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B Kinase Complex and Its Activation in Human Neutrophils 1
Pulmonary Division, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Québec, Canada
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Abstract |
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B/Rel proteins and IB-
in the nucleus. In this study, we show that in these cells, IB kinase (IKK), IKK
, and IKK
also partially localize to the nucleus, whereas IKK-related kinases (IKK
, TANK-binding kinase-1) are strictly cytoplasmic, and the NF-B-inducing kinase is strictly nuclear. Following neutrophil activation, IKK and IKK become transiently phosphorylated in both the cytoplasm and nucleus, whereas IKK transiently vanishes from both compartments in what appears to be an IKK-dependent process. These responses are paralleled by the degradation of IB-, and by the phosphorylation of RelA on serine 536, in both compartments. Although both proteins can be IKK substrates, inhibition of IKK prevented IB- phosphorylation, while that of RelA was mostly unaffected. Finally, we provide evidence that the nuclear IKK isoforms (, , ) associate with chromatin following neutrophil activation, which suggests a potential role in gene regulation. This is the first study to document IKK activation and the phosphorylation of NF-B/Rel proteins in primary neutrophils. More importantly, our findings unveil a hitherto unsuspected mode of activation for the IKK/IB signaling cascade within the cell nucleus. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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B/Rel proteins are ubiquitous transcription factors that play a central role in immunity and inflammation (reviewed in Refs. 1 and 2). In resting cells, NF-B complexes are typically sequestered in the cytoplasm through their interaction with inhibitory IB proteins (3, 4, 5, 6). In response to a wide array of stimuli, IB proteins become phosphorylated, leading to rapid ubiquitination and subsequent proteolysis by the 26S proteasome (7). This effectively frees the NF-B dimers, which can translocate to the nucleus to bind cognate B enhancer elements, and activate the transcription of several genes encoding (among others) inflammatory cytokines and chemokines, as well as IB- (8). Newly synthesized IB can shuttle into the nucleus, physically remove NF-B from DNA, and export the inactive NF-B/IB complex back to the cytoplasm to restore the latent state (9).
Most of the signals leading to NF-B activation converge on the activation of a high m.w. complex, the IB kinase (IKK)
3 signalosome (10), which comprises at least three subunits. The first two (IKK and IKK) are related catalytic subunits, whereas IKK is a regulatory subunit that serves as a scaffold for the former. Activation of the IKK complex involves the phosphorylation of serine residues within the activation loop of IKK and IKK, resulting in kinase activation (11, 12, 13, 14, 15, 16), although the main isoform that phosphorylates IB- appears to be IKK (17). In addition, it was described recently that both IKK and IKK can be recruited to the nucleus, where they associate with chromatin, a process that is required for the appropriate transcriptional activation of NF-B targets (18, 19, 20). Similarly, IKK has been demonstrated to translocate to the nucleus, where it interacts with CREB-binding protein to repress RelA-induced transcriptional activation (21). Finally, two IKK-related kinases, IKK (or IKKi) (22, 23) and TANK-binding kinase (TBK) 1 (also known as NAK or T2K) (24, 25, 26), have been implicated in NF-B activation. The first can phosphorylate IB- (23), while TBK-1 can activate IKK through direct phosphorylation and may induce IKK-mediated IB degradation and NF-B activation (25).
The manner in which IKK isoforms become phosphorylated remains unclear, but two models have been proposed. The first involves phosphorylation of the IKKs by a MEK kinase, such as NF-B-inducing kinase (NIK) (27, 28), MEK kinase 1 (29), and TBK-1 (25, 26). The other model suggests that IKK recruitment to the receptor complexes at the cell membrane results in its autophosphorylation and subsequent activation (7). A mounting body of evidence, however, suggests that the control of the NF-B pathway extends beyond the IKK-mediated regulation of IB/NF-B. Indeed, while the binding of NF-B to enhancer elements fulfills the basic requirements for promoter activation, the transcriptional activity of NF-B is further modulated by posttranscriptional modifications such as acetylation and/or phosphorylation of its subunits. For example, the phosphorylation of RelA greatly enhances its trans activation potential (reviewed in Ref. 30). The identity of the kinase involved remains elusive, but candidates include the catalytic subunit of protein kinase A (PKA), casein kinase II, IKK, IKK, TBK-1, and IKK (31, 32, 33, 34, 35, 36).
Neutrophils are best known for their role as professional phagocytes, but they can also express a wide array of cytokines and chemokines (reviewed in Ref. 37). Most of these mediators contain B (or B-like) motifs in their promoter region that are needed for transcriptional inducibility (38). Accordingly, we initially demonstrated that numerous agonists known for their ability to induce cytokine production in neutrophils can also activate NF-B in these cells (39, 40, 41, 42); these findings were later confirmed and extended by other investigators (reviewed in Ref. 43). Although NF-B activation in these cells is preceded by the phosphorylation and degradation of cytoplasmic IB- (39, 40, 41), and while it involves a nuclear accumulation of NF-B/Rel proteins (39, 44, 45, 46), a singular feature of neutrophils is that they constitutively express substantial amounts of NF-B/Rel proteins, and of their inhibitor, IB-, in the nucleus (39, 44). Moreover, neutrophil activation results in the degradation of IB- in both the cytoplasm and the nucleus (Ref. 44 and our unpublished data). These observations prompted us to investigate whether upstream components of the IKK signaling cascade might similarly be expressed and activated in the nucleus. We now report that IKK activation, IB- phosphorylation and degradation, and RelA phosphoryation all occur in both the nucleus and cytoplasm of human neutrophils. To our knowledge, this is the first study to document the activation of the IKK signaling cascade within the cell nucleus.
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Materials and Methods |
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Abs raised against NIK (sc-7211, sc-8417), TBK-1 (sc-9085), all IKK isoforms (sc-7606, sc-8014, sc-8330, sc-9914), and IB- (sc-371) were from Santa Cruz Biotechnology, whereas all phospho Abs (2687, 2694, 2689, and 3031) were from Cell Signaling Technology. Rabbit antisera raised against leukotriene A4 hydrolase and five lipoxygenase-activating protein were generously given by Dr. J. Evans of Merck Frosst Canada (Pointe-Claire, Quebec, Canada). Ficoll-Paque was from Pharmacia; endotoxin-free (<6 pg/ml) RPMI 1640 and FCS were from Wisent and HyClone, respectively. Recombinant cytokines were from R&D Systems, and LPS (from Escherichia coli 0111:B4) was from List Biological Laboratories. Cycloheximide, diisopropyl fluorophosphate (DFP), fMLP, leptomycin B, and PMSF were from Sigma-Aldrich. PGA1, PGE2, and 15-deoxy-PGJ2 were from Cayman Chemical. The protease inhibitors, aprotinin, AEBSF/Pefabbc, leupeptin, and pepstatin A were from Roche. Signaling pathway inhibitors (SB 203580, PD 98059, SP-600125, wortmannin, LY-294202, H89, 6-dichloro-1--D-ribofuranosylbenzimidazole, MG-132, leptomycin B) were from Calbiochem. All other reagents were of the highest available grade, and all buffers and solutions were prepared using pyrogen-free clinical grade water.
Cell isolation and culture
Neutrophils were isolated from the peripheral blood of healthy donors under endotoxin-free conditions by a modification of the method of Boyum (47), as described (48). Purified neutrophils were resuspended in RPMI 1640 supplemented with 10% FCS, at a final concentration of 5 x 106 cells/ml, and cultured in tissue culture grade plasticware at 37°C under a humidified 5% CO2 atmosphere (unless otherwise stated). As determined by Wright staining and nonspecific esterase cytochemistry, neutrophil suspensions consistently contained fewer than 0.5% monocytes or lymphocytes, and neutrophil viability exceeded 97% after up to 6 h in culture, as determined by trypan blue exclusion.
Denaturing electrophoreses and immunoblots
Cells were incubated at 37°C in the presence or absence of inhibitors or stimuli, as specified in figure legends. Incubations were stopped by adding equal volumes of ice-cold PBS supplemented with DFP (2 mM, final concentration) and phosphatase inhibitors (10 mM NaF, 1 mM Na3VO4, 10 mM Na4P2O7), before centrifugation at 300 x g for 5 min at 4°C. Cells were resuspended in ice-cold Relaxation buffer (10 mM PIPES, pH 7.30, 10 mM NaCl, 3.5 mM MgCl2, 0.5 mM EGTA, 0.5 mM EDTA, 1 mM DTT) supplemented with an antiprotease mixture (1 mM DFP, 1 mM PMSF, 1 mM AEBSF/Pefabbc, and 10 µg/ml each of aprotinin, leupeptin, and pepstatin A, final concentrations) and the aforementioned phosphatase inhibitors. Neutrophils were then disrupted by nitrogen cavitation, as described previously (39, 49). For whole-cell samples, a small aliquot was taken from the resulting cavitate for subsequent protein content determination. Boiling sample buffer was directly added to the samples, which were placed in boiling water for an additional 3 min. For cellular fractions, the cavitates were centrifuged for 10 min (1000 x g, 4°C); the resulting supernatants represent the cytoplasmic fractions, and the corresponding nuclear pellets were washed twice in Relaxation buffer containing the inhibitor mixture, and resuspended in the same volume as their cytoplasmic counterparts. After taking a small aliquot from each sample (for subsequent protein content determination), concentrated sample buffer (prewarmed at 95°C) was directly added to either cytoplasmic or nuclear fractions, before a 3-min incubation at 95°C. All samples were electrophoresed on denaturing gels according to the method of Laemmli (50); equal loading was ascertained by adjusting sample volumes based on their protein content. Following SDS-PAGE, proteins were transferred onto nitrocellulose membranes, which were stained with Ponceau Red, destained, and then processed for immunoblot analysis, as previously described (39).
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Results |
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Because neutrophil stimulation entails the degradation of IB- in both the nucleus and cytoplasm (Refs. 44 and 51 and our unpublished data), we examined whether upstream IKK cascade components are similarly localized. Resting neutrophils were therefore disrupted by nitrogen cavitation, and cell fractions were prepared. For comparison, autologous PBMCs were similarly prepared and analyzed in parallel. Fig. 1A shows that in neutrophils, the three constituent subunits of the IKK complex (IKK, IKK, IKK) distribute in both the cytoplasmic and nuclear compartments, whereas in autologous PBMC all three isoforms are cytoplasmic, as expected. Neutrophils were also found to weakly express the related IKK isoform, IKK, albeit only in the cytosol (Fig. 1A). The unexpected detection of several IKK isoforms in the nucleus of neutrophils prompted us to ascertain the purity of our subcellular fractions, which were analyzed for the presence of cytosolic and nuclear markers. As shown in Fig. 1B, strictly cytosolic proteins such as lactate dehydrogenase and leukotriene A4 hydrolase (52) were indeed exclusively detected in cytoplasmic fractions. Conversely, 5-lipoxygenase-activating protein, which localizes to the nuclear envelope in neutrophils (53), was only present in the nuclear fractions (Fig. 1B). Similarly, histone H3 was exclusively nuclear, as expected (Fig. 1B). Thus, we conclude that our nuclear and cytoplasmic fractions are reasonably exempt from cross-contamination. This is consistent with our previous studies using similar (although not identical) cell fractionation procedures (39, 49).
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B activators such as LPS and TNF did not affect the distribution of any IKK isoform for up to 6 h (Fig. 2A, and data not shown). Unexpectedly, however, both the nuclear and cytoplasmic levels of IKK were found to diminish substantially upon neutrophil stimulation (Fig. 2A). This phenomenon was usually detectable by 10 min (Fig. 2A) and most marked between 30 and 90 min (Fig. 2, A and B), but was also transient, insofar as IKK levels had usually replenished by 2–3 h (Fig. 2B, and data not shown). To determine whether the loss of IKK was due to proteolytic degradation, neutrophils were pretreated with the proteasome inhibitor, MG-132, before stimulation; alternatively, cells were pretreated with cycloheximide to determine whether IKK reappearance reflects de novo protein synthesis. As shown in Fig. 2C, MG-132 partially prevented the loss of IKK at the 45-min time point (lane 2 vs the forelast lane), but this interference was no longer evident by the time in which the IKK protein was reaccumulating (i.e., 90 min in the depicted experiment). A similar partial inhibition by MG-132 of IKK loss was also observed in neutrophils stimulated for 30 min (data not shown). By comparison, MG-132 completely prevented the degradation of IB-, a process known to be mediated by the proteasome, at all time points examined (Fig. 2C). Thus, the partial inhibition of IKK disappearance by MG-132 cannot be attributed to an incomplete inhibition of the proteasome, and further indicates that, in addition to the proteasome, other mechanisms are involved in the inducible loss of IKK. Similar results were obtained using another proteasome inhibitor, MG-262 (data not shown). Fig. 2C also shows that cycloheximide hindered the re-expression of IKK at later time points, while completely preventing that of IB-, indicating a potential role for de novo protein synthesis in these processes. In contrast to IKK, the levels of IKK and IKK were unaffected by cycloheximide for up to 4 h, whether in resting or activated cells, indicating a slow turnover (Fig. 2C, and data not shown). In unstimulated neutrophils, IKK levels were also unaltered by cycloheximide for up to 4 h (data not shown), suggesting a similarly slow turnover (provided that the cells are not activated).
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We next investigated whether the IKK complex becomes activated upon neutrophil stimulation. For this purpose, we monitored the phosphorylation state of individual IKK subunits, as well as the phosphorylation and degradation of the immediate IKK substrate, IB-. Additionally, we also examined whether another IKK substrate, RelA, becomes phosphorylated upon cell activation. As shown in Fig. 3A, neutrophil exposure to LPS caused a rapid and transient phosphorylation of what appeared to be IKK (lower band) and IKK (upper band) in both the cytoplasm and nucleus, which peaked by 10 min and gradually decreased thereafter, having returned to basal levels by 90 min (Fig. 3A, and data not shown). The phosphorylation of the upper band was always much stronger than that of the lower band, which initially suggested that IKK is more extensively phosphorylated than IKK. However, while the Ab used recognizes both IKK isoforms, and while the band pattern matches the expected behavior of phosphorylated IKK and species, immunodepletion of IKK from the samples did not affect the detection of either band (data not shown). This suggested that the two protein bands recognized by our dual Ab must principally represent IKK. The subsequent availability of an Ab specific for phospho-IKK revealed that an identical band pattern is observed using either this Ab or the dual-specificity Ab (Fig. 3B). Thus, it appears that LPS potently promotes the phosphorylation of IKK, but not that of IKK. Fig. 3A also shows that LPS induces the phosphorylation of IKK, IB-, and RelA with kinetics similar to that of IKK; again, these phenomena take place both in the nucleus and cytoplasm. When neutrophils were stimulated with TNF- instead of LPS, a very similar pattern was observed (Fig. 3C), in keeping with the fact that these two stimuli affect the NF-B/IB cascade in much the same way in neutrophils (39, 40).
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B cascade components between the cytoplasm and nucleus in human neutrophils
The presence of IKK isoforms and of IB- in both the cytoplasm and nucleus of neutrophils led us to investigate whether some of these proteins shuttle between these cell compartments, as demonstrated for IB- in other cell types (54, 55, 56). Neutrophils were therefore cultured in the presence or absence of leptomycin B (to block nuclear export), before stimulation with LPS or TNF for 4 h. As shown in Fig. 4A, leptomycin B failed to alter the cellular distribution of the three main IKK isoforms, whether in resting or activated cells, indicating that they do not shuttle between the cytoplasm and nucleus. In contrast, leptomycin treatment resulted in a moderate accumulation of IB- and RelA in the nucleus of neutrophils stimulated with LPS or TNF for 4 h (Fig. 4A). In resting cells, a similar shift in the distribution of IB- and RelA was not observed after 4 h of culture, and was only detectable after 8 h (Fig. 4A), indicating a very low rate of shuttling for IB- and RelA in unstimulated neutrophils. To determine whether leptomycin B might cause IB- and RelA to accumulate in the nucleus of activated neutrophils by mechanisms other than interference with nuclear export, we conducted time-course experiments at shorter stimulation times, i.e., under conditions in which intracellular shuttling is not detectable. As shown in Fig. 4B, leptomycin B delayed the inducible degradation of both cytoplasmic and nuclear IB-. This probably reflected an effect on the proteolysis of IB-, because leptomycin failed to affect the inducible phosphorylation of IKK or of IB- itself (Fig. 4B, and data not shown). Thus, leptomycin B appears to affect the NF-B cascade by acting on more than just nuclear export.
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We next determined whether some upstream activators of the IKK complex are expressed in neutrophils. As shown in Fig. 5A, neutrophils were found to express both NIK and TBK-1. On a per-cell basis, neutrophils contained less NIK, but much more TBK-1, than autologous PBMC (Fig. 5A). Although both proteins are reportedly cytoplasmic, NIK was restricted to the nucleus in neutrophils, whereas TBK-1 was cytoplasmic (Fig. 5B); the cellular distribution of these kinases was unaltered following cell stimulation with either LPS or TNF (Fig. 5B, and data not shown). In an effort to unveil other potential kinase(s) or pathway(s) involved in IKK activation, neutrophils were pretreated with inhibitors of MAPK pathways (p38 MAPK, MEK/ERK, JNK), of casein kinase II, of PI3K, of PKA, or with cyclopentenone PGs reputed to inhibit IKK (PGA1, 15-deoxy-PGJ2), before stimulation with LPS or TNF for 15 min, and immunoblot analysis of whole-cell cavitates. As shown in Fig. 6 for LPS-treated cells, the above inhibitors failed to significantly alter IKK subunit phosphorylation, IB- degradation, or RelA phosphorylation, with one notable exception. Indeed, PGA1 and 15-deoxy-PGJ2 (unlike the related prostanoid, PGE2) largely prevented IKK phosphorylation and IB- degradation, while marginally affecting RelA and IKK phosphorylation (Fig. 6, and data not shown). Interestingly, we also found that cyclopentenone PGs substantially hindered the inducible loss of IKK (Fig. 6B), indicating a role for IKK activation in this process. Nearly identical results were obtained when cytoplasmic and nuclear fractions were examined, or when TNF- was used instead of LPS (data not shown). These effects of the cyclopentenone PGs are in keeping with their demonstrated ability to inactivate IKK activity by a direct modification of cysteine 179 within IKK (57), and with the recent demonstration that 15-deoxy-PGJ2 blocks IB- degradation in human neutrophils (58).
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Because individual IKKs (, , ) were recently shown to associate with chromatin following cell stimulation with TNF (18), and because neutrophils constitutively express these proteins in the nucleus, we investigated whether IKKs might readily or inducibly associate to chromatin in these cells. To this end, nuclear fractions from resting or stimulated neutrophils were processed in various ways. To determine whether the IKKs bind to chromatin in much the same way as NF-B/Rel proteins, nuclei were submitted to salt extraction to yield the same nuclear extracts as those used for EMSA analyses, and compared with the residual nuclei (postextraction). In parallel, nuclei were deliberately lysed in high salt (800 mM NaCl). In a more aggressive approach, we sonicated the nuclei, a procedure known to shear chromatin and to disrupt nuclear membranes, which was used by the group that recently demonstrated the association of the IKKs with chromatin (18). As shown in Fig. 7A, only the latter procedure allowed for a partial dissociation of IKK proteins from isolated neutrophil nuclei; importantly, this only took place in stimulated cells. These results therefore constitute indirect evidence of an inducible association of the IKKs with chromatin in activated neutrophils. Because it was recently shown that at least IKK can phosphorylate histone H3 on serine 10 (18), we investigated whether IKK inhibition with cyclopentenone PGs would alter the phosphorylation of histone H3. Unexpectedly, we found that histone H3 is strongly phosphorylated in unstimulated neutrophils, and that LPS or TNF stimulation does not markedly enhance this phosphorylation over a time course spanning up to 60 min (Fig. 7B). Similarly, 15-deoxy-PGJ2 failed to affect constitutive histone H3 phosphorylation in either resting or activated neutrophils (Fig. 7C). Although these results do not exclude a role for chromatin-bound IKKs in neutrophils, they indicate that histone H3 is probably not a target of activated IKKs in these cells.
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Discussion |
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B- phosphorylation in primary human neutrophils. The rationale for focusing on these cells is 2-fold: neutrophils and their products (including many B-dependent cytokines/chemokines) are of undeniable physiological relevance, and their NF-B system features unusual characteristics. In particular, we and others showed that roughly half of the cellular content of p50, RelA, c-Rel, and IB- localizes to the nucleus in unstimulated neutrophils (39, 44), and that neutrophil activation results in the degradation of IB- in both the cytoplasm and the nucleus (Ref. 44 and this study). We now report that neutrophils represent a unique example of an inflammatory cell type in which the main events associated with NF-B activation (namely, IKK activation, IB- phosphorylation/degradation, and RelA phosphorylation) can all occur within the nucleus, in addition to the cytoplasm. Another intriguing feature of neutrophils is that their activation leads to the rapid and transient disappearance of IKK in both cellular compartments. Finally, we present evidence that nuclear IKK isoforms may inducibly associate with chromatin in activated neutrophils. Together, our findings add several novel and unexpected facets to the current understanding of how the NF-B pathway is activated.
Despite the fact that many aspects of NF-B activation in neutrophils have been investigated since our initial studies on the subject 8 years ago (39, 40, 41), the current study is the first to describe that RelA can undergo inducible phosphorylation in these cells, and that they express various components upstream of IB, such as functional IKK isoforms and the related kinases, TBK-1 and NIK. Unexpectedly, neutrophil activation by stimuli such as LPS or TNF led to the loss of cellular IKK, a phenomenon that to our knowledge is unprecedented, and whose purpose remains unclear. We showed that IKK loss can be partially prevented by proteasome inhibitors, which raises the possibility that the protein is somehow targeted for proteolytic degradation. That this proteolysis took place in both the cytoplasm and nucleus is consistent with the presence of the proteasome in both compartments. We also found that IKK inhibition (using cyclopentenone prostanoids) effectively prevented the inducible loss of IKK in stimulated neutrophils. Whether this is required for (or independent of) the proteasome-mediated degradation of IKK is currently under study. In any event, the rapid fall in IKK levels observed upon neutrophil stimulation indicates that to a great extent, the activated and functional IKK complex of neutrophils must mainly comprise IKK and IKK. Such a scenario is consistent with the recent demonstration that endogenous IKK-IKK subcomplexes exist in the cytosplasm of human monocytic THP-1 cells, and display increased IKK activity following cell stimulation with TNF (59). Similarly, immunodepletion studies showed the presence of IKK-IKK complexes activated by anti-CD3/CD28 in primary human T cells (60).
Neutrophil activation by physiological stimuli led to a robust IKK activation, as determined by the phosphorylation of its constituent subunits, and of its prototypical substrate, IB-. A notable exception was IKK, whose phosphorylation was undetectable under all conditions tested, be it on serines 176/180 (as shown in this study) or on threonine 23 (our unpublished data). In this regard, it is conceivable that the rapid fall in IKK levels occurring upon neutrophil stimulation contributed to the lack of detectable IKK phosphorylation. Whatever the case may be, the combined loss of IKK and its apparent lack of inducible phosphorylation suggest that the protein contributes little to the responses elicited by classical neutrophil stimuli (although it cannot be excluded that IKK might become phosphorylated under specific conditions). By contrast, neutrophil stimulation results in the phosphorylation of IKK on serines 177 and/or 181, and of IKK on serine 376. This is consistent with the fact that IKK phosphorylation enables the complexed kinase to phosphorylate substrates such as IB- and RelA. Similarly, studies performed using deletion mutants of IKK have shown that the C-terminal portion of the protein (i.e., the region being phosphorylated in our experiments) is needed for IKK activation via adapter proteins, as well as for downstream NF-B activation (61, 62). Accordingly, we observed that the onset of IKK/IKK phosphorylation faithfully mirrored the kinetics of IB- phosphorylation and degradation, RelA phosphorylation, and NF-B DNA-binding activity in neutrophils (this study and Refs. 39 , 40 , and 44). This strong correlation supports the notion that all of these responses are linked in neutrophils, as observed in many other cell types, albeit with one key variation, i.e., that the entire process can take place within the cell nucleus in human neutrophils.
In the particular case of RelA phosphorylation on serine 536, the link with IKK activation is probably not a direct one in neutrophils, despite the similar localization and activation kinetics of both proteins, and despite the fact that RelA increasingly appears to be a major IKK target. Indeed, specific inhibition of IKK with cyclopentenone PGs only had a modest effect on the inducible phosphorylation of RelA in LPS-treated cells, and minimally affected this response in TNF-treated neutrophils. Although the incomplete inhibition of RelA phosphorylation potentially leaves room for a (minor) participation of IKK in this process (at least in LPS-stimulated cells), other kinases clearly play a more prominent role. Among the kinases that have the potential to phosphorylate RelA in response to cell activation, casein kinase II and PKA can be excluded, because potent inhibitors of these two pathways failed to alter inducible RelA phosphorylation in neutrophils. Likewise, inhibitors of several other signaling cascades were ineffective. Thus, the identity of the kinase(s) responsible for the phosphorylation of RelA remains elusive at this stage. Because this occurs in both the cytoplasm and nucleus in neutrophils, the upstream kinase would ideally be present in both cell compartments. Alternatively, different kinases might phosphorylate RelA depending on its cellular localization. In keeping with such a scenario, neutrophils were found to express TBK-1 and IKK in the cytoplasm, and both kinases were recently shown to phosphorylate RelA on serine 536 (36, 63). To date, however, our data only offer circumstancial evidence for such a link, and provide no clues as to the identity of the kinase that phosphorylates RelA in the nucleus, thereby emphasizing the need for further studies.
The identity of the kinase(s) phosphorylating the IKK constituent subunits (be it in neutrophils or in other cell types) is similarly uncertain. In this regard, a wide array of signaling cascade inhibitors failed to significantly alter IKK activation in neutrophils, thereby yielding no clues as to which kinase (if any) is involved. Nevertheless, several upstream kinases have been proposed to act as IKK kinases. One such candidate is NIK, which has been shown to associate with, and to phosphorylate, both IKK and IKK (28, 64, 65). Another one is TBK-1, which can phosphorylate IKK (25, 63). Both were found to be differentially expressed in neutrophils relative to autologous PBMC, with the former less abundant, but strictly nuclear in neutrophils, while TBK-1 is cytosolic in both cell types (but far more abundant in neutrophils). However, it is not clear at this juncture whether NIK and TBK-1 become activated upon neutrophil stimulation, and whether their activation kinetics would match those of IKK subunit phosphorylation in both the cytoplasm and nucleus. Conversely, it is equally conceivable that neither NIK nor TBK-1 is involved in IKK activation; gene knockout studies have indeed raised some doubts as to whether NIK and TBK-1 actually function as IKK kinases (66, 67). Similarly, it has been proposed that IKK activation might stem from cross-phosphorylation of its constituent subunits upon their assembly into a signalosome complex (7). This represents an attractive concept as far as cytoplasmic IKK is concerned, especially because inhibition of IKK activity (with cyclopentenone prostanoids) markedly decreased IKK phosphorylation. In the case of nuclear IKK, however, the assembly of a signalosome in the absence of receptor-associated adaptor proteins is more difficult to envisage. Therefore, a role for a nuclear IKK kinase cannot be ruled out.
The occurrence of a nuclear NF-B activation cascade (such as the one described in this work for human neutrophils) conceivably allows for redundancy, yet the need for, or eventual benefit of, such redundancy is not obvious. Nevertheless, the very existence of a nuclear IKK/IB cascade raises a number of interrogations, such as whether it favors a constitutive association of IKK isoforms with chromatin following cell stimulation, as recently reported (18), and whether the IKKs might even constitutively bind chromatin in resting cells. In this regard, we presented indirect evidence of an inducible association of all three components of the IKK complex to chromatin. Such an association still awaits confirmation by other, more direct means, but is in keeping with observations made in the Jurkat lymphoid cell line following TNF stimulation (18), which suggest a role for the IKKs in gene regulation. Another interrogation stemming from the presence of constitutive nuclear IKK in neutrophils is whether there is some shuttling of components between the nucleus and cytoplasm. In this respect, nuclear export blockade experiments showed no detectable exchange of IKK isoforms between the cytosol and nucleus, be it in resting or activated neutrophils, so that the simultaneous activation of IKK in both compartments is likely to reflect parallel processes. These results also suggest that the inducible association of IKK isoforms with chromatin mainly involves nuclear IKKs. By contrast, the same experiments showed that RelA and IB- do shuttle to a limited extent in unstimulated neutrophils, in agreement with observations made in other cell types (9, 36, 55, 56, 68, 69, 70). This, however, required that neutrophils be cultured for at least 6–8 h, which is probably why a recent study failed to report this phenomenon in resting neutrophils (51). In activated neutrophils, IB-, p50, and RelA also accumulated in the nucleus of neutrophils cultured in the presence of leptomycin B. This was evident at earlier time points (2–4 h) and on a larger scale than in resting cells, supporting the notion that IB- and Rel protein shuttling is more pronounced in stimulated cells, as recently reported for IB- in neutrophils (51). In activated cells, we also found that leptomycin B interfered with IB- degradation (both in the cytoplasm and nucleus), without visibly affecting its resynthesis, thereby contributing to a larger accumulation of the protein (relative to cells activated in the absence of leptomycin). Why this accumulation preferentially distributes in the nucleus is less certain, but it is conceivable that the blocking of nuclear export leads to the nuclear retention of NF-B/Rel proteins (following NF-B activation), which in turn favors the nuclear distribution of newly synthesized IB-. We have finally shown that the delayed IB- degradation observed in leptomycin B-treated cells probably reflects interference with the proteolysis of IB-, as opposed to its phosphorylation, as IKK and IB- phosphorylation were unaffected by leptomycin B (this study and our unpublished data). In support of this conclusion, leptomycin B has indeed been reported to hinder the catalytic activity of the proteasome (54), which is believed to be responsible for the inducible degradation of IB-.
In conclusion, the demonstration of a nuclear IKK/IB cascade adds a new facet to our understanding of NF-B activation, and of neutrophil biology. Much remains to be learned about NF-B activation in neutrophils, which arguably represent a most interesting (and possibly unique) cellular model in which to study this fundamental process.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported by grants to P.P.M. from the Canadian Institutes of Health Research (Grant MOP 62705), the Canadian Foundation for Innovation (Grant 3946), and the Gouvernement du Québec. P.P.M. is a Scholar of the Medical Research Council of Canada. 
2 Address correspondence and reprint requests to Dr. Patrick P. McDonald, Pulmonary Division, Faculty of Medicine, Université de Sherbrooke, 3001 12e avenue Nord, pièce 4849, Sherbrooke, Québec, Canada J1H 5N4. E-mail address: patrick.mcdonald{at}USherbrooke.ca
3 Abbreviations used in this paper: IKK, IB kinase; DFP, diisopropyl fluorophosphate; NIK, NF-B-inducing kinase; PKA, protein kinase A; TBK, TANK-binding kinase.
Received for publication March 21, 2005. Accepted for publication May 21, 2005.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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0.5 x 106 cell equivalents, and 24 µg/lane for nuclear fractions, representing 
