Intracellular and extracellular innate immune signaling systems must be cross-regulated to allow appropriate cytokine responses. We previously found that activation of the NOD2/RIP2 intracellular innate immune signaling pathway causes the K63-linked ubiquitination of a novel site (K285) on the IKK scaffolding protein, NEMO (1
). Given this, we wanted to determine the effect of extracellular innate immune signaling pathways on K285 NEMO ubiquitination and the role of this ubiquitination event in mediating NF-κB signaling. To determine this ubiquitination site's effect on TLR signaling, NEMO-null MEFs (18
) were transduced with retrovirus expressing either empty vector, wt NEMO, or a form of NEMO that cannot be ubiquitinated on lysine 285 (K285R NEMO). Because MEFs have a well-described LPS/TLR4 response, selected pooled clones were exposed to 100 ng/ml of highly purified LPS (Invivogen) for 0, 15, 30, or 60 min. Western blotting showed that both K285R NEMO and wt NEMO were expressed in these stably transduced clones (Fig. ). Both active phospho-IKK and phospho-IκB were detected in wt NEMO-expressing MEFs weakly at 30 min and more strongly at 60 min. Neither the vector-only cell line nor the K285R NEMO cell line showed any activation of IKK or phosphorylation of IκB (Fig. ). As a control for LPS activity, phospho-p38 blotting showed similar activation between all three cell lines (Fig. , bottom 2 panels). In contrast to these results, when the same cells were treated with TNF, both the wt NEMO-reconstituted cells and the K285R NEMO-reconstituted cells showed activation of IκB (Fig. ). Surprisingly, the TNF response was consistently stronger in the K285R NEMO-reconstituted cells. This finding suggests that NEMO ubiquitination may determine a level of specificity to NF-κB activation in response to inflammatory agonists. To correlate these findings with the ubiquitination of NEMO, the wt NEMO cell line and the K285R cell line were either left untreated or were treated with 100 ng/ml LPS for 45 min. NEMO was immunoprecipitated under stringent washing conditions (RIPA buffer with 0.1% SDS and 1 M NaCl) and Western blotting was performed. The wt NEMO cell line showed increased NEMO ubiquitination in the LPS-treated cells; however, the K285R NEMO cell line failed to show any NEMO ubiquitination despite consistently larger amounts of NEMO immunoprecipitated (Fig. ). While the LPS used in these experiments is commercially available as “highly purified” (Invivogen), we could not rule out contamination by the NOD2 agonist MDP. For this reason, we treated the same MEFs with MDP and did not see IKK activation or NEMO ubiquitination (data not shown). In addition, we transduced these MEFs with NOD2 retrovirus (cell line generation shown in Fig. S1 in the supplemental material) and saw only a very minimal response to MDP stimulation (data not shown). These findings suggest that in addition to limited (if any) NOD2 present in the MEFs, the MEFs used in this study lack the ability to transport MDP into the cytoplasm. Thus, our LPS response is unlikely to be due to contaminating MDP. Taken together, these findings suggest that the TLR4 agonist, LPS, requires ubiquitination of NEMO at lysine 285 to optimally activate NF-κB signaling.
FIG. 1. NEMO ubiquitination at lysine 285 is required for optimal TLR4 signaling but not for optimal TNF signaling. (A) NEMO-null MEFs were infected with pBABE retrovirus expressing either vector alone, wt NEMO, or K285R NEMO. After 2 weeks of puromycin selection, (more ...)
TLR signaling involves the activation of the K63 E3 ligase TRAF6 to ultimately help activate the NF-κB signaling pathway (3
). Given that TRAF6 is a K63-specific E3 ligase in the TLR4 system, we wanted to determine if TRAF6 induced the ubiquitination of K285 on NEMO. Because lysine 399 (K399) is also ubiquitinated on NEMO in response to inflammatory stimuli (30
), we generated NEMO mutants with the conservative (lysine to arginine) mutations at lysine 285 (K285R), lysine 399 (K399R), or both lysine 285 and lysine 399 (K285/399R). These mutations allow the conservation of charge but do not allow these sites to ubiquitinated. These forms of NEMO were transfected into 293 cells along with HA-tagged ubiquitin and either TRAF2 (as a negative control) or TRAF6. IPs were performed under stringent washing conditions, and Western blotting was performed. TRAF6 strongly induced the ubiquitination of NEMO, and the pattern of ubiquitination shifted substantially in every case in which lysine 285 was mutated to an arginine (Fig. ). Mutation of K399 alone had little effect on ubiquitination, but mutation of both sites resulted in both decreased intensity and a shift in the pattern of the ubiquitination species of NEMO. These results indicate that, when overexpressed, TRAF6 can enhance ubiquitination of NEMO at multiple sites, including K285 and K399.
FIG. 2. TRAF6 ubiquitinates lysine 285 of NEMO, and this ubiquitination is required for optimal TRAF6-indued NF-κB activation. (A) myc-tagged wt NEMO or myc-tagged mutant NEMOs containing the conservative Lys-Arg mutations at two major NEMO ubiquitination (more ...)
To show the functional significance of these ubiquitination events, we transiently transfected each of the forms of NEMO into NEMO-null MEFs and determined their effect on TRAF6-induced NF-κB activity. In the presence of wt NEMO, TRAF6 caused an approximately eightfold activation of NF-κB. Both K399R NEMO and K285R NEMO were impaired in their ability to mediate TRAF6-induced NF-κB activity. This K399R effect is in accordance with previously published results (42
). The mutation of both K285R and K399R (K285 399R) substantially diminished TRAF6-induced NF-κB activity (Fig. ). The observation that mutation of K285 to arginine on NEMO completely blocks IκBα phosphorylation in response to LPS (Fig. ), but only partially blocks the NF-κB reporter response to TRAF6 overexpression (Fig. ), may be a consequence of nonphysiological ubiquitination events due to overexpression of TRAF6. Given this concern and to broaden the coverage of TLRs, we transfected the reconstituted NEMO cell lines with an NF-κB reporter construct and treated them with highly purified LPS, TNF, or the chemically synthesized TLR2 agonist PC. NF-κB activation was severely diminished under TLR4 or TLR2 stimulation with both K285R NEMO and K399R NEMO or the double mutant (Fig. ). In contrast, while TNF stimulation was reduced with K399R, it increased slightly in the presence of K285R and close-to-normal activity was seen in the presence of the K285R K399R double mutant (Fig. ). These findings closely mirror those seen in the signaling experiments of Fig. , in which a higher TNF signal is seen in the K285R NEMO-reconstituted cells. In addition, because the recombinant (bacterially produced) TNF and the chemically synthesized PC show different results and because the PC and LPS agonists show similar NEMO ubiquitination dependence, it is unlikely that the effect that we see is due to a contaminant in the preparation of these agonists.
These experiments suggest that TRAF6 can cause NEMO ubiquitination on the same residue of NEMO (K285) as that induced by the NOD2/RIP2 complex. To determine whether activation of NOD2 could activate TRAF6, NOD2 was transfected into cells with myc-tagged TRAF6 and HA-tagged ubiquitin. TRAF6 was immunoprecipitated under stringent conditions, and Western blotting was performed. TRAF6 shows greatly enhanced ubiquitination in the presence of coexpressed NOD2 (Fig. ). To determine if this effect was due to NOD2's binding partner, RIP2, RIP2 expression was inhibited by siRNA and TRAF6 ubiquitination experiments were performed. Upon transfection of TRAF6 and NOD2 into control siRNA-transfected cells, NOD2 continued to cause activation of TRAF6, while in the RIP2 siRNA-transfected cells, activation was greatly diminished (Fig. ). These results suggest that NOD2 activation can induce TRAF6 activation and that this activation is dependent on RIP2.
FIG. 3. NOD2 overexpression activates TRAF6 in a manner dependent on RIP2, and this activity is lost with Crohn's disease-associated alleles of NOD2. (A) HEK 293 cells were transfected with HA-tagged ubiquitin, myc-tagged TRAF6 (1 μg) or wt NOD2 (3 μg). (more ...)
Since we have previously shown that NOD2-induced NEMO ubiquitination is dependent on RIP2 and that this is lost with the Crohn's disease-associated polymorphisms of NOD2 (1
), we sought to determine whether the major Crohn's disease-associated polymorphism of NOD2 (L1007insC) lost the ability to activate TRAF6. Cells were transfected with myc-TRAF6, HA-ubiquitin, and either wt NOD2 or L1007insC NOD2. TRAF6 was immunoprecipitated, and Western blotting was performed using the indicated antibodies. Again, NOD2 strongly induced the ubiquitination of TRAF6, while this activity was greatly diminished in the L1007insC NOD2-transfected cells (Fig. ). In addition, when TRAF6 was cotransfected into cells with either NOD2 or L1007insC and NF-κB promoter activity was assayed, the additive effect on NF-κB activation was greatly diminished in the presence of L1007insC NOD2 (Fig. ). To determine the effect of an additional Crohn's disease-associated allele, D291N, on TRAF6's activation and as a control for a more general effect on the TRAF family of proteins, ubiquitination assays were performed using TRAF6 in the presence of wt NOD2, L1007insC NOD2, or D291N NOD2 or using TRAF2 in the presence of the same NOD2 variants. Again, NOD2 caused the strong ubiquitination of TRAF6, and this was lost when either the L1007insC allele or the D291N allele was used. NOD2 did cause minor ubiquitination of TRAF2; however, this was unaffected by the Crohn's disease-associated alleles (see Fig. S2 in the supplemental material). These findings suggest that NOD2 can activate TRAF6 in a disease-allele specific manner and suggest that the protein kinase RIP2 is required for this activity.
FIG. 4. MDP causes the activation of endogenous TRAF6. (A) THP-1 cells were stimulated with either MDP (10 μg/ml) or PC (1 μg/ml) or were left untreated. Cells were lysed in RIPA buffer and were immunopurified using antiubiquitin-Sepharose beads (more ...)
To test these findings in an endogenous manner, both THP-1 cells and RAW 264.7 macrophages were utilized. THP-1 cells were treated with the NOD2 agonist MDP. Lysates were generated and polyubiquitinated proteins were isolated using antiubiquitin columns (Pierce Biotechnology). Western blotting showed that both MDP and the TLR2 agonist PC could cause ubiquitination of TRAF6 (Fig. , upper blot). The reverse experiment could also be performed in a separate cell line and in a time-dependent manner. RAW 264.7 cells were treated with MDP for the indicated times. Lysates were generated, and TRAF6 IPs were performed. Western blotting was performed using either an anti-TRAF6 or an antiubiquitin antibody. MDP induced TRAF6 ubiquitination in a time-dependent manner (Fig. ). These results could be due to an autocrine/paracrine effect whereby NOD2 activation (either by transfection in 293 cells or by MDP stimulation in monocytes/macrophages) induces the release of cytokine, which then causes activation of TRAF6. Because the time course of TRAF6 activation is relatively acute in the monocytes/macrophages (see Fig. and Fig. ), this scenario is more likely in the transfected 293 cells. To test this possibility, 293 cells were transfected with NOD2 or with L1007insC. The next day, the medium was harvested from these cells and 1 ml of the medium was exposed to a cytokine array designed to test the cytokines present in the medium. In the NOD2-transfected cells, interleukin-8 (IL-8), macrophage chemoattractant protein 1 (MCP-1), angiopoietin, and vascular endothelial growth factor (VEGF) were at high levels (Fig. , bottom blot; for the relative position of each cytokine see Fig. S3 in the supplemental material). None of these cytokines has been shown to signal through TRAF6. Because we could not rule out levels of IL-1 below our detection limits, we applied the remaining conditioned medium from either NOD2- or L1007insC-transfected cells to 293 cells which had been previously transfected with myc-tagged TRAF6 and HA-ubiquitin for 30 min. Weak, but detectable TRAF6 ubiquitination was present; however, the levels of this ubiquitination were similar between cells treated with medium from NOD2-transfected cells and cells treated with medium from L1007insC-transfected cells (Fig. ). In addition, to compare the levels of TRAF6 ubiquitination in cells exposed to NOD2-transfected medium to TRAF6 ubiquitination in cells expressing both TRAF6 and NOD2, cells were also cotransfected with myc-TRAF6, wt NOD2 and HA-ubiquitin, or myc-TRAF6, L1007insC NOD2, and HA-ubiquitin and the levels of these transfectants of TRAF6 ubiquitination were compared to those of the medium-treated cells. In this overexposed blot (Fig. ), cells in which TRAF6 and NOD2 were cotransfected showed much higher levels of TRAF6 ubiquitination than cells exposed to conditioned medium from NOD2-transfected cells (Fig. ). While we cannot conclusively rule out an autocrine/paracrine effect, our results suggest that NOD2's effect on TRAF6 is more likely to be direct.
FIG. 7. RIP2 utilizes the same E2 as TRAF6 to cause NEMO ubiquitination. (A) RIP2 was transfected into 293 cells with the dominant-negative E2 ligase Ubc13 with myc-tagged K399R NEMO and HA-tagged ubiquitin. NEMO was immunoprecipitated, and Western blotting was (more ...)
The above data suggest that NOD2 can activate TRAF6 to affect NEMO ubiquitination. TRAF6 is a member of a family (currently containing seven members) of K63-specific E3 ubiquitin ligases that help coordinate inflammatory signaling, not only via TLRs, but also via TNF, CD40L, TRAIL, and other agonists (4
). Because of this complexity, we sought to determine whether TRAF6 was the only E3 ubiquitin ligase responsible for NOD2/RIP2-induced NEMO ubiquitination. To this end, we inhibited the expression of TRAF6 using two separate siRNAs. RIP2, K399R NEMO, and HA-tagged ubiquitin were transfected into cells previously transfected with either control siRNA or with TRAF6 siRNA. In these sets of experiments, K399R NEMO was utilized due to greatly decreased basal ubiquitination of NEMO (shown previously in reference 1
). TRAF6 expression was greatly inhibited (Fig. ), but RIP2-induced NEMO ubiquitination continued to be strong. To determine whether TRAF6 was required for NOD2-indued NF-κB activation, TRAF6 was again inhibited by siRNA. Either NOD2 or the active Blau syndrome form of NOD2 (R334W) was transfected into cells, and NF-κB activation was assayed. Inhibition of TRAF6 expression did not significantly affect NF-κB activation, while inhibition of RIP2 expression greatly decreased NOD2-induced NF-κB activation (Fig. ). Given that there are now seven published TRAF proteins and given that other E3 ligases have been reported to induce ubiquitination of NEMO (25
), it is not unexpected that while NOD2 can activate TRAF6, TRAF6 loss can be compensated for by other E3 ligases.
FIG. 5. TRAF6 is not the only E3 ubiquitin ligase responsible for NOD2/RIP2-induced NEMO ubiquitination. (A) TRAF6 expression was inhibited by siRNA (second blot from bottom) using two siRNAs designed to be unique to TRAF6, and RIP2-induced NEMO ubiquitination (more ...)
Because TRAF6 was not absolutely required for NOD2/RIP2-dependent NEMO ubiquitination, we considered a model in which the NOD2/RIP2 complex and TRAF6 utilize the same ubiquitin-dependent signaling components to activate IKK. To determine this, we took a biochemical approach. NEMO was immunopurified from cells transfected with either TRAF6 or RIP2. NEMO was eluted from the anti-myc- coupled protein G beads, and the eluate was subjected to SDS-PAGE followed by Coomassie staining. Stained bands were excised and subjected to mass spectrometry analysis. In both the TRAF6-transfected cells (Fig. ) and the RIP2-transfected cells (Fig. ), the TAK1/TAB kinase complex was present in the NEMO purification. This complex is activated by K63-linked ubiquitin chains (33
), and the binding to ubiquitinated NEMO would represent a mechanism by which TAK1 would have proximity to IKK such that it could phosphorylate IKK's activation loop. To test RIP2's dependence on TAK1 to activate IKK, HA-tagged IKK was transfected into cells with RIP2 and/or kinase-dead TAK1 (K63A). As a control for ubiquitin dependence, the transfection was also performed with the K63 deubiquitinase, CYLD, an inhibitor of RIP2-induced IKK activation (1
). IKK was immunoprecipitated, and Western blotting was performed utilizing an antibody that recognizes the phosphorylated activation loop of IKK. Figure shows that RIP2 activates IKK and that this activation is strongly inhibited by coexpression of kinase-dead TAK1. This set of experiments suggests that while TRAF6 is not the only E3 ligase downstream of the NOD2/RIP2 complex, TRAF6 and NOD2/RIP2 share a common ubiquitin-dependent signaling complex (TAK1/TAB) to activate IKK.
FIG. 6. RIP2 and TRAF6 utilize the TAK1/TAB complex of proteins to induce NEMO ubiquitination and IKK activation. (A) myc-tagged wt NEMO and Omni-tagged TRAF6 were transfected into cells. wt NEMO was immunopurified via four washes with modified RIPA buffer, four (more ...)
Given NOD2/RIP2's dependence on the TAK1/TAB complex and given that TRAF6 and TAK1 also require the K63-specific E2s Ubc13 and Uev1a to activate NF-κB (5
), we sought to determine whether the NOD2/RIP2 complex also required Ubc13 to signal to NF-κB. To this end, we transfected K399R NEMO (again to minimize background NEMO ubiquitination) with RIP2, HA-tagged ubiquitin, and/or dominant-negative Ubc13 (C87A). K399R NEMO was immunoprecipitated, and Western blotting was performed. RIP2 induced the ubiquitination of NEMO, and expression of dominant-negative Ubc13 strongly inhibited this ubiquitination (Fig. ). To determine the in vivo effect of loss of Ubc13 on NOD2/RIP2-induced NF-κB activation, two cell lines (RAW 264.7) stably expressing separate short hairpin RNAs (shRNAs) designed to inhibit the expression of Ubc13 were generated. These cells were treated with the NOD2 agonist MDP for the indicated times, and lysates were generated. Western blotting was performed with the indicated antibodies. Figure shows that both of these cell lines have limited expression of endogenous Ubc13. In both of these cell lines, MDP-induced NF-κB activity was substantially lower as judged by phospho-IκB in the Ubc13-knockdown cells (Fig. ). The same cell lines were then treated with MDP for 0, 30, 45, and 60 min. NEMO was immunoprecipitated under stringent washing conditions, and Western blotting was performed to determine whether Ubc13 loss correlated with lack of NEMO ubiquitination. In both of the Ubc13-knockdown cell lines, NEMO ubiquitination was substantially decreased, while NEMO ubiquitination was intact in the control cell line (Fig. ). Collectively, these results suggest that TRAF6 and NOD2/RIP2 share a common E2 complex to ubiquitinate NEMO and activate NF-κB.
If extracellular innate immune signaling (via TRAF6) and intracellular innate immune signaling (via NOD2/RIP2) utilize common ubiquitin-dependent molecular scaffolds to regulate cytokine release, then these two innate immune pathways could synergize to coordinate cytokine release. To test this, we utilized the chemically synthesized TLR2 agonist PC for experiments in THP-1 monocytes (a human cell line with high expression of NOD2) (11
). PC has the advantage of being chemically synthesized such that there is no contamination with MDP. We stimulated THP-1 cells with PC and/or the NOD2 agonist MDP. After exposure to these agonists, medium was collected and subjected to cytokine array analysis. Each of the 40 cytokines and their relative positions on the array are shown in Fig. S3 in the supplemental material. With no treatment, there was little cytokine release from the THP-1 cells. MDP caused only minor IL-8 release (Fig. , right top panel). This result is consistent with previous studies that have shown that MDP only causes slight cytokine release (19
). PC caused IL-8 and RANTES to be strongly upregulated, while treatment with both PC and MDP caused release of GRO, IL-6, MCP-1, and, to a lesser extent, TNF (Fig. , lower panels). Because we were concerned about the linearity of cytokine array analysis, these findings were further quantified by IL-6 enzyme-linked immunosorbent assays (ELISAs), which also showed an additive effect of PC and MDP (Fig. ), and by TaqMan real-time RT-PCR, which showed additive and synergistic increases of MCP-1 and IL-8 mRNA (Fig. ). To correlate these findings with NF-κB activation, macrophages were treated with 500 ng/ml PC, 10 μg/ml MDP, or both PC and MDP for 15, 30, or 60 min. NF-κB activation was monitored by IκB degradation and subsequent transcriptional activation. By itself, MDP caused a small amount of IκB degradation that peaked at 60 min. PC caused IκB degradation at 15 min, with undetectable IκB at 30 min. When both MDP and PC were added, there was no detectable IκB 15 min after treatment and there was increased IκB at 60 min (Fig. ). The phospho-IκB blot was consistent with these results. MDP alone caused phosphorylation of IκB at 30 and 60 min, the time when total IκB begins to decrease. PC caused earlier phosphorylation of IκB. When MDP and PC were added at the same time, there was no detectable phospho-IκB at 15 min due to the enhanced IκB degradation. In addition, the amount of IκB transcriptionally upregulated by 60 min was higher, as was the residual IKK activity (reflected by phospho-IκB) (Fig. ). To correlate these findings with NEMO ubiquitination, cells were treated with MDP, PC, or both MDP and PC for 0, 15, 30, and 60 min. Lysates were generated, and NEMO was immunoprecipitated. MDP alone caused NEMO ubiquitination at 30 and 60 min, while PC treatment caused a large degree of NEMO ubiquitination at 15 min but more limited NEMO ubiquitination at later time points (Fig. ). When MDP and PC were added together, NEMO ubiquitination occurred earlier (relative to MDP treatment alone) and was significantly prolonged (Fig. ). The results of these signaling experiments (Fig. ) correlate very well with the cytokine expression results of Fig. and suggest that rather than serving solely as a driver of an inflammatory response, MDP may be serving to modulate TLR signaling. These findings also provide further evidence for cross talk between extracellular and intracellular bacterial sensing systems.
FIG. 8. MDP, a NOD2 agonist, and PC, a TLR2 agonist, synergize to increase cytokine release, and this correlates with a prolonged NF-κB activation and with prolonged NEMO ubiquitination. (A) MDP and PC synergize to increase specific cytokine expression. (more ...)