TNF blockade in Crohn's disease (CD) and ulcerative colitis (UC) patients reveals that TNF plays a central role in disease pathogenesis. Here we examine the potential that TNF activates epithelial stem and progenitor cell (ISC/PC) β-catenin through intestinal epithelial cell (IEC) TNFR1 and TNFR2 signaling. Tissue sections and purified IEC protein isolates were analyzed using Akt-phosphorylated β-catenin (P-β-catenin552 or P-β-cat) Ab. Results: IHC staining of biopsy tissue from control (uninflamed) and inflamed CD/UC patients revealed that Pβ-cat levels increased 2.8-fold in untreated colitis. However, in anti-TNF treated patients selected for ongoing active inflammation, P-β-cat levels were reduced by over 50% (p host) revealed that T cell activation increased numbers of P-βcat-stained IECs by 110% in WT->WT BMC with no increases seen in anti-CD3-treated WT-> TNFR1/2 KO mice. These findings suggest epithelial TNFR signaling regulates IEC β-catenin activation. In studies comparing colitic IL-10 KO (colitis score >3/4) mice to WT healthy controls (n=5 in each group) mRNA from CD44+-sorted IECs was used for genomewide expression array analysis revealing that levels of NADPH oxidase 1 (Nox1) were significantly induced (3.22-fold, p-value: 3 x106). We next tested whether Nox1 may participate in TNF-mediated activation of β-catenin in ISC. We confirmed Nox1 mRNA induction in IECs from IL-10 KO colitis mice (17-fold). Furthermore Nox1 mRNA was induced in purified IECs by anti-CD3 mAb treatment of WT mice (6-fold). Importantly, Nox1 mRNA induction was reduced by over 50% in T cell stimulated TNF-R1/R2 KO mice. Lastly, mRNA from purified CD44+-sorted IECs from Nox1 KO mice revealed that baseline levels of cMyc, cyclinD1, Ascl2 and Lrig1 expression were reduced compared to WT mice. Conclusion: Together the data suggest that epithelial TNF receptor signaling is required for optimal levels of IEC β-catenin activation in patients with active colitis. Data in IL-10 ko and WT mice suggest that epithelial TNFR signaling induces Nox1 which has recently been shown to play a role in Wnt/β-catenin signaling in the intestine (Coant et al. MCB 2010). Together these data are consistent with the hypothesis that Nox1 is a key mediator of TNFinduced epithelial β-catenin activation in UC and CD.