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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.

No MeSH data available.


Role of Rac1-mediated F-actin formation in TLR4-induced VSMC migration. (A) VSMCs were subjected to different treatments (TE, LPS, IL-6, and in combination with anti-IL-6 or IL-12) for 24 h. Rhodamine-conjugated phalloidin staining was then performed to reveal actin stress fibers of VSMCs. The staining intensity was then quantified. p < 0.001 vs. LPS; p < 0.0001 vs. LPS + anti-IL6; (B,C) VSMCs were pretreated with Rac1 inhibitors NSC23766 (100 μM) for 30 min before stimulation with TE buffer, LPS or IL-6 for 24 h; (B) Migration assays were performed using transwell assays and PDGF-BB as a chemoattractant. p < 0.001 for TE buffer vs. NSC23766; (C) ELISA was performed to determine IL-6 levels in culture medium; (D) VSMC Phalloidin staining and quantitative analysis of staining intensity. p < 0.01 vs. LPS with TE buffer. Representative images of three independent experiments are shown in A and D. Scale bar, 5 μm. One-way ANOVA was used for statistical analyses in A–D.
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ijms-17-01394-f006: Role of Rac1-mediated F-actin formation in TLR4-induced VSMC migration. (A) VSMCs were subjected to different treatments (TE, LPS, IL-6, and in combination with anti-IL-6 or IL-12) for 24 h. Rhodamine-conjugated phalloidin staining was then performed to reveal actin stress fibers of VSMCs. The staining intensity was then quantified. p < 0.001 vs. LPS; p < 0.0001 vs. LPS + anti-IL6; (B,C) VSMCs were pretreated with Rac1 inhibitors NSC23766 (100 μM) for 30 min before stimulation with TE buffer, LPS or IL-6 for 24 h; (B) Migration assays were performed using transwell assays and PDGF-BB as a chemoattractant. p < 0.001 for TE buffer vs. NSC23766; (C) ELISA was performed to determine IL-6 levels in culture medium; (D) VSMC Phalloidin staining and quantitative analysis of staining intensity. p < 0.01 vs. LPS with TE buffer. Representative images of three independent experiments are shown in A and D. Scale bar, 5 μm. One-way ANOVA was used for statistical analyses in A–D.

Mentions: As actin cytoskeleton reorganization plays a key role in regulating cell migration, we thus examined actin polymerization of VSMCs after LPS stimulation. Rhodamine-conjugated phalloidin staining (for filamentous actin fiber (F-actin)) revealed that LPS significantly induced actin stress fiber formation (Figure 6A). Interestingly, IL-6- but not IL-12-neutralizing antibodies suppressed stress fiber formation (Figure 6A). This anti-IL-6 antibody-mediated suppression of LPS-induced F-actin organization in the VSMCs was restored by IL-6 administration (Figure 6A), indicating that LPS-induced F-actin formation in VSMCs is mediated via the release of IL-6 in the conditioned medium.


TLR4-Activated MAPK-IL-6 Axis Regulates Vascular Smooth Muscle Cell Function
Role of Rac1-mediated F-actin formation in TLR4-induced VSMC migration. (A) VSMCs were subjected to different treatments (TE, LPS, IL-6, and in combination with anti-IL-6 or IL-12) for 24 h. Rhodamine-conjugated phalloidin staining was then performed to reveal actin stress fibers of VSMCs. The staining intensity was then quantified. p < 0.001 vs. LPS; p < 0.0001 vs. LPS + anti-IL6; (B,C) VSMCs were pretreated with Rac1 inhibitors NSC23766 (100 μM) for 30 min before stimulation with TE buffer, LPS or IL-6 for 24 h; (B) Migration assays were performed using transwell assays and PDGF-BB as a chemoattractant. p < 0.001 for TE buffer vs. NSC23766; (C) ELISA was performed to determine IL-6 levels in culture medium; (D) VSMC Phalloidin staining and quantitative analysis of staining intensity. p < 0.01 vs. LPS with TE buffer. Representative images of three independent experiments are shown in A and D. Scale bar, 5 μm. One-way ANOVA was used for statistical analyses in A–D.
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ijms-17-01394-f006: Role of Rac1-mediated F-actin formation in TLR4-induced VSMC migration. (A) VSMCs were subjected to different treatments (TE, LPS, IL-6, and in combination with anti-IL-6 or IL-12) for 24 h. Rhodamine-conjugated phalloidin staining was then performed to reveal actin stress fibers of VSMCs. The staining intensity was then quantified. p < 0.001 vs. LPS; p < 0.0001 vs. LPS + anti-IL6; (B,C) VSMCs were pretreated with Rac1 inhibitors NSC23766 (100 μM) for 30 min before stimulation with TE buffer, LPS or IL-6 for 24 h; (B) Migration assays were performed using transwell assays and PDGF-BB as a chemoattractant. p < 0.001 for TE buffer vs. NSC23766; (C) ELISA was performed to determine IL-6 levels in culture medium; (D) VSMC Phalloidin staining and quantitative analysis of staining intensity. p < 0.01 vs. LPS with TE buffer. Representative images of three independent experiments are shown in A and D. Scale bar, 5 μm. One-way ANOVA was used for statistical analyses in A–D.
Mentions: As actin cytoskeleton reorganization plays a key role in regulating cell migration, we thus examined actin polymerization of VSMCs after LPS stimulation. Rhodamine-conjugated phalloidin staining (for filamentous actin fiber (F-actin)) revealed that LPS significantly induced actin stress fiber formation (Figure 6A). Interestingly, IL-6- but not IL-12-neutralizing antibodies suppressed stress fiber formation (Figure 6A). This anti-IL-6 antibody-mediated suppression of LPS-induced F-actin organization in the VSMCs was restored by IL-6 administration (Figure 6A), indicating that LPS-induced F-actin formation in VSMCs is mediated via the release of IL-6 in the conditioned medium.

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.