Involvement of c-Jun NH2 terminal kinase and p38MAPK in rapamycin-mediated inhibition of neointimal formation in rat carotid arteries.

OBJECTIVE: Rapamycin-coated stents in coronary artery lesions have recently been shown to be effective in inhibiting neointimal formation. However, little is known about the effects of rapamycin on mitogen-activated protein kinase (MAPK), which is an important signal for neointimal formation. Therefore, we examined the effects of rapamycin on MAPK and transcriptional factors in cultured human coronary artery smooth muscle cells (CASMC) and in balloon-injured rat carotid arteries. METHODS AND RESULTS: Activation of ERK, JNK, p38MAPK, AP-1, and NF-kB in coronary artery smooth muscle cells was increased by 2% fetal bovine serum. Ten nmol/L of rapamycin prevented the activation of JNK, p38MAPK, AP-1, and NF-kB (65%, 65%, 67%, and 26% respectively, P<0.01). In an in vivo study, remarkable neointimal formation was observed 14 days after injury. Coating Pluronic gel with 20 and 50 mug rapamycin around the injured artery significantly decreased the intimal area/medial area ratio, compared with vehicle (0.75 vs. 1.2, P<0.01). Rapamycin prevented the increase in activation of JNK, p38MAPK, AP-1, and NF-kB in injured artery (42%, 70%, 75%, and 60% respectively, P<0.05). CONCLUSIONS: Neointimal formation after balloon injury is inhibited by rapamycin, which is partially mediated by inhibition of JNK and p38MAPK, followed by AP-1 and NF-kB.

Role of c-Jun NH2-terminal kinase in G-protein-coupled receptor agonist-induced cardiac plasminogen activator inhibitor-1 expression.

Plasminogen activator inhibitor-1 (PAI-1) has been implicated as a contributing risk factor for cardiovascular disease. However, little is known about molecular mechanisms of cardiac PAI-1 gene expression. To elucidate these mechanisms, dominant negative mutants of c-Jun NH(2)-terminal kinase (JNK), p38MAPK, apoptosis signal-regulating kinase-1 (ASK-1) and c-Jun were overexpressed in rat neonatal ventricular cardiac myocytes and fibroblasts by adenovirus vector to abrogate the activation of the corresponding endogenous proteins. One hundred nmol/l of angiotensin II significantly enhanced the JNK and p38MAPK activities of cardiomyocytes (2.3-fold and 1.9-fold, P < 0.05) and fibroblasts (3.2-fold and 2.5-fold, P < 0.05). At 3 h after stimulation, angiotensin II was found to have significantly increased PAI-1 mRNA, by 5.2-fold in cardiomyocytes and by 9.7-fold in fibroblasts. Dominant negative mutants of JNK, ASK-1 and c-Jun significantly inhibited PAI-1 mRNA expression and protein synthesis in both cardiomyocytes and fibroblasts, whereas a dominant negative mutant of p38MAPK did not change this expression. Moreover, a dominant negative mutant of JNK also significantly prevented the induction of PAI-1 mRNA expression by 100 nmol/l endothelin-1 and 10 micromol/l phenylephrine. In conclusion, G-protein-coupled receptor agonist-induced PAI-1 expression is partially mediated through JNK activation.

Fibroblast growth factor-2 stimulates interleukin-6 secretion in human pancreatic periacinar myofibroblasts.

OBJECTIVES: Fibroblast growth factor-2 (FGF-2) plays an important role in the pathophysiology of acute and chronic pancreatitis. In the present study, to evaluate the proinflammatory nature of FGF-2, we investigated the effects of FGF-2 on IL-6 secretion in human pancreatic periacinar myofibroblasts. METHODS: IL-6 supernatant levels were determined by enzyme-linked immunosorbent assays (ELISA). IL-6 mRNA expression were determined by Northern blots and quantitative PCRs. Activated protein (AP)-1 DNA-binding activities were evaluated by electrophoretic gel mobility shift assays (EMSA). RESULTS: FGF-2 induced IL-6 release in a dose- and time-dependent manner. FGF-2 activity for IL-6 induction was the same as that of IL-17. The combination of FGF-2 and IL-17 exerted additive effects at mRNA and protein levels. FGF-2 induced AP-1 DNA-binding activity, but blockage of AP-1 signaling by adenovirus-mediated transfer of a dominant negative c-Jun gene did not affect FGF-2-induced IL-6 mRNA expression. FGF-2 rapidly induced activation of ERK1/2 and p38 MAP kinases, and specific inhibitors for these enzymes significantly reduced FGF-2-induced IL-6 release. CONCLUSION: In the pancreas, FGF-2 may not only play a role as a growth factor in tissue injury repair processes but also as an inducer of acute-phase response via stimulation of IL-6 release.

A specific chymase inhibitor, NK3201, suppresses bleomycin-induced pulmonary fibrosis in hamsters.

We evaluated whether a chymase inhibitor, 2-(5-formylamino-6-oxo-2-phenyl-1,6-dihydropyrimidine-1-yl)-N-[(3,4-dioxo-1-phenyl-7-(2-pyridyloxy))-2-heptyl]acetamide (NK3201), suppressed bleomycin-induced pulmonary fibrosis. Hamsters were orally administered NK3201 (30 mg/kg per day) or placebo, beginning 5 days before intratracheal instillation of bleomycin (10 mg/kg). Four weeks after the instillation of bleomycin, pulmonary chymase activity in placebo-treated hamsters was significantly higher than in control hamsters, whereas the activity in NK3201-treated hamsters was significantly lower than in placebo-treated hamsters. The ratio of fibrotic area to total area in NK3201-treated hamsters was significantly decreased to 54.0% of the ratio in placebo-treated hamsters. Therefore, NK3201 may be useful in the prevention of pulmonary fibrosis.

Effects of aldosterone receptor antagonist and angiotensin II type I receptor blocker on cardiac transcriptional factors and mRNA expression in rats with myocardial infarction.

BACKGROUND: Because the effects of an aldosterone receptor antagonist on transcriptional factors and mRNA expression have not been fully examined in myocardial infarction (MI), the present study examined the effects of spironolactone (SPIRO) and candesartan cilexitil (CAN) on activation of activator protein-1 (AP-1), nuclear factor-kappaB (NF-kappaB) and mRNA expression in the non-ischemic myocardium after MI. METHODS AND RESULTS: MI was induced by ligation of the coronary artery in Wistar rats, which were separated into (1) vehicle-treated group, (2) CAN-treated group (10 mg/kg per day), (3) SPIRO-treated group (100 mg/kg per day) and (4) CAN + SPIRO-treated group. The activity of both AP-1 and NF-kappaB was significantly increased at 4 weeks after MI. CAN or SPIRO significantly prevented the cardiac remodeling and activity of AP-1 and NF-kappaB. Furthermore, CAN + SPIRO prevented the cardiac remodeling and activation of AP-1 and NF-kappaB, compared with CAN or SPIRO alone. Myocardial atrial natriuretic peptide, brain natriuretic peptide, collagen I and III mRNAs were enhanced by MI, and CAN or SPIRO alone significantly reduced the mRNAs. CAN + SPIRO significantly prevented these mRNAs, compared with CAN or SPIRO alone. CONCLUSIONS: Both aldosterone and angiotensin II are involved in the myocardial transcriptional activation of AP-1, NF-kappaB and the cardiac remodeling-related mRNAs. The combination of CAN and SPIRO may be a potent therapeutic strategy for preventing cardiac remodeling after MI.

Overexpression or ablation of JNK in skeletal muscle has no effect on glycogen synthase activity.

c-Jun NH(2)-terminal kinase (JNK) is highly expressed in skeletal muscle and is robustly activated in response to muscle contraction. Little is known about the biological functions of JNK signaling in terminally differentiated muscle cells, although this protein has been proposed to regulate insulin-stimulated glycogen synthase activity in mouse skeletal muscle. To determine whether JNK signaling regulates contraction-stimulated glycogen synthase activation, we applied an electroporation technique to induce JNK overexpression (O/E) in mouse skeletal muscle. Ten days after electroporation, in situ muscle contraction increased JNK activity 2.6-fold in control muscles and 15-fold in the JNK O/E muscles. Despite the enormous activation of JNK activity in JNK O/E muscles, contraction resulted in similar increases in glycogen synthase activity in control and JNK O/E muscles. Consistent with these findings, basal and contraction-induced glycogen synthase activity was normal in muscles of both JNK1- and JNK2-deficient mice. JNK overexpression in muscle resulted in significant alterations in the basal phosphorylation state of several signaling proteins, such as extracellular signal-regulated kinase 1/2, p90 S6 kinase, glycogen synthase kinase 3, protein kinase B/Akt, and p70 S6 kinase, in the absence of changes in the expression of these proteins. These data suggest that JNK signaling regulates the phosphorylation state of several kinases in skeletal muscle. JNK activation is unlikely to be the major mechanism by which contractile activity increases glycogen synthase activity in skeletal muscle.

Induction of gicerin/CD146 in the rat carotid artery after balloon injury.

Gicerin is a cell adhesion molecule belonging to the immunoglobulin superfamily. It is reported that the human homologous molecule, CD146, is expressed in the endothelial cells. Here, we found that the expression of gicerin was increased in the rat carotid arteries after balloon injury. Immunohistochemical analysis demonstrated that the expression of gicerin protein was increased in the medial smooth muscle cells prior to the formation of neointima one week after the injury and was also increased in the luminal edge of the neointima after two weeks. We employed A10 cells, a cell line derived from rat aortic smooth muscle cell, and examined the effect of growth factors on the expression of gicerin, such as IGF-1, PDGF-BB, and bFGF. We found that IGF-1, but not PDGF-BB and bFGF, significantly increases the expression of gicerin protein in A10 cells. These suggest gicerin might be involved in the arteriosclerotic neointima formation in the artery.

Involvement of apoptosis signal-regulating kinase-1 on angiotensin II-induced monocyte chemoattractant protein-1 expression.

OBJECTIVE: Monocyte chemoattractant protein 1 (MCP-1) could contribute to enhanced leukocyte recruitment and activation resulting in chronic tissue damage. However, little is known about the molecular mechanisms of cardiac MCP-1 expression. To elucidate these molecular mechanisms, angiotensin II-induced expression of MCP-1 was examined in cultured rat neonatal ventricular cardiomyocytes and fibroblasts by adenovirus gene transfer. METHODS AND RESULTS: MCP-1 mRNA increased 3.6-fold in cardiac fibroblasts at 3 hours after 100 nmol/L angiotensin-II stimulation (P<0.01), whereas MCP-1 mRNA in cardiomyocytes was unchanged. Angiotensin II significantly enhanced JNK, p38MAPK, and nuclear factor-kappaB (NF-kappaB) activities of cardiac fibroblasts. Wild-type ASK-1 increased MCP-1 expression of cardiac fibroblasts, whereas dominant negative mutant of ASK-1 (DN-ASK), dominant negative mutant of p38MAPK (DN-p38MAPK), and pyrrolidine dithiocarbamate significantly inhibited such expression. The increased MCP-1 mRNA expression in wild-type ASK-1 transfected fibroblasts was inhibited by cotransfection with adenovirus expressing DN-p38MAPK. On the contrary, the decreased MCP-1 mRNA expression in DN-ASK transfected cells was increased by cotransfection with adenovirus expressing constitutively active MKK6. CONCLUSIONS: Angiotensin II induced MCP-1 gene expression in cardiac fibroblasts. The angiotensin II-induced activation of ASK-1 followed by p38MAPK and NF-kappaB signaling in cardiac fibroblasts is partially involved in myocardial MCP-1 expression.

Activation of apoptosis signal-regulating kinase 1 in injured artery and its critical role in neointimal hyperplasia.

BACKGROUND: Apoptosis signal-regulating kinase 1 (ASK1), recently identified as one of the mitogen-activated protein kinase kinase kinases, is activated by various extracellular stimuli and involved in a variety of cellular function. Therefore, we first examined the role of ASK1 in vascular remodeling. METHODS AND RESULTS: We used rat balloon injury model and cultured vascular smooth muscle cells (VSMCs). Arterial ASK1 activity was rapidly and dramatically increased after balloon injury. To specifically inhibit endogenous ASK1 activation, dominant-negative mutant of ASK1 (DN-ASK1) was transfected into rat carotid artery before balloon injury. Gene transfer of DN-ASK1 significantly prevented neointimal formation at 14 days after injury. Bromodeoxyuridine labeling index at 7 days after injury showed that DN-ASK1 remarkably suppressed VSMC proliferation in both the intima and the media. We also examined the role of ASK1 in cultured rat VSMCs. Infection with DN-ASK1 significantly attenuated serum-induced VSMC proliferation and migration. We also compared neointimal formation after cuff placement around the femoral artery between mice deficient in ASK1 (ASK1-/- mice) and wild-type (WT) mice. Neointimal formation at 28 days after cuff injury in ASK1-/- mice was significantly attenuated compared with WT mice. Furthermore, we compared the proliferation and migration of VSMCs isolated from ASK1-/- mice with WT mice. Both proliferation and migration of VSMCs from ASK1-/- mice were significantly attenuated compared with VSMCs from WT mice. CONCLUSIONS: ASK1 activation plays the key role in vascular intimal hyperplasia. ASK1 may provide the basis for the development of new therapeutic strategy for vascular diseases.

Apoptosis signal-regulating kinase 1 plays a pivotal role in angiotensin II-induced cardiac hypertrophy and remodeling.

Multiple lines of evidence establish that angiotensin II (Ang II) induces not only hypertension but also directly contributes to cardiac diseases. Apoptosis signal-regulating kinase 1 (ASK1), one of mitogen-activated protein kinase kinase kinases, plays a key role in stress-induced cellular responses. However, nothing is known about the role of ASK1 in cardiac hypertrophy and remodeling in vivo. In this study, by using mice deficient in ASK1 (ASK1-/- mice), we investigated the role of ASK1 in cardiac hypertrophy and remodeling induced by Ang II. Left ventricular (LV) ASK1 was activated by Ang II infusion in wild-type mice, which was mediated by angiotensin II type 1 receptor and superoxide. Although Ang II-induced hypertensive effect was comparable to wild-type and ASK1-/- mice, LV ASK1 activation by Ang II was not detectable in ASK1-/- mice, and p38 and c-Jun N-terminal kinase (JNK) activation was lesser in ASK-/- mice than in wild-type mice. Elevation of blood pressure by continuous Ang II infusion was comparable between ASK1-/- and wild-type mice. However, Ang II-induced cardiac hypertrophy and remodeling, including cardiomyocyte hypertrophy, cardiac hypertrophy-related mRNA upregulation, cardiomyocyte apoptosis, interstitial fibrosis, coronary arterial remodeling, and collagen gene upregulation, was significantly attenuated in ASK1-/- mice compared with wild-type mice. These results provided the first in vivo evidence that ASK1 is the critical signaling molecule for Ang II-induced cardiac hypertrophy and remodeling. Thus, ASK1 is proposed to be a potential therapeutic target for cardiac diseases.

Molecular mechanism and role of endothelial monocyte chemoattractant protein-1 induction by vascular endothelial growth factor.

OBJECTIVE: We investigated the role of monocyte chemoattractant protein-1 (MCP-1) in vascular endothelial growth factor (VEGF)-induced angiogenesis and vascular permeability and the underlying molecular mechanism of VEGF-induced endothelial MCP-1 expression in vitro and in vivo. METHODS AND RESULTS: We used an anti-MCP-1 neutralizing antibody for specific inhibition of MCP-1. VEGF increased tubule formation in the angiogenesis assay and vascular permeability in the Miles assay, and these effects were markedly inhibited by anti-MCP-1 antibody. Using a luciferase MCP-1 promoter-gene assay, we found that the activator protein-1 (AP-1) binding site of the MCP-1 promoter region contributes to the increase in MCP-1 promoter activity by VEGF. To specifically inhibit AP-1, we used recombinant adenovirus containing a dominant-negative c-Jun (Ad-DN-c-Jun). Ad-DN-c-Jun inhibited VEGF-induced endothelial MCP-1 mRNA expression and promoter activity in vitro. In vivo gene transfer of DN-c-Jun into rat carotid artery, with the hemagglutinating virus of the Japan liposome method, significantly blocked VEGF-induced MCP-1 and macrophage/monocyte (ED1) expression in endothelium. CONCLUSIONS: These results reveal that endothelial MCP-1 induced by VEGF seems to participate in angiogenesis, vascular leakage, or arteriosclerosis. AP-1 plays a critical role in the molecular mechanism underlying induction of MCP-1 by VEGF.

Stroke prevention: is it possible? If so, which antihypertensive agent should be used?

The importance of blood pressure control in stroke prevention is supported by a large body of clinical evidence. However, it is not known which antihypertensive agents are most effective in preventing stroke. As stroke is characterised by vascular remodelling and dysfunction and by effects on the cerebral circulation, selecting an agent that has a direct vascular protective effect beyond blood pressure control may be desirable. Results with the angiotensin receptor blockers (ARBs) in clinical trials (LIFE and SCOPE) support the findings in animal models that ARBs may be a promising therapeutic option for prevention of stroke and possibly, cognitive decline.

Regulation of IL-11 expression in intestinal myofibroblasts: role of c-Jun AP-1- and MAPK-dependent pathways.

IL-11 inhibits the activation of NF-kappaB and induces the Th2 polarization of CD4+ T cells. The clinical utility of IL-11 is being investigated in Crohn's disease. However, physiological secretion of IL-11 in the intestine remains unclear. In this study, we investigated IL-11 secretion in human intestinal subepithelial myofibroblasts (SEMFs). Intestinal SEMFs were isolated from the human colonic mucosa. IL-11 secretion and mRNA expression were determined by ELISA and Northern blot analysis. The activating protein (AP)-1-DNA binding activity was evaluated by EMSA. IL-11 secretion was induced by IL-1beta and transforming growth factor (TGF)-beta1. These were also observed at the mRNA level. The EMSAs demonstrated that both IL-1beta and TGF-beta1 induced AP-1 activation within 2 h after stimulation, and a blockade of AP-1 activation by the recombinant adenovirus containing a dominant negative c-Jun markedly reduced the IL-1beta- and TGF-beta1-induced IL-11 mRNA expression. IL-1beta and TGF-beta1 induced an activation of ERK p42/44 and p38 MAP kinases, and the MAP kinase inhibitors (SB-202190, PD-98059, and U-0216) significantly reduced the IL-1beta- and TGF-beta1-induced IL-11 secretion. The upregulation of IL-11 mRNA by IL-1beta- and TGF-beta1 was also mediated by a p38 MAP kinase-mediated mRNA stabilization. The combination of IL-1beta and TGF-beta1 additively enhanced IL-11 secretion. Intestinal SEMFs secreted IL-11 in response to IL-1beta- and TGF-beta1. Mucosal IL-11 secretion might be important as an anti-inflammatory response in the pathogenesis of intestinal inflammation.

Stress and vascular responses: mitogen-activated protein kinases and activator protein-1 as promising therapeutic targets of vascular remodeling.

Mitogen-activated protein kinases (MAP kinases), including extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38, play a central role in cellular responses by various stress stimuli such as cell proliferation, apoptosis, migration, or gene expression. Furthermore, activator protein-1 (AP-1), a transcription factor which can be activated by MAP kinases, also is involved in a variety of celllar responses, as well as MAP kinases. MAP kinases and AP-1 are significantly activated in vascular tissues by hypertension, angiotensin II, or balloon injury. We have made dominant negative mutants of MAP kinases or c-Jun, to specifically inhibit in vivo activation of MAP kinases or AP-1. Vascular gene transfer of each dominant negative mutant of MAP kinases or c-Jun prevents intimal hyperplasia after balloon injury, which is associated with the inhibition of smooth muscle cell proliferation in the intima and the media and probably also associated with inhibition of smooth muscle cell migration. However, in vitro findings on cultured vascular smooth muscle cells suggest that the molecular mechanism underlying inhibition of intimal hyperplasia may be different among each dominant negative mutant of MAP kinases and c-Jun. MAP kinases and c-Jun seem to be the promising therapeutic target for vascular remodeling.

Dominant negative c-Jun inhibits platelet-derived growth factor-directed migration by vascular smooth muscle cells.

The mitogen-activated protein (MAP) kinase pathways has been shown to be necessary for mitogen-stimulated proliferation, but its role in cell migration has not been fully understood. In this study, we investigated the possible contribution of signaling pathways through c-Jun in platelet-derived growth factor (PDGF)-BB directed cell migration in rat aortic vascular smooth muscle cells (VSMCs) infected with a recombinant adenovirus containing the dominant-negative c-Jun (Ad-DN-c-Jun). DN-c-Jun protein was expressed dose-dependently in VSMCs infected with Ad-DN-c-Jun. Expression of DN-c-Jun significantly inhibited VSMC migration induced by PDGF-BB. Our results provide the first evidence that signaling pathways through c-Jun participates in cell migration induced by PDGF-BB in addition to other MAP kinase pathways in VSMCs.

Activation of transcription factors AP-1 and NF-kappaB in chronic cyclosporine A nephrotoxicity: role in beneficial effects of magnesium supplementation.

BACKGROUND: It has been shown that the transcription factors activator protein (AP)-1 and nuclear factor (NF)-kappaB play a pivotal role in various renal diseases. We aimed to study their activations in chronic cyclosporine A (CsA) nephrotoxicity and evaluate the effect of magnesium (Mg) supplementation and blockade of the renin-angiotensin system (RAS), which are known to ameliorate CsA nephrotoxicity, on these transcription factors. METHODS: CsA (15 mg/kg/day) was administered subcutaneously daily to rats maintained on a low-sodium diet for 7, 14, and 28 days. DNA-binding activities of AP-1 and NF-kappaB in renal cortex were determined by electrophoretic mobility shift assay. RESULTS: DNA-binding activity of AP-1 and NF-kappaB started to increase at day 14 and further elevated at day 28 by CsA treatment. These activations were markedly attenuated when rats were maintained on a high-Mg diet. In contrast, angiotensin-converting enzyme inhibitor (ACEI) had no effect on CsA-induced AP-1 activation. CsA-induced activation of NF-kappaB was suppressed by ACEI at day 14, whereas such effect could not be observed at day 28. CONCLUSIONS: Renal cortical AP-1 and NF-kappaB DNA binding were activated in chronic CsA nephrotoxicity. These activations were induced largely by means of RAS-independent mechanisms. It is suggested that prevention of CsA-induced DNA-binding activation of these transcription factors is at least in part responsible for the beneficial effects of Mg supplementation on CsA nephrotoxicity.

A novel chymase inhibitor, 4-[1-([bis-(4-methyl-phenyl)-methyl]-carbamoyl)3-(2-ethoxy-benzyl)-4-oxo-azetidine-2-yloxy]-benzoic acid (BCEAB), suppressed cardiac fibrosis in cardiomyopathic hamsters.

Previously, we reported that levels of chymase activity and its mRNA in cardiac tissues were significantly increased along with progression of cardiac fibrosis in cardiomyopathic hamsters, but the involvement of chymase in the progression of fibrosis has been unclear. In cultured human fibroblasts, the concentration of transforming growth factor-beta in the supernatant of medium was significantly increased after injection of human chymase. Furthermore, human chymase dose dependently increased cell proliferation, and this chymase-dependent proliferation was completely suppressed by a chymase inhibitor, Suc-Val-Pro-Phe(p)(OPh)(2) (10 micro M) or an anti-transforming growth factor-beta antibody (100 micro g/ml). In this study, we used Bio14.6 and F1B hamsters as cardiomyopathic and control hamsters, respectively. Cardiomyopathic hamsters were orally administered a novel chymase inhibitor, 4-[1-([bis-(4-methylphenyl)-methyl]-carbamoyl)-3-(2-ethoxy-benzyl)-4-oxo-azetidine-2-yloxy]-benzoic acid (BCEAB; 100 mg/kg per day), or placebo from 5- to 45-week-old. In the placebo-treated group, the cardiac chymase activity in cardiomyopathic hamsters 45 weeks old was significantly increased compared with that in control hamsters. BCEAB significantly reduced the cardiac chymase activity. The indexes (+dP/dt and -dP/dt) of cardiac function were significantly improved by treatment with BCEAB. The mRNA levels of collagen I and collagen III in the placebo-treated hamsters were significantly reduced to 69.6 and 76.5% by treatment with BCEAB, respectively. The fibrotic area in cardiac tissues in the BCEAB-treated hamsters was significantly suppressed to 50.7% compared with that in the placebo-treated treated hamsters. Therefore, the activation of transforming growth factor-beta by chymase may play an important role in the progression of cardiac fibrosis and cardiac dysfunction in cardiomyopathy.

Role of JNK, p38, and ERK in platelet-derived growth factor-induced vascular proliferation, migration, and gene expression.

OBJECTIVE: We investigated the comparative roles of mitogen-activated protein (MAP) kinases, including c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38, in vascular smooth muscle cell (VSMC) proliferation, migration, and gene expression. METHODS AND RESULTS: VSMCs were infected with recombinant adenovirus containing dominant-negative mutants of ERK, p38, and JNK (Ad-DN-ERK, Ad-DN-p38, and Ad-DN-JNK, respectively) to specifically inhibit the respective MAP kinases and then stimulated with platelet-derived growth factor (PDGF)-BB. Ad-DN-ERK attenuated PDGF-BB-induced VSMC proliferation more potently than Ad-DN-p38 or Ad-DN-JNK, indicating the dominant role of ERK in VSMC proliferation. Ad-DN-ERK, Ad-DN-p38, and Ad-DN-JNK similarly inhibited PDGF-induced VSMC migration. Ad-DN-ERK and Ad-DN-JNK suppressed PDGF-BB-induced downregulation of cyclin-dependent kinase inhibitor p27Kip1, whereas Ad-DN-p38 decreased PDGF-BB-induced upregulation of p21Cip1. Ad-DN-ERK inhibited PDGF-BB-induced plasminogen activator inhibitor type-1 (PAI-1), monocyte chemoattractant protein-1, and transforming growth factor-beta1 expressions, Ad-DN-p38 blocked monocyte chemoattractant protein-1 and transforming growth factor-beta1 expression but not PAI-1, whereas Ad-DN-JNK suppressed only PAI-1 expression. Moreover, in vivo gene transfer of Ad-DN-p38 to rat carotid artery caused the inhibition of intimal hyperplasia by balloon injury, indicating the involvement of p38 in vascular remodeling in vivo. CONCLUSIONS: We propose that these 3 MAP kinases participate in vascular diseases via differential molecular mechanisms and are new therapeutic targets for treatment of vascular diseases.