Mitogen-Activated Protein Kinase Activation Is Involved in Platelet-Derived Growth Factor-Directed Migration by Vascular Smooth Muscle Cells
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Migration of vascular smooth muscle cells (VSMCs) is a crucial response to vascular injury resulting in neointima formation and atherosclerosis. Platelet-derived growth factor (PDGF-BB) functions as a potent chemoattractant for VSMCs and enhances these pathologies in the vasculature. However, little is known about the intracellular pathways that mediate VSMC migration. In the present study, we investigated the role of mitogen-activated protein kinase (MAPK) activation in this function, since PDGF-BB as well as other growth factors activate this pathway. Using an in-gel kinase assay, we observed that PD 98059, an inhibitor of MEK that activates MAP kinase, inhibited PDGF-BB-induced activation of ERK-1 and ERK-2 in cultured rat aortic smooth muscle cells in a concentration-dependent manner. In contrast, PDGF-mediated activation of intracellular calcium release was not affected by PD 98059. The chemotactic response of both rat aortic smooth muscle cells (RASMCs) and human umbilical vein smooth muscle cells (HUSMCs) toward PDGF-BB (10 ng/mL) was significantly reduced by PD 98059 (10 μmol/L) to 41.7±7.1% in RASMCs (P>.01) and to 47.2 ±5.3% in HUSMCs (P>.01). Similar inhibition was seen at 30 μmol/L, less at 1 μmol/L. To further confirm the specificity of these results implicating the MAPK pathway, an antisense oligodeoxynucleotide (ODN) directed against the initiation translation site of rat ERK-1 and ERK-2 mRNA was used to suppress MAP kinase synthesis and function in rat VSMCs. Liposomal transfection with 0.4 μmol/L antisense ODN reduced ERK-1 and ERK-2 protein by 65% (P>.01) after 48 hours. The chemotactic response to PDGF-BB (10 ng/mL) was reduced by 75% (P>.01) in rat VSMCs transfected with the same antisense ODN concentration. Sense and scrambled control ODNs (0.4 μmol/L) did not affect ERK-1 and ERK-2 protein concentrations or chemotaxis of VSMCs induced by PDGF-BB. These experiments provide the first evidence that activation of MAPK is a critical event in PDGF-mediated signal transduction regulating VSMC migration.
- FBS = fetal bovine serum
- HBKM = HEPES-buffered Krebs' medium
- IGF-1 = insulin-like growth factor
- MAP = mitogen-activated protein
- MAPK = mitogen-activated protein kinase
- MEK = mitogen-activated protein kinase kinase
- ODN = oligodeoxynucleotide
- PDGF = platelet-derived growth factor
- RAF = MEKK, MEK kinase
- RAS = guanine-nucleotide binding protein
- VSMC = vascular smooth muscle cell