Death receptors, Fas and TRAIL receptors, are involved in human osteoclast apoptosis

Survival and apoptosis are crucial aspects of the osteoclast life cycle. Although osteoclast survival has been extensively studied, little is known about the mechanisms involved in human osteoclast apoptosis. In the present study, cord blood monocytes (CBMs) were used as the source of human osteoclast precursors. When cultured in the presence of M-CSF and RANKL, CBMs formed multinucleated cells that expressed RANK and calcitonin receptor, and were able to resorb bone. These cells expressed TRAIL receptors (R1-R4). Surprisingly, although TRAIL-receptor expression was not detectable in osteoclasts from normal bone, osteoclasts from myeloma specimens did express TRAIL receptors to a variable extent. Significantly, we have shown for the first time that this pathway is indeed functional in human osteoclasts, and that apoptosis occurred and was significantly greater in the presence of TRAIL. In addition, we have shown that a Fas-activating antibody is also able to induce osteoclast apoptosis, as did TGFbeta, whereas the survival factor M-CSF decreased apoptosis. Overall, these findings suggest that death receptors, TRAIL receptors and Fas, could be involved in osteoclast apoptosis in humans.     

Homomeric and heteromeric interactions of the extracellular domains of death receptors and death decoy receptors

Death receptors (DRs) can induce apoptosis by oligomerization with TRAIL, whereas death decoy receptors (DcRs) cannot, due to their lack of functional intracellular death domains. However, it is not known whether DRs and DcRs can interact with one another to form oligomeric complexes prior to TRAIL binding. To address this issue, the extracellular domains (ECDs) of DR4 (sDR4), DR5 (sDR5), DcR1 (sDcR1), and DcR2 (sDcR2) were expressed in a soluble, monomeric form, and their binding interactions were quantified by surface plasmon resonance. The purified sDRs and sDcRs exhibited native-like secondary structure and bound to TRAIL with binding affinities in the nanomolar range (K(D)= approximately 10-62 nM), suggesting that they were properly folded and functional. The soluble receptors interacted homophilically and heterophilically with similar micromolar range affinities (K(D)= approximately 1-9 microM), with the exception that sDR5 did not interact with the sDcRs. Our results suggest that most DRs and DcRs can laterally interact through their ECDs to form homomeric and/or heteromeric complexes in the absence of TRAIL binding.     

The death receptor DR5 is involved in TRAIL-mediated human osteoclast apoptosis

The number and activity of osteoclasts (OCs) are critical for maintaining normal bone turnover. The number is determined by the rates of cell differentiation and death. TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, induces apoptosis by interacting with its death receptors, (DR4, DR5). However, its activity can be modulated by two decoy receptors, (DcR1 and DcR2). In this paper we show that TRAIL treatment causes reduced OC viability as well as an increased apoptotic OC number. Loss of nuclei integrity and derangement of the actin microfilament were also induced by TRAIL in OCs. Moreover, we demonstrated the expression of all TRAIL receptors in both precursors and differentiated OCs, and the upregulation of DR5 during OC differentiation. Interestingly, DcR2 was upregulated in the early stage of osteoclastogenesis and downregulated at the end of the differentiation process. We showed that DR5, upregulated by TRAIL, could be the mediator of TRAIL-induced OC apoptosis, since the addition of anti-DR5 neutralizing antibodies restores the OC viability previously reduced by TRAIL. Furthermore, the intracellular pathway induced by TRAIL in OCs involves caspase-8 and Bid activation. In conclusion, our data highlight an important role for the TRAIL/TRAIL receptor system in the regulation of OC apoptosis.     

LUCA-15/RBM5, a putative tumour suppressor, enhances multiple receptor-initiated death signals

LUCA-15/RBM5 is a putative tumour suppressor. The gene encodes a number of alternative RNA splice variants with differing abilities to either enhance or suppress apoptosis, and it is likely that this ability to modulate apoptosis is central to the putative tumour suppressor activity of LUCA-15. This report demonstrates for the first time that expression from the LUCA-15 locus modulates apoptosis triggered by the death-inducing ligand TRAIL. Using Jurkat T lymphoblastic leukemia cells, LUCA-15 expression was shown to enhance not only TRAIL but TNF-alpha- and Fas-mediated apoptosis. LUCA-15, therefore, has the ability to lower the apoptotic threshold of multiple receptor-initiated death-inducing signals. Of note, sensitisation of the Jurkat cells to TRAIL was shown to depend on new protein synthesis, since no enhancement of apoptosis was observed when cells were exposed to both TRAIL and the protein synthesis inhibitor cycloheximide. This result suggests that LUCA-15 does not act independently to regulate apoptosis, but modulates a process that requires additional, newly synthesized protein. These results may explain the putative role of LUCA-15 as a tumour suppressor, suggesting that lack of functional LUCA-15 could provide the means by which malignant T cells escape receptor-initiated apoptotic signals.     

Cell death in hematological tumors

Evasion of apoptosis is a hallmark of human cancers, for example in hematological malignancies. Apoptosis is an intrinsic cell death program that is crucial to maintain tissue homeostasis, for example in the hematopoietic system where there is a high turnover rate of cells. As a result, a decrease in the rate of apoptosis besides an increase in proliferation favors tumorigenesis as well as tumor progression. Further, the anti-leukemic action of current treatment approaches, including chemo-, radio- or immunotherapy, critically relies on intact cell death programs in cancer cells. Therefore, defects in apoptosis pathways are frequently associated with the resistance to anticancer therapies. In recent years, the identification and characterization of the molecules and pathways that are involved in the regulation and execution of cell death in leukemia and lymphoma cells have set the ground for the development of novel diagnostic tools and molecular therapeutics targeting apoptosis pathways in hematological malignancies.     

Death receptors 4 and 5 activate Nox1 NADPH oxidase through riboflavin kinase to induce reactive oxygen species-mediated apoptotic cell death

Stimulation of the proapoptotic tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, death receptors 4 (DR4) and 5 (DR5), conventionally induces caspase-dependent apoptosis in tumor cells. Here we report that stimulation of DR4 and/or DR5 by the agonistic protein KD548-Fc, an Fc-fused DR4/DR5 dual-specific Kringle domain variant, activates plasma membrane-associated Nox1 NADPH oxidase to generate superoxide anion and subsequently accumulates intracellular reactive oxygen species (ROS), leading to sustained c-Jun N-terminal kinase activation and eventual apoptotic cell death in human HeLa and Jurkat tumor cells. KD548-Fc treatment induces the formation of a DR4/DR5 signaling complex containing riboflavin kinase (RFK), Nox1, the Nox1 subunits (Rac1, Noxo1, and Noxa1), TNF receptor-associated death domain (TRADD), and TNF receptor-associated factor 2 (TRAF2). Depletion of RFK, but not the Nox1 subunits, TRADD and TRAF2, failed to recruit Nox1 and Rac1 to DR4 and DR5, demonstrating that RFK plays an essential role in linking DR4/DR5 with Nox1. Knockdown studies also reveal that RFK, TRADD, and TRAF2 play critical, intermediate, and negligible roles, respectively, in the KD548-Fc-mediated ROS accumulation and downstream signaling. Binding assays using recombinantly expressed proteins suggest that DR4/DR5 directly interact with cytosolic RFK through RFK-binding regions within the intracellular death domains, and TRADD stabilizes the DR4/DR5-RFK complex. Our results suggest that DR4 and DR5 have a capability to activate Nox1 by recruiting RFK, resulting in ROS-mediated apoptotic cell death in tumor cells.     

Generation of new TRAIL mutants DR5-A and DR5-B with improved selectivity to death receptor 5

TRAIL (tumor necrosis factor (TNF) related apoptosis-inducing ligand) has been introduced as an extrinsic pathway inducer of apoptosis that does not have the toxicities of Fas and TNF. However, the therapeutic potential of TRAIL is limited because of many primary tumor cells are resistant to TRAIL. Despite intensive investigations, little is known in regards to the mechanisms underlying TRAIL selectivity and efficiency. A major reason likely lies in the complexity of the interaction of TRAIL with its five receptors, of which only two DR4 and DR5 are death receptors. Binding of TRAIL with decoy receptors DcR1 and DcR2 or soluble receptor osteoprotegerin (OPG) fail to induce apoptosis. Here we describe design and expression in Escherichia coli of DR5-selective TRAIL variants DR5-A and DR5-B. The measurements of dissociation constants of these mutants with all five receptors show that they practically do not interact with DR4 and DcR1 and have highly reduced affinity to DcR2 and OPG receptors. These mutants are more effective than wild type TRAIL in induction of apoptosis in different cancer cell lines. In combination with the drugs targeted to cytoskeleton (taxol, cytochalasin D) the mutants of TRAIL induced apoptosis in resistant Hela cells overexpressing Bcl-2. The novel highly selective and effective DR5-A and DR5-B TRAIL variants will be useful in studies on the role of different receptors in TRAIL-induced apoptosis in sensitive and resistant cell lines. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

STI571 reduces TRAIL-induced apoptosis in colon cancer cells: c-Abl activation by the death receptor leads to stress kinase-dependent cell death

Background

In an effort to achieve better cancer therapies, we elucidated the combination cancer therapy of STI571 (an inhibitor of Bcr-Abl and clinically used for chronic myelogenous leukemia) and TNF-related apoptosis-inducing ligand (TRAIL, a developing antitumor agent) in leukemia, colon, and prostate cancer cells.

Methods

Colon cancer (HCT116, SW480), prostate cancer (PC3, LNCaP) and leukemia (K562) cells were treated with STI571 and TRAIL. Cell viability was determined by MTT assay and sub-G1 appearance. Protein expression and kinase phosphorylation were determined by Western blotting. c-Abl and p73 activities were inhibited by target-specific small interfering (si)RNA. In vitro kinase assay of c-Abl was conducted using CRK as a substrate.

Results

We found that STI571 exerts opposite effects on the antitumor activity of TRAIL. It enhanced cytotoxicity in TRAIL-treated K562 leukemia cells and reduced TRAIL-induced apoptosis in HCT116 and SW480 colon cancer cells, while having no effect on PC3 and LNCaP cells. In colon and prostate cancer cells, TRAIL caused c-Abl cleavage to the active form via a caspase pathway. Interestingly, JNK and p38 MAPK inhibitors effectively blocked TRAIL-induced toxicity in the colon, but not in prostate cancer cells. Next, we found that STI571 could attenuate TRAIL-induced c-Abl, JNK and p38 activation in HCT116 cells. In addition, siRNA targeting knockdown of c-Abl and p73 also reduced TRAIL-induced cytotoxicity, rendering HCT116 cells less responsive to stress kinase activation, and masking the cytoprotective effect of STI571.

Conclusions

All together we demonstrate a novel mediator role of p73 in activating the stress kinases p38 and JNK in the classical apoptotic pathway of TRAIL. TRAIL via caspase-dependent action can sequentially activate c-Abl, p73, and stress kinases, which contribute to apoptosis in colon cancer cells. Through the inhibition of c-Abl-mediated apoptotic p73 signaling, STI571 reduces the antitumor activity of TRAIL in colon cancer cells. Our results raise additional concerns when developing combination cancer therapy with TRAIL and STI571 in the future.     

Molecular mechanisms of programmed cell death

Programmed cell death is currently under active investigation. A recent meeting focused on the molecular machinery of programmed cell death and on its role in the pathogenesis of human diseases.     

Apoptotic potential of Fas-associated death domain on regulation of cell death regulatory protein cFLIP and death receptor mediated apoptosis in HEK 293T cells

Fas-associated death domain (FADD) is a common adaptor molecule which plays an important role in transduction of death receptor mediated apoptosis. The FADD provides DED motif for binding to both procaspase-8 and cFLIP molecules which executes death receptor mediated apoptosis. Dysregulated expression of FADD and cFLIP may contribute to inhibition of apoptosis and promote cell survival in cancer. Moreover elevated intracellular level of cFLIP competitively excludes the binding of procaspase-8 to the death effector domain (DED) of FADD at the DISC to block the activation of death receptor signaling required for apoptosis. Increasing evidence shows that defects in FADD protein expression are associated with progression of malignancies and resistance to apoptosis. Therefore, improved expression and function of FADD may provide new paradigms for regulation of cell proliferation and survival in cancer. In the present study, we have examined the potential of FADD in induction of apoptosis by overexpression of FADD in HEK 293T cells and validated further its consequences on the expression of pro and anti-apoptotic proteins besides initiation of death receptor mediated signaling. We have found deficient expression of FADD and elevated expression of cFLIP(L) in HEK 293T cells. Our results demonstrate that over expression of FADD attenuates the expression of anti-apoptotic protein cFLIP and activates the cascade of extrinsic caspases to execution of apoptosis in HEK 293T cells.     

ALS2CR7 (CDK15) attenuates TRAIL induced apoptosis by inducing phosphorylation of survivin Thr34

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered a promising agent for medical applications because it induces apoptosis selectively in a variety of cancer cells without toxicity to normal human cells. However, its therapeutic potential has been limited by the existence of several cancer cells with TRAIL resistance. TRAIL resistance results from a variety of mechanisms, which occur at various points in the cellular signaling pathways. In this study, we demonstrate that ALS2CR7 (CDK15) can mediate resistance to TRAIL. We also demonstrate that cell viability of TRAIL sensitive HCT116 and MDA-MB-231 cells increased after TRAIL treatment in ALS2CR7 transfected cancer cells compared with vector transfected cancer cells. Furthermore, cell viability was decreased by TRAIL treatment after knockdown with ALS2CR7 siRNA in TRAIL resistant HT29 and MCF-7 cells. We also show that the activated form of apoptotic proteins such as caspase-3, -8 and -9 and PARP increased after TRAIL treatment in the control group, but decreased in the ALS2CR7 transfected group. The expression of survival proteins such as bcl2 and survivin in TRAIL sensitive cancer cells increased in the ALS2CR7 transfected group, but decreased in TRAIL resistant cancer cells treated with ALS2CR7 siRNA. Other survival proteins such as FLIP and XIAP were not affected. ALS2CR7 appears to bind with only survivin, and not bcl2. The phospho-survivin (Thr34) critical in drug resistance was increased by transfection with ALS2CR7, but the expression of death receptors such as DR4 and DR5 was not affected. ALS2CR7 did not bind with any of the death receptors in our study. In summary, our results suggest that ALS2CR7 confers TRAIL resistance to cancer cells via phosphorylation of survivin.     

Screening of a novel octamer peptide, CNSCWSKD, that induces caspase-dependent cell death

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is known to induce apoptosis to various tumor cells but not in normal cells. We have screened cell death-inducing peptides from the extracellular domain sequence of TRAIL, using a peptide array. Peptides of higher activity were found through amino acid substitution, and the CNSCWSKD peptide induced >90% cell death in treated Jurkat cells. Features of apoptosis, such as DNA fragmentation, activation of caspase, phosphatidylserine externalization, chromatin condensation, and competition with TRAIL for binding to the death receptor (DR) 4 or DR5 were observed, suggesting that this peptide is a TRAIL mimic. Caspase-3 activation was observed in various tumor cells treated with this peptide as well as with TRAIL, while no activation was observed in human normal fibroblasts. The CNSCWSKD peptide is a potential candidate for use in cancer therapy.     

Nimbolide sensitizes human colon cancer cells to TRAIL through reactive oxygen species- and ERK-dependent up-regulation of death receptors, p53, and Bax

TNF-related apoptosis-inducing ligand (TRAIL) shows promise as a cancer treatment, but acquired tumor resistance to TRAIL is a roadblock. Here we investigated whether nimbolide, a limonoid, could sensitize human colon cancer cells to TRAIL. As indicated by assays that measure esterase activity, sub-G(1) fractions, mitochondrial activity, and activation of caspases, nimbolide potentiated the effect of TRAIL. This limonoid also enhanced expression of death receptors (DRs) DR5 and DR4 in cancer cells. Gene silencing of the receptors reduced the effect of limonoid on TRAIL-induced apoptosis. Using pharmacological inhibitors, we found that activation of ERK and p38 MAPK was required for DR up-regulation by nimbolide. Gene silencing of ERK abolished the enhancement of TRAIL-induced apoptosis. Moreover, our studies indicate that the limonoid induced reactive oxygen species production, which was required for ERK activation, up-regulation of DRs, and sensitization to TRAIL; these effects were mimicked by H(2)O(2). In addition, nimbolide down-regulated cell survival proteins, including I-FLICE, cIAP-1, cIAP-2, Bcl-2, Bcl-xL, survivin, and X-linked inhibitor of apoptosis protein, and up-regulated the pro-apoptotic proteins p53 and Bax. Interestingly, p53 and Bax up-regulation by nimbolide was required for sensitization to TRAIL but not for DR up-regulation. Overall, our results indicate that nimbolide can sensitize colon cancer cells to TRAIL-induced apoptosis through three distinct mechanisms: reactive oxygen species- and ERK-mediated up-regulation of DR5 and DR4, down-regulation of cell survival proteins, and up-regulation of p53 and Bax.     

Complestatin prevents apoptotic cell death: inhibition of a mitochondrial caspase pathway through AKT/PKB activation

Complestatin, a bicyclo hexapeptide from Streptomyces, was isolated as a possible regulator of neuronal cell death. In this study, we report an anti-apoptotic activity of complestatin and its underlying molecular mechanism. Complestatin blocked TRAIL (TNF-related apoptosis-inducing ligand)-induced apoptosis and activation of caspase-3 and -8 at micromolar concentration levels without inhibiting the catalytic activities of these caspases. Complestatin potently induced a rapid and sustained AKT/PKB activation and Bad phosphorylation, resulting in inhibition of mitochondrial cytochrome c release. These anti-apoptotic activities of complestatin were significantly abrogated in cells expressing dominant negative AKT/PKB. Taken together, our results suggest that complestatin prevents apoptotic cell death via AKT/PKB-dependent inhibition of the mitochondrial apoptosis signal pathway. The novel property of complestatin may be valuable for developing new pharmaceutical means that will control unwanted cell death.     

Playing the DISC: Turning on TRAIL death receptor-mediated apoptosis in cancer

Formation of the pro-apoptotic death-inducing signaling complex (DISC) can be initiated in cancer cells via binding of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to its two pro-apoptotic receptors, TRAIL receptor 1 (TRAIL-R1) and TRAIL-R2. Primary components of the DISC are trimerized TRAIL-R1/-R2, FADD, caspase 8 and caspase 10. The anti-apoptotic protein FLIP can also be recruited to the DISC to replace caspase 8 and form an inactive complex. Caspase 8/10 processing at the DISC triggers the caspase cascade, which eventually leads to apoptotic cell death. Besides TRAIL, TRAIL-R1- or TRAIL-R2-selective variants of TRAIL and agonistic antibodies have been designed. These ligands are of interest as anti-cancer agents since they selectively kill tumor cells. To increase tumor sensitivity to TRAIL death receptor-mediated apoptosis and to overcome drug resistance, TRAIL receptor ligands have already been combined with various therapies in preclinical models. In this review, we discuss factors influencing the initial steps of the TRAIL apoptosis signaling pathway, focusing on mechanisms modulating DISC assembly and caspase activation at the DISC. These insights will direct rational design of drug combinations with TRAIL receptor ligands to maximize DISC signaling.     

A new role of diacylglycerol kinase alpha on the secretion of lethal exosomes bearing Fas ligand during activation-induced cell death of T lymphocytes

Exosomes are small membrane vesicles that intracellularly accumulate into late or multivesicular endosomes (multivesicular bodies, MVB). Exosomes have a particular lipid and protein content, reflecting their origin as intraluminal vesicles of late endosomes. The stimulation of several hematopoietic cells induces the fusion of the limiting membrane of the MVB with the plasma membrane, leading to the release of exosomes towards the extracellular environment. In T lymphocytes, stimulation of the T cell receptor (TCR) induces the fusion of the MVBs with the plasma membrane and exosomes carrying several bio-active proteins are secreted. Among these proteins, the pro-apoptotic protein Fas ligand (FasL) is released as a non-proteolysed form (mFasL), associated to the exosomes. These mFasL-bearing exosomes may trigger the apoptosis of T lymphocytes. Here, we present evidences supporting a role of diacylglycerol kinase alpha (DGKalpha), a diacylglycerol (DAG)-consuming enzyme, on the secretion of exosomes carrying mFasL, and the subsequent activation-induced cell death (AICD) on a T cell line and primary T lymphoblasts.