(The FASEB Journal. 2008;22:lb431)
© 2008 FASEB
Promoter analysis shows that SMAD-3 and ATF-2 are involved in expression of IL-23p19 during Theiler’s virus-infection of RAW264.7 cells
Fahd M. Al-Salleeh and
Thomas M. Petro
Oral Biology, University of Nebraska Medical Center, Lincoln, NE
ABSTRACT
Interleukin (IL)-23p19/p40 promotes the development of Th17 effector cells and experimental autoimmune encephalomyelitis. To understand its expression we cloned the p19 promoter into the pGL3 luciferase vector. Sequence analysis of the cloned promoter region confirms identity with the mouse sequence Accession NT_039502. Using MatInspector, putative binding sites for the transcription factors IRF3(bp –734 to –731), IRF7(bp –533 to –525), ATF2(bp –571 to –568), SMAD3 (bp –584 to –581) and NF-
B (bp –215 to –209) were found in the p19 promoter region. Following transfection of p19pGL3 into RAW264.7 cells the promoter was responsive to challenge with Theiler’s virus (TMEV). Deletions upstream of the IRF3 site resulted in an 80% reduction in promoter activity. Site-mutations at the IRF3, SMAD3, ATF2, or NF-B sites, but not the IRF-7 site, resulted in 75% (IRF3 and SMAD3), 100% (ATF2), or 90% (NF-B) reduction in promoter activity. Following TMEV infection of RAW264.7 cells, immunublots show activation of IRF3, ATF2, and SMAD3, which was significantly reduced with specific short-hairpin (sh) RNA expression vectors. Transfection of shATF2 and shSMAD3, but not shIRF3 resulted in a 84% reduction in p19 promoter activity and p19 protein expression in RAW264.7 cells challenged with TMEV. These results suggest in addition to NF-B, that ATF2 and SMAD3 are essential transcription factors for IL-23 p19 expression.
