| 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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