title: A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90.
abstract: P2RX7 is an ATP-gated ion channel, which can also exhibit an open state with a considerably wider permeation. However, the functional significance of the movement of molecules through the large pore (LP) and the intracellular signaling events involved are not known. Here, analyzing the consequences of P2RX7 activation in primary myoblasts and myotubes from the Dmd(mdx) mouse model of Duchenne muscular dystrophy, we found ATP-induced P2RX7-dependent autophagic flux, leading to CASP3-CASP7-independent cell death. P2RX7-evoked autophagy was triggered by LP formation but not Ca(2+) influx or MAPK1-MAPK3 phosphorylation, 2 canonical P2RX7-evoked signals. Phosphoproteomics, protein expression inference and signaling pathway prediction analysis of P2RX7 signaling mediators pointed to HSPA2 and HSP90 proteins. Indeed, specific HSP90 inhibitors prevented LP formation, LC3-II accumulation, and cell death in myoblasts and myotubes but not in macrophages. Pharmacological blockade or genetic ablation of p2rx7 also proved protective against ATP-induced death of muscle cells, as did inhibition of autophagy with 3-MA. The functional significance of the P2RX7 LP is one of the great unknowns of purinergic signaling. Our data demonstrate a novel outcome--autophagy--and show that molecules entering through the LP can be targeted to phagophores. Moreover, we show that in muscles but not in macrophages, autophagy is needed for the formation of this LP. Given that P2RX7-dependent LP and HSP90 are critically interacting in the ATP-evoked autophagic death of dystrophic muscles, treatments targeting this axis could be of therapeutic benefit in this debilitating and incurable form of muscular dystrophy.
authors.affiliation: a Molecular Medicine Laboratory; Institute of Biomedical and Biomolecular Sciences; School of Pharmacy and Biomedical Sciences ; University of Portsmouth ; Portsmouth , UK.
authors.forename: Christopher N J
authors.initials: CN
authors.lastname: Young
authors.forename: Anthony
authors.initials: A
authors.lastname: Sinadinos
authors.forename: Alexis
authors.initials: A
authors.lastname: Lefebvre
authors.forename: Philippe
authors.initials: P
authors.lastname: Chan
authors.forename: Stephen
authors.initials: S
authors.lastname: Arkle
authors.forename: David
authors.initials: D
authors.lastname: Vaudry
authors.forename: Dariusz C
authors.initials: DC
authors.lastname: Gorecki
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects; D015220:Calcium Channels / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism; D008297:Male; D008810:Mice, Inbred C57BL; D018101:Mice, Inbred mdx; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008954:Models, Biological; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D009137:Muscular Dystrophy, Animal / Q000378:metabolism* / Q000473:pathology*; D032446:Myoblasts / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D010750:Phosphoproteins / Q000378:metabolism; D020543:Proteome / Q000378:metabolism; D058486:Receptors, Purinergic P2X7 / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D015220:Calcium Channels; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; C490434:Hspa2 protein, mouse; C480860:Map1lc3b protein, mouse; D008869:Microtubule-Associated Proteins; C546469:P2rx7 protein, mouse; D010750:Phosphoproteins; D020543:Proteome; D058486:Receptors, Purinergic P2X7; D000255:Adenosine Triphosphate
keywords: 3-MA, 3-methyladenine; ACTB, actin, β; ATP; BECN1, Beclin 1, autophagy-related; BzATP, 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate; CASP, caspase; DAPC, dystrophin associated protein complex; DMD; DMD, Duchenne muscular dystrophy; Dmdmdx p2rx7−/− double-mutant mouse model; Dmdmdx, C57BL/10ScSn-Dmdmdx/J mouse model of DMD; EtBr, ethidium bromide; GA, geldanamycin; HSP70; HSP90; HSP90, heat shock protein 90; HSPA2/HSP70, heat shock protein 2; LC3; LDH, lactate dehydrogenase; LP, large pore, P2RX7-dependent; LY, Lucifer Yellow; MAP1LC3B/LC3, microtubule-associated protein 1 light chain 3 β; MAPK, mitogen-activated protein kinase; P2RX7; P2RX7, purinergic receptor P2X, ligand-gated ion channel, 7; PtdIns3K, phosphatidylinositol 3-kinase, class III; Wt, C57BL/10ScSn wild-type mouse; autophagy; cell death; eATP, extracellular ATP; purinoceptors