Cell cycle-dependent regulation of FLIP levels and susceptibility to Fas-mediated apoptosis

Activation-induced cell death of peripheral T cells results from the interaction between Fas and Fas ligand. Resting peripheral T cells are resistant to Fas-induced apoptosis and become susceptible only after their activation. We have investigated the molecular mechanism mediating the sensitization of resting peripheral T cells to Fas-mediated apoptosis following TCR stimulation. TCR activation decreases the steady state protein levels of FLIP (FLICE-like inhibitory protein), an inhibitor of the Fas signaling pathway. Reconstitution of intracellular FLIP levels by the addition of a soluble HIV transactivator protein-FLIP chimera completely restores resistance to Fas-mediated apoptosis in TCR primary T cells. Inhibition of IL-2 production by cyclosporin A, or inhibition of IL-2 signaling by rapamycin or anti-IL-2 neutralizing Abs prevents the decrease in FLIP levels and confers resistance to Fas-mediated apoptosis following T cell activation. Using cell cycle-blocking agents, we demonstrate that activated T cells arrested in G1 phase contain high levels of FLIP protein, whereas activated T cells arrested in S phase have decreased FLIP protein levels. These findings link regulation of FLIP protein levels with cell cycle progression and provide an explanation for the increase in TCR-induced apoptosis observed during the S phase of the cell cycle.     

ARC contributes to the inhibitory effect of preconditioning on cardiomyocyte apoptosis

Inhibition of cardiomyocyte apoptosis plays a key role in preconditioning-triggered cardioprotection. However, the molecular mechanism(s) by which preconditioning inhibits apoptosis is not fully understood. Apoptosis repressor with caspase recruitment domain (ARC) possesses the ability to block hypoxia-induced cardiomyocyte apoptosis. We tested whether ARC contributes to the inhibitory effect of preconditioning on cardiomyocyte apoptosis. Cardiomyocytes from 1-day-old male Sprague-Dawley rats were preconditioned by exposing to 10 min of hypoxia, followed by 30 min of reoxygenation. Then, the preconditioned and non-preconditioned cardiomyocytes were exposed to 90 min of hypoxia followed by 120 min of reoxygenation. The results showed that preconditioning inhibited cell death induced by hypoxia and reoxygenation. Hypoxia and reoxygenation could induce a decrease of ARC protein levels. Intriguingly, preconditioning could maintain ARC protein levels. Inhibition of endogenous ARC expression by ARC antisense oligonucleotides reduced the inhibitory effect of preconditioning on apoptosis. Furthermore, preconditioning-induced suppression of the release of mitochondrial cytochrome c to cytosol and caspase-3 activation could be abolished by the inhibition of endogenous ARC expression using ARC antisense oligonucleotides. Conclusion: These data indicate that ARC participates in preconditioning-triggered cardioprotection by interfering with cytochrome c release and caspase-3 activation.     

Down-regulation of catalase and oxidative modification of protein kinase CK2 lead to the failure of apoptosis repressor with caspase recruitment domain to inhibit cardiomyocyte hypertrophy

Cardiac hypertrophy is regulated by a complex interplay of pro- and anti-hypertrophic factors. Here, we report a novel anti-hypertrophic pathway composed of catalase, protein kinase CK2 (CK2), and apoptosis repressor with caspase recruitment domain (ARC). Our results showed that ARC phosphorylation levels, CK2 activity, and catalase expression levels were decreased in the hearts of the angiotensinogen transgenic mice and in cardiomyocytes treated with the hypertrophic stimuli, including phenylephrine, tumor necrosis factor-alpha, and angiotensin II. To understand the role of ARC in hypertrophy, we observed that enforced expression of ARC could inhibit hypertrophy. Knockdown of endogenous ARC or inhibition of its phosphorylation could sensitize cardiomyocytes to undergoing hypertrophy. The phosphorylatable, but not the nonphosphorylatable, ARC could inhibit hypertrophy. Thus, ARC is able to inhibit hypertrophy in a phosphorylation-dependent manner. In exploring the molecular mechanism by which CK2 activity is reduced, we found that CK2 was carbonylated in angiotensinogen transgenic mice and in cardiomyocytes treated with the hypertrophic stimuli. The decrease in catalase expression led to an elevated level of reactive oxygen species. The latter oxidatively modified CK2, resulting in its carbonylation. CK2 lost its catalytic activity upon carbonylation. ARC is phosphorylated by CK2, and ARC phosphorylation levels were reduced as a consequence of the decrease of CK2 activity. To understand the molecular mechanism by which ARC inhibits hypertrophy, we observed that ARC could inhibit the activation of mitochondrial permeability transition. These results suggest that catalase, CK2, and ARC constitute an anti-hypertrophic pathway in the heart.     

The cardioprotective effect of postconditioning is mediated by ARC through inhibiting mitochondrial apoptotic pathway

Objective  Postconditioning protects the heart against ischemia/reperfusion injury by inhibiting cardiomyocyte apoptosis. However, the molecular mechanism by which postconditioning suppresses apoptosis remains to be fully understood. Apoptosis repressor with caspase recruitment domain (ARC) has been demonstrated to possess the ability to protect cardiomyocytes from apoptosis induced by ischemia/reperfusion. It is not yet clear as to whether ARC contributes to the inhibitory effect of postconditioning against cardiomyocyte apoptosis. Methods  The cultured cardiomyocytes from 1-day old male Sprague–Dawley rats were exposed to 3 h hypoxia followed by 3 h of reoxygenation. Cells were postconditioned by three cycles each of 5 min reoxygenation and 5 min hypoxia before 3 h of reoxygenation. Results  Hypoxia/reoxygenation led to a decrease of endogenous ARC protein levels. In contrast, postconditioning could block the reduction of endogenous ARC protein levels. Interestingly, inhibition of endogenous ARC expression by ARC antisense oligodeoxynucleotides reduced the inhibitory effect of postconditioning against apoptosis. Furthermore, our data showed that postconditioning suppressed the loss of mitochondrial membrane potential, Bax activation and the release of mitochondrial cytochrome c to cytosol. However, these inhibitory effects of postconditioning disappeared upon knockdown of endogenous ARC. Conclusion  Our data for the first time demonstrate that ARC plays an essential role in mediating the cardioprotective effect of postconditioning against apoptosis initiated by the mitochondrial pathway.     

ARC protects rat cardiomyocytes against oxidative stress through inhibition of caspase-2 mediated mitochondrial pathway

Apoptosis repressor with a CARD domain (ARC) has been demonstrated to protect heart cells against ischemia/reperfusion (I/R) injury. In this study, we investigated the mechanism by which ARC protects heart cells against oxidative stress. We monitored the extent of apoptosis and activity of multiple components of the intrinsic apoptotic pathway in rat cardiac myoblast cell line H9c2 with either reduced or increased expression of ARC during oxidative stress. Overexpression of ARC-inhibited oxidative stress-induced caspase-2/3 activation, cytochrome c release, and translocation of Bax to mitochondria. Furthermore, phosphorylation of ARC at threonine 149 was found to be critical to its function. ARC containing a T149A mutation failed to translocate to mitochondria, did not inhibit caspase-2 activation, and had a dominant negative effect against the protective effect of endogenous ARC during oxidative stress. In addition, wild-type ARC but not the T149A mutant inhibited cell death induced by overexpression of caspase-2. Using a yeast two-hybrid (YTH) screening approach and co-immunoprecipitation (Co-IP), we found that protein phosphatase 2C (PP2C) interacted with ARC and that PP2C mediated-dephosphorylation of ARC inhibited its anti-apoptotic activity. Eliminating either the N-terminal CARD domain or the C-terminal P/E domain also abolished the anti-apoptotic function of ARC, suggesting that full-length ARC is required for its apoptotic inhibition. These results indicate that ARC plays an important role in protection of H9c2 cells against oxidative stress-induced apoptosis by phosphorylation-dependent suppression of the mitochondria-mediated intrinsic pathway, partially initiated through the activation of caspase-2.     

Inhibition of both the extrinsic and intrinsic death pathways through nonhomotypic death-fold interactions

Death-fold domains constitute an evolutionarily conserved superfamily that mediates apoptotic signaling. These motifs, including CARD (caspase recruitment domain), DD (death domain), and DED (death effector domain), are believed to exert their effects solely through homotypic interactions. Herein we demonstrate that the CARD-containing protein ARC engages in nontraditional death-fold interactions to suppress both extrinsic and intrinsic death pathways. The extrinsic pathway is disrupted by heterotypic interactions between ARC's CARD and the DDs of Fas and FADD, which inhibit Fas-FADD binding and assembly of the death-inducing signaling complex (DISC). The intrinsic pathway is antagonized by ARC-Bax binding, involving ARC's CARD and the Bax C terminus. This inhibits Bax activation and translocation to the mitochondria. Knockdown of endogenous ARC facilitates DISC assembly and triggers spontaneous Bax activation and apoptosis. Conversely, physiological levels of ARC suppress these events. These studies establish a critical role for nonhomotypic death-fold interactions in the regulation of apoptosis.     

Regulation of apoptotic c-Jun N-terminal kinase signaling by a stabilization-based feed-forward loop

A sequential kinase cascade culminating in activation of c-Jun N-terminal kinases (JNKs) plays a fundamental role in promoting apoptotic death in many cellular contexts. The mechanisms by which this pathway is engaged in response to apoptotic stimuli and suppressed in viable cells are largely unknown. Here, we show that apoptotic stimuli increase endogenous cellular levels of pathway components, including POSH, mixed lineage kinases (MLKs), and JNK interacting protein 1, and that this effect occurs through protein stabilization and requires the presence of POSH as well as activation of MLKs and JNKs. Our findings suggest a self-amplifying, feed-forward loop mechanism by which apoptotic stimuli promote the stabilization of JNK pathway components, thereby contributing to cell death.     

Translational repression of MCL-1 couples stress-induced eIF2 alpha phosphorylation to mitochondrial apoptosis initiation

The integrated stress response (ISR) integrates a broad range of environmental and endogenous stress signals to the phosphorylation of the alpha-subunit of eukaryotic translation initiation factor 2 (eIF2 alpha). Although intense or prolonged activation of this pathway is known to induce apoptosis, the molecular mechanisms coupling stress-induced eIF2 alpha phosphorylation to the cell death machinery have remained incompletely understood. In this study, we characterized apoptosis initiation in response to classical activators of the ISR (tunicamycin, UVC, elevated osmotic pressure, arsenite). We found that all applied stress stimuli activated a mitochondrial pathway of apoptosis initiation. Rapid and selective down-regulation of the anti-apoptotic BCL-2 family protein MCL-1 preceded the activation of BAX, BAK, and caspases. Stabilization of MCL-1 blocked apoptosis initiation, while cells with reduced MCL-1 protein content were strongly sensitized to stress-induced apoptosis. Stress-induced elimination of MCL-1 occurred with unchanged protein turnover and independently of MCL-1 mRNA levels. In contrast, stress-induced phosphorylation of eIF2 alpha at Ser(51) was both essential and sufficient for the down-regulation of MCL-1 protein in stressed cells. These findings indicate that stress-induced phosphorylation of eIF2 alpha is directly coupled to mitochondrial apoptosis regulation via translational repression of MCL-1. Down-regulation of MCL-1 enables but not enforces apoptosis initiation in stressed cells.     

A novel role for the apoptosis inhibitor ARC in suppressing TNFα-induced regulated necrosis

TNFα signaling can promote apoptosis or a regulated form of necrosis. ARC (apoptosis repressor with CARD (caspase recruitment domain)) is an endogenous inhibitor of apoptosis that antagonizes both the extrinsic (death receptor) and intrinsic (mitochondrial/ER) apoptosis pathways. We discovered that ARC blocks not only apoptosis but also necrosis. TNFα-induced necrosis was abrogated by overexpression of wild-type ARC but not by a CARD mutant that is also defective for inhibition of apoptosis. Conversely, knockdown of ARC exacerbated TNFα-induced necrosis, an effect that was rescued by reconstitution with wild-type, but not CARD-defective, ARC. Similarly, depletion of ARC in vivo exacerbated necrosis caused by infection with vaccinia virus, which elicits severe tissue damage through this pathway, and sensitized mice to TNFα-induced systemic inflammatory response syndrome. The mechanism underlying these effects is an interaction of ARC with TNF receptor 1 that interferes with recruitment of RIP1, a critical mediator of TNFα-induced regulated necrosis. These findings extend the role of ARC from an apoptosis inhibitor to a regulator of the TNFα pathway and an inhibitor of TNFα-mediated regulated necrosis.     

An essential role of the NF-kappa B/Toll-like receptor pathway in induction of inflammatory and tissue-repair gene expression by necrotic cells

Tissue damage induced by infection or injury can result in necrosis, a mode of cell death characterized by induction of an inflammatory response. In contrast, cells dying by apoptosis do not induce inflammation. However, the reasons for underlying differences between these two modes of cell death in inducing inflammation are not known. Here we show that necrotic cells, but not apoptotic cells, activate NF-kappaB and induce expression of genes involved in inflammatory and tissue-repair responses, including neutrophil-specific chemokine genes KC and macrophage-inflammatory protein-2, in viable fibroblasts and macrophages. Intriguingly, NF-kappaB activation by necrotic cells was dependent on Toll-like receptor 2, a signaling pathway that induces inflammation in response to microbial agents. These results have identified a novel mechanism by which cell necrosis, but not apoptosis, can induce expression of genes involved in inflammation and tissue-repair responses. Furthermore, these results also demonstrate that the NF-kappaB/Toll-like receptor 2 pathway can be activated both by exogenous microbial agents and endogenous inflammatory stimuli.     

ARMER,apoptotic regulator in the membrane of the endoplasmic reticulum,a novel inhibitor of apoptosis

We have identified a novel protein, apoptotic regulator in the membrane of the endoplasmic reticulum (ARMER), which protects HT1080 fibrosarcoma cells from apoptosis induced by various stimuli. We demonstrate that ARMER is an endoplasmic reticulum (ER) integral membrane protein with four predicted transmembrane domains and a COOH-terminal KKXX ER retrieval motif. We used an inducible expression system (pIND) to study the effects of regulated ARMER overexpression. Cells in which ARMER was overexpressed exhibited protection from multiple apoptotic inducers including serum starvation, doxorubicin, UV irradiation, tumor necrosis factor alpha, and the ER stressors brefeldin A, tunicamycin, and thapsigargin. Analysis of the caspase proteolytic cascade reveals that ARMER inhibits proteolysis of the caspase-9-specific fluorogenic substrate LEHD-AFC as well as endogenous substrates downstream of caspase-9; however, it does not inhibit cytochrome c release or cleavage of caspase-9 itself. Apoptotic stimuli cause endogenous levels of ARMER protein and RNA to decrease, leading to cell death; however, sustaining ARMER protein levels through exogenous expression inhibits apoptosis. These data suggest that ARMER is a novel ER integral membrane protein which protects cells by inhibiting caspase-9 activity and reveal a possible role for ARMER in cell survival.     

Caspase-dependent cleavage of 170-kDa P-glycoprotein during apoptosis of human T-lymphoblastoid CEM cells

Multidrug resistance (MDR) mediated by the drug efflux protein, 170-kDa P-glycoprotein (P-gp), is one mechanism that tumor cells use to escape cell death induced by chemotherapeutic drugs. Moreover, evidence suggests that cell lines expressing high levels of 170-kDa P-gp are less sensitive to caspase-mediated apoptosis induced by a wide range of death stimuli, including Fas ligand, tumor necrosis factor, and ultraviolet irradiation. However, the fate of 170-kDa P-gp during apoptosis is unknown. In this study, we demonstrate for the first time that 170-kDa P-gp is cleaved during apoptosis of VBL100 human T-lymphoblastoid CEM cells. Apoptotic cell death was induced by LY294002 (a pharmacological inhibitor of the phosphoinositide 3-kinase/Akt survival pathway), H2O2, and Z-LEHD-FMK (a caspase-9 inhibitor which has been recently reported to induce apoptosis in CEM cells). Using an antibody to a common epitope present in both the third and the sixth extracellular loop of P-gp, two cleavage products were detected, with an apparent molecular weight of 80 and 85 kDa. DEVD-FMK (a caspase-3 inhibitor), but not VEID-CHO (a caspase-6 inhibitor), blocked 170-kDa P-gp cleavage. Recombinant caspase-3 was able to cleave in vitro 170-kDa P-gp yielding two fragments of equal size to those generated in vivo. Considering the size of the cleaved fragments and their reactivity with antibodies, which recognize either the N-half or the C-half region of the protein, it is conceivable that the cleavage occurs intracytoplasmically. Since 170-kDa P-gp has been reported to counteract apoptosis, its cleavage may be a mechanism aimed at blocking an important cell survival component.     

The Bcl-2 proteins Noxa and Bcl-xL co-ordinately regulate oxidative stress-induced apoptosis

Because the detailed molecular mechanisms by which oxidative stress induces apoptosis are not completely known, we investigated how the complex Bcl-2 protein network might regulate oxidative stress-induced apoptosis. Using MEFs (mouse embryonic fibroblasts), we found that the endogenous anti-apoptotic Bcl-2 protein Bcl-xL prevented apoptosis initiated by H(2)O(2). The BH3 (Bcl-2 homology 3)-only Bcl-2 protein Noxa was required for H(2)O(2)-induced cell death and was the single BH3-only Bcl-2 protein whose pro-apoptotic activity was completely antagonized by endogenous Bcl-xL. Upon H(2)O(2) treatment, Noxa mRNA displayed the greatest increase among BH3-only Bcl-2 proteins. Expression levels of the anti-apoptotic Bcl-2 protein Mcl-1 (myeloid cell leukaemia sequence 1), the primary binding target of Noxa, were reduced in H(2)O(2)-treated cells in a Noxa-dependent manner, and Mcl-1 overexpression was able to prevent H(2)O(2)-induced cell death in Bcl-xL-deficient MEF cells. Importantly, reduction of the expression of both Mcl-1 and Bcl-xL caused spontaneous cell death. These studies reveal a signalling pathway in which H(2)O(2) activates Noxa, leading to a decrease in Mcl-1 and subsequent cell death in the absence of Bcl-xL expression. The results of the present study indicate that both anti- and pro-apoptotic Bcl-2 proteins co-operate to regulate oxidative stress-induced apoptosis.     

Ceramide synthase-dependent ceramide generation and programmed cell death: involvement of salvage pathway in regulating postmitochondrial events

The sphingolipid ceramide has been widely implicated in the regulation of programmed cell death or apoptosis. The accumulation of ceramide has been demonstrated in a wide variety of experimental models of apoptosis and in response to a myriad of stimuli and cellular stresses. However, the detailed mechanisms of its generation and regulatory role during apoptosis are poorly understood. We sought to determine the regulation and roles of ceramide production in a model of ultraviolet light-C (UV-C)-induced programmed cell death. We found that UV-C irradiation induces the accumulation of multiple sphingolipid species including ceramide, dihydroceramide, sphingomyelin, and hexosylceramide. Late ceramide generation was also found to be regulated by Bcl-xL, Bak, and caspases. Surprisingly, inhibition of de novo synthesis using myriocin or fumonisin B1 resulted in decreased overall cellular ceramide levels basally and in response to UV-C, but only fumonisin B1 inhibited cell death, suggesting the presence of a ceramide synthase (CerS)-dependent, sphingosine-derived pool of ceramide in regulating programmed cell death. We found that this pool did not regulate the mitochondrial pathway, but it did partially regulate activation of caspase-7 and, more importantly, was necessary for late plasma membrane permeabilization. Attempting to identify the CerS responsible for this effect, we found that combined knockdown of CerS5 and CerS6 was able to decrease long-chain ceramide accumulation and plasma membrane permeabilization. These data identify a novel role for CerS and the sphingosine salvage pathway in regulating membrane permeability in the execution phase of programmed cell death.     

Staphylococcus aureus alpha-toxin induces apoptosis in peripheral blood mononuclear cells: role of endogenous tumour necrosis factor-alpha and the mitochondrial death pathway

Staphylococcus aureus infections can result in septic and toxic shock with depletion of immune cells and massive cytokine production. Recently, we showed that, in S. aureus-infected Jurkat T cells, alpha-toxin is the major mediator of caspase activation and apoptosis. Here, we investigated the mechanisms of cell death induced by alpha-toxin in peripheral blood mononuclear cells (MNC). We show that alpha-toxin is required and sufficient for S. aureus-induced cell death not only in transformed Jurkat T cells but also in MNC. Low alpha-toxin doses (3-30 ng ml-1) dose- and time-dependently induced apoptosis in both cell types, which was completely blocked by the caspase inhibitor zVAD-fmk. In Jurkat T cells and MNC, alpha-toxin induced the breakdown of the mitochondrial membrane potential and the intrinsic activation of caspase-3, -8 and -9. Interestingly, unlike in Jurkat T cells, apoptosis in MNC was additionally mediated by a caspase-9-independent component. MNC, but not Jurkat T cells, produced tumour necrosis factor (TNF)-alpha upon alpha-toxin stimulation. Blocking endogenous TNF-alpha with a TNF-alpha receptor antagonist partially decreased apoptosis in MNC. Our data therefore suggest that, whereas in Jurkat T cells apoptosis is solely mediated by the mitochondrial pathway, in MNC endogenous TNF-alpha and a death receptor-dependent pathway are also involved, which may contribute to depletion of immune cells during S. aureus infection.     

Programmed cell death by bcl-2-dependent and independent mechanisms in B lymphoma cells.

Programmed cell death (PCD) or apoptosis is a common form of cellular demise during embryogenesis, tumorigenesis and clonal selection in the immune system. The bcl-2 proto-oncogene has been recently implicated as a potential physiological regulator of the PCD pathway. Gene transfer studies have shown that overexpression of bcl-2 blocks apoptosis mediated by several stimuli in cultured cell lines and promotes the survival of B and T lymphocytes in transgenic mice. However, it remains unclear whether under normal conditions bcl-2 is responsible for controlling cell death. We have investigated the role of bcl-2 in the antimembrane IgM (mIgM)-induced apoptotic death of WEHI-231 B cell lymphoma, a model that mimics clonal deletion of immature B cells by antigen. Signalling of mIgM receptors triggered downregulation of both bcl-2 RNA and protein, and induced apoptosis in WEHI-231 B cells. This effect appeared to be specific since (i) the levels of beta 2-microglobulin and beta-actin RNA remain unchanged and (ii) signalling of the apoptosis-resistant B cell lymphoma line BAL-17 with anti-mu was not associated with downregulation of bcl-2 RNA. However, stable expression of bcl-2 by transfection did not rescue WEHI-231 B cells from apoptosis, yet WEHI-231 cells overexpressing bcl-2 were more resistant to programmed cell death induced by heat-shock.(ABSTRACT TRUNCATED AT 250 WORDS)