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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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TLR4 endogenous ligand HMGB1 induces IL-6 production and VSMC migration. (A) Serum HMGB1 concentrations in CAD patients (n = 227) and healthy subjects (n = 54). p < 0.001 vs. control; (B) Serum IL-6 concentrations in CAD patients (n = 227) and healthy subjects (n = 80) were measured by ELISA. p < 0.001 vs. control. Statistical analyses in A-B were performed using the t-test; (C) Correlation between serum HMGB1 and serum IL-6 in CAD patients (n = 227). Spearman’s rank correlation coefficient (r = 0.3102, p < 0.0001). All values are expressed as means ± SD; (D) Amounts of HMGB1 and IL-6 in the atherosclerotic tissue of CAD patients were measured by ELISA; (E,F) VSMCs were pretreated with plyB or CLI-095 for 30 min, then stimulated with HMGB1 (50 μg/mL) for 24 h; (E) IL-6 levels in culture supernatants were measured by ELISA; (F) VSMC migration was measured by the transwell assays. p < 0.001 vs. HMGB1. Data in E,F represent mean ± SD of three experiments. Statistical analyses in E–F were performed using the one-way ANOVA.
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ijms-17-01394-f007: TLR4 endogenous ligand HMGB1 induces IL-6 production and VSMC migration. (A) Serum HMGB1 concentrations in CAD patients (n = 227) and healthy subjects (n = 54). p < 0.001 vs. control; (B) Serum IL-6 concentrations in CAD patients (n = 227) and healthy subjects (n = 80) were measured by ELISA. p < 0.001 vs. control. Statistical analyses in A-B were performed using the t-test; (C) Correlation between serum HMGB1 and serum IL-6 in CAD patients (n = 227). Spearman’s rank correlation coefficient (r = 0.3102, p < 0.0001). All values are expressed as means ± SD; (D) Amounts of HMGB1 and IL-6 in the atherosclerotic tissue of CAD patients were measured by ELISA; (E,F) VSMCs were pretreated with plyB or CLI-095 for 30 min, then stimulated with HMGB1 (50 μg/mL) for 24 h; (E) IL-6 levels in culture supernatants were measured by ELISA; (F) VSMC migration was measured by the transwell assays. p < 0.001 vs. HMGB1. Data in E,F represent mean ± SD of three experiments. Statistical analyses in E–F were performed using the one-way ANOVA.

Mentions: Given that TLR4 induced IL-6 production in VSMCs, we investigated the clinical correlation between the TLR4 endogenous ligand, HMGB1, and IL-6 production in human patients with CAD. Serum HMGB1 (192.8 ± 33.24 pg/mL) and IL-6 (91.7 ± 13.5 pg/mL) levels in human CAD patients were significantly higher than those in the control subjects (HGB1: 2.5 ± 33.24 pg/mL; IL-6: 26.7 ± 6.42 pg/mL) (Figure 7A,B). The serum HMGB1 level was also found to be positively correlated with serum IL-6 level in the CAD patients (Figure 7C).


TLR4-Activated MAPK-IL-6 Axis Regulates Vascular Smooth Muscle Cell Function
TLR4 endogenous ligand HMGB1 induces IL-6 production and VSMC migration. (A) Serum HMGB1 concentrations in CAD patients (n = 227) and healthy subjects (n = 54). p < 0.001 vs. control; (B) Serum IL-6 concentrations in CAD patients (n = 227) and healthy subjects (n = 80) were measured by ELISA. p < 0.001 vs. control. Statistical analyses in A-B were performed using the t-test; (C) Correlation between serum HMGB1 and serum IL-6 in CAD patients (n = 227). Spearman’s rank correlation coefficient (r = 0.3102, p < 0.0001). All values are expressed as means ± SD; (D) Amounts of HMGB1 and IL-6 in the atherosclerotic tissue of CAD patients were measured by ELISA; (E,F) VSMCs were pretreated with plyB or CLI-095 for 30 min, then stimulated with HMGB1 (50 μg/mL) for 24 h; (E) IL-6 levels in culture supernatants were measured by ELISA; (F) VSMC migration was measured by the transwell assays. p < 0.001 vs. HMGB1. Data in E,F represent mean ± SD of three experiments. Statistical analyses in E–F were performed using the one-way ANOVA.
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Show All Figures
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ijms-17-01394-f007: TLR4 endogenous ligand HMGB1 induces IL-6 production and VSMC migration. (A) Serum HMGB1 concentrations in CAD patients (n = 227) and healthy subjects (n = 54). p < 0.001 vs. control; (B) Serum IL-6 concentrations in CAD patients (n = 227) and healthy subjects (n = 80) were measured by ELISA. p < 0.001 vs. control. Statistical analyses in A-B were performed using the t-test; (C) Correlation between serum HMGB1 and serum IL-6 in CAD patients (n = 227). Spearman’s rank correlation coefficient (r = 0.3102, p < 0.0001). All values are expressed as means ± SD; (D) Amounts of HMGB1 and IL-6 in the atherosclerotic tissue of CAD patients were measured by ELISA; (E,F) VSMCs were pretreated with plyB or CLI-095 for 30 min, then stimulated with HMGB1 (50 μg/mL) for 24 h; (E) IL-6 levels in culture supernatants were measured by ELISA; (F) VSMC migration was measured by the transwell assays. p < 0.001 vs. HMGB1. Data in E,F represent mean ± SD of three experiments. Statistical analyses in E–F were performed using the one-way ANOVA.
Mentions: Given that TLR4 induced IL-6 production in VSMCs, we investigated the clinical correlation between the TLR4 endogenous ligand, HMGB1, and IL-6 production in human patients with CAD. Serum HMGB1 (192.8 ± 33.24 pg/mL) and IL-6 (91.7 ± 13.5 pg/mL) levels in human CAD patients were significantly higher than those in the control subjects (HGB1: 2.5 ± 33.24 pg/mL; IL-6: 26.7 ± 6.42 pg/mL) (Figure 7A,B). The serum HMGB1 level was also found to be positively correlated with serum IL-6 level in the CAD patients (Figure 7C).

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