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

Activation of TLR4 signaling in vascular smooth muscle cells (VSMCs) induces IL-6 production. (A–F) IL-6 level in culture medium was measured using enzyme-linked immunosorbent assay (ELISA); (A) Serum-starved VSMCs were stimulated with various TLR ligands: 100 ng/mL lipopolysaccharide (LPS) (TLR4), 1 μg/mL pam3CSK4 (TLR2), 1 μg/mL Poly (I:C) (TLR3), 1 μg/mL CL087 (TLR7), or 1 μM CpG ODN (TLR9) for 24 h. After pretreating VSMCs with different concentrations of (B–D) polymyxin B (plyB; LPS inhibitor) or (E) CLI-095 (TLR4 inhibitor) for 30 min, the cells were stimulated with LPS (100 ng/mL) for 24 h (B–E) or 36 h (D); p < 0.001 for LPS + DMSO vs. LPS + ply or LPS + CLI-095. (F) LPS-treated VSMCs were further treated with anti-IgG, antibodies (5 μg/mL) against TLR2 or TLR4 for 24 h. p < 0.001 vs. anti-IgG. Data in A–F represent mean ± SD of three experiments. Statistical analyses were performed using the one-way analysis of variance (ANOVA); (G) VSMCs were pretreated with 5 μg/mL plyB or with different amounts of plyB for 30 min and then stimulated with TE buffer or LPS for the indicated times (left panel) or for 30 min (right panel). Cell lysates were subjected to Western blotting with antibodies against p38 mitogen-activated protein kinase (MAPK), phospho-p38 MAPK, ERK1/2, phospho-ERK1/2, Akt, phospho-Akt, JNK1/2, phospho-JNK1/2, or β-actin. A representative of three independent experiments is shown.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-17-01394-f001: Activation of TLR4 signaling in vascular smooth muscle cells (VSMCs) induces IL-6 production. (A–F) IL-6 level in culture medium was measured using enzyme-linked immunosorbent assay (ELISA); (A) Serum-starved VSMCs were stimulated with various TLR ligands: 100 ng/mL lipopolysaccharide (LPS) (TLR4), 1 μg/mL pam3CSK4 (TLR2), 1 μg/mL Poly (I:C) (TLR3), 1 μg/mL CL087 (TLR7), or 1 μM CpG ODN (TLR9) for 24 h. After pretreating VSMCs with different concentrations of (B–D) polymyxin B (plyB; LPS inhibitor) or (E) CLI-095 (TLR4 inhibitor) for 30 min, the cells were stimulated with LPS (100 ng/mL) for 24 h (B–E) or 36 h (D); p < 0.001 for LPS + DMSO vs. LPS + ply or LPS + CLI-095. (F) LPS-treated VSMCs were further treated with anti-IgG, antibodies (5 μg/mL) against TLR2 or TLR4 for 24 h. p < 0.001 vs. anti-IgG. Data in A–F represent mean ± SD of three experiments. Statistical analyses were performed using the one-way analysis of variance (ANOVA); (G) VSMCs were pretreated with 5 μg/mL plyB or with different amounts of plyB for 30 min and then stimulated with TE buffer or LPS for the indicated times (left panel) or for 30 min (right panel). Cell lysates were subjected to Western blotting with antibodies against p38 mitogen-activated protein kinase (MAPK), phospho-p38 MAPK, ERK1/2, phospho-ERK1/2, Akt, phospho-Akt, JNK1/2, phospho-JNK1/2, or β-actin. A representative of three independent experiments is shown.

Mentions: To investigate the role of TLR4 in the production of pro-inflammatory cytokines by VSMCs, quiesced VSMCs were stimulated with the TLR4 ligand, LPS, for 24 h, and the levels of pro-inflammatory cytokines (IL-6, IL-10, IL-12, and TNF-α) in the culture medium were measured. LPS substantially stimulated IL-6 production (Figure 1A) but did not stimulate the production of TNF-α, IL-10 or IL-12, (data not shown). As with LPS, the ligands of TLR2 (pam3CSK4) and TLR3 (Poly(I:C)) significantly induced IL-6 generation, while the agonists for TLR7/8 (CL087) and TLR9 (CpG ODN) did not (Figure 1A).


TLR4-Activated MAPK-IL-6 Axis Regulates Vascular Smooth Muscle Cell Function
Activation of TLR4 signaling in vascular smooth muscle cells (VSMCs) induces IL-6 production. (A–F) IL-6 level in culture medium was measured using enzyme-linked immunosorbent assay (ELISA); (A) Serum-starved VSMCs were stimulated with various TLR ligands: 100 ng/mL lipopolysaccharide (LPS) (TLR4), 1 μg/mL pam3CSK4 (TLR2), 1 μg/mL Poly (I:C) (TLR3), 1 μg/mL CL087 (TLR7), or 1 μM CpG ODN (TLR9) for 24 h. After pretreating VSMCs with different concentrations of (B–D) polymyxin B (plyB; LPS inhibitor) or (E) CLI-095 (TLR4 inhibitor) for 30 min, the cells were stimulated with LPS (100 ng/mL) for 24 h (B–E) or 36 h (D); p < 0.001 for LPS + DMSO vs. LPS + ply or LPS + CLI-095. (F) LPS-treated VSMCs were further treated with anti-IgG, antibodies (5 μg/mL) against TLR2 or TLR4 for 24 h. p < 0.001 vs. anti-IgG. Data in A–F represent mean ± SD of three experiments. Statistical analyses were performed using the one-way analysis of variance (ANOVA); (G) VSMCs were pretreated with 5 μg/mL plyB or with different amounts of plyB for 30 min and then stimulated with TE buffer or LPS for the indicated times (left panel) or for 30 min (right panel). Cell lysates were subjected to Western blotting with antibodies against p38 mitogen-activated protein kinase (MAPK), phospho-p38 MAPK, ERK1/2, phospho-ERK1/2, Akt, phospho-Akt, JNK1/2, phospho-JNK1/2, or β-actin. A representative of three independent experiments is shown.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-17-01394-f001: Activation of TLR4 signaling in vascular smooth muscle cells (VSMCs) induces IL-6 production. (A–F) IL-6 level in culture medium was measured using enzyme-linked immunosorbent assay (ELISA); (A) Serum-starved VSMCs were stimulated with various TLR ligands: 100 ng/mL lipopolysaccharide (LPS) (TLR4), 1 μg/mL pam3CSK4 (TLR2), 1 μg/mL Poly (I:C) (TLR3), 1 μg/mL CL087 (TLR7), or 1 μM CpG ODN (TLR9) for 24 h. After pretreating VSMCs with different concentrations of (B–D) polymyxin B (plyB; LPS inhibitor) or (E) CLI-095 (TLR4 inhibitor) for 30 min, the cells were stimulated with LPS (100 ng/mL) for 24 h (B–E) or 36 h (D); p < 0.001 for LPS + DMSO vs. LPS + ply or LPS + CLI-095. (F) LPS-treated VSMCs were further treated with anti-IgG, antibodies (5 μg/mL) against TLR2 or TLR4 for 24 h. p < 0.001 vs. anti-IgG. Data in A–F represent mean ± SD of three experiments. Statistical analyses were performed using the one-way analysis of variance (ANOVA); (G) VSMCs were pretreated with 5 μg/mL plyB or with different amounts of plyB for 30 min and then stimulated with TE buffer or LPS for the indicated times (left panel) or for 30 min (right panel). Cell lysates were subjected to Western blotting with antibodies against p38 mitogen-activated protein kinase (MAPK), phospho-p38 MAPK, ERK1/2, phospho-ERK1/2, Akt, phospho-Akt, JNK1/2, phospho-JNK1/2, or β-actin. A representative of three independent experiments is shown.
Mentions: To investigate the role of TLR4 in the production of pro-inflammatory cytokines by VSMCs, quiesced VSMCs were stimulated with the TLR4 ligand, LPS, for 24 h, and the levels of pro-inflammatory cytokines (IL-6, IL-10, IL-12, and TNF-α) in the culture medium were measured. LPS substantially stimulated IL-6 production (Figure 1A) but did not stimulate the production of TNF-α, IL-10 or IL-12, (data not shown). As with LPS, the ligands of TLR2 (pam3CSK4) and TLR3 (Poly(I:C)) significantly induced IL-6 generation, while the agonists for TLR7/8 (CL087) and TLR9 (CpG ODN) did not (Figure 1A).

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