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Abstract #25186 Published in IGR 12-1
Inhibition of RhoA signaling with increased Bves in trabecular meshwork cells
Russ PK; Kupperman AI; Presley SH; Haselton FR; Chang MSInvestigative Ophthalmology and Visual Science 2010; 51: 223-230
PURPOSE: Blood vessel epicardial substance (Bves) is a novel adhesion molecule that regulates tight junction (TJ) formation. TJs also modulate RhoA signaling, which has been implicated in outflow regulation. Given that Bves has been reported in multiple ocular tissues, the authors hypothesize that Bves plays a role in the regulation of RhoA signaling in trabecular meshwork (TM) cells. METHODS: Human TM cell lines NTM-5 and NTM-5 transfected to overexpress Bves (NTM-w) were evaluated for TJ formation, and levels of occludin, cingulin, and ZO-1 protein were compared. Assays of TJ function were carried out using diffusion of sodium fluorescein and transcellular electrical resistance (TER). Levels of activated RhoA were measured using FRET probes, and phosphorylation of myosin light chain (MLC-p), a downstream target of RhoA, was assessed by Western blot analysis. RESULTS: Overexpression of Bves led to increased TJ formation in NTM-5 cells. Increased TJ formation was confirmed by increased occludin, cingulin, and ZO-1 protein. Functionally, NTM-w cells showed decreased permeability and increased TER compared with NTM-5 cells, consistent with increased TJ formation. NTM-w cells also exhibited decreased levels of active RhoA and lower levels of MLC-p than did NTM-5 cells. These findings support a TJ role in RhoA signaling. CONCLUSIONS: Increased Bves in TM cells leads to increased TJ formation with decreased RhoA activation and decreased MLC-p. This is the first report of a regulatory pathway upstream of RhoA in TM cells. In TM tissue, RhoA has been implicated in outflow regulation; thus, Bves may be a key regulatory molecule in aqueous outflow.
Department of Biomedical Engineering, Vanderbilt University, Nashville, Tennessee 37232, USA.
Classification:
2.5.1 Trabecular meshwork (Part of: 2 Anatomical structures in glaucoma > 2.5 Meshwork)
3.6 Cellular biology (Part of: 3 Laboratory methods)