ZAS3 accentuates transforming growth factor β signaling in epithelial cells

In mammals, the ZAS family of transcription factors activates or represses transcription depending on the cellular context. In the current study, we explored the interaction between ZAS3 and TGF beta 1 signaling in epithelial cells using HEK293 cells and the intestinal epithelial cell line, RIE-1. Endogenous ZAS3 expression was detected in each cell line and the small intestine of mice. Additionally. endogenous ZAS3 expression was increased in both whole cell and nuclear lysates by TGF beta 1 and in vivo in TGF beta-overexpressing mice, indicating a potential interaction between ZAS3 and TOM. ZAS3 transfection enhanced TGF beta 1 activation of a luciferase reporter in both HEK293 and RIE-1 cells. Analysis of truncated ZAS3 constructs revealed a 155 amino acid, N-terminal sequence between amino acids 106 and 261 that was required for enhancement of TGF beta 1-mediated transcription. Co-immunoprecipitation experiments with nuclear extracts from TGF beta 1-stimulated HEK293 cells revealed an association between ZAS3 and the Smad complex. Additionally, transfected ZAS3 decreased the association between the Smad complex and the TGF beta transcriptional repressors Ski and SnoN, indicating a possible mechanism for the enhancement of transcription by exogenous ZAS3. These observations were confirmed by site-directed mutagenesis of ZAS domains homologous with Smad-interacting domains in Ski and SnoN. Finally, ZAS3 transfection enhanced the TGF beta 1-mediated induction of alpha-smooth muscle actin in HEK293 cells, indicating that ZAS3 plays a functional role in TGF beta signaling. In conclusion, we have identified an interaction between ZAS3 and Smad proteins that enhances TGF beta signaling. Since TGF beta signaling is primarily known as a negatively regulated pathway, the enhancement of signaling by ZAS3 has novel implications for understanding TGF beta biology. (C) 2010 Elsevier Inc. All rights reserved.