Effects of Wnt/beta-catenin and SMAD/TGF-beta crosstalk on cadherins in the trabecular meshwork

Purpose: We have shown cross-inhibition of the primary open angle glaucoma (POAG)-associated Wnt/beta-catenin and SMAD/TGFb signaling pathways in trabecular meshwork (TM) but the downstream effects of this crosstalk are unknown. Wnt transcription factor and cadherin accessory protein, beta-catenin, plays a role in cadherins junction stability. We studied the role of Wnt/beta-catenin and SMAD/TGFb signaling on cadherins junctions in the TM and TM cell adhesion. Methods: Confluent primary TM (NTM) cells (donated from Alcon) were treated for 2 or 24 hours with or without 100ng/mL Wnt3a or 5ng/mL TGFb2 and their membrane-bound protein, conditioned media, and total protein were isolated for western immunoblotting. Samples were probed for fibronectin (FN), PAI-1, p-Smad3, beta-catenin, GAPDH, Pan-, K-, or OB-cadherin. Some NTM cells were grown to 80% confluence then transfected with 0.3nM or 0.5nM K-cadherin, OB-cadherin, or non-targeting siRNA. Forty-eight or 72 hours after transfection, cells were harvested for western immunoblotting or immunofluorescence. NTM cells were plated for the Acea iCelligence system to determine cellular impedance with data collected every 30 minutes to establish baseline. After 24 hours, culture medium was replaced with transfection mixes as described. Cell impedance was continuously measured for an additional 96 hours. Bright field images of the cells were taken. Results: Wnt3a treatment resulted in increased K-cadherin expression. Co-treatment of Wnt3a and TGFb2 decreased the expression of K-cadherin. Three days after transfection with 0.5nM K-cadherin siRNA, decreased K-cadherin expression was observed in both whole cell lysate and membrane-bound fractions. Transfection with K-cadherin siRNA decreased NTM cellular impedance compared to non-targeting siRNA control. Conclusions: Crosstalk between Wnt/beta-catenin and SMAD/TGFb signaling pathways in the TM may regulate the expression of cadherins as well as NTM cell adhesion.