Identification of a novel distal control region upstream of the human steroidogenic acute regulatory protein (StAR) gene that participates in SF-1-dependent chromatin architecture.

StAR (steroidogenic acute regulatory protein) mediates the transport of cholesterol from the outer to the inner mitochondrial membrane, the process of which is the rate-limiting step for steroidogenesis. Transcriptional regulation of the proximal promoter of the human StAR gene has been well characterized, whereas analysis of its distal control region has not. Recently, we found that SF-1 (steroidogenic factor 1) induced the differentiation of mesenchymal stem cells (MSCs) into steroidogenic cells with the concomitant strong induction of StAR expression. Here, we show, using differentiated MSCs, that StAR expression is regulated by a novel distal control region. Using electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP) assays, we identified novel SF-1 binding sites between 3,000 and 3,400 bp upstream of StAR. A luciferase reporter assay revealed that the region worked as a strong regulator to exert maximal transcription of StAR. ChIP analysis of histone H3 revealed that upon SF-1 expression, nucleosome eviction took place at the SF-1 binding sites, not only in the promoter but also in the distal SF-1 binding sites. Chromosome conformation capture analysis revealed that the region upstream of StAR formed a chromatin loop both in the differentiated MSCs and in KGN cells, a human granulosa cell tumor cell line, where SF-1 is endogenously expressed. Finally, SF-1 knockdown resulted in disrupted formation of this chromatin loop in KGN cells. These results indicate that the novel distal control region participate in StAR activation through SF-1 dependent alterations of chromatin structure, including histone eviction and chromatin loop formation.

Cyp11b1 is induced in the murine gonad by luteinizing hormone/human chorionic gonadotropin and involved in the production of 11-ketotestosterone, a major fish androgen: conservation and evolution of the androgen metabolic pathway.

We have shown previously that Cyp11b1, an 11beta-hydroxylase responsible for glucocorticoid biosynthesis in the adrenal gland, was induced by cAMP in androgen-producing Leydig-like cells derived from mesenchymal stem cells. We found that Cyp11b1 was induced in male Leydig cells, or female theca cells, when human chorionic gonadotropin was administered in immature mice. Expression of Cyp11b1 in rodent gonads caused the production of 11-ketotestosterone (11-KT), a major fish androgen, which induces male differentiation or spermatogenesis in fish. As in teleosts, plasma concentrations of 11-KT were elevated in human chorionic gonadotropin-treated mice. In contrast to teleosts, however, plasma concentrations of 11-KT were similar in both sexes, despite levels of testosterone, a precursor substrate, being about 20 times higher in male mice. Because expression of 11beta-hydroxysteroid dehydrogenase type 2, was much higher in the mouse ovary than in the testis, conversion of testosterone into 11-KT may occur more efficiently in the ovary. In a luciferase reporter system that was responsive to and activated by androgens, 11-KT efficiently activated mammalian androgen receptor-mediated transactivation. Our results suggest that the androgen metabolic pathway is conserved between teleosts and mammals, despite sexual dominance and reproductive functions of 11-KT being altered during evolution.

Regulation of NGFI-B/Nur77 gene expression in the rat ovary and in leydig tumor cells MA-10.

NR4A1, also called NGFI-B in the rat, Nur77 in the mouse and TR3 in humans, belongs to the orphan nuclear steroid hormone receptor superfamily and is one of the immediate-early genes. In the endocrine organs, including the gonads, NGFI-B/Nur77 gene expression is rapidly induced by pituitary hormones. NGFI-B/Nur77 expression was found to be rapidly reduced by an estrogenic endocrine disrupter, diethylstilbestrol (DES) in theca interna cells of immature rat ovaries. DES treatment also triggered a rapid decrease of serum luteinizing hormone (LH) levels, suggesting that DES acts on the hypothalamo-pituitary axis to suppress LH secretion from the pituitary. The transcriptional regulation of NGFI-B/Nur77 by LH/human chorionic gonadotropin (hCG) or 8-bromoadenosine 3'-5'-cyclic monophosphate (8 Br-cAMP) was examined in mouse Leydig tumor cells MA-10. Luciferase assays using NGFI-B/Nur77 promoter constructs and electric mobility shift assays (EMSA) showed that NGFI-B/Nur77 gene expression was mediated through three of the four activator protein-1 (AP-1)-like sites, namely the -233 AP-1, -213 AP-1 and -69 AP-1 sites adjacent to the transcription start site of the NGFI-B/Nur77 promoter. We also demonstrated here that both the Jun family and cAMP-responsive element binding (CREB) proteins bind to the -233 AP-1 site, whereas the main binding protein to the -213 AP-1 site was CREB, and Jun family protein to the -69 AP-1 site, respectively. The rapid induction of NGFI-B/Nur77 gene expression by LH/hCG in MA-10 cells appears to be mediated by both CREB and Jun family proteins through the cAMP-protein kinase A (PKA) pathway.

Regulation of P450 oxidoreductase by gonadotropins in rat ovary and its effect on estrogen production.

BACKGROUND: P450 oxidoreductase (POR) catalyzes electron transfer to microsomal P450 enzymes. Its deficiency causes Antley-Bixler syndrome (ABS), and about half the patients with ABS have ambiguous genitalia and/or impaired steroidogenesis. POR mRNA expression is up-regulated when mesenchymal stem cells (MSCs) differentiate into steroidogenic cells, suggesting that the regulation of POR gene expression is important for steroidogenesis. In this context we examined the regulation of POR expression in ovarian granulosa cells by gonadotropins, and its possible role in steroidogenesis. METHODS: Changes in gene expression in MSCs during differentiation into steroidogenic cells were examined by DNA microarray analysis. Changes in mRNA and protein expression of POR in the rat ovary or in granulosa cells induced by gonadotropin treatment were examined by reverse transcription-polymerase chain reaction and western blotting. Effects of transient expression of wild-type or mutant (R457H or V492E) POR proteins on the production of estrone in COS-7 cells were examined in vitro. Effects of POR knockdown were also examined in estrogen producing cell-line, KGN cells. RESULTS: POR mRNA was induced in MSCs following transduction with the SF-1 retrovirus, and was further increased by cAMP treatment. Expression of POR mRNA, as well as Cyp19 mRNA, in the rat ovary were induced by equine chorionic gonadotropin and human chorionic gonadotropin. POR mRNA and protein were also induced by follicle stimulating hormone in primary cultured rat granulosa cells, and the induction pattern was similar to that for aromatase. Transient expression of POR in COS-7 cells, which expressed a constant amount of aromatase protein, greatly increased the rate of conversion of androstenedione to estrone, in a dose-dependent manner. The expression of mutant POR proteins (R457H or V492E), such as those found in ABS patients, had much less effect on aromatase activity than expression of wild-type POR proteins. Knockdown of endogenous POR protein in KGN human granulosa cells led to reduced estrone production, indicating that endogenous POR affected aromatase activity. CONCLUSION: We demonstrated that the expression of POR, together with that of aromatase, was regulated by gonadotropins, and that its induction could up-regulate aromatase activity in the ovary, resulting in a coordinated increase in estrogen production.

Diethylstilbestrol administration inhibits theca cell androgen and granulosa cell estrogen production in immature rat ovary.

Diethylstilbestrol (DES), a strong estrogenic compound, is well-known to affect the reproductive system. In this study, we investigated the effects of DES administration on gonadotropin levels and ovarian steroidogenesis in prepubertal rats. DES treatment acutely reduced serum LH levels, followed by a reduction in the expression of various steroidogenesis-related genes in theca cells. Serum FSH levels were almost unaffected by DES-treatment, even though Cyp19a1 expression was markedly reduced. Serum progesterone, testosterone and estradiol levels were also declined at this time. LH levels recovered from 12 h after DES-treatment and gradually increased until 96 h with a reduction of ERα expression observed in the pituitary. Steroidogenesis-related genes were also up-regulated during this time, except for Cyp17a1 and Cyp19a1. Consistent with observed gene expression pattern, serum testosterone and estradiol concentrations were maintained at lower levels, even though progesterone levels recovered. DES-treatment induced the inducible nitric oxide synthase (iNOS) in granulosa cells, and a nitric oxide generator markedly repressed Cyp19a1 expression in cultured granulosa cells. These results indicate that DES inhibits thecal androgen production via suppression of pituitary LH secretion and ovarian Cyp17a1 expression. In addition, DES represses Cyp19a1 expression by inducing iNOS gene expression for continuous inhibition of estrogen production in granulosa cells.

Allelic Imbalance of mRNA Associated with α2-HS Glycoprotein (Fetuin-A) Polymorphism.

Alpha 2-HS glycoprotein (AHSG), also designated as fetuin-A, exhibits polymorphism in population genetics consisting of two major alleles of AHSG(∗) 1 and AHSG(∗) 2. The serum level in the AHSG(∗) 1 homozygote is significantly higher than that of the AHSG(∗) 2 homozygote. This study examined the molecular mechanism for the cis-regulatory expression. To quantitate allele-specific mRNA in intra-assays of the heterozygote, RT-PCR method employing primers that were incorporated to the two closely located SNPs was developed. The respective magnitudes of AHSG(∗) 1 to AHSG(∗) 2 in the liver tissues and hepatic culture cells of PLC/PRF/5 were determined quantitatively as 2.5-fold and 6.2-fold. The mRNA expressional difference of two major alleles was observed, which is consistent with that in the serum level. The culture cells carried heterozygous genotypes in rs4917 and rs4918, but homozygous one in rs2248690. It was unlikely that the imbalance was derived from the SNP located in the promotor site. Furthermore, to investigate the effect of mRNA degradation, RNA synthesis in the cell culture was inhibited potently by the addition of actinomycin-D. No marked change was apparent between the two alleles. The results indicated that the cis-regulatory expressional difference is expected to occur at the level of transcription or splicing of mRNA.

Evaluation of the allele-sharing approach, known as the IBS method, in kinship analysis.

To infer relatedness from genetic data based on short tandem repeats, the exact method, in which shared allele frequencies are applied to relevant equations, has been conventionally used. An alternative approach is the IBS method that is based on the number of shared alleles between individuals. In the present study, the performance of the IBS method in pairwise kinship analysis was compared with the exact method using simulated data of 10,000 genotype pairs for 15 loci in the ABI Identifiler system. The likelihood ratio in allele-sharing of zero, one and two was calculated from joint probabilities based on allele frequencies of the Japanese population. Whereas the IBS method generally produced lower values of combined indices, smaller deviations of the distributions were evident. The threshold for identification of full siblings relative to non-relatives was comparable with that of the exact method, indicating that both inference powers were almost identical. The likelihood ratio in the IBS method depends on the heterozygosity at a locus, and heterozygosities of the 15 loci were consistent across various population groups, particularly in East Asians. The convenience of fixed LR values in the IBS method is beneficial for cases with uncertain allele frequencies and rare alleles.

Differentiation of mesenchymal stem cells and embryonic stem cells into steroidogenic cells using steroidogenic factor-1 and liver receptor homolog-1.

Previously, we have demonstrated that mesenchymal stem cells could be differentiated into steroidogenic cells through steroidogenic factor-1 and 8bromo-cAMP treatment. Use of liver receptor homolog-1, another of the nuclear receptor 5A family nuclear receptors, with 8bromo-cAMP also resulted in the differentiation of human mesenchymal stem cells into steroid hormone-producing cells. The same approaches could not be applied to other undifferentiated cells such as embryonic stem cells or embryonal carcinoma cells, because the over-expression of the nuclear receptor 5A family is cytotoxic to these cells. We established embryonic stem cells carrying tetracycline-regulated steroidogenic factor-1 gene at the ROSA26 locus. The embryonic stem cells were first differentiated into a mesenchymal cell lineage by culturing on collagen IV-coated dishes and treating with pulse exposures of retinoic acid before expression of steroidogenic factor-1. Although the untreated embryonic stem cells could not be converted into steroidogenic cells by expression of steroidogenic factor-1 in the absence of leukemia inhibitory factor due to inability of the cells to survive, the differentiated cells could be successfully converted into steroidogenic cells when expression of steroidogenic factor-1 was induced. They exhibited characteristics of adrenocortical-like cells and produced a large amount of corticosterone. These results indicated that pluripotent stem cells could be differentiated into steroidogenic cells by the nuclear receptor 5A family of protein via the mesenchymal cell lineage. This approach may provide a source of cells for future gene therapy for diseases caused by steroidogenesis deficiencies.

PPAR-gamma coactivator-1alpha regulates progesterone production in ovarian granulosa cells with SF-1 and LRH-1.

Previously, we demonstrated that bone marrow-derived mesenchymal stem cells (MSCs) differentiate into steroidogenic cells such as Leydig and adrenocortical cells by the introduction of steroidogenic factor-1 (SF-1) and treatment with cAMP. In this study, we employed the same approach to differentiate umbilical cord blood (UCB)-derived MSCs. Despite UCB-MSCs differentiating into steroidogenic cells, they exhibited characteristics of granulosa-luteal-like cells. We found that peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) was expressed and further induced by cAMP stimulation in UCB-MSCs. Consistent with these results, tissue-specific expression of Pgc-1alpha was observed in rat ovarian granulosa cells. PGC-1alpha binds to the NR5A family [SF-1 and liver receptor homolog-1 (LRH-1)] of proteins and markedly enhances their transcriptional activities. Reporter assays revealed that PGC-1alpha activated the promoter activities of SF-1 and LRH-1 target genes. Infection of KGN cells (a human cell line derived from granulosa cells) with adenoviruses expressing PGC-1alpha resulted in the induction of steroidogenesis-related genes and stimulation of progesterone production. PGC-1alpha also induced SF-1 and LRH-1, with the latter induced to a greater extent. Knockdown of Pgc-1alpha in cultured rat granulosa cells resulted in attenuation of gene expression as well as progesterone production. Transactivation of the NR5A family by PGC-1alpha was repressed by Dax-1. PGC-1alpha binds to the activation function 2 domain of NR5A proteins via its consensus LXXLL motif. These results indicate that PGC-1alpha is involved in progesterone production in ovarian granulosa cells by potentiating transcriptional activities of the NR5A family proteins.