Toll-like receptor 4 is expressed on intestinal stem cells and regulates their proliferation and apoptosis via the p53 up-regulated modulator of apoptosis

J Biol Chem. 2012 Oct 26;287(44):37296-308. doi: 10.1074/jbc.M112.375881. Epub 2012 Sep 6.


Factors regulating the proliferation and apoptosis of intestinal stem cells (ISCs) remain incompletely understood. Because ISCs exist among microbial ligands, immune receptors such as toll-like receptor 4 (TLR4) could play a role. We now hypothesize that ISCs express TLR4 and that the activation of TLR4 directly on the intestinal stem cells regulates their ability to proliferate or to undergo apoptosis. Using flow cytometry and fluorescent in situ hybridization for the intestinal stem cell marker Lgr5, we demonstrate that TLR4 is expressed on the Lgr5-positive intestinal stem cells. TLR4 activation reduced proliferation and increased apoptosis in ISCs both in vivo and in ISC organoids, a finding not observed in mice lacking TLR4 in the Lgr5-positive ISCs, confirming the in vivo significance of this effect. To define molecular mechanisms involved, TLR4 inhibited ISC proliferation and increased apoptosis via the p53-up-regulated modulator of apoptosis (PUMA), as TLR4 did not affect crypt proliferation or apoptosis in organoids or mice lacking PUMA. In vivo effects of TLR4 on ISCs required TIR-domain-containing adapter-inducing interferon-β (TRIF) but were independent of myeloid-differentiation primary response-gene 88 (MYD88) and TNFα. Physiological relevance was suggested, as TLR4 activation in necrotizing enterocolitis led to reduced proliferation and increased apoptosis of the intestinal crypts in a manner that could be reversed by inhibition of PUMA, both globally or restricted to the intestinal epithelium. These findings illustrate that TLR4 is expressed on ISCs where it regulates their proliferation and apoptosis through activation of PUMA and that TLR4 regulation of ISCs contributes to the pathogenesis of necrotizing enterocolitis.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adaptor Proteins, Vesicular Transport / metabolism
  • Animals
  • Apoptosis Regulatory Proteins / genetics
  • Apoptosis Regulatory Proteins / metabolism*
  • Apoptosis Regulatory Proteins / physiology
  • Apoptosis*
  • Cell Proliferation*
  • Enterocolitis, Necrotizing / metabolism
  • Enterocolitis, Necrotizing / pathology
  • Gene Knockout Techniques
  • Ileum / pathology
  • Intestinal Mucosa / immunology
  • Intestinal Mucosa / metabolism
  • Intestinal Mucosa / pathology*
  • Lipopolysaccharides / pharmacology
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Myeloid Differentiation Factor 88 / metabolism
  • Rats
  • Receptors, G-Protein-Coupled / metabolism
  • Receptors, Tumor Necrosis Factor, Type I / genetics
  • Receptors, Tumor Necrosis Factor, Type I / metabolism
  • Signal Transduction
  • Stem Cells / immunology
  • Stem Cells / metabolism*
  • Stem Cells / physiology
  • Toll-Like Receptor 4 / genetics
  • Toll-Like Receptor 4 / metabolism*
  • Transcriptional Activation
  • Tumor Necrosis Factor-alpha / genetics
  • Tumor Necrosis Factor-alpha / metabolism
  • Tumor Suppressor Proteins / genetics
  • Tumor Suppressor Proteins / metabolism*
  • Tumor Suppressor Proteins / physiology


  • Adaptor Proteins, Vesicular Transport
  • Apoptosis Regulatory Proteins
  • Lgr5 protein, mouse
  • Lipopolysaccharides
  • Myd88 protein, mouse
  • Myeloid Differentiation Factor 88
  • PUMA protein, mouse
  • Receptors, G-Protein-Coupled
  • Receptors, Tumor Necrosis Factor, Type I
  • TICAM-1 protein, mouse
  • Tlr4 protein, mouse
  • Tnfrsf1a protein, mouse
  • Toll-Like Receptor 4
  • Tumor Necrosis Factor-alpha
  • Tumor Suppressor Proteins