Activation of NF-kappa B Is Involved in the Survival of Osteoclasts Promoted by Interleukin-1*

Eijiro Jimi, Ichiro Nakamura, Tetsuro IkebeDagger , Shuichi Akiyama, Naoyuki Takahashi, and Tatsuo Suda§

From the Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555 Japan and the Dagger  Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

We previously reported that interleukin-1 (IL-1) promoted the survival of murine osteoclast-like cells (OCLs) formed in vitro and activated a transcription factor, NF-kappa B, of OCLs. The present study examined whether the activation of NF-kappa B is directly involved in the survival of OCLs promoted by IL-1. The expression of IL-1 type I receptor mRNA in OCLs was detected by the polymerase chain reaction amplification of reverse-transcribed mRNA. An electrophoretic mobility shift assay showed that IL-1 transiently activated NF-kappa B in the nuclei of the OCLs, and the maximal activation occurred at 30 min. The degradation of Ikappa Balpha coincided with the activation of NF-kappa B in the OCLs. The immunocytochemical study revealed that p65, a subunit of NF-kappa B, was translocated from the cytoplasm into almost all of the nuclei of the OCLs within 30 min after IL-1 stimulation. The purified OCLs spontaneously died via apoptosis, and IL-1 promoted the survival of OCLs by preventing their apoptosis. The pretreatment of purified OCLs with proteasome inhibitors suppressed the IL-1-induced activation of NF-kappa B and prevented the survival of OCLs supported by IL-1. When OCLs were pretreated with antisense oligodeoxynucleotides to p65 and p50 of NF-kappa B, the expression of respective mRNAs by OCLs was suppressed, and the IL-1-induced survival of OCLs was concomitantly inhibited. These results indicate that IL-1 promotes the survival of osteoclasts through the activation of NF-kappa B.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Osteoclasts are multinucleated giant cells responsible for bone resorption (1-3). Osteoclastic bone resorption consists of several important processes: the development of osteoclasts from hematopoietic progenitor cells, the attachment of osteoclasts to the bone surface, the formation of a ruffled border and clear zone, and the secretion of acids and lysosomal enzymes into the space beneath the ruffled border (2, 4). Osteoclasts are terminally differentiated cells with a limited life span. Although osteoclasts are believed to die soon after they play their role in bone (bone resorption), recent findings suggest that the survival of osteoclasts is tightly regulated by several factors (5-9). Two cytokines, macrophage colony stimulating factor (M-CSF,1 also called CSF-1) (5, 6) and interleukin 1 (IL-1) (6), and a calcium-regulating hormone, calcitonin (7), stimulate the survival of osteoclasts. In contrast, estrogen (8) and transforming growth factor-beta (TGF-beta ) (9) induce the apoptosis of osteoclasts. However, it is not yet known how the apoptosis and survival of osteoclasts is regulated by those factors.

Nuclear factor kappa B (NF-kappa B) is a ubiquitous transcription factor that regulates the expression of many genes involved in immune and inflammatory responses (10, 11). Conventional NF-kappa B is a heterodimer that consists of p50 and p65 subunits. The amino acid sequences of both subunits of NF-kappa B show a high homology to the Rel family, which includes c-Rel, Rel B, and p52, and they are now categorized as the NF-kappa B/Rel family (10, 11). The activity of NF-kappa B is strictly regulated by an inhibitor, Ikappa Balpha , that forms a complex with NF-kappa B and keeps NF-kappa B in the cytoplasm (10, 11). When cells receive signals that activate NF-kappa B, Ikappa Bs are phosphorylated and degraded through a ubiquitin/proteasome pathway. Multiple ubiquitin molecules attach to the phosphorylated Ikappa Bs, and then ubiquitinated Ikappa Bs are degraded by 26 S proteasome, an organella of intracellular protease complexes (11-14). The degradation of Ikappa Bs triggers the translocation of NF-kappa B from the cytoplasm into the nucleus. Thus, proteasome is believed to be a key enzyme which is involved in NF-kappa B activation (11-14).

Recently several lines of evidence have reported that activation of NF-kappa B is involved in cell survival (15-19) besides in immune responses and inflammation. We have also reported that IL-1 stimulates the survival of osteoclasts (6) and activates NF-kappa B in osteoclasts (20), separately. Therefore, in this report, we examined whether the effect of IL-1 on osteoclast survival is mediated by the activation of NF-kappa B. IL-1 transiently induced an activation of NF-kappa B in osteoclast-like cells (OCLs), which was concomitant with the degradation of Ikappa Balpha . The OCLs spontaneously died via apoptosis, which was markedly blocked by the addition of IL-1. The pretreatment of OCLs with either proteasome inhibitors or antisense oligodeoxynucleotides to p65 and p50 prevented the IL-1-induced survival of OCLs. These results indicate that IL-1 promotes the survival of osteoclasts by preventing apoptosis through the activation of NF-kappa B.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Antibodies and Chemicals-- Anti-human p65 (sc-109) and anti-human Ikappa Balpha rabbit polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and New England BioLabs (Lake Placid, NY), respectively. The peptide aldehydes N-acetyl-leucinyl-leucinyl-norleucinal-H (ALLN) and N-acetyl-leucinyl-leucinyl-methional (ALLM) were purchased from Wako Pure Chemical Co. (Osaka, Japan). Carbobenzoxyl-leucinyl-leucinyl-leucinal-H (ZLLLal), carbobenzoxyl-leucinyl-leucinal-H (ZLLal) were purchased from Peptide Institute Inc. (Osaka, Japan). Phenylmethylsulfonyl fluoride was purchased from Sigma. Recombinant human interleukin 1alpha (IL-1alpha ) and murine IL-1 receptor antagonist (IL-1ra) were obtained from R&D Systems (Minneapolis, MN).

Culture of Osteoclast-like Cells-- Osteoblasts obtained from the calvaria of newborn mice and bone marrow cells obtained from the tibiae of male mice were co-cultured in alpha -minimal essential medium (alpha -MEM) (Life Technologies, Inc., Grand Island, NY) containing 10% fetal bovine serum (FBS), 1alpha ,25-dihydroxyvitamin D3 (1alpha ,25(OH)2 D3) (10-8 M) and prostaglandin E2 (PGE2) (10-6 M) in 100-mm diameter dishes coated with collagen gels (Nitta Gelatin Co., Osaka, Japan). OCLs were formed within 6 days in culture and were removed from the dishes by treatment with 0.2% collagenase (Wako Pure Chemical Co.). The purity of OCLs in this fraction (crude OCL preparation) was about 5%. To further purify the OCLs, the crude OCL preparation was replated on culture dishes. After an 8-h culture, osteoblasts were removed with phosphate-buffered saline (PBS) containing 0.001% Pronase E (Calbiochem, La Jolla, CA) and 0.02% EDTA for 10 min at 37 °C according to the method described previously (20).

Polymerase Chain Reaction Amplification of Reverse-transcribed mRNA-- Total RNA from purified OCLs, crude OCLs and primary osteoblasts in culture dishes (60 mm-diameter), was extracted using Trizol solution (Life Technologies, Inc.). Five % of the first-strand cDNA pool was submitted to polymerase chain reaction (PCR) amplification using gene-specific PCR primers (see Table I) by standard PCR protocols. The PCR program was as follows: 30 cycles at 94 °C for 45 s, 60 °C for 45 s, and 72 °C for 2 min for IL-1 type I receptors (IL-1RI), calcitonin receptors (CTRs), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH); 40 cycles at 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min for osteocalcin; in a DNA thermal cycler (Program temperature control system, PC-700, Astec, Tokyo, Japan). The PCR products were separated by electrophoresis on 2% agarose gels and were visualized by ethidium bromide staining with ultraviolet light illumination.

                              
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Table I
PCR primers used for mRNA phenotyping analysis
The abbreviations are: IL-1RI, type I IL-1 receptor. Primer pairs used for cDNA amplification.

Electrophoretic Mobility Shift Assay-- Nuclear extracts were prepared according to the method described by Dignam et al. (21). The sequence of the NF-kappa B binding oligonucleotide used as a radioactive DNA probe was 5'-AGCTTGGGGACTTTCCGAG-3'. The Oct 1-binding oligonucleotide was 5'-TGTCGAATGCAAATCACTAGAA-3'. The DNA binding reaction was performed at room temperature in a volume of 20 µl, which contained the binding buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 4% glycerol, 100 mM NaCl, 5 mM dithiothreitol, 100 mg/ml bovine serum albumin), 3 µg of poly(dI-dC), 1 × 105 cpm of 32P-labeled probe, and 8 µg of nuclear proteins. After incubation for 15 min, the samples were electrophoresed on native 5% acrylamide, 0.25 × Tris borate-EDTA gels. The gels were dried and exposed to x-ray film.

Immunoblotting Analysis-- After purified OCL preparations were cultured for various periods in the presence of IL-1 (10 ng/ml), the cells were washed twice with ice-cold PBS and then lysed with an SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% bromphenol blue). The cell lysates (20 µg of protein) were resolved by 10% SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Millipore, Bedford, MA). After blocking with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T), the Ikappa B-alpha antibodies (1 µg/ml) were added in TBS-T containing 5% bovine serum albumin and visualized by an enhanced chemiluminescence assay (ECL) using reagents from Amersham Pharmacia Biotech (UK) and by exposure to x-ray film.

Immunofluorescence Microscopy-- For immunofluorescence analysis, OCLs were seeded onto sterile FBS-coated glass coverslips and purified by treatment with Pronase. After OCLs were purified, they were treated with or without IL-1 (10 ng/ml) for the indicated times and then fixed with 4% paraformaldehyde in PBS for 15 min, blocked with 5% skim milk in PBS for 15 min at room temperature, and incubated with 1 µg/ml polyclonal anti-p65 antibodies for 30 min at 37 °C. After extensive washes, the cells were incubated with FITC-conjugated anti-rabbit IgG (dilution 1:100) for 30 min at 37 °C. The cells were then washed and mounted in Immunon (Lipshaw, Pittsburgh, PA). The subcellular localization of FITC-labeled p65 was determined by fluorescence microscopy (Olympus BX-FLA, Osaka).

DNA Extraction and Electrophoretic Analysis-- DNA was prepared and analyzed by gel electrophoresis according to the method described by Bessho et al . (22). Briefly, purified OCLs were lysed by incubating them at 60 °C overnight in a digestion buffer containing 150 mM NaCl, 25 mM EDTA, 100 µg/ml proteinase K, and 0.2% SDS. The DNA was extracted twice with phenol/chloroform/isoamyl alchohol and once with chloroform and precipitated in ethanol with 150 mM CH3COONa, pH 5.2. The DNA was then dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and treated with 20 µg/ml RNase A. The procedures for DNA extraction and precipitation were repeated. Two µg of DNA was separated by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining with ultraviolet light illumination.

Antisense Oligodeoxynucleotides and Cell Cultures-- Synthetic phosphorothioate oligodeoxynucleotides (S-ODN) that include the ATG initiation codon of the cDNA for mouse p50 or p65 were used for the sense and antisense experiments (see Table II). Crude OCLs were incubated in the presence of 5 µM antisense or sense S-ODN/cationic liposomes (DOTAP, Boehringer Mannheim, Mannheim, Germany) as the S-ODN carrier in alpha -MEM. They were then cultured for 2 h, 5% FBS was added, and the OCLs were further cultured for 8 h. After OCLs were purified, they were incubated in the presence of the antisense or sense S-ODN/DOTAP in alpha -MEM. They were then cultured for 2 h, and 5% FBS was added. After incubation for 6 h, OCLs were treated with IL-1, and the incubation was continued for an additional 15 h. The expression of p50 or p65 mRNA was detected by RT-PCR using gene-specific PCR primers. The p50 primers (1) 5'-TCGGAGACTGGAGCCTGTGGTG-3' and (2) 5'-CCCTGCGTTGGATTTCGTGACT-3' (969-1551) define an amplicon of 604 base pairs. The p65 primers (1) 5'-GAAGAAGCGAGACCTGGAGCAA-3' and (2) 5'-GTTGATGGTGCTGAGGGATGCT-3' (423-1116) define an amplicon of 715 base pairs.

                              
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Table II
Phosphorothiooligodeoxynucleotides used in this study
The oligodeoxynucleotide sequences correspond to the 5' ends of respective mRNAs and include three or four nucleotides present upstream of the initiation codon.

Survival of OCLs-- The survival rate was measured as reported previously (6, 23). After OCLs were purified, some of the cultures were subjected to tartrate-resistant acid phosphatase (TRAP) staining. TRAP-positive multinucleated cells were counted as living OCLs. Other cultures were further incubated for indicated times in the presence or absence of IL-1. After incubation, the remaining OCLs were counted.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Expression of IL-1 Type I Receptor mRNA by OCLs-- We first examined whether OCLs express IL-1RI mRNA. Total RNA isolated from the cell preparations of purified OCLs, crude OCLs, and primary osteoblasts was analyzed by RT-PCR using primers specific for IL-1RI, CTR (a marker of osteoclasts), and osteocalcin (a marker of osteoblasts) (Table I). CTR mRNA was detected in the purified OCL and crude OCL preparations (Fig. 1A, 1st middle panel, lanes 1-4), whereas osteocalcin mRNA was detected in the crude OCL and osteoblast preparations (Fig. 1A, 2nd middle panel, lanes 3-6). Osteocalcin mRNA was not detected in the purified OCL preparations (Fig. 1A, 2nd middle panel, lanes 1 and 2) even after PCR was performed at 40 cycles, suggesting that there was no contamination with osteoblasts in the purified OCL preparations. IL-1RI mRNA was detected in all preparations (Fig. 1A, upper panel, lanes 1-6), indicating that OCLs express IL-1RI mRNA. Expression of GAPDH mRNA was used as a control (Fig. 1A, bottom panel, lanes 1-6).


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Fig. 1.   Expression of IL-1 type I receptors by osteoclast like-cells (OCLs) (A) and the effect of IL-1 receptor antagonist (IL-1ra) on the activation of NF-kappa B in OCLs treated with IL-1 (B). A, total RNA was extracted from two independent preparations of purified OCLs, crude OCLs and primary osteoblasts (POBs) and subjected to reverse transcription with a random primer. The first-strand cDNAs were submitted to PCR analysis for IL-1RI, CTR, osteocalcin, and GAPDH with the specific primer pairs shown in Table I. PCR was performed by 30 cycles for IL-1R, CTR, and GAPDH and by 40 cycles for osteocalcin. The PCR products were separated by electrophoresis on 2% agarose gels and visualized by ethidium bromide staining with ultraviolet light illumination. B, purified OCLs were pretreated with IL-1ra for 1 h, and incubated with vehicle or IL-1alpha for 30 min. The nuclear extract was prepared and analyzed by an EMSA with a kappa B wild oligonucleotide probe, as described under "Experimental Procedures."

To clarify whether IL-1 activates NF-kappa B of OCLs through their own IL-1RI, the effect of murine IL-1 receptor antagonist (IL-1ra) on the IL-1-induced activation of NF-kappa B was examined in the purified OCL preparations. The addition of IL-1 markedly activated NF-kappa B in OCLs within 30 min (Fig. 1B). The pretreatment of OCLs with IL-1ra suppressed NF-kappa B activity of OCLs induced by IL-1 (Fig. 1B). This result suggests that IL-1 activates NF-kappa B of OCLs through IL-1RI expressed by OCLs.

Degradation of Ikappa Balpha Triggers the Activation of NF-kappa B in OCLs-- A common feature of the regulation of NF-kappa B is their sequestration in the cytoplasm as an inactive complex with Ikappa Balpha . The activity of NF-kappa B was then compared with the dynamics of Ikappa Balpha in the purified OCLs after IL-1 stimulation. The activation of NF-kappa B induced by IL-1 was first detected within 5 min, attained a maximal level at 30 min, and declined thereafter. Ikappa Balpha rapidly disappeared upon IL-1 stimulation and reappeared after 30 min (Fig. 2A). The gene encoding Ikappa Balpha is one of the target genes of NF-kappa B (10, 11). When purified OCLs were pretreated with actinomycin D (an inhibitor of mRNA synthesis) and incubated with IL-1, the activation of NF-kappa B was first detected within 5 min, and the activity was prolonged up to 2 h (Fig. 2B). IL-1 also induced rapid disappearance of Ikappa Balpha in the actinomycin D-pretreated OCLs. However, the reappearance of Ikappa Balpha was not observed in the OCLs during the incubation for 2 h (Fig. 2B), suggesting that the degradation of Ikappa Balpha coincided with the activation of NF-kappa B in OCLs. Thus, IL-1 induces the degradation of Ikappa Balpha in OCLs, which triggers the activation of NF-kappa B in OCLs.


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Fig. 2.   Time course of changes in the activation of NF-kappa B and the levels of Ikappa Balpha in OCLs after IL-1alpha stimulation. Purified OCLs were pretreated with (B) or without (A) 2.5 µg/ml actinomycin D for 2 h, and further incubated with vehicle or IL-1alpha (10 ng/ml) for the indicated periods. NF-kappa B activity in the nuclear extract was determined by an EMSA, and the amount of Ikappa Balpha was determined by immunoblotting, as described under "Experimental Procedures."

Translocation of NF-kappa B into Multiple Nuclei of OCLs in Response to IL-1-- The localization of NF-kappa B into the nuclei of OCLs treated with IL-1 was also examined immunocytochemically using specific antibodies against p65 subunit. Before the IL-1 stimulation, p65 was distributed throughout the cytoplasm (Fig. 3A) and especially around the nuclei of OCLs, which were detected by staining with Hoechst 33342 (Fig. 3B). When purified OCLs were treated with IL-1 for 15 min (Fig. 3, C and D), p65 was translocated into some nuclei in OCLs, but the remaining nuclei of OCLs showed no accumulation of p65 at this time point. However, p65 was detected in most of the nuclei of OCLs after IL-1 stimulation for 30 min (Fig. 3, E and F). The immunoreactivity of p65 completely disappeared from the nuclei of the OCLs after stimulation for 90 min (Fig. 3, G and H). When nonimmune immunoglobulins were used as the first antibody, no specific immunolabeling was detected in the OCLs (data not shown). Thus, the immunocytochemical findings were well consistent with the findings by electrophoretic mobility shift assay (EMSA) for NF-kappa B in OCLs treated with IL-1.


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Fig. 3.   Translocation of p65, a subunit of NF-kappa B, into the nuclei of OCLs in response to IL-1alpha . Purified OCLs were treated with IL-1alpha (10 ng/ml) for 0 (A and B), 15 (C and D), 30 (E and F), or 90 min (G and H). Cells were then fixed and incubated with antibodies against p65 followed by FITC-conjugated anti-rabbit immunoglobulins. The subcellular localization of FITC-labeled p65 was observed by fluorescence microscopy (A, C, D, E, F, and G). To visualize the nuclei of the OCLs, the cells were further stained with Hoechst 33342 (B and H). The nuclear localization of the OCLs in panels A and G is shown in panels B and H, respectively. Before stimulation, p65 was localized in the cytoplasm, especially around the nuclei of the OCLs (A and B). At 15 min after IL-1alpha stimulation (C and D), p65 was found in some nuclei (arrows) but not other nuclei (arrowheads) of an OCL. At 30 min, most of the nuclei of OCLs accumulated p65 in response to IL-1alpha (E and F). The immunoreactivity of p65 disappeared from the nuclei of OCLs after IL-1alpha stimulation for 90 min (G). Bars = 50 µm.

Proteasome Inhibitors Block the Activation of NF-kappa B and the Survival of OCLs Promoted by IL-1-- Proteasome inhibitors were used to explore whether the activation of NF-kappa B is directly involved in the IL-1-induced survival of OCLs. The peptide aldehydes ALLN (calpain inhibitor I) and ZLLLal inhibit the proteolytic activity of proteasome (12). These inhibitors also suppress the protease activity of cathepsin B and calpain. Therefore, the structurally related compounds, ALLM (calpain inhibitor II) and ZLLal, which inhibit cathepsin B and calpain but not proteasome (12), were used as the controls. The pretreatment of OCLs with ALLN or ZLLLal prior to IL-1 addition markedly decreased the NF-kappa B activity induced by IL-1, whereas the pretreatment with ALLM or ZLLal did not (Fig. 4A). Neither ALLN nor ZLLLal affected the DNA-binding activity of another transcription factor, Oct-1, of the OCLs (Fig. 4A).


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Fig. 4.   Effects of proteasome inhibitors on NF-kappa B activation (A), survival (B), and DNA fragmentation (C) of purified OCLs treated with or without IL-1alpha . A, purified OCLs were pretreated with or without 10 µM ALLM, ALLN, ZLLal, or ZLLLaL for 1 h and further incubated with vehicle or IL-1alpha (10 ng/ml) for 30 min. An EMSA of the nuclear extracts was performed using oligonucleotide probes for NF-kappa B and Oct-1. B, purified OCLs were pretreated with or without 10 µM ALLM, ALLN, ZLLal, or ZLLLaL for 1 h, and further incubated with vehicle or IL-1alpha (10 ng/ml) for 24 h. The cells were then stained for TRAP, and TRAP-positive multinucleated cells were counted as OCLs. Similar results were obtained from three independent experiments. C, purified OCLs were pretreated with or without 10 µM concentration of either ZLLal or ZLLLaL for 1 h and further incubated with vehicle or IL-1alpha (10 ng/ml) for 18 h. Cells were lysed, and DNA was purified as described under "Experimental Procedures." Two µg of each DNA preparation were separated by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining with ultraviolet light illumination. lambda -StyI was used as the DNA molecular weight marker.

As reported previously (6), when purified OCLs were cultured for longer than 24 h, most died spontaneously, and the addition of IL-1 markedly supported the survival of the OCLs (Fig. 4B). The pretreatment of purified OCLs with ZLLLal, a proteasome inhibitor, completely prevented the IL-1-promoted survival of OCLs, but pretreatment with the control peptide, ZLLal, did not (Fig. 4B). Fragmentation of DNA was detected in the purified OCLs after culturing for 18 h (Fig. 4C, lane 2), suggesting that the death of OCLs was due to apoptosis. The treatment of OCLs with IL-1 inhibited ladder formation of DNA (Fig. 4C, lane 3). DNA fragmentation was also observed even in the presence of IL-1 in OCLs pretreated with ZLLLal (Fig. 4C, lane 5) but not with ZLLal (Fig. 4C, lane 4). These findings suggest that the the activation of NF-kappa B by IL-1 contributes greatly to the prevention of the apoptosis of OCLs.

Antisense Oligodeoxynucleotides to NF-kappa B Prevent the IL-1-induced Survival of OCLs-- S-ODNs to p50 and p65 were also used to confirm the notion that the activation of NF-kappa B is involved in the survival of OCLs promoted by IL-1. When purified OCLs were treated with the antisense S-ODNs to p50 or p65, expression of the respective mRNA by OCLs was suppressed (Fig. 5A). Treatment of OCLs with the sense S-ODNs showed no effect on the expression of p50 and p65 mRNA (Fig. 5A). The viability of OCLs treated with sense S-ODNs to p50 and p65 went down slightly when compared with that treated with IL-1 alone (p < 0.05, Fig. 5B). This indicates that the transfection of S-ODNs partially affects the viability of OCLs at a basal level. The survival of OCLs was markedly reduced even in the presence of IL-1 by pretreatment with antisense S-ODNs to p50 and p65, compared with sense S-ODNs to p50 and p65 (Fig. 5, B and C).


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Fig. 5.   Effects of antisense oligodeoxynucleotides to p50 and p65 subunits of NF-kappa B on the survival of OCLs. The nucleotide sequences of S-ODNs to murine p50 or p65 used in this study are shown in Table II. Crude OCL preparations plated on culture dishes were incubated for 10 h in the presence or absence of antisense S-ODNs (p50AS and p65AS) or sense S-ODNs (p50S and p65S) to p50 and p65. The cultures were then treated with Pronase to purify the OCLs. The purified OCLs were further incubated with or without the antisense or sense S-ODNs for 8 h. Total RNA was then isolated from some cultures to determine the expression of mRNAs to p50 and p65 (A). The expression of GAPDH mRNA was used as the control. The other cultures were further incubated with IL-1alpha (10 ng/ml) for an additional 15 h and then stained for TRAP. TRAP-positive multinucleated cells were counted as OCLs. Similar results were obtained from three independent sets of experiments. *, significantly different from the sense group; p < 0.01 (B). Panel C shows typical TRAP stainings of purified OCLs from the experiment described in panel B. Bar = 100 µm.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The present study clearly indicates that the activation of NF-kappa B is involved in the survival of OCLs promoted by IL-1. The OCLs formed in our co-cultures expressed IL-1 type I receptors, and IL-1 directly activated NF-kappa B of OCLs through the binding to the IL-1 type I receptors. The degradation of Ikappa Balpha appeared to trigger the activation of NF-kappa B in the OCLs because the level of Ikappa Balpha in OCLs varied inversely with the activation of NF-kappa B. The immunocytochemical study also showed that the nuclear translocation of p65 was well correlated with the activation of NF-kappa B detected by the EMSA. Almost all of the nuclei of OCLs accumulated NF-kappa B within 30 min in response to IL-1 and discharged NF-kappa B after the stimulation for 90 min. This suggests that all of the nuclei of OCLs are functionally active and also that the function of multiple nuclei in osteoclasts is harmoniously regulated by stimuli from outside of the cell. The experiments using proteasome inhibitors and antisense S-ODNs to NF-kappa B showed that the IL-1-promoted survival of OCLs was mediated by the activation of NF-kappa B. These results indicate that activation of NF-kappa B directly prevents the spontaneously occurring apoptosis of OCLs.

Although IL-1 is a potent bone-resorbing factor in vivo (24) and in vitro (25), it is still not fully understood how IL-1 regulates osteoclast development and function. We previously reported that IL-1 stimulated OCL formation in murine bone marrow cultures by a mechanism involving prostaglandin E2 synthesis (26). Osteoblasts or marrow stromal cells have been proposed to play an essential role in OCL formation induced by osteotropic factors including IL-1 (3). Thomson et al. (27) showed that IL-1 stimulates the pit-forming activity of isolated rat osteoclasts through soluble factors secreted by osteoblasts. However, neither contaminating osteoblasts nor nonspecific esterase-positive macrophages were detected in our purified OCL preparation (20). Using an in situ hybridization technique, Xu et al. (28) showed the expression of mRNAs to IL-1 types I and II receptors by osteoclasts in normal bone tissues in mice and rats and inflammatory bone tissues in rats. Yu and Ferrier (29) also obtained evidence that osteoclasts are one of the target cells of IL-1. These findings together with the present report strongly suggest that IL-1 also acts on osteoclasts directly through their IL-1 receptors and regulates their functions without the help of other stromal cells.

M-CSF (5, 6, 23) and calcitonin (7) have been shown to prevent the apoptosis of osteoclasts. We also reported that like IL-1, M-CSF strongly supported the survival of OCLs (6, 23). It was shown that the tyrosine phosphorylation of Ikappa Balpha represented a proteolysis-independent mechanism of NF-kappa B activation that directly coupled NF-kappa B to cellular tyrosine kinase (30). M-CSF, the receptors of which possess tyrosine kinase in the cytoplasmic domain, activated NF-kappa B of liver macrophages (Kupffer cells) of rats (31). However, M-CSF did not activate NF-kappa B of OCLs, and antisense S-ODNs to NF-kappa B failed to block the M-CSF-supported survival of OCLs in our culture system (data not shown). Furthermore, the pretreatment of actinomycin D suppressed the reappearance of Ikappa Balpha protein in the purified OCLs treated with IL-1. These results indicated that the IL-1-induced NF-kappa B activation was mediated by a proteolysis-dependent mechanism in OCLs. NF-kappa B of OCLs was not activated by the addition of calcitonin (20). In our preliminary experiments, IL-1 as well as M-CSF reduced the caspase activity in purified OCLs.2 These results suggest that signals other than the activation of NF-kappa B are involved in the survival of OCLs promoted by M-CSF and calcitonin.

Among the several factors examined, tumor necrosis factor alpha  (TNFalpha , 10 ng/ml) also activated NF-kappa B of OCLs and prevented their spontaneous apoptosis (data not shown). It was recently reported that a novel serine/threonine protein kinase, NF-kappa B-inducing kinase (NIK), which binds to TRAF2 (TNF receptor associated factor 2) and activates NF-kappa B, is a necessary component of an NF-kappa B-activating cascade common to TNFalpha and IL-1 signalings (32). More recently, Ikappa B kinase was identified which binds to NIK (33, 34). These findings suggest that OCLs also express functionally active TNFalpha receptors, and that serine/threonine protein kinases such as NIK and Ikappa B kinase are commonly involved in the activation of NF-kappa B in OCLs treated with IL-1 and TNFalpha .

On the contrary to the present report, it has been shown that the activation of NF-kappa B induces the apoptosis in certain cells (22, 35). Abbadie et al. (35) reported that the overexpression of c-Rel induced apoptosis of avian bone marrow cells. Bessho et al. (22) also showed that the treatment of human leukemia cells and thymocytes with a protease inhibitor, pyrrolidinedithiocarbamate (PDTC), which inhibited the activation of NF-kappa B, prevented their apoptosis. In addition, radiation or agents such as lipopolysaccharides and TNFalpha triggered the hydrolysis of membrane phospholipids to produce ceramide, which in turn activated NF-kappa B and induced apoptosis (11). It is not known at present if NF-kappa B is involved in promotion of apoptosis in certain cells and if also responsible for inhibition of apoptosis in other cells.

A novel function of NF-kappa B in apoptosis was recently found in Rel A (p65) knockout mice (15, 37). Beg et al. (36) showed that a considerable apoptosis occurred in hepatocytes in Rel A-deficient embryos. They also reported that TNFalpha induced the apoptosis of fibroblasts and macrophages derived from Rel A knockout mice, and transfection of Rel A into the cells rescued them from TNFalpha -induced apoptosis (15). Wang et al. (16) and Antwerp et al. (17) independently demonstrated the anti-apoptotic role of NF-kappa B using cell lines stably transfected with the dominant negative Ikappa Balpha . Liu et al. (18) also showed that three different responses to TNFalpha were mediated by TNFalpha receptor complex: the activation of Jun N-terminal kinase (JNK), the activation of NF-kappa B, and the induction of apoptosis. The activation of NF-kappa B protected the cells against TNFalpha -induced apoptosis. Ozaki et al. (19) also reported that NF-kappa B inhibitors pyrrolidinedithiocarbamate and choromethylketone stimulated apoptosis of rabbit mature osteoclasts, which resulted in the inhibition of bone resorption. These findings together with the present report indicate that the NF-kappa B signaling cascade is closely involved in the prevention of apoptosis of cells. Cell death therefore appears to be tightly regulated through a balance between apoptosis-inducing and apoptosis-preventing signals. The signaling cascade mediated by NF-kappa B may be important in the survival of osteoclasts.

Osteoclasts reported to have a short half-life undergo apoptosis within a few days (3, 5, 6, 23). Studying the control mechanism of limited life span of osteoclasts may be important for understanding the regulation of bone remodeling not only in vitro but also in vivo. Treatment of OCLs with IL-1 for 1 h could support their survival, which was evaluated 20 h later (data not shown). This indicates that NF-kappa B stimulates gene expression of long term effective proteins on the survival of OCLs. Identification of such proteins in the OCLs must be important in future studies. IL-1 has been implicated in increased bone loss in pathological conditions such as rheumatoid arthritis, lymphoma, and osteoporosis (37). The IL-1-induced survival of osteoclasts may also play an important role in the bone resorption stimulated by these pathological conditions. Further studies are required to determine a more detailed mechanism of involvement of NF-kappa B in the survival of osteoclasts and the gene expression of proteins that exert anti-apoptotic effects promoted by IL-1.

    ACKNOWLEDGEMENTS

We thank Drs. Hiroshi Takeuchi (Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Kyushu University), Nobuo Okahashi, and Tatsuji Nishihara (Department of Oral Science, The National Institute of Infectious Diseases) for helpful discussions and technical advice.

    FOOTNOTES

* This work was supported in part by Grants-in Aid (09771546 and 08557101) from the Ministry of Education, Science and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 813-3784-8162; Fax: 813-3784-5555; E-mail: suda{at}dent.showa-u.ac.jp.

1 The abbreviations used are: M-CSF, macrophage colony stimulating factor; TRAP, tartrate-resistant acid phosphatase; NF-kappa B, nuclear factor kappa B; IL, interleukin; ALLN, aldehydes N-acetyl-leucinyl-leucinyl-norleucinal-H; ALLM, N-acetyl-leucinyl-leucinyl-methional; ZLLLal, carbobenzoxyl-leucinyl-leucinyl-leucinal-H; ZLLal, carbobenzoxyl-leucinyl-leucinal-H; OCL, osteoclast-like cell; alpha -MEM, alpha -minimal essential medium; FBS, fetal bovine serum; RT-PCR, reverse-transcribed polymerase chain reaction; PBS, phosphate-buffered saline; CTR, calcitonin receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FITC, fluorescein isothiocyanate; S-ODN, synthetic phosphorothioate oligodeoxynucleotide; DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium salts; EMSA, electrophoretic mobility shift assay; TNFalpha , tumor necrosis factor alpha ; NIK, NF-kappa B-inducing kinase.

2 Okahashi, N., Koide, M., Jimi, E., Suda, T., and Nishihara, T., submitted for publication.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
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