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Hannah Webber, Abbot F Clark, Weiming Mao, North Texas Eye Research Institute; Wnt-Induced Cadherin Expression in the Trabecular Meshwork. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3286. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Primary Open Angle Glaucoma (POAG) is one of the leading causes of irreversible blindness in the world. Impaired Trabecular Meshwork (TM) structures and functions has been pinpointed as the cause of decreased aqueous outflow and therefore increased intraocular pressure (IOP), which is the most important causative risk factor of glaucoma. Our previous studies showed that the secreted Wnt antagonist SFRP1 is elevated in the glaucomatous TM (GTM). We also found that the canonical Wnt signaling pathway is inhibited in the glaucomatous TM. This inhibition leads to IOP elevation. However, how alteration in the Wnt pathway changes IOP is still not fully understood. In this study, we determined whether the canonical Wnt signaling pathway regulates cadherins junctions in TM cell cultures.
Six cadherin molecules were selected for this study because they show highest expression levels among all cadherins in the human TM (HTM) according to our published microarray data. We confirmed the expression of these 6 cadherins in HTM cell cultures by using immunocytochemistry (ICC) as well as western immunoblotting (WB). HTM cell cultures were treated with or without Wnt3a (100nM), SFRP1 (2uM), or a combination in DMEM-low-glucose, serum-free medium for 4, 24 or 48 hours. Treated cells were fixed with 4% ice-old paraformaldehyde for ICC or collected in cell lysis buffer for WB. For ICC staining, cells were probed with different types of anti-cadherin antibodies followed by corresponding secondary antibodies conjugated with Alex-488 or 568. Some cells were also stained with an anti-beta-catenin antibody.
All six cadherins, including OB, CDH19, K, E, N, and P cadherins, are expressed in the TM, which confirmed our microarray findings. Activation of the Wnt signaling pathway by Wnt3a recombinant protein increased the expression of E and K cadherins. Immunocytochemistry showed cadherin-junctions bridging between TM cells, and these junctions appeared to be enhanced upon Wnt3a treatment as well. Also, the distribution of E cadherins showed an actin stress-fiber like pattern while K cadherins displayed a microtubule-like pattern.
Wnt pathway activation by Wnt3a increased the expression of a subset of cadherins in the HTM. Based on the fact that the Wnt pathway inhibitor SFRP1 is elevated in the GTM, we speculate that cell-cell adhesion may be perturbed in the GTM, which contributes to IOP elevation.
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