A20 inhibits oxidized low-density lipoprotein-induced apoptosis through negative Fas/Fas ligand-dependent activation of caspase-8 and mitochondrial pathways in murine RAW264.7 macrophages.
A20 was originally characterized as a TNF-inducible gene in human umbilical vein endothelial cells. As an NF-kappaB target gene, A20 is also induced in many other cell types by a wide range of stimuli. Expression of A20 has been shown to protect from TNF-induced apoptosis and also functions via a negative-feedback loop to block NF-kappaB activation induced by TNF and other stimuli. To date, there are no reports on whether A20 can protect OxLDL-induced apoptosis in macrophages. For the first time we report that A20 expression blocks OxLDL-mediated cell toxicity and apoptosis. OxLDL induced the expression of Fas and FasL, and the subsequent caspase-8 cleavage and treatment with a neutralizing ZB4 anti-Fas antibody blocked apoptosis induced by OxLDL. Expression of dominant negative FADD efficiently prevented OxLDL-induced apoptosis and caspase-8 activation. A20 expression significantly attenuated the increased expression of Fas and FasL, and Fas-mediated apoptosis. These findings suggest that A20-mediated protection from OxLDL may occur at the level of Fas/FADD-caspase-8 and be FasL dependent. Treatment of RAW264.7 cells with OxLDL induces a series of time-dependent events, including the release of cytochrome c, Smac and Omi from the mitochondria to the cytosol, activation of caspase-9, -6, -2, and -3, which are blocked by A20 expression. No cleaved form of Bid was detected, even treatment with OxLDL for 48 h. Expression of dominant negative FADD also efficiently prevented OxLDL-induced the above apoptotic events. The release of cyto c, Smac and Omi from mitochondria to cytosol, activated by OxLDL treatment, and the activation of caspase-9 may not be a downstream event of caspase-8-mediated Bid cleavage. Therefore, the protective effect of A20 on mitochondrial apoptotic pathway activated by OxLDL may be dependent on FADD. A20 expression reversed OxLDL-mediated G(0)/G(1) stage arrest by maintaining the expression of cyclin B1, cyclin D1, and cyclin E, and p21 and p73. Thus, A20 expression blocks OxLDL-mediated apoptosis in murine RAW264.7 macrophages through disrupting Fas/FasL-dependent activation of caspase-8 and the mitochondria pathway.
Li
Hong-Liang
HL
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College Beijing, P.R. China.
Wang
Ai-Bing
AB
Zhang
Ran
R
Wei
Yu-Sheng
YS
Chen
Hou-Zao
HZ
She
Zhi-Gang
ZG
Huang
Yue
Y
Liu
De-Pei
DP
Liang
Chih-Chuan
CC
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003546:Cysteine Endopeptidases; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D053222:Fas Ligand Protein; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008077:Lipoproteins, LDL / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D008562:Membrane Glycoproteins; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D009687:Nuclear Proteins; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3; D048069:Tumor Necrosis Factors; D019014:fas Receptor / Q000378:metabolism*
C505661:FASLG protein, human; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008077:Lipoproteins, LDL; D008562:Membrane Glycoproteins; D009687:Nuclear Proteins; D011506:Proteins; D048069:Tumor Necrosis Factors; D019014:fas Receptor; C099252:oxidized low density lipoprotein; D007770:L-Lactate Dehydrogenase; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3; C505530:CASP8 protein, human; C505526:Casp8 protein, mouse; D053181:Caspase 8; D020169:Caspases; D003546:Cysteine Endopeptidases; C513261:Tnfaip3 protein, mouse