Purpose. well as by inhibitors of the canonical Smad2, -3, and -4 signaling and non-Smad JNK/AP-1 signaling pathways (< 0.05). Conclusions. Both Smad and non-Smad signaling pathways are involved in TGF-mediated induction, suggesting complex regulation of these important extracellular matrix cross-linking enzymes. Increased LOX activity may be at least partially responsible for TGF-mediated IOP elevation and increased aqueous humor outflow resistance. Glaucoma is a leading cause of irreversible visual impairment and blindness in the world, with primary open-angle glaucoma (POAG) being the major form.1,2 Elevated intraocular pressure (IOP) is a major risk factor for the development and progression of glaucoma,3,4 and this 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. The profibrotic cytokine TGF2 has been implicated in the pathogenesis of POAG.5 Levels of TGF2 are elevated in the aqueous humor6C8 and TM (Tovar-Vidalez T et al., manuscript submitted) PH-797804 of POAG patients. TM cells express TGF receptors, and TGF2 has direct effects on the TM.9 TGF2 has been shown to increase aqueous outflow resistance and to elevate IOP in perfusion cultured human and porcine eyes10C12 and in rodent eyes.13 TGF1 is elevated in the aqueous humor of patients with exfoliation glaucoma,14 suggesting that this TGF isoform is associated with the accumulation of exfoliation material, including ECM proteins, in the anterior segments of patients with this syndrome. TGF2 regulates ECM metabolism in TM cells and tissues. Rabbit Polyclonal to GPR12 This cytokine increases expression of a variety of ECM proteins, including fibronectin, collagen, elastin, and proteoglycans, as well as PAI-1 and TIMP-1, inhibitors that suppress proteolytic degradation of the ECM.15 In addition, TGF2 increases expression of the ECM cross-linking enzyme transglutaminase PH-797804 (TGM)-2 in TM cells.16 The combination of increased ECM synthesis, increased cross-linking, and decreased degradation causes increased ECM deposition in the TM, which may be responsible for the TGF2-mediated increased resistance to aqueous humor outflow. Similar changes occur in the TM PH-797804 of POAG patients, with increased levels of fibronectin,17 collagen,18 PAI-1,19 and TGM2.20 In addition to TGM2, there is a second important class of ECM cross-linking enzymes. The lysyl oxidase (and to -are associated with a significantly increased risk of exfoliation glaucoma,26,27 further suggesting potential roles for LOXs in glaucoma pathogenesis. The purpose of the present study was to determine (1) whether the and genes and proteins are expressed in human TM cells, (2) whether TGF induces gene expression and activity in the TM, and (3) which TGF signaling pathway(s) regulate expression in the TM. Methods TM Cell Culture Human TM cells were isolated from carefully dissected human TM tissue explants derived from patients with glaucoma or from normal donors and characterized as previously described.9 All donor tissues were obtained from regional eye banks and managed according to the guidelines in the Declaration of Helsinki for research involving human tissue. Isolated TM cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen-Gibco, Grand Island, NY) containing l-glutamine (0.292 mg/mL; Invitrogen-Gibco), penicillin (100 U/mL)/streptomycin (0.1 mg/mL; Invitrogen-Gibco), and 10% fetal bovine serum (Invitrogen-Gibco). TM Cell Treatments TM cells were grown to 100% confluence, and the medium was then replaced with serum-free medium for 24 hours before the treatments, to avoid the effects of serum proteins on the treatments. The cells were incubated with fresh medium containing specific signaling inhibitors for 1 to 12 hours before the addition of various concentrations of recombinant human TGF1, -2, or -3 proteins (R&D System, Minneapolis, MN). The small-molecule inhibitors LY364947 (5 M; Tocris Biosciences, Ellisville, MO) and SB431542 (5 M; Sigma-Aldrich, St. Louis, MO) were used to examine.