Chemokines, a large family of small chemoattractive cytokines, and their receptors play an integral role in the regulation of the immune response and homeostasis. The ability of chemokines to attract specific populations of immune cells sets them apart from other chemoattractants. Chemokines produced within the gastrointestinal mucosa, are critical players in directing the balance between physiological and pathophysiological inflammation in health, inflammatory bowel disease and the progression to colon cancer. In addition to the well-characterized role of chemokines in directed trafficking of immune cells to the gut mucosa, the expression of chemokine receptors on the cells of the epithelium makes them active participants in the chemokine signaling network. Recent findings demonstrate an important role for chemokines and chemokine receptors in epithelial barrier repair and maintenance as well as an intricate involvement in limiting metastasis of colonic carcinoma. Increased recognition of the association between barrier defects and inflammation and the subsequent progression to cancer in inflammatory bowel disease thus implicates chemokines as key regulators of mucosal homeostasis and disease pathogenesis.

Intestinal inflammation is exacerbated by defects in the epithelial barrier and subsequent infiltration of microbes and toxins into the underlying mucosa. Production of chemokines and antimicrobial peptides by an intact epithelium provide the first line of defense against invading organisms. In addition to its antimicrobial actions, human beta defensin-2 (HBD2) may also stimulate the migration of dendritic cells through binding the chemokine receptor CCR6. As human colonic epithelium expresses CCR6, we investigated the potential of HBD2 to stimulate intestinal epithelial migration. Using polarized human intestinal Caco2 and T84 cells and non-transformed IEC6 cells, HBD2 was equipotent to CCL20 in stimulating migration. Neutralizing antibodies confirmed HBD2 and CCL20 engagement to CCR6 were sufficient to induce epithelial cell migration. Consistent with restitution, motogenic concentrations of HBD2 and CCL20 did not induce proliferation. Stimulation with those CCR6 ligands leads to calcium mobilization and elevated active RhoA, phosphorylated myosin light chain, and F-actin accumulation. HBD2 and CCL20 were unable to stimulate migration in the presence of either Rho-kinase or phosphoinositide 3-kinase inhibitors or an intracellular calcium chelator. Together, these data indicate that the canonical wound healing regulatory pathway, along with calcium mobilization, regulates CCR6-directed epithelial cell migration. These findings expand the mechanistic role for chemokines and HBD2 in mucosal inflammation to include immunocyte trafficking and killing of microbes with the concomitant activation of restitutive migration and barrier repair.

Background & Aims-CXCL12 and CXCR4 signaling plays critical roles in development, homeostasis, and tumor metastasis. Previously we have shown that epigenetic silencing of CXCL12 in colorectal and mammary carcinoma promotes metastasis. Anoikis is an essential process of colonic epithelial turnover and limits the metastatic progression of carcinoma. We sought to determine the role for anoikis in limiting tumor metastasis upon re-expression of CXCL12 in human colorectal carcinoma cells.

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...

Barrier defects and/or alterations in the ability of the gut epithelium to repair itself are critical etiologic mechanisms of gastrointestinal disease. Our ongoing studies indicate that the chemokine receptor CXCR4 and its cognate ligand CXCL12 regulate intestinal epithelial barrier maturation and restitution in cell culture models. Gene deficient mice lacking CXCR4 expression specifically by the cells of the intestinal epithelium were used to test the hypothesis that CXCR4 regulates mucosal barrier integrity in vivo. Epithelial expression of CXCR4 was assessed by RT-PCR, Southern blot, Western blot and immunohistochemistry. In vivo wounding assays were performed by addition of 3% Dextran Sodium Sulfate in drinking water for 5 days. Intestinal damage and DAI scores were assessed by histological examination. ERK phosphorylation was assessed in vivo by immunoblot and immunofluorescence. CXCR4 knockdown cells were established using a lentiviral approach and ERK phosphorylation was assessed. Consistent with targeted roles in restitution, epithelium from patients with inflammatory bowel disease indicated that CXCR4 and CXCL12 expression was stable throughout the human colonic epithelium. Conditional CXCR4-deficient mice developed normally, with little phenotypic differences in epithelial morphology, proliferation, or migration. Re-epithelialization was absent in CXCR4 conditional knockout mice following acute dextran-sodium sulfate-induced inflammation. In contrast, heterozygous CXCR4 depleted mice displayed significant improvement in epithelial ulcer healing in acute and chronic inflammation. Mucosal injury repair was correlated with extracellular-regulated kinase (ERK)-1/2 activity and localization along the crypt-villus axis, with heterozygous mice characterized by increased ERK1/2 activation. Lentiviral depletion of CXCR4 in IEC6 cells similarly altered ERK1/2 activity and prevented chemokine stimulated migration. Together these data indicate that chemokine receptors participate in epithelial barrier responses through coordination of the ERK1/2 signaling pathway.