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

NF-kB对肾小球细胞基因表达的调控作用

• 批准号: 07670834
• 负责人: KAKIZAKI Yoshiki
• 金额: $ 1.54万
• 依托单位: HIROSAKI UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07670834/
• 关键词: 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

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

1.为了探讨NF-kappaB在牛肾小球内皮细胞中的激活机制，我们检测了蛋白激酶、氧自由基和AP-1转录因子的参与。电泳迁移率转移试验（EMSA）显示，tnf - α诱导的NF-kappaB活化被酪氨酸激酶抑制剂herbimycin a抑制，但不被其他酪氨酸激酶抑制剂（染料木素和tyrphostin）或蛋白激酶C抑制剂staurosporine抑制。抗氧化剂PDTC和c-jun/AP-1抑制剂姜黄素也能降低NF-kappaB的激活。核提取物经NF-kappaB和AP-1成分抗体处理后，应用于EMSA。抗jun抗体以及NF-kappaB p65和p50抗体降低了NF-kappaB与其位点的结合。此外，一种抗NF-kappaB p65抗体降低了AP-1与其位点的结合，这表明Jun和NF-kappaB p65分别是NF-kappaB- dna和AP-1- dna复合物形成所必需的，或者更重要的是它们在复合物内的存在。为了评估与MCP-1基因表达相关的细胞内信号，我们通过抑制研究检测了蛋白激酶和转录因子NF-kappaB和AP-1的参与。用蛋白激酶C抑制剂staurosporine和H-7或酪氨酸激酶抑制剂染料木素处理牛肾小球内皮细胞，然后刺激并提取RNA和核蛋白。Northern blot分析显示，tnf - α诱导的MCP-1 mRNA表达仅被染料木黄酮所抑制。在EMSA中，tnf - α诱导NF-kappaB和AP-1的激活。染料木黄酮降低了tnf - α诱导的AP-1的活化，而NF-kappaB的活化则没有，提示AP-1或其他因子调控了tnf - α诱导的MCP-1基因的表达。而NF-kappaB抑制剂PDTC和c-jun/AP-1抑制剂姜黄素均抑制MCP-1基因的表达，提示NF-kappaB和AP-1对MCP-1基因的表达是必需的。这些结果表明MCP-1基因的表达可能受多种转录因子的相互作用调控。少

项目成果

柿崎良樹: "培養糸球体内皮細胞における転写因子NF-_kBの活性化機序" 日本小児腎臓病学会雑誌. (印刷中).

Yoshiki Kakizaki：“培养肾小球内皮细胞中转录因子 NF-_kB 的激活机制”，日本小儿肾病学会杂志（正在出版）。