title: Promoter analysis reveals critical roles for SMAD-3 and ATF-2 in expression of IL-23 p19 in macrophages.
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-kappaB (nt -215 to -209) in the murine p19 promoter. The p19(prom) 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-kappaB, 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-beta1, which activates SMAD-3, was induced in RAW264.7 cells, BMM, and SPM by TMEV. Neutralizing Ab to TGF-beta1 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-kappaB, essential for IL-23 p19 expression.
authors.affiliation: Department of Oral Biology and Nebraska Center for Virology, University of Nebraska Medical Center, Lincoln, NE 68583, USA.
authors.forename: Fahd
authors.initials: F
authors.lastname: Al-Salleeh
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Petro
mesh_terms: D051698:Activating Transcription Factor 2 / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005260:Female; D015967:Gene Expression Regulation, Viral / Q000276:immunology; D006801:Humans; D050838:Interferon Regulatory Factor-3 / Q000378:metabolism / Q000502:physiology; D050839:Interferon Regulatory Factor-7 / Q000378:metabolism; D053760:Interleukin-23 Subunit p19 / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D008264:Macrophages / Q000276:immunology* / Q000378:metabolism* / Q000821:virology; D008562:Membrane Glycoproteins / Q000172:deficiency / Q000235:genetics / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000276:immunology*; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology; D051900:Smad3 Protein / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D017945:Theilovirus / Q000276:immunology; D051196:Toll-Like Receptor 3 / Q000172:deficiency / Q000235:genetics / Q000502:physiology; D051199:Toll-Like Receptor 7 / Q000172:deficiency / Q000235:genetics / Q000502:physiology
chemical_list: D051698:Activating Transcription Factor 2; C497106:Atf2 protein, mouse; D050838:Interferon Regulatory Factor-3; D050839:Interferon Regulatory Factor-7; D053760:Interleukin-23 Subunit p19; C494233:Irf3 protein, mouse; C494235:Irf7 protein, mouse; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; C519703:TLR3 protein, mouse; C477038:Tlr7 protein, mouse; D051196:Toll-Like Receptor 3; D051199:Toll-Like Receptor 7