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Department of Oral Biology and Nebraska Center for Virology, University of Nebraska Medical Center, Lincoln, NE 68583
IL-23 p19/p40, produced by macrophages and dendritic cells, is critical for development of Th17 in several autoimmune diseases. In this study, bone marrow-derived (BMM) and splenic macrophages (SPM) from SJL/J mice, susceptible to autoimmune demyelinating disease following Theiler’s virus (TMEV) infection, expressed IL-23 in response to TMEV. We identified potential binding sites for IFN response factor (IRF)-3 (nt –734 to –731), Sma- and Mad-related protein (SMAD)-3 (nt –584 to –581), activating transcription factor (ATF)-2 (nt –571 to –568), IRF-7 (nt –533 to-525), and NF-
B (nt –215 to –209) in the murine p19 promoter. The p19prom in the pGL3 promoter-reporter vector responded to TMEV or poly(I:C), a TLR3 agonist in the RAW264.7 macrophage cell line. Deletions upstream from the IRF-3 site and mutations at the IRF-3, SMAD-3, ATF-2, or NF-B, but not the IRF-7, sites significantly reduced promoter activity. ATF-2 or SMAD-3, but not IRF-3, short-hairpin RNA reduced p19 promoter activity and protein expression in RAW264.7 cells responding to TMEV. Chromosomal DNA immunoprecipitation assays revealed that SMAD-3 and ATF-2 bind to the endogenous p19 promoter in RAW264.7 cells and SJL/J SPM following challenge with TMEV. TGF-β1, which activates SMAD-3, was induced in RAW264.7 cells, BMM, and SPM by TMEV. Neutralizing Ab to TGF-β1 eliminated TMEV-induced IL-23 production and SMAD-3 activation in RAW264.7 cells, BMM, and SPM. Activation of ATF-2 was JNK, but not p38 or ERK MAPK dependent. Inhibition of the JNK, but also the ERK MAPK pathways decreased expression of p19. These results suggest that ATF-2 and SMAD-3 are transcription factors, which are, in addition to NF-B, essential for IL-23 p19 expression.
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1 This work was supported by a University of Nebraska Medical Center College of Dentistry Seed Grant and National Multiple Sclerosis Society Grant PP1423.
2 Address correspondence and reprint requests to Dr. Thomas M. Petro, Department of Oral Biology, University of Nebraska Medical Center, 40th and Holdrege Avenue, Lincoln, NE 68583-0740. E-mail address: tpetro{at}unmc.edu
3 Abbreviations used in this paper: MS, multiple sclerosis; ATF, activating transcription factor; BMM, bone marrow-derived macrophage; ChIP, chromosomal DNA immunoprecipitation; Ct, cycle threshold; IRF, IFN response factor; ORF, open reading frame; poly(I:C), polyinosine-polycytidylic acid; SCR, scrambled sequence; sh, short-hairpin; SMAD, Sma- and Mad-related protein; SPM, splenic macrophage; TMEV, Theiler’s murine encephalomyelitis virus.
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  W. Liu, X. Ouyang, J. Yang, J. Liu, Q. Li, Y. Gu, M. Fukata, T. Lin, J. C. He, M. Abreu, et al. AP-1 Activated by Toll-like Receptors Regulates Expression of IL-23 p19 J. Biol. Chem., September 4, 2009; 284(36): 24006 - 24016. [Abstract] [Full Text] [PDF]
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