title: Hypoxia triggers AMPK activation through reactive oxygen species-mediated activation of calcium release-activated calcium channels.
abstract: AMP-activated protein kinase (AMPK) is an energy sensor activated by increases in [AMP] or by oxidant stress (reactive oxygen species [ROS]). Hypoxia increases cellular ROS signaling, but the pathways underlying subsequent AMPK activation are not known. We tested the hypothesis that hypoxia activates AMPK by ROS-mediated opening of calcium release-activated calcium (CRAC) channels. Hypoxia (1.5% O(2)) augments cellular ROS as detected by the redox-sensitive green fluorescent protein (roGFP) but does not increase the [AMP]/[ATP] ratio. Increases in intracellular calcium during hypoxia were detected with Fura2 and the calcium-calmodulin fluorescence resonance energy transfer (FRET) sensor YC2.3. Antioxidant treatment or removal of extracellular calcium abrogates hypoxia-induced calcium signaling and subsequent AMPK phosphorylation during hypoxia. Oxidant stress triggers relocation of stromal interaction molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca(2+) sensor, to the plasma membrane. Knockdown of STIM1 by short interfering RNA (siRNA) attenuates the calcium responses to hypoxia and subsequent AMPK phosphorylation, while inhibition of L-type calcium channels has no effect. Knockdown of the AMPK upstream kinase LKB1 by siRNA does not prevent AMPK activation during hypoxia, but knockdown of CaMKKβ abolishes the AMPK response. These findings reveal that hypoxia can trigger AMPK activation in the apparent absence of increased [AMP] through ROS-dependent CRAC channel activation, leading to increases in cytosolic calcium that activate the AMPK upstream kinase CaMKKβ.
authors.affiliation: Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA.
authors.forename: Paul T
authors.initials: PT
authors.lastname: Mungai
authors.forename: Gregory B
authors.initials: GB
authors.lastname: Waypa
authors.forename: Amit
authors.initials: A
authors.lastname: Jairaman
authors.forename: Murali
authors.initials: M
authors.lastname: Prakriya
authors.forename: Danijela
authors.initials: D
authors.lastname: Dokic
authors.forename: Molly K
authors.initials: MK
authors.lastname: Ball
authors.forename: Paul T
authors.initials: PT
authors.lastname: Schumacker
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000111:Acetylcysteine / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase / Q000235:genetics / Q000378:metabolism*; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006801:Humans; D000860:Hypoxia; D008168:Lung / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D000071740:ORAI1 Protein; D034622:RNA Interference; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D000071737:Stromal Interaction Molecule 1; D019284:Thapsigargin / Q000494:pharmacology
chemical_list: D015220:Calcium Channels; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; D017382:Reactive Oxygen Species; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D000249:Adenosine Monophosphate; D019284:Thapsigargin; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D055372:AMP-Activated Protein Kinases; D002118:Calcium; D000111:Acetylcysteine