氧化-低密度脂蛋白诱导系膜细胞转录激活蛋白1活化的信号转导通路

Signal pathways involve in the stimulation of AP-1 binding activity induced by ox-LDL in mcsangial cells

目的 观察氧化-低密度脂蛋白(ox-LDL)诱导系膜细胞转录激活蛋白1(AP-1)活化的作用,探讨AP-1活化的上游信号转导机制。方法 大鼠肾小球系膜细胞随机分为正常细胞组,ox-LDL处理组和ox-LDL+蛋白激酶抑制剂组。采用Western blot测定丝裂原激活的蛋白激酶(MAPK)家族磷酸化水平,采用凝胶迁移率实验观察蛋白激酶抑制剂bisindolylmaleimide I、H89、genistein、PD98059及SB203580对ox-LDL诱导系膜细胞AP-1活性的影响。结果 ox-LDL呈浓度和时间依赖性激活JNK1/SAPK(stress activated protein kinase)、p38MAPK磷酸化(P<0.05),而ox-LDL对p44/42MAPK磷酸化无影响(P>0.05)。bisindolylmaleimide I浓度为50、100、200 nmol/L时均显著抑制ox-LDL诱导系膜细胞AP-1活性。H89剂量为0.5、5 μmol/L时,AP-1活性显著受抑制。genistein浓度为25、50 μmol/L时并不抑制ox-LDL诱导AP-1活性,浓度达100 μmol/L时则显著抑制ox-LDL诱导系膜细胞AP-1活性。SB203580和PD98059均不能抑制ox-LDL诱导AP-1活性。结论 PKC、PKA及PTK等多种蛋白激酶参与对ox-LDL诱导系膜细胞AP-1活性的调控。

Objective To investigate the possible role of protein kinase(PK) activity in the induction of AP-1 by ox-LDL in mesangial cells and to elucidate the upstream signal pathways involved in the ox-LDL-induced AP-1 binding. Methods Rat mesangial cells were randomly divided into the normal cells, ox-LDL-treated cells and PK inhibitor + ox-LDL-treated cells. Treatments with the PKC inhibitor bisindolylmaleimide I, the PKA inhibitor H89, the PTK inhibitor genistein (GEN), the MEKi inhibitor PD98059, or the p38 MAPK inhibitor SB203580 were applied prior to a 24-hour incubation of ox-LDL in mesangial cells. The phosphorylation of MAPK families was detected by Weatern blot analysis. AP-1 binding was determined by gel shift assay. Results Ox-LDL stimulated the phosphorylation of JNKi/SAPK and p38 MAPK( P < 0. 05), but did not increase the phosphorylation of p44/42 MAPK in mesangial cells ( P > 0. 05) . Bisindolylmaleimide I at 50, 100, 200 nmol/L appreciably reduced the ox-LDL-induced AP-1 binding. H89 at 0. 5, 5 umol/L significantly inhibited AP-1 binding by ox-LDL. GEN at 25, 50 umol/L did not reduce the AP-1 binding by ox-LDL, but when GEN rose up to 100 umol/L, the ox-LDL-induced AP-1 binding significantly decreased in mesangial cells. However, SB203580 and PD98059 did not reduce the ox-LDL-induced AP-1 binding in the present study. Conclusion Multiple protein kinases may involve in the stimulation of AP-1 by ox-LDL in mesangial cells.