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

1.通过蛋白激酶、氧自由基和AP-1转录因子的检测，探讨牛肾小球内皮细胞NF-κ B活化的机制。电泳迁移率变动分析（EMSA）显示，TNF-α诱导的NF-κ B激活抑制酪氨酸激酶抑制剂除莠霉素A，但不抑制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。抗氧化剂PDTC和c-jun/AP-1抑制剂姜黄素也降低了NF-κ B的激活。用抗NF-κ B和AP-1组分的抗体处理的核提取物应用于EMSA，NF-κ B与其位点的结合被抗Jun抗体以及NF-κ B p65和p50的抗体降低。此外，抗NF-κ B p65抗体减少AP-1与其位点的结合，表明NF-κ B-DNA和AP-1-DNA复合物的形成分别需要Jun和NF-κ B p65，或 ...更多信息 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-κ B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staurosporine和H-7或酪氨酸激酶抑制剂genistein处理牛肾小球内皮细胞。北方印迹分析显示，TNF-α诱导的MCP-1 mRNA表达仅被金雀异黄素废除。在EMSA中，TNF-α诱导NF-κ B和AP-1的活化。TNF-α诱导的AP-1的活化被genistein降低，而NF-κ B的活化则没有降低，这表明AP-1或其他因子对TNF-α诱导的MCP-1基因表达的调节。而NF-κ B抑制剂PDTC和c-jun/AP-1抑制剂姜黄素均能抑制MCP-1基因的表达，提示MCP-1基因的表达需要NF-κ B和AP-1的参与。这些结果表明，MCP-1基因的表达可能受到多种转录因子的相互作用。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