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

purposeTo clarify the primary oxidative stress response signaling pathways in trabecular meshwork (TM) cells and their effects on cell viability.

methodsPorcine TM cells were treated with 600 μM or 800 μM H₂O₂, 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 H₂O₂. Cell viability was assessed using propidium iodide and anticleaved caspase-3 antibody.

resultsTM cells treated with 600 μM H₂O₂ showed morphologic changes at 2 h that were partially recovered at 8 h after treatment. TM cells treated with 800 μM H₂O₂ did not recover, and the viability was significantly decreased. Both doses of H₂O₂ 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 H₂O₂. Of these three inhibitors, the PI3K and ERK1/2 inhibitors decreased TM cell viability under oxidative stress.

conclusionsIn 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 H₂O₂ treatment accompanied by actin cytoskeletal changes.