Previous investigations indicate that RhoGTP activation leading to regulation of MLC, with the cooperation of ROCK and MLCK, results in actomyosin bundling and F-actin accumulation and epithelial sheet migration. Although these key effectors are known to regulate, in part, epithelial migration, the actual cellular and biochemical mechanisms whereby extracellular immune mediators regulate those constituents to maintain and repair the innate epithelial barrier are not well understood.37-39
Further, while the chemokine receptor CXCR4 has been shown to participate in epithelial sheet migration in embryonic development,4,61
the cellular mechanisms regulating those processes have yet to be elucidated. Our data describe for the first time the cellular signaling mechanism for CXCR4-facilitated sheet migration in model epithelium and establish the CXCR4-CXCL12 signaling axis as a novel extracellular activator of the canonical wound healing pathway. Epithelial restitution of model epithelia, together with our previous reports demonstrating a role for CXCR4-CXCL12 signaling in epithelial chemotaxis28
of individual colonic carcinoma cells in vivo
, implicate this chemokine receptor-chemokine ligand pair in the health and homeostasis of the intestinal barrier.
Migratory processes of single cells have been well studied (reviewed in Sheetz et al62
) and are consistent with other reports showing that in leukocytes RhoGTPase regulates CXCR4-mediated chemotaxis or metastasis to sites of CXCL12 production.42,44
Our data demonstrate for the first time that CXCR4 signals RhoGTP in model intestinal epithelial cells and expand upon those reports by demonstrating CXCR4-mediated activation of Rho localization at the leading edge of migrating IEC-6 epithelial sheets.41,63
Recapitulation of these data in human polarized T84 monolayers demonstrates RhoGTP localization to the leading edge is not an artifact of either the rat IEC-6 cell line or the straight edge scrape wound. Since RhoGTP is known to be required for membrane protrusions in intestinal cell lines,64
we postulate CXCR4-mediated activation and localization of Rho at the leading edge of restitutive epithelial cells may facilitate formation of lamellae required for wound healing. As CXCR4 signaling is multifactorial and this receptor can signal through other GTPases such as Rac,65
we acknowledge that additional RhoGTPases, or down stream actin regulatory factors may also participate in CXCR4-mediated epithelial restitution.
Utilization of the pharmacological inhibitors Y27623 and Drev-PIK indicate that ROCK and MLCK are involved in CXCR4-mediated wound healing. ROCK is a well-characterized downstream effector of RhoGTP known to facilitate actomyosin bundling and contraction via phosphorylation of MLC, either directly or indirectly by inhibiting MYPT1.54
MLCK is the major kinase responsible for phosphorylation of MLC in intestinal cells41,57
and works in conjunction with MYPT1 to regulate activation of MLC. We determined that CXCL12 leads to MLC phosphorylation in IEC-6 cells and show that inhibition of either ROCK or MLCK abolished CXCR4-mediated wound healing to control levels. Our data are consistent with the notion that CXCL12 stimulates MLC phosphorylation via ROCK and MLCK, which, in turn, promotes restitution by facilitating accumulation of F-actin.
Studies using the specific CXCR4 inhibitor AMD3100 showed that CXCR4 stimulates actin polymerization in IEC-6 cells. This increase in F-actin accumulation at the wound edge of IEC-6 and T84 cells correlates with increased cell migration and parallels previous data showing F-actin rearrangement to the leading edge a hallmark event in epithelial wound healing.37,40,66
CXCL12-mediated accumulation of F-actin is activated after 20 min and parallels the activation of RhoGTP and pMLC. However, we also observed significant F-actin accumulation in CXCL12-stimulated monolayers 18 h after chemokine addition, a time point at which we show CXCL12 no longer activates pMLC suggesting that CXCR4-mediated F-actin accumulation may be regulated by more than one mechanism. For example, in leukocytes CXCR4 signals F-actin accumulation and migration via the LIM-kinase pathway,65
suggesting that LIMK and in turn cofilin-mediated inhibition of F-actin depolymerization67
may also regulate chemokine-mediated epithelial restitution.
Inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, radiation injury and enterocolitis have long been associated with epithelial permeability defects.68-70
Furthermore, mucosal healing is thought to be a major defect for people undergoing steroidal treatment for inflammatory bowel disease.71
The epithelium must be able to heal wounds rapidly that constantly afflict the intestine to prevent permeability defects and maintain health of this important innate immune barrier. Therefore, factors that contribute to wound healing are of clinical importance as possible therapeutics for permeability homeostasis. An array of cytokines and a subset of particular chemokines are upregulated during and participate in immunocyte infiltration into the gut mucosa.32,33,72
Notably an array of inflammatory chemokines including CXCL8, CCL2, and CCL5 are elevated in Crohn's disease and ulcerative colitis and likely direct the trafficking of monocytes and T lymphocytes into the gut mucosa.29-34
Mice deficient in the chemokine receptors CCR5,73
one of the receptors for CCL5, or alternatively, concomitant blockade of the chemokine receptors CCR2, receptor for CCL2, CCR5 and CXCR3 confers mucosal protection in dextran sodium sulfate-induced murine colitis through abrogation of leukocyte trafficking to the gut mucosa.36
Moreover, a recent report implicates gut T lymphocytes in exacerbating epithelial barrier defects and worsening gut inflammation in a murine model of colitis.74,75
Those reports ascribe a role for chemokines limited to the directed infiltration of the damage-provoking or, alternatively damage-exacerbating, immune cells into the gut mucosa in inflammatory bowel disease. However, our data endorse expanding that model and suggest a role for the immune surveillance chemokine and chemokine receptor pair CXCL12 and CXCR4 in the mucosal restitution steps subsequent to inflammatory injury.