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The Journal of Immunology, 2007, 179, 7868 -7875
Copyright © 2007 by The American Association of Immunologists, Inc.
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NF-{kappa}B-Inducing Kinase Regulates Cyclooxygenase 2 Gene Expression in Macrophages by Phosphorylation of PU.11

Anser C. Azim*, Xuerong Wang*, Gye Young Park*, Ruxana T. Sadikot*,{dagger}, Hongmei Cao{ddagger}, Biji Mathew*, Michael Atchison§, Richard B. van Breemen{ddagger}, Myungsoo Joo and John W. Christman2,*,{dagger}

* Department of Medicine and {dagger} Department of Medicinal Chemistry and Pharmacognosy, Section of Pulmonary, Critical Care, and Sleep Medicine, University of Illinois, Chicago, IL 60612; {ddagger} Jesse Brown Veterans Affairs Medical Center, Chicago, IL 60612; § Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104; and Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Selective expression of cyclooxygenase 2 (COX-2) by macrophages could have an important role in the pathobiology of inflammation. We reported a functional synergism between PU.1 and other transcription factors that contributes to COX-2 gene expression in macrophages. PU.1 resides in the nuclear compartment and is activated by phosphorylation to bind to cognate DNA elements containing a 5'-GGAA/T-3' motif, but the involved kinase has not been discovered. We tested the hypothesis that NF-{kappa}B-inducing kinase (NIK) regulates COX-2 gene expression in macrophages through inducible phosphorylation of PU.1. Our initial experiments showed an in vitro protein-protein binding interaction between myc-NIK and GST-PU.1. Purified myc-NIK had a strong in vitro kinase activity for purified GST-PU.1, and this activity and production of COX-2 protein is blocked by treatment with a nonspecific kinase inhibitor, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole. We used short interfering RNA to develop a stable NIK knockdown macrophage cell line that had an ~50% decrease in COX-2 protein production and decreased generation of PGD2, and this was correlated with decreased binding of activated PU.1 to the COX-2 promoter in response to treatment with endotoxin. These findings suggest a novel role for NIK in mediating COX-2 gene expression in endotoxin-treated macrophages by a mechanism that involves phosphorylation of PU.1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The PU.1 is a member of the Ets family of transcription factors that is expressed by neutrophils, B lymphocytes, and macrophages. Among these myeloid cell types, only mature macrophages have large amounts of immunoreactive PU.1 in the nuclear protein compartment. Although PU.1 also has an important role in differentiation of the mature macrophage phenotype, the role of PU.1 in regulation of inflammatory gene regulation in mature macrophages has not been completely defined. PU.1 is characterized by the presence of a DNA-binding domain that recognizes a core DNA element 5'-GGAA/T-3' (1, 2, 3, 4) PU.1 is potentially phosphorylated on five separate serine residues (Ser41, Ser45, Ser132, Ser133, and Ser148) and is an in vitro substrate for casein kinase II (CKII)3 (5). PU.1 is also phosphorylated at Ser41 and Ser142 by Ras-PI3K-dependent externally regulated kinase, Akt, and p38 MAPK-mediated pathways (6). We have recently reported (7) that PU.1 binds to the cyclooxygenase 2 promoter (COX-2p) in vitro in response to treatment with endotoxin and this leads to COX-2 gene expression. Our data indicate that binding of PU.1 to the COX-2 promoter is dependent on phosphorylation of PU.1 at Ser148 but the involved kinase, which was presumed to be CKII, was not identified.

The mechanism by which activated (phosphorylated) PU.1 regulates gene transcription, in general, appears to be context dependent through interactions with other transcription factors. For example, PU.1 regulates GATA-1 and GATA-2 transcription factors by directly interacting with and down-regulating their transcriptional activity. Other reports suggest that PU.1 inhibits acetylation of other transcription factors through protein-protein interactions with CREB-binding protein (CBP)/p300 (8, 9, 10, 11). PU.1 recruits binding of a second B cell-restricted NF, IFN regulatory factor 4 (NF-EM5,PIP), to a DNA site in the Ig {kappa} 3' enhancer (9). DNA binding by NF-EM5 requires a protein-protein interaction with PU.1 and specific DNA contacts. We have reported that activated PU.1 binds to the COX-2p in response to treatment of RAW cells with endotoxin. PU.1 binding to its cognate binding sequence in the COX-2p serves as anchor for CBP/p300, providing greater access of the acetylation machinery to C/EBP-β (7). Acetylation of C/EBP-β that is facilitated by the interaction between PU.1 and CBP/p300 leads to an accentuation of COX-2 gene expression in macrophages (7).

The NF-{kappa}B/Rel family has a central role in the inducible expression of a large number of genes involved in inflammation, host defense, cell survival, and proliferation (12, 13). The I{kappa}B kinase (IKK) complex (IKK-{alpha}β{gamma}) plays an important role in NF-{kappa}B activation. Two principal pathways for NF-{kappa}B activation have been characterized: a classical (canonical) and an alternative (noncanonical) pathway (14, 15, 16, 17). Under normal conditions, cells have NF-{kappa}B sequestered in the cytoplasm associated with inhibitory molecules like I{kappa}B{alpha}, I{kappa}Bβ, I{kappa}B{epsilon}, p105, and p100. This sequestration of NF-{kappa}B blocks its ability to bind to DNA. Cells respond to inflammatory cytokines, reactive oxygen species, and bacterial and viral cell components to initiate NF-{kappa}B signaling (18). IKK{alpha}, IKKβ. and IKK{gamma} play important roles in a phosphorylation-dependent manner to regulate I{kappa}B species and their subsequent degradation by the proteasome.

NF-{kappa}B-inducing kinase (NIK) is a serine/threonine protein kinase and has been shown to interact with TNFR-associated factor 2, IKK{alpha}, and IKKβ (19, 20) It has been suggested that NIK participates in signaling events initiated by specific inducers that activate both the classical and alternative pathway in B lymphocytes (20). NIK has been shown to mediate lymphotoxin β-induced phosphorylation of RelA at serine 536 and to phosphorylate and regulate RelB and RelC (21, 22). Recent studies have suggested that components of the NF-{kappa}B signaling pathway go through nuclear localization in activated and nonactivated cells (23, 24, 25). We have reported that NIK predominantly resides in the cytoplasm of unstimulated macrophages and rapidly translocates to the nuclear compartment in response to endotoxin treatment. We noticed that the timing of nuclear translocation of NIK was similar to the activation and binding of PU.1 to the COX-2p, raising the possibility that NIK has a role in phosphorylation of PU.1 (25).

PGs are produced by the action of COX on arachidonic acid released from the membrane phospholipids by phospholipases (26, 27). PGs play an important physiologic role in inflammation by participating in recruitment of proinflammatory cells, maintenance of vascular permeability, and regulation of vasodilation (28, 29). COX-1 is usually constitutively expressed, whereas COX-2 is rapidly and transiently induced in response to a variety of stimuli (LPS, cytokines, growth factors, and hormones) and some are critical regulators of macrophages. Promoter analysis of the COX-2p shows the presence of several transcription factor-binding motifs, like PU.1, CREB, NF-{kappa}B, and C/EBP-β, that result in cell-selective COX-2 gene expression (7).

In this article, we examined whether NIK regulates PU.1 transcriptional activity by phosphorylation of PU.1. We provide evidence that there is a protein-protein interaction between NIK and PU.1 and that NIK has in vitro kinase activity for PU.1. We further demonstrate that NIK knockdown macrophages have reduced binding of activated PU.1 to the COX-2p and decreased COX-2 gene expression and production of PGD2. In our experiments, activation of the NF-{kappa}B and p38 pathways in NIK knockdown macrophages was not affected. In our cellular system, CKII did not play an in vivo role in phosphorylation of PU.1, although we were able to confirm the findings of others that it has in vitro kinase activity. These data indicate that macrophage expression of COX-2 is regulated by NIK through a novel mechanism that involves phosphorylation of PU.1. Because PU.1 is not expressed by nonmyeloid cell types, NIK-mediated phosphorylation of PU.1 could allow a highly selective inhibition of COX-2 in macrophages that could potentially spare COX-2 gene expression in structural and mesenchymal cell types. The exact end product of COX-2 is dependent on the cellular source. Macrophages and mast cells produce PGD2 which is proinflammatory, epithelial cells produced PGE2 which is counterinflammatory, platelets produce thromboxane, which is a vasoconstrictor, and endothelial cells produce prostacyclin, which is a vasodilator. Each of these different prostanoid products signals through unique receptors to result in a specific phenotype (30). Specific interdiction in macrophage COX-2 gene expression could disrupt the complicated balance of eicosanoids when multiple cellular sources are concomitantly stimulated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture

A murine macrophage cell line RAW 264.7 (American Type Culture Collection) was maintained in DMEM supplemented with 10% FBS (HyClone). The tissue culture medium was supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml; Invitrogen Life Technologies). HEK293 cells (American Type Culture Collection) were also cultured in the same medium and were used for transfection studies at 70% confluency.

Reagents

Abs for NIK and PU.1 (rabbit polyclonal) were purchased from Santa Cruz Biotechnology and Ab for COX-2 was obtained from (rabbit polyclonal from Cayman Chemical). CKII Abs and CKII inhibitor 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) were purchased from Calbiochem.

Constructs

Myc-tagged wild-type NIK were gifts from Tularik. GST-PU.1 was a gift from Dr. N. Yaseen (Northwestern University, Chicago, IL). GST-PU.1 was purified as described elsewhere (31). Fusion proteins were expressed in the Escherichia coli strains of DH5 and HB101. Fusion proteins containing GST were purified by affinity chromatography. Proteolysis of fusion proteins was reduced by adding a protease inhibitor mixture in the solubilization and washing buffers. In most of the studies, we have used Pierce cell lysis buffer that we supplemented with protease inhibitors to avoid proteolytic degradation of proteins.

Chromatin immunoprecipitation (ChIP) assay

PU.1 binding to the COX-2p was analyzed by PCR with two sets of oligonucleotides as described earlier (7). The first set covered the distal COX-2 binding site 5'-TAGGAAGATATCCAACACTAT3' and 5'-TAGTTCCATGAAAGACTTCAA-3') and produced a 408-bp PCR product. The second set (5'-CTAATTCCACCAGTACAGATG-3' and 5'-ACTAGGCGAGACTCAGCGAAC-3') of oligonucleotide sites produced a 275-bp PCR product encompassing the proximal binding site. The PCR conditions were 94°C for 30 s, 54°C for 30 s, and 68°C for 30 s with 30 cycles of reactions as described previously (7). PCR for the input was performed with 100 ng of genomic DNA.

Myc-NIK immunoprecipitation and Western blot

HEK-293 cells (~70% confluency) were transfected with 2–4 µg/10-cm2 wild-type Myc-NIK plasmid DNA. We have used either GenePORTER2 (Gene Therapy Systems) or LipofectAMINE (Invitrogen Life Technologies) as specified by the manufacturer. After 48 h, transfected cells were used for either immunoprecipitation or immunofluorescent imaging. We used polyclonal and mAbs (New England Biolabs) for immunoprecipitating NIK for the kinase experiment. In all of the experiments, cell lysis buffer from Pierce was used. After immunoprecipitation, the beads were washed with TBS containing 0.05% Tween 20 (pH 7.4). Samples were preloaded with protein A-agarose beads before immunoprecipitation with the appropriate Ab. Immunocomplexes bound to protein A-agarose beads were washed in immunoprecipitation buffer four times and once in PBS. The protein samples were separated by 12% or 10% SDS-PAGE after boiling in Laemmli buffer. Western blots were performed as described elsewhere (31). After the transfer, nitrocellulose membranes were blocked overnight at 4°C with 5% skim milk in PBST (PBS and 0.1% Tween 20) and incubated with the appropriate primary and secondary Abs in PBST containing 1% skim milk for 1 h each. The bands were visualized by using the Amersham ECL kit (Amersham International).

NIK kinase assay

CKII and NIK assays were performed in CKII assay buffer. Briefly, glutathione-Sepharose beads containing GST and GST-PU.1 fusion proteins (50 µl) were suspended in 100 µl of CKII assay buffer (200 mM NaCl, 10 mM MgCl2, and 25 mM Tris-HCl (pH 7.4), and 10 µM ATP) in the presence of 2–5 µCi of [32P]ATP. The kinase reaction was started by adding 1 unit of CKII (Calbiochem) and incubating samples at 37°C for 30 min. The beads were washed with Triton buffer (1% Triton X-100, 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1 mM PMSF) four times and separated by 10% SDS-PAGE, dried, and exposed to x-ray film.

Establishment of NIK knockdown cell line

We have generated a NIK knockdown cell line by using short interfering RNA target sequences against the mouse NIK gene using BLOCK-iT RNAi Designer software (Invitrogen Life Technologies) (25). Three test sequences were selected, corresponding to nucleotide positions 1142, 1437, and 2292. The most active sequences of these, targets 2 and 3, are based on the sequence 5'-GCTACAGCACTGGAGATAGA-3' and 5'-GGGTCCTGCTTACTGAGAAAC-3', respectively. We annealed primers and cloned them into pSIREN-RetroQ (BD Clontech) downstream of the human U6 promoter. RAW cells were transfected with LipofectAMINE-2000 (Invitrogen Life Technologies) and selected in the condition of 2.5 µg/ml puromycin for 1 mo. Of 36 clones, the line that expressed the least NIK was used in the present studies as judged by immunoreactive NIK by Western blots of whole-cell RAW cell lysates.

Measurement of PGD2

RAW 264.7 and short interfering RNA (siRNA) NIK knockdown cells were treated with 500 ng/ml LPS and grown to 70% confluency. Supernatant samples were collected at 0, 1, 2, 4, 8, and 16 h. These supernatants were frozen for later measurement of PGD2 using liquid chromatography-mass spectroscopy-mass spectroscopy.

Statistical analysis

Our statistical analyses were performed with GraphPad InStat using a nonpaired t test or ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
There is protein-protein interaction between GST-PU.1 and Myc-NIK

Recent studies suggest that NIK and PU.1 are both localized in the nucleus in endotoxin-treated RAW cells. PU.1 is exclusively found in the nucleus, whereas NIK translocates from the cytoplasmic to the nuclear compartment in response to treatment with endotoxin (25). We have expressed ectopic Myc-NIK as a fusion protein in HEK-293 cells, and the presence of abundant immunoreactive Myc-NIK was detected in cell lysates by using anti-myc Abs (data not shown). This Myc-NIK-enriched lysate was used to determine whether there is a protein-protein interaction between NIK and PU.1. GST-PU.1 was expressed in bacteria and purified by affinity chromatography using glutathione-Sepharose 4B beads. GST-PU.1 or GST alone was mixed with the Myc-NIK- enriched or controlled lysates, incubated with glutathione-Sepharose 4B beads, extensively washed, and then the pulled-down proteins were separated by 10% SDS-PAGE and immunoblotted with anti-Myc Abs. As shown, immunoreactive Myc-NIK that was pulled down with GST-PU.1 was detected as an upper and lower band (Fig. 1A, lane 3). These data suggest that there is a direct or indirect association between GST-PU.1 and Myc-NIK. In contrast, we were unable to detect any binding of immunoreactive Myc-NIK to GST alone (Fig. 1A, lane 1), and endogenous NIK, in nontransfected control HEK-293 cell lysates, did not bind to GST-PU.1 (Fig. 1A, lane 2). We used another source of anti-NIK Abs from Santa Cruz Biotechnology to test our immunoprecipitates and the results were similar and showed a doublet banding pattern. Because NIK should be a single rather than double band, we examined immunoreactivity to the anti-myc tag on the N terminus and compared this to an Ab raised to the C terminus of NIK. These data (Fig. 1B) show that the lower band that binds to the GST-PU.1 is a C-terminal truncation degradation product of Myc-NIK. As shown, the anti-Myc Ab recognizes two separate bands that contain the immunoreactive Myc tag on the N terminus of the full-length myc-NIK fusion protein. In contrast, a C terminus anti-NIK Ab failed to detect the lower band of degraded NIK, suggesting that the lower band is a C-terminal truncation degradation product of the myc-NIK fusion protein that is missing the Myc-NIK-containing N terminus. This may be the result of contamination due to bacterial proteases that are not completely inhibited by the protease mixture used in the lysis buffer.


Figure 1
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  		FIGURE 1. GST-PU.1 binds to Myc NIK. A, Bacterially expressed GST and GST-PU.1 glutathione-Sepharose beads were mixed with equal amounts of protein lysates from Myc-NIK-transfected HEK-293 lysates and untransfected cell lysates. The beads were harvested after 2 h and subsequently washed with TBST buffer several times and an equal amount of protein was separated on 10% SDS-PAGE and subsequently probed and immunoblotted using anti-Myc Abs. Myc-NIK binding was compared with GST and GST-PU.1. B, Western blot showing the presence of Myc-NIK and degraded NIK in the Myc-NIK and GST-PU.1 mix supernatant. The right panel fails to show the ~65-kDa band when probed with a peptide-specific C terminus Ab, whereas the anti-Myc Ab shows both the 65- and 94-kDa bands. C, We coexpressed Myc-NIK and GFP-PU.1 in HEK-293 cells. We immunoprecipitated GFP-PU.1 with anti-PU.1 Abs from HEK-293 cells that were transfected with both Myc-NIK and GFP-PU.1 vectors. We detected the presence of PU.1 and NIK in the immunoprecipitate. The left panel shows the presence of GFP-PU.1 in lanes 2 and 3 (left panel) that was detected with an anti-GFP Ab. The anti- PU.1 Abs show the 55-kDa band in the middle panel. Finally, we show that anti-Myc Abs recognize only NIK but not PU.1 (lane 3, far right panel in C) and the presence of the IgG band is indicated by an arrow. These experiments were repeated two times with similar results.

 
GFP-PU.1 and Myc-NIK immunoprecipitate together

To verify that there is an interaction between PU.1 and NIK, we performed a coimmunoprecipitation experiment using cell lysate from HEK-293 cells that coexpressed GFP-PU.1 and Myc-NIK together (Fig. 1C). GFP-PU.1 was immunoprecipitated with anti-PU.1 Ab, separated on 10% SDS-PAGE gels, and subsequently probed with anti-GFP (Fig. 1C, left panel), anti-PU.1 (Fig. 1C, middle panel), and anti-Myc Abs (Fig. 1C, far right panel). As shown, we were able to detect immunoreactive Myc-NIK as a doublet (Fig. 1 C, lane 3, left panel) and were able to coimmunoprecipitate GFP-PU.1 (Fig. 1C, lane 3, left and middle panels). These experiments indicate that GFP-PU.1 binds to Myc-NIK. Thus, we were able to show a protein-protein interaction between NIK and PU.1 by both a GST pull-down and a coimmunoprecipitation experiment. GFP-PU.1 runs as a 55- to 59-kDa protein and it was very difficult to clearly separate the IgG H chain from the immunoprecipitated GFP-PU.1. The location of the GFP-PU.1 fusion protein is indicated by an arrow in the left panel and by two arrows in the middle panel of Fig. 1C.

GST-PU.1 is phosphorylated in vitro by Myc-NIK-enriched lysate

Next, we determined whether Myc-NIK-enriched lysates had in vitro kinase activity for GST-PU.1. As shown in Fig. 2A, Myc-NIK-enriched lysates (data not shown) from HEK-293 cells (Fig. 2A, lanes 3 and 4), but not lysates from nontransfected cells (Fig. 2A, lanes 1 and 2) contained kinase activity for GST-PU.1 (Fig. 2A, lane 4) but not GST alone (Fig. 2A, lanes 1 and 3). The presence of the immunoreactive GST-PU.1 is shown as a 55-kDa band in the lower panel of Fig. 2A and is present only in lanes 2 and 4. In some experiments, we were able to detect a very slight level of kinase activity in control lysates, presumably due to endogenous kinase activity, but NIK-enriched lysates consistently showed very strong kinase activity for GST-PU.1. As a positive control (data not shown), purified CKII had similar phosphorylation activity for GST-PU.1 compared with the Myc-NIK-enriched lysates. As shown in Fig. 2B, we noticed that the PU.1 kinase activity of the overexpressed Myc-NIK lysate was abolished by DRB, a CKII inhibitor. This experiment suggested a possible in vitro role of either CKII or NIK in PU.1 phosphorylation.


Figure 2
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  		FIGURE 2. Kinase experiment showing that Myc-NIK-enriched total cellular lysate was able to phosphorylate GST-PU.1. A, GST and GST-PU.1 glutathione beads were mixed with control lysate and HEK-293 lysate that was transfected with Myc-NIK vector. The glutathione beads (equal amount of protein) were incubated with lysates for 1 h in the presence of protein kinase-specific buffer and [{gamma}-32P]ATP and subsequently separated on 12% agarose gel and the gel was exposed to x-ray films. Lower panel shows the presence of GST-PU.1 in lanes 2 and 4 for correlation with the data in the upper panel. B, A second kinase experiment showing phosphorylation of GST-PU.1 from Myc-NIK lysate that was totally blocked and abolished because of the presence of 50 µM DRB, a nonspecific kinase inhibitor. We repeated these experiments more than three times.

 
A possible confounding issue is that our total cell lysates contained abundant amounts of immunoreactive CKII, which is known to have kinase activity for PU.1. It is already known that RAW cells contain abundant CKII in the cytoplasmic compartment and we confirmed the findings of others that CKII can phosphorylate PU.1 (data not shown). To separate the kinase activity of NIK from CKII, we examined the presence of immunoreactive CKII in the supernatant and in the immunoprecipitated Myc-NIK bead complex (Fig. 3A). As expected, abundant immunoreactive CKII was detected in the supernatant of Myc-NIK-enriched (Fig. 3A, lane 1) and control (Fig. 3A, lane 2) cell lysates. However, we were unable to detect any immunoreactive CKII in the immunoprecipitated, beaded fraction of Myc-NIK-enriched or control cell (Fig. 3A, lanes 3 and 4, respectively). Thus, the immunoprecipitated NIK used in the experiment show that our immunoprecipitated material did not contain any detectable immunoreactive CKII. The blot was probed with CKII Abs.


Figure 3
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  		FIGURE 3. Immunoprecipitated Myc-NIK phosphorylates GST-PU.1. A, Western blot showing that Myc-NIK-enriched beads are not contaminated with CKII. NIK was immunoprecipitated using anti-myc Abs. The NIK-enriched beads and the supernatants were probed for the presence of CKII. The Western blot was probed with CKII and anti-myc Abs together. B, Myc-NIK was immunoprecipitated from HEK-293 cells that were transfected with the Myc-NIK vector. The middle panel shows the presence of similar amounts of GST-PU.1 in both lanes 1 and 2. This experiment shows that PU.1 is also a substrate for NIK. The right panel shows that immunoprecipitated NIK fails to phosphorylate the Ser148 PU.1 mutant, suggesting possible kinase specificity.

 
To improve the specificity of these experiments, we determined whether purified immunoprecipitated Myc-NIK has kinase activity for purified GST-PU.1 (Fig. 3B). As shown above, this immunoprecipitated material was free from any CKII contamination. As shown in Fig. 3A, immunoprecipitated Myc-NIK (lane 2, left panel) contained strong kinase activity for GST-PU.1 (lane 2) but not for GST-PU.1 alone in the absence of immunoprecipitated NIK (lane 1, left panel). As shown in the middle panel of Fig. 3B, relatively equal amounts of GST-PU.1 were present in the reaction mixture. both lanes by immunoblotting. The NIK kinase activity was blocked by the presence of 50 µM DRB, a kinase inhibitor that also blocks CKII activity (data not shown). Interestingly, immunoprecipitated myc-NIK was unable to phosphorylate mutant-PU.1 (Ser148-alanine) compared with CKII (Fig. 3B, right panel). These experiments indicate that NIK is directly or indirectly involved in phosphorylation of PU.1 and that the kinase activity is not effective in phosphorylating PU.1 that is mutated at the Ser148 residue. In contrast, CKII can phosphorylate either wild type or the mutant PU.1. Thus, NIK is remarkable specific for the Ser148 residue of PU.1 that is necessary for transcriptional activity.

NIK knockdown RAW 264.7 cells that express 80–90% less NIK

We previously showed that treatment of wild-type RAW cells with endotoxin results in binding of PU.1 to a proximal and distal cETS cognate binding sites in the COX-2p. We have shown that binding of PU.1 to the COX-2p is highly dependent on phosphorylation of PU.1 at Ser148. To determine whether NIK is involved in phosphorylation-dependent activation of PU.1-binding activity to the COX-2p, we constructed a stable line of RAW cells where NIK protein production was reduced by siRNA technology. As shown, in Fig. 4A, there was a 90% reduction in the production of immunoreactive NIK in the NIK siRNA stable cell line compared with wild-type RAW cells. Fig. 4B shows relative NIK expression in the NIK siRNA stable cell line that has been used in these studies.


Figure 4
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  		FIGURE 4. Establishment of NIK knockdown cell line. A, We designed siRNA target sequences to the mouse NIK gene. The cell lysate was probed for the presence of NIK using anti-NIK Abs. The NIK-deficient cells expressed 80–90% less NIK in RAW 264.7 cells after siRNA treatment. B, Densitometric quantitation of NIK in NIK-deficient RAW 264.7 cells.

 
Next, we determined the time course for phosphorylation of p38 MAPK (Fig. 5A, upper panel) and degradation of I{kappa}B{alpha} (Fig. 5A, middle panel) in RAW cells in response to treatment with endotoxin in the NIK knockdown (Fig. 5A, lanes 1–5) and wild-type RAW cells (Fig. 5A, lanes 6–10). As shown, there was a nearly identical time course for phosphorylation of p38 MAPK in the two cells lines that peaked between 30 and 45 min (Fig. 5A, row A, lanes 3 and 4 and 8 and 9, respectively). Degradation of I{kappa}B{alpha} occurred in both the wild-type (Fig. 5A, middle panel, lanes 1–5) and NIK knockdown RAW cells (Fig. 5A, middle panel, lanes 6–10); however, there this was slightly delayed with a nadir at 30 min in the NIK knockdown RAW cells (Fig. 5A, middle panel, lane 8) compared with 15 min (Fig. 5A, middle panel, lane 2) in the wild-type RAW cells. Full recovery of total cellular I{kappa}B{alpha} was evident in both cell types by 2 h (Fig. 5A, lanes 5 and 10, respectively). These data suggest that loss of NIK does not affect either of these signaling pathways in response to treatment with endotoxin.


Figure 5
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  		FIGURE 5. Endotoxin treatment and immunoblot showing that the p38 MAPK pathway and I{kappa}B{alpha} degradation were not affected by loss of NIK in siRNA-treated RAW 264.7 cells. Wild-type RAW cells and NIK-deficient cells were stimulated with 100 ng/ml LPS and cells lysates were separated and frozen in lysis buffer containing protease inhibitor mixture. A, The blots were probed with anti-p38 phospho-Abs and anti-I{kappa}B{alpha} Abs. The same blot was probed for β-actin for protein control. ChIP assays with Ab against PU.1 and semiquantitative PCR using primers for the proximal PU.1 site (B) and for the distal site (C) in the COX-2p. RAW cells were treated with endotoxin (100 ng/ml) for various time points (0–60 min), PU.1 was immunoprecipitated with specific Ab. and binding to the promoter region was detected by PCR primers for the COX-2p PU.1-binding DNA motif. These experiments were done twice. D, Another ChIP assay with Ab against PU.1 and semiquantitative PCR using primers for the proximal PU.1 site over a longer time course of 0–6 h. Binding of PU.1 to the COX-2p was not detected at any time point in siRNA NIK knockdown cells.

 
PU.1 binding to the COX-2p is reduced in NIK knockdown RAW cells

To show that PU.1 binding to the COX-2p was affected by NIK deficiency, we performed the ChIP assay. The COX-2p has two c-Ets binding sites located in COX-2p region between sequences –725 and –734 (distal) and –320 and –329 (proximal) (11). We treated wild-type RAW and NIK knockdown RAW cells with 1 µg/ml LPS for 0–60 min and performed the ChIP assay as previously reported (11). The cells were fixed with formaldehyde and the samples were sonicated to get ~200-bp fragments. As shown in Fig. 5, there was reduced binding to the proximal cETS site (Fig. 5B) of the COX-2p in the NIK knockdown RAW cells (Fig. 5B, lanes 4–6) compared with wild-type RAW cells (Fig. 5B, lanes 1–3). For the proximal cEts site (Fig. 5A), in response to treatment with endotoxin, abundant PU.1 was recruited to the COX-2p by 30 and 60 min (Fig. 5A, lanes 2 and 3) in the wild-type RAW cells compared with very scant binding to the proximal cEts site at these time points in the NIK knockdown RAW cells (Fig. 5A, lanes 5 and 6). For the distal cEts site, activated PU.1 was recruited to the COX-2p only at the 60-min time point (Fig. 5C, lane 3) and this was not detected in the NIK knockdown RAW cells at this time point (Fig. 5C, lane 6). To show whether the lack of PU.1 binding is persistent over a longer time course, we performed a ChIP assay (as shown in Fig. 5D) from 0 to 6 h. In this experiment, we detected a very slight binding of PU.1 to the COX-2p in wild-type cells but none was detected in the siRNA NIK knockdown cells. After 1 h, PU.1 binding to COX-2p was reduced and was not detected at the 6-h time point in the wild-type cells. We were unable to detect any binding of PU.1 to the COX-2p in the siRNA NIK knockdown cells, which is consistent with lack of phosphorylation of PU.1 This binding pattern for activated PU.1 to the COX-2p is identical to our previous publication (7). These data indicate that binding of activated PU.1, a process known to be dependent on phosphorylation of PU.1 at Ser148, to the proximal and distal c-Ets binding sites in the COX-2p is dependent on NIK because it is markedly reduced in NIK knockdown RAW cells that have <10% of the wild-type amounts of NIK. This finding is consistent with a prominent role for NIK in phosphorylation of PU.1.

LPS-induced COX-2 expression is affected by decreased expression of NIK

To determine the physiologic consequence of our findings, we measured whether NIK deficiency affects COX-2 gene expression in NIK knockdown RAW cells compared with wild-type RAW cells (Fig. 6A). Equal numbers of NIK knockdown RAW cells and wild-type RAW cells were treated with 100 ng/ml LPS for 1 to 6 h. As shown, there was a marked reduction in immunoreactive COX-2 production by NIK knockdown RAW cells compared with that of wild-type RAW cells that is especially evident at the 2-h time point. Treatment with endotoxin had no effect on the amount of immunoreactive NIK in the two cell types but, of course, this was greatly reduced in the NIK knockdown RAW cells compared with the wild-type RAW cells. We also examined the time course for the production of PGD2 by NIK knockdown RAW cells and wild-type RAW cells (Fig. 6B) by measuring PGD2 in culture supernatant using a sensitive and highly specific assay that employed liquid chromatography in conjunction with mass spectrometry. As shown, there was a 3-fold decrease in the production of PGD2 by the NIK knockdown RAW cells compared with the wild-type RAW cells by 6 h of treatment with endotoxin. Thus, there was reduced production of COX-2 protein production and enzymatic activity in NIK knockdown RAW cells compared with wild-type RAW cells and this correlated with decreased binding of activated PU.1 to the COX-2p.


Figure 6
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  		FIGURE 6. COX-2 protein expression in RAW 264.7 cells and NIK-deficient RAW 264.7 cells. A, Immunoblot showing wild-type (WT) RAW cells and NIK-deficient RAW cell lysates that were treated with endotoxin (100 ng/ml) for various time points and probed with COX-2, NIK, and actin Abs. B, PGD2 quantitation from wild-type and NIK-deficient RAW 264.7 cell medium that was stimulated with endotoxin for various time points and then analyzed by a highly specific liquid chromatography-mass spectroscopy-mass spectroscopy method to determine the PGD2 level in cellular supernatants. Data are mean ± SE (n = 3, for each condition). These experiments were repeated twice and the best possible blot is presented here.

 
Our data are summarized as a schematic depicting our hypothesis (Fig. 7) that NIK is the specific kinase that phosphorylates PU.1 upon endotoxin stimulation and regulates COX-2p activity for COX-2 expression and PGD2 synthesis. This shows a novel NIK-mediated synergistic pathway for COX-2p gene regulation.


Figure 7
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  		FIGURE 7. A schematic depicting our proposed hypothesis that NIK is the specific kinase that phosphorylates PU.1 upon endotoxin stimulation. Phosphorylated PU.1 in turn regulates COX-2p activity for COX-2 expression and PGD2 synthesis. This shows a novel NIK-mediated synergistic pathway for COX-2p gene regulation.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
PU.1 interacts with many transcription factors, including IFN regulatory factor 4 (NF-EM5/PIP, c-JUN, and GATA-1 (9, 32), to result in combinatorial transcriptional regulation of gene expression. In many cases, phosphorylation of PU.1 is necessary for regulation of gene transcription (6, 7, 33, 34), like COX-2 and TLR4 (7, 11), which in turn modulates interaction with cofactors, other transcription factors, trans-activating activity, or DNA-binding activity. Little is known about the specific kinase that phosphorylates PU.1, although there are several reports that CKII has in vitro kinase activity for PU.1 (5, 9). There are no in vivo experimental data that clearly show that CKII phosphorylates PU.1 in response to treatment with endotoxin. Since CKII is a cytosolic kinase and activation of PU.1 by phosphorylation is confined to the nuclear compartment, we hypothesized that NIK that translocates to the nuclear compartment has a prominent role in phosphorylation of PU.1 in response to treatment with endotoxin.

In this study, we report, for the first time, that there is a protein-protein interaction between PU.1 and NIK and that immunoprecipitated NIK is able to phosphorylate PU.1. We tested the NIK immunoprecipitates for CKII contamination, but CKII Abs failed to detect any CKII presence in our immunoprecipitates. This suggests that the complex that was brought down by anti-Myc Abs is not contaminated with immunoreactive CKII to account for the PU.1 kinase activity. In these studies, we are not able to totally exclude the possibility that immunoprecipitated NIK acts in conjunction with an unknown kinase or cofactor that might be present in the immunoprecipitated complex. To evaluate the necessity of NIK in regulating PU.1 activity, we generated a macrophage cell line that has ~90% less NIK by using siRNA technology. The NIK-deficient cell line produced less COX-2 in response to treatment with endotoxin and this was correlated with decreased production of PGD2. We have previously reported that there is recruitment of activated PU.1 to the COX-2p that is dependent on phosphorylation of PU.1 and, in this report, we found that recruitment of activated PU.1 to the COX-2p is decreased in the NIK knockdown macrophages. In addition to this report that NIK is an important kinase for PU.1, we have also reported that NIK regulates COX-2 gene expression through modification of nucleosomal structure and through phosphorylation of endogenous IKK{alpha} and phosphorylation of histones in macrophages (25). Thus, NIK affects COX-2 gene expression through two independent mechanisms, phosphorylation of PU.1, which has a combinatorial role in regulating COX-2 gene transcription, and phosphorylation of IKK{alpha}, which has a role in phosphorylation of histone H3 and regulation of chromosomal structure and the availability of the COX-2p for binding of transcription factors. This suggests that nuclear translocation and activation of NIK, in response to treatment with endotoxin, is a critical point for regulation of COX-2 gene expression in macrophages.

We have ruled out involvement by CKII, although we were unable to detect immunoreactive CKII in the anti-Myc immunoprecipitates for the Myc-NIK fusion molecule. It is interesting to note that CKII phosphorylates the C terminus of IkB{alpha}, which is also phosphorylated by IKK{alpha} and IKKβ. Interestingly, the immunoprecipitated NIK was unable to phosphorylate the Ser148 mutant PU.1 but CKII phosphorylates both wild-type and the mutant PU.1. Cells that are stimulated with growth factors have been shown to have increased CKII activity, but these studies failed to demonstrate translocation of CKII from the cytoplasm to the nuclear compartment in response to treatment with endotoxin, and no studies have demonstrated a protein-protein interaction between CKII and PU.1 or NIK. Others have suggested that CKII is involved in phosphorylation of PU.1 because it contains a purported CKII consensus sequence. We were able to confirm that CKII has DRB- sensitive kinase action for PU.1 in vitro but this does not seem to be activated in vivo by treatment with endotoxin because the NIK knockdown RAW cells, with normal CKII levels, failed to activate PU.1 to bind to the COX-2p. To evaluate the necessity of NIK in regulating PU.1 activity, we generated a macrophage cell line that has ~90% less NIK by using siRNA technology. The NIK-deficient cell line produced less COX-2 in response to treatment with endotoxin and this was correlated with decreased production of PGD2. We have previously reported that recruitment of PU.1 to the COX-2p is dependent on phosphorylation of PU.1 and, in this report, we found that recruitment of activated PU.1 to the COX-2p is decreased in the NIK knockdown macrophages.

In summary, our study suggests that NIK translocates to the nucleus in response to endotoxin treatment of RAW cells and plays an important role in regulating PU.1 phosphorylation and its binding to the COX-2 gene promoter. Our data indicate that induction of COX-2 gene expression in response to treatment with endotoxin is, at least partially, dependent upon NIK-mediated PU.1 phosphorylation. PU.1 binds to the promoter of COX-2 on the two separate cognate binding sites in a NIK-dependent pathway. Involvement of NIK and PU.1 in COX-2 expression is potentially an important point of interdiction that could limit COX-2 gene expression in macrophages without affecting COX-2 gene expression in cells where PU.1 is not involved.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Department of Veterans Affairs and National Institutes of Health Grants HL 075557 and HL 66196. Back

2 Address correspondence and reprint requests to Dr. John William Christman, Section of Pulmonary, Critical Care, and Sleep Medicine, University of Illinois, 840 South Wood Street, Chicago, IL 60612. E-mail address: jwc{at}uic.edu Back

3 Abbreviations used in this paper: CKII, casein; CBP, CREB-binding protein; NIK, NF-{kappa}B-inducing kinase; COX, cyclooxygenase; IKK, I{kappa}B kinase; NIK, NF-{kappa}B-inducing kinase; DRB, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole; ChIP, chromatin immunoprecipitation; siRNA, short interfering RNA. Back

Received for publication March 8, 2007. Accepted for publication September 14, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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