Toll-like receptor-4 inhibits enterocyte proliferation via impaired beta-catenin signaling in necrotizing enterocolitis.
OBJECTIVE
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 beta-catenin in regulating proliferation of many cell types, we hypothesize that TLR4 impairs enterocyte proliferation in NEC via impaired beta-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. beta-Catenin signaling was assessed by cell fractionation or immunoconfocal microscopy to detect its nuclear translocation. Activation and inhibition of beta-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. beta-Catenin activation reversed these effects in vitro. To determine the mechanisms involved, TLR4 activation phosphorylated the upstream inhibitory kinase GSK3beta, causing beta-catenin degradation. NEC in both mouse and humans was associated with decreased beta-catenin and increased mucosal GSK3beta expression. Strikingly, the inhibition of enterocyte beta-catenin signaling in NEC could be reversed, and enterocyte proliferation restored, through adenoviral-mediated inhibition of TLR4 signaling in the small intestinal mucosa.


CONCLUSIONS
We now report a novel pathway linking TLR4 with inhibition of beta-catenin signaling via GSK3beta 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.
Division of Pediatric Surgery, Department of Surgery, Children's Hospital of Pittsburgh and University of Pittsburgh, Pittsburgh, Pennsylvania 15224, USA.
Chhinder P
CP
Sodhi
Xia-Hua
XH
Shi
Ward M
WM
Richardson
Zachary S
ZS
Grant
Richard A
RA
Shapiro
Thomas
T
Prindle
Maria
M
Branca
Anthony
A
Russo
Steven C
SC
Gribar
Congrong
C
Ma
David J
DJ
Hackam
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D003106:Colon / Q000473:pathology; D020345:Enterocolitis, Necrotizing / Q000378:metabolism* / Q000473:pathology; D020895:Enterocytes / Q000166:cytology* / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D007082:Ileum / Q000473:pathology; D007231:Infant, Newborn; D007413:Intestinal Mucosa / Q000378:metabolism / Q000473:pathology; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008809:Mice, Inbred C3H; D008817:Mice, Mutant Strains; D015398:Signal Transduction / Q000502:physiology; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D051176:beta Catenin / Q000378:metabolism*
C495267:CTNNB1 protein, mouse; D008070:Lipopolysaccharides; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D051176:beta Catenin; C082161:lipopolysaccharide A; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3