P-183 Kindlin 1and Kindlin 2 Regulate Adhesion and Migration of Colonic Epithelial Cells

Abstract

The Intestinal epithelial barrier plays an important role in the pathophysiology of inflammatory bowel disease (IBD). Integrity and barrier properties of the intestinal epithelium are determined by specialized adhesive structures such as intercellular junctions and focal adhesions, which respectively mediate cell-cell and cell-matrix interactions. Disassembly of epithelial junctions and matrix adhesion mediates disruption of the gut barrier during acute inflammation, whereas restoration of these adhesion structures is critical for epithelial restitution. Kindlins are unique cytosolic adaptor proteins that are enriched in both epithelial junctions and matrix adhesions. They have multiple binding partners including major classes of integrins and different cytoskeletal regulators. Importantly, mutations in the kindlin-1 gene result in Kindler syndrome, a genetic disorder associated with severe colitis, and deletion of kindlin-2 gene in mice is embryonically lethal. Despite these dramatic phenotypes caused by kindlin dysfunctions, little is known regarding the role of these adaptor proteins in regulation of the intestinal mucosal barrier disruption and restitution. This study was performed using a well-differentiated clone of HT29 human colonic epithelial cell line (HT29cF8). Transient downregulation of kindlin isoforms was achieved using RNA interference. Stable overexpression of kindlins 1 and 2 was achieved using a lentiviral expression system. Cell lines were cultured either on collagen-coated, permeable polycarbonate filters (0.4 mm pore size), or on 6-well plastic plates, for permeability, adhesion and motility assays. Barrier function of epithelial cell monolayers was determined via transepithelial electrical resistance (TEER) measurements Focal adhesions and cytoskeleton were visualized by immunofluorescence labeling and confocal microscopy. Overexpression of either kindlin 1 and/or kindlin 2 resulted in a significant increase in TEER, while siRNA-mediated knockdown of either isoform caused a substantial increase in paracellular permeability. Interestingly, both overexpression and depletion of kindlin 1 attenuated collective cell migration as determined by wound closure assay. By contrast, downregulation of kindlin 2 expression did not affect cell motility. Analysis of cell adhesion and spreading on a collagen matrix revealed significant attenuation of matrix adhesion and spreading of kindlin 1 and kindlin 2 overexpressing cells. These effects correlate well with a marked reduction of the phosphorylated form of focal adhesion protein, paxillin. Additionally, kindlin 1 overexpression regulated an increased level of non-muscle myosin IIB, which is indicative of increased contractile forces. Our data highlights kindlins as important regulators of barrier properties and restitution of human intestinal epithelium.