Role of TGFβ/Smad signaling in gremlin induction of human trabecular meshwork extracellular matrix proteins

Glaucoma is a leading cause of irreversible visual impairment and blindness in the world, with primary open-angle glaucoma (POAG) being the major form of glaucoma.1,2 Elevated intraocular pressure (IOP) is a major risk factor for the development and progression of glaucoma.3,4 Ocular hypertension is due to increased aqueous humor outflow resistance in the trabecular meshwork (TM) and is associated with increased deposition of extracellular matrix (ECM) material within the TM. Transforming growth factor (TGF)-β2 levels are elevated in the aqueous humor5–7 and TM (Tovar-Vidales T et al. submitted for publication) of POAG patients. Trabecular meshwork cells express TGFβ2 receptors, and TGFβ2 has several effects on the TM.8 It has been shown to increase aqueous outflow resistance and elevate IOP in perfusion-cultured human and porcine eyes,9–11 as well as in rodent eyes.12 TGFβ2 modulates ECM metabolism in TM cells and tissues. This cytokine increases expression of a variety of ECM proteins, including fibronectin (FN), collagen (COL), elastin (ELN), and proteoglycans, as well as levels of plasminogen activator inhibitor-1 (PAI1) and tissue inhibitor of metalloproteinase-1 (TIMP1), inhibitors that suppress proteolytic degradation of the ECM.13 In addition, TGFβ2 increased expression of the ECM cross-linking enzymes transglutaminase-2 (TGM2)14 and the lysyl oxidases (LOXs).15 We have previously reported that TM cells express several members of the bone morphogenetic protein (BMP) family, including BMP ligands (BMP2, BMP4, BMP5, and BMP7), receptors (BMPR1a, BMPR1b, and BMPR2), and the antagonists gremlin, follistatin, and norrin.16,17 BMPs are members of the TGFβ superfamily of proteins that control multiple functions in a variety of cell types.18,19 BMP4 and BMP7 block the TGFβ2-induction of a variety of ECM proteins, including fibronectin-1, collagen IV and VI, TSP-1, and PAI1.19,20 Several structurally distinct BMP antagonists tightly regulate BMP cellular activity. BMP antagonists like gremlin directly bind BMP ligands and block BMP binding to their receptors.21,22 We have reported that there are greater levels of gremlin in glaucomatous TM cells and tissues.19 Gremlin antagonizes BMP4 inhibition of TGFβ2-induced ECM proteins like FN and PAI1 in TM cells and elevates IOP in perfusion-cultured human anterior segments.19 It is a highly conserved 20.7-kDa glycoprotein that heterodimerizes with BMP-2, -4, and -7 and plays a key role in regulating multiple cellular functions both during early development and in adult tissue homeostasis.22,23 Gremlin may potentiate the profibrotic effects of TGFβ2 by blocking the BMP4 regulation of TGFβ2 activity. However, whether gremlin alone can induce fibrosis-like activities in cultured TM cells is currently unknown, and the potential signaling mechanisms involved have not been characterized. The purpose of the present study was to determine (1) whether gremlin induces ECM genes and proteins in cultured TM cells and (2) what signaling mechanisms are involved in gremlin induction of ECM genes and proteins.