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,¶,1
Departments of
Physiology and
* Community Occupational and Family Medicine, Yong Loo Lin School of Medicine, and
¶ NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore
1Correspondence: Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Bldg. MD9, Level 3, Singapore 117597, Singapore. E-mail: phssp{at}nus.edu.sg
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ABSTRACT |
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 TOP ABSTRACT ROS AND OXIDATIVE STRESSTNF AND TNFR SUPERFAMILYEFFECT OF ROS ON...ROS AND TNF-INDUCED NF-{kappa}B...ROS AS THE KEY...ROS IN TNFR1-MEDIATED APOPTOTIC...ROS IN DEATH RECEPTOR-MEDIATED...ROS IN CD95(Fas/Apo1)-MEDIATED...CONCLUDING REMARKSREFERENCES |
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is the founding member of the 19 different proteins that have so far been identified within this family. TNF family members exert their biological effects through the TNF receptor (TNFR) superfamily of cell surface receptors that share a stretch of 
80 amino acids within their cytoplasmic region, the death domain (DD), critical for recruiting the death machinery. Work over the last decade has unraveled critical signaling networks involved in TNFR-induced cell death, specifically using the constitutively expressed TNFR1 as a prototype. Of particular interest is the intermediary role of intracellular reactive oxygen species (ROS) in signal transduction after ligation of the TNFR1. With the increasing understanding of the of death receptor signaling pathways, the exact role of ROS in TNF-induced execution is now believed to be far more complicated than originally thought. Recently, some important discoveries have underscored the critical role of ROS in TNF signaling, notably in TNF-mediated activation of nuclear factor-
B (NF-B) and c-Jun N-terminal kinase (c-Jun NH2-terminal kinase, JNK), as well as in cell death (apoptotic and necrotic) pathways. Here we attempt to review the existing knowledge on the involvement of ROS in death receptor signaling using TNF-TNFR1 as the model system, specifically addressing the involvement of intracellular ROS in TNF-induced cell death and in TNF-induced activation of NF-B and JNK and their crosstalk.—Shen, H-M., Pervaiz, S. TNF receptor superfamily-induced cell death: redox-dependent execution.
Key Words: oxidative stress • reactive oxygen species • death receptor • TRAF
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ROS AND OXIDATIVE STRESS |
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TOPABSTRACTROS AND OXIDATIVE STRESSTNF AND TNFR SUPERFAMILYEFFECT OF ROS ON...ROS AND TNF-INDUCED NF-{kappa}B...ROS AS THE KEY...ROS IN TNFR1-MEDIATED APOPTOTIC...ROS IN DEATH RECEPTOR-MEDIATED...ROS IN CD95(Fas/Apo1)-MEDIATED...CONCLUDING REMARKSREFERENCES |
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, 2)
. ROS usually include superoxide anion (O2.–), hydrogen peroxide (H2O2), and the highly reactive by-product of H2O2, hydroxyl radicals (.OH), that are capable of reacting with and damaging DNA, proteins, and lipids. Aside from the damaging activity of ROS, low levels of intracellular ROS have also been identified as second messengers involved in a variety of signaling pathways and serve as transcription regulators (3
, 4)
. These diverse activities of intracellular ROS in initiating and/or amplifying death signals or in the regulation of apoptosis have been well studied and extensively reviewed (5
6
7
8
9)
. More recent data seem to suggest that O2.– may affect pathways involved in cell death and proliferation in a way distinct from H2O2 (10
11
12
13
14
15
16
17)
.
Of particular interest is the involvement of ROS in death receptor-initiated signaling pathways, specifically in tumor necrosis factor alpha-tumor necrosis factor receptor 1 (TNF-TNFR1) pathway. With the increasing understanding of the intracellular death circuitry initiated by TNF and death receptors, the exact role of ROS in TNF signaling pathway is now believed to be far more complicated than originally thought. Recently, some important discoveries have further highlighted the critical role of ROS in TNF signaling, notably in TNF-mediated activation of NF-B and JNK, as well as in cell death (apoptotic and necrotic) pathways.
In this review, using TNF-TNFR1 as the model system, we attempt to address the role of ROS and oxidative stress in cell death triggered upon ligation of the death receptors. The focus will be on the 1) involvement of intracellular ROS in TNF-induced cell death, including both apoptosis and necrosis, and 2) intermediary role of ROS in TNF-induced activation of NF-B and JNK and their crosstalk. A brief discussion on the involvement of ROS in cell death induced by other death receptor ligands, such as FasL (CD95L/Apo1L) and TNF-related apoptosis-inducing ligand (TRAIL), is also included.
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TNF AND TNFR SUPERFAMILY |
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TOPABSTRACTROS AND OXIDATIVE STRESSTNF AND TNFR SUPERFAMILYEFFECT OF ROS ON...ROS AND TNF-INDUCED NF-{kappa}B...ROS AS THE KEY...ROS IN TNFR1-MEDIATED APOPTOTIC...ROS IN DEATH RECEPTOR-MEDIATED...ROS IN CD95(Fas/Apo1)-MEDIATED...CONCLUDING REMARKSREFERENCES |
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, 19)
. TNF is the founding member of the 19 different proteins so far identified within this family. Other important signaling molecules include CD95L/Apo1L/FasL, TRAIL, and lymphotoxin. TNF family members exert their biological effects through the TNFR superfamily of cell surface receptors (18
, 20)
. Some of these receptors share a stretch of 80 amino acids within their cytoplasmic region, the death domain (DD), which is critical for recruiting the death machinery after ligation of the receptors. Work over the last decade has unraveled pivotal signaling networks involved in death receptor-induced cell death; in this regard, the TNFR1 has been the focus of many studies as a prototype for members within the TNFR superfamily.
TNFR1 is constitutively expressed in most cell types, and multiple experimental approaches have confirmed that TNFR1 mediates majority of the biological effects attributed to TNF (19
, 21)
. The binding of TNF to TNFR1 triggers a series of intracellular events initiated by the recruitment of a key adaptor protein TNFR1-associated death domain protein (TRADD) to the receptor complex (22)
. Downstream of TRADD, two signaling complexes are formed (23)
: 1) the plasma membrane-bound complex (complex I) consisting of TNFR1, TRADD, the receptor interacting protein (RIP), and TNF receptor-associated factor 2 (TRAF2), leading to rapid activation of NF-B and the mitogen-activated protein kinases (MAPK) pathways, and 2) the cytoplasmic complex (complex II) containing TRADD, RIP, FAS-associated death domain protein (FADD), and caspase 8, essential for TNF-induced apoptosis through a caspase cascade. In most cells the combination of the above signaling pathways determines the diverse biological activities of TNF, including cell growth, development, oncogenesis, inflammation, stress-induced signaling, and cell death (19
, 21
, 24)
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EFFECT OF ROS ON TNF-INDUCED JNK ACTIVATION
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TOPABSTRACTROS AND OXIDATIVE STRESSTNF AND TNFR SUPERFAMILYEFFECT OF ROS ON...ROS AND TNF-INDUCED NF-{kappa}B...ROS AS THE KEY...ROS IN TNFR1-MEDIATED APOPTOTIC...ROS IN DEATH RECEPTOR-MEDIATED...ROS IN CD95(Fas/Apo1)-MEDIATED...CONCLUDING REMARKSREFERENCES |
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and ROS are potent activators of JNK (also known as stress-activated protein kinases) when they are applied to cells individually. Moreover, it has been well established that ROS play a critical role in TNF-mediated JNK activation (25
, 26)
. JNK form an important subgroup of MAPK with diverse cellular functions such as cell proliferation, differentiation, and apoptosis (27)
. Similar to other members of the MAPK family, JNK activation is mediated by the mammalian MAPK module comprised of MAP kinase kinase kinase (MAPKKK), MAP kinase kinase (MPAKK), and MAPK. JNK is phosphorylated and activated by two MAPKKs (JNNK1/MKK4/SEK1 and JNKK2/MKK7), which possess dual specificity on Thr183 and Tyr185. Upstream of JNKK1 and JNKK2, several MAPKKKs have been identified, including MEK kinase 1 (MEKK1), apoptosis signal-regulating kinase (ASK1), mixed-lineage kinase (MLK), transforming growth factor-beta-associated kinase (TAK1), and TPL2/Cot (28)
. Although the physiological relevance of all these MAPKKKs in controlling JNK activation is not completely clear, studies of genetic deletion of these genes have suggested that different stimuli may act through different MAPKKKs to initiate the JNK signaling pathway (29
30
31)
.
It is now recognized that there are two phases of JNK activation mediated by two different activation pathways in TNF-treated cells: the earlier and transient activation of JNK is mediated by TRAF2 (32)
, while the delayed and persistent activation of JNK is mediated by ROS (33
, 34)
. Although the conventional dogma places ROS upstream of JNK activation, it is noteworthy that a recent study points to a positive feedback loop between JNK activation and ROS production; JNK contributes to TNF-stimulated ROS production; which in turn induces JNK activation (34)
. This conclusion is based on observations that TNF-induced elevated ROS level was found only in wild-type (WT) mouse fibroblasts but not in jnk–/– cells (34)
. Given these reports, it is plausible that JNK activation and ROS production, two reciprocally amplifying events, work in tandem to trigger TNF-induced cell death.
Molecular mechanisms of ROS-mediated JNK activation
Having linked ROS to JNK activation, the challenge over the years has been to decipher the molecular mechanism(s) involved in this pathway upon exposure to TNF. A number of candidate signaling pathways have been implicated, and among them the most well-understood pathway involves ASK1, a ubiquitously expressed MAPKKK that activates both JNK and p38 by phosphorylating and activating respective MAPKKs (JNKK1/MKK4, JNKK2/MKK7, MKK3 and MKK6) (35
, 36)
. In the effort to elucidate the mechanism of ASK1 activation, Saitoh et al. identified an important relationship between thioredoxin, an important cellular redox regulatory protein (37)
, and ASK1 (38)
. These data show that the activity of ASK1 depends on the redox status of thioredoxin, and in its reduced form thioredoxin is capable of binding to ASK1 and blocking its kinase activity. Conversely, in the presence of ROS the oxidized thioredoxin dissociates from ASK1, thereby inducing oligomerization and subsequent phosphorylation of a critical threonine residue within the active loop of ASK1 (38
39
40)
. These findings provide strong evidence linking ROS and oxidative stress to ASK1 activation. Other functions of thioredoxin on ASK1 have also been observed. For instance, thioredoxin is capable of promoting ASK1 ubiquitination and degradation to inhibit ASK1-mediated JNK activation and apoptosis (41)
. Therefore, it is believed that the ROS-thioredoxin-ASK1 system serves as the molecular switch that converts redox signal to JNK kinase activation.
In addition to the regulatory role of thioredoxin on ASK1, other mechanisms controlling ROS-mediated ASK1 activation have been studied. There is evidence showing that ROS are able to activate ASK1 through its dissociation with the docking protein 14-3-3 (42)
. A recent study by Karin and colleagues has provided a new mechanism of ROS-mediated JNK activation downstream of TNFR1 (43)
. In their study, an enhanced level of ROS in TNF-treated cells is able to block the function of MAPK phosphatase (MKP) via oxidizing a critical cysteine residue in its catalytic domain. The diminished MKP thus leads to persistent activation of JNK and cell death.
In summary, ROS act as important coactivators in TNFR1-mediated JNK activation, and the sustained JNK activation constitutes one of the key events in TNF-induced cell death. The involvement of ROS-c-Jun NH2-terminal kinase (JNK) in TNFR1-mediated programmed cell death (including both apoptotic and necrotic cell death) will be readdressed later in this review.
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ROS AND TNF-INDUCED NF-B ACTIVATION
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TOPABSTRACTROS AND OXIDATIVE STRESSTNF AND TNFR SUPERFAMILYEFFECT OF ROS ON...ROS AND TNF-INDUCED NF-{kappa}B...ROS AS THE KEY...ROS IN TNFR1-MEDIATED APOPTOTIC...ROS IN DEATH RECEPTOR-MEDIATED...ROS IN CD95(Fas/Apo1)-MEDIATED...CONCLUDING REMARKSREFERENCES |
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B is one of the key regulatory mechanisms involved in controlling transcription of number of genes that are critical in immune function, inflammation, cell proliferation, and cell death (44
, 45)
. NF-B exists as homodimers or heterodimers of Rel proteins, characterized by the presence of a Rel homology domain required for specific DNA binding, dimerization, and nuclear localization. In unstimulated cells, NF-B is sequestered in the cytoplasm by its inhibitory proteins, IB. Upon stimulation, activation of IB kinases (IKK) leads to IB phosphorylation, ubiquitination, and degradation to liberate the NF-B dimers. Subsequently the freed NK-B proteins translocate to the nucleus and bind to the responsive elements in the target genes to activate transcription.
During TNFR1 signaling, NF-B is one of the principal signaling pathways, and the activation of IKK requires some of the key adaptor proteins such as TRADD, TRAF2, and RIP (44
, 45)
. There is substantial controversy with respect to the role/involvement of intracellular ROS in TNF-mediated NF-B activation. One school of thought supports a role for intracellular ROS in TNF-mediated NF-B activation (46
47
48)
, corroborated by the inhibitory effect of the mitochondrial-specific antioxidant MitoVitE in human monocytic cell line U937 and human T cell line Jurkat (46)
. On the other hand, there are reports contending that ROS do not activate NF-B but, on the contrary, suppress NF-B activation triggered by TNF (49
50
51
52)
. The mechanism of inhibition is associated with direct oxidation of the active cysteine residues in the IKK complex (50
, 51)
. The latter was reinforced by a recent study with more conclusive evidence to dissociate the link between ROS and NF-B activation (53)
. In that study, N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), two antioxidants often used to block NF-B activation, suppressed the TNF-mediated NF-B pathway via mechanism(s) independent of their antioxidant activity. Moreover, endogenous ROS produced through Rac-NADPH oxidase failed to activate NF-B signaling, and instead reduced the magnitude of its activation (53)
. Taken together, the emerging consensus is that ROS are unlikely to be a general modulator in NF-B activation, at least in the TNF signaling pathways (54
, 55)
Effect of NF-B on TNF-induced ROS production
While the jury is still out on the role of ROS in TNF-induced NF-B activation, there is convincing evidence that NF-B in turn has a significant impact on ROS production and accumulation in TNF-treated cells; NF-B acts as a suppressor of intracellular ROS formation in response to TNF. Supporting this, TNF-induced elevated level of ROS was found only in TRAF2-TRAF5 double knockout or p65 knockout mouse embryonic fibroblasts (MEF), and not in wild-type (WT) cells (33)
, indicating that TRAF-mediated NF-B may have an important role in reducing ROS accumulation upon TNF stimulation. Similar results have been reported in IKKß knockout MEF cells challenged with arsenic (56)
. These data beg the question: How does NF-B block TNF-stimulated ROS production and accumulation? One possible mechanism could be that some of the key antioxidant enzymes/proteins are under NF-B regulation, and thenceforth deficiency of NF-B could weaken the antioxidant defense system, leading to intracellular accumulation of ROS. The antioxidant function of NF-B was confirmed by a genome-wide microarray analysis showing that up-regulation of antioxidant enzymes via the NF-B pathway is crucial for elimination of ROS produced in TNF-treated cells (57)
. It has been well established that manganese superoxide dismutase (SOD) (MnSOD), the mitochondrial specific form of SOD with important function in eliminating O2– derived from the mitochondrial respiratory chain, is one of the target genes under the transcriptional control of NF-B (58
, 59)
. Moreover, NF-B-dependent expression of MnSOD is responsible for the resistance to TNF-induced apoptosis, probably via the clearance of mitochondrial-derived ROS (58
, 60
61
62
63
64)
.
In addition to MnSOD, a recent study by Pham et al. has identified another novel mechanism by which NF-B executes its antioxidant function (65)
. In that study, ferritin heavy chain (FHC), the primary iron storage factor highly capable of suppressing ROS accumulation through iron sequestration, prevented sustained JNK activation and apoptosis triggered by TNF. Whereas induction of MnSOD promotes the dismutation of O2– to H2O2, it is plausible that FHC-regulated depletion of intracellular free iron helps to prevent the formation of highly reactive hydroxyl radical through the Fenton reaction, and thus to assist the disposal of H2O2 by catalase or peroxidases (66)
. Therefore, it is now believed that the antioxidant function of NF-B is one of the underlying mechanisms responsible for its antiapoptotic role in TNFR1 signaling (67)
.
On the other hand, some recent studies have suggested NF-B may indirectly block mitochondrial ROS production. Ricci et al. have provided convincing evidence that activated caspases act directly on the respiratory complexes of mitochondria to disrupt mitochondrial function and enhance the generation of ROS (68
, 69)
. The main antiapoptotic function of NF-B is based on its transcriptional regulation of many antiapoptotic genes such as XIAP and c-FLIP, which have a direct inhibitory effect on caspase activation (21)
. It thus appears that activation of NF-B can indirectly suppress mitochondria-derived ROS production via blockage of caspase activation.
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ROS AS THE KEY MEDIATOR IN THE CROSSTALK BETWEEN NF-B AND JNK
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TOPABSTRACTROS AND OXIDATIVE STRESSTNF AND TNFR SUPERFAMILYEFFECT OF ROS ON...ROS AND TNF-INDUCED NF-{kappa}B...ROS AS THE KEY...ROS IN TNFR1-MEDIATED APOPTOTIC...ROS IN DEATH RECEPTOR-MEDIATED...ROS IN CD95(Fas/Apo1)-MEDIATED...CONCLUDING REMARKSREFERENCES |
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B, the two key signaling events downstream of TNFR1, has attracted a great deal of interest in recent years. Two earlier studies reported the suppressive effect of NF-B on JNK activation pathway (70
, 71)
. In normal cells, TNF induces an earlier and transient JNK activation, whereas in cells that are deficient in NF-B activation JNK activation is enhanced and sustained, suggesting that NF-B functions as a potent JNK inhibitor. More important, these studies also demonstrated that the antiapoptotic function of NF-B is achieved at least in part through its ability to suppress the prolonged activation of JNK.
The intriguing question is, How does NF-B suppress TNF-induced JNK activation and apoptosis? Two NF-B target genes, XIAP (71)
and GADD45ß (70
, 72)
, have been identified as the mediators regulating the crosstalk between these two key signaling pathways. However, the inhibitory effects of these two proteins on JNK activation are questionable and not consistent with earlier reports. For instance, GADD45ß is known to be an activator of MTK1, one of the MAPKKK responsible for JNK activation (73
, 74)
; genetic disruption of xiap or gadd45ß did not affect JNK activation (75
, 76)
.
In the search for key mediators of the interplay between NF-B and JNK in cells treated with TNF, the role of ROS has emerged (67
, 77
78
79)
. The intermediacy of ROS in the crosstalk between JNK and NF-B is summarized in Fig. 1
: 1) TNF-induced increase in intracellular ROS is responsible for sustained JNK activation, as well as impaired NF-B activation; 2) NF-B regulates the expression of several key antioxidants enzymes or proteins, such as MnSOD and FHC, to eliminate ROS, thus serving as a negative feedback loop; and 3) activated JNK is capable of promoting ROS production, thus forming a positive feedback loop between JNK and ROS.
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The involvement of ROS in the interaction between JNK and NF-B affects cell fate downstream of TNF stimulation, and NF-B is the principal antiapoptotic mechanism and sustained JNK is a critical proapoptotic factor (21
, 80
, 81)
. Therefore, it appears that ROS delivers a "one-two" punch during TNF-induced programmed cell death by suppressing antiapoptotic NF-B and activating proapoptotic JNK. Most of these effects of ROS downstream of TNF signaling have been reported in systems using murine fibroblasts, and so it remains to be further investigated whether the intermediary role of ROS between JNK and NF-B could be extended to other cell types, particularly cancer cells. To that end, there is evidence to link an increase in JNK activation to apoptotic death in oncogenically transformed NIH 3T3 cells (82)
. However, it remains to be determined whether ROS play a similar role in cell death induced by other stimuli, capable of activating NF-B and JNK simultaneously. Understanding the critical role of ROS in the crosstalk between NF-B and JNK and cell death could have potential implications not only for developing better and more effective therapies, but also provides ideas for reviewing the current clinical practices in light of the diverse roles of ROS in the biology of disease.
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ROS IN TNFR1-MEDIATED APOPTOTIC CELL DEATH |
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TOPABSTRACTROS AND OXIDATIVE STRESSTNF AND TNFR SUPERFAMILYEFFECT OF ROS ON...ROS AND TNF-INDUCED NF-{kappa}B...ROS AS THE KEY...ROS IN TNFR1-MEDIATED APOPTOTIC...ROS IN DEATH RECEPTOR-MEDIATED...ROS IN CD95(Fas/Apo1)-MEDIATED...CONCLUDING REMARKSREFERENCES |
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-induced apoptosis has been extensively studied and reviewed (83
84
85
86)
. The initial evidence pointing to involvement of ROS in TNF-induced apoptosis was based on observations that stimulation with TNF results in a rapid increase of intracellular ROS levels (87
88
89)
. This was further supported by findings that antioxidants, such as N-acetylcysteine (NAC), and increased expression of SOD and thioredoxin were capable of protecting against TNF cytotoxicity (58
, 64
, 88
, 90)
. Subsequent studies have identified mitochondria as the main source of intracellular ROS production on treatment with TNF (91
92
93)
. The role of nonmitochondrial sources of ROS, such as Rac1-NADPH oxidase and cytosolic phospholipase A2-linked cascade (21
, 94
95
96)
are relatively less well understood.
The question of how the death signal generated at the cell membrane results in ROS production from the mitochondria remains unanswered. A recent report provides evidence that TNF-mediated JNK activation could be the mechanism controlling ROS production from the mitochondria (34)
. In that study, TNF-induced ROS production was observed only in WT murine fibroblasts, but not in the JNK1 and JNK2 double knockout counterparts. Indeed, such an observation provides credence to earlier reports linking TNFR-associated factors (TRAF) to ROS production in TNF-treated cells (97)
and the fact that TRAF2 is a critical adaptor protein in TNF-induced JNK activation (98
, 99)
. Further circumstantial evidence to support a role for mitochondria in ROS generation downstream of TNF signaling is the observation that Bcl-2, a mitochondrial antiapoptotic protein, is one of the main molecular targets of JNK (27)
. Although the mechanism of JNK-induced mitochondrial ROS production is not well understood, one possible site could be the mitochondrial electron transport chain, which has been implicated as a source of intracellular ROS in TNF-stimulated cells (92)
.
Similar to TNF, some recent studies provide strong evidence to support a proapoptotic role of ROS in TRAIL-induced signaling. First, there is significant ROS accumulation in TRAIL-treated cancer cells, and scavenging of ROS by antioxidants is able to abrogate caspase activation and apoptosis induced by TRAIL (100
, 101)
. Second, enhanced ROS production from mitochondria is the likely mechanism underlying the sensitizing activity of carbonyl cyanide m-chlorophenylhydrazone (CCCP), a classic uncoupler of oxidative phosphorylation, to TRAIL-induced apoptosis (102
, 103)
. It is still not clear how ROS exert their proapoptotic effect on TRAIL-induced signaling pathway. One possible mechanism could be up-regulation of the death receptors by ROS, as shown by a recent report with DR5 (104)
. Apparently more work is needed to understand whether the redox-sensitive mechanisms operative in the TNF signaling pathways affect TRAIL-mediated death signaling in a similar manner.
Evidence supporting the role of ROS in TNF-induced cell death appears to be overwhelming; however, there are contradictory reports demonstrating an inhibitory effect of ROS on TNF-induced cell death (46
, 105
, 106)
. The latter could be attributed to the intriguing finding that ROS from different intracellular sources could elicit different effects. For instance, ROS generated from the Rac1-NADPH oxidase protect, whereas mitochondria-derived ROS promote, TNF-induced apoptosis (107)
. Three critical points may help clarify the discrepant role of ROS in TNF-mediated cell death. First, the level and duration of ROS production on TNF exposure may be critical. The central mediators of apoptosis, caspases, could be inhibited in a prooxidant milieu (108)
. Unfortunately, the actual ROS levels were rarely quantified in most of the studies discussed above, and so it remains to be determined whether the endogenously generated ROS in TNF-treated cells could reach the concentrations commonly added exogenously for the induction of cell death. Second, the emerging evidence linking ROS to TNF-induced nonapoptotic (caspase-independent) or necrotic cell death, and not so much to receptor-induced apoptotic signaling (33
, 34
, 43
, 109)
. This warrants development of sensitive techniques to differentiate the two forms of cell death in order to study the effect of ROS, downstream of TNF stimulation. Third, enhanced ROS production on signaling through cell death receptors, such as TNFR1, ought to be looked at in the context of the diverse biological effects elicited on receptor ligation
n particular, JNK and NF-B activation.
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ROS IN DEATH RECEPTOR-MEDIATED NECROTIC CELL DEATH |
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TOPABSTRACTROS AND OXIDATIVE STRESSTNF AND TNFR SUPERFAMILYEFFECT OF ROS ON...ROS AND TNF-INDUCED NF-{kappa}B...ROS AS THE KEY...ROS IN TNFR1-MEDIATED APOPTOTIC...ROS IN DEATH RECEPTOR-MEDIATED...ROS IN CD95(Fas/Apo1)-MEDIATED...CONCLUDING REMARKSREFERENCES |
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, 110
, 111)
.
Death receptor-mediated necrotic cell death was first established in one particular cell type, the murine L929 fibrosarcoma cell line (112
, 113)
, and subsequently in other cell types such as mouse embryonic fibroblasts (MEFs), human T lymphocytes, and neutrophils (33
, 34
, 43
, 109
, 114
, 115)
. Although the molecular mechanisms involved in death receptor-mediated necrosis are relatively blurred compared to death receptor-mediated apoptosis, some key molecules have been identified. The current level of understanding is that both the apoptotic and necrotic machineries coexist in the cell, and the final format of cell death mediated by death receptor is determined by the 1) nature of the stimulus, 2) availability of active caspase(s), 3) interaction of several key signaling molecules such as FADD, RIP, TRAF2, and JNK, and 4) presence of ROS and oxidative stress (91
, 110
, 111)
. Among these, ROS seem to play a key role as the action of ROS is closely related to other mechanisms leading to necrotic cell death. To that end, studies by Vandenabeele and colleagues have provided convincing evidence that mitochondria-originated ROS are the main mediating factor in TNF- and CD95L-induced necrosis (91
, 110)
. As discussed earlier, the catalytic activity of caspases is susceptible to redox regulation, and elevated intracellular ROS is capable of inducing necrosis by suppressing caspase activation (5
6
7
8
9)
. The latter is supported by the remarkable finding that the presence of the general caspase inhibitor (zVAD) induces a 1000-fold increase in sensitivity to TNF-induced necrosis in L929 cells (113)
, suggesting antinecrotic or prosurvival role of caspases. Moreover, the caspase inhibition further leads to enhanced ROS production (112)
, indicating a positive feedback mechanism to promote death receptor-induced necrosis. Thus, it is plausible that one of the underlying mechanisms of action for ROS to promote necrosis is the inhibition of caspase activation.
ROS and RIP
RIP is one of the key components of the TNFR1 signaling complex. In the last several years RIP has emerged as a crucial sensor and integrator of cellular stress (116)
. An early study has demonstrated that RIP is required for necrosis induced by FasL, TNF, and TRAIL in T lymphocytes (114)
. Similar results were also found in virus-infected and TNF-treated Jurkat cells (117)
. Here again, the evidence seems to support a critical role of ROS downstream of RIP in CD95(Fas/Apo1)- and TNFR1-induced necrotic cell death; increase in ROS levels and necrosis was only observed in WT MEF cells, but not in RIP–/– cells in response to TNF treatment (109)
. RIP has also been found to play an essential role in necrotic cell death (in MEF cells) induced upon exogenous addition of H2O2 (118)
, further supporting the notion that RIP and ROS act together in regulating necrotic cell death. A critical question that remains to be answered is how RIP affect the intracellular ROS level. A recent report provided some clues by demonstrating the direct inhibitory effect of RIP on mitochondrial function: in TNF-treated cells RIP disrupts mitochondrial adenine nucleotide translocase (ANT)-dependent ADP transport (119)
. It is thus possible that RIP may directly promote ROS production from mitochondria. Such a hypothesis helps to explain the early findings that TNF fails to enhance intracellular ROS level in RIP–/– cells (109).
ROS and JNK
Over the years, experimental evidence has consolidated the proapoptotic role of JNK in the TNFR1 signaling pathway (80)
. More recent reports demonstrate that prolonged JNK activation is also involved in TNF-induced necrotic cell death (33
, 34)
. As discussed in an earlier section, elevated levels of ROS mediate prolonged JNK activation (33)
, and activated JNK further promotes ROS production from mitochondria (34)
, forming a positive feedback loop. It is thus believed that in certain cell types, such as T cells and MEF cells, ROS and JNK work together to divert TNF-triggered cell death from apoptotic to necrotic. However, most current studies are limited to some selected cell types such as L929, Jurkat, and MEF cells; therefore it remains to be investigated whether the above mechanism is also applicable to other cell types, such as solid tumors. If so, it may provide another possible way to enhance the sensitivity of cancer cells to death stimuli.
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ROS IN CD95(Fas/Apo1)-MEDIATED APOPTOSIS |
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TOPABSTRACTROS AND OXIDATIVE STRESSTNF AND TNFR SUPERFAMILYEFFECT OF ROS ON...ROS AND TNF-INDUCED NF-{kappa}B...ROS AS THE KEY...ROS IN TNFR1-MEDIATED APOPTOTIC...ROS IN DEATH RECEPTOR-MEDIATED...ROS IN CD95(Fas/Apo1)-MEDIATED...CONCLUDING REMARKSREFERENCES |
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, 121)
. Downstream of receptor ligation, two signaling pathways have been identified: one where the ligation results in early and robust activation of the initiator caspase 8 through the formation death-inducing signaling complex (DISC), and another where the DISC assembly and caspase 8 activation is rather weak and hence requires mitochondrial amplification factors to efficiently activate downstream effector caspases (122)
. Similar to the controversial role of ROS in TNFR1-initiated cell death pathway, it is also a highly debatable issue whether ROS play an anti- or proapoptotic role in CD95-induced cell death.
There is experimental evidence implicating ROS in CD95-mediated cell death in some systems; CD95 ligation promotes intracellular ROS production (123
124
125
126
127)
and various antioxidants such as N-acetyl-L-cysteine (NAC), SOD overexpression or its mimic, catalase, block CD95-induced apoptosis (124
, 126
, 128
129
130
131)
. Moreover, induction of ROS and oxidative stress are also reported to be the underlying mechanisms involved in the sensitizing effect of the chemotherapeutic agent camptothecin on CD95-mediated apoptosis in glioblastoma cells (132)
. As for the intracellular source of ROS generated on CD95-mediated signaling, both the NADPH oxidase (127
, 133)
and the mitcochondrial electron transport chain (123
, 126
, 134)
have been implicated in different cell types.
Attempts have been made to elucidate the underlying mechanisms responsible for the proapoptotic role of ROS in CD95-mediated cell death. One possibility is that ROS are able to up-regulate the cell surface expression of CD95 (134
, 135)
. Another explanation is based on the damaging effect of ROS on mitochondria: ROS promote apoptosome formation to facilitate CD95-mediated apoptosis in type II cells (126)
.
Against the backdrop of observations supporting a facilitative role of ROS in CD95-mediated apoptosis, Clement and Stamenkovic were the first to report that an increase in intracellular O2– inhibited CD95-mediated death signaling (13)
. Subsequent studies have supported such a notion by showing an inverse correlation between ROS production and susceptibility to CD95-mediated apoptosis (136
137
138
139)
. For example, decreased ROS production from mitochondria by mitochondrial ATP synthase inhibitor oligomycin or the mitochondrial uncoupler FCCP is correlated with enhanced apoptosis in CD95L-treated Jurkat T cells (136)
. The molecular mechanisms by which ROS inhibit CD95-mediated cell death are poorly understood. Of note, most of the above studies were carried out in type II cells, such as Jurkat cells, in which the mitochondria are closely involved, thus providing the possibility that ROS exert their antiapoptotic function at the mitochondrial level. Such a hypothesis is clearly supported by a recent study in which ROS up-regulate the expression of antiapoptotic Bcl-2 family members, which attenuate CD95-mediated apoptosis in type II cells (137)
. However, a contrasting picture is presented in another study that links the antiapoptotic activity of Bcl-2 to its prooxidant activity (10)
. Furthermore, in light of the fact that the inhibitory effect of O2– on death signaling is not exclusive to death receptor-induced apoptosis (10
, 12
, 15
, 140)
, it appears that a slight prooxidant state provides a survival advantage via mechanisms that may be shared by diverse death stimuli.
Taken together, it is intriguing that both the pro- and antiapoptotic roles of ROS on CD95-mediated apoptosis are exerted at the mitochondrial level. Further elucidation of the molecular mechanisms by which ROS exert the pro- or antiapoptotic function will help to clarify pathways/mechanism(s) regulating CD95-mediated apoptosis.
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CONCLUDING REMARKS |
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TOPABSTRACTROS AND OXIDATIVE STRESSTNF AND TNFR SUPERFAMILYEFFECT OF ROS ON...ROS AND TNF-INDUCED NF-{kappa}B...ROS AS THE KEY...ROS IN TNFR1-MEDIATED APOPTOTIC...ROS IN DEATH RECEPTOR-MEDIATED...ROS IN CD95(Fas/Apo1)-MEDIATED...CONCLUDING REMARKSREFERENCES |
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ACKNOWLEDGMENTS |
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Received for publication December 19, 2005. Accepted for publication March 31, 2006.
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REFERENCES |
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TOPABSTRACTROS AND OXIDATIVE STRESSTNF AND TNFR SUPERFAMILYEFFECT OF ROS ON...ROS AND TNF-INDUCED NF-{kappa}B...ROS AS THE KEY...ROS IN TNFR1-MEDIATED APOPTOTIC...ROS IN DEATH RECEPTOR-MEDIATED...ROS IN CD95(Fas/Apo1)-MEDIATED...CONCLUDING REMARKSREFERENCES |
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