Gastroenterology

Gastroenterology

Volume 138, Issue 1, January 2010, Pages 185-196
Gastroenterology

Basic—Alimentary Tract
Toll-Like Receptor-4 Inhibits Enterocyte Proliferation via Impaired β-Catenin Signaling in Necrotizing Enterocolitis

https://doi.org/10.1053/j.gastro.2009.09.045Get rights and content

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.

Section snippets

Cell Culture, Mice, and Reagents

IEC-6 cells enterocytes were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were maintained as described.7 Immortalized primary murine embryonic fibroblasts were isolated from 13-day-old TLR4–wild-type and TLR4-mutant embryos and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine, and 10 μg blasticidin/mL (InvivoGen, San Diego, CA). LPS (99% pure) was purified from Escherichia coli O127:B8 by gel filtration

TLR4 Activation Inhibits Enterocyte Proliferation In Vitro and In Vivo

We first sought to assess the effects of TLR4 activation with LPS on enterocyte proliferation in vitro and in vivo. As shown in Figure 1, LPS caused a dose-dependent reduction in the extent of enterocyte proliferation, as determined by colorimetric XTT assay (Figure 1A), and reduced expression of the cell proliferation marker pCNA by SDS-PAGE (Figure 1B) and confocal microscopy (note the loss of green pCNA staining with increased concentration of LPS in Figure 1C). There was no effect of LPS on

Discussion

We now describe the identification of a novel and potentially important link between TLR4 signaling and activation of the AKT-GSK3β pathway leading to the inhibition of enterocyte proliferation, an effect that plays a role in the pathogenesis of NEC. The current findings are consistent with previous work indicating that enterocyte proliferation may be inhibited after endotoxin exposure,18, 19 yet extend these observations by providing both mechanistic insights and physiologic relevance. In

Acknowledgments

C.P.S. and X.S. contributed equally to this work.

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    Conflicts of interest The authors disclose no conflicts.

    Funding D.J.H. is supported by R01GM078238 and RO1DK08752 from the National Institutes of Health, and from The Hartwell Foundation.

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