The role of canonical Wnt signaling and K cadherin in the regulation of intraocular pressure

Purpose: Primary open angle glaucoma (POAG) is the most prevalent form of glaucoma and has been associated with pathological changes in the trabecular meshwork (TM), the primary site of aqueous humor outflow in the eye. We have found that inhibition of canonical Wnt signaling in the TM raises intraocular pressure (IOP), and restoration of Wnt signaling normalizes IOP, though the mechanisms by which Wnt signaling maintains TM homeostasis are unknown. We hypothesize that the canonical Wnt signaling pathway in the TM regulates IOP via cadherins junctions. Materials and methods: We studied five cadherin isoforms abundant in the TM as shown by exome sequencing of normal and glaucomatous human TM (NTM and GTM, respectively) tissues. For in vitro studies, NTM cells (gift from Novartis) were treated with or without recombinant 100ng/ml Wnt3a or 1ug/ml sFRP-1 or both for 4-48 hours. Membrane protein fractions were isolated for western immunoblotting (WB) and probed for the cadherin isoforms. TM cells were also immunostained for cadherin isoforms or β-catenin. RNA was extracted from TM cells for cDNA synthesis and qPCR analysis of cadherins. Ad5.CMV recombinant adenoviruses encoding E cadherin, K cadherin, and/or sFRP-1 were injected unilaterally into the eyes of 4-6 month old female BALB/cJ mice (n=6). Conscious IOP of both eyes was then non-invasively measured for up to 35 days. Results: WB showed that Wnt3a TM cell membrane associated K-cadherin, which was inhibited with the addition of the Wnt antagonist sFRP-1. Immunostaining showed that -catenin accumulated on TM cell membrane upon Wnt3a treatment, and filopodia-like connections formed between TM cells. qPCR showed that Wnt3a also significantly increased K cadherin expression (n=3, p Conclusion: Our results suggested that cadherins play a role in the regulation of TM homeostasis and IOP via the Wnt signaling pathway.