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Brief Bone Morphogenetic Protein 2 Treatment of Glucocorticoid-inhibited MC3T3-E1 Osteoblasts Rescues Commitment-associated Cell Cycle and Mineralization without Alteration of Runx2

2003; Elsevier BV; Volume: 278; Issue: 45 Linguagem: Inglês

10.1074/jbc.m306730200

ISSN

1083-351X

Autores

Cynthia A. Luppen, Nathalie Leclerc, Tommy Noh, Artem Barski, Arvinder Khokhar, Adele L. Boskey, Elisheva Smith, Baruch Frenkel,

Tópico(s)

Bone and Dental Protein Studies

Resumo

Glucocorticoids (GCs) inhibit bone formation in vivo. In MC3T3-E1 osteoblasts, chronic administration of 1 μm dexamethasone (DEX) starting at confluency results in >98% inhibition of bone morphogenetic protein 2 (BMP-2) expression and apatite mineral deposition. Here, it is shown that brief exposure to recombinant human BMP-2 (rhBMP-2), as short as 6 h, is sufficient to induce irreversible commitment to mineralization in DEX-treated cultures. RhBMP-2 dose dependently rescued mineralization but not collagen accumulation in DEX-inhibited cultures. The selective restoration of mineralization was evident in the higher mineral to matrix ratios of DEX/rhBMP-2 co-treated cultures compared with control. We tested the involvement of the runt-related transcription factor 2 (Runx2) in the DEX inhibition and rhBMP-2 rescue of mineralization. Surprisingly, DEX did not decrease Runx2 DNA binding activity, transactivation, or association with the endogenous osteocalcin gene promoter. Furthermore, the rhBMP-2 rescue did not involve Runx2 stimulation, suggesting an important role for factors other than Runx2 in BMP-2 action. Finally, we studied the differentiation-related cell cycle, which persists during commitment to mineralization in untreated cultures, but is inhibited along with mineralization in DEX-treated cultures. Although both rhBMP-2 alone and DEX alone were antimitogenic, rhBMP-2 stimulated this cell cycle in DEX-inhibited cultures. In conclusion, brief rhBMP-2 treatment restores mineralization in DEX-inhibited MC3T3-E1 osteoblasts via a mechanism different from Runx2 stimulation. This restoration may be functionally related to the accompanying rescue of the differentiation-related cell cycle. The efficacy of short term BMP-2 treatment supports the potential of short-lived BMP vectors for skeletal therapy in both traditional and gene therapeutic approaches. Glucocorticoids (GCs) inhibit bone formation in vivo. In MC3T3-E1 osteoblasts, chronic administration of 1 μm dexamethasone (DEX) starting at confluency results in >98% inhibition of bone morphogenetic protein 2 (BMP-2) expression and apatite mineral deposition. Here, it is shown that brief exposure to recombinant human BMP-2 (rhBMP-2), as short as 6 h, is sufficient to induce irreversible commitment to mineralization in DEX-treated cultures. RhBMP-2 dose dependently rescued mineralization but not collagen accumulation in DEX-inhibited cultures. The selective restoration of mineralization was evident in the higher mineral to matrix ratios of DEX/rhBMP-2 co-treated cultures compared with control. We tested the involvement of the runt-related transcription factor 2 (Runx2) in the DEX inhibition and rhBMP-2 rescue of mineralization. Surprisingly, DEX did not decrease Runx2 DNA binding activity, transactivation, or association with the endogenous osteocalcin gene promoter. Furthermore, the rhBMP-2 rescue did not involve Runx2 stimulation, suggesting an important role for factors other than Runx2 in BMP-2 action. Finally, we studied the differentiation-related cell cycle, which persists during commitment to mineralization in untreated cultures, but is inhibited along with mineralization in DEX-treated cultures. Although both rhBMP-2 alone and DEX alone were antimitogenic, rhBMP-2 stimulated this cell cycle in DEX-inhibited cultures. In conclusion, brief rhBMP-2 treatment restores mineralization in DEX-inhibited MC3T3-E1 osteoblasts via a mechanism different from Runx2 stimulation. This restoration may be functionally related to the accompanying rescue of the differentiation-related cell cycle. The efficacy of short term BMP-2 treatment supports the potential of short-lived BMP vectors for skeletal therapy in both traditional and gene therapeutic approaches. Glucocorticoids (GCs) 1The abbreviations used are: GC, glucocorticoid; DEX, dexamethasone; BMP-2, bone morphogenetic protein 2; rhBMP-2, recombinant human bone morphogenetic protein 2; ChIP, chromatin immunoprecipitation; FTIR, Fourier transform infrared spectroscopy; ALP, alkaline phosphatase; EMSA, electrophoretic mobility shift assay.1The abbreviations used are: GC, glucocorticoid; DEX, dexamethasone; BMP-2, bone morphogenetic protein 2; rhBMP-2, recombinant human bone morphogenetic protein 2; ChIP, chromatin immunoprecipitation; FTIR, Fourier transform infrared spectroscopy; ALP, alkaline phosphatase; EMSA, electrophoretic mobility shift assay. are potent anti-inflammatory agents for the treatment of diseases such as rheumatoid arthritis, systemic lupus erythematosus, asthma, and some types of cancer. A major side effect of GC treatment is rapid bone loss and increased risk for fracture (1Van Staa T.P. Leufkens H.G. Abenhaim L. Zhang B. Cooper C. J. Bone Miner. 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Diverse cellular and molecular mechanisms contribute to GC-mediated inhibition of osteoblastic bone formation (reviewed in Refs. 3Canalis E. Delany A.M. Ann. N. Y. Acad. Sci. 2002; 966: 73-81Crossref PubMed Scopus (296) Google Scholar and 4Lukert B. Markus R. Feldman D. Kelsey J. Osteoporosis. Academic Press, San Diego1997: 801-820Google Scholar), including: (a) attenuation of osteoblast proliferation (7Chen T.L. Cone C.M. Feldman D. Endocrinology. 1983; 112: 1739-1745Crossref PubMed Scopus (61) Google Scholar, 8Chevalley T. Strong D.D. Mohan S. Baylink D. Linkhart T.A. Eur. J. Endocrinol. 1996; 134: 591-601Crossref PubMed Scopus (58) Google Scholar, 9Kamalia N. McCulloch C.A. Tenebaum H.C. Limeback H. Blood. 1992; 79: 320-326Crossref PubMed Google Scholar, 10Lian J.B. Shalhoub V. Aslam F. Frenkel B. Green J. Hamrah M. Stein G.S. Stein J.L. Endocrinology. 1997; 138: 2117-2127Crossref PubMed Google Scholar) and especially of a differentiation-related cell cycle (11Smith E. Redman R.A. 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Bone. 1996; 19: 1S-12SCrossref PubMed Scopus (399) Google Scholar, 16Luppen C.A. Smith E. Spevak L. Boskey A.L. Frenkel B. J. Bone Miner. Res. 2003; 18: 1186-1197Crossref PubMed Scopus (98) Google Scholar); and (e) inhibition of the osteoblast master transcription factor Runx2, also known as core-binding factor α1 and AML3 (17Chang D.J. Ji C. Kim K.K. Casinghino S. McCarthy T.L. Centrella M. J. Biol. Chem. 1998; 273: 4892-4896Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Investigation of the adverse effects of GCs on osteoblasts has been hampered by the positive actions of these same agents, which are primarily seen in vitro. In some isolated osteoblast cultures, the positive versus the negative effects occur at physiological versus pharmacological concentrations, respectively (18Ishida Y. Heersche J.N. J. Bone Miner. Res. 1998; 13: 1822-1826Crossref PubMed Scopus (140) Google Scholar). However, many investigators have observed positive effects in vitro even with pharmacological GC concentrations (19Tenenbaum H.C. Heersche J.N. Endocrinology. 1985; 117: 2211-2217Crossref PubMed Scopus (105) Google Scholar, 20Leboy P.S. Beresford J.N. Devlin C. Owen M.E. J. Cell. Physiol. 1991; 146: 370-378Crossref PubMed Scopus (294) Google Scholar, 21Shalhoub V. Conlon D. Tassinari M. Quinn C. Partridge N. Stein G.S. Lian J.B. J. Cell. Biochem. 1992; 50: 425-440Crossref PubMed Scopus (196) Google Scholar). The present study utilizes the MC3T3-E1 osteoblast culture system, under conditions that support differentiation, evidenced by the robust deposition of bone-like collagenous matrix with crystalline apatite (11Smith E. Redman R.A. Logg C.R. Coetzee G.A. Kasahara N. Frenkel B. J. Biol. Chem. 2000; 275: 19992-20001Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 16Luppen C.A. Smith E. Spevak L. Boskey A.L. Frenkel B. J. Bone Miner. Res. 2003; 18: 1186-1197Crossref PubMed Scopus (98) Google Scholar). Moreover, in these cultures, administration of the synthetic GC dexamethasone (DEX) at pharmacological concentrations of 0.1–1 μm strongly inhibits collagen accumulation and mineralization (11Smith E. Redman R.A. Logg C.R. Coetzee G.A. Kasahara N. Frenkel B. J. Biol. Chem. 2000; 275: 19992-20001Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 16Luppen C.A. Smith E. Spevak L. Boskey A.L. Frenkel B. J. Bone Miner. Res. 2003; 18: 1186-1197Crossref PubMed Scopus (98) Google Scholar). We have previously shown that the DEX inhibition of mineralization occurs only during a commitment stage that is characterized by a cobblestone culture morphology and a uniquely controlled cell cycle (12Smith E. Coetzee G.A. Frenkel B. J. Biol. Chem. 2002; 277: 18191-18197Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Additionally, DEX inhibits this commitment-associated cell cycle, possibly revealing a mechanism for the DEX inhibition of nodule formation and mineralization (11Smith E. Redman R.A. Logg C.R. Coetzee G.A. Kasahara N. Frenkel B. J. Biol. Chem. 2000; 275: 19992-20001Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 16Luppen C.A. Smith E. Spevak L. Boskey A.L. Frenkel B. J. Bone Miner. Res. 2003; 18: 1186-1197Crossref PubMed Scopus (98) Google Scholar). BMPs are potent promoters of osteoblast differentiation and bone formation (22Urist M.R. Science. 1965; 150: 893-899Crossref PubMed Scopus (4479) Google Scholar, 23Wozney J.M. Rosen V. Celeste A.J. Mitsock L.M. Whitters M.J. Kriz R.W. Hewick R.M. Wang E.A. Science. 1988; 242: 1528-1534Crossref PubMed Scopus (3345) Google Scholar, 24Wang E.A. Rosen V. Cordes P. Hewick R.M. Kriz M.J. Luxenberg D.P. Sibley B.S. Wozney J.M. Proc. Natl. Acad. Sci. U. S. 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Res. 2003; 18: 1186-1197Crossref PubMed Scopus (98) Google Scholar). This inhibition appears to play a pivotal role in the overall adverse effect of GCs because administration of exogenous rhBMP-2 along with DEX counteracted the inhibitory effect of DEX on mineralization (16Luppen C.A. Smith E. Spevak L. Boskey A.L. Frenkel B. J. Bone Miner. Res. 2003; 18: 1186-1197Crossref PubMed Scopus (98) Google Scholar). However, nodule formation, characteristic of osteoblast differentiation in vitro, was not rescued by rhBMP-2 in the DEX-inhibited cultures (16Luppen C.A. Smith E. Spevak L. Boskey A.L. Frenkel B. J. Bone Miner. Res. 2003; 18: 1186-1197Crossref PubMed Scopus (98) Google Scholar). Furthermore, although collagen mRNA was induced by rhBMP-2 in the DEX-treated cultures, the DEX inhibition of extracellular collagen fibril accumulation was not reversed by rhBMP-2 (16Luppen C.A. Smith E. Spevak L. Boskey A.L. Frenkel B. J. Bone Miner. Res. 2003; 18: 1186-1197Crossref PubMed Scopus (98) Google Scholar). The inhibitory effect of GCs on mineralization in MC3T3-E1 cultures is reversible (11Smith E. Redman R.A. Logg C.R. Coetzee G.A. Kasahara N. Frenkel B. J. Biol. Chem. 2000; 275: 19992-20001Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Therefore, we began the present study by asking if rhBMP-2 could rescue mineralization in DEX-treated cultures when administered after DEX. Also, because GCs cannot inhibit mineralization when administered after the commitment stage described above, we asked if brief rhBMP-2 exposure of chronically DEX-treated cultures could elicit a commitment process sufficient for mineralization. Furthermore, we asked if a rescue mediated by brief exposure to rhBMP-2 is associated with recapitulation of the commitment-associated cell cycle as observed in untreated cultures. Additionally, because GCs inhibit Runx2 in calvarial and bone marrow stromal osteoblast cultures (17Chang D.J. Ji C. Kim K.K. Casinghino S. McCarthy T.L. Centrella M. J. Biol. Chem. 1998; 273: 4892-4896Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 28Pereira R.C. Delany A.M. Canalis E. Bone. 2002; 30: 685-691Crossref PubMed Scopus (134) Google Scholar), and because BMPs induce Runx2 (29Gori F. Thomas T. Hicok K.C. Spelsberg T.C. Riggs B.L. J. Bone Miner. Res. 1999; 14: 1522-1535Crossref PubMed Scopus (262) Google Scholar, 30Lee K.S. Kim H.J. Li Q.L. Chi X.Z. Ueta C. Komori T. Wozney J.M. Kim E.G. Choi J.Y. Ryoo H.M. Bae S.C. Mol. Cell. Biol. 2000; 20: 8783-8792Crossref PubMed Scopus (758) Google Scholar), we tested the involvement of this osteoblast master transcription factor in the rhBMP-2 rescue. We report that brief exposure to rhBMP-2 indeed restores both a differentiation-related cell cycle and mineralization in GC-treated MC3T3-E1 cultures. Moreover, both the cell cycle and mineralization are rescued even when exposure to rhBMP-2 commences after DEX treatment has begun. Surprisingly, the rhBMP-2 rescue is not associated with increased Runx2 activity. Reagents—To maintain the MC3T3-E1 cell line, α-minimum essential medium and penicillin/streptomycin were obtained from Invitrogen Corp. (Carlsbad, CA). Individual lots of fetal bovine serum, also from Invitrogen, were selected based on their ability to support mineralization. The lot used in the current study was somewhat less osteogenic than that used in our recent study (16Luppen C.A. Smith E. Spevak L. Boskey A.L. Frenkel B. J. Bone Miner. Res. 2003; 18: 1186-1197Crossref PubMed Scopus (98) Google Scholar), resulting in delayed mineralization (day 14 versus day 10 in our recent study), as well as stimulation of the control cultures by rhBMP-2. Ascorbic acid, β-glycerophosphate, and dexamethasone were from Sigma. RhBMP-2 was generously provided by Wyeth Research (Cambridge, MA). Cell culture dishes were purchased from Corning Inc. (Corning, NY). The biochemical assays were conducted using Sigma diagnostics kit 587 for calcium, Sigma diagnostics kit 104-LL for alkaline phosphatase, diaminobenzoic acid for DNA (Sigma), MicroBCA (Pierce) for protein, and Sircol® assay kit (Biocolor Ltd., Newtonabbey, North Ireland) for collagen. The histological calcium assay was conducted with Alizarin Red (Sigma). Runx2 antibodies for electromobility supershift assays were from Oncogene Research (San Diego, CA). [γ-32P]ATP for probe labeling was from Amersham Biosciences. Runx2 antibodies (sc-10758X), preimmune rabbit IgG, and Protein A/G Plus-agarose beads for chromatin immunoprecipitation (ChIP) were from Santa Cruz Biotechnology (Santa Cruz, CA). The protein A/G-agarose beads were preblocked with 1 mg/ml salmon sperm DNA and 1 mg/ml bovine serum albumin prior to use in the ChIP assays. The luciferase assay system was purchased from Promega Corp. Luciferase constructs based on osteocalcin promoter sequences were generously provided by Dr. Gerard Karsenty (Baylor College of Medicine, Houston, TX). For cell cycle analysis, propidium iodide and ribonuclease A were obtained from Sigma. Cell Culture—A robustly mineralizing subclone of the MC3T3-E1 cell line that has been previously described was used in this study (11Smith E. Redman R.A. Logg C.R. Coetzee G.A. Kasahara N. Frenkel B. J. Biol. Chem. 2000; 275: 19992-20001Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Cells were plated at 30,000 cells/cm2 in 12-well plates for histological and biochemical assays, 6-well plates for transfection, and 100-mm plates for cell cycle, Fourier transform infrared spectroscopy (FTIR), Northern, reverse transcriptase-PCR, electromobility shift and chromatin immunoprecipitation experiments. Cells were maintained in α-minimum essential medium supplemented with 10% fetal bovine serum and 1.5% penicillin/streptomycin. Starting at 80% confluency (typically day 3, after plating on day 0), the culture medium was supplemented with 50 μg/ml ascorbic acid and 10 mm β-glycerophosphate. In some experiments that are not shown, calcium deposition was measured in DEX-treated cultures after brief or chronic exposure to rhBMP-2, all in the presence of 5 mm instead of 10 mm β-glycerophosphate, and the results were essentially unchanged. Histological Demonstration of Calcium Deposition—Culture wells were washed once in phosphate-buffered saline and fixed for 1 h at 4 °C in 70% ethyl alcohol. Calcium deposits were stained for 10 min at room temperature with Alizarin Red solution (40 mm, pH 4.2) that was filtered through Whatman paper prior to application. Nonspecific staining was removed by several washes in water. Biochemical Assays—Extracts for biochemical assays were collected by scraping the contents of each well in 10 mm Tris saline buffer (pH 7.2) containing 0.2% Triton X-100. The extracts were tested for alkaline phosphatase activity using p-nitrophenyl phosphate as substrate. To measure calcium and DNA, aliquots of the extracts were acid-hydrolyzed (final concentration 0.5 n HCl) prior to processing. The spectrophotometric analyses of the protein (562 nm), alkaline phosphatase (ALP) (410 nm), and calcium (575 nm) assays were conducted in 96-well plates using a PowerWaveX microplate scanning spectrophotometer (Biotek Instruments, Winooski, VT). The fluorometric DNA assay was conducted using diaminobenzoic acid essentially according to a published protocol, 2P. G. Held (2001) Lab Division Applications Detail, www.biotek.com. using excitation wavelength 400 ± 15 nm and emission wavelength 485 ± 10 nm on a FLx800 fluorescence microplate reader (Biotek Instruments). For collagen accumulation, cell layers were scraped in PBS, centrifuged, and hydrolyzed in 0.5 m acetic acid for 18 h at 4° C. The acid extracts were reacted with Sircol® reagent, and the collagen-bound dye was quantitated according to the manufacturer's protocol. FTIR—Extracellular matrix composition was analyzed by FTIR. Cell layers were collected in ammoniated water (50 mm ammonium bicarbonate, pH 8.0), lyophilized, and analyzed as potassium bromide (KBr) pellets on a Bio-Rad FTS 40-A spectrometer (Bio-Rad). The spectral data were baseline corrected and analyzed using GRAMS/386 software (Galactic Industries, Salem, NH) as previously described (32Kato Y. Boskey A. Spevak L. Dallas M. Hori M. Bonewald L.F. J. Bone Miner. Res. 2001; 16: 1622-1633Crossref PubMed Scopus (180) Google Scholar). The mineral to matrix ratio was derived from the areas of phosphate (900 to 1200 cm-1) and protein amide I (1585 to 1720 cm-1) absorbance. Crystallinity, or crystal maturity, was evaluated based on the intensity ratios of the 1030:1020 sub-bands of the phosphate ν1,ν3 absorbance spectrum (33Boskey A.L. Spevak L. Paschalis E. Doty S.B. McKee M.D. Calcif. Tissue Int. 2002; 71: 145-154Crossref PubMed Scopus (244) Google Scholar). The presence of crystalline apatite was verified by a split phosphate ν2 band (500 to 635 cm-1). RNA Analyses—Total cellular RNA was isolated using TRIzol reagent (Invitrogen) and osteocalcin gene expression was evaluated as previously described (16Luppen C.A. Smith E. Spevak L. Boskey A.L. Frenkel B. J. Bone Miner. Res. 2003; 18: 1186-1197Crossref PubMed Scopus (98) Google Scholar). For Northern analysis, 15 μg of total RNA was separated in a 1% agarose-formaldehyde gel, transferred onto Hybond-N+ membrane (Amersham Biosciences), and hybridized with a 300-bp mouse osteocalcin probe excised from pBGP with BamHI and EcoRI. Prehybridization and hybridization were performed in 50 mm sodium phosphate buffer (pH 7.4) containing 50% formamide, 5× SSC, 10× Denhardt's solution, 1% SDS, and 0.1 mg/ml denatured salmon sperm DNA. The blots were washed extensively in buffer containing 2× SSC and 0.1% SDS at room temperature and then at 50 °C and exposed to film overnight. For reverse transcriptase-PCR, single-stranded cDNA was synthesized from the Trizol-extracted, DNase-treated RNA. A 187-bp osteocalcin cDNA fragment was PCR amplified as described previously under conditions providing close to linear signal as a function of input (16Luppen C.A. Smith E. Spevak L. Boskey A.L. Frenkel B. J. Bone Miner. Res. 2003; 18: 1186-1197Crossref PubMed Scopus (98) Google Scholar). Runx2 Electromobility Shift Assay (EMSA)—Cultures for whole cell extract preparation were washed with phosphate-buffered saline, and the cell layers were scraped and centrifuged at 3,000 rpm for 5 min at 4 °C. Cell pellets were resuspended in 1.5 packed cell volumes of lysis buffer (100 mm Hepes, pH 7.5, 500 mm KCl, 5 mm MgCl2, 0.5 mm EDTA, 28% glycerol) containing protease and phosphatase inhibitors (5 mm NaF, 0.1 mm Na3VO4, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and 20 μm MG132). The cells were further subjected to successive passes through 18.5-, 20.5-, and then 23-gauge needles, followed by centrifugation at 14,000 rpm for 30 min at 4 °C to remove cell debris. The supernatant was snap frozen and stored at -80 °C. EMSA was performed with 15 μg of whole cell extract and 80 fmol of an end-labeled 23-base pair oligonucleotide probe containing the Runx2-binding OSE2 site from the mouse osteocalcin gene 2 (OG2) promoter (34Ducy P. Karsenty G. Mol. Cell. Biol. 1995; 15: 1858-1869Crossref PubMed Scopus (521) Google Scholar). The cell extract was initially preincubated on ice for 10 min in the presence of 100 mm KCl with 1 μg of salmon sperm DNA and, when indicated, unlabeled oligonucleotides or antibodies. Probe binding reaction (final volume: 20 μl) was then performed in 20 mm Hepes buffer (pH 7.5) containing (final concentrations) 50 mm KCl, 1 mm MgCl2, 2 mm EDTA, and 2.8% glycerol for 10 min on ice followed by 15 min at room temperature. The protein-DNA complexes were then resolved in a 0.25× TBE native polyacrylamide (5%) gel containing 5% glycerol. Transient Transfection and Luciferase Assay—MC3T3-E1 cells were plated as above (day 0) and transiently transfected on day 1 using calcium phosphate co-precipitation as described previously (11Smith E. Redman R.A. Logg C.R. Coetzee G.A. Kasahara N. Frenkel B. J. Biol. Chem. 2000; 275: 19992-20001Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). DEX (1 μm) and/or rhBMP-2 (100 ng/ml) treatment commenced as cultures became confluent on day 4 and lasted 48 h. Cells were then lysed and luciferase activity was determined using an MLX microtiter plate luminometer (Dynex Technologies, Chantilly, VA). ChIP—ChIP was performed essentially as previously described (35Boyd K.E. Wells J. Gutman J. Bartley S.M. Farnham P.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13887-13892Crossref PubMed Scopus (246) Google Scholar). Cross-linking was performed using 1% formaldehyde (10 min, 25 °C) and was stopped by adding glycine to a final concentration of 0.125 m. Cells were swelled in hypotonic buffer: 10 mm HEPES, 10 mm KCl, 1.5 mm MgCl2, and protease inhibitor mixture (Complete Mini, Roche Applied Science). Nuclei were lysed in 50 mm Tris-HCl buffer (pH 8.1) containing 1% SDS, 10 mm EDTA, and protease inhibitors. Resulting chromatin solution was sonicated using a Virsonic 60 sonicator (VirTis Co., Gardiner, NY; 4 pulses at 4 watts, 10 s each) and centrifuged at 16,000 × g for 10 min to remove cell debris. At this point, samples were diluted to adjust the absorbance to 0.25 A 260 units and 100 μl was further diluted 10 times with IP buffer (16.7 mm Tris-HCl, pH 8.1, 0.01% SDS, 1.1% Triton X-100, 1.2 mm EDTA, 167 mm NaCl) and protease inhibitors. Preclearing was performed with 2 μg of preimmune rabbit IgG and 100 μl of protein A/G-agarose beads. Following overnight incubation with 10 μg of Runx2 antibodies, complexes were precipitated with 30 μl of protein A/G-agarose and the beads were sequentially washed with IP buffer, high salt buffer (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-HCl, pH 8.1, 500 mm NaCl), Sarcosyl buffer (0.2% Sarcosyl, 2 mm EDTA, 50 mm Tris-HCl, pH 8.1), and TE. Complexes were eluted twice with 250 μl of elution buffer (1% SDS, 0.1 m NaHCO3) and cross-links were reversed by incubation at 65 °C for 4 h. The DNA was then deproteinized and purified on QIAquick PCR purification column (Qiagen, Valencia, CA). The osteocalcin -264/-7 promoter fragment was amplified with the primers 5′-gagagcacacagtaggagtggtggag and 5′-tccagcatccagtagcatttatatcg. The insulin -246/-4 promoter fragment was amplified as a negative control with the primers 5′-tggatgcccaccagctttatagtcc and 5′-aactggttcatcaggccatctggtc. PCR were performed within a close to linear range under conditions that yield comparable signals for osteocalcin and insulin when genomic DNA is used as template. Cell Cycle Analysis—Cell cycle profiles were determined as previously described (11Smith E. Redman R.A. Logg C.R. Coetzee G.A. Kasahara N. Frenkel B. J. Biol. Chem. 2000; 275: 19992-20001Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Briefly, cells were trypsinized and collected by centrifugation (4000 rpm, 4 °C, 15 min) in phosphate-buffered saline. Following centrifugation, the cells were resuspended in 100% ethyl alcohol and kept at -20 °C. The ethyl alcohol was removed by centrifugation (4000 rpm, 4 °C, 15 min) and the cells were resuspended in Hanks' balanced buffer solution containing 20 μg/ml propidium iodide and 100 μg/ml DNase-free ribonuclease A. Following 30 min incubation at room temperature, the stained cells were analyzed by flow cytometry using the EPICS™ XL-MCL analyzer (Beckman Coulter, Fullerton, CA) and the percentages of cells in the G1, S, and G2/M phases of the cell cycle were determined using MultiCycle analysis software (Phoenix Flow Systems, San Diego, CA). Statistical Analysis—Results from each quantitative assay were analyzed to test four effects: (i) the effect of DEX in the absence of BMP-2; (ii) the effect of BMP-2 in the absence of DEX; (iii) the effect of BMP-2 in the presence of DEX and (iv) the effect of chronic versus brief rhBMP-2 treatment for each condition. Mean ± S.D. were compared using the Student's t test and the differences were considered significant when p ≤ 0.05. Inhibition of Mineralization by Chronic DEX Treatment Is Corrected by Chronic BMP-2 Treatment Commencing 48 h after DEX—We have previously shown that rhBMP-2 counteracts GC-mediated inhibition of mineralization in MC3T3-E1 osteoblast cultures (16Luppen C.A. Smith E. Spevak L. Boskey A.L. Frenkel B. J. Bone Miner. Res. 2003; 18: 1186-1197Crossref PubMed Scopus (98) Google Scholar). In that study the negative regulator, DEX, and the positive regulator, rhBMP-2, were administered together commencing on day 3. The ability of rhBMP-2 to rescue mineralization in cultures that have already been exposed to DEX for a substantial period of time was not tested. Therefore, in the current study, MC3T3-E1 cells were treated with DEX starting on day 3 and rhBMP-2 was added 48 h later (day 5). DEX and rhBMP-2 treatments continued throughout the rest of the experiment. Analysis of calcium deposition on day 14, by either Alizarin Red staining (Fig. 1A) or biochemical assay (Fig. 1B), showed that 1 μm DEX completely inhibited mineralization while rhBMP-2, at either 10 or 100 ng/ml, dramatically counteracted the DEX inhibition. A time course experiment (Fig. 1B) revealed that the rhBMP-2-rescued mineralization was even accelerated as compared with that of control cultures. Furthermore, FTIR analysis of day 14 cultures showed that the crystallinity of the rescued mineral was comparable with that of control; the intensity ratio between the 1030 and 1020 phosphate sub-bands ranged between 1.057 and 1.068 in the control cultures and between 1.051 and 1.088 in the cultures rescued with ei

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