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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.


Related in: MedlinePlus

IL-6 mediates TLR4 signaling-induced VSMC migration. (A) VSMCs were treated with TE buffer or LPS and conditioned medium collected. Migration assays were then performed using conditioned medium (with or without anti-IL-6, anti-IL-12 antibodies (5 μg/mL) or recombinant IL-6 (50 ng/mL)) as chemoattractants. Migrated cells were then counted 24 h later. p < 0.01 vs. LPS-CM or LPS-CM + anti-IL-6; (B) Serum-starved VSMCs were stimulated with TE buffer or LPS with or without anti-IL-6, anti-IL-12 antibodies or IL-6 (50 ng/mL) for 24 h and migration assays performed as in Figure 2A. p < 0.001 vs. LPS or LPS + anti-IL-6; (C) VSMCs (in the presence or absence of plyB, CLI-095 or IL-6) were treated with TE buffer or LPS for 24 h. VSMC migration was then measured by the transwell assays. p < 0.001 vs. LPS + ply or LPS + CLI-095. Data in (A–C) represent mean ± SD of three experiments. Statistical analyses were performed using the one-way ANOVA.
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ijms-17-01394-f003: IL-6 mediates TLR4 signaling-induced VSMC migration. (A) VSMCs were treated with TE buffer or LPS and conditioned medium collected. Migration assays were then performed using conditioned medium (with or without anti-IL-6, anti-IL-12 antibodies (5 μg/mL) or recombinant IL-6 (50 ng/mL)) as chemoattractants. Migrated cells were then counted 24 h later. p < 0.01 vs. LPS-CM or LPS-CM + anti-IL-6; (B) Serum-starved VSMCs were stimulated with TE buffer or LPS with or without anti-IL-6, anti-IL-12 antibodies or IL-6 (50 ng/mL) for 24 h and migration assays performed as in Figure 2A. p < 0.001 vs. LPS or LPS + anti-IL-6; (C) VSMCs (in the presence or absence of plyB, CLI-095 or IL-6) were treated with TE buffer or LPS for 24 h. VSMC migration was then measured by the transwell assays. p < 0.001 vs. LPS + ply or LPS + CLI-095. Data in (A–C) represent mean ± SD of three experiments. Statistical analyses were performed using the one-way ANOVA.

Mentions: The involvement of IL-6 in regulating VSMC migration has been reported [19,25]. Given that TLR4 inhibition attenuated IL-6 levels and VSMC migration (Figure 1 and Figure 2), it is likely that IL-6 is released into conditioned medium to function as a soluble factor. To test this, we evaluated whether IL-6-neutralizing antibodies could affect the migration-inducing activity of LPS-CM. LPS-CM-induced VSMC migration was significantly inhibited by IL-6- but not IL-12-neutralizing antibodies. This inhibitory effect of anti-IL-6 antibody was restored by administration of exogenous recombinant mouse IL-6 (Figure 3A). To further confirm this finding, IL-6-neutralizing antibodies were added prior to LPS stimulation. Neutralizing antibodies against IL-6, but not IL-12, abrogated LPS-induced VSMC migration (Figure 3B). Furthermore, exogenous recombinant mouse IL-6 restored LPS-mediated VSMC migration that was inhibited by either polymyxin B or CLI-095 (Figure 3C). Collectively, our data suggest that IL-6 in the extracellular milieu mediates TLR4 activation-induced VSMC migration.


TLR4-Activated MAPK-IL-6 Axis Regulates Vascular Smooth Muscle Cell Function
IL-6 mediates TLR4 signaling-induced VSMC migration. (A) VSMCs were treated with TE buffer or LPS and conditioned medium collected. Migration assays were then performed using conditioned medium (with or without anti-IL-6, anti-IL-12 antibodies (5 μg/mL) or recombinant IL-6 (50 ng/mL)) as chemoattractants. Migrated cells were then counted 24 h later. p < 0.01 vs. LPS-CM or LPS-CM + anti-IL-6; (B) Serum-starved VSMCs were stimulated with TE buffer or LPS with or without anti-IL-6, anti-IL-12 antibodies or IL-6 (50 ng/mL) for 24 h and migration assays performed as in Figure 2A. p < 0.001 vs. LPS or LPS + anti-IL-6; (C) VSMCs (in the presence or absence of plyB, CLI-095 or IL-6) were treated with TE buffer or LPS for 24 h. VSMC migration was then measured by the transwell assays. p < 0.001 vs. LPS + ply or LPS + CLI-095. Data in (A–C) represent mean ± SD of three experiments. Statistical analyses were performed using the one-way ANOVA.
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ijms-17-01394-f003: IL-6 mediates TLR4 signaling-induced VSMC migration. (A) VSMCs were treated with TE buffer or LPS and conditioned medium collected. Migration assays were then performed using conditioned medium (with or without anti-IL-6, anti-IL-12 antibodies (5 μg/mL) or recombinant IL-6 (50 ng/mL)) as chemoattractants. Migrated cells were then counted 24 h later. p < 0.01 vs. LPS-CM or LPS-CM + anti-IL-6; (B) Serum-starved VSMCs were stimulated with TE buffer or LPS with or without anti-IL-6, anti-IL-12 antibodies or IL-6 (50 ng/mL) for 24 h and migration assays performed as in Figure 2A. p < 0.001 vs. LPS or LPS + anti-IL-6; (C) VSMCs (in the presence or absence of plyB, CLI-095 or IL-6) were treated with TE buffer or LPS for 24 h. VSMC migration was then measured by the transwell assays. p < 0.001 vs. LPS + ply or LPS + CLI-095. Data in (A–C) represent mean ± SD of three experiments. Statistical analyses were performed using the one-way ANOVA.
Mentions: The involvement of IL-6 in regulating VSMC migration has been reported [19,25]. Given that TLR4 inhibition attenuated IL-6 levels and VSMC migration (Figure 1 and Figure 2), it is likely that IL-6 is released into conditioned medium to function as a soluble factor. To test this, we evaluated whether IL-6-neutralizing antibodies could affect the migration-inducing activity of LPS-CM. LPS-CM-induced VSMC migration was significantly inhibited by IL-6- but not IL-12-neutralizing antibodies. This inhibitory effect of anti-IL-6 antibody was restored by administration of exogenous recombinant mouse IL-6 (Figure 3A). To further confirm this finding, IL-6-neutralizing antibodies were added prior to LPS stimulation. Neutralizing antibodies against IL-6, but not IL-12, abrogated LPS-induced VSMC migration (Figure 3B). Furthermore, exogenous recombinant mouse IL-6 restored LPS-mediated VSMC migration that was inhibited by either polymyxin B or CLI-095 (Figure 3C). Collectively, our data suggest that IL-6 in the extracellular milieu mediates TLR4 activation-induced VSMC migration.

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