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Activation of NF-κB Is Involved in the Survival of Osteoclasts Promoted by Interleukin-1

1998; Elsevier BV; Volume: 273; Issue: 15 Linguagem: Inglês

10.1074/jbc.273.15.8799

1083-351X

Eijiro Jimi, Ichiro Nakamura, Tetsuro Ikebe, Shuichi Akiyama, Naoyuki Takahashi, Tatsuo Suda,

Immune Response and Inflammation

We previously reported that interleukin-1 (IL-1) promoted the survival of murine osteoclast-like cells (OCLs) formedin vitro and activated a transcription factor, NF-κB, of OCLs. The present study examined whether the activation of NF-κB 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-κB in the nuclei of the OCLs, and the maximal activation occurred at 30 min. The degradation of IκBα coincided with the activation of NF-κB in the OCLs. The immunocytochemical study revealed that p65, a subunit of NF-κB, 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-κB and prevented the survival of OCLs supported by IL-1. When OCLs were pretreated with antisense oligodeoxynucleotides to p65 and p50 of NF-κB, 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-κB. We previously reported that interleukin-1 (IL-1) promoted the survival of murine osteoclast-like cells (OCLs) formedin vitro and activated a transcription factor, NF-κB, of OCLs. The present study examined whether the activation of NF-κB 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-κB in the nuclei of the OCLs, and the maximal activation occurred at 30 min. The degradation of IκBα coincided with the activation of NF-κB in the OCLs. The immunocytochemical study revealed that p65, a subunit of NF-κB, 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-κB and prevented the survival of OCLs supported by IL-1. When OCLs were pretreated with antisense oligodeoxynucleotides to p65 and p50 of NF-κB, 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-κB. Osteoclasts are multinucleated giant cells responsible for bone resorption (1Chambers T.J. Revell P.A. Fuller K. Athanasou N.A. J. Cell Sci. 1984; 66: 383-399Crossref PubMed Google Scholar, 2Baron R. Neff L. Louvard D. Courtoy P.J. J. Cell Biol. 1985; 101: 2210-2222Crossref PubMed Scopus (596) Google Scholar, 3Suda T. Takahashi N. Martin T.J. Endocr. Rev. 1995; 4: 266-270Google Scholar). 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 (2Baron R. Neff L. Louvard D. Courtoy P.J. J. Cell Biol. 1985; 101: 2210-2222Crossref PubMed Scopus (596) Google Scholar, 4Nakamura I. Takahashi N. Sasaki T. Jimi E. Kurokawa T. Suda T. J. Bone Miner. Res. 1996; 11: 1873-1879Crossref PubMed Scopus (93) Google Scholar). 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 (5Fuller K. Owens J.M. Jagger C.J. Wilson A. Moss R. Chambers T.J. J. Exp. Med. 1993; 178: 1733-1744Crossref PubMed Scopus (312) Google Scholar, 6Jimi E. Shuto T. Koga T. Endocrinology. 1995; 136: 808-811Crossref PubMed Google Scholar, 7Selander K.S. Härkönen P.L. Valve E. Mönkkönen J. Hannuniemi R. Väänänen K.H. Mol. Cell. Endocrinol. 1996; 122: 119-129Crossref PubMed Scopus (53) Google Scholar, 8Kameda T. Mano H. Yuasa T. Mori Y. Miyazawa K. Shiokawa M. Nakamaru Y. Hiroi E. Hiura K. Kameda A. Yang N.N. Hakeda Y. Kumegawa M. J. Exp. Med. 1997; 186: 495-497Crossref Scopus (378) Google Scholar, 9Hughes D.E. Dai A. Tiffee J.C. Li H.H. Mundy G.R. Boyce B.F. Nat. Med. 1996; 2: 1132-1136Crossref PubMed Scopus (702) Google Scholar). Two cytokines, macrophage colony stimulating factor (M-CSF, 1The abbreviations used are: M-CSF, macrophage colony stimulating factor; TRAP, tartrate-resistant acid phosphatase; NF-κB, nuclear factor κB; IL, interleukin; ALLN, aldehydesN-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; α-MEM, α-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; TNFα, tumor necrosis factor α; NIK, NF-κB-inducing kinase. also called CSF-1) (5Fuller K. Owens J.M. Jagger C.J. Wilson A. Moss R. Chambers T.J. J. Exp. Med. 1993; 178: 1733-1744Crossref PubMed Scopus (312) Google Scholar, 6Jimi E. Shuto T. Koga T. Endocrinology. 1995; 136: 808-811Crossref PubMed Google Scholar) and interleukin 1 (IL-1) (6Jimi E. Shuto T. Koga T. Endocrinology. 1995; 136: 808-811Crossref PubMed Google Scholar), and a calcium-regulating hormone, calcitonin (7Selander K.S. Härkönen P.L. Valve E. Mönkkönen J. Hannuniemi R. Väänänen K.H. Mol. Cell. Endocrinol. 1996; 122: 119-129Crossref PubMed Scopus (53) Google Scholar), stimulate the survival of osteoclasts. In contrast, estrogen (8Kameda T. Mano H. Yuasa T. Mori Y. Miyazawa K. Shiokawa M. Nakamaru Y. Hiroi E. Hiura K. Kameda A. Yang N.N. Hakeda Y. Kumegawa M. J. Exp. Med. 1997; 186: 495-497Crossref Scopus (378) Google Scholar) and transforming growth factor-β (TGF-β) (9Hughes D.E. Dai A. Tiffee J.C. Li H.H. Mundy G.R. Boyce B.F. Nat. Med. 1996; 2: 1132-1136Crossref PubMed Scopus (702) Google Scholar) 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 κB (NF-κB) is a ubiquitous transcription factor that regulates the expression of many genes involved in immune and inflammatory responses (10Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell. Biol. 1994; 10: 405-455Crossref PubMed Scopus (2015) Google Scholar, 11Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5579) Google Scholar). Conventional NF-κB is a heterodimer that consists of p50 and p65 subunits. The amino acid sequences of both subunits of NF-κB show a high homology to the Rel family, which includes c-Rel, Rel B, and p52, and they are now categorized as the NF-κB/Rel family (10Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell. Biol. 1994; 10: 405-455Crossref PubMed Scopus (2015) Google Scholar, 11Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5579) Google Scholar). The activity of NF-κB is strictly regulated by an inhibitor, IκBα, that forms a complex with NF-κB and keeps NF-κB in the cytoplasm (10Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell. Biol. 1994; 10: 405-455Crossref PubMed Scopus (2015) Google Scholar, 11Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5579) Google Scholar). When cells receive signals that activate NF-κB, IκBs are phosphorylated and degraded through a ubiquitin/proteasome pathway. Multiple ubiquitin molecules attach to the phosphorylated IκBs, and then ubiquitinated IκBs are degraded by 26 S proteasome, an organella of intracellular protease complexes (11Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5579) Google Scholar, 12Read M.A. Neish A.S. Luscinskas F.W. Palombella V.J. Maniatis T. Collins T. Immunity. 1995; 2: 493-506Abstract Full Text PDF PubMed Scopus (310) Google Scholar, 13Orian A. Whiteside S. Israël A. Stancovski I. Schwartz A.L. Ciechanover A. J. Biol. Chem. 1995; 270: 21707-21714Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 14Chen Z.J. Parent L. Maniatis T. Cell. 1996; 84: 853-862Abstract Full Text Full Text PDF PubMed Scopus (871) Google Scholar). The degradation of IκBs triggers the translocation of NF-κB from the cytoplasm into the nucleus. Thus, proteasome is believed to be a key enzyme which is involved in NF-κB activation (11Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5579) Google Scholar, 12Read M.A. Neish A.S. Luscinskas F.W. Palombella V.J. Maniatis T. Collins T. Immunity. 1995; 2: 493-506Abstract Full Text PDF PubMed Scopus (310) Google Scholar, 13Orian A. Whiteside S. Israël A. Stancovski I. Schwartz A.L. Ciechanover A. J. Biol. Chem. 1995; 270: 21707-21714Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 14Chen Z.J. Parent L. Maniatis T. Cell. 1996; 84: 853-862Abstract Full Text Full Text PDF PubMed Scopus (871) Google Scholar). Recently several lines of evidence have reported that activation of NF-κB is involved in cell survival (15Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2935) Google Scholar, 16Wang C.Y. Mayo M.W. Baldwin A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2512) Google Scholar, 17Antwerp D.J.V. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2449) Google Scholar, 18Liu Z. Hsu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1783) Google Scholar, 19Ozaki K. Takeda H. Iwahashi H. Kitano S. Hanazawa S. FEBS Lett. 1997; 410: 297-300Crossref PubMed Scopus (72) Google Scholar) besides in immune responses and inflammation. We have also reported that IL-1 stimulates the survival of osteoclasts (6Jimi E. Shuto T. Koga T. Endocrinology. 1995; 136: 808-811Crossref PubMed Google Scholar) and activates NF-κB in osteoclasts (20Jimi E. Ikebe T. Takahashi N. Hirata M. Suda T. Koga T. J. Biol. Chem. 1996; 271: 4605-4608Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), separately. Therefore, in this report, we examined whether the effect of IL-1 on osteoclast survival is mediated by the activation of NF-κB. IL-1 transiently induced an activation of NF-κB in osteoclast-like cells (OCLs), which was concomitant with the degradation of IκBα. 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-κB. Anti-human p65 (sc-109) and anti-human IκBα rabbit polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and New England BioLabs (Lake Placid, NY), respectively. The peptide aldehydesN-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 1α (IL-1α) and murine IL-1 receptor antagonist (IL-1ra) were obtained from R&D Systems (Minneapolis, MN). Osteoblasts obtained from the calvaria of newborn mice and bone marrow cells obtained from the tibiae of male mice were co-cultured in α-minimal essential medium (α-MEM) (Life Technologies, Inc., Grand Island, NY) containing 10% fetal bovine serum (FBS), 1α,25-dihydroxyvitamin D3(1α,25(OH)2 D3) (10−8m) and prostaglandin E2 (PGE2) (10−6m) 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 (20Jimi E. Ikebe T. Takahashi N. Hirata M. Suda T. Koga T. J. Biol. Chem. 1996; 271: 4605-4608Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). 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.Table IPCR primers used for mRNA phenotyping analysisTarget mRNAPrimer sequencesNucleotideIL-1 RI5′-AAATAATGAGTTACCCGAGGTCCAGGTCCAGTGG-3′686–13955′-AGGCATGTATGTCTTTCCATCTGAAGC-3′CTR5′-CAAGGCACGGACAATGTTGAGAAG-3′1023–15865′-TTTCAAGAACCTTAGCTGCCAGAG-3′Osteocalcin5′-CAAGTCCCACACAGCAGCTT-3′8–3785′-AAAGCCGAGCTGCCAGAGTT-3′GAPDH5′-TGAAGGTCGGTGTGAACGGATTTGGC-351–10335′-CATGTAGGCCATGAGGTCCACCAC-3′ Open table in a new tab Nuclear extracts were prepared according to the method described by Dignam et al.(21Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids. Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9160) Google Scholar). The sequence of the NF-κB 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 of32P-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. 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 IκB-α 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. 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 was prepared and analyzed by gel electrophoresis according to the method described by Bessho et al . (22Bessho R. Matsubara K. Kubota M. Kuwakado K. Hirota H. Wakazono Y. Lin Y.W. Okuda A. Kawai M. Nishikomori R. Heike T. Biochem. Pharmacol. 1994; 48: 1883-1889Crossref PubMed Scopus (202) Google Scholar). 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 mmTris-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. 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 α-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 α-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 (1Chambers T.J. Revell P.A. Fuller K. Athanasou N.A. J. Cell Sci. 1984; 66: 383-399Crossref PubMed Google Scholar) 5′-TCGGAGACTGGAGCCTGTGGTG-3′ and (2Baron R. Neff L. Louvard D. Courtoy P.J. J. Cell Biol. 1985; 101: 2210-2222Crossref PubMed Scopus (596) Google Scholar) 5′-CCCTGCGTTGGATTTCGTGACT-3′ (969–1551) define an amplicon of 604 base pairs. The p65 primers (1Chambers T.J. Revell P.A. Fuller K. Athanasou N.A. J. Cell Sci. 1984; 66: 383-399Crossref PubMed Google Scholar) 5′-GAAGAAGCGAGACCTGGAGCAA-3′ and (2Baron R. Neff L. Louvard D. Courtoy P.J. J. Cell Biol. 1985; 101: 2210-2222Crossref PubMed Scopus (596) Google Scholar) 5′-GTTGATGGTGCTGAGGGATGCT-3′ (423–1116) define an amplicon of 715 base pairs.Table IIPhosphorothiooligodeoxynucleotides used in this studyGeneSequencesNucleotidep50 sense5′-CTGCGCATCTTCACCATGGCA-3′277–297p50 antisense5′-TGCCATGGTGAAGATGCGCAG-3′p65 sense5′-GGGACCCTGACCATGGACGAT-3′49–69p65 antisense5′-ATCGTCCATGGTCAGGGTCCC-3′ Open table in a new tab The survival rate was measured as reported previously (6Jimi E. Shuto T. Koga T. Endocrinology. 1995; 136: 808-811Crossref PubMed Google Scholar, 23Okahashi N. Nakamura I. Jimi E. Koide M. Suda T. Nishihara T. J. Bone Miner. Res. 1997; 12: 1116-1123Crossref PubMed Scopus (65) Google Scholar). 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. 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. 1 A, 1st middle panel, lanes 1–4), whereas osteocalcin mRNA was detected in the crude OCL and osteoblast preparations (Fig. 1 A, 2nd middle panel, lanes 3–6). Osteocalcin mRNA was not detected in the purified OCL preparations (Fig. 1 A, 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. 1 A, upper panel, lanes 1–6), indicating that OCLs express IL-1RI mRNA. Expression of GAPDH mRNA was used as a control (Fig. 1 A, bottom panel, lanes 1–6). To clarify whether IL-1 activates NF-κB 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-κB was examined in the purified OCL preparations. The addition of IL-1 markedly activated NF-κB in OCLs within 30 min (Fig. 1B). The pretreatment of OCLs with IL-1ra suppressed NF-κB activity of OCLs induced by IL-1 (Fig. 1 B). This result suggests that IL-1 activates NF-κB of OCLs through IL-1RI expressed by OCLs. A common feature of the regulation of NF-κB is their sequestration in the cytoplasm as an inactive complex with IκBα. The activity of NF-κB was then compared with the dynamics of IκBα in the purified OCLs after IL-1 stimulation. The activation of NF-κB induced by IL-1 was first detected within 5 min, attained a maximal level at 30 min, and declined thereafter. IκBα rapidly disappeared upon IL-1 stimulation and reappeared after 30 min (Fig. 2 A). The gene encoding IκBα is one of the target genes of NF-κB (10Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell. Biol. 1994; 10: 405-455Crossref PubMed Scopus (2015) Google Scholar, 11Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5579) Google Scholar). When purified OCLs were pretreated with actinomycin D (an inhibitor of mRNA synthesis) and incubated with IL-1, the activation of NF-κB was first detected within 5 min, and the activity was prolonged up to 2 h (Fig. 2 B). IL-1 also induced rapid disappearance of IκBα in the actinomycin D-pretreated OCLs. However, the reappearance of IκBα was not observed in the OCLs during the incubation for 2 h (Fig. 2 B), suggesting that the degradation of IκBα coincided with the activation of NF-κB in OCLs. Thus, IL-1 induces the degradation of IκBα in OCLs, which triggers the activation of NF-κB in OCLs. The localization of NF-κB 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. 3 A) and especially around the nuclei of OCLs, which were detected by staining with Hoechst 33342 (Fig. 3 B). 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-κB in OCLs treated with IL-1. Proteasome inhibitors were used to explore whether the activation of NF-κB 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 (12Read M.A. Neish A.S. Luscinskas F.W. Palombella V.J. Maniatis T. Collins T. Immunity. 1995; 2: 493-506Abstract Full Text PDF PubMed Scopus (310) Google Scholar). 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 (12Read M.A. Neish A.S. Luscinskas F.W. Palombella V.J. Maniatis T. Collins T. Immunity. 1995; 2: 493-506Abstract Full Text PDF PubMed Scopus (310) Google Scholar), were used as the controls. The pretreatment of OCLs with ALLN or ZLLLal prior to IL-1 addition markedly decreased the NF-κB activity induced by IL-1, whereas the pretreatment with ALLM or ZLLal did not (Fig. 4 A). Neither ALLN nor ZLLLal affected the DNA-binding activity of another transcription factor, Oct-1, of the OCLs (Fig. 4 A). As reported previously (6Jimi E. Shuto T. Koga T. Endocrinology. 1995; 136: 808-811Crossref PubMed Google Scholar), 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. 4 B). 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. 4 B). Fragmentation of DNA was detected in the purified OCLs after culturing for 18 h (Fig. 4 C, 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. 4 C, lane 3). DNA fragmentation was also observed even in the presence of IL-1 in OCLs pretreated with ZLLLal (Fig. 4 C, lane 5) but not with ZLLal (Fig. 4 C, lane 4). These findings suggest that the the activation of NF-κB by IL-1 contributes greatly to the prevention of the apoptosis of OCLs. S-ODNs to p50 and p65 were also used to confirm the notion that the activation of NF-κB 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. 5 A). Treatment of OCLs with the sense S-ODNs showed no effect on the expression of p50 and p65 mRNA (Fig. 5 A). 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. 5 B). 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). The present study clearly indicates that the activation of NF-κB 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-κB of OCLs through the binding to the IL-1 type I receptors. The degradation of IκBα appeared to trigger the activation of NF-κB in the OCLs because the level of IκBα in OCLs varied inversely with the activation of NF-κB. The immunocytochemical study also showed that the nuclear translocation of p65 was well correlated with the activation of NF-κB detected by the EMSA. Almost all of the nuclei of OCLs accumulated NF-κB within 30 min in response to IL-1 and discharged NF-κB 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-κB showed that the IL-1-promoted survival of OCLs was mediated by the activation of NF-κB. These results indicate that activation of NF-κB directly prevents the spontaneously occurring apoptosis of OCLs. Although IL-1 is a potent bone-resorbing factor in vivo (24Boyce B.F. Aufdemorte T.B. Garrett I.R. Yates A.J.P. Mundy G.R. Endocrinology. 1989; 125: 1142-1150Crossref PubMed Scopus (318) Google Scholar) and in vitro (25Gowen M. Wood D.D. Ihrie E.J. McGuire M.K.B. Russel R.G.G. 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However, neither contaminating osteoblasts nor nonspecific esterase-positive macrophages were detected in our purified OCL preparation (20Jimi E. Ikebe T. Takahashi N. Hirata M. Suda T. Koga T. J. Biol. Chem. 1996; 271: 4605-4608Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Using an in situhybridization technique, Xu et al. (28Xu L.X. Kukita T. Nakano Y. Yu Hao. Hotokebuchi T. Kuratani T. Iijima T. Koga T. Lab. Invest. 1996; 75: 677-687PubMed Google Scholar) 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 (29Yu H. Ferrier J. Biochem. Biophys. Res. Commum. 1993; 191: 343-350Crossref PubMed Scopus (14) Google Scholar) also obtained evidence that osteoclasts are one of the target cells of IL-1. 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However, M-CSF did not activate NF-κB of OCLs, and antisense S-ODNs to NF-κB 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 IκBα protein in the purified OCLs treated with IL-1. These results indicated that the IL-1-induced NF-κB activation was mediated by a proteolysis-dependent mechanism in OCLs. NF-κB of OCLs was not activated by the addition of calcitonin (20Jimi E. Ikebe T. Takahashi N. Hirata M. Suda T. Koga T. J. Biol. Chem. 1996; 271: 4605-4608Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). In our preliminary experiments, IL-1 as well as M-CSF reduced the caspase activity in purified OCLs. 2Okahashi, N., Koide, M., Jimi, E., Suda, T., and Nishihara, T., submitted for publication. These results suggest that signals other than the activation of NF-κB are involved in the survival of OCLs promoted by M-CSF and calcitonin. Among the several factors examined, tumor necrosis factor α (TNFα, 10 ng/ml) also activated NF-κB of OCLs and prevented their spontaneous apoptosis (data not shown). It was recently reported that a novel serine/threonine protein kinase, NF-κB-inducing kinase (NIK), which binds to TRAF2 (TNF receptor associated factor 2) and activates NF-κB, is a necessary component of an NF-κB-activating cascade common to TNFα and IL-1 signalings (32Malinin N.L Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1165) Google Scholar). More recently, IκB kinase was identified which binds to NIK (33Régnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1072) Google Scholar, 34DiDonate J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1913) Google Scholar). 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FEBS Lett. 1997; 410: 297-300Crossref PubMed Scopus (72) Google Scholar) also reported that NF-κB 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-κB 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-κB may be important in the survival of osteoclasts. Osteoclasts reported to have a short half-life undergo apoptosis within a few days (3Suda T. Takahashi N. Martin T.J. Endocr. Rev. 1995; 4: 266-270Google Scholar, 5Fuller K. Owens J.M. Jagger C.J. Wilson A. Moss R. Chambers T.J. J. Exp. Med. 1993; 178: 1733-1744Crossref PubMed Scopus (312) Google Scholar, 6Jimi E. Shuto T. Koga T. Endocrinology. 1995; 136: 808-811Crossref PubMed Google Scholar, 23Okahashi N. Nakamura I. Jimi E. Koide M. Suda T. Nishihara T. J. Bone Miner. Res. 1997; 12: 1116-1123Crossref PubMed Scopus (65) Google Scholar). 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 alsoin 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-κB 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 (37Dinarello C.A. FASEB J. 1994; 8: 1314-1325Crossref PubMed Google Scholar). 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-κB in the survival of osteoclasts and the gene expression of proteins that exert anti-apoptotic effects promoted by IL-1. 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.