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BIBTEX RIS

Structural Coupling of Smad and Runx2 for Execution of the BMP2 Osteogenic Signal

2008; Elsevier BV; Volume: 283; Issue: 13 Linguagem: Inglês

10.1074/jbc.m705578200

ISSN

1083-351X

Autores

Amjad Javed, Jong-Sup Bae, Faiza Afzal, Soraya Gutiérrez, Jitesh Pratap, Sayyed K. Zaidi, Yang Lou, André J. van Wijnen, Janet L. Stein, Gary S. Stein, Jane B. Lian,

Tópico(s)

Bone health and treatments

Resumo

Two regulatory pathways, bone morphogenetic protein (BMP)/transforming growth factor-β (TGFβ) and the transcription factor RUNX2, are required for bone formation in vivo. Here we show the interdependent requirement of these pathways to induce an osteogenic program. A panel of Runx2 deletion and point mutants was used to examine RUNX2-SMAD protein-protein interaction and the biological consequences on BMP2-induced osteogenic signaling determined in Runx2 null cells. These cells do not respond to BMP2 signal in the absence of Runx2. We established that a triple mutation in the C-terminal domain of RUNX2, HTY (426-428), disrupts the RUNX2-SMAD interaction, is deficient in its ability to integrate the BMP2/TGFβ signal on promoter reporter assays, and is only marginally functional in promoting early stages of osteoblast differentiation. Furthermore, the HTY mutation overlaps the unique nuclear matrix targeting signal of Runx factors and exhibits reduced subnuclear targeting. Thus, formation of a RUNX2-SMAD osteogenic complex and subnuclear targeting are structurally and functionally inseparable. Our results establish the critical residues of RUNX2 for execution and completion of BMP2 signaling for osteoblastogenesis through a mechanism that requires RUNX2-SMAD transcriptional activity. Two regulatory pathways, bone morphogenetic protein (BMP)/transforming growth factor-β (TGFβ) and the transcription factor RUNX2, are required for bone formation in vivo. Here we show the interdependent requirement of these pathways to induce an osteogenic program. A panel of Runx2 deletion and point mutants was used to examine RUNX2-SMAD protein-protein interaction and the biological consequences on BMP2-induced osteogenic signaling determined in Runx2 null cells. These cells do not respond to BMP2 signal in the absence of Runx2. We established that a triple mutation in the C-terminal domain of RUNX2, HTY (426-428), disrupts the RUNX2-SMAD interaction, is deficient in its ability to integrate the BMP2/TGFβ signal on promoter reporter assays, and is only marginally functional in promoting early stages of osteoblast differentiation. Furthermore, the HTY mutation overlaps the unique nuclear matrix targeting signal of Runx factors and exhibits reduced subnuclear targeting. Thus, formation of a RUNX2-SMAD osteogenic complex and subnuclear targeting are structurally and functionally inseparable. Our results establish the critical residues of RUNX2 for execution and completion of BMP2 signaling for osteoblastogenesis through a mechanism that requires RUNX2-SMAD transcriptional activity. Skeletal development and bone formation require coordinated activities of multiple signaling pathways that include bone morphogenetic protein 2 (BMP2) 5The abbreviations used are:BMPbone morphogenetic proteinTGFβtransforming growth factor-βRunx2runt-related transcription factor 2RHDrunt homology domainSMIDSMAD interacting domainALPalkaline phosphataseNMTSnuclear matrix targeting signalαMEMα minimal essential mediumHAhemagglutininm.o.i.multiplicity of infectionWTwild type. and transforming growth factor-β (TGFβ). Transduction of these signals results in the activation of target genes that are essential for bone development. Specific receptor-regulated SMADs (R-SMADs) serve as substrates for the BMP and TGFβ/activin/Nodal receptors. SMAD-1, -2, -3, and -5 transduce, whereas Smad-4 serves as a common partner for all R-SMADs to provide the DNA binding property (1Liu F. Hata A. Baker J.C. Doody J. Carcamo J. Harland R.M. Massague J. Nature. 1996; 381: 620-623Crossref PubMed Scopus (591) Google Scholar, 2Massague J. Seoane J. Wotton D. Genes Dev. 2005; 19: 2783-2810Crossref PubMed Scopus (1939) Google Scholar). The structural and functional domains of SMAD proteins are well characterized with binding sites for SMAD ubiquitination-related factor (SMURF) ubiquitin ligases, and phosphorylation sites for several classes of protein kinases (3Massague J. Clin. Adv. Hematol. Oncol. 2003; 1: 576-577PubMed Google Scholar). The MH2 domain mediates interactions with transcriptional activators and repressors for signal transduction; including co-regulators of skeletal development (4Li X. Nie S. Chang C. Qiu T. Cao X. Exp. Cell Res. 2006; 312: 854-864Crossref PubMed Scopus (42) Google Scholar, 5Haag J. Aigner T. Arthritis Rheum. 2006; 54: 3878-3884Crossref PubMed Scopus (15) Google Scholar, 6Hendy G.N. Kaji H. Sowa H. Lebrun J.J. Canaff L. Horm. Metab Res. 2005; 37: 375-379Crossref PubMed Scopus (68) Google Scholar, 7Suzuki A. 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Gene Expr. 2004; 14: 1-41Crossref PubMed Google Scholar, 10Schroeder T.M. Jensen E.D. Westendorf J.J. Birth Defects Res. C. Embryo. Today. 2005; 75: 213-225Crossref PubMed Scopus (249) Google Scholar, 11Yang X. Karsenty G. Trends Mol. Med. 2002; 8: 340-345Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 12Franceschi R.T. Xiao G. J. Cell. Biochem. 2003; 88: 446-454Crossref PubMed Scopus (463) Google Scholar). Mutations in the human RUNX2 cause cleidocranial dysplasia (13Zhang Y.W. Yasui N. Ito K. Huang G. Fujii M. Hanai J. Nogami H. Ochi T. Miyazono K. Ito Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10549-10554Crossref PubMed Scopus (311) Google Scholar, 14Otto F. Kanegane H. Mundlos S. Hum. Mutat. 2002; 19: 209-216Crossref PubMed Scopus (236) Google Scholar). Targeted disruption of Runx2 in mice results in the maturational arrest of osteoblasts and a complete lack of mineralized bone (15Komori 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 (3678) Google Scholar, 16Otto F. Thornell A.P. Crompton T. Denzel A. Gilmour K.C. Rosewell I.R. Stamp G.W.H. Beddington R.S.P. Mundlos S. Olsen B.R. Selby P.B. Owen M.J. Cell. 1997; 89: 765-771Abstract Full Text Full Text PDF PubMed Scopus (2430) Google Scholar, 17Choi J.-Y. Pratap J. Javed A. Zaidi S.K. Xing L. Balint E. Dalamangas S. Boyce B. van Wijnen A.J. Lian J.B. Stein J.L. Jones S.N. Stein G.S. Proc. Natl. Acad. Sci., U. S. A. 2001; 98: 8650-8655Crossref PubMed Scopus (245) Google Scholar). The gene regulatory properties of RUNX factors are mediated not only by DNA binding to cognate elements, but also through the formation of selective co-regulatory protein interactions with co-activator and co-repressor proteins (9Lian J.B. Javed A. Zaidi S.K. Lengner C. Montecino M. van Wijnen A.J. Stein J.L. Stein G.S. Crit. Rev. Eukaryot. Gene Expr. 2004; 14: 1-41Crossref PubMed Google Scholar, 10Schroeder T.M. Jensen E.D. Westendorf J.J. Birth Defects Res. C. Embryo. Today. 2005; 75: 213-225Crossref PubMed Scopus (249) Google Scholar). The C terminus of RUNX proteins contains a 31-amino acid nuclear matrix targeting signal (NMTS), an essential well conserved functional domain, required to direct RUNX to distinct nuclear matrix-associated sites within the nucleus that support gene expression (18Tang L. Guo B. Javed A. Choi J.-Y. Hiebert S. Lian J.B. van Wijnen A.J. Stein J.L. Stein G.S. Zhou G.W. J. Biol. Chem. 1999; 274: 33580-33586Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 19Javed A. Guo B. Hiebert S. Choi J.-Y. Green J. Zhao S.-C. Osborne M.A. Stifani S. Stein J.L. Lian J.B. van Wijnen A.J. Stein G.S. J. Cell Sci. 2000; 113: 2221-2231Crossref PubMed Google Scholar, 20Zaidi S.K. Javed A. Pratap J. Schroeder T.M. Westendorf J. Lian J.B. van Wijnen A.J. Stein G.S. Stein J.L. J. Cell. Physiol. 2006; 209: 935-942Crossref PubMed Scopus (35) Google Scholar). The tight association of RUNX proteins with the nuclear matrix provides a platform for assembly of multicomponent regulatory complexes that control both activation and repression of genes during cell fate determination and differentiation (9Lian J.B. Javed A. Zaidi S.K. Lengner C. Montecino M. van Wijnen A.J. Stein J.L. Stein G.S. Crit. Rev. Eukaryot. Gene Expr. 2004; 14: 1-41Crossref PubMed Google Scholar, 21Young D.W. Hassan M.Q. Pratap J. Galindo M. Zaidi S.K. Lee S. Yang X. Xie R. Underwood J. Furcinitti P. Imbalzano A.N. Penman S. Nickerson J.A. Montecino M.A. Lian J.B. Stein J.L. van Wijnen A.J. Stein G.S. Nature. 2007; 445: 442-446Crossref PubMed Scopus (196) Google Scholar, 22Javed A. Barnes G.L. Jassanya B.O. Stein J.L. Gerstenfeld L. Lian J.B. Stein G.S. Mol. Cell. Biol. 2001; 21: 2891-2905Crossref PubMed Scopus (164) Google Scholar, 23Afzal F. Pratap J. Ito K. Ito Y. Stein J.L. van Wijnen A.J. Stein G.S. Lian J.B. Javed A. J. Cell. Physiol. 2005; 204: 63-72Crossref PubMed Scopus (127) Google Scholar). Among the co-regulatory proteins interacting with the RUNX2 C terminus and recruited to RUNX2 subnuclear domains are mediators of developmental signals, which include the TGFβ- and BMP-induced SMADs (13Zhang Y.W. Yasui N. Ito K. Huang G. Fujii M. Hanai J. Nogami H. Ochi T. Miyazono K. Ito Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10549-10554Crossref PubMed Scopus (311) Google Scholar, 24Zaidi S.K. Sullivan A.J. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. Proc. Natl. Acad. Sci., U. S. A. 2002; 99: 8048-8053Crossref PubMed Scopus (188) Google Scholar, 25Lee 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 (762) Google Scholar). The biological significance of the RUNX2 C terminus is revealed by a knock-in mutation in which mice lacking the C terminus fail to develop a mineralized skeleton (17Choi J.-Y. Pratap J. Javed A. Zaidi S.K. Xing L. Balint E. Dalamangas S. Boyce B. van Wijnen A.J. Lian J.B. Stein J.L. Jones S.N. Stein G.S. Proc. Natl. Acad. Sci., U. S. A. 2001; 98: 8650-8655Crossref PubMed Scopus (245) Google Scholar), confirming the importance of this region for in vivo osteogenesis. Particularly relevant to bone formation, early studies showed that BMP2-treated Runx2 null cells could not induce the complete osteoblast differentiation program (15Komori 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 (3678) Google Scholar, 26Young D.W. Pratap J. Javed A. Weiner B. Ohkawa Y. van Wijnen A. Montecino M. Stein G.S. Stein J.L. Imbalzano A.N. Lian J.B. J. Cell. Biochem. 2005; 94: 720-730Crossref PubMed Scopus (83) Google Scholar, 27Bae J.S. Gutierrez S. Narla R. Pratap J. Devados R. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. Javed A. J. Cell. Biochem. 2007; 100: 434-449Crossref PubMed Scopus (70) Google Scholar). RUNX2 has been shown to interact with SMAD proteins (13Zhang Y.W. Yasui N. Ito K. Huang G. Fujii M. Hanai J. Nogami H. Ochi T. Miyazono K. Ito Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10549-10554Crossref PubMed Scopus (311) Google Scholar, 28Miyazono K. Maeda S. Imamura T. Cytokine Growth Factor Rev. 2005; 16: 251-263Crossref PubMed Scopus (716) Google Scholar, 29Leboy P. Grasso-Knight G. D'Angelo M. Volk S.W. Lian J.V. Drissi H. Stein G.S. Adams S.L. J. Bone Joint Surg. Am. 2001; 83: S15-S22PubMed Google Scholar, 30Lee K.S. Hong S.H. Bae S.C. Oncogene. 2002; 21: 7156-7163Crossref PubMed Scopus (280) Google Scholar, 31Xiao G. Gopalkrishnan R.V. Jiang D. Reith E. Benson M.D. Franceschi R. J. Bone Miner. Res. 2002; 17: 101-110Crossref PubMed Scopus (410) Google Scholar, 32Phimphilai M. Zhao Z. Boules H. Roca H. Franceschi R.T. J. Bone Miner. Res. 2006; 21: 637-646Crossref PubMed Scopus (306) Google Scholar) and recruit SMADs to subnuclear sites of active transcription (24Zaidi S.K. Sullivan A.J. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. Proc. Natl. Acad. Sci., U. S. A. 2002; 99: 8048-8053Crossref PubMed Scopus (188) Google Scholar). Deletion mutant studies have identified a RUNX2-SMAD interacting domain (SMID) in the C terminus (23Afzal F. Pratap J. Ito K. Ito Y. Stein J.L. van Wijnen A.J. Stein G.S. Lian J.B. Javed A. J. Cell. Physiol. 2005; 204: 63-72Crossref PubMed Scopus (127) Google Scholar) that overlaps the NMTS, but definitive proof that RUNX2-SMAD interaction is essential for osteoblastogenesis has yet to be established. Only by disruption of the RUNX2-SMAD interaction in the context of the entire protein can the hypothesis that RUNX2 is required to mediate the BMP2 osteogenic signal, be tested. In these studies, by site-directed mutagenesis, we defined the specific RUNX2 amino acids required for physical and functional interaction with either BMP- or TGFβ-responsive SMADs. Three residues (HTY, 426-428) positioned in the carboxyl end of the RUNX2 SMID contribute to the osteogenic activity of RUNX2, formation of the RUNX2-SMAD complex, and integration of the BMP/TGFβ signal. The HTY mutant RUNX2 protein retains DNA binding and transcriptional activity but has impaired subnuclear targeting and no ability to bind SMADs, thus preventing transduction of a BMP2-mediated osteogenic function. These findings of minimal amino acid requirements for both RUNX2-SMAD interactions and the targeting of a RUNX2-SMAD functional complex to subnuclear domains provide compelling evidence for a structural coupling of SMADs with RUNX2 that is essential for execution and completion of BMP2 osteogenic signal. Cell Cultures—Human cervical carcinoma HeLa cells were cultured and maintained as previously described (23Afzal F. Pratap J. Ito K. Ito Y. Stein J.L. van Wijnen A.J. Stein G.S. Lian J.B. Javed A. J. Cell. Physiol. 2005; 204: 63-72Crossref PubMed Scopus (127) Google Scholar). Runx2 null cells were isolated from calvarial tissue of 17.5-day-old mouse embryos. Cells were maintained in modified Eagle's medium (αMEM) containing 10% fetal bovine serum, and penicillin G (100 units/ml), and streptomycin (100 μg/ml) at 37 °C in a humidified atmosphere of 5% CO2 in air. Cells were stably transfected with expression vector of mouse telomerase using FuGENE 6 regents (Roche Applied Science) and selected against G418 for 2 weeks. Detailed description and morphological characteristic of the established Runx2 null cell lines are reported elsewhere (27Bae J.S. Gutierrez S. Narla R. Pratap J. Devados R. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. Javed A. J. Cell. Biochem. 2007; 100: 434-449Crossref PubMed Scopus (70) Google Scholar). Plasmids and Adenoviral Constructs—The BMP2- and TGFβ-responsive FLAG-tagged receptor SMAD (SMAD2 and 3) constructs, and the 3×TBRE-Luciferase and 6×Runx-Luciferase plasmids are reported previously (23Afzal F. Pratap J. Ito K. Ito Y. Stein J.L. van Wijnen A.J. Stein G.S. Lian J.B. Javed A. J. Cell. Physiol. 2005; 204: 63-72Crossref PubMed Scopus (127) Google Scholar). Expression construct of hemagglutinin (HA)-tagged RUNX2, deletion Δ391, Y428A, and R398A&Y428A are described earlier (20Zaidi S.K. Javed A. Pratap J. Schroeder T.M. Westendorf J. Lian J.B. van Wijnen A.J. Stein G.S. Stein J.L. J. Cell. Physiol. 2006; 209: 935-942Crossref PubMed Scopus (35) Google Scholar, 23Afzal F. Pratap J. Ito K. Ito Y. Stein J.L. van Wijnen A.J. Stein G.S. Lian J.B. Javed A. J. Cell. Physiol. 2005; 204: 63-72Crossref PubMed Scopus (127) Google Scholar). HA-tagged RUNX2 point mutants H426A, T427A, HT426, 427AA, FTY405-407AAA, HTY426-428AAA, and FTYHTY405-407,426-428AAAAAA were generated with a two-step PCR approach. In the first step two independent but overlapping PCR products were generated using wild-type Runx2 cDNA as template. For PCR product 1, the common forward primer contained an ApaI site, 5′-GAACTGGGCCCTTTTTCAGACCCCAG-3′, whereas the mutant specific reverse primers were 5′-GTGGTGGCAGGTACGTGGCGTAGTGAGTG-3′ for H426A, 5′-GTGGTGGCAGGTACGCGTGGTAGTGAGTG-3′ for T427A, 5′-GTGGTGGCAGGTACGCGGCGTAGTGAGTG-3′, for HT426,427AA, 5′-GTGGTGGCAGTGCCGCGGCGTAGTGAGTG-3′ for HTY426-428AAA, and 5′-CTGGCGGGGTTGCTGCAGCGGTGGCTGGG-3′ for FTY405-407AAA. For PCR product 2, the mutant-specific forward primers were 5′-CACTCACTACGCCACGTACCTGCCACCAC-3′ for H426A, 5′-CACTCACTACCACGCGTACCTGCCACCAC-3′ for T427A, 5′-CACTCACTACGCCGCGTACCTGCCACCAC-3′ for HT426,427AA, 5′-CACTCACTACGCCGCGGCACTGCCACCAC-3′ for HTY426-428AAA, and 5′-CCCAGCCACCGCTGCAGCAACCCCGCCAG-3′ for FTY405-407AAA with the common reverse primer containing an XhoI site and stop codon, 5′-TTTCTCGAGTCAATATGGCCG-3′. Both PCR products for each mutant plasmid were purified and combined as template to generate a full-length PCR product containing the respective mutation. For the full-length PCR reactions the forward primer with ApaI site and the reverse primer with XhoI site are the same as above. The final products carrying the respective mutations were sequentially digested with ApaI/XhoI and ligated into the similarly digested HA-RUNX2 vector. A similar two step PCR approach was implied to generate RUNX2 expression plasmid carrying six alanine mutations (FTYHTY405-407,426-428AAAAAA). FTY405-407AAA plasmid was used as template, and the primer pairs are as described above for HTY426-428AAA. The presence of mutated sequences and the in-frame ligation of all the plasmids were confirmed by automated sequencing using an internal primer. Adenovirus expressing wild-type and C-terminal deletion mutant (Δ391) of RUNX2 are described previously (23Afzal F. Pratap J. Ito K. Ito Y. Stein J.L. van Wijnen A.J. Stein G.S. Lian J.B. Javed A. J. Cell. Physiol. 2005; 204: 63-72Crossref PubMed Scopus (127) Google Scholar). The Y428A and HTY426-428AAA mutant RUNX2 adenoviruses were generated by using the Adenovator system (Qbiogene, Carlsbad, CA). Briefly, HA-tagged mutant Runx2 cDNA were amplified by PCR using a common forward primer, 5′-CTTGGAAGATCTTTACCATGGATCTGTACGACGATGACGATAAG-3′, and a common reverse primer, 5′-CCTGAAAGATCTGCCCTCTAGATCAATATGG-3′, containing an engineered BglII restriction site. PCR products were digested with BglII enzyme and cloned into similarly digested pAdenoVator-CMV-IRES-GFP (Qbiogene). Presence of mutation and the integrity of the reading frame of the positive clone were confirmed by automated DNA sequencing. The expression and transcriptional activity of all pAdeno Vator constructs were examined in HeLa cells. The plasmids were then linearized with PmeI digestion and co-transformed into BJ5183 Escherichia coli cells along with the viral DNA plasmid pAdenoVator ΔE1/ΔE3. Recombinants were selected for kanamycin resistance and screened by restriction enzyme analysis. The recombinant adenoviral constructs were subsequently cleaved with PacI to expose its inverted terminal repeats and transfected into QBI-293A cells to produce viral particles. The virus particles were recovered from cells by three freeze/thaw cycles and further amplified. Fourth amplification containing high titer viral particles was purified by CsCl2 gradient and used for subsequent infections. The following multiplicity of infections (m.o.i.) was used for wild-type and point mutants of Runx2 adenovirus to maintain an equal level of expression and low cytotoxicity in Runx2 null cells (wild type, 50 m.o.i.; HTY, 60 m.o.i.; Y428A, 70 m.o.i.; and Δ391, 37 m.o.i.). Transient Transfection and Luciferase Assays—All transient transfections were performed using SuperFect transfection reagent (Qiagen Inc). For promoter reporter assays, HeLa cells plated in 6-well dishes were transfected at 40% confluency with varying concentrations (250-500 ng) of Runx2 expression vectors, 0.5 μg of receptor SMADs, 500 ng of the multimerized Runx (6xRunx) or 3xTBRE promoter fused with luciferase gene, and 100 ng of Renilla luciferase reporter construct. Briefly, DNA-lipid complexes were formed in serum-free Dulbecco's modified Eagle's medium by mixing the indicated DNA amount with 7 μl of SuperFect reagent and incubating for 15 min at room temperature. The complexes were diluted in 1 ml of complete Dulbecco's modified Eagle's medium and overlaid on pre-washed cells. After 3 h, DNA-lipid complexes were removed, washed once with 1× phosphate-buffered saline, fed with fresh medium, and cultured for an additional 18 h in the presence or absence of 100 ng/ml BMP2. For TGFβ-responsive Smads, treatment with TGFβ (5 ng/ml) was carried out 6 h prior to harvesting. Cells were lysed in reporter lysis buffer, and luciferase activity was determined using a Dual Luciferase Reporter Assay kit (Promega, Madison, WI). Results were obtained from at least three independent experiments with triplicate samples. Adenoviral Infections—Runx2 null cells were plated at a density of 0.3 × 106 cells per well of a 6-well plate. Cultures at 70% confluence were infected with the Runx2 (WT, Y428A, HTY, and Δ391) or β-galactosidase expressing control adenovirus in serum-free αMEM. After 90 min, cells were washed in αMEM without serum followed by addition of complete αMEM. Cells were cultured in the presence or absence of BMP2 (100 ng/ml) for an additional 12 days. Two days post infection cells were fed with osteogenic media (αMEM containing β-glycerophosphate and ascorbic acid). Media were changed every other day, and cells were subsequently harvested at indicated days for isolation of RNA or fixed in 2% formaldehyde in cacodylic buffer for ALP histology. RNA Isolation and Quantitative PCR Analysis—RNA was isolated from control and adenovirus-reconstituted Runx2 null cells at different days of culture in the presence and absence of BMP2. Cells were briefly washed with phosphate-buffered saline and lysed with TRIzol reagent (Invitrogen) to isolate total RNA according to the manufacturer's protocol. RNA was treated with DNase I to remove any DNA contamination and purified using a DNA-free RNA column kit (Zymo Research, Orange, CA). SuperScript first strand synthesis kit (Invitrogen) was used to reverse transcribed 1 μg of RNA. cDNA was then subjected to real-time PCR to measure relative transcript levels using SYBR Green Master Mix (Bio-Rad) and gene-specific primers. Specificity of primers was initially verified by dissociation/melting curve for the amplicons. Transcript levels were normalized to glyceraldehyde-3-phosphate dehydrogenase levels in respective samples. The primers used for amplification were reported previously (27Bae J.S. Gutierrez S. Narla R. Pratap J. Devados R. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. Javed A. J. Cell. Biochem. 2007; 100: 434-449Crossref PubMed Scopus (70) Google Scholar). ALP Histology—Cells infected with indicated adenoviruses and cultured in the presence or absence of BMP2 for 4, 8, and 12 days were washed twice in 0.1 m cacodylic buffer and subsequently fixed in 2% formaldehyde in cacodylic buffer for 10 min at room temperature. After washing with 0.1 m cacodylic buffer, the following reagents all purchased from Sigma-Aldrich were added for the histological stain: Napthol AS-Mx phosphate disodium salt (25 mg), N,N-dimethyl formamide (1.4 ml), 0.2 m Tris maleate buffer (25 ml), and Fast red salt (50 mg). The total volume was made up to 50 ml by the addition of distilled water, and the solution was filter-sterilized and immediately added to the wells. The plates were incubated at 37 °C for 30 min or until color development was observed. The wells were then rinsed with distilled water, and stained cells were visualized using an inverted microscope. Immunoprecipitation and Western Blots—HeLa cells were plated at a density of 0.7 × 106 cells per 100-mm plates. Cultures at 60% confluence were transfected with 5 μg each of RUNX2 (WT or mutant protein) and SMAD1 or -3 expression plasmids using SuperFect reagent. BMP2 (100 ng/ml) treatments were carried out 3 h post-transfection, whereas those for TGFβ (5 ng/ml) were carried out 6 h before harvesting. Cell were harvested 20-24 h following transfection in phosphate-buffered saline containing 25 μm proteasome inhibitor MG132 (Sigma-Aldrich) and Complete protease inhibitor mixture (Roche Applied Science). Cells were lysed by sonication at 10% for 10 s, and this process was repeated 5 times. Lysates clear of cellular debris were collected by centrifugation at 3000 rpm for 4 min, and 10% of the total lysate was saved as input. Remaining lysates were combined with 15 μg of species matched normal IgG, rabbit polyclonal Runx2 (M70, Santa Cruz Biotechnology) or mouse monoclonal FLAG (Sigma-Aldrich) antibody, and incubated on a rotating wheel for 1 h at 4 °C. Immunoprecipitates were collected with Protein A/G-agarose beads (Santa Cruz Biotechnology) and washed four times with phosphate-buffered saline containing 25 μm MG132 and protease inhibitor mixture. Total cell lysates or immunoprecipitates were fractionated by electrophoresis on a 12% SDS-polyacrylamide gel, transferred onto Immobilon P membranes (Millipore). Western blots were probed with monoclonal antibodies against Runx2 and FLAG and visualized with appropriate horseradish peroxidase-conjugated secondary antibody and chemiluminescence. Isolation of Nuclear Extracts and Electrophoretic Mobility Shift Assay—HeLa cells plated in 100-mm culture dishes were transfected with 5 μg of wild-type and mutant Runx2 expression plasmid and pcDNA3.1 as empty vector control. Cells were harvested 30 h later for isolation of nuclear extracts essentially as described previously (22Javed A. Barnes G.L. Jassanya B.O. Stein J.L. Gerstenfeld L. Lian J.B. Stein G.S. Mol. Cell. Biol. 2001; 21: 2891-2905Crossref PubMed Scopus (164) Google Scholar). Concentrations of nuclear protein were determined by Bradford assay, and vials were flash frozen and stored at -75 °C till required. Oligonucleotides representing a consensus Runx binding site was used for electrophoretic mobility shift assay and are reported earlier (22Javed A. Barnes G.L. Jassanya B.O. Stein J.L. Gerstenfeld L. Lian J.B. Stein G.S. Mol. Cell. Biol. 2001; 21: 2891-2905Crossref PubMed Scopus (164) Google Scholar). Preparation and radioactive labeling of the oligonucleotide probe is essentially as described previously (22Javed A. Barnes G.L. Jassanya B.O. Stein J.L. Gerstenfeld L. Lian J.B. Stein G.S. Mol. Cell. Biol. 2001; 21: 2891-2905Crossref PubMed Scopus (164) Google Scholar). Nuclear extracts (10 μg) from wild-type and mutated RUNX2 were incubated with labeled probe for 20 min at 22 °C and loaded onto a 4% nondenaturing polyacrylamide gel. Dried gels were exposed to film for autoradiography. In Situ Immunofluorescence—HeLa cells were plated at a density of 8 × 104 cells/well on gelatin-coated coverslips in 6-well culture dishes. Cells at 40% confluency were transfected with 1 μg each of Runx2 (WT or mutant), FLAG-tagged BMP2-responsive Smad1 and cultured for 18 h. BMP2 (100 ng/ml) was added 3 h post-transfection. Cells were processed for in situ immunofluorescence as whole cell preparations as described previously (19Javed A. Guo B. Hiebert S. Choi J.-Y. Green J. Zhao S.-C. Osborne M.A. Stifani S. Stein J.L. Lian J.B. van Wijnen A.J. Stein G.S. J. Cell Sci. 2000; 113: 2221-2231Crossref PubMed Google Scholar). RUNX2 was detected by a rabbit polyclonal antibody (Santa Cruz Biotechnology) and SMAD with a mouse monoclonal antibody against FLAG epitope (Sigma-Aldrich) at a dilution of 1:400 each. Secondary antibodies used were anti-rabbit Alexa 488 and anti-mouse Alexa 568 (Molecular Probes) at a dilution of 1:800. Images were captured using a Zeis Axioplan microscope interfaced with a charge-coupled device camera and analyzed with MetaMorph software (Universal Imaging Inc). For analysis of in situ co-localization, confocal microscopy was performed. A Leica TCS-SP inverted confocal microscope equipped with argon-helium and neon lasers with specific excitation lines was used (Bannockburn, IL). Ten randomly selected cells (five each from two independent cover slips) with equal expression levels of both proteins were used. Cell images with 0.25-μm section in respective wavelength were captured using similar exposure time. Two-dimensional progression was obtained, and numbers of Runx2 and SMAD foci per nucleus were counted using Leica Lite 2.0 and MetaMorph Imaging Software. The percent overlap was counted from total foci expressing WT and HTY mutant proteins. Average numbers of foci pooled from ten independent cells that exhibited colocalization are presented. Specific Residues within the SMID/NMTS Domain Define RUNX2-SMAD Interactions—Studies were initiated to test the hypothesis that a critical role of RUNX2 in inducing bone formation and in regulating the progression of osteoblast differentiation depends on its interaction with SMAD proteins in response to BMP2 and TGFβ signaling. We previously documented by C-terminal deletion studies the presence of a SMID in RUNX2, which overlaps the well characterized nuclear matrix targeting signal (NMTS) as illustrated in Fig. 1A. We first mutated the FTY and HTY residues that form the tips of loops 1 and 2 in the NMTS/SMID, respectively. These residues are conserved among other family members and species (18Tang L. Guo B. Javed A. Choi J.-Y. Hiebert S. Lian J.B. van Wijnen A.J. Stein J.L. Stein G.S. Zhou G.W. J. Biol. Chem. 1999; 274: 33580

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