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M. S. Chang, S.-H. Presley, P. K. Russ; Extracellular Domain of Bves Critical for Human Corneal Epithelial Cell Adhesion. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1971.
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The highly uniform architecture of corneal epithelium is maintained through cell-cell adhesions. Cell adhesion junctions, tight junctions (TJ) and adherens junctions (AJ), are also signaling structures, important in cellular communication. We previously reported that a novel adhesion molecule Bves (blood vessel epicardial substance) regulates TJ and AJ formation. Bves also modulates TJ associated Rho and AJ associated Wnt signaling. However, very little is known regarding how Bves coordinates cell adhesion. We postulate that the extracellular domain of Bves is critical in regulating TJ and AJ formation in corneal epithelial cells.
To test our hypothesis, antibodies against the extracellular domain of Bves were produced. In addition, human corneal epithelial (HCE) cells were stably transfected to express a mutant Bves without the extracellular domain (N41). Normal HCE cells were cultured in the presence of Bves antibodies against either the extracellular (EC) or intracellular (IC) domain. Antibodies were applied to cell cultures at both low and full confluence. N41 cell growth, morphology, and adhesion formation were compared to normal and cells transfected with an empty vector.
HCE cells treated with IC antibodies exhibit no changes in growth, morphology, or cell behavior compared to untreated HCE cells. However, EC antibodies treatment induced delayed monolayer formation in a dose dependent manner. Protein levels of occludin and e-cadherin were reduced, indicating decreased formation of TJs and AJs. These findings are similar to the N41 cells, which also exhibited delayed monolayer formation and decreased occludin and e-caherin levels. EC antibody exposure in fully confluent cultures induced HCE cells to become less uniform. Immunofluorescent localization analyses revealed that cells within the monolayer were dissociating, as indicated by changes in staining for AJ and TJ proteins. E-cadherin, β-catenin, ZO-1, and occludin staining in IC antibody treated cultures were observed at the cell membrane, outlining borders of adherent cells. With EC antibody exposure, cell borders are split, and localization of these proteins are seen at cell membranes that have dissociated from each other.
These finding indicate that the extracellular domain of Bves is critical for both initiation and maintenance of TJs and AJs formation in HCE cells. Furthermore, these findings are suggestive that Bves acts as a "sensor" protein, allowing cells to detect presence of neighboring cells and regulating cell-cell adhesions.
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