Gremlin Induces Ocular Hypertension in Mice Through Smad3-Dependent Signaling

Abstract

PURPOSE. Transforming growth factor-b2 induces extracellular matrix (ECM) remodeling, which likely contributes to the defective function of the trabecular meshwork (TM) leading to glaucomatous ocular hypertension. Bone morphogenetic proteins (BMPs) inhibit these profibrotic effects of TGFb2. The BMP antagonist gremlin is elevated in glaucomatous TM cells and increases IOP in an ex vivo perfusion culture model. The purpose of this study was to determine whether gremlin regulates ECM proteins in the TM, signals through the Smad3-dependent pathway, and induces ocular hypertension in mice.METHODS. Ad5.Gremlin or Ad5.TGFb2 was injected intravitreally into one eye of each mouse. Intraocular pressure measurements were taken using a TonoLab tonometer. Gremlin, TGFb2, fibronectin (FN), and collagen-1 (Col-1) expression in the TM was determined by immunofluorescence, Western immunoblot, and quantitative (q)PCR analyses.RESULTS. Ad5.Gremlin or Ad5.TGFb2 each caused significant IOP elevation in mice.Immunofluorescence and Western blot analysis demonstrated that gremlin and TGFb2 reciprocally increased the expression of each other, and both increased FN expression in the TM and surrounding tissues. Ad5.Gremlin elevated IOP and increased Fn and Col-1 gene expression in the TM of Smad3 wild-type (WT) mice, but had no effect in Smad3 HET or Smad3 KO mice.CONCLUSIONS. Our results demonstrate that intravitreal injections of either Ad5.Gremlin or Ad5.TGFb2 elevate IOP and upregulate the ECM protein FN in the TM of mice. These data show that gremlin signals through the Smad3-dependent pathway in the TM to elevate IOP. We determined for the first time gremlin's role in inducing ocular hypertension in an in vivo model system. Keywords: gremlin, glaucoma, trabecular meshwork, TGFb2, intraocular pressure E levated IOP is a well-known causative risk factor for both the development and progression of glaucoma. Intraocular pressure is regulated by aqueous humor (AH) production in the ciliary body and drainage from the eye via the trabecular meshwork (TM) and uveoscleral pathway. The role of the TM and the surrounding extracellular matrix (ECM) in IOP regulation has been extensively studied. Primary open-angle glaucoma (POAG) is associated with changes in the ECM composition within the TM, increased AH outflow resistance, and elevated IOP. 1,2Transforming growth factor-b2 signaling pathway has been shown to be an important regulator of ECM proteins in the TM, 3-9 and TGFb2 has been found to be elevated in the AH and TM of POAG patients. 7,10-13 Transforming growth factor-b2 has also been shown to cause ocular hypertension in both ex vivo anterior segment perfusion organ culture models, 14,15 and by overexpression of a bioactivated form of TGFb2 in mouse eyes. 4,16 Transforming growth factor-b2 is known to regulate the expression of ECM proteins through the canonical Smad pathway as well as noncanonical signaling pathways. [17][18][19][20] We have previously demonstrated that TGFb2 signals through the canonical Smad and non-Smad pathways an...

The many faces of the trabecular meshwork cell

“…TM cells express bone morphogentic proteins (BMPs) and BMP receptors (Wordinger et al, 2002), which normally antagonize TGFβ2 signaling (Wordinger et al, 2007, Fuchshofer et al, 2007). However, expression of the BMP antagonist gremlin is elevated in GTM cells, and over-expression of gremlin elevates IOP in perfusion cultured human anterior segments (Wordinger et al, 2007) and in mouse eyes (McDowell et al, 2015). TGFβ2 increases gremlin expression and gremlin increases TGFβ2 expression in cultured TM cells (Sethi et al, 2011a), creating a profibrotic environment.…”

Effects of Salidroside on Trabecular Meshwork Cell Extracellular Matrix Expression and Mouse Intraocular Pressure

“…A suspension of Ad.hTGF-b2 226/228 (6 3 10 7 pfu) in a volume of 2 lL was injected intravitreally using a Hamilton (Reno, NV, USA) glass micro-syringe fitted with a 1-inch, 33gauge needle with a 108 bevel, as described previously. 15,25,26 Each injection was made over the course of approximately 30 seconds. The needle was then left in place for an additional 1 minute before being rapidly withdrawn.…”

BMP and Activin Membrane Bound Inhibitor Regulates the Extracellular Matrix in the Trabecular Meshwork

“… 11 – 17 We and others have demonstrated previously that TGFβ2 elevates IOP in the anterior segment perfusion organ culture models 14 , 18 and Ad5.TGFβ2 induces ocular hypertension in mice. 19 – 21 It is well known that TM cells express and secrete TGFβ2. 22 TGFβ2-induced changes to the TM occur through the canonical SMAD and non-SMAD signaling pathways, and the canonical SMAD pathway is essential for TGFβ2-induced ocular hypertension in mice.…”

“…Bone morphogenetic proteins (BMPs) are a family of growth factors involved in regulation of the ECM. BMPs can suppress TGFβ2-induced ECM deposition, 16 , 24 the BMP antagonist gremlin elevates IOP in perfusion cultured anterior segments, 24 and overexpression of gremlin in mouse eyes 20 and BMP2 in rats 25 causes ocular hypertension, suggesting that BMP signaling is required for regulating outflow. Similar to TGFβ2, BMP signaling requires two types of transmembrane serine/threonine receptors, type I (BMPR-I) and type II (BMPR-II).…”