Correspondence to: Sakae Tanaka, Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel:81-3-3815-5411 ext 3375 Fax:81-3-3818-4082 E-mail:tanakas-ort{at}h.u-tokyo.ac.jp.
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Abstract |
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To examine the role of mitogen-activated protein kinase and nuclear factor kappa B (NF-B) pathways on osteoclast survival and activation, we constructed adenovirus vectors carrying various mutants of signaling molecules: dominant negative Ras (RasDN), constitutively active MEK1 (MEKCA), dominant negative I
B kinase 2 (IKKDN), and constitutively active IKK2 (IKKCA). Inhibiting ERK activity by RasDN overexpression rapidly induced the apoptosis of osteoclast-like cells (OCLs) formed in vitro, whereas ERK activation after the introduction of MEKCA remarkably lengthened their survival by preventing spontaneous apoptosis. Neither inhibition nor activation of ERK affected the bone-resorbing activity of OCLs. Inhibition of NF-
B pathway with IKKDN virus suppressed the pit-forming activity of OCLs and NF-
B activation by IKKCA expression upregulated it without affecting their survival. Interleukin 1
(IL-1
) strongly induced ERK activation as well as NF-
B activation. RasDN virus partially inhibited ERK activation, and OCL survival promoted by IL-1
. Inhibiting NF-
B activation by IKKDN virus significantly suppressed the pit-forming activity enhanced by IL-1
. These results indicate that ERK and NF-
B regulate different aspects of osteoclast activation: ERK is responsible for osteoclast survival, whereas NF-
B regulates osteoclast activation for bone resorption.
Key Words:
osteoclast, adenovirus, apoptosis, Ras, NF-B
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Introduction |
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Osteoclasts, multinucleated giant cells responsible for bone resorption, are terminally differentiated cells with a short life span. Recent findings suggest that the survival of osteoclasts is precisely regulated through interactions with supporting cells as well as by the action of several cytokines (
The small GTP-binding protein Ras is ubiquitously found in eukaryotic organisms and functions as an inducer of various intracellular signaling pathways including cell proliferation and differentiation (
Nuclear factor kappa B (NF-B) is a transcription factor that regulates the expression of many genes involved in immune and inflammatory responses (
B is controlled by sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit, I
B. A large multiprotein complex, the I
B kinase (IKK) signalsome, was found to contain a cytokine-inducible I
B kinase activity that phosphorylates I
B-
and I
B-ß. The functional IKK complex contains IKK1 (IKK
), IKK2 (IKKß), and IKK
, and both IKK1 and IKK2 appear to make an essential contribution to I
B phosphorylation.
B activation induced by TNF-
, whereas Ser177 and Ser181 to Glu mutant induced constitutive activation of NF-
B. It was recently reported that mice deficient in both the p50 and p52 subunits of NF-
B fail to generate mature osteoclasts and exhibit severe osteopetrosis (
B pathways in the survival of osteoclasts promoted by interleukin 1 (IL-1). These results suggest that the NF-
B pathway is important for both the differentiation of osteoclast precursors and the survival of mature osteoclasts.
We recently reported that adenoviral vectors are useful for modulating osteoclast function by transducing foreign genes into osteoclasts (B pathways in the survival and activation of osteoclasts, and found that the ERK activation is essential in maintaining osteoclast survival as is the NF-
B pathway for upregulation of bone-resorbing activity of osteoclasts.
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Materials and Methods |
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Animals and Chemicals
Newborn ddY mice and 8-wk-old male ddY mice were purchased from Shizuoka Laboratories Animal Center. -Modified minimum essential medium (
MEM) and DME were purchased from GIBCO BRL and Life Technologies Inc., and FBS was purchased from Cell Culture Laboratory. Bacterial collagenase and 1
,25-dihydroxyvitamin D3 (1
,25(OH)2D3) were purchased from Wako Pure Chemical Co., and dispase from Godo Shusei Co. MEK inhibitor PD98059, antiphospho-ERK and antiI
B-
antibodies were purchased from New England Biolabs, Inc., antiv-src antibody (clone 327) from Oncogene Research Products, antiFlag antibody from Sigma Chemicals, and antiNF-
B (p65) antibody from Santa Cruz Biotechnology. AntiRas, antiMEK1, and antiERK antibodies were obtained from Transduction Laboratories. Other chemicals and reagents used in this study were of analytical grade.
Cells and Cell Cultures
Mouse primary osteoblastic cells were obtained from 1-d-old ddY mouse calvaria by enzymatic digestion, and bone marrow cells were from tibiae of 8-wk-old male ddY mice (,25(OH)2D3 and 1 µM PGE2 as previously reported (
MEM, and treated with 5 ml of
MEM containing 0.1% collagenase and 0.2% dispase for 10 min to remove osteoblastic cells. The purity of OCLs and their precursors was >90% at the final preparation (
Constructs and Gene Transduction
The recombinant adenovirus vector carrying either dominant negative Ras (Ser17 to Asn, AxRasDN), constitutively active MEK1 (Ser218 and Ser222 to Glu, AxMEKCA), dominant negative IKK2 (also called IKKß) (Ser177 and Ser181 to Ala, AxIKKDN), or constitutively active IKK2 (Ser177 and Ser181 to Glu, AxIKKCA) gene under the control of the CAG (cytomegalovirus IE enhancer + chicken ß-actin promoter + rabbit ß-globin poly (A)+ signal) promoter was constructed by homologous recombination between the expression cosmid cassette and the parental virus genome in 293 cells as described (MEM containing the recombinant adenoviruses for 1 h at 37°C at an indicated MOI. The cells were washed twice with PBS and further incubated with
MEM/10%FBS at 37°C. Experiments were performed 24 h after the infection.
Western Blotting
All extraction procedures were performed at 4°C or on ice. Cells were washed with ice-cold PBS, and then lysed by adding TNE buffer (1% NP-40, 10 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1 mM EDTA, 2 mM Na3VO4, 10 mM NaF, and 10 µg/ml aprotinin). The lysates were clarified by centrifugation at 15,000 rpm for 20 min. An equal amount (20 µg) of protein was subjected to 8% SDS-PAGE under a reducing condition, transferred electrophoretically onto a nitrocellulose membrane, and probed sequentially with an appropriate primary antibody followed by secondary antibodies coupled with HRP (Promega Corp.). Immunoreactive proteins were visualized using ECL Western blotting detection reagents (Amersham Co.) following the procedures recommended by the supplier. The blots were stripped by incubating for 20 min in stripping buffer (2% SDS, 100 mM 2-mercaptoethanol, 62.5 mM Tris-HCl, pH 6.7) at 50°C and reprobed by other antibodies.
Immunoprecipitation and In Vitro Kinase Assay
Treated cell cultures were washed twice with ice-cold PBS and lysed in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 12 mM ß-glycerophosphate, 5 mM EGTA, 0.5% deoxycholate, 3 mM DTT, 10 mM NaF, 1 mM Na3VO4, 2 µM leupeptin, 20 µg/ml aprotinin, and 1 mM PMSF). After 30 min on ice, cell lysates were cleared by centrifugation at 12,000 g for 20 min. The protein concentration in each sample was quantified by the Bradford method, and immunoprecipitation was performed by incubating 200 µl of lysate with 2 µg of antiERK2 (p42) antibody (Santa Cruz Biotechnology) for 1 h, and then adding 20 µl of protein Aagarose. After incubation for 1 h at 4°C with end-over-end mixing, the immunocomplex was recovered by centrifugation and washed with washing buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EGTA, 2 mM DTT, and 1 mM PMSF) twice. Kinase activity was assayed for 20 min at 37°C in the presence of 6 µg substrate (MBP), 30 µM ATP, and 20 µCi -[32P] ATP in 55 µl assay buffer (20 mM Tris-HCl, pH 7.5, and 20 mM MgCl2). After completion of kinase assays, the protein was resolved by SDS-PAGE, and the gels were dried and subjected to autoradiography. The relative activity of ERK2 was quantified by measuring the radioactivity of phosphorus-32 incorporated into MBP.
DNA Extraction and Electrophoretic Analysis
DNA was prepared and analyzed by gel electrophoresis according to the method described previously (
TUNEL Assay
Cells undergoing apoptosis were identified by means of the TdT-mediated dUTP-dioxigenin nick-end labeling (TUNEL) method, which specifically labels the 3'-hydroxyl terminal of DNA strand breaks. For the TUNEL procedure, all agents, including buffers, were part of a kit (apoptosis in situ detection kit; Wako Pure Chemical Co.); the staining procedure was carried out according to the manufacturer's recommendation. Negative controls included omission of TdT. Positive controls included treatment of the samples with DNase I. Apoptotic cells were recognized by their dark nuclear staining (TUNEL-positive), and nuclei of nonapoptotic cells were visualized by staining with methyl green.
Survival of OCLs
The survival rate was measured as reported (
Immunofluorescence Microscopy
For immunofluorescence analysis, cells were plated on sterile FBS-coated glass coverslips and purified by treatment with MEM containing 0.1% collagenase and 0.2% dispase. After purification, OCLs were preincubated for 20 min at 37°C in
MEM without FBS and further incubated in
MEM with IL-1
(10 ng/ml) for indicated times, fixed in PBS containing 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 in PBS for 5 min, and then blocked with 5% skim milk in PBS for 20 min at room temperature. Cells were sequentially stained with antip65 antibody followed by Cy5-conjugated anti-rabbit IgG (Amersham Co.) as previously reported (
Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared as described previously (B was provided by Dr. Jun-Ichiro Inoue (The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan). The DNA binding reaction was performed at room temperature in a volume of 20 µl, which contained the binding buffer (25 mM Tris-HCl, pH 7.5, 1.5 mM EDTA, 7.5% glycerol, 75 mM NaCl, 1.5 mM DTT, 0.3% NP-40, and 1 mg/ml BSA), 1 µg of poly (dI-dC), 105 cpm of 32P-labeled probe, and 8 µg of nuclear proteins. After incubation for 30 min, the samples were fractionated on a 4% polyacrylamide gel and visualized by exposing dried gel to film.
Pit Formation Assay
Functionally active OCLs formed in cocultures were obtained by a collagen gel culture system as described by ,25(OH)2D3 and 1 µM PGE2 on culture dishes (6 cm) coated with 0.2% collagen gel matrix. OCLs 24 h after the infection were recovered by digesting the collagen gel with 0.2% collagenase for 20 min at 37°C. The cells released from the dishes were collected by centrifugation at 250 g for 5 min and resuspended in 5 ml of
MEM containing 10% FBS. An aliquot of the crude OCL preparation was transferred onto dentine slices and cultured for an additional 12 h. After 12 h of incubation, the medium was removed and 1 M NH4OH was added to the wells for 30 min. Adherent cells were removed from the dentine slices by ultrasonication, and the resorption pits were visualized by staining with 1% toluidine blue. The resorbed area was measured using an image analysis system (SYSTEM SUPPLY) linked to a light microscope (Nikon).
Statistical Analysis
Each series of experiments was repeated at least three times. The results obtained from a typical experiment were expressed as the means ± SD. Significant differences were determined using factorial analysis of variance (ANOVA).
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Results |
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Downregulation of Ras Inhibits Osteoclast Survival
Recently, we have reported that a replication-deficient adenovirus vector that contains a reporter gene encoding ß-galactosidase efficiently infected human OCLs derived from human giant cell tumors and mouse OCLs formed in vitro (MEM containing 10% FBS to determine their survival. After the removal of osteoblastic cells, ~70% of OCLs infected with or without Ax1w1 died within 18 h (Figure 1D and Figure E). In contrast, almost all the RasDN-expressed OCLs disappeared within 18 h (Figure 1D and Figure E), indicating a critical role of Ras in the survival of osteoclasts.
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The Deteriorative Effect of RasDN Overexpression Is Reversed by AxMEKCA Infection
We next examined the signaling pathways working downstream of Ras in osteoclasts. As shown in the top and middle panels of Figure 1 C, overexpression of RasDN clearly inhibited the basal activity of ERK in OCLs and ERK activity was ~90% downregulated in RasDN-expressed OCLs at an MOI of 100. To determine whether or not modulating ERK activity could affect the survival of OCLs, we used PD98059, a specific inhibitor of ERK activation that does not affect the activation of other MAPKs (
To examine whether or not ERK is the main signaling pathway working downstream of Ras in osteoclasts, we investigated the survival of OCLs that were coinfected with AxRasDN and AxMEKCA. Coinfected OCLs expressed RasDN and MEKCA at the similar level with the cells infected with either RasDN or MEKCA (Figure 2 A), and the deteriorative effect of AxRasDN on OCLs was completely rescued by coinfection with AxMEKCA (Figure 2 B). Nor did the treatment with PD98059, which suppresses the intrinsic MEK activation, inhibit the survival of MEKCA-expressed OCLs (Figure 2 B). We also found that the expression of RasDN did not alter the activity of JNK/SAPK or p38 in OCLs (data not shown). Taken together, these findings clearly demonstrate that the Ras/ERK pathway is an important component of survival signal in osteoclasts.
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RasDN Expression Causes Apoptosis of Osteoclasts
We next determined if the reduction of OCL survival by expression of RasDN was due to apoptosis. After purification, uninfected OCLs or OCLs infected with Ax1w1, AxRasDN, or AxMEKCA at an MOI of 100 were cultured for 12 or 24 h. The cells were harvested, and genomic DNA was isolated and resolved on 1.5% agarose gel. After 12 h of culturing, DNA fragmentation was hardly detectable in uninfected OCLs or OCLs infected with Ax1w1, whereas OCLs infected with AxRasDN clearly showed DNA fragmentation (Figure 3 A). This fragmentation was observed even in uninfected OCLs or OCLs infected with Ax1w1 after 24 h of culture, and the expression of MEKCA markedly inhibited the fragmentation (Figure 3 A). Almost all the nuclei of MEKCA-expressed OCLs, 24 h after the purification, were stained uniformly with Hoechst 33258, which indicates that the cells were alive and the nuclei were intact, whereas expression of RasDN caused condensation of >80% of nuclei 12 h after purification (Figure 3 B). We further determined that ~80% of the nuclei infected with AxRasDN underwent apoptosis 12 h after the purification as shown in Figure 3 C by TUNEL staining. In contrast, positive TUNEL staining was hardly observed in MEKCA-expressed OCLs even 24 h after the purification (Figure 3 C). These findings suggest that Ras/ERK pathways contribute greatly to preventing apoptosis of osteoclasts.
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IL-1 Activates ERK in OCLs
IL-1 is a proinflammatory cytokine that strongly promotes the survival of osteoclasts by preventing their apoptosis (
treatment on ERK activity in OCLs. After purification, Ax1w1-infected OCLs and RasDN-expressed OCLs were preincubated for 20 min at 37°C in
MEM without FBS and further incubated in
MEM with IL-1
(10 ng/ml) for various periods. As shown in the top panel of Figure 4 A, IL-1
stimulated a rapid and transient increase in ERK activity in OCLs. ERK activation in response to IL-1
was transient, reaching the highest level after 1530 min of stimulation and returning to the basal level after 60 min. Stimulation of ERK activity was not accompanied by a change in the expression level of ERK protein (Figure 4 A, middle). As previously reported by
treatment strongly promoted the survival of OCLs (Figure 4 B). Interestingly, the expression of RasDN in OCLs only partially inhibited the ERK stimulation induced by IL-1
, as shown in the top panel of Figure 4 A, and partially suppressed the promotion of their survival induced by IL-1
(Figure 4 B).
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RasDN Expression Does Not Affect the IL-1induced Nuclear Translocation of NF-
B in OCLs
Because activation of NF-B was reported to be involved in the survival of osteoclasts promoted by IL-1
(
B in response to IL-1
. NF-
B is present in the cytosol in an inactive state, complexed with the inhibitory I
B proteins. Activation occurs via phosphorylation of I
B-
at Ser 32 and 36, which promotes the degradation of I
B-
, resulting in the dissociation and nuclear translocation of active NF-
B (
B-
expression rapidly decreased upon IL-1
stimulation and returned to the basal level after 60 min of stimulation in Ax1w1-infected OCLs. The bottom panel of Figure 4 A shows that RasDN expression in OCLs did not affect the kinetics of the degradation of I
B-
by IL-1
. The change in the localization of NF-
B in OCLs treated with IL-1
was also examined immunocytochemically using specific antibodies against the p65 subunit of NF-
B. Before stimulation, p65 was diffusely distributed throughout the cytoplasm. Consistent with the previous report (
for 30 min, p65 was detected in most nuclei of the cells. Overexpression of RasDN did not affect the kinetics of nuclear translocation of NF-
B in OCLs (Figure 4 D). Consistent with the results of the immunocytochemistry, electrophoretic mobility shift assay (EMSA) showed that NF-
B binding activity was not impaired in RasDN-overexpressed OCLs (Figure 4 C).
Modulation of NF-B Activity Did Not Affect the Survival of OCLs
To further examine the role of NF-B pathway in osteoclasts, we constructed adenovirus vectors encoding dominant negative IKK2 (IKKDN) and constitutively active IKK2 (IKKCA) with Flag tag, and investigated their effects on OCLs. Figure 5 A demonstrates the clear induction of IKKDN and IKKCA in infected OCLs in an MOI-dependent manner. Figure 5 B shows that the degradation of I
B-
induced by IL-1
treatment was strongly blocked by IKKDN expression. Immunocytochemical studies showed that IKKDN expression inhibited nuclear translocation of p65 in IL-1
stimulated OCLs (Figure 5 D), and <25% of the nuclei of OCLs were positively stained for p65 even after 30 min of IL-1
stimulation. In contrast, >60% of the nuclei of AxIkkCA-infected OCLs were positively stained without any stimulation (Figure 5 D). EMSA demonstrated that NF-
B activation was inhibited in IKKDN-expressed OCLs treated with IL-1
. Conversely, slight constitutive NF-
B activation was observed in IKKCA-expressed OCLs (Figure 5 C). As shown in Figure 6, neither IKKDN nor IKKCA virus infection significantly changed the survival of OCLs with or without IL-1
stimulation.
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NF-B Pathway Is Involved in Osteoclast Activation for Bone Resorption
We next examined the role of ERK pathways and NF-B pathways on bone-resorbing activity of OCLs using the adenovirus vectors described above. This activity of OCLs was quantified by measuring the pit area formed on dentine slices as previously reported (
strongly promotes not only the survival of osteoclasts, but also their bone resorbing activity (
. As shown in Figure 7 B, IL-1
treatment strongly enhanced the OCL pit-forming activity, and inhibiting NF-
B activation by the IKKDN virus significantly suppressed the pit-forming activity enhanced by IL-1
.
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Colony-stimulating Factor 1 (CSF-1) Activates ERK, but Not NF-B in OCLs
Because CSF-1 (also called macrophage colony-stimulating factor) has been reported to prolong the survival of OCLs without upregulating their bone resorbing activity, we examined the effect of CSF-1 treatment on ERK and NF-B activity in OCLs. As shown in the top panel of Figure 8 A, ERK activation in response to CSF-1 was transient, reaching the highest level after 215 min of stimulation and returning to the basal level after 60 min. Stimulation of ERK activity was not accompanied by a change in the expression level of ERK protein (Figure 8 A, middle). As previously reported (
B-
(Figure 8 A, bottom) and activate NF-
B (Figure 8 B). In addition, OCL bone-resorbing activity remained unchanged despite CSF-1 stimulation (Figure 8 D). These results strongly support our findings that ERK and NF-
B pathways are involved in osteoclast survival and activation for bone resorption, respectively.
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Discussion |
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The Ras superfamily of GTPases is implicated in a number of cellular physiological processes including cell proliferation and differentiation, and plays important roles in the organization of cellular architecture and regulation of membrane dynamism in addition to cell cycle control (
The pleiotropic functions of the Ras protein are now explained by its capability to activate multiple downstream pathways (, which strongly promotes OCL survival, rapidly stimulates ERK activation in OCLs. It should be noted that overexpression of RasDN only partially inhibited the ERK activation and suppressed the prolonged survival induced by IL-1
, whereas PD98059 strongly eliminated the effect of IL-1
(data not shown), suggesting that IL-1
stimulates ERK activity via Ras-independent pathways as well. These results clearly demonstrate that ERK activation is indispensable for the survival of osteoclasts, and that Ras is at least partially involved in this process.
It remains elusive how ERK activation stimulates OCL survival. In fibroblasts, ERK activation is followed by phosphorylation of transcription factors and induction of immediate early gene expression. The dimeric transcription factor AP-1 (activator protein-1) is a major target of cell growth, differentiation, and stress signaling pathways ( stimulation increased c-Fos, Fra-2, c-Jun, and JunB expression (data not shown), suggesting that AP-1 activation may be involved in the survival signaling in osteoclasts. Further study will be necessary to elucidate the detail of osteoclast survival signaling.
B is involved in the survival of osteoclasts promoted by IL-1
. They clearly showed the nuclear translocation of NF-
B and degradation of I
B in OCLs in response to IL-1
treatment. The survival of OCLs supported by IL-1
was suppressed by pretreatment with proteasome inhibitors, which inhibits NF-
B activation by preventing I
B degradation, or with antisense oligonucleotides to NF-
B components (
B on the survival of OCLs. Consistent with the previous report, IL-1
treatment induced rapid nuclear translocation of NF-
B in OCLs and degradation of I
B. However, overexpression of neither RasDN nor MEKCA changed the localization of NF-
B in the presence or absence of IL-1
. In addition, either inhibition of NF-
B activity by overexpression of dominant negative IKK (IKKDN) or sustained activation of NF-
B by transducing constitutively active IKK (IKKCA) had little effect on the survival of OCLs with or without IL-1
stimulation. We cannot fully explain the reason for the discrepancy between our results and those of the previous report, but it is possible that proteasome inhibitors and antisense oligonucleotides may possess some undesirable effects on OCLs unrelated to NF-
B inhibition.
However, NF-B has an important role in the bone-resorbing activity of osteoclasts because adenovirus vector-induced IKKDN expression significantly suppressed the pit-forming activity of OCLs and IKKCA expression enhanced it, although these two viruses had no effect on OCL survival. Members of the TNF receptor family and the IL-1 receptor associated either directly or indirectly with TNF receptor-associated factors (TRAFs), adaptor proteins that recruit and activate downstream signaling transducers (
B by signaling via NIK and IKK, but it also activates ERK in a Ras-independent manner (
can promote survival and activity of osteoclasts simultaneously via ERK and NF-
B activation, both of which are mediated by TRAF6. Our present study proposes the reciprocal role of ERK and NF-
B pathways in the survival and activation of osteoclasts. Further investigation of these pathways in osteoclasts will give us new insight into the molecular mechanism regulating bone resorption.
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Footnotes |
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1 Abbreviations used in this paper: -MEM,
-modified minimum essential medium; CSF, colony stimulating factor; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal-regulated kinase; IKK, I
B kinase; IL-1
, interleukin 1
; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; MOI, multiplicity of infection; NF-
B, nuclear factor kappa B; OCLs, osteoclast-like cells; TUNEL, Tdt-mediated dUTP dioxigenin nick-end labeling.
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Acknowledgements |
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R. Yamaguchi (Department of Orthopaedic Surgery, The University of Tokyo) provided expert technical assistance. We thank I. Saito, J. Miyazaki, and J.-I. Inoue (The Institute of Medical Science, The University of Tokyo) for gifts of the recombinant adenovirus vector system, the CAG promoter, and the NF-B binding oligonucleotide, respectively.
This work was in part supported by a grant from the Japan Orthopaedic and Traumatology Foundation Inc. (No. 0089; Alcare Award) and Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan to S. Tanaka and from Bristol-Myers Squibb/Zimmer Unrestricted Research Grants and Health Science Research Grants from Ministry of Health and Welfare to K. Nakamura.
Submitted: 12 July 1999
Revised: 7 December 1999
Accepted: 7 December 1999
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References |
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