Closing the gap: identification of human 3-ketosteroid reductase,the last unknown enzyme of mammalian cholesterol biosynthesis

The protein encoded by the HSD17B7 gene was originally described as a prolactin receptor-associated protein and as 17beta-hydroxysteroid dehydrogenase (HSD) type 7. Its ability to synthesize 17beta-estradiol in vitro has been reported previously. However, we demonstrate that HSD17B7 is the ortholog of the yeast 3-ketosteroid reductase Erg27p and converts zymosterone to zymosterol in vitro, using reduced nicotinamide adenine dinucleotide phosphate as cofactor. Expression of human and murine HSD17B7 in an Erg27p-deficient yeast strain complements the 3-ketosteroid reductase deficiency of the cells and restores growth on sterol-deficient medium. A fusion of HSD17B7 with green fluorescent protein is located in the endoplasmic reticulum, the site of postsqualene cholesterogenesis. Further critical evidence for a role of HSD17B7 in cholesterol metabolism is provided by the observation that its murine ortholog is a member of the same highly distinct embryonic synexpression group as hydroxymethyl-glutaryl-coenzyme A reductase, the rate-limiting enzyme of sterol biogenesis, and is specifically expressed in tissues that are involved in the pathogenesis of congenital cholesterol-deficiency disorders. We conclude that HSD17B7 participates in postsqualene cholesterol biosynthesis, thus completing the molecular cloning of all genes of this central metabolic pathway. In its function as the 3-ketosteroid reductase of cholesterol biosynthesis, HSD17B7 is a novel candidate for inborn errors of cholesterol metabolism.     

Prolactin receptor-associated protein/17beta-hydroxysteroid dehydrogenase type 7 gene (Hsd17b7) plays a crucial role in embryonic development and fetal survival

Our laboratory has previously cloned and purified a protein named PRAP (prolactin receptor-associated protein) that was shown to be a novel 17beta-hydroxysteroid dehydrogenase (HSD) enzyme with dual activity. This enzyme, renamed HSD17B7 or PRAP/17beta-HSD7, converts estrone to estradiol and is also involved in cholesterol biosynthesis. The major site of its expression is the corpus luteum of a great number of species including rodents and humans. To examine the functional significance of HSD17B7 in pregnancy, we generated a knockout mouse model with targeted deletions of exons 1-4 of this gene. We anticipated a mouse with a severe fertility defect due to its inability to regulate estrogen levels during pregnancy. The heterozygous mutant mice are normal in their development and gross anatomy. The females cycle normally, and both male and female are fertile with normal litter size. To our surprise, the breeding of heterozygous mice yielded no viable HSD17B7 null mice. However, we found HSD17B7 null embryo alive in utero on d 8.5 and d 9.5. By d 10.5, the fetuses grow and suffer from severe brain malformation and heart defect. Because the brain depends on in situ cholesterol biosynthesis for its development beginning at d 10, the major cause of fetal death appears to be due to the cholesterol synthetic activity of this enzyme. By ablating HSD17B7 function, we have uncovered, in vivo, an important requirement for this enzyme during fetal development.     

Functional analysis of cholesterol biosynthesis by RNA interference

Inborn errors of cholesterol biosynthesis caused by dysfunctionality of single enzymes are known to cause severe malformation syndromes like X-linked chondrodysplasia punctata (CDPX2), CHILD syndrome or Smith–Lemli–Opitz-syndrome (SLOS). In this study we established the method of RNA interference (RNAi) for analyzing the molecular mechanisms underlying disrupted cholesterol biosynthesis. For different genes involved in the cholesterol biosynthesis pathway-NAD(P) dependent steroid dehydrogenase-like (NSDHL), 17-beta hydroxysteroid dehydrogenase type 7 (HSD17B7) and emopamil binding protein (EBP)-shRNA sequences were designed and tested for their effectiveness. For a better comparability of the experiments and to avoid different transfection efficiencies, examined shRNA sequences which reached a knock down of at least 80% were stably transfected in a HeLa cell line with a tetracycline-regulated expression (HeLa T-REx). These stable transfected cell lines represent novel tools for the analysis of cholesterol biosynthesis.     

环腺苷一磷酸对人Ⅰ型17β羟类固醇脱氢酶在绒癌细胞系中表达的调节作用

由HSD17B1基因编码的人Ⅰ型17β-羟类固醇脱氢酶(17β-hydroxysteroid dehydrogenasetype 1,简称Ⅰ型17HSD)催化雌酮与雌二醇之间的转化。本文研究环腺苷一磷酸简称(cAM-P)对该酶在培养的绒癌细胞系(JAR和JEG-3)中表达的调节作用。用8-bromo-cAMP处理两种绒癌细胞后,观察到在伴随1.3 kbⅠ型17 HSDmRNA表达的同时,Ⅰ型17 HSD蛋白浓度也显著上升。标记基因分析表明,cAMP可诱导HSD 17 B1基因启动子在JAR和JEG-3细胞系中的转录活性,参与调节这一诱导作用的区域位于HSD 17 B1基因编码区上游-659至-550处。凝胶阻滞实验显示这一区域可同JAR、JEG-3、T-47 D和HeLa细胞核抽提物形成特异的DNA-蛋白复合物。本结果首次证实cAMP激活HSD 17 B1基因启动子在绒癌细胞中的转录。     

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

Two 17β-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.     

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

Two 17β-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.     

Sex steroid hormone metabolism takes place in human ocular cells

Steroids are potentially important mediators in the pathophysiology of ocular diseases. In this study, we report on the gene expression in the human eye of a group of enzymes, the 17beta-hydroxysteroid dehydrogenases (17HSDs), involved in the biosynthesis and inactivation of sex steroid hormones. In the eye, the ciliary epithelium, a neuroendocrine secretory epithelium, co-expresses the highest levels of 17HSD2 and 5 mRNAs, and in lesser level 17HSD7 mRNA. The regulation of gene expression of these enzymes was investigated in vitro in cell lines, ODM-C4 and chronic open glaucoma (GCE), used as cell models of the human ciliary epithelium. The estrogen, 17beta-estradiol (10(-7) M) and androgen agonist, R1881 (10(-8) M) elicited in ODM-C4 and GCE cells over a 24 h time course a robust up-regulation of 17HSD7 mRNA expression. 17HSD2 was up-regulated by estradiol in ODM-C4 cells, but not in GCE cells. Under steady-state conditions, ODM-C4 cells exhibited a predominant 17HSD2 oxidative enzymatic activity. In contrast, 17HSD2 activity was low or absent in GCE cells. Our collective data suggest that cultured human ciliary epithelial cells are able to metabolize estrogen, androgen and progesterone, and that 17HSD2 and 7 in these cells are sex steroid hormone-responsive genes and 17HSD7 is responsible to keep on intra/paracrine estrogenic milieu.     

Divergent interactions involving the oxidosqualene cyclase and the steroid-3-ketoreductase in the sterol biosynthetic pathway of mammals and yeasts

In mammals and yeasts, oxidosqualene cyclase (OSC) catalyzes the formation of lanosterol, the first cyclic intermediate in sterol biosynthesis. We used a murine myeloma cell line (NS0), deficient in the 17β-hydroxysteroid dehydrogenase type 7 (HSD17B7), as a model to study the potential interaction of the HSD17B7 with the OSC in mammals. HSD17B7 is the orthologue of the yeast steroid-3-ketoreductase (ERG27), an enzyme of ergosterol biosynthesis that plays a protective role towards OSC. Tracer experiments with NS0 cells showed that OSC is fully active in these mammalian cells, suggesting that in mammals the ketosteroid reductase is not required for OSC activity. Mouse and human HSD17B7 were overexpressed in ERG27-deletant yeast cells, and recombinant strains were tested for (i) the ability to grow on different media, (ii) steroid-3-ketoreductase activity, and (iii) OSC activity. Recombinant strains grew more slowly than the control yeast ERG27-overexpressing strain on sterol-deficient media, whereas the growth rate was normal on media supplemented with a 3-ketoreductase substrate. The full enzymatic functionality of mammalian steroid-3-ketoreductase expressed in yeast along with the lack of (yeast) OSC activity point to an inability of the mammalian reductase to assist yeast OSC. Results demonstrate that in mammals, unlike in yeast, OSC and steroid-3-ketoreductase are non-interacting proteins.     

Lack of association between common polymorphisms in the 17beta-hydroxysteroid dehydrogenase type V gene (HSD17B5) and precocious pubarche

BACKGROUND: 17beta-Hydroxysteroid dehydrogenase (type V; HSD17B5) is a key enzyme involved in testosterone production in females. A single nucleotide polymorphism (SNP) in the promoter region of its gene was recently found to be associated with polycystic ovary syndrome (PCOS) and its related hyperandrogenaemia. Precocious pubarche (PP) is a clinical entity pointing to adrenal androgen excess from mid-childhood onward and is associated with ovarian androgen excess from puberty onward. It is therefore a strong risk factor for PCOS. METHODS: To investigate associations between this promoter SNP along with three exonic SNPs (one non-synonymous and two synonymous) from the same gene, and PP, a case-control study was performed in 190 girls with PP (84 of which were also tested for functional ovarian hyperandrogenism) from Barcelona, Spain and 71 healthy controls. Clinical features and hormone concentrations relevant to hyperandrogenism were compared by HSD17B5 genotype and haplotype. RESULTS: Neither HSD17B5 genotypes nor haplotype were associated with PP, or subsequent androgen excess in girls from Barcelona (all P>0.05). CONCLUSIONS: HSD17B5 SNPs predicted to have functional effects do not appear to be a risk factor for PP in girls from Barcelona, despite these girls being at high risk of developing androgen excess in adulthood.     

Polymorphism of the pig 17beta-hydroxysteroid dehydrogenase type1 (HSD17B1) gene and its association with reproductive traits

17beta-Hydroxysteroid dehydrogenase type 1 (HSD17B1) is a key enzyme of 17beta-estradiol biosynthesis, which might play an important role in follicular development of the ovary. In this study, we isolated the complete coding sequence of porcine HSD17B1 gene and its unique intron sequences of porcine HSD17B1 gene, identified a single nucleotide polymorphism (SNP: A/C) in intron 4, and developed a PCR-MvaI-RFLP genotyping assay. Association of the SNP and litter size was assessed in two populations (purebred Large White and a experimental synthetic Line (DIV) sows). Statistical analysis demonstrated that, in the first parity, AC animals in experimental synthetic Line (DIV) sows had 0.52 more piglets born compared to the CC animals (P<0.05). In the all parities, pigs with the AA genotype had an additional 1.11 and 0.96 piglets born alive compared to the CC animals (P<0.05) in both experimental synthetic Line (DIV) and purebred Large White, respectively. Experimental synthetic Line (DIV) sows inheriting the AC genotype had additional 0.84 piglets born alive compared to the CC animals (P<0.01) in all parities. In addition, significant additive effect of -0.55+/-0.24 piglets/litter and -0.48+/-0.22 piglets/litter on piglet born alive was detected in both experimental synthetic Line (DIV) sows and purebred Large White lines (P<0.05), respectively. Therefore, HSD17B1 gene was significantly associated with litter size in two populations and could be a useful molecular marker in selection for increasing litter size in pigs.     

Activation of androgens by hydroxysteroid (17beta) dehydrogenase 1 in vivo as a cause of prenatal masculinization and ovarian benign serous cystadenomas

Hydroxysteroid (17beta) dehydrogenases (HSD17Bs) belong to the short-chain dehydrogenase/reductase family consisting of a diverse pool of enzymes with oxidoreductase activity. HSD17B enzymes catalyze the conversion between 17-keto and 17-hydroxy steroids, either activating or inactivating sex steroids. Previous studies have demonstrated a role for human HSD17B1 enzyme in estradiol (E2) biosynthesis both in gonads and extragonadal steroid target tissues and various estrogen-dependent diseases. In the present study, five transgenic (TG) mouse lines universally overexpressing human HSD17B1 were generated and characterized at fetal and adult ages, especially to study the enzyme function in vivo. Activity measurements in vivo indicated that in addition to activating estrone to E2, the enzyme is able to significantly reduce androstenedione to testosterone, and TG females presented increased testosterone concentration preceding birth. As a consequence, TG females suffered from several phenotypic features typical to enhanced fetal androgen exposure. Furthermore, the ovaries developed androgen-dependent ovarian benign serous cystadenomas at adulthood. Androgen dependency of the phenotypes was confirmed by rescuing them by antiandrogen treatment, or by transplanting wild-type ovaries to the TG females. In conclusion, the data evidently show that, in addition to activating estrone to E2, human HSD17B1 enhances androgen action in vivo. Thus, the relative amounts of androgenic and estrogenic substrates available partially determine the physiological function of the enzyme in vivo. The novel function observed for human HSD17B1 is likely to open new possibilities also for the use of HSD17B1-inhibitors as drugs against androgen-related dysfunctions in females.