Abstract
BackgroundOxidative stress is a major factor in retinal pigment epithelium (RPE) cells injury that contributes to age-related macular degeneration (AMD). NaIO3 is an oxidative toxic agent and its selective RPE cell damage makes it as a reproducible model of AMD. Although NaIO3 is an oxidative stress inducer, the roles of ROS in NaIO3-elicited signaling pathways and cell viability have not been elucidated, and the effect of NaIO3 on autophagy in RPE cells remains elusive.MethodsIn human ARPE-19 cells, we used Annexin V/PI staining to determine cell viability, immunoblotting to determine protein expression and signaling cascades, confocal microscopy to determine mitochondrial dynamics and mitophagy, and Seahorse analysis to determine mitochondrial oxidative phosphorylation.ResultsWe found that NaIO3 can dramatically induce cytosolic but not mitochondrial ROS production. NaIO3 can also activate ERK, p38, JNK and Akt, increase LC3II expression, induce Drp-1 phosphorylation and mitochondrial fission, but inhibit mitochondrial respiration. Confocal microscopic data indicated a synergism of NaIO3 and bafilomycin A1 on LC3 punctate formation, indicating the induction of autophagy. Using cytosolic ROS antioxidant NAC, we found that p38 and JNK are downstream signals of ROS and involve in NaIO3-induced cytotoxicity but not in mitochondrial dynamics, while ROS is also involved in LC3II expression. Unexpectedly NAC treatment upon NaIO3 stimulation leads to an enhancement of mitochondrial fragmentation and cell death. Moreover, inhibition of autophagy and Akt further enhances cell susceptibility to NaIO3.ConclusionsWe conclude that NaIO3-induced oxidative stress and cytosolic ROS production exert multiple signaling pathways that coordinate to control cell death in RPE cells. ROS-dependent p38 and JNK activation lead to cytotoxicity, while ROS-mediated autophagy and mitochondrial dynamic balance counteract the cell death mechanisms induced by NaIO3 in RPE cells.
Highlights
Age-related macular degeneration (AMD) is characterized by progressive degenerative change of the macula of the retina, resulting in loss of central vision
In this study using human retinal pigment epithelium (RPE) cell line ARPE-19, we found that NaIO3 can induce cytosolic reactive oxygen species (ROS) production, which regulates mitochondrial dynamics, mediates autophagy and activates stress kinases (p38 MAPK and JNK)
NaIO3-induced mixed type cell death in ARPE-19 cells is accompanied by ROS production and mitochondrial dysfunction Before addressing the signaling cascades that mediate the cell death caused by NaIO3, we first clarified the death mode
Summary
Age-related macular degeneration (AMD) is characterized by progressive degenerative change of the macula of the retina, resulting in loss of central vision. The pathophysiology of AMD is not well understood, one of the risk factors associated with it is oxidative stress injury of the RPE [2, 3]. The aged retina is characterized by increased levels of reactive oxygen species (ROS), impaired autophagy, excessive energy consumption and DNA damage, all of which contribute to the degeneration of RPE cells and link to AMD pathogenesis [5,6,7]. Oxidative stress is a major factor in retinal pigment epithelium (RPE) cells injury that contributes to age-related macular degeneration (AMD). NaIO3 is an oxidative stress inducer, the roles of ROS in NaIO3-elicited signaling pathways and cell viability have not been elucidated, and the effect of NaIO3 on autophagy in RPE cells remains elusive
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