Title: Concerted Action of Smad and CREB-binding Protein Regulates Bone Morphogenetic Protein-2-stimulated Osteoblastic Colony-stimulating Factor-1 Expression
Abstract: Bone remodeling depends upon proper osteoblast and osteoclast function. Bone morphogenetic protein-2 (BMP-2) stimulates differentiation of osteoblasts from pluripotent precursors. Osteoclast formation depends on the concerted action of osteoblast-derived receptor activator of NF-κB ligand and colony-stimulating factor-1 (CSF-1). BMP-2 stimulates receptor activator of NF-κB ligand expression. However, the effect of BMP-2 on CSF-1 expression has not been studied. We investigated the role of BMP-2 in CSF-1 expression in osteogenic C2C12 cells. Incubation of C2C12 cells with BMP-2 supported osteoclastogenesis of spleen cells with a concomitant increase in expression of CSF-1 mRNA and protein. To determine the mechanism, we identified a BMP-responsive element between –627 bp and –509 bp in the CSF-1 promoter. DNase I footprint analysis revealed the presence of consensus Smad binding motif in this region. Electrophoretic mobility shift assay showed BMP-2-stimulated binding of proteins to this motif. Mutation of core sequence as well as its 5′- and 3′-flanking sequences abolished the DNA-protein interaction resulting in inhibition of CSF-1 transcription. Supershift analysis detects the presence of Smads 1, 5, and 4 and the transcriptional coactivator CREB-binding protein in the BMP-responsive element-protein complex. In addition, Smads 1 and 5 alone or in combination with Smad 4 increased CSF-1 transcription. Furthermore, CREB-binding protein markedly increased transcription of CSF-1. These data represent the first evidence that BMP-2 increases the osteoclastogenic CSF-1 expression by a transcriptional mechanism using the canonical Smad pathway and provide a mechanism for BMP-2-induced osteoclast differentiation. Bone remodeling depends upon proper osteoblast and osteoclast function. Bone morphogenetic protein-2 (BMP-2) stimulates differentiation of osteoblasts from pluripotent precursors. Osteoclast formation depends on the concerted action of osteoblast-derived receptor activator of NF-κB ligand and colony-stimulating factor-1 (CSF-1). BMP-2 stimulates receptor activator of NF-κB ligand expression. However, the effect of BMP-2 on CSF-1 expression has not been studied. We investigated the role of BMP-2 in CSF-1 expression in osteogenic C2C12 cells. Incubation of C2C12 cells with BMP-2 supported osteoclastogenesis of spleen cells with a concomitant increase in expression of CSF-1 mRNA and protein. To determine the mechanism, we identified a BMP-responsive element between –627 bp and –509 bp in the CSF-1 promoter. DNase I footprint analysis revealed the presence of consensus Smad binding motif in this region. Electrophoretic mobility shift assay showed BMP-2-stimulated binding of proteins to this motif. Mutation of core sequence as well as its 5′- and 3′-flanking sequences abolished the DNA-protein interaction resulting in inhibition of CSF-1 transcription. Supershift analysis detects the presence of Smads 1, 5, and 4 and the transcriptional coactivator CREB-binding protein in the BMP-responsive element-protein complex. In addition, Smads 1 and 5 alone or in combination with Smad 4 increased CSF-1 transcription. Furthermore, CREB-binding protein markedly increased transcription of CSF-1. These data represent the first evidence that BMP-2 increases the osteoclastogenic CSF-1 expression by a transcriptional mechanism using the canonical Smad pathway and provide a mechanism for BMP-2-induced osteoclast differentiation. Bone morphogenetic proteins (BMPs) 3The abbreviations used are: BMP-2, bone morphogenetic protein-2; BMPR, bone morphogenetic protein receptor; BMPR I, bone morphogenetic protein receptor type I; BMPR II, bone morphogenetic protein receptor type II; CSF-1, macrophage colony-stimulating factor; EMSA, electrophoretic mobility shift assay; BRE, BMP-2 response element; W T, wild type; MT, mutant; CBP, CREB-binding protein; CREB, cAMP-response element-binding protein; E1A, adenovirus 5 early protein 1A; RANKL, receptor activator of NF-κB ligand; R-Smad, receptor-regulated Smad; Co-Smad, common Smad; DMEM, Dulbecco's modified Eagle's medium; TRAP, tartrate-resistant acid phosphatase.3The abbreviations used are: BMP-2, bone morphogenetic protein-2; BMPR, bone morphogenetic protein receptor; BMPR I, bone morphogenetic protein receptor type I; BMPR II, bone morphogenetic protein receptor type II; CSF-1, macrophage colony-stimulating factor; EMSA, electrophoretic mobility shift assay; BRE, BMP-2 response element; W T, wild type; MT, mutant; CBP, CREB-binding protein; CREB, cAMP-response element-binding protein; E1A, adenovirus 5 early protein 1A; RANKL, receptor activator of NF-κB ligand; R-Smad, receptor-regulated Smad; Co-Smad, common Smad; DMEM, Dulbecco's modified Eagle's medium; TRAP, tartrate-resistant acid phosphatase. have been shown to induce osteogenic activities in animals (1.Wozney J.M. 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Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (477) Google Scholar). Smads are downstream molecules that are activated by BMP-like ligands. They exist as monomers in the absence of ligand stimulation. Receptor-regulated Smads (R-Smads) are directly phosphorylated by BMPR I and subsequently associate with common Smad (Co-Smad). Heteromeric complexes then translocate into the nucleus and regulate transcription of target genes in association with other nuclear proteins (30.Massague J. Chen Y.G. Genes Dev. 2000; 14: 627-644Crossref PubMed Google Scholar, 31.Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (477) Google Scholar). BMPR I activates Smads 1, 5, and 8 (R-Smads), which in turn heterodimerize with Smad 4 (Co-Smad) to transduce BMP-mediated intracellular signals (30.Massague J. Chen Y.G. Genes Dev. 2000; 14: 627-644Crossref PubMed Google Scholar, 31.Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (477) Google Scholar). Smad binding elements have been identified to contain a consensus sequence CAGACA (CAGA sequences) in the promoters of the genes that are activated by Smad signaling (32.Jonk L.J. Itoh S. Heldin C.H. ten Dijke P. Kruijer W. J. Biol. Chem. 1998; 273: 21145-21152Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar). BMPR and their downstream signaling molecules are expressed in osteoclasts, isolated from rabbit long bones, and BMP-2 stimulates the bone-resorbing activity of these osteoclasts (33.Kaneko H. Arakawa T. Mano H. Kaneda T. Ogasawara A. Nakagawa M. Toyama Y. Yabe Y. Kumegawa M. Hakeda Y. Bone. 2000; 27: 479-486Crossref PubMed Scopus (274) Google Scholar). BMPR I mRNA was found to be expressed in granulocyte macrophage-colony stimulating factor-supported hematopoietic blast cells (34.Kanatani M. Sugimoto T. Kaji H. Kobayashi T. Nishiyama K. Fukase M. Kumegawa M. Chihara K. J. Bone Miner. Res. 1995; 10: 1681-1690Crossref PubMed Scopus (191) Google Scholar). It was recently reported that bone marrow macrophages and purified mature osteoclasts express BMPRIA (35.Itoh K. Udagawa N. Katagiri T. Iemura S. Ueno N. Yasuda H. Higashio K. Quinn J.M. Gillespie M.T. Martin T.J. Suda T. Takahashi N. Endocrinology. 2001; 142: 3656-3662Crossref PubMed Scopus (166) Google Scholar). BMP-2 can stimulate parathyroid hormone-induced osteoclast formation, which was inhibited by BMP-2 antagonist noggin (36.Abe E. Yamamoto M. Taguchi Y. Lecka-Czernik B. O'Brien C.A. Economides A.N. Stahl N. Jilka R.L. Manolagas S.C. J. Bone Miner. Res. 2000; 15: 663-673Crossref PubMed Scopus (274) Google Scholar). BMP-2 was found to increase RANKL expression in murine osteoclast cultures and enhance osteoclast formation by interleukin-1α (37.Koide M. Murase Y. Yamato K. Noguchi T. Okahashi N. Nishihara T. Biochem. Biophys. Res. Commun. 1999; 259: 97-102Crossref PubMed Scopus (87) Google Scholar). 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Our goal for this study was to analyze the role of BMP-2 in CSF-1 gene expression and subsequent osteoclast differentiation. In this report, we show that BMP-2 induces expression of CSF-1 mRNA and protein in the pluripotent C2C12 cells that differentiate into osteoblasts upon BMP-2 stimulation. We identified a BMP-2-responsive element (BRE) in the CSF-1 promoter. We show that upon BMP-2 stimulation CBP associates with BMP-specific Smads. We provide evidence that BRE interacts with BMP-specific Smads and the transcription cofactor CBP to regulate CSF-1 expression. In addition, we show that BMP-2-stimulated C2C12 cells can support multinucleated TRAP-positive osteoclast formation in a coculture system. Taken together, our data provide a molecular mechanism of CSF-1 expression in response to BMP-2 to support osteoclast survival and differentiation. Furthermore, this is the first report of the regulation of CSF-1 production in osteoblasts by BMP-2 during osteoclastogenesis. Also this report unravels the mechanism by which BMP-2 induces CSF-1 transcription. Materials—Recombinant BMP-2 was a gift from Anthony Celeste (Wyeth Research, Cambridge, MA). Tissue culture reagents and Lipofectamine Plus were obtained from Invitrogen. pGL3 luciferase plasmids (pGL3 basic and pGL3 promoter) and dual luciferase assay kits were purchased from Promega Inc. (Madison, WI). A nuclear fraction extraction kit was purchased from Pierce. Antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Plasmids expressing Smads 1, 4, and the constitutively active BMP receptors were kindly provided by Dr. Miyazono (University of Tokyo, Japan). Smad 5 expression plasmid was kindly provided by Dr. Nishimura (Osaka University, Japan). CBP expression plasmid was a gift from Dr. Christopher Glass (University of California at San Diego). RNAzol B was purchased from Biotex Laboratories (Houston, TX). Nylon filters were bought from Schleicher and Schuell (Keene, NH). A CSF-1 ELISA assay kit was purchased from R & D systems (Minneapolis, MN). A nuclear extract preparation kit and poly(dI·dC) were purchased from Pierce, and DNase I was purchased from Sigma-Aldrich. Cell Culture—C2C12 cells were purchased from American Type Culture Collection (Manassas, VA). C2C12 cells undergo osteoblastic differentiation in the presence of BMP-2. The cells were grown in DMEM medium containing 10% fetal calf serum and 1% penicillin and streptomycin. For driving osteoblastic differentiation the cells are routinely grown up to 70% confluency and treated with 300 ng/ml recombinant BMP-2 in serum-free DMEM for the indicated periods of time. RNA Preparation and Northern Analysis—RNA was isolated from C2C12 cells cultured in 10-cm tissue culture plates in serum-free DMEM containing 300 ng/ml BMP-2 for 6, 12, 24, and 48 h. Control cells were cultured for similar time periods in serum-free DMEM without BMP-2 in the culture medium. At the indicated time points the cells were harvested, and RNA was isolated using 5 ml of RNAzol B followed by chloroform extraction and precipitation of RNA with isopropanol (38.Ghosh-Choudhury N. Windle J.J. Koop B.A. Harris M.A. Guerrero D.L. Wozney J.M. Mundy G.R. Harris S.E. Endocrinology. 1996; 137: 331-339Crossref PubMed Scopus (82) Google Scholar). 20 μg of RNA was separated by electrophoresis according to size in denaturing agarose gels and transferred to Nylon filters. The filters were hybridized with CSF-1 and glyceraldehyde-3-phosphate dehydrogenase cDNA probes (38.Ghosh-Choudhury N. Windle J.J. Koop B.A. Harris M.A. Guerrero D.L. Wozney J.M. Mundy G.R. Harris S.E. Endocrinology. 1996; 137: 331-339Crossref PubMed Scopus (82) Google Scholar). Northern analysis was repeated three times with different RNA isolations. CSF-1 ELISA Assay—CSF-1 levels in C2C12 cells treated with BMP-2 for 0, 24, 36, and 48 h were assayed using the Quantikine enzyme-linked immunoassay kit according to the manufacturer's instructions. CSF-1 concentrations were calculated from a standard curve (31.2–2000 pg/ml) using log-log linear regression (39.Abboud S.L. Woodruff K. Liu C. Shen V. Ghosh-Choudhury N. Endocrinology. 2002; 143: 1942-1949Crossref PubMed Scopus (49) Google Scholar). DNaseI Footprint Assay—Nuclear extract was prepared from C2C12 and fetal rat calvarial osteoblast cells as described earlier following the protocol specified by the vendor (40.Ghosh-Choudhury N. Abboud S.L. Mahimainathan L. Chandrasekar B. Choudhury G.G. J. Biol. Chem. 2003; 278: 21998-22005Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The DNA fragment spanning a region, –627 to –509 bp, of CSF-1 promoter was cloned into plasmid vector. Purified DNA fragment was end-labeled using [α-32P]ATP. The DNaseI footprinting was carried out as follows: C2C12 or fetal rat calvarial nuclear proteins were allowed to bind the labeled DNA fragment at 25 °C for 15 min in the presence of the binding buffer (50 mm NaCl, 0.1 mm EDTA, 20 mm Hepes/potassium hydroxide (KOH), pH 7.5, 0.5 mm dithiothreitol, and 10% glycerol) and 2 μg of poly(dI·dC). The DNaseI treatment was then carried out for 45 s using 5 μl of 1:10,000-fold diluted DNaseI at 25 °C in the presence of 5 μl of 50 mm MgCl2 and 5 μl of 10 mm CaCl2. The DNaseI was immediately inactivated by using 100 μl of stop solution (0.375% SDS, 15 mm EDTA, 100 mm NaCl, and 100 mm Tris·Cl, pH 7.6) followed by proteinase K digestion and phenolchloroform extraction, ethanol precipitation, and electrophoresis on a 7% sequencing gel. A DNaseI protection assay (footprint assay) was repeated four times. Electrophoretic Mobility Shift Assay—C2C12 cells were serum-starved for 24 h and then treated with recombinant BMP-2 for an additional 24 h in serum-free DMEM. Nuclear extracts were prepared as described above. The oligonucleotide probe spanning the BMP-2 response element in CSF-1 promoter was prepared by annealing the oligonucleotide 5′-GGA GGG AGC AAG CCA ATC TGC AAA CCC CGG GTT AAG GGC G-3′ with its complementary strand. The core consensus Smad binding sequence is underlined. The mutant oligonucleotides were designed by (a) mutating the core binding sequence ATC TGC to gga TtC (MT), (b) mutating DNA sequences 3′ to the core binding site CGG GTT to att cGg (3′-MT), and (c) mutating 5′ sequence to the core binding site GGG AGC to ata Atg (5′-MT). The double-stranded oligonucleotides were labeled using [γ-32P]ATP and T4 polynucleotide kinase (40.Ghosh-Choudhury N. Abboud S.L. Mahimainathan L. Chandrasekar B. Choudhury G.G. J. Biol. Chem. 2003; 278: 21998-22005Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The EMSA was performed using 5 μg of nuclear extract as described previously (40.Ghosh-Choudhury N. Abboud S.L. Mahimainathan L. Chandrasekar B. Choudhury G.G. J. Biol. Chem. 2003; 278: 21998-22005Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 41.Choudhury G.G. Ghosh-Choudhury N. Abboud H.E. J. Clin. Invest. 1998; 101: 2751-2760Crossref PubMed Google Scholar, 42.Choudhury G.G. J. Biol. Chem. 2004; 279: 27399-27409Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). For determining the specificity of DNA-protein interaction the nuclear extract was incubated with increasing amounts of cold oligonucleotide before the addition of the radioactive probe. Specific antibodies were added to the nuclear extract to perform supershift analysis followed by incubation with radioactive probe as described previously (40.Ghosh-Choudhury N. Abboud S.L. Mahimainathan L. Chandrasekar B. Choudhury G.G. J. Biol. Chem. 2003; 278: 21998-22005Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). EMSA performed with unlabeled oligonucleotides, antibodies, and mutant oligonucleotides were repeated at least three to five times each with different preparations of nuclear extracts. Transfection and Luciferase Assay—Sequential 5′ deletion constructs of CSF-1 promoter driving the firefly luciferase reporter gene were transfected in C2C12 cells using Lipofectamine Plus reagent as described previously (40.Ghosh-Choudhury N. Abboud S.L. Mahimainathan L. Chandrasekar B. Choudhury G.G. J. Biol. Chem. 2003; 278: 21998-22005Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). To correct transfection efficiency a cytomegalo-virus promoter-driven Renilla luciferase plasmid was cotransfected in the transient transfection assays. Luciferase activities were assayed using a dual luciferase assay kit. Site-directed Mutagenesis—The BRE sequences were mutated using a QuikChange II site-directed mutagenesis kit from Stratagene (La Jolla, CA). The oligonucleotide used for mutating the core CAGA sequences was 5′-GGA GGG AGC AAG CCA gga TtC AAA CCC CGG GTT AAG GGC G-3′. Lowercase letters indicate mutated sequences. The reactions were carried out exactly as directed by the vendor's instruction manual. Coculture Assay for TRAP-positive Multinucleated Osteoclast Formation—Coculture of C2C12 and mouse spleen cells was done as described by Otsuka et al. (43.Otsuka E. Notoya M. Hagiwara H. Calcif. Tissue Int. 2003; 73: 72-77Crossref PubMed Scopus (42) Google Scholar). In brief, 104 C2C12 cells were cultured in 24-well tissue culture plates in α-minimal essential medium. After 24 h, 106 mouse spleen cells were added per well to C2C12 cells in the presence of 10–8 m 1,25-dihydroxyvitamin D3 (BIOMOL, Plymouth Meeting, PA), 10–7 m dexamethasone, and 300 ng/ml recombinant human BMP-2 or bovine serum albumin (for vehicle control). Fresh medium was replenished every 2 days. After 6 days adherent cells were fixed in 10% formalin for 5 min and then treated with 1:1 mixture of ethanol and acetone for 1 min. Cultures were then dried and stained for TRAP activity using acid phosphatase kit and Fast Garnet dye (Sigma). TRAP-positive multinucleated cells (with three or more nuclei) were photographed using a light microscope (Nikon, Japan). Coimmunoprecipitation Assay—C2C12 cells were stimulated with 300 ng/ml BMP-2 for 24 h or left untreated for control. Cell lysates were prepared using radioimmune precipitation buffer as described earlier (44.Ghosh-Choudhury N. Abboud S.L. Nishimura R. Celeste A. Mahimainathan L. Choudhury G.G. J. Biol. Chem. 2002; 277: 33361-33368Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Supernatants of cell lysates were immunoprecipitated with antibody against CBP, and the proteins were separated by SDS gel electrophoresis, as described earlier (40.Ghosh-Choudhury N. Abboud S.L. Mahimainathan L. Chandrasekar B. Choudhury G.G. J. Biol. Chem. 2003; 278: 21998-22005Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The immunoprecipitated proteins were then immunoblotted with antibody against Smad 1/Smad5 (Smad 1/5) (40.Ghosh-Choudhury N. Abboud S.L. Mahimainathan L. Chandrasekar B. Choudhury G.G. J. Biol. Chem. 2003; 278: 21998-22005Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 44.Ghosh-Choudhury N. Abboud S.L. Nishimura R. Celeste A. Mahimainathan L. Choudhury G.G. J. Biol. Chem. 2002; 277: 33361-33368Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Statistical Analysis of Data—To determine the significance of the data we have used analysis of variance following Student-Newman-Keuls comparison. Significance level was considered at a p value of <0.05. BMP-2 Stimulates Osteoclast Differentiation, CSF-1 mRNA Expression, and Protein Secretion—In vitro osteoclast differentiation requires coculturing hematopoietic cells and mouse osteoblast or stromal cells. BMP-2 has been shown to induce osteoblast phenotype in the pluripotent mesenchymal precursor cell line C2C12 (10.Katagiri T. Yamaguchi A. Komaki M. Abe E. Takahashi N. Ikeda T. Rosen V. Wozney J.M. Fujisawa-Sehara A. Suda T. J. Cell Biol. 1994; 127: 1755-1766Crossref PubMed Scopus (1295) Google Scholar). We tested whether C2C12 cells can support osteoclast formation in the presence of BMP-2 by coculturing C2C12 cells and mouse spleen cells in the presence of 1,25-dihydroxyvitamin D3 and dexamethasone with or without BMP-2. C2C12 cells treated with BMP-2 supported TRAP-positive multinucleated osteoclast formation, whereas only huge myotube formation was detected in the absence of BMP-2 (Fig. 1A). Osteoclast formation essentially depends on production of RANKL and CSF-1 by cocultured osteoblast cells. Recently BMP-2 has been shown to induce RANKL expression in C2C12 cells (43.Otsuka E. Notoya M. Hagiwara H. Calcif. Tissue Int. 2003; 73: 72-77Crossref PubMed Scopus (42) Google Scholar). In this study we tested the effect of BMP-2 on CSF-1 gene expression using C2C12 cells as our model system. We examined the effect of BMP-2 on CSF-1 expression. Incubation of C2C12 cells with BMP-2 increased expression of CSF-1 mRNA in a time-dependent manner (Fig. 1B). To confirm this observation, we tested CSF-1 protein abundance in the conditioned medium of C2C12 cells using an ELISA. Incubation of C2C12 cells with BMP-2 increased secretion of CSF-1 protein in the medium in a time-dependent manner (Fig. 1C). In complete accordance with the mRNA data, CSF-1 protein secretion was optimally increased by 24 h of BMP-2 treatment (3-fold) and remained elevated at 36 and 48 h following BMP-2 treatment. These data provide the first direct evidence that BMP-2 stimulates CSF-1 protein expression in osteoblasts. Identification of a BMP-responsive Region in CSF-1 Promoter—To understand the mechanism underlying CSF-1 gene regulation, we first used a CS