Bves Overexpression in Trabecular Meshwork Cells Inhibits RhoA Signaling
Patricia K Russ 1,
Asher I. Kupperman 2,
Sai-Han Presley 3,
Frederick R Haselton 1,
and
Min S. Chang 4*
1 Biomedical Engineering, Vanderbilt University, Nashville, Tennessee, United States
2 School of Medicine, Vanderbilt University, Nashville, Tennessee, United States
3 Dept. of Ophthalmology, Vanderbilt University Medical Center, Nashville, Tennessee, United States
4 Biomedical Engineering, Vanderbilt University, Nashville, Tennessee, United States; Dept. of Ophthalmology, Vanderbilt University Medical Center, Nashville, Tennessee, United States
* To whom correspondence should be addressed. E-mail: min.chang{at}vanderbilt.edu.
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Abstract |
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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. Since Bves has been reported in multiple ocular tissues, we hypothesize that Bves plays a role in regulation of RhoA signaling in 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.
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 to NTM-5 cells, consistent with increased TJ formation. NTM-w cells also
exhibited decreased levels of active RhoA and lower levels of MLC-p compared to
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, and thus Bves may be a key regulatory molecule in aqueous outflow.
Key Words:
aqueous flow, cell adhesion, cell culture, glaucoma anterior segment, intercellular adhesion molecules, trabecular meshwork
