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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-mediated IL-6 production is involved in LPS-induced VSMC migration. (A) Serum-starved VSMCs were stimulated with LPS for the indicated times; (B,C) VSMCs were pretreated with different amounts of polymyxin B (B); SB202190 or U0126 (C) for 30 min and then stimulated with LPS for 30 min. Cell lysates were subjected to Western blotting with antibodies for CREB, phospho-CREB, NF-κB p65, phospho-NF-κB p65 (Ser536) or β-actin; (D–F) VSMCs were transfected with the indicated siRNAs for 24 h and then serum depleted for 24 h; (D) Cells were incubated with LPS for 30 min. Cell lysates were immunoblotted with antibodies for CREB, phospho-CREB, phospho-p38, phospho-ERK or β-actin; (E) Cells were stimulated with LPS for 24 h and IL-6 level in culture supernatants and CREB level in cell lysates were measured by ELISA and western blot, respectively; (F) Quiesced VSMCs were incubated with LPS for 24 h and migration assays performed as in Figure 2A. p < 0.001 vs. LPS + scRNA. The experiments in A–D were repeated three times with similar results. Data in E–F represent mean ± SD of three experiments. Statistical analyses were performed using the one-way ANOVA.
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ijms-17-01394-f005: CREB-mediated IL-6 production is involved in LPS-induced VSMC migration. (A) Serum-starved VSMCs were stimulated with LPS for the indicated times; (B,C) VSMCs were pretreated with different amounts of polymyxin B (B); SB202190 or U0126 (C) for 30 min and then stimulated with LPS for 30 min. Cell lysates were subjected to Western blotting with antibodies for CREB, phospho-CREB, NF-κB p65, phospho-NF-κB p65 (Ser536) or β-actin; (D–F) VSMCs were transfected with the indicated siRNAs for 24 h and then serum depleted for 24 h; (D) Cells were incubated with LPS for 30 min. Cell lysates were immunoblotted with antibodies for CREB, phospho-CREB, phospho-p38, phospho-ERK or β-actin; (E) Cells were stimulated with LPS for 24 h and IL-6 level in culture supernatants and CREB level in cell lysates were measured by ELISA and western blot, respectively; (F) Quiesced VSMCs were incubated with LPS for 24 h and migration assays performed as in Figure 2A. p < 0.001 vs. LPS + scRNA. The experiments in A–D were repeated three times with similar results. Data in E–F represent mean ± SD of three experiments. Statistical analyses were performed using the one-way ANOVA.

Mentions: Several cis-elements and cognate transcription factors such as CREB and NF-κB in the promoter region of IL-6 gene controls IL-6 transcription [26]. Therefore, we wondered whether LPS activated these two transcription factors in VSMCs. In our previous experiment, LPS induced the phosphorylation of CREB and NF-κB p65 (Ser536) in VSMCs within 10 min, which lasted for at least 30 min (Figure 5A), and this induction was abrogated by polymyxin B (Figure 5B). Further, since our results (Figure 4) indicate that both p38 MAPK and ERK1/2 signaling were involved in LPS-induced IL-6 production, we reasoned that these pathways might mediate LPS-induced activation of CREB and NF-κB p65. Interestingly, inhibiting p38 MAPK or ERK1/2 signaling significantly reduced CREB but not NF-κB p65 (Ser536) phosphorylation (Figure 5C), indicating that p38 MAPK and ERK1/2 signaling mediates LPS-stimulated CREB phosphorylation.


TLR4-Activated MAPK-IL-6 Axis Regulates Vascular Smooth Muscle Cell Function
CREB-mediated IL-6 production is involved in LPS-induced VSMC migration. (A) Serum-starved VSMCs were stimulated with LPS for the indicated times; (B,C) VSMCs were pretreated with different amounts of polymyxin B (B); SB202190 or U0126 (C) for 30 min and then stimulated with LPS for 30 min. Cell lysates were subjected to Western blotting with antibodies for CREB, phospho-CREB, NF-κB p65, phospho-NF-κB p65 (Ser536) or β-actin; (D–F) VSMCs were transfected with the indicated siRNAs for 24 h and then serum depleted for 24 h; (D) Cells were incubated with LPS for 30 min. Cell lysates were immunoblotted with antibodies for CREB, phospho-CREB, phospho-p38, phospho-ERK or β-actin; (E) Cells were stimulated with LPS for 24 h and IL-6 level in culture supernatants and CREB level in cell lysates were measured by ELISA and western blot, respectively; (F) Quiesced VSMCs were incubated with LPS for 24 h and migration assays performed as in Figure 2A. p < 0.001 vs. LPS + scRNA. The experiments in A–D were repeated three times with similar results. Data in E–F represent mean ± SD of three experiments. Statistical analyses were performed using the one-way ANOVA.
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Related In: Results  -  Collection

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ijms-17-01394-f005: CREB-mediated IL-6 production is involved in LPS-induced VSMC migration. (A) Serum-starved VSMCs were stimulated with LPS for the indicated times; (B,C) VSMCs were pretreated with different amounts of polymyxin B (B); SB202190 or U0126 (C) for 30 min and then stimulated with LPS for 30 min. Cell lysates were subjected to Western blotting with antibodies for CREB, phospho-CREB, NF-κB p65, phospho-NF-κB p65 (Ser536) or β-actin; (D–F) VSMCs were transfected with the indicated siRNAs for 24 h and then serum depleted for 24 h; (D) Cells were incubated with LPS for 30 min. Cell lysates were immunoblotted with antibodies for CREB, phospho-CREB, phospho-p38, phospho-ERK or β-actin; (E) Cells were stimulated with LPS for 24 h and IL-6 level in culture supernatants and CREB level in cell lysates were measured by ELISA and western blot, respectively; (F) Quiesced VSMCs were incubated with LPS for 24 h and migration assays performed as in Figure 2A. p < 0.001 vs. LPS + scRNA. The experiments in A–D were repeated three times with similar results. Data in E–F represent mean ± SD of three experiments. Statistical analyses were performed using the one-way ANOVA.
Mentions: Several cis-elements and cognate transcription factors such as CREB and NF-κB in the promoter region of IL-6 gene controls IL-6 transcription [26]. Therefore, we wondered whether LPS activated these two transcription factors in VSMCs. In our previous experiment, LPS induced the phosphorylation of CREB and NF-κB p65 (Ser536) in VSMCs within 10 min, which lasted for at least 30 min (Figure 5A), and this induction was abrogated by polymyxin B (Figure 5B). Further, since our results (Figure 4) indicate that both p38 MAPK and ERK1/2 signaling were involved in LPS-induced IL-6 production, we reasoned that these pathways might mediate LPS-induced activation of CREB and NF-κB p65. Interestingly, inhibiting p38 MAPK or ERK1/2 signaling significantly reduced CREB but not NF-κB p65 (Ser536) phosphorylation (Figure 5C), indicating that p38 MAPK and ERK1/2 signaling mediates LPS-stimulated CREB phosphorylation.

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