Inhibition of YAP/TAZ-TEAD activity induces cytotrophoblast differentiation into syncytiotrophoblast in human trophoblast.

During placentation, placental cytotrophoblast (CT) cells differentiate into syncytiotrophoblast (ST) cells and extravillous trophoblast (EVT) cells. In the placenta, the expression of various genes is regulated by the Hippo pathway through a transcription complex, Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ)-TEA domain transcription factor (TEAD) (YAP/TAZ-TEAD) activity. YAP/TAZ-TEAD activity is controlled by multiple factors and signaling, such as cAMP signaling. cAMP signaling is believed to be involved in the regulation of trophoblast function but is not yet fully understood. Here we showed that YAP/TAZ-TEAD expressions and their activities were altered by cAMP stimulation in BeWo cells, a human choriocarcinoma cell line. The repression of YAP/TAZ-TEAD activity induced the expression of ST-specific genes without cAMP stimulation, and transduction of constitutively active YAP, i.e. YAP-5SA, resulted in the repression of 8Br-cAMP-induced expressions of ST-specific genes in a TEAD-dependent manner. We also investigated the role of YAP/TAZ-TEAD in maintaining CT cells and their differentiation into ST and EVT cells using human trophoblast stem (TS) cells. YAP/TAZ-TEAD activity was involved in maintaining the stemness of TS cells. Induction or repression of YAP/TAZ-TEAD activity resulted in marked changes in the expression of ST-specific genes. Using primary CT cells, which spontaneously differentiate into ST-like cells, the effects of YAP-5SA transduction were investigated, and the expression of ST-specific genes was found to be repressed. These results indicate that the inhibition of YAP/TAZ-TEAD activity, with or without cAMP stimulation, is essential for the differentiation of CT cells into ST cells.

Development of a novel and rapid measurement system for growth differentiation factor-15, progranulin, and osteopontin in uterine sarcoma.

Uterine sarcomas are rare and aggressive gynecologic tumors with poor prognosis; therefore, early diagnosis is crucial for therapy. However, it is very difficult to distinguish uterine sarcomas from leiomyomas which are common benign uterine tumors. Therefore, the development of a diagnostic method that utilizes reliable biomarkers to distinguish uterine sarcomas from leiomyomas is important so as to identify the rare tumors. The candidate factors as novel biomarkers were searched for in public databases and a pilot study was performed for confirmation. Growth differentiation factor-15 (GDF15), progranulin, and osteopontin were identified as candidate biomarkers for diagnosing uterine sarcoma. Thus, developing a rapid and easy method to measure these factors could help establish a screening system for uterine sarcomas. In this study, we developed a novel measurement system for these factors using a compact chemical luminescence immunological automatic analyzer POCubeTM. This assay system, which is based on the flow-through membrane immunoassay, completes the whole process and generates results within 15 min. Serum concentrations of these factors measured via POCubeTM correlated well with those measured using enzyme-linked immunosorbent assay (r = 0.994 for GDF15, r = 0.992 for progranulin, and r = 0.976 for osteopontin). The POCubeTM system provides rapid and easy measurement of these factors, thereby facilitating uterine sarcoma diagnosis.

Transcriptional Regulation of Ovarian Steroidogenic Genes: Recent Findings Obtained from Stem Cell-Derived Steroidogenic Cells.

Ovaries represent one of the primary steroidogenic organs, producing estrogen and progesterone under the regulation of gonadotropins during the estrous cycle. Gonadotropins fluctuate the expression of various steroidogenesis-related genes, such as those encoding steroidogenic enzymes, cholesterol deliverer, and electronic transporter. Steroidogenic factor-1 (SF-1)/adrenal 4-binding protein (Ad4BP)/NR5A1 and liver receptor homolog-1 (LRH-1) play important roles in these phenomena via transcriptional regulation. With the aid of cAMP, SF-1/Ad4BP and LRH-1 can induce the differentiation of stem cells into steroidogenic cells. This model is a useful tool for studying the molecular mechanisms of steroidogenesis. In this article, we will provide insight into the transcriptional regulation of steroidogenesis-related genes in ovaries that are revealed from stem cell-derived steroidogenic cells. Using the cells derived from the model, novel SF-1/Ad4BP- and LRH-1-regulated genes were identified by combined DNA microarray and promoter tiling array analyses. The interaction of SF-1/Ad4BP and LRH-1 with transcriptional regulators in the regulation of ovarian steroidogenesis was also revealed.

Cyclooxygenase-2 is acutely induced by CCAAT/enhancer-binding protein ß to produce prostaglandin E 2 and F 2α following gonadotropin stimulation in Leydig cells.

Cyclooxygenase 2 (COX-2) is an inducible rate-limiting enzyme for prostanoid production. Because COX-2 represents one of the inducible genes in mouse mesenchymal stem cells upon differentiation into Leydig cells, we investigated COX-2 expression and production of prostaglandin (PG) in Leydig cells. Although COX-2 was undetectable in mouse testis, it was transiently induced in Leydig cells by human chorionic gonadotropin (hCG) administration. Consistent with the finding that Leydig cells expressed aldo-keto reductase 1B7 (PGF synthase) and PGE synthase 2, induction of COX-2 by hCG caused a marked increase in testicular PGF 2α and PGE 2 levels. Using mouse Leydig cell tumor-derived MA-10 cells as a model, it was indicated by reporter assays and electron mobility shift assays that transcription of the COX-2 gene was activated by CCAAT/enhancer-binding protein ß (C/EBPß) with cAMP-stimulation. C/EBPß expression was induced by cAMP-stimulation, whereas expression of C/EBP homolog protein (CHOP) was robustly downregulated. Transfection of CHOP expression plasmid inhibited cAMP-induced COX-2 promoter activity. In addition, CHOP reduced constitutive COX-2 expression in other mouse Leydig cell tumor-derived TM3 cells. These results indicate that COX-2 is induced in Leydig cells by activation of C/EBPß via reduction of CHOP expression upon gonadotropin-stimulation to produce PGF 2α and PGE 2 .

Angiotensin II promotes pulmonary metastasis of melanoma through the activation of adhesion molecules in vascular endothelial cells.

Hypertension is considered as one of the cancer progressive factors, and often found comorbidity in cancer patients. Renin-angiotensin system (RAS) plays an important role in the regulation of blood pressure, and angiotensin II (Ang II) is well known pressor peptide associated with RAS. Ang II has been reported to accelerate progression and metastasis of cancer cells. However, its precise mechanisms have not been fully understood. In this study, we sought to elucidate the mechanisms by which Ang II exacerbates hematogenous metastasis in mouse melanoma cells, focusing the adhesion pathway in vascular endothelial cells. For this purpose, B16/F10 mouse melanoma cells, which do not express the Ang II type 1 receptor (AT1R), were intravenously injected into C57BL/6 mice. Two weeks after cell injection, the number of lung metastatic colonies was significantly higher in the Ang II-treated group (1 µg/kg/min) than in the vehicle-treated group. The AT1R blocker valsartan (40 mg/kg/day), but not the calcium channel blocker amlodipine (5 or 10 mg/kg/day), significantly suppressed the effect of Ang II. In endothelium-specific Agtr1a knockout mice, Ang II-mediated acceleration of lung metastases of melanoma cells was significantly diminished. Ang II treatment significantly increased E-selectin mRNA expression in vascular endothelial cells collected from lung tissues, and thus promoted adherence of melanoma cells to the vascular endothelium. Ang II-accelerated lung metastases of melanoma cells were also suppressed by treatment with anti-E-selectin antibody (20 mg/kg). Taken together, Ang II-treatment exacerbates hematogenous cancer metastasis by promoting E-selectin-mediated adhesion of cancer cells to vascular endothelial cells.

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.

Induction of steroidogenic cells from adult stem cells and pluripotent stem cells [Review].

Steroid hormones are mainly produced in adrenal glands and gonads. Because steroid hormones play vital roles in various physiological processes, replacement of deficient steroid hormones by hormone replacement therapy (HRT) is necessary for patients with adrenal and gonadal failure. In addition to HRT, tissue regeneration using stem cells is predicted to provide novel therapy. Among various stem cell types, mesenchymal stem cells can be differentiated into steroidogenic cells following ectopic expression of nuclear receptor (NR) 5A subfamily proteins, steroidogenic factor-1 (also known as adrenal 4 binding protein) and liver receptor homolog-1, with the aid of cAMP signaling. Conversely, these approaches cannot be applied to pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, because of poor survival following cytotoxic expression of NR5A subfamily proteins. However, if pluripotent stem cells are first differentiated through mesenchymal lineage, they can also be differentiated into steroidogenic cells via NR5A subfamily protein expression. This approach offers a potential suitable cells for future regenerative medicine and gene therapy for diseases caused by steroidogenesis deficiencies. It represents a powerful tool to investigate the molecular mechanisms involved in steroidogenesis. This article highlights our own and current research on the induction of steroidogenic cells from various stem cells. We also discuss the future direction of their clinical application.

11-Ketotestosterone Is a Major Androgen Produced in Human Gonads.

CONTEXT: 11-ketotestosterone (11-KT) is a novel class of active androgen. However, the detail of its synthesis remains unknown for humans. OBJECTIVE: The objective of this study was to clarify the production and properties of 11-KT in human. Design, Participants, and Methods: Expression of cytochrome P450 and 11ß-hydroxysteroid dehydrogenase types 1 and 2 (key enzymes involved in the synthesis of 11-KT) were investigated in human gonads. The production of 11-KT was investigated in Leydig cells. Plasma concentrations of testosterone and 11-KT were measured in 10 women and 10 men of reproductive age. Investigation of its properties was performed using breast cancer-derived MCF-7 cells. RESULTS: Cytochrome P450 and 11ß-hydroxysteroid dehydrogenase types 1 and 2 were detected in Leydig cells and theca cells. Leydig cells produced 11-KT, and relatively high levels of plasma 11-KT were measured in both men and women. There was no sexual dimorphism in the plasma levels of 11-KT, even though testosterone levels were more than 20 times higher in men than in women. It is noteworthy that the levels of testosterone and 11-KT were similar in women. In a luciferase reporter system, 11-KT activated human androgen receptor-mediated transactivation. Conversely, 11-KT did not activate estrogen receptor-mediated transactivation in aromatase-expressed MCF-7 cells, whereas testosterone did following conversion to estrogen. 11-KT did not affect the estrogen/estrogen receptor -mediated cell proliferation of MCF-7 cells. Furthermore, it significantly inhibited cell proliferation when androgen receptor was transfected into MCF-7 cells. CONCLUSIONS: The current study indicates that 11-KT is produced in the gonads and represents a major androgen in human. It can potentially serve as a nonaromatizable androgen.

Regulation of Steroidogenesis, Development, and Cell Differentiation by Steroidogenic Factor-1 and Liver Receptor Homolog-1.

Steroidogenic factor-1 (SF-1) and liver receptor homolog-1 (LRH-1) belong to the nuclear receptor superfamily and are categorized as orphan receptors. In addition to other nuclear receptors, these play roles in various physiological phenomena by regulating the transcription of target genes. Both factors share very similar structures and exhibit common functions. Of these, the roles of SF-1 and LRH-1 in steroidogenesis are the most important, especially that of SF-1, which was originally discovered and named to reflect such roles. SF-1 and LRH-1 are essential for steroid hormone production in gonads and adrenal glands through the regulation of various steroidogenesis-related genes. As SF-1 is also necessary for the development of gonads and adrenal glands, it is also considered a master regulator of steroidogenesis. Recent studies have clearly demonstrated that LRH-1 also represents another master regulator of steroidogenesis, which similarly to SF-1, can induce differentiation of non-steroidogenic stem cells into steroidogenic cells. Here, we review the functions of both factors in these steroidogenesis-related phenomena.

Establishment and characterization of a novel orthotopic mouse model for human uterine sarcoma with different metastatic potentials.

Uterine sarcomas are rare and aggressive gynecologic tumors with a poor prognosis because of recurrence and metastasis. However, the mechanisms of uterine sarcoma metastasis are largely unknown. To investigate this mechanism, we developed a novel uterine sarcoma tissue-derived orthotopic and metastatic model in KSN nude mice using a green fluorescent protein stably expressed uterine sarcoma cell line, MES-SA. Histological analysis showed that all orthotopic primary tumors were undifferentiated sarcoma. Primary tumors were characterized by high (18)F-fluorodeoxyglucose uptake with a positive correlation to the number of pulmonary metastases. In addition, we generated uterine sarcoma cell sublines with high or low metastatic potentials by serial in vivo selection. Microarray analysis between orthotopic tumors with high and low metastatic potentials revealed differential expression of genes related to cell proliferation and migration (TNNT1, COL1A2, and ZIC1). Our model would be useful to compensate for the limited clinical cases of uterine sarcoma and to investigate the molecular mechanisms of metastatic uterine sarcoma.

Transcriptional regulation of genes related to progesterone production.

Steroid hormones are synthesized from cholesterol in various tissues, mainly in the adrenal glands and gonads. Because these lipid-soluble steroid hormones immediately diffuse through the cells in which they are produced, their secretion directly reflects the activity of the genes related to their production. Progesterone is important not only for luteinization and maintenance of pregnancy, but also as a substrate for most other steroids. Steroidogenic acute regulatory protein (STAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and 3ß-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3ß-HSD) are well-known proteins essential for progesterone production. In addition to them, glutathione S-transferase A1-1 and A3-3 are shown to exert Δ(5)-Δ(4) isomerization activity to produce progesterone in a cooperative fashion with 3ß-HSD. 5-Aminolevulinic acid synthase 1, ferredoxin 1, and ferredoxin reductase also play a role in steroidogenesis as accessory factors. Members of the nuclear receptor 5A (NR5A) family (steroidogenic factor 1 and liver receptor homolog 1) play a crucial role in the transcriptional regulation of these genes. The NR5A family activates these genes by binding to NR5A responsive elements present within their promoter regions, as well as to the elements far from their promoters. In addition, various NR5A-interacting proteins including peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear receptor subfamily 0, group B, member 1 (DAX-1), and CCAAT/enhancer-binding proteins (C/EBP) are involved in the transcription of NR5A target genes and regulate the transcription either positively or negatively under both basal and tropic hormone-stimulated conditions. In this review, we describe the transcriptional regulation of genes related to progesterone production.

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 ß as components of SF-1 nuclear complex. In this review, we will describe novel roles of the newly identified genes for steroidogenesis.

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.

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.

C/EBPß (CCAAT/enhancer-binding protein ß) 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/EBPß (CCAAT/enhancer-binding protein ß), which is an essential transcription factor for ovulation and luteinization, as the transcriptional mechanisms of C/EBPß working together with SF-1 are poorly understood. C/EBPß 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, 3ß- and steroid δ-isomerase 2) expression. EMSA and ChIP assays revealed novel C/EBPß-binding sites in the upstream regions of the HSD3B2 and CYP11A1 genes. These interactions were enhanced by cAMP stimulation. Luciferase assays showed that C/EBPß-responsive regions were found in each promoter and C/EBPß is involved in the cAMP-induced transcriptional activity of these genes together with SF-1. These results indicate that C/EBPß is an important mediator of progesterone production by working together with SF-1, especially under tropic hormone-stimulated conditions.

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.

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 Δ(5)-Δ(4) isomerization are thought to be enzymatic properties of 3ß-hydroxysteroid dehydrogenase (3ß-HSD). Here, we demonstrated that, in steroidogenic cells, the hGSTA1 and hGSTA3 gene products catalyze Δ(5)-Δ(4) isomerization in a coordinated fashion with 3ß-HSD II to produce progesterone or Δ(4)-androstenedione from their Δ(5)-precursors. Thus, hGSTA1 and hGSTA3 gene products are new members of steroidogenesis working as Δ(5)-Δ(4) isomerases.

Dysregulation of ovarian follicular development in female rat: LH decreases FSH sensitivity during preantral-early antral transition.

Several clinical studies have shown a correlation of hypersecretion of LH and polycystic ovary syndrome (PCOS), infertility, and miscarriage in women, suggesting that chronically elevated LH impairs fertility. Growth arrest of small antral follicles in PCOS is also assumed to be associated with an abnormal endocrine environment involving increased LH stimulation, a hyperandrogenic milieu, and subsequent dysregulated FSH action in the ovarian follicles. In this study, we examined whether and how LH modulates follicular development and steroid production during preantral-early antral follicle transition by using a rat preantral follicle culture system. LH augments testosterone and estradiol production in preantral follicles via up-regulating mRNA abundance of CYP17A1 and CYP19A1. LH promotes rat preantral follicle growth, and the follicular size reaches that of early antral follicles in vitro, a response attenuated by the specific androgen receptor antagonist and a targeted disruption of androgen receptor gene. Sustained follicle stimulation by LH, but not by androgen, decreases FSH receptor mRNA levels and FSH receptor signaling and inhibits FSH-induced follicular growth. The data suggest that LH promotes preantral-early antral transition via the increased synthesis and growth-promoting action of androgen. However, chronic LH stimulation impairs FSH-dependent antral follicle growth by suppressing granulosa cell FSHR expression via the modulation of intraovarian regulators, including LH-induced thecal factors.

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.

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.