Date of Award

5-1-2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Graduate School of Biomedical Sciences

First Advisor

Weiming Mao

Second Advisor

Abbot F. Clark

Third Advisor

Iok-Hou Pang

Abstract

Ocular hypertension is the greatest causative risk factor of primary open angle glaucoma (POAG), the most prevalent subset of age-related glaucoma. Wnt signaling antagonist sFRP1 is increased in the trabecular meshwork (TM) of patients with POAG and induces ocular hypertension in human ex vivo eyes and in mice, which is resolved upon downstream Wnt/β-catenin signaling activation. The molecular mechanisms behind this remain unknown. β-catenin plays a role as an accessory protein to classical cadherin cytosolic domains, connecting these cell-cell adhesion proteins to the actin cytoskeleton. In other cell types, Wnt/β-catenin signaling crosstalks with the TGFβ/SMAD pathway, which is overactive in the POAG TM and is implicated in ocular hypertension. Our hypothesis is that the Wnt/β-catenin signaling pathway maintains TM cell adhesion and intraocular pressure by stabilizing cadherins junctions on the TM cell membrane and by inhibiting the POAG-related TGFβ/SMAD pathway. We used primary or transformed human non-glaucomatous TM (NTM) cells for all molecular and cell-based studies. NTM cells were treated with reporter viruses to study DNA binding element activity, recombinant protein to modulate Wnt/β-catenin or TGFβ/SMAD pathways, or siRNA to knockdown pathway mediators or cadherins. After treatment, NTM nucleic acid or protein was isolated or probed for Wnt/β-catenin or TGFβ signaling markers or cadherins. Some NTM cells were also plated for Real Time Cell Analysis (RTCA) cell impedance assays. Ad5.CMV recombinant adenoviruses encoding K-cadherin and/or sFRP1 were injected into BALB/cJ mouse eyes. Conscious IOP was assessed for up to 35 days. We found that Wnt/β-catenin signaling cross-inhibits TGFβ signaling in a β-catenin and Smad4-dependent manner. This cross-inhibition resulted in a decreased K-cadherin and fibronectin expression. Wnt/β-catenin signaling also enhanced mRNA, protein, and membrane-bound levels of K-cadherin, the most highly expressed cadherin isoform in the TM. In vivo, K-cadherin reduced the ocular hypertensive effects of sFRP1. RTCA assays showed that Wnt/β-catenin signaling and K-cadherin are responsible for maintenance of TM cell adhesion. Wnt/β-catenin signaling is responsible for intraocular pressure maintenance through increased expression of K-cadherin-mediated TM cell adhesion and through inhibition of TGFβ/SMAD signaling.

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