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J.M. Gonzalez, D.M. P. Peters; Heparin II (Hep II) binding Domain of Fibronectin Disrupts Cell–Cell Junctions in Human Trabecular Meshwork Cells . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4423.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine if the Hep II domain could increase outflow facility in human eye organ cultures by disrupting cadherin/ß–catenin cell–cell junctions in trabecular meshwork cells (HTM). Methods: Serum starved HTM cells and transformed HTM cells (TM–1) were treated for 24 hours with increasing concentrations of a recombinant Hep II domain previously shown to increase outflow facility (Santas, et al., IOVS. 44:4796, 2003). Disruptions in cadherin/ß–catenin cell–cell junctions were determined using both immunofluorescence microscopy and immunoprecipitation techniques. Phalloidin was used to detect the formation of actin filaments while anti–ß–catenin and anti–cadherin antibodies were used to detect the formation of cell–cell junctions. In some experiments, cells were treated with serum (to trigger actin polymerization) or H–7 (a serine threonine kinase inhibitor) for 1 hour prior to treatment with the Hep II domain to determine the signaling mechanism used to regulate cell–cell junctions. Cell attachment assays were also done to detect the effect of the Hep II domain on cell–matrix contacts. Results: Immunofluorescence and immunoprecipitation studies indicated that the Hep II domain disrupted cell–cell junctions and caused a dissociation of the cadherin/ß–catenin complex. Concomitant with the dissociation of the cadherin/ß–catenin complex in the presence of the Hep II domain, a disassembly of actin filaments was also observed. The disruption of the cadherin/ß–catenin complex occurred in a dose–dependent fashion and was reversible when the Hep II domain was removed. Removal of the Hep II domain, however, did not lead to a re–assembly of actin filaments, unless serum was also present. When H–7 was added in the presence of the Hep II domain, it enhanced the effect of the Hep II domain on cell–cell junctions. The concentration of Hep II needed to disrupt cell–cell junctions was much lower than that needed to disrupt cell–matrix contacts and the disruption in cadherin/ß–catenin complexes was not observed in cells lifted off the plate with a dissociation buffer. Conclusions: Treatment of the trabecular meshwork with the Hep II domain could lead to a disruption in cell–cell contacts. The effect on cell–cell contacts was not due to a disruption of cell–matrix contacts and may be mechanistically similar to the treatment of HTM cells with H–7. This could explain how the Hep II domain could enhance outflow facility in human eye organ cultures.
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