TRAIL Receptor 1/2 (Death Receptor 4/5, DR4/5)
The tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL, Apo2L) identified in the middle of 1990s (Pitti et al. 1996; Wiley et al. 1995) was given its name due to its ability of inducing apoptosis and its high homology to other TNF member ligands. Then, its first receptor, death receptor 4 (DR4, TRAIL-R1), was identified by searching an EST database for sequences related to the TNF receptor-1 (TNFR1) death domain (DD) (Pan et al. 1997b). Thereafter, by virtue of its sequence homology to the DR4 DD, death receptor 5 (DR5, TRAIL-R2) was also cloned independently by two groups (MacFarlane et al. 1997; Walczak et al. 1997). Both DR4 and DR5 are belonging to members of the TNF receptor superfamily (TNFRSF). However, unlikely with TNFRSF receptors containing the intracellular DD, such as TNFR1 and CD95, DR4 and DR5 are capable of transmitting tumor cell-selective apoptosis signaling upon activation by ligation with the cognate ligand of TRAIL (Ashkenazi 2008; Johnstone et al. 2008). DR4 and DR5 share a sequence identity of 58%, and their fucntions are largely redundant. However, recent studies with the receptor-specific monoclonal antibodies (mAbs) and TRAIL mutants have shown that some tumor cells undergo apoptotic cell death through signaling by either pro-apoptotic DR4 or DR5, but not both (Sung et al. 2009).
The two additional membrane receptors, decoy receptor 1 (DcR1/TRAIL-R3/TNFRSF10C/TRID) and decoy receptor 2 (DcR2/TRAIL-R4/TNFRSF10D/TRUNDD), were also identified by sequence homology search with the extracellular domain of DR4 and DR5 (MacFarlane et al. 1997; Marsters et al. 1997; Pan et al. 1997a). DcR1 completely lacks the intracellular DD and is anchored to the membrane via a glycophosphatidyl inositol (GPI) tail, whereas DcR2 is a type I transmembrane protein, but contains a truncated DD (Marsters et al. 1997). Due to the lack of functional DD, the two decoy receptors are not capable of inducing apoptosis. However, they may protect cells against TRAIL-mediated apoptosis by competing with DR4 and DR5 for binding to the ligand (Ashkenazi 2008; Johnstone et al. 2008). In addition to these four cell-surface expressed receptors, a soluble receptor called osteoprotegerin (OPG) was identified to bind to TRAIL with low affinity (Emery et al. 1998).
Structural Features of DR4 and DR5
The extracellular domain DR5 protein forms a compact 3:3 complex with TRAIL, where TRAIL forms a central homotrimer around which three DR5 molecules snuggled into long crevices between pairs of monomers of the homotrimeric ligand (Cha et al. 2000). There has been no report yet about tertiary structures of DR4 alone or in complex with TRAIL.
Cell and Tissue Expression of DR4 and DR5
Cellular surface expression of DR4 and DR5 has been reported for a broad spectrum of cancer lines and/or primary tumor cells isolated from tissues, including lung, blood, skin, bone, breast, ovary, thyroid, and the brain (Fox et al. 2010; Johnstone et al. 2008). Weak, but detectable DR4 and/or DR5 expression has been identified by flow cytometry on the surface of normal cell types, including hepatocytes, keratinocytes, astrocytes, and osteoblasts (Fox et al. 2010; Johnstone et al. 2008). Noticeably, however, there has been no definite correlation between expression levels of DR4 and/or DR5 at both the mRNA and protein level, and the susceptibility to apoptosis by the agonist treatments (Ashkenazi 2008; Fox et al. 2010; Johnstone et al. 2008). Although most tumor cells co-express DR4 and DR5 to some degree, DR5 is expressed more widely by both tumor cells and normal tissue than DR4 (Johnstone et al. 2008). A specific physiological role for either DR4 or DR5 has yet to be defined, although the existence of two death receptors for the same ligand might suggest an essential role in tissue homeostasis.
DR4- and DR5-Mediated Signaling
Caspase-Dependent Apoptosis Signaling
Binding of trimeric or multimeric TRAIL to the pro-apoptotic receptors results in the receptor clustering to activate the intracellular DD and recruit Fas-associated death domain (FADD) and then the initiator caspases, caspase-8 and/or -10, similarly to what occurs when Fas (CD95) is activated (reviewed in Gonzalvez and Ashkenazi (2010)). This primary signaling complex, consisting of ligand, receptor, FADD, and apical caspase(s), is called the death-inducing signaling complex (DISC) (Gonzalvez and Ashkenazi 2010). Activated caspase-8/10 by the DISC formation triggers the extrinsic pathway by directly activating the downstream effector caspases, such as caspase-3, -6, and -7, which in turn cleave many cellular substrates to exert apoptosis (Fig. 3). The extrinsic pathway can be amplified by cross-talk with the intrinsic pathway, the link of which is Bid cleavage by activated caspase-8 (Ashkenazi 2008; Johnstone et al. 2008). Cleaved Bid (tBid) induces oligomerisation of pro-apoptotic Bax and/or Bak, leading to the release of cytochrome c and Smac/Diablo from mitochondria into cytoplasm and activation of caspase-9 (Ashkenazi 2008; Johnstone et al. 2008). The mitochondrial pathway eventually activates the effector caspases to execute apoptotic cell death. In some cells (designated Type I cells), caspase-8 activation is sufficient to activate the effector caspases to execute apoptosis via the extrinsic pathway, whereas, in other cells (designated Type II cells), amplification of the extrinsic pathway through the intrinsic (mitochondrial) pathway is needed to commit the cells to apoptotic cell death (Pennarun et al. 2010; Sung et al. 2009).
Activation of the initiator caspase-8/10 at DISC can be inhibited, subsequently decreasing the apoptosis signaling through the extrinsic and/or intrinsic pathways. The anti-apoptotic protein of cellular FLICE-inhibitory protein (c-FLIP) can also be recruited to the DISC to replace caspase-8, forming inactive DISC to inhibit the following downstream signaling. Furthermore, DcR1 and DcR2 might form ineffective DISC via heteromeric complex formation with DR4 and/DR5, leading to blocking the following apoptosis signaling (Fox et al. 2010; Pennarun et al. 2010).
In addition to the cognate ligand of recombinant TRAIL, a number of agonistic monoclonal antibodies (mAbs) have been developed by targeting DR4 or DR5, which also induce cell death in various types of tumors in vitro and in vivo (reviewed in Ashkenazi 2008; Fox et al. 2010; Johnstone et al. 2008). More recently, alternative protein scaffold based on human Kringle domain (Lee et al. 2010) and peptides (Pavet et al. 2010) isolated against DR4 or DR5 have been reported to specifically bind to activate DR4 and/or DR5, leading to tumor cell-selective apoptotic cell death. Some of them are now under various phases of clinical trials (Fox et al. 2010; Johnstone et al. 2008). The cell death mechanism of most agonistic mAbs against DR4 or DR5 has been reported to be similar to that of TRAIL, inducing caspase-dependent apoptotic cell death through forming the canonical DISC in the intracellular domain of DR4 and/or DR5 (Ashkenazi 2008).
The receptor-specific agonistic mAb studies demonstrated that, while various solid tumors were more sensitive to DR5-mediated apoptosis than DR4-induced apoptosis, primary chronic lymphocytic leukemia (CLL) cells underwent apoptotic cell death almost exclusively through DR4, not DR5 (MacFarlane et al. 2005), indicative of distinct functions of DR4 and DR5 depending on the specific type.
Kinase Activation Signaling
In addition to caspase activation by forming the canonical DISC, TRAIL stimulation of DR4 and/or DR5 can activate several kinase pathways, including nuclear factor (NF)-κB (NF-κB), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK), either by forming a secondary signaling complex in the downstream of DISC or by forming different signaling complex at the intracellular domain (Gonzalvez and Ashkenazi 2010; Pennarun et al. 2010).
Mühlenbeck et al. (2000) showed that TRAIL stimulation can lead to JNK activation, which occurs in a cell type-specific manner. TRAIL-induced JNK activation was dependent on caspase activation in HeLa cells, whereas it was not in Kym-1 cells, indicative of the two independent pathways leading to JNK activation. Further, JNK activation was independent of FADD in HeLa cells, suggesting another adaptor molecule to DR4/DR5 might be involved in the JNK activation. The group also showed that DR4 and DR5 have different capabilities for stimulating the JNK pathway: DR4 can signal NF-κB activation and apoptosis, whereas DR5 can signal NF-κB activation, apoptosis, and JNK activation (Muhlenbeck et al. 2000). However, detailed studies to analyze the molecular components involved in the signaling pathway had not been done.
Lin et al. (2000) had shown that RIP1 was essential for TRAIL-induced NF-κB and JNK activation, but not required for the apoptosis. Askenazi group (Varfolomeev et al. 2005) had shown in detail that TRAIL stimulation in HT1080 human fibrosarcoma cells leads to activations of kinase pathways by promoting the association of a secondary signaling complex II at the downstream of the primary DISC assembly (Fig. 3). The signaling complex II retained the DISC components FADD and caspase-8, but recruited several additional factors, such as Receptor interacting protein 1 (RIP1), TNF receptor-associated factor 2 (TRAF2), and I-κB kinase subunit gamma (IKKγ/NEMO). TRAIL stimulation of JNK and p38 further depended on receptor interacting protein 1 (RIP1) and TRAF2, whereas the inhibitor of κB kinase (IKK) activation required IKKγ/NEMO. More recently, Jin and El-Deiry (2006) showed that TRAIL can induce formation of a complex II containing FADD, RIP, IKKα, and caspase-8 and -10, leading to activation of caspase-8. Thus they showed that TRADD and caspase-10 can be recruited to the signaling complex II.
Secchiero et al. (2003) also showed that TRAIL could activate the protein kinase B (PKB/Akt) and extracellular signal-regulated kinase 1//2 (ERK1/2), without NF-κB, p38 and JNK activations in primary human umbilical vein endothelial cells (HUVECs) and aortic endothelial cells, thereby promoting the cell survival. However, the detailed molecular mechanisms are not completely understood.
Ohtsuka et al. (2003) also reported that, using agonistic anti-DR4 2E12 and anti-DR5 TRA-8 mAbs, both DR4 and DR5 have a capability to activate JNK and p38, which was mediated by MAPK kinase 4 (MKK4). The JNK/p38 signaling activated the mitochondrial apoptosis pathways, leading to caspase-dependent apoptotic cell death of breast MDA-MB-231 cell lines. However, they did not examine the signaling complex recruited to each receptor after mAb stimulation. Sah et al. (2003) reported that JNK activation is required for sensitization of prostate PC3 cells to TRAIL-induced apoptosis by translation inhibitors in cells that are otherwise TRAIL-resistant.
Zheng group (Chen et al. 2009) showed that, while TRAIL induces only caspase-dependent cell death, an anti-DR5 agonistic mAb, AD5-10, induces both caspase-dependent and caspase-independent cell death in Jurkat cells. AD5-10-mediated stimulation of DR5 generated reactive oxygen species (ROS) accumulation, which subsequently evoked sustained activation of JNK, loss of mitochondrial membrane potential, and release of apoptosis-inducing factor (AIF) and endonuclease G (Endo G) from mitochondria into the cytosol and then nucleus in Jurkat cells (Fig. 4; Chen et al. 2009). Immunoprecipitation experiments showed that AD5-10 induced assembly of DISCs containing DR5, FADD, caspase-8, and RIP in wild-type Jurkat cells. Moreover, a dominant-negative form of JNK enhanced NF-κB activation, suppressed caspase-8 recruitment in DISCs. However, how DR5 stimulation by AD5-10 induced ROS accumulation and how JNK activation impairs the function of mitochondria remain to be investigated.
Caspase-Independent Autophagy Signaling
Autophagic cell death has been involved in physiological cell death during development and reported in cancer cells treated with chemicals or irradiation. TRAIL also can induce caspase-independent autophagic cell death in normal epithelial cells and the breast cancer cell line MCF-10A (Mills et al. 2004), implicating that DR4 and/or DR5 are involved in the autophagic cell death as well as apoptosis. Park et al. (2007) recently reported that an agonistic antibody, single chain variable fragment (scFv) HW1, which specifically binds to DR5, triggered autophagic cell death of cancer cells dominantly through JNK pathway in a caspase-independent manner (Fig. 4). Analysis of the signaling complex induced by HW1 binding to DR5 exhibits the recruitment of TNF receptor (TNFR)-associated death domain (TRADD) and TRAF2 to the receptor, but not FADD, caspase-8, or RIP, which is distinct from the canonical DISC induced by TRAIL. Analyses of upstream kinase(s) for JNK activation upon HW1 binding to DR5 revealed that JNK activation was most likely mediated by TRAF2-MKK4/MKK7-dependent signaling cascade (Park et al. 2009). DR5-stimulated JNK activation by HW1 resulted in upregulation of Beclin-1 expression, Bcl-2 phosphorylation, and p53 phosphorylation, suggesting that these pro-autophagic signaling pathways are involved in the autophagic cell death (Park et al. 2009). The DR5-mediated autophagy signaling sensitized both TRAIL-sensitive and TRAIL-resistant tumor cells with much less cytotoxicity on normal cells (Park et al. 2007, 2009), providing a new strategy for the elimination of cancer cells through the nonapoptotic cell death.
Resistance to DR4- and DR5-Mediated Apoptotic Cell Death Signaling
Many highly malignant tumor cells (>50%) even expressing DR4 and/or DR5 remain resistant to TRAIL-induced and/or anti-DR4/DR5 agonistic mAb-mediated apoptosis, the underlying mechanism of which has been poorly understood and varies with the cellular context (Ashkenazi 2008; Fox et al. 2010; Johnstone et al. 2008). Potential resistance mechanisms are numerous and include loss/mutation of death receptors, overexpression of decoy receptors, overexpression of cellular FLICE inhibitory protein (c-FLIP), absence of proper O-glycosylation in the receptors, and/or complex downstream regulation of the extrinsic and intrinsic apoptotic pathways (Gonzalvez and Ashkenazi 2010).
The agonists against DR4 and/or DR5, including recombinant TRAIL and mAbs, have demonstrated anti-cancer activities both as monotherapy and in combination with anti-cancer agents in preclinical and clinical studies with no apparent systemic toxicity (Ashkenazi 2008; Fox et al. 2010; Johnstone et al. 2008). All of the receptor agonists in clinic have been reported to induce tumor cell death through the caspase-dependent apoptosis (Ashkenazi 2008; Johnstone et al. 2008). Depending on the external stimuli and specific cell types, however, DR4 and/or DR5 can also transduce multiple cellular signaling pathways in a caspase-dependent or caspase-independent manner, including JNK, p38, and NF-?B signaling. These distinct signaling can exert ROS-mediated apoptosis or autophagic cell death, although the components of which remain largely undefined. Recent clinical data of TRAIL and agonistic mAbs have shown much less tumor killing activities in patients than those observed in preclinical studies. Accordingly, deep understanding of the diverse molecular signaling mechanisms mediated by DR4 and/or DR5 and their cross-talks leading to a final outcome of the stimulation will be essential to design next-generation agonists targeting DR4 and/or DR5.
- Muhlenbeck F, Schneider P, Bodmer JL, Schwenzer R, Hauser A, Schubert G, et al. The tumor necrosis factor-related apoptosis-inducing ligand receptors TRAIL-R1 and TRAIL-R2 have distinct cross-linking requirements for initiation of apoptosis and are non-redundant in JNK activation. J Biol Chem. 2000;275:32208–13.PubMedCrossRefGoogle Scholar
- Sah NK, Munshi A, Kurland JF, McDonnell TJ, Su B, Meyn RE. Translation inhibitors sensitize prostate cancer cells to apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by activating c-Jun N-terminal kinase. J Biol Chem. 2003;278:20593–602.PubMedCrossRefGoogle Scholar