Inositol hexakisphosphate kinases promote autophagy

We and other authors have previously reported that increasing cellular diphosphoinositol pentakisphosphate (InsP₇) levels increases cell sensitivity to cell death. In the present study, we elucidated the relationship between inositol hexakisphosphate kinases (InsP₆Ks), which form InsP₇, and autophagy using InsP₆Ks overexpression and disruption systems. A large number of autophagosomes were induced in cells transfected with InsP₆Ks, as revealed by the conversion of LC3-I to LC3-II, which was examined using immunoblotting, immunocytochemistry, and immuno-electron microscopy for LC3; consequently, the rate of cell death was higher among these cells than among cells transfected with a control vector, as shown using propidium iodide staining. However, the reduction of InsP₆Ks levels using RNAi suppressed the formation of autophagosomes. Moreover, the number of autophagosomes and the rate of cell death were significantly higher among cells transfected with InsP₆Ks subjected to staurosporine-induced stress than among cells transfected with InsP₆Ks subjected to normal conditions. The cell death induced by InsP₆Ks was not completely suppressed by z-VAD-fmk, a pan-caspase inhibitor. The phosphorylation of mammalian target of rapamycin (mTOR) was also depressed in cells overexpressing InsP₆Ks, suggesting that the mTOR pathway regulates autophagosomes generated by InsP₆Ks. These findings imply that InsP₆Ks promote autophagy and induce caspase-independent cell death. This phenomenon opens a new pathway of autophagy via InsP₆Ks.