ROLE OF NF-kB ON THE REGULATION OF GENE EXPRESSION IN RENAL GLDMERULAR CELLS

Research Project

Project/Area Number	07670834
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	Pediatrics
Research Institution	HIROSAKI UNIVERSITY
Principal Investigator	KAKIZAKI Yoshiki HIROSAKI UNIVERSITY, UNIVERSITY HOSPTAL,LECTEURER, 医学部・附属病院, 講師 (40160973)
Co-Investigator(Kenkyū-buntansha)	WAGA Shinobu HIROSAKI UNIVERSITY,SCHOOL OF MEDICINE,ASSOCIATE Prof., 医学部, 助教授 (10167744)
Project Period (FY)	1995 – 1996
Project Status	Completed (Fiscal Year 1996)
Budget Amount *help	¥2,400,000 (Direct Cost: ¥2,400,000) Fiscal Year 1996: ¥900,000 (Direct Cost: ¥900,000) Fiscal Year 1995: ¥1,500,000 (Direct Cost: ¥1,500,000)
Keywords	GLOMERULAR ENDOTHELIAL CELLS / NF-kappaB / AP-1 / MCP-1 / GENE EXPRESSION / NFkB / MCP-1遺伝子 / 糸球体メサンギウム細胞 / NF-κB
Research Abstract	1.To explore the mechanisms of NF-kappaB activation in bovine glomerular endothelial cells, we examined the involvement of protein kinases, oxygen radicals, and AP-1 transcription factor. Electrophoretic mobility shift assay (EMSA) revealed that TNF-alpha-induced activation of NF-kappaB was inhibited by a tyrosine kinase inhibitor herbimycin A,but not by other tyrosine kinase inhibitors (genistein and tyrphostin) or a protein kinase C inhibitor staurosporine. An anti-oxidant PDTC and a c-jun/AP-1 inhibitor curcumin also reduced NF-kappaB activation. Nuclear extracts treated with antibodies to NF-kappaB and to AP-1 components were applied to EMSA.The binding of NF-kappaB to its site was reduced by an anti-Jun antibody as well as by antibodies to NF-kappaB p65 and to p50. In addition, an anti-NF-kappaB p65 antibody reduced the binding of AP-1 to its site, suggesting the requirement of Jun and NF-kappaB p65 for the formation of the NF-kappaB-DNA and the AP-1-DNA complex, respectively, or … More the presence of them within the complex. 2.To evaluate the intracellular signals associated with MCP-1 gene expression, we examined the involvement of protein kinases and transcription factors NF-kappaB and AP-1 by inhibition studies. Bovine glomerular endothelial cells were treated with protein kinase C inhibitors staurosporine and H-7, or a tyrosine kinase inhibitor genistein, before stimulation and extraction of RNA and nuclear protein. Northern blot analysis revealed that TNF-alpha-induced MCP-1 mRNA expression was abrogated only by genistein. In EMSA,TNF-alpha induced the activation of NF-kappaB and AP-1. TNF-alpha-induced activation of AP-1 was reduced by genistein, whereas that of NF-kappaB was not, suggesting the regulation by AP-1 or other factor of TNF-alpha-induced MCP-1 gene expression. However, both of NF-kappaB inhibitor PDTC and c-jun/AP-1 inhibitor curcumin inhibited MCP-1 gene expression, suggesting the requirement of NF-kappaB and AP-1 for the expression of MCP-1 gene. These results indicate that MCP-1 gene expression may be regulated by the interaction of multiple transcription factors. Less