Inhibition of Chronic Endoplasmic Reticulum Stress Prevents TGFβ2-induced ECM Accumulation in Human Trabecular Meshwork Cells

Purpose: TGFβ2 is known to be involved in the pathogenesis of primary open angle glaucoma. TGFβ2-induced ECM accumulation is associated with glaucomatous trabecular meshwork (TM) damage and IOP elevation. Previously, we have demonstrated that dexamethasone-induced abnormal ECM accumulation leads to ER stress in TM. Here, we investigated whether TGFβ2 induces ER stress in human TM cells and whether chronic ER stress plays a pathological role in dysfunction of TM cells. Methods: Transformed GTM3 cells or primary human TM cells were treated with vehicle or recombinant TGFβ2 (5ng/mL) to determine its effect on expression of chronic ER stress markers and ECM proteins using Western blot (WB) and immunostaining. Further, chronic ER stress induced ATF4 or CHOP were knock down in these cells using CRISPR/Cas9 technology. The endogenous ATF4 activity was also inhibited by transducing with ad5 ATF4􀀀RK followed by TGFβ2 treatment. Results: WB analysis demonstrated TGFβ2 induced ER stress as evident from increased levels of GRP78, GRP94, ATF4 and CHOP proteins. Immunostaining revealed that TGFβ2 treatment increased fibronectin and collagen IV levels and its colocalization with ER stress markers (KDEL and calreticulin), suggesting TGFβ2-induced ECM proteins are associated with ER stress. Knockdown of key chronic ER stress transcriptional factors, ATF4 or CHOP prevented TGFβ2-induced ECM deposition and also reduced ER stress in TM cells. Treatment of TM cells with small molecule, PBA also inhibited TGFβ2-induced fibronectin deposition. Conclusion: These data indicate that TGFβ2 induces chronic ER stress, which is associated with increased ECM accumulation.