TGFβ2 induces chronic endoplasmic reticulum stress in trabecular meshwork cells.

Purpose: TGFβ2-induced extracellular matrix (ECM) accumulation in trabecular meshwork (TM) is associated with aqueous humor outflow resistance and IOP elevation. Recently, we have demonstrated that abnormal ECM accumulation leads to endoplasmic reticulum (ER) stress in TM. Here, we examined whether TGFβ2 induces ER stress in human TM cells. Methods: GTM3 or primary human TM cells (n=2) were treated with vehicle or recombinant TGFβ2 (5 ng/ml) in 0.5% FBS containing DMEM medium for 3 days & 7 days respectively. ER stress markers (Grp78, Grp94, ATF4 and CHOP) and ECM proteins (Fibronectin & Collagen IV) were examined by Western blot and immunostaining. GTM3 cells were transfected with plasmids expressing CRISPR-Cas9 targeting ATF4 or CHOP and subsequently treated with TGFβ2 for 48 hours. Cellular lysates were examined for ER stress and ECM proteins. Results: Western blot analysis demonstrated that TGFβ2 treatment led to ER stress as evident from increased levels of Grp78, Grp94, ATF4 and CHOP proteins compared to the vehicle treatment. TGFβ2-induced ER stress markers were also associated with ECM protein (fibronectin and Collagen IV) levels. Moreover, TGFβ2 treatment increased fibronectin staining and its colocalization with ER stress markers, 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 GTM3 cells. Conclusions: Our preliminary findings clearly indicate that TGFβ2 can directly induce chronic ER stress in cultured human TM cells.