Kaoru Miyamoto

Androgen/androgen receptor pathway regulates expression of the genes for cyclooxygenase-2 and amphiregulin in periovulatory granulosa cells☆

It is well known that the androgen/androgen receptor (AR) pathway is involved in both male and female fertility in mammals. AR knockout female mice are reported to exhibit various abnormalities in follicle development, and a subfertile phenotype. In exogenous gonadotropin-induced superovulation, serum androgen levels were robustly elevated in female mice at the periovulatory stage after human chorionic gonadotropin (hCG) treatment. At this stage, ovarian AR proteins were strongly expressed in cumulus cells. Because these results suggested that the androgen/AR pathway is involved in ovulation, we investigated the expression of ovulation-related genes in the mouse ovary treated with the nonaromatizable androgen, 5α-dihydrotestosterone (DHT). DHT treatment induced the expression of the genes for cyclooxyganase-2 (Cox-2 or prostaglandin endoperoxidase synthase 2) and the epidermal growth factor-like factor, amphiregulin (Areg), in the ovary, whereas their hCG-induced expression was suppressed by the AR antagonist flutamide. These genes were also induced by DHT in AR-expressing primary granulosa and granulosa tumor-derived cells. Reporter assays, electrophoretic shift mobility assays and chromatin immunoprecipitation assays demonstrated that androgen response sequence(s) existing upstream of each gene were responsible for androgen responsiveness and were occupied by the AR in periovulatory granulosa cells. Our results suggest that the androgen/AR pathway is involved in the ovulatory process via expression of the Cox-2 and Areg genes in periovulatory granulosa cells.

Human glutathione S-transferase A (GSTA) family genes are regulated by steroidogenic factor 1 (SF-1) and are involved in steroidogenesis

Steroidogenic factor 1 (SF‐1) is a master regulator for steroidogenesis. In this study, we identified novel SF‐1 target genes using a genome‐wide promoter tiling array and a DNA microarray. SF‐1 was found to regulate human glutathione S‐transferase A (GSTA) family genes (hGSTA1–hGSTA4), a superfamily of detoxification enzymes clustered on chromosome 6p12. All hGSTA genes were up‐regulated by transduction of SF‐1 into human mesenchymal stem cells, while knockdown of endogenous SF‐1 in H295R cells down‐regulated all hGSTA genes. Chromatin immunoprecipitation assays, however, revealed that SF‐1 bound directly to the promoters of hGSTA3 and weakly of hGSTA4. Chromosome conformation capture assays revealed that the coordinated expression of the genes was based on changes in higher‐order chromatin structure triggered by SF‐1, which enables the formation of long‐range interactions, at least between hGSTA1 and hGSTA3 gene promoters. In steroidogenesis, dehydrogenation of the 3‐hydroxy group and subsequent Δ5‐Δ4 isomerization are thought to be enzymatic properties of 3β‐hydroxysteroid dehydrogenase (3β‐HSD). Here, we demonstrated that, in steroidogenic cells, the hGSTA1 and hGSTA3 gene products catalyze Δ5‐Δ4 isomerization in a coordinated fashion with 3β‐HSD II to produce progesterone or Δ4‐androstenedione from their Δ5‐precursors. Thus, hGSTA1 and hGSTA3 gene products are new members of steroidogenesis working as Δ5‐Δ4 isomerases.—Matsumura, T., Imamichi, Y., Mizutani, T., Ju, Y., Yazawa, T., Kawabe, S., Kanno, M., Ayabe, T., Katsumata, N., Fukami, M., Inatani, M., Akagi, Y., Umezawa, A., Ogata, T., Miyamoto, K., Human glutathione S‐transferase A (GSTA) family genes are regulated by steroidogenic factor 1 (SF‐1) and are involved in steroidogenesis. FASEB J. 27, 3198–3208 (2013). www.fasebj.org

Transcriptional regulation of human ferredoxin reductase through an intronic enhancer in steroidogenic cells.

Ferredoxin reductase (FDXR, also known as adrenodoxin reductase) is a mitochondrial flavoprotein that transfers electrons from NADPH to mitochondrial cytochrome P450 enzymes, mediating the function of an iron-sulfur cluster protein, ferredoxin. FDXR functions in various metabolic processes including steroidogenesis. It is well known that multiple steroidogenic enzymes are regulated by a transcription factor steroidogenic factor-1 (SF-1, also known as Ad4BP). Previously, we have shown that SF-1 transduction causes human mesenchymal stem cell differentiation into steroidogenic cells. Genome-wide analysis of differentiated cells, using a combination of DNA microarray and promoter tiling array analyses, showed that FDXR is a novel SF-1 target gene. In this study, the transcriptional regulatory mechanism of FDXR was examined in steroidogenic cells. A chromatin immunoprecipitation assay revealed that a novel SF-1 binding region was located within intron 2 of the human FDXR gene. Luciferase reporter assays showed that FDXR transcription was activated through the novel SF-1 binding site within intron 2. Endogenous SF-1 knockdown in human adrenocortical H295R and KGN cells decreased FDXR expression. In H295R cells, strong binding of two histone markers of active enhancers, histones H3K27ac and H3K4me2, were detected near the SF-1 binding site within intron 2. Furthermore, the binding of these histone markers was decreased concurrent with SF-1 knockdown in H295R cells. These results indicated that abundant FDXR expression in these steroidogenic cells was maintained through SF-1 binding to the intronic enhancer of the FDXR gene.

Nuclear Receptor 5A (NR5A) Family Regulates 5-Aminolevulinic Acid Synthase 1 (ALAS1) Gene Expression in Steroidogenic Cells

5-Aminolevulinic acid synthase 1 (ALAS1) is a rate-limiting enzyme for heme biosynthesis in mammals. Heme is essential for the catalytic activities of P450 enzymes including steroid metabolic enzymes. Nuclear receptor 5A (NR5A) family proteins, steroidogenic factor-1 (SF-1), and liver receptor homolog-1 (LRH-1) play pivotal roles in regulation of steroidogenic enzymes. Recently, we showed that expression of SF-1/LRH-1 induces differentiation of mesenchymal stem cells into steroidogenic cells. In this study, genome-wide analysis revealed that ALAS1 was a novel SF-1-target gene in differentiated mesenchymal stem cells. Chromatin immunoprecipitation and reporter assays revealed that SF-1/LRH-1 up-regulated ALAS1 gene transcription in steroidogenic cells via binding to a 3.5-kb upstream region of ALAS1. The ALAS1 gene was up-regulated by overexpression of SF-1/LRH-1 in steroidogenic cells and down-regulated by knockdown of SF-1 in these cells. Peroxisome proliferator-activated receptor-γ coactivator-1α, a coactivator of nuclear receptors, also strongly coactivated expression of NR5A-target genes. Reporter analysis revealed that peroxisome proliferator-activated receptor-γ coactivator-1α strongly augmented ALAS1 gene transcription caused by SF-1 binding to the 3.5-kb upstream region. Finally knockdown of ALAS1 resulted in reduced progesterone production by steroidogenic cells. These results indicate that ALAS1 is a novel NR5A-target gene and participates in steroid hormone production.

C/EBPβ (CCAAT/enhancer-binding protein β) mediates progesterone production through transcriptional regulation in co-operation with SF-1 (steroidogenic factor-1)

The transcription factor SF-1 (steroidogenic factor-1) is a master regulator of steroidogenesis. Previously, we have found that SF-1 induces the differentiation of mesenchymal stem cells into steroidogenic cells. To elucidate the molecular mechanisms of SF-1-mediated functions, we attempted to identify protein components of the SF-1 nuclear protein complex in differentiated cells. SF-1 immunoaffinity chromatography followed by MS/MS analysis was performed, and 24 proteins were identified. Among these proteins, we focused on C/EBPβ (CCAAT/enhancer-binding protein β), which is an essential transcription factor for ovulation and luteinization, as the transcriptional mechanisms of C/EBPβ working together with SF-1 are poorly understood. C/EBPβ knockdown attenuated cAMP-induced progesterone production in granulosa tumour-derived KGN cells by altering STAR (steroidogenic acute regulatory protein), CYP11A1 (cytochrome P450, family 11, subfamily A, polypeptide 1) and HSD3B2 (hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 2) expression. EMSA and ChIP assays revealed novel C/EBPβ-binding sites in the upstream regions of the HSD3B2 and CYP11A1 genes. These interactions were enhanced by cAMP stimulation. Luciferase assays showed that C/EBPβ-responsive regions were found in each promoter and C/EBPβ is involved in the cAMP-induced transcriptional activity of these genes together with SF-1. These results indicate that C/EBPβ is an important mediator of progesterone production by working together with SF-1, especially under tropic hormone-stimulated conditions.

Transcriptional regulation of human ferredoxin 1 in ovarian granulosa cells.

Ferredoxin 1 (FDX1; adrenodoxin) is an iron-sulfur protein that is involved in various metabolic processes, including steroid hormone synthesis in mammalian tissues. We investigated the transcriptional regulation of FDX1 in ovarian granulosa cells. Previously, we reported that the NR5A family, including steroidogenic factor-1 (SF-1) and liver receptor homolog-1 could induce differentiation of human mesenchymal stem cells (hMSCs) into steroidogenic cells. A ChIP assay showed that SF-1 could bind to the FDX1 promoter in differentiated hMSCs. Luciferase reporter assays showed that transcription of FDX1 was synergistically activated by the NR5A family and 8Br-cAMP treatment through two SF-1 binding sites and a CRE-like sequence in a human ovarian granulosa cell line, KGN. Knockdown of FDX1 attenuated progesterone production in KGN cells. These results indicate transcription of FDX1 is regulated by the NR5A family and cAMP signaling, and participates in steroid hormone production in ovarian granulosa cells.

A Novel Isoform of Liver Receptor Homolog-1 Is Regulated by Steroidogenic Factor-1 and the Specificity Protein Family in Ovarian Granulosa Cells

Liver receptor homolog-1 (LRH-1) is a member of the nuclear receptor 5A (NR5A) subfamily. It is expressed in granulosa cells of the ovary and is involved in steroidogenesis and ovulation. To reveal the transcriptional regulatory mechanism of LRH-1, we determined its transcription start site in the ovary using KGN cells, a human granulosa cell tumor cell line. 5-rapid amplification of cDNA ends PCR revealed that human ovarian LRH-1 was transcribed from a novel transcription start site, termed exon 2o, located 41 bp upstream of the reported exon 2. The novel LRH-1 isoform was expressed in the human ovary but not the liver. Promoter analysis and an EMSA indicated that a steroidogenic factor-1 (SF-1) binding site and a GC box upstream of exon 2o were required for promoter activity, and that SF-1 and specificity protein (Sp)-1/3 bind to the respective regions in ovarian granulosa cells. In KGN cells, transfection of SF-1 increased ovarian LRH-1 promoter activity and SF-1-dependent reporter activity was further enhanced when peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) was cotransfected. In Drosophila SL2 cells, Sp1 was more effective than Sp3 in enhancing promoter activity, and co-transfection of the NR5A-family synergistically increased activity. Infection with adenoviruses expressing SF-1 or PGC-1α induced LRH-1 expression in KGN cells. These results indicate that the expression of human LRH-1 is regulated in a tissue-specific manner, and that the novel promoter region is controlled by the Sp-family, NR5A-family and PGC-1α in ovarian granulosa cells in a coordinated fashion.

Identification of novel steroidogenic factor 1 (SF-1)-target genes and components of the SF-1 nuclear complex.

Steroidogenic factor 1 (SF-1) is a master regulator of adrenal and reproductive development and function. Although SF-1 was identified as a transcriptional regulator for steroid metabolic enzymes, it has been shown that SF-1 also regulates other genes that are involved in various cellular processes. Previously, we showed that introduction of SF-1 into mesenchymal stem cells resulted in the differentiation of these cells to the steroidogenic lineage. By using this method of differentiation, we performed comprehensive analyses to identify the novel SF-1-target genes and components of the SF-1 nuclear complex. Genome-wide analyses with promoter tiling array and DNA microarray identified 10 genes as novel SF-1-target genes including glutathione S-transferase A family, 5-aminolevulinic acid synthase 1 and ferredoxin reductase. Using SF-1 immuno-affinity chromatography of nuclear proteins followed by MS/MS analysis, we identified 24 proteins including CCAAT/enhancer-binding protein β as components of SF-1 nuclear complex. In this review, we will describe novel roles of the newly identified genes for steroidogenesis.

Differentiation of mesenchymal stem cells into gonad and adrenal steroidogenic cells.

Hormone replacement therapy is necessary for patients with adrenal and gonadal failure. Steroid hormone treatment is also employed in aging people for sex hormone deficiency. These patients undergo such therapies, which have associated risks, for their entire life. Stem cells represent an innovative tool for tissue regeneration and the possibility of solving these problems. Among various stem cell types, mesenchymal stem cells have the potential to differentiate into steroidogenic cells both in vivo and in vitro. In particular, they can effectively be differentiated into steroidogenic cells by expressing nuclear receptor 5A subfamily proteins (steroidogenic factor-1 and liver receptor homolog-1) with the aid of cAMP. This approach will provide a source of cells for future regenerative medicine for the treatment of diseases caused by steroidogenesis deficiencies. It can also represent a useful tool for studying the molecular mechanisms of steroidogenesis and its related diseases.

Sex-determining region Y-box 2 and GA-binding proteins regulate the transcription of liver receptor homolog-1 in early embryonic cells

Pluripotent stem cells maintain their pluripotency and undifferentiated status through a network of transcription factors. Liver receptor homolog-1 (Lrh-1) is one of these, and regulates the expression of other important transcription factors such as Oct-3/4 and Nanog. In early embryo and embryonic stem (ES) cells, Lrh-1 is transcribed using a unique promoter. In this study, we investigated the transcriptional regulation of embryonic Lrh-1 using ES and embryonal carcinoma F9 cells. Reporter assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays demonstrated that Sox2 and Gabp proteins bind to the promoter region of embryonic Lrh-1, and are necessary for its activation. The Sox2 site showed strong promoter activity and affinity for protein binding. Upon differentiation into the parietal endoderm by retinoic acid and cAMP, Lrh-1 promoter activity and transcripts were markedly reduced within 24 h. At the same time, Sox2 and Gabp binding to the promoter region of Lrh-1 were decreased, followed by a reduction of their expression. These results indicate that embryonic Lrh-1 expression is regulated by both Sox2 and Gabp. Our study presents new insights into the transcription factor network of pluripotent stem cells.