Inhibition of TRAIL-induced apoptosis and forced internalization of TRAIL receptor 1 by adenovirus proteins

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis through two receptors, TRAIL-R1 (also known as death receptor 4) and TRAIL-R2 (also known as death receptor 5), that are members of the TNF receptor superfamily of death domain-containing receptors. We show that human adenovirus type 5 encodes three proteins, named RID (previously named E3-10.4K/14.5K), E3-14.7K, and E1B-19K, that independently inhibit TRAIL-induced apoptosis of infected human cells. This conclusion was derived from studies using wild-type adenovirus, adenovirus replication-competent mutants that lack one or more of the RID, E3-14.7K, and E1B-19K genes, and adenovirus E1-minus replication-defective vectors that express all E3 genes, RID plus E3-14.7K only, RID only, or E3-14.7K only. RID inhibits TRAIL-induced apoptosis when cells are sensitized to TRAIL either by adenovirus infection or treatment with cycloheximide. RID induces the internalization of TRAIL-R1 from the cell surface, as shown by flow cytometry and indirect immunofluorescence for TRAIL-R1. TRAIL-R1 was internalized in distinct vesicles which are very likely to be endosomes and lysosomes. TRAIL-R1 is degraded, as indicated by the disappearance of the TRAIL-R1 immunofluorescence signal. Degradation was inhibited by bafilomycin A1, a drug that prevents acidification of vesicles and the sorting of receptors from late endosomes to lysosomes, implying that degradation occurs in lysosomes. RID was also shown previously to internalize and degrade another death domain receptor, Fas, and to prevent apoptosis through Fas and the TNF receptor. RID was shown previously to force the internalization and degradation of the epidermal growth factor receptor. E1B-19K was shown previously to block apoptosis through Fas, and both E1B-19K and E3-14.7K were found to prevent apoptosis through the TNF receptor. These findings suggest that the receptors for TRAIL, Fas ligand, and TNF play a role in limiting virus infections. The ability of adenovirus to inhibit killing through these receptors may prolong acute and persistent infections.     

Enhanced TRAIL sensitivity by E1A expression in human cancer and normal cell lines: inhibition by adenovirus E1B19K and E3 proteins

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily of cytokines that induces apoptosis in a variety of cancer cells, but not in normal cells. However, more and more tumor cells remain resistant to TRAIL, which limited its application for cancer therapy. Expression of the adenovirus serotype 5 (Ad5) E1A sensitizes tumor cells to apoptosis by TNF-alpha, Fas-ligand, and TRAIL. Here we asked whether E1A overcomes this resistance and enhances TRAIL-induced apoptosis in the tumor cells. Our results revealed that the tumor cell lines, HeLa and HepG2, with infection by Ad-E1A, were highly sensitive to TRAIL-induced apoptosis. Importantly, we found that in normal primary human lung fibroblast cells (HLF) TRAIL is capable of inducing apoptosis in combination with E1A as efficiently as in some tumor cell lines. The adenovirus type 5 encoding proteins, E1B19K and E3 gene products, have been shown to inhibit E1A and TRAIL-induced apoptosis of HLF cells by using the recombinant adenovirus AdDeltaE1B55K, with mutation of E1B55K, containing E1B19K and complete E3 region. Further results demonstrated that the expression of DR5 and TRAIL was down-regulated in the AdDeltaE1B55K co-infected HLF cells. These findings suggest that TRAIL may play an important role in limiting virus infections and the ability of adenovirus to inhibit killing may prolong acute and persistent infections. The results from this study have also suggested the possibility that the combination of E1A with TRAIL could be used in the treatment of human malignancy, or in the selection of the optimal adenovirus mutant as effective delivering vector for cancer therapy.     

Three adenovirus E3 proteins cooperate to evade apoptosis by tumor necrosis factor-related apoptosis-inducing ligand receptor-1 and -2

Adenovirus encodes multiple gene products that regulate proapoptotic cellular responses to viral infection mediated by both the innate and adaptive immune systems. The E3-10.4K and 14.5K gene products are known to modulate the death receptor Fas. In this study, we demonstrate that an additional viral E3 protein, 6.7K, functions in the specific modulation of the two death receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The 6.7K protein is expressed on the cell surface and forms a complex with the 10.4K and 14.5K proteins, and this complex is sufficient to induce down-modulation of TRAIL receptor-1 and -2 from the cell surface and reverse the sensitivity of infected cells to TRAIL-mediated apoptosis. Down-modulation of TRAIL-R2 by the E3 complex is dependent on the cytoplasmic tail of the receptor, but the death domain alone is not sufficient. These results identify a mechanism for viral modulation of TRAIL receptor-mediated apoptosis and suggest the E3 protein complex has evolved to regulate the signaling of selected cytokine receptors.     

Adenovirus genes that modulate the sensitivity of virus-infected cells to lysis by TNF

TNF is a key inflammatory cytokine with antiviral properties. Human adenoviruses encode several intracellular proteins that mediate the effects of TNF. Expression of the adenovirus immediate early E1A proteins induces viral genes and a host of cellular genes, drives G₀ cells into S-phase, and induces apoptosis and susceptibility to TNF-induced apoptosis. The adenovirus E1B-19K protein inhibits both E1A- and TNF-induced apoptosis. The E3-14.7K protein and the E3-10.4K/14.5K complex of proteins inhibit TNF- but not E1A-induced apoptosis. The E3 14.7K and 10.4K/14.5K proteins inhibit TNF activation of cytosolic phospholipase A₂ (cPLA₂), which may explain how they inhibit TNF cytolysis. Since eicosinoids produced from arachidonic acid (the product of cPLA₂) are potent mediators of inflammation, the E3 proteins may block the inflammatory response to adenovirus infection. These adenovirus proteins should be novel tools to understand adenovirus pathogenesis, TNF signal transduction, and TNF cytolysis.     

The adenovirus E3-6.7K protein adopts diverse membrane topologies following posttranslational translocation

The E3 region of adenovirus codes for several membrane proteins, most of which are involved in immune evasion and prevention of host cell apoptosis. We explored the topology and targeting mechanisms of E3-6.7K, the most recently described member of this group, by using an in vitro translation system supplemented with microsomes. Here, we present evidence that E3-6.7K, one of the smallest signal-anchor proteins known, translocates across the membrane of the endoplasmic reticulum in a posttranslational, ribosome-independent, yet ATP-dependent manner, reminiscent of the translocation of tail-anchored proteins. Our analysis also demonstrated that E3-6.7K could achieve several distinct topological fates. In addition to the previously postulated type III orientation (N-luminal/C-cytoplasmic, termed NtmE3-6.7K), we detected a tail-anchored form adopting the opposite orientation (N-cytoplasmic/C-luminal, termed CtmE3-6.7K) as well as the possibility of a fully translocated form (N and C termini are both translocated, termed NCE3-6.7K). Due to the translocation of a positively charged domain, both the CtmE3-6.7K and NCE3-6.7K topologies of E3-6.7K constitute exceptions to the "positive inside" rule. The NtmE3-6.7K and NCE3-6.7K are the first examples of posttranslationally translocated proteins in higher eukaryotes that are not tail anchored. Distinct topological forms were also found in transfected cells, as both N and C termini of E3-6.7K were detected on the extracellular surface of transfected cells. The demonstration of unexpected topological forms and translocation mechanisms for E3-6.7K defies conventional thinking about membrane protein topogenesis and advises that both the mode of targeting and topology of signal-anchor proteins should be determined experimentally.     

Adenovirus E3 14.7K protein functions in the absence of other adenovirus proteins to protect transfected cells from tumor necrosis factor cytolysis.

A 14,700-kDa protein (14.7K) encoded by the E3 region of adenovirus has been shown to protect adenovirus-infected mouse C3HA cells from lysis by tumor necrosis factor (TNF) (L. R. Gooding, L. W. Elmore, A. E. Tollefson, H. A. Brady, and W. S. M. Wold, Cell 53:341-346, 1988). These infected cells are sensitized to TNF by expression of the adenovirus E1A proteins (P. Duerksen-Hughes, W. S. M. Wold, and L. R. Gooding, J. Immunol. 143:4193-4200, 1989). In this study we show that 14.7K suppresses TNF cytolysis independently of adenovirus infection. Mouse C3HA and C127 cells were transfected with the 14.7K gene controlled by the mouse metallothionein promoter, and permanent 14.7K-expressing cell lines were tested for sensitivity to TNF cytolysis. Transfected cells which were sensitized to TNF either by inhibitors of protein synthesis, microfilament-destabilizing agents, or adenovirus infection were found to be resistant to TNF cytolysis. Two monoclonal antibodies were isolated and used to quantitate 14.7K in transfected and infected cells. Enzyme-linked immunosorbent assay (ELISA) analysis with these monoclonal antibodies and 14.7K immunoblots showed that 14.7K expression can be induced with cadmium in C3HA and C127 transfectants. The 14.7K induction correlated with a dose-dependent decrease in sensitivity to TNF cytotoxicity. The 14.7K protein does not substantially alter cell surface TNF receptor numbers or affinity on C3HA mouse fibroblasts, as determined by Scatchard analysis of 125I-TNF binding. The 14.7K protein also does not alter TNF signal transduction in general, because TNF induction of cell surface class I major histocompatibility complex molecules on 14.7K transfectants was unmodified. Our findings indicate that the adenovirus 14.7K protein functions as a specific inhibitor of TNF cytolysis in the absence of other adenovirus proteins and thus is a unique tool to study the mechanism of TNF cytotoxicity.     

The adenovirus E3-10.4K/14.5K complex mediates loss of cell surface Fas (CD95) and resistance to Fas-induced apoptosis.

Cytotoxic T cells use Fas (CD95), a member of the tumor necrosis factor (TNF) receptor superfamily, to eliminate virus-infected cells by activation of the apoptotic pathway for cell death. The adenovirus E3 region encodes several proteins that modify immune defenses, including TNF-dependent cell death, which may allow this virus to establish a persistent infection. Here we show that, as an early event during infection, the adenovirus E3-10.4K/14.5K complex selectively induces loss of Fas surface expression and blocks Fas-induced apoptosis of virus-infected cells. Loss of surface Fas occurs within the first 4 h postinfection and is not due to decreased production of Fas protein. The decrease in surface Fas is distinct from the 10.4K/14.5K-mediated loss of the epidermal growth factor receptor on the same cells, because intracellular stores of Fas are not affected. Further, 10.4K/14.5K, which was previously shown to protect against TNF cytolysis, does not induce a loss of TNF receptor, indicating that this complex mediates more than one function to block host defense mechanisms. These results suggest yet another mechanism by which adenovirus modulates host cytotoxic responses that may contribute to persistent infection by human adenoviruses.     

Identification of a new murine tumor necrosis factor receptor locus that contains two novel murine receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)

Tumor necrosis factor (TNF) ligand and receptor superfamily members play critical roles in diverse developmental and pathological settings. In search for novel TNF superfamily members, we identified a murine chromosomal locus that contains three new TNF receptor-related genes. Sequence alignments suggest that the ligand binding regions of these murine TNF receptor homologues, mTNFRH1, -2 and -3, are most homologous to those of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors. By using a number of in vitro ligand-receptor binding assays, we demonstrate that mTNFRH1 and -2, but not mTNFRH3, bind murine TRAIL, suggesting that they are indeed TRAIL receptors. This notion is further supported by our demonstration that both mTNFRH1:Fc and mTNFRH2:Fc fusion proteins inhibited mTRAIL-induced apoptosis of Jurkat cells. Unlike the only other known murine TRAIL receptor mTRAILR2, however, neither mTNFRH2 nor mTNFRH3 has a cytoplasmic region containing the well characterized death domain motif. Coupled with our observation that overexpression of mTNFRH1 and -2 in 293T cells neither induces apoptosis nor triggers NFkappaB activation, we propose that the mTnfrh1 and mTnfrh2 genes encode the first described murine decoy receptors for TRAIL, and we renamed them mDcTrailr1 and -r2, respectively. Interestingly, the overall sequence structures of mDcTRAILR1 and -R2 are quite distinct from those of the known human decoy TRAIL receptors, suggesting that the presence of TRAIL decoy receptors represents a more recent evolutionary event.     

FADD is required for DR4- and DR5-mediated apoptosis: lack of trail-induced apoptosis in FADD-deficient mouse embryonic fibroblasts

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a member of the tumor necrosis factor family that can kill a wide variety of tumor cells but not normal cells. TRAIL-induced apoptosis in humans is mediated by its receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2). What constitutes the signaling molecules downstream of these receptors, however, remains highly controversial. Using the FADD dominant negative molecule, several groups have reached different conclusions with respect to the role of FADD in TRAIL-induced apoptosis. More recently, using FADD-deficient (-/-) mouse embryonic fibroblasts, Yeh et al. (Yeh, W.-C., Pompa, J. L., McCurrach, M. E., Shu, H.-B., Elia, A. J., Shahinian, A., Ng, M., Wakeham, A., Khoo, W., Mitchell, K., El-Deiry, W. S., Lowe, S. W., Goeddel, D. V., and Mak, T. W. (1998) Science 279, 1954-1958) concluded that DR4 utilizes a FADD-independent apoptotic pathway. The latter experiment, however, involved transient overexpression, which often leads to nonspecific aggregation of death domain-containing receptors. To address this issue in a more physiological setting, we stably transfected mouse DR4/5, human DR4, or human DR5 into FADD(-/-) mouse embryonic fibroblast cells. We showed that FADD(-/-) MEF cells stably transfected with TRAIL receptors are resistant to TRAIL-mediated cell death. In contrast, TRAIL receptors stably transfected into heterozygous FADD(+/-) cells or FADD(-/-) cells reconstituted with a FADD retroviral construct are sensitive to the TRAIL cytotoxic effect. We conclude that FADD is required for DR4- and DR5-mediated apoptosis.     

TNF-related apoptosis-inducing ligand (TRAIL) and erythropoiesis: a role for PKC epsilon

The regulation of the hematopoietic stem cell pool size and the processes of cell differentiation along the hematopoietic lineages involve apoptosis. Among the different factors with a recognized activity on blood progenitor cells, TRAIL - a member of the TNF family of cytokines - has an emerging role in the modulation of normal hematopoiesis.PKC(epsilon) levels are regulated by EPO in differentiating erythroid progenitors and control the protection against the apoptogenic effect of TRAIL. EPO-induced erythroid CD34 cells are insensitive to the apoptogenic effect of TRAIL between day 0 and day 3, due to the lack of specific surface receptors expression. Death receptors appear after day 3 of differentiation and consequently erythroid cells become sensitive to TRAIL up to day 9/10, when the EPO-driven up-regulation of PKC epsilon intracellular levels inhibits the TRAIL-mediated apoptosis, via Bcl-2. In the time interval between day 3 and 9, therefore, the number of erythroid progenitors can be limited by the presence of soluble or membrane-bound TRAIL present in the bone marrow microenvironment.     

Roscovitine sensitizes breast cancer cells to TRAIL-induced apoptosis through a pleiotropic mechanism

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/APO2L) is a member of the TNF gene superfamily that induces apoptosis upon engagement of cognate death receptors. While TRAIL is relatively non-toxic to normal cells, it selectively induces apoptosis in many transformed cells. Nevertheless, breast tumor cells are particularly resistant to the effects of TRAIL. Here we report that, in combination with the cyclin-dependent kinase inhibitor roscovitine, exposure to TRAIL induced marked apoptosis in the majority of TRAIL-resistant breast cancer cell lines examined. Roscovitine facilitated TRAIL death-inducing signaling complex formation and the activation of caspase-8. The cFLIP(L) and cFLIP(S) FLICE-inhibitory proteins were significantly down-regulated following exposure to roscovitine and, indeed, the knockdown of cFLIP isoforms by siRNA sensitized breast tumor cells to TRAIL-induced apoptosis. In addition, we demonstrate that roscovitine strongly suppressed Mcl-1 expression and up-regulated E2F1 protein levels in breast tumor cells. Significantly, the silencing of Mcl-1 by siRNA sensitized breast tumor cells to TRAIL-induced apoptosis. Furthermore, the knockdown of E2F1 protein by siRNA reduced the sensitizing effect of roscovitine in TRAIL-induced apoptosis. In summary, our results reveal a pleitropic mechanism for the pro-apoptotic influence of roscovitine, highlighting its potential as an antitumor agent in breast cancer in combination with TRAIL.     

Snake venom toxin from Vipera lebetina turanica sensitizes cancer cells to TRAIL through ROS- and JNK-mediated upregulation of death receptors and downregulation of survival proteins

We investigated whether snake venom toxin (SVT) from Vipera lebetina turanica enhances the apoptosis ability of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in cancer cells. TRAIL inhibited HCT116 cell growth in a dose-dependent manner; however, this reduction did not occur in TRAIL resistant HT-29, A549 and HepG2 cells with an even higher dose of TRAIL. SVT, but not TRAIL enhanced expression of cell death receptor (DR) in TRAIL resistant cancer cells in a dose-dependent manner. A combination of SVT with TRAIL significantly inhibited cell growth of TRAIL resistant HT-29, A549 and HepG2 cells. Consistent with cell growth inhibition, the expression of TRAIL receptors; DR4 and DR5 was significantly increased as well as apoptosis related proteins such as cleaved caspase-3, -8, -9 and Bax. However, the expression of survival proteins (e.g., cFLIP, survivin, XIAP and Bcl2) was suppressed by the combination treatment of SVT and TRAIL. Depletion of DR4 or DR5 by small interfering RNA significantly reversed the cell growth inhibitory and apoptosis blocking effects of SVT in HCT116 and HT-29 cells. Pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the reactive oxygen species (ROS) scavenger N-acetylcysteine reduced the SVT and TRAIL-induced upregulation of DR4 and DR5 expression, expression of the apoptosis related protein such as caspase-3 and-9, as well as cell growth inhibitory effects. The collective results suggest that SVT facilitates TRAIL-induced apoptosis in cancer cells through up-regulation of the TRAIL receptors; DR4 and DR5 via ROS/JNK pathway signals.     

E3-14.7K is recruited to TNF-receptor 1 and blocks TNF cytolysis independent from interaction with optineurin

Escape from the host immune system is essential for intracellular pathogens. The adenoviral protein E3-14.7K (14.7K) is known as a general inhibitor of tumor necrosis factor (TNF)-induced apoptosis. It efficiently blocks TNF-receptor 1 (TNFR1) internalization but the underlying molecular mechanism still remains elusive. Direct interaction of 14.7K and/or associated proteins with the TNFR1 complex has been discussed although to date not proven. In our study, we provide for the first time evidence for recruitment of 14.7K and the 14.7K interacting protein optineurin to TNFR1. Various functions have been implicated for optineurin such as regulation of receptor endocytosis, vesicle trafficking, regulation of the nuclear factor κB (NF-κB) pathway and antiviral signaling. We therefore hypothesized that binding of optineurin to 14.7K and recruitment of both proteins to the TNFR1 complex is essential for protection against TNF-induced cytotoxic effects. To precisely dissect the individual role of 14.7K and optineurin, we generated and characterized a 14.7K mutant that does not confer TNF-resistance but is still able to interact with optineurin. In H1299 and KB cells expressing 14.7K wild-type protein, neither decrease in cell viability nor cleavage of caspases was observed upon stimulation with TNF. In sharp contrast, cells expressing the non-protective mutant of 14.7K displayed reduced viability and cleavage of initiator and effector caspases upon TNF treatment, indicating ongoing apoptotic cell death. Knockdown of optineurin in 14.7K expressing cells did not alter the protective effect as measured by cell viability and caspase activation. Taken together, we conclude that optineurin despite its substantial role in vesicular trafficking, endocytosis of cell surface receptors and recruitment to the TNFR1 complex is dispensable for the 14.7K-mediated protection against TNF-induced apoptosis.     

Enhanced proliferation and increased IFN-gamma production in T cells by signal transduced through TNF-related apoptosis-inducing ligand

TNF-related apoptosis-inducing ligand (TRAIL, also called Apo2L), a novel member of TNF superfamily, induces apoptosis in transformed cell lines of diverse origin. TRAIL is expressed in most of the cells, and the expression is up-regulated in activated T cells. Four receptors for TRAIL have been identified, and there is complex interplay between TRAIL and TRAIL receptors in vivo. The actual biological function of TRAIL/TRAIL receptor is still not clear. Growing evidence has demonstrated that members of TNF superfamily transduce signals after engagement with their receptors. Cross-linking of TRAIL by plate-bound rTRAIL receptor, death receptor 4-Fc fusion protein enhanced T cell proliferation and increased IFN-gamma production in conjunction with immobilized suboptimal anti-CD3 stimulation in mouse splenocytes. The increase of T cell proliferation by death receptor 4-Fc was dose dependent, and this effect could be blocked by soluble rTRAIL proteins, indicating the occurrence of reverse signaling through TRAIL on T cell. The enhanced secretion of IFN-gamma mediated via TRAIL could be blocked by SB203580, a p38 mitogen-activated protein kinase-specific inhibitor. Thus, in addition to its role in inducing apoptosis by binding to the death receptors, TRAIL itself can enhance T cell proliferation after TCR engagement and signal the augmentation of IFN-gamma secretion via a p38-dependent pathway. This provides another example of reverse signaling by a member of TNF superfamily. In conclusion, our data suggest that TRAIL can itself transduce a reverse signal, and this may shed light on the biological function of TRAIL.     

Downregulation of endogenous STAT3 augments tumoricidal activity of interleukin 15 activated dendritic cell against lymphoma and leukemia via TRAIL

Effector functions in tumor resistance by dendritic cells (DCs) are less well characterized. In this study, we describe that the murine DCs upon stimulation with recombinant IL-15 in vitro or in vivo, expresses TNF superfamily member TRAIL which mediates cytotoxicity and growth inhibition against a murine lymphoma called Dalton lymphoma (DL) via apoptosis. Presence of tumor lysate or intact tumor cells significantly reduces the DC mediated tumoricidal effect, possibly via masking and down-regulating TRAIL in DCs. The antitumor effect of DC derived TRAIL was further augmented by deactivation of STAT3 in tumor cells by cucurbitacin I, which makes it more susceptible to DC derived TRAIL Treatment of tumor cells with cucurbitacin I upregulates TRAIL receptor expression in addition to activation of caspases. Compared to naïve DCs, DCs from tumor bearing mice are significantly impaired in TRAIL expression and consequent antitumor functions against DL which was partially restored by activation with IL-15 or LPS. Priming with recombinant IL-15 prolongs the survival of tumor bearing mice treated with cucurbitacin I. Naïve peripheral blood DCs derived from chronic myeloid leukemia (CML) patients have significant impairment in expression of TRAIL and consequent tumoricidal properties against TRAIL sensitive lymphoma cell lines and primary tumor cells compared to normal control.