p53 independent induction of PUMA mediates intestinal apoptosis in response to ischaemia-reperfusion.
BACKGROUND
The small intestine is highly sensitive to ischaemia-reperfusion (I/R) induced injury which is associated with high morbidity and mortality. Apoptosis, or programmed cell death, is a major mode of cell death occurring during I/R induced injury. However, the mechanisms by which I/R cause apoptosis in the small intestine are poorly understood. p53 upregulated modulator of apoptosis (PUMA) is a p53 downstream target and a member of the BH3-only group of Bcl-2 family proteins. It has been shown that PUMA plays an essential role in apoptosis induced by a variety of stimuli in different tissues through a mitochondrial pathway.


OBJECTIVE
The role of PUMA in I/R induced injury and apoptosis in the small intestine was investigated. The mechanisms by which PUMA is regulated in I/R induced intestinal apoptosis were also studied.


METHODS
Ischaemia was induced by superior mesenteric artery occlusion in the mouse small intestine. Induction of PUMA in response to ischaemia alone, or ischaemia followed by reperfusion (I/R), was examined. I/R induced intestinal apoptosis and injury were compared between PUMA knockout and wild-type mice. The mechanisms of I/R induced and PUMA mediated apoptosis were investigated through analysis of caspase activation, cytosolic release of mitochondrial cytochrome c and alterations of the proapoptotic Bcl-2 family proteins Bax and Bak. To determine whether PUMA is induced by reactive oxygen species and/or reactive nitrogen species generated by I/R, superoxide dismutase (SOD) and N-nitro-l-arginine methyl ester (L-NAME) were used to treat animals before I/R. To determine whether p53 is involved in regulating PUMA during I/R induced apoptosis, PUMA induction and apoptosis in response to I/R were examined in p53 knockout mice.


RESULTS
PUMA was markedly induced following I/R in the mucosa of the mouse small intestine. I/R induced intestinal apoptosis was significantly attenuated in PUMA knockout mice compared with that in wild-type mice. I/R induced caspase 3 activation, cytochrome c release, Bax mitochondrial translocation and Bak multimerisation were also inhibited in PUMA knockout mice. SOD or L-NAME significantly blunted I/R induced PUMA expression and apoptosis. Furthermore, I/R induced PUMA expression and apoptosis in the small intestine were not affected in the p53 knockout mice.


CONCLUSIONS
Our data demonstrated that PUMA is activated by oxidative stress in response to I/R to promote p53 independent apoptosis in the small intestine through the mitochondrial pathway. Inhibition of PUMA is potentially useful for protecting against I/R induced intestinal injury and apoptosis.
Wu
Bin
B
University of Pittsburgh Cancer Institute, Hillman Cancer Center, Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
Qiu
Wei
W
Wang
Peng
P
Yu
Hui
H
Cheng
Tao
T
Zambetti
Gerard P
GP
Zhang
Lin
L
Yu
Jian
J
D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D053148:Caspase 3 / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation; D007421:Intestine, Small / Q000098:blood supply*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018384:Oxidative Stress; D015427:Reperfusion Injury / Q000378:metabolism / Q000473:pathology* / Q000517:prevention & control; D016159:Tumor Suppressor Protein p53 / Q000502:physiology; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; C494773:Bak1 protein, mouse; C494827:Bax protein, mouse; C488588:PUMA protein, mouse; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; D053148:Caspase 3