Differential inhibition of TRAIL-mediated DR5-DISC formation by decoy receptors 1 and 2

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family that induces cancer cell death by apoptosis with some selectivity. TRAIL-induced apoptosis is mediated by the transmembrane receptors death receptor 4 (DR4) (also known as TRAIL-R1) and DR5 (TRAIL-R2). TRAIL can also bind decoy receptor 1 (DcR1) (TRAIL-R3) and DcR2 (TRAIL-R4) that fail to induce apoptosis since they lack and have a truncated cytoplasmic death domain, respectively. In addition, DcR1 and DcR2 inhibit DR4- and DR5-mediated, TRAIL-induced apoptosis and we demonstrate here that this occurs through distinct mechanisms. While DcR1 prevents the assembly of the death-inducing signaling complex (DISC) by titrating TRAIL within lipid rafts, DcR2 is corecruited with DR5 within the DISC, where it inhibits initiator caspase activation. In addition, DcR2 prevents DR4 recruitment within the DR5 DISC. The specificity of DcR1- and DcR2-mediated TRAIL inhibition reveals an additional level of complexity for the regulation of TRAIL signaling.     

Expression of tumor necrosis factor-alpha-related apoptosis-inducing ligand and its receptors in rat testis during development

Tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor-alpha family of cytokines that is known to induce apoptosis upon binding to its death domain-containing receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2. Two additional TRAIL receptors, DcR1/TRAIL-R3 and DcR2/TRAIL-R4, lack functional death domains and act as decoy receptors for TRAIL. In this study, the presence of TRAIL and its receptors was investigated in the rat testis during development. TRAIL and its receptors were immunolocalized to the different testicular cell types. TRAIL and its receptors were also identified in the rat testis in terms of protein and mRNA. Our immunohistochemical studies indicate that TRAIL, DR5/TRAIL-R2, and DcR2-TRAIL-R4 are detected in Leydig cells, whereas ligand and all receptors are localized in germ cells. TRAIL was permanently immunodetected in germ cells from the fetal stage to adulthood, whereas its receptors were immunolocalized exclusively in postmeiotic germ cells. The expression of TRAIL and receptor mRNAs was consistent with the immunodetection of TRAIL and receptor proteins. Indeed, TRAIL ligand mRNA was also identified in the rat testis from the fetal stage to adulthood. The mRNAs of the death receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2, were weakly detected during the perinatal period and increased from the pubertal stage to adulthood. The mRNAs of the decoy receptors, DcR1 and DcR2, were present in the rat testis at all ages studied, but the DcR2/TRAIL-R4 mRNa level was higher from the pubertal period to adulthood. Together, the present findings demonstrate that 1) TRAIL and its receptors are expressed in the testis during normal development, and 2) TRAIL protein is present in the different germ cell types, whereas its receptors were predominantly detected in the postmeiotic germ cells.     

The effect of cisplatin on cytotoxicity of anticancer cytokine TRAIL and its receptor-selective mutant variant DR5-B<Superscript>1</Superscript>

Cytokine TRAIL selectively induces apoptosis in vitro and in vivo in tumor cells without affecting normal cells, but its therapeutic application is limited, since many primary tumors are insensitive to TRAIL. To improve the efficiency of TRAIL, we have previously developed TRAIL mutant variant DR5-B, which binds the apoptosis-inducing death receptor DR5 as efficiently as wild type TRAIL, but shows almost no affinity to other receptors. In this study, we investigated the effect of the chemotherapeutic agent cisplatin on the cytotoxicity of TRAIL variants in 12 tumor cell lines of various origin. Cisplatin effectively enhances the cytotoxic activity of TRAIL preparations. The synergistic effect is most pronounced in the prostate cancer cell lines, where the combined effect exceeds the sum of the separate effects by more than 2 times. The cytotoxicity of DR5-B variant is significantly higher compared to wild-type TRAIL in combination with cisplatin in 9 of 12 tumor cell lines.     

TNF-alpha-related apoptosis-inducing ligand decoy receptor DcR2 is targeted by androgen action in the rat ventral prostate

The apoptotic cell death process in the prostate is known to be under the control of androgens. Tumor necrosis factor-alpha (TNF-alpha)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF-alpha family of cytokines, known to induce apoptosis upon binding to its death domain-containing receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2. Two additional TRAIL receptors, DcR1/TRAIL-R3 and DcR2/TRAIL-R4, lack functional death domains and act as decoy receptors for TRAIL. In this study, we examined whether TRAIL and cellular receptors expression was targeted by androgens during the apoptotic cell death process in the hormone sensitive ventral prostate. The role of androgens was investigated using two sets of experiment. (1) Androgen deprivation associated with an apoptotic process resulted in a decrease in DcR2 mRNA and protein expression in the ventral prostate 3 days after castration. Testosterone administration to castrated adult rats prevented the decrease in DcR2 mRNA and protein levels in the ventral prostate. In contrast, DcR2 expression was modified, neither in the dorsolateral nor in the anterior prostate following castration. No changes were observed in DR4, DR5, DcR1, and TRAIL mRNA and protein levels in prostate after castration. (2) A specific decrease in DcR2 expression was observed in the ventral prostate after treatment of rats with the anti-androgen flutamide. Together, the present results suggest that testosterone specifically controls DcR2 expression in the adult rat ventral prostate. Androgen withdrawal, by reducing DcR2 expression, might leave the cells vulnerable to cell death signals generated by TRAIL via its functional receptors.     

Arf and Rho GAP adapter protein ARAP1 participates in the mobilization of TRAIL-R1/DR4 to the plasma membrane

TRAIL, a ligand of the TNFα family, induces upon binding to its pro-death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 the apoptosis of cancer cells. Activated receptors incite the formation of the Death-Inducing Signaling Complex followed by the activation of the downstream apoptotic signaling. TRAIL-induced apoptosis is regulated at multiple levels, one of them being the presence and relative number of TRAIL pro- and anti-apoptotic receptors on the cytoplasmic membrane. In a yeast two-hybrid search for proteins that interact with the intracellular part (ICP) of DR4, we picked ARAP1, an adapter protein with ArfGAP and RhoGAP activities. In yeast, DR4(ICP) interacts with the alternatively spliced ARAP1 lacking 11 amino acids from the PH5 domain. Transfected ARAP1 co-precipitates with DR4 and co-localizes with it in the endoplasmic reticulum/Golgi, at the cytoplasmic membrane and in early endosomes of TRAIL-treated cells. ARAP1 knockdown significantly compromises the localization of DR4 at the cell surface of several tumor cell lines and slows down their TRAIL-induced death. ARAP1 overexpressed in HEL cells does not affect their TRAIL-induced apoptosis or the membrane localization of DR4, but it enhances the cell-surface presentation of phosphatidyl serine. Our data indicate that ARAP1 is likely involved in the regulation of the cell-specific trafficking of DR4 and might thus affect the efficacy of TRAIL-induced apoptosis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

TRAIL pathway components and their putative role in granulosa cell apoptosis in the human ovary

Extensive apoptotic oocyte reduction occurs during fetal ovarian development. The regulatory pathways responsible for oocyte selection to programmed cell death are, however, poorly understood. The aim of this study was to investigate the potential involvement of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 and decoy receptors TRAIL-R3/DcR1 and TRAIL-R4/DcR2 in the apoptotic process characterizing human fetal and adult ovaries. For this purpose, in situ hybridization and immunohistochemistry were applied to human fetal and adult ovarian samples to study the mRNA and protein expression of TRAIL pathway components, and a human granulosa cell tumor-derived cell line (KGN) was used to elucidate functional effects of TRAIL on apoptosis. TRAIL was expressed in human fetal ovary from the 11th week until term. The pro-apoptotic TRAIL-R2/DR5 and the anti-apoptotic TRAIL-R4/DcR2 were also expressed in human ovaries throughout the fetal period. Among the different ovarian cell types, these TRAIL pathway components were mainly localized in the oocytes, and their expression increased towards term. Expression of TRAIL-R1/DR4 and TRAIL-R3/DcR1 was negligible in all of the fetal ovaries studied. Adult ovaries expressed TRAIL, TRAIL-R2/DR5, TRAIL-R3/DcR1 and TRAIL-R4/DcR2 in granulosa cells and oocytes of small primary/secondary follicles as well as in granulosa and theca cells of more developed antral follicles. In KGN cells, TRAIL efficiently induced apoptosis in a dose-dependent manner, and this was blocked by a caspase inhibitor. The results indicate a role of the TRAIL pathway components in the regulation of granulosa cell apoptosis in in vitro and suggest that these factors may have a role in regulating ovarian apoptosis also in vivo.     

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.     

Curcumin enhances the apoptosis-inducing potential of TRAIL in prostate cancer cells: molecular mechanisms of apoptosis, migration and angiogenesis

Background

We have recently shown that curcumin (a diferuloylmethane) inhibits growth and induces apoptosis, and also demonstrated that TRAIL induces apoptosis by binding to specific cell surface death receptors in prostate cancer cells. The objectives of this paper were to investigate the molecular mechanisms by which curcumin enhanced the apoptosis-inducing potential of TRAIL in prostate cancer cells.

Results

Curcumin enhanced the apoptosis-inducing potential of TRAIL in androgen-unresponsive PC-3 cells and sensitized androgen-responsive TRAIL-resistant LNCaP cells. Curcumin inhibited the expressions of Bcl-2, Bcl-X_L, survivin and XIAP, and induced the expressions Bax, Bak, PUMA, Bim, and Noxa and death receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5) in both cell lines. Overexpression of dominant negative FADD inhibited the interactive effects of curcumin and TRAIL on apoptosis. Treatment of these cells with curcumin resulted in activation of caspase-3, and caspase-9, and drop in mitochondrial membrane potential, and these events were further enhanced when combined with TRAIL. Curcumin inhibited capillary tube formation and migration of HUVEC cells and these effects were further enhanced in the presence of MEK1/2 inhibitor PD98059.

Conclusion

The ability of curcumin to inhibit capillary tube formation and cell migration, and enhance the therapeutic potential of TRAIL suggests that curcumin alone or in combination with TRAIL can be used for prostate cancer prevention and/or therapy.     

TRAIL induces apoptosis but not necroptosis in colorectal and pancreatic cancer cells preferentially via the TRAIL-R2/DR5 receptor

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine that can trigger apoptosis in many types of human cancer cells via engagement of its two pro-apoptotic receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). TRAIL can also activate several other signaling pathways such as activation of stress kinases, canonical NF-κB signaling and necroptosis. Though both receptors are ubiquitously expressed, their relative participation in TRAIL-induced signaling is still largely unknown. To analyze TRAIL receptor-specific signaling, we prepared Strep-tagged, trimerized variants of recombinant human TRAIL with high affinity for either DR4 or DR5 receptor. Using these receptor-specific ligands, we examined the contribution of individual pro-apoptotic receptors to TRAIL-induced signaling pathways. We found that in TRAIL-resistant colorectal HT-29 cells but not in pancreatic PANC-1 cancer cells, DISC formation and initial caspase-8 processing proceeds comparably via both DR4- and DR5-activated signaling. TRAIL-induced apoptosis, enhanced by the inhibitor of the Bcl-2 family ABT-737, or by the translation inhibitor homoharringtonine, proceeded in both cell lines predominantly via the DR5 receptor. ShRNA-mediated downregulation of DR4 or DR5 receptors in HT-29 cells also pointed to a stronger contribution of DR5 in TRAIL-induced apoptosis. In contrast to apoptosis, necroptotic signaling was activated similarly by both DR4- or DR5-specific ligands. Activation of auxiliary signaling pathways involving NF-κB or stress kinases proceeded under apoptotic conditions mainly in a DR5-dependent manner, while these signaling pathways were during necroptosis similarly activated by either of these ligands. Our study provides the first systematic insight into DR4 ?/DR5-specific signaling in colorectal and pancreatic cancer cells.     

Regulation in the targeting of TRAIL receptor 1 to cell surface via GODZ for TRAIL sensitivity in tumor cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5), promote the selective clearing of various malignancies by inducing apoptosis, holding the promise as a potent therapeutic agent for anticancer. Though DR4 and DR5 have high sequence similarity, differential regulation of both receptors in human tumor cells remains largely unexplored. Here, we repot that golgi-specific Asp-His-His-Cys (DHHC) zinc finger protein (GODZ) regulates TRAIL/DR4-mediated apoptosis. Using the SOS protein recruitment-yeast two-hybrid screening, we isolated GODZ that interacted with the death domain of DR4. GODZ binds to DR4, but not to DR5, through the DHHC and the C-terminal transmembrane domain. Expression level of GODZ affects apoptosis of tumor cells triggered by TRAIL, but not that induced by TNF-α/cycloheximide (CHX) or DNA-damaging drugs. In parallel, GODZ functions to localize DR4 to the plasma membrane (PM) via DHHC motif. Also, introduction of mutation into the cysteine-rich motif of DR4 results in its mistargeting and attenuates TRAIL- or GODZ-mediated apoptosis. Interestingly, GODZ expression is highly downregulated in Hep-3B tumor cells, which show resistance to TRAIL. However, reconstitution of GODZ expression enhances the targeting of DR4 to cell surface and sensitizes Hep-3B cells to TRAIL. Taken together, these data establish that GODZ is a novel DR4-selective regulator responsible for targeting of DR4 to the PM, and thereby for TRAIL-induced apoptosis.     

Sensitivity to TRAIL/APO-2L-mediated apoptosis in human renal cell carcinomas and its enhancement by topotecan

TRAIL (APO-2L) is a newly identified member of the TNF family and induces apoptosis in cancer cells without affecting most non-neoplastic cells, both in vitro and in vivo. Our study focused on the expression and function of TRAIL and its receptors in renal cell carcinoma (RCC) cell lines of all major histological types. Here, we demonstrate that all RCC cell lines express TRAIL as well as the death-inducing receptors TRAIL-R1 (DR4) and TRAIL-R2 (Killer/DR5). Exposure to TRAIL induced apoptosis in 10 of 16 RCC cell lines. Remarkably, five of six TRAIL-resistant RCC cell lines exhibited high levels of TRAIL expression. Topotecan, a novel topoisomerase I inhibitor, induced upregulation of TRAIL-R2 as well as downregulation of TRAIL. Neutralization of TRAIL with recombinant soluble TRAIL-R1-Fc and TRAIL-R2-Fc failed to inhibit topotecan-induced apoptosis indicating that topotecan-induced cell death can occur in a TRAIL-independent fashion. However, exposure to topotecan resulted in an enhancement of TRAIL-induced apoptosis in all primarily TRAIL-resistant RCC cell lines. This synergistic effect of cotreatment with Topotecan and TRAIL may provide the basis for a new therapeutic approach to induce apoptosis in otherwise unresponsive RCC.     

Direct binding of Fas-associated death domain (FADD) to the tumor necrosis factor-related apoptosis-inducing ligand receptor DR5 is regulated by the death effector domain of FADD

Members of the tumor necrosis factor superfamily of receptors induce apoptosis by recruiting adaptor molecules through death domain interactions. The central adaptor molecule for these receptors is the death domain-containing protein Fas-associated death domain (FADD). FADD binds a death domain on a receptor or additional adaptor and recruits caspases to the activated receptor. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signals apoptosis through two receptors, DR4 and DR5. Although there is much interest in TRAIL, the mechanism by which FADD is recruited to the TRAIL receptors is not clear. Using a reverse two-hybrid system we previously identified mutations in the death effector domain of FADD that prevented binding to Fas/CD95. Here we show that these mutations also prevent binding to DR5. FADD-deficient Jurkat cells stably expressing these FADD mutations did not transduce TRAIL or Fas/CD95 signaling. Second site compensating mutations that restore binding to and signaling through Fas/CD95 and DR5 were also in the death effector domain. We conclude that in contrast to current models where the death domain of FADD functions independently of the death effector domain, the death effector domain of FADD comes into direct contact with both TRAIL and Fas/CD95 receptors.     

Fas-associated protein with death domain (FADD)-independent recruitment of c-FLIPL to death receptor 5

Here we show a novel mechanism by which FLICE-like inhibitory protein (c-FLIP) regulates apoptosis induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and one of its receptors, DR5. c-FLIP is a critical regulator of the TNF family of cytokine receptor signaling. c-FLIP has been postulated to prevent formation of the competent death-inducing signaling complex (DISC) in a ligand-dependent manner, through its interaction with FADD and/or caspase-8. In order to identify regulators of TRAIL function, we used the intracellular death domain (DD) of DR5 as a target to screen a phage-displayed combinatorial peptide library. The DD of DR5 selected from the library a peptide that showed sequence similarity to a stretch of amino acids in the C terminus of c-FLIP(L). The phage-displayed peptide selectively interacted with the DD of DR5 in in vitro binding assays. Similarly, full-length c-FLIP (c-FLIP(L)) and the C-terminal p12 domain of c-FLIP interacted with DR5 both in in vitro pull-down assays and in mammalian cells. This interaction was independent of TRAIL. To the contrary, TRAIL treatment released c-FLIP(L) from DR5, permitting the recruitment of FADD to the active DR5 signaling complex. By employing FADD-deficient Jurkat cells, we demonstrate that DR5 and c-FLIP(L) interact in a FADD-independent manner. Moreover, we show that a cellular membrane permeable version of the peptide corresponding to the DR5 binding domain of c-FLIP induces apoptosis in mammalian cells. Taken together, these findings indicate that c-FLIP interacts with the DD of DR5, thus preventing death (L)signaling by DR5 prior to the formation of an active DISC. Because TRAIL and DR5 are ubiquitously expressed, the interaction of c-FLIP(L) and DR5 indicates a mechanism by which tumor selective apoptosis can be achieved through protecting normal cells from undergoing death receptor-induced apoptosis.     

TRAIL-R2 (DR5) mediates apoptosis of synovial fibroblasts in rheumatoid arthritis

TRAIL has been proposed as an anti-inflammatory cytokine in animal models of rheumatoid arthritis (RA). Using two agonistic mAbs specific for TRAIL-R1 (DR4) and TRAIL-R2 (DR5), we examined the expression and function of these death receptors in RA synovial fibroblast cells. The synovial tissues and primary synovial fibroblast cells isolated from patients with RA, but not those isolated from patients with osteoarthritis, selectively expressed high levels of cell surface DR5 and were highly susceptible to anti-DR5 Ab (TRA-8)-mediated apoptosis. In contrast, RA synoviocytes did not show increased expression of TRAIL-R1 (DR4), nor was there any difference in expression of Fas between RA and osteoarthritis synovial cells. In vitro TRA-8 induced apoptosis of RA synovial cells and inhibited production of matrix metalloproteinases induced by pro-inflammatory cytokines. In vivo TRA-8 effectively inhibited hypercellularity of a SV40-transformed RA synovial cell line and completely prevented bone erosion and cartilage destruction induced by these cells. These results indicate that increased DR5 expression and susceptibility to DR5-mediated apoptosis are characteristic of the proliferating synovial cells in RA. As highly proliferative transformed-appearing RA synovial cells play a crucial role in bone erosion and cartilage destruction in RA, the specific targeting of DR5 on RA synovial cells with an agonistic anti-DR5 Ab may be a potential therapy for RA.     

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.     

TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL.

TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines and induces apoptosis in a wide variety of cells. Based on homology searching of a private database, a receptor for TRAIL (DR4 or TRAIL-R1) was recently identified. Here we report the identification of a distinct receptor for TRAIL, TRAIL-R2, by ligand-based affinity purification and subsequent molecular cloning. TRAIL-R2 was purified independently as the only receptor for TRAIL detectable on the surface of two different human cell lines that undergo apoptosis upon stimulation with TRAIL. TRAIL-R2 contains two extracellular cysteine-rich repeats, typical for TNF receptor (TNFR) family members, and a cytoplasmic death domain. TRAIL binds to recombinant cell-surface-expressed TRAIL-R2, and TRAIL-induced apoptosis is inhibited by a TRAIL-R2-Fc fusion protein. TRAIL-R2 mRNA is widely expressed and the gene encoding TRAIL-R2 is located on human chromosome 8p22-21. Like TRAIL-R1, TRAIL-R2 engages a caspase-dependent apoptotic pathway but, in contrast to TRAIL-R1, TRAIL-R2 mediates apoptosis via the intracellular adaptor molecule FADD/MORT1. The existence of two distinct receptors for the same ligand suggests an unexpected complexity to TRAIL biology, reminiscent of dual receptors for TNF, the canonical member of this family.