Matti Poutanen

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.

Origin of Substrate Specificity of Human and Rat 17β-Hydroxysteroid Dehydrogenase Type 1, Using Chimeric Enzymes and Site-Directed Substitutions1

Human 17beta-hydroxysteroid dehydrogenase (17-HSD) type 1 predominantly catalyzes the 17beta-reduction of estrone to estradiol. The present results, however, show that rat 17-HSD type 1 equally uses both estrone and androstenedione as substrates. Analyzing the activity of various rat/human chimeric enzymes indicated that the region between amino acids 148 and 268 is responsible for the difference in substrate specificity, which is in line with the structural data showing that the recognition end of the active site is primarily at residues 185-230. The enzymes are highly conserved between amino acids 148-191, and the data indicate that in this region Asn152HisAsp153Glu and Pro187Ala variations are most closely related to the differential steroid specificity. The structural analyses furthermore suggested that the presence of His instead of Asn at position 152 of the human enzyme might result in considerable rearrangement of the loop located close to the beta-face of the A- and B-rings of the bound substrate, and that the Pro187Ala variation could modify the flexible region involved in substrate recognition and access of the substrate to the active site. Altogether, our results indicate that the Asn152His and Pro187Ala variations, together with several amino acid variations at the recognition end of the catalytic cleft built by residues 190-230, alter the structure of the active site of rat 17-HSD type 1 to one more favorable to an androgenic substrate.

17β-hydroxysteroid dehydrogenases in normal human mammary epithelial cells and breast tissue

Abstract17β‐hydroxysteroid dehydrogenase activity represents a group of several isoenzymes (17HSDs) that catalyze the interconversion between highly active 17β‐hydroxy‐ and low activity 17‐ketosteroids and thereby regulate the biological activity of sex steroids. The present study was carried out to characterize the expression of 17HSD isoenzymes in human mammary epithelial cells and breast tissue. In normal breast tissues 17HSD types 1 and 2 mRNAs were both evenly expressed in glandular epithelium. In two human mammary epithelial cell lines, mRNAs for 17HSD types 1, 2 and 4 were detected. In enzyme activity measurements only oxidative 17HSD activity, corresponding to either type 2 or type 4 enzyme, was present. The role of 17HSD type 4 in estrogen metabolism was further investigated, using several cell lines originating from various tissues. No correlation between the presence of 17HSD type 4 mRNA and 17HSD activity in different cultured cell lines was detected. Instead, oxidative 17HSD activity appeared in cell lines where 17HSD type 2 was expressed and reductive 17HSD activity was present in cells expressing 17HSD type 1. These data strongly suggest that in mammary epithelial cell lines the oxidative activity is due to type 2 17HSD and that oxidation of 17β‐hydroxysteroids is not the primary activity of the 17HSD type 4 enzyme.

Expression of 17β-Hydroxysteroid Dehydrogenase Type 1 and Type 2, P450 Aromatase, and 20α-Hydroxysteroid Dehydrogenase Enzymes in Immature, Mature, and Pregnant Rats1

In the present study, we evaluated the expression and regulation of 17β-hydroxysteroid dehydrogenase (17HSD) type 1 and type 2, cytochrome P450 aromatase (P450arom), and 20α-hydroxysteroid dehydrogenase (20HSD) in mature and pregnant rats. Immunohistochemical analysis of rat 17HSD type 1 showed that the enzyme is exclusively expressed in the granulosa cells of developing, healthy, primary, secondary, and tertiary follicles at all stages of the estrous cycle and pregnancy, and is not detected in the corpora lutea. The data showed that the amount of the enzyme expressed in the follicle increases as follicular maturation progresses and is highest in tertiary and Graafian follicles. However, Northern blot analysis of total RNA from whole ovaries showed a rather constitutive expression of the 17HSD type 1 enzyme. It is evident that compared with P450arom, 17HSD type 1 is more widely expressed in the follicles during the various maturational stages of folliculogenesis. Hence, the data indicate distinct localiza...

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.

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

17β‐hydroxysteroid dehydrogenase (17HSD) type 1 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 1 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 HSD‐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 1 in breast‐cancer cells may therefore be decisive with regard to estrogen exposure and the estrogen‐responsive growth of breast‐cancer tissues.

Ontogeny of 17β-hydroxysteroid dehydrogenase type 2 mRNA expression in the developing mouse placenta and fetus

17beta-Hydroxysteroid dehydrogenase type 2 (17HSD type 2) catalyzes the inactivation of estradiol, testosterone and dihydrotestosterone into biologically less active 17-keto forms. Our recent Northern analysis indicated that the enzyme is expressed both in mouse placenta and fetus. The present data indicate that in the placenta the distribution of enzyme expression changes during pregnancy. In the choriovitelline placenta (day 8) 17HSD type 2 was expressed both in mural and polar giant cells. Later, on days 9-12.5, the mRNA was also detected in the junctional zone, and in late gestation (days 14.5-17.5), 17HSD type 2 mRNA was predominantly expressed only at the labyrinth region. In the fetus, 17HSD type 2 expression appears in the liver on day 11. At day 12 the expression was strongly increased in the liver, and at the same time moderate mRNA expression was also detected in the esophagus and intestine. In these tissues, high constitutive expression of 17HSD type 2 was then maintained throughout pregnancy. At later stages of development (days 15-16) the mRNA was, furthermore, detected in epithelial cells of the stomach, tongue, oropharynx and nasopharynx as well as in the kidney. We conclude that the expression pattern of 17HSD type 2 in the developing placenta and fetus suggests a role for the enzyme in maintaining a barrier to the transfer of active 17-hydroxy forms of sex steroids between the fetus and maternal circulation.

Steroid Biosynthetic Enzymes: 17 β Hydroxysteroid Dehydrogenase

Polyclonal antibodies produced against human placental 17 β-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. Immunostalning was also present in cultured human granulosa cells and in about half of the endometrial and breast carcinoma specimens investigated. Prog...

Growth factors and phorbol-12-myristate-13-acetate modulate the follicle-stimulating hormone- and cyclic adenosine-3′,5′-monophosphate-dependent regulation of 17β-hydroxysteroid dehydrogenase type 1 expression in rat granulosa cells

Abstract In the present study primary cultures of rat granulosa cells obtained from diethylstilbestrol (DES)-primed immature rats were used to study the regulation of 17β-hydroxysteroid dehydrogenase (17HSD) activity and type 1 expression via protein kinase A (PKA)- and C (PKC)-dependent pathways, and by several autocrine and/or paracrine growth factors. Follicle-stimulating hormone (FSH), 8-bromo-cyclic adenosine-3′,5′-monophosphate (8-Br-cAMP) and transforming growth factor β1 (TGFβ1) strongly enhanced 17HSD activity and type 1 expression. The stimulatory effects of FSH and 8-Br-cAMP were further potentiated by TGFβ1. In contrast, neither phorbol-12-myristate-13-acetate (PMA), epidermal growth factor (EGF), transforming growth factor α (TGFα) nor fibroblast growth factor (bFGF) affected 17HSD activity or type 1 expression when given alone. However, they effectively neutralized the stimulatory effects of 8-Br-cAMP and FSH. The present data suggest that, in rat granulosa cells 17HSD type 1 expression is primarily induced by FSH acting via PKA-dependent pathway and the extent of the induction is modulated by PKC-dependent inhibition and autocrine/paracrine growth factors present in the ovary.

Activin-A, but not inhibin, regulates 17β-hydroxysteroid dehydrogenase type 1 activity and expression in cultured rat granulosa cells

17beta-Hydroxysteroid dehydrogenase type 1 (17HSD type 1) catalyzes the reduction of estrone (E(1)) to biologically more active estradiol (E(2)). In the present study, the effect of activin, inhibin, and follistatin on 17HSD activity and 17HSD type 1 expression in cultured, unluteinized rat granulosa cells was examined. Furthermore, the effects of these hormones on 17HSD type 1 expression were compared with the expression of P450 aromatase (P450arom). Rat granulosa cells were pre-incubated in serum-free media for 3 days, followed by a 2-day treatment with activin, inhibin, follistatin and 8-Br-cAMP. Activin in increasing concentrations appeared to effect a dose-dependent increase in 17HSD activity. In addition, increasing concentrations of activin also increased 17HSD type 1 mRNA expression. Addition of 8-Br-cAMP at concentrations of 0.25 and 1.5 mmol/l together with activin significantly augmented the stimulatory effects of activin alone in the cultured cells. Neither inhibin, nor follistatin, either alone or in combination with 8-Br-cAMP, had any notable effects on 17HSD activity and 17HSD type 1 expression. Preincubation of activin with increasing concentrations of follistatin significantly diminished the stimulatory effect of activin. In the presence of follistatin, activin did not significantly increase the 8-Br-cAMP-induced 17HSD activity and 17HSD type 1 expression. The culturing of granulosa cells in the presence or the absence of inhibin or follistatin with or without 8-Br-cAMP did not alter the effect of these peptides on P450arom expression in rat granulosa cells as judged by Northern blot analysis of total RNA. However, cAMP-induced P450arom expression was enhanced by activin treatment, except when follistatin was present. This is in line with the suggested role of follistatin as an activin-binding protein, which limits the bioavailability of activin to its membrane receptors. Thus, the results support the notion of a paracrine/autocrine role of activin in follicular steroidogenesis of growing follicles.