Because TRAIL selectively induces apoptosis in tumor or transformed cells but not in normal cells, it has shown great potential to be a valuable tumor therapeutic agent (2
). Like other members of the TNF superfamily, TRAIL has other biological functions such as activating transcription factor NF-κB and JNKs (14
). Although much effort has been made to investigate the biological functions of TRAIL since it was discovered, the molecular mechanism of TRAIL signaling is still largely unknown. Recently, it was suggested that TRAF2, an important effector of TNF signaling, was involved in both NF-κB and JNK activation induced by overexpression of TRAIL receptors (14
). In our study, we reported that another critical effector of TNF signaling, RIP, plays a critical role in TRAIL-induced activation of both IKK and JNK. We also found that while overexpression of the dominant negative mutant TRAF2 blocked TRAIL-induced IKK and JNK activation, the absence of TRAF2 affected TRAIL-induced JNK activation but had little effect on IKK activation. In addition, we also demonstrated that neither RIP nor TRAF2 was required for TRAIL-induced apoptosis.
The death domain kinase RIP is an essential effector for TNF-induced NF-κB activation (20
). It has been suggested that RIP plays a similar role in DR3/Apo3-mediated NF-κB activation (1
). Here we provided evidence that RIP is also essential in TRAIL-induced NF-κB activation. The dominant negative mutant of RIP efficiently blocked TRAIL-induced IKK activation. In RIP−/−
cell lines, no IKK activity was detected following TRAIL treatment. Furthermore, we found that RIP was present in the TRAIL-R1 complex, whose formation is TRAIL dependent. However, because RIP does not directly interact with TRAIL-R1 (Fig. A), it seems that the recruitment of RIP requires additional adapter molecules. In previous overexpression experiments, it was shown that the presence of TRADD and FADD resulted in the interaction of TRAIL-R1 and RIP (5
). Our results have confirmed this observation (Fig. A). But since TRADD and FADD were not found in the TRAIL-R1 complex (Fig. B), they may not be the molecules that mediate the endogenous TRAIL-R1–RIP interaction. Consistent with this possibility, it has been shown that FADD is not required for TRAIL-R1-mediated apoptosis (25
). Therefore, it is possible that the recruitment of RIP to TRAIL receptors is mediated by other death domain-containing factors.
Previous studies involving overexpression of the dominant negative mutant RIP had shown that RIP was involved in TNF-induced JNK activation (23
). However, a study using RIP knockout mice showed that RIP had little effect on TNF-induced JNK activation (20
). In this study, however, we found that RIP was required for TRAIL-induced JNK activation. Overexpression of the dominant negative mutant RIP completely abolished TRAIL-induced JNK activation (Fig. ). In addition, TRAIL-induced JNK activation was greatly decreased in RIP−/−
cells (Fig. ). These data suggested that RIP is involved in IKK and JNK activation by TRAIL treatment. Therefore, RIP is a critical effector in TRAIL signaling.
TRAF2 was initially identified as a component of the TNF-R2 complex and was also found in the TNF-R1 signaling complex (23
). Previous studies involving overexpression of TRAF2 and its dominant negative mutant had shown that TRAF2 played a critical role in TNF-induced NF-κB and JNK activation. However, the aforementioned study with a genetic approach reported that removal of TRAF2 caused the diminishment of TNF-induced JNK activation and had only a minor effect on TNF-induced NF-κB activation (51
). In this study, we found that TRAF2 had a similar effect on TRAIL signaling: the absence of TRAF2 severely affected TRAIL-induced JNK activation but had no detectable effect on IKK activation (Fig. and ). But in the overexpression experiments, we found that TRAIL-induced activation of both IKK and JNK was blocked by the dominant negative mutant of TRAF2 (Fig. ). This observation is consistent with a recent report (14
). One possibility is that other TRAF proteins, such as TRAF5, may replace the function of TRAF2 to mediate TRAIL-induced IKK activation. Therefore, the effect of the absence of TRAF2 on TRAIL-induced IKK activation might be minimized by the presence of other TRAF proteins. However, when the dominant negative mutant of TRAF2 is overexpressed, it might also block the function of other TRAF proteins; as a result, overexpression of the dominant negative mutant of TRAF2 inhibits TRAIL-induced IKK activation. Further studies are necessary to elucidate the role of TRAF proteins in TRAIL-induced IKK activation.
It has been reported that FADD is dispensable for TRAIL-induced apoptosis although it is essential for TNF- and Fas-mediated cell death (25
). But because overexpression of dominant negative FADD efficiently blocked TRAIL-induced apoptosis (45
), it is possible that a FADD-like death factor mediates TRAIL-induced cell death. In this study, we demonstrated that neither RIP nor TRAF2 is required for TRAIL-induced apoptosis (Fig. ). Although JNK activation is essential for cells to undergo apoptosis in some circumstances, it is unlikely that JNK activation is involved in TRAIL-induced apoptosis since TRAF2−/−
cells died to the same extent as wt fibroblasts. On the other hand, because NF-κB activation provides an antiapoptotic effect (4
), RIP-mediated NF-κB activation following TRAIL treatment may protect cells against TRAIL-induced apoptosis. Unfortunately, because RIP−/−
fibroblasts are insensitive to TRAIL treatment and CHX is necessary to induce death of RIP−/−
cells, we failed to evaluate the antiapoptotic effect of NF-κB activation in TRAIL-induced apoptosis with those fibroblasts. However, we found in a previous study that RIP was cleaved by caspase-8 in Fas-, TNF-, and TRAIL-induced apoptosis (22
). Importantly, the cleavage of RIP abolished its ability to efficiently activate NF-κB. Therefore, NF-κB activation may also be antiapoptotic in response to TRAIL treatment. This possibility is further supported by the observation that inhibition of NF-κB activation sensitized several types of tumor cells to TRAIL treatment (16
Taken together, the results of our study shed some light on the molecular mechanisms of TRAIL signaling. We demonstrated that both RIP and TRAF2 are important effectors of TRAIL signaling. In addition, neither RIP nor TRAF2 is required for TRAIL-induced apoptosis. Because TRAIL has been pursued as a potential cancer therapy, knowledge of TRAIL signaling will accelerate this process and help in developing new strategies for improving its therapeutic value.