Promoter analysis reveals critical roles for SMAD-3 and ATF-2 in expression of IL-23 p19 in macrophages

Fahd Al-Salleeh, Thomas M Petro

Research output: Contribution to journalArticle

37 Scopus citations

Abstract

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-κB (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-κB, 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-κB, essential for IL-23 p19 expression.

Original languageEnglish (US)
Pages (from-to)4523-4533
Number of pages11
JournalJournal of Immunology
Volume181
Issue number7
DOIs
Publication statusPublished - Oct 1 2008

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ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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