzyme characterization and tissue distribution, it is plausible to attribute crucial roles to 17beta-HSDs in the gonadal steroidogenesis of teleosts.     

17Beta-HSD type 5 expression and the emergence of differentiated epithelial Type II cells in fetal lung: a novel role for androgen during the surge of surfactant

Lung maturation is delayed in male fetuses compared to female fetuses. This has been attributed to higher levels of androgens in the male lung. We previously showed that the genes encoding for the 17beta-hydroxysteroid dehydrogenase (HSD) type 5 (conversion of androstenedione in testosterone) and type 2 (the opposite reaction) are, respectively, expressed in the human epithelial Type II (PTII)-like A549 cells and in human lung fibroblasts. Here, we aim to explain the physiological relevance of androgen synthesis by PTII cells. We showed that 17beta-HSD type 2 and type 5 genes are both up-regulated in correlation with the emergence of mature PTII cells in both male and female developing lungs of the fetal mouse. In contrast, the androgen receptor gene is expressed at similar levels in both sexes with no temporal regulation. In conclusion, the expression profile of the 17beta-HSD type 5 gene does not explain the presence of higher levels of androgens in the male fetal lung but that androgen synthesis must be a normal characteristic of mature PTII cells for both sexes. The production of androgens after the emergence of mature PTII cells should negatively regulate PTII cell maturation and thus, a novel and normal role for androgens in cell reprogramming is proposed.     

Structure and function of human 17beta-hydroxysteroid dehydrogenases

17Beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyze the NAD(P)(H) dependent oxidoreduction at C17 oxo/beta-hydroxyl groups of androgen and estrogen hormones. This reversible reaction constitutes an important pre-receptor control mechanism for nuclear receptor ligands, since the conversion "switches" between the 17beta-OH receptor ligands and their inactive 17-oxo metabolites. At present, 14 mammalian 17beta-HSDs are described, of which at least 11 exist within the human genome, encoded by different genes. The enzymes differ in their expression pattern, nucleotide cofactor preference, steroid substrate specificity and subcellular localization, and thus constitute a complex system ensuring cell-specific adaptation and regulation of sex steroid hormone levels. Broad and overlapping substrate specificities with enzymes involved in lipid metabolism suggest interactions of several 17beta-HSDs with other metabolic pathways. Several 17beta-HSDs enzymes constitute promising drug targets, of particular importance in cancer, metabolic diseases, neurodegeneration and possibly immunity.     

A link between lung androgen metabolism and the emergence of mature epithelial type II cells

Lung maturation is delayed in male fetuses compared with female fetuses, which has been attributed to higher levels of androgens in the male lung. Our previous studies demonstrated that the genes encoding for the 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) type 5 (androstenedione --> testosterone) and type 2 (the opposite reaction) are expressed in human epithelial type II (PTII)-like A549 cells and in human lung fibroblasts, respectively. Here, we aim to explain the physiological relevance of androgen synthesis by PTII cells. We showed that both 17 beta-HSD type 2 and type 5 genes are upregulated in correlation with the emergence of mature PTII cells in both male and female developing lungs of the mouse. In contrast, the androgen receptor gene is expressed equally in both sexes with no temporal regulation. We conclude that the expression profile of the 17 beta-HSD type 5 gene does not explain the presence of higher levels of androgen in the male fetal lung, but that androgen synthesis must be a normal feature of mature PTII cells for both sexes. The production of androgens after the emergence of mature PTII cells should negatively regulate PTII cell maturation and, thus, a role for androgens in cell reprogramming is suggested.     

Characterization of a human 20alpha-hydroxysteroid dehydrogenase

It has been suggested that 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) is a T-cell differentiation marker in mice. In the human, this enzyme has generally been associated with types 1 and 2 17beta-HSDs, which belong to the short-chain alcohol dehydrogenase family, whereas the rat, rabbit, pig and bovine 20alpha-HSDs are members of the aldoketo reductase superfamily, which also includes the 3alpha-HSD family. In this study, we report the cloning, from a human skin cDNA library, of a cDNA that shows, after transfection into human embryonic kidney (HEK-293) cells, high 20alpha-HSD activity but negligible 3alpha- and 17beta-hydroxysteroid dehydrogenase activities. A comparison of the amino acid sequence of the human 20alpha-HSD with those of other related 20alpha- and 3alpha-HSDs indicates that the human 20alpha-HSD shares 79.9, 68.7 and 52.3% identity with rabbit, rat and bovine 20alpha-HSDs, whereas it shows 97, 84 and 65% identity with human type 3, type 1 and rat 3alpha-HSDs. In contrast, the enzyme shares only 15.2 and 15.0% identity with type 1 and type 2 human 17beta-HSDs. DNA analysis predicts a protein of 323 amino acids, with a calculated molecular weight of 36 767 Da. In intact transfected cells, the human 20alpha-HSD preferentially catalyzes the reduction of progesterone to 20alpha-hydroxyprogesterone with a K(m) value of 0.6 microM, the reverse reaction (oxidation) being negligible. In a cell cytosolic preparation, the enzyme could use both NADPH and NADH as cofactors, but NADPH, which gave 4-fold lower K(m) values, was preferred. We detected the expression of 20alpha-HSD mRNA in liver, prostate, testis, adrenal, brain, uterus and mammary-gland tissues and in human keratinocyte (HaCaT) cells. The present study clearly indicates that the genuine human 20alpha-HSD belongs to the aldoketo reductase family, like the 20alpha-HSDs from other species.     

Inhibitory effects of fluorine-substituted estrogens on the activity of 17beta-hydroxysteroid dehydrogenases

In search for new inhibitors of human 17beta-hydroxysteroid dehydrogenase type 1 (h17beta-HSD1) a specific group of steroids with interesting properties including novel compounds was investigated. Several estratriene derivatives with fluorine-substitution in position 17 of the steroidal scaffold were synthesised and tested in vitro towards recombinant h17beta-HSD1, 2, 4, 5 and 7. Moderate, mostly unselective inhibitors of h17beta-HSD1 and h17beta-HSD2 and a selective inhibitor of h17beta-HSD5 were identified. The structure-activity relationship with respect to inhibitory strengths and selectivity of these compounds on five h17beta-HSDs is discussed.     

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.     

Isoform-specific regulation of uridine diphosphate-glucuronosyltransferase 2B enzymes in the human prostate: differential consequences for androgen and bioactive lipid inactivation

Androgens as well as monohydroxy-fatty acids are implicated in the pathogenesis of prostate cancer. Like a huge variety of endo- and xenobiotics, they are eliminated as glucuronide conjugates formed by uridine diphosphate-glucuronosyltransferase (UGT) enzymes. In the present study, we observe that treatment of the prostate cancer cells LNCaP with natural and synthetic androgens, IL-1alpha, or epidermal growth factor (EGF) differently modulates the glucuronidation of androgen and bioactive lipid metabolites. Indeed, glucuronidation of 5alpha-androstane-3alpha,17beta-diol and 13-hydroxyoctadecadienoic acid was drastically reduced, whereas 12-hydroxyeicosatetraenoic acid conjugation by UGT was increased after androgen treatment. These effects reflected the reduction of UGT2B10, -B15, and -B17 enzyme expression, and the activation of the UGT2B11 gene. In human prostate epithelial cells, only UGT2B11 and -B15 mRNAs are detected and are regulated by androgens in a similar manner as in LNCaP cells. In LNCaP cells, IL-1alpha and EGF also regulate UGT2B expression in an isoform-specific manner; IL-1alpha induced UGT2B10 and reduced UGT2B17, while having no effects on UGT2B11 mRNA levels. EGF treatment resulted in a decreased UGT2B17 expression, whereas UGT2B10 and -B11 mRNA remained at their basal levels. Overall, these results demonstrate that in the human prostate, androgens do not only affect their own inactivation but also influence the levels of monohydroxy-fatty acids by regulating the expression of UGT2B enzymes in an isoform-specific manner. These differential effects of androgens, IL-1alpha, and EGF on lipid metabolism likely constitute an additional mechanism by which these endogenous factors promote prostate cancer development.     

Androgen inactivation in human lung fibroblasts: variations in levels of 17 beta-hydroxysteroid dehydrogenase type 2 and 5 alpha-reductase activity compatible with androgen inactivation

Androgens delay lung maturation through their action on lung fibroblasts. Knowing that testosterone is secreted by the lung epithelial-like cell line A-549, we have studied the metabolism of androgens by several human lung diploid fibroblasts cell lines. No 17-ketosteroid reductase activity was detected. In contrast, testosterone was transformed mainly into androstenedione and androstanedione with no 5 alpha-dihydrotestosterone formed, indicating the presence of 17 beta- hydroxysteroid dehydrogenase (HSD) type 2 and 5 alpha-reductase activities. The eight cell lines analyzed had either a low or high 17 beta-HSD type 2 activity level. No correlation between these levels and the sex or age stage of cells was established, but Northern blot analysis of human lung RNA samples of five adult subjects revealed very similar variations between subjects in the level of 17 beta-HSD type 2 mRNA. The 5 alpha-reductase activity had a marked substrate preference for androstenedione, the product of 17 beta-HSD type 2. When tritiated testosterone was used as substrate, only barely detectable levels of 5 alpha-dihydrotestosterone were observed by HPLC in the presence of the 17 beta-HSD type 2 inhibitor EM-919. The use of unlabeled testosterone or of the antiandrogen hydroxyflutamide demonstrated that the tritiated testosterone substrate itself had no effect on levels of 5 alpha-reduction. In fact, in these cells, 5 alpha-reductase has no significant activity on testosterone, but it further converts the product of 17 beta-HSD type 2, thus playing a role with 17 beta-HSD type 2 in androgen inactivation. Because androgens delay lung maturation and surfactant synthesis by their action on lung fibroblasts, it is of particular interest to find that the steroid metabolism of these lung fibroblast cells is oriented toward androgen inactivation. Because lung fibroblasts of subjects with low 17 beta-HSD type 2 expression levels are likely to be exposed to higher levels of androgens, an allelic variation of the 17 beta-HSD-2 gene is suspected, which would result in familial incidence of respiratory distress. This is in line with reported cases of familial incidence of respiratory distress.     

Multifunctionality of human 17beta-hydroxysteroid dehydrogenases

17Beta-hydroxysteroid dehydrogenases (17beta-HSDs) belong to the family of short chain dehydrogenases/reductases (SDRs) and aldoketo-reductases (AKRs). Some of the enzymes were discovered and named due to their enzymatic activity on steroid substrates or according to their sequence homology to other 17beta-HSDs. During characterisation of these enzymes it turned out that their substrate specificity is broader than first expected and key functions of some 17beta-HSDs in vivo are probably not in steroid metabolism but in basic metabolic pathways. The issue of such multifunctionality is the topic of this review.     

In situ androgen producing enzymes in human prostate cancer

Androgens have been proposed to be actively produced in situ in human prostate cancer. These locally produced androgens have also been considered to play important roles in the pathogenesis and development of prostate cancer. Therefore, it is important to examine the status of this in situ androgen metabolism and/or synthesis in detail in order to improve the clinical response to hormonal therapy in patients diagnosed with prostate cancer. Several studies have previously demonstrated the expression of androgen-producing enzymes such as 5alpha-reductase types 1 and 2, and 17beta-hydroxysteroid dehydrogenase type 5 (17beta-HSD5), in human prostate carcinoma cells. However, their biological significance has remained largely unknown. In this study, we evaluated the immunoreactivities of these steroidogenic enzymes in human prostate cancer obtained from surgery (n = 70), and correlated the findings with clinicopathological features of the patients. 17Beta-HSD5 immunoreactivity was detected in 54 cases (77%), 5alpha-reductase type 1 in 51 cases (73%) and 5alpha-reductase type 2 in 39 cases (56%). 5Alpha-reductase type 2 immunoreactivity was significantly correlated with that of androgen receptor (AR), and 17beta-HSD5 positive cases were significantly associated with clinical stage (TNM stage pT3 vs pT2). These data all suggest that androgen-producing enzymes, such as 5alpha-reductase type 1 and type 2, and 17beta-HSD5 are expressed in a majority of prostate cancers, and are involved in the local production and actions of androgens in prostate cancers.     

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 primate adrenal zona reticularis is defined by expression of cytochrome b5, 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17) and NADPH-cytochrome P450 reductase (reductase) but not 3beta-hydroxysteroid dehydrogenase/delta5-4 isomerase (3beta-HSD).

Biochemical studies suggest that 17,20-lyase activity, and thus efficient synthesis of androgens by human P450c17, requires both reductase and the accessory protein cytochrome b5. Since the human and primate zona reticularis (ZR) secrete androgens, the expression of these proteins, and of 3beta-HSD, was investigated by immunocytochemistry in the adrenal cortex of the mature rhesus macaque. Cytochrome b5 expression was restricted to the cells of the ZR which appeared deficient in 3beta-HSD. However, both P450c17 and reductase were evident throughout the zona fasciculata. These data provide essential evidence in support of a functional role for cytochrome b5 in the regional control of 17alpha-hydroxylase and 17,20-lyase activities of P450c17 and thereby adrenal C19 steroid secretion by the primate adrenal gland.