title: Hyperactivation of the mammalian degenerin MDEG promotes caspase-8 activation and apoptosis.
abstract: Intracellular calcium overload plays a critical role in numerous pathological syndromes such as heart failure, brain ischemia, and stroke. Hyperactivation of the acid-sensing ion channels including degenerin/epithelial amiloride-sensitive sodium (DEG/ENaC) channels has been shown to elevate intracellular calcium and cause subsequent neuronal cell death that is independent of the canonical Egl-1/Ced-9/Ced-4/Ced-3 apoptotic pathway in Caenorhabditis elegans. In mammalian cells, hyperactivation of the DEG/ENaC channels can also lead to cell death, although the underlying mechanism remains largely unknown. Here, we use a tetracycline-inducible system to express the hyperactivation mutant of a mammalian DEG/ENaC channel protein, MDEG G430F, in murine kidney epithelial cells deficient in the key mitochondrial apoptotic proteins Bax and Bak. Remarkably, expression of MDEG G430F induces increased intracellular calcium, reactive oxygen species (ROS) production, and cell death. The MDEG G430F-induced cell death is blocked by the intracellular calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl ester), ROS scavengers, and the caspase inhibitor z-VAD-fmk (where z and fmk are benzyloxycarbonyl and fluoromethyl ketone). Mechanistically, the intracellular calcium overload and ROS increase lead to the inhibition of proteasomal and autophagic protein degradation, which promotes the accumulation of protein aggregates containing caspase-8 and subsequent caspase-8 activation. As protein aggregation upon the inhibition of proteasomal and autophagic degradation pathways is mediated by the ubiquitin-binding protein SQSTM1/p62 and the autophagy-related protein LC3, silencing of p62 and LC3 protects cells from MDEG G430F-induced cell death. Our results uncover a new mechanism of caspase-8-mediated apoptosis induced by intracellular calcium overload that is dependent on the autophagy-related proteins LC3 and p62 upon hyperactivation of DEG/ENaC channels.
authors.affiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York 11794, USA.
authors.forename: Ji-An
authors.initials: JA
authors.lastname: Pan
authors.forename: Yongjun
authors.initials: Y
authors.lastname: Fan
authors.forename: Rajesh Kumar
authors.initials: RK
authors.lastname: Gandhirajan
authors.forename: Muniswamy
authors.initials: M
authors.lastname: Madesh
authors.forename: Wei-Xing
authors.initials: WX
authors.lastname: Zong
mesh_terms: D062565:Acid Sensing Ion Channels / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D053181:Caspase 8 / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D015640:Ion Channel Gating / Q000187:drug effects; D008322:Mammals / Q000378:metabolism*; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D025821:Polyubiquitin / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D062565:Acid Sensing Ion Channels; C568425:Asic2 protein, rat; C490981:Gtf2h1 protein, mouse; C480860:Map1lc3b protein, mouse; D008869:Microtubule-Associated Proteins; D050505:Mutant Proteins; D061988:Proteasome Inhibitors; D017382:Reactive Oxygen Species; D014157:Transcription Factors; D025821:Polyubiquitin; D051758:Transcription Factor TFIIH; D053181:Caspase 8; D046988:Proteasome Endopeptidase Complex; D002118:Calcium