The zinc finger protein A20 is encoded by an immediate early response gene and acts as an inhibitor of NF-κB– dependent gene expression induced by different stimuli including TNF and IL-1 (Cooper et al., 1996
; Jäättelä et al., 1996
; Song et al., 1996
). Here we show that the TNF-induced expression of GM-CSF and IL-6, as well as the TNF-induced expression of a luciferase reporter gene that is expressed under control of the complete hIL-6 promoter or the minimal hIL-6 promoter preceded by 3 NF-κB recognition sequences, are clearly inhibited in L929sA cells stably transfected with A20. These results are consistent with the fact that NF-κB is required for IL-6 and GM-CSF gene transcription (Schreck et al., 1990; Zhang et al., 1990
). Surprisingly, gel retardation assays revealed that overexpression of A20 had no effect on the TNF-induced nuclear translocation and DNA binding of NF-κB. Also the constitution of the NF-κB complex was not altered in cells overexpressing A20, and consisted in both cases of a p65 and a p50 subunit as revealed by gel supershift assays (data not shown). Therefore, the inhibition of NF-κB– dependent gene expression by A20 cannot be explained by an A20-induced alteration in the subunits of NF-κB. Ferran et al. (1998)
showed recently that A20 acts upstream of IκB degradation and prevented the nuclear translocation of NF-κB. The reason for the discrepancy with our results is still unclear. Because activation of NF-κB is an early response after stimulation with TNF, we analyzed NF-κB translocation at 5, 15, and 30 min after TNF stimulation, whereas results of Ferran et al. were obtained 2 h after TNF stimulation. The latter is quite late and might already be regulated by secondary factors that are A20-sensitive. Moreover, NF-κB activation at later times is also regulated by TNF-induced negative regulatory proteins such as IκBα and A20 whose expression is itself under the control of NF-κB, further raising the complexity of NF-κB activation at later time points (Krikos et al., 1992
; LeBail et al., 1993
). Alternatively, we cannot exclude cell type–dependent differences.
Until now, the mechanism by which A20 blocks the activation of NF-κB–dependent gene regulation was not known. Up to now, the NIK–IκB kinase pathway has been assumed to be responsible for NF-κB activation upon TNF treatment. However, our finding that A20 has no effect on the translocation of NF-κB to the nucleus argues against this pathway as the target for A20. Moreover we demonstrated that A20 could inhibit RIP- and TRAF2- but not NIK-induced NF-κB–dependent reporter gene activation, suggesting that A20 interferes with NF-κB activation upstream of NIK. These conclusions were confirmed by the inability of A20 to block Tax-induced NF-κB activation since the latter was shown to activate NF-κB by directly interfering with the downstream kinases NIK, IKKα, and IKKβ, independent of TRAF2 (Chu et al., 1998
; Uhlik et al., 1998
; Yin et al., 1998
The above results suggest that TNF-induced NF-κB– dependent gene activation requires at least two different pathways: an NIK-mediated pathway leading to translocation of NF-κB to the nucleus, and an NIK-independent pathway leading to transactivation of NF-κB. Our results demonstrate that A20 specifically interferes with the NF-κB transactivation pathway. A20 has also been shown to interact with the TNF receptor–associated protein TRAF2 (Song et al., 1996
). Interestingly, TRAF2 as well as some other members of the TRAF protein family, including TRAF5 and TRAF6, were shown to play a positive role in NF-κB activation induced by different cytokines, such as TNF, IL-1, and CD40 ligand, via their interaction with the NF-κB inducing kinase NIK (Cao et al., 1996
; Malinin et al., 1997
). The fact that A20 directly associates with TRAF2, as well as our observation that A20 not only prevents NF-κB activation by TNF but also by IL-1 and CD40 overexpression, points to a role of a TRAF-mediated signaling pathway as a target for A20. However, gene knockout studies have recently shown that TRAF2 is not absolutely required for NF-κB activation by TNF, although this probably is a consequence of redundancy within the TRAF protein family (Yeh et al., 1997
). Alternatively, RIP may be more important than TRAF2 in mediating activation of NF-κB upon TNF stimulation (Kelliher et al., 1998
The nature of the RIP/TRAF–initiated NF-κB transactivation signal is still unclear. Recently, an important role in the transactivating potential of NF-κB upon TNF stimulation was demonstrated for p38 MAP kinase (Beyaert et al., 1996
; Bergmann et al., 1998
; Vanden Berghe et al., 1998). This kinase becomes activated by stimulation of cells with TNF as well as by overexpression of TRAF2. In contrast, overexpression of NIK did not induce the phosphorylation of p38 MAP kinase, indicating that a separate pathway initiating at TRAF2 leads to the activation of p38 MAP kinase (Carpentier et al., 1998
). Similar to the effect of A20 overexpression, inhibition of p38 MAP kinase with the specific inhibitor SB203580 also prevented NF-κB– dependent gene expression without altering the translocation of NF-κB to the nucleus (Beyaert et al., 1996
; Bergmann et al., 1998
). As these results suggested that A20 might interfere with the TRAF2-p38 MAP kinase pathway, we investigated if A20 was able to prevent the TNF-induced activation of p38 MAP kinase. However, no significant effect of A20 on p38 MAP kinase phosphorylation, which is a marker for its activation, could be observed. Although these results indicate that A20 does not act upstream of p38 MAP kinase activation, it is still possible that A20 interferes with the TRAF2-p38 MAP kinase/ NF-κB transactivation pathway downstream of p38 MAP kinase. The validation of this possibility awaits the identification of the p38 MAP kinase substrate that is involved in NF-κB transactivation. Alternatively, our results might also fit with the existence of another RIP- or TRAF2-initiated pathway that contributes to NF-κB–dependent transcription, and which is blocked by A20.
Our observation that A20 also prevents NF-κB activation by TPA indicates that the A20-sensitive pathway might also be activated by protein kinase C, at least in some cell lines. Similar results were obtained by Cooper et al. (1996)
. In contrast, stable expression of A20 has been reported to be unable to prevent TPA-induced NF-κB activation in breast carcinoma MCF cells (Jäättelä et al., 1996
). These controversial results might reflect a cell type specific effect or differences in A20 expression levels upon stable and transient transfection. It should be mentioned that a role for protein kinase C in TNF-induced NF-κB transactivation has been suggested recently based on the inhibition with a protein kinase C inhibitor (Bergmann et al., 1998
). Whether protein kinase C functions downstream of TRAF2 or in a totally separate pathway remains to be established. In any case, the protein kinase C–mediated pathway sensitive to A20 seems to regulate specifically NF-κB–dependent gene expression, as TPA-induced transcription that is controlled by an AP1-responsive element or a SRE was not sensitive to A20. The latter result, as well as our finding that Tax-mediated NF-κB activation is not affected by A20, also excludes an aspecific effect of A20 on the general transcription machinery.
The transcription activating potential of NF-κB has been primarily attributed to the p65 subunit, whose transactivating potential resides in its COOH-terminal portion (Ballard et al., 1992
; Schmitz et al., 1994
). Furthermore, the p65 subunit becomes phosphorylated during the activation of NF-κB upon TNF stimulation (Naumann and Scheidereit, 1994
; Schmitz et al., 1995
). Indeed, a p65 phosphorylating activity was found in the IκB kinase complex (Mercurio et al., 1997
). Moreover, it was also shown that IκB is associated with the protein kinase A catalytic subunit that can phosphorylate the p65 in its rel homology domain resulting in enhanced activity of NF-κB (Zhong et al., 1997
). Recently, p65 phosphorylation was shown to promote an interaction between p65 and the coactivators CBP/p300 (Zhong et al., 1998
). The latter were previously shown to synergistically enhance the transcription activating potential of NF-κB (Gerritsen et al., 1997
; Perkins et al., 1997
). However, it is unlikely that A20 interferes with protein kinase A or another signaling pathway leading to the engagement of the coactivators CBP/p300 in the transactivation of NF-κB because we were unable to rescue NF-κB activation from A20 inhibition by overexpression of CBP/p300. Moreover, activation of the protein kinase A catalytic subunit requires degradation of IκB and the activation of the NIK–IκB kinase pathway which is, however, not modulated by A20. Also, A20 did not interfere with NF-κB–dependent gene expression obtained by overexpression of the p65 subunit as such (Cooper et al., 1996
In endothelial cells, TRAF2 has been recently shown to translocate to the nucleus, where it might directly regulate transcription (Min et al., 1998
). Because A20 can bind to TRAF2 (Song et al., 1996
), and exclusively resides in the cytosol, A20 might prevent nuclear localization of TRAF2.
Screening of a cDNA library for A20 interacting proteins by the yeast two-hybrid system has revealed some isoforms of the 14-3-3 proteins that interact with the COOH-terminal zinc finger domain of A20 (Vincenz and Dixit, 1996
; De Valck et al., 1997
). 14-3-3 proteins were shown to function as adapter proteins between A20 and c-Raf. Moreover, 14-3-3 also functioned as a chaperone in these studies (Vincenz and Dixit, 1996
). However, by mutation analysis we previously demonstrated that the interaction of 14-3-3 proteins with A20 is not involved in the effect of A20 on NF-κB activation (De Valck et al., 1997
). By the yeast two-hybrid screening system, we also identified ABIN as a novel A20-interacting leucine zipper protein. The interaction of ABIN with A20 was confirmed in human cells and shown to map to the functional COOH-terminal zinc finger–containing domain of A20. Upon overexpression, ABIN potently inhibits NF-κB activation induced by TNF. Furthermore, also ABIN interferes with TNF-induced NF-κB activation at the level of RIP/ TRAF2. Therefore, the ability of A20 to block TNF-mediated NF-κB activation is likely to involve the binding of the NF-κB inhibitory protein ABIN to the COOH-terminal zinc finger domain of A20. Moreover, the fact that A20 can also interact via its NH2
-terminal domain with TRAF1 and TRAF2 (Song et al., 1996
) suggests that A20 can recruit ABIN to the TRAF2 complex in the TNF signaling pathway.
In conclusion, A20 appears to prevent NF-κB–dependent gene expression by specifically interfering in the cytosol with a novel RIP/TRAF2–initiated transactivation pathway, thus inhibiting the TNF-induced expression of several cytokines and proinflammatory proteins. Since A20 also inhibits NF-κB activation by IL-1 and CD40, which all signal to NF-κB activation via members of the TRAF family, further identification of TRAF-mediated NF-κB transactivation signals may provide means of achieving more specific antiinflammatory treatments.