Takashi Yazawa

PPAR-γ Coactivator-1α 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.

Early Growth Response Gene-1 Regulates the Expression of the Rat Luteinizing Hormone Receptor Gene

Abstract LH receptor gene expression is primarily regulated via specific interactions of trans-acting proteins and cis-acting DNA sequences in the upstream region of the gene. In this study, we report, using luciferase assays, that the region between −171 and −137 base pairs (bp) is essential for basal expression of the rat LH receptor gene. To identify factors that interact with the region between −171 and −137 bp and regulate expression of the gene, a rat granulosa cell cDNA library was screened using a yeast one-hybrid system. A positive clone, isolated by the screening, encodes a transcription factor early growth response gene-1 (Egr-1). To determine the sequence to which Egr-1 protein binds, electrophoretic mobility shift assay (EMSA) was employed. The Egr-1 protein was produced by an in vitro transcription/translation system using a full-length rat Egr-1 cDNA. The upstream region between −171 and −137 bp contains 2 overlapping Egr-1 consensus sequences. The EMSA revealed that Egr-1 binds independently to both sites. The overexpression of Egr-1 in MA-10 cells caused an approximately 2-fold increase in reporter luciferase activity. However, no induction of the luciferase activity was observed when luciferase constructs that lacked or had mutations in either or both of the Egr-1 sites were used, indicating that Egr-1 positively regulates LH receptor gene expression. In differentiated granulosa cells that had been pretreated with FSH for 48 h, the levels of both mRNA and Egr-1 protein were induced by hCG or cAMP, reaching maximal levels approximately 1.5 h after treatment and then returning to basal levels 8 h thereafter. No Egr-1 mRNA or protein was detected in undifferentiated granulosa cells, even after stimulation with 8-bromoadenosine-cAMP. These results suggest that Egr-1 functions only in luteinized granulosa cells after stimulation with hCG or cAMP. In conclusion, the findings demonstrate that Egr-1 actually binds to the regulatory upstream region of the LH receptor gene and positively regulates receptor gene expression. In addition, Egr-1 expression was observed only in luteinized granulosa cells after stimulation with hCG or cAMP. The present study provides further support to the hypothesis that Egr-1 plays important roles in the pituitary-gonadal axis.

Functional analysis and the molecular dissection of zinc-fingers and homeoboxes 1 (ZHX1).

Zinc-fingers and homeoboxes 1 (ZHX1) is a protein that interacts with the activation domain of the A subunit of nuclear factor-Y. The function of ZHX1, as a transcription factor, was characterized and their domains were mapped. To determine the nuclear localization signal, expression vectors, in which various truncated forms of ZHX1 were fused to the C-terminal of green fluorescence protein (GFP), were transfected into human embryonic kidney (HEK) 293 cells. All GFP-ZHX1 fusion proteins including an arginine-rich region that corresponds to the amino acid sequence between 734 and 768 were localized in the nuclei. A dimerization domain of the ZHX1 was also mapped using protein-protein interaction assays. The homeodomain (HD) 1 consisting of the amino acid sequence between 272 and 432 of ZHX1 was necessary and sufficient for dimerization. Lastly, the transcriptional activity of ZHX1 was examined using a mammalian one-hybrid system. ZHX1, fused to the C-terminal of the GAL4 DNA-binding domain, was co-transfected with luciferase reporter plasmids with or without five copies of the GAL4-binding site into HEK293 cells. The luciferase activity was decreased in both concentration- and GAL4-binding site-dependent manner. The acidic region corresponding to the amino acid sequence between 831 and 873 was a repressor domain and dimerization was prerequisited for full repressor activity.

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. Mol. Reprod. Dev. 75: 931–939, 2008.

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.

Rat zinc-fingers and homeoboxes 1 (ZHX1), a nuclear factor-YA-interacting nuclear protein, forms a homodimer

Zinc-fingers and homeoboxes 1 (ZHX1) is a protein which interacts with the activation domain of the A subunit of nuclear factor-Y. To analyze the physiological role(s) of ZHX1, we searched ZHX1-interacting protein(s) using a yeast two-hybrid system. The rat counterpart of ZHX1 cDNAs was cloned from an ovarian granulosa cell complementary DNA (cDNA) library, indicating that ZHX1 is able to form a homodimer. An analysis of the nucleotide sequence and its deduced amino acid sequence show that rat ZHX1 consists of 873 amino acid residues. Northern blot analysis shows that ZHX1 messenger RNA is expressed ubiquitously and that the level in the ovary are not regulated by gonadotropins. Furthermore, transfection experiments with green fluorescence protein (GFP) expression vectors into human embryonic kidney HEK293 cells reveal that full-length ZHX1 fused to the GFP is localized in the nuclei. Thus, we report on the molecular cloning, expression and characterization of full-length rat ZHX1 cDNA.

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

Cloning and Functional Expression of an E Box-Binding Protein from Rat Granulosa Cells

Abstract Ovarian granulosa cells undergo cell growth and cytodifferentiation during follicular maturation. In a number of tissues, the gene expression that is responsible for the cytodifferentiation is largely dependent on E box(es) located upstream of the responsible genes. In this study, we report on the cloning of cDNA(s) encoding E box (5′-CACGTG-3′)-binding protein from a rat granulosa cell cDNA library using a yeast one-hybrid system. When multiple E box sequences were used as target, we obtained a positive clone that encodes the rat homologue of upstream stimulatory factor 2 (USF2). An analysis of the nucleotide sequence and its deduced amino acid sequence reveals that rat USF2 protein consists of 346 amino acid residues and belongs to the basic helix-loop-helix/leucine zipper protein family. Northern blot analysis shows that rat USF2 mRNA exists as multiple forms between 1.6 and 2.2 kilobases. The size of the cloned insert was identical to that of the transcript of maximal length. Electrophoretic mobility shift assays showed that in vitro-translated rat USF2 specifically binds to the E box. In addition, cotransfection experiments with luciferase-reporter constructs in HepG2 cells reveal that the overexpression of rat USF2 leads to an increase of luciferase activity in the E box sequence-dependent manner. Thus, we report molecular cloning, expression, and functional characterization of full-length rat USF2 cDNA.

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.

Gene Expression of Basic Helix-Loop-Helix Transcription Factor, SHARP-2, Is Regulated by Gonadotropins in the Rat Ovary and MA-10 Cells

Abstract Basic helix-loop-helix (bHLH) proteins regulate transcription from the E box sequence (5′-CANNTG-3′) located in the regulatory region of most gene promoters. The rat enhancer of split- and hairy-related protein 2 (SHARP-2) is a member of the bHLH protein family. To analyze the possible role of SHARP-2 in the rat ovary, the regulation of the expression of the SHARP-2 gene was examined, and the SHARP-2 protein was characterized. Northern blot analysis revealed that the level of SHARP-2 mRNA abruptly and temporarily increases as the result of the action of LH, i.e., eCG or hCG treatment alone or hCG after eCG treatment, in the rat ovary, as indicated by the treatment of primary cultured rat granulosa cells with hCG after FSH treatment or of mouse Leydig MA-10 cells with hCG or 8-bromoadenosine 3′,5′-cyclic monophosphate. An in situ hybridization analysis showed that eCG treatment increases the level of the SHARP-2 transcript in theca interna cells and that hCG treatment, after the administration of eCG, increases the level of the SHARP-2 transcript in granulosa cells. Furthermore, transfection experiments with green fluorescence protein (GFP) expression vectors into primary cultured granulosa cells and MA-10 cells revealed that the entire coding sequence of SHARP-2 fused to the GFP is localized in the nucleus. The transcriptional activity of SHARP-2 also was examined using transient DNA transfection experiments. When an expression vector encoding the full length of SHARP-2 was cotransfected with thymidine kinase promoter-luciferase reporter plasmids, with or without E box sequences, into MA-10 cells, the luciferase activity was decreased in an E box-dependent manner. We conclude that the level of SHARP-2 mRNA is regulated by gonadotropins and that SHARP-2 functions as a transcriptional repressor localized in the nucleus.