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The function of the gene encoding human 17beta-hydroxysteroid dehydrogenase (17HSD) type 1, the hHSD17B1 gene, is regulated by a cell-specific enhancer at position -662 to -392. The adjacent hHSD17BP1 gene, whose function is not known, contains an analogous region in its 5'-flanking region. The identity between the hHSD17B1 enhancer and the hHSD17BP1 equivalent is as high as 98%, i.e. they differ by only five nucleotides. Results from reporter gene analyses showed that the hHSD17BP1 analog, a pseudoenhancer, has only 10% the activity of the hHSD17B1 enhancer. Furthermore, the results indicate that the reduced function of the pseudoenhancer is a consequence of the presence of G and A at positions -480 and -486, whereas the hHSD17B1 enhancer contains -480C and -486G. In addition, three protected areas were localized to regions -495/-485 (FP1), -544/-528 (FP2), and -589/-571 (FP3) in deoxyribonuclease I footprinting analysis of the hHSD17B1 enhancer. Replacement of the footprinted regions with a nonsense sequence demonstrated that the FP2 region is the most critical for enhancer activity. Mutations of FP2 or a short palindromic region within it led to almost complete abolishment of enhancer activity. We have identified several subelements that are essential for appropriate function of the hHSD17B1 enhancer. The results also show that the hHSD17B1 and hHSD17BP1 genes operate differently despite the high homology between them.

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.

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 Sp1 motif. Finally, GATA factors, GATA-3 in particular, were demonstrated to bind their cognate sequence in the HSD17B1 silencer region, and mutations introduced into the GATA-binding site increased transcriptional activity to the level seen in constructs not containing the silencer element. Thus, GATA-3 seems to prevent transcription in the constructs, and hence, the GATA motif also may operate as a negative control element for HSD17B1 transcription.

In the present study, we evaluated the expression and regulation of 17beta-hydroxysteroid dehydrogenase (17HSD) type 1 and type 2, cytochrome P450 aromatase (P450arom), and 20alpha-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 localization, expression, and regulation patterns for 17HSD type 1 and P450arom during the rat estrous cycle and pregnancy. Furthermore, compared with the two estradiol biosynthetic enzymes, a different expression pattern was detected for 20HSD messenger RNA. During the estrous cycle the enzyme was detected in the ovaries throughout the cycle, and in the ovaries of pregnant animals the enzyme showed an expression pattern the opposite of that observed for P450arom. Rat 17HSD type 2, not detected in the ovaries, was constitutively expressed in both female and male liver and small intestine in 21-day-old fetuses up to 6-week-old mature animals. Similarly, in these tissues the enzyme was constitutively expressed in normal cycling and pregnant animals, but it showed increasing expression in the placenta as pregnancy advanced. The relatively constitutive expression of the enzyme at all physiological stages of the animals suggests a general role for the enzyme in the inactivation of circulating sex steroids.

Human 17 beta-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-3 cells, whereas no such phenomenon was observed in T47D cells. In JEG-3 cells, simultaneous administration of RAs with TPA and EGF maximally resulted in approximately 40- and 20-fold increases in 17HSD type 1 messenger RNA expression, respectively. The present data indicate that RAs may stimulate estradiol biosynthesis by regulating 17HSD type 1 expression in certain breast cancer and choriocarcinoma cells. The results suggest that interaction of multiple regulatory pathways is involved in maintaining high 17HSD type 1 expression in the placenta. In addition, regulation of 17HSD type 1 expression may be different in trophoblast cells from that in breast epithelial cells.