HOME
Return to Table of Contents
010 TGFbeta ENHANCES SEQUESTERING OF STAT1 BY PROTEIN INHIBITOR OF ACTIVATED STAT 1 (PIAS1) AND SUPPRESSES IFNgamma-STAT1 DEPENDENT GENE TRANSCRIPTION IN EPITHELIA C Reardon, D McKay Interferon-gamma (IFNgamma) and transforming growth factor-beta (TGFbeta) are important regulators of mucosal immunity that typically function in opposition to each other. Here we assessed if TGFbeta could attenuate IFNgamma elicited signal transducer and activation of transcription-1 (STAT1) signaling.
Gastrointestinal Research Group, University of Calgary, Calgary, Alberta
METHODS: Model epithelial cell lines (HEp-2, HT-29, T84) or mononuclear cells (THP-1 cell line, human blood mononuclear cells) were pretreated with TGFbeta (1ng/ml; 0-60min), prior to IFNgamma exposure (20ng/ml; 30min). After cytokine stimulation, STAT1 DNA-binding, phosphorylation, methylation, and binding to PIAS1 were assessed. Some epithelia were transfected with an expression plasmid encoding the inhibitory SMAD7 protein to block TGFbeta-SMAD signaling, or nucleofected with siRNA directed against PIAS1.
RESULTS: Epithelia, but not mononuclear cells, pretreated with TGFbeta were hypo-responsive to IFNgamma stimulation as indicated by reduced expression of STAT1-regulated genes (eg, interferon response factor-1) and reduced STAT1 DNA binding on electrophoretic mobility shift assays. However, STAT1 tyrosine701-, serine727-phosphorylation, and nuclear recruitment of STAT1 were not significantly different in IFNgamma ± TGFbeta treated cells, indicating that the effects of TGFbeta are downstream of IFNgamma receptor-Janus kinase-STAT1 interaction. Antagonism of IFNgamma-STAT1 signaling requires intact TGFbeta signaling, as evidenced by restoration of STAT1 DNA binding in TGFbeta pre-treated cells over-expressing SMAD7. This antagonism by TGFbeta occurs through enhanced sequestering of STAT1 by pre-existing PIAS1 in epithelia as evidenced by increased co-immunoprecipitation, and restoration of STAT1 DNA binding in epithelia nucleofected with PIAS1 siRNA. Sequestering of STAT1 by PIAS1 is also independent of TGFbeta induced alteration of STAT1 methylation, as no methylation was observed.
CONCLUSIONS: These results demonstrate that TGFbeta rapidly suppresses IFNgamma-driven STAT1 signaling by reducing DNA-binding through enhanced sequestering of PIAS1, without affecting STAT1 activation; an event that may be specific to epithelia and represent a novel mode of action of TGFbeta. Defining cytokine intracellular crosstalk has the potential to highlight novel approaches to blocking the effects of pro-inflammatory cytokines and enhance the effects of cytokines with reparatory properties.
Funded by the CIHR and NSERC