Study On The Role Of Lysosomes In Cadmium - Induced Autophagy Of WRL - 68 Cells

Our previous study has shown that the cadmium (Cd) in low concentrations (<10 μM) induces autophagy in WRL-68 cells, but does not induce apoptosis. Up to now, there has been clearly that lysosomes were involved in autophagy, but how autophagy affects the activation of lysosomal function is not clear yet. Our research was to explore the relationship between Cd-induced autophagy and lysosomal activation in WRL-68 cells. There are four parts in our research. Lysosomal activation induced by Cd in WRL-68 cells is autophagosome-dependent. Activation of lysosomal function depends on autophagosome-lysosome fusion. Autophagosome-lysosome fusion is inhibited by a rise of pH of acidic compartments. The intracellular calcium ion levels affect lysosomal activation.In the present study, we used cell culture, transmission electron microscope (TEM), western blot analysis, confocal microscope and fluorescence microscope to detect the expression of LC3B-II, mature cathepsin L and the colocalization of autophagosome and lysosome under Cd in low concentrations (<10 μM). We used flow cytometry (FCM) to detect the relationship between lysosomal activation and the intracellular calcium ion levels. The following are our study results:1. Autophagosome-dependent lysosomal activation induced by Cd in WRL-68 cellsWRL-68 cells showed autophagy after exposure to CdCl2 in low doses (0-10 μM) for 12 h. We find the cytoplasm was full of autophagosomes containing visible cytoplasmic contents under transmission electron microscope. But when co-treated with the 3-MA (an autophagy inhibitor), the expression of the mature cathepsin L and LC3B-II/I was decreased. We demonstrated that the activation of lysosomal function might be leaded by autophagy, when autophagy was inhibited, lysosome enzyme activity was decreased.2. Activation of lysosomal function depends on autophagosome-lysosome fusion in WRL-68 cellsWRL-68 cells showed autophagy after exposure to Cd in low doses. When cells were treated with Cd, the autophagosome-lysosome fusion was markedly increased, but there was no significant influence co-treated with thapsigargin (TG). TG effectively reduced the increase of the mature cathepsin L activity, but markedly increased the accumulation of LC3B-II in the cells under Cd, and significantly reduced the colocalization of LAMP2 and GFP-LC3. We demonstrated that deletion or blockage of the autophagosome-lysosome fusion process effectively diminished lysosomal activation, which suggests that lysosomal activation occurring in the course of autophagy is dependent on autophagosome-lysosome fusion.3. Autophagosome-lysosome fusion inhibited by arise of pH of acidic compartments in WRL-68 cellsWRL-68 cells showed autophagy after exposure to Cd in low doses. Significantly increase of LysoTracker staining was observed in WRL-68 cells treated with Cd. However, when the cells co-treated with CQ, the LysoTracker staining was markedly abolished under various treatments. CQ also effectively prevented the mature cathepsin L enzyme activity induced by Cd. The treatments led to increased LC3B-Ⅱ protein levels. But the GFP-LC3 and LAMP2 colocalization was markedly decreased. This suggests that increasing pH in acidic compartments in WRL-68 cells inhibits the autophagosome-lysosome fusion.4. The relationship between the intracellular calcium levels and lysosomal activation in WRL-68 cellsWRL-68 cells showed autophagy after exposure to Cd in low doses. The respective composites with LysoTracker puncta revealed stark increase in lysosome numbers in WRL-68 cells treated with 8 μM Cd, and significant decrease co-treated with CQ but no marked decrease co-treated with 2-APB. We also found that the intracellular Ca2+ levels were significantly decreased when WRL-68 cells were treated with CQ or 2-APB. GFP-LC3 and LAMP2 colocalization was markedly decreased, too. These indicated that although the intracellular Ca2+ levels did not markedly affect the pH in acidic compartments, lysosomal pH had a profound effect on the intracellular Ca2+ levels.The present study has shown that the Cd in low concentrations (<10 μM) induces autophagy in WRL-68 cells. This paper has mainly explored the effect of autophagy on the activation of lysosomal function and such a process depends on three critical factors: occurrence of autophagosome-lysosome fusion, the pH of acidic compartments and the intracellular Ca2+ levels in WRL-68 cells.