The role of Wnt signaling in treating glucocorticoid-induced glaucoma

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PROJECT SUMMARY/ABSTRACT Glucocorticoid (GC)-induced ocular hypertension (OHT) and glaucoma (GIG) occur in ~40% of the general population and ~90% primary open angle glaucoma (POAG) patients. Our published studies and preliminary data show that canonical Wnt signaling activation inhibits glucocorticoid receptor signaling and GC-induced OHT. We hypothesize that activation of canonical Wnt signaling inhibits GIG, and this inhibition requires nucle- ar β-catenin, glucocorticoid receptor (GR), and epigenetic modification enzymes including HDAC1 and MeCP2. In this supplemental application, our objective is to identify new epigenetic mechanisms of and therapeutic compounds for glucocorticoid-induced OHT/glaucoma. Our rationale is that Wnt activation is a potential ap- proach to prevent and treat OHT/GIG, and studying GIG will help us to better understand primary open angle glaucoma. We propose two specific aims. SA 1: Determine whether G-quadruplex is regulated by the glucocor- ticoid receptor (GR) signaling pathway in the trabecular meshwork (TM). Recently, emerging evidence showed the importance of a DNA secondary structure, G-quadruplex (G4) in many biological processes. We hypothe- size that GCs enhance G4 formation in the TM. We will treat primary human trabecular meshwork (pHTM) cells from glucocorticoid responders or non-responders with or without 100nM DEX or 0.1% ethanol for 1 day. The cells will be used for immunoprecipitation against G4 chromatin, and purified DNA containing G4 will be used for DNA sequencing. At least 3 glucocorticoid responder and non-responder TM cell strains will be tested. We expect to find that glucocorticoid-responder TM cells have more G4 structures and these G4s are associated with key genes such as cell survival and ECM metabolism. We also expect to find that glucocorticoids enhance G4 formation and this enhancement is greater in responder TM cells. SA 2: Identify novel Wnt signaling path- way activation compounds with high potency in inhibiting GR signaling in the TM. We have found that the acti- vation of the Wnt signaling pathway using a small molecule compound CHIR is effective in the inhibition of GR signaling. Our preliminary data from the parent R01 also showed that CHIR did not compromise DEX’s anti- inflammatory effects in mouse eyes with endotoxin-induced uveitis. However, CHIR needs to be applied at high concentrations. Therefore, it is important to find more potent small molecule Wnt signaling activators. The pro- posed study is significant because it provides new molecular mechanisms as well as therapeutic targets for the prevention and treatment of glucocorticoid-induced OHT/glaucoma. The findings, especially from SA2, will have translational applications. It is also innovative because the role of G4 in the TM, glaucoma, and glucocor- ticoid receptor signaling, to our best knowledge, is completely unknown. Our future studies will focus on 1) whether G4 can be inhibited by Wnt signaling; 2) validation of the downstream target of glucocorticoid-induced G4 in the TM; and 3) testing the compound hits in vivo.