Y-27632 increased the permeability of the SCE-cell monolayer in association with disruption of the tight junction, F-actin depolymerization, and changes in various cell functions, including calcium transfer.
PURPOSE. We investigated the involvement of the RhoA/Rho kinase (ROCK) signal transduction pathway in dexamethasone (DEX)-induced changes in aqueous outflow.METHODS. Using trabecular meshwork (TM) and Schlemm's canal endothelial (SCE) cells, RhoA activation was evaluated with a pull-down assay and myosin light chain phosphorylation was evaluated by Western blot analysis. Outflow facility was measured in perfused porcine anterior segment organ cultures treated with DEX and/or Y-27632, a selective ROCK inhibitor. The barrier function of the cultured cells on a micropore filter was evaluated by measuring the transendothelial electrical resistance. Collagen, fibronectin, and integrin mRNA expression levels were evaluated by quantitative realtime RT-PCR. RESULTS.Relative RhoA activities increased following stimulation with 100 nM DEX in TM and SCE cells. Perfusion with DEX decreased outflow facility by 31.9 6 14.3% compared to controls at 24 hours, but not by 50 lM Y-27632 in addition to DEX. The transendothelial electrical resistance of the SCE cell monolayer was increased by 48.6 6 6.4% and 5.3 6 5.0% following DEX treatments without and with 10 lM Y-27632, respectively, compared to controls. In TM cells, the mRNA expressions of COL4A1 and fibronectin were increased significantly by DEX treatment, but combined treatment with Y-27632 and DEX significantly inhibited the increase in COL4A1and fibronectin expression.CONCLUSIONS. Activation of the Rho/ROCK pathway in SCE cells contributes to the mechanism of DEX-induced changes in aqueous outflow. (Invest Ophthalmol Vis Sci. 2012;53:7097-7108) DOI:10.1167/iovs.12-9989 S teroid-induced ocular hypertension is an important clinical problem related to the use of glucocorticoid therapy for many types of disorders. Already in 1963, Armaly reported that the widely used glucocorticoid dexamethasone (DEX) induces changes in intraocular pressure (IOP) and fluid dynamics in normal eyes, and that DEX-induced IOP elevation is observed more frequently in the patients with glaucoma. 1-3 IOP levels often are quite high in patients with steroid-induced ocular hypertension, and prolonged IOP elevation sometimes results in the loss of visual function. Topical administration of triamcinolone acetonide (TA), a synthetic corticosteroid, is considered a useful therapeutic modality for the management of intraocular inflammatory and/or proliferative diseases. The increasing number of patients treated with TA by intravitreous and/or sub-Tenon injections, however, has attracted the attention of ophthalmologists because of the potential risk of steroid-induced ocular hypertension. 4,5The mechanisms underlying steroid-induced ocular hypertension are associated with abnormal changes in the conventional outflow pathway caused by steroid administration. Some researchers attribute the decreased outflow to an accumulation of extracellular matrix (ECM) components, such as glycosaminoglycans, collagens, fibronectin, and elastin. [6][7][8][9] Others have proposed that the decreased outflow is due to decrea...
PURPOSE. To investigate the roles of Yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ), the major effector molecules of the Hippo pathway, in TGFb2-mediated conjunctival fibrosis.METHODS. Primary human conjunctival fibroblasts were treated with TGF-b2. The expression of YAP/TAZ was examined by Western blot analyses and immunocytochemistry. The expression of fibrotic proteins and genes were evaluated by Western blot analyses and quantitative real-time PCR, respectively. The effects of YAP/TAZ on fibrotic changes were examined by knockdown experiments and the YAP/TAZ inhibitor, verteporfin.RESULTS. TGF-b2 stabilized YAP/TAZ and subsequently activated Smad2/3, which led to the transcription of fibrotic genes in human primary conjunctival fibroblasts. These fibrotic genes were differently regulated by YAP/TAZ. Notably, a-smooth muscle actin, fibronectin, collagen I, and collagen IV were primarily regulated by YAP. In contrast, CCN family proteins (CTGF and CYR61) depended on both YAP and TAZ. Mechanistically, YAP/TAZ were located in close proximity to Smad2/3, and in particular, YAP was required for TGF-b2-mediated phosphorylation and the nuclear translocation of Smad2/3. Furthermore, a YAP/TAZ inhibitor markedly suppressed TGF-b2-mediated fibrotic changes in conjunctival fibroblasts.CONCLUSIONS. YAP/TAZ acted as a molecular hub of TGF-b2 signaling in a cellular model of conjunctival fibrosis. Moreover, verteporfin, a YAP/TAZ inhibitor exerted potent antifibrosis effects by suppressing TGF-b2-YAP/TAZ-Smad signaling. Our study highlights YAP/TAZ as essential regulators of conjunctival fibrosis and shows that inhibition of YAP/TAZ might potentially improve the outcomes of glaucoma filtration surgery.
Glaucoma is an age-related neurodegenerative disease of retinal ganglion cells, and appropriate turnover of the extracellular matrix in the trabecular meshwork is important in its pathology. Here, we report the effects of Rho-associated kinase (ROCK) and p38 MAP kinase on transforming growth factor (TGF)-β2–induced type I collagen production in human trabecular meshwork cells. TGF-β2 increased RhoA activity, actin polymerization, and myosin light chain 2 phosphorylation. These effects were significantly inhibited by Y-27632, but not SB203580. TGF-β2 also increased promoter activity, mRNA synthesis, and protein expression of COL1A2. These effects were significantly inhibited by SB203580, but not Y-27632. Additionally, Y-27632 did not significantly inhibit TGF-β2–induced promoter activation, or phosphorylation or nuclear translocation of Smad2/3, whereas SB203580 partially suppressed these processes. Collectively, TGF-β2–induced production of type 1 collagen is suppressed by p38 inhibition and accompanied by partial inactivation of Smad2/3, in human trabecular meshwork cells.
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