Title

c-Jun n-terminal kinase activity protects cardiac myocytes from reoxygenation-mediated apoptosis

Date of Award

2002

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Molecular and Cellular Pharmacology

First Committee Member

Keith A. Webster, Committee Chair

Abstract

Reperfusion injury occurs when oxygenated blood is returned to previously ischemic tissue. It involves the generation and release of reactive oxygen that activates numerous signaling pathways including the initiation of cell death. Exposure of isolated cardiac myocytes to chronic hypoxia followed by reoxygenation results in the early activation of c-Jun N-terminal kinase (JNK) and death by apoptosis of approximately 33.3 +/- 0.8% of the myocytes. Although JNK activation has been described in a number of models of ischemia-reperfusion, neither the pathway of activation nor the contributions of JNK activation to myocyte survival have been established. In this dissertation I describe components of the signaling pathway and the activation of JNK by reoxygenation correlating with myocyte survival.Transfection of myocytes with plasmid or adenoviral vectors encoding JNK pathway modulators supported the protective role for JNK as long as ATP remained near control levels. Introduction of JNK inhibitory mutants increased the level of apoptosis by almost 2-fold compared with control cultures maintained aerobically or subjected to hypoxia and reoxygenation. JNK activation by reoxygenation was blocked by dominant negative (dn)TAK-1, and dnTAK-1 stimulated apoptosis by an amount equivalent to dnJNK. DnRac-1 and dnCdc42 blocked JNK activation by reoxygenation and also increased the rates of apoptosis. Rac-1 activity increased within 1 minute of reoxygenation and peaked with a >4-fold induction after 5 minutes, consistent with its possible role as an upstream activator in the signal transduction of JNK. Hypoxia-reoxygenation mediated biphasic activations (2.6 and 2.9-fold) of p38 MAP kinase, and there was a small increase of TNF-alpha secretion, but treatments with the p38-specific inhibitor SB203580 or saturating levels of a TNF-alpha blocking antibody provided only partial protection against apoptosis. These results suggest that JNK activation is protective in this model depending on normal ATP levels, TAK-1, Rac-1, and Cdc42 are intermediates, and the pathway is largely independent of p38 or secreted TNF-alpha.

Keywords

Biology, Molecular; Biology, Cell; Health Sciences, Pharmacology; Health Sciences, Medicine and Surgery

Link to Full Text

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