CXCR4 and CXCR7 Mediate TFF3-Induced Cell Migration Independently From the ERK1/2 Signaling Pathway.

Abstract

Trefoil factor family (TFF) peptides, and in particular TFF3, are characteristic secretory products of mucous epithelia that promote antiapoptosis, epithelial migration, restitution, and wound healing. For a long time, a receptor for TFF3 had not yet been identified. However, the chemokine receptor CXCR4 has been described as a low affinity receptor for TFF2. Additionally, CXCR7, which is able to heterodimerize with CXCR4, has also been discussed as a potential TFF2 receptor. Since there are distinct structural similarities between the three known TFF peptides, this study evaluated whether CXCR4 and CXCR7 may also act as putative TFF3 receptors. We evaluated the expression of both CXCR4 and CXCR7 in samples of human ocular surface tissues and cell lines, using RT-PCR, immunohistochemistry, and Western blot analysis. Furthermore, we studied possible binding interactions between TFF3 and the receptor proteins in an x-ray structure-based modeling system. Functional studies of TFF3-CXCR4/CXCR7 interaction were accomplished by cell culture-based migration assays, flow cytometry, and evaluation of activation of the mitogen-activated protein (MAP) kinase signaling cascade. We detected both receptors at mRNA and protein level in all analyzed ocular surface tissues, and in lesser amount in ocular surface cell lines. X-ray structure-based modeling revealed CXCR4 and CXCR7 dimers as possible binding partners to TFF3. Cell culture-based assays revealed enhanced cell migration under TFF3 stimulation in a conjunctival epithelial cell line, which was completely suppressed by blocking CXCR4 and/or CXCR7. Flow cytometry showed increased proliferation rates after TFF3 treatment, while blocking both receptors had no effect on this increase. Trefoil factor family 3 also activated the MAP kinase signaling cascade independently from receptor activity. Dimers CXCR4 and CXCR7 are involved in TFF3-dependent activation of cell migration, but not cell proliferation. The ERK1/2 pathway is activated in the process, but not influenced by CXCR4 or CXCR7. These results implicate a dependence of TFF3 activity as to cell migration on the chemokine receptors CXCR4 and CXCR7 at the ocular surface.