p53 independent induction of PUMA mediates intestinal apoptosis in response to ischaemia-reperfusion

Gut. 2007 May;56(5):645-54. doi: 10.1136/gut.2006.101683. Epub 2006 Nov 24.

Abstract

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.

Aims: 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis Regulatory Proteins
  • Apoptosis*
  • Caspase 3 / metabolism
  • Cytochromes c / metabolism
  • DNA Fragmentation
  • Intestine, Small / blood supply*
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Oxidative Stress
  • Reperfusion Injury / metabolism
  • Reperfusion Injury / pathology*
  • Reperfusion Injury / prevention & control
  • Tumor Suppressor Protein p53 / physiology
  • Tumor Suppressor Proteins / genetics
  • Tumor Suppressor Proteins / metabolism
  • Tumor Suppressor Proteins / physiology*
  • bcl-2 Homologous Antagonist-Killer Protein / metabolism
  • bcl-2-Associated X Protein / metabolism

Substances

  • Apoptosis Regulatory Proteins
  • Bak1 protein, mouse
  • Bax protein, mouse
  • PUMA protein, mouse
  • Tumor Suppressor Protein p53
  • Tumor Suppressor Proteins
  • bcl-2 Homologous Antagonist-Killer Protein
  • bcl-2-Associated X Protein
  • Cytochromes c
  • Caspase 3