title: Trypsin stimulates proteinase-activated receptor-2-dependent and -independent activation of mitogen-activated protein kinases.
abstract: 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.
authors.affiliation: Department of Physiology and Pharmacology, University of Strathclyde, Royal College, Glasgow, U.K.
authors.forename: C M
authors.initials: CM
authors.lastname: Belham
authors.forename: R J
authors.initials: RJ
authors.lastname: Tate
authors.forename: P H
authors.initials: PH
authors.lastname: Scott
authors.forename: A D
authors.initials: AD
authors.lastname: Pemberton
authors.forename: H R
authors.initials: HR
authors.lastname: Miller
authors.forename: R M
authors.initials: RM
authors.lastname: Wadsworth
authors.forename: G W
authors.initials: GW
authors.lastname: Gould
authors.forename: R
authors.initials: R
authors.lastname: Plevin
mesh_terms: D000818:Animals; D001011:Aorta; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002417:Cattle; D005576:Colforsin / Q000494:pharmacology; D004789:Enzyme Activation / Q000502:physiology*; D005347:Fibroblasts; D048031:JNK Mitogen-Activated Protein Kinases; D008168:Lung; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D009130:Muscle, Smooth / Q000201:enzymology; D009842:Oligopeptides / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D044464:Receptor, PAR-2; D011956:Receptors, Cell Surface / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology*; D014361:Trypsin Inhibitors / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases
chemical_list: D009842:Oligopeptides; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D044464:Receptor, PAR-2; D011956:Receptors, Cell Surface; D014361:Trypsin Inhibitors; C099570:seryl-leucyl-isoleucyl-glycyl-arginyl-leucine; D005576:Colforsin; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D014357:Trypsin