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P. K. Russ, S.-H. Presley, R. Haselton, M. Chang; Tight Junction Associated RhoA Signaling Through Modulation of GEF-H1 in TM Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6094.
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While TM cells in vivo do not form a significant barrier to fluid flow, they do express tight junction (TJ) molecules. Blood Vessel Epicardial Substance (Bves) is an adhesion molecule that regulates TJ formation. In addition to their role in cell-cell adhesion, TJs also modulate RhoA signaling, which has been implicated in outflow regulation. We hypothesize that TJs in TM cells regulate RhoA signaling through TJ sequestration of GEF-H1, an activator of RhoA.
TM cell lines from non-glaucomatous donors (NTM-5) and NTM-5 cells transfected to over-express Bves (NTM-w) were evaluated for TJ formation. Western blotting and immunolocalization of TJ proteins were performed in the two cell lines. Levels of active Rho were detected using FRET probes, and phosphorylated myosin light chain (MLC-p), a downstream target of RhoA, was assessed by Western blot.
Overexpression of Bves leads to increased TJ formation in NTM-5 cells. Increased TJ formation was confirmed by increased TJ protein expression. Immunostaining of TJ proteins ZO-1, occludin, and GEF-H1 appears increased at cell-cell junctions in NTM-w cells compared to NTM-5 cells. NTM-w cells also exhibited decreased levels of active RhoA and lower levels of MLC-p compared to NTM-5 cells. These findings are in agreement with the role of GEF-H1 in RhoA signaling.
Increased Bves in TM cells leads to increased TJ formation and increased sequestration of GEF-H1 at cell-cell junctions, leading to decreased active RhoA. In TM tissue, RhoA has been implicated in outflow regulation. Therefore, Bves working through GEF-H1 sequestration at TJs potentially serves as a key regulatory molecule in aqueous outflow.
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