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M. S. Chang, J.-L. Yang, S.-H. Presley, F. R. Haselton; Coordination of Cellular Motility, Proliferation and Differentiation by Bves During Corneal Wound Healing. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5037.
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Coordination of cellular migration, proliferation, and differentiation during corneal wound healing is critical. Tight junctions (TJ) have been shown to be important regulators of these cellular functions by sequestering GEF-H1, an activator of RhoA, and ZONAB, a transcription factor. Evidence is now provided that Bves (blood vessel epicardial substance), a novel adhesion molecule that regulates TJ formation, can also modulate Rho activation and transcriptional activity in a culture model of corneal healing.
Stably transfected clones overexpressing a full length (WTX) and a dominant negative truncated Bves (DN) were generated from a human corneal epithelial cell line (HCE). Clonal isolates were compared for differences in growth rate, cell cycling (flow cytometry), cellular polarization as indicated by TER (transepithelial electrical resistance), RhoA activation (FRET probes), and ZONAB transcription activity (luciferase reporter system). In addition, wounding assays were performed on the confluent WTX, HCE, and DN cultures to assess cell migration and repair of the epithelial monolayer.
In wounding assays, WTX cells "healed" the slowest and DN cells the quickest. WTX and HCE cells exhibited well organized monolayers. However, the WTX monolayers demonstrated increased TJ formation with an approximate doubling of TER compared to HCE. In contrast, the DN cells exhibited a fibroblast like appearance, with impaired monolayer formation and increased motility. There were differences in RhoA activation which may account for differences seen in cellular motility and morphology. RhoA activation was highest in DN cells and lowest in WTX cells. Growth characteristics also differed, with WTX cells displaying the longest doubling time and DN cells the shortest. By flow cytometry, WTX cells appeared to be blocked in S phase. Finally, the transcriptional activity of ZONAB differed, with DN cells exhibiting highest activity and WTX lowest. ZONAB has been shown to regulate transcription of cell cycle activators, including cyclin D and PCNA.
Our findings show that Bves plays a role in monolayer formation during corneal epithelial healing, with increased Bves leading to increased TJ formation. This in turn leads to decreased RhoA activation and decreased ZONAB transcriptional activity. By modulating RhoA and ZONAB activity, cellular motility, proliferation, and differentiation are coordinated within the healing monolayer.
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