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Cells 2018, 7(12), 239;

The ER Stress Inducer l-Azetidine-2-Carboxylic Acid Elevates the Levels of Phospho-eIF2α and of LC3-II in a Ca2+-Dependent Manner

Laboratory for Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine & Leuven Kanker Instituut, KU Leuven, Campus Gasthuisberg O/N-1 B-802, Herestraat 49, BE-3000 Leuven, Belgium
Centre for Molecular Medicine Norway, Nordic EMBL Partnership for Molecular Medicine, University of Oslo, P.O. Box 1137 Blindern, N-0318 Oslo, Norway
Author to whom correspondence should be addressed.
Received: 31 October 2018 / Revised: 27 November 2018 / Accepted: 28 November 2018 / Published: 30 November 2018
(This article belongs to the Special Issue Cellular Stress Response in Health and Disease)
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Accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR) to reduce protein load and restore homeostasis, including via induction of autophagy. We used the proline analogue l-azetidine-2-carboxylic acid (AZC) to induce ER stress, and assessed its effect on autophagy and Ca2+ homeostasis. Treatment with 5 mM AZC did not induce poly adenosine diphosphate ribose polymerase (PARP) cleavage while levels of binding immunoglobulin protein (BiP) and phosphorylated eukaryotic translation initiation factor 2α (eIF2α) increased and those of activating transcription factor 6 (ATF6) decreased, indicating activation of the protein kinase RNA-like ER kinase (PERK) and the ATF6 arms of the UPR but not of apoptosis. AZC treatment in combination with bafilomycin A1 (Baf A1) led to elevated levels of the lipidated form of the autophagy marker microtubule-associated protein light chain 3 (LC3), pointing to activation of autophagy. Using the specific PERK inhibitor AMG PERK 44, we could deduce that activation of the PERK branch is required for the AZC-induced lipidation of LC3. Moreover, both the levels of phospho-eIF2α and of lipidated LC3 were strongly reduced when cells were co-treated with the intracellular Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraaceticacid tetra(acetoxy-methyl) ester (BAPTA-AM) but not when co-treated with the Na+/K+ ATPase inhibitor ouabain, suggesting an essential role of Ca2+ in AZC-induced activation of the PERK arm of the UPR and LC3 lipidation. Finally, AZC did not trigger Ca2+ release from the ER though appeared to decrease the cytosolic Ca2+ rise induced by thapsigargin while also decreasing the time constant for Ca2+ clearance. The ER Ca2+ store content and mitochondrial Ca2+ uptake however remained unaffected. View Full-Text
Keywords: autophagy; ER stress; UPR; PERK; Ca2+; l-azetidine-2-carboxylic acid autophagy; ER stress; UPR; PERK; Ca2+; l-azetidine-2-carboxylic acid

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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Roest, G.; Hesemans, E.; Welkenhuyzen, K.; Luyten, T.; Engedal, N.; Bultynck, G.; Parys, J.B. The ER Stress Inducer l-Azetidine-2-Carboxylic Acid Elevates the Levels of Phospho-eIF2α and of LC3-II in a Ca2+-Dependent Manner. Cells 2018, 7, 239.

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