Elsevier

Life Sciences

Volume 87, Issues 11–12, 11 September 2010, Pages 367-374
Life Sciences

C-reactive protein triggers inflammatory responses partly via TLR4/IRF3/NF-κB signaling pathway in rat vascular smooth muscle cells

https://doi.org/10.1016/j.lfs.2010.07.012Get rights and content

Abstract

Aims

C-reactive protein (CRP) plays an important role in the inflammatory process of atherosclerosis. Toll-like receptor 4 (TLR4) participates in atherogenesis by mediating the inflammatory responses. The aim of this experiment was to investigate the pro-inflammatory effects and mechanisms of CRP in rat vascular smooth muscle cells (VSMCs), especially focusing on the effects of CRP on IL-6 and peroxisome proliferator-activated receptor γ (PPARγ), and TLR4-dependent signal pathway.

Main methods

Rat VSMCs were cultured, and CRP was used as a stimulant for IL-6 and peroxisome proliferator-activated receptor γ (PPARγ). IL-6 level in the culture supernatant was measured by ELISA, and mRNA and protein expressions were assayed by quantitative real-time PCR and western blot, respectively. RNA interference was used to assess the roles of TLR4 and interferon regulatory factor 3 (IRF3) in the pro-inflammatory signal pathway of CRP.

Key findings

CRP stimulated IL-6 secretion, and inhibited mRNA and protein expression of PPARγ in VSMCs in a concentration-dependent manner. Additionally, CRP induced TLR4 expression, promoted nuclear translocation of NF-κB (p65), and augmented IκBα phosphorylation in VSMCs. Taken together, CRP induces the inflammatory responses through increasing IL-6 generation and reducing PPARγ expression in VSMCs, which is mediated by TLR4/IRF3/NF-κB signal pathway.

Significance

CRP is able to stimulate IL-6 production and to inhibit PPARγ expression in VSMCs via MyD88-independent TLR4 signaling pathway (TLR4/IRF3/NF-κB). These provide the novel evidence for the pro-inflammatory action of CRP involved in atherogenesis.

Introduction

Inflammation is recognized as a major contributor to the initiation and progression of atherosclerosis (Libby et al. 2002). Although it is uncertain whether C-reactive protein (CRP) is causally related to atherosclerosis as a marker of cardiovascular disease risk, increasing data show that CRP exerts a crucial role in vascular inflammation (Ridker et al., 2002, Speidl et al., 2002), and promotes atherosclerosis and its acute complications (Lagrand et al., 1999, Verma et al., 2006). Several lines of evidence demonstrate that CRP is not only an inflammatory marker but also an inflammatory mediator acting on vascular cells. Recent research suggests that vascular smooth muscle cells (VSMCs) may be a target of CRP for its pro-inflammatory and pro-atherogenic actions. In fact, CRP has the ability to trigger the inflammatory responses in VSMCs by activating NF-κB and inducing the production of pro-inflammatory cytokines and chemokines (Hattori et al. 2003). These indicate a strong association between CRP, inflammation and atherosclerosis.

Toll-like receptors (TLRs), as principal sensors of the innate immune system, provide a mechanistic link between inflammation and atherosclerosis (Edfeldt et al., 2002, Michelsen et al., 2004). Much data demonstrate that TLR4 is expressed at high levels in human atherosclerotic lesions, and regulates the inflammatory responses via MyD88- and TRIF-dependent signaling pathways (Beutler, 2004, Tobias and Curtiss, 2005, Faure et al., 2000). Actually, TLR4 activation under lipopolysaccharide (LPS) stimulation is very important for the production of inflammatory cytokines such as TNF-α and IL-6 in VSMCs (Medzhitov et al. 1997), and for the inflammatory signals associated with atherosclerosis (Yang et al., 2005, Li et al., 2007, Frantz et al., 2007). Therefore, we assume that TLR4 mediates the pro-inflammatory action of CRP to participate in pathogenesis of atherosclerosis.

Although roles of CRP in atherogenesis have been implicated, little information is available regarding its pro-inflammatory mechanisms in VSMCs. The aim of this experiment was to investigate the effects of CRP on IL-6 and peroxisome proliferator-activated receptor γ (PPARγ), and TLR4-related signal pathway in rat VSMCs.

Section snippets

Reagents

Recombinant human CRP and polymyxin B (PMB) sulfate were purchased from Calbiochem (San Diego, CA, USA). LPS from Escherichia coli 0111:B4 and pyrrolidine dithiocarbamate (PDTC) were provided by Sigma (St Louis, MO, USA). Rat IL-6 ELISA kit was ordered from Bender (Bender MedSystems, CA, USA). Antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TLR4 and NF-κB (p65) were bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-PPARγ antibody was provided by Abcam (Abcam,

CRP increases IL-6 production and decreases PPARγ expression in VSMCs

To observe if CRP modulates IL-6 generation and PPARγ expression in VSMCs, the cells were exposed to the different concentrations of CRP or 100 ng/ml LPS for the indicated time. The result from Fig. 1A showed that the unstimulated VSMCs exhibited a low IL-6 level. However, LPS increased IL-6 secretion in a time-dependent manner in VSMCs. Similarly, CRP at 10, 25 and 50 μg/ml also potently stimulated VSMCs to release IL-6 in concentration- and time-dependent ways. In addition, CRP

Discussion

It is known that higher circulating CRP levels are associated with coronary heart disease incidence and mortality rates in prospective studies. But, the role of CRP in cardiovascular disease risk remains controversial. A recent extensive meta-analysis finds that CRP is a marker and unlikely to contribute directly to cardiovascular disease as a pathogenic factor (Kaptoge et al. 2010). However, much data have demonstrated that CRP itself is indeed a promoter of atherosclerosis and its acute

Conclusion

The study demonstrates that CRP is able to stimulate IL-6 production and to inhibit PPARγ expression in VSMCs via MyD88-independent TLR4 signaling pathway (TLR4/IRF3/NF-κB). These provide the novel evidence for the pro-inflammatory action of CRP involved in atherogenesis.

Conflict of interest statement

None.

Acknowledgement

This study was supported by a grant from National Natural Science Foundation of China to Juntian Liu (No. 30772567).

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