Protein kinase C and JN K mediate lysophosphatidic acid-induced monocyte chemo-attractant protein-1 expression in human fetal lung fibroblasts

目的:; 研究蛋白激酶C(PKC)和c-Jun氨基末端激酶(JNK)在溶血磷脂酸(LPA)诱导人肺成纤维细胞(HLF-1)单核细胞趋化蛋白-1(MCP-1; )表达中的作用。方法:培养HLF-1细胞,以不同浓度(0、1、3和10 mumol/L)的LPA处理细胞不同时间(0.5、6、12和24; h)后,ELISA检测细胞培养基上清液中MCP-1的蛋白表达,荧光定量PCR检测MCP-1; mRNA的表达水平。用不同浓度的PKC抑制剂Bisindolylmaleimide I(0、0.1、1和10; mumol/L)或JNK抑制剂SP600125(0、0.1、1和10 mumol/L)预孵育细胞30 min后,用LPA(10; mumol/L)刺激2或6 h后,ELISA方法检测细胞培养基上清液中MCP-1的蛋白表达,荧光定量PCR检测MCP-1; mRNA的表达水平。用PKC抑制剂Bisindolylmaleimide I(1 mumol/L)预孵育30 min,以浓度为10; mumol/L的LPA处理细胞不同时间(0、5、30和60 min)后,Western; blot检测c-Jun蛋白磷酸化水平。结果:LPA可诱导HLF-1细胞MCP-1蛋白释放,并呈剂量效应和时间效应关系。LPA的浓度为10; mumol/L时,HLF-1细胞释放MCP-1蛋白的量是对照组的2.4倍(P<0.05);细胞在LPA处理24; h后,MCP-1蛋白的释放量较对照组增加约1倍(P<0.05)。LPA可诱导HLF-1细胞MCP-1 mRNA的表达并呈时间效应,LPA处理2; h后,MCP-1 mRNA表达水平是对照组的5.3倍(P<0.05)。PKC抑制剂Bisindolylmaleimide; I和JNK抑制剂SP600125均可显著抑制LPA诱导的HLF-1细胞MCP-1; mRNA表达及MCP-1蛋白释放。Bisindolylmaleimide I的浓度为1; mumol/L时可阻断LPA诱导的HLF-1细胞MCP-1蛋白释放量的60%(P<0.05),浓度为3 mumol/L时对MCP-1; mRNA表达有明显抑制效果,抑制率达40%(P<0.05);而SP600125的浓度为1; mumol/L时可阻断LPA诱导的HLF-1细胞MCP-1蛋白释放量的78%(P<0.05),对MCP-1; mRNA表达有明显抑制效果,抑制率达87%(P<0.05)。10; mumol/L的LPA可显著诱导HLF-1细胞c-Jun磷酸化,同时PKC抑制剂Bisindolylmaleimide I(1; mumol/L)可显著抑制LPA(10; mumol/L)诱导的HLF-1细胞c-Jun磷酸化。结论:PKC与JNK通路均参与LPA诱导HLF-1细胞MCP-1的表达。OBJECTIVE: To investigate the role of protein kinase C (PKC) and c-Jun; N-terminal kinase (JNK) in modulating lysophosphatidic acid; (LPA)-induced monocyte chemo-attractant protein-1 (MCP-1) expression in; human fetal lung fibroblasts (HLF-1). METHODS:Cultured human lung; fibroblasts (HLF-1) were incubated with LPA(0,1,3 and 10 mumol/L) for; 0.5,6,12 and 24 h. ELISA was used to detect MCP-1 protein levels in the; supernatants,and quantitative real-time PCR (qPCR) for MCP-1 mRNA levels; in the cell lysate. In addition,cells were pre-incubated with PKC; inhibitor bisindolylmaleimide I (0,0.1,1 and 10 mumol/L) or JNK; inhibitor SP600125 (0,0.1,1 and 10 mumol/L) for 30 min,and then treated; with LPA (10 mumol/L) for 2 or 6 h,and ELISA and qPCR assays were; conducted. Cells were also pre-incubated with PKC inhibitor; bisindolylmaleimide I (1 mumol/L) for 30 min,and then treated with LPA; (10 mumol/L) for 30 min. C-Jun N-terminal phosphorylation levels in the; cell lysate were measured by Western blot. RESULTS:LPA stimulated MCP-1; protein expression in dose-and time-dependent manners. The MCP-1 protein; production in the LPA (10 mumol/L) treated group was 2.4 times higher; than that in the untreated group (P<0.01). MCP-1 protein production in; the LPA (10 mumol/L) treated group for 24 h was 1 time higher than that; in the untreated group (P<0.01). LPA stimulated MCP-1 mRNA expression in; a time-dependent manner. The MCP-1 mRNA expression in the LPA (10; mumol/L) treated group for 2 h was 5.3 times higher than that in the; untreated group (P<0.01). PKC inhibitors bisindolylmaleimide I and JNK; SP600125 clearly inhibited LPA-induced MCP-l mRNA and protein; expressions. The LPA (10 mumol/L)-induced MCP-1 protein production was; reduced by 60% with bisindolylmaleimide I(1 mumol/L) compared with that; in the control group (P<0.05). With 3 mumol/L of bisindolylmaleimide; I,the induced MCP-1 mRNA expression was reduced by 40% (P<0.05). The LPA; (10 mumol/L)-induced MCP-1 protein production was reduced by 78% with; SP600125 (1 mumol/L) (P<0.05). The 10 mumol/L LPA-induced MCP-1 mRNA; expression was reduced by 40% with SP600125 (1 mumol/L) (P<0.05). The; activity of JNK was enhanced by LPA in HLF-1 while PKC inhibitors; bisindolylmaleimide I (1 mumol/L) suppressed the activity of JNK which; was induced by LPA(10 mumol/L). CONCLUSION:PKC and JNK mediated; LPA-induced MCP-1 expression in HLF-1.国家自然科学基金项目; 厦门大学中央高校基本业务费专项资金项目; 福建省自然科学基金青年项目; 福建省卫生系统中青年骨干人才培养项目;; 厦门市科技惠民计划项

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