Belham C M, Tate R J, Scott P H, Pemberton A D, Miller H R, Wadsworth R M, Gould G W, Plevin R
Department of Physiology and Pharmacology, University of Strathclyde, Royal College, Glasgow, U.K.
Biochem J. 1996 Dec 15;320 ( Pt 3)(Pt 3):939-46. doi: 10.1042/bj3200939.
We have examined protease-mediated activation of the mitogen-activated protein (MAP) kinase cascade in rat aortic smooth-muscle cells and bovine pulmonary arterial fibroblasts. Exposure of smooth-muscle cells to trypsin evoked rapid and transient activation of c-Raf-1, MAP kinase kinase 1 and 2 and MAP kinase that was sensitive to inhibition by soybean trypsin inhibitor. The actions of trypsin were closely mimicked by the proteinase-activated receptor 2 (PAR-2)-activating peptide sequence SLIGRL but not LSIGRL. Peak MAP kinase activation in response to both trypsin and SLIGRL was also dependent on concentration, with EC50 values of 12.1 +/- 3.4 nM and 62.5 +/- 4.5 microM respectively. Under conditions where MAP kinase activation by SLIGRL was completely desensitized by prior exposure of smooth-muscle cells to the peptide, trypsin-stimulated MAP kinase activity was markedly attenuated (78.9 +/- 15.1% desensitization), whereas the response to thrombin was only marginally affected (16.6 +/- 12.1% desensitization). Trypsin and SLIGRL also weakly stimulated the activation of the MAP kinase homologue p38 in smooth-muscle cells without any detectable activation of c-Jun N-terminal kinase. Strong activation of the MAP kinase cascade and modest activation of p38 by trypsin were also observed in fibroblasts, although in this cell type these effects were not mimicked by SLIGRL nor by the thrombin receptor-activating peptide SFLLRNPNDKYEPF. Reverse transcriptase-PCR analysis confirmed the presence of PAR-2 mRNA in smooth-muscle cells but not fibroblasts. Our results suggest that in vascular smooth-muscle cells, trypsin stimulates the activation of the MAP kinase cascade relatively selectively, in a manner consistent with an interaction with the recently described PAR-2. Activation of MAP kinase by trypsin in vascular fibroblasts, however, seems to be independent of PAR-2 and occurs by an undefined mechanism possibly involving novel receptor species.
我们研究了蛋白酶介导的丝裂原活化蛋白（MAP）激酶级联反应在大鼠主动脉平滑肌细胞和牛肺动脉成纤维细胞中的激活情况。将平滑肌细胞暴露于胰蛋白酶会引发c-Raf-1、MAP激酶激酶1和2以及MAP激酶的快速短暂激活，这种激活对大豆胰蛋白酶抑制剂的抑制敏感。胰蛋白酶的作用被蛋白酶激活受体2（PAR-2）激活肽序列SLIGRL紧密模拟，但不被LSIGRL模拟。对胰蛋白酶和SLIGRL的反应中，MAP激酶激活的峰值也依赖于浓度，其EC50值分别为12.1±3.4 nM和62.5±4.5 μM。在平滑肌细胞预先暴露于该肽而使SLIGRL激活MAP激酶完全脱敏的条件下，胰蛋白酶刺激的MAP激酶活性明显减弱（脱敏78.9±15.1%），而对凝血酶的反应仅受到轻微影响（脱敏16.6±12.1%）。胰蛋白酶和SLIGRL还微弱地刺激了平滑肌细胞中MAP激酶同源物p38的激活，而未检测到c-Jun N末端激酶的激活。在成纤维细胞中也观察到胰蛋白酶对MAP激酶级联反应的强烈激活和对p38的适度激活，尽管在这种细胞类型中，这些作用既不被SLIGRL模拟，也不被凝血酶受体激活肽SFLLRNPNDKYEPF模拟。逆转录聚合酶链反应分析证实平滑肌细胞中存在PAR-2 mRNA，而成纤维细胞中不存在。我们的结果表明，在血管平滑肌细胞中胰蛋白酶相对选择性地刺激MAP激酶级联反应的激活，其方式与最近描述的与PAR-2的相互作用一致。然而，在血管成纤维细胞中，胰蛋白酶激活MAP激酶似乎与PAR-2无关，并且通过一种可能涉及新型受体种类的未定义机制发生。
