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P. A. Knepper, R. M. Beverley, R. D. McCarty, M. J. Nolan, B. Y. J. T. Yue, J. R. Samples; Wnt Signaling and Trabecular Meshwork Lipid Rafts. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4875.
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Wnt proteins interact with Frizzled receptors and members of the low-density-lipoprotein receptor (LRP). LRP serves as an endocytosis receptor for -2-macroglobulin, a mediator of retinal ganglion cell death in glaucoma and also for amyloid precursor protein, a mediator of selected cell death in Alzheimer’s disease. Secreted frizzled-related protein-1 (sFRP-1) is an antagonist of Wnt signaling that causes a decreased outflow facility in ex vivo perfusion-of cultured human eyes. To explore possible signaling mechanisms by which sFRP-1 decreases outflow facility, we challenged trabecular meshwork (TM) cells with exogenous sFRP-1 and evaluated its effects on cell viability and its downstream effects on Wnt pathway of LRP-6 (canonical) and CD44 (non-canonical) content in caveolin/lipid rafts.
Human TM cells cultured in the presence 2.5, 5.0 and 10.0 ug of recombinant human sFRP-1 (R & D Systems) were tested for cell survival. Caveolin enriched lipid rafts were isolated using ice cold Triton X-100 and then separated on an OptiPrep density gradient (Sigma D1556). The preparation was centrifuged at 200,000 x g for 18 hrs; nine 1.0 ml fractions were pipetted from the top (lightest) to bottom (heaviest). Each fraction was analyzed for protein content, resolved by SDS polyacrylamide electrophoresis, and immunoblotted with anti-caveolin-1, CD44, and LRP-6 antibodies.
There was a statistically significant (P<0.001) dose dependent decrease of cell viability in TM cells treated with sFRP-1. The distribution of 22-kDa caveolin-1 in sFRP-1 treated TM cells was in fractions 3 through 7 with the strongest intensity in fractions 5 and 6. The amount of caveolin-1 in sFRP-1 treated TM cells was considerably greater, approximately two-fold. The distribution of CD44 in sFRP-1 treated TM cells remained in fractions 1 through 9 but with an increased content of 32 kDa CD44 in fraction 5 compared to controls. The distribution of LRP-6 in sFRP-1 treated TM cells was in fractions 4 through 9 and decreased compared to control TM cells.
This is the first demonstration of the effects of exogenous sFRP-1 on the downstream signaling of caveolin, CD44 and LRP-6 in TM cells. sFRP-1 increased the cell caveolin-1 content, changed caveolin-1 density gradient distribution, decreased LRP-6 content and increased sCD44. These results indicate that caveolin-1, CD44 and/or LRP-6 are downstream effectors of sFRP-1.
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