p53 independent induction of PUMA mediates intestinal apoptosis in response to ischaemia–reperfusion
- 1University of Pittsburgh Cancer Institute, Hillman Cancer Center, Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
- 2University of Pittsburgh Cancer Institute, Hillman Cancer Center, Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
- 3University of Pittsburgh Cancer Institute, Hillman Cancer Center, Department of Radiation Oncology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
- 4Department of Biochemistry, St Jude Children’s Research Hospital, Memphis, Tennessee, USA
- Correspondence to:
Dr Jian Yu or Dr Lin Zhang
Hillman Cancer Center Research Pavilion, 5117 Centre Ave, Pittsburgh, PA 15213, USA; or
- Accepted 10 October 2006
- Revised 21 September 2006
- Published Online First 24 November 2006
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.
- I/R, ischaemia–reperfusion
- L-NAME, N-nitro-L-arginine methyl ester
- PBS, phosphate buffered saline
- PCR, polymerase chain reaction
- PUMA, p53 upregulated modulator of apoptosis
- RNS, reactive nitrogen species
- ROS, reactive oxygen species
- SOD, superoxide dismutase
- TUNEL, terminal deoxynucleotidyl transferase mediated deoxyuridinetriphosphate nick end labelling
Published Online First 23 November 2006
Funding: This work was supported in part by the General Motors (GM) Cancer Research Foundation, the V Foundation for Cancer Research (LZ), the Flight Attendant Medical Research Institute (FAMRI) and the Alliance for Cancer Gene Therapy (ACGT) (JY).
Competing interests: None.