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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.


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CREB activation is essential for HMGB1-induced IL-6 production and migration of VSMCs. (A,B) VSMCs were pretreated with different concentrations of the indicated inhibitors for 30 min and then stimulated with HMGB1 for 24 h; (A) ELISA was used to measure IL-6 levels in culture supernatants; (B) Transwell migration assays were performed to measure VSMC migration. p < 0.001 vs. HMGB1; (C–E) VSMCs were transfected with the indicated siRNAs for 24 h and then serum depleted for 24 h; (C) Cells stimulated with HMGB1 for 30 min and cell lysates prepared. Western blot analysis was then performed with antibodies to detect phospho-CREB or β-actin; (D) Culture supernatants from cells incubated with HMGB1 for 24 h was prepared and IL-6 levels measured by ELISA. p < 0.001 vs. HMGB1 + scRNA; (E) Quiesced VSMCs were incubated with HMGB1 for 24 h and migration assays performed as in Figure 2A. p < 0.001 vs. HMGB1 + scRNA. Data in A–E represent mean ± SD of three experiments. Statistical analyses were performed using the one-way ANOVA; (F) Immunohistochemistry on a mouse aortic arch stained with antibodies for p-CREB as well as HMGB1 and counterstained with hematoxylins. Scale bar, 50 μm.
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ijms-17-01394-f008: CREB activation is essential for HMGB1-induced IL-6 production and migration of VSMCs. (A,B) VSMCs were pretreated with different concentrations of the indicated inhibitors for 30 min and then stimulated with HMGB1 for 24 h; (A) ELISA was used to measure IL-6 levels in culture supernatants; (B) Transwell migration assays were performed to measure VSMC migration. p < 0.001 vs. HMGB1; (C–E) VSMCs were transfected with the indicated siRNAs for 24 h and then serum depleted for 24 h; (C) Cells stimulated with HMGB1 for 30 min and cell lysates prepared. Western blot analysis was then performed with antibodies to detect phospho-CREB or β-actin; (D) Culture supernatants from cells incubated with HMGB1 for 24 h was prepared and IL-6 levels measured by ELISA. p < 0.001 vs. HMGB1 + scRNA; (E) Quiesced VSMCs were incubated with HMGB1 for 24 h and migration assays performed as in Figure 2A. p < 0.001 vs. HMGB1 + scRNA. Data in A–E represent mean ± SD of three experiments. Statistical analyses were performed using the one-way ANOVA; (F) Immunohistochemistry on a mouse aortic arch stained with antibodies for p-CREB as well as HMGB1 and counterstained with hematoxylins. Scale bar, 50 μm.

Mentions: Additionally, HMGB1-induced IL-6 production and VSMC migration were suppressed by treatment with the p38 inhibitor, SB203580, and the ERK1/2 inhibitor, PD98059, suggesting that HMGB1-induced IL-6 production and VSMC migration were signaled via the TLR4-driven p38 MAPK- and ERK1/2-dependent pathways (Figure 8A,B). Since CREB activation is required for LPS/TLR4-mediated IL-6 production and VSMC migration, as demonstrated in Figure 5E,F, we next investigated whether CREB activation was essential for HMGB1-induced IL-6 production and VSMC migration. CREB siRNA2 decreased HMGB1-induced phosphorylated CREB level in the VSMCs (Figure 8C), accompanied by inhibition of HMGB1-induced IL-6 production and VSMC migration (Figure 8D,E).


TLR4-Activated MAPK-IL-6 Axis Regulates Vascular Smooth Muscle Cell Function
CREB activation is essential for HMGB1-induced IL-6 production and migration of VSMCs. (A,B) VSMCs were pretreated with different concentrations of the indicated inhibitors for 30 min and then stimulated with HMGB1 for 24 h; (A) ELISA was used to measure IL-6 levels in culture supernatants; (B) Transwell migration assays were performed to measure VSMC migration. p < 0.001 vs. HMGB1; (C–E) VSMCs were transfected with the indicated siRNAs for 24 h and then serum depleted for 24 h; (C) Cells stimulated with HMGB1 for 30 min and cell lysates prepared. Western blot analysis was then performed with antibodies to detect phospho-CREB or β-actin; (D) Culture supernatants from cells incubated with HMGB1 for 24 h was prepared and IL-6 levels measured by ELISA. p < 0.001 vs. HMGB1 + scRNA; (E) Quiesced VSMCs were incubated with HMGB1 for 24 h and migration assays performed as in Figure 2A. p < 0.001 vs. HMGB1 + scRNA. Data in A–E represent mean ± SD of three experiments. Statistical analyses were performed using the one-way ANOVA; (F) Immunohistochemistry on a mouse aortic arch stained with antibodies for p-CREB as well as HMGB1 and counterstained with hematoxylins. Scale bar, 50 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037674&req=5

ijms-17-01394-f008: CREB activation is essential for HMGB1-induced IL-6 production and migration of VSMCs. (A,B) VSMCs were pretreated with different concentrations of the indicated inhibitors for 30 min and then stimulated with HMGB1 for 24 h; (A) ELISA was used to measure IL-6 levels in culture supernatants; (B) Transwell migration assays were performed to measure VSMC migration. p < 0.001 vs. HMGB1; (C–E) VSMCs were transfected with the indicated siRNAs for 24 h and then serum depleted for 24 h; (C) Cells stimulated with HMGB1 for 30 min and cell lysates prepared. Western blot analysis was then performed with antibodies to detect phospho-CREB or β-actin; (D) Culture supernatants from cells incubated with HMGB1 for 24 h was prepared and IL-6 levels measured by ELISA. p < 0.001 vs. HMGB1 + scRNA; (E) Quiesced VSMCs were incubated with HMGB1 for 24 h and migration assays performed as in Figure 2A. p < 0.001 vs. HMGB1 + scRNA. Data in A–E represent mean ± SD of three experiments. Statistical analyses were performed using the one-way ANOVA; (F) Immunohistochemistry on a mouse aortic arch stained with antibodies for p-CREB as well as HMGB1 and counterstained with hematoxylins. Scale bar, 50 μm.
Mentions: Additionally, HMGB1-induced IL-6 production and VSMC migration were suppressed by treatment with the p38 inhibitor, SB203580, and the ERK1/2 inhibitor, PD98059, suggesting that HMGB1-induced IL-6 production and VSMC migration were signaled via the TLR4-driven p38 MAPK- and ERK1/2-dependent pathways (Figure 8A,B). Since CREB activation is required for LPS/TLR4-mediated IL-6 production and VSMC migration, as demonstrated in Figure 5E,F, we next investigated whether CREB activation was essential for HMGB1-induced IL-6 production and VSMC migration. CREB siRNA2 decreased HMGB1-induced phosphorylated CREB level in the VSMCs (Figure 8C), accompanied by inhibition of HMGB1-induced IL-6 production and VSMC migration (Figure 8D,E).

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