The Vitamin D Receptor, Runx2, and the Notch Signaling Pathway Cooperate in the Transcriptional Regulation of Osteopontin
2005; Elsevier BV; Volume: 280; Issue: 49 Linguagem: Inglês
10.1074/jbc.m504166200
ISSN1083-351X
Autores Tópico(s)Bone Metabolism and Diseases
ResumoOsteopontin (OPN), a glycosylated phosphoprotein that binds calcium, is present in bone extracellular matrix and has been reported to modulate both mineralization and bone resorption. Targeted disruption in mice of the vitamin D receptor (VDR) or Runx2 results in marked inhibition of OPN expression in osteoblasts. In this study, we addressed possible cross-talk between VDR and Runx2 in regulating OPN transcription. 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) or Runx2 stimulated OPN transcription (mouse OPN promoter -777/+79) 2–3-fold. However, coexpression of Runx2 and VDR in COS-7 cells and treatment with 1,25(OH)2D3 resulted in an 8-fold induction of OPN transcription, indicating for the first time functional cooperation between Runx2 and VDR in the regulation of OPN transcription. In ROS 17/2.8 and MC3T3-E1 cells that contain endogenous Runx2, AML-1/ETO, which acts as a repressor of Runx2, significantly inhibited 1,25(OH)2D3 induction of OPN transcription, OPN mRNA, and protein expression. Both a Runx2 site (-136/-130) and the vitamin D response element (-757/-743) in the OPN promoter are needed for cooperative activation. Chromatin immunoprecipitation analyses showed that 1,25(OH)2D3 can enhance VDR and Runx2 recruitment on the OPN promoter, further indicating cooperation between these two factors in the regulation of OPN. In osteoblastic cells, Hes-1, a downstream factor of the Notch signaling pathway, was found to enhance basal and 1,25(OH)2D3-induced OPN transcription. This enhancement was inhibited by AML-1/ETO, an inhibitor of Runx2. Immunoprecipitation assays indicated that Hes-1 and Runx2 interact and that 1,25(OH)2D3 can enhance this interaction. Taken together, these findings define novel mechanisms involving the intersection of three pathways, Runx2, 1,25(OH)2D3, and Notch signaling, that play a major role in the regulation of OPN in osteoblastic cells and therefore in the process of bone remodeling. Osteopontin (OPN), a glycosylated phosphoprotein that binds calcium, is present in bone extracellular matrix and has been reported to modulate both mineralization and bone resorption. Targeted disruption in mice of the vitamin D receptor (VDR) or Runx2 results in marked inhibition of OPN expression in osteoblasts. In this study, we addressed possible cross-talk between VDR and Runx2 in regulating OPN transcription. 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) or Runx2 stimulated OPN transcription (mouse OPN promoter -777/+79) 2–3-fold. However, coexpression of Runx2 and VDR in COS-7 cells and treatment with 1,25(OH)2D3 resulted in an 8-fold induction of OPN transcription, indicating for the first time functional cooperation between Runx2 and VDR in the regulation of OPN transcription. In ROS 17/2.8 and MC3T3-E1 cells that contain endogenous Runx2, AML-1/ETO, which acts as a repressor of Runx2, significantly inhibited 1,25(OH)2D3 induction of OPN transcription, OPN mRNA, and protein expression. Both a Runx2 site (-136/-130) and the vitamin D response element (-757/-743) in the OPN promoter are needed for cooperative activation. Chromatin immunoprecipitation analyses showed that 1,25(OH)2D3 can enhance VDR and Runx2 recruitment on the OPN promoter, further indicating cooperation between these two factors in the regulation of OPN. In osteoblastic cells, Hes-1, a downstream factor of the Notch signaling pathway, was found to enhance basal and 1,25(OH)2D3-induced OPN transcription. This enhancement was inhibited by AML-1/ETO, an inhibitor of Runx2. Immunoprecipitation assays indicated that Hes-1 and Runx2 interact and that 1,25(OH)2D3 can enhance this interaction. Taken together, these findings define novel mechanisms involving the intersection of three pathways, Runx2, 1,25(OH)2D3, and Notch signaling, that play a major role in the regulation of OPN in osteoblastic cells and therefore in the process of bone remodeling. Osteopontin (OPN) 2The abbreviations used are: OPNosteopontin1,25(OH)2D31,25-dihydroxyvitamin D3OCosteocalcinVDRvitamin D receptorFBSfetal bovine serumPBSphosphate-buffered salineChIPchromatin immunoprecipitationVDREvitamin D response elementhVDRhuman VDRWTwild typeBSPbone sialoproteinPipes1,4-piperazinediethanesulfonic acid.2The abbreviations used are: OPNosteopontin1,25(OH)2D31,25-dihydroxyvitamin D3OCosteocalcinVDRvitamin D receptorFBSfetal bovine serumPBSphosphate-buffered salineChIPchromatin immunoprecipitationVDREvitamin D response elementhVDRhuman VDRWTwild typeBSPbone sialoproteinPipes1,4-piperazinediethanesulfonic acid. is a sialic acid-rich glycosylated phosphoprotein, comprising about 2% of the noncollagenous protein in bone (1Denhardt D.T. Giachelli C.M. Rittling S.R. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 723-749Crossref PubMed Scopus (306) Google Scholar, 2McKee M.D. Nanci A. Connect. Tissue Res. 1996; 35: 197-205Crossref PubMed Scopus (144) Google Scholar). OPN is produced by osteoblasts when they form bone matrix (1Denhardt D.T. Giachelli C.M. Rittling S.R. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 723-749Crossref PubMed Scopus (306) Google Scholar, 2McKee M.D. Nanci A. Connect. Tissue Res. 1996; 35: 197-205Crossref PubMed Scopus (144) Google Scholar). OPN is an extracellular matrix protein that contains arginine-glycine-aspartate (RGD) integrin binding motifs and promotes attachment of bone cells to the bone surface through binding to OPN receptors such as the αvβ3 integrin and CD44 (1Denhardt D.T. Giachelli C.M. Rittling S.R. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 723-749Crossref PubMed Scopus (306) Google Scholar, 2McKee M.D. Nanci A. Connect. Tissue Res. 1996; 35: 197-205Crossref PubMed Scopus (144) Google Scholar, 3Chellaiah M.A. Kizer N. Biswas R. Alvarez U. Strauss-Schoenberger J. Rifas L. Rittling S.R. Denhardt D.T. Hruska K.A. Mol. Biol. Cell. 2003; 14: 173-189Crossref PubMed Scopus (187) Google Scholar). OPN has been suggested to be involved in the attachment of osteoclasts during bone resorption, to play a role in osteogenesis by attachment of osteoblasts when they form bone matrix, and to act to regulate crystal size during bone mineralization (2McKee M.D. Nanci A. Connect. Tissue Res. 1996; 35: 197-205Crossref PubMed Scopus (144) Google Scholar). In addition, OPN has been suggested to be a mediator of bone remodeling in response to mechanical strain (4Terai K. Takano-Yamamoto T. Ohba Y. Hiura K. Sugimoto M. Sato M. Kawahata H. Inaguma N. Kitamura Y. Nomura S. J. Bone Miner. Res. 1999; 14: 839-849Crossref PubMed Scopus (195) Google Scholar). OPN null mice are resistant to mineral loss and bone resorption upon estrogen deprivation and have impaired activation of osteoclasts (3Chellaiah M.A. Kizer N. Biswas R. Alvarez U. Strauss-Schoenberger J. Rifas L. Rittling S.R. Denhardt D.T. Hruska K.A. Mol. Biol. Cell. 2003; 14: 173-189Crossref PubMed Scopus (187) Google Scholar, 5Yoshitake H. Rittling S.R. Denhardt D.T. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8156-8160Crossref PubMed Scopus (312) Google Scholar, 6Ihara H. Denhardt D.T. Furuya K. Yamashita T. Muguruma Y. Tsuji K. Hruska K.A. Higashio K. Enomoto S. Nifuji A. Rittling S.R. Noda M. J. Biol. Chem. 2001; 276: 13065-13071Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 7Shapses S.A. Cifuentes M. Spevak L. Chowdhury H. Brittingham J. Boskey A.L. Denhardt D.T. Calcif. Tissue Int. 2003; 73: 86-92Crossref PubMed Scopus (62) Google Scholar). Also, vascularization and resorption of bone discs have been reported to be significantly impaired in the absence of OPN (8Asou Y. Rittling S.R. Yoshitake H. Tsuji K. Shinomiya K. Nifuji A. Denhardt D.T. Noda M. Endocrinology. 2001; 142: 1325-1332Crossref PubMed Scopus (140) Google Scholar). Although recent studies using OPN null mice have provided new insight into the role of OPN in vivo in bone metabolism, the factors that affect the regulation of OPN are not yet clearly defined. 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the active form of vitamin D, is a major calcitropic hormone involved in calcium homeostasis (9Christakos S. Bilezikian J.P. Raisz L.G. Rodan G.A. Principles of Bone Biology. Academic Press, San Diego, CA2002: 573-586Google Scholar). One of its functions in bone is to regulate the synthesis of the bone calcium-binding proteins osteocalcin (OC) and OPN (9Christakos S. Bilezikian J.P. Raisz L.G. Rodan G.A. Principles of Bone Biology. Academic Press, San Diego, CA2002: 573-586Google Scholar). 1,25(OH)2D3 modulates the expression of these genes through transcriptional regulation. The actions of 1,25(OH)2D3 are mediated through the vitamin D receptor (VDR). Liganded VDR heterodimerizes with the retinoid X receptor and interacts with a vitamin D response element (VDRE). The VDRE in the mouse OPN promoter (at -757/-743) is a perfect direct repeat of the motif GGTTCA spaced by three nucleotides (10Noda M. Vogel R.L. Craig A.M. Prahl J. DeLuca H.F. Denhardt D.T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9995-9999Crossref PubMed Scopus (434) Google Scholar). Transcription proceeds through the interaction of VDR with coactivators and coregulators, including SRC-1/NcoA1, SRC-2/GRIP-1 (GR-interacting protein)/NcoA2, SRC-3/ACTR, and the multisubunit DRIP (vitamin D receptor-interacting protein) complex (11Rachez C. Freedman L.P. Gene (Amst.). 2000; 246: 9-21Crossref PubMed Scopus (282) Google Scholar). Although a VDRE has been identified in the mouse OPN promoter (10Noda M. Vogel R.L. Craig A.M. Prahl J. DeLuca H.F. Denhardt D.T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9995-9999Crossref PubMed Scopus (434) Google Scholar) and VDR null mice show marked inhibition of OPN expression in osteoblasts (12Yoshizawa T. Handa Y. Uematsu Y. Takeda S. Sekine K. Yoshihara Y. Kawakami T. Arioka K. Sato H. Uchiyama Y. Masushige S. Fukamizu A. Matsumoto T. Kato S. Nat. Genet. 1997; 16: 391-396Crossref PubMed Scopus (965) Google Scholar), the exact mechanisms, including protein-protein and protein-DNA interactions, involved in 1,25(OH)2D3-regulated OPN transcription are not well understood.Runx2/Cbfa1 is a member of the runt/Cbfa family of transcription factors that was first identified as an osteoblast-specific transcription factor and a regulator of osteoblast differentiation (13Komori T. Yagi H. Nomura S. Yamaguchi A. Sasaki K. Deguchi K. Shimizu Y. Bronson R.T. Gao Y.H. Inada M. Sato M. Okamoto R. Kitamura Y. Yoshiki S. Kishimoto T. Cell. 1997; 89: 755-764Abstract Full Text Full Text PDF PubMed Scopus (3596) Google Scholar, 14Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3604) Google Scholar). Runx2 -/- mice die shortly after birth and show a complete lack of mineralized bone tissue (13Komori T. Yagi H. Nomura S. Yamaguchi A. Sasaki K. Deguchi K. Shimizu Y. Bronson R.T. Gao Y.H. Inada M. Sato M. Okamoto R. Kitamura Y. Yoshiki S. Kishimoto T. Cell. 1997; 89: 755-764Abstract Full Text Full Text PDF PubMed Scopus (3596) Google Scholar, 14Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3604) Google Scholar). Marked decreases in the expression of osteopontin and osteocalcin are observed in Runx2 -/- mice, indicating the regulation of these genes by Runx2 (13Komori T. Yagi H. Nomura S. Yamaguchi A. Sasaki K. Deguchi K. Shimizu Y. Bronson R.T. Gao Y.H. Inada M. Sato M. Okamoto R. Kitamura Y. Yoshiki S. Kishimoto T. Cell. 1997; 89: 755-764Abstract Full Text Full Text PDF PubMed Scopus (3596) Google Scholar). Three Runx2 binding motifs have been identified in the rat OC promoter (15Javed A. Gutierrez S. Montecino M. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. Mol. Cell. Biol. 1999; 19: 7491-7500Crossref PubMed Scopus (129) Google Scholar). In addition, Runx2 has been shown to play a key role in the 1,25(OH)2D3 regulation of rat OC (15Javed A. Gutierrez S. Montecino M. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. Mol. Cell. Biol. 1999; 19: 7491-7500Crossref PubMed Scopus (129) Google Scholar, 16Paredes R. Arriagada G. Cruzat F. Villagra A. Olate J. Zaidi K. van Wijnen A. Lian J.B. Stein G.S. Stein J.L. Montecino M. Mol. Cell. Biol. 2004; 24: 8847-8861Crossref PubMed Scopus (120) Google Scholar). However, it is not yet known whether a similar cooperation occurs between VDR and Runx2 in the regulation of OPN.Hes-1 (Hairy and enhancer of split homologue-1), a downstream target of the Notch signaling pathway, is a helix-loop-helix transcription factor that has been reported to play a role in developmental processes, including myogenesis and neurogenesis (17Sasai Y. Kageyama R. Tagawa Y. Shigemoto R. Nakanishi S. Genes Dev. 1992; 6: 2620-2634Crossref PubMed Scopus (576) Google Scholar). The expression of the Hes-1 gene is widely detected in embryos as well as adults (17Sasai Y. Kageyama R. Tagawa Y. Shigemoto R. Nakanishi S. Genes Dev. 1992; 6: 2620-2634Crossref PubMed Scopus (576) Google Scholar). Hes-1 is also expressed in osteoblastic cells (18Matsue M. Kageyama R. Denhardt D.T. Noda M. Bone. 1997; 20: 329-334Crossref PubMed Scopus (32) Google Scholar). Hes-1 is coexpressed with Runx2 in osteoblastic cells, and Runx2 and Hes-1 physically interact (19McLarren K.W. Theriault F.M. Stifani S. J. Biol. Chem. 2001; 276: 1578-1584Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 20McLarren K.W. Lo R. Grbavec D. Thirunavukkarasu K. Karsenty G. Stifani S. J. Biol. Chem. 2000; 275: 530-538Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). In addition, studies in Drosophila indicate that runt and hairy contribute to common transcriptional regulatory events (21Jimenez G. Pinchin S.M. Ish-Horowicz D. EMBO J. 1996; 15: 7088-7098Crossref PubMed Scopus (42) Google Scholar, 22Tsai C. Gergen P. Development. 1995; 121: 453-462Crossref PubMed Google Scholar). Due to the relationship between Hes-1 and Runx2 and the suggested role of Runx2 in OPN regulation (13Komori T. Yagi H. Nomura S. Yamaguchi A. Sasaki K. Deguchi K. Shimizu Y. Bronson R.T. Gao Y.H. Inada M. Sato M. Okamoto R. Kitamura Y. Yoshiki S. Kishimoto T. Cell. 1997; 89: 755-764Abstract Full Text Full Text PDF PubMed Scopus (3596) Google Scholar, 14Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3604) Google Scholar, 19McLarren K.W. Theriault F.M. Stifani S. J. Biol. Chem. 2001; 276: 1578-1584Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 20McLarren K.W. Lo R. Grbavec D. Thirunavukkarasu K. Karsenty G. Stifani S. J. Biol. Chem. 2000; 275: 530-538Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 23Sato M. Morii E. Komori T. Kawahata H. Sugimoto M. Terai K. Shimizu H. Yasui T. Ogihara H. Yasui N. Ochi T. Kitamura Y. Ito Y. Nomura S. Oncogene. 1998; 17: 1517-1525Crossref PubMed Scopus (238) Google Scholar), we tested the possibility that Hes-1 may cooperate with Runx2 in the regulation of OPN. Our findings define, for the first time, novel mechanisms involving the intersection of Runx2, 1,25(OH)2D3 and Notch signaling that are involved in the regulation of the OPN gene.EXPERIMENTAL PROCEDURESMaterials—[γ-32P]ATP (3,000 Ci (111 TBq)/mmol), nylon membrane, and the enhanced chemiluminescent detection system (ECL) were purchased from PerkinElmer Life Sciences. Dulbecco's modified Eagle's medium plus Ham's F-12 nutrient mixture, Dulbecco's modified Eagle's medium, fetal bovine serum (FBS), and PSN antibiotic mixture were purchased from Invitrogen. α-Minimal essential medium was purchased from Sigma. VDR antiserum (C-20), mouse OPN antiserum (P-18), and histone deacetylase-1 antiserum (H-51) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Runx2 antiserum was purchased from Oncogene Research Products (San Diego, CA). The antiserum reacting to Hes-1 (a gift from T. Sudo, Kamakura, Japan) was produced by immunizing rabbits with a fusion protein consisting of the C-terminal 19 amino acids (SPSSGSSLTSDSMWRPWRN) of mouse Hes-1 coupled to keyhole limpet hemocyanin. 1,25-Dihydroxyvitamin D3 was a generous gift from Dr. Milan Uskokovic (Hoffmann-LaRoche, Nutley, NJ).Cell Culture—COS-7 African green monkey kidney cells were obtained from the American Type Culture Collection (Manassas, VA) and were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS. ROS17/2.8 cells (a gift of S. Rodan and G. Rodan (Merck)) were maintained in Dulbecco's modified Eagle's medium/F-12 medium supplemented with 5% FBS, 1% PSN. MC3T3-E1 cells (Riken Cell Bank, Tsukuba, Japan) were cultured in α-minimal essential medium supplemented with 10% FBS, 1% PSN. All cells were cultured in a humidified atmosphere of 95% air, 5% CO2 at 37 °C. Cells were seeded at 70–80% confluence 24 h before experiments. Treatments with 1,25(OH)2D3 were performed in medium supplemented with 2% charcoal-stripped serum.Transient Transfection and Dual Luciferase Assay—The mouse osteopontin promoter (-777/+79) firefly luciferase reporter construct was kindly provided by D. Denhardt (Rutgers University, Piscataway, NJ). pCMV-Runx2 was a gift of G. Karsenty (Baylor College of Medicine, Houston, TX), and pCMV-AML-1/ETO expression vector was from S. W. Hiebert (Vanderbilt University School of Medicine, Nashville, TN). pcDNA3-Hes1 expression vector was a gift from Dr. S. Stifani (McGill University, Montreal, Canada). Cells were seeded in a 24-well culture dish 24 h prior to transfection at 70% confluence. Cells in each well were transfected using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. Empty vectors were used to keep the total DNA concentration the same. Efficiency of transfection, as assessed by green fluorescent protein cotransfection and subsequent visualization, was estimated at 60–70%. 1,25(OH)2D3 (10-8 m) or TSA (15 nm) was added to cells 24 h post-transfection for another 24 h. Cells were washed twice with phosphate-buffered saline (PBS) and harvested by incubating with 1× passive lysis buffer, supplied by the Dual-Luciferase reporter assay kit (Promega). The luciferase activity assay was performed according to the protocol of the manufacturer and normalized to values for pRL-TK-Renilla luciferase. For all transcription studies, OPN promoter activity (firefly/Renilla luciferase) is represented as -fold induction by comparison with basal levels (basal levels refer to levels of OPN promoter activity in cells transfected with vector alone and treated with vehicle).Site-directed Mutagenesis—Mutant mouse OPN promoter (-777/+79) luciferase reporter constructs were generated by site directed mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The oligonucleotides used to generate the Runx2 mutated site (shown in lowercase) were as follows: 5′-CCT TTT TTT TTT TTT AAg aAC AAA ACC AGA GGA GG-3′ (top strand) and 5′-CCT CCT CTG GTT TTG Ttc TTA AAA AAA AAA AAA GG-3′ (lower strand). The oligonucleotides used to generate the VDRE mutated site (shown in lowercase) were as follows: 5′-CAG AGC AAC AAG Gcc CAC GAG GTT CAC GTC-3′ (top strand) and 5′-GAC GTG AAC CTC GTG ggC CTT GTT GCT CTG-3′ (bottom strand).Northern Blot Analysis—ROS17/2.8 cells or MC3T3-E1 cells, plated at 70% confluence in 100-mm tissue culture dishes, were transfected using Lipofectamine 2000 reagent, with AML-1/ETO or Hes-1 expression vector or vector alone. 24 h after transfection, cells were treated for 24 h with 1,25(OH)2D3 (10-8 m) or vehicle control. The treated cells were then harvested by trypsinization, pelleted, and washed with PBS. Total RNA was isolated by RNA-bee RNA extraction solution (Tel-Test, Friendswood, TX) and precipitated by chloroform and isopropyl alcohol. 20 μg of total RNA from each sample was used for Northern blot analysis as previously described (24Barletta F. Freedman L.P. Christakos S. Mol. Endocrinol. 2002; 16: 301-314Crossref PubMed Scopus (61) Google Scholar). 32P-Labeled cDNA was prepared using the Random Primers DNA labeling system (Invitrogen) according to the random primer method (25Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocol in Molecular Biology. John Wiley & Sons, Inc., New York2005: 6.3.1-6.3.4Google Scholar). The mouse osteopontin cDNA was generated by HindIII digestion and was a gift from D. Denhardt (Rutgers University, Piscataway, NJ). The β-actin cDNA was purchased from Clontech. The blots were hybridized with the 32P-labeled mouse OPN cDNA probes for 16 h at 42 °C, washed, air-dried and exposed to Eastman Kodak Co. BIOMAX MR film at -80 °C for 1 day. The same blots were stripped and probed with 32P-labeled β-actin cDNA. Autoradiograms were analyzed by densitometric scanning using the Dual-Wavelength Flying Spot Scanner. The relative optical density obtained using the OPN probe was divided by the relative optical density obtained after probing with β-actin to normalize for sample variation.OPN Western Blot Analysis—MC3T3-E1 cells, plated at 70% confluence in 100-mm tissue culture dishes, were transfected with vector alone or pCMV-AML1/ETO and treated with vehicle or 1,25(OH)2D3 (10-8 m) for 24 h and harvested by trypsinization. For Western blot analysis, 50 μg of protein from total cell lysates was loaded onto a 10% SDS-polyacrylamide gel and separated by electrophoresis. Protein was transferred onto a polyvinylidene difluoride membrane (Bio-Rad). Membranes were incubated overnight at 4 °C with mouse OPN polyclonal antibody (P-18; Santa Cruz Biotechnology) at a 1:1000 dilution in PBS containing 5% nonfat milk. The membrane was washed with PBS and incubated for 1 h with the corresponding secondary antibody conjugated with horseradish peroxidase. The enhanced chemiluminescent Western blotting detection system (PerkinElmer Life Sciences) was used to detect the antigen-antibody complex.Chromatin Immunoprecipitation (ChIP) Assay—MC3T3-E1 cells were cultured in α-minimal essential medium supplemented with 10% FBS to 95% confluence prior to the experiment and then treated in α-minimal essential medium supplemented with 2% charcoal-stripped serum under the conditions and for the times indicated. Treated cells were used for the ChIP assay (26Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Scopus (1436) Google Scholar, 27Yamamoto H. Shevde N.K. Warrier A. Plum L.A. DeLuca H.F. Pike J.W. J. Biol. Chem. 2003; 278: 31756-31765Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Briefly, cells were first washed with PBS and subjected to a cross-link reaction with 1% formaldehyde for 15 min. The cross-link reaction was stopped by adding glycine to a final concentration of 0.125 m. Cells were washed with ice-cold PBS twice. The cells were collected by scraping and lysed sequentially in 5 mm Pipes, pH 8.0, 85 mm KCl, 0.5% Nonidet P-40 and then in 1% SDS, 10 mm EDTA, 50 mm Tris-HCl, pH 8.1, for 20 min individually. The chromatin pellets were sonicated to an average DNA size of 500 bp DNA (assessed by 1% agarose gel electrophoresis) using a Fisher model 100 sonic dismembranator at a power setting of 1. The sonicated extract was centrifuged for 10 min at maximum speed and then diluted into ChIP dilution buffer (16.7 mm Tris-HCl, pH 8.1, 150 mm NaCl, 0.01% SDS, 1.1% Triton X-100, 1.2 mm EDTA). Immunoprecipitations were performed at 4 °C overnight with the indicated antibody overnight. After a 1-h incubation with salmon sperm DNA and bovine serum albumin-pretreated Zysorbin (Zymed Laboratories Inc., San Francisco, CA), the precipitates were collected by centrifugation. Precipitates were washed sequentially in buffer I (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-HCl, pH 8.1, 150 mm NaCl), buffer II (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-HCl, pH 8.1, 500 mm NaCl), buffer III (0.25 m LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mm EDTA, 10 mm Tris-HCl, pH 8.1), and TE buffer (10 mm Tris, 1 mm EDTA) twice. The protein-DNA was then eluted by using 1% SDS and 0.1 m NaHCO3 for 15 min twice. Cross-links were reversed by incubating at 65 °C overnight in elution buffer with 0.2 m NaCl. DNA fragments were purified using Qiagen QIAquick PCR purification kits (Valencia, CA) and subjected to PCR using the primers designed to amplify fragments of murine osteopontin promoter VDRE motif (upper, 5′-ACC ACC TCT TCT GCT CTA TAT GGC-3′; lower, 5′-TGA CAC TTG AAC TAT GCA GCC GC-3′) and the primers designed to amplify the Runx2 motif (upper, 5′-TTC CGG GAT TCT AAA TGC AGT CTA-3′; lower, 5′-CTC CCA GAA TTT AAA TGC TGG TCC-3′). PCR analysis was carried out in the linear range of DNA amplification. PCR products were resolved in 5% TBE acrylamide gel and visualized using ethidium bromide staining. DNA acquired prior to precipitation was collected and used as the input. 10% of input was used for PCR evaluation.In re-ChIP experiments, complexes were eluted by incubation for 30 min at 37°C in 60 μl of elution buffer containing 10 mm dithiothreitol. The eluted samples were diluted 50 times with ChIP dilution buffer and subjected again to the ChIP procedure with specific antibodies.Nuclear Extracts—Cells were washed with cold PBS twice, harvested by scraping, pelleted by centrifuging at 4,000 rpm for 4 min. The pellets were washed and lysed in hypotonic buffer containing 10 mm HEPES (pH 7.4), 1.5 mm MgCl2, 10 mm KCl, 0.5 mm dithiothreitol, phosphatase inhibitors (0.5 mm phenylmethylsulfonyl fluoride, 1 mg/ml pepstatin A, 2 mg/ml leupeptin, 2 mg/ml aprotinin), and 1% Triton X-100. Nuclei were pelleted at 4,000 rpm for 4 min, and cytoplasmic supernatants were separated. Nuclei were resuspended in hypertonic buffer containing 0.42 mm NaCl, 0.2 mm EDTA, 25% glycerol, and the phosphatase and protease inhibitors indicated above. After a 2-h incubation at 4 °C, nuclear soluble proteins were collected by centrifuging at 13,000 rpm for 10 min. Protein concentration of the supernatant was measured by the method of Bradford (28Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213377) Google Scholar), and aliquots were stored at -80 °C.Immunoprecipitation—To examine the association of Runx2 and Hes-1 in the presence or absence of 1,25(OH)2D3 coimmunoprecipitation experiments were done. Nuclear extracts were prepared as indicated above from ROS17/2.8 cells or MC3T3-E1 cells, and protein concentration was detected by the Bradford method (28Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213377) Google Scholar). 500 μg of each preparation was used for immunoprecipitation with the addition of 4 μg of Hes-1 antiserum or 4 μg of Runx2 antiserum in the presence or absence of 1,25(OH)2D3 (10-8 m) for 24 h at 4 °C. Then 30 μl of protein A-Sepharose 4 Fast Flow Beads (Amersham Biosciences) were added to each sample, and, after further incubation by rotating at 4 °C for 1 h, the immunoprecipitated complex was collected by centrifuging at 3,000 rpm for 5 min. The complex was separated by 12% SDS-PAGE and probed with Runx2 antibody or Hes-1 antibody. Immunoprecipitation experiments were also done as described above using COS-7 cells transfected with VDR and treated with 1,25(OH)2D3 (10-8 m for 24 h) and cotransfected with vector alone (pCMV) or 2 μg of pCMV-Runx2 to examine the association of Hes-1 with histone deacetylase-1 in the presence or absence of Runx2. For these studies, 500 μg of nuclear extract was used for immunoprecipitation with the addition of 4 μg of histone deacetylase-1 antiserum followed by the addition of protein A-Sepharose 4 Fast Flow Beads incubated and collected by centrifugation as described above. The complex was separated by 12% SDS-PAGE and probed with Hes-1 antibody.Statistical Analysis—Results are expressed as the mean ± S.E., and significance was determined by analysis with Student's t test for two-group comparison or analysis of variance for multiple group comparison.RESULTSRunx2 Cooperates with VDR in Regulating OPN—Targeted disruption in mice of VDR or Runx2 results in a marked inhibition of OPN expression in osteoblasts (12Yoshizawa T. Handa Y. Uematsu Y. Takeda S. Sekine K. Yoshihara Y. Kawakami T. Arioka K. Sato H. Uchiyama Y. Masushige S. Fukamizu A. Matsumoto T. Kato S. Nat. Genet. 1997; 16: 391-396Crossref PubMed Scopus (965) Google Scholar, 13Komori T. Yagi H. Nomura S. Yamaguchi A. Sasaki K. Deguchi K. Shimizu Y. Bronson R.T. Gao Y.H. Inada M. Sato M. Okamoto R. Kitamura Y. Yoshiki S. Kishimoto T. Cell. 1997; 89: 755-764Abstract Full Text Full Text PDF PubMed Scopus (3596) Google Scholar). In order to address possible cross-talk between VDR and Runx2 in regulating OPN transcription, studies were done using COS-7 cells (that lack endogenous VDR and Runx2) transfected with the mouse OPN promoter (-777/+79; VDRE -757/-743) and hVDR and/or Runx2 expression vectors. In 1,25(OH)2D3-treated (10-8 m for 24 h) VDR-transfected COS-7 cells, OPN transcription was induced 3.0 ± 0.4-fold. OPN transcription was induced 2.4 ± 0.1-fold by cotransfection of Runx2 expression vector in the absence of 1,25(OH)2D3 (Fig. 1). Coexpression of Runx2 and VDR and treatment with 1,25(OH)2D3 (10-8 m 24 h) resulted in an 8.3 ± 0.8-fold induction of OPN transcription (Fig. 1), suggesting functional cooperation between Runx2 and VDR in the regulation of OPN.The chimeric protein AML-1/ETO can efficiently block Runx2-mediated transcriptional activation (29Meyers S. Lenny N. Hiebert S.W. Mol. Cell. Biol. 1995; 15: 1974-1982Crossref PubMed Scopus (349) Google Scholar). In COS-7 cells, the enhancement of the inductive action by 1,25(OH)2D3 and Runx2 was inhibited by AML-1/ETO in a dose-dependent manner (Fig. 2A). In ROS17/2.8 cells and MC3T3-E1 cells that contain endogenous Runx2, AML-1/ETO significantly diminished the 1,25(OH)2D3 induction of OPN transcription (Fig. 2, B and C), further indicating cooperation between Runx2 and VDR in the regulation
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