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 µM 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.
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December 1996
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Research Article|
December 15 1996
Trypsin stimulates proteinase-activated receptor-2-dependent and -independent activation of mitogen-activated protein kinases
Christopher M. BELHAM;
Christopher M. BELHAM
‖
*Department of Physiology and Pharmacology, University of Strathclyde, Royal College, 204 George Street, Glasgow G1 1XW, U.K.
‖To whom correspondence should be addressed.
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Rothwelle J. TATE;
Rothwelle J. TATE
†Department of Molecular Biology Laboratory, University of Strathclyde, Royal College, 204 George Street, Glasgow G1 1XW, U.K.
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Pamela H. SCOTT;
Pamela H. SCOTT
*Department of Physiology and Pharmacology, University of Strathclyde, Royal College, 204 George Street, Glasgow G1 1XW, U.K.
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Alan D. PEMBERTON;
Alan D. PEMBERTON
‡Department of Veterinary Clinical Studies, Royal School of Veterinary Studies, University of Edinburgh, Veterinary Field Station, Easter Bush, Roslin, Midlothian EH25 9RG, U.K.
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Hugh R. P. MILLER;
Hugh R. P. MILLER
‡Department of Veterinary Clinical Studies, Royal School of Veterinary Studies, University of Edinburgh, Veterinary Field Station, Easter Bush, Roslin, Midlothian EH25 9RG, U.K.
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Roger M. WADSWORTH;
Roger M. WADSWORTH
*Department of Physiology and Pharmacology, University of Strathclyde, Royal College, 204 George Street, Glasgow G1 1XW, U.K.
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Gwyn W. GOULD;
Gwyn W. GOULD
§Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, University Avenue, Glasgow G12 8QQ, U.K.
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Robin PLEVIN
Robin PLEVIN
*Department of Physiology and Pharmacology, University of Strathclyde, Royal College, 204 George Street, Glasgow G1 1XW, U.K.
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Publisher: Portland Press Ltd
Received:
April 24 1996
Revision Received:
August 15 1996
Accepted:
August 27 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 320 (3): 939–946.
Article history
Received:
April 24 1996
Revision Received:
August 15 1996
Accepted:
August 27 1996
Citation
Christopher M. BELHAM, Rothwelle J. TATE, Pamela H. SCOTT, Alan D. PEMBERTON, Hugh R. P. MILLER, Roger M. WADSWORTH, Gwyn W. GOULD, Robin PLEVIN; Trypsin stimulates proteinase-activated receptor-2-dependent and -independent activation of mitogen-activated protein kinases. Biochem J 15 December 1996; 320 (3): 939–946. doi: https://doi.org/10.1042/bj3200939
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