title: Endotoxin inhibits intestinal epithelial restitution through activation of Rho-GTPase and increased focal adhesions.
abstract: Diseases of gut inflammation such as neonatal necrotizing enterocolitis (NEC) result after an injury to the mucosal lining of the intestine, leading to translocation of bacteria and endotoxin (lipopolysaccharide). Intestinal mucosal defects are repaired by the process of intestinal restitution, during which enterocytes migrate from healthy areas to sites of injury. In an animal model of NEC, we determined that intestinal restitution was significantly impaired compared with control animals. We therefore sought to determine the mechanisms governing enterocyte migration under basal conditions and after an endotoxin challenge. Here we show that the cytoskeletal reorganization and stress fiber formation required for migration in IEC-6 enterocytes requires RhoA. Enterocytes were found to express the endotoxin receptor Toll-like receptor 4, which served to bind and internalize lipopolysaccharide. Strikingly, endotoxin treatment significantly inhibited intestinal restitution, as measured by impaired IEC-6 cell migration across a scraped wound. Lipopolysaccharide was found to increase RhoA activity in a phosphatidylinositol 3-kinase-dependent manner, leading to an increase in phosphorylation of focal adhesion kinase and an enhanced number of focal adhesions. Importantly, endotoxin caused a progressive, RhoA-dependent increase in cell matrix tension/contractility, which correlated with the observed impairment in enterocyte migration. We therefore conclude that endotoxin inhibits enterocyte migration through a RhoA-dependent increase in focal adhesions and enhanced cell adhesiveness, which may participate in the impaired restitution observed in experimental NEC.
authors.affiliation: Division of Pediatric Surgery, Department of Surgery, Children's Hospital of Pittsburgh and University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: Selma
authors.initials: S
authors.lastname: Cetin
authors.forename: Henri R
authors.initials: HR
authors.lastname: Ford
authors.forename: Laura R
authors.initials: LR
authors.lastname: Sysko
authors.forename: Charu
authors.initials: C
authors.lastname: Agarwal
authors.forename: James
authors.initials: J
authors.lastname: Wang
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Neal
authors.forename: Catherine
authors.initials: C
authors.lastname: Baty
authors.forename: Gerard
authors.initials: G
authors.lastname: Apodaca
authors.forename: David J
authors.initials: DJ
authors.lastname: Hackam
mesh_terms: D000818:Animals; D002448:Cell Adhesion; D002460:Cell Line; D002465:Cell Movement; D002467:Cell Nucleus / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004731:Endotoxins / Q000378:metabolism*; D020895:Enterocytes / Q000378:metabolism; D004848:Epithelium / Q000378:metabolism*; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D022001:Focal Adhesions; D007249:Inflammation; D008070:Lipopolysaccharides / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism; D013997:Time Factors; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014162:Transfection; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D004731:Endotoxins; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; C495347:Tlr4 protein, rat; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; C496052:Ptk2 protein, rat; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein