Abstract
Purpose :
Glucocorticoids (GCs) induce glaucoma (GIG) in a subpopulation of patients. GCs function mainly via the glucocorticoid receptor (GR) signaling pathway which causes excessive ECM deposition and increased TM stiffness. These changes contribute to elevated intraocular pressure (IOP) and GIG. Our published data showed that the β-catenin/canonical Wnt signaling genes are differentially regulated in GC responder and non-responder bovine TM using RNA sequencing, which suggests the role of β-catenin/canonical Wnt signaling in GIG. Here we determined the effect of β-catenin/canonical Wnt signaling activation and inhibition on the TM and eyes exposed to GCs.
Methods :
GTM3 and primary human TM (HTM) cells were treated with dexamethasone (DEX) and with or without recombinant proteins or small molecular GSK3β inhibitors (Wnt activators). Some TM cells were transfected or transduced with siRNA or adenovirus to knockdown or overexpress the genes of interest. Plasmids or lentiviral luciferase reporter vectors were used to determine Wnt or GR signaling. qPCR and western immunoblotting were used to determine gene expression. C57BL6/j mice were used to study IOP changes.
Results :
In GTM3 and HTM cells, activation of canonical Wnt signaling using Wnt3a inhibited GR signaling while inhibition of canonical Wnt signaling using sFRP1 enhanced GR signaling. Similar inhibition was observed by knocking down Dkk1, another Wnt pathway inhibitor using siRNA, and this inhibition was dose dependent. Activation of β-catenin/Wnt signaling using small molecule GSK3β inhibitors inhibited DEX-induced extracellular matrix proteins in HTM cells. In contrast, DEX did not activate β-catenin/Wnt signaling using luciferase assays. We found that axin2, a frequently used Wnt reporter gene, was only slightly upregulated (~20%) by DEX but it was independent of β-catenin (with siRNA knockdown), suggesting 1) axin2 is not a suitable marker in DEX-β-catenin/Wnt crosstalk study; and 2) DEX did not activate β-catenin/Wnt signaling. In a mouse GIG model, we found that intravitreal injection of adenovirus expressing Wnt3a inhibited DEX-induced OHT.
Conclusions :
Our studies suggest that activation of the β-catenin/Wnt pathway would be a promising approach to prevent or inhibit GC-induced OHT.
This is a 2020 ARVO Annual Meeting abstract.