title: Resveratrol-induced autophagy is dependent on IP3Rs and on cytosolic Ca2.
abstract: 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.
authors.affiliation: KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine and Leuven Kanker Instituut, Campus Gasthuisberg O/N-I box 802, Herestraat 49, 3000 Leuven, Belgium.
authors.forename: Tomas
authors.initials: T
authors.lastname: Luyten
authors.affiliation: KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine and Leuven Kanker Instituut, Campus Gasthuisberg O/N-I box 802, Herestraat 49, 3000 Leuven, Belgium.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Welkenhuyzen
authors.affiliation: KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine and Leuven Kanker Instituut, Campus Gasthuisberg O/N-I box 802, Herestraat 49, 3000 Leuven, Belgium.
authors.forename: Gemma
authors.initials: G
authors.lastname: Roest
authors.affiliation: KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine and Leuven Kanker Instituut, Campus Gasthuisberg O/N-I box 802, Herestraat 49, 3000 Leuven, Belgium.
authors.forename: Elzbieta
authors.initials: E
authors.lastname: Kania
authors.affiliation: University of Rochester, Department of Pharmacology and Physiology, Rochester, NY 14642, USA.
authors.forename: Liwei
authors.initials: L
authors.lastname: Wang
authors.affiliation: KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine and Leuven Kanker Instituut, Campus Gasthuisberg O/N-I box 802, Herestraat 49, 3000 Leuven, Belgium.
authors.forename: Mart
authors.initials: M
authors.lastname: Bittremieux
authors.affiliation: University of Rochester, Department of Pharmacology and Physiology, Rochester, NY 14642, USA.
authors.forename: David I
authors.initials: DI
authors.lastname: Yule
authors.affiliation: KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine and Leuven Kanker Instituut, Campus Gasthuisberg O/N-I box 802, Herestraat 49, 3000 Leuven, Belgium. Electronic address: jan.parys@kuleuven.be.
authors.forename: Jan B
authors.initials: JB
authors.lastname: Parys
authors.affiliation: KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine and Leuven Kanker Instituut, Campus Gasthuisberg O/N-I box 802, Herestraat 49, 3000 Leuven, Belgium. Electronic address: geert.bultynck@kuleuven.be.
authors.forename: Geert
authors.initials: G
authors.lastname: Bultynck
mesh_terms: D001343:Autophagy / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism*; D000077185:Resveratrol; D013267:Stilbenes / Q000494:pharmacology*
chemical_list: D053496:Inositol 1,4,5-Trisphosphate Receptors; D013267:Stilbenes; D000077185:Resveratrol; D002118:Calcium
keywords: Autophagy; Cytosolic Ca(2+); HEK 3KO; Inositol 1,4,5-trisphosphate; Inositol 1,4,5-trisphosphate receptor; Resveratrol