Cl. Belham et al., TRYPSIN STIMULATES PROTEINASE-ACTIVATED RECEPTOR-2-DEPENDENT AND RECEPTOR-2-INDEPENDENT ACTIVATION OF MITOGEN-ACTIVATED PROTEIN-KINASES, Biochemical journal, 320, 1996, pp. 939-946
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.