The relationship between Cd‐induced autophagy and lysosomal activation in WRL‐68 cells

This study shows that Cd induces autophagy in the human's embryonic normal liver cell line (WRL‐68). The expression of LC3B‐II and the mature cathepsin L were analyzed by Western blotting. The autophagosomes and lysosomes were directly visualized by electron microscopy and confocal microscopy analysis in Cd‐exposed WRL‐68 cells. In this study, we first found that autophagy induced the activation of lysosomal function in WRL‐68 cells. The lysosomal activation was markedly decreased when the cells were co‐treated with 3‐MA (an inhibitor of autophagy). Secondly, we provided the evidence that the activation of lysosomal function depended on autophagosome–lysosome fusion. The colocalization of lysosome‐associated membrane protein‐2 (LAMP2) and GFP‐LC3 was significantly reduced, when they were treated with thapsigargin (an inhibitor of autophagosome–lysosome fusion). 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. Thirdly, we provided evidence that the activation of lysosomal function was associated with lysosomal acid. We investigated the relationship between autophagosome–lysosome fusion and pH in acidic compartments by visualizing fusion process in WRL‐68 cells. This suggests that increasing pH in acidic compartments in WRL‐68 cells inhibits the autophagosome–lysosome fusion. Finally, we found that the activation of lysosomal function was associated with Ca 2+ stores and the intracellular Ca 2+ channels or pumps were possibly pH‐dependent. Copyright © 2015 John Wiley & Sons, Ltd.