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

• DOI: 10.1074/jbc.273.15.8799
• Publication Date: 2002-07-26T15:13:03Z
• Authors: Eijiro Jimi; Ichiro Nakamura; Tetsuro Ikebe; Shuichi Akiyama; Naoyuki Takahashi; Tatsuo Suda
• Keywords: 0301 basic medicine; Proteasome Endopeptidase Complex; Osteoblasts; Cell Survival; NF-kappa B; Osteoclasts; Apoptosis; Bone Marrow Cells; Cysteine Proteinase Inhibitors; Oligonucleotides, Antisense; Polymerase Chain Reaction; Cysteine Endopeptidases; Mice; 03 medical and health sciences; Animals, Newborn; Oligodeoxyribonucleotides; Multienzyme Complexes; Dactinomycin; Animals; RNA, Messenger; DNA Primers; Interleukin-1

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, OCLs. The present study examined whether activation NF-κB is directly involved in OCLs by IL-1. expression IL-1 type I receptor mRNA was detected polymerase chain reaction amplification reverse-transcribed mRNA. An electrophoretic mobility shift assay showed transiently nuclei OCLs, maximal occurred at 30 min. degradation IκBα coincided with immunocytochemical revealed p65, subunit translocated from cytoplasm into almost all within min after stimulation. purified spontaneously died via apoptosis, preventing their apoptosis. pretreatment proteasome inhibitors suppressed IL-1-induced prevented supported When were pretreated antisense oligodeoxynucleotides to p65 p50 respective mRNAs suppressed, concomitantly inhibited. These results indicate promotes osteoclasts through NF-κB. Osteoclasts are multinucleated giant 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. Biol. 1985; 101: 2210-2222Crossref Scopus (596) 3Suda T. Takahashi N. Martin Endocr. Rev. 1995; 4: 266-270Google Scholar). Osteoclastic consists several important processes: development hematopoietic progenitor cells, attachment surface, formation ruffled border clear zone, secretion acids lysosomal enzymes space beneath (2Baron 4Nakamura I. Sasaki Jimi E. Kurokawa Suda Bone Miner. Res. 1996; 11: 1873-1879Crossref (93) terminally differentiated limited life span. Although believed die soon they play role (bone resorption), recent findings suggest tightly regulated factors (5Fuller Owens J.M. Jagger C.J. Wilson A. Moss Chambers Exp. Med. 1993; 178: 1733-1744Crossref (312) 6Jimi Shuto Koga Endocrinology. 136: 808-811Crossref 7Selander K.S. Härkönen P.L. Valve Mönkkönen Hannuniemi Väänänen K.H. Mol. Cell. Endocrinol. 122: 119-129Crossref (53) 8Kameda Mano H. Yuasa Mori Y. Miyazawa Shiokawa M. Nakamaru Hiroi Hiura Kameda Yang N.N. Hakeda Kumegawa 1997; 186: 495-497Crossref (378) 9Hughes D.E. Dai Tiffee J.C. Li H.H. Mundy G.R. Boyce B.F. Nat. 2: 1132-1136Crossref (702) Two cytokines, macrophage colony stimulating factor (M-CSF, 1The abbreviations used are: M-CSF, factor; TRAP, tartrate-resistant acid phosphatase; nuclear 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, cell; α-MEM, α-minimal essential medium; FBS, fetal bovine serum; RT-PCR, 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, assay; TNFα, tumor necrosis α; NIK, NF-κB-inducing kinase. also called CSF-1) Scholar) interleukin 1 (6Jimi Scholar), calcium-regulating hormone, (7Selander stimulate osteoclasts. In contrast, estrogen (8Kameda transforming growth factor-β (TGF-β) (9Hughes induce apoptosis However, it not yet known how those factors. Nuclear κB (NF-κB) ubiquitous regulates many genes immune inflammatory responses (10Siebenlist U. Franzoso G. Brown Annu. 1994; 10: 405-455Crossref (2015) 11Baldwin A.S. Immunol. 14: 649-681Crossref (5579) Conventional heterodimer subunits. amino sequences both subunits show high homology Rel family, which includes c-Rel, B, p52, now categorized as NF-κB/Rel family activity strictly an inhibitor, IκBα, forms complex keeps receive signals activate IκBs phosphorylated degraded ubiquitin/proteasome pathway. Multiple ubiquitin molecules attach IκBs, then ubiquitinated 26 S proteasome, organella intracellular protease complexes (11Baldwin 12Read M.A. Neish Luscinskas F.W. Palombella V.J. Maniatis Collins Immunity. 493-506Abstract Full Text PDF (310) 13Orian Whiteside S. Israël Stancovski Schwartz A.L. Ciechanover Chem. 270: 21707-21714Abstract (131) 14Chen Z.J. Parent 84: 853-862Abstract (871) triggers translocation nucleus. Thus, be key enzyme Recently lines evidence have cell (15Beg A.A. Baltimore Science. 274: 782-784Crossref (2935) 16Wang C.Y. Mayo M.W. Baldwin 784-787Crossref (2512) 17Antwerp D.J.V. S.J. Kafri Green D.R. Verma I.M. 787-789Crossref (2449) 18Liu Z. Hsu Goeddel D.V. Karin 87: 565-576Abstract (1783) 19Ozaki Takeda Iwahashi Kitano Hanazawa FEBS Lett. 410: 297-300Crossref (72) besides inflammation. stimulates activates (20Jimi Ikebe Hirata 271: 4605-4608Abstract (74) separately. Therefore, this report, we effect on osteoclast mediated induced (OCLs), concomitant IκBα. markedly blocked addition either or Anti-human (sc-109) anti-human rabbit polyclonal antibodies purchased Santa Cruz Biotechnology (Santa Cruz, CA) New England BioLabs (Lake Placid, NY), respectively. peptide aldehydesN-acetyl-leucinyl-leucinyl-norleucinal-H (ALLN) N-acetyl-leucinyl-leucinyl-methional (ALLM) Wako Pure Chemical Co. (Osaka, Japan). Carbobenzoxyl-leucinyl-leucinyl-leucinal-H (ZLLLal), carbobenzoxyl-leucinyl-leucinal-H (ZLLal) Peptide Institute Inc. Phenylmethylsulfonyl fluoride Sigma. Recombinant human 1α (IL-1α) antagonist (IL-1ra) obtained R&D Systems (Minneapolis, MN). Osteoblasts calvaria newborn mice marrow tibiae male co-cultured medium (α-MEM) (Life Technologies, Inc., Grand Island, NY) containing 10% serum (FBS), 1α,25-dihydroxyvitamin D3(1α,25(OH)2 D3) (10−8m) prostaglandin E2 (PGE2) (10−6m) 100-mm diameter dishes coated collagen gels (Nitta Gelatin Co., Osaka, formed 6 days culture removed treatment 0.2% collagenase (Wako Co.). purity fraction (crude OCL preparation) about 5%. To further purify crude preparation replated dishes. After 8-h culture, osteoblasts saline (PBS) 0.001% Pronase E (Calbiochem, La Jolla, 0.02% EDTA 10 37 °C according method described Total RNA primary (60 mm-diameter), extracted using Trizol solution Inc.). Five % first-strand cDNA pool submitted (PCR) gene-specific PCR primers (see Table I) standard protocols. program follows: cycles 94 45 s, 60 72 2 receptors (IL-1RI), (CTRs), dehydrogenase (GAPDH); 40 min, osteocalcin; DNA thermal cycler (Program temperature control system, PC-700, Astec, Tokyo, products separated electrophoresis 2% agarose visualized ethidium bromide staining ultraviolet light illumination.Table IPCR 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 new tab extracts prepared Dignam et al.(21Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids. 1983; 1475-1489Crossref (9160) sequence binding oligonucleotide radioactive probe 5′-AGCTTGGGGACTTTCCGAG-3′. Oct 1-binding 5′-TGTCGAATGCAAATCACTAGAA-3′. performed room volume 20 μl, contained buffer (10 mm Tris-HCl, pH 7.5, EDTA, 4% glycerol, 100 NaCl, 5 dithiothreitol, mg/ml albumin), 3 μg poly(dI-dC), × 105 cpm of32P-labeled probe, 8 proteins. incubation 15 samples electrophoresed native 5% acrylamide, 0.25 Tris borate-EDTA gels. dried exposed x-ray film. preparations cultured various periods presence ng/ml), washed twice ice-cold PBS lysed SDS sample (62.5 6.8, SDS, 50 0.1% bromphenol blue). lysates (20 protein) resolved SDS-polyacrylamide gel transferred onto PVDF membranes (Millipore, Bedford, MA). blocking skim milk Tris-buffered Tween (TBS-T), IκB-α (1 μg/ml) added TBS-T albumin enhanced chemiluminescence (ECL) reagents Amersham Pharmacia Biotech (UK) exposure For immunofluorescence analysis, seeded sterile FBS-coated glass coverslips Pronase. purified, treated without ng/ml) indicated times fixed paraformaldehyde temperature, incubated μg/ml anti-p65 °C. extensive washes, FITC-conjugated anti-rabbit IgG (dilution 1:100) mounted Immunon (Lipshaw, Pittsburgh, PA). subcellular localization FITC-labeled determined fluorescence microscopy (Olympus BX-FLA, Osaka). analyzed Bessho al . (22Bessho Matsubara Kubota Kuwakado Hirota Wakazono Lin Y.W. Okuda Kawai Nishikomori Heike Biochem. Pharmacol. 48: 1883-1889Crossref (202) Briefly, incubating them overnight digestion 150 25 proteinase K, SDS. phenol/chloroform/isoamyl alchohol once chloroform precipitated ethanol CH3COONa, 5.2. dissolved TE mmTris-HCl, 8.0) RNase procedures extraction precipitation repeated. illumination. Synthetic (S-ODN) include ATG initiation codon mouse sense experiments II). Crude μm S-ODN/cationic liposomes (DOTAP, Boehringer Mannheim, Germany) S-ODN carrier α-MEM. They h, FBS added, h. S-ODN/DOTAP added. IL-1, continued additional RT-PCR primers. 5′-TCGGAGACTGGAGCCTGTGGTG-3′ 5′-CCCTGCGTTGGATTTCGTGACT-3′ (969–1551) define amplicon 604 base pairs. 5′-GAAGAAGCGAGACCTGGAGCAA-3′ 5′-GTTGATGGTGCTGAGGGATGCT-3′ (423–1116) 715 pairs.Table IIPhosphorothiooligodeoxynucleotides studyGeneSequencesNucleotidep50 sense5′-CTGCGCATCTTCACCATGGCA-3′277–297p50 antisense5′-TGCCATGGTGAAGATGCGCAG-3′p65 sense5′-GGGACCCTGACCATGGACGAT-3′49–69p65 antisense5′-ATCGTCCATGGTCAGGGTCCC-3′ rate measured 23Okahashi Nakamura Koide Nishihara 12: 1116-1123Crossref (65) some cultures subjected phosphatase (TRAP) staining. TRAP-positive counted living Other absence incubation, remaining counted. first express IL-1RI isolated specific IL-1RI, CTR (a marker osteoclasts), osteocalcin osteoblasts) (Table I). (Fig. A, 1st middle panel, lanes 1–4), whereas osteoblast 2nd 3–6). Osteocalcin 2) even cycles, suggesting there no contamination preparations. upper 1–6), indicating Expression GAPDH bottom 1–6). clarify own 1B). IL-1ra B). This result suggests expressed A common feature regulation sequestration inactive compared dynamics attained level declined thereafter. rapidly disappeared upon stimulation reappeared A). gene encoding one target actinomycin D (an inhibitor synthesis) prolonged up h rapid disappearance D-pretreated reappearance observed during B), induces immunocytochemically against subunit. Before stimulation, distributed throughout A) especially around Hoechst 33342 3, C D), but accumulation time point. most 3,E F). immunoreactivity completely 90 G H). nonimmune immunoglobulins antibody, immunolabeling (data shown). well consistent (EMSA) Proteasome explore aldehydes ALLN (calpain ZLLLal inhibit proteolytic (12Read suppress cathepsin B calpain. structurally related compounds, ALLM II) calpain controls. prior decreased ZLLal did 4 Neither nor affected DNA-binding another Oct-1, As when longer than 24 spontaneously, IL-1-promoted peptide, Fragmentation culturing 18 C, lane 2), death due inhibited ladder 3). fragmentation 5) 4). contributes greatly prevention S-ODNs confirm notion Treatment viability went down slightly alone (p < 0.05, Fig. indicates transfection partially affects basal level. reduced 5, C). clearly our co-cultures receptors, receptors. appeared trigger because varied inversely correlated EMSA. Almost accumulated response discharged functionally active function multiple harmoniously stimuli outside cell. prevents occurring potent bone-resorbing vivo (24Boyce Aufdemorte T.B. Garrett I.R. Yates A.J.P. 1989; 125: 1142-1150Crossref (318) (25Gowen Wood D.D. Ihrie E.J. McGuire M.K.B. Russel R.G.G. Nature. 306: 378-380Crossref (832) still fully understood function. stimulated mechanism involving synthesis (26Akatsu Udagawa Imamura Yamaguchi Sato Nagata 1991; 6: 183-190Crossref (215) stromal been proposed osteotropic including (3Suda Thomson al. (27Thomson B.M. Saklatvala 1986; 164: 104-112Crossref (328) pit-forming rat soluble secreted osteoblasts. neither contaminating nonspecific esterase-positive macrophages Using situhybridization technique, Xu (28Xu L.X. Kukita Nakano Yu Hao. Hotokebuchi Kuratani Iijima Lab. Invest. 75: 677-687PubMed types II normal tissues rats rats. Ferrier (29Yu Biophys. Commum. 191: 343-350Crossref (14) together report strongly acts functions help other cells. M-CSF shown prevent like It tyrosine phosphorylation represented proteolysis-independent coupled cellular kinase (30Imbert V. Rupec R.A. Livolsi Pahl H.L. Traenckner E.B. Mueller-Dieckmann C. Farahifar Auberger P. Baeuerle Peyron J.F. 86: 787-798Abstract (628) possess cytoplasmic domain, liver (Kupffer cells) (31Tran-Thi T.A. Decker Hepatology. 22: 613-619Crossref failed block M-CSF-supported system Furthermore, protein proteolysis-dependent preliminary experiments, caspase 2Okahashi, N., Koide, M., Jimi, E., Suda, T., Nishihara, publication. calcitonin. Among examined, α (TNFα, spontaneous recently novel serine/threonine kinase, (NIK), binds TRAF2 (TNF associated necessary component NF-κB-activating cascade TNFα signalings (32Malinin N.L Boldin M.P. Kovalenko A.V. Wallach 385: 540-544Crossref (1165) More recently, IκB identified NIK (33Régnier C.H. Song H.Y. Gao X. Cao Rothe 90: 373-383Abstract (1072) 34DiDonate J.A. Hayakawa Rothwarf D.M. Zandi 388: 548-554Crossref (1913) kinases such commonly TNFα. On contrary has certain 35Abbadie Kabrun Bouali F. Smardova Stéhelin Vandenbunder B. Enrietto 899-912Abstract (220) Abbadie (35Abbadie overexpression c-Rel avian leukemia thymocytes pyrrolidinedithiocarbamate (PDTC), addition, radiation agents lipopolysaccharides triggered hydrolysis membrane phospholipids produce ceramide, turn if promotion inhibition found (p65) knockout 37Dinarello C.A. FASEB 8: 1314-1325Crossref Beg (36Beg Sha W.C. Bronson Ghosh 376: 167-170Crossref (1638) considerable hepatocytes A-deficient embryos. fibroblasts derived mice, rescued TNFα-induced Wang (16Wang Antwerp (17Antwerp independently demonstrated anti-apoptotic stably transfected dominant negative Liu (18Liu three different complex: Jun N-terminal (JNK), induction protected Ozakiet (19Ozaki choromethylketone mature osteoclasts, resulted resorption. signaling closely therefore appears balance between apoptosis-inducing apoptosis-preventing signals. may short half-life undergo few 5Fuller Studying span understanding remodeling only alsoin vivo. could support survival, evaluated later long term effective proteins Identification must future studies. implicated increased loss pathological conditions rheumatoid arthritis, lymphoma, osteoporosis (37Dinarello these conditions. Further studies required determine more detailed involvement exert effects thank Drs. Hiroshi Takeuchi (Department Oral Maxillofacial Surgery, Faculty Dentistry, Kyushu University), Nobuo Okahashi, Tatsuji Science, National Infectious Diseases) helpful discussions technical advice.