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.
ABSTRACT 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.
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ABSTRACT: CD40/CD40 ligand (CD40L) signaling is a potent activator of endothelial cells (ECs) and promoter of atherosclerosis. In this study, we investigate whether A20 (a gene we have shown to be antiinflammatory and antiapoptotic in ECs) can protect from CD40/CD40L-mediated EC activation. Overexpression of CD40, in a transient transfection system, activates the transcription factor NF-kappaB and upregulates IkappaBalpha, E-selectin, and tissue factor (TF) reporter activity. Coexpression of A20 inhibits NF-kappaB and upregulation of IkappaBalpha and E-Selectin but not TF, suggesting that CD40 induces TF in a non-NF-kappaB-dependent manner. In human coronary artery ECs (HCAECs), adenovirus-mediated overexpression of A20 blocks physiological, CD40-induced activation of NF-kappaB, upstream of IkappaBalpha degradation (Western blot) and subsequently upregulation of ICAM-1, VCAM-1, and E-selectin (flow cytometry). Although A20 does not block TF transcription its expression in HCAECs inhibits TF induction (colorimetric assay and RT-PCR) by blunting CD40 upregulation. We demonstrate that CD40 signaling induces apoptosis in a proinflammatory microenvironment. A20 overexpression protects from CD40-mediated EC apoptosis (DNA content analysis and trypan blue exclusion). We also demonstrate that signaling through CD40L activates NF-kappaB and induces apoptosis in ECs, both of which are inhibited by A20 overexpression. A20 works at multiple levels to protect ECs from CD40/CD40L mediated activation and apoptosis. A20-based therapy could be beneficial for the treatment of vascular diseases such as atherosclerosis and transplant-associated vasculopathy.Circulation 10/2003; 108(9):1113-8. · 15.20 Impact Factor
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ABSTRACT: X-linked inhibitor-of-apoptosis protein (XIAP) interacts with caspase-9 and inhibits its activity, whereas Smac (also known as DIABLO) relieves this inhibition through interaction with XIAP. Here we show that XIAP associates with the active caspase-9-Apaf-1 holoenzyme complex through binding to the amino terminus of the linker peptide on the small subunit of caspase-9, which becomes exposed after proteolytic processing of procaspase-9 at Asp315. Supporting this observation, point mutations that abrogate the proteolytic processing but not the catalytic activity of caspase-9, or deletion of the linker peptide, prevented caspase-9 association with XIAP and its concomitant inhibition. We note that the N-terminal four residues of caspase-9 linker peptide share significant homology with the N-terminal tetra-peptide in mature Smac and in the Drosophila proteins Hid/Grim/Reaper, defining a conserved class of IAP-binding motifs. Consistent with this finding, binding of the caspase-9 linker peptide and Smac to the BIR3 domain of XIAP is mutually exclusive, suggesting that Smac potentiates caspase-9 activity by disrupting the interaction of the linker peptide of caspase-9 with BIR3. Our studies reveal a mechanism in which binding to the BIR3 domain by two conserved peptides, one from Smac and the other one from caspase-9, has opposing effects on caspase activity and apoptosis.Nature 04/2001; 410(6824):112-6. · 38.60 Impact Factor
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ABSTRACT: Oxidized low-density lipoproteins (oxLDL) play a critical role in atherogenesis. One oxidative pathway of LDL involves myeloperoxidase, which catalyzes the production of hypochlorous acid (HOCl) in monocytes. We investigated the apoptotic mechanism induced by oxLDL, generated by HOCl treatment of native LDL, in human monocytic U937 cell line. The involvement of the mitochondrial apoptotic pathway was analyzed in Bcl-2-overexpressing clones, generated from U937 cells. HOCl-oxLDL induced in U937 cells (i) a marked caspase-dependent increase of apoptosis, (ii) a loss of mitochondrial membrane potential, (iii) a specific activation of caspase-2, -3, -8, and -9, and (iv) a similar degree of apoptosis in presence or absence of anti-Fas and anti-TNF-R1 antibodies. Moreover, the degree of HOCl-oxLDL-induced caspase-3 and -8 activation, and apoptosis was significantly reduced in U937/Bcl-2 cells, with no activation of caspase-9. By contrast, Cu-oxLDL-mediated apoptosis in U937 cells involved exclusively the mitochondrial pathway. In conclusion, the mechanism of HOCl-oxLDL-induced apoptosis in monocytic U937 cells involves the two pathways of apical caspase activation: (i) death receptor-mediated caspase-8 and (ii) mitochondria-mediated caspase-9. This converges in the activation of executing caspases, including caspase-3, and apoptosis. The interference of Bcl-2 overexpression with HOCl-oxLDL-induced apoptosis suggests the importance of mitochondrial involvement in this apoptotic mechanism.Free Radical Biology and Medicine 10/2003; 35(6):603-15. · 5.27 Impact Factor