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M. J. Nolan, T. Koga, B. Y. J. T. Yue, M. C. Giovingo, A. Shepard, A. F. Clark, M. B. Wax, J. R. Samples, P. A. Knepper; Caveolin-1 Expression in Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1627.
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Caveolin-1, a 22-kDa, intergral membrane protein forming oligomers in cells, is a principal component of caveolae in lipid rafts and is also important in regulating vesicle trafficking through cells. Caveolin-1 is involved, consequently, in signal transduction, tight junction formation, endocytosis, transcytosis, as well serving as a mechanosensor in endothelial cells. Lipid-modified proteins such as endothelial nitric oxide synthase and the Src family of kinases can target to caveolae and interact with caveolin-1 to regulate signal transduction. The purpose of this study was to determine whether trabecular meshwork (TM) cells express caveolin-1 and if caveolin-1 expression is influenced by dexamethasone (Dex) treatment.
Human TM cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal calf serum (FCS) until confluent, washed twice with PBS, and incubated in DMEM containing 0.1% FCS and 100 µM Dex for 1, 24 and 96 hours. The media was aspirated; the cells were washed with cold PBS, subjected to lysis buffer (Sigma CS0750) containing 1% Triton X-100, and separated by Optiprep density gradient (Sigma D1556). The preparation was centrifuged at 200,000 x g for 18 hrs; nine 0.5 ml fractions were pipetted from the top (lightest) to bottom (heaviest). Each fraction was analyzed for protein content, resolved by SDS polyarylamide electrophoresis, and immunoblotted with mouse monoclonal anti-caveolin-1 antibody (Sigma).
The distribution of 22-kDa caveolin-1 in TM cells was in fractions 4 through 7 and the strongest intensity was in fraction 5; a high molecular weight (>300-kDa) was observed in fractions 5 through 7. The distribution of 22-kDa caveolin-1 in Dex-treated TM cells was in fractions 5 through 7 and the strongest intensity was in fractions 5 and 6, with minor amounts in fraction 8 and 9; a high molecular weight (>300-kDa) was also observed in fractions 5 through 7. The amount of caveolin-1 in Dex-treated TM cells was considerably greater, approximately two-fold. Similarly, the amount of caveolin-1 in the media was also increased in the Dex-treated TM cells.
This is the first demonstration of caveolin-1 in TM cells. Dex treatment increased the cell caveolin-1 content and changed caveolin-1 density gradient distribution as well as increasing the secretion of media caveolin-1. These results indicate that caveolin-1 may play an important role in TM barrier function, cell signaling, and endocytosis.
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