CXCL12 activation of CXCR4 regulates mucosal host defense through stimulation of epithelial cell migration and promotion of intestinal barrier integrity

Abstract

Intestinal epithelial cell migration plays a key role in gastrointestinal mucosal barrier formation, enterocyte development, differentiation, turnover, wound healing, and adenocarcinoma metastasis. Chemokines, through engagement of their corresponding receptors, are potent mediators of directed cell migration and are critical in the establishment and regulation of innate and adaptive immune responses. The aim of this study was to define the role for the chemokine CXCL12 and its sole cognate receptor CXCR4 in regulating intestinal epithelial cell migration and to determine its impact on barrier integrity. CXCL12 stimulated the dose-dependent chemotactic migration of human T84 colonic epithelial cells. Epithelial cell migration was inhibited by CXCR4 neutralizing antibody, pertussis toxin, LY-294002, and PD-98059, thereby implicating Galpha(i), phosphatidylinositol 3-kinase (PI3-kinase), and the ERK1/2 MAP kinase pathways in CXCR4-specific signaling. CXCL12 was also shown to increase barrier integrity, as defined by transepithelial resistance and paracellular flux across differentiating T84 monolayers. To determine whether CXCL12 regulated epithelial restitution, we used the normal nontransformed intestinal epithelial cell-6 (IEC-6) wound healing model. By using RT-PCR, immunoblot analysis, and immunofluorescence microscopy, we first showed expression of both CXCR4 and its ligand by IEC-6 cells. We then demonstrated that CXCL12 activated comparable signaling mechanisms to stimulate epithelial migration in the absence of proliferation in wounded IEC-6 monolayers. Taken together, these data indicate that CXCL12 signaling via CXCR4 directs intestinal epithelial cell migration, barrier maturation, and restitution, consistent with an important mechanistic role for these molecules in mucosal barrier integrity and innate host defense.

Epigenetic silencing of CXCL12 increases the metastatic potential of mammary carcinoma cells

“…Further, decreased calcium flux observed in CXCL12 re-expressing cells correlated with a diminished in vitro chemotactic response to exogenous recombinant CXCL12 (Figure 8c). Consistent with our prior analyses (Smith et al, 2005) calcium flux in 231-luc cells was dependent upon CXCR4 signaling as pretreatment with either CXCR4 antagonist AMD3100 or pertussis toxin markedly decreased the response (data not shown). Our data demonstrating decreased calcium flux and decreased breast cancer cell chemotaxis in response to CXCL12 is supported by previous data in B cells (Brauweiler et al, 2007).…”

“…In agreement with the changes in cell cycle, thymidine incorporation was similarly increased in those CXCL12 re-expressing cells compared to eGFP control cells (Figure 6c). Increased cellular proliferation induced by CXCL12 re-expression in mammary carcinoma cells is in stark contrast to our previous observations in intestinal epithelial cell lines (Smith et al, 2005). Moreover, the apoptotic response we had previously described in HT29 colorectal cancer cells following re-expression of CXCL12 (Wendt et al, 2006) was not observed in mammary carcinoma cells following reestablishment of ligand expression (Figure 6d).…”

“…In agreement with the epithelial cells lining the human gastrointestinal lumen (Agace et al, 2000;Smith et al, 2005;Wendt et al, 2006) CXCL12 was strongly and consistently expressed by epithelial cells lining mammary ducts (Figure 1a). In contrast, immunohistochemical analysis of primary mammary carcinomas revealed CXCL12 was minimally and variably expressed by tumor cells (Figure 1a).…”

“…Protein detection by immunoblot and immunohistochemistry was conducted as previously described (Smith et al, 2005;Wendt et al, 2006). Cell surface CXCR4 was detected using fluorescein isothiocyanate-conjugated antibodies specific for human CXCR4 (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions and quantified by flow cytometry.…”

“…The constitutive chemokine ligand CXCL12 and its cognate receptor CXCR4 are evolutionarily conserved and essential for life in mice (Nagasawa et al, 1996a, b). Homeostatic roles for CXCL12 signaling through CXCR4 in various physiological compartments have been described, including B-cell development, gastrointestinal vascularization, heart and cerebral development, as well as mucosal epithelial wound healing (Nagasawa et al, 1996a;Tachibana et al, 1998;Zou et al, 1998;Heidemann et al, 2003;Smith et al, 2005;Moyer et al, 2007). Thus, CXCL12 and CXCR4 are widely expressed throughout the body and serve diverse physiological roles depending on the tissue or cell type.…”