RelB-induced Expression of Cot, an MAP3K Family Member, Rescues RANKL-induced Osteoclastogenesis in Alymphoplasia Mice by Promoting NF-κB2 Processing by IKKα^*

Animals

Mice (8 weeks old) that were heterozygous for aly (aly/+) were purchased from Nippon Clea (Tokyo, Japan). Heterozygous males and females were bred to obtain homozygous aly/aly mice, and the offspring were screened using PCR (22). NF-κB2-deficient (NF-κB2^−/−) and RelB-deficient (RelB^−/−) mice were obtained from the Leibniz-Institute for Age Research at the Fritz-Lipmann-Institute (Jena, Germany). All mice were maintained at the Animal Resource Center, and experimental procedures were approved by the Animal Care and Use Committee of Kyushu Dental University (approval number 10-021).

Reagents

GST-RANKL was kindly provided by the Oriental Yeast Co., Ltd. (Shiga, Japan), and recombinant human M-CSF was purchased from PeproTech Inc. (Rocky Hill, NJ). The anti-RelB (sc-226), IκBα (sc-371), phosphorylated IKKα (sc-101706), HDAC1 (sc-7872), and Cot (sc-720) antibodies and Cot shRNA (sc-35096-SH) were purchased from Santa Cruz Biotechnology, Inc. The anti-NF-κB2 (catalog no. 4882), phosphorylated NF-κB2 (catalog no. 4810), phosphorylated IKKα/β (catalog no. 2681), Akt (catalog no. 2920), and phosphorylated Akt (catalog no. 9271) antibodies were obtained from Cell Signaling (Beverly, MA). The anti-NFATc1 (ab25916) antibodies and anti-p65 antibodies (SA-171) were purchased from Abcam (Cambridge, UK) and Biomol (Plymouth Meeting, PA), respectively. The anti-FLAG M5 and β-actin antibodies were purchased from Sigma-Aldrich.

Cell Culture

BMMs were prepared as osteoclast precursors from 5–8-week-old male WT, aly/aly, NF-κB2^−/−, or RelB^−/− mice. Bone marrow cells were obtained from mouse tibias and suspended in 96-well plates for 16 h in the presence of M-CSF (100 ng/ml) in α-minimal essential medium containing 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin. Non-adherent cells were harvested and further cultured for 2 days with M-CSF (100 ng/ml). More than 90% of the adherent cells that expressed macrophage-specific antigens, such as Mac-1, Moma-2, and F4/80, were used as BMMs (23). BMMs were cultured for 3 days with RANKL (100 ng/ml). Cultures were fixed with 3.7% formaldehyde, and osteoclasts were detected by staining for tartrate-resistant acid phosphatase (TRAP). TRAP-positive (TRAP⁺) multinucleated cells that had more than five nuclei were observed using a microscope and counted as osteoclasts. The 293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 5% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin.

Fractionation, Western Blot Analysis, and Immunoprecipitation

BMMs treated with RANKL for the indicated time were lysed in Tris-buffered saline (20 mm Tris-HCl and 200 mm NaCl) containing 1% Triton X-100 and protease inhibitors (aprotinin, pepstatin, dithiothreitol, and leupeptin). The cytosolic and nuclear extracts were prepared as described previously (23). For immunoprecipitation, an equal amount of protein was immunoprecipitated with anti-IKKα antibodies for 2 h; then protein A-Sepharose beads were added, and the samples were incubated for another 1 h. The immunoprecipitates were washed three times with lysis buffer and extracted in SDS sample buffer. The lysates or immunoprecipitates were resolved by 10% SDS-PAGE, transferred to Immobilon-P membranes (Millipore, Billerica, MA), and immunoblotted with individual antibodies. Then the membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). The immunoreactive proteins were visualized using ECL (Amersham Biosciences) and analyzed using a Luminescent image analyzer (Fujifilm, Tokyo, Japan).

Retroviral Transduction

The retroviral vector pMX-IRES-EGFP and Plat-E cells were kindly provided by Dr. T. Kitamura (University of Tokyo) (24, 25). The following constructs were cloned into the pMX-IRES-EGFP vector: the wild-type IKKα; a mutant form of IKKα (IKKαAA) that is not phosphorylated by NIK, with serine to alanine mutations at residues 176 and 180; a mutant form of IKKα (IKKαAAT23A) that is not phosphorylated by NIK or Akt, with serine to alanine mutations at residues 176 and 180 and a threonine to alanine mutation at residue 23; the dominant negative form of IKKα (IKKαKM), with a lysine to methionine mutation at residue 44; wild-type IKKα with a FLAG tag; wild-type p100; and p100ΔGRR, which cannot be processed to p52 (21). The constitutively activated form of Akt (AktCA), which lacks amino acids 4–129 and has an HA tag; the dominant negative form of Akt (AktDN), which has a lysine residue 179 to methionine mutation; a wild type, HA-tagged Akt; and Cot expression vectors were obtained from Addgene (Cambridge MA). Retrovirus packaging was performed by transfecting the plasmids into Plate-E cells using the Genejuice (Merck) transfection reagent. The supernatants of all retroviral vector-transfected Plat-E cells were used to infect primary BMMs in the presence of 8 μg/ml Polybrene.

Real-time PCR Analysis

Total RNA of BMMs or osteoclasts from WT or aly/aly mice was prepared using TRIzol reagent (Invitrogen). Two micrograms of total RNA was used to synthesize first-strand cDNA using SuperScript II transcriptase and random primers (Invitrogen). Real-time PCR was performed using the SYBR Green PCR Master Mix and a 7300 real-time PCR system (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. The experimental results were fit to a standard curve that was generated by amplifying serially diluted products under the same PCR conditions. GAPDH expression served as an internal control. The primer sequences were as follows: Cot, 5′-cttgcatttgcaaaccatgc-3′ (forward) and 5′-ggaacaaggagaacatccga-3′ (reverse); GAPDH, 5′-aactttggcattgtggaagg-3′ (forward) and 5′-acacattggggtaggaaca-3′ (reverse).

Construction of Reporter Plasmids and Luciferase Assays

To construct the luciferase reporter plasmid, a 0.5-kb fragment upstream from the transcription initiation site of the mouse Cot gene was cloned by a standard PCR method using PrimeSTAR DNA polymerase (Takara Shuzo Co., Siga, Japan) and mouse genomic DNA as a template. It was then subcloned into the HindIII and BamHI recognition sites of the pfLuc-basic vector from Dr. Sankar Ghosh (Columbia University) (26). Genejuice was used to transfect 293 cells with the reporter plasmids. The luciferase activity was measured using a Dual-Luciferase reporter assay system (Promega, Madison, WI).

Chromatin Immunoprecipitation (ChIP)

ChIP was performed with a ChIP assay kit (Upstate Biotechnology, Waltham, MA) according to the manufacturer's instructions, using antibodies against p65, RelB, NF-κB2, and normal IgG. The purified DNA was analyzed by PCR using primers that detect sequences within the region of Cot promoter that harbors the NF-κB binding NF-κB-responsive element. The primer pairs containing the distal NF-κB-responsive element were 5′-aggatctaaaaccgcaaact-3′ (forward) and 5-cttttccatttcccttttct-3′ (reverse). The primers containing the proximal NF-κB-responsive element were 5′-ccaccgacacagaacactca-3′ (forward) and 5′-gtcttgcgcaaggtgttttc-3′ (reverse).

Data Analysis

Comparisons were made using factorial analysis of variance. When significant F values were detected, Fisher's protected least significant difference post hoc test was performed to compare each of the groups. The data were expressed as the mean ± S.D., and p values of p < 0.05 were considered significant.

The Translocation of RelB into the Nucleus Correlates with RANKL-induced Osteoclastogenesis

We previously reported that alymphoplasia (aly/aly) mice, in which the processing of p100 to p52 does not occur due to an inactive form of NIK, showed an osteopetrotic phenotype with significantly reduced osteoclast numbers (14, 15). In contrast, the bone phenotype of NF-κB2^−/− mice, which lacks p100 and p52, was normal, and BMMs differentiated to osteoclasts, similar to WT BMMs; this result may be due to compensation for the lack of NF-κB2 by NF-κB1 (15). BMMs from WT, aly/aly, or NF-κB2^−/− mice were treated with RANKL (100 ng/ml) for 6 days in the presence of M-CSF (100 ng/ml). As reported previously (15), numerous osteoclasts were formed from BMMs in WT and NF-κB2^−/− mice, but few osteoclasts were formed from aly/aly BMMs (Fig. 1, A and B). Therefore, we hypothesized that the residual active p52 moiety, together with its binding partner RelB, eventually translocates into the nucleus to exert transcriptional activity. When BMMs from WT, aly/aly, or NF-κB2^−/− mice were treated with RANKL for the indicated time, RANKL led to translocation of p65 to the nucleus; the maximal level of this translocation occurred at 30 min, and the degradation of IκBα coincided with the translocation of p65 in all BMMs (Fig. 1C). The translocation of RelB was observed within 6 h after RANKL stimulation in WT BMMs. RANKL transiently led to the translocation of RelB with kinetics similar to that of p65 translocation in NF-κB2^−/− BMMs, which suggests that the RelB-p50 heterodimer translocates into the nucleus after RANKL stimulation in NF-κB2^−/− BMMs (27). However, RelB translocation by RANKL stimulation was not observed in aly/aly BMMs (Fig. 1C). The HDAC1 expression levels were comparable in all nuclear extracts from WT, aly/aly, and NF-κB2^−/− mice. These results suggested that the translocation of RelB into the nucleus correlates with RANKL-induced osteoclastogenesis.

The Overexpression of RelB Rescues RANKL-induced Osteoclastogenesis in aly/aly BMMs

To confirm that the translocation of RelB is important for RANKL-induced osteoclastogenesis, we transfected p65 or RelB into BMMs from aly/aly mice. The overexpression of p65 failed to rescue RANKL-induced osteoclastogenesis in aly/aly BMMs (Fig. 2, A–C). In contrast, overexpression of RelB rescued RANKL-induced osteoclastogenesis in aly/aly mice (Fig. 2, A and B). Furthermore, the overexpression of RelB induced NF-κB2 processing and NFATc1 expression (Fig. 2C).

We previously reported that the osteoclastogenesis induced by RANKL correlated well with the ratio of p52 to p100 (15). As reported previously, overexpression of p100ΔGRR, which cannot be processed to p52 (28), strongly inhibited RANKL-induced osteoclastogenesis in NF-κB2^−/− BMMs (Fig. 3, A and B). To examine whether NF-κB2 processing or the nuclear translocation of RelB in the nucleus is more important for RANKL-induced osteoclastogenesis, we transfected RelB into BMMs from NF-κB2^−/− mice in the presence of p100ΔGRR. The overexpression of RelB failed to restore RANKL-induced osteoclastogenesis in NF-κB2^−/− BMMs in the presence of p100ΔGRR, which suggested that NF-κB2 processing was more important for RANKL-induced osteoclastogenesis than RelB nuclear translocation (Fig. 3, A–C).

NIK phosphorylation of IKKα at serine residues 176 and 180 is required for downstream signaling (29, 30). Therefore, we mutated these serine residues to alanine (IKKαAA) and then transfected BMMs from WT mice with either IKKαAA or IKKαAA and RelB. Although the overexpression of IKKαAA suppressed RANKL-induced osteoclastogenesis by suppressing NF-κB2 processing and NFATc1 induction in WT BMMs, the overexpression of RelB restored these inhibitions by IKKαAA (Fig. 3, D–F). These results strongly suggested that the overexpression of RelB induced p100 processing by a mechanism that was independent of these NIK-phosphorylated sites of IKKα.

The Overexpression of RelB Induces Expression of Cot, Which Contributes to the Processing of NF-κB2

To further examine the NIK-independent molecular mechanism by which RelB overexpression rescued RANKL-induced osteoclastogenesis in aly/aly BMMs, we performed microarray analysis using RelB-transfected BMMs. Among the several genes that were up-regulated, we focused on Cot (cancer Osaka thyroid), which is an MAP3K family member (19) that activates NF-κB. BMMs from either WT or aly/aly mice were transfected with or without RelB and then treated with RANKL. RANKL induced Cot expression in the WT BMMs but not in the aly/aly BMMs (Fig. 4A). However, the overexpression of RelB strongly induced the expression of Cot (Fig. 4A) in WT and aly/aly BMMs (Fig. 4A).

To confirm that RANKL induces Cot expression in WT and aly/aly BMMs, BMMs from either WT or aly/aly mice were treated with RANKL for the indicated time. RANKL transiently induced expression of Cot at 30 min and further induced Cot from 6 to 48 h after RANKL stimulation in WT BMMs. The induction of Cot attained a maximal level 30 min after RANKL stimulation and declined thereafter in aly/aly BMMs (Fig. 4B). As reported previously (15), NF-κB2 processing and NFATc1 induction were impaired in aly/aly BMMs compared with WT BMMs (Fig. 4B).

To examine the regulatory mechanism by which RelB induces Cot expression, we constructed a series of luciferase reporter plasmids from Cot0.5-luc, which carries a 0.5-kb fragment upstream of the transcription initiation site of the mouse Cot promoter harboring the NF-κB-responsive element. Furthermore, we generated an additional pair of deletion plasmids with 1 or no NF-κB-responsive elements, named Cot0.3-luc and Cot0.1-luc, respectively. We then transfected 293 cells with each of these luciferase reporter plasmids, with or without p65, RelB, or RelB and p52. The expression of p65 significantly stimulated Cot0.5-luc. Although RelB alone failed to stimulate Cot0.5-luc, RelB moderately stimulated Cot0.5-luc in the presence of p52 (Fig. 4C). RelB, but not p65, stimulated Cot0.3-luc. This activity was further enhanced in the presence of p52. Neither p65 nor RelB stimulated Cot0.1-luc in the presence or absence of p52 (Fig. 4C). These data indicate that the distal NF-κB-responsive element was activated mainly by the classical NF-κB pathway and that the proximal NF-κB-responsive element of the Cot promoter was activated by the alternative NF-κB pathway.

ChIP analyses confirmed that p65 was only recruited to the distal NF-κB-responsive element and not the proximal NF-κB-responsive element of the Cot promoter after 30 min of RANKL stimulation in BMMs isolated from both WT and aly/aly mice. RelB and p52 were recruited to both the distal and proximal NF-κB-responsive elements of the Cot promoter after 12 h of RANKL stimulation in BMMs isolated from WT mice. However, RelB and p52 failed to bind to the proximal NF-κB-responsive element of the Cot promoter in BMMs isolated from aly/aly or RelB^−/− mice (Fig. 4D). These results suggest that the alternative NF-κB pathway is involved in the induction and/or maintenance of Cot expression.

To further examine the involvement of RelB-induced Cot expression in RANKL-induced osteoclastogenesis, we knocked down Cot expression using an shRNA that specifically targeted the expression of Cot. Although the overexpression of RelB moderately enhanced RANKL-induced osteoclastogenesis, the knockdown of Cot expression in WT BMMs suppressed RANKL-induced osteoclastogenesis in the presence of RelB (Fig. 5, A and B). In aly/aly BMMs, knocking down of Cot expression significantly suppressed the RANKL-induced osteoclastogenesis that was induced by RelB overexpression (Fig. 5, A and B). It has been reported that IKKα phosphorylates NF-κB2 at serine residues 865 and 869 (Ser-865/869) (13, 32). In our study, RelB overexpression induced phosphorylation and processing of NF-κB2 and expression of NFATc1 in aly/aly BMMs, and knocking down Cot completely inhibited these effects, suggesting that RelB-induced Cot expression triggers the phosphorylation and processing of NF-κB2 and restores RANKL-induced osteoclastogenesis in aly/aly BMMs (Fig. 5).

We then confirmed that the RelB-dependent expression of Cot was critical for RANKL-induced osteoclastogenesis using RelB^−/− BMMs. BMMs from WT or RelB^−/− mice were treated with RANKL for the indicated time, and the expression levels of Cot were examined. Similar to aly/aly BMMs, RANKL transiently induced the expression of Cot at 30 min, but not between 6 and 48 h, after RANKL stimulation in RelB^−/− BMMs, suggesting that the second wave of Cot induction depends on RelB (Fig. 6A). NF-κB2 processing and NFATc1 induction were also impaired in RelB^−/− BMMs compared with in WT BMMs (Fig. 6A).

Few osteoclasts were formed in the RelB^−/− BMMs after RANKL stimulation, and the processing of NF-κB2 was barely detectable (Fig. 6, B and C). The overexpression of RelB in RelB^−/− BMMs fully rescued RANKL-induced osteoclastogenesis (Fig. 6, B and C). The induction of Cot expression and NF-κB2 processing was also observed in RelB-overexpressing RelB^−/− BMMs (Fig. 6D). In contrast, knocking down Cot suppressed RANKL-induced osteoclastogenesis and the processing of NF-κB2, even in the presence of RelB (Fig. 6). In addition, overexpression of Cot partially rescued RANKL-induced osteoclastogenesis and the processing of NF-κB2 (Fig. 6, B–D). These results strongly indicated that the RelB-dependent expression of Cot was critical for RANKL-induced osteoclastogenesis by the alternative NF-κB pathway.

RelB-induced Cot Phosphorylates Threonine 23 of IKKα, Which Induces Processing of NF-κB2

We then examined whether the Cot-induced processing of NF-κB2 depended on IKKα kinase activity. Knocking down IKKα expression failed to restore the RANKL-induced osteoclastogenesis that was induced by RelB overexpression as well as the inhibition of processing of NF-κB2 and NFATc1 expression in aly/aly BMMs (Fig. 7, A–C).

WT BMMs transfected with empty, IKKαWT, IKKαAA, or IKKαKM (kinase-dead) and with or without Cot were treated with RANKL and M-CSF for 6 days. Overexpression of IKKα WT enhanced RANKL-induced osteoclastogenesis by autophosphorylating IKKα at serine 180, compared with control, and overexpression of IKKαWT with Cot further enhanced RANKL-induced osteoclastogenesis by further phosphorylating Thr-23 of IKKα (Fig. 7, D and E). Overexpression of IKKαAA alone markedly suppressed osteoclastogenesis compared with control or IKKαWT (Fig. 7, D and E). In contrast, overexpression of Cot in the presence of IKKαAA recovered osteoclastogenesis compared with IKKαAA alone. Furthermore, overexpression of Cot strongly induced the phosphorylation of IKKα at Thr-23 and the phosphorylation (Ser-865/869) and processing of NF-κB2 (Fig. 7F). To further examine the importance of Cot-induced IKKα phosphorylation at Thr-23, we generated an additional construct, IKKαAAT23A. The overexpression of Cot failed to rescue RANKL-induced osteoclastogenesis because the phosphorylation (Ser-865/869) and processing of NF-κB2 was inhibited (Fig. 7, G–I). Although overexpression of Cot resulted in phosphorylation of IKKα at Thr-23, it failed to induce phosphorylation and processing of NF-κB2 and osteoclastogenesis in the presence of IKKαKM (Fig. 7, D–F). These results strongly indicate that phosphorylation of IKKα at Thr-23 and its kinase activity are important for NF-κB2 processing and osteoclastogenesis by the overexpression of Cot.

Cot and Akt Cooperatively Induce the Processing of NF-κB2 and RANKL-induced Osteoclastogenesis

Previous reports showed that Akt phosphorylates IKKα at Thr-23 (33) and that Akt and Cot physiologically associate and functionally cooperate (34). Thus, we examined the relationship between Akt and Cot during RelB-induced osteoclastogenesis in WT or aly/aly BMMs. First, BMMs isolated from WT or aly/aly mice were transfected with RelB with or without a constitutively activated form of Akt (AktCA) or a dominant-negative form of Akt (AktDN) and were then treated with RANKL and M-CSF for 6 days. In WT BMMs, RelB overexpression slightly enhanced RANKL-induced osteoclastogenesis. The co-expression of AktCA and RelB further enhanced RANKL-induced osteoclastogenesis. As described above, RelB overexpression in aly/aly BMMs rescued RANKL-induced osteoclastogenesis. The co-expression of AktCA and RelB further enhanced RANKL-induced osteoclastogenesis in these cells (Fig. 8, A and B). However, Cot knockdown moderately suppressed these effects in WT BMMs and significantly suppressed the AktCA and RelB-dependent rescue of RANKL-induced osteoclastogenesis in aly/aly BMMs (Fig. 8, A and B). The phosphorylation and processing of NF-κB2 induced by RelB overexpression in both WT and aly/aly BMMs was further enhanced by the co-expression of AktCA and RelB (Fig. 8C). In contrast, AktDN expression strongly inhibited RelB-induced osteoclastogenesis and the phosphorylation and processing of NF-κB2 in WT and aly/aly BMMs (Fig. 8, A–C).

To further investigate the relationship between Cot and Akt, BMMs isolated from WT or aly/aly mice were transfected with shCot, with or without AktCA, and then treated with RANKL and M-CSF for 6 days. AktCA moderately enhanced RANKL-induced osteoclastogenesis by enriching the phosphorylation (Ser-865/869) and processing of NF-κB2, whereas Cot knockdown inhibited RANKL-induced osteoclastogenesis in WT BMMs, even in the presence of AktCA (Fig. 8, D–F). The overexpression of AktCA alone failed to promote RANKL-induced osteoclastogenesis in aly/aly cells, possibly due to the markedly reduced expression of Cot in aly/aly BMMs compared with WT BMMs (Fig. 8, D and E).

The slightly enhanced osteoclastogenesis caused by Cot overexpression in WT BMMs was suppressed in the presence AktDN (Fig. 8, G and H). Cot overexpression promoted RANKL-induced osteoclastogenesis by enhancing the phosphorylation (Ser-865/869) and processing of NF-κB2 and increasing NFATc1 expression, whereas co-transfection of Cot and AktDN negated these effects (Fig. 8, G–I). These results suggest that RelB-induced Cot collaborates with Akt to regulate NF-κB2 processing and RANKL-induced osteoclastogenesis.