Background & Aims: Necrotizing enterocolitis (NEC), the leading cause of gastrointestinal death from gastrointestinal disease in preterm infants, is characterized by exaggerated TLR4 signaling and decreased enterocyte proliferation through unknown mechanisms. Given the importance of β-catenin in regulating proliferation of many cell types, we hypothesize that TLR4 impairs enterocyte proliferation in NEC via impaired β-catenin signaling. Methods: Enterocyte proliferation was detected in IEC-6 cells or in ileum or colon from wild-type, TLR4-mutant, or TLR4-/- mice after induction of NEC or endotoxemia. β-Catenin signaling was assessed by cell fractionation or immunoconfocal microscopy to detect its nuclear translocation. Activation and inhibition of β-catenin were achieved via cDNA or small interfering RNA, respectively. TLR4 in the intestinal mucosa was inhibited with adenoviruses expressing dominant-negative TLR4. Results: TLR4 activation significantly impaired enterocyte proliferation in the ileum but not colon in newborn but not adult mice and in IEC-6 enterocytes. β-Catenin activation reversed these effects in vitro. To determine the mechanisms involved, TLR4 activation phosphorylated the upstream inhibitory kinase GSK3β, causing β-catenin degradation. NEC in both mouse and humans was associated with decreased β-catenin and increased mucosal GSK3β expression. Strikingly, the inhibition of enterocyte β-catenin signaling in NEC could be reversed, and enterocyte proliferation restored, through adenoviral-mediated inhibition of TLR4 signaling in the small intestinal mucosa. Conclusion: We now report a novel pathway linking TLR4 with inhibition of β-catenin signaling via GSK3β activation, leading to reduced enterocyte proliferation in vitro and in vivo. These data provide additional insights into the pathogenesis of diseases of intestinal inflammation such as NEC.
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