IFN-Gamma induziert Apoptose durch Inhibierung des Wnt/beta-Catenin-Signalwegs in intestianler Entzündung

Abstract

Increased epithelial apoptosis induced by pro-inflammatory cytokines contributes to the breakdown of the intestinal epithelial barrier, which plays a central role in the pathogenesis of inflammatory bowel disease (IBD). Since the Wnt-signaling pathway has been implicated to play a role in epithelial proliferation and differentiation we examined contribution of this pathway to IFN-γ-induced intestinal epithelial apoptosis. For these studies we utilized complementary in vitro cell culture and in vivo DSS-induced colitis models. We observed upregulation of an antagonist of the Wnt signaling pathway referred to as Dickkopf Homolog 1 (DKK-1) in inflamed intestinal mucosa. Since IFN-γ is upregulated in inflamed intestinal mucosa of IBD patients, we examined the role of DKK-1 in IFN-γ-induced epithelial apoptosis. Apoptosis in intestinal epithelial cells (IECs) incubated with human IFN-γ (100 U/mL) for 72 hrs was confirmed by analysis of caspase-3 and cleavage of PARP by western blotting and immunofluorescence labeling/confocal microscopy. IECs undergoing apoptosis showed a 3 fold upregulation of DKK-1, its receptor Kremen1, and a significant downregulation of its coreceptor LRP6 compared to controls. Furthermore, INF-γ induced upregulation of p53 which is known to regulate DKK-1 expression. Overexpression of p53 in IECs resulted in a significant upregulation of DKK-1 expression compared to controls, and this effect was reversed by expression of dominant negative p53. The ability of INF-γ to induce apoptosis was prevented by using a functional inhibitory antibody to DKK-1 and a glycogen synthase kinase-3 (GSK-3β - downstream target of DKK-1) inhibitor. As an in vivo correlate, we observed significant upregulation of DKK-1 in colonic mucosa and plasma from DSS-treated mice and in colonic mucosa from Crohn’s disease patients. These findings support a novel role for the Wnt pathway inhibitor, DKK-1 in mediating IFN-γ induced IEC apoptosis. In conclusion, exposure to IFN-γ induces p53 and DKK-1 secretion and engagement of DKK-1 to its cell surface receptors Kremen1 and LRP-6. Subsequently, the APC-axin-GSK-3-β complex is activated resulting in phosphorylation and degradation of β-catenin thereby inhibiting cell proliferation and stimulating cell death. These findings represent the first report demonstrating participation of the Wnt signaling pathway in apoptosis induced by a pro-inflammatory cytokine. (Supported by grants from the German Research Foundation - La 2359/1-1 and the NIH - DK-55679, DK - 61379).