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Science 325 (5938): 332-336

Copyright © 2009 by the American Association for the Advancement of Science

RIP3, an Energy Metabolism Regulator That Switches TNF-Induced Cell Death from Apoptosis to Necrosis

Duan-Wu Zhang1, Jing Shao1, Juan Lin1, Na Zhang1, Bao-Ju Lu2, Sheng-Cai Lin1, Meng-Qiu Dong2, and Jiahuai Han1,*

1 Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China.
2 National Institute of Biological Sciences, Beijing 102206, China.


Figure 1
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Fig. 1. Effects of RIP3 on type of cell death. (A) Viabilities of A cells treated with medium (Ctrl), zVAD (20 µM), TNF (30 ng/ml), or TNF+zVAD. (B) Viabilities of N cells treated as described in (A). (C) Immunoblotting analysis with antibody to RIP3. (D) Viabilities of N cells treated with or without (–) a lentiviral vector encoding LacZ shRNA (Ctrl) or RIP3-shRNAs for 48 hours then stimulated with or without TNF or TNF+zVAD for 24 hours. RIP3 protein amounts in these cells were analyzed by means of immunoblotting 48 hours after infection. (E) Viabilities of A cells infected with a lentivirus encoding nothing (vector) or RIP3 for 36 hours then stimulated with or without TNF or TNF+zVAD for 24 hours. RIP3 protein amounts were analyzed 36 hours after infection. (F) Viabilities of wild-type and RIP3–/– mouse peritoneal macrophages treated with medium (Ctrl), zVAD, TNF, TNF+zVAD, LPS (100 ng/ml), or LPS+zVAD for 24 hours. Data are the mean ± SD of triplicates [(A), (B), and (D) to (F)] and are representative of two to five experiments [(A) to (F)].

 

Figure 2
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Fig. 2. Role of RIP3 and RIP1 in apoptosis and necrosis. (A) Viabilities of N cells treated with or without (–) a lentiviral vector encoding control or RIP1-shRNAs for 48 hours then stimulated with or without TNF or TNF+zVAD for 24 hours. RIP1 protein amounts were analyzed by means of immunoblotting 48 hours after infection. (B) Wild-type, RIP3–/–, RIP1–/–*, and RIP1–/–RIP3d MEF cells infected with a lentiviral vector encoding RIP1 or RIP3, treated with or without zVAD. Viabilities and RIP1 and RIP3 expression were analyzed 36 hours after infection. (C) N and A cells were treated and analyzed as described in (B). (D) RIP1 and RIP3 expression levels and viabilities of N cells infected with lentiviral vectors expressing control shRNA and RIP3; RIP1-shRNA and RIP3; control shRNA and RIP1; or RIP3-shRNA and RIP1 and treated with or without zVAD. Data are the mean ± SD of triplicates and are representative of two to three experiments [(A) to (D)].

 

Figure 3
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Fig. 3. Activation of PYGL by RIP3 and the involvement of PYGL in necrosis. (A) A stable HA-PYGL–expressing N cell line was treated with TNF+zVAD for various time intervals. The cell lysates and antibody-to-RIP3 immunoprecipitates were immunoblotted with antibodies to HA and RIP3. (B) Same as in (A), except the treatments were nothing, zVAD, TNF, or TNF+zVAD. (C) PYGL activity after flag-PYGL was incubated with 0.25 mM adenosine monophosphate (AMP) for 10 min then with or without recombinant GST-RIP3 or GST-RIP3-K51A (inactive mutant) in a kinase buffer for 30 min at 30°C. (D) PYGL activity in N cells infected with a lentiviral vector expressing control shRNA or RIP3 shRNA for 48 hours, treated with nothing, TNF, or TNF+zVAD for 2 hours. (E) PYGL activity in wild-type and RIP3–/– mouse peritoneal macrophages, treated with nothing, TNF+zVAD, or LPS+zVAD for 2 hours. (F) Viabilities of N cells infected with a lentivirus expressing control shRNA or PYGL shRNAs for 48 hours then stimulated with or without TNF+zVAD for 24 hours. PYGL mRNA levels were determined by means of real-time polymerase chain reaction 48 hours after infection. Data are the mean ± SD of triplicates [(C) to (F)] and are representative of two experiments [(A) to (F)].

 

Figure 4
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Fig. 4. Activation of PYGL, GLUL, and GLUD1 by RIP3 contributes to TNF+zVAD–induced ROS production. (A) ROS levels in N cells treated with a lentiviral vector expressing control shRNA or RIP3-shRNAs for 48 hours then stimulated with TNF+zVAD for 12 hours. (B) ROS levels in A cells infected with or without a lentivirus encoding nothing or RIP3 for 36 hours then stimulated with TNF+zVAD for 6 hours. (C) ROS levels in N cells infected with a lentivirus expressing control shRNA, PYGL-shRNA, GLUL-shRNA, or GLUD1-shRNA for 48 hours then stimulated with TNF+zVAD for 12 hours. (D) Proposed mechanism for RIP3 in necrosis. When RIP3 is absent, RIP1-mediated cell death is apoptotic. High levels of RIP3 switch apoptosis to necrosis in some cell systems. RIP3 activates PYGL, increasing the availability of energy substrate glucose; RIP3 also activates GLUL and GLUD1, increasing Glu and Gln consumption as energy substrates. These lead to an increase in energy metabolism and subsequent overproduction of the oxidative metabolism product, ROS. ROS is at least in part responsible for RIP3-mediated necrosis.

 


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