Docosahexaenoic acid and eicosapentaenoic acid reduceC-reactive protein expression and STAT3 activation in IL-6-treated HepG2 cells

Tzu-Ming Wang • Shu-Chen Hsieh •

Jaw-Wen Chen • An-Na Chiang

Received: 27 August 2012 / Accepted: 24 January 2013 / Published online: 30 January 2013

� Springer Science+Business Media New York 2013

Abstract C-reactive protein (CRP), an acute phase pro-

tein in humans, is predominantly produced by hepatocytes

in response to interleukin-6 (IL-6). Several epidemiological

studies have reported that dietary intake of n-3 polyunsat-

urated fatty acids (n-3 PUFAs) is inversely associated with

serum CRP concentration. However, the molecular mech-

anism by which n-3 PUFAs reduce the serum CRP level in

HepG2 cells remains unclear. The aims of this study were

to examine the effect of the n-3 PUFAs, docosahexaenoic

acid (DHA), and eicosapentaenoic acid (EPA), on the

modulation of IL-6-induced CRP expression and to explore

its possible mechanisms. We demonstrated that DHA and

EPA inhibited IL-6-induced CRP protein and mRNA

expression, as well as reduced CRP promoter activity in

HepG2 cells. Knockdown of Signal Transducer and Acti-

vator of Transcription 3 (STAT3) and CCAAT box/

Enhancer-Binding Protein b (C/EBPb) by small interfering

RNAs (siRNAs) significantly decreased IL-6-induced CRP

promoter activity. Gel electrophoresis mobility shift assays

(EMSA) showed that pretreatment with DHA and EPA

decreased IL-6-induced STAT3 DNA binding activity but

not C/EBPb. By western blot analysis, DHA and EPA

inhibited IL-6-induced STAT3 phosphorylation but not

ERK1/2 or C/EBPb. The suppression of the phosphoryla-

tion of STAT3 by DHA and EPA was further verified by

immunofluorescence staining. Taken together, our results

demonstrate that DHA and EPA are able to reduce IL-6-

induced CRP expression in HepG2 cells via an inhibition

of STAT3 activation. This mechanism, which explains the

inhibitory effect of n-3 PUFAs on the CRP expression,

provides new insights into the beneficial anti-inflammatory

effect of n-3 PUFAs.

Keywords n-3 PUFAs � C-reactive protein � IL-6 �STAT3 � HepG2 cells

Introduction

Dietary supplementation of n-3 polyunsaturated fatty acids

(n-3 PUFAs) has been reported to alleviate the symptoms

of several chronic inflammatory diseases, such as athero-

sclerosis, rheumatoid arthritis, and inflammatory bowel

disease [1–4]. Docosahexaenoic acid (DHA, C22:6n-3) and

eicosapentaenoic acid (EPA, C20:5n-3) are two abundant

n-3 PUFAs that are mainly found in fish oil. Several studies

have shown that the protective effects of n-3 PUFAs are

mediated by diverse mechanisms [5, 6]. Inhibition of the

inflammatory mediators has been considered to be one of

the mechanisms that contribute to the beneficial effects of

n-3 PUFAs.

C-reactive protein (CRP) is an acute phase protein that

is synthesized predominantly by hepatocytes. Synthesis

and secretion of CRP is often increased during inflamma-

tory states and infection [7]. Elevation of plasma CRP

levels also appears to play a critical role in the pathogen-

esis of atherosclerosis [8, 9]. Increasing evidence suggests

that CRP may serve as a biomarker for a higher risk of

T.-M. Wang � A.-N. Chiang (&)

Institute of Biochemistry and Molecular Biology,

National Yang-Ming University, Taipei 112, Taiwan

e-mail: anchia@ym.edu.tw

S.-C. Hsieh

Institute of Food Science and Technology,

National Taiwan University, Taipei 112, Taiwan

J.-W. Chen

Division of Cardiology, Department of Medicine, Taipei

Veterans General Hospital, and Institute of Pharmacology,

National Yang-Ming University, Taipei 112, Taiwan

123

Mol Cell Biochem (2013) 377:97–106

DOI 10.1007/s11010-013-1574-1

cardiovascular disease [10, 11]. Animal studies reported

that CRP can accelerate the formation of atherosclerotic

lesions in apolipoprotein E (apoE)-deficient mice [12]. It

has also been found that increased levels of circulating

CRP are associated with enhanced CRP secretion by

hepatocytes [13]. Nevertheless, the regulation of CRP

expression in hepatocytes and the mechanisms underlying

this still remain poorly understand.

Several epidemiological studies have shown an inverse

relationship between n-3 PUFAs intake and circulating CRP

levels [14–19]. It is reasonable that n-3 PUFAs possess anti-

inflammatory properties that have the ability to inhibit CRP

production. However, the molecular mechanism by which

n-3 PUFAs regulate CRP expression remains unclear.

Studies have shown that the synthesis of CRP by hepatocytes

is mainly regulated by interleukin-6 (IL-6) and transcription

factor Signal Transducer and Activator of Transcription 3

(STAT3) [20–22]. In addition, CCAAT box/Enhancer-

Binding Protein b (C/EBPb) and C/EBPd also seem to play a

role in the regulation of CRP expression [23, 24]. We thus

hypothesized that n-3 PUFAs might repress IL-6-induced

CRP gene expression via the STAT3, C/EBPb, and/or

C/EBPd pathways.

The aim of this study was to investigate the signaling

pathway by which DHA and EPA regulate IL-6-induced

CRP production in HepG2 cells. The involvement of

transcription factors STAT3, C/EBPb, and/or C/EBPd in

CRP gene regulation by n-3 PUFAs is also evaluated. We

conclude that n-3 PUFAs regulate CRP expression by

repressing the binding of STAT3 to the STAT3 responsive

element in the promoter region of the CRP gene. However,

activation of C/EBPb is not affected by n-3 PUFAs.

Materials and methods

Materials

DHA, EPA, cis-linoleic acid (LA), and arachidonic acid

(AA) were obtained from Cayman Chemical Co (Ann

Arbor, MI, USA). Recombinant human IL-6 was purchased

from R&D Systems (Minneapolis, MN, USA). Antibodies

against phospho-STAT3 (Try705), STAT3, phospho-C/EBPb(Thr235), phospho-ERK1/2 (Thr202/Tyr204), and ERK1/2 were

obtained from Cell Signaling Technology (Beverly, MA,

USA). Antibody recognizing STAT3 (C-20) was purchased

from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-

bodies against a-tubulin and C/EBPb were purchased from

Abcam (Cambridge, UK). Antibody recognizing C/EBPdwas obtained from Rockland (Gilbertsville, PA, USA).

Antibodies against CRP, albumin, and all other chemi-

cals were purchased from Sigma-Aldrich (St. Louis, MO,

USA).

Cell culture

HepG2 cells were cultured in DMEM medium (Hyclone

Laboratories, Logan, UT, USA) supplemented with 10 % (v/v)

heat-inactivated FBS (PAA-Laboratories GmbH, Pasching,

Austria), 100 U/ml penicillin, 100 lg/ml streptomycin, 0.25

lg/ml amphotericin B, 0.3 mg/ml L-glutamine, 0.1 mM non-

essential amino acids (Grand Island, NY, USA), and incubated

at 37 �C in a humidified atmosphere containing 5 % CO2. The

stock solution of PUFAs (100 mM) was dissolved in 100 %

ethanol. For the experiments, the stock solution of PUFAs were

mixed with fatty acid-free bovine serum albumin at a 4:1 molar

ratio and adjusted to a final concentration of 100 lM in the

complete culture medium.

Western blot analysis

At the end of each treatment, HepG2 cells were lysed with lysis

buffer containing 1 % Tween 20, 0.1 % SDS, 20 mM Tris–

HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, phosphatase

inhibitor and complete protease inhibitor cocktail (Roche

Applied Science, Mannheim, Germany). Cell lysates were

collected after centrifugation at 12,0009g for 10 min at 4 �C.

The protein concentrations were determined using the Bradford

assay (Bio-Rad, Hercules, CA, USA). Equal amount of proteins

were subjected to 10 % SDS-PAGE and then transferred onto

polyvinylidene difluoride membrane (Millipore, Billerica, MA,

USA) after gel electrophoresis. The immunoblots were blocked

with 5 % non-fat milk for 1 h and then incubated with primary

antibodies overnight at 4 �C. After washing, the transferred

blots were incubated with horseradish peroxidase (HRP)-con-

jugated secondary antibodies for 1 h at 4 �C. The bound IgG

was visualized using an enhanced chemiluminescence detec-

tion kit system (PerkinElmer, Shelton, CT, USA), and quanti-

fied by ImageQuant 5.2 software (Healthcare Bio-Sciences,

Pennsylvania, USA). The blots were then stripped for further

probing with albumin or a-tubulin antibodies as an internal

control.

Enzyme-linked immunosorbent assay

The supernatants of the cell cultures were collected for

measurement of secreted CRP production using a sandwich

ELISA. Briefly, capture antibody was coated on a 96-well

plate at 4 �C overnight using coating buffer [100 mM

Na2CO3/NaHCO3 (pH 9.6)]. The plate was blocked with

1 % bovine serum albumin at room temperature for 1 h

and washed thrice with TBS. The culture supernatants

containing 20 lg of sample protein were added to each

well and incubated at 4 �C overnight. The plate was washed

and then incubated with secondary antibody at room

temperature for 2 h, followed by HRP-labeled goat anti-

mouse IgG for 30 min. After washing, the HRP substrate

98 Mol Cell Biochem (2013) 377:97–106

123

o-phenylenediamine dihydrochloride (1 mg/ml in 0.1 M

citrate buffer, pH 5.0 containing 0.001 % v/v of 30 %

H2O2) was added to each well and finally color develop-

ment after 5 min was measured at a wavelength of 405 nm

using a microplate reader (Multiskan Spectrum, Thermo

Fisher Scientific, Vantaa, Finland).

Plasmid construct, transfection, and luciferase assay

The human CRP promoter fragment from -300/?1 was

amplified using the primers 50-CCGACGCGTACCCA

GATGGCCACTCGTTTAATATGTTACC-30 and 50-CCT

AGATCTAGAGCTACCTCCTCCTGCCTGG-30 which

contain MluI and BglII restriction sites. The PCR products

were cloned into the luciferase reporter pGL3 basic vector

(Promega, Madison, WI, USA), and the DNA sequences

were verified by sequencing of the clones. HepG2 cells

were seeded at 1 9 105 cells/well in 24-well plates for 24 h

before transfection. For transient transfection, 1 lg of the

CRP reporter plasmid or pGL3 plasmid was transfected

along with 0.1 lg of the internal control cytomegalovirus-

b-galactosidase vector (pCMV-b-Gal) using Lipofetamine

2000TM reagent (Invitrogen, Carlsbad, CA, USA) according

to the manufacturer’s instructions. After 6 h of transfection,

PUFAs (100 lM) were added to the cells for 24 h before

stimulation with IL-6 (10 ng/ml) for another 24 h. Cells

were harvested and lysed with lysis buffer [70 mM

K2HPO4, 55 mM Tris–HCl (pH 7.8), 2.1 mM MgCl2,

0.7 mM dithiothreitol, 0.1 % NP-40 (v/v), and protease

inhibitor cocktail]. Cell extracts were collected after cen-

trifugation at 12,0009g for 10 min at 4 �C. Then, 30 ll of

the cell extract, 100 ll of luciferase assay reagent [43 mM

glycylglycine (pH 7.8), 22 mM MgSO4, 2.4 mM EDTA,

1 mM dithiothreitol, 0.4 mg/ml BSA, 7.4 mM ATP], and

20 ll of 0.5 mM luciferin substrate were added to each well

of the 96-well microtiter plate. The luciferase activity was

measured using a luminometer (Perkin Elmer, Turku, Fin-

land) and transfection efficiency was normalized against the

b-galactosidase activity. All values are expressed as fold

induction relative to basal activity.

RNA interference

Double-stranded small interfering RNAs (siRNAs) target-

ing STAT3, C/EBPb, and C/EBPd were obtained from the

National RNAi Core Facility at the Institute of Molecular

Biology (Academia Sinica, Taipei, Taiwan). Negative

control GFP siRNA was purchased from Qiagen (Chats-

worth, CA, USA). HepG2 cells were co-transfected with

1 lg of siRNA, 1 lg of CRP reporter plasmid, and 0.1 lg

of pCMV-b-Gal using Lipofetamine 2000TM reagent

for 24 h. Transfection mixtures were removed and fresh

complete DMEM medium was added to each well. Cells were

incubated in DMEM medium for 24 h and then stimulated

with IL-6 for another 24 h. Finally, the cells were lysed in lysis

buffer, and luciferase activity was measured as described

previously.

Reverse transcription-polymerase chain reaction (RT-

PCR) and real-time quantitative PCR

Total RNA was isolated from cells using the TRI Reagent

(Invitrogen, Carlsbad, CA, USA) according to the manufac-

turer’s protocol. A sample of 2 lg RNA was converted into

cDNA using the reverse transcriptase (Invitrogen, Carlsbad,

USA) with oligo-dT as the primer. The RT-PCR was per-

formed on a Peltier Thermal Cycler (Model PTC-200, MJ

Research, Inc., Waltham, MA, USA). The PCR program was

95 �C for 2 min followed by 32 cycles of 95 �C for 30 s, 60 �C

for 30 s, and 72 �C for 1 min; and then the final extension was

72 �C for 10 min. The sequences of the PCR primers were as

follow: CRP: forward, 50-CCTATGTATCCCTCAAAGCA-30;reverse, 50-CCCACAGTGTATCCCTCTT-30. Porphobilinogen

deaminase (PBGD): forward, 50-AGGATGGGCAACTG

TACC-30; reverse, 50-GTTTTGGCTCCTTTGCTCAG-30.Real-time quantitative PCR was performed with SYBR

green master mixture (Qiagen, Valencia, CA, USA) in a

LightCycler Carousel-Based System (Roche). Reaction

condition was 95 �C for 10 min, followed by 40 cycles of

95 �C for 20 s, 60 �C for 20 s, and 72 �C for 20 s. The

primers were used as follows: CRP: forward, 50-ACTTC

CTATGTATCCCTCAAAG-30; reverse, 50-CTCATTGTC

TTGTCTCTTGGT-30. Glyceraldehyde-3-phosphate dehy-

drogenase (GAPDH): forward, 50-GAAGGTGAAGGTCG

GAGTC-30; reverse, 50-GAAGATGGTGATGGGATTTC-30.Quantification of CRP mRNA was calculated by the Ct

method (ratio = 2 - (Ct(CRP) - Ct(GAPDH))) as descri-

bed previously by Patel et al. [25].

Immunofluorescence microscopy

HepG2 cells were treated with PUFAs (100 lM) for 24 h

before stimulation by IL-6 (10 ng/ml) for 30 min on poly

(L-lysine)-coated glass coverslips in 6-well plates. Then, cells

were fixed in 5 % paraformaldehyde for 15 min and perme-

abilized with 0.2 % Triton X-100 for 5 min. After washing,

coverslips were blocked with 5 % bovine serum albumin for 1 h

at 37 �C and then incubated with anti-phospho-STAT3 (Try705)

primary antibody (1:200) for 24 h at 4 �C. The coverslips were

washed and incubated with Alexa Fluor-594-conjugated sec-

ondary antibody (1:200) (Molecular Probes, Eugene, Oregon,

USA) for 1 h at room temperature. The cells were counterstained

with 40,6-diamidino-2-phenylindole (DAPI) to identify the

nuclei. Fluorescence was visualized using an Olympus FV1000

confocal laser scanning biological microscope. Colocalization of

Mol Cell Biochem (2013) 377:97–106 99

123

fluorescein and DAPI staining were performed by overlay

projection.

Electrophoretic mobility shift assay

Nuclear extracts were prepared from HepG2 cells in hypertonic

buffer containing 20 mM HEPES (pH 7.4), 10 mM KCl,

1 mM MgCl2, 0.5 % NP-40, 0.5 mM dithiothreitol supple-

mented with complete protease inhibitor cocktail. Following

centrifugation at 3,0009g for 5 min at 4 �C, the pellets were

resuspended in ice-cold extraction buffer containing 20 mM

HEPES (pH 7.4), 0.4 M NaCl, 1 mM MgCl2, 10 mM KCl,

0.5 mM dithiothreitol, 20 % glycerol supplemented with

complete protease inhibitor cocktail. The pellets were incu-

bated on ice for 30 min and cell debris was removed by

centrifugation at 12,0009g for 10 min at 4 �C. The oligo-

nucleotides containing the STAT3-binding site or C/EBPb-

binding site derived from the CRP promoter used in the

EMSAs were: 50-GATCTGCTTCCCGAACGT-30 and 50-TA

CATAGTGGCGCAAACTCCC-30, respectively. Comple-

mentary oligonucleotides were annealed and labeled with

[a-32P]dCTP by a fill-in reaction using the Klenow fragment

of DNA polymerase I. For each EMSA reactions, 5 lg of

nuclear extracts incubated in 20 mM HEPES (pH 7.4),

30 lM MgCl2, 50 mM NaCl, 5 mM DTT, 0.1 mg/ml BSA,

and 10 % glycerol, with the addition of 1 9 105 cpm of

oligonucleotide probes, in a final volume of 20 ll. After

incubation at room temperature for 30 min, samples were

resolved by electrophoresis on a 5 % non-denaturing poly-

acrylamide gel. For the competition experiments, unlabeled

annealed oligonucleotides were added at 5- to 20-fold molar

excess for each competition assay. For the supershift assay,

antibodies against STAT3 (C-20), phospho-C/EBPb (Thr235),

or C/EBPd were added to the reaction mixture for 1 h at room

temperature before addition of the labeled oligonucleotides.

Statistical analysis

Data are presented as mean ± SEM from at least three

independent experiments. Multiple comparisons were ana-

lyzed by one-way analysis of variance combined with the

post hoc Fisher’s least significance difference (LSD) test.

The value for P \ 0.05 was considered statistically signif-

icant between means of two groups.

Results

DHA and EPA inhibit IL-6-induced CRP protein

release in the medium of HepG2 cells culture

HepG2 cells were incubated with various concentrations

(25, 50, 100 lM) of PUFAs (DHA, EPA, LA, or AA) for

24 h and then stimulated with IL-6 (10 ng/ml) for an addi-

tional 24 h. The levels of CRP protein in the medium from

the cells were determined by western blot analysis as shown

in Fig. 1a–d. IL-6 significantly induced CRP release into the

culture medium compared to the control cells. In contrast,

pretreatment of HepG2 cells with DHA and EPA decreased

IL-6-induced CRP secretion in a dose-dependent manner,

whereas incubation with LA and AA (25–100 lM) had no

effect. We also determined the effect of the PUFAs on CRP

secretion by ELISA. As shown in Fig. 1e, IL-6-induced CRP

secretion to the culture medium was inhibited by DHA and

EPA, while treatment with LA did not affect CRP secretion.

DHA and EPA inhibit the expression of CRP mRNA

in HepG2 cells

We next investigated whether the inhibitory effect of DHA

and EPA on IL-6-induced CRP expression is at transcrip-

tional level. The mRNA level of CRP was examined by

semi-quantitative RT-PCR (Fig. 2a) and real-time PCR

(Fig. 2b). The results demonstrate that IL-6 induced CRP

mRNA expression, while DHA and EPA significantly

suppressed the induction. In contrast, LA did not affect

CRP mRNA expression. Based on a previous study, the

300-bp promoter fragment of the CRP gene, which con-

tains several regulatory elements, is known to play an

important role in IL-6-induced CRP promoter activity [26].

Based on this, HepG2 cells were transiently transfected

with a pGL3-basic luciferase vector containing the 300-bp

promoter fragment of the CRP gene (CRP-luc), or an

empty pGL3-basic vector (pGL3-luc). As shown in Fig. 2c,

IL-6 induced CRP promoter activity in the CRP-luc

transfected cells, and this induction was significantly

inhibited by DHA and EPA. However, LA did not display

any inhibitory effect. Taken together, these findings indi-

cate that the inhibitory effect of DHA and EPA on IL-6-

induced CRP expression occurs at the transcriptional level.

STAT3 and CEBP/b mediate IL-6-induced CRP

expression

To investigate the potential mechanisms underlying the

IL-6-induced CRP expression in HepG2 cells, we used

siRNAs to knock-down independently STAT3, C/EBPb,

and C/EBPd by transfecting individual siRNAs to HepG2

cells. The efficacy of siRNAs in gene knockdown is shown

in Fig. 3a. Inhibition of STAT3 and C/EBPb expression

significantly suppressed IL-6-induced CRP promoter activity.

However, silencing of C/EBPd expression had no suppressive

effect on IL-6-induced CRP promoter activity (Fig. 3b).

These findings provide evidence that STAT3 and C/EBPbplay essential roles in IL-6-enhanced CRP expression in

HepG2 cells.

100 Mol Cell Biochem (2013) 377:97–106

123

Effect of PUFAs on phosphorylation of STAT3,

C/EBPb, and ERK1/2

Next, we investigated the effect of PUFAs on the IL-6-

induced phosphorylation of STAT3, C/EBPb and ERK1/2

by western blot analysis. As shown in Fig. 4a, DHA and

EPA inhibited IL-6-induced STAT3 phosphorylation. LA

did not have suppressive effect on STAT3 phosphorylation.

In contrast, DHA, EPA, LA, and AA did not display any

inhibitory effect on C/EBPb phosphorylation (Fig. 4b).

Previous studies have reported that ERK1/2 is the upstream

kinase of C/EBPb [27, 28]. Our data show that DHA, EPA,

and LA also did not participate in regulation of the IL-6-

induced ERK1/2 phosphorylation (Fig. 4c). To confirm

the inhibitory effect of DHA and EPA on IL-6-induced

STAT3 phosphorylation, we examined the phosphorylated

STAT3 by immunofluorescence imaging confocal micros-

copy analysis. As shown in Fig. 5, DHA and EPA signif-

icantly suppressed the IL-6-induced nuclear translocation

of phosphorylated STAT3, while LA had no similar

inhibitory effect.

Effect of PUFAs on the binding of STAT3 and C/EBPbto the cis-elements of CRP promoter

Since we have shown that activation of both STAT3 and

C/EBPb are required for IL-6-induced CRP expression, we

therefore examined whether the binding of these tran-

scription factors to the cis-elements of CRP promoter

Fig. 1 Effect of polyunsaturated fatty acids on the IL-6-induced CRP

protein release. HepG2 cells were pretreated with A docosahexaenoic

acid (DHA); B ecosapentaenoic acid (EPA); C cis-linoleic acid (LA);

and D arachidonic acid (AA) at various concentrations as indicated

for 24 h and then stimulated with IL-6 (10 ng/ml) for another 24 h.

The CRP release in cell medium was determined by western blot

analysis. The effects of PUFAs at 100 lM on CRP production in the

medium were determined by ELISA (E). Data represent mean ± -

SEM from three independent experiments and the bars labeled with

different letters (a, b) indicate a significant difference (P \ 0.05)

Mol Cell Biochem (2013) 377:97–106 101

123

region is regulated by the different PUFAs. Treatment of

HepG2 cells with IL-6 results in activation of STAT3 as

indicated by a gel shift assay (Fig. 6a). DHA and EPA

almost abolished the IL-6-induced STAT3 activation,

whereas LA and AA did not have a similar inhibitory

effect. We next investigated the effect of PUFAs on IL-6-

induced activation of C/EBPb. There was a significant

increase in binding of C/EBPb to its responsive site in

HepG2 cells challenged with IL-6. However, DHA, EPA,

LA, and AA did not show any inhibitory effect on the

interaction between C/EBPb and its binding site (Fig. 6b).

Competition assays with unlabeled probes confirmed

specificity of the STAT3 and C/EBPb binding. To examine

whether the transcription factor binding of CRP promoter

region is regulated by DHA and EPA, supershift assays

were performed using the specific antibodies. As shown in

Fig. 6a, the STAT3-DNA complex was shifted by the

STAT antibody, while no supershifted band was observed

using antibodies against C/EBPb and C/EBPd as the con-

trol. The band intensities were diminished by using anti-

body against C/EBPb, though the supershifted band of

C/EBPb was not prominent. The control antibodies against

STAT3 and C/EBPd did not attenuate the band intensities

or elicit supershift bands (Fig. 6b, lanes 11 and 12). The

results from Fig. 6 indicate that DHA and EPA inhibit

IL-6-induced CRP gene expression through activation of

STAT3, but not via C/EBPb activation.

Discussion

Circulating CRP is produced primarily by hepatocytes and

has been recognized as a risk marker to predict cardio-

vascular related diseases, especially in the development of

atherosclerosis [8–11]. Elevated plasma CRP levels are

also associated with the increased inflammatory status [7].

IL-6 has been considered to play a role in the induction of

CRP expression [20, 21]. In the present study, DHA and

EPA are able to reduce the IL-6-induced CRP protein

expression, while LA and AA do not affect CRP protein

expression in HepG2 cells. Down-regulation of CRP expres-

sion by n-3 PUFAs may therefore contribute to the pre-

vention of cardiovascular disease beyond merely a risk

factor correlation. This study provides valuable insight that

should help atherosclerotic prevention from a dietary

perspective.

DHA and EPA inhibit IL-6-induced CRP mRNA

expression and promoter activity, suggesting that the reg-

ulation of DHA and EPA on CRP gene expression is at the

transcriptional level. Overexpressed STAT3 has been shown

to transactivate CRP-chloramphenicol acetyltransferase

reporter constructs in response to IL-6 challenge through

binding to the CRP-acute phase response element in Hep3B

cells [21]. As for the transcription mechanism, Nishikawa

et al. [22] reported that transcriptional complex formation of

c-Fos, STAT3, and hepatocyte NF-1a contributes to the

Fig. 2 DHA and EPA inhibit IL-6-induced CRP transcription.

HepG2 cells were treated for 24 h with PUFAs (100 lM) prior to

the addition of IL-6 (10 ng/ml) for another 4 h. A The expression of

CRP mRNA was determined by semi-quantitative RT-PCR and

B real-time quantitative RT-PCR. The expression of PBGD and

GAPDH mRNA were used as internal controls for the semi-

quantitative RT-PCR and for the real-time quantitative RT-PCR,

respectively. Data are represented as mean ± SEM of four indepen-

dent experiments. The bars labeled with different letters (a, b) are

significantly different (P \ 0.05). HepG2 cells were transiently

transfected with 1 lg of CRP reporter plasmid and 0.1 lg of

pCMV-b-Gal for 6 h. The transfected cells were incubated with

individual PUFAs (100 lM) for 24 h, and then stimulated with IL-6

(10 ng/ml) for another 24 h. C The luciferase activity was assayed

and normalized against b-galactosidase activity. Data are represented

as mean ± SEM of four independent experiments. The bars labeled

with different letters (a, b, c) are significantly different (P \ 0.05)

102 Mol Cell Biochem (2013) 377:97–106

123

synergistic induction of CRP gene expression by IL-1 plus

IL-6 stimulation in Hep3B cells. However, their studies do

not clarify whether n-3 PUFAs regulate CRP gene expres-

sion through those transcription factors. In the present study,

we found that knockdown of STAT3 by siRNA abolished

IL-6-induced CRP promoter activity. It clearly shows that

STAT3 plays an important role in regulating CRP gene

transcription in HepG2 cells. STAT3 is an acute-phase

response factor activated by phosphorylation at Tyr705,

such activation leads to IL-6 regulation of many acute-phase

protein genes [29]. It has established that IL-6 induces

intracellular signaling through activation of STAT dimer-

ization and nuclear translocation [30, 31]. The promoter

region of CRP gene also contains IL-6 responsive elements

which interact with the transcription factors of the C/EBP

family. C/EBPd/NF-IL-6b has been identified to be the

major IL-6 responsive elements in the nuclei of the Hep3B

cells [23]. NF-IL-6 is a member of the basic leucine zipper

family transcription factors which is involved in the regu-

lation of immune and inflammatory responsive genes [27].

Another report by Li and Goldman [24] demonstrate that

HNF-1a and HNF-3/Octamer-like factors synergistically

mediate the IL-6-induced CRP gene expression in a human

hepatoma (PLC/PRF/5) cell culture system. In contrast, we

found that knockdown of C/EBPb by siRNA suppressed IL-

6-induced CRP promoter activity, suggesting that C/EBPbplays a crucial role in regulating CRP gene transcription in

HepG2 cells. Nevertheless, our results show that knockdown

of C/EBPd by siRNA does not affect CRP gene transcription

in HepG2 cells. It is possible that the mechanism for CRP

gene regulation varies with different cell types. Based on our

data, IL-6 enhances CRP gene expression through activation

of STAT3 and C/EBPb, but only STAT3 is involved in the

n-3 PUFA suppression of IL-6-enhanced CRP gene

expression in HepG2 cells.

Various n-3 PUFAs can exert anti-inflammatory activity

through a variety of mechanisms, including modulation of

lipid rafts [32], alteration of redox signaling [33], and

activation of peroxisome proliferator-activated receptor-

gamma (PPAR-c) [34]. An increase in the amount of DHA

and EPA in membranes alters the pattern of production of

eicosanoids and resolvins [35, 36]. Growing evidence sug-

gests that some of the anti-inflammatory effects of n-3

PUFAs may result from suppression of gene expression via

actions on intracellular signaling pathways that, in turn,

affect the activation of transcription factors such as NF-jB

or STAT3 [37–41]. Phosphorylation of STAT3 at Tyr705 is

required for nuclear translocation and transcription activa-

tion [42]. Our results show that DHA and EPA abolished

IL-6-induced Tyr705 phosphorylation of STAT3. Phos-

phorylation of C/EBPb at Thr235 specifically by ERK2 is a

key determinant of its capacity for trans-activation of pro-

inflammatory genes [31]. Moreover, phosphorylation of

ERK1/2 at Thr202/Tyr204 is necessary for kinase activation

[43, 44]. Kaur et al. [45] reported that specific inhibition of

the ERK1/2 pathway abolished IL-6 and IL-1b-induced

CRP expression in Hep3B cells. Our findings show that

DHA and EPA do not affect Thr235 phosphorylation of

C/EBPb and Thr202/Tyr204 phosphorylation of ERK1/2.

In this study, we further confirm the effect of DHA and EPA

on STAT3 activation and CRP gene regulation by EMSA.

Based on our findings, it is clear that DHA and EPA inhibit

IL-6-induced CRP expression through a suppression of the

activation of STAT3 but not through a suppression of the

activation of C/EBPb.

In conclusion, the data from the present study suggest

that the marine n-3 PUFAs DHA and EPA have an inhibi-

tory effect on IL-6-stimulated CRP expression. The atten-

uation of CRP activation by n-3 PUFAs is mediated through

phosphorylation of STAT3, which in turn suppresses the

binding of STAT3 to its response element present in the

CRP promoter. Our results provide additional information

on the molecular mechanisms by which n-3 PUFAs affect

IL-6-induced CRP expression in HepG2 cells. These find-

ings provide a clear explanation for the reduced circulating

Fig. 3 Effect of siRNAs targeting STAT3, C/EBPb, and C/EBPdgenes on IL-6-induced CRP promoter activity. HepG2 cells were

cotransfected with 1 lg of CRP reporter plasmid, 0.1 lg of pCMV-b-

Gal and 1 lg of indicated siRNA expression vector for 24 h. After

transfection, cells were incubated in complete DMEM for another

24 h. A Knockdown of STAT3, C/EBPb, and C/EBPd was confirmed

by western blot analysis. a-Tubulin served as a control. Cytokine

stimulation was performed with IL-6 (10 ng/ml) for 24 h. B The

luciferase activity was measured and normalized against b-galacto-

sidase activity. Results represent mean ± SEM from four indepen-

dent experiments and the bars labeled with different letters (a, b) are

significantly different (P \ 0.05)

Mol Cell Biochem (2013) 377:97–106 103

123

Fig. 4 DHA and EPA inhibit IL-6-induced phosphorylation of

STAT3 but have no inhibitory effect on ERK1/2 and C/EBPbphosphorylation. HepG2 cells were incubated with PUFAs (100 lM)

for 24 h before the addition of IL-6 (10 ng/ml) for 30 min, then the

cell extracts were prepared and the phosphorylation levels of STAT3

(A), C/EBPb (B), and ERK1/2 (C) were measured by western blot

analysis. The intensity of protein bands normalized against a-tubulin,

and data are shown as the fold of the control. Data are presented

as mean ± SEM of at least three independent experiments and the

bars labeled with different letters (a, b) are significantly different

(P \ 0.05)

Fig. 5 DHA and EPA inhibit IL-6-induced nuclear translocation of

phosphorylated STAT3. HepG2 cells were pretreated with individual

PUFAs (100 lM) for 24 h, and then treated with IL-6 (10 ng/ml) for

another 30 min. Cells were fixed with 4 % paraformaldehyde and

reacted with the phospho-STAT3 (Try705) antibody. The nuclei were

stained with 40,6-diamidino-2-phenylindole (DAPI). Images were

obtained by using a confocal laser scanning biological microscope.

Scale bar 10 lm. The results are representative of three separate

experiments

104 Mol Cell Biochem (2013) 377:97–106

123

CRP levels in subjects treating with n-3 PUFAs and thus

may help to better understand their beneficial effects during

the development and progression of cardiovascular diseases

or progression of inflammatory disorders.

Acknowledgments This study was supported by grants from the

VGHUST Joint Research Program Tsou’s Foundation and Aim for

the Top University Plan, Ministry of Education (101AC-P504), Tai-

wan, ROC.

References

1. Mozaffarian D, Wu JHY (2011) Omega-3 fatty acids and car-

diovascular disease: effects on risk factors, molecular pathways,

and clinical events. J Am Coll Cardiol 58:2047–2067

2. De Caterina R (2011) n-3 fatty acids in cardiovascular disease.

N Engl J Med 364:2439–2450

3. Rontoyanni VG, Sfikakis PP, Kitas GD, Protogerou AD (2012)

Marine n-3 fatty acids for cardiovascular risk reduction and

disease control in rheumatoid arthritis: ‘‘kill two birds with one

stone’’? Curr Pharm Des 18:1531–1542

4. Bassaganya-Riera J, Hontecillas R (2010) Dietary conjugated

linoleic acid and n-3 polyunsaturated fatty acids in inflammatory

bowel disease. Curr Opin Clin Nutr Metab Care 13:569–573

5. Calder PC (2012) Mechanisms of action of (n-3) fatty acids.

J Nutr 142:592S–5929S

6. Adkins Y, Kelley DS (2010) Mechanisms underlying the car-

dioprotective effects of omega-3 polyunsaturated fatty acids.

J Nutr Biochem 21:781–792

7. Black S, Kushner I, Samols D (2004) C-reactive protein. J Biol

Chem 279:48487–48490

8. Momiyama Y, Ohmori R, Fayad ZA, Kihara T, Tanaka N, Kato

R, Taniguchi H, Nagata M, Nakamura H, Ohsuzu F (2010)

Associations between plasma C-reactive protein levels and the

severities of coronary and aortic atherosclerosis. J Atheroscler

Thromb 17:460–467

9. Bisoendial RJ, Boekholdt SM, Vergeer M, Stroes ES, Kastelein

JJ (2010) C-reactive protein is a mediator of cardiovascular dis-

ease. Eur Heart J 31:2087–2091

10. Buckley DI, Fu R, Freeman M, Rogers K, Helfand M (2009)

C-reactive protein as a risk factor for coronary heart disease: a

systematic review and meta-analyses for the U.S. Preventive

Services Task Force. Ann Intern Med 151:483–495

11. Abd TT, Eapen DJ, Bajpai A, Goyal A, Dollar A, Sperling L

(2011) The role of C-reactive protein as a risk predictor of cor-

onary atherosclerosis: implications from the JUPITER trial. Curr

Atheroscler Rep 13:154–161

12. Paul A, Ko KW, Li L, Yechoor V, McCrory MA, Szalai AJ, Chan

L (2004) C-reactive protein accelerates the progression of ath-

erosclerosis in apolipoprotein E-deficient mice. Circulation 109:

647–655

13. Pepys MB, Hirschfield GM (2003) C-reactive protein: a critical

update. J Clin Invest 111:1805–1812

14. Ferruci L, Cherubini A, Bandinelli S, Bartali B, Corsi A, Lau-

retani F, Martin A, Andres-Lacueva C, Senin U, Guralnik JM

(2006) Relationship of plasma polyunsaturated fatty acids to

circulating inflammatory markers. J Clin Endocrinol Metab

91:439–446

15. Tsitouras PD, Gucciardo F, Salbe AD, Heward C, Harman SM

(2008) High omega-3 fat intake improves insulin sensitivity and

reduces CRP and IL6, but does not affect other endocrine axes in

healthy older adults. Horm Metab Res 40:199–205

16. Micallef MA, Munro IA, Garg ML (2009) An inverse relation-

ship between plasma n-3 fatty acids and C-reactive protein in

healthy individuals. Eur J Clin Nutr 63:1154–1156

Fig. 6 DHA and EPA inhibit IL-6-induced activation of STAT3 but

have no significant inhibitory effect on IL-6-induced activation of

C/EBPb. HepG2 cells were pretreated with PUFAs (100 lM) for

24 h, and then stimulated with IL-6 (10 ng/ml) for 30 min. Nuclear

extracts were coincubated with 32P-labeled STAT3 oligonucleotide

(A) or C/EBPb oligonucleotide (B), and analyzed by electrophoretic

mobility shift assay. Lane 1 nuclear extracts from non-stimulated

cells; lane 2 nuclear extracts from HepG2 cells stimulated with IL-6;

lanes 3–6 nuclear extracts from PUFAs-treated cells; lanes 7–9nuclear extracts from IL-6-treated cells in the presence of unlabelled

STAT3 or C/EBPb probes; lanes 10–12 nuclear extracts were

incubated with specific antibodies against STAT3, phospho-C/EBPb,

and C/EBPd, respectively. All results are representative of three

independent experiments

Mol Cell Biochem (2013) 377:97–106 105

123

17. Farzaneh-Far R, Harris WS, Garg S, Na B, Whooley MA (2009)

Inverse association of erythrocyte n-3 fatty acid levels with

inflammatory biomarkers in patients with stable coronary artery

disease: the Heart and Soul Study. Atherosclerosis 205:538–543

18. He K, Liu K, Daviglus ML, Jenny NS, Mayer-Davis E, Jiang R,

Steffen L, Siscovick D, Tsai M, Herrington D (2009) Associa-

tions of dietary long-chain n-3 polyunsaturated fatty acids and

fish with biomarkers of inflammation and endothelial activation

(from the Multi-Ethnic Study of Atherosclerosis MESA). Am J

Cardiol 103:1238–1243

19. Kelley DS, Siegel D, Fedor DM, Adkins Y, Mackey BE (2009) DHA

supplementation decreases serum C-reactive protein and other markers

of inflammation in hypertriglyceridemic men. J Nutr 139:495–501

20. Castell JV, Gomez-Lechion MJ, David M, Fabra R, Trullenque R,

Heinrich PC (1990) Acute-phase response of human hepatocytes:

regulation of acute-phase protein synthesis by interleukin-6.

Hepatology 12:1179–1186

21. Zhang D, Sun M, Samols D, Kushner I (1996) STAT3 partici-

pates in transcriptional activation of the C-reactive protein gene

by interleukin-6. J Biol Chem 271:9503–9509

22. Nishikawa T, Hagihara K, Serada S, Isobe T, Matsumura A, Song

J, Tanaka T, Kawase I, Naka T, Yoshizaki K (2008) Transcrip-

tional complex formation of c-Fos, STAT3, and hepatocyte NF-1

alpha is essential for cytokine-driven C-reactive protein gene

expression. J Immunol 180:3492–3501

23. Ramji DP, Vitelli A, Tronche F, Cortese R, Ciliberto G (1993) The

two C/EBP isoforms, IL-6DBP/NF-IL6 and C/EBP delta/NF-IL6

beta, are induced by IL-6 to promote acute phase gene transcrip-

tion via different mechanisms. Nucleic Acids Res 21:289–294

24. Li SP, Goldman ND (1996) Regulation of human C-reactive

protein gene expression by two synergistic IL-6 responsive ele-

ments. Biochemistry 35:9060–9068

25. Patel DN, King CA, Bailey SR, Holt JW, Venkatachalam K,

Agrawal A, Valente AJ, Chandrasekar B (2007) Interleukin-17

stimulates C-reactive protein expression in hepatocytes and

smooth muscle cells via p38 MAPK and ERK1/2-dependent

NF-jB and C/EBPb activation. J Biol Chem 282:27229–27238

26. Li SP, Liu TY, Goldman ND (1990) cis-acting elements

responsible for interleukin-6 inducible C-reactive protein gene

expression. J Biol Chem 265:4136–4142

27. Nakajima T, Kinoshita S, Sasagawa T, Sasaki K, Naruto M,

Kishimoto T, Akira S (1993) Phosphorylation at threonine-235 by a

ras-dependent mitogen-activated protein kinase cascade is essential for

transcription factor NF-IL6. Proc Natl Acad Sci USA 90:2207–2211

28. Hanlon M, Sturgill TW, Sealy L (2001) ERK2- and p90 (Rsk2)-

dependent pathways regulate the CCAAT/enhancer-binding

protein-beta interaction with serum response factor. J Biol Chem

276:38449–38456

29. Heinrich PC, Behrmann I, Haan S, Hermanns HM, Muller-Newen

G, Schaper F (2003) Principles of interleukin (IL)-6-type cyto-

kine signalling and its regulation. Biochem J 374:1–20

30. Hoey T, Grusby MJ (1999) STATs as mediators of cytokine

induced responses. Adv Immunol 71:145–160

31. Sun H, Zhang Y, Gao P, Li Q, Sun Y, Zhang J, Xu C (2011) Adipo-

nectin reduces C-reactive protein expression and downregulates

STAT3 phosphorylation induced by IL-6 in HepG2 cells. Mol Cell

Biochem 347:183–189

32. McMurray DN, Bonilla DL, Chapkin RS (2011) n-3 Fatty acids

uniquely affect anti-microbial resistance and immune cell plasma

membrane organization. Chem Phys Lipids 164:626–635

33. Arab K, Rossary A, Flourie F, Tourneur Y, Steghens JP (2006)

Docosahexaenoic acid enhances the antioxidant response of

human fibroblasts by upregulating gamma-glutamyl-cysteinyl

ligase and glutathione reductase. Br J Nutr 95:18–26

34. Draper E, Reynolds CM, Canavan M, Mills KH, Loscher CE, Roche

HM (2011) Omega-3 fatty acids attenuate dendritic cell function via

NF-jB independent of PPARc. J Nutr Biochem 22:784–790

35. Norris PC, Dennis EA (2012) Omega-3 fatty acids cause dramatic

changes in TLR4 and purinergic eicosanoid signaling. Proc Natl

Acad Sci USA 109:8517–8522

36. Bannenberg G, Serhan CN (2010) Specialized pro-resolving lipid

mediators in the inflammatory response: an update. Biochim

Biophys Acta 1801:1260–1273

37. Wang TM, Chen CJ, Lee TS, Chao HY, Wu WH, Hsieh SC, Sheu

HH, Chiang AN (2011) Docosahexaenoic acid attenuates VCAM-1

expression and NF-jB activation in TNF-a-treated human aortic

endothelial cells. J Nutr Biochem 22:187–194

38. Rahman M, Kundu JK, Shin JW, Na HK, Surh YJ (2011) Doco-

sahexaenoic acid inhibits UVB-induced activation of NF-jB and

expression of COX-2 and NOX-4 in HR-1 hairless mouse skin by

blocking MSK1 signaling. PLoS ONE 6:e28065

39. Diaz Encarnacion MM, Warner GM, Cheng J, Gray CE, Nath

KA, Grande JP (2011) n-3 Fatty acids block TNF-a-stimulated

MCP-1 expression in rat mesangial cells. Am J Physiol Renal

Physiol 300:F1142–F1151

40. Wu MH, Tsai YT, Hua KT, Chang KC, Kuo ML, Lin MT (2012)

Eicosapentaenoic acid and docosahexaenoic acid inhibit macro-

phage-induced gastric cancer cell migration by attenuating the

expression of matrix metalloproteinase 10. J Nutr Biochem. doi:

10.1016/j.jnutbio.2011.09.004

41. Monk JM, Kim W, Callaway E, Turk HF, Foreman JE, Peters JM,

He W, Weeks B, Alaniz RC, McMurray DN, Chapkin RS (2012)

Immunomodulatory action of dietary fish oil and targeted dele-

tion of intestinal epithelial cell PPARd in inflammation-induced

colon carcinogenesis. Am J Physiol Gastrointest Liver Physiol

302:G153–G167

42. Reich NC (2009) STAT3 revs up the powerhouse. Sci Signal

2:pe61

43. Roux PP, Blenis J (2004) ERK and p38 MAPK-activated protein

kinases: a family of protein kinases with diverse biological

functions. Microbiol Mol Biol Rev 68:320–344

44. Giachini FR, Sullivan JC, Lima VV, Carneiro FS, Fortes ZB,

Pollock DM, Carvalho MH, Webb RC, Tostes RC (2010)

Extracellular signal-regulated kinase 1/2 activation, via down-

regulation of mitogen-activated protein kinase phosphatase 1,

mediates sex differences in desoxycorticosterone acetate-salt

hypertension vascular reactivity. Hypertension 55:172–179

45. Kaur G, Rao LV, Agrawal A, Pendurthi UR (2007) Effect of wine

phenolics on cytokine-induced C-reactive protein expression.

J Thromb Haemost 5:1309–1317

106 Mol Cell Biochem (2013) 377:97–106

123