| Project/Area Number |
09671185
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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 |
Kidney internal medicine
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| Research Institution | TOKYO WOMEN'S MEDICAL UNIVERSITY |
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Principal Investigator |
KAWASHIMA Akira TOKYO WOMEN'S MEDICAL UNIVERSITY,SCHOOL OF MEDICINE, INSTRUCTOR, 医学部, 助手 (20224769)
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| Co-Investigator(Kenkyū-buntansha) |
UCHIDA Keiko TOKYO WOMEN'S MEDICAL UNIVERSITY,SCHOOL OF MEDICINE, INSTRUCTOR, 医学部, 助手 (60246478)
NITTA Kosaku TOKYO WOMEN'S MEDICAL UNIVERSITY,SCHOOL OF MEDICINE, INSTRUCTOR, 医学部, 助手 (50241071)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1998: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1997: ¥800,000 (Direct Cost: ¥800,000)
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| Keywords | AUTOREGULATION / TRANSCRIPTION / G-PROTEIN / LLC-PK1 CELL / Sp1 / 緊糸球体 / メサンギウム細胞 / 増殖 / 転写因子 |
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| Research Abstract |
A previous report demonstrated that Gαi-2 is regulated by steroid hormone. A mutation of the Gαi-2 gene that decreases GTPase activity produces the oncogene gip2. Autoregulation of this gene should be critical to prevent overgrowth of the cells. We detected Gai-2 protein in glomerular cells by immunostaining and immunoblotting. Then, we tried to transfect Gαi-2 gene into mesangial cells using calcium phosphate precipitation method or lipofection method. However, it was insufficient for the following experiments. Thus, we used LLC-PK1 cells in place of mesangial cells. In the cells transfected with 5'-flanking sequences fused to firefly luciferase cDNA reporter gene, we demonstrated a 24 % transcriptional repression of Gαi-2 gene after overexpressing Gαi-2 protein and a 88 % repression of gip2 protein when gip2 protein is overexpressed. Deletion analysis of the reporter gene indicated that the site of repression in gip2 transfected cells occurred in the region of the Gαi-2 gene promoter. Utilizing the 27-bp DNA segment as a probe in mobility shift assay, we identified a complex with decreased binding in gip2 induced cells. This DNA segment containing tow GC boxes was recognized specifically by a transcription factor Sp1. Antibody to Sp1 added to the mobility shift assay supershifted the complex. These studies demonstrate that overexpression of Gαi-2 protein represses Gαi-2 gene transcription by a reduction of transcription factor Sp1, and suggest the possibility of an autoregulation mechanism of Gαi-2 transcription.
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