The regulation of autophagy by calcium signals: Do we have a consensus?

Macroautophagy (hereafter called 'autophagy') is a cellular process for degrading and recycling cellular constituents, and for maintenance of cell function. Autophagy initiates via vesicular engulfment of cellular materials and culminates in their degradation via lysosomal hydrolases, with the whole process often being termed 'autophagic flux'. Autophagy is a multi-step pathway requiring the interplay of numerous scaffolding and signalling molecules. In particular, orthologs of the family of ∼30 autophagy-regulating (Atg) proteins that were first characterised in yeast play essential roles in the initiation and processing of autophagic vesicles in mammalian cells. The serine/threonine kinase mTOR (mechanistic target of rapamycin) is a master regulator of the canonical autophagic response of cells to nutrient starvation. In addition, AMP-activated protein kinase (AMPK), which is a key sensor of cellular energy status, can trigger autophagy by inhibiting mTOR, or by phosphorylating other downstream targets. Calcium (Ca2+) has been implicated in autophagic signalling pathways encompassing both mTOR and AMPK, as well as in autophagy seemingly not involving these kinases. Numerous studies have shown that cytosolic Ca2+ signals can trigger autophagy. Moreover, introduction of an exogenous chelator to prevent cytosolic Ca2+ signals inhibits autophagy in response to many different stimuli, with suggestions that buffering Ca2+ affects not only the triggering of autophagy, but also proximal and distal steps during autophagic flux. Observations such as these indicate that Ca2+ plays an essential role as a pro-autophagic signal. However, cellular Ca2+ signals can exert anti-autophagic actions too. For example, Ca2+ channel blockers induce autophagy due to the loss of autophagy-suppressing Ca2+ signals. In addition, the sequestration of Ca2+ by mitochondria during physiological signalling appears necessary to maintain cellular bio-energetics, thereby suppressing AMPK-dependent autophagy. This article attempts to provide an integrated overview of the evidence for the proposed roles of various Ca2+ signals, Ca2+ channels and Ca2+ sources in controlling autophagic flux.

Bootman MD ，Chehab T ，Bultynck G ，Parys JB ，Rietdorf K ... -  《-》

Mitochondrial Ca(2+) signals in autophagy.

Macroautophagy (autophagy) is a lysosomal degradation pathway that is conserved from yeast to humans that plays an important role in recycling cellular constituents in all cells. A number of protein complexes and signaling pathways impinge on the regulation of autophagy, with the mammalian target of rapamycin (mTOR) as the central player in the canonical pathway. Cytoplasmic Ca(2+) signaling also regulates autophagy, with both activating and inhibitory effects, mediated by the canonical as well as non-canonical pathways. Here we review this regulation, with a focus on the role of an mTOR-independent pathway that involves the inositol trisphosphate receptor (InsP(3)R) Ca(2+) release channel and Ca(2+) signaling to mitochondria. Constitutive InsP(3)R Ca(2+) transfer to mitochondria is required for autophagy suppression in cells in nutrient-replete media. In its absence, cells become metabolically compromised due to insufficient production of reducing equivalents to support oxidative phosphorylation. Absence of this Ca(2+) transfer to mitochondria results in activation of AMPK, which activates mTOR-independent pro-survival autophagy. Constitutive InsP(3)R Ca(2+) release to mitochondria is an essential cellular process that is required for efficient mitochondrial respiration, maintenance of normal cell bioenergetics and suppression of autophagy.

Cárdenas C ，Foskett JK 《-》

Resveratrol-induced autophagy is dependent on IP(3)Rs and on cytosolic Ca(2).

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.

Luyten T ，Welkenhuyzen K ，Roest G ，Kania E ，Wang L ，Bittremieux M ，Yule DI ，Parys JB ，Bultynck G ... -  《BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH》

IP(3) Receptor-Mediated Calcium Signaling and Its Role in Autophagy in Cancer.

Kania E ，Roest G ，Vervliet T ，Parys JB ，Bultynck G ... -  《Frontiers in Oncology》

AMPK-independent induction of autophagy by cytosolic Ca2+ increase.

Autophagy is a eukaryotic lysosomal bulk degradation system initiated by cytosolic cargo sequestration in autophagosomes. The Ser/Thr kinase mTOR has been shown to constitute a central role in controlling the initiation of autophagy by integrating multiple nutrient-dependent signaling pathways that crucially involves the activity of PI3K class III to generate the phosphoinositide PI(3)P. Recent reports demonstrate that the increase in cytosolic Ca(2+) can induce autophagy by inhibition of mTOR via the CaMKK-alpha/beta-mediated activation of AMPK. Here we demonstrate that Ca(2+) signaling can additionally induce autophagy independently of the Ca(2+)-mediated activation of AMPK. First, by LC3-II protein monitoring in the absence or presence of lysosomal inhibitors we confirm that the elevation of cytosolic Ca(2+) induces autophagosome generation and does not merely block autophagosome degradation. Further, we demonstrate that Ca(2+)-chelation strongly inhibits autophagy in human, mouse and chicken cells. Strikingly, we found that the PI(3)P-binding protein WIPI-1 (Atg18) responds to the increase of cytosolic Ca(2+) by localizing to autophagosomal membranes (WIPI-1 puncta) and that Ca(2+)-chelation inhibits WIPI-1 puncta formation, although PI(3)P-generation is not generally affected by these Ca(2+) flux modifications. Importantly, using AMPK-alpha1(-/-)alpha2(-/-) MEFs we show that thapsigargin application triggers autophagy in the absence of AMPK and does not involve complete mTOR inhibition, as detected by p70S6K phosphorylation. In addition, STO-609-mediated CaMKK-alpha/beta inhibition decreased the level of thapsigargin-induced autophagy only in AMPK-positive cells. We suggest that apart from reported AMPK-dependent regulation of autophagic degradation, an AMPK-independent pathway triggers Ca(2+)-mediated autophagy, involving the PI(3)P-effector protein WIPI-1 and LC3.

Grotemeier A ，Alers S ，Pfisterer SG ，Paasch F ，Daubrawa M ，Dieterle A ，Viollet B ，Wesselborg S ，Proikas-Cezanne T ，Stork B ... -  《-》