转录活化蛋白在人肾小球系膜细胞表达金属蛋白酶组织抑制物1中的信号转导作用

STAT mediated thrombin induction of tissue inhibitor of metalloproteinase1 in human mesangial cells

目的 探讨凝血酶对体外培养的人肾小球系膜细胞表达基质金属蛋白酶组织抑制物 1(TIMP1)的影响以及信号转导转录活化蛋白 (STAT)信号转导途径对这一过程的介导机制。方法 分离并传代培养人肾小球系膜细胞。在无血清条件下 ,采用不同浓度凝血酶 (0 5、1 5和 4 5U/ml)刺激后提取细胞总RNA ,进行TIMP1的Northern杂交分析。制备细胞核蛋白提取物 ,应用凝胶阻滞试验(EMSA)测定STAT DNA结合活性变化。分别应用凝血酶特异性抑活物 水蛭素和STAT1、STAT3反义核酸转染技术进行阻断分析。结果 在体外培养的人系膜细胞 ,基础状态下可以表达一定水平的TIMP1mRNA和STAT DNA结合活性。不同浓度凝血酶 (0 5、1 5、4 5U/ml)刺激后TIMP1mRNA转录水平分别是对照组的 0 4、1 4和 3 7倍 ;在凝血酶作用下 ,细胞TIMP1mRNA表达水平与STAT DNA结合活性呈一致性变化 ;凝血酶特异性抑活物 水蛭素可同时抑制TIMP1mRNA表达水平及STAT DNA结合活性 ;细胞转染STAT1和STAT3反义寡核苷酸后在降低STAT DNA结合活性的同时 ,阻断凝血酶对TIMP1mRNA表达的诱导作用。结论 STAT参与了凝血酶诱导人肾小球系膜细胞TIMP1的基因表达过程。

Objective To depict the role of signal transducers and activators of transcription (STAT) pathway in thrombin induction of tissue inhibitor of matrix metalloproteinase 1 (TIMP1) in human mesangial cells. Methods Human mesangial cells were isolated, seeded in RPMI1640, and stimulated with thrombin (0.5, 1.5, and 4.5 U/ml). Northern blot and electrophoretic mobility shift assay (EMSA) were employed to determine the levels of TIMP1 mRNA and STAT DNA binding activity in the cells, respectively. In order to confirm the role of STAT in this cell event, STAT 1 and STAT 3 antisense oligonucleotides were employed to suppress their intra cellular activity. Results Cultured human mesangial cells expressed basal TIMP1 mRNA, and thrombin promoted TIMP1 gene transcription in dose dependent manner. Hirudin, a specific inhibitor of thrombin, blocked thrombin induced TIMP1 gene expression. Thrombin promoted STAT DNA binding activity in a manner parallel to TIMP1 mRNA expression, which was inhibited by its inhibitor, hirudin again. STAT1 and STAT3 antisense oligonucleotides inhibited both STAT DNA binding activity and TIMP1 mRNA expression in mesangial cells. Conclusion STAT mediates the thrombin induced expression of TIMP1 gene in cultured human mesangial cells.