Site-directed Mutagenesis Identifies Amino Acid Residues Associated with the Dehydrogenase and Isomerase Activities of Human Type I (Placental) 3β-Hydroxysteroid Dehydrogenase/Isomerase

3β-Hydroxysteroid dehydrogenase/steroid Δ^{5 → 4}-isomerase (3β-HSD/isomerase) was expressed by baculovirus in Spodoptera fungiperda (Sf9) insect cells from cDNA sequences encoding human wild-type I (placental) and the human type I mutants - H261R, Y253F and Y253,254F. Western blots of SDS-polyacrylamide gels showed that the baculovirus-infected Sf9 cells expressed the immunoreactive wild-type, H261R, Y253F or Y253,254F protein that co-migrated with purified placental 3β-HSD/isomerase (monomeric M_r=42,000 Da). The wild-type, H261R and Y253F enzymes were each purified as a single, homogeneous protein from a suspension of the Sf9 cells (5.01). In kinetic studies with purified enzyme, the H261R mutant enzyme had no 3β-HSD activity, whereas the K_m and V_max values of the isomerase substrate were similar to the values obtained with the wild-type and native enzymes. The V_max (88 nmol/min/mg) for the conversion of 5-androstene-3,17-dione to androstenedione by the Y253F isomerase activity was 7.0-fold less than the mean V_max (620 nmol/min/mg) measured for the isomerase activity of the wild-type and native placental enzymes. In microsomal preparations, isomerase activity was completely abolished in the Y253,254F mutant enzyme, but Y253,254F had 45% of the 3β-HSD activity of the wild-type enzyme. In contrast, the purified Y253F, wild-type and native enzymes had similar V_max values for substrate oxidation by the 3β-HSD activity. The 3β-HSD activities of the Y253F, Y253,254F and wild-type enzymes reduced NAD⁺ with similar kinetic values. Although NADH activated the isomerase activities of the H261R and wild-type enzymes with similar kinetics, the activation of the isomerase activity of H261R by NAD⁺ was dramatically decreased. Based on these kinetic measurements, His²⁶¹ appears to be a critical amino acid residue for the 3β-HSD activity, and Tyr²⁵³ or Tyr²⁵⁴ participates in the isomerase activity of human type I (placental) enzyme.     

The murine 3β-hydroxysteroid dehydrogenase multigene family: Structure, function and tissue-specific expression

The classical form of the enzyme 5-ene-3β-hydroxysteroid dehydrogenase/isomerase (3βHSD), expressed in adrenal glands and gonads, catalyzes the conversion of 5-ene-3β-hydroxysteroids to 4-ene-3-ketosteroids, an essential step in the biosynthesis of all active steroid hormones. To date, four distinct mouse 3βHSD cDNAs have been isolated and characterized. These cDNAs are expressed in a tissue-specific manner and encode proteins of two functional classes. Mouse 3βHSD I and III function as 3β-hydroxysteroid dehydrogenases and 5-en→4-en isomerases using NAD⁺ as a cofactor. The enzymatic function of 3βHSD II has not been completely characterized. Mouse 3βHSD IV functions only as a 3-ketosteroid reductase using NADPH as a cofactor. The predicted amino acid sequences of the four isoforms exhibit a high degree of identity. Forms II and III are 85 and 83% homologous to form I. Form IV is most distant from the other three with 77 and 73% sequence identity to I and III, respectively. 3βHSD I is expressed in the gonads and adrenal glands of the adult mouse. 3βHSD II and III are expressed in the kidney and liver with the expression of form II greater in kidney and form III greater in liver. Form IV is expressed exclusively in the kidney. Although the amino acid composition of forms I, III and IV predicts proteins of the same molecular weight, the proteins have different mobilities on SDS-polyacrylamide gel electrophoresis. This characteristic allows for differential identification of the expressed proteins. The four structural genes encoding the different isoforms are closely linked within a segment of mouse chromosome 3 that is conserved on human chromosome 1.     

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.     

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.     

Bovine adrenal 3beta-hydroxysteroid dehydrogenase (E.C. 1.1.1. 145)/5-ene-4-ene isomerase (E.C. 5.3.3.1): characterization and its inhibition by isoflavones

The isoflavones daidzein, genistein, biochanin A and formononetin inhibit potently and preferentially the γ-isozymes of mammalian alcohol dehydrogenase (γγ-ADH), the only ADH isozyme that catalyzes the oxidation of 3β-hydroxysteroids. Based on these results, we proposed that these isoflavones might also act on other enzymes involved in 3β-hydroxysteroid metabolism. Recently, we showed that they indeed are potent inhibitors of a bacterial β-hydroxysteroid dehydrogenase (β-HSD). To extend this finding to the mammalian systems, we hereby purified, characterized and studied the effects of isoflavones and structurally related compounds on, a bovine adrenal 3β-hydroxysteroid dehydrogenase (3β-HSD). This enzyme catalyzes the oxidation of 3β-hydroxysteroids but not 3-, 11β- or 17β-hydroxysteroids. The same enzyme also catalyzes 5-ene-4-ene isomerization, converting 5-pregnen 3, 20-dione to progesterone. The K_m values of its dehydrogenase activity determined for a list of 3β-hydroxysteroid substrates are similar (1 to 2 μM) and that of its isomerase activity, determined with 5-pregnen 3, 20-dione as a substrate, is 10 μM. The k_cat value determined for its isomerase activity (18.2 min⁻¹) is also higher than that for its dehydrogenase activity (1.4–2.4 min⁻¹). A survey of more than 30 isoflavones and structurally related compounds revealed that daidzein, genistein, biochanin A and formononetin inhibit both the dehydrogenase and isomerase activity of this enzyme. Inhibition is potent and concentration dependent. IC₅₀ values determined for these compounds range from 0.4 to 11 μM, within the plasma and urine concentration ranges of daidzein and genistein of individuals on vegetarian diet or semi-vegetarian diet. These results suggest that dietary isoflavones may exert their biological effects by inhibiting the action of 3β-HSD, a key enzyme of neurosteroid and/or steroid hormone biosynthesis.     

Dual subcellular localization of the 3β-hydroxysteroid dehydrogenase isomerase: Characterization of the mitochondrial enzyme in the bovine adrenal cortex

The enzyme 3β-hydroxysteroid dehydrogenase isomerase (3β-HSD/I) in an essential step in the biosynthesis of steroid such as progesterone, mineralo- and gluco-corticoids, estrogens and androgens in steroidogenic tissues. It is considered to be mainly localized in microsomes; however, 3β-HSD/I activity has also been described to be associated with mitochondrial preparations. In this study, we examined the subcellular distribution of 3β-HSD/I in bovine adrenocortical tissue and we characterized the catalytic properties of the enzyme present in the various cell compartments. About 30% of the total 3β-HSD/I activity was found to remain tightly associated with the purified mitochondrial pellet. The 3β-HSD/I and 3-ketoreductase activities were found in microsomes as well as in mitochondria. The 3β-HSD/I associated with the mitochondrial fraction did not required addition of exogenous NAD⁺. When the pyridine nucleotide was reduced ollowing addition of substrate of the tricarboxyllic acids cycle, the mitochondrial 3β-HSD/I activity decreased, suggesting that the enzyme utilizes NAD⁺ available from the matrix space. By contrast, the microsomal enzyme was inactive in the absence of exogenous NAD⁺. Submitochondrial fraction disclosed that 3β-HSD/I was associated (i) with the inner membrane and (ii) with a particulate fraction sedimenting in a density gradient between inner and outer membranes. This fraction was characterized as contact sites between the two membranes. 3β-HSD/I specific activity was much higher in this fraction than in the inner mitochondrial membrane. Altogether, these observations suggest that these mitochondrial intermembrane contact sites may represent a spacial organization of functional significance, facilitating both the access of cholesterol to the inner membrane where cytochrome P-450_scc is located and the rapid transformation of its product, pregnenolone, to progesterone, through 3β-HSD/I activity.     

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.     

Purification and characterization of 3β-hydroxysteroid-dehydrogenase/isomerase from bovine adrenal cortex

The formation of 4-ene-3-ketosteroids from 3β-hydroxy-5-ene precursors is an obligatory step in the biosynthesis of hormonal steroids such as glucocorticoids, mineralocorticoids, estrogens and androgens. In the adrenal cortex, pregnenolone, 17-hydroxy-pregnenolone and dehydroisoandrosterone are converted to progesterone, 17-hydroxy-progesterone and androstenedione, respectively, by the enzymatic system 3β-hydroxy-5-ene steroid dehydrogenase and 3-keto-5-ene steroid isomerase (3β-HSD/I).

The present work reports a two step purification procedure which yields an homogenous preparation of 3β-HSD/I from bovine adrenal cortex. It uses solubilization of the microsomal proteins followed by two chromatographic steps, i.e. DEAE-cellulose and heparine-sepharose columns. The enzyme was obtained as an homogeneous protein exhibiting an apparent molecular size of 45 kDa upon SDS-gel electrophoresis and of 81 kDa upon gel filtration. The purified enzyme exhibits both the 5-ene-3β-ol steroid dehydrogenase and isomerase activities in contrast to previous work using a more complex procedure which yielded a final preparation having lost its isomerase activity [Hiwatashi et al., Biochem. J. 98 (1985) 1519–1525]. N-terminal aminoacid (29 residues) sequence of the purified protein was determined and was found identical to that predicted from the nucleic acid sequence of the recently identified enzyme cDNA [Zhas et al. FEBS Lett. 259 (1989) 153–157].     

Regulation of expression of the 3β-hydroxysteroid dehydrogenases of human placenta and fetal adrenal

The appropriate expression of 3β-hydroxysteroid dehydrogenase/Δ^5→4-isomerase (3β-HSD) is vital for mammalian reproduction, fetal growth and life maintenance. Several isoforms of 3β-HSD, the products of separate genes, have been identified in various species including man. Current investigations are targeted toward defining the processes that regulate the levels of specific isoforms in various steroidogenic tissues of man. High levels of expression of 3β-HSD were observed in placental tissues. It has been generally considered that the multinucleated syncytiotrophoblastic cells are the principal sites of 3β-HSD expression and, moreover, that 3β-HSD expression is intimately associated with cyclic AMP-promoted formation of syncytia. Herein we report the presence of 3β-HSD immunoreactive and mRNA species in uninucleate cytotrophoblasts in the chorion laeve, similar to that in syncytia but not cytotrophoblast placenta. In vitro, 3β-HSD levels in chorion laeve cytotrophoblasts were not increased with time nor after treatment with adenylate cyclase activators, whereas villous cytotrophoblasts spontaneously demonstrated progressive, increased 3β-HSD expression. Moreover, 3β-HSD synthesis appeared to precede morphologic syncytial formation. Thus high steroidogenic enzyme expression in placenta is not necessarily closely linked to formation of syncytia. Both Western immunoblot and enzymic activity analyses also indicated that the 3β-HSD expressed in these cytotrophoblastic populations was the 3β-HSD type I gene product (M_r, 45K) and not 3β-HSD type II (M_r, 44K) expressed in fetal testis. In cultures of fetal zone and definitive zone cell of human fetal adrenal, 3β-HSD expression was not detected until ACTH was added. ACTH, likely acting in a cyclic AMP-dependent process, induced 3β-HSD type II activity and mRNA expression. The higher level of 3β-HSD mRNA in definitive zone compared with fetal zone cells was associated with parallel increases in cortisol secretion relative to dehydroepiandrosterone sulfate formation.     

Structure, function and tissue-specific gene expression of 3β-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase enzymes in classical and peripheral intracrine steroidogenic tissues

The membrane-bound enzyme 3β-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3β-HSD) catalyses an essential step in the transformation of all 5-pregnen-3β-ol and 5-androsten-3β-ol steroids into the corresponding 3-keto-4-ene-steroids, namely progesterone as well as all the precursors of androgens, estrogens, glucocorticoids and mineralocorticoids. We have recently characterized two types of human 3β-HSD cDNA clones and the corresponding genes which encode type I and II 3β-HSD isoenzymes of 372 and 371 amino acids, respectively, and share 93.5% homology. The human 3β-HSD genes containing 4 exons were assigned by in situ hybridization to the p11-p13 region of the short arm of chromosome 1. Human type I 3β-HSD is the almost exclusive mRNA species present in the placenta and skin while the human type II is the predominant mRNA species in the adrenals, ovaries and testes. The type I protein possesses higher 3β-HSD activity than type II. We elucidated the structures of three types of rat 3β-HSD cDNAs as well that of one type of 3β-HSD from bovine and macaque ovary λgt11 cDNA libraries, which all encode a 372 amino acid protein. The rat type I and II 3β-HSD proteins expressed in the adrenals, gonads and adipose tissue share 93.8% homology. Transient expression of human type I and II as well as rat type I and II 3β-HSD cDNAs in HeLa human cervical carcinoma cells reveals that 3β-ol dehydrogenase and 5-ene-4-ene isomerase activities reside within a single protein. These expressed 3β-HSD proteins convert 3β-hydroxy-5-ene-steroids into 3-keto-4-ene derivatives and catalyze the interconversion of 3β-hydroxy and 3-keto-5α-androstane steroids. By site-directed mutagenesis, we demonstrated that the lower activity of expressed rat type II compared to rat type I 3β-HSD is due to a change of four residues probably involved in a membrane-spanning domain. When homogenates from cells transfected with a plasmid vector containing rat type I 3β-HSD is incubated in the presence of dihydrotestosterone (DHT) using NAD? as co-factor, 5α-androstanedione was formed (A-dione), indicating an intrinsic androgenic 17β-hydroxysteroid dehydrogenase (17β-HSD) activity of this 3β-HSD. We cloned a third type of rat cDNA encoding a predicted type III 3β-HSD specifically expressed in the rat liver, which shares 80% similarity with the two other isoenzymes. Transient expression in human HeLa cells reveals that the type III isoenzyme does not display oxidative activity for the classical substrates of 3β-HSD. However, in common with the type I enzyme, it converts A-dione and DHT to the corresponding 3β-hydroxysteroids, thus showing an exclusive 3-ketosteroid reductase activity. When NADPH is used as co-factor, the affinity for DHT of the type III enzyme becomes 10-fold higher than that of the type I. Rat type III mRNA was below the detection limit in intact female liver. Following hypophysectomy, its concentration increased to 55% of the values measured in intact or hypophysectomized male rats, an increase which can be blocked by administration of ovine prolactin (oPRL). Treatment with oPRL for 10 days starting 15 days after hypophysectomy markedly decreased ovarian 3β-HSD mRNA accumulation accompanied by a similar decrease in 3β-HSD activity and protein levels. Treatment with the gonadotropin hCG reversed the potent inhibitory effect of oPRL on these parameters and stimulated 3β-HSD mRNA levels in ovarian interstitial cells. These data indicate that the presence of multiple 3β-HSD isoenzymes offers the possibility of tissue-specific expression and regulation of this enzymatic activity that plays an essential role in the biosynthesis of all hormonal steroids in classical as well as peripheral intracrine steroidogenic tissues.     

Molecular genetics of androgenic 17β-Hydroxysteroid dehydrogenases

17β-Hydroxysteroid dehydrogenase (17β-HSD) type 2 catalyzes the NAD⁺-dependent oxidation of androgens, estrogens and progestins, predominantly in the secretory endometrium, placenta, liver and small intestine. 17β-HSD type 3 catalyzes the NADPH-dependent conversion of androstenedione to testosterone in the testis, and the genetic disease 17β-HSD deficiency is caused by mutations in the 17β-HSD3 gene.     

Structure and tissue-specific expression of 3ß-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase genes in human and rat classical and peripheral steroidogenic tissues

The enzyme 3ß-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3ß-HSD) catalyzes the oxidation and isomerization of 5-ene-3ß-hydroxypregnene and 5-ene-hydroxyandrostene steroid precursors into the corresponding 4-ene-ketosteroids necessary for the formation of all classes of steroid hormones. We have recently characterized two types of human 3ß-HSD cDNA clones and the corresponding genes which encode deduced proteins of 371 and 372 amino acids, respectively, and share 93.5% homology. The human 3ß-HSD genes containing 4 exons were assigned by in situ hybridization to the p11–p13 region of the short arm of chromosome 1. We have also recently elucidated the structure of three types of rat 3ß-HSD cDNAs as well as that of one type of 3ß-HSD from bovine and macaque ovary λgt11 cDNA libraries which all encode 372 amino acid proteins. The human type I 3ß-HSD is the almost exclusive mRNA species detected in the placenta and skin, while the human type II is the predominant mRNA species in the adrenals, ovaries and testes. The predicted rat type I and type II 3ß-HSD proteins expressed in adrenals, gonads and adipose tissue share 94% homology while they share 80% similarity with the liver-specific type III 3ß-HSD. Transient expression of human type I and type II as well as rat type I and type II 3ß-HSD cDNAs in Hela human cervical carcinoma cells reveals that 3ß-ol dehydrogenase and 5-ene-4-ene isomerase activities reside within a single protein and these cDNAs encode functional 3ß-HSD proteins that are capable of converting 3ß-hydroxy-5-ene-steroids into 3-keto-4-ene derivatives as well as the interconversion of 3ß-hydroxy and 3-keto-5-androstane steroids. We have found that the rat type III mRNA species was below the detection limit in intact female liver while, following hypophysectomy, its accumulation increased to 55% of the levels measured in intact or HYPOX male rats, an increase which can be blocked by administration of ovine prolactin (oPRL). In addition, in female rats, treatment with oPRL for 10 days starting 15 days after HYPOX, markedly decreased ovarian 3ß-HSD mRNA accumulation accompanied by a similar decrease in 3ß-HSD activity and protein levels. Treatment with the gonadotropin hCG reversed the potent inhibitory effect of oPRL on these parameters and stimulated 3ß-HSD mRNA levels in ovarian interstitial cells. In intact females, hCG exerted marked trophic effects on rat corpora lutea with an increase in total ovarian 3ß-HSD expression and activity. We have also shown that treatment with hCG for 15 days in intact male rats caused a marked increase in testicular 3ß-HSD expression and activity while glucocorticoids exerted inhibitory effects on these parameters. We have also observed that the ontogeny of 3ß-HSD expression in human and rat adrenal gland, testis and ovary is closely correlated with steroid hormone biosynthesis, thus suggesting that regulation of the expression of 3ß-HSD is a limiting step in the biosynthesis of steroids in these tissues.     

Molecular basis of human 3β-hydroxysteroid dehydrogenase deficiency

The enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD) catalyses an essential step in the biosynthesis of all classes of steroid hormones. Classical 3β-HSD deficiency is responsible for CAHII, a severe form of congenital adrenal hyperplasia (CAH) that impairs steroidogenesis in both the adrenals and gonads. Newborns affected by 3β-HSD deficiency exhibit signs and symptoms of adrenal insufficiency of varying degrees associated with pseudohermaphroditism in males, whereas females exhibit normal sexual differentiation or mild virilization. Elevated ratios of 5-ene-to 4-ene-steroids appear as the best biological parameter for the diagnosis of 3β-HSD deficiency. The nonclassical form has been suggested to be related to an allelic variant of the classical form of 3β-HSD as described for steroid 21-hydroxylase deficiency. To elucidate the molecular basis of the classical form of 3β-HSD deficiency, we have analysed the structure of the highly homologous type I and II 3β-HSD genes in 12 male pseudohermaphrodite 3β-HSD deficient patients as well as in four female patients. 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 finding of a normal type I 3β-HSD gene provides the explanation for the intact peripheral intracrine steroidogenesis in these patients and increased androgen manifestations at puberty. The influence of the detected mutations on enzymatic activity was assessed by in vitro expression analysis of mutant enzymes generated by site-directed mutagenesis in COS-1 cells. The mutant type II 3β-HSD enzymes carrying mutations detected in patients affected by the salt-losing form exhibit no detectable activity in intact tranfected cells, whereas those with mutations found in nonsalt-loser index cases have some residual activity ranging from 1–10% compared to the wild-type enzyme. Although in general, our findings provide a molecular explanation for the enzymatic heterogeneity ranging from the severe salt-losing form to the clinically inapparent salt-wasting form of the disease, we have observed that the mutant L108W or P186L enzymes found in a compound heterozygote male presenting the salt-wasting form of the disease, has some residual activity (1%) similar to that observed for the mutant N100S enzyme detected in an homozygous male patient suffering from a nonsalt-losing form of this disorder. Unlike the classical 3β-HSD deficiency, our study in women presenting nonclassical 3β-HSD deficiency strongly suggests that this disorder is not due to a mutant type II 3β-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.     

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.     

Effect of nomegestrol acetate on estrone-sulfatase and 17β-hydroxysteroid dehydrogenase activities in human breast cancer cells

It is well recognized that estradiol (E₂) is one of the most important hormones supporting the growth and evolution of breast cancer. Consequently, to block this hormone before it enters the cancer cell or in the cell itself, has been one of the main targets in recent years. In the present study we explored the effect of the progestin, nomegestrol acetate, on the estrone sulfatase and 17β-hydroxy-steroid dehydrogenase (17β-HSD) activities of MCF-7 and T-47D human breast cancer cells. Using physiological doses of estrone sulfate (E₁S: 5 × 10⁻⁹ M), nomegestrol acetate blocked very significantly the conversion of E₁S to E₂. In the MCF-7 cells, using concentrations of 5 × 10⁻⁶ M and 5 × 10⁻⁵ M of nomegestrol acetate, the decrease of E₁S to E₂ was, respectively, −43% and −77%. The values were, respectively, −60% and −71% for the T-47D cells. Using E₁S at 2 × 10⁻⁶ M and nomegestrol acetate at 10⁻⁵ M, a direct inhibitory effect on the enzyme of −36% and −18% was obtained with the cell homogenate of the MCF-7 and T-47D cells, respectively. In another series of studies, it was observed that after 24 h incubation of a physiological concentration of estrone (E₁: 5 × 10⁻⁹ M) this estrogen is converted in a great proportion to E₂. Nomegestrol acetate inhibits this transformation by −35% and −85% at 5 × 10⁻⁷ M and 5 × 10⁻⁵ M, respectively in T-47D cells; whereas in the MCF-7 cells the inhibitory effect is only significant, −48%, at 5 × 10⁻⁵ M concentration of nomegestrol acetate. It is concluded that nomegestrol acetate in the hormone-dependent MCF-7 and T-47D breast cancer cells significantly inhibits the estrone sulfatase and 17β-HSD activities which converts E₁S to the biologically active estrogen estradiol. This inhibition provoked by this progestin on the enzymes involved in the biosynthesis of E₂ can open new clinical possibilities in breast cancer therapy.