Limits...
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

Role of TLR4 in VSMC migration. (A) VSMCs (in the presence or absence of anti-TLR2 or anti-TLR4 antibodies (5 μg/mL)) were treated with TE buffer or LPS for 24 h; (B) Quiesced VSMCs were stimulated with TE buffer, LPS or P3C4 in the presence or absence of plyB or CLI-095 for 24 h. Migration assays were then performed with PDGF-BB as a chemoattractant. p < 0.001 vs. LPS or LPS + DMSO; (C) Serum-starved VSMCs were seeded into the upper chambers of transwell plates and stimulated with TE buffer or LPS for 24 h. Migration assays were then performed with or without PDGF-BB as a chemoattractant. p < 0.001 for LPS vs. TE. p < 0.05 for LPS vs. LPS + PDGF; (D) Conditioned medium from VSMCs stimulated with TE buffer, LPS or P3C4 with or without plyB or CLI-095 for 24 h was collected and added to the lower chamber as a chemoattractant in the transwell migration assays. PDGF-BB was used as a positive control. p < 0.01 vs. LPS; (E) Serial dilutions of conditioned medium (CM) from VSMCs stimulated with LPS (LPS-CM) were prepared by diluting with TE-treated CM (TE-CM) and added to the lower chambers; VSMCs were plated onto top chambers of transwells. After 24 h incubation, VSMC migration was determined by the transwell assay. p < 0.01 vs. 0x/10x; (F) Checkerboard assays. CM from VSMCs treated with TE buffer (TE-CM) or LPS (LPS-CM) for 24 h was added either to the upper or lower chamber of the transwell plates for VSMC migration assays. p < 0.01 vs. TE-CM/TE-CM. Data in A–F represent mean ± SD of three experiments. Statistical analyses were performed using the one-way ANOVA.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5037674&req=5

ijms-17-01394-f002: Role of TLR4 in VSMC migration. (A) VSMCs (in the presence or absence of anti-TLR2 or anti-TLR4 antibodies (5 μg/mL)) were treated with TE buffer or LPS for 24 h; (B) Quiesced VSMCs were stimulated with TE buffer, LPS or P3C4 in the presence or absence of plyB or CLI-095 for 24 h. Migration assays were then performed with PDGF-BB as a chemoattractant. p < 0.001 vs. LPS or LPS + DMSO; (C) Serum-starved VSMCs were seeded into the upper chambers of transwell plates and stimulated with TE buffer or LPS for 24 h. Migration assays were then performed with or without PDGF-BB as a chemoattractant. p < 0.001 for LPS vs. TE. p < 0.05 for LPS vs. LPS + PDGF; (D) Conditioned medium from VSMCs stimulated with TE buffer, LPS or P3C4 with or without plyB or CLI-095 for 24 h was collected and added to the lower chamber as a chemoattractant in the transwell migration assays. PDGF-BB was used as a positive control. p < 0.01 vs. LPS; (E) Serial dilutions of conditioned medium (CM) from VSMCs stimulated with LPS (LPS-CM) were prepared by diluting with TE-treated CM (TE-CM) and added to the lower chambers; VSMCs were plated onto top chambers of transwells. After 24 h incubation, VSMC migration was determined by the transwell assay. p < 0.01 vs. 0x/10x; (F) Checkerboard assays. CM from VSMCs treated with TE buffer (TE-CM) or LPS (LPS-CM) for 24 h was added either to the upper or lower chamber of the transwell plates for VSMC migration assays. p < 0.01 vs. TE-CM/TE-CM. Data in A–F represent mean ± SD of three experiments. Statistical analyses were performed using the one-way ANOVA.

Mentions: VSMC migration is a key event in atherosclerosis progression [9,10]. We wondered if LPS stimulates VSMC migration. LPS markedly increased VSMC migration as compared with that observed with endotoxin-free TE buffer (Figure 2A). We next used the anti-TLR4 neutralization assay to characterize the potential role of TLR4 in LPS-induced VSMC migration. Anti-TLR4, but not anti-TLR2 antibodies, reduced LPS-induced VSMC migration (Figure 2A). Additionally, LPS-mediated VSMC migration was suppressed by treatment with polymyxin B and CLI-095, while pam3CSK4-induced VSMC migration was unaffected by the two inhibitors (Figure 2B), suggesting that TLR4 is required for VSMC migration induced by LPS. Next, to gain mechanistic insights of LPS-induced migration, we used TE buffer or PDGF-BB as chemoattractant to assay for random and directed migration, respectively. The directed migration of TE-treated VSMCs was induced by PDGF-BB as a chemoattractant placed in the bottom chambers (Figure 2C), indicating that VSMCs can respond to chemoattractant stimulation. When TE buffer was placed in the bottom chambers, the random migration of LPS-treated VSMCs was 1.6-fold that of TE-treated cells (Figure 2C), while PDGF-BB placed in the bottom chambers further enhanced the migration of VSMCs treated with LPS (Figure 2C). Taken together, these results indicate that the enriched migration of VSMCs following LPS stimulation was due to augmented random and directed migration.


TLR4-Activated MAPK-IL-6 Axis Regulates Vascular Smooth Muscle Cell Function
Role of TLR4 in VSMC migration. (A) VSMCs (in the presence or absence of anti-TLR2 or anti-TLR4 antibodies (5 μg/mL)) were treated with TE buffer or LPS for 24 h; (B) Quiesced VSMCs were stimulated with TE buffer, LPS or P3C4 in the presence or absence of plyB or CLI-095 for 24 h. Migration assays were then performed with PDGF-BB as a chemoattractant. p < 0.001 vs. LPS or LPS + DMSO; (C) Serum-starved VSMCs were seeded into the upper chambers of transwell plates and stimulated with TE buffer or LPS for 24 h. Migration assays were then performed with or without PDGF-BB as a chemoattractant. p < 0.001 for LPS vs. TE. p < 0.05 for LPS vs. LPS + PDGF; (D) Conditioned medium from VSMCs stimulated with TE buffer, LPS or P3C4 with or without plyB or CLI-095 for 24 h was collected and added to the lower chamber as a chemoattractant in the transwell migration assays. PDGF-BB was used as a positive control. p < 0.01 vs. LPS; (E) Serial dilutions of conditioned medium (CM) from VSMCs stimulated with LPS (LPS-CM) were prepared by diluting with TE-treated CM (TE-CM) and added to the lower chambers; VSMCs were plated onto top chambers of transwells. After 24 h incubation, VSMC migration was determined by the transwell assay. p < 0.01 vs. 0x/10x; (F) Checkerboard assays. CM from VSMCs treated with TE buffer (TE-CM) or LPS (LPS-CM) for 24 h was added either to the upper or lower chamber of the transwell plates for VSMC migration assays. p < 0.01 vs. TE-CM/TE-CM. Data in A–F represent mean ± SD of three experiments. Statistical analyses were performed using the one-way ANOVA.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037674&req=5

ijms-17-01394-f002: Role of TLR4 in VSMC migration. (A) VSMCs (in the presence or absence of anti-TLR2 or anti-TLR4 antibodies (5 μg/mL)) were treated with TE buffer or LPS for 24 h; (B) Quiesced VSMCs were stimulated with TE buffer, LPS or P3C4 in the presence or absence of plyB or CLI-095 for 24 h. Migration assays were then performed with PDGF-BB as a chemoattractant. p < 0.001 vs. LPS or LPS + DMSO; (C) Serum-starved VSMCs were seeded into the upper chambers of transwell plates and stimulated with TE buffer or LPS for 24 h. Migration assays were then performed with or without PDGF-BB as a chemoattractant. p < 0.001 for LPS vs. TE. p < 0.05 for LPS vs. LPS + PDGF; (D) Conditioned medium from VSMCs stimulated with TE buffer, LPS or P3C4 with or without plyB or CLI-095 for 24 h was collected and added to the lower chamber as a chemoattractant in the transwell migration assays. PDGF-BB was used as a positive control. p < 0.01 vs. LPS; (E) Serial dilutions of conditioned medium (CM) from VSMCs stimulated with LPS (LPS-CM) were prepared by diluting with TE-treated CM (TE-CM) and added to the lower chambers; VSMCs were plated onto top chambers of transwells. After 24 h incubation, VSMC migration was determined by the transwell assay. p < 0.01 vs. 0x/10x; (F) Checkerboard assays. CM from VSMCs treated with TE buffer (TE-CM) or LPS (LPS-CM) for 24 h was added either to the upper or lower chamber of the transwell plates for VSMC migration assays. p < 0.01 vs. TE-CM/TE-CM. Data in A–F represent mean ± SD of three experiments. Statistical analyses were performed using the one-way ANOVA.
Mentions: VSMC migration is a key event in atherosclerosis progression [9,10]. We wondered if LPS stimulates VSMC migration. LPS markedly increased VSMC migration as compared with that observed with endotoxin-free TE buffer (Figure 2A). We next used the anti-TLR4 neutralization assay to characterize the potential role of TLR4 in LPS-induced VSMC migration. Anti-TLR4, but not anti-TLR2 antibodies, reduced LPS-induced VSMC migration (Figure 2A). Additionally, LPS-mediated VSMC migration was suppressed by treatment with polymyxin B and CLI-095, while pam3CSK4-induced VSMC migration was unaffected by the two inhibitors (Figure 2B), suggesting that TLR4 is required for VSMC migration induced by LPS. Next, to gain mechanistic insights of LPS-induced migration, we used TE buffer or PDGF-BB as chemoattractant to assay for random and directed migration, respectively. The directed migration of TE-treated VSMCs was induced by PDGF-BB as a chemoattractant placed in the bottom chambers (Figure 2C), indicating that VSMCs can respond to chemoattractant stimulation. When TE buffer was placed in the bottom chambers, the random migration of LPS-treated VSMCs was 1.6-fold that of TE-treated cells (Figure 2C), while PDGF-BB placed in the bottom chambers further enhanced the migration of VSMCs treated with LPS (Figure 2C). Taken together, these results indicate that the enriched migration of VSMCs following LPS stimulation was due to augmented random and directed 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