Abstract
Purpose::
Bves (blood vessel epicardial substance) is a novel adhesion molecule which regulates formation of tight junctions (TJ) in human corneal epithelial cells (HCE). When Bves expression is disrupted, HCE undertake a mesenchymal appearance and do not establish cell-cell contacts. These observations led to the hypothesis that Bves regulates epithelial-mesenchymal transition (EMT) in HCE by modulating TJ and associated signaling pathways.
Methods::
To test our hypothesis, stably transfected clones overexpressing a full length (WTX) and dominant negative truncated Bves (DN) were generated from a HCE cell line. Isolates were analyzed for alterations in cytoskeleton (immunofluorescence staining), TJ formation (Westerns), cell growth characteristics (soft agar assay, flow cytometry), cell motility (time lapse microscopy), and cell morphology. In addition, co-immunoprecipitation (IP) studies were performed to determine alterations in the TJ protein interactions. Finally, state of Rho activation was determined using Raichu FRET probes.
Results::
DN had impaired monolayer formation with loss of epithelial morphology. DN also exhibited accelerated cell cycling, increased colony formation in soft agar, and increased cell motility. WTX formed an organized monolayer with decreased cell cycling, decreased soft agar colony formation, and reduced cell motility: essentially opposite of DN. The DN cells expressed vimentin as the main intermediate cytoskeletal filament, while WTX retained cytokeratin expression. These results indicate that DN cells have transformed to a mesenchymal phenotype while WTX remain epithelial. TJ associated proteins, ZO1, cingulin, and GEF-H1, co- immunoprecipitated with Bves in protein isolates from WTX and HCE, but not from DN. There were reduced levels of ZO-1 and cingulin in the DN while levels of GEF-H1 were similar. This indicates less TJ bound GEF-H1, free to active RhoA in DN. This was verified by higer state of RhoA activation in the DN cells as indicated by a 7 fold increase in FRET.
Conclusions::
When Bves function is disrupted, HCE undertake a mesenchymal phenotype as shown by changes in cellular morphology, increased cellular motility, increased proliferation within a 3-D matrix, expression of mesenchymal markers, and loss of TJ formation. The molecular mechanism for Bves in EMT is likely through modulation of Rho activation. These data indicate that Bves may be an important regulator of EMT in epithelial cells.
Keywords: cornea: basic science • cell adhesions/cell junctions • EMT (epithelial mesenchymal transition)