TGF Beta 2 and Gremlin Signaling Pathways Regulate Extracellular Matrix Changes in TM Cells: Implications for Glaucoma

Purpose: Glaucoma is a leading cause of blindness worldwide. The leading risk factor is elevated intraocular pressure (IOP) due to fibrotic changes in the trabecular meshwork (TM) tissue. The profibrotic cytokine TGFβ2 is elevated in the aqueous humor and TM of glaucomatous eyes. TGFβ2-mediated extracellular matrix (ECM) deposition in the TM appears to be responsible for increased IOP in ex vivo and in vivo models. Bone morphogenetic proteins (BMPs) inhibit TGFβ regulation of ECM, and elevated levels of the BMP antagonist gremlin in the glaucomatous TM restores the fibrotic response of TGFβ2. Lysyl oxidase (LOX) is a collagen and elastin polymer crosslinking enzyme, and recent genome wide association studies showed that SNPs in LOXL1, a LOX family member, significantly increased the risk of developing exfoliation glaucoma. The overall aim of my work is to delineate the signaling pathways involved in TGFβ2 and gremlin alteration of the TM ECM and to evaluate the potential role of the LOX family of cross-linking enzymes in this TM ECM remodeling. Methods: Human TM cells were cultured in the presence or absence of recombinant human TGFβ (0.1-10 ng/ml) or mouse gremlin (100-5000 ng/ml) for 1-72 hours, and total RNA or protein lysates and conditioned medium were harvested from the cells. Effects of gremlin treatment on ECM gene and protein expression were assayed by qRT-PCR and western immunoblotting, respectively. TGFβ2- and gremlin-treated TM cells were also examined for Smad2/3, p38, and JNK activation using western immunoblotting. Cells were treated with ii Smad3 inhibitor SIS3 (5 uM), TGFβ receptor inhibitors LY364947 and SB431542 (5 uM), JNK inhibitor SP600125 (10 uM), and AP-1 inhibitor SR11342 (5 uM) with or without TGFβ2/gremlin, to examine the involvement of the TGFβ/Smad signaling pathway. Results: TGFβ2 and gremlin induced expression of each other in TM cells. TGFβ2 activated both Smad and non-Smad pathways and strongly induced mRNA and protein expression of all 5 LOX genes. Using a novel LOX activity assay, we observed greater ECM crosslinking in TGFβ- treated cells. Gremlin elevated ECM gene and protein expression via the Smad signaling pathway. Conclusions: The TGFβ induction of LOXs and gremlin-induction of ECM proteins highlight the complex interplay of Smad and the non-Smad signaling in regulating the TGFβ response. TGFβ2 and gremlin are profibrotic in a feed forward loop. The TGFβ response in TM cells involves evasion of BMP inhibition by gremlin induction and also ECM crosslinking through the LOX enzymes.