Abstract
Macroautophagy, a tightly orchestrated intracellular process for bulk degradation of cytoplasmic proteins or organelles, is believed to be essential for cell survival or death in response to stress conditions. Recent observations indicate that autophagy is an adaptive response in cells subjected to prolonged hypoxia. However, the signaling mechanisms that activate autophagy under acute hypoxic stress are not clearly understood. In this study, we show that acute hypoxic stress by treatment with 1% O(2) or desferroxamine, a hypoxia-mimetic agent, of cells renders a rapid induction of LC3-II level changes and green fluorescent protein-LC3 puncta accumulation, hallmarks of autophagic processing, and that this process involves protein kinase Cdelta (PKCdelta), and occurs prior to the induction of BNIP3 (Bcl-2/adenovirus E1B 19-kDa interacting protein 3). Interestingly, hypoxic stress leads to a rapid and transient activation of JNK in Pa-4 or mouse embryo fibroblast cells. Acute hypoxic stress-induced changes in LC3-II level and JNK activation are attenuated in Pa-4 cells by dominant negative PKCdeltaKD or in mouse embryo fibroblast/PKCdelta-null cells. Intriguingly, the requirement of PKCdelta is not apparent for starvation-induced autophagy. The importance of PKCdelta in hypoxic stress-induced adaptive responses is further supported by our findings that inhibition of PKCdelta-facilitated autophagy by 3-methyladenine or Atg5 knock-out renders a greater prevalence of cell death following prolonged desferroxamine treatment, whereas PKCdelta- or JNK1-deficient cells exhibit resistance to extended hypoxic exposure. These results uncover dual roles of PKCdelta-dependent signaling in the cell fate determination upon hypoxic exposure.
Highlights
Macroautophagy, selfcannibalization to degrade cells’ own constituents, including their organelles, is induced by certain environmental cues, such as starvation, heat shock, and hypoxia [1]
We show that acute hypoxic stress by treatment with 1% O2 or desferroxamine, a hypoxia-mimetic agent, of cells renders a rapid induction of LC3-II level changes and green fluorescent protein-LC3 puncta accumulation, hallmarks of autophagic processing, and that this process involves protein kinase C␦ (PKC␦), and occurs prior to the induction of BNIP3 (Bcl-2/adenovirus E1B 19-kDa interacting protein 3)
PKC␦ Is Required for DFO-induced Autophagy—To examine whether or not PKC␦ is involved in DFO-induced autophagic process, we demonstrated that the time-dependent changes in LC3-II level are almost completely abrogated in DFO-treated Pa-4/ PKC␦KD cells (Fig. 2A, left)
Summary
E1, ubiquitin-activating enzyme, E2, ubiquitin carrier protein; PKC␦, protein kinase C␦; DFO, desferroxamine; 3-MA, 3-methyladenine; CQ, chloroquine; EBSS, Earle’s balanced salt solution without phenol red; PI3K, phosphoinositide 3-kinase; AMPK, AMP-activated protein kinase; MTOR, mammalian target of rapamycin; WT, wild type; MEF, mouse embryo fibroblast(s); JNK, Jun N-terminal kinase; JNKi, JNK peptide inhibitor; GFP, green fluorescent protein; PBS, phosphatebuffered saline; FACS, fluorescence-activated cell sorter; ER, endoplasmic reticulum. We demonstrate a novel pathway by which acute hypoxic stress utilizes a rapid activation of the PKC␦ signaling pathway to release Beclin-1 from Bcl-2, leading to autophagy induction
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