Trypsin stimulates proteinase-activated receptor-2-dependent and -independent activation of mitogen-activated protein kinases

摘要

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 tiypsin inhibitor. The actions of trypsin were closely mimicked by the proteinase-activated receptor 2 (PAR-2)-activating peptide sequence SLIGRL butnot LSIGRL Peak MAP kinase activation in response to both trypsin and SLIGRL was also dependent on concentration, with EC_(50), values of 12.1±3.4 nM and 62,5 ± 4 5 μM respectively Under conditions where MAP kinase activation by SLIGRL was completelydesensitized by prior exposure of smooth-muscle cells to the peptide, trypsln-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). Tiypsin and SLIGRL also weakly stimulated the activation of the MAP kiaase homologue p.38 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 p38by 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 tianscriptase-PCR analysis confirmed the presence of PAR-2 mRNA insmooth-muscle cells but not fibroblasts. Our results suggest that in vascular smooth-muscle cells, tiypsin 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.