TGFβ2 induces Chronic Endoplasmic Reticulum Stress in Trabecular Meshwork cells

Purpose:TGFβ2 induced extracellular matrix(ECM)accumulation is known to be associated with glaucomatous trabecular meshwork(TM)damage and IOP elevation. Previously, we have demonstrated that abnormal ECM accumulation leads to endoplasmic reticulum(ER)stress in TM. Here, we investigated whether TGFβ2 induces ER stress in TM cells and whether chronic ER stress plays a pathological role in dysfunction of TM cells. Methods:Primary human TM cells were treated with vehicle or recombinant TGFβ2(5ng/mL) to determine its effect on chronic ER stress markers(GRP78,ATF4 and CHOP)and ECM proteins(Fibronectin and Collagen-I/IV). Chronic ER stress-induced ATF4/CHOP were genetically knocked-down using targeted CRISPR/Cas9 expression plasmids, or by transducing with ad5-ATF4deltaRK that inhibits endogenous ATF4 activity. The effect on IOP of intravitreal gene delivery of active-TGFβ2 viral-vector in Chop-/- and C57BL/6J mice was evaluated. Pharmacological inhibition of ER stress using ATF4/CHOP inhibitor ISRIB and chemical chaperon sodium4phenylbutyrate(PBA) was also determined on TGFβ2-treated TM cells. Results:Westernblot and immunostaining demonstrated that TGFβ2 induced chronic ER stress markers along with increased levels of ECM proteins, suggesting TGFβ2-induced ECM deposition is associated with ER stress. Knockdown of key transcriptional factors, ATF4/CHOP, and ISRIB treatment prevented TGFβ2-induced ECM expression and reduced ER stress in TM cells. Moreover, activeTGFβ2 viral delivery caused no IOP elevation in Chop-/-mice compared to control C57BL/6J mice. Treatment of TM cells with PBA also inhibited TGFβ2-induced fibronectin deposition via induced expression and activation of MMP2/9. Conclusion:This study indicates that TGFβ2 induces chronic ER stress, which is associated with increased ECM accumulation.