Abstract
Purpose :
Elevated TGFβ2 is found in the aqueous humor and trabecular meshwork (TM) of glaucoma patients. TGFβ2 induces excessive accumulation of extracellular matrix, alters cytoskeleton, and increases intraocular pressure in various experimental models. We previously reported that the Wnt signaling is able to inhibit TGFβ signaling in the TM at transcriptional level. Here, we determined if small molecule Wnt signaling activators are able to inhibit TGFβ2 signaling.
Methods :
GTM3 cells were transfected with Wnt signaling reporter luciferase vectors. The cells were treated with different GSK3β inhibitors (Wnt activators) at different concentrations, and luciferase levels were measured using a plate reader. After determination of the optimal concentration, TM cells were transfected or transduced with TGFβ signaling reporter luciferase vectors. These cells were treated with 5ng/ml TGFβ2 with or without GSK3β inhibitors, and luciferase levels were measured. Primary human TM (pHTM) cells were also treated with or without TGFβ2 and/or GSK3β inhibitors to study fibronectin and collagen I using Western immunoblotting and immunofluorescent microscopy.
Results :
We found that the optimal concentration of the GSK3β inhibitors in TM cells was 1uM for 6-bromoindirubin-3’-oxime (BIO), 10uM for SB216763 (SB), and 5uM for CHIR-99021 (CHIR). Also, these compounds were more potent compared to Wnt3a recombinant proteins in Wnt signaling activation. At these concentrations, they inhibited TGFβ2-induced TGFβ signaling activation in the TM. They also inhibited TGFβ2-induced expression of fibronectin and collagen I in pHTM cells.
Conclusions :
Small molecule Wnt signaling activators inhibit TGFβ signaling in the TM and may have potentials for therapeutic use in treating glaucoma.
This is a 2021 ARVO Annual Meeting abstract.