TRYPSIN STIMULATES PROTEINASE-ACTIVATED RECEPTOR-2-DEPENDENT AND RECEPTOR-2-INDEPENDENT ACTIVATION OF MITOGEN-ACTIVATED PROTEIN-KINASES

We have examined protease-mediated activation of the mitogen-activated protein (MAP) kinase cascade in rat aortic smooth-muscle cells and bo vine pulmonary arterial fibroblasts. Exposure of smooth-muscle cells t o 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 soy bean trypsin inhibitor. The actions of-trypsin were closely mimicked b y the proteinase-activated receptor 2 (PAR-2)-activating peptide seque nce SLIGRL but not LSIGRL. Peak MAP kinase activation in response to b oth trypsin and SLIGRL was also dependent on concentration, with EC(50 ) values of 12.1+/-3.4 nM and 62.5+/-4.5 mu M respectively. Under cond itions where MAP kinase activation by SLIGRL was completely desensitiz ed by prior exposure of smooth-muscle cells to the peptide, trypsin-st imulated MAP kinase activity was markedly attenuated (78.9+/-15.1% des ensitization), whereas the response to thrombin was only marginally af fected (16.6+/-12.1% desensitization). Trypsin and SLIGRL also weakly stimulated the activation of the MAP kinase homologue p38 in smooth-mu scle cells without any detectable activation of c-Jun N-terminal kinas e. Strong activation of the MAP kinase cascade and modest activation o f p38 by trypsin were also observed in fibroblasts, although in this c ell 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-muscl e cells, trypsin stimulates the activation of the MAP kinase cascade r elatively 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 oc curs by an undefined mechanism possibly involving novel receptor speci es.