Oxidative stress response signaling pathways in trabecular meshwork cells and their effects on cell viability

PURPOSE: To clarify the primary oxidative stress response signaling pathways in trabecular meshwork (TM) cells and their effects on cell viability. METHODS: Porcine TM cells were treated with 600 μM or 800 μM H(2)O(2), and their time-dependent morphologic changes were observed. Phosphorylation of protein kinase B (Akt), extracellular regulated kinase (ERK)1/2, p38, and c-Jun NH2-terminal kinase (JNK) was evaluated by western blot analysis. The intracellular localization of NFκB was evaluated by western blot analysis. One-hour pretreatments with LY294002, U0126, and SB203580, with the inhibitors of PI3K, ERK1/2, and p38, respectively, were conducted to evaluate the roles of these molecules in the cellular reaction against H(2)O(2). Cell viability was assessed using propidium iodide and anticleaved caspase-3 antibody. RESULTS: TM cells treated with 600 μM H(2)O(2) showed morphologic changes at 2 h that were partially recovered at 8 h after treatment. TM cells treated with 800 μM H(2)O(2) did not recover, and the viability was significantly decreased. Both doses of H(2)O(2) activated Akt, ERK1/2, and p38 in TM cells at 20 min after treatment, but not JNK or NFкB until 1 h after treatment. Inhibitors of PI3K, ERK1/2, and p38 suppressed recovery from the morphologic changes induced by 600 μM H(2)O(2). Of these three inhibitors, the PI3K and ERK1/2 inhibitors decreased TM cell viability under oxidative stress. CONCLUSIONS: In TM cells, the PI3K-Akt, ERK, and p38 signaling pathways are primary oxidative stress response pathways involved in the mechanism of recovery from cellular morphologic changes induced by H(2)O(2) treatment accompanied by actin cytoskeletal changes.