Inhibition of RhoA signaling with increased Bves in trabecular meshwork cells.
OBJECTIVE
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.
Russ
Patricia K
PK
Department of Biomedical Engineering, Vanderbilt University, Nashville, Tennessee 37232, USA.
Kupperman
Asher I
AI
Presley
Sai-Han
SH
Haselton
Frederick R
FR
Chang
Min S
MS
D030161:Avian Proteins / Q000502:physiology*; D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000502:physiology*; D002460:Cell Line; D017097:Electric Impedance; D019793:Fluorescein / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism; D009124:Muscle Proteins / Q000502:physiology*; D018994:Myosin Light Chains / Q000378:metabolism; D062793:Occludin; D010539:Permeability; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D015398:Signal Transduction; D019108:Tight Junctions / Q000378:metabolism; D014129:Trabecular Meshwork / Q000378:metabolism*; D014162:Transfection; D062826:Zonula Occludens-1 Protein; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D030161:Avian Proteins; C498788:Bves protein, Gallus gallus; C061407:CGN protein, human; D015815:Cell Adhesion Molecules; D008565:Membrane Proteins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D018994:Myosin Light Chains; C568985:OCLN protein, human; D062793:Occludin; D010750:Phosphoproteins; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; D020742:rhoA GTP-Binding Protein; D019793:Fluorescein