C-reactive protein triggers inflammatory responses partly via TLR4/IRF3/NF-κB
signaling pathway in rat vascular smooth muscle cells
Na Liua, Jun-Tian Liua,⁎, Yuan-Yuan Jib, Pei-Pei Lua
aDepartment of Pharmacology, Xi'an Jiaotong University School of Medicine, Xi'an 710061, China
bScientific Research Center, The Second Affiliated Hospital, Xi'an Jiaotong University School of Medicine, Xi'an 710004, China
a b s t r a c ta r t i c l ei n f o
Received 22 April 2010
Accepted 20 July 2010
Toll-like receptor 4
Vascular smooth muscle cell
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.
© 2010 Elsevier Inc. All rights reserved.
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
its acute complications (Lagrandet 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
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
Toll-likereceptors(TLRs), asprincipal sensorsof theinnate 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 athero-
sclerotic 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 produc-
tion 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.
Life Sciences 87 (2010) 367–374
⁎ Corresponding author. Postbox 58, Xi'an Jiaotong University School of Medicine,
76 West Yanta Road, Xi'an 710061, China. Tel.: +86 29 82655188; fax: +86 29
0024-3205/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
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Materials and methods
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
antibodywas provided by Abcam (Abcam, Cambridge,UK).Antibodies to
interferon regulatory factor 3 (IRF3) and IκBα were from Cell Signaling
Technology (Beverly, MA, USA). The small-interfering RNA (siRNA) for
TLR4 (NM_019178) and IRF3 (NM_001006969), siCONTROL Non-target-
ing siRNA (a negative control siRNA, NC siRNA) (D-001210-02-20), siGlo
RISC-free siRNA (D-001600-01) and DharmaFECT 2 transfection reagent
(T-2002-01) were obtained from Dharmacon (Lafayette, CO, USA).
For our experiments, sodium azide was removed from CRP with
biospin columns (Bio-Rad, Marnesla-Coquette, France).Endotoxin level
in CRP was tested using chromogenic Limulus amebocyte lysate assay
(WinKQCL, Bio-Whittaker, USA) (Ong et al. 2006). The average
endotoxin level in CRP was ~0.006 endotoxin unit/ml, which was not
sufficient to induce TLR4 expression. The purity of CRP was N99%, as
confirmed by 12% SDS-PAGE.
The investigation conforms to the Guide for the Care and Use of
Laboratory Animals published by the US National Institutes of Health
(NIH Publication No. 85-23, revised in 1996). Male Sprague–Dawley
(SD) rats were obtained from the Laboratory Animal Center of Xi'an
Jiaotong University School of Medicine. VSMCs were prepared from the
thoracic aorta of 2–3-month-old male SD rats by the explant method
(Ji et al. 2009). The cells were grown in DMEM supplemented with 10%
FBS in a 5% CO2atmosphere at 37 °C. Finally, identity of VSMCs was
Primer sequence used for PCR.
Gene Primer sequence Accession number
5′-TAA GGG AGA TCG GCT GGC TG-3′
5′-TGA TGG AGA GGT CCC CAA GG-3′
Fig. 1. CRP increases IL-6 generation and decreases PPARγ expression in VSMCs. (A) After incubation of the cells with the different concentrations of CRP or LPS for the indicated time,
IL-6 level in the supernatant was quantified by ELISA. (B and C) VSMCs were stimulated by the different concentrations of CRP or LPS for 9 h and then, PPARγ expression was
determined by quantitative real-time PCR and western blot. Data are expressed as mean±SD from three different experiments. *Pb0.05, **Pb0.01, ***Pb0.001 vs. control.
N. Liu et al. / Life Sciences 87 (2010) 367–374
estimated with the immunocytochemical staining for α-actin. The cells
at passage 3–6 were used for the experiments. After the cells were
grown to confluence, the medium was changed to serum-free medium
for an additional 24 h culture before the experiments.
ELISA for IL-6
VSMCs were incubated in 6-well plates, and stimulated with the
various indicated agents for the different time. After the treatment, the
culturesupernatant wascollected,and centrifuged at1500 rpm for5 min
to remove any particulate material. IL-6 level in the supernatant was
measured with ELISA kit according to the manufacturer's instructions.
RNA isolation and quantitative real-time PCR
Total RNA of VSMCs was extracted with TRIzol reagent kit
(Invitrogen, USA) and cDNA was synthesized using Revert Aid™ First
Strand cDNA synthesis kit (Fermentas, Germany). Real-time PCR was
performed with the SYBR® Premix Ex Taq™ II kit (Takara, Japan) on an
iCycler iQ™5 thermocycler (Bio-Rad, USA). The samples were run in
triplicate. Traditional PCR was performed according to the manufac-
turer's instructions. GAPDH was used as an internal control. Primers
were designed with Beacon Designer 4.0 (Premier Biosoft, USA)
(see Table 1 for the sequences).
Total lysates and nuclear extracts of the cells were extracted as
suggested in the previous study (Zambrano et al. 1998). Equal amount
(20 μg) of protein was subjected to 12% SDS-PAGE gel, and transferred
USA). The membranes were blocked with 5% nonfat dry milk in
Tris-buffered saline containing 0.1% Tween 20, and incubated with the
specific antibodies against PPARγ (1:200 dilution),TLR4 (1:200 dilution),
and GAPDH (1:400 dilution). Reagents for strengthening chemilumines-
cence (Pierce, USA) were applied to the blots, and the light signals were
Fig. 2. CRP induces TLR4 expression in VSMCs. (A and B) Following treatment of the cells with the different concentrations of CRP or LPS for 9 h, TLR4 expression was evaluated by
quantitative real-time PCR and western blot. (C and D) The cells were preincubated with polymyxin B (PMB) for 1 h prior to stimulation with CRP or LPS for 9 h and then, mRNA and
protein expression of TLR4 was observed. Data are expressed as mean±SD from three different experiments. *Pb0.05, **Pb0.01 vs. control;##Pb0.01 vs. LPS.
N. Liu et al. / Life Sciences 87 (2010) 367–374
detected by X-ray film. Optical density of the bands was scanned, and
quantified with Gel Doc 2000 (Bio-Rad, USA).
Forty to fifty percent of confluent VSMCs were transiently
2 transfection reagent following the manufacturer's protocols. The
transfection efficiency was determined by measuring uptake of siGlo
RISC-free siRNA 48 h after transfection, and approximately 70% of the
cells werepositiveforsiGlo RISC-freesiRNA. And the silencingeffects of
TLR4 and IRF3 were monitored by quantitative real-time PCR and
western blot 48 h after transfection. All assays were performed at least
48 h after RNA transfection.
Data were shown as mean±SD. Statistical evaluation was per-
formed by Student t test and one- or two-way ANOVA followed by
Dunnett test. Value of Pb0.05 was considered statistically significant.
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 concentration-dependently inhibited PPARγ expression in VSMCs
in mRNA and protein levels along with LPS (Fig. 1B and C).
CRP induces TLR4 expression in VSMCs
To examine whether CRP affects TLR4 expression in VSMCs, the
cells were incubated with the different concentrations of CRP or
100 ng/ml LPS for 9 h and then, TLR4 expression was determined with
quantitative real-time PCR and western blot. The analyses of mRNA
and protein displayed that CRP increased TLR4 expressionin VSMCs in
Fig. 3. CRP regulatesIL-6productionand PPARγ expression via TLR4 inVSMCs. (Aand B)After VSMCs were transfectedwith TLR4 siRNA (100 nM) orNC siRNA for48 h,TLR4 expression
was determined by quantitative real-time PCR and western blot. (C–E) After exposure of the transfected cells with TLR4 siRNA (100 nM) or NC siRNA to CRP for 9 h, IL-6 production and
PPARγ expression were measured. Data are expressed as mean±SD from three different experiments.$$Pb0.01,$$$Pb0.001 vs. NC siRNA.
N. Liu et al. / Life Sciences 87 (2010) 367–374
a concentration-dependent manner (Fig. 2A and B). To eliminate the
possibility for misinterpretation of our results by endotoxin contam-
ination in CRP, we also observed the effect of PMB, a well-known
pharmacological LPS scavenger, on CRP-induced up-regulation of
TLR4 in VSMCs. As expected, PMB (25 μg/ml) significantly reduced
LPS-induced TLR4 expression in mRNA and protein levels, but did not
affect CRP-induced TLR4 expression in VSMCs (Fig. 2C and D). The
results suggest that CRP is able to directly stimulate TLR4 expression
CRP regulates IL-6 secretion and PPARγ expression via TLR4 in VSMCs
To certify that TLR4 mediates CRP-induced IL-6 generation and
PPARγ down-regulation in VSMCs, the cells were stimulated with CRP
(25 μg/ml) for 9 h after transfection with TLR4 siRNA (100 nM) or NC
siRNA for 48 h. As seen from Fig. 3A and B, knockdown of TLR4 with
special siRNA obviously blocked mRNA and protein expression of TLR4,
the cells with NC siRNA did not affect the stimulatory effect of CRP on
IL-6 and the inhibitory effect of CRP on PPARγ. But, TLR4 siRNA
significantly antagonized CRP-induced generation of IL-6, and substan-
tially reversed the inhibitory effect of CRP on mRNA and protein
expression of PPARγ in VSMCs (Fig. 3C–E). These manifest that TLR4 is
involved in CRP-modulated IL-6 production and PPARγ expression in
IRF3 mediates CRP-induced NF-κB activation in VSMCs
important for the inflammatory responses in many cell types, we
evaluated the role of IRF3 in CRP-induced NF-κB activation in VSMCs.
The results found that siRNA to IRF3 effectively depressed IRF3
expression in mRNA and protein levels, and silencing efficiency of
IRF3 was about 66% (Fig. 4A and B). The result in Fig. 4C revealed that
CRP promoted nuclear translocation of NF-κB (p65) in VSMCs, and IRF3
knockdown effectively suppressed the CRP-induced nuclear transloca-
tion of NF-κB, thus verifying CRP-induced NF-κB activation in VSMCs
It is known that NF-κB activation requires the phosphorylation and
degradation of IκBα, a natural inhibitor of NF-κB (Ghosh and Karin
2002). Therefore, we further observed the effect of CRP on IκBα.
The result found that CRP augmented IκBα phosphorylation (Fig. 4D),
and IRF3 siRNA significantly blunted the effect, indicating that IκBα
phosphorylation is possibly necessary for IRF3-facilitated NF-κB
activation in CRP-stimulated VSMCs.
CRP modulates IL-6 generation and PPARγ expression via TLR4/IRF3/NF-κB
signal pathway in VSMCs
To explore the signal pathway responsible to CRP-modulated IL-6
and PPARγ, VSMCs were stimulated with CRP (25 μg/ml) for 9 h after
pretreatment with PMB (25 μg/ml) for 1 h, IRF3 siRNA (100 nM) for
Fig. 4. CRPactivatesNF-κBthroughIRF3inVSMCs.(AandB)AfterVSMCsweretransfectedwithIRF3siRNA (100 nM) orNCsiRNAfor48 h,IRF3 expression wasobservedbyquantitative
real-timePCRandwesternblot.(CandD)AfterthetransfectedcellswithIRF3siRNA(100 nM)orNCsiRNAwerestimulatedbyCRPfor9 h,p65-NF-κBandIκBαexpressionsweredetected
using western blot. Data are expressed as mean±SD from three different experiments.$$Pb0.01,$$$Pb0.001 vs. NC siRNA.
N. Liu et al. / Life Sciences 87 (2010) 367–374
48 h or PDTC (100 μΜ) for 2 h alone. As shown in Fig. 5, PMB was not
capable of changing IL-6 secretion and PPARγ expression in VSMCs in
response to CRP. However, IRF3 siRNA and PDTC obviously inhibited
CRP-stimulated IL-6 generation, and increased protein expression of
PPARγ in VSMCs compared with CRP alone. Considering the data from
Figs. 4 and 5 together, it is concluded that CRP regulates IL-6 secretion
and PPARγ expression through the TLR4/IRF3/NF-κB signal pathway
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 andunlikelytocontributedirectlyto cardiovasculardiseaseasa
pathogenic factor (Kaptoge et al. 2010). However, much data have
levels of CRP activate coagulation and inflammation (Bisoendial et al.
2005) and predict risk of myocardial infarction and thromboembolic
stroke in male volunteers (Ridker et al. 1998). These suggest that CRP is
not only a well-known marker of cardiovascular disease, but also
probably a mediator of atherosclerotic disease.
Previous studies showed that rat CRP does not activate rat
complement, whereas human CRP activates both rat and human
complement (de Beer et al. 1982). Administration of human CRP to rats
is an excellent model for the actions of endogenous human CRP (Griselli
and Herbert 1999; Gill et al. 2004). Inhibition of CRP in rats is a valid
therapeutic strategy that may prove informative about the physiological
and pathological roles of human CRP (Pepys et al. 2006). Furthermore,
highly purified CRP from human serum promotes ox-LDL uptake and
MMP production in Wistar rats. Because ox-LDL uptake by macrophages
instability, this study provides a novel in vivo evidence for the role of CRP
in atherosclerosis (Singh et al. 2008). Both recombinant human CRP and
highly purified CRPfrom humanserum causeNF-κB and AP-1 activations
which lead to generations of MCP-1, IL-6 and iNOS in rat VSMCs (Hattori
et al. 2003). Thus, CRP may play a role in atherogenesis by activating
Recent research indicates that CRP circulates as a pentamer (pCRP) in
plasma, and monomeric CRP (mCRP) is deposited in human aortic and
mCRP has a pro-inflammatory and pro-thrombotic property (Eisenhardt
(pCRP) or highly purified CRP (pCRP) from human serum trigger
inflammatory response by activating NF-κB signal transduction pathway
in endothelial cells (Verma et al. 2003) and VSMCs (Hattori et al. 2003),
and inducing monocyte activation and recruitment to endothelial cells
(Woollard et al. 2002). In addition, recombinant human CRP promotes
VSMCs migration and proliferation, neointimal formation, and increases
productions of ROS, collagen and elastin via AT1R in vitro and in vivo
(Wanget al. 2003).Thesedata demonstratethat pCRPfunctions asa pro-
inflammatory and pro-atherosclerotic factor.
Inflammation plays an important role throughout the development
of atherosclerosis. As a potential cardiovascular risk factor, CRP
participates in atherogenesis by inducing inflammatory responses. In
the study, recombinant human CRP was used to further probe its pro-
inflammatory action and mechanisms in rat VSMCs. IL-6 is a multifunc-
tional inflammatory cytokine, and implicated in pathogenesis of
atherosclerosis as well (Baggio et al. 1998; Wassmann et al. 2004). It is
reported that IL-6 is expressed in animal and human atherosclerotic
lesions (Seino et al. 1994; Sukovich et al. 1998; Ikeda et al. 1992), and
may induce CRP expression in human coronary artery smooth muscle
importantly, CRP is also able to stimulate IL-6 generation in human
saphenous vein endothelial cells, which partly contributes to its pro-
inflammatory and pro-atherosclerotic actions (Verma et al. 2002).
Although the existing evidence suggests the interaction between IL-6
at the concentrations used remarkably stimulated VSMCs to secret IL-6.
From this result, we are tempted to speculate that CRP may trigger the
vascular inflammatory responses involved in the initiation and
progression of atherosclerosis by stimulating IL-6 generation in VSMCs.
PPARs are a family of ligand-activated transcription factors, including
PPARα, PPARγ, and PPARδ (Ricote et al. 1998). PPARγ activation
negatively regulates the inflammatory responses through repressing
NF-κB, NFAT, STAT and AP-1 target genes in response to a variety of
Fig. 5. CRP modulates IL-6 generation and PPARγ expression by TLR4/IRF3/NF-κB signal
pathway in VSMCs. The cells were stimulated with CRP for 9 h after pretreatment with
PMBfor1 h,IRF3siRNAfor48 horPDTCfor2 halone.Then,IL-6level(A)wasmeasuredby
ELISA and protein expression of PPARγ (B) was evaluated by western blot. Data are
expressed as mean±SD from three different experiments. **Pb0.01, ***Pb0.001
vs. control;#Pb0.05,##Pb0.01 vs. CRP.
N. Liu et al. / Life Sciences 87 (2010) 367–374
inflammatory stimuli, including cytokines and TLR ligands (Duval et al.
2002; Marx et al. 2004; Welch et al. 2003; Ogawa et al. 2005). Although
many factors participate in the execution of PPARγ activity, very little is
known about the regulation of PPARγ activity by CRP. In this study, we
found that CRP remarkably down-regulated PPARγ expression in VSMCs,
demonstrating that CRP also induces inflammation in VSMCs through
decreasing anti-inflammatory activity of PPARγ.
Recent reports indicate that TLR4 participates in mediating the
inflammatory responses in vascular diseases (Frantz et al. 2007). TLR4
expression is markedly increased in human atherosclerotic lesions
(Edfeldt et al. 2002). TLR4 ligand may cause the expressions of several
inflammatory cytokines in VSMCs by activating NF-κB (Yang et al.
2005; Li et al. 2007). So, we inferred that TLR4 possibly mediated the
pro-inflammatory effect of CRP in VSMCs. The results from analyses of
mRNA and protein displayed that CRP indeed up-regulated TLR4
expression in VSMCs. Moreover, this effect is not related to endotoxin
contamination in CRP, as pretreatment of the cells with LPS scavenger
PMB prior to CRP stimulation did not affect the stimulatory effect of
CRP on TLR4 expression. Further investigation confirms that TLR4
mediatesCRP-induced IL-6 productionandPPARγdown-regulation in
VSMCs, as indicated by the evidence that TLR4 siRNA almostabolished
CRP-induced IL-6 generation, and substantially antagonized the
inhibitory effect of CRP on PPARγ expression.
It is well known that IRF3 is a downstream molecular of TLR4, and
cytokines (Navarro and David 1999; Yoneyama et al. 1998). TLR4/IRF3-
dependent signal pathway culminates in the delayed activation of NF-
genes (TakedaandAkira 2005;Hanetal. 2004;Rheeand Hwang2000).
In the experiment, CRP-induced IκBα phosphorylation and subsequent
NF-κB (p65) nuclear translocation were conspicuously suppressed in
IRF3 siRNA-transfected VSMCs, this providing a proof that IRF3 is a
critical component for CRP-induced NF-κB activation in VSMCs. Further
experiments exhibited that both IRF3 siRNA and NF-κB inhibitor PDTC
apparently depressed IL-6 production, and enhancedPPARγ expression
in CRP-stimulated VSMCs. These support that TLR4/IRF3/NF-κB signal
pathway mediates the pro-inflammatory effect of CRP in VSMCs.
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
This study was supported by a grant from National Natural Science
Foundation of China to Juntian Liu (No. 30772567).
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