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J. Biol. Chem., Vol. 279, Issue 23, 24592-24600, June 4, 2004

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Endotoxin Inhibits Intestinal Epithelial Restitution through Activation of Rho-GTPase and Increased Focal Adhesions^*

Selma Cetin, Henri R. Ford, Laura R. Sysko¶||, Charu Agarwal**, James Wang**, Matthew D. Neal, Catherine Baty¶||, Gerard Apodaca||, and David J. Hackam||¶¶

From the Division of Pediatric Surgery, Department of Surgery, Children's Hospital of Pittsburgh and University of Pittsburgh, Pittsburgh, Pennsylvania 15213 and the ^¶Center of Biologic Imaging, the ^||Department of Cell Biology and Physiology, the ^**Department of Orthopedic Surgery, Department of Surgery, and the Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213

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.

Received for publication, December 12, 2003 , and in revised form, March 5, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by National Institutes of Health (NIH) Grants R01 AI-49473-01 and R01-AI-14032-20.

Supported by NIH Grant RO1 DK51970-06.

^¶¶ Supported by NIH Grant K08 GM65583-01. To whom correspondence should be addressed: Division of Pediatric Surgery, Rm. 4A-486 DeSoto Wing, Children's Hospital of Pittsburgh, 3705 Fifth Ave., Pittsburgh, PA 15213. Tel.: 412-692-8735; Fax: 412-692-8299; E-mail: david.hackam{at}chp.edu.

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