Splice variants of mIAP1 have an enhanced ability to inhibit apoptosis

c-IAP1 is a member of the Inhibitor of Apoptosis protein family. Functions ascribed to c-IAP1 include inhibition of apoptosis and activation of NF-kappaB. Herein, we show that murine c-IAP1 (mIAP1) undergoes alterative splicing, generating two truncated proteins; one that lacks the CARD and RING domains (mIAP1-DeltaCARDDeltaRING) and the other that lacks only the CARD domain (mIAP1-DeltaCARD). mIAP1-DeltaCARDDeltaRING mRNA is expressed at 2-3% of the levels of full-length mIAP1 (FL-mIAP1) in mouse tissues, yet it encodes a protein that accumulates at 50-fold higher levels than the FL-mIAP1 in cultured cells. This protein has an enhanced ability to inhibit Bax-induced apoptosis, but does not activate an NF-kappaB reporter. In contrast to mIAP1-DeltaCARDDeltaRING, the mIAP1-DeltaCARD mRNA displays distinct tissue variation, ranging from 5% to 15% of the FL-mIAP1 mRNA levels and its levels increase in the mammary gland during involution. This isoform also has enhanced anti-apoptotic activity, but diminished NF-kappaB activation. In summary, mIAP1 is alternatively spliced, generating protein isoforms with distinct functional characteristics.     

Ubiquitin binding modulates IAP antagonist-stimulated proteasomal degradation of c-IAP1 and c-IAP2(1)

A family of anti-apoptotic regulators known as IAP (inhibitor of apoptosis) proteins interact with multiple cellular partners and inhibit apoptosis induced by a variety of stimuli. c-IAP (cellular IAP) 1 and 2 are recruited to TNFR1 (tumour necrosis factor receptor 1)-associated signalling complexes, where they mediate receptor-induced NF-kappaB (nuclear factor kappaB) activation. Additionally, through their E3 ubiquitin ligase activities, c-IAP1 and c-IAP2 promote proteasomal degradation of NIK (NF-kappaB-inducing kinase) and regulate the non-canonical NF-kappaB pathway. In the present paper, we describe a novel ubiquitin-binding domain of IAPs. The UBA (ubiquitin-associated) domain of IAPs is located between the BIR (baculovirus IAP repeat) domains and the CARD (caspase activation and recruitment domain) or the RING (really interesting new gene) domain of c-IAP1 and c-IAP2 or XIAP (X-linked IAP) respectively. The c-IAP1 UBA domain binds mono-ubiquitin and Lys(48)- and Lys(63)-linked polyubiquitin chains with low-micromolar affinities as determined by surface plasmon resonance or isothermal titration calorimetry. NMR analysis of the c-IAP1 UBA domain-ubiquitin interaction reveals that this UBA domain binds the classical hydrophobic patch surrounding Ile(44) of ubiquitin. Mutations of critical amino acid residues in the highly conserved MGF (Met-Gly-Phe) binding loop of the UBA domain completely abrogate ubiquitin binding. These mutations in the UBA domain do not overtly affect the ubiquitin ligase activity of c-IAP1 or the participation of c-IAP1 and c-IAP2 in the TNFR1 signalling complex. Treatment of cells with IAP antagonists leads to proteasomal degradation of c-IAP1 and c-IAP2. Deletion or mutation of the UBA domain decreases this degradation, probably by diminishing the interaction of the c-IAPs with the proteasome. These results suggest that ubiquitin binding may be an important mechanism for rapid turnover of auto-ubiquitinated c-IAP1 and c-IAP2.     

c-IAP1 and c-IAP2 are critical mediators of tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation

The inhibitor of apoptosis (IAP) proteins are a family of anti-apoptotic regulators found in viruses and metazoans. c-IAP1 and c-IAP2 are recruited to tumor necrosis factor receptor 1 (TNFR1)-associated complexes where they can regulate receptor-mediated signaling. Both c-IAP1 and c-IAP2 have been implicated in TNFalpha-stimulated NF-kappaB activation. However, individual c-IAP1 and c-IAP2 gene knock-outs in mice did not reveal changes in TNF signaling pathways, and the phenotype of a combined deficiency of c-IAPs has yet to be reported. Here we investigate the role of c-IAP1 and c-IAP2 in TNFalpha-stimulated activation of NF-kappaB. We demonstrate that TNFalpha-induced NF-kappaB activation is severely diminished in the absence of both c-IAP proteins. In addition, combined absence of c-IAP1 and c-IAP2 rendered cells sensitive to TNFalpha-induced cell death. Using cells with genetic ablation of c-IAP1 or cells where the c-IAP proteins were eliminated using IAP antagonists, we show that TNFalpha-induced RIP1 ubiquitination is abrogated in the absence of c-IAPs. Furthermore, we reconstitute the ubiquitination process with purified components in vitro and demonstrate that c-IAP1, in collaboration with the ubiquitin conjugating enzyme (E2) enzyme UbcH5a, mediates polymerization of Lys-63-linked chains on RIP1. Therefore, c-IAP1 and c-IAP2 are required for TNFalpha-stimulated RIP1 ubiquitination and NF-kappaB activation.     

c-IAP1 and c-IAP2 Redundancy Differs between T and B Cells

Cellular Inhibitors of Apoptosis 1 and 2 (c-IAP1 and c-IAP2) are ubiquitin protein ligases (E3s) that constitutively ubiquitinate and induce proteasomal-mediated degradation of NF-κB Inducing Kinase (NIK) and repress non-canonical NF-κB activation. Mice expressing an E3-inactive c-IAP2 mutant (c-IAP2^H570A) have constitutive activation of non-canonical NF-κB, resulting in B cell hyperplasia and T cell costimulation-independence. If, and if so to what extent, c-IAP1 and c-IAP2 are redundant in NF-κB regulation in these mice is not known. Here we have generated mice expressing a mutant c-IAP1 that lacks E3 activity (c-IAP1^H582A). These mice were phenotypically normal and did not have constitutive NF-κB activation in B cells or MEFs. siRNA-mediated knockdown of c-IAP2 showed that accumulated c-IAP2, resulting from lack of c-IAP1-dependent degradation, compensated for absent c-IAP1 E3 activity. Surprisingly, c-IAP1^H582A T cells had a lower p100/p52 ratio than wild type T cells, and in the absence of costimulation proliferated to a degree intermediate between wild type and c-IAP2^H570A T cells. Therefore, although c-IAP1 and c-IAP2 both can repress constitutive NF-κB activation, the relative importance of each varies according to cell type.     

The inhibitor of apoptosis protein fusion c-IAP2.MALT1 stimulates NF-kappaB activation independently of TRAF1 AND TRAF2

The inhibitors of apoptosis (IAPs) are a family of cell death inhibitors found in viruses and metazoans. All members of the IAP family have at least one baculovirus IAP repeat (BIR) motif that is essential for their anti-apoptotic activity. The t(11, 18)(q21;q21) translocation fuses the BIR domains of c-IAP2 with the paracaspase/MALT1 (mucosa-associated lymphoid tissue) protein, a critical mediator of T cell receptor-stimulated activation of NF-kappaB. The c-IAP2.MALT1 fusion protein constitutively activates the NF-kappaB pathway, and this is considered critical to malignant B cell transformation and lymphoma progression. The BIR domains of c-IAP1 and c-IAP2 interact with tumor necrosis factor receptor-associated factors 1 and 2 (TRAF1 and TRAF2). Here we investigated the importance of TRAF1 and TRAF2 for c-IAP2.MALT1-stimulated NF-kappaB activation. We identified a novel epitope within the BIR1 domains of c-IAP1 and c-IAP2 that is crucial for their physical interaction with TRAF1 and TRAF2. The c-IAP2.MALT1 fusion protein associates with TRAF1 and TRAF2 using the same binding site. We explored the functional relevance of this interaction and established that binding to TRAF1 and TRAF2 is not required for c-IAP2.MALT1-stimulated NF-kappaB activation. Furthermore, gene ablation of TRAF2 or combined down-regulation of TRAF1 and TRAF2 did not affect c-IAP2.MALT1-stimulated signaling. However, TRAF1/2-binding mutants of c-IAP2.MALT1 still oligomerize and activate NF-kappaB, suggesting that oligomerization might be important for signaling of the fusion protein. Therefore, the t(11, 18)(q21;q21) translocation creating the c-IAP2.MALT1 fusion protein activates NF-kappaB and contributes to human malignancy in the absence of signaling adaptors that might otherwise regulate its activity.     

Receptor interacting protein is ubiquitinated by cellular inhibitor of apoptosis proteins (c-IAP1 and c-IAP2) in vitro

Receptor interacting protein (RIP) is recruited to tumor necrosis factor-alpha receptor 1 (TNFR1) complex upon stimulation and plays a crucial role in the receptor-mediated NF-kappaB activation. Among the components of the TNFR1 complex are proteins that possess ubiquitin-protein isopeptide ligase (E3) activities, such as TNFR1-associated factor 2 (TRAF2), cellular inhibitor of apoptosis proteins (c-IAPs) namely, c-IAP1 and c-IAP2. Here, we showed that ectopically expressed RIP is ubiquitinated, and either the intermediate or death domain of RIP is required for this modification. Expression of c-IAP1 and c-IAP2 decreased the steady-state level of RIP, which was blocked by inhibition of the 26S proteasome. RIP degradation requires intact c-IAP2 containing the RING domain. Our in vitro ubiquitination assay revealed that while TRAF2 had no effect, both c-IAP1 and c-IAP2-mediated RIP ubiquitination with similar efficiency, indicating that c-IAPs can function as E3 toward RIP.     

c-IAP1 cooperates with Myc by acting as a ubiquitin ligase for Mad1

c-IAP1, a member of the inhibitor of apoptosis protein (IAP) family and a RING finger ubiquitin ligase (E3), has been proposed to be an important oncogene. In many types of cancers, the levels of c-IAP1 are upregulated, which contributes positively to tumorigenesis. However, the mechanism by which c-IAP1 promotes tumorigenesis has proven elusive. Although proteins in the IAP family may function as caspase inhibitors, c-IAP1 was shown to be a poor inhibitor of caspases. Here we show that c-IAP1 catalyzes ubiquitination of Max-dimerization protein-1 (Mad1), a cellular antagonist of Myc. Ubiquitination of Mad1 by c-IAP1 accelerates its degradation by the 26S proteasome pathway, and this reduction of the Mad1 levels cooperates with Myc to promote cell proliferation. Our results demonstrate that c-IAP1 exerts its oncogenic functions by promoting the degradation of an important negative regulator in the Myc pathway.     

Distinctive effects of the cellular inhibitor of apoptosis protein c-IAP2 through stabilization by XIAP in glioblastoma multiforme cells

Inhibitor of apoptosis proteins (IAPs) are extensively involved in NFκB signaling pathways. Regulation of c-IAP2 turnover by other proteins was investigated in glioblastoma multiforme (GBM) cells in the present study. When overexpressed, X-linked IAP (XIAP) enhanced expression of ectopic c-IAP2, but not c-IAP1, and endogenous c-IAP2 levels were reduced once XIAP expression was silenced. TNFα stimulation substantially increased c-IAP2 expression, and this upregulation was impaired by suppression of XIAP. Similarly, when XIAP was limiting due to severe hypoxic conditions, c-IAP2 levels were downregulated. These data together indicate that XIAP is an important regulator responsible for stabilization of c-IAP2 levels under different conditions. Protein interactions occur through binding of BIR2 and BIR3 domains of c-IAP2 with the RING finger of XIAP. XIAP inhibition of c-IAP2 auto-degradation was dependent on this physical interaction, and it was independent of XIAP E3 ligase activity. Global c-IAP2 ubiquitination was not affected by XIAP, although c-IAP2 levels were significantly increased. A CARD-RING-containing fragment of c-IAP2 was found to target XIAP for proteasome-independent degradation, but it was unable to sensitize GBM cells to chemo-reagents. The XIAP-stabilized c-IAP2 was found to enhance IκB-α phosphorylation on serines 32 and 36, and to antagonize XIAP-induced increase in mature Smac and Bcl10. Taken together, our data identify a distinctive role of c-IAP2 as stabilizer of XIAP, which is likely involved in regulation of NFκB activation and apoptosis in GBM cells.     

Enhanced Cytoprotective Effects of the Inhibitor of Apoptosis Protein Cellular IAP1 through Stabilization with TRAF2

Inhibitor of apoptosis (IAP) proteins are key regulators of intracellular signaling that interact with tumor necrosis factor (TNF) receptor superfamily members as well as proapoptotic molecules such as Smac/DIABLO and caspases. Whereas the X-linked IAP is an established caspase inhibitor, the protective mechanisms utilized by the cellular IAP (c-IAP) proteins are less clear because c-IAPs bind to but do not inhibit the enzymatic activities of caspases. In this study, c-IAPs are shown to be highly unstable molecules that undergo autoubiquitination. The autoubiquitination of c-IAP1 is blocked upon coexpression with TNF receptor-associated factor (TRAF) 2, and this is achieved by inhibition of the E3 ubiquitin ligase activity intrinsic to the RING of c-IAP1. Consistent with these observations, loss of TRAF2 results in a decrease in c-IAP1 levels. Stabilized c-IAP1 was found to sequester and prevent Smac/DIABLO from antagonizing X-linked IAP and protect against cell death. Therefore, this study describes an intriguing cytoprotective mechanism utilized by c-IAP1 and provides critical insight into how IAP proteins function to alter the apoptotic threshold.The inhibitors of apoptosis (IAPs)² are an evolutionarily conserved gene family described originally as encoding cell death inhibitors. IAP proteins have subsequently been found to participate in a variety of additional intracellular signaling processes (1), and it has become evident that IAP proteins are versatile molecules playing numerous distinct roles within the cell. Although a more complete understanding of these additional functions for IAP proteins is emerging, the distinct mechanisms utilized by some IAP proteins to function in their originally defined roles as cell death inhibitors remain unclear.Members of the IAP family are characterized by the presence of 1–3 tandem repeats of an ∼70-residue baculovirus IAP repeat domain (2). The baculovirus IAP repeat domains of many IAP proteins have been shown to be the region within IAP proteins that associates with caspases and other proapoptotic molecules (3, 4). IAP proteins have remarkably different apoptotic inhibitory abilities. For example, X-linked IAP (XIAP) is a highly potent cell death inhibitor (5) and is thought to be the only mammalian IAP protein that directly inhibits the enzymatic activities of caspases (2–4, 6). Although cellular IAP1 and -2 (c-IAP1 and c-IAP2) are anti-apoptotic proteins that can bind to caspase-7 and -9, they do not inhibit the enzymatic activities of these caspases (2, 6).Many IAP proteins, including c-IAP1 and c-IAP2, contain a carboxyl-terminal RING domain that can function as an E3 ubiquitin ligase (7). The E3 ubiquitin ligase activity of the RING domain in c-IAP1 and c-IAP2 was previously shown to negatively regulate the apoptotic inhibitory properties of c-IAP proteins and to promote autoubiquitination and degradation of c-IAP1 (8, 9), thus hindering attempts to define the cellular properties of this protein.A specialized property of the c-IAP proteins is their involvement in tumor necrosis family (TNF) signaling (10–12). Both c-IAP1 and c-IAP2 were discovered in a biochemical screen for factors associated with the type 2 TNF receptor. This association was found to be indirect and bridged by interactions with TNF receptor-associated factors (TRAFs), most notably TRAF1 and TRAF2 (11). Though the consequences of the association between TRAF2 and c-IAP1 on TNF-mediated signaling have been investigated (12), less is known about the functional significance of the association between TRAF2 and c-IAP1 on cell death inhibition. Because both c-IAP1 and TRAF2 possess E3 ubiquitin ligase activity in their respective RING domains, it seemed that the association between these molecules might impact the protective properties of c-IAP1 and alter the apoptotic threshold.In this study, the role of TRAF2 in c-IAP1 stability and how the association of TRAF2 with c-IAP1 affects the apoptotic inhibitory properties of c-IAP1 were examined. The presence of TRAF2 greatly enhanced the stability of c-IAP1, and these data suggest that the interaction between TRAF2 and c-IAP1 inhibits the E3 ubiquitin ligase activity intrinsic to the RING domain of c-IAP1. Using stabilized c-IAP1, the anti-apoptotic activity of c-IAP1 was characterized, and it was found that c-IAP1 suppresses apoptosis to a degree comparable with XIAP. Furthermore, we show that c-IAP1 functions to prevent the IAP antagonist, Smac/DIABLO (13, 14), from interfering with XIAP inhibition of caspases. Together, this study demonstrates that although c-IAP1 does not directly inhibit caspase activity, stabilized c-IAP1 can sequester Smac/DIABLO, prevent Smac/DIABLO from antagonizing XIAP, and inhibit cell death.     

Interaction of heat-shock protein 90 beta isoform (HSP90 beta) with cellular inhibitor of apoptosis 1 (c-IAP1) is required for cell differentiation

Members of the inhibitor of apoptosis protein (IAP) family have demonstrated functions in cell death, cell signalling, cell migration and mitosis. Several of them are E3 enzymes in the ubiquitination of proteins that leads to their degradation by the proteosomal machinery. We previously reported that one of them, cellular inhibitor of apoptosis protein-1 (c-IAP1), migrated from the nucleus to the surface of the Golgi apparatus in cells undergoing differentiation. Here, we show that c-IAP1 is a client protein of the stress protein HSP90 beta. In three distinct cellular models, the two proteins interact and migrate from the nucleus to the cytoplasm along the differentiation process through a leptomycin B-sensitive pathway. Inhibition of HSP90 proteins by small chemical molecules and specific depletion of HSP90 beta isoform by siRNA both lead to auto-ubiquitination of c-IAP1 and its degradation by the proteasome machinery. This chaperone function of HSP90 towards c-IAP1 is specific of its beta isoform as specific depletion of HSP90alpha does not affect c-IAP1 content. Chemical inhibition of HSP90 or siRNA-mediated depletion of HSP90 beta both inhibit cell differentiation, which can be reproduced by siRNA-mediated depletion of c-IAP1. Altogether, these results suggest that HSP90 beta prevents auto-ubiquitination and degradation of its client protein c-IAP1, whose depletion would be sufficient to inhibit cell differentiation.     

Tumor necrosis factor receptor 2 signaling induces selective c-IAP1-dependent ASK1 ubiquitination and terminates mitogen-activated protein kinase signaling

TRAF2 and ASK1 play essential roles in tumor necrosis factor alpha (TNF-alpha)-induced mitogen-activated protein kinase signaling. Stimulation through TNF receptor 2 (TNFR2) leads to TRAF2 ubiquitination and subsequent proteasomal degradation. Here we show that TNFR2 signaling also leads to selective ASK1 ubiquitination and degradation in proteasomes. c-IAP1 was identified as the ubiquitin protein ligase for ASK1 ubiquitination, and studies with primary B cells from c-IAP1 knock-out animals revealed that c-IAP1 is required for TNFR2-induced TRAF2 and ASK1 degradation. Moreover, in the absence of c-IAP1 TNFR2-mediated p38 and JNK activation was prolonged. Thus, the ubiquitin protein ligase activity of c-IAP1 is responsible for regulating the duration of TNF signaling in primary cells expressing TNFR2.     

c-IAP1 is cleaved by caspases to produce a proapoptotic C-terminal fragment

Although human c-IAP1 and c-IAP2 have been reported to possess antiapoptotic activity against a variety of stimuli in several mammalian cell types, we observed that full-length c-IAP1 and c-IAP2 failed to protect cells from apoptosis induced by Bax overexpression, tumor necrosis factor alpha treatment or Sindbis virus infection. However, deletion of the C-terminal RING domains of c-IAP1 and c-IAP2 restored antiapoptotic activity, indicating that this region negatively regulates the antiapoptotic function of the N-terminal BIR domain. This finding is consistent with the observation by others that the spacer region and RING domain of c-IAP1 functions as an E3 ligase, promoting autoubiquitination and degradation of c-IAP1. In addition, we found that c-IAP1 is cleaved during apoptosis to 52- and 35-kDa fragments. Both fragments contain the C-terminal end of c-IAP1 including the RING finger. In vitro cleavage of c-IAP1 with apoptotic cell extracts or with purified recombinant caspase-3 produced similar fragments. Furthermore, transfection of cells with the spacer-RING domain alone suppressed the antiapoptotic function of the N-terminal BIR domain of c-IAP1 and induced apoptosis. Optimal death-inducing activity of the spacer-RING required both the spacer region and the zinc-binding RING domain of c-IAP1 but did not require the caspase recruitment domain located within the spacer region. To the contrary, deletion of the caspase recruitment domain increased proapoptotic activity, apparently by stabilizing the C-terminal fragment.     

TNF-alpha induced c-IAP1/TRAF2 complex translocation to a Ubc6-containing compartment and TRAF2 ubiquitination

Signaling through tumor necrosis factor receptor 2 (TNF-R2) results in ubiquitination of TRAF2 by the E3 c-IAP1. In this report, we confirm that TRAF2 translocates to a Triton X-100 (TX)-insoluble compartment upon TNF-R2 engagement. Moreover, TRAF2 ubiquitination occurs in this compartment, from which TRAF2 is degraded in a proteasome-dependent manner. Confocal microscopy demonstrated that the TX-insoluble compartment is perinuclear and co-localizes with endoplasmic reticulum (ER) markers. The ER transmembrane Ubc6 bound to c-IAP1 and served as a cognate E2 for c-IAP1's E3 activity in vitro. Furthermore, Ubc6 co-localized with translocated TRAF2/c-IAP1 in the ER-associated compartment in vivo, and a catalytically inactive Ubc6 mutant inhibited TNF-alpha-induced, TNF-R2-dependent TRAF2 degradation. These results indicate that upon TNF-R2 signaling, translocation of TRAF2 and c-IAP1 to an ER-associated, Ubc6-containing perinuclear compartment is required for the ubiquitination of TRAF2 by c-IAP1. Therefore, the ER plays a key role in the TNF-R-mediated signal transduction cascade by acting as a site of assembly for E2/E3/substrate complexes.     

Non-canonical NF-κB activation and abnormal B cell accumulation in mice expressing ubiquitin protein ligase-inactive c-IAP2

Chromosomal translocations between loci encoding MALT1 and c-IAP2 are common in MALT lymphomas. The resulting fusion proteins lack the c-IAP2 RING domain, the region responsible for its ubiquitin protein ligase (E3) activity. Ectopic expression of the fusion protein activates the canonical NF-κB signaling cascade, but how it does so is controversial and how it promotes MALT lymphoma is unknown. Considering recent reports implicating c-IAP1 and c-IAP2 E3 activity in repression of non-canonical NF-κB signaling, we asked if the c-IAP2/MALT fusion protein can initiate non-canonical NF-κB activation. Here we show that in addition to canonical activation, the fusion protein stabilizes NIK and activates non-canonical NF-κB. Canonical but not non-canonical activation depended on MALT1 paracaspase activity, and expression of E3-inactive c-IAP2 activated non-canonical NF-κB. Mice in which endogenous c-IAP2 was replaced with an E3-inactive mutant accumulated abnormal B cells with elevated non-canonical NF-κB and had increased numbers of B cells with a marginal zone phenotype, gut-associated lymphoid hyperplasia, and other features of MALT lymphoma. Thus, the c-IAP2/MALT1 fusion protein activates NF-κB by two distinct mechanisms, and loss of c-IAP2 E3 activity in vivo is sufficient to induce abnormalities common to MALT lymphoma.     

15-deoxy-delta 12,14-prostaglandin J2 and laminar fluid shear stress stabilize c-IAP1 in vascular endothelial cells

Laminar shear stress strongly inhibits vascular endothelial cell apoptosis by unknown mechanisms. We reported that shear stress stimulates endothelial cells to produce 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) by elevating the expression level of lipocalin-type prostaglandin D synthase. To investigate the role of 15d-PGJ2 produced in the vascular wall, we examined the effect of 15d-PGJ2 on endothelial cell apoptosis. We induced apoptosis in human umbilical vein endothelial cells (HUVECs) by growth factor deprivation. 15d-PGJ2 strongly inhibited DNA ladder formation, nuclear fragmentation, and caspase-3-like activity in HUVECs. To elucidate the mechanism by which 15d-PGJ2 inhibits endothelial cell apoptosis, we examined expression of the inhibitor of apoptosis proteins (IAP) cellular-IAP1 (c-IAP1), c-IAP2, x-linked IAP, and survivin in HUVECs. In parallel with the inhibition of apoptosis, 15d-PGJ2 elevated the expression level of c-IAP1 protein in a dose- and time-dependent manner without changing the mRNA level. Laminar shear stress also induced c-IAP1 expression. Chase experiments with the use of cycloheximide revealed that 15d-PGJ2 and shear stress both inhibited the proteolytic degradation of c-IAP1 protein. These results suggested that 15d-PGJ2 inhibits endothelial cell apoptosis through, at least in part, c-IAP1 protein stabilization. This mechanism might be involved in the antiapoptotic effect of laminar shear stress.     

c-IAP1 blocks TNFalpha-mediated cytotoxicity upstream of caspase-dependent and -independent mitochondrial events in human leukemic cells

Tumor necrosis factor-alpha (TNFalpha) mediates cytochrome c release from mitochondria, loss of mitochondrial membrane potential (DeltaPsim) and apoptosis in sensitive leukemic cells. In the present study, by using the human leukemic U937 cell line, we demonstrate that the cytochrome c release is caspase-8-dependent and can be blocked by an inhibitor of caspase-8, Z-Ile-Glu (OMe)-Thr-Asp(OMe)-fluoromethyl ketone (Z-IETD.fmk), or a pan caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD.fmk). However, TNFalpha-mediated loss of DeltaPsim was not inhibited by caspase inhibitors. The apoptotic process was blocked by either Z-IETD.fmk or Z-VAD.fmk in cells with lower DeltaPsim. U937 cells with stable transfection of the cellular inhibitor of apoptosis protein 1 (c-IAP1) are resistant to TNFalpha-induced activation of caspases, Bid cleavage, cytochrome c release and DeltaPsim collapse. In addition, both c-IAP1 and XIAP were not up-regulated upon prolonged exposure to TNFalpha. In contrast, there was a caspase-dependent cleavage of XIAP, but not c-IAP1, during treatment with TNFalpha for 7 days. These results demonstrate that c-IAP1 blocks TNFalpha signaling at a level controlling both activation of caspase-8 and a signal to cause loss of DeltaPsim. The sensitive U937 cell line failed to acquire resistance and gain a self-protecting advantage against apoptosis, upon induction of c-IAP1 expression.