| 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)
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| 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
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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
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