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Transcriptional Complex Formation of c-Fos, STAT3, and Hepatocyte NF-1 Is Essential for Cytokine-Driven C-Reactive Protein Gene Expression¹

Teppei Nishikawa^2,*, Keisuke Hagihara^2,, Satoshi Serada, Tomoyasu Isobe^*, Atsumi Matsumura, Jian Song^*, Toshio Tanaka, Ichiro Kawase, Tetsuji Naka and Kazuyuki Yoshizaki^3,*,

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

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-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, 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 forms a complex on the CRP gene promoter. Because human fetus liver cells failed to express c-Fos/STAT3/HNF-1 showed no CRP production, transcriptional complex formation of c-Fos/STAT3/HNF-1 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.

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.

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

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

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.