TY - JOUR
T1 - Hyperosmotic stress activates p65/RelB NFκB in cultured cardiomyocytes with dichotomic actions on caspase activation and cell death
AU - Eisner, Verónica
AU - Quiroga, Clara
AU - Criollo, Alfredo
AU - Eltit, José Miguel
AU - Chiong, Mario
AU - Parra, Valentina
AU - Hidalgo, Karla
AU - Toro, Barbra
AU - Díaz-Araya, Guillermo
AU - Lavandero, Sergio
N1 - Funding Information:
We are indebted to Dr. M. Karin (University of California, San Diego) and Dr. X. Zhou (University of North Carolina, Chapel Hill) for their donation of 2×NFκB reporter gene and AdIκBα adenovirus, respectively. We specially thank Dr. P. Bull for her help on discussing our work, Dr. M.R. Bono for her help on FACS experiments and F. Albornoz for his technical assistance. This work was supported in part by FONDECYT Grant 1010246, FONDAP Grant 15010006, SOCHICAR Grant. V.E. A.C. and C.Q. hold a fellowship from CONICYT, Chile.
PY - 2006/6/12
Y1 - 2006/6/12
N2 - NFκB is a participant in the process whereby cells adapt to stress. We have evaluated the activation of NFκB 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 IKKα/β as well as degradation of IκBα. All five members of the NFκB family were identified in cardiomyocytes. Analysis of the subcellular distribution of NFκB isoforms in response to hyperosmotic stress showed parallel migration of p65 and RelB from the cytosol to the nucleus. Measurement of the binding of NFκB to the consensus DNA κB-site binding by EMSA revealed an oscillatory profile with maximum binding 1, 2 and 6 h 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 NFκB to DNA. Hyperosmotic stress also resulted in activation of the NFκB-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 NFκB with AdIκBα, an IκBα 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 NFκB isoforms and NFκB mediates caspase 9 activation in cardiomyocytes. However cell death triggered by hyperosmotic stress was caspase- and NFκB-independent.
AB - NFκB is a participant in the process whereby cells adapt to stress. We have evaluated the activation of NFκB 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 IKKα/β as well as degradation of IκBα. All five members of the NFκB family were identified in cardiomyocytes. Analysis of the subcellular distribution of NFκB isoforms in response to hyperosmotic stress showed parallel migration of p65 and RelB from the cytosol to the nucleus. Measurement of the binding of NFκB to the consensus DNA κB-site binding by EMSA revealed an oscillatory profile with maximum binding 1, 2 and 6 h 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 NFκB to DNA. Hyperosmotic stress also resulted in activation of the NFκB-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 NFκB with AdIκBα, an IκBα 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 NFκB isoforms and NFκB mediates caspase 9 activation in cardiomyocytes. However cell death triggered by hyperosmotic stress was caspase- and NFκB-independent.
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U2 - 10.1016/j.febslet.2006.05.023
DO - 10.1016/j.febslet.2006.05.023
M3 - Article
C2 - 16716309
AN - SCOPUS:33646927823
SN - 0014-5793
VL - 580
SP - 3469
EP - 3476
JO - FEBS Letters
JF - FEBS Letters
IS - 14
ER -