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Defining the intracellular redox‐dependent protein signaling networks between cell‐junctions and focal adhesions during intestinal mucosal repair

Mucosal wounds and inflammation of the human intestine are hallmark features of intestinal bowel disease. It is the coordinated intestinal epithelial cell spreading and migration at the wound edge that facilitates mucosal wound closure in a process known as restitution. During restitution, intestinal epithelial cells spread and migrate as a sheet along focal adhesions, or points of basal cellular contact with the extracellular matrix. Cell‐to‐cell contacts of migrating cells are maintained by junction proteins. As restitution begins, epithelial cells produce reactive oxygen species (ROS) by NOX‐1 enzyme. Over time immune cells infiltrate the inflamed and damaged mucosa and increase the oxidative stress by depositing even larger quantities of ROS around and onto the damaged epithelium, leading to cysteine oxidation of a wide variety of cellular and extracellular proteins. As intestinal epithelial cells spread and migrate as a sheet to close the wound, the cellular junctions and the focal adhesions must somehow communicate through a cross‐talk that maintains directional migration and barrier properties. Little is known about the cross‐talk between cell junctions and focal adhesions, or how ROS regulates this mucosal repair program. We have previously reported redox‐dependent subcellular changes of the focal adhesion protein, Cas, localizing to cellular junctions within the epithelium at the edges of mucosal wounds. We hypothesize those redox‐dependent changes in protein signaling drive the mucosal repair program by cysteine‐oxidation and subsequent alteration in kinase/phosphatase activity that produces changes in cell function, and we have sought to elucidate the profile of this protein network. We show here that epithelial cell Cas localization is indeed dependent on oxidation by ROS generated by immune cells during mucosal repair, and provide evidence that similar redox conditions also regulate the cell border accumulation of AKT and FAK. Remarkably, pharmacological inhibition of FAK increased barrier function. Furthermore, we have employed a novel proteomics technique, called Isotope‐Coded Affinity Tag (ICAT) that quantifies the level of proteome‐wide cysteine oxidation in order to further define these signaling modules. Our analysis has revealed redox‐dependent protein signaling driven by MAPK/PAK2/TLN1/actin cytoskeleton interactions, which might regulate Cas, AKT and FAK localization to junctions during mucosal repair. Collectively, these data shed new light on the redox‐dependent junction/focal adhesion cross‐talk during intestinal restitution.