Our studies have revealed the ability of TRAM/TRIF-dependent signals to regulate TNF-α mRNA translation. Our findings help to explain why both Mal/MyD88- and TRAM/TRIF-dependent signals are required for normal TNF-α expression in response to stimulation with LPS [5
]. Based on our data and existing information in the literature, the major consequence of Mal/MyD88-transduced signals appears to be the transcriptional upregulation of the TNF-α gene via effects on transcription factors such as NF-κB, while TRAM/TRIF-dependent signals act in a complementary fashion to activate the MK2 kinase and thus promote the translation of the TNF-α mRNA (fig. ). The influence of TRAM/TRIF signaling on TNF-α translation appears to be mediated via effects on regulatory elements in the 3′ UTR. These elements are known to interact with RNA-binding proteins that modulate translation [13
], and the signals transduced by the TRAM/TRIF pathway may alter the binding and/or function of these proteins. Our results also indicate that the 34 nucleotide minimal ARE in the TNF-α 3′ UTR is not sufficient to mediate the effects of TRAM on translation (fig. ), suggesting the involvement of other regions of the 3′ UTR. Although the ARE plays a major role in translational regulation, there are other cis-
acting elements in the TNF-α 3′ UTR that have been implicated in this process [13
]. The effect of TRAM-dependent signals on TNF-α translation may be mediated by such elements. TRAM deficiency also probably affects aspects of TNF-α expression other than translation since we found that the levels of TNF-α mRNA in the TRAM knockout BMDM were significantly lower than those in wild-type BMDM 6 h after LPS stimulation (fig. ). This would not be surprising given that several of the proteins that interact with the TNF-α 3′ UTR influence transcript stability in addition to translation, and that MK2 regulates the function of such proteins [13
]. It should also be noted, as mentioned earlier, that the TRAM/TRIF pathway does contribute to NF-κB activation, albeit in a relatively minor and delayed fashion, and thus, could play a role in regulating TNF-α expression at the level of transcription [11
]. Moreover, based on a combination of experimental and computational modeling data, Covert et al. [28
] have suggested that TRAM/TRIF-dependent signals can function in an NF-κB-independent, IRF3-dependent fashion, to induce the production of small amounts of TNF-α, which then acts in an autocrine manner to cause the delayed activation of NF-κB and thereby promote further TNF-α transcription. Thus, the TRAM/TRIF pathway can influence TNF-α expression by multiple mechanisms.
Fig. 8 A model of the role of TLR4 signaling pathways in regulating TNF-α expression. Mal/MyD88-dependent signals are required for the transcriptional upregulation of the TNF-α gene. In addition to a minor contribution to transcription of the (more ...)
While our experiments were in progress, Gais et al. [29
] reported that BMDM, as well as bone marrow-derived dendritic cells, from the TRIF mutant Lps2
mouse strain had impairments in LPS-induced activation of the MK2 kinase and TNF-α translation. Our findings complement those of Gais et al. [29
] by showing that TRAM, which mediates recruitment of TRIF to the TLR4 cytoplasmic domain [6
], is also required for LPS-induced MK2 activation and TNF-α translation. Furthermore, our data provide additional mechanistic insight by showing that TRAM influences translation in a TIR domain-dependent fashion (fig. ) and that cis-
regulatory elements outside the minimal ARE in the TNF-α 3′ UTR are involved in the translational regulatory effects (fig. ). The requirement for the TRAM TIR domain, which is needed for binding to TRIF [7
], and the phenotype of the Lps2
BMDM (fig. ) [29
], strongly support the notion that regulation of TNF-α translation is carried out by TRAM and TRIF acting together (fig. ).
Although our results (fig. ), as well those of Gais et al. [29
], indicate a requirement for TRIF in TNF-α translation, we were somewhat surprised to find that transfection of TRIF into HEK293T cells did not increase the translation of the TNF-α 3′ UTR-dependent luciferase reporter (fig. ). This observation indicates that transient expression of TRIF in HEK293T cells is not sufficient to activate TNF-α translation even though expression of TRAM under the same conditions is sufficient for this function. We do not have a definitive explanation for these findings. Given that TRAM and TRIF are both required for the effect on TNF-α translation and that HEK293T cells do not express transcripts for either of these proteins (E.T., B.J.C., unpublished data), one possible explanation is that HEK293T cells may express a protein that is able to substitute functionally for TRIF but no protein that can substitute for TRAM. Another possibility is that TRAM may be able to achieve the ‘activated’ conformation required to upregulate TNF-α translation when expressed in HEK293T cells, whereas TRIF may require additional factors (presumably present in the environment of LPS-activated macrophages but not in HEK293T cells) to attain this state. Further studies will be required to clarify these issues.
Although lack of TRAM or functional TRIF resulted in a reduction in TNF-α protein production without decrease in the mRNA (fig. , ), the deficiencies of these proteins impaired IL-6 expression at the level of both protein and mRNA (fig. ). This result suggests that whereas a significant effect of TRAM/TRIF-transduced signals on TNF-α expression appears to be on translation, the effect on IL-6 expression may be largely at the level of transcription or post-transcriptional mRNA stability, with any influence on translation obscured by the changes in the amounts of mRNA. This idea would be consistent with the fact that although the IL-6 3′ UTR does contain an ARE, there are clear differences from the TNF-α 3′ UTR, indicative of the expression being influenced by distinct regulatory mechanisms [30
]. Our observation that the ARE is not sufficient to mediate the translational effects of TRAM (fig. ) would also be consistent with differential effects of TRAM/TRIF-dependent signals on TNF-α versus IL-6.
Like the observations of Gais et al. [29
] on TRIF mutant macrophages, we found that TRAM deficiency had differential effects on LPS-induced TNF-α expression in peritoneal macrophages versus BMDM. While the absence of TRAM in BMDM significantly reduced TNF-α protein production without affecting mRNA levels (fig. ), TRAM-deficient peritoneal macrophages showed equivalent reductions in both mRNA and protein (fig. ). The difference in behavior between the peritoneal macrophages and the BMDM could reflect a cell type-specific involvement of the TRAM/TRIF pathway in TNF-α translation. Alternatively, TRAM/TRIF-dependent signals could be required for TNF-α translation in both cell types, but an additional requirement for these signals in controlling levels of the mRNA in peritoneal macrophages could mask the effects on translation in these cells.
The requirement for TRAM and TRIF for normal production of TNF-α appears to be specific to TLR4: other TLRs, such as TLR2 and TLR9, are able to induce normal expression of TNF-α mRNA and protein even though they only activate MyD88-dependent signals and are not connected to the TRAM/TRIF pathway [15
]. The explanation for this unique dependence of TLR4 on TRAM and TRIF for the induction of TNF-α is not clear. TLR4 is distinguished from other members of its family by the fact that it signals from 2 different subcellular locations: it activates the Mal/MyD88 pathway from the plasma membrane, while its interaction with TRAM and TRIF requires internalization into an endosomal compartment [10
]. It has recently been suggested that these processes may involve separate populations of TLR4 molecules [32
], and perhaps the existence of these 2 distinct receptor pools may make the signals activated by either one of them insufficient to induce normal expression of TNF-α. Further investigation will be required to clarify this issue and to elucidate the newly discovered role for the TRAM/TRIF pathway in cytokine translation.