Abstract
Purpose :
Elevated intraocular pressure (IOP) is the primary risk factor for primary open-angle glaucoma (POAG). Elevated IOP is due to glaucomatous changes in the trabecular meshwork (TM) including excessive synthesis and deposition of extracellular matrix (ECM) proteins, formation of cross-linked actin networks (CLANs), and loss of TM cells. The existence of chronic endoplasmic reticulum (ER) stress and elevated Dkk1(a Wnt pathway inhibitor) in the glaucomatous TM has been reported. They both induce pathological changes in TM and elevate IOP in mouse and/or human eyes. However, the relationship between chronic ER stress and the Wnt signaling pathway in the glaucomatous TM is not clear. Here, we show that inhibition of the Wnt signaling pathway induces ER stress in the TM.
Methods :
Primary HTM cells were treated with or without DKK1, Thapsigargin, and/or CHIR. Whole-cell lysates (WCL) were used for western immunoblotting (WB) analysis. Some cells were used to extract cytosolic and nuclear fractions for co-immunoprecipitation (Co-IP) assays. Some cells were transfected with non-target siRNA or β-catenin siRNA for knockdown studies and WCL was used for WB analysis. Luciferase assays were performed in the TCF-GTM3 cell line to study Wnt signaling activity.
Results :
Dkk1 treatment induced ER stress markers (ATF6, IRE1α, and CHOP), increased phosphorylation of pP38 and pJNK, as well as induced pro-apoptotic markers (BAK, BAX) in pHTM cells. In contrast, CHIR (a canonical Wnt activator) showed downregulation of ER stress markers, decreased phosphorylation of pP38, pJNK, reduced pro-apoptotic markers, as well as increased nuclear translocation of β-catenin, ATF4, and CHOP proteins. Since β-catenin is a key mediator of the Wnt signaling pathway, we knocked down β-catenin using siRNA and observed an upregulation of the ER stress markers IRE1α and CHOP, similar to Dkk1 treatment. Furthermore, Co-IP assays showed that CHIR promoted protein-protein interaction between ATF4, CHOP, and β-catenin in pHTM cells. In the TCF-GTM3 reporter cells, Thapsigargin (an ER stress inducer) inhibited Wnt signaling activity, and this inhibition was blocked by co-treatment with CHIR.
Conclusions :
Our results show that inhibition of the Wnt signaling pathway induced ER stress in the pHTM while activation of the Wnt signaling pathway alleviated ER stress.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.