Loss of FFAR2 promotes colon cancer by epigenetic dysregulation of inflammation suppressors.

Free fatty acid receptor 2 (FFAR2, also named GPR43), is activated by short-chain fatty acids (SCFAs), such as butyrate, that are produced when gut bacteria ferment dietary fiber. FFAR2 has been suggested to regulate colonic inflammation, which is a major risk factor for the development of colon cancer and is also linked to epigenetic dysregulation in colon carcinogenesis. The current study assessed whether FFAR2, acting as an epigenetic regulator, protects against colon carcinogenesis. To mimic the mild inflammation that promotes human colon cancer, we treated mice with dextran sodium sulfate (DSS) overnight, which avoids excessive inflammation but induces mild inflammation that promotes colon carcinogenesis in the ApcMin/+ and the azoxymethane (AOM)-treated mice. Our results showed that FFAR2 deficiency promotes the development of colon adenoma in the ApcMin/+ /DSS mice and the progression of adenoma to adenocarcinoma in the AOM/DSS mice. FFAR2's downstream cAMP-PKA-CREB pathway was enhanced, leading to overexpression of histone deacetylases (HDACs) in the FFAR2-deficient mice. ChIP-qPCR analysis revealed differential binding of H3K27me3 and H3K4me3 histone marks onto the promoter regions of inflammation suppressors (e.g., sfrp1, dkk3, socs1), resulting in decreased expression of these genes in the FFAR2-deficient mice. Also, more neutrophils infiltrated into tumors and colon lamina propria of the FFAR2-deficient mice. Depletion of neutrophils blocked the progression of colon tumors. In addition, FFAR2 is required for butyrate to suppress HDAC expression and hypermethylation of inflammation suppressors. Therefore, our results suggest that FFAR2 is an epigenetic tumor suppressor that acts at multiple stages of colon carcinogenesis.

E-cadherin is critical for collective sheet migration and is regulated by the chemokine CXCL12 protein during restitution.

Chemokines and other immune mediators enhance epithelial barrier repair. The intestinal barrier is established by highly regulated cell-cell contacts between epithelial cells. The goal of these studies was to define the role for the chemokine CXCL12 in regulating E-cadherin during collective sheet migration during epithelial restitution. Mechanisms regulating E-cadherin were investigated using Caco2(BBE) and IEC-6 model epithelia. Genetic knockdown confirmed a critical role for E-cadherin in in vitro restitution and in vivo wound repair. During restitution, both CXCL12 and TGF-ß1 tightened the monolayer by decreasing the paracellular space between migrating epithelial cells. However, CXCL12 differed from TGF-ß1 by stimulating the significant increase in E-cadherin membrane localization during restitution. Chemokine-stimulated relocalization of E-cadherin was paralleled by an increase in barrier integrity of polarized epithelium during restitution. CXCL12 activation of its cognate receptor CXCR4 stimulated E-cadherin localization and monolayer tightening through Rho-associated protein kinase activation and F-actin reorganization. These data demonstrate a key role for E-cadherin in intestinal epithelial restitution.

Calcium mobilization triggered by the chemokine CXCL12 regulates migration in wounded intestinal epithelial monolayers.

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 (PLCbeta3) 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 p130(Cas). 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.

Chemokine stimulation promotes enterocyte migration through laminin-specific integrins.

Intestinal homeostasis is regulated in part by the single cell layer of the mucosal epithelium. This physical barrier is a prominent part of the innate immune system and possesses an intrinsic ability to heal damage and limit infection. The restitutive epithelial migration phase of healing requires dynamic integrin adhesion to the extracellular matrix. Previously, we have shown that the homeostatic chemokine CXCL12 utilizes intracellular calcium to increase enterocyte migration on laminin. The aim of these studies was to investigate integrin specificity and, in turn, functional responses elicited by CXCL12 stimulation. Analysis of cellular adhesion and spreading revealed CXCL12 preferentially activated laminin-specific integrins compared with collagen IV-binding integrins. Laminin-specific cell adhesion and spreading elicited by CXCL12 was dependent on intracellular calcium. CXCL12 increased activated ß1-integrins on the surface of epithelial cells compared with untreated cells. RT-PCR confirmed expression of the laminin-binding integrins-α3ß1, -α6ß1, and -α6ß4. Interestingly, shRNA-mediated depletion of laminin-specific α3- or α6-integrin subunits revealed differential functions. α3-Integrin knockdown reduced basal as well as inducible restitution. Depletion of α6-integrin specifically abolished CXCL12-stimulated, but not TGF-ß1 or basal, migration. Depletion with either shα3-integrin or shα6-integrin prevented CXCL12-evoked cell spreading. Our data indicate that CXCL12 stimulates the inside-out activation of laminin-specific integrins to promote cell migratory functions. Together, our findings support the notion that extracellular mediators within the gastrointestinal mucosa coordinate cell-matrix interactions during epithelial restitution.