副腎皮質腫瘍における核内受容体によるホルモン産生異常機構の検討
| Project/Area Number |
14571072
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Endocrinology
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| Research Institution | Keio University |
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Principal Investigator |
SHIBATA Hiretaka Keio University, Health Center, Assistant Professor, 保健管理センター, 専任講師 (20245484)
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| Co-Investigator(Kenkyū-buntansha) |
栗原 勲 慶應義塾大学, 医学部, 助手 (90338038)
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| Project Period (FY) |
2002 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2004: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2003: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2002: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | COUP-TF / coactivator / Ubc9 / PIAS1 / aldosterone / CYP11B2 / CYP17 / corepressor / 副腎皮質 |
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| Research Abstract |
Aldosterone synthase (CYP11B2) is involved in the final steps of aldosterone biosynthesis and expressed exclusively in the adrenal zona glomerulosa cells. Using electrophoretic mobility shift assay, we demonstrate that COUP-TFI binds to the -129/-114 element (Ad5) of human CYP11B2 promoter. Transient transfection in H295R adrenal cells demonstrated that COUP-TFI enhanced CYP11B2 reporter activity. However, the reporter construct with mutated Ad5 sequences showed reduced basal and COUP-TFI-enhanced activity, suggesting that binding of COUP-TFI to Ad5 is important for CYP11B2 transactivation. To elucidate molecular mechanisms of COUP-TFI-mediated activity, we subsequently screened for COUP-TFI-interacting proteins from a human adrenal cDNA library using a yeast two-hybrid system and identified Ubc9 and PIAS1 which have small ubiquitin-related modifier-1 (SUMO- 1) conjugase and ligase activities, respectively. The co-immunoprecipitation assays confirmed that COUP-TFI forms a complex with Ubc9 and PIAS1 in mammalian cells. Immunohistochemistry showed that Ubc9 and PIAS1 are markedly expressed in rat adrenal glomerulosa cells. Co-expression of Ubc9 and PIAS1 synergistically enhanced the COUP-TFI-mediated CYP11B2 reporter activity, indicating that both proteins function as coactivators of COUP-TFI. However, SUMOylation defective mutants, Ubc9 (C93S) and PIAS1 (C351S), continued to function as coactivators of COUP-TFI, indicating that SUMOylation activity are separable from coactivator ability. In addition, chromatin immunoprecipitation assays demonstrated that ectopically expressed COUP-TFI, Ubc9 and PIAS1 were recruited to an endogenous CYP11B2 promoter. Moreover, reduction of Ubc9 or PIAS1 protein levels by small interfering RNA inhibited the CYP11B2 transactivation by COUP-TFI. Our data support a physiological role of Ubc9 and PIAS1 as transcriptional coactivators in COUP-TFI-mediated CYP11B2 transcription.
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Report
(4 results)
Research Products
(30 results)
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[Journal Article] Spironolactone2004
Author(s)
Hirotaka Shibata
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Journal Title
Evidence of clinical-related endocrinology and metabolic diseases
Pages: 97-99
Description
「研究成果報告書概要(欧文)」より
Related Report
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