598 Transforming Growth Factor-Beta Signaling on Dendritic Cells Regulates Bacterial Communities and Gut Homeostasis

Abstract

Background: Transcription factor Zinc-finger Binding Protein-89 (ZBP-89, ZNF148) expression is regulated by butyrate and forms protein-protein complexes with tumor suppressor factors, e.g. p53, p300, ataxia-telangiectasia mutated (ATM). To study the role of ZBP-89 in vivo, we generated a conditional knockout in the intestine and colon (ZBP-89ΔInt). Mice exhibited increased morbidity and mortality when challenged with Salmonella typhimurium, in part due to reduced tryptophan hydroxylase 1(Tph1) expression and reduced colonic antimicrobial peptide secretion (defensins). We found that β-catenin cooperates with ZBP89 to induce tryptophan hydroxylase 1(Tph1) expression in enterochromaffin cells. Aim: To determinewhether ZBP-89 interacts directly with β-catenin to prevent colonic transformation. Methods: ZBP-89ΔInt and WT littermates were treated with 7.4mg/kg azoxymethane (AOM) and water containing 2% dextran sulfate sodium (DSS). After 3 cycles, mice were sacrificed 5 weeks later for tumor evaluation, mRNA and histological analysis. Expression of β-cateninTCF gene targets was determined by rt-qPCR on colonic lysates. The ZBP-89 expression vector was co-transfected with or without β-catenin into HEK293 or SW480, a human colorectal cell line with the TCF reporter plasmid TOPFLASH to assess direct regulation of Wnt-β-catenin-TCF transcriptional activity. To demonstrate the association of ZBP-89 with β-catenin, cell lysates of SW480 were used to perform co-immunoprecipitation. Results: We observed 100% increase in tumor incidence and size in the distal colon and rectum of ZBP-89ΔInt compared to WT mice after the administration of AOM/DSS, N=16 mice/group (P<0.05). mRNA for cyclinD1, c-myc and Axin2 was significantly increased 3-fold in the ZBP89ΔInt mice after AOM/DSS treatment. Ectopic expression of ZBP-89 mitigated induction of the TOPFLASH reporter by β-catenin by 60% in both HEK293 and SW480 cell lines. Coimmunoprecipitation/western blotting showed that ZBP-89 and β-catenin formed a complex in cytoplasmic and nuclear extracts. Confocal microscopy demonstrated that ZBP-89 colocalized with β-catenin in the nucleus. In addition to protein-protein interactions, we found that knockdown of ZBP-89 by siRNA reduced β-catenin protein levels, suggesting that ZBP89 regulates β-catenin expression. Indeed, in silico analysis revealed a putative ZBP-89 binding site in the human and mouse β-catenin promoter at -83 to -78. Conclusion: Collectively, the data suggests that ZBP-98 modulates β-catenin activity and subsequently the induction of Wnt-β-catenin-TCF pathway in vivo and in vitro. Pull-down assays demonstrated that ZBP-89 modulates β-catenin activity through both protein-protein interactions and possibly transcriptional regulation of β-catenin expression, providing a possible mechanism by which this transcription factor inhibits colonic tumor formation.