Calcium Mobilization Triggered by the Chemokine CXCL12 Regulates Migration in Wounded Intestinal Epithelial Monolayers

Abstract

Restitution of intestinal epithelial barrier damage involves the coordinated remodeling of focal adhesions in actively migrating enterocytes. Defining the extracellular mediators and the intracellular signaling pathways regulating those dynamic processes is a key step in developing restitution-targeted therapies. Previously we have determined that activation of the chemokine receptor CXCR4 by the cognate ligand CXCL12 enhances intestinal epithelial restitution through reorganization of the actin cytoskeleton. The aim of these studies was to investigate the role of calcium effectors in CXCL12-mediated restitution. CXCL12 stimulated release of intracellular calcium in a dose-dependent manner. Inhibition of intracellular calcium flux impaired CXCL12-mediated migration of IEC-6 and CaCo2 cells. Pharmacological blockade and specific shRNA depletion of the phospholipase-C (PLC␤3) isoform attenuated CXCL12-enhanced migration, linking receptor activation with intracellular calcium flux. Immunoblot analyses demonstrated CXCL12 activated the calcium-regulated focal adhesion protein proline-rich tyrosine kinase-2 (Pyk2) and the effector proteins paxillin and p130Cas . Interruption of Pyk2 signaling potently blocked CXCL12-induced wound closure. CXCL12-stimulated epithelial cell migration was enhanced on laminin and abrogated by intracellular calcium chelation. These results suggest CXCL12 regulates restitution through calcium-activated Pyk2 localized to active focal adhesions. Calcium signaling pathways may therefore provide a novel avenue for enhancing barrier repair.A battery of molecules stimulate epithelial restitution in vitro and in vivo, including cytokines, luminal peptides, probiotics, and as we have reported, chemokines (1-11). Restitution in vivo is dependent on several factors absent from cellculture model systems, including mucin-producing goblet cells, extracellular matrix-producing fibroblasts, immune cells, and the luminal microbiota. Within that complex environment, deletion of genes specifically within the intestinal epithelium has proven useful in deciphering roles for transforming growth factor-␤1 (TGF-␤1) 3 receptor, epidermal growth factor receptor, cadherin, laminin, and Vav in integrity and repair of the gut mucosa (12-16). More recent reports have begun to link mucosal fibroblasts and T cells with key roles in injury repair (17)(18)(19)(20)(21)(22). Despite these findings, the mechanisms by which those molecules elicit their functions, in either reductionist cell-culture models, or complex in vivo systems, remain incompletely characterized.Chemokines are abundantly and ubiquitously produced host defense molecules that participate in activation and directional trafficking of leukocytes. The chemokine receptors CXCR4, CCR5, CCR6, and CX 3 CR1 are expressed by the cells of the human intestinal epithelium (23-26). Chemokines produced by intestinal epithelial cells play an important role in orchestrating physiological and pathological inflammation, consistent with a role in amplifying intestinal inflamm...

Chemokine stimulation promotes enterocyte migration through laminin-specific integrins

“…In vitro, basal restitution and cell adhesion are enhanced by the presence of ECM proteins, with collagen isoforms promoting the highest migration and adhesion followed by laminin and fibronectin (4). Furthermore, the intrinsic migration potential inherent to restitution is increased when intestinal epithelial cells are cultured on laminin (1). Together, these data support a model in which ECM proteins regulate migration through outside-in integrin signaling.…”

“…We have previously verified that restitution of model intestinal epithelia is increased on laminin, a migration pattern that was further enhanced following stimulation with CXCL12, but not TGF-␤1 (1). In addition, CXCL12-induced migration was dependent on intracellular calcium (1). CXCL12 stimulation enhances basal restitution and therefore could be a potential therapeutic for intestinal wound repair.…”

“…The nontransformed rat small intestinal epithelial cell line IEC-6 was purchased and maintained as previously described (1,56). The human colonic carcinoma cell line Caco2 subclone BBe (Caco2-BBe) was a kind gift from Dr. Jerrold Turner (University of Chicago) and was maintained in DMEM (4 g/l glucose) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (Omega Scientific, Tarzana, CA), 2 mM L-glutamine, and 15 mM HEPES (Invitrogen).…”

“…Restitution occurs within minutes to hours after injury and is therefore minimally influenced by proliferation (32,42). This inherent ability to migrate across the injured mucosa, a process we classify as basal restitution, can be increased in vitro by multiple growth factors including transforming growth factor (TGF)-␤1, epidermal growth factor (EGF), trefoil factor (4,12,15,41,45) and, as we have shown, the chemokines CXCL12 and CCL20 (1,43,56,61). Restitution occurs along the exposed and newly deposited basement membrane (32,45) and requires dynamic cellular adhesion and cytoskeletal remodeling within the moving enterocyte (10,39,44,50).…”

“…Together, these data support a model in which ECM proteins regulate migration through outside-in integrin signaling. Although an abundance of data indicate that extracellular stimulants enhance epithelial migration (1,4,12,15,41,43,45,56,61), roles for those effectors in inside-out regulation of integrins and cell-matrix interactions during restitution remain poorly characterized.…”