Hyperosmotic stress activates p65/RelB NFkappaB in cultured cardiomyocytes with dichotomic actions on caspase activation and cell death.
NFkappaB is a participant in the process whereby cells adapt to stress. We have evaluated the activation of NFkappaB pathway by hyperosmotic stress in cultured cardiomyocytes and its role in the activation of caspase and cell death. Exposure of cultured rat cardiomyocytes to hyperosmotic conditions induced phosphorylation of IKKalpha/beta as well as degradation of IkappaBalpha. All five members of the NFkappaB family were identified in cardiomyocytes. Analysis of the subcellular distribution of NFkappaB isoforms in response to hyperosmotic stress showed parallel migration of p65 and RelB from the cytosol to the nucleus. Measurement of the binding of NFkappaB to the consensus DNA kappaB-site binding by EMSA revealed an oscillatory profile with maximum binding 1, 2 and 6h after initiation of the hyperosmotic stress. Supershift analysis revealed that p65 and RelB (but not p50, p52 or cRel) were involved in the binding of NFkappaB to DNA. Hyperosmotic stress also resulted in activation of the NFkappaB-lux reporter gene, transient activation of caspases 9 and 3 and phosphatidylserine externalization. The effect on cell viability was not prevented by ZVAD (a general caspase inhibitor). Blockade of NFkappaB with AdIkappaBalpha, an IkappaBalpha dominant negative overexpressing adenovirus, prevented activation of caspase 9 (more than that caspase 3) but did not affect cell death in hyperosmotically stressed cardiomyocytes. We conclude that hyperosmotic stress activates p65 and RelB NFkappaB isoforms and NFkappaB mediates caspase 9 activation in cardiomyocytes. However cell death triggered by hyperosmotic stress was caspase- and NFkappaB-independent.
Eisner
Verónica
V
Faculty of Chemical and Pharmaceutical Sciences, University of Chile, Santiago.
Quiroga
Clara
C
Criollo
Alfredo
A
Eltit
José Miguel
JM
Chiong
Mario
M
Parra
Valentina
V
Hidalgo
Karla
K
Toro
Barbra
B
Díaz-Araya
Guillermo
G
Lavandero
Sergio
S
D000818:Animals; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism*; D002478:Cells, Cultured; D004789:Enzyme Activation; D009206:Myocardium / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D009997:Osmotic Pressure; D051381:Rats; D051841:Transcription Factor RelB / Q000378:metabolism*
C497595:Relb protein, rat; D051841:Transcription Factor RelB; D020169:Caspases