Substrate specificities of peroxisomal members of short-chain alcohol dehydrogenase superfamily: expression and characterization of dehydrogenase part of Candida tropicalis multifunctional enzyme.

In addition to several other enzymes, the short-chain alcohol dehydrogenase superfamily includes a group of peroxisomal multifunctional enzymes involved in fatty acid and cholesterol side-chain beta-oxidation. Mammalian peroxisomal multifunctional enzyme type 2 (perMFE-2) is a 2-enoyl-CoA hydratase-2/(R)-3-hydroxyacyl-CoA dehydrogenase. As has been shown previously, perMFE-2 hydrates (24E)-3alpha,7alpha, 12alpha-trihydroxy-5beta-cholest-24-enoyl-CoA to (24R, 25R)-3alpha, 7alpha,12alpha,24xi-tetrahydroxy-5beta-choles tanoyl-CoA, which has been characterized as a physiological intermediate in cholic acid synthesis. Out of four possible stereoisomers of 3alpha,7alpha, 12alpha,24xi-tetrahydroxy-5beta-cholestanoyl-CoA , the mammalian perMFE-2 dehydrogenates only the (24R,25R)-isomer. The yeast peroxisomal multifunctional enzyme (MFE) was first described as 2-enoyl-CoA hydratase-2/(R)-3-hydroxyacyl-CoA dehydrogenase. To investigate the stereospecificity of yeast peroxisomal MFE, the two dehydrogenase domains of C. tropicalis MFE were expressed in E. coli as a 65 kDa recombinant protein. This protein catalyzes the dehydrogenation of straight-chain (R)-3-hydroxyacyl-CoAs, but it is devoid of (S)-3-hydroxyacyl-CoA dehydrogenase and 2-enoyl-CoA hydratase activities. The protein dehydrogenates (24R,25R)- and (24R, 25S)-isomers of 3alpha,7alpha, 12alpha, 24xi-tetrahydroxy-5beta-cholestanoyl-CoA. Interestingly, the protein also shows 17beta-estradiol dehydrogenase activity. As a monofunctional (R)-specific 3-hydroxyacyl-CoA dehydrogenase is currently unavailable, this recombinant enzyme can be used to study the stereochemistry of bile acid synthesis.

Characterization of molecular and catalytic properties of intact and truncated human 17beta-hydroxysteroid dehydrogenase type 2 enzymes: intracellular localization of the wild-type enzyme in the endoplasmic reticulum.

Human 17beta-hydroxysteroid dehydrogenase (17HSD) type 2 is a widely distributed enzyme that primarily converts the highly active 17beta-hydroxysteroids to their inactive keto forms. In the present study, full-length human 17HSD type 2 was localized in the endoplasmic reticulum using a double immunofluorescence labeling technique. As a consequence of its strong membrane interaction, full-length human 17HSD type 2 could not be solubilized as a biologically active form in vitro. However, by deleting the first 29 amino acids from the N-terminus, we were able to purify a catalytically active enzyme from the cytosolic fraction of Sf9 insect cells. Biochemical and catalytic properties of the purified truncated human 17HSD type 2 protein confirm its suitability for structure-function analyses of the enzyme. Both intact and truncated 17HSD type 2 enzymes efficiently catalyzed the oxidation of estradiol, testosterone, dihydrotestosterone, androstenediol, and 20alpha-dihydroprogesterone. The oxidation of estradiol brought about by human 17HSD type 2 was effectively inhibited by several other steroidal compounds, such as 2-hydroxyestradiol, 5beta-androstan-3alpha,17beta-diol, 5alpha-androstan-3alpha,17beta-diol, and 5alpha-androstan-3beta,17beta-diol. The broad substrate specificity of human 17HSD type 2 together with its predominant oxidative activity and intracellular location, as observed in this study, indicate the physiological role of the enzyme to be primarily an inactivator of highly active 17beta-hydroxysteroids.

Human 17beta-hydroxysteroid dehydrogenase type 2 messenger ribonucleic acid expression and localization in term placenta and in endometrium during the menstrual cycle.

According to the current hypothesis, 17beta-hydroxysteroid dehydrogenases (17HSDs) regulate the extent of estrogen influence in the endometrium by converting estradiol (E2) locally into a biologically less active sex steroid, estrone (E1), and vice versa. Recently, we have shown that both 17HSD type 1 and type 2 are expressed in the human endometrium, and in the present work, using in situ hybridization, we show that 17HSD type 2 is localized in the glandular epithelial cells as previously shown for the type 1 enzyme, but in contrast to type 1, the expression of type 2 is highest at the end of the cycle. Hence, we hypothesize that the differential expression of the two 17HSD enzymes, with opposite activities in same cell types, could modulate intracellular E2 concentrations during the end of the luteal phase of the menstrual cycle. We further analyzed the expression of 17HSD type 1 and type 2 mRNAs in term human placenta. Expression of 17HSD type 1 mRNA was detected in the syncytiotrophoblasts, and signals for type 2 mRNA were found inside the villi, corresponding to cytotrophoblasts. The expression of 17HSD type 2 in the placenta may serve to maintain the presence of inactive sex steroids and attenuate the formation of biologically potent androgens and estrogens.

Mouse 17 beta-hydroxysteroid dehydrogenase type 2 mRNA is predominantly expressed in hepatocytes and in surface epithelial cells of the gastrointestinal and urinary tracts.

17 beta-Hydroxysteroid dehydrogenase (17HSD) type 2 efficiently catalyzes the conversion of the high activity 17 beta-hydroxy forms of sex steroids into less potent 17-ketosteroids. In the present study in situ hybridization was utilized to analyze the cellular localization of 17HSD type 2 expression in adult male and female mice. The data indicate that 17HSD type 2 mRNA is expressed in several epithelial cell layers, including both absorptive and secretory epithelia as well as protective epithelium. In both males and females, strong expression of 17HSD type 2 was particularly detected in epithelial cells of the gastrointestinal and urinary tracts. The mRNA was expressed in the stratified squamous epithelium of the esophagus, and surface epithelial cells of the stomach, small intestine and colon. The hepatocytes of the liver and the thick limbs of the loops of Henle in the kidneys, as well as the epithelium of the urinary bladder, also showed strong expression of 17HSD type 2 mRNA in both male and female mice. In the genital tracts, low 17HSD type 2 expression was detected in the seminiferous tubules, the uterine epithelial cells and the surface epithelium of the ovary. Expression of the mRNA was also detected in the sebaceous glands of the skin. The results indicate that in both male and female mice, 17HSD type 2 is expressed mainly in the various epithelial cell types of the gastrointestinal and urinary tracts, and therefore suggest a role for the enzyme in steroid inactivation in a range of tissues and cell types not considered as classical sex steroid target tissues.

Cloning of mouse 17beta-hydroxysteroid dehydrogenase type 2, and analysing expression of the mRNAs for types 1, 2, 3, 4 and 5 in mouse embryos and adult tissues.

17beta-Hydroxysteroid dehydrogenases (17HSDs) are responsible for the conversion of low-activity sex steroids to more potent forms, and vice versa. 17HSD activity is essential for the biosynthesis of sex steroids in the gonads, and it is also one of the key factors regulating the availability of active ligands for sex-steroid receptors in various extragonadal tissues. In this study, we have characterized mouse 17HSD type 2 cDNA, and analysed the relative expression of 17HSD types 1, 2, 3, 4 and 5 mRNAs in mouse embryos and adult male and female tissues. The cDNA characterized has a open reading frame of 1146 bp, and encodes a protein of 381 amino acids with a predicted molecular mass of 41837 kDa. Northern-blot analysis of adult mouse tissues revealed that, of the different 17HSDs, the type 2 enzyme is most abundantly expressed. High expression of the enzyme, which oxidizes both testosterone and oestradiol, in several large organs of both sexes indicates that it is the isoform having the most substantial role in the metabolism of sex steroids. Interestingly, four of the five 17HSD enzymes were also detected by Northern blots of whole mouse embryos, and each of the enzymes showed a unique pattern of expression. The oestradiol-synthesizing type 1 enzyme predominates in early days of development embryonic day 7, but after that the oxidative type 2 enzyme becomes the predominant form of all 17HSDs. The data therefore suggest that there is transient oestradiol production in the early days of embryonic development, after which inactivation of sex steroids predominates in the fetus and placenta.

Peroxisomal multifunctional enzyme of beta-oxidation metabolizing D-3-hydroxyacyl-CoA esters in rat liver: molecular cloning, expression and characterization.

In the present study we have cloned and characterized a novel rat peroxisomal multifunctional enzyme (MFE) named perMFE-II. The purified 2-enoyl-CoA hydratase 2 with an M(r) of 31500 from rat liver [Malila, Siivari, Mäkelä, Jalonen, Latipää, Kunau and Hiltunen (1993) J. Biol. Chem. 268, 21578-21585] was subjected to tryptic fragmentation and the resulting peptides were isolated and sequenced. Surprisingly, the full-length cDNA, amplified by PCR, had an open reading frame of 2205 bp encoding a polypeptide with a predicted M(r) of 79,331 and contained a potential peroxisomal targeting signal in the C-terminus (Ala-Lys-Leu). The sequenced peptide fragments of hydratase 2 gave a full match in the middle portion of the cDNA-derived amino acid sequence. The predicted amino acid sequence showed a high degree of similarity with pig 17 beta-hydroxysteroid dehydrogenase type IV and MFE of yeast peroxisomal beta-oxidation. Recombinant perMFE-II (produced in Pichia pastoris) had 2-enoyl-CoA hydratase 2 and D-specific 3-hydroxyacyl-CoA dehydrogenase activities and was catalytically active with several straight-chain trans-2-enoyl-CoA, 2-methyltetradecenoyl-CoA and pristenoyl-CoA esters. The results showed that in addition to an earlier described multifunctional isomerase-hydratase-dehydrogenase enzyme from rat liver peroxisomes (perMFE-I), another MFE exists in rat liver peroxisomes. They both catalyse sequential hydratase and dehydrogenase reactions of beta-oxidation but through reciprocal stereochemical courses.

Characterization of estrogen-dependent growth of cultured MCF-7 human breast-cancer cells expressing 17beta-hydroxysteroid dehydrogenase type 1.

17beta-hydroxysteroid dehydrogenase (17HSD) type I converts the weakly active estrogen, estrone, into highly active estradiol. In addition to being essential for gonadal estradiol biosynthesis, the enzyme is also expressed in a significant proportion of breast tumors. In order to study the role of the enzyme in estrogen-dependent growth of breast cancer, MCF-7 breast-cancer cells stably expressing human 17HSD type I were generated. In control MCF-7 cells a very low 17HSD activity was observed and, in line with its low estrogenic activity, estrone was devoid of the growth-enhancing effect of estradiol. The presence of the enzyme in the stably transfected MCF-7 cells resulted in a rapid conversion of estrone into estradiol but did not alter the estrogen-receptor concentration in the cells. However, in transfected cells, estrone had a growth-promoting effect practically identical to that of estradiol. The presence or absence of 17HSD type I in breast-cancer cells may therefore be decisive with regard to estrogen exposure and the estrogen-responsive growth of breast-cancer tissues.

Human 17 beta-hydroxysteroid dehydrogenase type 1 and type 2 isoenzymes have opposite activities in cultured cells and characteristic cell- and tissue-specific expression.

17 beta-Hydroxysteroid dehydrogenase (17HSD) isoenzymes catalyse the interconversion between highly active 17 beta-hydroxy- and low-activity 17-keto-steroids and thereby regulate the biological activity of sex steroids. The present study was carried out to characterize 17HSD activity and the expression of 17HSD type 1 and 2 isoenzymes in several human cell types and tissues. The data indicate that in cultured cells the direction of 17HSD activity is exclusively determined by the expression of these distinct isoenzymes. The intracellular environment could not modulate the direction of the enzyme activities in any of the cell types analysed. 17HSD type 1 acts as a reductase converting oestrone into oestradiol, whereas 17HSD type 2 possesses oxidative activity inactivating oestradiol by converting it into oestrone. The data, furthermore, suggest that of the two 17HSD type 1 mRNAs (1.3 and 2.3 kb), expression of the 1.3 kb mRNA is related to enzyme concentration in all the cell types studied. This mRNA is principally expressed in cells of placental and ovarian origin, but is also present in malignant breast epithelial cells. In contrast, 17HSD type 2 is more widely expressed. It is present in several oestradiol-metabolizing tissues as well as in some target cells of sex steroid action. The opposite reaction directions observed in the cultured cells, together with differences in the distribution of the isoenzymes, suggest that type 1 is involved in oestradiol production in females while type 2 plays a role in the inactivation of this sex steroid in peripheral tissues, both in females and in males. However, some examples exist of simultaneous expression of both enzymes in the same cell type or tissue.

Site-directed mutagenesis of the putative active site of human 17 beta-hydroxysteroid dehydrogenase type 1.

Several amino acid residues (Cys54, Tyr155, His210, His213 and His221) at a putative catalytic site of human 17 beta-hydroxysteroid dehydrogenase type 1 were mutated to Ala. Replacement of His221 by Ala remarkably reduced the catalytic activity, which resulted from a change of both the Km and the Vmax. values of the enzyme. Compared with the wild-type enzyme, the catalytic efficiency of the His221-->Ala mutant was reduced 20-fold for the oxidative reaction and 11-fold for the reductive reaction. With similar mutations at His210 or His213, no notable effects on the catalytic properties of the enzyme were detected. However, a simultaneous mutation of these amino acid residues decreased the Vmax. values of both oxidation and reduction by about 50% from those measured for the wild-type enzyme. Although Cys54 has been localized in the cofactor-binding region of the enzyme, a Cys54-->Ala mutation did not lead to changes in the enzymic activity. The most dramatic effects on the catalytic properties of the enzyme were achieved by mutating Tyr155, which resulted in an almost completely inactivation of the enzyme. The decreased enzymic activities of the Tyr155-->Ala, His210-->Ala + His213-->Ala and His221-->Ala mutations were also reflected in a reduced immunoreactivity of the enzymes. The results thus suggest that the lower catalytic efficiency of the mutant enzymes is due to an exchange of catalytically important amino acid residues and/or remarkable alterations in the three-dimensional structure of the enzyme. The recently detected polymorphisms (Ala237<-->Val and Ser312<-->Gly) were not found to affect either the catalytic or the immunological properties of the type 1 enzyme.

Expression of 17 beta-hydroxysteroid dehydrogenase in human granulosa cells: correlation with follicular size, cytochrome P450 aromatase activity and oestradiol production.

The aim of this study was to examine the expression and regulation of type 1 17 beta-hydroxysteroid dehydrogenase (type 1 17-HSD) enzyme protein and mRNA, and 17-HSD activity in human granulosa cells. The cells were obtained from patients taking part in an in vitro fertilization programme. The cells from each patient were divided into two groups: cells obtained from preovulatory follicles (LGC = granulosa cells from large follicles > or = 18 mm in diameter), and cells from other visible follicles (SGC = granulosa cells from small follicles, less than 15 mm in diameter). The identity of 17-HSD enzyme protein expressed in human granulosa cells with placental cytosolic 17-HSD (type 1 17-HSD) was assessed by immunoblot analysis using polyclonal antibodies, and the enzyme was immunolocalized in the cytoplasm of granulosa cells. Type 1 17-HSD protein concentration, 17-HSD and cytochrome P450 aromatase (P450arom) activities and oestradiol (OE2) production in cells from LGC were significantly lower than the corresponding values obtained in SGC in the same patient (paired t-test). The type 1 17-HSD protein concentration, 17-HSD activity and P450arom activity were 140 +/- 16% (mean +/- S.E.M.), 121 +/- 22% and 113 +/- 26% higher in cells from SGC, which was also reflected in a 70 +/- 12% higher OE2 production in these cells. In freshly isolated cells from LGC or SGC, a high correlation between 17-HSD and P450arom activities was observed (r = 0.93, P < 0.001). In long-term cultured cells, type 1 17-HSD was stably expressed at least until day 9, while P450arom expression decreased. In addition, treatments with gonadotrophins did not affect type 1 17-HSD protein concentration and 17-HSD activity. In contrast to this, both P450arom activity and OE2 production were significantly increased (P < 0.05). The data, therefore, suggest that type 1 17-HSD and P450arom are expressed in parallel during the latest stages of follicular maturation but, in cultured granulosa-luteal cells, the enzymes are regulated by distinct mechanisms.

Expression of 17 beta-hydroxysteroid dehydrogenase in the rat ovary during follicular development and luteinization induced with pregnant mare serum gonadotrophin and human chorionic gonadotrophin.

Antibodies against human placental 17 beta-hydroxysteroid dehydrogenase (17-HSD) and 17-HSD cDNA were used to study the expression of the corresponding enzyme in the immature rat ovary during follicular development and luteinization, which were induced by treating the animals with pregnant mare serum gonadotrophin (PMSG) or with PMSG followed by human chorionic gonadotrophin (hCG). Immuno-blot analysis indicated that the M(r) of the 17-HSD expressed in rat granulosa cells was 35,000, as previously shown for the human placental enzyme. In immunohistochemical studies of untreated immature rat ovaries, only the granulosa cells from small antral follicles were stained. One day after PMSG treatment, strong expression of 17-HSD was observed in the granulosa cells of growing Graafian follicles. A marked decrease in enzyme expression was observed in preovulatory follicles on day 2 of PMSG treatment, starting from the basal layers of granulosa cells and progressing toward the luminal cells. No 17-HSD expression was detected in luteinized follicles or corpora lutea 22 h after hCG injection. The stroma and theca cells were negative for 17-HSD staining. In Northern hybridization analyses, two 17-HSD mRNAs were detected (1.4 and 1.7 kb). The strongest expression for both mRNAs was detected after 1 day of PMSG treatment, coinciding with maximal immunostaining of the enzyme protein. Down-regulation of 17-HSD observed by immunohistochemistry was reflected in a similar decrease in mRNA expression and the signals were almost undetectable 22 h after hCG injection. Our data suggest that 17-HSD expression in rat granulosa cells is up-regulated during follicular development and, thereafter, the enzyme expression is down-regulated during luteinization.

Steroid biosynthetic enzymes: 17 beta-hydroxysteroid dehydrogenase.

Polyclonal antibodies produced against human placental 17 beta-hydroxysteroid dehydrogenase (17HSD), purified to homogeneity, and the corresponding cDNA for the enzyme were used to study the expression of 17HSD in a number of human tissues using various immunological methods together with RNA hybridization techniques. In addition, two 17HSD genes and their putative regulatory elements were sequenced. Immunoblotting analysis showed that the placental-type enzyme is expressed in granulosa-luteal cells, breast cancer tissue and breast cancer cell lines. An immunologically identical antigen was also detected in normal and carcinomatous human endometrium. The same antiserum, following affinity purification, was used for immunohistochemical studies of the endometrium and breast tissue, whereupon staining of the cytoplasm of the epithelial cells alone was observed. Immunostaining was also present in cultured human granulosa cells and in about half of the endometrial and breast carcinoma specimens investigated. Progesterone induction of the 17HSD enzyme protein was demonstrated in the human endometrium during the secretory phase of the menstrual cycle and in one breast cancer cell line (T-47D) following progestin treatment. There are at least two mRNAs for placental 17HSD (1.3 kb, 2.3 kb). RNA hybridization analysis of various breast cancer cell lines showed that the 1.3 kb mRNA was most closely associated with enzyme protein expression and was also the only form responding to progesterone induction. We conclude that placental-type 17HSD is also expressed in some other human tissues, both steroid-synthesizing and steroid-responding, and that the mRNA and enzyme protein are induced by progesterone. The availability of the sequence of 17HSD genes and surrounding regions allows us to study the sequences responsible for the expression and regulation of 17HSD.