蛋白激酶C和JNK在溶血磷脂酸诱导人肺成纤维细胞MCP-1表达中的作用

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 μmol/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 μmol/L）或JNK抑制剂SP600125（0、0.1、1和10 μmol/L）预孵育细胞30 min后，用LPA（10 μmol/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 μmol/L）预孵育30 min，以浓度为10 μmol/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 μmol/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 μmol/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 μmol/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 μmol/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 μmol/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 μmol/L）可显著抑制LPA（10 μmol/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。

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

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 μmol/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 μmol/L) or JNK inhibitor SP600125 (0,0.1,1 and 10 μmol/L) for 30 min,and then treated with LPA (10 μmol/L) for 2 or 6 h,and ELISA and qPCR assays were conducted. Cells were also pre-incubated with PKC inhibitor bisindolylmaleimide I (1 μmol/L) for 30 min,and then treated with LPA (10 μmol/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 μmol/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 μmol/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 μmol/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 μmol/L)-induced MCP-1 protein production was reduced by 60% with bisindolylmaleimide I(1 μmol/L) compared with that in the control group (P < 0.05). With 3 μmol/L of bisindolylmaleimide I,the induced MCP-1 mRNA expression was reduced by 40% (P < 0.05). The LPA (10 μmol/L)-induced MCP-1 protein production was reduced by 78% with SP600125 (1 μmol/L) (P < 0.05). The 10 μmol/L LPA-induced MCP-1 mRNA expression was reduced by 40% with SP600125 (1 μmol/L) (P < 0.05). The activity of JNK was enhanced by LPA in HLF-1 while PKC inhibitors bisindolylmaleimide I (1 μmol/L) suppressed the activity of JNK which was induced by LPA(10 μmol/L). CONCLUSION: PKC and JNK mediated LPA-induced MCP-1 expression in HLF-1.