Abstract-We previously reported that extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK), belonging to mitogen-activated protein kinases, are rapidly activated in balloon-injured artery. Therefore, we examined the role of these kinase activations in neointimal formation by using an in vivo gene transfer technique. We made the dominant-negative mutants of ERK (DN-ERK) and JNK (DN-JNK) to specifically inhibit endogenous ERK and JNK activation, respectively. Before balloon injury, these mutants were transfected into rat carotid artery using the hemagglutinating virus of Japan liposome method. In vivo transfection of DN-ERK and DN-JNK significantly suppressed the activation of ERK and JNK, respectively, after balloon injury, confirming successful expression of the transfected genes. Neointimal formation at 14 and 28 days after injury was prevented by gene transfer of DN-ERK or DN-JNK. Furthermore, bromodeoxyuridine labeling index and total cell-counting analysis at 7 days showed that either DN-ERK or DN-JNK remarkably suppressed smooth muscle cell (SMC) proliferation in both the intima and the media after injury. Gene transfer of wild-type ERK (W-ERK) or JNK (W-JNK) significantly enhanced neointimal hyperplasia at 14 days after injury. Furthermore, DN-ERK and DN-JNK significantly suppressed serum-induced SMC proliferation in vitro. We obtained the first evidence that in vivo gene transfer of DN-ERK or DN-JNK prevented neointimal formation in balloon-injured artery by inhibiting SMC proliferation. Thus, ERK and JNK activation triggers SMC proliferation, leading to neointimal formation. These kinases may be the new therapeutic targets for prevention of vascular diseases.
Abstract-Although platelet-derived growth factor (PDGF)-BB is thought to participate in vascular disorders, the mechanism of PDGF-induced vascular smooth muscle cell (SMC) proliferation is not fully understood. This study was undertaken to examine the role of c-Jun in PDGF-BB-induced proliferation of rat aortic SMCs. PDGF-BB (10 ng/mL) significantly increased activator protein (AP)-1 DNA binding activity in SMCs, followed by the increase in [ 3 H]thymidine incorporation and cell number. SMCs were infected with recombinant adenovirus containing TAM67, a dominant-negative c-Jun lacking the transactivation domain of wild c-Jun (Ad-DN-c-Jun), to inhibit endogenous AP-1. Ad-DN-c-Jun, which specifically blocked AP-1 transcriptional activity, significantly inhibited PDGF-BB-induced increases in [ 3 H]thymidine incorporation or cell number. As shown by flow cytometric analysis, Ad-DN-c-Jun inhibited PDGF-BB-induced entrance of SMCs into S phase, leading to a G 1 arrest. Ad-DN-c-Jun attenuated PDGF-BB-induced downregulation of p27 Kip1 , as shown by Western blot analysis, and the prevented PDGF-BB-induced decrease in cyclin E/cyclin-dependent kinase 2 complex-associated p27 Kip1 , as shown by immunoprecipitation study. Furthermore, protein kinase assay showed that Ad-DN-c-Jun blocked PDGF-BB-induced activation of cyclin-dependent kinase 2. Our results provide the first evidence that dominant-negative c-Jun inhibits PDGF-BB-induced vascular SMC proliferation by preventing the downregulation of p27 Key Words: platelet-derived growth factor Ⅲ smooth muscle cells Ⅲ c-Jun Ⅲ gene transfer Ⅲ proliferation P latelet-derived growth factor (PDGF)-BB is known as a multifunctional growth factor capable of stimulating various quiescent cells to proliferate. Accumulating evidence indicates that PDGF-BB is involved in various vascular diseases by stimulating vascular smooth muscle cell (SMC) proliferation, as reviewed. 1 However, the mechanism underlying PDGF-induced vascular SMC proliferation is not fully understood. In vascular proliferative diseases, entry into the progression of vascular cells through the cell cycle is thought to be a key event, 2 and PDGF-BB, through activation of the -PDGF receptor, is a critical mediator of the lesion formation after vascular injury. 3 Therefore, detailed investigation into the molecular mechanism underlying PDGF-BBinduced vascular SMC proliferation is essential not only to elucidate the mechanism of vascular diseases but also to develop a new therapeutic strategy for vascular diseases.The mitogenic action of PDGF-BB is initiated by its interaction with PDGF-BB receptor. Receptor-mediated relocation of cytoplasmic proteins to the inner surface of the plasma membrane, accompanied by subsequent tyrosine phosphorylation, results in the stimulation of multiple signaling pathways, including activation of SHP-2, Src, phospholipase C-␥, Ras, protein kinase A, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase, ultimately leading to DNA synthesis, as reviewed. 4,5 PDGF also induces t...
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