The role of Bax/Bcl-2 and pro-caspase peptides in hypoxia/reperfusion-dependent regulation of MAPK(ERK): discordant proteomic effect of MAPK(p38).
BACKGROUND
The molecular regulation of MAPKs and apoptosis was investigated in a model of hypoxia-tolerance. Survival of neurons in Chrysemys picta bellii, an anoxia-tolerant turtle, involves a reduction in energy metabolism. The biochemical/physiological mechanisms of anoxia tolerance have been examined at the level of ion transport and ATP turnover. However, changes in the phosphorylation state of key enzymes and kinases, mainly, MAPKs, may occur during anoxia, thereby reversible protein phosphorylation could be a critical factor and major mechanism of metabolic reorganization for enduring anaerobiosis.


METHODS
If a turtle were to undergo hypoxia akin to that experienced in its native habitat, it was placed in a glass aquarium filled with water to within a half inch of the top. After the turtle was anesthetized, through extended hypoxia or anesthesia, the animal was sacrificed by decapitation. The brain was then excised and placed in anoxic artificial cerebrospinal fluid. Total protein extraction was performed by homogenizing brain in a buffer, followed by threonine and tyrosine phosphorylation determination of MAPKs, and caspase activity.


RESULTS
MAPK(p38) was decreased after reoxygenation following 1 day and 1 week hypoxia. The effect of hypoxia on the phosphorylation of MAPK(ERK) was biphasic: Enhancement at 5h and inhibition at 6 weeks. Pro-caspases 8/9 were unchanged by hypoxia until increasing at 6 weeks. Both pro-caspases were upregulated by reoxygenation at 1 day or 6 weeks hypoxia. Neither hypoxia nor reoxygenation induced the cleavage of pro-caspases 8/9 into p20 and p10, respectively. Furthermore, hypoxia induced Bax at 3 days and 1 week, and reoxygenation increased Bax #8776; 4-fold at 1 day. Although the expression of Bcl-2 was slightly increased by hypoxia, [Bcl-2] was 3-4-fold smaller in comparison with Bax.


CONCLUSIONS
These results indicate that hypoxia up-regulates MAPK(ERK) but not MAPK(p38;) hypoxia/reperfusion increases the expression of caspases and pro-apoptotic cofactors. The patterns of MAPK regulation suggest the significance of these kinases in cellular adaptation to oxygen deprivation with biomedical correlations, and thereby identify novel natural responsive signaling cofactors in Chrysemys picta bellii with potential pharmacologic and clinical applications.
Haddad
John J
JJ
Molecular Signaling Research Group, Division of Biological Sciences, Department of Biology, Faculty of Arts and Sciences, Lebanese International University, Beirut, Lebanon. john.haddad@yahoo.co.uk
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D053181:Caspase 8 / Q000502:physiology*; D053453:Caspase 9 / Q000502:physiology*; D020925:Hypoxia-Ischemia, Brain / Q000503:physiopathology*; D020935:MAP Kinase Signaling System / Q000502:physiology; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology*; D015427:Reperfusion Injury / Q000503:physiopathology*; D014426:Turtles; D051028:bcl-2-Associated X Protein / Q000502:physiology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D048051:p38 Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D053181:Caspase 8; D053453:Caspase 9