Preventing hypoxia/reoxygenation damage to hepatocytes by p66(shc) ablation: up-regulation of anti-oxidant and anti-apoptotic proteins.
OBJECTIVE
Ischemia/reperfusion damage to the liver remains a serious concern in many clinical situations. Major mechanisms for this certainly include oxidative stress.


METHODS
The effects of ablating the p66 isoform of ShcA (p66(shc)) on hypoxia/reoxygenation (H/R)-induced oxidative stress and cell injury in hepatocytes were investigated.


RESULTS
Immediately after reoxygenation, AML12 cells were clearly under oxidative stress; many cells underwent apoptosis. However, knockdown of p66(shc) by specific RNAi markedly decreased cellular oxidative stress and H/R-induced apoptosis, as well as conferring resistance to H(2)O(2) insult. These data suggest that prevention of apoptosis conferred by ablation of p66(shc) results from changed ROS-scavenging, but not inhibition of ROS generation. These data were also confirmed in fibroblasts from p66(shc) knockout mice. Anti-oxidant molecules, such as MnSOD and Ref-1 and the anti-apoptotic molecule Bcl-xL were up-regulated, and pro-apoptotic FLICE was down-regulated, by ablation of p66(shc). Interestingly, catalase expression was not affected in p66(shc)-knockdown-AML12 cells although it is a major target in other cell types.


CONCLUSIONS
Our findings suggest that in hepatocytes, ablation of p66(shc) is cytoprotective against H/R-induced oxidative stress, with MnSOD and Ref-1 playing critical roles, and with up-regulation of Bcl-xL and down-regulation of FLICE contributing jointly to preventing cells from undergoing oxidant-induced apoptosis.
Haga
Sanae
S
Department of Surgery, Hokkaido University School of Medicine, Sapporo, Hokkaido, Japan.
Terui
Keita
K
Fukai
Moto
M
Oikawa
Yuko
Y
Irani
Kaikobad
K
Furukawa
Hiroyuki
H
Todo
Satoru
S
Ozaki
Michitaka
M
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D053181:Caspase 8 / Q000235:genetics / Q000378:metabolism; D015687:Cell Hypoxia / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics / Q000378:metabolism*; D004195:Disease Models, Animal; D015536:Down-Regulation / Q000235:genetics / Q000502:physiology; D022781:Hepatocytes / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000009:adverse effects / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D018384:Oxidative Stress / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D012313:RNA / Q000494:pharmacology; D015427:Reperfusion Injury / Q000235:genetics / Q000378:metabolism / Q000517:prevention & control*; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation / Q000235:genetics / Q000502:physiology*; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; C494812:Bcl2l1 protein, mouse; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D051020:bcl-X Protein; D012313:RNA; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; C505526:Casp8 protein, mouse; D053181:Caspase 8; C486216:Apex1 protein, mouse; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase