Abl regulates smooth muscle cell proliferation by modulating actin dynamics and ERK1/2 activation

Abl is a nonreceptor tyrosine kinase that has a role in regulating migration and adhesion of nonmuscle cells as well as smooth muscle contraction. The role of Abl in smooth muscle cell proliferation has not been investigated. In this study, treatment with endothelin-1 (ET-1) and platelet-derived growth factor (PDGF) increased Abl phosphorylation at Tyr(412) (an indication of Abl activation) in vascular smooth muscle cells. To assess the role of Abl in smooth muscle cell proliferation, we generated stable Abl knockdown cells by using lentivirus-mediated RNA interference. ET-1- and PDGF-induced cell proliferation was attenuated in Abl knockdown cells compared with cells expressing control shRNA and uninfected cells. Abl silencing also arrested cell cycle progression from G(0)/G(1) to S phase. Furthermore, activation of smooth muscle cells with ET-1 and PDGF induced phosphorylation of ERK1/2 and Akt. Abl knockdown attenuated ERK1/2 phosphorylation in smooth muscle cells stimulated with ET-1 and PDGF. However, Akt phosphorylation upon stimulation with ET-1 and PDGF was not reduced. Because Abl is known to regulate actin polymerization in smooth muscle, we also evaluated the effects of inhibition of actin polymerization on phosphorylation of ERK1/2. Pretreatment with the actin polymerization inhibitor latrunculin-A also blocked ERK1/2 phosphorylation during activation with ET-1 and PDGF. The results suggest that Abl may regulate smooth muscle cell proliferation by modulating actin dynamics and ERK1/2 phosphorylation during mitogenic activation.     

Evidence for modulation of smooth muscle force by the p38 MAP kinase/HSP27 pathway

Mitogen-activated protein (MAP) kinases signal to proteins that could modify smooth muscle contraction. Caldesmon is a substrate for extracellular signal-related kinases (ERK) and p38 MAP kinases in vitro and has been suggested to modulate actin-myosin interaction and contraction. Heat shock protein 27 (HSP27) is downstream of p38 MAP kinases presumably participating in the sustained phase of muscle contraction. We tested the role of caldesmon and HSP27 phosphorylation in the contractile response of vascular smooth muscle by using inhibitors of both MAP kinase pathways. In intact smooth muscle, PD-098059 abolished endothelin-1 (ET-1)-stimulated phosphorylation of ERK MAP kinases and caldesmon, but p38 MAP kinase activation and contractile response remained unaffected. SB-203580 reduced muscle contraction and inhibited p38 MAP kinase and HSP27 phosphorylation but had no effect on ERK MAP kinase and caldesmon phosphorylation. In permeabilized muscle fibers, SB-203580 and a polyclonal anti-HSP27 antibody attenuated ET-1-dependent contraction, whereas PD-098059 had no effect. These results suggest that ERK MAP kinases phosphorylate caldesmon in vivo but that activation of this pathway is unnecessary for force development. The generation of maximal force may be modulated by the p38 MAP kinase/HSP27 pathway.     

Role of 15-hydroxyeicosatetraenoic acid in phosphorylation of ERK1/2 and caldesmon in pulmonary arterial smooth muscle cells

We have reported that 15-hydroxyeicosatetraenoic acid (15-HETE) induces pulmonary artery (PA) contraction in rats exposed to hypoxia by activating extracellular signal-regulated kinase 1/2 (ERK1/2). In this study, we investigated the characteristics of 15-HETE mediating phosphorylation of ERK1/2 and caldesmon in rat pulmonary arterial smooth muscle cells (PASMCs). Our data showed that 15-HETE upregulated ERK1/2 phosphorylation in a dose-dependent manner, which could be blocked by ERK pathway inhibitors U0126 and PD98059. ERK1/2 phosphorylation was attenuated by inhibiting endogenous 15-HETE formation with lipoxygenase inhibitor, cinnamyl 3,4-dihydroxy-[alpha]-cyanocinnamate (CDC), in both normoxic and hypoxic PASMCs. ERK1/2 phosphorylation in response to 15-HETE was detected in cytosol as well as in nucleus and phosphorylatd ERK1/2 partly translocated into nucleus, which could be blocked by PD98059. In addition, caldesmon was phosphorylated in 15-HETE-stimulated cells; this could be inhibited by PD98059. These data demonstrated that 15-HETE is associated with ERK1/2 activation and caldesmon phosphorylation in PASMCs and that 15-HETE is at least partly involved in mediating activation of hypoxia-initiated ERK pathway, possibly leading to hypoxic pulmonary vasoconstriction.     

Receptor heterodimerization: essential mechanism for platelet-derived growth factor-induced epidermal growth factor receptor transactivation

Previous studies showed that the epidermal growth factor receptor (EGFR) can be transactivated by platelet-derived growth factor (PDGF) stimulation and that EGFR transactivation is required for PDGF-stimulated cell migration. To investigate the mechanism for cross talk between the PDGF beta receptor (PDGFbetaR) and the EGFR, we stimulated rat aortic vascular smooth muscle cells (VSMC) with 20 ng of PDGF/ml. Transactivation of the EGFR, defined by receptor tyrosine phosphorylation, occurred with the same time course as PDGFbetaR activation. Basal formation of PDGFbetaR-EGFR heterodimers was shown by coimmunoprecipitation studies, and interestingly, disruption of this receptor heterodimer abolished EGFR transactivation. Breakdown of the heterodimer was observed when VSMC were pretreated with antioxidants or with a Src family kinase inhibitor. Disruption of heterodimers decreased ERK1 and ERK2 activation by PDGF. Although PDGF-induced PDGFbetaR activation was abolished after pretreatment with 1 microM AG1295 (a specific PDGF receptor kinase inhibitor), EGFR transactivation was still observed, indicating that PDGFbetaR kinase activity is not required. In conclusion, our data demonstrate that the PDGFbetaR and the EGFR form PDGFbetaR-EGFR heterodimers basally, and we suggest that heterodimers represent a novel signaling complex which plays an important role in PDGF signal transduction.     

Endothelin-1 Augments Na+/H+ Exchange Activity in Murine Pulmonary Arterial Smooth Muscle Cells via Rho Kinase

Excessive production of endothelin-1 (ET-1), a potent vasoconstrictor, occurs with several forms of pulmonary hypertension. In addition to modulating vasomotor tone, ET-1 can potentiate pulmonary arterial smooth muscle cell (PASMC) growth and migration, both of which contribute to the vascular remodeling that occurs during the development of pulmonary hypertension. It is well established that changes in cell proliferation and migration in PASMCs are associated with alkalinization of intracellular pH (pH_i), typically due to activation of Na⁺/H⁺ exchange (NHE). In the systemic vasculature, ET-1 increases pH_i, Na⁺/H⁺ exchange activity and stimulates cell growth via a mechanism dependent on protein kinase C (PKC). These results, coupled with data describing elevated levels of ET-1 in hypertensive animals/humans, suggest that ET-1 may play an important role in modulating pH_i and smooth muscle growth in the lung; however, the effect of ET-1 on basal pH_i and NHE activity has yet to be examined in PASMCs. Thus, we used fluorescent microscopy in transiently (3–5 days) cultured rat PASMCs and the pH-sensitive dye, BCECF-AM, to measure changes in basal pH_i and NHE activity induced by increasing concentrations of ET-1 (10⁻¹⁰ to 10⁻⁸ M). We found that application of exogenous ET-1 increased pH_i and NHE activity in PASMCs and that the ET-1-induced augmentation of NHE was prevented in PASMCs pretreated with an inhibitor of Rho kinase, but not inhibitors of PKC. Moreover, direct activation of PKC had no effect on pH_i or NHE activity in PASMCs. Our results indicate that ET-1 can modulate pH homeostasis in PASMCs via a signaling pathway that includes Rho kinase and that, in contrast to systemic vascular smooth muscle, activation of PKC does not appear to be an important regulator of PASMC pH_i.     

Coupling of M(2) muscarinic receptors to ERK MAP kinases and caldesmon phosphorylation in colonic smooth muscle

Coupling of M(2) and M(3) muscarinic receptors to activation of mitogen-activated protein (MAP) kinases and phosphorylation of caldesmon was studied in canine colonic smooth muscle strips in which M(3) receptors were selectively inactivated by N, N-dimethyl-4-piperidinyl diphenylacetate (4-DAMP) mustard (40 nM). ACh elicited activation of extracellular signal-regulated kinase (ERK) 1, ERK2, and p38 MAP kinases in control muscles and increased phosphorylation of caldesmon (Ser(789)), a putative downstream target of MAP kinases. Alkylation of M(3) receptors with 4-DAMP had only a modest inhibitory effect on ERK activation, p38 MAP kinase activation, and caldesmon phosphorylation. Subsequent treatment with 1 microM AF-DX 116 completely prevented activation of ERK and p38 MAP kinase and prevented caldesmon phosphorylation. Caldesmon phosphorylation was blocked by the MAP kinase/ERK kinase inhibitor PD-98509 but not by the p38 MAP kinase inhibitor SB-203580. These results indicate that colonic smooth muscle M(2) receptors are coupled to ERK and p38 MAP kinases. Activation of ERK, but not p38 MAP kinases, results in phosphorylation of caldesmon in vivo, which is a novel function for M(2) receptor activation in smooth muscle.     

PKC-delta-dependent pathways contribute to PDGF-stimulated ERK1/2 activation in vascular smooth muscle

Platelet-derived growth factor (PDGF) is an important regulator of vascular smooth muscle (VSM) cell growth and migration and has been identified as a key mediator of neointima formation resulting from vascular injury. PDGF exerts its effects, in part, through activation of ERK1/2. Previously, we reported that PKC-delta, specifically compared with PKC-alpha, mediated phorbol ester- and ATP-dependent activation of ERK1/2 in VSM cells. The purpose of this study was to determine whether PKC-delta was involved in PDGF-dependent activation of ERK1/2 in VSM cells. The addition of PDGF resulted in the activation, and Src family kinase-dependent tyrosine phosphorylation, of PKC-delta. Treatment with rottlerin (0.1-10 microM), a selective PKC-delta inhibitor, or adenoviral overexpression of kinase-negative PKC-delta significantly attenuated PDGF-induced activation of ERK1/2. The effects of the PKC-delta inhibitors decreased with increasing concentrations of activator PDGF. Interestingly, treatment with Go6976 (0.1-3 microM), a selective inhibitor of cPKCs, or adenoviral overexpression of kinase-negative PKC-alpha also inhibited PDGF-stimulated ERK1/2. Furthermore, inhibition of cPKC activity with Go6976 or overexpression of kinase-negative PKC-alpha attenuated PKC-delta activation and tyrosine phosphorylation in response to PDGF. These studies indicate involvement of both PKC-delta and PKC-alpha isozymes in PDGF-stimulated signaling in VSM and suggest an unexpected role for PKC-alpha in the regulation of PKC-delta activity.     

PI 3-kinases and Src kinases regulate spreading and migration of cultured VSMCs

Pulmonary artery smooth muscle cell (PASMC)adhesion, spreading, and migration depend on matrix-stimulatedreorganization of focal adhesions. Platelet-derived growth factor(PDGF) activates intracellular signal transduction cascades that alsoregulate adhesion, spreading, and migration, but the signalingmolecules involved in these events are poorly defined. We hypothesizedthat phosphatidylinositol (PI) 3-kinases and Src tyrosine kinasestranslate matrix and PDGF-initiated signals into cell motility. Inexperiments with cultured canine PASMCs, inhibition of PI 3-kinaseswith wortmannin (0.3 µM) and LY-294002 (50 µM) and of Src kinasewith PP1 (30 µM) did not decrease spontaneous (nonstimulated) orPDGF-stimulated (10 ng/ml) adhesion onto collagen. PI 3-kinase and Srckinase activities, however, were necessary for cell spreading: PP1inhibited cell spreading and Src Tyr-418 phosphorylation in aconcentration-dependent manner. Inhibition of PI 3-kinase and Srcpartially reduced cell migration, while at 10 and 30 µM, PP1eliminated migration, likely due to inhibition of PDGF receptors. Inconclusion, both PI 3-kinases and Src tyrosine kinases are componentsof pathways that mediate spreading and migration of cultured PASMCs on collagen.

     

Platelet-derived growth factor-induced alterations in vinculin distribution in porcine vascular smooth muscle cells

Exposure of porcine vascular smooth muscle cells to platelet-derived growth factor (PDGF; 18-180 ng/ml) but not epidermal growth factor (EGF; 30 ng/ml), somatomedin C (SmC; 30 ng/ml), or insulin (10 microM), results in a rapid, reversible, time- and concentration-dependent disappearance of vinculin staining in adhesion plaques; actin-containing stress fibers also become disrupted following exposure of cells to PDGF. Disappearance of vinculin staining from adhesion plaques is also caused by 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 200-400 nM), though the time course of the disappearance of vinculin staining under these conditions takes longer than in cells exposed to PDGF. The PDGF-induced removal of vinculin from adhesion plaques was inhibited in a concentration-dependent fashion by 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate (TMB-8; 0.25-4 microM) and leupepetin (2-300 microM), and by n-alpha-tosyl-L-lysine chloromethylketone (TLCK; 100 microM) and trifluoperazine (TFP; 2.5 microM). Addition of PDGF to vascular smooth muscle cells caused a rapid, transient increase in cytosolic free calcium, from a basal resting level of 146 +/- 6.9 nM (SEM, n = 62) to 414 +/- 34 nM (SEM, n = 22) as determined using the calcium-sensitive indicator Fura-2 and Digitized Video Microscopy. This increase in cellular calcium preceded the disappearance of vinculin from adhesion plaques and was partially blocked by pretreatment of cells with TMB-8 but not leupeptin. This rise in cytosolic free calcium was found to occur in approximately 80% of the sample population and displayed both spatial and temporal subcellular heterogeneity. Exposure of cells to TPA (100 nM) did not result in a change in cytosolic free calcium. Both PDGF (20 ng/ml) and TPA (100 nM) caused cytosolic alkalinization which occurred after PDGF-induced disruption of vinculin from adhesion plaques, as determined using the pH-sensitive indicator BCECF and Digitized Video Microscopy. PDGF stimulated DNA synthesis and vinculin disruption in a similar dose-dependent fashion. Both could be inhibited by leupeptin or TMB-8. These results suggest that 1) exposure of vascular smooth muscle cells to PDGF is associated with the disruption of vinculin from adhesion plaques, 2) PDGF-induced vinculin disruption is regulated by an increase in cytosolic calcium (but not cytosolic alkalinization), and involves proteolysis; 3) activation of protein kinase C also causes vinculin removal from adhesion plaques but by a calcium-independent mechanism, and 4) the cellular response to PDGF-stimulated increases in cytosolic free calcium is heterogeneous.(ABSTRACT TRUNCATED AT 400 WORDS)     

Platelet-derived growth factor-BB and lysophosphatidic acid distinctly regulate hepatic myofibroblast migration through focal adhesion kinase

Although hepatic myofibroblast (HMF) migration contributes to the development of fibrosis, the mechanisms coordinating this movement are uncertain. We determined the effects of lysophosphatidic acid (LPA) and platelet-derived growth factor-BB (PDGF) on actin polymerization, FAK tyrosine phosphorylation, and migration of cultured human HMFs. LPA (0.4-100 microM) stimulated migration, FAK tyrosine phosphorylation, and stress fiber assembly with a sigmoidal dose response. PDGF (1-250 ng/ml) stimulated migration, FAK tyrosine phosphorylation, and actin polymerization with a bell-shape dose-response characterized by a maximum at 10-25 ng/ml. Concentrations of cytochalasin D, which abolished FAK tyrosine phosphorylation, also blocked LPA- and PDGF-induced migration. A dose of 1-10 ng/ml PDGF acted synergistically with LPA (10 microM) to stimulate FAK tyrosine phosphorylation and migration, whereas higher concentrations of PDGF (100-250 ng/ml) inhibited FAK tyrosine phosphorylation and migration in response to LPA (10 microM). These data indicate that PDGF and LPA coordinately govern the migration of HMFs by differentially regulating FAK and suggest a novel model in which PDGF, acting as an amplifier/attenuator of LPA-induced signaling, facilitates HMF accumulation within injured areas of the liver.     

非磷酸化肌球蛋白在血小板衍生生长因子诱导的血管平滑肌细胞迁移中的作用

为了阐明非磷酸化肌球蛋白在平滑肌细胞迁移中的作用,研究探讨了非磷酸化肌球蛋白是否介导了血小板衍生生长因子(PDGF)诱导豚鼠脑基底动脉平滑肌细胞(GbaSM-4)的迁移。研究结果显示,20ng/ml以下剂量的PDGF可诱导GbaSM-4细胞发生迁移,此时肌球蛋白轻链(MLC20)磷酸化水平无变化。该迁移作用可被肌球蛋白特异性抑制剂blebbistatin所拮抗。应用RNA干扰技术抑制肌球蛋白轻链激酶表达,经免疫印迹检测经果显示,MLC20的磷酸化水平发生了显著下降;但对PDGF诱导的迁移作用无影响;在RNA干扰后blebbistatin也可抑制其迁移作用。体外ATP酶活性测定结果显示,blebbistatin对从平滑肌中提取的非磷酸化肌球蛋白的ATP酶活性有明显的抑制作用,其主要作用位点位于肌球蛋白头的头部S1。上述结果提示,非磷酸化的肌球蛋白参与了PDGF诱导的平滑肌细胞迁移。     

Tyrosine kinase receptor activation inhibits NPR-C in lung arterial smooth muscle cells

We have previously demonstrated that expression of the atrial natriuretic peptide (ANP) clearance receptor (NPR-C) is reduced selectively in the lung of rats and mice exposed to hypoxia but not in pulmonary arterial smooth muscle cells (PASMCs) cultured under hypoxic conditions. The current study tested the hypothesis that hypoxia-responsive growth factors, fibroblast growth factors (FGF-1 and FGF-2) and platelet-derived growth factor-BB (PDGF-BB), that activate tyrosine kinase receptors can reduce expression of NPR-C in PASMCs independent of environmental oxygen tension. Growth-arrested rat PASMCs were incubated under hypoxic conditions (1% O2) for 24 h; with FGF-1, FGF-2, or PDGF-BB (0.1-20 ng/ml for 1-24 h); or with ANG II (1-100 nM), endothelin-1 (ET-1, 0.1 microM), ANP (0.1 microM), sodium nitroprusside (SNP, 0.1 microM), or 8-bromo-cGMP (0.1 mM) for 24 h under normoxic conditions. Steady-state NPR-C mRNA levels were assessed by Northern blot analysis. FGF-1, FGF-2, and PDGF-BB induced dose- and time-dependent reduction of NPR-C mRNA expression within 1 h at a threshold concentration of 1 ng/ml; hypoxia, ANG II, ET-1, ANP, SNP, or cGMP did not decrease NPR-C mRNA levels in PASMCs under the above conditions. Downregulation of NPR-C expression by FGF-1, FGF-2, and PDGF-BB was inhibited by the selective FGF-1 receptor tyrosine kinase inhibitor PD-166866 and mitogen-activated protein/extracellular signal-regulated kinase inhibitors U-0126 and PD-98059. These results indicate that activation of tyrosine kinase receptors by hypoxia-responsive growth factors, but neither hypoxia per se nor activation of G protein-coupled receptors, inhibits NPR-C gene expression in PASMCs. These results suggest that FGF-1, FGF-2, and PDGF-BB play a role in the signal transduction pathway linking hypoxia to altered NPR-C expression in lung.     

Role of platelet-derived growth factor-BB (PDGF-BB) in human pulmonary artery smooth muscle cell proliferation

Abstract

Pulmonary arterial hypertension (PAH) is a vascular remodeling disease characterized by enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs) and suppressed apoptosis. Platelet-derived growth factor (PDGF) is a potent mitogen involved in cell proliferation and migration. PDGF-BB induces the proliferation and migration of PASMCs and has been proposed to be a key mediator in the progression of PAH. Previous studies have shown that PDGF and its receptor are substantially elevated in lung tissues and PASMCs isolated from patients and animals with PAH, but the underlying mechanisms are still poorly manifested. MAP kinases, including extracellular signal-regulated kinase1/2 (ERK1/2), c-Jun NH2-terminal kinase1/2 (JNK1/2), and p38 are the key intracellular signals for stimuli-induced cell proliferation, survival, and apoptosis. Therefore, the purpose of this study is to determine whether PDGF-BB on cell proliferation process is mediated through the MAP kinases pathway in human PASMCs (HPASMCs). Our results showed PDGF-BB-induced proliferating cell nuclear antigen (PCNA), Cyclin A and Cyclin E expression in a concentration-dependent manner. The expression levels of phosphorylated JNK (p-JNK) was upregulated with 20?ng/ml PDGF-BB treatment, while PDGF-BB could not increase phosphorylated ERK1/2 (p-ERK1/2) and p-38 (p-p38) expression. The effects of PDGF-BB on cell proliferation and survival were weakened after the administration of antagonist of the JNK pathway or si-JNK. In addition, PDGF-BB protected against the loss of mitochondrial membrane potentials evoked by serum deprivation (SD) in a JNK-dependent manner. These results suggest that PDGF-BB promotes HPASMCs proliferation and survival, which is likely to be mediated via the JNK pathway.     

Platelet-derived growth factor and heparin-like glycosaminoglycans regulate thrombospondin synthesis and deposition in the matrix by smooth muscle cells

Platelet-derived growth factor (PDGF), a smooth muscle cell (SMC) mitogen, and heparin-like glycosaminoglycans, known inhibitors of SMC growth and migration, were found to regulate thrombospondin synthesis and matrix deposition by cultured rat aortic SMC. The synthesis and distribution of thrombospondin was examined in growth-arrested SMCs, in PDGF-stimulated SMCs, and in heparin-treated SMCs using metabolic labeling and immunofluorescence techniques. Thrombospondin synthesis in response to purified PDGF occurred within 1 h after addition of growth factor to growth-arrested SMCs, peaked at 2 h, and returned to baseline levels by 5 h. The induction of synthesis of thrombospondin by PDGF was dose dependent, with a maximal effect observed at 2.5 ng/ml. Actinomycin D (2 micrograms/ml) inhibited thrombospondin induction by PDGF, suggesting a requirement for new RNA synthesis. In the presence of heparin and related polyanions, the incorporation of thrombospondin into the SMC extracellular matrix was markedly reduced. This effect was dose dependent with a maximal effect observed at a heparin concentration of 1 microgram/ml. Heparin did not affect the ability of SMCs to synthesize thrombospondin in response to PDGF. We interpret these data to suggest a role for thrombospondin in the SMC proliferative response to PDGF and in the regulation of SMC growth and migration by glycosaminoglycans.     

Caldesmon phosphorylation is catalyzed by two kinases in permeabilized and intact vascular smooth muscle

Smooth muscle contraction is initiated by myosin light chain (MLC) phosphorylation catalyzed by the Ca(2+) dependent MLC kinase. However, many aspects of smooth muscle contraction cannot be accounted for by MLC phosphorylation. One hypothesis that has received experimental support involves the thin filament protein caldesmon. Caldesmon inhibits myosin ATPase activity; phosphorylation of caldesmon relieves this inhibitory effect. The primary candidates for catalysis of caldesmon phosphorylation are the p42/p44 ERK MAP kinases. However, we and others have shown that inhibition of the ERK MAP kinases has no effect on many smooth muscles. The goal of this study was to determine if evidence for a second endogenous caldesmon kinase may be obtained. We used Triton X-100 skinned and intact tissues of the swine carotid artery to address this goal. Caldesmon phosphorylation was evident in resting and Ca(2+) stimulated Triton X-100 skinned fibers. Ca(2+)-dependent caldesmon phosphorylation was partially sensitive to the ERK MAP kinase inhibitor PD98059, whereas all caldesmon phosphorylation was sensitive to the general kinase inhibitor, staurosporine. Histamine increased caldesmon phosphorylation levels in intact swine carotid artery, which was sensitive to both PD98059 and staurosporine. Histamine increased ERK MAP kinase activity, which was reversed by PD98059, staurosporine, and EGTA. Histamine-induced contractions were inhibited by staurosporine but not by PD98059. We interpret these results to suggest that although ERK MAP kinases catalyze caldesmon phosphorylation, a second staurosporine sensitive kinase is also important in caldesmon phosphorylation and it is this pathway that may be more important in contractile regulation.     

Insulin inhibits PDGF-directed VSMC migration via NO/ cGMP increase of MKP-1 and its inactivation of MAPKs

In this study, we examined the roleof insulin in the control of vascular smooth muscle cell (VSMC)migration in the normal vasculature. Platelet-derived growth factor(PDGF) increased VSMC migration, which was inhibited by pretreatmentwith insulin in a dose-dependent manner. Insulin also caused a 60%decrease in PDGF-stimulated mitogen-activated protein kinase (MAPK)phosphorylation and activation. Insulin inhibition of MAPK wasaccompanied by a rapid induction of MAPK phosphatase (MKP-1), whichinactivates MAPKs by dephosphorylation. Pretreatment with inhibitors ofthe nitric oxide (NO)/cGMP pathway, blocked insulin-induced MKP-1 expression and restored PDGF-stimulated MAPK activation and migration. In contrast, adenoviral infection of VSMCs with MKP-1 or cGMP-dependent protein kinase I (cGK I), the downstream effector of cGMPsignaling, blocked the activation of MAPK and prevented PDGF-directedVSMC migration. Expression of antisense MKP-1 RNA prevented insulin's inhibitory effect and restored PDGF-directed VSMC migration and MAPKphosphorylation. We conclude that insulin inhibition of VSMC migrationmay be mediated in part by NO/cGMP/cGK I induction of MKP-1 andconsequent inactivation of MAPKs.

     

Urokinase potentiates PDGF-induced chemotaxis of human airway smooth muscle cells

We investigated the chemotactic action of PDGF and urokinase on human airway smooth muscle (HASM) cells in culture. Cells were put in collagen-coated transwells with 8-micro m perforations, incubated for 4 h with test compounds, then fixed, stained, and counted as migrated nuclei by microscopy. Cells from all culture conditions showed some basal migration (migration in the absence of stimuli during the assay), but cells preincubated for 24 h in 10% FBS or 20 ng/ml PDGF showed higher basal migration than cells quiesced in 1% FBS. PDGF(BB), PDGF(AA), and PDGF(AB) were all chemotactic when added during the assay. PDGF chemotaxis was blocked by the phosphatidyl 3'-kinase inhibitor LY-294002, the MEK inhibitor U-0126, PGE(2), formoterol, pertussis toxin, and the Rho kinase inhibitor Y-27632. Urokinase alone had no stimulatory effect on migration of quiescent cells but caused a dose-dependent potentiation of chemotaxis toward PDGF. Urokinase also potentiated the elevated basal migration of cells pretreated in 10% FBS or PDGF. This potentiating effect of urokinase appears to be novel. We conclude that PDGF and similar cytokines may be important factors in airway remodeling by redistribution of smooth muscle cells during inflammation and that urokinase may be important in potentiating the response.     

Effects of platelet-contained growth factors (PDGF, EGF, IGF-I, and TGF-β) on DNA synthesis in porcine aortic smooth muscle cells in culture

Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and transforming growth factor-β (TGF-β) are potent mitogens present in human platelets. Since they are likely to be released simultaneously at the site of vessel injury, their combined effects on vascular smooth muscle cells are more relevant physiologically than their individual actions. Therefore, we added various concentrations of growth factors to quiescent porcine aortic smooth muscle cells cultured in lowserum (0.5%) medium and measured the amount of [³H]thymidine incorporated into DNA. Effect of TGF-β alone was concentration-dependent: stimulatory (1.5-fold increase over the basal) at 0.025 ng/ml and inhibitory at 0.1 ng/ml. Effects of the other three growth factors on DNA synthesis were only stimulatory; their maximally effective concentrations were 20 ng/ml for PDGF (eightfold over the basal), 40 ng/ml for EGF (sixfold increase), and 20 ng/ml for IGF-I (fourfold increase). When PDGF, EGF, and IGF-I were added at submaximally effective concentrations, their effects were additive. TGF-β at 1 ng/ml inhibited at least 50% of the effects of 20 ng/ml EGF and of 10 ng/ml IGF-I, whereas inhibition of the effect of 10 ng/ml PDGF required 10 ng/ml of TGF-β. The concentration of TGF-β needed to inhibit 50% of the combined effect of EGF, IGF-1, and PDGF was 5 ng/ml. These results show complex interrelationships between the growth factors contained in the α-granules of human platelets in their effects on porcine aortic smooth muscle cells.     

Differential effects of imatinib on PDGF-induced proliferation and PDGF receptor signaling in human arterial and venous smooth muscle cells

Platelet-derived growth factor (PDGF) has been implicated in smooth muscle cell (SMC) proliferation, a key event in the development of myointimal hyperplasia in vascular grafts. Recent evidence suggests that the PDGF receptor (PDGFR) tyrosine kinase inhibitor, imatinib, can prevent arterial proliferative diseases. Because hyperplasia is far more common at the venous anastomosis than the arterial anastomosis in vascular grafts, we investigated whether imatinib also inhibited venous SMC (VSMC) proliferation, and examined possible differences in its mechanism of action between VSMC and arterial SMC (ASMC). Human ASMC and VSMC were stimulated with PDGF-AB, in the presence or absence of imatinib (0.1-10 microM). Proliferation was assayed using the 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, while PDGFR, Akt and ERK1/2-mitogen activated protein kinase (MAPK) signaling pathways were investigated by immunoblotting. The proliferative response to PDGF at 50 and 100 ng/ml was 32 and 43% greater, respectively, in VSMC than in ASMC. Similarly, PDGF-stimulated proliferation was more sensitive to inhibition by imatinib in VSMC than ASMC (IC(50) = 0.05 microM vs. 0.4 microM; P < 0.01). Imatinib also more effectively inhibited PDGF-induced phosphorylation of PDGFRbeta and Akt in VSMC, compared to ASMC. These data highlight inherent pharmacodynamic differences between VSMC and ASMC in receptor and cell signaling functions and suggest that imatinib therapy may be useful for the prevention of venous stenosis in vascular grafts.     

Inhibition of MEK1,2/ERK mitogenic pathway by estrogen with antiproliferative properties in rat aortic smooth muscle cells

Proliferation and migration of vascular smooth muscle cells (VSMCs) are believed to contribute significantly to intimal thickening in atheroscleosis, restenosis, and venous bypass graft disease. Estrogen inhibits proliferation and migration of VSMCs. However, antiproliferative mechanisms of estrogen were not well elucidated yet. In this study, we investigated the antiproliferative effect of estrogen to determine whether the transduction signals and protooncogenes were affected in rat aortic smooth muscle cells (RASMCs). Estrogen inhibited the proliferative response stimulated by 5% fetal bovine serum (FBS) dose-dependently in RASMCs (IC50: 40 nM). In 0.5% serum-treated RASMCs, estrogen dramatically inhibited the activity of extracellular signal-regulated kinases (ERK) followed by inhibition of MEK1,2 activity in dose-dependent manner without affecting the other mitogen-activating protein kinases (MAPKs), c-jun N-terminal kinases (JNK) and p38. Induction of Elk-1 mRNA was significantly reduced dose-dependently up to 100 nM of estrogen. These results indicate that the antiproliferative effects of estrogen in RASMCs involved ERK inhibition followed by the inactivation of MEK1,2 and downregulation of Elk-1 expression.