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

1.为了探讨牛肾小球内皮细胞核因子-kappaB活化的机制，我们检测了蛋白激酶、氧自由基和AP-1转录因子的参与。凝胶迁移率改变分析(EMSA)显示，酪氨酸激酶抑制剂赫比霉素A可抑制肿瘤坏死因子-α诱导的核因子-kappaB的激活，而其他酪氨酸激酶抑制剂(染料木素和酪氨酸蛋白)或蛋白激酶C抑制剂星形孢子素则不能抑制其激活。抗氧化剂PDTC和c-jun/AP-1抑制剂姜黄素也降低了核因子-kappaB的活性。用抗核因子-kappaB和抗AP-1组分的抗体处理核提取液，用抗Jun抗体和抗p65和p50抗体减少核转录因子-kappaB与其结合部位的结合。此外，抗核因子-kappaB p65抗体减少了AP-1与其位点的结合，表明Jun和NF-kappaB p65分别是形成核因子-kappaB-DNA和AP-1-DNA复合体或…所必需的2.为了评估与MCP-1基因表达相关的细胞内信号，我们通过抑制实验检测了蛋白激酶和转录因子NF-kappaB和AP-1的参与。在刺激和提取RNA和核蛋白之前，用蛋白激酶C抑制剂星形孢子素和H-7或酪氨酸激酶抑制剂金雀异黄素处理牛肾小球内皮细胞。Northern印迹分析显示，只有金雀异黄素能抑制肿瘤坏死因子-α诱导的单核细胞趋化蛋白-1mRNA的表达。在EMSA中，肿瘤坏死因子-α诱导核因子-kappaB和AP-1的活化。金雀异黄素可降低肿瘤坏死因子-α诱导的AP-1的活化，而对核因子-kappaB的激活无影响，提示AP-1或其他因子对肿瘤坏死因子-α诱导的单核细胞趋化蛋白-1基因表达有调节作用。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基因的表达有调节作用。这些结果表明，MCP-1基因的表达可能受多个转录因子的相互作用调节。较少

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