Oxidative stress and calpain inhibition induce alpha B-crystallin phosphorylation via p38-MAPK and calcium signalling pathways in H9c2 cells.
We investigated the response of alphaB-crystallin to oxidative stress and calpain inhibition in an attempt to elucidate the signalling pathways mediating its phosphorylation. Given the high expression levels of alphaB-crystallin in cardiac muscle one can evaluate the significance of its participation in preservation of homeostasis under adverse conditions. H9c2 cardiac myoblasts were used as our experimental model since their response reflects the signal transduction pathways activated by stress conditions in the myocardium. Thus, in H9c2 cells treated with H2O2 the mechanism regulating alphaB-crystallin phosphorylation was found to involve p38-MAPK/MSK1 as well as intracellular free calcium levels. Our immunocytochemical experiments demonstrated phosphorylated alphaB-crystallin to be co-localized with tubulin, potentially preserving cytoskeletal architecture under these interventions. In H9c2 cells treated with calpain inhibitors (ALLN, ALLM) alphaB-crystallin exhibited a p38-MAPK- and [Ca 2+](i)-dependent phosphorylation pattern since the latter was ablated in the presence of the selective p38-MAPK inhibitor SB203580 and calcium chelator BAPTA-AM. Calpain activity repression ultimately led to apoptosis confirmed by PARP fragmentation and chromatin condensation. However, the apoptotic pathway activated by ALLM and ALLN differed, underlying the diverse transduction mechanisms stimulated. In addition to this, an anti-apoptotic role for phospho-alphaB-crystallin was verified by confirmation of its interaction with pro-caspase 3, hindering its cleavage and subsequent activation. Collectively, our findings underline alphaB-crystallin crucial role as a participant of cardiac cells early response to stressful stimuli compromising their survival.
Aggeli
Ioanna-Katerina S
IK
Department of Animal and Human Physiology, School of Biology, Faculty of Sciences, University of Athens, Panepistimioupolis Ilissia, 157 84 Athens, Greece.
Beis
Isidoros
I
Gaitanaki
Catherine
C
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D020013:Calcium Signaling / Q000187:drug effects; D002154:Calpain / Q000037:antagonists & inhibitors*; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D009842:Oligopeptides / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000378:metabolism; D013997:Time Factors; D014404:Tubulin / Q000378:metabolism; D038203:alpha-Crystallin B Chain / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D007976:Leupeptins; D009842:Oligopeptides; D014404:Tubulin; D038203:alpha-Crystallin B Chain; C063261:acetylleucyl-leucyl-norleucinal; C069861:calpain inhibitor 2; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D010768:Phosphoserine; D004533:Egtazic Acid; D045304:Cytochromes c; D006861:Hydrogen Peroxide; D011065:Poly(ADP-ribose) Polymerases; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C116919:mitogen and stress-activated protein kinase 1; D048051:p38 Mitogen-Activated Protein Kinases; D002154:Calpain; D053148:Caspase 3