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The Journal of Immunology, 2008, 180, 3492 -3501
Copyright © 2008 by The American Association of Immunologists, Inc.
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Transcriptional Complex Formation of c-Fos, STAT3, and Hepatocyte NF-1{alpha} Is Essential for Cytokine-Driven C-Reactive Protein Gene Expression1

Teppei Nishikawa2,*, Keisuke Hagihara2,{dagger}, Satoshi Serada§, Tomoyasu Isobe*, Atsumi Matsumura{ddagger}, Jian Song*, Toshio Tanaka{dagger}, Ichiro Kawase{dagger}, Tetsuji Naka§ and Kazuyuki Yoshizaki3,*,{ddagger}

* Health Care Center, Osaka University, {dagger} Department of Respiratory Medicine, Allergy, Rheumatic Diseases, Osaka University Graduate School of Medicine, {ddagger} School of Medicine, Osaka University Graduate School of Medicine, and § Laboratory for Immune Signal, National Institute of Biomedical Innovation, Osaka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
C-reactive protein (CRP) is a sensitive marker and mediator of inflammation, whereas IL-6 blocking therapy can normalize serum levels of CRP in chronic inflammatory diseases. We investigated the precise synergistic induction mechanism of CRP gene expression by IL-1 and IL-6 in Hep3B cells. In the early induction phase, IL-1 inhibited IL-6-mediated CRP gene expression, and NF-{kappa}B p65 inhibited the luciferase activity of pGL3-CRP by IL-1 plus IL-6 even in the presence of overexpressed STAT3. In the late induction phase, we focused on JNK and p38 activated by IL-1. SP600125 reduced the expression of the CRP gene induced by IL-1 plus IL-6. Unexpectedly, overexpression of c-Fos dramatically enhanced the luciferase activity by IL-1 and IL-6 even though the CRP gene has no AP-1 response element (RE) in its promoter. The augmentative effect of c-Fos required the presence of STAT3 and 3'-hepatocyte NF-1 (HNF-1) RE, which were eliminated by dominant negative STAT3 and HNF-1{alpha}, respectively. SB203580 inhibited the phosphorylation of c-Fos enhanced by IL-1 plus IL-6, and diminished expression of the CRP gene. Immunoprecipitation, Western blot analysis, the Supershift assay using a CRP oligonucleotide containing STAT3 and 3'-HNF-1 RE, and the chromatin immunoprecipitation assay demonstrated that c-Fos/STAT3/HNF-1{alpha} forms a complex on the CRP gene promoter. Because human fetus liver cells failed to express c-Fos/STAT3/HNF-1{alpha} showed no CRP production, transcriptional complex formation of c-Fos/STAT3/HNF-1{alpha} is essential for the synergistic induction of CRP gene expression by IL-1 plus IL-6. Our findings fully explain the clinical results of IL-6 blocking therapy and are expected to contribute to the development of a therapeutic strategy for chronic inflammatory diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The C-reactive protein (CRP)4 is a major acute phase protein in humans and increases under acute or chronic inflammatory conditions, such as bacterial infection, trauma, myocardial infarction, rheumatoid arthritis, Castleman’s disease, and other conditions (1, 2). CRP has several effects on immune function. CRP activates the classical complement pathway and binds to phagocytic cells through Fc{gamma}RI and Fc{gamma}RII. CRP is protective in mouse models of bacterial infection by various species (1, 2). Recent research supports the notion that atherosclerosis is, in part, an inflammatory disease. It has been reported that activated immune cells in atherosclerotic plaque release proinflammatory cytokines, which then produce acute-phase reactants (3, 4). Various clinical findings have shown that minor elevation of CRP and serum amyloid A (SAA) is predictive of cardiovascular disease (1, 2, 3, 4, 5, 6). Recently, CRP has been reported to have various effects on proinflammatory development by accelerating aortic atherosclerosis in Apo-E-deficient mice (7), down-regulating endothelial NO synthase in mice (8), and promoting monocyte-platelet aggregation (9).

In addition, the serum level of CRP has been reported to be associated with aging, with CRP levels in elderly adults being significantly higher than in young adults (10, 11), whereas the cord serum level of CRP is significantly lower than that in normal healthy adults (11, 12). CRP is known not to be a sensitive marker for neonatal infection even though IL-1R antagonist and IL-6 are elevated in neonatal sepsis (12, 13, 14). Furthermore, it is well known that IL-6 and IL-1 have a synergistic effect on the induction of CRP and SAA gene expression (1, 15), but the mechanism underlying these increases and inductions remains unclear.

As for the transcription mechanism of CRP, some studies have discussed the possibility that NF-{kappa}B p50 may be involved in the CRP gene promoter via the nonconsensus {kappa}B site overlapping the proximal C/EBP binding site (16, 17, 18, 19). However, these experimental results do not fully explain our clinical findings of IL-6 blockade, anti-IL-6R Ab, normalized serum levels of CRP, and SAA in chronic inflammatory diseases (20, 21). Thus, NF-{kappa}B p50 seems to be important but not sufficient for CRP induction.

It has been further reported that STAT3 and C/EBPβ, which are the main transcription factors activated by IL-6, induce CRP gene expression via binding to their consensus sequence on the CRP gene promoter (22, 23). Hepatocyte NF-1 (HNF-1), the liver-enriched homeodomain-containing transcription factor, cytokine-independently binds to its consensus sequence and regulates the expression of the CRP gene as well as that of other hepatic genes (22, 24). However, the synergistic induction mechanism of CRP gene expression by IL-1 plus IL-6 has not been identified because NF-{kappa}B and AP-1, the latter being the main transcription factor activated by the IL-1 signaling pathway, have no consensus sequence in the CRP gene promoter.

We previously reported on the induction mechanism of SAA gene expression based on the findings of our clinical trials. Our results demonstrated that IL-6 plays a critical role in the synergistic induction of the SAA gene by IL-6, IL-1, and TNF-{alpha} (25), and that the formation of a complex comprising STAT3, NF-{kappa}B p65, and p300 is essential for the synergistic induction of the SAA gene by IL-1 plus IL-6 stimulation, even though the SAA gene promoter lacks a consensus STAT3 response element (RE) (26).

In this study, we investigated how transcriptional complex formation contributes to the synergistic induction of CRP gene expression by IL-1 plus IL-6. Furthermore, we investigated a role for the AP-1 transcription factor in the regulation of CRP gene expression because it is activated by IL-1 and has been shown to regulate the basal and inducible transcription activity of several proinflammatory genes via binding to the AP-1 site, also known as the TPA-RE (5'-TGAG/CTCA-3'). Interaction between AP-1 and other transcription factors has also been reported (27, 28). For example, the interaction between STAT3 and c-Jun was found to be necessary for the maximal activation of the {alpha}2-macroglobulin promoter (29). Leu et al. (30) reported that HNF-1{alpha} coordinates the interaction of STAT3 and c-Fos, which in turn induces the synergistic transcriptional regulation of the insulin-like growth factor binding protein-1 promoter. We were able to confirm that CRP production in human fetal liver cells was much lower than that in the human hepatoma Hep3B cells due to the low expression level of c-Fos, STAT3, or HNF-1{alpha} and that these differences were normalized by overexpression of c-Fos, STAT3, and HNF-1{alpha}. In this study, we were able to confirm that transcriptional complex formation of c-Fos, STAT3, and HNF-1{alpha} is essential for cytokine-driven CRP gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents and cell culture

Recombinant human IL-6 was provided by Ajinomoto and used at a final concentration of 10 ng/ml, whereas recombinant human IL-1β was purchased from BioSource International and used at a concentration of 10 ng/ml. SP600125 and SB203580 were purchased from Calbiochem, and the human hepatoma cell line Hep3B was donated by the Cell Resource Center for Biomedical Research (Tohoku University, Sendai, Japan). The fetal liver cell line WRL-68 was purchased from American Type Culture Collection (31), whereas primary fetal liver cells were obtained from Dainippon-Sumitomo Pharmaceutical (32, 33). These hepatocytes were grown in DMEM (except for primary fetal liver cells grown in CS-C medium) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, and 50 U/ml penicillin-streptomycin.

Plasmid construction

The 942 bp upstream region of human CRP and 18 bp of exon 1 were amplified with the MluI and BglII restriction sites introduced at the 5' and 3' ends, respectively. The 5' primer was 5'-TCGACGCGTGGAAAGAGTAGAATCAGATTATCCTGAC-3', and the 3' primer was 5'-TCCTAGATCTCTTGCCTTAGAGCTACCTCC-3'. The PCR product was then inserted into a pGL3-Basic vector (Promega). 5'-HNF-1 RE (–170/–58), STAT3 RE (–112/–105), and 3'-HNF-1 RE (–73/–61) of the pGL3-CRP (–942/+18) were deleted or mutated with the QuikChange Site-Directed Mutagenesis kit (Stratagene), according to the manufacturer’s instructions. pcDNA3.1 NF-{kappa}B p65 and pcDNA3.1 NF-{kappa}B p50 were constructed by means of PCR-cloning using the Marathon cDNA amplification kit (Clontech Laboratories) and verified by DNA sequencing. pEF-BOS wild-type (wt) STAT3 and pEF-BOS dominant negative STAT3 Y705F were provided by Dr. S. Akira (Research Institute for Microbial Diseases, Osaka University, Osaka, Japan) (26), pRSV-c-Jun, and pEF-Flag-c-Fos by Dr. K. Nakajima (Osaka City University, Osaka, Japan) (34), and pcDNA3.1 HNF-1{alpha} and pcDNA3.1 dominant negative HNF-1{alpha} by Dr. K. Yamagata (Graduate School of Medicine, Osaka University, Osaka, Japan) (35).

Transfection and luciferase assay

Hepatocyte cells were seeded at 2 x 105 cells/well in 6-well plates, and after 24 h the cells were transfected with 1 µg of plasmid DNA by means of Fugene 6 (Roche Molecular Biochemicals) according to the manufacturer’s instructions. The cells were then stimulated with various combinations of cytokines 48 h after transfection, lysed in passive lysate buffer, and assayed with the Dual-Luciferase Reporter Assay System (Promega) as specified by the manufacturer. Luciferase activity was normalized with the activity of Renilla luciferase. Assays were conducted in triplicate, and the experiments were repeated at least three times.

Real-time quantitative RT-PCR

Hepatocyte cells were seeded at 5 x 105 cells/well in 6-well plates and stimulated with various combinations of cytokines for the following 72 h in a subconfluent state. Total RNA was isolated from the cells by means of QiaShredder spin columns and RNeasy Mini kit (Qiagen). cDNA was synthesized with M-MLV reverse transcriptase (Promega) in 25-µl reactions containing 2 µg of total RNA.

Quantitative real-time PCR (TaqMan PCR) was performed in duplicate with the ABI PRISM 7000 Sequence Detection System (Applied Biosystems). The PCR primers/probe combination of CRP was based on the one described by Jabs et al. (36). PCR primer sequences were the following: CRP (forward) 5'-GAACTTTCAGCCGAATACATCTTTT-3', (reverse) 5'-CCTTCCTCGACATGTCTGTCT-3', (probe) 5'-VIC- CAGGCCCTTGTATCACTGGCAGCAGG-TAMRA-3'.

The PCR was performed for a final volume of 25 µl, including 12.5 µl TaqMan Universal PCR Master Mix (Applied Biosystems). Thermal cycle parameters were performed for 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of amplification with 15 s at 95°C for denaturation, and 1 min at 62°C for elongation. PCR products of CRP ligated to plasmids with the Original TA Cloning kit (Invitrogen Life Technologies) were adopted as standards, and β2-microglobulin (β2M) was used as an internal control to correct the rate of CRP expression (25). The relative rates of CRP expression represent a comparison with the expression levels in an unstimulated control sample.

Nuclear extracts and EMSA

Nuclear extracts were prepared from WRL-68 cells and primary fetal hepatocyte cells as previously described (26), and nuclear extracts from Hep3B cells were prepared according to the method in Long et al. (37). We assessed equal protein loading by using the DC protein assay kit (Bio-Rad). Briefly, binding reactions used 5 µg of nuclear extracts in 20 mM HEPES (pH 7.9), 1 mM MgCl2, 0.1 mM EGTA, 4% Ficoll, 0.5 mM DTT, 40 mM KCl, and 5 ng/µl polydeoxyinosinic-polydeoxycytidylic acid, with the addition of 5 fmol of 5'-biotin end-labeled oligonucleotide, for a final volume of 20 µl. After incubation at 25°C for 15 min, the complexes were detected with a LightShift Chemiluminescent EMSA kit (Pierce). CRP oligonucleotide (–123/–51) was synthesized by using Sigma Genosys. For the Supershift assay, Abs against phospho-c-Fos (Calbiochem), HNF-1{alpha} (Santa Cruz Biotechnology), and phospho-STAT3 Ser727 (6E4; Cell Signaling Technology) were added to the reaction mixture before the addition of the 5'-biotin end-labeled oligonucleotides.

Immunoprecipitation and Western blot analysis

Nuclear extracts (200–300 µg) obtained 120 min after cytokine stimulation were mixed with Abs overnight at 4°C. We assessed equal protein loading by using the DC protein assay kit (Bio-Rad). Immunocomplexes were precipitated with protein A-Sepharose beads (Amersham Biosciences), and washed four times with lysis buffer. All precipitated proteins were then resolved with 7.5% SDS-PAGE followed by Western blot analysis. We used the following Abs: anti-STAT3 (C20), anti-HNF-1{alpha} (C19), anti-c-Jun, anti-c-Fos (Santa Cruz Biotechnology) (4), anti-phospho-STAT3 Tyr705, anti-phospho-c-Jun (Cell Signaling Technology), and anti-phospho-c-Fos Ser374 (Calbiochem).

Chromatin immunoprecipitation (ChIP) assay

ChIP analysis was performed with a magnetic bead-based ChIP-IT Express Magnetic Chromatin Immunoprecipitation kit (Active Motif) according to the manufacturer’s instructions. Briefly, Hep3B cells were seeded at 1.5 x 105 cells/ml in a 10-cm dish, and after 72 h the cells were stimulated with IL-6 (10 ng/ml) or IL-1β (10 ng/ml) for 120 min. The cells were then cross-linked with 1% formaldehyde for 10 min. Sucrose density-purified nuclear pellets were obtained as previously described (37). The chromatin was sheared with an enzymatic shearing mix for 10 min at 37°C. Immunoprecipitation was performed overnight at 4°C with 1 µg of specific Abs and a negative control with anti-rabbit IgG. We used the following Abs: anti-STAT3 (C20) (Santa Cruz Biotechnology), anti-HNF-1{alpha} (C19) (Santa Cruz Biotechnology), and anti-c-Fos (4) (Santa Cruz Biotechnology). Immunoprecipitates were recovered with magnetic protein G-coated beads and DNA was purified by means of Amersham PCR DNA and the Gel Band Purification kit (Amersham Biosciences). DNA fragments in the range from 200 to 1500 bp were analyzed by PCR using a pair of primers ((forward) 5'-GAAATAATTTTGCTTCCCCTCTTCCC-3' and (reverse) 5'-TCCTAGATCTCTTGCCTTAGAGCTACCTCC-3') spanning the putative STAT3 and HNF-1{alpha} response elements in the CRP promoter (–133/+18), or a pair of primers for β-actin ((forward) 5'-TGCCTAGGTCACCCACTAATG-3' and (reverse) 5'-GTGGCCCGTGATGAAGGCTA-3') (38). The 2 µl from a 20 µl of DNA extraction was amplified for 40 cycles of 98°C for 10 s, 55°C for 30 s, and 72°C for 30 s.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IL-1 has inhibitory or synergistic effects on IL-6-mediated induction of CRP gene expression

To investigate the effect of the cross-talk mechanism between IL-1 and IL-6 on the expression of the CRP gene, we first established a real-time quantitative RT-PCR assay of CRP following IL-1, IL-6, or IL-1 plus IL-6 stimulation of Hep3B cells. From 3 to 24 h of the assay time, IL-6 alone induced an ~6- to 10-fold increase in the mRNA level of CRP, but IL-1 alone had little effect on the induction of CRP. However, the effect of IL-6 was reduced at 3 h and enhanced at 12 and 24 h by IL-1 stimulation (Fig. 1A). To confirm these results, we conducted a promoter assay of the CRP gene after cytokine stimulation using pGL3-CRP (–942/+18) (pGL3-CRP) transfected into the Hep3B cells. We obtained almost the same results, that is, IL-1 reduced the expression of the CRP gene induced by IL-6 at 3 h and increased it 12 and 24 h after stimulation (Fig. 1B). The time course study thus demonstrates that IL-1 has an inhibitory effect (early induction phase) and a synergistic effect (late induction phase) on IL-6-mediated induction of CRP gene expression. Next, we investigated the mechanism of the inhibitory effect of IL-1 on CRP gene expression 3 h after cytokine stimulation.


Figure 1
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  		FIGURE 1. Time course study of CRP mRNA and transcriptional expression. Hep3B cells were stimulated with IL-6 (10 ng/ml) and/or IL-1β (10 ng/ml). CRP gene expression was determined 3, 6, 12, and 24 h after cytokine stimulation. A, CRP mRNA was measured with a real-time quantitative RT-PCR assay system. The relative expressions of CRP mRNA were corrected with β2M. Data show mean + SD of duplicate measurement for the same assay. B, Hep3B cells were transfected with 1 µg of pGL3-CRP (–942/+18). Relative luciferase activity is expressed as mean + SD of triplicate measurements for the same assay. Representative data of at least three independent experiments are shown with similar patterns. *, p < 0.05; **, p < 0.01. CRP gene expression by IL-6 compared with that of IL-1 plus IL-6 using Student’s t test.

 
NF-{kappa}B p65 inhibits CRP gene expression 3 h after cytokine stimulation

IL-1 has two main signaling pathways, the NF-{kappa}B and the MAPK pathway. It is well known that NF-{kappa}B is rapidly activated by IL-1 15–30 min after stimulation. We examined whether NF-{kappa}B p65 or NF-{kappa}B p50 affects the induction of CRP 3 h after stimulation. Unexpectedly, coexpression of pcDNA3.1-NF-{kappa}B p65, but not of pcDNA3.1-NF-{kappa}B p50, inhibited the luciferase activity of pGL3-CRP in a dose-dependent manner (Fig. 2, A and B). We reported previously that direct interaction between STAT3 and NF-{kappa}B p65 is essential for the synergistic induction of the SAA gene by IL-1 plus IL-6 (26). The SAA gene possesses an NF-{kappa}B RE but STAT3 RE does not; however, STAT3 interacts with the 3' site of NF-{kappa}B RE on the SAA gene promoter by forming a complex with NF-{kappa}B p65. In contrast, the CRP gene promoter contains STAT3 RE but not NF-{kappa}B RE. We investigated whether NF-{kappa}B p65 interacts with STAT3 for expression of the CRP gene by IL-1 plus IL-6 and found that coexpression of pEF-BOS wt-STAT3 approximately doubled the luciferase activity of pGL3-CRP after IL-1 plus IL-6 stimulation, and that this increase was inhibited by pcDNA3.1 NF-{kappa}B p65 in a dose-dependent manner (Fig. 2C). We previously reported that STAT3 forms a complex with NF-{kappa}B p65 following IL-1 plus IL-6 stimulation using nuclear extracts of HepG2 cells (26). We performed immunoprecipitation and Western blot analysis of STAT3 and NF-{kappa}B p65 using nuclear extracts of Hep3B cells. Fig. 2D clearly shows that STAT3 interacts with NF-{kappa}B p65 for 30 min following IL-1 plus IL-6 stimulation. These findings indicate that NF-{kappa}B p65 inhibits expression of the CRP gene by forming a complex with STAT3. Next, we examined the synergistic induction mechanism of the CRP gene 12 and 24 h after IL-1 plus IL-6 stimulation.


Figure 2
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  		FIGURE 2. NF-{kappa}B p65 inhibits IL-6-induced CRP gene expression 3 h after cytokine stimulation. A, Hep3B cells were transfected with pGL3-CRP (1.0 µg) and pcDNA3.1-NF-{kappa}B p65 (wt-NF-{kappa}B p65) (0.1–0.2 µg). *, p < 0.01; **, p < 0.001 for CRP gene expression by IL-1 plus IL-6 compared with mock using Student’s t test. B, Hep3B cells were transfected with pGL3-CRP (1.0 µg) and pcDNA3.1-NF-{kappa}B p50 (wt-NF-{kappa}B p50) (0.5–1.0 µg). n.s., Not significant, for CRP gene expression by IL-1 plus IL-6 compared with mock using Student’s t test. C, Hep3B cells were cotransfected with 0.5 µg of pGL3-CRP, pEF-BOS wt-STAT3 (wt-STAT3) (0.5 µg), and pcDNA3.1-NF-{kappa}B p65 (0.1–0.2 µg). Cytokine stimulation was performed with IL-6, IL-1β, or both for 3 h. Relative luciferase activity is expressed as mean + SD of triplicate cultures and transfections. *, p < 0.01; n.s., Not significant, for CRP gene expression by IL-1 plus IL-6 compared with STAT3 alone using Student’s t test. D, Nuclear extracts of Hep3B cells treated with IL-1 plus IL-6 for 30 min were immunoprecipitated with the anti-STAT3 Ab. Results of Western blot analysis of immunoprecipitates with anti-STAT3 and anti-NF-{kappa}B p65 Abs. Endogenous STAT3 interacted with NF-{kappa}B p65 following IL-1 plus IL-6 treatment.

 
SP600125 and SB203580 suppresses CRP gene expression 12 and 24 h after cytokine stimulation

To investigate the late-phase induction mechanism of the CRP gene, we investigated whether JNK and p38, which is another main signaling pathway of IL-1, affect CRP gene expression. Hep3B cells were preincubated for 30 min with 20 µM of the JNK inhibitor SP600125 (39) and 10 µM of the p38 inhibitor SB203580 (40) before cytokine stimulation. Fig. 3A shows that SP600125 and SB203580 reduced the mRNA level of CRP in Hep3B cells 12 h after IL-6 or IL-1 plus IL-6 stimulation. Similarly, these inhibitors lowered the luciferase activity of pGL3-CRP 24 h after induction by IL-6 or IL-1 plus IL-6 (Fig. 3B). These results suggest that JNK and p38 contribute to the expression of the CRP gene 12 and 24 h after IL-1 plus IL-6 stimulation. It is well known that JNK enhances the transcriptional activity of AP-1 (41). Next, we sought to determine whether c-Jun or c-Fos, which comprises AP-1, contributes to CRP gene expression by IL-1 plus IL-6.


Figure 3
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  		FIGURE 3. JNK inhibitor SP600125 and p38 inhibitor SB203580 inhibits CRP gene expression 12 and 24 h after cytokine stimulation. Hep3B cells were preincubated for 30 min with DMSO, 20 µM SP600125 and 10 µM SB203580, and then exposed to IL-6, IL-1β or both. A, CRP mRNA was determined with a real-time quantitative RT-PCR assay system 12 h after cytokine stimulation. Data show mean + SD of duplicate measurements for the same assay. B, Luciferase activity of pGL3-CRP (1.0 µg) was assayed 24 h after cytokine stimulation. Relative luciferase activity is expressed as mean + SD of triplicate measurements for the same assay. Representative data of at least three independent experiments are shown with similar patterns. *, p < 0.05; **, p < 0.01, for CRP gene expression by IL-6 or IL-1 plus IL-6 with inhibitor compared with IL-6 or IL-1 plus IL-6 with DMSO using Student’s t test.

 
c-Fos augments CRP gene expression 24 h after cytokine stimulation

We investigated whether coexpression of pRSV-c-Jun (wt-c-Jun) or pEF-Flag-c-Fos (wt-c-Fos) augments the luciferase activity of pGL3-CRP 24 h after IL-6 or IL-1 plus IL-6 stimulation. It was found that wt-c-Jun approximately doubles or triples luciferase activity after IL-6 stimulation, but little enhancement was detected after IL-1 plus IL-6 stimulation. In contrast, wt-c-Fos dramatically enhanced transcriptional activity of the CRP gene after IL-6 and IL-1 plus IL-6 stimulation (Fig. 4A).


Figure 4
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  		FIGURE 4. c-Fos augments CRP gene expression 24 h after cytokine stimulation via STAT3 RE and 3'-HNF-1 RE. A, A total of 50 ng of pRSV-c-Jun (wt-c-Jun) or pEF-Flag-c-Fos (wt-c-Fos) was cotransfected with 0.5 µg of pGL3-CRP in Hep3B cells. *, p < 0.05; **, p < 0.01; {dagger}, p < 0.001, for CRP gene expression by IL-6 or IL-1 plus IL-6 compared with mock using Student’s t test. B, Time course study of the phosphorylation of c-Jun or c-Fos after cytokine stimulation is shown. Nuclear extracts of Hep3B cells treated with IL-6, IL-1β, or IL-6 plus IL-1β (IL-1 + 6) were analyzed at the times indicated from 15 min to 16 h after stimulation by means of Western blotting of phospho-c-Jun (P-c-Jun) and phospho-c-Fos (P-c-Fos) with the specified Abs. C, 50 ng of wt-c-Fos was cotransfected with 0.5 µg of pGL3-CRP 5'HNF-1 RE (–170/–58) deleted mutant ({Delta}5'HNF-1 RE), pGL3-CRP STAT3 RE (–112/–105) deleted mutant ({Delta}STAT3 RE), and pGL3-CRP 3'HNF-1 RE (–73/–61) deleted mutant ({Delta}3'HNF-1 RE). The wt-c-Fos could not enhance the transcriptional activity of CRP without STAT3 RE or 3'HNF-1 RE. Cytokine stimulation was performed with IL-6, IL-1, or both for 24 h. Relative luciferase activity is expressed as mean + SD of triplicate measurements for the same assay. Representative data of at least three independent experiments are shown with similar patterns. *, p < 0.05; {dagger}, p < 0.001, for CRP gene expression by IL-1 plus IL-6 (pGL3-CRP mutants + wt-c-Fos) compared with IL-1 plus IL-6 (pGL3-CRP + wt-c-Fos) using Student’s t test.

 
We next conducted a time course study of the phosphorylation of c-Jun or c-Fos after cytokine stimulation. The phosphorylation of c-Jun induced by IL-1 or IL-1 plus IL-6 showed its peak intensity from 15–60 min and returned to background level at 120 min. In contrast, the phosphorylation of c-Fos induced by IL-1 plus IL-6 reached its peak intensity at 15 min and continued at this level until 120 min, whereas enhancement was also more evident than that induced by IL-6 or IL-1 alone (Fig. 4B). These results indicate that c-Fos plays an important role in the late induction phase of the CRP gene. Because no typical AP-1 consensus sequence has been identified in the human CRP gene promoter, it needs to be determined how c-Fos is involved in the transcriptional activation of CRP gene. Leu et al. (30) reported that interaction among STAT3, AP-1, and HNF-1{alpha} is important for the amplification of hepatic gene expression. Furthermore, it was reported that two response elements, HNF-1 RE and STAT3 RE, in the proximal region of the CRP promoter are necessary for the expression of the CRP gene (22, 23, 24). To examine the interaction among STAT3, AP-1, and HNF-1{alpha} in the CRP gene promoter, we created a pGL3-CRP 5'-HNF-1 RE (–170/–58) deleted mutant (pGL3-CRP {Delta}5'-HNF-1 RE), a pGL3-CRP STAT3 RE (–112/–105) deleted mutant (pGL3-CRP {Delta}STAT3 RE), and a pGL3-CRP 3'-HNF-1 RE (–73/–61) deleted mutant (pGL3-CRP {Delta}3'-HNF-1 RE). As shown in Fig. 4C, coexpression of wt-c-Fos almost tripled the transcriptional activity of pGL3-CRP and pGL3-CRP {Delta}5'-HNF-1 RE, but did not enhance the transcriptional activity of pGL3-CRP {Delta}STAT3 RE or pGL3-CRP {Delta}3'-HNF-1 RE (Fig. 4C). These results suggest that c-Fos may interact with STAT3 RE and 3'-HNF-1 RE in the transcriptional activity of CRP.

c-Fos forms a complex with STAT3 and HNF-1{alpha} on the CRP gene promoter

To examine the interaction between c-Fos and STAT3 on the CRP promoter, we performed a luciferase assay using pGL3-CRP cotransfected with wt-STAT3 and wt-c-Fos, and found that coexpression with wt-STAT3 alone or wt-c-Fos alone almost doubled the luciferase activity of pGL3-CRP, but that coexpression with both wt STAT3 and wt-c-Fos dramatically enhanced the luciferase activity by a factor of about four. In addition, pEF-BOS dominant negative STAT3 Y705F eliminated the luciferase activity of pGL3-CRP even in the presence of wt-c-Fos (Fig. 5A). These results indicate that the augmentation effect of c-Fos on the expression of CRP is dependent on the activation of STAT3.


Figure 5
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  		FIGURE 5. Interaction of c-Fos with STAT3 and HNF-1{alpha} induces synergistic induction of CRP gene expression. A, Hep3B cells were transfected with pGL3-CRP (0.5 µg), wt-STAT3 (0.5 µg), or pEF-BOS dominant negative STAT3 Y705F (dn-STAT3) (0.5 µg), and with or without wt-c-Fos (0.5 µg), or with both. B, Hep3B cells were transfected with pGL3-CRP (0.5 µg), pcDNA3.1 HNF-1{alpha} (wt-HNF-1{alpha}) (2.5 ng), or pcDNA3.1 dominant negative HNF-1{alpha} (dn-HNF-1{alpha}) (0.5 µg), and with or without wt-c-Fos (50 ng). Cytokine stimulation was performed with IL-6, IL-1β, or a combination for 24 h. Relative luciferase activity is expressed as mean + SD of triplicate measurements for the same assay. Representative data of at least three independent experiments are shown with similar patterns. *, p < 0.01; **, p < 0.001; n.s., Not significant, for CRP gene expression by IL-1 plus IL-6 compared with mock using Student’s t test. C, Nuclear extracts of Hep3B cells treated with IL-1 plus IL-6 for 120 min were immunoprecipitated with the anti-c-Fos Ab. Results of Western blots of immunoprecipitates with anti-STAT3, anti-HNF-1{alpha}, and anti-c-Fos Abs. Endogenous c-Fos interacted with STAT3 and HNF-1{alpha} following IL-1 plus IL-6 treatment.

 
We next used pcDNA3.1 HNF-1{alpha} (wt-HNF-1{alpha}) and pcDNA3.1 dominant negative HNF-1{alpha} to examine whether the interaction between c-Fos and HNF-1{alpha} contributes to transcriptional activity of the CRP gene. Coexpression with wt-HNF-1{alpha} alone did not increase the luciferase activity of pGL3-CRP, but coexpression with dominant negative HNF-1{alpha} prevented the luciferase activity of pGL3-CRP induced by wt-c-Fos (Fig. 5B). This observation shows that HNF-1{alpha} is needed for transcriptional activity of the CRP gene induced by c-Fos.

We postulated formation of a complex with c-Fos, STAT3, and HNF-1{alpha} contributes to the transcriptional augmentation of the CRP gene. To examine the validity of our hypothesis, we performed immunoprecipitation and Western blot analysis of c-Fos, STAT3, and HNF-1{alpha}. Nuclear extracts of Hep3B cells were immunoprecipitated with anti-c-Fos Ab, after which the immunoprecipitates were blotted against STAT3, HNF-1{alpha}, and c-Fos. Fig. 5C clearly shows that c-Fos is associated with STAT3 and HNF-1{alpha} following IL-1 plus IL-6 treatment.

Next, we performed an EMSA using the oligonucleotide which contains STAT3 RE and 3'-HNF-1 RE located between –123 and –51 in the CRP promoter. We previously used such a long oligonucleotide human SAA1 promoter to examine the formation of the heteromeric complex of STAT3 and NF-{kappa}B p65 (26). We first performed a 16-h time course study of the effect of IL-6, IL-1, or IL-1 plus IL-6 stimulation and detected three major nucleoprotein complexes after cytokine stimulation. IL-6 somewhat induced complex I, which reached its peak intensity at 16 h, while IL-1 produced two peak intensities of complex I at 60 min and 16 h, which was further enhanced by IL-1 plus IL-6 at 120 min and 16 h. Complex II was induced without cytokine stimulation, and complex III after stimulation by either of the cytokines (Fig. 6A). These results indicate that complex I is associated with the augmentation of transcriptional activation by IL-1 plus IL-6. We next performed Supershift analyses using anti-HNF-1{alpha}, anti-phospho-STAT3 Ser727 (26), and anti-phospho-c-Fos Ab to identify the components of complex I. As shown in Fig. 6B, these analyses clearly demonstrated that complex I is composed of c-Fos, STAT3, and HNF-1{alpha}. In addition, we confirmed these results by using ChIP for evaluation of the actual transcription factors binding to the CRP promoter in vivo. ChIP assays were performed with chromatin prepared from Hep3B cells. Oligonucleotide primers designed to amplify the CRP promoter region (–133/+18) containing the STAT3 RE and 3'-HNF-1 RE were used for PCR on DNA purified after chromatin immunoprecipitation. STAT3 was found to be recruited to the CRP promoter region in response to IL-6 or IL-1 plus IL-6, whereas HNF-1{alpha} was cytokine-independently recruited and c-Fos was recruited only by IL-1 plus IL-6 (Fig. 6C). We also demonstrated that c-Fos forms a complex with STAT3 and HNF-1{alpha} on the CRP gene promoter in response to IL-1 and IL-6 stimulation. However, the question remains why p38 inhibitor SB203580 reduced the mRNA level and promoter activity of CRP after IL-6 or IL-1 IL-6 stimulation. We also investigated how p38 contributes to expression of the CRP gene after IL-1 plus IL-6 stimulation. It has been reported that SB203580 inhibits the expression of c-Fos induced by IL-1 (40) and that c-Fos is phosphorylated by p38 (42). We were able to confirm that SB203580 inhibits the nuclear translocation of c-Fos by blocking the phosphorylation of c-Fos induced by IL-1 plus IL-6 (Fig. 6D), which suggests that p38 affects CRP gene expression via the phosphorylation of c-Fos.


Figure 6
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  		FIGURE 6. c-Fos forms a transcriptional complex with STAT3 and HNF-1{alpha} on the CRP gene promoter. A, EMSA was performed with 5 µg of nuclear extracts from Hep3B cells using the CRP (–123/–51) oligonucleotide probe containing STAT3 RE and 3'-HNF-1 RE. Cytokine stimulation was performed with IL-6, IL-1β or both from 15 min to 16 h at the time indicated. B, In the Supershift assay, each of the specific Abs against phospho-STAT3 Ser727, HNF-1{alpha}, phospho-c-Fos Ser374, and c-Jun was added to the reactions. IL-1 plus IL-6 stimulation was performed for 120 min. C, ChIP assays demonstrate recruitment pattern of STAT3, c-Fos, and HNF-1{alpha} on the CRP promoter (–133/+18) from Hep3B cells treated with IL-6 (10 ng/ml), IL-1 (0.1 ng/ml) or a combination for 120 min. Assays used anti-STAT3 (C20), anti-HNF-1{alpha} (C19), and a anti-c-Fos (4 ) Ab. Chromatin immunoprecipitates were analyzed for the CRP promoter (–133/+18) and β-actin. D, Hep3B cells were preincubated for 30 min with 10 µM SB203580 and then treated with IL-1 plus IL-6. Nuclear extracts were analyzed by Western blotting with anti-c-Fos and anti-phospho-c-Fos Abs 120 min after treatment.

 
Transcriptional complex of c-Fos, STAT3, and HNF-1{alpha} plays an essential role in the cytokine-driven CRP gene expression

To summarize our experimental results, transcriptional complex formation of c-Fos, STAT3, and HNF-1{alpha} was found to play a critical role in the late-phase induction of the CRP gene after IL-1 plus IL-6 stimulation. However, the question remains as to how the expression level of c-Fos, STAT3, and HNF-1{alpha} is involved in cytokine-driven CRP gene expression. It has been reported that the serum level of CRP is related to aging (10, 11), and that the expression of CRP in the cord serum is significantly lower than in normal healthy adults (11, 12). Finally, we examined CRP gene expression in primary human fetal liver hepatocytes (32, 33) and the human fetal liver cell line WRL-68 (31). As shown in Fig. 7A, the mRNA level of the CRP gene in primary fetal liver cells and WRL-68 cells was not generated after any form of cytokine stimulation as much as it was in Hep3B cells. This was followed by Western blot analysis to examine the expression levels of HNF-1{alpha}, STAT3 and phospho-STAT3, c-Fos and phospho-c-Fos induced by IL-1 plus IL-6 stimulation using the nuclear extracts of primary fetal liver cells and WRL-68 cells. HNF-1{alpha} was below the level of detection in both cells, STAT3 and phospho-STAT3 was detected in WRL-68 cells but not in primary fetal liver cells, whereas c-Fos was detected in both cells, whereas a slight presence of phospho-c-Fos was detected in primary fetal liver cells but not in WRL-68 cells (Fig. 7B). The expression levels of c-Fos, STAT3, and HNF-1{alpha} in primary fetal liver cells and WRL-68 cells thus appear to be different from those in Hep3B cells. We next investigated whether luciferase activity of pGL3-CRP in primary fetal liver cells and WRL-68 cells is affected by overexpression of wt-STAT3, wt-c-Fos, and wt-HNF-1{alpha}. In WRL-68 cells, wt-HNF-1{alpha} alone showed only a slight effect on the luciferase activity of pGL3-CRP, but coexpression of wt-HNF-1{alpha} and wt-c-Fos was seen to induce CRP gene expression. Moreover, the combination of wt-STAT3, wt-c-Fos, and wt-HNF-1{alpha} dramatically enhanced expression of the CRP gene by IL-1 plus IL-6 (Fig. 7C). These results clearly demonstrate that transcriptional complex formation of c-Fos, STAT3, and HNF-1{alpha} plays an essential role in cytokine-driven CRP gene expression.


Figure 7
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  		FIGURE 7. Transcriptional complex of c-Fos, STAT3, and HNF-1{alpha} plays an essential role in cytokine-driven CRP gene expression. A, CRP mRNA was measured with a real-time quantitative RT-PCR assay system after cytokine stimulation in Hep3B cells, WRL-68 human fetal liver cell line cells, and primary human fetal hepatocytes (Hc). Data show mean + SD of duplicate measurements for the same assay. PCR products of CRP and β2M were electrophoresed as shown in the bottom panel. n.d., Not detected. B, Nuclear extracts of Hep3B cells, WRL-68 cells, and primary human fetal hepatocytes treated with IL-1 were analyzed with Western blotting of HNF-1{alpha}, STAT3, and phospho-STAT3 Tyr705 (P-STAT3), c-Fos and phospho-c-Fos, and c-Jun and phospho-c-Jun with the corresponding specific Abs 120 min after treatment. C, WRL-68 cells were transfected with pGL3-CRP (0.5 µg), wt-STAT3 (0.5 µg), wt-c-Fos (50 ng), or wt-HNF-1{alpha} (2.5 ng). Cytokine stimulation was performed with IL-6, IL-1β, or a combination of both for 24 h. Relative luciferase activity is expressed as mean + SD of triplicate measurements for the same assay. Representative data of at least three independent experiments are shown with similar patterns. *, p < 0.05; **, p < 0.001; n.s., Not significant, for CRP gene expression by IL-1 plus IL-6 compared with mock using Student’s t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
CRP has been identified as a mediator of inflammation and a predictor of cardiovascular disease (1, 2, 3, 4, 5, 6). In this study, we have been able to demonstrate that there are two phases in the transcription mechanism of cytokine-driven CRP gene expression.

In the early induction phase, IL-1 inhibited the effect of IL-6 on CRP expression by forming a complex with NF-{kappa}B p65 and STAT3, and we showed that the inhibitory effect of IL-1 was augmented by NF-{kappa}B p65 in a dose-dependent manner (Fig. 2A). In contrast, luciferase activity induced by overexpression of STAT3 was reduced in a dose-dependent manner by NF-{kappa}B p65 (Fig. 2C). Moreover, we confirmed that STAT3 interacts with NF-{kappa}B p65 in Hep3B cells (Fig. 2D). Cha-Molstad et al. (16) reported that NF-{kappa}B p65 eliminated the induction of a –157/+3 CRP-Luc construct 24 h after IL-1 plus IL-6 stimulation. This inhibitory effect of NF-{kappa}B p65 did not result from the competitive binding to the nonconsensus {kappa}B site in which NF-{kappa}B p50 is involved (17). In addition, Yoshida et al. (43) reported that IL-1 inhibits the induction by IL-6 of a Luc-construct containing the {gamma}-IFN activation sequence, a classical consensus sequence of STAT3. They also found that NF-{kappa}B p65 inhibits the induction of the {gamma}-IFN activation sequence Luc construct. Because the {gamma}-IFN activation sequence site is located in the CRP gene promoter, these reported findings strongly support our experimental results for the early induction phase of CRP.

We previously reported that the interaction between NF-{kappa}B p65 and STAT3 is essential for the synergistic induction of SAA gene expression by IL-1 plus IL-6 stimulation (26). The SAA gene possesses NF-{kappa}B RE, but not consensus STAT3 RE, in its promoter. The formation of the NF-{kappa}B p65 and STAT3 complex imparts STAT3 binding affinity to around the NF-{kappa}B RE of SAA gene promoter (26). Taken together, these results indicate that the interaction between NF-{kappa}B p65 and STAT3 has two opposite roles. One is an inhibitory effect on the {gamma}-IFN activation sequence site, the other a synergistic effect on NF-{kappa}B RE. Further study is needed, however, to clarify the mechanism of interaction between NF-{kappa}B p65 and STAT3.

NF-{kappa}B p50 has been reported to be involved in the promoter of the CRP gene via the nonconsensus {kappa}B site overlapping the proximal C/EBP binding site (16, 17, 18, 19). However, the working model used in these studies is not consistent with the findings of our clinical trial of IL-6 blocking therapy (20, 21). In fact, we found that NF-{kappa}B p50 had no inhibitory effect on expression of the CRP gene in our system (Fig. 2B). We performed our luciferase assay 3 h after cytokine stimulation without overexpression of C/EBPβ. In the other studies, promoter assays were performed in the presence of C/EBPβ overexpression. It is well known that C/EBPβ is induced by LPS or IL-6 (44), whereas the effect of NF-{kappa}B p50 on CRP expression is thought to depend on the amount of C/EBPβ. Under our assay conditions, the amount of C/EBPβ may have been insufficient for NF-{kappa}B p50 to have an effect. In addition, it has been reported that activation of NF-{kappa}B by cytokines is gradually attenuated (45, 46). Therefore, NF-{kappa}B may be mainly involved in the early induction phase of CRP expression.

In the late induction phase, we demonstrated that transcriptional complex formation of c-Fos, STAT3, and HNF-1{alpha} is essential for the synergistic induction of CRP gene expression by IL-1 plus IL-6, thus making this the first documented evidence that AP-1, especially c-Fos, is involved in the expression of CRP. In Fig. 8, we present a schematic explanatory model of the mechanism of human CRP gene expression induction by IL-1 plus IL-6 stimulation.


Figure 8
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  		FIGURE 8. Schematic model of cytokine-driven transcriptional activity of human CRP gene. A, In the early induction phase, NF-{kappa}B p65 forms a complex with STAT3, which may inhibit expression of the CRP gene. B, In the late phase induction, cytokine stimulation causes the formation of a heteromeric complex with c-Fos, STAT3, and HNF-1{alpha}, which in turn induces synergistic expression of the CRP gene.

 
JNK inhibitor SP600125 reduced CRP gene expression by IL-1 plus IL-6 (Fig. 3, A and B). JNK is well known as an enhancer of the transcriptional activity of AP-1 (41). We first expected that the interaction between STAT3 and c-Jun, previously reported by Zhang et al. (29), would play a critical role in the induction of CRP, but it did not.

Leu et al. (30) reported that complex formation of c-Fos, STAT3, and HNF-1{alpha} is required for the maximal expression of the insulin-like growth factor binding protein-1 promoter via the HNF-1 site. It has been suggested that liver-specific genes encoding HNF-1 site, such as CRP, fibrinogen and others, are regulated by c-Fos, STAT3, and HNF-1{alpha} (30). The CRP gene, like the insulin-like growth factor binding protein-1 gene, has no AP-1 RE in its promoter. Our study showed that overexpression of c-Fos, but not of c-Jun, dramatically enhanced the luciferase activity of pGL3-CRP 24 h after the start of treatment with IL-1 plus IL-6 (Fig. 4A). This activity was then synergistically enhanced by wt-STAT3 or wt-HNF-1{alpha}, and eliminated by dominant negative STAT3 or dominant negative HNF-1{alpha} (Fig. 5, A and B). We demonstrated by means of immunoprecipitation and Western blot analyses (Fig. 5C), Supershift assay (Fig. 6B), and ChIP assay (Fig. 6C) that c-Fos, STAT3, and HNF-1{alpha} form a complex on the CRP gene promoter.

In addition, we were able to clarify the mechanism of p38 involvement in CRP gene expression. p38 specific inhibitor SB203580 reduced the induction of CRP gene expression by IL-1 plus IL-6 (Fig. 3, A and B), which is consistent with the finding reported by Westra et al. (47) that CRP production was diminished by another p38-specific inhibitor RWJ67657 in hepatoma cell lines containing Hep3B. Recently, it has been reported that the transactivation domain of c-Fos is phosphorylated by UV-induced p38 MAPK, which mediates AP-1 activity (42). We found that the phosphorylation of c-Fos is more obviously enhanced by IL-1 plus IL-6 than by IL-6 or IL-1 alone (Fig. 4B). In addition, we also found that SB203580 inhibited the phosphorylation and nuclear translocation of c-Fos induced by IL-1 plus IL-6 stimulation (Fig. 6D). These reports and our findings therefore indicate that the phosphorylation of c-Fos induced by p38 is important for the full activation of CRP gene expression in the late induction phase.

We further showed that CRP production in human fetus liver is significantly lower than that in Hep3B cells due to the expression level of c-Fos, STAT3, and HNF-1{alpha}. Liver development is characterized by various gene expressions such as {alpha}-fetoprotein and albumin. In situ hybridization studies using mice have demonstrated that, before liver organization when HNF-1{alpha} is not yet expressed, albumin gene expression was not detectable in prehepatic cells (48). The primary human fetal hepatocytes have been used for analysis of TNF-{alpha}-induced apoptosis (32, 33), and the WRL-68 cells of a human fetal liver cell line has been used in cytotoxicity tests (31). In our study, HNF-1{alpha} was not detected by Western blot analysis in either cell type, which indicates that these cells have the characteristics of prehepatic cells. Overexpression of wt-c-Fos, wt-STAT3, and wt-HNF-1{alpha} restored the luciferase activity of pGL3-CRP induced by IL-1 plus IL-6 in WRL-68 cells (Fig. 7C). It is thus reasonable to postulate that CRP expression is the phenotypic marker of mature liver cells, which can explain our findings that the expression level of CRP in cord serum is significantly lower than in normal healthy adults (11, 12) and that CRP is not a sensitive marker for neonatal infection even though IL-6 is increased in neonatal sepsis (12, 13, 14). However, a limitation of our study is that we were not able to show whether the expression of the acute phase protein depends on the development of liver cells in vivo. Further study is therefore needed to determine the validity of our hypothesis.

In conclusion, we were able to demonstrate that transcriptional complex formation of c-Fos, STAT3, and HNF-1{alpha} plays an essential role in cytokine-driven CRP gene expression. This explains why anti-IL-6R Ab therapy normalizes serum levels of CRP in chronic inflammatory diseases, such as rheumatoid arthritis, Castleman’s disease, and so on (23, 24). The findings of previous studies and our study point to the existence of two important interaction mechanisms. One is of STAT3 and c-Fos, and the other of STAT3 and NF-{kappa}B p65 (26), indicating that STAT3 plays a pivotal role in the induction of acute phase proteins. Moreover, CRP is considered to be a mediator of proinflammatory development (7, 8, 9). In this context, our findings indicate that interaction between STAT3 and c-Fos should be considered a novel therapeutic target for chronic inflammatory diseases such as rheumatoid arthritis and cardiovascular disease.


   Acknowledgments
 
We thank Drs. Koichi Nakajima and Kazuya Yamagata for providing the plasmids. We also appreciate the skillful secretarial assistance by A. Okajima and K. Umetani.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Ministry of Health, Labor and Welfare of Japan, and the Osaka Foundation for Promotion of Clinical Immunology. Back

2 T.N. and K.H. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Kazuyuki Yoshizaki, Health Care Center, Osaka University, 2-1 Yamada-Oka, Suita-City, Osaka 565-0871, Japan. E-mail address: kyoshizaki{at}hpc.cmc.osaka-u.ac.jp Back

4 Abbreviations used in this paper: CRP, C-reactive protein; HNF-1, hepatocyte NF-1; SAA, serum amyloid A; RE, response element; ChIP, chromatin immunoprecipitation; β2M, β2-microglobulin; wt, wild type. Back

Received for publication November 28, 2007. Accepted for publication December 18, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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