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Mizutani T, Orisaka M, Kawabe S, Morichika R, Uesaka M, Yoshida Y. YAP/TAZ-TEAD is a novel transcriptional regulator of genes encoding steroidogenic enzymes in rat granulosa cells and KGN cells. Mol Cell Endocrinol 2023; 559:111808. [PMID: 36309205 DOI: 10.1016/j.mce.2022.111808] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/04/2022] [Revised: 10/14/2022] [Accepted: 10/18/2022] [Indexed: 11/09/2022]
Abstract
Steroidogenesis in ovarian granulosa cells is regulated by the follicle-stimulating hormone (FSH) via transcriptional regulation of its related genes. We herein showed the involvement of the Hippo pathway in this regulation. In KGN granulosa cell, repression of YAP/TAZ activity induced the expression of CYP11A1, HSD3B2, and CYP19A1 in a TEAD-dependent manner without cAMP stimulation. A selective inhibitor of p38 MAP kinase, suppressed YAP/TAZ knockdown-indued the expression of these genes, suggesting this signal could be involved. The expression of these genes was induced by 8Br-cAMP, whereas that of CYR61 and ADATS1, typical YAP/TAZ-TEAD target genes, was suppressed, suggesting that the cellular signaling of cAMP reduced YAP/TAZ-TEAD activity. The constitutively active mutant YAP canceled the FSH- and 8Br-cAMP-mediated induction of these genes in primary rat granulosa and KGN cells, respectively. Moreover, regulation of steroidogenesis-related genes by YAP/TAZ-TEAD was independent of steroidogenic factor 1, a master gene regulator of steroidogenesis. These results suggest that YAP/TAZ-TEAD is a negative regulator of steroidogenesis and that suppression of YAP/TAZ-TEAD activity by FSH is involved in ovarian steroidogenesis.
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Affiliation(s)
- Tetsuya Mizutani
- Department of Nursing, Faculty of Nursing and Welfare Sciences, Fukui Prefectural University, Japan.
| | - Makoto Orisaka
- Department of Obstetrics and Gynecology, Faculty of Medical Sciences, University of Fukui, Japan
| | - Shinya Kawabe
- Department of Food Science and Technology, National Fisheries University, Japan
| | - Ririko Morichika
- Department of Nursing, Faculty of Nursing and Welfare Sciences, Fukui Prefectural University, Japan
| | - Miki Uesaka
- Department of Obstetrics and Gynecology, Faculty of Medical Sciences, University of Fukui, Japan
| | - Yoshio Yoshida
- Department of Obstetrics and Gynecology, Faculty of Medical Sciences, University of Fukui, Japan
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Abobaker H, Omer NA, Hu Y, Idriss AA, Zhao R. In ovo injection of betaine promotes adrenal steroidogenesis in pre-hatched chicken fetuses. Poult Sci 2022; 101:101871. [PMID: 35487119 PMCID: PMC9170934 DOI: 10.1016/j.psj.2022.101871] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2022] [Revised: 03/09/2022] [Accepted: 03/14/2022] [Indexed: 12/05/2022] Open
Abstract
Corticosterone is critical for the maturation and survival of chicken fetus around hatching. Betaine is used as a feed additive in poultry industry to promote growth and mitigate stress. However, it remains unknown whether betaine could affect adrenal corticosterone synthesis in pre-hatching chicken fetuses. In this study, betaine (2.5 mg/egg) was injected into developing chicken fetuses at d 11 of incubation (E11) and its impact on adrenal steroidogenesis was investigated at day 19 (E19). Plasma corticosterone concentration was significantly (P < 0.05) elevated in betaine-treated fetuses, together with increased adrenal expression of melanocortin 2 receptor and steroidogenic acute regulatory protein. Accordingly, the corticosterone biosynthetic enzymes, such as cytochrome P450 family 11 subfamily A member 1, 3β-hydroxysteroid dehydrogenase and cytochrome P450 family 21 subfamily A member 2, as well as cholesterol biosynthesis or regulation-related genes, such as sterol regulatory element-binding protein 1, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase and low-density lipoprotein receptor, were all significantly (P < 0.05) upregulated in betaine group. Meanwhile, steroidogenic factor-1 and glucocorticoid receptor were significantly (P < 0.05) enhanced, whereas expression of dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome gene, a nuclear receptor known as a repressor of adrenal steroidogenesis, was significantly (P < 0.05) downregulated. Betaine significantly (P < 0.05) increased adrenal expression of genes involved in one-carbon metabolism and DNA methylation, such as S-adenosyl homocysteine hydrolase, betaine-homocysteine-methyltransferase, methionine adenosyl transferase and DNA methyltransferases, yet the promoter regions of most steroidogenic genes were significantly (P < 0.05) hypomethylated. These results indicate that in ovo injection of betaine promotes adrenal glucocorticoid synthesis in chicken fetuses before hatching, which involves alterations in DNA methylation.
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Analyses of Molecular Characteristics and Enzymatic Activities of Ovine HSD17B3. Animals (Basel) 2021; 11:ani11102876. [PMID: 34679897 PMCID: PMC8532638 DOI: 10.3390/ani11102876] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2021] [Revised: 09/20/2021] [Accepted: 09/21/2021] [Indexed: 11/16/2022] Open
Abstract
17β-hydroxysteroid dehydrogenase type 3 (HSD17B3) converts androstenedione (A4) into testosterone (T), which regulates sex steroid production. Because various mutations of the HSD17B3 gene cause disorder of sex differentiation (DSD) in multiple mammalian species, it is very important to reveal the molecular characteristics of this gene in various species. Here, we revealed the open reading frame of the ovine HSD17B3 gene. Enzymatic activities of ovine HSD17B3 and HSD17B1 for converting A4 to T were detected using ovine androgen receptor-mediated transactivation in reporter assays. Although HSD17B3 also converted estrone to estradiol, this activity was much weaker than those of HSD17B1. Although ovine HSD17B3 has an amino acid sequence that is conserved compared with other mammalian species, it possesses two amino acid substitutions that are consistent with the reported variants of human HSD17B3. Substitutions of these amino acids in ovine HSD17B3 for those in human did not affect the enzymatic activities. However, enzymatic activities declined upon missense mutations of the HSD17B3 gene associated with 46,XY DSD, affecting amino acids that are conserved between these two species. The present study provides basic information and tools to investigate the molecular mechanisms behind DSD not only in ovine, but also in various mammalian species.
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Yazawa T, Imamichi Y, Yuhki KI, Uwada J, Mikami D, Shimada M, Miyamoto K, Kitano T, Takahashi S, Sekiguchi T, Suzuki N, Rafiqul Islam Khan M, Ushikubi F, Umezawa A, Taniguchi T. Cyclooxygenase-2 is acutely induced by CCAAT/enhancer-binding protein β to produce prostaglandin E 2 and F 2α following gonadotropin stimulation in Leydig cells. Mol Reprod Dev 2019; 86:786-797. [PMID: 31087493 DOI: 10.1002/mrd.23163] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2018] [Revised: 04/01/2019] [Accepted: 04/13/2019] [Indexed: 12/14/2022]
Abstract
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 β (C/EBPβ) with cAMP-stimulation. C/EBPβ 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/EBPβ via reduction of CHOP expression upon gonadotropin-stimulation to produce PGF 2α and PGE 2 .
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Affiliation(s)
- Takashi Yazawa
- Department of Biochemistry, Asahikawa Medical University, Asahikawa, Japan
| | - Yoshitaka Imamichi
- Department of Pharmacology, Asahikawa Medical University, Asahikawa, Japan
| | - Koh-Ichi Yuhki
- Department of Pharmacology, Asahikawa Medical University, Asahikawa, Japan
| | - Junsuke Uwada
- Department of Biochemistry, Asahikawa Medical University, Asahikawa, Japan
| | - Daisuke Mikami
- Department of Nephrology, Asahikawa Medical University, Asahikawa, Japan
| | - Masayuki Shimada
- Laboratory of Reproductive Endocrinology, Graduate School of Biosphere Sciences, Hiroshima University, Higashi-Hiroshima, Japan
| | - Kaoru Miyamoto
- Department of Biochemistry, Faculty of Medical Sciences, University of Fukui, Fukui, Japan
| | - Takeshi Kitano
- Department of Materials and Life Science, Graduate School of Science and Technology, Kumamoto University, Kumamoto, Japan
| | - Satoru Takahashi
- Department of Pediatrics, Asahikawa Medical University, Asahikawa, Japan
| | - Toshio Sekiguchi
- The Noto Marine Laboratory, Division of Marine Environmental Studies, Institute of Nature and Environmental Technology, Kanazawa University, Ishikawa, Japan
| | - Nobuo Suzuki
- The Noto Marine Laboratory, Division of Marine Environmental Studies, Institute of Nature and Environmental Technology, Kanazawa University, Ishikawa, Japan
| | - Md Rafiqul Islam Khan
- Department of Biochemistry, Asahikawa Medical University, Asahikawa, Japan.,Department of Pharmacy, University of Rajshahi, Rajshahi, Bangladesh
| | - Fumitaka Ushikubi
- Department of Pharmacology, Asahikawa Medical University, Asahikawa, Japan
| | - Akihiro Umezawa
- Department of Reproductive Biology, Center for Regenerative Medicine, National Research Institute for Child Health and Development, Tokyo, Japan
| | - Takanobu Taniguchi
- Department of Biochemistry, Asahikawa Medical University, Asahikawa, Japan
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Transcriptional Regulation of Ovarian Steroidogenic Genes: Recent Findings Obtained from Stem Cell-Derived Steroidogenic Cells. BIOMED RESEARCH INTERNATIONAL 2019; 2019:8973076. [PMID: 31058195 PMCID: PMC6463655 DOI: 10.1155/2019/8973076] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/11/2018] [Revised: 10/15/2018] [Accepted: 02/03/2019] [Indexed: 12/16/2022]
Abstract
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.
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Mackeh R, Marr AK, Fadda A, Kino T. C2H2-Type Zinc Finger Proteins: Evolutionarily Old and New Partners of the Nuclear Hormone Receptors. NUCLEAR RECEPTOR SIGNALING 2018; 15:1550762918801071. [PMID: 30718982 PMCID: PMC6348741 DOI: 10.1177/1550762918801071] [Citation(s) in RCA: 31] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/20/2016] [Accepted: 02/02/2017] [Indexed: 12/21/2022]
Abstract
Nuclear hormone receptors (NRs) are evolutionarily conserved ligand-dependent
transcription factors. They are essential for human life, mediating the actions
of lipophilic molecules, such as steroid hormones and metabolites of fatty acid,
cholesterol, and external toxic compounds. The C2H2-type zinc finger proteins
(ZNFs) form the largest family of the transcription factors in humans and are
characterized by multiple, tandemly arranged zinc fingers. Many of the C2H2-type
ZNFs are conserved throughout evolution, suggesting their involvement in
preserved biological activities, such as general transcriptional regulation and
development/differentiation of organs/tissues observed in the early embryonic
phase. However, some C2H2-type ZNFs, such as those with the Krüppel-associated
box (KRAB) domain, appeared relatively late in evolution and have significantly
increased family members in mammals including humans, possibly modulating their
complicated transcriptional network and/or supporting the morphological
development/functions specific to them. Such evolutional characteristics of the
C2H2-type ZNFs indicate that these molecules influence the NR functions
conserved through evolution, whereas some also adjust them to meet with specific
needs of higher organisms. We review the interaction between NRs and C2H2-type
ZNFs by focusing on some of the latter molecules.
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Roy S, Gandra D, Seger C, Biswas A, Kushnir VA, Gleicher N, Kumar TR, Sen A. Oocyte-Derived Factors (GDF9 and BMP15) and FSH Regulate AMH Expression Via Modulation of H3K27AC in Granulosa Cells. Endocrinology 2018; 159:3433-3445. [PMID: 30060157 PMCID: PMC6112599 DOI: 10.1210/en.2018-00609] [Citation(s) in RCA: 64] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/28/2018] [Accepted: 07/20/2018] [Indexed: 12/15/2022]
Abstract
Anti-Müllerian hormone (AMH) produced by ovarian granulosa cells (GCs) plays a crucial role in ovarian function. It is used as a diagnostic and/or prognostic marker of fertility as well as for pathophysiological conditions in women. In this study, we investigated the underlying mechanism for regulation of AMH expression in GCs using primary mouse GCs and a human GC tumor-derived KGN cell line. We find that growth differentiation factor 9 (GDF9) and bone morphogenetic factor 15 (BMP15) together (GDF9 + BMP15), but not when tested separately, significantly induce AMH expression in vitro and in vivo (serum AMH). Our results show that GDF9 + BMP15 through the PI3K/Akt and Smad2/3 pathways synergistically recruit the coactivator p300 on the AMH promoter region that promotes acetylation of histone 3 lysine 27 (H3K27ac), facilitating AMH/Amh expression. Intriguingly, we also find that FSH inhibits GDF9 + BMP15-induced increase of AMH/Amh expression. This inhibition occurs through FSH-induced protein kinase A/SF1-mediated expression of gonadotropin inducible ovarian transcription factor 1, a transcriptional repressor, that recruits histone deacetylase 2 to deacetylate H3K27ac, resulting in the suppression of AMH/Amh expression. Furthermore, we report that ovarian Amh mRNA levels are significantly higher in Fshβ-null mice (Fshβ-/-) compared with those in wild-type (WT) mice. In addition, ovarian Amh mRNA levels are restored in Fshβ-null mice expressing a human WT FSHβ transgene (FSHβ-/-hFSHβWT). Our study provides a mechanistic insight into the regulation of AMH expression that has many implications in female reproduction/fertility.
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Affiliation(s)
- Sambit Roy
- Reproductive and Developmental Sciences Program, Department of Animal Sciences, Michigan State University, East Lansing, Michigan
| | - Divya Gandra
- Reproductive and Developmental Sciences Program, Department of Animal Sciences, Michigan State University, East Lansing, Michigan
| | - Christina Seger
- Division of Endocrinology and Metabolism, Department of Medicine, University of Rochester Medical Center, Rochester, New York
| | - Anindita Biswas
- Reproductive and Developmental Sciences Program, Department of Animal Sciences, Michigan State University, East Lansing, Michigan
| | | | - Norbert Gleicher
- Center for Human Reproduction, New York, New York
- Stem Cell Biology and Molecular Embryology Laboratory, The Rockefeller University, New York, New York
- Department of Obstetrics and Gynecology, Vienna University of Medicine, Vienna, Austria
| | - T Rajendra Kumar
- Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Colorado Anschutz, Denver, Colorado
| | - Aritro Sen
- Reproductive and Developmental Sciences Program, Department of Animal Sciences, Michigan State University, East Lansing, Michigan
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Effect of ACTH and hCG on the Expression of Gonadotropin-Inducible Ovarian Transcription Factor 1 ( Giot1) Gene in the Rat Adrenal Gland. Int J Mol Sci 2018; 19:ijms19082285. [PMID: 30081524 PMCID: PMC6121328 DOI: 10.3390/ijms19082285] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2018] [Revised: 07/26/2018] [Accepted: 08/01/2018] [Indexed: 01/17/2023] Open
Abstract
Gonadotropin-inducible ovarian transcription factor-1 (Giot1) belongs to a family of fast-responsive genes, and gonadotropins rapidly induce its expression in steroidogenic cells of ovaries and testes of rats. Gonadal Giot1 gene expression is regulated by cyclic adenosine monophosphate (cAMP) -dependent protein kinase A pathway, with essential role of orphan nuclear receptor NR4A1 transcription factor (nuclear receptor subfamily 4, group A, member 1). A recent study reports that Giot1 is also expressed in adrenals, however, the mechanism of its regulation in adrenal gland is yet to be identified. Therefore, the aim of this study was to characterise the changes in Giot1 gene expression in male and female rat adrenals using wide range of in vivo and in vitro experimental models. Special emphasis was directed at the Giot1 gene regulation by ACTH and gonadotropin. In our study, we found that ACTH rapidly stimulates Giot1 expression both in vivo and in vitro. However, gonadotropin does not affect the adrenal Giot1 gene expression, presumably due to the low expression of gonadotropin receptor in adrenals. Both testosterone and estradiol administered in vivo had inhibitory effect on Giot1 gene expression in the adrenals of post-gonadectomized adult rats. Further, our studies revealed that the intracellular mechanism of Giot1 gene regulation in rat adrenals is similar to that of gonads. As in the case of gonads, the expression of Giot1 in adrenal gland is regulated by cAMP-dependent signaling pathway with essential role of the NR4A1 transcription factor. The results of our studies suggest that Giot1 may be involved in the regulation of rat adrenocortical steroidogenesis.
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Yazawa T, Imamichi Y, Miyamoto K, Khan MRI, Uwada J, Umezawa A, Taniguchi T. Induction of steroidogenic cells from adult stem cells and pluripotent stem cells [Review]. Endocr J 2016; 63:943-951. [PMID: 27681884 DOI: 10.1507/endocrj.ej16-0373] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
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.
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Affiliation(s)
- Takashi Yazawa
- Department of Biochemistry, Asahikawa Medical University, Asahikawa 078-8510, Japan
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Protein Kinase A: A Master Kinase of Granulosa Cell Differentiation. Sci Rep 2016; 6:28132. [PMID: 27324437 PMCID: PMC4914995 DOI: 10.1038/srep28132] [Citation(s) in RCA: 47] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2015] [Accepted: 06/01/2016] [Indexed: 12/16/2022] Open
Abstract
Activation of protein kinase A (PKA) by follicle stimulating hormone (FSH) transduces the signal that drives differentiation of ovarian granulosa cells (GCs). An unresolved question is whether PKA is sufficient to initiate the complex program of GC responses to FSH. We compared signaling pathways and gene expression profiles of GCs stimulated with FSH or expressing PKA-CQR, a constitutively active mutant of PKA. Both FSH and PKA-CQR stimulated the phosphorylation of proteins known to be involved in GC differentiation including CREB, ß-catenin, AKT, p42/44 MAPK, GAB2, GSK-3ß, FOXO1, and YAP. In contrast, FSH stimulated the phosphorylation of p38 MAP kinase but PKA-CQR did not. Microarray analysis revealed that 85% of transcripts that were up-regulated by FSH were increased to a comparable extent by PKA-CQR and of the transcripts that were down-regulated by FSH, 76% were also down-regulated by PKA-CQR. Transcripts regulated similarly by FSH and PKA-CQR are involved in steroidogenesis and differentiation, while transcripts more robustly up-regulated by PKA-CQR are involved in ovulation. Thus, PKA, under the conditions of our experimental approach appears to function as a master upstream kinase that is sufficient to initiate the complex pattern of intracellular signaling pathway and gene expression profiles that accompany GC differentiation.
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Hunzicker-Dunn M, Mayo K. Gonadotropin Signaling in the Ovary. KNOBIL AND NEILL'S PHYSIOLOGY OF REPRODUCTION 2015:895-945. [DOI: 10.1016/b978-0-12-397175-3.00020-x] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/05/2025]
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Mizutani T, Ishikane S, Kawabe S, Umezawa A, Miyamoto K. Transcriptional regulation of genes related to progesterone production. Endocr J 2015; 62:757-63. [PMID: 26135521 DOI: 10.1507/endocrj.ej15-0260] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
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 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3β-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 3β-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.
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Affiliation(s)
- Tetsuya Mizutani
- Department of Biochemistry, Faculty of Medical Sciences, University of Fukui, Fukui 910-1193, Japan
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Role of Orphan Nuclear Receptor DAX-1/NR0B1 in Development, Physiology, and Disease. ACTA ACUST UNITED AC 2014. [DOI: 10.1155/2014/582749] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
DAX-1/NR0B1 is an unusual orphan receptor that has a pivotal role in the development and function of steroidogenic tissues and of the reproductive axis. Recent studies have also indicated that this transcription factor has an important function in stem cell biology and in several types of cancer. Here I critically review the most important findings on the role of DAX-1 in development, physiology, and disease of endocrine tissues since the cloning of its gene twenty years ago.
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Kanno M, Yazawa T, Kawabe S, Imamichi Y, Usami Y, Ju Y, Matsumura T, Mizutani T, Fujieda S, Miyamoto K. Sex-determining region Y-box 2 and GA-binding proteins regulate the transcription of liver receptor homolog-1 in early embryonic cells. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2014; 1839:406-14. [DOI: 10.1016/j.bbagrm.2014.03.016] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/11/2013] [Revised: 03/26/2014] [Accepted: 03/27/2014] [Indexed: 01/08/2023]
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Yazawa T, Imamichi Y, Miyamoto K, Umezawa A, Taniguchi T. Differentiation of mesenchymal stem cells into gonad and adrenal steroidogenic cells. World J Stem Cells 2014; 6:203-212. [PMID: 24772247 PMCID: PMC3999778 DOI: 10.4252/wjsc.v6.i2.203] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/02/2013] [Revised: 12/24/2013] [Accepted: 01/20/2014] [Indexed: 02/06/2023] Open
Abstract
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.
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Orisaka M, Hattori K, Fukuda S, Mizutani T, Miyamoto K, Sato T, Tsang BK, Kotsuji F, Yoshida Y. Dysregulation of ovarian follicular development in female rat: LH decreases FSH sensitivity during preantral-early antral transition. Endocrinology 2013; 154:2870-80. [PMID: 23709086 DOI: 10.1210/en.2012-2173] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
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.
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Affiliation(s)
- Makoto Orisaka
- Department of Obstetrics and Gynecology, University of Fukui, Fukui, Japan 910-1193.
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17
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Imamichi Y, Mizutani T, Ju Y, Matsumura T, Kawabe S, Kanno M, Yazawa T, Miyamoto K. Transcriptional regulation of human ferredoxin 1 in ovarian granulosa cells. Mol Cell Endocrinol 2013; 370:1-10. [PMID: 23435367 DOI: 10.1016/j.mce.2013.02.012] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/19/2012] [Revised: 01/30/2013] [Accepted: 02/13/2013] [Indexed: 01/20/2023]
Abstract
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.
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Affiliation(s)
- Yoshitaka Imamichi
- Department of Biochemistry, Faculty of Medical Sciences, University of Fukui, Fukui 910-1193, Japan
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18
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Yazawa T, Kawabe S, Kanno M, Mizutani T, Imamichi Y, Ju Y, Matsumura T, Yamazaki Y, Usami Y, Kuribayashi M, Shimada M, Kitano T, Umezawa A, Miyamoto K. Androgen/androgen receptor pathway regulates expression of the genes for cyclooxygenase-2 and amphiregulin in periovulatory granulosa cells. Mol Cell Endocrinol 2013; 369:42-51. [PMID: 23415714 DOI: 10.1016/j.mce.2013.02.004] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/04/2012] [Revised: 12/26/2012] [Accepted: 02/05/2013] [Indexed: 01/30/2023]
Abstract
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.
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Affiliation(s)
- Takashi Yazawa
- Department of Biochemistry, Faculty of Medical Sciences, University of Fukui, Matsuoka, Fukui, Japan.
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19
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Kawabe S, Yazawa T, Kanno M, Usami Y, Mizutani T, Imamichi Y, Ju Y, Matsumura T, Orisaka M, Miyamoto K. A novel isoform of liver receptor homolog-1 is regulated by steroidogenic factor-1 and the specificity protein family in ovarian granulosa cells. Endocrinology 2013; 154:1648-60. [PMID: 23471216 DOI: 10.1210/en.2012-2008] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
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.
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Affiliation(s)
- Shinya Kawabe
- Department of Biochemistry, University of Fukui, Fukui 910-1193, Japan
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20
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Chen J, Zhang Y, Tang Z, Mao J, Kuang Z, Qin C, Li W. Production of recombinant orange-spotted grouper (Epinephelus coioides) follicle-stimulating hormone (FSH) in single-chain form and dimer form by Pichia pastoris and their biological activities. Gen Comp Endocrinol 2012; 178:237-49. [PMID: 22684083 DOI: 10.1016/j.ygcen.2012.05.009] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/16/2012] [Revised: 05/24/2012] [Accepted: 05/29/2012] [Indexed: 11/21/2022]
Abstract
FSH is a key regulator of steroidogenesis and gonadal growth in teleosts. However, function of FSH is elusive in grouper due to the lack of purified and native FSH. In the present study, we reported production of bioactive orange-spotted grouper (Epinephelus coioides) FSH in dimer form and single-chain form by Pichia pastoris. Dimer form of recombinant grouper FSH (rgFSHba) was accomplished by co-expressing mature FSHb-subunit and a-subunit genes. Fusion of mature FSHb-subunit and a-subunit genes together linking with a polypeptide (4×(Gly-Ser)-Gly-Thr) gene generated single-chain form of recombinant grouper FSH (rgFSHb-a). Recombinant grouper common α-subunit (rgCga) and FSHb-subunit (rgFSHb) were also separately produced. Recombinant proteins were verified by Western blot and mass spectrometry assays, and characterized by deglycosylation analysis. Deglycosylation assay suggested that glycosylation of recombinant FSH mainly occurred on common a-subunit. Bioactivities of recombinant proteins were initially evaluated by activating grouper FSH receptor, and further demonstrated by incubating ovarian fragments of adult grouper and intraperitoneal injection in juvenile female grouper. Two forms of recombinant FSH presented similar biological activities of activating FSH receptor and stimulating in vitro testosterone (T) and estradiol-17β (E2) secretion, though the dimer form functioned slightly weaker than the single-chain form. However, injections of rgFSHb-a or rgFSHba could significantly increase serum T and E2 levels, induce early ovarian development, reduce hypothalamic gnrh1 mRNA level, and increase hypothalamic cyp19a1b mRNA level. Data in this study suggested that recombinant gonadotropin could be produced in dimer form or single-chain form by P. pastoris, and FSH could regulate steroidogenesis and early ovarian development in juvenile grouper.
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Affiliation(s)
- Jun Chen
- State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals and Guangdong Provincial Key Laboratory for Aquatic Economic Animals, Sun Yat-Sen University, Guangzhou 510275, China
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21
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Yazawa T, Kawabe S, Inaoka Y, Okada R, Mizutani T, Imamichi Y, Ju Y, Yamazaki Y, Usami Y, Kuribayashi M, Umezawa A, Miyamoto K. Differentiation of mesenchymal stem cells and embryonic stem cells into steroidogenic cells using steroidogenic factor-1 and liver receptor homolog-1. Mol Cell Endocrinol 2011; 336:127-32. [PMID: 21129436 DOI: 10.1016/j.mce.2010.11.025] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/31/2010] [Revised: 11/23/2010] [Accepted: 11/23/2010] [Indexed: 12/19/2022]
Abstract
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.
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Affiliation(s)
- Takashi Yazawa
- Department of Biochemistry, Faculty of Medical Sciences, University of Fukui, Shimoaizuki 23, Matsuoka, Eiheiji-cho, Fukui 910-1193, Japan.
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22
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Mizutani T, Yazawa T, Ju Y, Imamichi Y, Uesaka M, Inaoka Y, Matsuura K, Kamiki Y, Oki M, Umezawa A, Miyamoto K. 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. J Biol Chem 2010; 285:28240-51. [PMID: 20601698 DOI: 10.1074/jbc.m110.129510] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
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.
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Affiliation(s)
- Tetsuya Mizutani
- Department of Biochemistry, Faculty of Medical Sciences, University of Fukui, Fukui 910-1193, Japan
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23
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Yazawa T, Inaoka Y, Okada R, Mizutani T, Yamazaki Y, Usami Y, Kuribayashi M, Orisaka M, Umezawa A, Miyamoto K. PPAR-gamma coactivator-1alpha regulates progesterone production in ovarian granulosa cells with SF-1 and LRH-1. Mol Endocrinol 2010; 24:485-96. [PMID: 20133449 PMCID: PMC5419099 DOI: 10.1210/me.2009-0352] [Citation(s) in RCA: 81] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2009] [Accepted: 12/30/2009] [Indexed: 12/16/2022] Open
Abstract
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.
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Affiliation(s)
- Takashi Yazawa
- Department of Biochemistry, Faculty of Medical Sciences, University of Fukui, Shimoaizuki 23-3, Matsuoka, Eiheiji-cho, Fukui 910-1193, Japan
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24
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Mono-(2-ethylhexyl) phthalate induces NR4A subfamily and GIOT-1 gene expression, and suppresses CYP19 expression in human granulosa-like tumor cell line KGN. Toxicol Lett 2009; 191:353-9. [DOI: 10.1016/j.toxlet.2009.10.004] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2009] [Revised: 10/05/2009] [Accepted: 10/05/2009] [Indexed: 11/19/2022]
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25
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Yazawa T, Inanoka Y, Mizutani T, Kuribayashi M, Umezawa A, Miyamoto K. Liver receptor homolog-1 regulates the transcription of steroidogenic enzymes and induces the differentiation of mesenchymal stem cells into steroidogenic cells. Endocrinology 2009; 150:3885-93. [PMID: 19359379 DOI: 10.1210/en.2008-1310] [Citation(s) in RCA: 57] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Steroidogenic factor-1 (SF-1, also known as Ad4BP) has been demonstrated to be a primary transcriptional regulator of steroidogenic-related genes. However, mRNA for liver receptor homolog-1 (LRH-1), which together with SF-1, belongs to the NR5A nuclear receptor family, is expressed at much higher levels than SF-1 mRNA in the human gonad. In our previous studies, we demonstrated that SF-1 induced the differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into steroidogenic cells such as Leydig or adrenocortical cells. The introduction of LRH-1 into human MSCs (hMSCs) with the aid of cAMP also induced the expression of steroidogenic enzymes, including CYP17, and their differentiation into steroid hormone-producing cells. Promoter analysis, EMSA, and chromatin immunoprecipitation assay using LRH-1-transduced hMSCs indicated that three LRH-1 binding sites were responsible for CYP17 transactivation. Immunohistochemical studies showed that LRH-1 protein was expressed in human Leydig cells. The CYP17 promoter region was highly methylated in hMSCs, whereas it was demethylated by the introduction of LRH-1 and cAMP treatment. These results indicate that LRH-1 could represent another key regulator of the steroidogenic lineage in MSCs and play a vital role in steroid hormone production in human Leydig cells.
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MESH Headings
- Adrenal Glands/metabolism
- Animals
- Cell Differentiation/genetics
- Cell Line
- Chromatin Immunoprecipitation
- Cyclic AMP/pharmacology
- DAX-1 Orphan Nuclear Receptor
- DNA-Binding Proteins/genetics
- DNA-Binding Proteins/physiology
- Electrophoretic Mobility Shift Assay
- Female
- Gonads/metabolism
- Humans
- Immunohistochemistry
- Male
- Mesenchymal Stem Cells/cytology
- Mesenchymal Stem Cells/metabolism
- Mice
- Mice, Mutant Strains
- Nuclear Receptor Subfamily 4, Group A, Member 1
- Receptors, Cytoplasmic and Nuclear/genetics
- Receptors, Cytoplasmic and Nuclear/physiology
- Receptors, Retinoic Acid/genetics
- Receptors, Retinoic Acid/physiology
- Receptors, Steroid/genetics
- Receptors, Steroid/physiology
- Repressor Proteins/genetics
- Repressor Proteins/physiology
- Reverse Transcriptase Polymerase Chain Reaction
- Steroid 17-alpha-Hydroxylase/genetics
- Steroidogenic Factor 1/genetics
- Steroidogenic Factor 1/physiology
- Steroids/biosynthesis
- Transduction, Genetic
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Affiliation(s)
- Takashi Yazawa
- Department of Biochemistry, Faculty of Medical Sciences, University of Fukui, Eiheiji-cho, Fukui 910-1193 Japan
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26
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Lu C, Yang W, Chen M, Liu T, Yang J, Tan P, Li L, Hu X, Fan C, Hu Z, Liu Y. Inhibin A inhibits follicle-stimulating hormone (FSH) action by suppressing its receptor expression in cultured rat granulosa cells. Mol Cell Endocrinol 2009; 298:48-56. [PMID: 18992787 DOI: 10.1016/j.mce.2008.09.039] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/16/2008] [Revised: 08/24/2008] [Accepted: 09/29/2008] [Indexed: 10/21/2022]
Abstract
Inhibin has long been considered as a suppresser of follicle-stimulating hormone (FSH) secretion from anterior pituitary through pituitary-gonad negative feedback to regulate follicle development. We demonstrated that addition of inhibin A could significantly suppress FSH-induced FSHR mRNA level in cultured rat granulosa cells (GCs) measured by real-time PCR. The inhibin A exerted its action mainly by inhibiting FSHR promoter activity. Furthermore, exogenous inhibin A could dramatically decrease FSH-induced P450arom and P450scc level and suppress progesterone and estradiol production in the cultured GCs, but it did not decrease forskolin-induced steroidogenesis, indicating that the inhibitory effect of inhibin A on FSH action may be upstream of cAMP signaling. Inhibin A was also capable of suppressing FSH-induced expression of steroidogenic factor 1 (SF-1) and androgen receptor, but stimulating DAX-1 expression in the culture. Our study has provided new evidence to show that inhibin A is capable of feedback antagonizing FSH action on GCs by reducing FSHR expression at ovarian level via a short feedback loop. Transcriptional factor receptors, such as SF-1, AR and DAX-1 were involved in this regulation.
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Affiliation(s)
- Cuiling Lu
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Chaoyang District, Beijing 100101, China
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27
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Coactivation of estrogen receptor beta by gonadotropin-induced cofactor GIOT-4. Mol Cell Biol 2008; 29:83-92. [PMID: 18981223 DOI: 10.1128/mcb.00884-08] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
Estrogen exerts its diverse effects through two subtypes of estrogen receptors (ER), ERalpha and ERbeta. Each subtype has its own distinct function and expression pattern in its target tissues. Little, however, is known about the transcriptional regulatory mechanism of ERbeta in the major ERbeta-expressing tissues. Using biochemical methods, we identified and described a novel ERbeta coactivator. This protein, designated GIOT-4, was biochemically purified from 293F cells. It coactivated ERbeta in ovarian granulosa cells. GIOT-4 expression was induced by stimulation with follicle-stimulating hormone (FSH). GIOT-4 recruited an SWI/SNF-type complex in a ligand-independent manner to ERbeta as an ER subtype-specific physical bridging factor and induced subsequent histone modifications in the ERbeta target gene promoters in a human ovarian granulosa cell line (KGN). Indeed, two ERbeta-specific target genes were upregulated by FSH at a specific stage of a normal ovulatory cycle in intact mice. These findings imply the presence of a novel regulatory convergence between the gonadotropin signaling cascade and ERbeta-mediated transcription in the ovary.
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28
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Johari H, Parhizkar Z, Talebi E. Effects of adenine on the pituitary-gonad axis in newborns rats. Pak J Biol Sci 2008; 11:2413-2417. [PMID: 19137851 DOI: 10.3923/pjbs.2008.2413.2417] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/27/2023]
Abstract
The present study was undertaken to investigate effects of the adenine on the hypothalamus-pituitary-gonad axis and changes in blood hormone concentration such as FSH, LH, progesterone and estrogen in newborn female rats. Adenine is a common organic base and its concentration variations caused by foods, has various effects on the body metabolic systems. In present study, fifty newborns rats were used divided into five groups, of 10s, including control I, control II which received solvent (normal saline) only and three experimental groups which received 50, 100 and 200 mg body weight adenine respectively. All the animals were kept under same condition with plenty food and water and treated Intra Peritoneally (IP) during days 2-16 after birth. At the end of experiment, all the animals were weighed, their ovaries were removed and blood samples were taken for hormone analysis. The results showed that dose dependent adenine solution significantly reduced the body and ovarian weight on 30 and 70 days after birth. In addition adenine led into no significant difference in concentration of FSH and LH in the experimental groups relative to the control on 30th day of life. But on the 70th day, the levels of these hormones raised significantly in the experimental groups. Furthermore, the adenine solution significantly increased the levels of progesterone and estrogen hormones in the experimental groups relative to the control on the 30th day, while decreased their concentration significantly on the 70th day. This situation has close similarities to metabolic disorders present in human caused by excessive use of adenine. High amounts consumption of adenine in can lead into hormone abnormality, weight loss and metabolic anomalies.
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Affiliation(s)
- H Johari
- Islamic Azad University of Darab, Islamic Azad University Street, P.O. Box 74817-83143, Darab, Fars, Iran
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29
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Yazawa T, Uesaka M, Inaoka Y, Mizutani T, Sekiguchi T, Kajitani T, Kitano T, Umezawa A, Miyamoto K. 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. Endocrinology 2008; 149:1786-92. [PMID: 18162527 DOI: 10.1210/en.2007-1015] [Citation(s) in RCA: 73] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
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.
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Affiliation(s)
- Takashi Yazawa
- Department of Biochemistry, Faculty of Medical Sciences, University of Fukui, Shimoaizuki, Matsuoka, Eiheiji-cho, Fukui, Japan
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30
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Tsuchiya Y, Nakajima M, Takagi S, Katoh M, Zheng W, Jefcoate CR, Yokoi T. Binding of steroidogenic factor-1 to the regulatory region might not be critical for transcriptional regulation of the human CYP1B1 gene. J Biochem 2007; 139:527-34. [PMID: 16567417 DOI: 10.1093/jb/mvj055] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
Cytochrome P450 (CYP) 1B1, which catalyzes 17beta-estradiol 4-hydroxylation, is expressed in steroid-related tissues including ovary, testis, and adrenal gland. Generally, the expressions of steroidogenic CYPs are transcriptionally regulated by steroidogenic factor-1 (SF-1) and cAMP response element (CRE) binding protein (CREB). In the present study, we examined the possibility that the human CYP1B1 gene might be regulated by SF-1 and CREB. Gel shift analyses revealed that in vitro translated SF-1 can bind to the putative SF-1 binding sites, SF-1a (at -1722) and SF-1b (at -2474), on the CYP1B1 gene. In vitro translated CREB barely binds to the putative SF-1 binding sites. Luciferase analysis revealed that a reporter plasmid, pGL3 (-2623/+25), containing the SF-1a and SF-1b elements is transactivated by the concomitant co-expression of SF-1 and protein kinase A (PKA). However, the transcriptional activity is induced by PKA alone. Mutations in the SF-1a and SF-1b elements did not affect the luciferase activity. Thus, the binding of SF-1 to the putative SF-1 binding sites of the human CYP1B1 gene might not be essential for transcriptional regulation. Interestingly, deletion and mutation analyses indicated that the PKA signaling pathway is involved in the xenobiotic responsive element (XRE)-mediated transactivation of the human CYP1B1 gene.
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Affiliation(s)
- Yuki Tsuchiya
- Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kakuma-machi, Kanazawa 920-1192
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31
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He PJ, Hirata M, Yamauchi N, Hashimoto S, Hattori MA. Gonadotropic regulation of circadian clockwork in rat granulosa cells. Mol Cell Biochem 2007; 302:111-8. [PMID: 17483911 DOI: 10.1007/s11010-007-9432-7] [Citation(s) in RCA: 56] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2006] [Accepted: 02/09/2007] [Indexed: 10/23/2022]
Abstract
The circadian clock is responsible for the generation of circadian rhythms in hormonal secretion and metabolism. These peripheral clocks could be reset by various cues in order to adapt to environmental variations. The ovary can be characterized as having highly dynamic physiology regulated by gonadotropins. Here, we aimed to address the status of circadian clock in the ovary, and to explore how gonadotropins could regulate clockwork in granulosa cells (GCs). To this end, we mainly utilized the immunohistochemistry, RT-PCR, and real-time monitoring of gene expression methods. PER1 protein was constantly abundant across the daily cycle in the GCs of immature ovaries. In contrast, PER1 protein level was obviously cyclic through the circadian cycle in the luteal cells of pubertal ovaries. In addition, both FSH and LH induced Per1 expression in cultured immature and mature GCs, respectively. The promoter analysis revealed that the Per1 expression was mediated by the cAMP response element binding protein. In cultured transgenic GCs, both FSH and LH also induced the circadian oscillation of Per2. However, the Per2 oscillation promoted by FSH quickly dampened within only one cycle, whereas the Per2 oscillation promoted by LH was persistently maintained. Collectively, these findings strongly suggest that both FSH and LH play an important role in regulating circadian clock in the ovary; however, they might exert differential actions on the clockwork in vivo due to each specific role within ovarian physiology.
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Affiliation(s)
- Pei-Jian He
- Laboratory of Reproductive Physiology and Biotechnology, Department of Animal and Marine Bioresource Sciences, Graduate School of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
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Qiu J, Yao S, Hindmarch C, Antunes V, Paton J, Murphy D. Transcription factor expression in the hypothalamo-neurohypophyseal system of the dehydrated rat: upregulation of gonadotrophin inducible transcription factor 1 mRNA is mediated by cAMP-dependent protein kinase A. J Neurosci 2007; 27:2196-203. [PMID: 17329416 PMCID: PMC6673476 DOI: 10.1523/jneurosci.5420-06.2007] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2006] [Revised: 01/15/2007] [Accepted: 01/15/2007] [Indexed: 11/21/2022] Open
Abstract
The supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamo-neurohypophyseal system (HNS) undergo a dramatic function-related plasticity during dehydration. We hypothesize that alterations in steady-state transcript levels might be partially responsible for this remodeling. In turn, regulation of transcript abundance might be mediated by transcription factors. We used microarrays to identify changes in the expression of mRNAs encoding transcription factors in response to water deprivation in the SON. We observed downregulation of 10 and upregulation of 28 transcription factor transcripts. For five of the upregulated mRNAs, namely gonadotropin inducible ovarian transcription factor 1 (Giot1), Giot2, cAMP-responsive element binding protein 3-like 1, CCAAT/enhancer binding protein beta, and activating transcription factor 4, in situ hybridization was used to confirm the array results, demonstrating a significant increase in expression in SON and PVN magnocellular neurons (MCNs) after 3 d of water deprivation and, in some cases, upregulation in parvocellular PVN neurons. Using a viral vector expressing a potent inhibitor of cAMP-dependent protein kinase A (PKA), we show that the osmotically induced increase in the abundance of transcripts encoding Giot1 is mediated in vivo by the PKA pathway. We thus suggest that signaling pathways activated by dehydration in MCNs mediate transcription factor gene activation, which, in turn, regulate target genes that mediate HNS remodeling.
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Affiliation(s)
- Jing Qiu
- Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol BS1 3NY, United Kingdom, and
| | - Song Yao
- Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol BS1 3NY, United Kingdom, and
| | - Charles Hindmarch
- Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol BS1 3NY, United Kingdom, and
| | - Vagner Antunes
- Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol BS1 3NY, United Kingdom, and
| | - Julian Paton
- Department of Physiology, Bristol Heart Institute, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom
| | - David Murphy
- Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol BS1 3NY, United Kingdom, and
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Saxena D, Escamilla-Hernandez R, Little-Ihrig L, Zeleznik AJ. Liver receptor homolog-1 and steroidogenic factor-1 have similar actions on rat granulosa cell steroidogenesis. Endocrinology 2007; 148:726-34. [PMID: 17095585 DOI: 10.1210/en.2006-0108] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Granulosa cells express the closely related orphan nuclear receptors steroidogenic factor-1 (SF-1) and liver receptor homolog-1 (LRH-1). To determine whether SF-1 and LRH-1 have differential effects on steroid production, we compared the effects of overexpressing LRH-1 and SF-1 on estrogen and progesterone production by undifferentiated rat granulosa cells. Adenovirus mediated overexpression of LRH-1 or SF-1 had qualitatively similar effects. Neither LRH-1 nor SF-1 alone stimulated estrogen or progesterone production, but when combined with FSH and testosterone, each significantly augmented progesterone production and mRNAs for cholesterol side-chain cleavage enzyme and 3beta-hydroxysteroid dehydrogenase above that observed with FSH alone, with SF-1 being more effective than LRH-1. LRH-1 did not augment FSH-stimulated estrogen production, whereas SF-1 produced only a slight ( approximately 30%) augmentation of FSH-stimulated estrogen production. The stimulatory actions of both were reduced by overexpression of dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1. Expression of either LRH-1 or SF-1 together with constitutively active protein kinase B in the absence of FSH stimulated progesterone production and mRNAs for 3beta-hydroxysteroid dehydrogenase and cholesterol side-chain cleavage enzyme but did not stimulate estrogen production or mRNA for aromatase. These findings demonstrate that LRH-1 and SF-1 have qualitatively similar actions on FSH-stimulated estrogen and progesterone production, which would suggest that these factors may have overlapping actions in the regulation of steroidogenesis that accompanies granulosa cell differentiation.
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Affiliation(s)
- Deeksha Saxena
- Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 830 Scaife Hall, 3500 Terrace Street, Pittsburgh, Pennsylvania 15261, USA
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Abstract
The corpus luteum (CL) is one of the few endocrine glands that forms from the remains of another organ and whose function and survival are limited in scope and time. The CL is the site of rapid remodeling, growth, differentiation, and death of cells originating from granulosa, theca, capillaries, and fibroblasts. The apparent raison d'etre of the CL is the production of progesterone, and all the structural and functional features of this gland are geared toward this end. Because of its unique importance for successful pregnancies, the mammals have evolved a complex series of checks and balances that maintains progesterone at appropriate levels throughout gestation. The formation, maintenance, regression, and steroidogenesis of the CL are among the most significant and closely regulated events in mammalian reproduction. During pregnancy, the fate of the CL depends on the interplay of ovarian, pituitary, and placental regulators. At the end of its life span, the CL undergoes a process of regression leading to its disappearance from the ovary and allowing the initiation of a new cycle. The generation of transgenic, knockout and knockin mice and the development of innovative technologies have revealed a novel role of several molecules in the reprogramming of granulosa cells into luteal cells and in the hormonal and molecular control of the function and demise of the CL. The current review highlights our knowledge on these key molecular events in rodents.
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Affiliation(s)
- Carlos Stocco
- Department of Obstetrics, Gynecology and Reproductive Science, Yale University School of Medicine, New Haven, CT 06510, USA
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35
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Cui M, Li W, Liu W, Yang K, Pang Y, Haoran L. Production of recombinant orange-spotted grouper (Epinephelus coioides) luteinizing hormone in insect cells by the baculovirus expression system and its biological effect. Biol Reprod 2006; 76:74-84. [PMID: 17021348 DOI: 10.1095/biolreprod.105.050484] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/01/2022] Open
Abstract
The cDNA sequence encoding orange-spotted grouper lhb (LHbeta) and cga (GTHalpha) subunits were cocloned into baculovirus transfer vectors and expressed in insect Sf9 cells. The results showed that two bands of 15.6 kDa and 11.4 kDa could be detected by SDS-PAGE and a band of 30 kDa could be detected by native PAGE. The recombinant grouper Lh (rgLh) could stimulate the secretion of testosterone (T) and estradiol-17beta (E2) from the gonad in a static incubation system in a time-dependent, but not a dose-dependent, manner. Using in vivo bioassay, the mRNA levels of two aromatases (cyp19a1a [P450aromA] and cyp19a1b [P450aromB]), gnrh (GnRH), lhb, and cga in the pituitary, gonad, and hypothalamus were determined in different groups of orange-spotted groupers treated respectively with rgLh, human chorionic gonadotropin (hCG), and a culture medium of insect cells transformed with an expression vector without lhb and cga subunits. The mRNA levels of cyp19a1a and cyp19a1b rose dramatically after injecting rgLh intraperitoneally, which was consistent with the secretion of sex steroid hormones. Interestingly, the mRNA levels of gnrh dropped in the pituitary, hypothalamus, and gonad, and the mRNA levels of lhb and cga in the pituitary of the experimental group expressed at a higher level than that of the hCG group. These results are in accord with the long positive feedback loop of Lh on gonad sex steroid hormones and the short negative feedback loop of Lh on gnrh mRNA levels. These results indicate that the rgLh is successfully expressed by the baculovirus-insect expression system and that the rgLh has biological activity.
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Affiliation(s)
- Miao Cui
- State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou 510275, People's Republic of China
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36
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Andrieu T, Féral C, Joubert M, Benhaim A, Mittre H. The absence of a functional nuclear receptor element A (NREA) in the promoter II of the aromatase P450 gene in rabbit granulosa cells. J Steroid Biochem Mol Biol 2006; 101:127-35. [PMID: 16901689 DOI: 10.1016/j.jsbmb.2006.06.007] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Aromatase protein is synthesized in response to gonadotropins that activate expression of their target genes via the cAMP second messenger system. The -882/+103 bp region of the rabbit ovarian promoter (PII) was ligated to a luciferase vector and transfected into granulosa cells to elucidated the mechanism by which cAMP stimulates transcription. Deletions and mutational experiments indicate that (i) a cAMP-response element-like sequence (CLS) present at -208 to -200 bp is the main element required for the activation of the rabbit PII by cAMP and that (ii) both nuclear receptor element sites; NREA (-133/-126 bp) and NREB (-188/-181 bp) do not participate to the cAMP-dependent activity of the PII. The replacement of the specific rabbit NREA site by the human NREA site increases two-fold the cAMP response and indicates that trans-activating factors are present in rabbit granulosa cells. This study shows for the first time an efficient aromatase transcription occurs in granulosa cells in absence of a consensus NREA site. In addition a comparative study has been performed on the sheep aromatase promoter where sites deviate from rabbit. Mutagenesis experiments suggest that some of them are involved in the cAMP-induced response of the rabbit PII.
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Affiliation(s)
- Thomas Andrieu
- Laboratoire de Biochimie, EA 2608-USC INRA 2006, Université, Esplanade de la Paix, 14032 Caen Cedex, France.
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Yazawa T, Mizutani T, Yamada K, Kawata H, Sekiguchi T, Yoshino M, Kajitani T, Shou Z, Umezawa A, Miyamoto K. Differentiation of adult stem cells derived from bone marrow stroma into Leydig or adrenocortical cells. Endocrinology 2006; 147:4104-11. [PMID: 16728492 DOI: 10.1210/en.2006-0162] [Citation(s) in RCA: 111] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Adult stem cells from bone marrow, referred to as mesenchymal stem cells or marrow stromal cells (MSCs), are defined as pluripotent cells and have the ability to differentiate into multiple mesodermal cells. In this study, we investigated whether MSCs from rat, mouse, and human are able to differentiate into steroidogenic cells. When transplanted into immature rat testes, adherent marrow-derived cells (including MSCs) were found to be engrafted and differentiate into steroidogenic cells that were indistinguishable from Leydig cells. Isolated murine MSCs transfected with green fluorescence protein driven by the promoter of P450 side-chain cleaving enzyme gene (CYP11A), a steroidogenic cell-specific gene, were used to detect steroidogenic cell production in vitro. During in vitro differentiation, green fluorescence protein-positive cells, which had characteristics similar to those of Leydig cells, were found. Stable transfection of murine MSCs with a transcription factor, steroidogenic factor-1, followed by treatment with cAMP almost recapitulated the properties of Leydig cells, including the production of testosterone. Transfection of human MSCs with steroidogenic factor-1 also led to their conversion to steroidogenic cells, but they appeared to be glucocorticoid- rather than testosterone-producing cells. These results indicate that MSCs represent a useful source of stem cells for producing steroidogenic cells that may provide basis for their use in cell and gene therapy.
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Affiliation(s)
- Takashi Yazawa
- Department of Biochemistry, Faculty of Medical Sciences, University of Fukui, Matsuoka-cho, Fukui 910-1193, Japan
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38
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Hunzicker-Dunn M, Maizels ET. FSH signaling pathways in immature granulosa cells that regulate target gene expression: branching out from protein kinase A. Cell Signal 2006; 18:1351-9. [PMID: 16616457 PMCID: PMC1564187 DOI: 10.1016/j.cellsig.2006.02.011] [Citation(s) in RCA: 282] [Impact Index Per Article: 14.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2006] [Accepted: 02/20/2006] [Indexed: 11/19/2022]
Abstract
Follicle-stimulating hormone (FSH) is necessary and sufficient to induce maturation of ovarian follicles to a mature, preovulatory phenotype in the intact animal, resulting in the generation of mature eggs and production of estrogen. FSH accomplishes these actions by inducing a complex pattern of gene expression in target granulosa cells that is regulated by input from many different signaling cascades, including those for the extracellular regulated kinases (ERKs), p38 mitogen-activated protein kinases (MAPKs), and phosphatidylinositol-3 kinase (PI3K). The upstream kinase that appears to be responsible for initiating all of the signaling that regulates gene expression in these epithelial cells is protein kinase A (PKA). PKA not only signals to directly phosphorylate transcription factors like cAMP response element binding protein and to promote chromatin remodeling by phosphorylating histone H3, this versatile kinase also enhances the activity of the p38 MAPK, ERK, and PI3K pathways. Additionally, accumulating evidence suggests that activation of a single signaling cascade downstream of PKA is not sufficient to activate target gene expression. Rather, cross-talk between and among signaling cascades is required. We will review the signaling cascades activated by FSH in granulosa cells and how these cascades contribute to the regulation of select target gene expression.
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Affiliation(s)
- Mary Hunzicker-Dunn
- Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
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Song KH, Park YY, Kee HJ, Hong CY, Lee YS, Ahn SW, Kim HJ, Lee K, Kook H, Lee IK, Choi HS. Orphan nuclear receptor Nur77 induces zinc finger protein GIOT-1 gene expression, and GIOT-1 acts as a novel corepressor of orphan nuclear receptor SF-1 via recruitment of HDAC2. J Biol Chem 2006; 281:15605-14. [PMID: 16595694 DOI: 10.1074/jbc.m505937200] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Kruppel-associated box (KRAB) domain-containing proteins consist of potential transcriptional repression modules. Previously, gonadotropin-inducible ovarian transcription factor-1 (GIOT-1) was identified as a novel KRAB-containing zinc finger protein and shown to have transcriptional repression activity. Here, we demonstrate that orphan nuclear receptor Nur77 regulates GIOT-1 gene expression in testicular Leydig cell lines and that GIOT-1 acts as a novel corepressor of the orphan nuclear receptor steroidogenic factor 1 (SF-1). Mutation analysis of the GIOT-1 promoter and overexpression analysis of dominant-negative Nur77 revealed that luteinizing hormone activates GIOT-1 gene expression through Nur77. Electrophoretic mobility shift and chromatin immunoprecipitation assays showed that Nur77 directly binds to the GIOT-1 promoter. GIOT-1 represses the SF-1 transactivation, and specific interaction between GIOT-1 and SF-1 was observed. We also demonstrate an interaction between GIOT-1 and histone deacetylase 2 (HDAC2). GIOT-1-mediated transrepression was recovered by down-regulation of HDAC2 expression with small interfering RNA of HDAC2. Knock down of the endogenous GIOT-1 results in significant enhancement of CYP17 expression in Leydig cells. In conclusion, this study of cross-talk between GIOT-1 and orphan nuclear receptors will provide new insights into the role of KRAB-containing zinc finger proteins in nuclear receptor action.
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Affiliation(s)
- Kwang-Hoon Song
- Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea
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40
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Zheng W, Jefcoate CR. Steroidogenic factor-1 interacts with cAMP response element-binding protein to mediate cAMP stimulation of CYP1B1 via a far upstream enhancer. Mol Pharmacol 2005; 67:499-512. [PMID: 15523052 DOI: 10.1124/mol.104.005504] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
CYP1B1 activates polycyclic aromatic hydrocarbon carcinogens in cAMP-regulated tissues such as the adrenal, ovary, and testis. A 27-fold cAMP stimulation of the CYP1B1-luciferase reporter in Y-1 adrenal cells depends entirely on a far upstream enhancer region (FUER; -5298 to -5110). Cooperative participation of multiple steroidogenic factor 1 (SF-1) elements with the downstream cAMP response element (CRE) in FUER is essential for both basal and cAMP-stimulated activities of FUER. Basal and induced activities were similarly lowered by DAX-1, an SF-1 suppressor, and raised by steroid receptor coactivator 1, an SF-1 coactivator. cAMP response element-binding protein (CREB)-binding protein (CBP) that interacts preferentially with the phosphorylated-CREB increased the cAMP-induced FUER. 10T1/2 cells and human embryonic kidney (HEK)293 cells do not express SF-1. Introduction of exogenous SF-1 generated cAMP stimulation of the FUER in 10T1/2 fibroblasts. The same transfection only increased basal activity of FUER in HEK293 cells, despite presence of active CREB in cells. HEK293 cells therefore remain deficient in additional factor(s) critical to the cAMP stimulation of CYP1B1. Mutations of the protein kinase A (PKA) and the mitogen-activated protein kinase phosphorylation sites (Ser-430 and Ser-203) on SF-1 had no effect on the SF-1-dependent FUER stimulation in Y-1 and 10T1/2 cells. This contrasts with loss of activity with mutation of CREB at PKA phosphorylation site (Ser-133). SF-1 phosphorylation at these sites is therefore not essential for the cAMP stimulation and the cooperation with CREB. cAMP-enhanced activation protein 1 (AP-1) and stimulatory protein 1 (Sp1) complexes in the proximal promoter region contributed substantially to both basal and cAMP-stimulated FUER activity. Chromatin immunoprecipitation from primary rat adrenal cells demonstrated cAMP stimulation of histone acetylation proximal to, respectively, the FUER and AP-1 sites of CYP1B1.
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Affiliation(s)
- Wenchao Zheng
- Department of Pharmacology, University of Wisconsin, 1300 University Avenue, Madison, WI 53706, USA
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41
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Park Y, Maizels ET, Feiger ZJ, Alam H, Peters CA, Woodruff TK, Unterman TG, Lee EJ, Jameson JL, Hunzicker-Dunn M. Induction of cyclin D2 in rat granulosa cells requires FSH-dependent relief from FOXO1 repression coupled with positive signals from Smad. J Biol Chem 2004; 280:9135-48. [PMID: 15613482 PMCID: PMC1564190 DOI: 10.1074/jbc.m409486200] [Citation(s) in RCA: 137] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Ovarian follicles undergo exponential growth in response to follicle-stimulating hormone (FSH), largely as a result of the proliferation of granulosa cells (GCs). In vitro under serum-free conditions, rat GCs differentiate in response to FSH but do not proliferate unless activin is also present. In the presence of FSH plus activin, GCs exhibit enhanced expression of cyclin D2 as well as inhibin-alpha, aromatase, steroidogenic factor-1 (SF-1), cholesterol side chain (SCC), and epiregulin. In this report we sought to identify the signaling pathways by which FSH and activin promote GC proliferation and differentiation. Our results show that these responses are associated with prolonged Akt phosphorylation relative to time-matched controls and are dependent on phosphatidylinositol 3-kinase (PI 3-kinase) and Smad2/3 signaling, based on the ability of the PI 3-kinase inhibitor LY294002 or infection with adenoviral dominant negative Smad3 (DN-Smad3) mutant to attenuate induction of cyclin D2, inhibin-alpha, aromatase, SCC, SF-1, and epiregulin. The DN-Smad3 mutant also abolished prolonged Akt phosphorylation stimulated by FSH plus activin 24 h post-treatment. Infection with the adenoviral constitutively active forkhead box-containing protein, O subfamily (FOXO)1 mutant suppressed induction of cyclin D2, aromatase, inhibin-alpha, SF-1, and epiregulin. Transient transfections of GCs with constitutively active FOXO1 mutant also suppressed cyclin D2, inhibin-alpha, and epiregulin promoter-reporter activities. Chromatin immunoprecipitation results demonstrate in vivo the association of FOXO1 with the cyclin D2 promoter in untreated GCs and release of FOXO1 from the cyclin D2 promoter upon addition of FSH plus activin. These results suggest that proliferation and differentiation of GCs in response to FSH plus activin requires both removal of FOXO1-dependent repression and positive signaling from Smad2/3.
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Affiliation(s)
- Youngkyu Park
- From the Departments of Cell and Molecular Biology and
| | | | | | - Hena Alam
- From the Departments of Cell and Molecular Biology and
| | | | - Teresa K. Woodruff
- Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois 60208, and the
| | - Terry G. Unterman
- Department of Medicine, University of Illinois College of Medicine and Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois 60612
| | - Eun Jig Lee
- Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois 60208, and the
| | - J. Larry Jameson
- Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois 60208, and the
| | - Mary Hunzicker-Dunn
- From the Departments of Cell and Molecular Biology and
- ** To whom correspondence should be addressed: Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611. Tel.: 312-503-8940; Fax: 312-503-0566; E-mail:
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Hirsh L, Ben-Ami I, Freimann S, Dantes A, Tajima K, Kotsuji F, Amsterdam A. Desensitization to gonadotropic hormones: a model system for the regulation of a G-protein-coupled receptor with 7-transmembrane spanning regions. Biochem Biophys Res Commun 2004; 326:1-6. [PMID: 15567144 DOI: 10.1016/j.bbrc.2004.10.168] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2004] [Indexed: 11/24/2022]
Abstract
Gonadotropic hormone, luteinizing hormone, and follicle-stimulating hormone exert their effect via activation of G-coupled receptors, which activate the hormone sensitive adenylyl cyclase, protein kinase A, and cyclic AMP responsive elements. This activation leads to specific de novo synthesis of steroidogenic factors and steroidogenic enzymes. In normal cells and following activation of this signaling pathway, desensitization period will be followed. This down-regulation, which was studied in detail for the last three decays, was found to take place at various steps of these signal transduction pathways as well as at different kinetics. A common and diverse feature of the mechanism of desensitization in other G-coupled-7-transmembrane receptor system is also discussed.
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Affiliation(s)
- Liron Hirsh
- Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel
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43
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Iyer AK, McCabe ERB. Molecular mechanisms of DAX1 action. Mol Genet Metab 2004; 83:60-73. [PMID: 15464421 DOI: 10.1016/j.ymgme.2004.07.018] [Citation(s) in RCA: 141] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/25/2004] [Revised: 07/12/2004] [Accepted: 07/13/2004] [Indexed: 11/24/2022]
Abstract
DAX1 (dosage sensitive sex reversal (DSS), adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1) encoded by the gene NR0B1, is an unusual orphan nuclear receptor that when mutated causes AHC with associated hypogonadotropic hypogonadism (HH), and when duplicated causes DSS. DAX1 expression has been shown in all regions of the hypothalamic-pituitary-adrenal-gonadal (HPAG) axis during development and in adult tissues, suggesting a critical role for DAX1 in the normal development and function of this axis. Steroidogenic factor 1 (SF1, NR5A1) knockout mice show similar developmental defects as AHC and HH patients, but paradoxically, DAX1 is a negative coregulator of SF1 transactivation. The function of DAX1 as an antagonist of SF1 in gonadal development is consistent with the fact that in humans, duplication of the region of the X chromosome containing DAX1 causes a similar phenotype as mutations in SF1. However, how disruption of DAX1 leads to adrenal, hypothalamic, and pituitary developmental defects similar to SF1 disruption remains to be clarified. The exact mechanism of DAX1 action in each of these tissues during adulthood and critical stages of development are not fully understood. Recent evidence suggests a broader functional role for DAX1 as a negative coregulator of estrogen receptor (ER, NR3A1-2), liver receptor homologue-1 (LRH-1, NR5A2), androgen receptor (AR, NR3C4), and progesterone receptor (PR, NR3C3), each by distinct repression mechanisms. DAX1 may have pleiotropic roles in addition to its function as a negative regulator of steroidogenesis during the development and adult function of the HPAG axis.
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MESH Headings
- Animals
- DAX-1 Orphan Nuclear Receptor
- DNA-Binding Proteins/genetics
- DNA-Binding Proteins/metabolism
- DNA-Binding Proteins/physiology
- Female
- Gene Expression Regulation, Developmental
- Homeodomain Proteins
- Humans
- Hypothalamo-Hypophyseal System/physiology
- Male
- Mice
- Ovary/growth & development
- Ovary/physiology
- Pituitary-Adrenal System/physiology
- Receptors, Androgen/genetics
- Receptors, Androgen/metabolism
- Receptors, Cytoplasmic and Nuclear/genetics
- Receptors, Cytoplasmic and Nuclear/metabolism
- Receptors, Estrogen/genetics
- Receptors, Estrogen/metabolism
- Receptors, Progesterone/genetics
- Receptors, Progesterone/metabolism
- Receptors, Retinoic Acid/physiology
- Repressor Proteins/physiology
- Sex Determination Processes
- Steroidogenic Factor 1
- Testis/growth & development
- Testis/physiology
- Transcription Factors/genetics
- Transcription Factors/metabolism
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Affiliation(s)
- Anita K Iyer
- Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
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Saxena D, Safi R, Little-Ihrig L, Zeleznik AJ. Liver receptor homolog-1 stimulates the progesterone biosynthetic pathway during follicle-stimulating hormone-induced granulosa cell differentiation. Endocrinology 2004; 145:3821-9. [PMID: 15117876 DOI: 10.1210/en.2004-0423] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
FSH-stimulated granulosa cell differentiation is associated with the induction of the LH receptor (LHr) as well as induction of the estrogen and progesterone biosynthetic pathways. Although activation of the cAMP-protein kinase A pathway is sufficient to stimulate progesterone production, additional pathways are required for the induction of the LHr and p450 aromatase. The orphan nuclear receptor, liver receptor homolog-1 (LRH-1), is expressed in granulosa cells and has been shown to synergize with the cAMP signaling system to regulate the gonadal type II aromatase promoter in transient transfection assays. To determine whether LRH-1 can interact with the cAMP pathway in the induction of aromatase and the LHr, we examined the effects of an adenoviral vector that directs the expression of human LRH-1 (Ad-LRH-1) on FSH-stimulated granulosa cell differentiation. Infection of undifferentiated granulosa cells with LRH-1 alone had no effect on estrogen production, progesterone production, or the expression of the LHr. However, combination of FSH stimulation and Ad-LRH-1 infection led to significantly greater progesterone production and increases in mRNA for p450 side-chain cleavage and 3beta-hydroxysteroid dehydrogenase than granulosa cells stimulated by FSH alone. However, infection with Ad-LRH-1 did not stimulate estradiol production or increases in mRNA for p450 aromatase or the LHr above that seen with FSH treatment alone. Moreover, infection with Ad-LRH-1 was able to overcome H-89 inhibition of FSH-stimulated progesterone but not estrogen production. Collectively, these observations support a direct role for LRH-1 in the induction of the progesterone but not the estrogen biosynthetic pathway during granulosa cell differentiation.
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Affiliation(s)
- Deeksha Saxena
- Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA
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Kajitani T, Mizutani T, Yamada K, Yazawa T, Sekiguchi T, Yoshino M, Kawata H, Miyamoto K. Cloning and characterization of granulosa cell high-mobility group (HMG)-box protein-1, a novel HMG-box transcriptional regulator strongly expressed in rat ovarian granulosa cells. Endocrinology 2004; 145:2307-18. [PMID: 14764631 DOI: 10.1210/en.2003-1343] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Specific events in the ovary are dependent on gene expression in the tissue. By screening a rat ovarian granulosa cell cDNA library, a cDNA clone encoding a novel transcription factor-like protein containing a high-mobility group-box, referred to as granulosa cell high-mobility group-box protein-1 (GCX-1), was identified. The expression of GCX-1 is restricted to the hypothalamus, pituitary, testis, uterus, and ovary but was not detected in the adrenal gland. An in situ hybridization study revealed that the expression of GCX-1 was restricted to granulosa cell layers in early-stage follicles, and the expression was very low in large antral follicles and the corpus luteum, but localized expression in the testis or pituitary was not clear. Endogenous GCX-1 protein in the granulosa cells was identified by a Western blot analysis, and an analysis using the green fluorescence protein-GCX-1 fusion protein revealed that the GCX-1 protein was localized in the cell nucleus. GAL4 fusion protein-based assays demonstrated that GCX-1 is a potent transcriptional activator, and its putative transactivation domain was mapped to the region between amino acid residues 25 and 63 from the N terminus. These data strongly suggest that GCX-1 is likely a novel transcriptional activator that is exclusively expressed in reproductive tissues involving the hypothalamo-pituitary-gonadal axis, and functions as a specific regulator of follicular development, and may also participate in other specific events related to reproduction, particularly in the female.
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Affiliation(s)
- Takashi Kajitani
- Department of Biochemistry, Fukui Medical University, Matsuoka, Fukui, 910-1193, Japan
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Fayard E, Schoonjans K, Annicotte JS, Auwerx J. Liver receptor homolog 1 controls the expression of carboxyl ester lipase. J Biol Chem 2003; 278:35725-31. [PMID: 12853459 DOI: 10.1074/jbc.m302370200] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
The orphan nuclear receptor liver receptor homolog 1 (LRH-1) plays a central role in cholesterol homeostasis by regulating a number of hepatic and intestinal genes critical for reverse cholesterol transport and bile acid homeostasis. Herein, we describe the identification of carboxyl ester lipase (CEL) as a novel target of LRH-1 in pancreas, a tissue in which LRH-1 is abundantly expressed. In situ hybridization and gene expression studies demonstrate that both LRH-1 and CEL are co-expressed and confined to the exocrine pancreas. LRH-1 interacts with a consensus LRH-1 response element in the human CEL promoter, which is perfectly conserved in the rat gene, and induces CEL promoter activity in cotransfection assays. As reported for other LRH-1 target genes, the nuclear receptor short heterodimer partner represses LRH-1-induced CEL promoter activity. Chromatin immunoprecipitation demonstrates that binding of LRH-1 to the CEL promoter increases histone H4 acetylation corresponding with the activation of endogenous CEL gene transcription. Our data, identifying CEL as the first pancreatic LRH-1 target gene, indicate that LRH-1 is an important player in enterohepatic cholesterol homeostasis.
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Affiliation(s)
- Elisabeth Fayard
- Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS/INSERM/Université Louis Pasteur, B.P. 10142, F-67404 Illkirch, C.U. de Strasbourg, France
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