Cellular metastasis is the most detrimental step in carcinoma disease progression, yet the mechanisms that regulate this process are poorly understood. CXCL12 and its receptor CXCR4 are co-expressed in several tissues and cell types throughout the body and play essential roles in development. Disruption of either gene causes embryonic lethality due to similar defects. Post-natally, CXCL12 signaling has a wide range of effects on CXCR4-expressing cells, including the directed migration of leukocytes, lymphocytes and hematopoietic stem cells. Recently, this signaling axis has also been described as an important regulator of directed carcinoma cell metastasis. We show herein that while CXCR4 expression remains consistent, constitutive colonic epithelial expression of CXCL12 is silenced by DNA hypermethylation in primary colorectal carcinomas as well as colorectal carcinomaderived cell lines. Inhibition of DNA methyltransferase (Dnmt) enzymes with 5-aza-2 0 -deoxycytidine or genetic ablation of both Dnmt1 and Dnmt3b prevented promoter methylation and restored CXCL12 expression. Re-expression of functional, endogenous CXCL12 in colorectal carcinoma cells dramatically reduced metastatic tumor formation in mice, as well as foci formation in soft agar. Decreased metastasis was correlated with increased caspase activity in cells re-expressing CXCL12. These data constitute the unique observation that silencing CXCL12 within colonic carcinoma cells greatly enhances their metastatic potential.

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.

The worldwide emergence of epidemic strains of Clostridium difficile linked to increased disease severity and mortality has resulted in greater research efforts toward determining the virulence factors and pathogenesis mechanisms used by this organism to cause disease. C. difficile is an opportunist pathogen that employs many factors to infect and damage the host, often with devastating consequences. This review will focus on the role of the 2 major virulence factors, toxin A (TcdA) and toxin B (TcdB), as well as the role of other putative virulence factors, such as binary toxin, in C. difficile-mediated infection. Consideration is given to the importance of spores in both the initiation of disease and disease recurrence and also to the role that surface proteins play in host interactions.

Chemokines are critical regulatory factors that direct migration, proliferation and maturation of receptor expressing target cells within gut mucosa. The aim of the present study was to define the cellular mechanisms whereby engagement of the essential chemokine CXCL12 to CXCR4 regulates restitutive epithelial cell migration. Non-transformed IEC-6 cells or polarized T84 epithelial monolayers were wounded and F-actin accumulation assessed using fluorescence microscopy and flow cytometry. Immunoblot analysis, pull-down assays, fluorescence microscopy and wound healing assays defined activation of Rho, Rho-kinase (ROCK), and myosin light chain (MLC) and the role for those Rho effectors in CXCL12-regulated epithelial restitution. CXCL12 increased RhoGTP and F-actin localization to the leading edge of wounded IEC-6 and T84 monolayers. CXCL12 congruently stimulated an increase in active MLC that was inhibited by blockade of ROCK and myosin light chain kinase and regulated epithelial migration. Our data in model intestinal epithelia suggest CXCR4 and CXCL12 may function as an autocrine and paracrine mucosal signaling network regulating the competency of the epithelial barrier to withstand injury and mediate repair following damage.