A Small Chemical Chaperone, Sodium 4 Phenylbutyrate Inhibits TGFβ2-Induced ECM Remodeling in Human TM Cells

Purpose: The pathological mechanisms underlying increased outflow resistance at the trabecular meshwork (TM) that is responsible for elevating intraocular pressure (IOP) have not been fully delineated. We have previously shown that progressive accumulation of unfolded proteins and endoplasmic reticulum (ER) stress play an important role in the pathophysiology of glaucomatous TM damage in myocilin-associated POAG. However, it is not understood whether other glaucoma factors lead to similar pathological ER stress in the TM, leading to IOP elevation. Transforming growth factor β2 (TGFβ2) is known to induce abnormal extracellular matrix (ECM) deposition in the TM cells, which may be responsible for IOP elevation. Here, we examined whether TGFβ2 induces ER stress and whether reducing ER stress via a small chemical chaperone, sodium 4-phenylbutyrate (PBA) reduces TGFβ2–induced ECM remodeling. Methods: Human GTM-3 cells or primary TM cells (n=2) were treated with TGFβ2 (5ng/ml) with or without 5mM PBA for 48 hours. Total cellular lysates, conditioned medium, and fixed cells were examined for ECM and ER stress markers by Western blotting and immunostaining. We also used RT-PCR to demonstrate XBP1 splicing in response to TGFβ2 treatment. Results: TGFβ2 increased synthesis and deposition of ECM proteins in GTM-3 and primary TM cells. TGFβ2 induced ER stress as evident from increased ER chaperones and splicing of XBP-1. Treatment of TM cells with TGFβ2 and PBA demonstrated reduced synthesis and deposition of ECM and ER stress markers as evident from reduced fibronectin, GRP78, GRP94 and CHOP. Conclusions: Our studies suggest that TGFβ2 induces abnormal ECM accumulation and ER stress, and PBA may reduce TGFβ2-induced IOP elevation by decreasing abnormal ECM accumulation and reducing ER stress.