Analysis and characteristics of multiple types of human 17beta-hydroxysteroid dehydrogenase

Androgens and estrogens are not only synthesized in the gonads but also in peripheral target tissues. Accordingly, recent molecular cloning has allowed us to identify multiple types of 17β-hydroxysteroid dehydrogenases (17β-HSD), the key and exclusive enzymes involved in the formation and inactivation of sex steroids. However, only one form, namely, type 3 17β-HSD, is responsible for pseudohermaphroditism in deficient boys. To date, seven human 17β-HSDs have been isolated and characterized. Although they catalyze substrates having a similar structure, 17β-HSDs have very low homology. In intact cells in culture, these enzymes catalyze the reaction in a unidirectional way — types 1, 3, 5 and 7 catalyze the reductive reaction, while types 2, 4 and 8 catalyze the oxidative reaction. It is noteworthy that rat type 6 17β-HSD also catalyzes the reaction in the oxidative direction. In this report, we analyze the different characteristics of the multiple types of human 17β-HSD.     

Intrinsic sterol- and phosphatidylcholine transfer activities of 17β-hydroxysteroid dehydrogenase type IV

Previous studies have shown that the 80 kDa 17β-hydroxysteroid dehydrogenase (17β-HSD) type IV comprises distinct domains, including an N-terminal region related to the short chain alcohol dehydrogenase multigene family and a C-terminal part related to the lipid transfer protein sterol carrier protein 2 (SCP2). In this study, we have investigated whether the SCP2-related part of the 80 kDa protein leads to an intrinsic sterol and phospholipid transfer activity, as shown earlier for the 60 kDa SCP2-related peroxisomal 3-ketoacyl CoA thiolase with intrinsic sterol and phospholipid transfer activity called sterol carrier protein x (SCPx). Our results indicate that a fraction rich in the 80 kDa form of 17β-HSD type IV exhibits high transfer activities for 7-dehydrocholesterol and phosphatidylcholine. In addition, a purified recombinant peptide derived from the SCP2-related domain of the 17β-HSD type IV has about 30% of the transfer activities for 7-dehydrocholesterol and phosphatidylcholine seen with purified recombinant human SCP2. We conclude that the 80 kDa type IV 17β-HSD represents a potentially multifunctional protein with intrinsic in vitro sterol and phospholipid transfer activity in addition to its enzymatic activity.     

Expression and characterization of isoforms of 3β-hydroxysteroid dehydrogenase/Δ-isomerase in the hamster

The enzyme 3β-hydroxysteroid dehydrogenase/Δ^5→4-isomerase (3β-HSD) is essential for the production of all classes of steroid hormones. Multiple isozymes of this enzyme have been demonstrated in the kidney and liver of both the rat and the mouse, although the function of the enzyme in these tissues is unknown. We have characterized three isozymes of 3β-HSD expressed in various tissues of the hamster. Both western and northern blot analyses demonstrated very high levels of 3β-HSD in the adrenal, kidney and male liver. Conversely, there were extremely low levels of enzyme expression in the female liver. cDNA libraries prepared from RNA isolated from hamster adrenal, kidney and liver were screened with a full-length cDNA encoding human type 1 3β-HSD. Separate cDNAs encoding three isoforms of 3β-HSD were isolated from these libraries. To examine the properties of the isoforms, the cDNAs were ligated into expression vectors for over-expression in 293 human fetal kidney cells. The type 1 isoform, isolated from an adrenal cDNA library, was identified as a high-affinity 3β-hydroxysteroid dehydrogenase. A separate isoform, designated type 2, was isolated from the kidney, and this was also a high-affinity dehydrogenase/isomerase. Two cDNAs were isolated from the liver, one identical in sequence to type 2 of the kidney, and a distinct cDNA encoding an isoform designated type 3. The type 3 3β-HSD possessed no steroid dehydrogenase activity but was found to function as a 3-ketosteroid reductase. Thus male hamster liver expresses a high-affinity 3β-HSD (type 2) and a 3-ketosteroid reductase (type 3), whereas the kidney of both sexes express the type 2 3β-HSD isoform. These differ from the type 1 3β-HSD expressed in the adrenal cortex.     

Structure-function relationships and molecular genetics of the 3β-hydroxysteroid dehydrogenase gene family

The isoenzymes of the 3β-hydroxysteroid dehydrogenase/5-ene-4-ene-isomerase (3β-HSD) gene family catalyse the transformation of all 5-ene-3β-hydroxysteroids into the corresponding 4-ene-3-keto-steroids and are responsible for the interconversion of 3β-hydroxy- and 3-keto-5-androstane steroids. The two human 3β-HSD genes and the three related pseudogenes are located on the chromosome 1p13.1 region, close to the centromeric marker D1Z5. The 3β-HSD isoenzymes prefer NAD⁺ to NADP⁺ as cofactor with the exception of the rat liver type III and mouse kidney type IV, which both prefer NADPH as cofactor for their specific 3-ketosteroid reductase activity due to the presence of Tyr³⁶ in the rat type III and of Phe³⁶ in mouse type IV enzymes instead of Asp³⁶ found in other 3β-HSD isoenzymes. The rat types I and IV, bovine and guinea pig 3β-HSD proteins possess an intrinsic 17β-HSD activity psecific to 5-androstane 17β-ol steroids, thus suggesting that such “secondary” activity is specifically responsible for controlling the bioavailability of the active androgen DHT. To elucidate the molecular basis of classical form of 3β-HSD deficiency, the structures of the types I and II 3β-HSD genes in 12 male pseudohermaphrodite 3β-HSD deficient patients as well as in four female patients were analyzed. The 14 different point mutations characterized were all detected in the type II 3β-HSD gene, which is the gene predominantly expressed in the adrenals and gonads, while no mutation was detected in the type I 3β-HSD gene predominantly expressed in the placenta and peripheral tissues. The mutant type II 3β-HSD enzymes carrying mutations detected in patients affected by the salt-losing form exhibit no detectable activity in intact transfected cells, at the exception of L108W and P186L proteins, which have some residual activity (1%). Mutations found in nonsalt-loser patients have some residual activity ranging from 1 to 10% compared to the wild-type enzyme. Characterization of mutant proteins provides unique information on the structure-function relationships of the 3β-HSD superfamily.     

Kinetic analysis of enzymic activities: Prediction of multiple forms of 17β-hydroxysteroid dehydrogenase

An overview of the application of kinetic methods to the delineation of 17β-hydroxysteroid dehydrogenase (17β-HSD) heterogeneity in mammalian tissues is presented. Early studies of 17β-HSD activity in animal liver and kidney subcellular fractions were suggestive of multiple forms of the enzyme. Subsequently, detailed characterization of activity in cytosol and subcellular membrane fractions of human placenta, with particular emphasis on inhibition kinetics, yielded evidence of two kinetically-differing forms of 17β-HSD in that organ. Gene cloning and transfection experiments have confirmed the identity of these two proteins as products of separate genes. 17β-HSD type 1 is a cytosolic enzyme highly specific for C₁₈ steroids such as 17β-estradiol (E₂) and estrone (E₁). 17β-HSD type 2 is a membrane bound enzyme reactive with testosterone (T) and androstenedione (A), as well as E₂ and E₁. Useful parameters for the detection of multiple forms of 17β-HSD appear to be the E₂/T activity ratio, NAD/NADP activity ratios, steroid inhibitor specificity and inhibition patterns over a wide range of putative inhibitor concentrations. Evaluation of these parameters for microsomes from samples of human breast tissue suggests the presence of 17β-HSD type 2. The 17β-HSD enzymology of human testis microsomes appears to differ from placenta. Analysis of human ovary indicates granulosa cells are particularly enriched in the type 1 enzyme with type 2-like activity in stroma/theca. Mouse ovary appears to contain forms of 17β-HSD which differ from 17β-HSD type 1 and type 2 in their kinetic properties.     

Expression of human 17β-hydroxysteroid dehydrogenase in mammalian cells

The insert of 1278 bp containing the entire coding region of cDNA encoding human 17β-hydroxysteroid dehydrogenase (17β-HSD) was inserted into a pHS1 vector and expressed in HeLA human cervical carcinoma cells and COS-1 monkey kidney tumor cells. Western blot analysis indicated that the expressed protein migrates at the same position as the purified enzyme and is recognized by the antibody raised against purified human placental 17β-HSD. The expressed enzyme efficiently catalyzes the interconversion of estrone and estradiol while dehydroepiandrosterone and 5-androstene-3β,17β-diol are interconverted at a lower rate. The present data suggest the existence of two 17β-HSDs.     

Human 17β-hydroxysteroid dehydrogenase: overproduction using a baculovirus expression system and characterization

Estrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD) plays a pivotal role in the synthesis of estrogens. We overproduced human placental estrogenic 17β-HSD using a baculovirus expression system for the study of the enzyme mechanism. A cDNA encoding the entire open reading frame of human 17β-HSD was inserted into the genome of Autographa californica nuclear polyhedrosis virus and expressed in Spodoptera frugiperda (Sf9) insect cells. Metabolic labeling and Western blot analysis using polyclonal antibodies raised against native human 17β-HSD indicated that a molecule with an apparent mass of 35 kDa was maximally expressed 60 h after infection. At that time interval, intracellular 17β-HSD activity reached 0.26 U/mg of protein in crude homogenate, about 70 times the level measured in human placenta. Purification of recombinant 17β-HSD was achieved by a single affinity fast liquid protein chromatography step yielding 24 mg of purified 17β-HSD protein per liter of suspension culture, with a specific activity of about 8 μmol/min/mg of protein for conversion of estradiol into estrone, at pH 9.2. In addition, the recombinant protein purified from infected Sf9 cells was assembled as a dimer with molecular mass and specific activity identical to those of the enzyme purified directly from placenta. The present data show that the baculovirus expression system can provide active 17β-HSD that is functionally identical to its natural counterpart and easy to purify in quantities suitable for its physico-chemical studies.     

The molecular biology of androgenic 17β-hydroxysteroid dehydrogenases

The enzyme 17β-hydroxysteroid dehydrogenase (17β-HSD) catalyzes the 17β-oxidation/reduction of C₁₈- and C₁₉-steroids in a variety of tissues. Three human genes encoding isozymes of 17β-HSD, designated 17β-HSD types 1, 2 and 3 have been cloned. 17β-HSD type 1 (also referred to as estradiol 17β-dehydrogenase) catalyzes the conversion of estrone to estradiol, primarily in the ovary and placenta. The 17β-HSD type 2 is expressed to high levels in the liver, secretory endometrium and placenta. The type 2 isozyme catalyzes the oxidation of androgens and estrogens equally efficiently. Also, the enzyme possesses 20-HSD activity demonstrated by its ability to convert 20-dihydro-progesterone to progesterone. Testicular 17β-HSD type 3 catalyzes the conversion of androstenedione to testosterone, dehydroepiandrosterone to 5-androstenediol and estrone to estradiol. The 17β-HSD3 gene is mutated in male pseudohermaphrodites with the genetic disease 17β-HSD deficiency.     

The human 11β-hydroxysteroid dehydrogenase type II enzyme: Comparisons with other species and localization to the distal nephron

Effective glucocorticoid inactivation is currently thought to be an indispensable feature of mineralocorticoid target cells. The enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD) inactivates glucocorticoids and prevents them from binding to the non-selective mineralocorticoid receptor. In the kidney it is the NAD dependent high affinity isoform (11β-HSD2) which is thought to endow specificity on the receptor. The recet cloning of the human, sheep and rabbit 11β-HSD2 enzymes permits a comparison of the enzyme from the three species. Human and rabbit enzymes are 87% identical and of similar length, while the human and sheep enzymes have only 75% identity. The last 12 residues in all three species were found to be highly divergent, but most of the ovine dishomology can be accounted for by the deletion of a single nucleotide toward the C-terminus of the protein resulting in a shift in reading frame generating a protein 27 residues longer than the human isoform. Numerous other deletions were also observed in this region of the sheep cDNA sequence. Furthermore, the rabbit cDNA also displayed a large degree of dishomology with the human sequence a short distance downstream from the termination codon. Conserved overlapping cytoplasmic translocation signals were observed in all three species, suggesting a topology whereby the enzyme is anchored into the endoplasmic reticulum by multiple hydrophobic regions in the N-terminus and the bulk of the 11β-HSD2 peptide is sited in the cytoplasm. A polyclonal antibody generated against the C-terminus of human 11β-HSD2 was used to localize the enzyme within the kidney. A high level of immunoreactivity was observed in distal tubules and collecting ducts, localizing the enzyme to the same part of the nephron as the mineralocorticoid receptor. Moderate levels of staining were also seen in vascular smooth muscle cells. These results support the notion that 11β-HSD2 is an autocrine protector of the mineralocorticoid receptor and that it plays an important role in cardiovascular homeostatic mechanisms.     

Placental 11β-hydroxysteroid dehydrogenase and the programming of hypertension

Excessive foetal exposure to glucocorticoids retards growth and “programmes” adult hypertension in rats. Placental 11β-hydroxysteroid dehydrogenase (11β-HSD), which catalyses the conversion of corticosterone and cortisol to inert 11 keto-products, normally protects the foetus from excess maternal glucocorticoids. In both rats and humans there is considerable natural variation in placental 11β-HSD, and enzyme activity correlates with birth weight. Moreover, inhibition of placental 11β-HSD in the rat reduces birth weight and produces hypertensive adult offspring, many months after prenatal treatment with enzyme inhibitors; these effects are dependent upon maternal adrenal products. These data suggest that placental 11β-HSD, by regulating foetal exposure to maternal glucocorticoids, crucially determines foeto-placental growth and the programming of hypertension. Maternal protein restriction during pregnancy also produces hypertensive offspring and selectively attenuates placental 11β-HSD activity. Thus, deficiency of the placental barrier to maternal glucocorticoids may represent a common pathway between the maternal environment and foeto-placental programming of later disease. These data may, at least in part, explain the human epidemiological observations linking early life events to the risk of subsequent hypertension. The recent characterization, purification and cDNA cloning of a distinct human placental 11β-HSD (type 2) will aid the further study of these intriguing findings.     

Distribution of 17β-hydroxysteroid dehydrogenase gene expression and activity in rat and human tissues

The interconversion of estrone (E₁) and 17β-estradiol (E₂), androstenedione (4-ene-dione) and testosterone (T), as well as dehydroepiandrosterone and androst-5-ene-3β,17β-diol is catalyzed by 17β-hydroxysteroid dehydrogenase (17β-HSD). The enzyme 17β-HSD thus plays an essential role in the formation of all active androgens and estrogens in gonadal as well as extragonadal tissues. The present study investigates the tissue distribution of 17β-HSD activity in the male and female rat as well as in some human tissues and the distribution of 17β-HSD mRNA in some human tissues. Enzymatic activity was measured using ¹⁴C-labeled E₁, E₂, 4-ene-dione and T as substrates. Such enzymatic activity was demonstrated in all 17 rat tissues examined for both androgenic and estrogenic substrates. While the liver had the highestlevel of 17β-HSD activity, low but significant levels of E₂ as well as T formation were found in rat brain, heart, pancreas and thymus. The oxidative pathway (E₂→E₁, T→4-ene-dione) was favored over the reverse reaction in almost all rat tissues while in the human, almost equal rates were found in most of the 15 tissues examined. The widespread distribution of 17β-HSD in rat and human tissues clearly indicates the importance of this enzyme in peripheral sex steroid formation or intracrinology.     

Role of 17β-hydroxysteroid dehydrogenase type 1 in endocrine and intracrine estradiol biosynthesis

Enzymes with 17β-hydroxysteroid dehydrogenase (17β-HSD) activity catalyse reactions between the low-active female sex steroid, estrone, and the more potent estradiol, for example. 17β-HSD activity is essential for glandular (endocrine) sex hormone biosynthesis, but it is also present in several extra-gonadal tissues. Hence, 17β-HSD enzymes also take part in local (intracrine) estradiol production in the target tissues of estrogen action. Four distinct 17β-HSD isozymes have been characterized so far, and the data strongly suggests that different 17β-HSD isozymes have distinct roles in endocrine and intracrine metabolism of sex steroids. Current data suggest that 17β-HSD type 1 is the principal isoenzyme involved in glandular estradiol production both in humans and rodents. During ovarian follicular development and luteinization, rat 17β-HSD type 1 is regulated by gonadotropins, and the effects of gonadotropins are modulated by steroid hormones and paracrine growth factors. Human 17β-HSD type 1 favors the reduction reaction, thereby converting estrone to estradiol both in vitro and in cultured cells. Hence, the enzymatic properties of the enzyme are also in line with its suggested role in estradiol biosynthesis. Interestingly, 17β-HSD type 1 is also expressed in certain target tissues of estrogen action such as normal and malignant human breast and endometrium. Hence, 17β-HSD type 1 could be one of the factors leading to a relatively high tissue/plasma ratio of estradiol in breast cancer tissues of postmenopausal women. We conclude that 17β-HSD type 1 has a central role in regulating the circulating estradiol concentration as well as its local production in estrogen target cells.     

Differential expression of 11beta-hydroxysteroid dehydrogenase types 1 and 2 mRNA and glucocorticoid receptor protein during mouse embryonic development

Accumulating evidence suggests that the actions of glucocorticoids in target tissues are critically determined by the expression of not only the glucocorticoid receptor (GR) but also the glucocorticoid-metabolizing enzymes, known as 11β-hydroxysteroid dehydrogenase types 1 and 2 (11β-HSD1 and 11β-HSD2). To gain insight into the role of glucocorticoids in fetal development, the expression patterns of the two distinct 11β-HSD isozymes and GR were studied in the mouse embryo from embryonic day 12.5 (E12.5, TERM = E19) to postnatal day 0.5 (P0.5) by in situ hybridization and immunohistochemistry, respectively. 11β-HSD1 mRNA was detected in the heart as early as E12.5 and maintained thereafter. In the lung and liver, 11β-HSD1 mRNA was first detected between E14.5 and E16.5, increased to high levels towards term and maintained after birth. Relatively low levels of 11β-HSD1 mRNA were also detected in the kidney, adrenal glands and gastrointestinal tract at E18.5. However, the mRNA for 11β-HSD1 was undetectable in all other embryonic tissues including the brain. In contrast, kidney was the only organ that expressed appreciable levels of 11β-HSD2 mRNA during embryonic life. The level of 11β-HSD2 mRNA in the kidney increased dramatically in the newborn, which coincided with expression of 11β-HSD2 mRNA in the whisker follicle, tooth and salivary gland. Distinct from the profiles of 11β-HSD1 and 11β-HSD2 mRNA, GR protein was detectable in all tissues at all ages studied except for the thymus, salivary gland, and bone. Taken together, the present study demonstrates that tissue- and developmentally-stage specific expression of 11β-HSD1 and 11β-HSD2 as well as GR occurs in the developing mouse embryo, thus highlighting the importance of these two enzymes and GR in regulating glucocorticoid-mediated maturational events in specific tissues during murine embryonic development.     

Local estradiol metabolism in osteoblast- and osteoclast-like cells

Bone is an estradiol-responsive tissue. Estrogen withdrawal during the menopause causes loss of bone mass and clinically relevant osteoporosis in a third of all women. Sufficient or impaired local production, as well as degradation of estradiol in cells present in the bone microenvironment might be an important mechanism of rescue or might contribute to the development of osteoporosis, respectively. We therefore investigated aromatase and 17β-hydroxysteroid dehydrogenase type IV (17β-HSD IV) expression in osteoblast- and osteoclast-like cells. Aromatase mRNA was increasingly expressed in myeloid THP 1 cells differentiated along the monocyte/phagocyte pathway exploiting vitamin D and either granulocyte-macrophage-stimulating factor (GMCSF) or macrophage-stimulating factor (MCSF). In long-term cultures, when sequentially exposed to vitamin D (days 0–21) and GMCSF (days 5–10) and plated on collagen, the amount of expression of aromatase mRNA steadily increased along with the increasing expression of osteopontin mRNA, ν integrin mRNA, c-fms (MCSF-receptor) mRNA and multinucleated cells developing. The conversion of estradiol from testosterone (10⁻⁷ M/1) in the supernatants of dishes mirrored changes in aromatase mRNA expression and by day 21 rose to 30,000 ng/10⁷ cells/24 h. 17β-HSD IV mRNA expression was abundant in undifferentiated THP 1 cells and was decreased to approximately 50% by day 21. Unstimulated SV-40 immortalized fetal osteoblasts did not express aromatase mRNA, but the expression was stimulated by the addition of the phorbol ester phorbol myristate acetate (PMA). Unstimulated osteoblasts from primary cultures did not express aromatase mRNA. Osteoblast-like osteosarcoma cells MG 63 expressed faint levels of aromatase mRNA in contrast to the osteosarcoma cell line HOS 58. 17β-HSD IV mRNA was expressed in fetal osteoblasts as well as in osteoblasts from primary culture, MG 63 and HOS 58 cells. In summary, we can show the expression of estradiol metabolizing enzymes in cells which are present in the bone microenvironment. Impaired aromatase expression and/or enhanced expression of 17β-HSD IV may contribute to the pathogenesis of osteoporosis.     

Inhibition of human and rat 11beta-hydroxysteroid dehydrogenase type 1 by 18beta-glycyrrhetinic acid derivatives

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) plays an important role in regulating the cortisol availability to bind to corticosteroid receptors within specific tissue. Recent advances in understanding the molecular mechanisms of metabolic syndrome indicate that elevation of cortisol levels within specific tissues through the action of 11β-HSD1 could contribute to the pathogenesis of this disease. Therefore, selective inhibitors of 11β-HSD1 have been investigated as potential treatments for metabolic diseases, such as diabetes mellitus type 2 or obesity. Here we report the discovery and synthesis of some 18β-glycyrrhetinic acid (18β-GA) derivatives (2–5) and their inhibitory activities against rat hepatic11β-HSD1 and rat renal 11β-HSD2. Once the selectivity over the rat type 2 enzyme was established, these compounds’ ability to inhibit human 11β-HSD1 was also evaluated using both radioimmunoassay (RIA) and homogeneous time resolved fluorescence (HTRF) methods. The 11-modified 18β-GA derivatives 2 and 3 with apparent selectivity for rat 11β-HSD1 showed a high percentage inhibition for human microsomal 11β-HSD1 at 10 μM and exhibited IC₅₀ values of 400 and 1100 nM, respectively. The side chain modified 18β-GA derivatives 4 and 5, although showing selectivity for rat 11β-HSD1 inhibited human microsomal 11β-HSD1 with IC₅₀ values in the low micromolar range.