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Transcriptional Induction of FosB/ΔFosB Gene by Mechanical Stress in Osteoblasts

2004; Elsevier BV; Volume: 279; Issue: 48 Linguagem: Inglês

10.1074/jbc.m404096200

1083-351X

Daisuke Inoue, Shinsuke Kido, Toshio Matsumoto,

Bone health and treatments

Mechanical stress to bone plays a critical role in maintaining bone mass and strength. However, the molecular mechanism of mechanical stress-induced bone formation is not fully understood. In the present study, we demonstrate that FosB and its spliced variant ΔFosB, which is known to increase bone mass by stimulating bone formation in vivo, is rapidly induced by mechanical loading in mouse hind limb bone in vivo and by fluid shear stress (FSS) in mouse calvarial osteoblasts in vitro both at the mRNA and protein levels. FSS induction of FosB/ΔFosB gene expression was dependent on gadlinium-sensitive Ca2+ influx and subsequent activation of ERK1/2. Analysis of the mouse FosB/ΔFosB gene upstream regulatory region with luciferase reporter gene assays revealed that the FosB/ΔFosB induction by FSS occurred at the transcriptional level and was conferred by a short fragment from –603 to –327. DNA precipitation assays and DNA decoy experiments indicated that ERK-dependent activation of CREB binding to a CRE/AP-1 like element (designated “CRE2”) at the position of –413 largely contributed to the transcriptional effects of FSS. These results suggest that ΔFosB participates in mechanical stress-induced intracellular signaling cascades that activate the osteogenic program in osteoblasts. Mechanical stress to bone plays a critical role in maintaining bone mass and strength. However, the molecular mechanism of mechanical stress-induced bone formation is not fully understood. In the present study, we demonstrate that FosB and its spliced variant ΔFosB, which is known to increase bone mass by stimulating bone formation in vivo, is rapidly induced by mechanical loading in mouse hind limb bone in vivo and by fluid shear stress (FSS) in mouse calvarial osteoblasts in vitro both at the mRNA and protein levels. FSS induction of FosB/ΔFosB gene expression was dependent on gadlinium-sensitive Ca2+ influx and subsequent activation of ERK1/2. Analysis of the mouse FosB/ΔFosB gene upstream regulatory region with luciferase reporter gene assays revealed that the FosB/ΔFosB induction by FSS occurred at the transcriptional level and was conferred by a short fragment from –603 to –327. DNA precipitation assays and DNA decoy experiments indicated that ERK-dependent activation of CREB binding to a CRE/AP-1 like element (designated “CRE2”) at the position of –413 largely contributed to the transcriptional effects of FSS. These results suggest that ΔFosB participates in mechanical stress-induced intracellular signaling cascades that activate the osteogenic program in osteoblasts. Mechanical stress to bone plays a critical role in maintaining bone mass and strength. Reduced mechanical loading because of long-term bed rest or immobilization, or microgravity conditions in space, has been shown to cause significant bone loss and mineral changes. Evidence from human and animal studies has indicated that impaired bone formation significantly contributes to such unloading associated osteoporosis (1Morey E.R. Baylink D.J. Science. 1978; 201: 1138-1141Crossref PubMed Scopus (417) Google Scholar, 2Schneider V. Oganov V. LeBlanc A. Rakmonov A. Taggart L. Bakulin A. Huntoon C. Grigoriev A. Varonin L. Acta Astronaut. 1995; 36: 463-466Crossref PubMed Scopus (52) Google Scholar, 3Inoue M. Tanaka H. Moriwake T. Oka M. Sekiguchi C. Seino Y. Bone (N.Y.). 2000; 26: 281-286Crossref PubMed Scopus (83) Google Scholar). Therefore, it is an important task for us to elucidate the mechanism by which mechanical loading induces bone formation to understand the pathophysiology and to establish an effective treatment of osteoporosis caused by reduced mechanical loading.Bone cells have the ability to sense mechanical loading to activate multiple intracellular signaling pathways and transcription of various genes. Although the molecular device and the cellular repertoire in bone responsible for sensing such mechanical stimuli in vivo has not been precisely determined, it has been proposed that mechanotransduction in bone is mediated by changes in extracellular fluid flow caused by dynamic loading, which engenders fluid shear stress (FSS) 1The abbreviations used are: FSS, fluid shear stress; AP-1, activator protein-1; CREB, cyclic AMP response element-binding protein; ERK, extracellular signal-regulated kinase; COX, cyclooxygenase; SRE, serum response element; RT, reverse transcription; CDODN, circular dumbbell decoy oligonucleotides; BAPTA-AM, 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid, tetraacetoxymethyl ester; MEM, minimal essential medium.1The abbreviations used are: FSS, fluid shear stress; AP-1, activator protein-1; CREB, cyclic AMP response element-binding protein; ERK, extracellular signal-regulated kinase; COX, cyclooxygenase; SRE, serum response element; RT, reverse transcription; CDODN, circular dumbbell decoy oligonucleotides; BAPTA-AM, 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid, tetraacetoxymethyl ester; MEM, minimal essential medium. to bone cells, particularly osteocytes that are a terminally differentiated form of osteoblasts embedded in calcified bone (4Burr D.B. Robling A.G. Turner C.H. Bone (N.Y.). 2002; 30: 781-786Crossref PubMed Scopus (325) Google Scholar, 5Knothe Tate M.L. J. Biomech. 2003; 36: 1409-1424Crossref PubMed Scopus (250) Google Scholar). Consistently, FSS has been shown to elicit a divergent array of intracellular signaling pathways including intracellular calcium rise, activation of enzymes such as protein kinase B/Akt, mitogen-activated protein kinase family, and activation of transcription factors such as AP-1 (activator protein-1) and CREB (cyclic AMP response element-binding protein) in cells of the osteoblast lineage (6Mikuni-Takagaki Y. J. Bone Miner. Metab. 1999; 17: 57-60Crossref PubMed Scopus (104) Google Scholar, 7Nomura S. Takano-Yamamoto T. Matrix Biol. 2000; 19: 91-96Crossref PubMed Scopus (218) Google Scholar, 8Ehrlich P.J. Lanyon L.E. Osteoporos. Int. 2002; 13: 688-700Crossref PubMed Scopus (371) Google Scholar). Downstream of such signaling events is induction of various gene expression including extracellular matrix proteins such as type I collagen and osteopontin, and growth factors such as insulin-like growth factor-I (7Nomura S. Takano-Yamamoto T. Matrix Biol. 2000; 19: 91-96Crossref PubMed Scopus (218) Google Scholar). However, the molecular mechanisms as well as relevance to bone formation of such gene induction by mechanical stress in bone cells are largely unknown.One of the earliest transcriptional events caused by mechanical loading in bone cells is induction of c-Fos (9Lean J.M. Mackay A.G. Chow J.W. Chambers T.J. Am. J. Physiol. 1996; 270: E937-E945PubMed Google Scholar), a protooncogene that belongs to the AP-1 transcription factor family consisting of three Fos and four Jun family members. As a prototype of “immediate early genes,” c-Fos expression is known to be rapidly and transiently induced by various stimuli including serum and growth factors (10Karin M. Liu Z. Zandi E. Curr. Opin. Cell Biol. 1997; 9: 240-246Crossref PubMed Scopus (2281) Google Scholar, 11Treisman R. EMBO J. 1995; 14: 4905-4913Crossref PubMed Scopus (346) Google Scholar, 12Whitmarsh A.J. Davis R.J. J. Mol. Med. 1996; 74: 589-607Crossref PubMed Scopus (1382) Google Scholar). Induction of c-Fos expression by mechanical force has also been demonstrated in non-bone cells such as cardiac, muscle, and endothelial cells (13Li C. Xu Q. Cell. Signal. 2000; 12: 435-445Crossref PubMed Scopus (257) Google Scholar). Because mechanical loading induces several AP-1-responsive genes presumably involved in bone formation, c-Fos has been assumed to play a role in induction of such AP-1 target genes, thereby contributing to mechanical stress-induced bone formation. However, ubiquitous overexpression of c-Fos in transgenic mice resulted in development of osteosarcoma without evidence for increased bone formation (14Grigoriadis A.E. Schellander K. Wang Z.Q. Wagner E.F. J. Cell Biol. 1993; 122: 685-701Crossref PubMed Scopus (283) Google Scholar). Therefore, although c-Fos may stimulate proliferation and/or survival of the osteoblast lineage cells, it is not able to induce bone formation when overexpressed alone in bone cells.Another “immediate early gene” type member of the Fos family is FosB. Recently, transgenic mice overexpressing ΔFosB (15Mumberg D. Lucibello F.C. Schuermann M. Muller R. Genes Dev. 1991; 5: 1212-1223Crossref PubMed Scopus (110) Google Scholar, 16Yen J. Wisdom R.M. Tratner I. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5077-5081Crossref PubMed Scopus (127) Google Scholar, 17Nakabeppu Y. Nathans D. Cell. 1991; 64: 751-759Abstract Full Text PDF PubMed Scopus (350) Google Scholar), a short splicing isoform of FosB gene, have been reported to exhibit a progressive increase in bone mass because of enhanced bone formation (18Sabatakos G. Sims N.A. Chen J. Aoki K. Kelz M.B. Amling M. Bouali Y. Mukhopadhyay K. Ford K. Nestler E.J. Baron R. Nat. Med. 2000; 6: 985-990Crossref PubMed Scopus (291) Google Scholar). Expression of ΔFosB was observed within the osteoblast lineage and regulated in a differentiation-associated manner, and the effects of ΔFosB overexpression on osteoblasts appeared to be cell-autonomous and reversible (19Kveiborg M. Chiusaroli R. Sims N.A. Wu M. Sabatakos G. Horne W.C. Baron R. Endocrinology. 2002; 143: 4304-4309Crossref PubMed Scopus (24) Google Scholar, 20Sims N.A. Sabatakos G. Chen J.S. Kelz M.B. Nestler E.J. Baron R. Bone (N.Y.). 2002; 30: 32-39Crossref PubMed Scopus (23) Google Scholar). These results indicate that expression of ΔFosB alone is sufficient to induce bone formation, and further imply that ΔFosB may be involved in signaling pathways induced by osteogenic stimuli such as mechanical loading.In the present study, we investigated a potential role of ΔFosB in mechanical stress-induced bone formation. The results indicated that FosB/ΔFosB gene expression in bone cells was induced by mechanical stress both in vitro and in vivo. The induction occurred in a manner dependent on gadolinium (Gd3+)-sensitive Ca2+ influx and ERK (extracellular signal-regulated kinase) but independent of prostaglandin production, and involved a transcriptional mechanism with a major contribution of CREB. Our results suggest that ΔFosB acts as a downstream effector of mechanical loading and participates in mechanical stress-induced osteogenic signaling pathways.EXPERIMENTAL PROCEDURESMaterials—A selective cyclooxygenase (COX)-2 inhibitor, JTE-522 (21Matsushita M. Masaki M. Yagi Y. Tanaka T. Wakitani K. Inflamm. Res. 1997; 46: 461-466Crossref PubMed Scopus (52) Google Scholar, 22Ono K. Akatsu T. Murakami T. Kitamura R. Yamamoto M. Shinomiya N. Rokutanda M. Sasaki T. Amizuka N. Ozawa H. Nagata N. Kugai N. J. Bone Miner. Res. 2002; 17: 774-781Crossref PubMed Scopus (57) Google Scholar), was a kind gift from Japan Tobacco Inc. (Osaka, Japan). Indomethacin, nifedipine (L-type Ca2+ channel blocker), BAPTA-AM (intracellular Ca2+ chelator), A23187 (calcium ionophore), bucladesine (dibutyryl cAMP), U0126 (ERK inhibitor), and SB203580 (p38 kinase inhibitor) were purchased from Calbiochem. pAP1-Luc, pCRE-Luc, and pSRE-Luc plasmids, which are cis-reporter plasmids containing tandem consensus AP-1-binding sites, CRE (cAMP-response element), and c-Fos-derived SRE (serum response element), respectively, were from Invitrogen. All chemicals for dual-luciferase reporter assays were from Promega (Madison, WI). Antibody against FosB/ΔFosB was from Santa Cruz Biotechnology (San Diego, CA). Polyclonal antibodies against ERK1/2 and activated (phospho-) ERK1/2 were from Promega, and anti-CREB and phospho-CREB antibodies were from New England Biolabs (Beverly, MA). All DNA modifying enzymes used in this study were from New England Biolabs Inc. and the other reagents were from Sigma unless otherwise indicated.Experimental Animals—For in vivo experiments, 7–9-week-old ICR male mice were purchased from Charles River Japan (Tokyo, Japan), and were acclimatized for 1 week. The body weight of the mice ranged from 20 to 25 g. All mice were allowed free access to food and water, housed in stainless cages in an air-conditioned environment (temperature: 24–25 °C, humidity: 50–55%) that was illuminated from 8:00 a.m. to 8:00 p.m. The experimental protocols in the current study have been approved by the Institutional Animal Care and Oversight Committee according to the guideline principles in the Care and Use of Animals. Mechanical unloading by tail suspension and the following reloading was performed according to the procedures described before (23Sakata T. Sakai A. Tsurukami H. Okimoto N. Okazaki Y. Ikeda S. Norimura T. Nakamura T. J. Bone Miner. Res. 1999; 14: 1596-1604Crossref PubMed Scopus (78) Google Scholar, 24Matsumoto T. Nakayama K. Kodama Y. Fuse H. Nakamura T. Fukumoto S. Bone (N.Y.). 1998; 22: 89S-93SCrossref PubMed Scopus (64) Google Scholar) with slight modifications. Mice were anesthetized with ether and unloaded by suspending tails with the hind limbs being lifted off from the ground. After tail suspension for 4 days, mice were forced to run in rotating cages for the indicated times for reloading, sacrificed, and analyzed for gene expression.Cell Culture—Primary osteoblasts were prepared from calvariae of newborn ICR mice by sequential digestion with 0.1% (w/v) type IA collagenase and 0.2% (w/v) dispase as previously described (25Inoue D. Santiago P. Horne W.C. Baron R. J. Biol. Chem. 1997; 272: 25386-25393Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). The osteoblasts were cultured in α-MEM supplemented with 10% fetal bovine serum (fetal bovine serum) (Sigma), penicillin/streptomycin (Invitrogen) for 48 h. Before induction experiments, 1 × 105/ml osteoblasts were plated on culture dishes, grown to 70–80% confluence, and serum-deprived in α-MEM with 1% fetal bovine serum for 24 h. For mechanical loading in vitro, osteoblasts were exposed to FSS by placing 6-well culture dishes on a horizontal shaking apparatus fixed inside the culture incubator. All the experiments in the present study were performed at 100–120 rpm. The shear stress force in our system was estimated to be slightly less than that produced by 200 rpm with a cone viscometer, which was ∼2 pascal at the edge (26Sakai K. Mohtai M. Shida J. Harimaya K. Benvenuti S. Brandi M.L. Kukita T. Iwamoto Y. J. Bone Miner. Res. 1999; 14: 2089-2098Crossref PubMed Scopus (33) Google Scholar), and thus theoretically stayed within the physiological range.Adenovirus Infection—A recombinant adenoviral vector for constitutively active MEK1 (MEKCA) was a kind gift from Dr. Sakae Tanaka (University of Tokyo). Primary osteoblasts at passage two were infected with MEKCA adenoviruses at a multiplicity of infection varying from 0 to 100. At 48 h after infection, cells were harvested and examined for expression of FosB/ΔFosB and other proteins by Western blot analysis.RNA Analysis—Total RNA was extracted from tibia and femur of the mice or cultured primary osteoblasts using TRIzol reagents (Invitrogen), and mRNA expression for various genes was determined by reverse transcription-polymerase chain reaction (RT-PCR) or ribonuclease (RNase) protection assays. For RT-PCR, 1 μg of total RNA was reverse-transcribed using SuperScript II RT™ (Invitrogen) and random primers (Promega Corp., Madison, WI). One of a 20-μl RT reaction was used for PCR analysis. Primer sets used for amplification were as follows: sense, 5′-aaaaggcagagctggagtcgg-3′, and antisense, 5′-tgtacgaagggctaacaacgg-3′ for mouse FosB, which amplifies both the short spliced ΔFosB isoform and the long FosB transcripts; and sense, 5′-tgtcttcaccaccatggagaagg-3′, and antisense, 5′-gtggatgcagggatgatgttctg-3′, for mouse GAPDH gene. The PCR cycle numbers were 28 for FosB/ΔFosB and 22 for GAPDH, which were determined so that quantitative information was not lost. Amplified products were separated on 1.5–2.0% agarose gels and stained with ethidium bromide for visualization. For the RNase protection assay, a fragment of mouse FosB cDNA (nucleotides 1480–2051) was subcloned into pBlueScript SKII(+) (Stratagene, La Jolla, CA). The resultant plasmid was linearized and purified with Gel Extraction Kit (Qiagen Inc., Valencia, CA) and in vitro transcribed to generate a cRNA probe using the MAXIscript in Vitro Transcription Kit (Ambion Inc., Austin, TX). A control template for mouse β-actin was purchased from Ambion. RNase protection assays were performed using the RPAII Kit (Ambion) as previously described (27Inoue D. Reid M. Lum L. Kratzschmar J. Weskamp G. Myung Y.M. Baron R. Blobel C.P. J. Biol. Chem. 1998; 273: 4180-4187Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Briefly, RNA samples were incubated with [α-32P]UTP-labeled cRNA probes at 42 °C for 18 h and digested with an RNase A/T1 mixture. Non-digested RNA was precipitated, separated on a 6% polyacrylamide gel containing 8 m urea, transferred to 3MM Whatman filter paper, dried, and autoradiographed.Protein Analysis—Preparation of nuclear extracts was described previously (25Inoue D. Santiago P. Horne W.C. Baron R. J. Biol. Chem. 1997; 272: 25386-25393Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Briefly, 1–2 × 107 cells were collected, washed twice with ice-cold phosphate-buffered saline, incubated in ice-cold hypotonic buffer (10 mm HEPES-KOH, pH 7.2, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 0.75 mm spermidine, 0.15 mm spermine, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 1× protease inhibitor mixture (Sigma), and 10 mm Na2MoO4) for 10 min, vortexed for 10 s, and centrifuged at 15,000 × g at 4 °C for 60 s. The pellet was resuspended in NLB buffer (20 mm HEPES-KOH, pH 7.2, 0.4 m NaCl, 1 mm EDTA, 1mm EGTA, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 1× Protease inhibitor mixture (Sigma), and 10 mm Na2MoO4), placed on ice for 15 min, and centrifuged at 4 °C at 15,000 × g for 10 min. The supernatant was saved as nuclear extracts. Total cell lysates were prepared using 1× Cell Lysis Buffer (Cell Signaling Technology Inc., Beverly, MA) according to the manufacturer's instructions. The nuclear extracts and total cell lysates were stored at –80 °C until use. For Western blot analysis, 30 μg of protein was separated on a 5–20% gradient SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore Corp.). The membrane was rinsed and blocked with 5% nonfat skim milk or 2% bovine serum albumin (fraction V from Sigma) in Tris-buffered saline (TBS) with 0.1% Tween 20 for 2 h at room temperature, blotted sequentially with primary antibodies, then with a horseradish-conjugated secondary antibody for 1 h, and the protein bands were visualized with an ECL detection system (Amersham Biosciences).Cloning of the Mouse FosB Gene Promoter—Fragments of mouse FosB gene (GenBank™ number AF093624) upstream regulatory region from nucleotides –1000 to +307 (1.0K), –603 to +307 (0.6K), and –327 to +307 (0.3K) (with the transcription start site being numbered +1) were cloned from a mouse genomic library (Toyobo Co., Ltd., Osaka, Japan) by PCR with the following primer sets: sense, 5′-acgggtaccttagtctccggttcggagaac-3′, and antisense, 5′-acgaagcttttagtctccggttcggagaac-3′, for 1.0K; sense, 5′-ctaattgcgtcacaggaactcc-3′, and antisense, acgaagcttttagtctccggttcggagaac-3′ for 0.6K; and sense, 5′-agcggtacccagggtcacactatgggcaggt-3′, and antisense, 5′-acgaagcttttagtctccggttcggagaa-3′, for 0.3K. PCR products were purified, digested with KpnI/XhoI (1.0K and 0.3K) or SmaI/XhoI) (0.6K), and subcloned into a luciferase reporter plasmid pGL3-basic (Promega, Madison, WI), which lacks eukaryotic promoter and enhancer sequences. From the luciferase vector containing the longest 1.0-kb promoter, a series of mutant plasmids were generated by site-directed mutagenesis using the QuikChange™ Site-directed Mutagenesis Kit (STRATAGENE) with mutagenic primers including 5′-ccttatatCC-3′ for SRE mutation (g547c and g548c) and 5′-tgGCAca-3′ for CRE2 mutation (c556g, g557c, and t558a). All the constructs were verified by sequencing.Dual Luciferase Assay—Osteoblasts were seeded in 6-well culture plates at 50% confluence and cultured in α-MEM supplemented with 10% fetal bovine serum. After 18–24 h, the cells were co-transfected with 1 μg of chimeric luciferase reporter plasmids and 0.025 μg of pRL-TK Renilla luciferase plasmids (Promega) using GenePorter 2 Transfection Reagents (Gene Therapy Systems, Inc., San Diego, CA) in Opti-MEM supplemented with 1% fetal bovine serum. At 6 h after transfection, the medium was replaced by 2 ml of Opti-MEM supplemented with 1% fetal bovine serum, and the cells were incubated for an additional 24 h. The transfected cells were then treated with various reagents and/or exposed to FSS by placing the culture plates on a shaking apparatus at 100 to 120 rpm. For dual luciferase assays, the cells were washed twice with PBS and lysed with 100 μl of passive cell lysis buffer (Promega). Luciferase activities were measured with luminometer (ATTO, Tokyo, Japan) by mixing 50 μl of luciferase substrate solution (Promega) with 10 μl of cell lysates. Transcriptional activity was normalized for Renilla luciferase activity or protein concentrations.Electrophoretic Mobility Shift Assay—Electrophoretic mobility shift assay was performed as previously described (25Inoue D. Santiago P. Horne W.C. Baron R. J. Biol. Chem. 1997; 272: 25386-25393Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Radiolabeled double-stranded oligonucleotide probes were prepared by annealing complementary oligonucleotides end-labeled with [α-32P]dATP and DNA polymerase I fragment (Klenow) (New England Biolabs). The labeled probes were purified by Sephadex G-25 columns (Quick Spin Columns, Roche Molecular Biochemicals), diluted with distilled water, and 3 × 104 cpm was incubated with nuclear extracts (10 μg) and 4 μg of poly(dI-dC)·poly(dI-dC) in a total volume of 20 μl of 1/2 × NLB buffer at 4 °C for 30 min. For competition analysis, a hundred times molar excess of cold oligonucleotide probes were added to the electrophoretic mobility shift assay reaction. Resolution was accomplished by electrophoresing 15 μl of the reaction mixture on 4.5 to 5.0% polyacrylamide gels using 0.25× TBE buffer (22.3 mm Tris-HCl, 22.3 mm boric acid, and 0.25 mm EDTA, pH 8.0). Gels were then transferred onto 3MM filter paper and dried, and protein-DNA complexes were visualized by autoradiography.DNA Precipitation Assay—Biotinylated double-stranded oligonucleotide probes containing three tandem repeats of SRE/CRE2 (–437 to –401) or CRE1 (–488 to –461) sequences were prepared by annealing complementary oligonucleotides followed by ethanol precipitation. The annealed probes (100 pmol) were incubated with 100 μg of nuclear extracts and 15 μg of poly(dI-dC)·poly(dI-dC) in 500 μl of NLB100 buffer (20 mm HEPES-KOH, pH 7.2, 0.1 m NaCl, 1 mm EDTA, 1 mm EGTA, 0.5 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, protease inhibitor mixture (Sigma), 20 mm sodium fluoride, and 1 mm VO4 for 4 °C with gentle agitation, and then with streptavidine-conjugated magnetic beads (Magnotex-SA, TaKaRa, Japan) at 4 °C for 30 min. DNA-protein complexes bound to magnetic beads were collected by placing the reaction tubes on a magnetic stand and washed with ice-cold NLB100 buffer for four times. After the final wash, beads were collected and resupended in 2× sample buffer. Sequence-specific DNA-bound proteins were separated on a SDS-polyacrylamide gel and detected by Western blotting. For competition analysis, 100 times molar excess of non-labeled biotinylated double-stranded oligonucleotide probes were added to the DNA-protein reactions.Decoy Oligonucleotides—An SRE/CRE2 “circular dumbbell” decoy oligonucleotide (CDODN) (28Ahn J.D. Kim C.H. Magae J. Kim Y.H. Kim H.J. Park K.K. Hong S. Park K.G. Lee I.K. Chang Y.C. Biochem. Biophys. Res. Commun. 2003; 310: 1048-1053Crossref PubMed Scopus (31) Google Scholar, 29Lee I.K. Ahn J.D. Kim H.S. Park J.Y. Lee K.U. Curr. Drug Targets. 2003; 4: 619-623Crossref PubMed Scopus (27) Google Scholar) containing the SRE/CRE2 sequences in the FosB promoter region (5′-ataaggagctcgcctttttttggcgagctccttatatggctaattgcgtcacaggtttttttcctgtgacgcaattagccat-3′) was synthesized to form a double-stranded DNA oligomer with the both ends hinged by a stretch of seven Ts. This structure has been demonstrated to confer resistance to endogenous nucleases (29Lee I.K. Ahn J.D. Kim H.S. Park J.Y. Lee K.U. Curr. Drug Targets. 2003; 4: 619-623Crossref PubMed Scopus (27) Google Scholar). As controls, we also synthesized mutant CDODNs including: Scramble, 5′-taacgactcggtagtttttttctaccgagtcgttaattgcctgcggcagtggatgtttttttcatccactgccgcaggcaat-3′; SREmt, 5′-ataaCCagctcgcctttttttggcgagctGGttatatggctaattgcgtcacaggtttttttcctgtgacgcaattagccat-3′; and CRE2mt, 5′-ataaggagctcgcctttttttggcgagctccttatatggctaattgGCAcacaggtttttttcctgtgTGCcaattagccat-3′. After self-annealing and ligation, the CDODNs were introduced into osteoblasts by GenePorter 2 Transfection Reagents according to the manufacturer's instruction.RESULTSIn Vivo Mechanical Forces and in Vitro FSS to Osteoblasts Induce FosB/ΔFosB Gene Expression at Both the mRNA and Protein Levels—We first investigated whether or not expression of the FosB/ΔFosB gene in bone is induced by mechanical loading in vivo. Seven- to 9-week-old ICR male mice were tail suspended for 4 days to reduce the background expression, and then mechanically reloaded in rotating cages. As shown in Fig. 1A, expression of (long) FosB and ΔFosB mRNA in tibiae and femurs was undetectable in tail-suspended mice, but was induced as early as 30 min after mechanical reloading and reached the maximum within 2 h. In the in vitro experiments shown in Fig. 1B, primary osteoblasts derived from newborn mouse calvariae were subjected to FSS on a shaking apparatus. As a result, we observed that FosB/ΔFosB mRNA was induced by FSS in primary osteoblasts, recapitulating the in vivo induction in reloaded mice. These results are consistent with an assumption that the in vivo mechanical forces caused FSS to bone cells of the osteoblast lineage, leading to increased expression of FosB/ΔFosB. To confirm that the induction of FosB/ΔFosB mRNA by FSS leads to an increase in the amount of their protein products, nuclear extracts were obtained from cells that had been exposed to FSS and were analyzed for FosB/ΔFosB protein expression by Western blotting. The results indicated that FosB (50 kDa) and ΔFosB (32 kDa) proteins were both induced either by mechanical loading in vivo or FSS in vitro in a time-dependent manner (Fig. 1, C and D). These findings were similarly observed in other osteoblastic cell lines such as a murine calvarial cell line, MC3T3-E1, and a murine bone marrow stromal cell line, ST-2 (data not shown). Therefore, induction of FosB/ΔFosB gene expression by mechanical stress occurs at both the mRNA and protein levels in cells of the osteoblast lineage.Induction of FosB/ΔFosB mRNA Is Independent of Prostaglandin Production—Previous reports suggest that prostaglandins are important mediators of mechanical stress-induced bone formation. Mechanical stress has been shown to cause activation of a constitutive type of prostaglandin G/H synthase (COX-1) and transcriptional up-regulation of an inducible isoform (COX-2) (8Ehrlich P.J. Lanyon L.E. Osteoporos. Int. 2002; 13: 688-700Crossref PubMed Scopus (371) Google Scholar, 30Ogasawara A. Arakawa T. Kaneda T. Takuma T. Sato T. Kaneko H. Kumegawa M. Hakeda Y. J. Biol. Chem. 2001; 276: 7048-7054Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), and both events would lead to increased production of prostaglandins, especially the E series that have been shown to induce c-Fos (31Weinreb M. Rutledge S.J. Rodan G.A. Bone (N. Y.). 1997; 20: 347-353Crossref PubMed Scopus (37) Google Scholar). To determine whether FSS-induced FosB/ΔFosB expression is mediated by prostaglandins, tail-suspended mice were subcutaneously injected with indomethacin, a general COX inhibitor (both COX-1 and COX-2), prior to reloading, and FosB/ΔFosB mRNA induction in tibiae and femurs was analyzed by RNase protection assay. As shown in Fig. 2A, FosB/ΔFosB mRNA induction was not blocked by indomethacin. Similarly, pretreatment with either indomethacin or a selective COX-2 inhibitor, JTE-522, had no major effects on FSS-induced FosB/ΔFosB expression in osteoblasts in vitro (Fig. 2B). These results indicated that mechanical stress-induced FosB/ΔFosB induction was independent of prostaglandin production both in vivo and in vitro.Fig. 2Induction of FosB/ΔFosB mRNA is independent of prostaglandin production.A, 7–9-week-old ICR male mice were first tail suspended for 4 days and orally given 10 mg/kg of indomethacin or vehicle. After 1 h, mice were reloaded in a rotating cage for 30 min and sacrificed, and expression of FosB/ΔFosB and β-actin mRNA in tibiae and femurs was analyzed by RNase protection assay. B, mouse calvarial osteoblasts were pretreated with 1 mm indomethacin or a selective COX-2 inhibitor, JTE-522, 30 min prior to FSS. Cells were then exposed to FSS for 30 min, and mRNA expression of FosB/ΔFosB and GAPDH as an internal control was examined by RT-PCR.View Large Image Figure ViewerDownload (PPT)FSS-induced FosB/ΔFosB mRNA Expression Is Dependent on ERK—We then attempted to delineate the intracellular signaling pathways leading to FosB/ΔFosB induction. We first investigated involvement of the mitogen-activated protein kinase family members including ERK, p38 kinase, and c-Jun N-terminal kinase, which have been suggested to link FSS to various gene inductions (32Takahashi M. Ishida T. Traub O. Corson M.A. Berk B.C. J. Vasc. Res. 1997; 34: 212-219Crossref PubMed Scopus (156)