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