Hellevi Peltoketo

Complete amino acid sequence of human placental 17β‐hydroxysteroid dehydrogenase deduced from cDNA

CDNA clones for 17β‐hydroxysteroid dehydrogenase (17‐HSD; EC 1.1.1.62) were isolated from a placental λgt11 expression library using polyclonal antibodies against placental 17‐HSD. The largest cDNA contained 1325 nucleotides, consisting of a short 5′‐noncoding segment, a coding segment of 987 nucleotides terminated by a TAA codon, and a 329 nucleotide long 3′‐noncoding segment. The open reading frame encoded a polypeptide of 327 amino acid residues with a predicted M r of 34853. The amino acid sequence of 23 N‐terminal amino acids determined from purified 17‐HSD agreed with the sequence deduced from cDNA. The deduced amino acid sequence also contained two peptides previously characterized from the proposed catalytic area of placental 17‐HSD.

Production, purification, and functional analysis of recombinant human and mouse 17β-hydroxysteroid dehydrogenase type 7 ☆

17beta-Hydroxysteroid dehydrogenases (17HSDs) have a central role in the regulation of the biological activity of sex steroid hormones. There is increasing evidence that in addition to their importance in gonads, these hormones also have substantial metabolic roles in a variety of peripheral tissues. In the present study, the cDNA of human 17HSD type 7 was cloned. In silico, the gene corresponding to the cDNA was localized on chromosome 1q23, close to the locus of hereditary prostate cancer 1 (HPC1) (1q24-25) and primary open-angle glaucoma (GLC1A) (1q23-25). Further, a pseudogene was found on chromosome 1q44, close to the locus of predisposing for early-onset prostate cancer (PCaP) (1q42.2-43). Both human (h17HSD7) and mouse 17HSD type 7 (m17HSD7) were for the first time produced as recombinant proteins and purified for functional analyses. Further, kinetic parameters and specific activities were described. h17HSD7 converted estrone (E1) to a more potent estrogen, estradiol (E2), and dihydrotestosterone (DHT), a potent androgen, to an estrogenic metabolite 5alpha-androstane-3beta, 17beta-diol (3betaA-diol) equally, thereby catalyzing the reduction of the keto group in either 17- or 3-position of the substrate. Minor 3betaHSD-like activity towards progesterone (P) and 20-hydroxyprogesterone (20-OH-P), leading to the inactivation of P by h17HSD7, was also detected. m17HSD7 efficiently catalyzed the reaction from E1 to E2 and moderately converted DHT to an inactive metabolite 5alpha-androstane-3alpha,17beta-diol (3alphaA-diol) and to an even lesser degree 3betaA-diol. The mouse enzyme did not metabolize P or 20-OH-P. The expression of 17HSD type 7 was observed widely in human tissues, most distinctly in adrenal gland, liver, lung, and thymus. Based on the enzymatic characteristics and tissue distribution, we conclude that h17HSD7 might be an intracrine regulator of steroid metabolism, fortifying the estrogenic milieu in peripheral tissues.

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

Enzymes with 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity catalyse reactions between the low-active female sex steroid, estrone, and the more potent estradiol, for example. 17 beta-HSD activity is essential for glandular (endocrine) sex hormone biosynthesis, but it is also present in several extra-gonadal tissues. Hence, 17 beta-HSD enzymes also take part in local (intracrine) estradiol production in the target tissues of estrogen action. Four distinct 17 beta-HSD isozymes have been characterized so far, and the data strongly suggests that different 17 beta-HSD isozymes have distinct roles in endocrine and intracrine metabolism of sex steroids. Current data suggest that 17 beta-HSD type 1 is the principal isoenzyme involved in glandular estradiol production both in humans and rodents. During ovarian follicular development and luteinization, rat 17 beta-HSD type 1 is regulated by gonadotropins, and the effects of gonadotropins are modulated by steroid hormones and paracrine growth factors. Human 17 beta-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, 17 beta-HSD type 1 is also expressed in certain target tissues of estrogen action such as normal and malignant human breast and endometrium. Hence, 17 beta-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 17 beta-HSD type 1 has a central role in regulating the circulating estradiol concentration as well as its local production in estrogen target cells.

Induction of ureter branching as a response to Wnt-2b signaling during early kidney organogenesis.

Epithelial–mesenchymal tissue interactions play a central role in vertebrate organogenesis, but the molecular mediators and mechanisms of these morphogenetic interactions are still not well characterized. We report here on the expression pattern of Wnt‐2b during mouse organogenesis and on tests of its function in epithelial– mesenchymal interactions during kidney development. Wnt‐2b is expressed in numerous developing organs in the mouse embryo, including the kidney, lung, salivary gland, gut, pancreas, adrenal gland, and genital tubercle. Additional sites of expression include the branchial arches and craniofacial placodes such as the eye and ear. The data suggest that the expression of Wnt‐2b is associated with organs regulated by epithelial–mesenchymal interactions. It is typically localized in the capsular epithelium or peripheral mesenchymal cells of organ rudiments, e.g., the perinephric mesenchymal cells in the region of the presumptive renal stroma in the developing kidney at E11.5. Functional studies of the kidney demonstrate that cells expressing Wnt‐2b are not capable of inducing tubule formation but instead stimulate ureter development. Incubation of isolated ureteric buds on such cells supports bud growth and branching. In addition, recombination of Wnt‐2b‐pretreated ureteric bud tissue with isolated nephrogenic mesenchyme results in a recovery of organogenesis and the expression of epithelial genes within the reconstituted organ explant. Lithium, a known activator of Wnt signaling (Hedgepeth et al. [ 1997 ] Dev Biol 185:82–91), is also sufficient to promote ureter branching in the reconstituted kidney in a comparable manner to Wnt‐2b signaling, whereas Wnt‐4, which induces tubules, neither supports the growth of a ureteric bud nor leads to reconstitution of the ureteric bud with the kidney mesenchyme. We conclude that Wnt‐2b may act in the mouse kidney as an early mesenchymal signal controlling morphogenesis of epithelial tissue, and that the Wnt pathway may regulate ureter branching directly. In addition, Wnt signals in the kidney differ qualitatively and are specific to either the epithelial ureteric bud or the kidney mesenchyme.

The Proximal Promoter Region of the Gene Encoding Human 17β-Hydroxysteroid Dehydrogenase Type 1 Contains GATA, AP-2, and Sp1 Response Elements: Analysis of Promoter Function in Choriocarcinoma Cells1

The 5′-flanking region from −78 to +9 in the HSD17B1 gene serves as a promoter, and an HSD17B1 silencer element is located in position −113 to −78. In the present studies, we have characterized three regulatory elements in the proximal 5′-flanking regions of the gene, using electrophoretic mobility shift assays and reporter gene analysis. First, nuclear factors recognized by antibodies against Sp1 and Sp3 were found to bind the Sp1 motif in the region from −52 to −43. Mutation of the Sp1-binding site decreased the promoter activity to 30% in JEG-3 cells and to 60% in JAR cells, suggesting that binding to the Sp1 motif has a substantial role in the complete functioning of the HSD17B1 promoter. Second, the binding of AP-2 to its motif in the region from −62 to −53 led to reduced binding of Sp1 and Sp3, and furthermore, mutation of the AP-2 element increased promoter activity to 260% in JEG-3 cells. The data thus implied that AP-2 can repress the function of the HSD17B1 promoter by preventing binding to the ...

Human familial and sporadic breast cancer : analysis of the coding regions of the 17β-hydroxysteroid dehydrogenase 2 gene (EDH17B2) using a single-strand conformation polymorphism assay

Abstract17β-Hydroxysteroid dehydrogenase (17HSD) is one of the key enzymes in estrogen metabolism, catalyzing the reversible reaction between estradiol and the less active estrogen, estrone. The gene encoding this enzyme, EDH17B2, has been mapped to chromosome 17, region q12–q21, in the vicinity of BRCA1, an as yet unidentified gene that appears to be involved in familial breast cancer and in familial ovarian cancer. The possibility that EDH17B2 gene is the same as BRCA1 was tested by screening for mutations in the coding regions of EDH17B2, using a polymerase chain reaction/single-strand conformation polymorphism method. An A→G transition creating a new BstUI site at exon 6 was the only frequent sequence alteration found in the coding region of the gene. This mutation also led to an amino acid substitution of serine to glycine at position 312 (312S→312G) in the 17HSD protein. Since the nucleotide change was detected both in specimens from patients with familial or sporadic cancer and in control samples, and at similar rates, this mutation appears to be of a polymorphic nature. In addition, a rare polymorphism located at intron 5 was detected. This C→T substitution creates a BbvI site and is not thought to have any effect on 17HSD activity. The results indicate that there are no major alterations in the coding areas of EDH17B2 and thus studies testing the hypothesis that EDH17B2 may be the same as BRCA1 should be extended to the promoter and regulatory elements of EDH17B2.

Two 17β-hydroxysteroid dehydrogenases (17HSDs) of estradiol biosynthesis: 17HSD type 1 and type 7☆

Two 17beta-hydroxysteroid dehydrogenases (17HSDs), type 1 and type 7, are enzymes of estradiol biosynthesis, in addition to which rodent type 1 enzymes are also able to catalyze androgens. Both of the 17HSDs are abundantly expressed in ovaries, the type 1 enzyme in granulosa cells and type 7 in luteinized cells. The expression of 17HSD7, which has also been described as a prolactin receptor-associated protein (PRAP), is particularly up-regulated in corpus luteum during the second half of rodent pregnancy. A moderate or slight signal for mouse 17HSD7/PRAP mRNA has also been demonstrated in samples of placenta and mammary gland, for example. Human, but not rodent, 17HSD1 is expressed in placenta, breast epithelium and endometrium in addition to ovaries. A cell-specific enhancer, silencer and promoter in the hHSD17B1 gene participate in the regulation of type 1 enzyme expression. The enhancer consists of several subunits, including a retinoic acid response element, the silencer has a binding motif for GATA factors, and the proximal promoter contains adjacent and competing AP-2 and Sp binding sites.

Retinoic Acids Increase 17β-Hydroxysteroid Dehydrogenase Type 1 Expression in JEG-3 and T47D Cells, but the Stimulation Is Potentiated by Epidermal Growth Factor, 12-O-Tetradecanoylphorbol-13-Acetate, and Cyclic Adenosine 3′,5′-Monophosphate Only in JEG-3 Cells1

Human 17β-hydroxysteroid dehydrogenase type 1 (17HSD type 1) primarily catalyzes the reduction of low activity estrone to high activity estradiol in ovarian granulosa cells and placental trophoblasts. 17HSD type 1 is also present in certain peripheral tissues, such as breast tissue. In the present study we investigated the effects of retinoic acids (RAs) together with other stimuli known to modulate estradiol production and/or cell growth on expression of 17HSD type 1 in JEG-3 choriocarcinoma cells and estrogen-responsive T47D breast cancer cells. Treatment of cultured JEG-3 and T47D cells with all-trans-RA and 9-cis-RA increased reductive 17HSD activity and 17HSD type 1 messenger RNA expression severalfold in both cell lines. On the other hand, epidermal growth factor (EGF), Ca ionophore, the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA), and cAMP elevated 17HSD type 1 expression only in JEG-3 cells. Correspondingly, the effects of RAs were potentiated by EGF, TPA, and cAMP in JEG...

Immunological measurement of human 17β-hydroxysteroid dehydrogenase

Abstract Human placental 17β-hydroxysteroid dehydrogenase (17-HSD) was purified to apparent homogeneity using ammonium sulfate precipitation and chromatography on Red-Agarose and DEAE-Sepharose columns. Electrophoresis on polyacrylamide gels under denaturing conditions and using silver staining showed a single protein with an apparent molecular weight of 37,800. Antibodies to the purified protein were raised in rabbits and were found by immunoblotting to be specific to 17-HSD. A sensitive radioimmunoassay was established using 125I-labeled 17-HSD as a tracer, an appropriate dilution of the antibody, and a kaolin-coupled double antibody for separating the antibody-bound and free fractions. The detection limit of the assay was approximately 150 pg/tube (1.5 μg/l). The cytosol fraction (105,000 g) of term placental tissue contained approximately 0.7 mg of 17-HSD per gram of protein, and the concentrations of 17-HSD measured by immunoassay and enzymatic activity proved to be strictly parallel in different partly purified placental preparations. The supernatants from centrifugations of human endometrial homogenates at 800 g and 105,000 g (after detergent treatment) displayed cross-reactivity with the antibody. The mean concentration of the cross-reacting substance in the radioimmunoassay was 14.1 μg/g protein (range 2–62.3) in specimens taken on different days in the cycle. These concentrations showed a significant correlation with the 17-HSD activities measured in the endometrial specimens (r = 0.722, P

Generation of an allele to inactivate Wnt4 gene function conditionally in the mouse.

The Wnt family member Wnt4 is critical for the development of several organs, including the kidney, gonad, and adrenal, mammary, and pituitary glands. To study its potential postnatal functions, we generated a floxed Wnt4 allele. A single loxP site was targeted to the second intron, while a loxP‐Neo‐loxP cassette was placed 3′ from Exon 5. The floxed Neo cassette was subsequently removed by crossing with MeuCre40 transgenic mice. The Wnt4 gene was specifically inactivated with CAGCre and another Wnt4 allele, Wnt4EGFPCre, in which the Cre is driven by the endogenous Wnt4 promoter. Deletion of Wnt4 gene function with CAGCre impaired kidney development, as is the case with the conventional knockout. Similarly, the Wnt4EGFPCre‐mediated inactivation of Wnt4 function considerably reduced the amount of Wnt4 transcripts, led to a severe defect in kidney development, and caused the female embryos to undergo partial sex reversal to males. All in all, the floxed Wnt4 allele serves as a useful tool for studying the roles of Wnt4 signaling during the life cycle. genesis 47:782–788, 2009.