Tumor necrosis factor alpha inhibits insulin-induced mitogenic signaling in vascular smooth muscle cells

Tumor necrosis factor alpha (TNFalpha) interferes with insulin signaling in adipose tissue and may promote insulin resistance. Insulin binding to the insulin receptor (IR) triggers its autophosphorylation, resulting in phosphorylation of Shc and the downstream activation of p42/p44 extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2), which mediates insulin-induced proliferation in vascular smooth muscle cells (VSMC). Since insulin resistance is a risk factor for vascular disease, we examined the effects of TNFalpha on mitogenic signaling by insulin. In rat aortic VSMC, insulin induced rapid phosphorylation of the IR and Shc and caused a 5.3-fold increase in activated, phosphorylated ERK1/2 at 10 min. Insulin induced a biphasic ERK1/2 activation with a transient peak at 10 min and a sustained late phase after 2 h. Preincubation (30-120 min) with TNFalpha had no effect on insulin-induced IR phosphorylation. In contrast, TNFalpha transiently suppressed insulin-induced ERK1/2 activation. Insulin-induced phosphorylation of Shc was inhibited by TNFalpha in a similar pattern. Since mitogenic signaling by insulin in VSMC requires ERK1/2 activation, we examined the effect of TNFalpha on insulin-induced proliferation. Insulin alone induced a 3.4-fold increase in DNA synthesis, which TNFalpha inhibited by 48%. TNFalpha alone was not mitogenic. Inhibition of ERK1/2 activation with PD98059 also inhibited insulin-stimulated DNA synthesis by 57%. TNFalpha did not inhibit platelet-derived growth factor-induced ERK1/2 activation or DNA synthesis in VSMC. Thus, TNFalpha selectively interferes with insulin-induced mitogenic signaling by inhibiting the phosphorylation of Shc and the downstream activation of ERK1/2.     

The association between integrin-associated protein and SHPS-1 regulates insulin-like growth factor-I receptor signaling in vascular smooth muscle cells

Growth factor signaling is usually analyzed in isolation without considering the effect of ligand occupancy of transmembrane proteins other than the growth factor receptors themselves. In smooth muscle cells, the transmembrane protein Src homology 2 domain containing protein tyrosine phosphatase substrate-1 (SHPS-1) has been shown to be an important regulator of insulin-like growth factor-I (IGF-I) signaling. SHPS-1 is phosphorylated in response to IGF-I, leading to recruitment of Src homology 2 domain tyrosine phosphatase (SHP-2). Subsequently, SHP-2 is transferred to IGF-I receptor and regulates the duration of IGF-I receptor phosphorylation. Whether ligand occupancy of SHPS-1 influences SHPS-1 phosphorylation or SHP-2 recruitment, thereby altering growth factor signaling, is unknown. Previous studies have shown that integrin associated protein (IAP) associates with SHPS-1. We undertook these studies to determine whether this interaction controlled SHPS-1 phosphorylation and/or SHP-2 recruitment and thereby regulated IGF-I signaling. Disruption of IAP-SHPS-1 binding, by using an IAP monoclonal antibody or cells expressing mutant forms of IAP that did not bind to SHPS-1, inhibited IGF-I-stimulated SHPS-1 phosphorylation and SHP-2 recruitment. This was associated with a lack of SHP-2 transfer to IGF-I receptor and sustained receptor phosphorylation. This resulted in an inability of IGF-I to stimulate sustained mitogen-activated protein kinase activation, cell proliferation, and cell migration. The effect was specific for IGF-I because disruption of the IAP-SHPS-1 interaction had no effect on platelet-derived growth factor-stimulated SHPS-1 phosphorylation or cell migration. In summary, our results show that 1) ligand occupancy of SHPS-1 is a key determinant of its ability to be phosphorylated after IGF-I stimulation, and 2) the interaction between IAP and SHPS-1 is an important regulator of IGF-I signaling because disruption of the results in impaired SHP-2 recruitment and subsequent inhibition of IGF-I-stimulated cell proliferation and migration.     

Redox-dependent MAP kinase signaling by Ang II in vascular smooth muscle cells: role of receptor tyrosine kinase transactivation

We investigated the role of receptor tyrosine kinases in Ang II-stimulated generation of reactive oxygen species (ROS) and assessed whether MAP kinase signaling by Ang II is mediated via redox-sensitive pathways. Production of ROS and activation of NADPH oxidase were determined by DCFDA (dichlorodihydrofluorescein diacetate; 2 micromol/L) fluorescence and lucigenin (5 micromol/L) chemiluminescence, respectively, in rat vascular smooth muscle cells (VSMC). Phosphorylation of ERK1/2, p38MAP kinase and ERK5 was determined by immunoblotting. The role of insulin-like growth factor-1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) was assessed with the antagonists AG1024 and AG1478, respectively. ROS bioavailability was manipulated with Tiron (10(-5) mol/L), an intracellular scavenger, and diphenylene iodinium (DPI; 10(-6) mol/L), an NADPH oxidase inhibitor. Ang II stimulated NADPH oxidase activity and dose-dependently increased ROS production (p < 0.05). These actions were reduced by AG1024 and AG1478. Ang II-induced ERK1/2 phosphorylation (276% of control) was decreased by AG1478 and AG1024. Neither DPI nor tiron influenced Ang II-stimulated ERK1/2 activity. Ang II increased phosphorylation of p38 MAP kinase (204% of control) and ERK5 (278% of control). These effects were reduced by AG1024 and AG1478 and almost abolished by DPI and tiron. Thus Ang II stimulates production of NADPH-inducible ROS partially through transactivation of IGF-1R and EGFR. Inhibition of receptor tyrosine kinases and reduced ROS bioavaliability attenuated Ang II-induced phosphorylation of p38 MAP kinase and ERK5, but not of ERK1/2. These findings suggest that Ang II activates p38MAP kinase and ERK5 via redox-dependent cascades that are regulated by IGF-1R and EGFR transactivation. ERK1/2 regulation by Ang II is via redox-insensitive pathways.     

血管平滑肌细胞在动脉粥样硬化中的作用

动脉粥样硬化的发生发展是一个复杂的过程,涉及到多种细胞及细胞因子的相互作用.平滑肌细胞作为血管壁的重要成分,调节着血管的收缩舒张功能,同时也分泌多种细胞因子及细胞间质;它的生物学行为对动脉粥样硬化的发生、发展及最终的结局产生着重要的影响.本文就平滑肌细胞的生物学行为的变化及其在动脉粥样硬化的不同发展阶段的作用进行综述.     

Src family tyrosine kinases participate in insulin-like growth factor I mitogenic signaling in 3T3-L1 cells.

Insulin-like growth factor-I (IGF-I) stimulates proliferation and differentiation of many cell types, including preadipocytes. We have previously shown that IGF-I stimulates proliferation of 3T3-L1 preadipocytes through activation of the extracellular regulated kinase (ERK)-1 and -2 mitogen-activated protein kinase (MAPK) pathway, and that IGF-I-stimulated MAPK is predominantly downstream of Shc, not IRS-1 phosphorylation. The Src family of nonreceptor tyrosine kinases has been shown to mediate the mitogenic effects of other growth factors that also activate Shc and the ERK-1 and -2 MAPKs. Although Src family kinases (SFK) have been implicated in IGF-I action, no specific role for SFKs in IGF-I regulation of mitogenesis has been previously demonstrated. We studied the role of SFKs in IGF-I mitogenic signaling in 3T3-L1 preadipocytes. The SFK-selective inhibitor PP1 completely inhibited both IGF-I-stimulated DNA synthesis and MAPK activation in proliferating 3T3-L1 cells. PP1 inhibited IGF-I phosphorylation of Shc but not of IRS-1. In addition, IGF-I activation of MAPK was inhibited in proliferating cells transiently transfected with a dominant-negative c-Src. Finally, the kinetics of SFK and MAPK activation by IGF-I suggest that SFKs may act upstream of MAPK. IGF-I activation of SFK members c-Src and Fyn occurred within 1 min of treatment, and activity was back to baseline by 10 min. Our previous studies found that IGF-I activation of MAPK peaked at 5 min and was also back to baseline by 10 min. Our results are the first to demonstrate that SFKs mediate IGF-I mitogenic signaling in 3T3-L1 cells and add to the growing body of evidence that SFKs play a crucial role in IGF-I action.     

Leukocyte antigen-related deficiency enhances insulin-like growth factor-1 signaling in vascular smooth muscle cells and promotes neointima formation in response to vascular injury

Increase in the expression of leukocyte antigen-related (LAR) protein causes insulin resistance, an important contributor to atherosclerosis. However, the function of LAR in atherosclerosis is not known. To address whether LAR is important in the response of vascular cells to atherogenic stimuli, we investigated cell proliferation, migration, and insulin-like growth factor-1 receptor (IGF-1R) signaling in wild-type and LAR(-/-) mouse vascular smooth muscle cells (VSMC) treated with IGF-1. Absence of LAR significantly enhanced proliferation and migration of VSMC compared with wild-type cells after IGF-1 treatment. U0126 and LY249002, specific inhibitors of MAPK/ERK kinase (MEK) and phosphoinositide 3-kinase, respectively, inhibited IGF-1-induced DNA synthesis and migration in both wild-type and LAR(-/-) VSMC. IGF-1 markedly enhanced IGF-1R phosphorylation in both wild-type and LAR(-/-) VSMC, but the phosphorylation was 90% higher in knock-out cells compared with wild-type cells. Absence of LAR enhanced phosphorylation of insulin receptor substrate-1 and insulin receptor substrate-1-associated phosphoinositide 3-kinase activity in VSMC treated with IGF-1. IGF-1-induced phosphorylation of ERK1/2 also increased significantly in LAR(-/-) VSMC compared with wild-type cells. Furthermore, LAR directly binds to IGF-1R in glutathione S-transferase-LAR pull-down and IGF-1R immunoprecipitation experiments and recombinant LAR dephosphorylates IGF-1R in vitro. Neointima formation in response to arterial injury and IGF-1R phosphorylation in neointima increased significantly in LAR(-/-) mice compared with wild-type mice. A significant decrease in body weight, fasting insulin, and IGF-1 levels were observed in LAR(-/-) mice compared with wild-type mice. Together, these data indicate that LAR regulates IGF-1R signaling in VSMC and dysregulation of this phosphatase may lead to VSMC hyperplasia.     

The Ang II-induced growth of vascular smooth muscle cells involves a phospholipase D-mediated signaling mechanism

Angiotensin (Ang) II acts as a mitogen in vascular smooth muscle cells (VSMC) via the activation of multiple signaling cascades, including phospholipase C, tyrosine kinase, and mitogen-activated protein kinase pathways. However, increasing evidence supports signal-activated phospholipases A(2) and D (PLD) as additional mechanisms. Stimulation of PLD results in phosphatidic acid (PA) formation, and PA has been linked to cell growth. However, the direct involvement of PA or its metabolite diacylglycerol (DAG) in Ang II-induced growth is unclear. PLD activity was measured in cultured rat VSMC prelabeled with [(3)H]oleic acid, while the incorporation of [(3)H]thymidine was used to monitor growth. We have previously reported the Ang II-dependent, AT(1)-coupled stimulation of PLD and growth in VSMC. Here, we show that Ang II (100 nM) and exogenous PLD (0.1-100 units/mL; Streptomyces chromofuscus) stimulated thymidine incorporation (43-208% above control). PA (100 nM-1 microM) also increased thymidine incorporation to 135% of control. Propranolol (100 nM-10 microM), which inhibits PA phosphohydrolase, blocked the growth stimulated by Ang II, PLD, or PA by as much as 95%, an effect not shared by other beta-adrenergic antagonists. Propranolol also increased the production of PA in the presence of Ang II by 320% and reduced DAG and arachidonic acid (AA) accumulation. The DAG lipase inhibitor RHC-80267 (1-10 microM) increased Ang II-induced DAG production, while attenuating thymidine incorporation and release of AA. Thus, it appears that activation of PLD, formation of PA, conversion of PA to DAG, and metabolism of DAG comprise an important signaling cascade in Ang II-induced growth of VSMC.     

Modulation of ANP-C receptor signaling by arginine-vasopressin in A-10 vascular smooth muscle cells: role of protein kinase C

We have previously shown that pretreatment of A-10 vascular smooth muscle cells (VSMC) with angiotensin II (Ang II) attenuated atrial natriuretic peptide receptor-C (ANP-C)-mediated inhibition of adenylyl cyclase without altering [125I]ANP binding. In the present studies, we have investigated the modulation of ANP-C receptor signaling by arginine-vasopressin (AVP). Pretreatment of A-10 VSMC with AVP for 24h resulted in a reduction in ANP receptor binding activity by about 50% (B(max); control cells, 22.9+/-2.5 fmol/mg protein, AVP-treated cells, 11.4+/-1.2 fmol/mg protein). In addition, the expression of ANP-C receptor as determined by immunoblotting was also decreased by about 50% by AVP treatment, which was prevented by GF109203X, an inhibitor of protein kinase C (PKC). The decreased expression of ANP-C receptor was reflected in an attenuation of ANP-C receptor-mediated inhibition of adenylyl cyclase. C-ANP(4-23) [des(Gln(18),Ser(19),Gln(20),Leu(21),Gly(22))ANP(4-23)-NH(2)], a ring deleted peptide of ANP that interacts specifically with ANP-C receptor, inhibited adenylyl cyclase activity by about 30% in control cells, which was completely attenuated in AVP-treated cells. This attenuated inhibition was significantly restored by GF 109203X. In addition, AVP treatment augmented the levels of Gialpha-2 and Gialpha-3 proteins; however, the Gi functions were completely attenuated. The increased expression of Gialpha proteins induced by AVP was inhibited by GF109203X as well as by actinomycin D treatments. In addition, AVP treatment also enhanced the expression of Gsalpha protein and Gsalpha-mediated stimulation of adenylyl cyclase by GTPgammaS, N-ethylcarboxamide adenosine (NECA), and forskolin (FSK), whereas the levels of Gbeta were not altered by AVP treatment. These results indicate that AVP-induced PKC signaling may be responsible for the down-regulation of ANP-C receptor that results in the attenuation of C-ANP(4-23)-mediated inhibition of adenylyl cyclase activity, and suggest a cross-talk between vasopressin V(1) and ANP-C receptor-mediated signaling pathways.     

Hydrogen peroxide inhibits insulin signaling in vascular smooth muscle cells

Both insulin resistance and reactive oxygen species (ROS) have been reported to play essential pathophysiological roles in cardiovascular diseases, such as hypertension and atherosclerosis. However, the mechanistic link between ROS, such as H2O2 and insulin resistance in the vasculature, remains undetermined. Akt, a Ser/Thr kinase, mediates various biological responses induced by insulin. In this study, we examined the effects of H2O2 on Akt activation in the insulin-signaling pathway in vascular smooth muscle cells (VSMCs). In VSMCs, insulin stimulates Akt phosphorylation at Ser473. Pretreatment with H2O2 concentration- and time-dependently inhibited insulin-induced Akt phosphorylation with significant inhibition observed at 50 microM for 10 min. A ROS inducer, diamide, also inhibited insulin-induced Akt phosphorylation. In addition, H2O2 inhibited insulin receptor binding partially and inhibited insulin receptor autophosphorylation almost completely. However, pretreatment with a protein kinase C inhibitor, GF109203X (2 microM), for 30 min did not block the inhibitory effects of H2O2 on insulin-induced Akt phosphorylation, suggesting that protein kinase C is not involved in the inhibition by H2O2. We conclude that ROS inhibit a critical insulin signal transduction component required for Akt activation in VSMCs, suggesting potential cellular mechanisms of insulin resistance, which would require verification in vivo.     

Role of SHPS-1 in the regulation of insulin-like growth factor I-stimulated Shc and mitogen-activated protein kinase activation in vascular smooth muscle cells

Insulin-like growth factor I (IGF-I) stimulates smooth muscle cell (SMC) proliferation, and the mitogen-activated protein kinase (MAPK) pathway plays an important role in mediating IGF-I-induced mitogenic signaling. Our prior studies have shown that recruitment of Src homology 2 domain tyrosine phosphatase (SHP-2) to the membrane scaffolding protein Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1) is required for IGF-I-dependent MAPK activation. The current studies were undertaken to define the upstream signaling components that are required for IGF-I-stimulated MAPK activation and the role of SHPS-1 in regulating this process. The results show that IGF-I-induced Shc phosphorylation and its subsequent binding to Grb2 is required for sustained phosphorylation of MAPK and increased cell proliferation in SMCs. Furthermore, for Shc to be phosphorylated in response to IGF-I requires that Shc must associate with SHPS-1 and this association is mediated in part by SHP-2. Preincubation of cells with a peptide that contains a phospho-tyrosine binding motif sequence derived from SHPS-1 inhibited IGF-I-stimulated SHP-2 transfer to SHPS-1, the association of Shc with SHPS-1, and IGF-I-dependent Shc phosphorylation. Expression of an SHPS-1 mutant that did not bind to Shc or SHP-2 resulted in decreased Shc and MAPK phosphorylation in response to IGF-I. In addition, SMCs expressing a mutant form of the beta3 subunit of the alphaVbeta3, which results in impairment of SHP-2 transfer to SHPS-1, also showed attenuated IGF-I-dependent Shc and MAPK phosphorylation. Further analysis showed that Shc and SHP-2 can be coimmunoprecipitated after IGF-I stimulation. A cell-permeable peptide that contained a polyproline sequence from Shc selectively inhibited Shc/SHP-2 association and impaired Shc but not SHP-2 binding to SHPS-1. Exposure to this peptide also inhibited IGF-I-stimulated Shc and MAPK phosphorylation. Cells expressing a mutant form of Shc with the four prolines substituted with alanines showed no Shc/SHPS-1 association in response to IGF-I. We conclude that SHPS-1 functions as an anchor protein that recruits both Shc and SHP-2 and that their recruitment is necessary for IGF-I-dependent Shc phosphorylation, which is required for an optimal mitogenic response in SMCs.     

TNFalpha inhibits insulin's antiapoptotic signaling in vascular smooth muscle cells

Mutations in TIGR/MYOC (myocilin), a secretory protein of unknown function, have been recently linked to glaucoma. Most known mutations map to the C-terminus, an olfactomedin-like domain. We have previously shown that, in contrast to the wild-type, a truncated form of myocilin lacking the olfactomedin domain is not secreted. In this study, we present evidence that the mutant protein is not correctly processed in the endoplasmic reticulum (ER) and accumulates into insoluble aggregates. In addition, we show that the presence of increasing amounts of mutant protein induces a fraction of the soluble, native myocilin to move to the insoluble fraction. Given the importance of such protein aggregates in the etiology of several aging-related diseases, we propose that olfactomedin-defective mutants might contribute to the pathology of glaucoma through a mechanism involving intracellular accumulation of misfolded proteins.     

Insulin receptor substrate-1/SHP-2 interaction, a phenotype-dependent switching machinery of insulin-like growth factor-I signaling in vascular smooth muscle cells

Insulin-like growth factor-I (IGF-I) plays a role in mutually exclusive processes such as proliferation and differentiation in a variety of cell types. IGF-I is a potent mitogen and motogen for dedifferentiated vascular smooth muscle cells (VSMCs) in vivo and in vitro. However, in differentiated VSMCs, IGF-I is only required for maintaining the differentiated phenotype. Here we investigated the VSMC phenotype-dependent signaling and biological processes triggered by IGF-I. In differentiated VSMCs, IGF-I activated a protein-tyrosine phosphatase, SHP-2, recruited by insulin receptor substrate-1 (IRS-1). The activated SHP-2 then dephosphorylated IRS-1 Tyr(P)-895, resulting in blockade of the pathways from IRS-1/Grb2/Sos to the ERK and p38 MAPK. Conversely, such negative regulation was silent in dedifferentiated VSMCs, where IGF-I activated both MAPKs via IRS-1/Grb2/Sos interaction-linked Ras activation, leading to proliferation and migration. Thus, our present results demonstrate that the IRS-1/SHP-2 interaction acts as a switch controlling VSMC phenotype-dependent IGF-I-induced signaling pathways and biological processes, and this mechanism is likely to be applicable to other cells.     

Proinflammatory phenotype of vascular smooth muscle cells: role of efficient Toll-like receptor 4 signaling

Recent evidence supports a role of Toll-like receptor (TLR) signaling in the development of atherosclerotic lesions. In this study, we tested whether TLR4 signaling promotes a proinflammatory phenotype in human and mouse arterial smooth muscle cells (SMC), characterized by increased cytokine and chemokine synthesis and increased TLR expression. Human arterial SMC were found to express mRNA encoding TLR4 and the TLR4-associated molecules MD-2 and CD14 but not TLR2 mRNA. Mouse aortic SMC, on the other hand, expressed both TLR2 and TLR4 mRNA constitutively. Human SMC derived from the coronary artery, but not those from the pulmonary artery, were found to express cell surface-associated CD14. Low concentrations (ng/ml) of Escherichia coli LPS, the prototypical TLR4 agonist, markedly stimulated extracellular regulated kinase 1/2 (ERK1/2) activity, induced release of monocyte-chemoattractant protein-1 (MCP-1) and interleukin (IL)-6, and stimulated IL-1alpha expression in human aortic SMC, and exogenous CD14 enhanced these effects. Expression of a dominant negative form of TLR4 in human SMC attenuated LPS-induced ERK1/2 and MCP-1 release. LPS was a potent inducer of NF-kappaB activity, ERK1/2 phosphorylation, MCP-1 release, and TLR2 mRNA expression in wild-type mice but not in TLR4-signaling deficient mouse aortic SMC. These studies show that TLR4 signaling promotes a proinflammatory phenotype in vascular smooth muscle cells (VSMC) and suggest that VSMC may potentially play an active role in vascular inflammation via the release of chemokines, proinflammatory cytokines, and increased expression of TLR2.     

Regulation of RGS2 and second messenger signaling in vascular smooth muscle cells by cGMP-dependent protein kinase

RGS2, a GTPase-activating protein (GAP) for G(q)alpha, regulates vascular relaxation and blood pressure. RGS2 can be phosphorylated by type Ialpha cGMP-dependent protein kinase (cGKIalpha), increasing its GAP activity. To understand how RGS2 and cGKIalpha regulate vascular smooth muscle signaling and function, we identified signaling pathways that are controlled by cGMP in an RGS2-dependent manner and discovered new mechanisms whereby cGK activity regulates RGS2. We show that RGS2 regulates vasoconstrictor-stimulated Ca(2+) store release, capacitative Ca(2+) entry, and noncapacitative Ca(2+) entry and that RGS2 is required for cGMP-mediated inhibition of vasoconstrictor-elicited phospholipase Cbeta activation, Ca(2+) store release, and capacitative Ca(2+) entry. RGS2 is degraded in vascular smooth muscle cells via the proteasome. Inhibition of cGK activity blunts RGS2 degradation. However, inactivation of the cGKIalpha phosphorylation sites in RGS2 does not stabilize the protein, suggesting that cGK activity regulates RGS2 degradation by other mechanisms. cGK activation promotes association of RGS2 with the plasma membrane by a mechanism requiring its cGKIalpha phosphorylation sites. By regulating GAP activity, plasma membrane association, and degradation, cGKIalpha therefore may control a cycle of RGS2 activation and inactivation. By diminishing cGK activity, endothelial dysfunction may impair RGS2 activation, thereby blunting vascular relaxation and contributing to hypertension.