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 vascular smooth muscle cell (VSMC) proliferation are not completely understood. Therefore, we investigated the effect of OX-LDL on cell proliferation associated with a specific pattern of mitogen-activated protein kinase (MAPK) by [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in canine cultured VSMCs. OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in VSMCs. Pretreatment of these cells with pertussis toxin (PTX) for 24 hours attenuated the OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation, indicating that these responses were mediated through a receptor 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 inhibitor genistein for 1h, substantially reduced the [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in response to OX-LDL. Removal of Ca(2+) by addition of BAPTA/AM plus EGTA significantly inhibited OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation, indicating the requirement of Ca(2+) for these responses. OX-LDL-induced [3H]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 mutants of Ras (RasN17) and Raf (Raf-301) completely suppressed MEK1/2 and p42/p44 MAPK activation induced by OX-LDL and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. Taken together, these results suggest that the mitogenic effect of OX-LDL is mediated through a PTX-sensitive G-protein-coupled receptor that involves the activation o Ras/Raf/MEK/MAPK pathway similar to those of PDGF-BB in canine cultured VSMCs.
Yang
C M
CM
Cellular and Molecular Pharmacology Laboratory, Department of Pharmacology, College of Medicine, Chang Gung University, Kwei-San, Taiwan. chuenmao@mail.cgu.edu.tw
Chiu
C T
CT
Wang
C C
CC
Chien
C S
CS
Hsiao
L D
LD
Lin
C C
CC
Tu
M T
MT
Pan
S L
SL
D000818:Animals; D001011:Aorta; D002455:Cell Division; D002478:Cells, Cultured; D004285:Dogs; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000494:pharmacology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000502:physiology*; D037342:Pertussis Toxin; D010766:Phosphorylation; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D008077:Lipoproteins, LDL; D010566:Virulence Factors, Bordetella; C099252:oxidized low density lipoprotein; D037342:Pertussis Toxin; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D019204:GTP-Binding Proteins