RESUMO
Reactive oxygen species have been implicated in processes involving cellular damage and subsequent cell death, especially in organs such as the eye that are constantly exposed to excitatory signals. However, recent studies have shown that oxidant species can also act as intracellular signalling molecules promoting cell survival, but little is known about this mechanism in the retina. The present study demonstrates for the first time that hydrogen peroxide (H2O2) is generated rapidly and acts as a pro-survival signal in response to a variety of apoptotic stimuli in retina-derived 661W cells and in the retinal ganglion cell line RGC-5. Focussing on 661Ws and serum deprivation, we systematically investigated pro-survival and pro-death pathways and discovered that the rapid and transient burst of H2O2 activates the AKT survival pathway. Activation of the apoptotic machinery takes place following the decline of H2O2 to basal levels. To substantiate this proposed pro-survival role of H2O2, we inhibited the oxidant burst, which exacerbated cell death. Conversely, maintenance of the oxidant signal using exogenous H2O2 enhanced cell survival. Overall, the results presented in this study provide evidence for a novel role of H2O2 in mediating survival of retinal cells in response to apoptotic stimuli.
Assuntos
Apoptose , Peróxido de Hidrogênio/metabolismo , Retina/metabolismo , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo , Animais , Benzopiranos/farmacologia , Calpaína/metabolismo , Linhagem Celular , Sobrevivência Celular , Glicoproteínas de Membrana/metabolismo , Camundongos , NADPH Oxidase 2 , NADPH Oxidase 4 , NADPH Oxidases/metabolismo , Oniocompostos/farmacologia , Oxirredução , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Retina/efeitos dos fármacos , Células Ganglionares da Retina/efeitos dos fármacos , Transdução de SinaisRESUMO
The interaction of soluble forms of the human cation-independent insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-IIR) with IGFs and mannosylated ligands was analyzed in real time. IGF-IIR proteins containing domains 1-15, 10-13, 11-13, or 11-12 were combined with rat CD4 domains 3 and 4. Following transient expression in 293T cells, secreted protein was immobilized onto biosensor chips. beta-Glucuronidase and latent transforming growth factor-beta1 bound only to domains 1-15. IGF-II bound to all constructs except a control, which contained a point mutation in domain 11. The affinity of domains 1-15, 10-13, 11-13, and 11-12 to IGF-II were 14, 120, 100, and 450 nm, respectively. Our data suggest that domain 13 acts as an enhancer of IGF-II affinity by slowing the rate of dissociation, but additional enhancement by domains other than 10-13 also occurs. As the receptor functions to transport ligands from either the trans-Golgi network or extracellular space to the endosomes, the interaction of IGF-IIR extracellular domains with IGF-II was analyzed over a pH range of 5.0-7.4. The constructs behaved differently in response to pH and in recovery after low pH exposure, suggesting that pH stability of the extracellular domains depends on domains other than 10-13.