RESUMO
AMP-activated protein kinase (AMPK) signaling activation can inhibit Ultra-violet (UV) radiation (UVR)-induced retinal pigment epithelium (RPE) cell injuries. LB-100 is a novel inhibitor of protein phosphatase 2A (PP2A), the AMPKα1 phosphatase. Here, our results demonstrated that LB-100 significantly inhibited UVR-induced viability reduction, cell death and apoptosis in established ARPE-19â¯cells and primary murine RPE cells. LB-100 activated AMPK, nicotinamide adenine dinucleotide phosphate (NADPH) and Nrf2 (NF-E2-related factor 2) signalings, inhibiting UVR-induced oxidative injuries and DNA damage in RPE cells. Conversely, AMPK inhibition, by AMPKα1-shRNA, -CRISPR/Cas9 knockout or -T172A mutation, almost blocked LB-100-induced RPE cytoprotection against UVR. Importantly, CRISPR/Cas9-mediated PP2A knockout mimicked and nullified LB-100-induced anti-UVR activity in RPE cells. Collectively, these results show that PP2A inhibition by LB-100 protects RPE cells from UVR via activation of AMPK signaling.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Piperazinas/farmacologia , Proteína Fosfatase 2/genética , Protetores Solares/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Ativação Enzimática , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Edição de Genes , Regulação da Expressão Gênica , Humanos , Camundongos , NADP/metabolismo , Cultura Primária de Células , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/efeitos da radiação , Transdução de Sinais , Raios Ultravioleta/efeitos adversosRESUMO
Ultra-violet (UV) radiation (UVR) causes significant oxidative injury to retinal pigment epithelium (RPE) cells. Obacunone is a highly oxygenated triterpenoid limonoid compound with various pharmacological properties. Its potential effect in RPE cells has not been studied thus far. Here in ARPE-19 cells and primary murine RPE cells, obacunone potently inhibited UVR-induced reactive oxygen species accumulation, mitochondrial depolarization, lipid peroxidation and single strand DNA accumulation. UVR-induced RPE cell death and apoptosis were largely alleviated by obacunone. Obacunone activated Nrf2 signaling cascade in RPE cells, causing Keap1-Nrf2 disassociation, Nrf2 protein stabilization and nuclear translocation. It promoted transcription and expression of antioxidant responsive element-dependent genes. Nrf2 silencing or CRISPR/Cas9-induced Nrf2 knockout almost reversed obacunone-induced RPE cytoprotection against UVR. Forced activation of Nrf2 cascade, by Keap1 knockout, similarly protected RPE cells from UVR. Importantly, obacunone failed to offer further RPE cytoprotection against UVR in Keap1-knockout cells. In vivo, intravitreal injection of obacunone largely inhibited light-induced retinal damage. Collectively, obacunone protects RPE cells from UVR-induced oxidative injury through activation of Nrf2 signaling cascade.