NIBP, a Novel NIK and IKKβ-binding Protein That Enhances NF-κB Activation*

The transcription factor NF-κB plays an important role in both physiological and pathological events in the central nervous system. Nevertheless, the mechanisms of NF-κB-mediated regulation of gene expression, and the signaling molecules participating in the NF-κB pathway in the central nervous system are, to date, poorly understood. To identify such molecules, we conducted a yeast two-hybrid screen of a human brain cDNA library using NIK as bait. As a result, we identified a novel NIK and IKKβ binding protein designated NIBP that is mainly expressed in brain, muscle, heart, and kidney. Interestingly, low levels of expression were detected in immune tissues such as spleen, thymus, and peripheral blood leukocytes, where NF-κB is known to modulate immune function. We demonstrated by immunohistochemistry that NIBP expression in the brain is localized to neurons. NIBP physically interacts with NIK, IKKβ, but not IKKα or IKKγ. NIBP overexpression potentiates tumor necrosis factor-α-induced NF-κB activation through increased phosphorylation of the IKK complex and its downstream IκBα and p65 substrates. Finally, knockdown of NIBP expression by small interfering RNA reduces tumor necrosis factor-α-induced NF-κB activation, prevents nerve growth factor-induced neuronal differentiation, and decreases Bcl-xL gene expression in PC12 cells. Our data demonstrate that NIBP, by interacting with NIK and IKKβ, is a new enhancer of the cytokine-induced NF-κB signaling pathway. Because of its neuronal expression, we propose that NIBP may be a potential target for modulating the NF-κB signaling cascade in neuronal pathologies dependent upon abnormal activation of this pathway.

NF-B is a multipotent transcription factor that regulates the expression of numerous genes involved in a wide array of biological responses such as inflammation, immunity, apoptosis, and synaptic plasticity (1)(2)(3)(4). In mammals, the NF-B family of transcription factors consists of five members: p65 (or RelA), RelB, c-Rel, NF-B1 (or p105, precursor of p50), and NF-B2 (or p100, precursor of p52). To date, two principal pathways for NF-B activation have been characterized, a classical and an alternative pathway (5)(6)(7). The classical pathway is triggered by stimuli such as tumor necrosis factor ␣ (TNF␣), 1 IL-1, CD40 ligand, and lymphotoxin-␤ (8). Activation of this pathway depends on the IB kinase (IKK) signalsome, which consists of at least two catalytic subunits (IKK␣ or IKK1, and IKK␤ or IKK2) and a regulatory subunit (IKK␥ or NEMO). The activated IKK complex phosphorylates the inhibitor proteins of NF-B (IBs) to induce their ubiquitination and degradation, resulting in the translocation of NF-B dimers (mainly p65/ p50) to the nucleus and transcriptional activation of specific target genes. This pathway is crucial for the activation of innate immunity and inflammation. The alternative pathway is activated by lymphotoxin-␤ (8 -10), CD40 ligand (11), and B cell-activating factor (12). It relies on the phosphorylation of IKK␣ homodimers by NF-B inducing kinase (NIK) to induce p100 processing and nuclear translocation of RelB/p52 dimers (7). This alternative pathway is necessary for secondary lymphoid organ development, maturation of B cells, and adaptive humoral immunity.
NF-B is involved in the regulation of both physiological and pathological processes. Synaptic stimulation activates NF-B, which then participates in long-term potentiation, a process associated with memory formation (13). In addition, NF-B has anti-apoptotic properties in neurons because blocking its activation increases their sensitivity to TNF␣-induced apoptosis (14,15). Induction of NF-B is also associated with several neurological diseases such as Parkinsons (16), Alzheimers (17), AIDS dementia (18,19), and spinal cord injury (20,21). NIK also appears to perform specific functions in the central nervous system and has been implicated in the activation of both the classical and alternative pathways.
NIK was originally identified as a serine/threonine protein kinase related to the mitogen-activated protein kinase (MAP3K) that interacts with TNF receptor-associated factor 2 (TRAF2), IKK␣, IKK␤, and can strongly activate NF-B follow-ing TNF/NGF receptor family stimulation (22). Although earlier overexpression studies using mutant NIK pointed to a crucial role for NIK in TNF␣-induced NF-B activation, later studies using NIK knock-out mice challenged this view, finding NIK to be an essential component only of the alternative pathway. More recent data, however, indicated that NIK participates in signaling events initiated by specific inducers that activate both the classical and alternative pathways (23).
The first demonstration of the function of NIK in the central nervous system was the observation that Aly mice, in which a naturally occurring mutation in the alymphoplasia allele causes NIK to be non-functional, displayed progressive neurological abnormalities leading to hind limb paralysis (24). Moreover, in PC12 cells, NIK promotes neurite formation and prevents apoptosis (25). Nevertheless, the regulatory mechanisms of NIK and, in general, of the NF-B signaling pathway in the central nervous system are not well understood. To investigate these mechanisms, we performed a yeast two-hybrid screen of a brain cDNA library with NIK as bait. As a result, we identified a novel protein designated NIBP (for NIK and IKK␤ binding protein), which interacts directly with NIK and IKK␤ and is expressed in neurons. NIBP functions as an enhancer of cytokine-mediated NF-B activation and IKK kinase activity. Finally, preliminary in vitro studies suggest that NIBP may be a regulator of Bcl-xL gene expression as well as neuronal differentiation, because it is required for NGF-induced neurite extension in PC12 cells.

EXPERIMENTAL PROCEDURES
Yeast Two-hybrid Screening-Yeast two-hybrid assay was performed with the Matchmaker Two-hybrid System II (Clontech, Palo Alto, CA). The bait, encoding the N-terminal domain (amino acids 1-145) of NIK, was inserted into the GAL4 DNA binding domain in the yeast expression vector pBridge (Clontech) as previously described (26). The yeast strain G1945 was transformed with pBridge-NIK followed by the pACT2 expression vector that contained a human brain cDNA library fused to the GAL4 transactivation domain.
Northern Blot-Tissue-specific expression of NIBP mRNA was examined by hybridization of a human multiple tissue Northern blot containing 2 g of poly(A) ϩ RNA (Clontech) with a PCR-produced, [ 32 P]dCTP-labeled 630-bp probe. Hybridizations were conducted in the ExpressHyb hybridization solution (Clontech) at 65°C for 2 h according to the manufacturer's protocol. The same blots were then stripped and hybridized with a 32 P-labeled 300-bp ␤-actin probe as control.
Immunohistochemistry-Fifteen-m cryostat sections from mouse brain and spinal cord were incubated overnight with anti-NIBP (1: 1000). Antibody labeling was visualized with 3,3Ј-diaminobenzidine staining using the Vector Elite ABC kit (Vector Laboratories, Burlingame, CA) according to manufacturer's instructions.
Glutathione S-Transferase (GST) Pull-down Assay-The C-terminal portion (211 amino acids) of NIBP was cloned into the pGEX-4T-2 expression vector (Amersham Biosciences) to generate a GST-NIBP(cd) fusion protein, which was incubated with in vitro translated NIK, IKK␣, IKK␥, and truncated IKK␤. In vitro translation was performed with the TNT® System (Promega, Madison, WI) in the presence of [ 35 S]methionine. Truncated IKK␤ (288 amino acids from the C terminus) was generated by EcoRI digestion. In vitro translation mixture was incubated overnight at 4°C with bacterially expressed GST fusion proteins coupled to 20 l of glutathione-Sepharose beads. Following multiple washes (wash buffer: 20 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl, 0.5% Nonidet P-40), GST-bound proteins were eluted with SDS-PAGE sample buffer, resolved on a 10% polyacrylamide gel, and detected by autoradiography.
Immunoprecipitation and Immunoblotting-Immunoprecipitation and Western blot experiments were performed according to standard procedures as previously described (31).
Reporter Gene Assay-The chemiluminescent reporter gene assay for the combined detection of luciferase and ␤-galactosidase activity was performed with the Dual-Light Combined Reporter Gene Assay System from Applied Biosystems (Foster City, CA) according to the manufacturer's protocol. Luciferase activity was normalized to ␤-galactosidase. Four separate experiments were conducted and, in each experiment, data were calculated as the average Ϯ S.E. of triplicate samples.
Electrophoretic Mobility Shift Assay-Nuclear extracts were prepared according to Dignam et al. (32). Electrophoretic mobility shift assays were carried out as previously described (20). Briefly, doublestranded NF-B consensus oligonucleotides (5Ј-AGTTGAGGGGACTT-TCCCAGGC-3Ј; Promega) were 32 P-labeled with T4 polynucleotide kinase (Promega), and added to binding reactions in the presence of poly(dI-dC):poly(dI-dC) (Sigma), herring sperm DNA (Invitrogen), and nuclear extracts. Equal amounts of extracts, varying from 5 to 20 g/sample, were loaded in each binding reaction. After a 30-min incubation at room temperature, samples were loaded onto a pre-electrophoresed 0.5ϫ Tris-borate EDTA buffer, 6% polyacrylamide gel, and run at 150 V for ϳ1.5 h. Gels were then fixed and dried, and autoradiograms obtained.
PC12 Cell Differentiation-PC12 cells were infected with siRNA lentivirus for 4 weeks prior to differentiation experiments. Cells were plated on collagen-coated 24-well dishes at a density of 10 4 cells/well. Twenty-four hours later, cells were treated with NGF (100 ng/ml). Three days after treatment, the fluorescence of enhanced green fluorescent protein in infected cells was observed.

RESULTS
Cloning and Characterization of NIBP-To investigate the regulatory mechanisms controlling NF-B signaling in the central nervous system, we performed a yeast two-hybrid screen of a human brain cDNA library using as bait for the N-terminal portion of NIK (amino acids 1-145). From 3 ϫ 10 6 yeast transformants, 40 clones were both histidine and ␤-galactosidase positive. Sequence analysis of the positive clones revealed the presence of several known gene products encoding for proteins previously reported as NIK interacting partners (e.g. TRAF3 (36) and TRAF2 (37)). In addition, several novel clones were identified, one of which, TNAP, is a negative regulator of cytokine-induced NF-B activation (26). In the present study we report the identification of a 211-amino acid C-terminal portion of a novel protein, NIBP. Data base analysis showed that NIBP is identical to GenBank sequence KIAA1882 (TULARIC GENE 1), the function of which had not been described. Human NIBP, a 1246-amino acid peptide (139.4 kDa), is found on chromosome 8q24.3, contains 23 exons, and shares a high degree of homology (80 -90%) with rat and mouse sequences. Northern blot analysis of human tissues indicated that NIBP is expressed at high levels in muscle and kidney, and at lower levels in brain, heart, and placenta (Fig. 1a). At least two different transcripts were detected in muscle, kidney, liver, and heart, suggesting that two or more isoforms of NIBP may exist. Interestingly, NIBP transcripts were absent or only weakly detectable in immune organs and cells such as thymus, spleen, and periph-eral blood leukocytes, where the NF-B signaling pathway is known to play key regulatory roles. Immunostaining of mouse brain and spinal cord sections with an antibody raised against a peptide sequence conserved between mouse, rat, and human NIBP indicated the presence of NIBP in neuronal cells. Strong NIBP staining was observed in both the cell bodies and processes of neurons of the pyramidal layer of the cortex (Fig. 1b,  A and B), in spinal cord motor neurons (Fig. 1b, C and D), and white matter neurons (data not shown). Positive NIBP immunolabeling was also detected in primary neurons from mouse cortex and colocalized with the neuronal-specific marker MAP2 (Fig. 1c).
NIBP Interacts with NIK and IKK␤ and but Not IKK␣ or IKK␥-To confirm the interaction between NIBP and NIK in a mammalian system, Myc-tagged NIK and FLAG-tagged NIBP C-terminal 211 amino acid domains (NIBP(cd)) were coexpressed in HEK293T cells. As shown in Fig. 2a, NIBP(cd) was coimmunoprecipitated with NIK, indicating that they are interacting partners in the same signaling complex. Because NIK interacts with and activates the IKK complex (37,38), we tested the hypothesis that NIBP could also interact with subunits of the IKK complex. Indeed, immunoprecipitation analysis of HEK293T cells cotransfected with Myc-NIBP and FLAG-IKK␣, FLAG-IKK␤, or FLAG-IKK␥ showed that NIBP interacts strongly with IKK␤, weakly with IKK␣, and does not interact with IKK␥ (Fig. 2b). The strong interaction between IKK␤ and NIBP could also be detected endogenously in mouse brain lysate following immunoprecipitation with the rabbit polyclonal anti-NIBP antibody (Fig. 2c). Likewise, NIK was also found to interact with NIBP (Fig. 2c), supporting the results obtained in the overexpression system (Fig. 2b). No interaction was detected with either IKK␣ or IKK␥ (Fig. 2c), suggesting that the weak interaction detected between NIBP and IKK␣ in HEK293T cells (Fig. 2b) could be an artifact of the overexpression system. It is noteworthy that we could detect endogenous NIK in brain lysates only following immunoprecipitation with NIBP (Fig. 2c). Endogenous NIBP was also detected following reciprocal immunoprecipitation of mouse brain lysate with an anti-IKK␣/␤ and two anti-NIK antibodies (Fig. 2d). We ruled out that the interaction between NIBP and IKK␣/␤ could be because of an interaction solely with IKK␣, because no NIBP was detected when brain lysate was immunoprecipitated with a specific anti-IKK␣ antibody (Fig. 2d). In support of the physiological relevance of such interactions, these findings were replicated in other systems, such as in PC12 cells (Fig. 6a) and MCF7 cells (data not shown). By using the GST pull-down assay we determined that NIBP binding to NIK and IKK␤ is direct. Interestingly, it appears that the interaction between NIK and NIBP is enhanced in the presence of IKK␤ (Fig. 2e). Finally, no binding was detected with either IKK␣ or IKK␥, in agreement with our previous results in endogenous mouse brain (Fig. 2e).
NIBP Potentiates Cytokine-induced NF-B Activation-To investigate the specific function of NIBP within the NF-B cascade, we tested the hypothesis that NIBP regulates cytokine-induced NF-B activation by its interaction with IKK␤, a key intermediate in this pathway (39).
TNF␣-induced NF-B activation was assayed in HEK293T cells overexpressing NIBP and a specific NF-B-luciferase reporter construct. NIBP overexpression in untreated cells did not affect basal levels of NF-B activation. However, in cells treated with increasing concentrations of TNF␣ (0.1-100 ng/ ml), NIBP potentiated NF-B activation. Similar results were obtained following treatment with IL-1␤ (data not shown). NIBP did not affect IFN␥-induced interferon response factor-1dependent gene expression, suggesting that the potentiation of gene expression by NIBP is specific to the NF-B pathway (Fig. 3b).
The ability of NIBP to enhance TNF␣-induced NF-B activation was further assessed by electrophoretic mobility shift assays. Following TNF␣ treatment, NF-B DNA binding activity was markedly increased in the presence of NIBP compared with control, with a maximum peak at 15 and 30 min (Fig. 3c). This correlated with an increased degradation of the inhibitor of NF-B, IB␣, at the same time points of TNF␣ treatment (Fig. 3d). Taken together, these data demonstrated that overexpression of NIBP enhances cytokineinduced NF-B activation.
To determine whether endogenous NIBP modulates NF-B activation, we knocked down NIBP expression using a lentiviral vector-mediated siRNA approach (Fig. 4). We generated three siRNA constructs directed against the N-terminal (NR), middle (MR), and C-terminal (CR) regions of NIBP. To assess the efficacy and specificity of the siRNA constructs, lentiviral vector-siRNA-transduced HEK293T cells were transiently transfected with FLAG-NIBP, and ectopic and endogenous NIBP expression were measured (Fig. 4d). Among the siRNA constructs tested, NR markedly reduced and CR completely abolished both ectopic and endogenous expression of NIBP in HEK293T cells (Fig. 4d). Based on this result, we used the CR siRNA construct in subsequent knockdown experiments.
Knockdown of endogenous NIBP significantly reduced TNF␣-induced NF-B-dependent luciferase reporter gene expression (Fig. 4e). Because TNF␣ induces NF-B activation through the TRAF2-NIK-IKK pathway, we tested which steps of this pathway were the targets of NIBP by overexpressing these proteins in HEK293T cells where NIBP expression was abolished by siRNA (Fig. 4f). Knockdown of endogenous NIBP reduced IKK␤-and NIK-mediated NF-B activation (Fig. 4f), confirming that NIBP affects the function of NIK and IKK␤. As expected, because IKK␤ is downstream of TRAF2, TRAF2mediated NF-B activation was also reduced. Because overexpression of IKK␣ did not markedly stimulate NF-B activation, we cannot conclude whether NIBP affects its function (Fig. 4f).
To demonstrate that NIBP affects the function of NIK and the IKK complex, we examined the level of phosphorylation of IB␣ and p65, which are key downstream targets of NIK-IKK in the cytokine-induced NF-B activation pathway (40,41). Overexpression of NIBP enhanced TNF␣ and IL-1␤-induced phosphorylation of IB␣ and p65 (Fig. 5a, left). In addition, we  (IgG lane). b, lysates from HEK293T cells cotransfected with Myc-NIBP and FLAG-IKK␣, FLAG-IKK␤, or FLAG-IKK␥ were immunoprecipitated with anti-FLAG and probed with an anti-NIBP antibody. NIBP was coimmunoprecipitated with IKK␤ and IKK␣, but not IKK␥. c, brain lysates were immunoprecipitated with anti-NIBP and analyzed by Western blot with anti-IKK␣, anti-IKK␤, anti-IKK␥, anti-NIK, or anti-NIBP antibodies. Two different anti-NIK antibodies were used to confirm the identification of endogenous NIK. One representative blot (IB) is shown. IKK␤ and NIK were coimmunoprecipitated with NIBP. d, mouse brain lysate was immunoprecipitated with anti-NIK, anti-IKK␣/␤, and anti-IKK␣ antibodies, and analyzed by Western blot with anti-NIBP, anti-NIK, anti-IKK␤, or anti-IKK␣ antibodies. NIBP was coimmunoprecipitated with NIK and IKK␤. e, GST-pull down assay to evaluate direct protein-protein interactions. Purified GST or GST-NIBP(cd) fusion protein (Coomassie staining, left) was incubated with in vitro translated [ 35 S]methionine-labeled full-length NIK, IKK␣, IKK␥, or truncated IKK␤ and analyzed by autoradiography. GST-NIBP interacted directly with IKK␤ and NIK, and when cold IKK␤ was added to 35 S-labeled NIK, the interaction of NIBP with NIK was enhanced (right panel). evaluated IB␣ and p65 phosphorylation following overexpression of IKK␣, IKK␤, or IKK␣/␤. IKK␣ overexpression did not induce IB␣ phosphorylation in the presence or absence of overexpressed NIBP (Fig. 5a, right), in agreement with the luciferase reporter gene assay data (Fig. 4f). In contrast, overexpression of IKK␤ markedly induced IB␣ phosphorylation, which was further potentiated in the presence of overexpressed NIBP (Fig. 5a, right). This is in agreement with our previous results showing that the absence of NIBP strongly reduces IKK␤-induced NF-B activation (Fig. 4f). When coexpressed, IKK␣ and IKK␤ activated IB␣ phosphorylation (Fig. 5a,  right). This was enhanced in the presence of NIBP, although to a lesser extent than with IKK␤ alone. This could be because of an inhibitory effect of IKK␣ on IKK␤ kinase activity (42). IKK␣ overexpression stimulated p65 phosphorylation only when coexpressed with NIBP (Fig. 5a, right). In this case, NIBP could be recruiting IKK␤ and/or NIK, which would be responsible for p65 phosphorylation (40,43). As previously reported (43), overexpression of IKK␤ alone induced p65 phosphorylation (Fig. 5a,  right). This was significantly enhanced by coexpression of NIBP, suggesting that NIBP may play a role in modulating IKK␤ kinase activity. When coexpressed, IKK␣ and IKK␤ induced a much higher degree of p65 phosphorylation than when expressed singularly, indicating a possible synergistic cooperation between the two kinases. We were unable to detect any further increase in phosphorylation of p65 in the presence of NIBP, possibly because of a saturation of the system (Fig. 5a,  right). Because it has been reported that IKK kinase activity is dependent on the phosphorylation of the activation loop of IKK␣ and IKK␤ (44,45), we evaluated whether this was the mechanism of NIBP-mediated potentiation of IKK kinase activity. We measured IKK␣ and IKK␤ phosphorylation with a phospho-specific antibody recognizing the activation loop of these kinases in HEK293T cells overexpressing IKK␣, IKK␤, or IKK␣/␤ (Fig. 5b). IKK␣ overexpression did not induce IKK␣ phosphorylation, whereas IKK␤ overexpression stimulated IKK␤ phosphorylation (45) (Fig. 5b). When IKK␣ and IKK␤ were coexpressed, an increase in phosphorylation of both IKK␣ and IKK␤ was observed (42) (Fig. 5b). Coexpression of IKK␣ with NIBP had no effect on the phosphorylation state of IKK␣ (Fig.  5b, right). However, IKK␣ and IKK␤ displayed an increased phosphorylation when coexpressed with NIBP (Fig. 5b, right). We next assessed the effect of NIBP on the phosphorylation of endogenous IKK␣ and IKK␤. In the absence of TNF␣ stimulation, NIBP increased the phosphorylation of IKK␣ and IKK␤ (Fig. 5c). HEK293 cells treated with TNF␣ exhibited a timedependent increase in IKK␣ and IKK␤ phosphorylation that was potentiated in the presence of overexpressed NIBP (Fig.  5c). Taken together, these data suggest that NIBP may be recruiting kinases such as NIK to the IKK complex, and therefore act like an adaptor protein.
Interestingly, our immunohistochemical studies in the central nervous system indicated that NIBP is predominantly expressed in neurons (Fig. 1b), suggesting that NIBP might perform a unique modulatory function in the neuronal NF-B signaling pathway. To address this question, we performed a series of experiments in the PC12 neuronal cell line that endogenously expresses NIBP (Fig. 6, a and b). As previously demonstrated in HEK293T cells and brain extracts (Fig. 2), NIBP interacts with both IKK␤ and NIK in PC12 cells (Fig. 6a). Based on recent studies demonstrating the involvement of NIK and NF-B in NGF-induced PC12 survival and neurite outgrowth (25,35,46,47), we evaluated NIBP function in NGFinduced PC12 gene expression and differentiation. After verifying that the NIBP(CR) siRNA construct effectively abolished NIBP protein expression (Fig. 6b), we assessed the expression of Bcl-xL, a survival gene induced by NGF and known to be regulated by NF-B in PC12 cells. As predicted, knockdown of NIBP dramatically reduced NGF-stimulated Bcl-xL gene expression, which was restored following overexpression of NIBP (Fig. 6c). In unstimulated cells, overexpression of NIBP markedly up-regulated Bcl-xL, mimicking the effect of NGF treatment (Fig. 6c). These data suggest that NIBP functions as a downstream component of the NGF pathway in PC12 cells. , NF-B-luciferase or interferon response factor-1-luciferase reporter vectors, and a ␤-galactosidase vector. Cells were treated with increasing concentrations of TNF␣ or IFN␥ for 6 h, and luciferase and ␤-galactosidase activity (for normalization) were assessed. Data are expressed as relative luciferase activity compared with control, and represent the mean Ϯ S.E. of four independent experiments run in triplicate. *, p Ͻ 0.05, one-way analysis of variance and Tukey test. c, HEK293T cells were transfected with FLAG-NIBP or empty vector, and NF-B DNA binding activity was measured by electrophoretic mobility shift assay following treatment with TNF␣ (10 ng/ml) for the indicated time periods (minutes). ns, nonspecific binding. d, Kinetics of IB␣ degradation in HEK293T cells treated with TNF␣ (10 ng/ml, 0 -60 min), in the absence or presence of overexpressed NIBP.

DISCUSSION
Although NF-B was initially discovered and characterized as a transcription factor required for B-cell-specific gene expression, further studies demonstrated that it is ubiquitously expressed and serves as a regulator of the expression of a wide variety of genes in many organs and tissues. In recent years, a large body of evidence has implicated NF-B in the regulation of both physiological and pathological processes in the central nervous system (48 -50). Nevertheless, little is known about the specific regulatory mechanisms of NF-B function in this system. To address this issue, we used a yeast two-hybrid approach and identified NIBP, a novel NF-B regulatory protein, which directly interacts with NIK and IKK␤ and is required for cytokine-induced NF-B activation.
Coimmunoprecipitation experiments with both overexpressed and endogenously expressed NIBP demonstrated the interaction of NIBP not only with NIK, originally chosen as bait in the yeast two-hybrid screening, but also with IKK␤, one of the members of the IKK complex. No interaction was detected with IKK␣ or IKK␥. These results point at a role of NIBP in the regulation of the classical NF-B pathway, in which, upon cytokine stimulation, IKK␤ is activated and phosphorylates IB proteins releasing NF-B dimers to the nucleus (51). To further corroborate this function, we demonstrated that NIBP is necessary for TNF␣-induced NF-B activation, which requires the recruitment of the classical NF-B signaling cascade (7). Indeed, when NIBP expression is abolished, TNF␣induced NF-B-dependent gene expression is reduced, as is the phosphorylation level of IB␣ and p65, two downstream targets of IKK␤. This suggests that NIBP modulates the function of IKK␤ by affecting its kinase activity. Interestingly, the interaction of NIBP with NIK is enhanced in the presence of IKK␤, suggesting that these three proteins are likely components of the same multimeric signalsome. This leads us to speculate that NIBP may act as a scaffolding protein and possibly interact with other members of the NF-B signaling cascade that we have not yet investigated. NIBP shares some similar functions with several other scaffolding molecules of the IKK complex that have been previously identified (e.g. IKAP (52), ELKS (53), and TRUSS (54)). In contrast to IKAP, ELKS, and TRUSS, however, NIBP does not interact with IKK␣ and IKK␥. This may indicate that the role of NIBP as a scaffold is even more specialized, and therefore that NIBP is recruited to the NF-B signaling cascade only following certain stimuli in specific cell types.
Even though NIBP does not appear to interact with IKK␣ or to affect its kinase activity, we cannot discard the possibility of a role of NIBP in the regulation of the alternative pathway. To specifically address this issue, we are currently evaluating NIBP function following stimulation with known activators of the alternative pathway (e.g. B cell-activating factor and lymphotoxin-␤).
Interestingly, NIBP is detectable only at low levels in immune organs, in which NF-B is known to perform important biological functions. On the other hand, it is highly expressed in non-immune organs such as muscle, kidney, heart, and brain. It is relevant to point out that NIBP expression in the central nervous system appears to be restricted to neurons, suggesting a highly specialized function for this protein in the modulation of NF-B signaling in this cell type. Indeed, to adapt a ubiquitous pathway, such as the NF-B pathway, to specific cell requirements, additional molecules like adaptors, scaffolds, activators, and inhibitors, specifically expressed in certain cell types, are necessary. Based on our results, we hypothesize that NIBP could represent one such molecule, selectively placed along the neuronal NF-B signaling cascade to confer specificity to the activation of NF-B in the central nervous system. That NIBP is necessary for NGF-induced neurite extension in PC12 cells further supports this hypothesis. The absence of NIBP completely prevents the ability of PC12 cells to differentiate when exposed to NGF, indicating that NIBP is an essential component of the signaling machinery required for this specific function. Our results are in agreement with earlier studies by Foehr and colleagues (25), demonstrating that PC12 differentiation is dependent on NF-B signaling, and specifically on NIK activation, which in turn leads to downstream phosphorylation and activation of the ERK1/ ERK2 MAPK pathway. Based on this evidence we can speculate that NIBP participates in this mechanism by interacting with NIK and IKK␤ and possibly by activating the ERK1/ERK2 MAPK pathway.
To further confirm the critical role of NIBP in PC12 cells, we evaluated its ability to modulate the expression of the antiapoptotic gene Bcl-xL. Bcl-xL is required for the survival of many peripheral and central neurons during development and its induction in PC12 cells following NGF treatment is dependent upon NF-B activation (35). By both overexpression and knockdown experiments we demonstrated that NIBP is required for NGF-induced Bcl-xL gene expression in PC12 cells. This indicates an essential role of NIBP within the NF-B pathway not only in the differentiation but also the survival of these cells, leading us to speculate that similar functions may be performed by NIBP in vivo in different neuronal populations. Ultimately, we will be able to fully appreciate these functions in an NIBP-deficient mouse, which is currently being generated in our laboratory. Taking advantage of such a model, FIG. 5. NIBP enhances cytokine-induced phosphorylation of IB␣ and p65. a, left panel, HEK293T cells transfected with NIBP or empty vector (control) and treated with TNF␣ (10 ng/ml TNF␣, 10 min) or IL-1␤ (100 ng/ml, 10 min). Right panel, HEK293T cells transfected with IKK␣, IKK␤, or IKK␣/␤ in the presence or absence of overexpressed NIBP. In both experiments, phosphorylation levels of IB␣ and p65 were evaluated by Western blot with specific anti-phospho-IB␣ (Ser-32/36) and anti-phospho-p65 (Ser-536) antibodies. As controls, the same blots were reprobed for ␤-actin and total IB␣ and p65. b, HEK293T cells were cotransfected with NIBP and IKK␣, IKK␤, or IKK␣/␤, and analyzed by Western blot with an anti-phospho-IKK␣/␤ (Ser-176/180) specific antibody. c, HEK293T cells transfected with NIBP or empty vector (Con) and either untreated or treated with TNF␣ (10 ng/ml, 2 or 5 min). Following immunoprecipitation (IP) with an anti-IKK␣ antibody, lysates were analyzed by Western blot (IB) with an anti-phospho-IKK␣/␤ antibody. As a control, the same blots were probed for anti-IKK␣.
we hope to better understand NF-B regulatory mechanisms in neurons. This knowledge could be valuable from a therapeutic viewpoint, as NF-B activity has been implicated in the pathophysiology of various neurodegenerative disorders.
FIG. 6. NIBP regulates NGF-induced gene expression and differentiation in PC12 cells. a, PC12 cell lysates were immunoprecipitated with an anti-NIBP antibody or control IgG, and analyzed by Western blot with anti-IKK␣, anti-IKK␤, anti-IKK␥, and anti-NIK antibodies. NIBP interacted with IKK␤ and NIK, but not with IKK␣ or IKK␥. b, PC12 cells were transduced with CR siRNA or empty vector and analyzed by Western blot with anti-NIBP. c, Bcl-xL gene expression was assessed by reverse transcriptase-PCR in PC12 cells in the presence or absence of NGF stimulation. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as a control. d, control (Con) lentivirus, NR, MR, and CR siRNA-infected PC12 cells were treated with NGF (100 ng/ml). Three days following treatment, neurite extension was evaluated by fluorescent microscopy in lentivirus-transduced cells expressing enhanced green fluorescent protein.