Activation of mitogen-activated protein kinase by oxidized low-density lipoprotein in canine cultured vascular smooth muscle cells

Oxidized low-density lipoprotein (OX-LDL) contributes significantly to the development of atherosclerosis. However, the mechanisms of OX-LDL-induced v ascular smooth muscle cell (VSMC) proliferation are not completely understo od. Therefore, we investigated the effect of OX-LDL on cell proliferation a ssociated with a specific pattern of mitogen-activated protein kinase (MAPK ) by [H-3]thymidine incorporation and p42/p44 MAPK phosphorylation in canin e cultured VSMCs. OX-LDL-induced [H-3]thymidine incorporation and p42/p44 M APK phosphorylation in a time- and concentration-dependent manner in VSMCs. Pretreatment of these cells with pertussis toxin (PTX) for 24 hours attenu ated the OX-LDL-induced [H-3]thymidine incorporation and p42/p44 MAPK phosp horylation, indicating that these responses were mediated through a recepto r coupled to a PTX-sensitive G protein. In cells pretreated with PMA for 24 h and with either the PKC inhibitor staurosporine or the tyrosine kinase i nhibitor genistein for Ih, substantially reduced the [H-3]thymidine incorpo ration and p42/p44 MARK phosphorylation in response to OX-LDL. Removal of C a2+ by addition of BAPTA/AM plus EGTA significantly inhibited OX-LDL-induce d [H-3]thymidine incorporation and p42/p44 MAPK phosphorylation, indicating the requirement of Ca2+ for these responses. OX-LDL-induced [H-3]thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 MAPK). Furthermore, we also showed that overexpression of dominant negative mutant s of Ras (RasN17) and Raf (Raf-301) completely suppressed MEK1/2 and p42/p4 4 MAPK activation induced by OX-LDL and PDGF-BB, indicating that Ras and Ra f may be required for activation of these kinases, Taken together, these re sults suggest that the mitogenic effect of OX-LDL is mediated through a PTX -sensitive G-protein-coupled receptor that involves the activation o Ras/Ra f/MEK/MAPK pathway similar to those of PDGF-BB in canine cultured VSMCs. (C ) 2000 Elsevier Science Inc. All rights reserved.