Resveratrol-induced autophagy is dependent on IP₃Rs and on cytosolic Ca²

Previous work revealed that intracellular Ca²⁺ signals and the inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R) are essential to increase autophagic flux in response to mTOR inhibition, induced by either nutrient starvation or rapamycin treatment. Here, we investigated whether autophagy induced by resveratrol, a polyphenolic phytochemical reported to trigger autophagy in a non-canonical way, also requires IP₃Rs and Ca²⁺ signaling. Resveratrol augmented autophagic flux in a time-dependent manner in HeLa cells. Importantly, autophagy induced by resveratrol (80μM, 2h) was completely abolished in the presence of 10μM BAPTA-AM, an intracellular Ca²⁺-chelating agent. To elucidate the IP₃R's role in this process, we employed the recently established HEK 3KO cells lacking all three IP₃R isoforms. In contrast to the HEK293 wt cells and to HEK 3KO cells re-expressing IP₃R1, autophagic responses in HEK 3KO cells exposed to resveratrol were severely impaired. These altered autophagic responses could not be attributed to alterations in the mTOR/p70S6K pathway, since resveratrol-induced inhibition of S6 phosphorylation was not abrogated by chelating cytosolic Ca²⁺ or by knocking out IP₃Rs. Finally, we investigated whether resveratrol by itself induced Ca²⁺ release. In permeabilized HeLa cells, resveratrol neither affected the sarco- and endoplasmic reticulum Ca²⁺ ATPase (SERCA) activity nor the IP₃-induced Ca²⁺ release nor the basal Ca²⁺ leak from the ER. Also, prolonged (4 h) treatment with 100μM resveratrol did not affect subsequent IP₃-induced Ca²⁺ release. However, in intact HeLa cells, although resveratrol did not elicit cytosolic Ca²⁺ signals by itself, it acutely decreased the ER Ca²⁺-store content irrespective of the presence or absence of IP₃Rs, leading to a dampened agonist-induced Ca²⁺ signaling. In conclusion, these results reveal that IP₃Rs and cytosolic Ca²⁺ signaling are fundamentally important for driving autophagic flux, not only in response to mTOR inhibition but also in response to non-canonical autophagy inducers like resveratrol. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.