Caspase-10-dependent cell death in Fas/CD95 signalling is not abrogated by caspase inhibitor zVAD-fmk

Background

Upon CD95/Fas ligation, the initiator caspase-8 is known to activate effector caspases leading to apoptosis. In the presence of zVAD-fmk, a broad-spectrum caspase inhibitor, Fas engagement can also trigger an alternative, non-apoptotic caspase-independent form of cell death, which is initiated by RIP1. Controversy exists as to the ability of caspase-10 to mediate cell death in response to FasL (CD95L or CD178). Herein, the role of caspase-10 in FasL-induced cell death has been re-evaluated.

Methodology and Principal Findings

The present study shows that FasL-induced cell death was completely impaired in caspase-8- and caspase-10-doubly deficient (I9-2e) Jurkat leukaemia T-cell lines. Over-expressing of either caspase-8 or caspase-10 in I9-2e cells triggered cell death and restored sensitivity to FasL, further arguing for a role of both initiator caspases in Fas apoptotic signalling. In the presence of zVAD-fmk, FasL triggered an alternative form of cell death similarly in wild-type (A3) and in caspase-8-deficient Jurkat cells expressing endogenous caspase-10 (clone I9-2d). Cell death initiated by Fas stimulation in the presence of zVAD-fmk was abrogated in I9-2e cells as well as in HeLa cells, which did not express endogenous caspase-10, indicating that caspase-10 somewhat participates in this alternative form of cell death. Noteworthy, ectopic expression of caspase-10 in I9-2e and HeLa cells restored the ability of FasL to trigger cell death in the presence of zVAD-fmk. As a matter of fact, FasL-triggered caspase-10 processing still occurred in the presence of zVAD-fmk.

Conclusions and Significance

Altogether, these data provide genetic evidence for the involvement of initiator caspase-10 in FasL-induced cell death and indicate that zVAD-fmk does not abrogate caspase-10 processing and cytotoxicity in Fas signalling. Our study also questions the existence of an alternative caspase-independent cell death pathway in Fas signalling.     

Caspase-10 sensitizes breast carcinoma cells to TRAIL-induced but not tumor necrosis factor-induced apoptosis in a caspase-3-dependent manner

Although signaling by death receptors involves the recruitment of common components into their death-inducing signaling complexes (DISCs), apoptosis susceptibility of various tumor cells to each individual receptor differs quite dramatically. Recently it was shown that, besides caspase-8, caspase-10 is also recruited to the DISCs, but its function in death receptor signaling remains unknown. Here we show that expression of caspase-10 sensitizes MCF-7 breast carcinoma cells to TRAIL- but not tumor necrosis factor (TNF)-induced apoptosis. This sensitization is most obvious at low TRAIL concentrations or when apoptosis is assessed at early time points. Caspase-10-mediated sensitization for TRAIL-induced apoptosis appears to be dependent on caspase-3, as expression of caspase-10 in MCF-7/casp-3 cells but not in caspase-3-deficient MCF-7 cells overcomes TRAIL resistance. Interestingly, neutralization of TRAIL receptor 2 (TRAIL-R2), but not TRAIL-R1, impaired apoptosis in a caspase-10-dependent manner, indicating that caspase-10 enhances TRAIL-R2-induced cell death. Furthermore, whereas processing of caspase-10 was delayed in TNF-treated cells, TRAIL triggered a very rapid activation of caspase-10 and -3. Therefore, we propose a model in which caspase-10 is a crucial component during TRAIL-mediated apoptosis that in addition actively requires caspase-3. This might be especially important in systems where only low TRAIL concentrations are supplied that are not sufficient for the fast recruitment of caspase-8 to the DISC.     

Phosphatidylinositol 3'-kinase blocks CD95 aggregation and caspase-8 cleavage at the death-inducing signaling complex by modulating lateral diffusion of CD95

Activation of phosphatidylinositol 3'-kinase (PI 3'-K) after ligation of CD3 protects Th2 cells from CD95-mediated apoptosis. Here we show that protection is achieved by inhibition of the formation of CD95 aggregates and consequent activation of caspase-8. Inhibition of aggregate formation is mediated by changes in the actin cytoskeleton, which in turn inhibit lateral diffusion of CD95, reducing its diffusion coefficient, D, 10-fold. After cytochalasin D treatment of stimulated cells, the lateral diffusion of CD95 increases to the value measured on unstimulated cells, and CD95 molecules aggregate to process caspase-8 and mediate apoptosis. Regulation of functional receptor formation by modulating lateral diffusion is a novel mechanism for controlling receptor activity.     

TRAIL receptor and CD95 signal to mitochondria via FADD,caspase-8/10, Bid,and Bax but differentially regulate events downstream from truncated Bid

The death receptor ligand TRAIL arouses much interest for clinical application. We found that TRAIL receptor could induce cytochrome c (Cyt c) release from mitochondria in cells that failed to respond to CD95. Therefore, we examined whether these two closely related death receptors use different intermediates to convey the apoptotic signal to mitochondria. Dominant negative FADD, FLIP(L), or a Bid mutant lacking cleavage sites for caspase-8/10 completely inhibited Cyt c release in response to either receptor. Depletion of Bid from TRAIL- or CD95-activated cytosols blocked their capacity to mediate Cyt c release from mitochondria in vitro, whereas Bax depletion reduced it. We conclude that FADD, caspase-8/10, and caspase-cleaved Bid are required for TRAIL receptor and CD95 signaling to mitochondria, whereas Bax is a common accessory. In vitro, caspase-8 treatment of cytosol from CD95-resistant cells permitted generation of truncated Bid and its association with mitochondria. However, this cytosol impaired the ability of truncated Bid to liberate Cyt c from exogenous mitochondria. We conclude that the TRAIL receptor can bypass or neutralize the activity of cytosolic factor that blocks truncated Bid function. This may benefit the capacity of TRAIL to break apoptosis resistance in tumor cells.     

Cellular FLICE-inhibitory protein (cFLIP) isoforms block CD95- and TRAIL death receptor-induced gene induction irrespective of processing of caspase-8 or cFLIP in the death-inducing signaling complex

Death receptors (DRs) induce apoptosis but also stimulate proinflammatory "non-apoptotic" signaling (e.g. NF-κB and mitogen-activated protein kinase (MAPK) activation) and inhibit distinct steps of DR-activated maturation of procaspase-8. To examine whether isoforms of cellular FLIP (cFLIP) or its cleavage products differentially regulate DR signaling, we established HaCaT cells expressing cFLIP(S), cFLIP(L), or mutants of cFLIP(L) (cFLIP(D376N) and cFLIP(p43)). cFLIP variants blocked TRAIL- and CD95L-induced apoptosis, but the cleavage pattern of caspase-8 in the death inducing signaling complex was different: cFLIP(L) induced processing of caspase-8 to the p43/41 fragments irrespective of cFLIP cleavage. cFLIP(S) or cFLIP(p43) blocked procaspase-8 cleavage. Analyzing non-apoptotic signaling pathways, we found that TRAIL and CD95L activate JNK and p38 within 15 min. cFLIP variants and different caspase inhibitors blocked late death ligand-induced JNK or p38 MAPK activation suggesting that these responses are secondary to cell death. cFLIP isoforms/mutants also blocked death ligand-mediated gene induction of CXCL-8 (IL-8). Knockdown of caspase-8 fully suppressed apoptotic and non-apoptotic signaling. Knockdown of cFLIP isoforms in primary human keratinocytes enhanced CD95L- and TRAIL-induced NF-κB activation, and JNK and p38 activation, underscoring the regulatory role of cFLIP for these DR-mediated signals. Whereas the presence of caspase-8 is critical for apoptotic and non-apoptotic signaling, cFLIP isoforms are potent inhibitors of TRAIL- and CD95L-induced apoptosis, NF-κB activation, and the late JNK and p38 MAPK activation. cFLIP-mediated inhibition of CD95 and TRAIL DR could be of crucial importance during keratinocyte skin carcinogenesis and for the activation of innate and/or adaptive immune responses triggered by DR activation in the skin.     

Caspase-3 is a component of Fas death-inducing signaling complex in lipid rafts and its activity is required for complete caspase-8 activation during Fas-mediated cell death

Since its discovery, caspase-8 has been placed at the apex of the proteolytic cascade triggered by death receptor (DR) cross-linking. Because of its capacity to interact with the cytoplasmic portion of DR, it has been suggested that caspase-8 acts independently of other caspases in the initiation of Fas and other DR signaling. In this study, we demonstrate that in Jurkat cells, caspase-3 cleavage is an early step during Fas-induced apoptosis. We show that caspase-3 processing into its p20 occurs rapidly after Fas cross-linking, in the absence of mitochondrial depolarization and caspase-9 activation. Moreover, caspase-3 is present in lipid rafts of untreated Jurkat cells and peripheral T lymphocytes. Caspase-3, caspase-8, and Fas-associated death domain are further recruited to lipid rafts of Jurkat cells following anti-Fas treatment. Fas immunoprecipitation reveals that caspase-3 is a component of the death-inducing signaling complex, suggesting that this cysteine protease is in close proximity to caspase-8. Furthermore, transduction of Jurkat cells with a caspase-3 dominant-negative form inhibits caspase-8 processing and results in inhibition of apoptosis, suggesting that caspase-3 activity is required for caspase-8 activation. Overall, these findings support a model whereby caspase-3 is a component of the death-inducing signaling complex located in lipid rafts, and as such, is involved in the amplification of caspase-8 activity by the mitochondrion.     

c-FLIP(L) is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis

Activation of the caspase cascade is a pivotal step in apoptosis and can occur via death adaptor-mediated homo-oligomerization of initiator procaspases. Here we show that c-FLIP(L), a protease-deficient caspase homolog widely regarded as an apoptosis inhibitor, is enriched in the CD95 death-inducing signaling complex (DISC) and potently promotes procaspase-8 activation through hetero-dimerization. c-FLIP(L) exerts its effect through its protease-like domain, which associates efficiently with the procaspase-8 protease domain and induces the enzymatic activity of the zymogen. Ectopic expression of c-FLIP(L) at physiologically relevant levels enhances procaspase-8 processing in the CD95 DISC and promotes apoptosis, while a decrease of c-FLIP(L) expression results in inhibition of apoptosis. c-FLIP(L) acts as an apoptosis inhibitor only at high ectopic expression levels. Thus, c-FLIP(L) defines a novel type of caspase regulator, distinct from the death adaptors, that can either promote or inhibit apoptosis.     

Caspase-8 activation and bid cleavage contribute to MCF7 cellular execution in a caspase-3-dependent manner during staurosporine-mediated apoptosis

There are at least two distinct classes of caspases, initiators (e.g. caspases-8, -9, and -10) and effectors (e.g. caspase-3). Furthermore, it is believed that there are two distinct primary apoptotic signaling pathways, one of which is mediated by death receptors controlled by caspases-8/10, and the other by the release of cytochrome c and activation of a caspase-9/Apaf1/cytochrome c apoptosome. However, several recent reports have demonstrated that caspase-8, and its substrate Bid, are frequently activated in response to certain apoptotic stimuli in a death receptor-independent manner. These results suggest that significant cross-talk may exist between these two distinct signaling arms, allowing each to take advantage of elements unique to the other. Here we provide evidence that activation of caspase-8, and subsequent Bid cleavage, does indeed participate in cytochrome c-mediated apoptosis, at least in certain circumstances and cell types. Furthermore, the participation of activated caspase-3 is essential for activation of caspase-8 and Bid processing to occur. Although caspase-8 activation is not required for the execution of a cytochrome c-mediated death signal, we found that it greatly shortens the execution time. Thus, caspase-8 involvement in cytochrome c-mediated cell death may help to amplify weaker death signals and ensure that apoptosis occurs within a certain time frame.     

Interleukin-15 is a potent survival factor in the prevention of spontaneous but not CD95-induced apoptosis in CD4 and CD8 T lymphocytes of HIV-infected individuals. Correlation with its ability to increase BCL-2 expression

IL-15 shares many biological properties with IL-2, a cytokine whose administration to HIV-infected individuals has been effective in enhancing depleted CD4 T lymphocyte numbers. The present study examined whether exogenous IL-15 could protect lymphocytes of HIV-infected individuals from spontaneous apoptosis, associated with growth factor deprivation, and CD95-induced apoptosis, which is believed to play a major role in T lymphocyte loss and HIV pathogenesis. Although IL-15, like IL-2, failed to inhibit CD95-induced lymphocyte apoptosis in vitro, IL-15 was found to act as a potent survival factor in the prevention of spontaneous apoptosis. The greater enhancement of lymphocyte survival, promoted by IL-15 as compared with IL-2 when used at an equivalent concentration, was associated with higher up-regulation of bcl-2 expression. In addition, IL-15 was more potent than IL-2 in stimulating lymphocyte proliferation. Despite the strong ability of IL-15 to promote both lymphocyte survival and proliferation, the increases in representation and total numbers of viable cells induced by IL-15 were not higher than those induced by IL-2. This appears to be associated with the greater ability of IL-15 to activate lymphocytes and increase their apoptosis-susceptibility. Therefore, lymphocyte loss occurring by growth factor deprivation in HIV infection may be potentially prevented by IL-15, although its benefits for survival need to be closely assessed against its ability to augment lymphocyte activation.     

Membrane expression of DR4, DR5 and caspase-8 levels, but not Mcl-1, determine sensitivity of human myeloma cells to Apo2L/TRAIL

The improved recombinant form of the death ligand Apo2L/TRAIL (Apo2L/TRAIL.0) is not cytotoxic for normal human cells and is a good candidate for the therapy of multiple myeloma (MM), a B-cell neoplasia that remains incurable. We have analyzed the molecular determinants of myeloma sensitivity to Apo2L/TRAIL.0 in a number of MM cell lines, the mechanisms of resistance and a possible way of overcoming it. Expression of one death receptor for Apo2L/TRAIL (DR4 or DR5) is sufficient to transduce death signals, though DR5 was more efficient when both receptors were present. Membrane expression of decoy receptors (DcR1, DcR2) and intracellular levels of c-FLIP(L), XIAP and Mcl-1 were not predictive of resistance to Apo2L/TRAIL. Inhibition of Mcl-1 degradation did not prevent Apo2L/TRAIL-induced apoptosis. In IM-9 cells, resistance was associated to a reduced caspase-8 expression. U266 cells, though expressing significant levels of DR4 and caspase-8, were nevertheless resistant to Apo2L/TRAIL. This resistance could be overcome by co-treatment with valproic acid (VPA), a histone deacetylase inhibitor. VPA caused the redistribution of DR4 to plasma membrane lipid rafts and restored DR4 signaling. Overexpression of Mcl-1 in U266 cells did not prevent Apo2L/TRAIL cytotoxicity in VPA-sensitized cells. These results, taken together, support the possible use of Apo2L/TRAIL.0 in the treatment of MM.     

ICAM-1 contributes to but is not essential for tumor antigen cross-priming and CD8+ T cell-mediated tumor rejection in vivo

ICAM-1 has been described to provide both adhesion and costimulatory functions during T cell activation. In the setting of antitumor immunity, ICAM-1/LFA-1 interactions could be important at the level of T cell priming by APCs in draining lymph nodes as well as for transendothelial migration and tumor cell recognition at the tumor site. To determine the contribution of ICAM-1 to tumor rejection in vivo, we performed adoptive transfer of 2C TCR-transgenic/RAG2(-/-) T cells into TCRalpha(-/-) vs ICAM(-/-)/TCRalpha(-/-) recipient animals. ICAM-1-deficient mice successfully rejected HTR.C tumors expressing Ld recognized by the 2C TCR, albeit with a kinetic delay. Inasmuch as HTR.C tumor cells themselves express ICAM-1, a second model was pursued using B16-F10 melanoma cells that lack ICAM-1 expression. These cells were transduced to express the SIYRYYGL peptide recognized by the 2C TCR in the context of Kb, which is cross-presented by APCs in H-2b mice in vivo. These tumors also grew more slowly but were eventually rejected by the majority of ICAM-1(-/-)/TCRalpha(-/-) recipients. Delayed rejection in ICAM-1(-/-) mice was associated with diminished T cell priming as assessed by ELISPOT. In contrast, T cell penetration into the tumor was comparable in wild-type and ICAM-1(-/-) hosts, and adoptively transferred primed effector 2C cells rejected normally in ICAM-1(-/-) recipients. Our results suggest that ICAM-1 contributes to but is not absolutely required for CD8+ T cell-mediated tumor rejection in vivo and dominantly acts at the level of priming rather than the effector phase of the antitumor immune response.     

Lrp5 is not required for the proliferative response of osteoblasts to strain but regulates proliferation and apoptosis in a cell autonomous manner

Although Lrp5 is known to be an important contributor to the mechanisms regulating bone mass, its precise role remains unclear. The aim of this study was to establish whether mutations in Lrp5 are associated with differences in the growth and/or apoptosis of osteoblast-like cells and their proliferative response to mechanical strain in vitro. Primary osteoblast-like cells were derived from cortical bone of adult mice lacking functional Lrp5 (Lrp5(-/-)), those heterozygous for the human G171V High Bone Mass (HBM) mutation (LRP5(G171V)) and their WT littermates (WT(Lrp5), WT(HBM)). Osteoblast proliferation over time was significantly higher in cultures of cells from LRP5(G171V) mice compared to their WT(HBM) littermates, and lower in Lrp5(-/-) cells. Cells from female LRP5(G171V) mice grew more rapidly than those from males, whereas cells from female Lrp5(-/-) mice grew more slowly than those from males. Apoptosis induced by serum withdrawal was significantly higher in cultures from Lrp5(-/-) mice than in those from WT(HBM) or LRP5(G171V) mice. Exposure to a single short period of dynamic mechanical strain was associated with a significant increase in cell number but this response was unaffected by genotype which also did not change the 'threshold' at which cells responded to strain. In conclusion, the data presented here suggest that Lrp5 loss and gain of function mutations result in cell-autonomous alterations in osteoblast proliferation and apoptosis but do not alter the proliferative response of osteoblasts to mechanical strain in vitro.     

Protein kinase C delta is not activated by caspase-3 and its inhibition is sufficient to induce apoptosis in the colon cancer line, COLO 205

Activation of protein kinase C delta (PKCdelta) is believed to be pro-apoptotic. PKCdelta is reported to be reduced in colon cancers. Using a colon cancer cell line, COLO 205, we have examined the roles of PKCdelta in apoptosis and of caspase-3 in the activation and inhibition of PKCdelta. PKCdelta activation with bistratene A and its inhibition with rottlerin induced apoptosis. Effects of PKC activators and inhibitors were additive, suggesting that PKCdelta down-regulation was responsible for the effects on apoptosis. Different apoptotic pathways induced PKCdelta cleavage, but the fragment produced was inactive in kinase assays. Caspase-3 inhibition did not block DNA fragmentation or PKCdelta proteolysis despite blocking intracellular caspase-3 activity. Calpain inhibition with calpeptin did not prevent TPA-induced PKCdelta cleavage. We conclude that in colonocytes, inhibition of PKCdelta is sufficient to lead to caspase-3-independent apoptosis. Caspase-3 does not cleave PKCdelta to an active form, nor does caspase-3 inhibition block apoptosis.     

Evidence that the respiratory syncytial virus polymerase is recruited to nucleotides 1 to 11 at the 3' end of the nucleocapsid and can scan to access internal signals

The 3'-terminal end of the respiratory syncytial virus genomic RNA contains a 44-nucleotide leader (Le) region adjoining the gene start signal of the first gene. Previous mapping studies demonstrated that there is a promoter located at the 3' end of Le, which can signal initiation of antigenome synthesis. The aim of this study was to investigate the role of the 3' terminus of the RNA template in (i) promoter recognition and (ii) determining the initiation site for antigenome synthesis. A panel of minigenomes containing additional sequence at the 3' end of the Le were analyzed for their ability to direct antigenome and mRNA synthesis. Minigenomes containing heterologous extensions of 6 nucleotides or more were unable to support efficient RNA synthesis. However, the activity of a minigenome with a 56-nucleotide extension could be restored by insertion of Le nucleotides 1 to 11 or 1 to 13 at the 3' end, indicating that these nucleotides, in conjunction with the 3' terminus, are sufficient to recruit polymerase to the template. Northern blot and 5' rapid amplification of cDNA ends analysis of antigenome RNA indicated that antigenome initiation occurred at the first position of Le, irrespective of the terminal extension. This finding demonstrates that the 3' terminus of the RNA is not necessary for determining the antigenome initiation site. Data are presented which suggest that following recruitment to a promoter at the 3' end of Le, the polymerase is able to scan and respond to a promoter signal embedded within the RNA template.     

Rrp8p is a yeast nucleolar protein functionally linked to Gar1p and involved in pre-rRNA cleavage at site A2

Chemical modifications and processing of the 18S, 5.8S, and 25S ribosomal RNAs from the 35S pre-ribosomal RNA depend on an important set of small nucleolar ribonucleoprotein particles (snoRNPs). Genetic depletion of yeast Gar1p, an essential common component of H/ACA snoRNPs, leads to inhibition of uridine isomerizations to pseudo-uridines on the 35S pre-rRNA and of the early pre-rRNA cleavages at sites A1 and A2, resulting in a loss of mature 18S rRNA synthesis. To identify Gar1p functional partners, we screened for mutations that are synthetically lethal with a gar1 mutant allele encoding a Gar1p mutant protein lacking its two glycine/arginine-rich (GAR) domains. We identified a previously uncharacterized Saccharomyces cerevisiae open reading frame, YDR083W (now designated RRP8), that encodes a highly conserved protein containing motifs found in methyltransferases. Rrp8p localizes to the nucleolus. A yeast strain lacking this protein is viable at 30 degrees C but displays strong growth impairment at lower temperatures. In this strain, cleavage of the pre-rRNA at site A2 is strongly affected whereas cleavages at sites A0 and A1 are only slightly inhibited or delayed.     

Dextromethorphan potentiates morphine-induced antinociception at both spinal and supraspinal sites but is not related to the descending serotoninergic or adrenergic pathways

Morphine is a strong and widely used opioid analgesic in pain management, but some adverse effects limit its clinical use at high doses. The clinically available non-opioid antitussive, dextromethorphan (DM) can potentiate the analgesic effect of morphine and decrease the dose of morphine in acute postoperative pain. However, the mechanism underlying this synergistic phenomenon is still not clear. To examine if the potentiation by DM occurs through the descending pain-inhibitory pathways, ketanserin (a 5-HT₂ receptor antagonist) and yohimbine (an ₂-adrenergic receptor antagonist) were employed and found to have no significant effect on the potentiation by DM. Using local delivery of drugs in rats in the present study, potentiation of morphine-induced antinociception by DM was observed via both intrathecal and intracerebroventricular routes, suggesting that both spinal and supraspinal sites are involved. This suggests that the potentiation of morphine-induced antinociception by DM is not mediated by the serotoninergic or adrenergic descending pain-inhibitory pathways. The present results are consistent with findings in clinical studies, which showed that DM can effectively decrease the consumption of morphine in patients suffering from pain. Since DM has excellent clinical potential as a synergistic agent with morphine, further investigating and clarifying the possible pharmacological mechanism of DM are of great importance for future studies.     

ts1, a temperature-sensitive mutant of Moloney murine leukemia virus TB, can infect both CD4+ and CD8+ T cells but requires CD4+ T cells in order to cause paralysis and immunodeficiency.

When neonatal FVB/N mice were inoculated with ts1, a temperature-sensitive mutant of Moloney murine leukemia virus TB, they developed a progressive bilateral hindlimb paralysis and immunodeficiency leading to death 4 to 6 weeks after inoculation. T lymphocytes have been shown to be primarily responsible for this ts1-induced syndrome. Here we compare the role played by each subset of T lymphocytes, i.e., CD4+ and CD8+ T cells, in disease development. Mice were depleted of a specific subset for the first 10 days of their lives by using either anti-CD4 or anti-CD8 monoclonal antibodies in vivo. Disease development in these mice was then monitored. Depletion of CD4+ T cells significantly attenuated the ts1-induced syndrome: virus replication was decreased, disease latency was extended, and death was prevented in 60% of the mice. Similar treatment with anti-CD8 antibody had almost no effect on disease progression. However, when depletion was begun 2 weeks after neonatal ts1 inoculation, CD4+ T cell depletion did not affect disease development. ts1 infected CD4+ and CD8+ T lymphocytes equally well in vivo, as shown by flow cytometric analysis, but virus replication was restricted primarily to the CD4+ subset of T cells, as found by in vitro assay. Hence, CD4+ T lymphocytes play an important role in the development of ts1-induced paralysis and immunodeficiency. The mechanism of this CD4+ T-cell-mediated disease production by ts1 is not clear; however, increased replication of ts1 in the CD4+ T cells, especially in the early stages of the disease, seems to play a crucial role.