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TLR4-Activated MAPK-IL-6 Axis Regulates Vascular Smooth Muscle Cell Function

View Article: PubMed Central - PubMed

ABSTRACT

Migration of vascular smooth muscle cells (VSMCs) into the intima is considered to be a vital event in the pathophysiology of atherosclerosis. Despite substantial evidence supporting the pathogenic role of Toll-like receptor 4 (TLR4) in the progression of atherogenesis, its function in the regulation of VSMC migration remains unclear. The goal of the present study was to elucidate the mechanism by which TLR4 regulates VSMC migration. Inhibitor experiments revealed that TLR4-induced IL-6 secretion and VSMC migration were mediated via the concerted actions of MyD88 and TRIF on the activation of p38 MAPK and ERK1/2 signaling. Neutralizing anti-IL-6 antibodies abrogated TLR4-driven VSMC migration and F-actin polymerization. Blockade of p38 MAPK or ERK1/2 signaling cascade inhibited TLR4 agonist-mediated activation of cAMP response element binding protein (CREB). Moreover, siRNA-mediated suppression of CREB production repressed TLR4-induced IL-6 production and VSMC migration. Rac-1 inhibitor suppressed TLR4-driven VSMC migration but not IL-6 production. Importantly, the serum level of IL-6 and TLR4 endogenous ligand HMGB1 was significantly higher in patients with coronary artery diseases (CAD) than in healthy subjects. Serum HMGB1 level was positively correlated with serum IL-6 level in CAD patients. The expression of both HMGB1 and IL-6 was clearly detected in the atherosclerotic tissue of the CAD patients. Additionally, there was a positive association between p-CREB and HMGB1 in mouse atherosclerotic tissue. Based on our findings, we concluded that, upon ligand binding, TLR4 activates p38 MAPK and ERK1/2 signaling through MyD88 and TRIF in VSMCs. These signaling pathways subsequently coordinate an additive augmentation of CREB-driven IL-6 production, which in turn triggers Rac-1-mediated actin cytoskeleton to promote VSMC migration.

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Related in: MedlinePlus

TLR4-mediated IL-6 production and VSMC migration depend on p38 MAPK and ERK1/2 pathway-mediated by Myd88 or TRIF. VSMCs were pretreated with pepinh-MyD88 (Myd88 signaling inhibitor) or pepinh-TRIF (TRIF signaling inhibitor) for 30 min, then stimulated with LPS for 24 h. (A) IL-6 levels in culture supernatants were measured by ELISA. p < 0.001 vs. LPS; (B) VSMC migration was measured by the transwell assays. p < 0.05 vs. LPS; (C–F) VSMCs were pretreated with various concentrations of the indicated inhibitors (SB202190 or SB203580 for p38; U0126 for ERK1/2; SP600125 for JNK; or LY294002 for PI3K) for 30 min and then stimulated with LPS for 24 h; (C) IL-6 levels in culture supernatants were measured by ELISA. p < 0.001 or 0.05 vs. LPS; (D) MTT assay was used to determine cell viability. p < 0.001 vs. LPS with SP600125; p < 0.01 vs. LPS with LY294002; (E) VSMC migration was measured by the transwell assays; (F) IL-6 levels in culture supernatants were measured by ELISA. p < 0.001 vs. LPS + dimethyl sulfoxide (DMSO). Data in A–F represent mean ± SD of three experiments. Statistical analyses were performed using the one-way ANOVA.
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ijms-17-01394-f004: TLR4-mediated IL-6 production and VSMC migration depend on p38 MAPK and ERK1/2 pathway-mediated by Myd88 or TRIF. VSMCs were pretreated with pepinh-MyD88 (Myd88 signaling inhibitor) or pepinh-TRIF (TRIF signaling inhibitor) for 30 min, then stimulated with LPS for 24 h. (A) IL-6 levels in culture supernatants were measured by ELISA. p < 0.001 vs. LPS; (B) VSMC migration was measured by the transwell assays. p < 0.05 vs. LPS; (C–F) VSMCs were pretreated with various concentrations of the indicated inhibitors (SB202190 or SB203580 for p38; U0126 for ERK1/2; SP600125 for JNK; or LY294002 for PI3K) for 30 min and then stimulated with LPS for 24 h; (C) IL-6 levels in culture supernatants were measured by ELISA. p < 0.001 or 0.05 vs. LPS; (D) MTT assay was used to determine cell viability. p < 0.001 vs. LPS with SP600125; p < 0.01 vs. LPS with LY294002; (E) VSMC migration was measured by the transwell assays; (F) IL-6 levels in culture supernatants were measured by ELISA. p < 0.001 vs. LPS + dimethyl sulfoxide (DMSO). Data in A–F represent mean ± SD of three experiments. Statistical analyses were performed using the one-way ANOVA.

Mentions: Since TLR4-mediated cellular effects signal through adaptor protein MyD88 and TRIF [23], we used well-characterized peptide inhibitors to examine the roles of the MyD88 and TRIF pathways in TLR4-induced IL-6 production in the VSMCs. Peptide blockers of MyD88 and TRIF, but not control peptide blockers, significantly suppressed IL-6 production and VSMC migration induced by LPS (Figure 4A,B) without affecting VSMC viability (data not shown), suggesting that both MyD88 and TRIF pathways contribute to LPS/TLR4-mediated cellular effects in VSMCs.


TLR4-Activated MAPK-IL-6 Axis Regulates Vascular Smooth Muscle Cell Function
TLR4-mediated IL-6 production and VSMC migration depend on p38 MAPK and ERK1/2 pathway-mediated by Myd88 or TRIF. VSMCs were pretreated with pepinh-MyD88 (Myd88 signaling inhibitor) or pepinh-TRIF (TRIF signaling inhibitor) for 30 min, then stimulated with LPS for 24 h. (A) IL-6 levels in culture supernatants were measured by ELISA. p < 0.001 vs. LPS; (B) VSMC migration was measured by the transwell assays. p < 0.05 vs. LPS; (C–F) VSMCs were pretreated with various concentrations of the indicated inhibitors (SB202190 or SB203580 for p38; U0126 for ERK1/2; SP600125 for JNK; or LY294002 for PI3K) for 30 min and then stimulated with LPS for 24 h; (C) IL-6 levels in culture supernatants were measured by ELISA. p < 0.001 or 0.05 vs. LPS; (D) MTT assay was used to determine cell viability. p < 0.001 vs. LPS with SP600125; p < 0.01 vs. LPS with LY294002; (E) VSMC migration was measured by the transwell assays; (F) IL-6 levels in culture supernatants were measured by ELISA. p < 0.001 vs. LPS + dimethyl sulfoxide (DMSO). Data in A–F represent mean ± SD of three experiments. Statistical analyses were performed using the one-way ANOVA.
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ijms-17-01394-f004: TLR4-mediated IL-6 production and VSMC migration depend on p38 MAPK and ERK1/2 pathway-mediated by Myd88 or TRIF. VSMCs were pretreated with pepinh-MyD88 (Myd88 signaling inhibitor) or pepinh-TRIF (TRIF signaling inhibitor) for 30 min, then stimulated with LPS for 24 h. (A) IL-6 levels in culture supernatants were measured by ELISA. p < 0.001 vs. LPS; (B) VSMC migration was measured by the transwell assays. p < 0.05 vs. LPS; (C–F) VSMCs were pretreated with various concentrations of the indicated inhibitors (SB202190 or SB203580 for p38; U0126 for ERK1/2; SP600125 for JNK; or LY294002 for PI3K) for 30 min and then stimulated with LPS for 24 h; (C) IL-6 levels in culture supernatants were measured by ELISA. p < 0.001 or 0.05 vs. LPS; (D) MTT assay was used to determine cell viability. p < 0.001 vs. LPS with SP600125; p < 0.01 vs. LPS with LY294002; (E) VSMC migration was measured by the transwell assays; (F) IL-6 levels in culture supernatants were measured by ELISA. p < 0.001 vs. LPS + dimethyl sulfoxide (DMSO). Data in A–F represent mean ± SD of three experiments. Statistical analyses were performed using the one-way ANOVA.
Mentions: Since TLR4-mediated cellular effects signal through adaptor protein MyD88 and TRIF [23], we used well-characterized peptide inhibitors to examine the roles of the MyD88 and TRIF pathways in TLR4-induced IL-6 production in the VSMCs. Peptide blockers of MyD88 and TRIF, but not control peptide blockers, significantly suppressed IL-6 production and VSMC migration induced by LPS (Figure 4A,B) without affecting VSMC viability (data not shown), suggesting that both MyD88 and TRIF pathways contribute to LPS/TLR4-mediated cellular effects in VSMCs.

View Article: PubMed Central - PubMed

ABSTRACT

Migration of vascular smooth muscle cells (VSMCs) into the intima is considered to be a vital event in the pathophysiology of atherosclerosis. Despite substantial evidence supporting the pathogenic role of Toll-like receptor 4 (TLR4) in the progression of atherogenesis, its function in the regulation of VSMC migration remains unclear. The goal of the present study was to elucidate the mechanism by which TLR4 regulates VSMC migration. Inhibitor experiments revealed that TLR4-induced IL-6 secretion and VSMC migration were mediated via the concerted actions of MyD88 and TRIF on the activation of p38 MAPK and ERK1/2 signaling. Neutralizing anti-IL-6 antibodies abrogated TLR4-driven VSMC migration and F-actin polymerization. Blockade of p38 MAPK or ERK1/2 signaling cascade inhibited TLR4 agonist-mediated activation of cAMP response element binding protein (CREB). Moreover, siRNA-mediated suppression of CREB production repressed TLR4-induced IL-6 production and VSMC migration. Rac-1 inhibitor suppressed TLR4-driven VSMC migration but not IL-6 production. Importantly, the serum level of IL-6 and TLR4 endogenous ligand HMGB1 was significantly higher in patients with coronary artery diseases (CAD) than in healthy subjects. Serum HMGB1 level was positively correlated with serum IL-6 level in CAD patients. The expression of both HMGB1 and IL-6 was clearly detected in the atherosclerotic tissue of the CAD patients. Additionally, there was a positive association between p-CREB and HMGB1 in mouse atherosclerotic tissue. Based on our findings, we concluded that, upon ligand binding, TLR4 activates p38 MAPK and ERK1/2 signaling through MyD88 and TRIF in VSMCs. These signaling pathways subsequently coordinate an additive augmentation of CREB-driven IL-6 production, which in turn triggers Rac-1-mediated actin cytoskeleton to promote VSMC migration.

No MeSH data available.


Related in: MedlinePlus