Intracellular calcium has a dual role in the regulation of autophagy

Autophagy, a cellular self-eating process, is important for eukaryotic survival and involves degradation of misfolded proteins, dysfunctional organelles and pathogens through the action of lysosomal hydrolases. Autophagy also has a housekeeping role in cellular turnover, and is substantially increased in response to nutrient starvation. Mechanistic target of rapamycin (mTOR), a protein kinase that controls cell growth, is a key regulator of autophagy, and pharmacological inhibition of mTOR using rapamycin triggers autophagy. For over two decades, intracellular calcium has been established as an important regulator of autophagy. However, the mechanisms by which calcium regulates autophagy are unclear. A convenient method for assessing cellular autophagy in living cells is to monitor the generation of autophagic vesicles (AVs) using fluorescent reporters. In this study, HeLa cells stably expressing GFP-tagged microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) were used to quantitate the formation of AVs in response to various stimuli. Using genetic and pharmacological tools, preventing calcium release from inositol 1,4,5-trisphosphate receptors (InsP3Rs) resulted in enhanced autophagy. The inhibition of InsP3R-mediated calcium release evoked a similar extent of autophagy as that triggered by inhibition of mTOR using rapamycin. Plausibly, the release of calcium from InsP3Rs is required to stimulate mitochondrial respiration and ATP production, and thereby prevent induction of autophagy by limiting the activity of mTOR. To test this hypothesis, we inhibited mitochondrial respiration with oligomycin and antimycin and found an increase in autophagy. The results of our study are consistent with the proposal that the flux of calcium from InsP3Rs to the mitochondrial matrix is necessary to stimulate ATP production and thereby avoid induction of autophagy. We have also found that loading cells with the calcium chelator BAPTA-AM did not stimulate autophagy, but inhibited autophagy evoked by rapamycin. Similarly, blocking SERCA pumps with CPA in a calcium-free environment did not stimulate autophagy, but prevented the induction of autophagy by rapamycin. Conversely, increasing cytosolic calcium levels with ionomycin elevated autophagy significantly. These results suggest that calcium is needed to induce autophagy, and that non-specific calcium signals arising from ionophore-mediated calcium transport can activate autophagy, yet physiologically occurring calcium signals (via InsP3Rs) prevent autophagy