2006 Fiscal Year Final Research Report Summary
Regulation of aldosterone synthesis by nuclear receptor Nurr1
| Project/Area Number |
17590966
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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 Hirotaka Keio University, Health Center, Assistant Professor, 保健管理センター, 専任講師 (20245484)
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| Project Period (FY) |
2005 – 2006
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| Keywords | aldosterone / Nurr1 / aldosterone synthase / signal transduction |
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| Research Abstract |
1.Screening of Nurr1-interacitng proteins
1)Generation of adrenal adeoma cDNA library and yeast two-hybrid screening
Complementary DNAs were prepared by mRNA preparation followed by reverse transcription with random oligomers from aldosterone-producing adenomas. EcoRI and XhoI linker oligonucleotides were added at both ends of the individual cDNAs. We then performed yeast two-hybrid screening with Gal4 DNA-binding domain-fused Nurr1 as a bait from the cDNA library. We then identified Ubc9 and PIAS1 as Nurr1-interacitng proteins.
2)Effects of Nurr1 on aldosterone synthase CYP11B2 gene promoter activity
Several cis-elements of the human CYP11B2 promtoer such as Ad5 and NBRE elements were reported. We performed the CYP11B2 promoter-linked luciferase reporter assays in human adrenocortical H295R cells. COUP-TFI, COUP-TFII, Nurr1,and NGFI-B significantly increased the CYP11B2 reporter activity in a dose-dependent manner, whereas SF-1 suppressed the activity.
3)Significance of Ad5 and NBRE elemen
… More
ts of the CYP11B2 gene promoter
We subsequently investigated the significance of the Ad5 and NBRE elements of the CYP11B2 promoter activity utilizing reporters in which Ad5 and/or NBRE elements were mutated. Mutation of the Ad5 element almost completely disrupted the reporter activity. Mutation of the NBRE elements reduced the reporter activity by 75%. Therefore, both Ad5 and NBRE elements play important roles in CYP11B2 promoter activity.
2.Effects of Ubc9 and PIAS1 on Nurr1-mediated transcriptional activation
Nurr1 activated the CYP11B2 promoter activity. Coexpression of Ubc9 or PIAS1 further potentiated the CYP11B2 reporter activity. In addition, sumoylation-inactive mutants Ubc9(C93S) and PIAS1(C351S) similarly potentiated the CYP11B2 promoter activity, suggesting that sumoylation activity is not required for coactivation of the CYP11B2 promoter activity. Although sumoylation of nuclear receptors generally represses transcriptional activity, both Ubc9 and PIAS1 may have new function of coactivation activity for Nurr1 in H295R cells. The molecular nature of the novel functions remain to be clarified in the future. Less
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