TGF-beta suppresses IFN-gamma-STAT1-dependent gene transcription by enhancing STAT1-PIAS1 interactions in epithelia but not monocytes/macrophages.
IFN-gamma and TGF-beta are important regulators of mucosal immunity, typically functioning in opposition to each other. In this study, we assessed whether TGF-beta could modulate IFN-gamma-induced STAT1 signaling. Model epithelial cell lines (HEp-2, HT-29, and T84) or monocytes/macrophages (THP-1 cell line, human blood mononuclear cells) were pretreated with TGF-beta (1 ng/ml; 5-60 min), followed by IFN-gamma exposure (20 ng/ml; 30 min), and then STAT1 transcriptional activity, DNA-binding activity, phosphorylation, and methylation were assessed. Some epithelia were transfected with an expression plasmid encoding SMAD7 to block TGF-beta-SMAD signaling. Epithelia, but not macrophages, pretreated with TGF-beta were hyporesponsive to IFN-gamma stimulation as indicated by reduced expression of four STAT1-regulated genes and reduced STAT1 DNA binding on EMSA. However, STAT1 Tyr(701)-, Ser(727) phosphorylation, and nuclear recruitment of STAT1 were not significantly different in IFN-gamma with or without TGF-beta-treated cells, indicating that the effects of TGF-beta are downstream of IFN-gammaR-JAK-STAT1 interaction. The TGF-beta effect was not dependent on ERK1/2, p38, or JNK activation but was prevented by overexpression of the inhibitory SMAD7 protein. Additional studies suggest that TGF-beta blockade of IFN-gamma activity in epithelia is via enhanced sequestering of STAT1 by pre-existing protein inhibitor of activated STAT1. These results demonstrate that TGF-beta rapidly suppresses IFN-gamma-driven STAT1 signaling by reducing DNA binding via promotion of STAT1-protein inhibitor of activated STAT1 interactions and not inhibition of STAT1 activation; an event that may be specific to epithelia and represent a novel mode of action of TGF-beta.
Reardon
Colin
C
Gastrointestinal Research Group, Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Drive Northwest, Calgary, Alberta, Canada.
McKay
Derek M
DM
D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004847:Epithelial Cells / Q000187:drug effects* / Q000276:immunology; D005786:Gene Expression Regulation; D006801:Humans; D007371:Interferon-gamma / Q000037:antagonists & inhibitors / Q000494:pharmacology; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects / Q000276:immunology; D008745:Methylation; D009000:Monocytes / Q000187:drug effects / Q000276:immunology; D010766:Phosphorylation; D050828:Protein Inhibitors of Activated STAT / Q000378:metabolism*; D050794:STAT1 Transcription Factor / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D051785:Smad Proteins / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
C106529:PIAS1 protein, human; D050828:Protein Inhibitors of Activated STAT; D050794:STAT1 Transcription Factor; D051785:Smad Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D016212:Transforming Growth Factor beta; D007371:Interferon-gamma; D004247:DNA; D048670:MAP Kinase Kinase 4