AP-1在转录水平调控氧化低密度脂蛋白诱导的转化生长因子-β_1表达

Activating protein-1 complex regulates oxidized low density lipoprotein-induced overproduction of rat TGF-β_1 at transcriptional level

目的 了解转录激活蛋白 1(AP 1)是否参与调控氧化低密度脂蛋白 (Ox LDL)诱导转化生长因子 β1(TGF β1)基因的表达 ,探讨Ox LDL激活AP 1可能的信号转导途径。方法 首先利用SD大鼠TGF β1启动子缺失体 荧光素酶 (luciferase)报告基因瞬时转染系膜细胞并检测虫荧光素酶相对活性 ,找出可能的应答区域 ;然后对AP 1结合位点进行突变并加入c Jun/AP 1抑制剂 ,比较启动子活性的变化情况。利用Western印迹检测了Ox LDL对系膜细胞p38有丝分裂原激活的蛋白激酶 (MAPK)总蛋白和磷酸化水平的影响。最后 ,用凝胶阻滞法 (EMSA)观察在特异性的蛋白激酶抑制剂处理下的AP 1活性的变化。结果 对Ox LDL刺激的应答 ,TGF β1启动子 - 15 5 0到 - 84 5之间是一个正调控区域 ,- 6 2 9到 - 4 2 2之间则是一个负调控区。AP 1结合位点突变和c Jun/AP 1抑制剂均导致TGF β1启动子活性显著降低。在Ox LDL刺激下p38MPAK表现出的不是量的变化 ,而是其磷酸化水平的增加。PKC抑制剂明显地抑制了Ox LDL诱导的AP 1活性 ;与之相反 ,p38MPAK抑制剂则对AP 1活性无显著影响。结论 在大鼠肾系膜细胞 ,AP 1在转录水平参与调控Ox LDL诱导TGF β1表达的过程 ;且Ox LDL可能部分是通过PKC信号转导途径激活AP 1中的c Jun蛋白 ,再由c Jun/AP 1从?

Objective To clarify whether the activating protein-1 (AP-1) is involved in the transcriptional regulation of expression of oxidized low density lipoprotein (Ox-LDL) induced transforming growth factor (TGF)-β 1 gene and further to investigate the possible signal transduction pathway in the cultured mesangial cells (MsCs). Methods Firstly, a deletion analysis of SD rat renal mesangial TGF-β 1 promoter (pRT)--luciferase reporter genes was adopted to identify the possible regulatory regions responding to Ox-LDL. With mutation of AP-1 binding site and addition of the c-Jun/AP-1 inhibitor curcumin, the changes of relative luciferase of pRT stimulated by Ox-LDL were assayed, respectively. Futhermore, the phosphorylation of p38MAPK was detected by Western blotting. Pre-treatment with the different specific kinase inhibitors, the activities of AP-1 induced by Ox-LDL were determined by electrophoretic mobility shift assay (EMSA). Results Relative luciferase activity showed two regions responding to stimulation by Ox-LDL in rat TGF-β 1 promoter: one positive regulatory region (-1 550 to -845) containing an accurate AP-1 binding site, and one negative ( -629 to -422) regulatory region. Both mutation of AP-1 binding site and addition of curcumin markedly decrease of activity of TGF-β 1 promoter. The phosphorylation, but not total protein, of p38MAPK was significantly enhanced by Ox-LDL stimulation. The inhibitor of PKC remarkably reduced the activity of AP-1 induced by Ox-LDL. Reversely, the activity of AP-1 didn′t significantly change with the inhibitor of p38MAPK. Conclusion In the cultured rat mesangial cells, the AP-1 complex regulates Ox-LDL induced-overproduction of TGF-β 1 at the transcrptional level. Furthermore, the functional and structural results showed that Ox-LDL regulates rat TGF-β 1 gene expression through AP-1 binding site and c-Jun/AP-1 complex, it gives rise to the involvement of protein kinase C, but not p38MAPK, in Ox-LDL-induced TGF-β 1 gene expression.