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Cbfa1/RUNX2 Directs Specific Expression of the Sclerosteosis Gene (SOST)

2004; Elsevier BV; Volume: 279; Issue: 14 Linguagem: Inglês

10.1074/jbc.m306249200

1083-351X

Brad Sevetson, Scott L. Taylor, Yang Pan,

Bone health and osteoporosis research

Loss-of-function mutations in the sclerosteosis gene (SOST) cause a rare sclerosing bone dysplasia characterized by skeletal overgrowth. Cbfa1/RUNX2 is a key transcriptional regulator of osteoblast function. Here we link these two pathways by demonstrating, via gel shift and transient transfection analyses, that Cbfa1 binding to the proximal SOST promoter contributes to differential SOST expression in two osteosarcoma cell lines. Additionally, an E-box binding motif in the 1.8-kb proximal SOST promoter appears to be functional in SAOS-2 cells, but does not account for SAOS-specific expression of SOST. The regulation of SOST expression by Cbfa1 suggests a potential role for the sclerosteosis gene in homeostatic regulation of osteoblast differentiation and function. Furthermore, the juxtaposition of Cbfa1, E-box, and C/EBP binding sites in the SOST proximal promoter bears an intriguing resemblance to the promoter for osteocalcin, another osteoblast-specific gene with a loss-of-function phenotype of bone overgrowth. Loss-of-function mutations in the sclerosteosis gene (SOST) cause a rare sclerosing bone dysplasia characterized by skeletal overgrowth. Cbfa1/RUNX2 is a key transcriptional regulator of osteoblast function. Here we link these two pathways by demonstrating, via gel shift and transient transfection analyses, that Cbfa1 binding to the proximal SOST promoter contributes to differential SOST expression in two osteosarcoma cell lines. Additionally, an E-box binding motif in the 1.8-kb proximal SOST promoter appears to be functional in SAOS-2 cells, but does not account for SAOS-specific expression of SOST. The regulation of SOST expression by Cbfa1 suggests a potential role for the sclerosteosis gene in homeostatic regulation of osteoblast differentiation and function. Furthermore, the juxtaposition of Cbfa1, E-box, and C/EBP binding sites in the SOST proximal promoter bears an intriguing resemblance to the promoter for osteocalcin, another osteoblast-specific gene with a loss-of-function phenotype of bone overgrowth. Sclerosteosis is a rare, progressive disorder characterized by general skeletal overgrowth (1Truswell A.S. J. Bone Joint Surg. 1958; 40: 208-218Google Scholar). Symptoms of sclerosteosis include gigantism, entrapment of cranial nerves, increased intracranial pressure due to widening of the calvarium of the skull, and increased thickness and density of both trabecular and cortical bone (2Beighton P. J. Med. Genet. 1988; 25: 200-203Google Scholar, 3Epstein S. Hamersma H. Beighton P. S. Afr. Med. J. 1979; 55: 1105-1110Google Scholar, 4Stein S. Witkop c. Hill S. Fallon M. Viernstein L. Gucer G. McKeever P. Long D. Altman J. Miller N.R. Teitelbaum S.L. Schlesinger S. Neurology. 1983; 33: 267-277Google Scholar). The disease is inherited in an autosomal recessive manner (5Beighton P. Davidson J. Durr L. Hamersma H. Clin. Genet. 1977; 11: 1-7Google Scholar), and has been mapped to null mutations in sclerostin, or SOST (6Balemans W. Ebeling M. Patel N. Van Hul E. Olson P. Dioszegi M. Lacza C. Wuyts W. Van Den Ende J. Willems P. Paes-Alves A.F. Hill S. Bueno M. Ramos F.J. Tacconi P. Dikkers F.G. Stratakis C. Lindpaintner K. Vickery B. Foernzler D. Van Hul W. Hum. Mol. Genet. 2001; 10: 537-543Google Scholar, 7Brunkow M.E. Gardner J.C. Van Ness J. Paeper B.W. Kovacevich B.R. Proll S. Skonier J.E. Zhao L. Sabo P.J. Fu Y. Alisch R.S. Gillett L. Colbert T. Tacconi P. Galas D. Hamersma H. Beighton P. Mulligan J. Am. J. Hum. Genet. 2001; 68: 577-589Google Scholar). The SOST gene is expressed in osteoblast cells and encodes a secreted 213 amino acid polypeptide with homology to the DAN family. Because DAN family proteins are secreted TGF-β 1The abbreviations used are: TGF-β, transforming growth factor β; CCD, cleidocranial dysplasia; CSE, conserved sequence element; BMP, bone morphogenetic protein; EMSA, electrophoretic mobility shift assay. antagonists (8Pearce J.J. Penny G. Rossant J. Dev. Biol. 1999; 209: 98-110Google Scholar, 9Hsu D.R. Economides A.N. Wang X. Eimon P.M. Harland R.M. Mol. Cell. 1998; 1: 673-683Google Scholar), SOST may repress bone growth by antagonizing TGF-β or BMP function. Cbfa1/RUNX2 is a sequence-specific DNA-binding protein whose consensus element RACCRCW (10Javed A. Gutierrez S. Montecino M. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. Mol. Cell. Biol. 1999; 19: 7491-7500Google Scholar) is found in the promoters of a variety of genes related to osteoblast differentiation or function, including osteocalcin, osteopontin, bone sialoprotein, and type I collagen (11Merriman H.L. van Wijnen A.J. Hiebert S. Bidwell J.P. Fey E. Lian J. Stein J. Stein G.S. Biochemistry. 1995; 34: 13125-13132Google Scholar, 12Geoffroy V. Ducy P. Karsenty G. J. Biol. Chem. 1995; 270: 30973-30979Google Scholar, 13Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Google Scholar, 14Ducy P. Karsenty G. Mol. Cell. Biol. 1995; 15: 1858-1869Google Scholar, 15Banerjee C. Hiebert S.W. Stein J.L. Lian J.B. Stein G.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4968-4973Google Scholar, 16Harada H. Tagashira S. Fujiwara M. Ogawa S. Katsumata T. Yamaguchi A. Komori T. Nakatsuka M. J. Biol. Chem. 1999; 274: 6972-6978Google Scholar). Homozygous deletion of mouse Cbfa1 leads to a complete absence of bone formation due to an arrest in osteoblast maturation that prevents endochondral ossification (17Otto F. Thornell A.P. Crompton T. Denzel A. Gilmour K.C. Rosewell I.R. Stamp G.W. Beddington R.S. Mundlos S. Olsen B.R. Selby P.B. Owen M.J. Cell. 1997; 89: 765-771Google Scholar, 18Komori 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: 755-764Google Scholar). Mice heterozygous for Cbfa1 develop bone abnormalities strikingly similar to those of patients suffering from the heritable genetic disorder cleidocranial dysplasia (CCD), an observation, which led to the discovery that Cbfa1 haploinsufficiency is the cause of CCD (17Otto F. Thornell A.P. Crompton T. Denzel A. Gilmour K.C. Rosewell I.R. Stamp G.W. Beddington R.S. Mundlos S. Olsen B.R. Selby P.B. Owen M.J. Cell. 1997; 89: 765-771Google Scholar, 19Lee B. Thirunavukkarasu K. Zhou L. Pastore L. Baldini A. Hecht J. Geoffroy V. Ducy P. Karsenty G. Nat. Genet. 1997; 16: 307-310Google Scholar, 20Mundlos S. Otto F. Mundlos C. Mulliken J.B. Aylsworth A.S. Albright S. Lindhout D. Cole W.G. Henn W. Knoll J.H. Owen M.J. Mertelsmann R. Zabel B.U. Olsen B.R. Cell. 1997; 89: 773-779Google Scholar). Although Cbfa1 was originally implicated in osteoblast differentiation, a further role for Cbfa1 in mature bone was demonstrated by creation of a transgenic mouse line expressing a dominant negative form of Cbfa1 using the bone-specific osteocalcin promoter. The inactivity of this promoter in pre-osteoblasts allowed these animals to develop a normal skeleton, but they later developed an osteopenic phenotype due to decreased bone formation, indicating that Cbfa1 plays a role in postnatal osteoblast function as well (21Ducy P. Starbuck M. Priemel M. Shen J. Pinero G. Geoffroy V. Amling M. Karsenty G. Genes Dev. 1999; 13: 1025-1036Google Scholar). Surprisingly, an osteopenic phenotype also developed in transgenic mice overexpressing wild-type Cbfa1 from the pro-α (I) collagen promoter. This phenotype, caused by a late stage blockage of osteoblast maturation that resulted in a dramatic decrease in the number of osteocytes, suggests that Cbfa1 negatively regulates a late stage of osteoblast/osteocyte development (22Liu W. Toyosawa S. Furuichi T. Kanatani N. Yoshida C. Liu Y. Himeno M. Narai S. Yamaguchi A. Komori T. J. Cell Biol. 2001; 155: 157-166Google Scholar). Among the target genes of Cbfa1/RUNX2 is osteocalcin, an osteoblast-specific gene that is the most highly expressed non-collagenous protein in bone (23Gallop P.M. Lian J.B. Hauschka P.V. N. Engl. J. Med. 1980; 302: 1460-1466Google Scholar). Mice in which both osteocalcin genes were deleted showed an increased rate of bone formation, implicating osteocalcin as a negative regulator of bone growth, although the mechanism has not been fully defined (24Ducy P. Desbois C. Boyce B. Pinero G. Story B. Dunstan C. Smith E. Bonadio J. Goldstein S. Gundberg C. Bradley A. Karsenty G. Nature. 1996; 382: 448-452Google Scholar). The osteocalcin promoter features three Cbfa consensus motifs, and studies have shown Cbfa1 to be important for both basal and vitamin D-regulated osteocalcin transcription (10Javed A. Gutierrez S. Montecino M. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. Mol. Cell. Biol. 1999; 19: 7491-7500Google Scholar). A C/EBP binding motif in the proximal osteocalcin promoter also has been shown to be functional, and the C/EBPβ protein has been shown to synergistically cooperate with Cbfa1 at this promoter through a direct interaction (25Gutierrez S. Javed A. Tennant D.K. van Rees M. Montecino M. Stein G.S. Stein J.L. Lian J.B. J. Biol. Chem. 2002; 277: 1316-1323Google Scholar). E-box consensus binding sites are present in the osteocalcin proximal promoter as well, and one particular E-box appears functionally important, with mutations reducing transcriptional activity by greater than 50% in the rat ROS17/2.8 osteoblastic osteosarcoma cell line (26Tamura M. Noda M. J. Cell Biol. 1994; 126: 773-782Google Scholar). To gain insight into the pathway by which the newly discovered SOST gene might inhibit osteoblast differentiation and/or activity, we use a comparative genomics approach to identify conserved elements in the mouse and human noncoding sequences near the SOST genomic locus. A 1.8-kb fragment of the proximal promoter is well conserved between humans and mice and is active in the SAOS-2 but not the MG-63 human osteosarcoma cell line, paralleling the pattern of SOST expression in these cells. Promoter deletion analysis demonstrates that a 140-bp element just upstream of the SOST gene accounts for this transcriptional activity. The active region contains two E-boxes, a C/EBP binding site, and a Cbfa1 binding site. Mutational and gel mobility shift analyses demonstrate that transactivation by Cbfa1 accounts for the difference in SOST expression between the two cell lines. These observations are further supported by the expression of Cbfa1 in SAOS-2 cells but not MG-63, and the ability of transfected Cbfa1 to stimulate a SOST reporter in MG-63 cells. An upstream E-box is functional and may be a target of MyoD regulation, but does not account for the difference in SOST expression between these two cell lines. Our results identify SOST as a novel target gene for Cbfa1, and suggest a possible feedback inhibition role for the SOST protein in maintaining Cbfa1 at levels appropriate for osteoblast function and/or development. SOST Genomic Sequence Analysis—Approximately 200 kb of human genomic sequence from chromosome 17q12-q21 2Celera Human Genome Database, release R25, unpublished data. containing SOST and bounded proximally by MEOX1 and distally by DUSP3, was compared with the syntenic region from the mouse genome 2Celera Human Genome Database, release R25, unpublished data. in an attempt to identify conserved sequence elements (CSEs) in non-coding sequence that could serve as a starting point for the identification of factors that affect the regulation of SOST. A CSE is defined as a 100 bp or greater sequence with minimum 50% identity. Both the human and mouse sequences were masked for repeats prior to comparison using Repeat-Masker. 3A. G. A. Smit and P. Green, unpublished results and repeatmasker.genome.washington.edu/cgi-bin/RepeatMasker. Alignment of the human and mouse sequences was estimated using BLASTn (29Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Google Scholar) with default parameters. The alignment was scanned with a 100-bp window, and the percent identity within the window was calculated and plotted using VISTA (30Dubchak I. Brudno M. Loots G.G. Pachter L. Mayor C. Rubin E.M. Frazer K.A. Genome Res. 2000; 10: 1304-1306Google Scholar, 31Mayor C. Brudno M. Schwartz J.R. Poliakov A. Rubin E.M. Frazer K.A. Pachter L.S. Dubchak I. Bioinformatics. 2000; 16: 1046-1047Google Scholar). A 15-kb region of the human genome containing SOST is represented in Fig. 1A. Reporter and Expression Constructs—The SOST promoter sequence selected for further study spans nucleotides -2000 to -190 of the SOST gene locus relative to the position (+1) of the initiation methionine for the SOST open reading frame (6Balemans W. Ebeling M. Patel N. Van Hul E. Olson P. Dioszegi M. Lacza C. Wuyts W. Van Den Ende J. Willems P. Paes-Alves A.F. Hill S. Bueno M. Ramos F.J. Tacconi P. Dikkers F.G. Stratakis C. Lindpaintner K. Vickery B. Foernzler D. Van Hul W. Hum. Mol. Genet. 2001; 10: 537-543Google Scholar). The sequence was amplified from human genomic DNA (Promega) by PCR using the following 5′ and 3′ primers, respectively: 5′-TCTCCCCCGGGTGTGGATCATTTAGAGGTTCAAG-3′ and 5′-GCCCTAGATCTCCCAAAGACTTCTCCTCTAGCTC-3′. The resulting PCR product was digested with SmaI and BglII and inserted into the pGL2-Basic luciferase reporter vector (Promega) digested at the same sites. PCR was carried out using HotStarTaq master mix (Qiagen) with the following conditions: 95 °C for 15 min, followed by 30 cycles of 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 3 min. Large-scale deletion mutations of the SOST promoter were constructed by digestion at native restriction sites (MluI, HpaI, SphI, HindIII, BamHI, EcoRV), treatment with Klenow or T4 DNA polymerase to fill in overhangs, digestion with BglII, and insertion into pGL2-Basic (Promega) digested with SmaI and BglII. Finer-scale deletions and point mutations involving the SOST promoter fragment between the EcoRV and BglII sites were generated using a PCR-based approach with oligonucleotides spanning the ends of the desired promoter sequence and bearing BglII, EcoRV, or SmaI restriction sites. Oligonucleotides used to generate mutations in transcription factor binding sites were as follows (mutated nucleotides are underlined). Upstream E-box mutant primer: 5′-GCTCCCCCGGGCCAGGTAAACGGAGGTGCCGGAGAGCAGG-3′. Downstream E-box mutant primer (reverse orientation): 5′-GCGGAAGATCTCT-CCGTTTACACCCAGAGAGAGGGGGCGTGTGA-3. Cbfa1 site mutant primer: 5′-GCTCCGATATCTGAAGTAAAGTGCCGCCAGCACGTGGGAG-3′. Similar primers, but with longer flanking 5′- and 3′-sequences, were used to mutate the Cbfa1 and Ebox sites in the full-length SOST-luc promoter via PCR-based mutagenesis. Expression constructs for human MyoD1 and Cbfa1/RUNX2 were created using the mammalian expression vector pDC409, which has been previously described (32Giri J.G. Ahdieh M. Eisenman J. Shanebeck K. Grabstein K. Kumaki S. Namen A. Park L.S. Cosman D. Anderson D. EMBO J. 1994; 13: 2822-2830Google Scholar). MyoD1 cDNA was generated by PCR using 5′-CCGTAAACGTCGACGCCATGGAGCTACTGTCGCCACC-3′ as a forward primer and 5′-CGCCTAGATCTTCAGAGCACCTGGTATATCGG-3′ as a reverse primer, using HotStarTaq (Qiagen) as described above for 25 cycles with the IMAGE clone 2961494 (Research Genetics) as template. Cbfa1 cDNA was generated by PCR using SAOS-2 cDNA as a template with 5′-CCGACGGTCGACACCATGCGTATTCCCGTAGATCCG-3′ as a forward primer and 5′-CCGGAAGATCTTCAATATGGTCGCCAAACAGATTC-3′ as a reverse primer using the Advantage GC PCR kit (Clontech) with 1 m GC Melt reagent under the following conditions: 95 °C for 5 min, followed by 40 cycles of 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min. Cbfa1 sequences recovered from SAOS-2 cDNA included both the wild-type gene and an alternatively spliced variant lacking Exon 4, as described in the text. All constructs were verified by sequencing. Transfection, Histology, and Cell Culture Conditions—The SAOS-2 and MG-63 human osteosarcoma cell lines were purchased from the American Type Culture Collection and maintained in Minimal Essential Medium (Eagle) supplemented with 2 mml-glutamine, Earle's BSS, heat-inactivated fetal bovine serum (10%) and penicillin at 37 °C in a humidified atmosphere of 5% CO2. SAOS-2 and MG-63 cells were fixed and stained for alkaline phosphatase activity using the Leukocyte Alkaline Phosphatase kit (Sigma Diagnostics, part 85L-3R) and counterstained with Harris hematoxylin (Sigma). For transfection, cells were plated at a density of 2.5 × 106 cells/well in 6-well tissue culture plates (Corning). After 24 h, cells were transfected in duplicate with a mixture of FuGENE 6 Transfection Reagent (Roche Applied Science), MEM, and 1 μg DNA/well, including 20 ng/well of the pSEAP2-control secreted alkaline phosphatase vector (Clontech). p-Bluescript (Stratagene) was used when needed to provide a final total of 1 μg DNA per well. 48 h after transfection, cell supernatants (100 μl) were harvested and assayed using a SEAP Chemiluminescence Detection Kit (Clontech). Cell lysates were harvested using the Bright-Glo Luciferase Assays System (Promega). SEAP and luciferase samples were transferred to white opaque 96-well plates (Costar) and assayed using a MicroBeta Trilux luminescence counter (Wallac). Luciferase values were normalized to SEAP data to correct for well-to-well variations in transfection efficiency. Each construct shown was tested in a minimum of two experiments with consistent results. Cell Treatment, RNA Harvest, and Semi-Quantitative RT-PCR— SAOS-2 and MG-63 cells were plated at 106 cells/plate on 10-cm plates (Corning) and treated with either 1,25-(OH)2-vitamin D3 (Calbiochem) at 10-7m, Osteogenesis Induction Medium (OIM; see below), both vitamin D3 and OIM, or neither for 72 h prior to harvest. OIM consisted of: 0.1 μm dexamethasone (Sigma), 50 μm ascorbic acid 2-phosphate (Sigma), and 10 mm β-glycerophosphate (Sigma). RNA was prepared using the RNeasy Midi kit (Qiagen) according to the manufacturer's instructions, and its quality was evaluated using an Agilent 2100 Bioanalyzer. Double-stranded cDNA was prepared with a Super Script Choice Kit (Invitrogen) using 1 μg of input RNA and 50 ng of random hexamers. RT-PCR of MyoD family members was performed using the following intron-spanning primers: for MyoD, 5′-AAGTAAATGAGGCCTTTGAGACAC-3′ and 5′-CGATGCTGGACAGGTAGTCTAGG-3′; for Myogenin, 5′-AGTGCCATCCAGTACATCGAG-3′ and 5′-TGGGTTAACCTTACATGGATGAG-3′; for Myf5, 5′-AACTACTATAGCCTGCCGGGAC-3′ and 5′-CCTTCTTCGTCCTGTGTATTAGG-3′; for Myf6, 5′-GAGCGCCATCAGCTATATTGAG-3′ and 5′-ATACTTGCTCCTCCTTCCTTAGC-3′. PCR reactions were carried out for 40 cycles using the Advantage GC PCR kit (Clontech) with 1 m GC Melt reagent as described above. For positive control samples, cDNA from human heart (Clontech) and human fetal skeletal muscle (Clontech) libraries was combined. Real-Time Quantitative RT-PCR—RNA samples were treated with DNase (Ambion, Austin, TX), and reverse-transcribed using TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions using random hexamers. Samples were distributed on plates at either 5 or 20 ng per well and run in triplicate. TaqMan primer/probe sets were designed using Primer Express software (Applied Biosystems, Foster City, CA) and were as follows: huSOST 5′ oligo, 5′-CACCACCCCTTTGAGACCAA-3′; huSOST 3′ oligo, 5′-GGTCACGTAGCGGGTGAAGT; huSOST TaqMan probe 5′-CTGCCGCGAGCTG-3′; huCbfa1 5′ oligo, 5′-CCCGTGGCCTTCAAGGT-3′; huCbfa1 3′ oligo, 5′-TGACAGTAACCACAGTCCCATCTG-3′; huCbfa1 TaqMan probe, 5′-ACCTCTCCGAGGGCTA-3′. Forward and reverse primer concentrations for human SOST and Cbfa1 were optimized and determined to be 300, 900, and 300, 50 nm each; the 6-FAM labeled TaqMan probe was used at 200 nm. Forward and reverse primer concentrations for the housekeeping gene HPRT were 300 nm each; the VIC-labeled TaqMan probe was used at 200 nm. Multiplex TaqMan PCR reactions were set up in 25-μl volumes with TaqMan Universal PCR Master Mix on an Applied Biosystems Prism 7700 Sequence Detection System. Threshold cycle values (CT) were determined using Sequence Detector software version 1.7a (Applied Biosystems) and transformed to 2-ΔCT for relative expression compared with HPRT. Nuclear Extract Preparation and Electrophoretic Mobility Shift Analysis—Nuclear extracts were prepared from SAOS-2 and MG-63 cells growing on 10-cm plates at a density similar to that used in transfection experiments (2.1 × 106 cells/plate) using a Nuclear Extract Kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions. Protein concentrations of nuclear extracts were determined using the Coomassie Plus Protein Assay reagent (Pierce), and extracts were aliquoted and stored at -80 °C until use. Probes for the electrophoretic mobility shift assay (EMSA) were end-labeled using 2 μl of Redivue [γ-32P]ATP (10 mCi/ml) (Amersham Biosciences) and 1 μl of T4 polynucleotide kinase (New England Biolabs) and purified over G-25 Sephadex Quick Spin Columns (Roche Applied Sciences). The probe spanning nucleotides -331 to -257 in the SOST promoter was generated by PCR using the Advantage GC PCR kit (Clontech) with 1 m GC Melt reagent for 27 cycles with the following primers: 5′-ATCTGAAAACCACAGCCGCCA-3′ and 5′-AGAGGGGGCGTGTGAGGC-3′. The 75-nt probe was purified by agarose gel electrophoresis and recovered using the Qiaex II Gel Extraction kit (Qiagen). Smaller probes encompassing regions A–D as designated in Fig. 3C were generated by heating complementary nucleotides in a solution of 50 mm Tris-HCl, pH 8, and 10 mm MgCl2 for 5 min in a heat block at 95 °C followed by slow cooling to room temperature. Top strand probe sequences used were as follows: probe A, 5′-ATCTGAAAACCACAGCCG-3′; probe B, 5′-CCAGCACGTGGGAGGTGCC-3′; probe C, 5′-CCGGAGAGCAGGCTTGGGCC-3′; probe D, 5′-CCTTGCCTCACACGCCCCCT-3′. The probe B competitor top strand probe with a mutant MyoD site (underlined) was: 5′-CCAGGTAAACGGAGGTGCC-3′. Labeling reactions used 0.5 pmol of PCR-generated probe or 1.75 pmol annealed oligonucleotides. For the EMSA binding reactions, 12,000 cpm labeled probe (∼70 fmol) was incubated 20 min at room temperature in Gel Shift Binding Buffer (Promega) with 2 μg of SAOS-2 or MG-63 nuclear extract in a total reaction volume of 10 μl prior to electrophoresis on a 6% DNA Retardation Gel (Invitrogen) for 20 min at 280Vat4 °C. Gels were briefly fixed and then dried and exposed to film (Kodak) at -80 for exposure for 10 to 120 min. For competition experiments, unlabeled annealed oligonucleotides (1.75 pmol) were preincubated for 10 min with nuclear extract before probe addition. Unlabeled oligonucleotides were present at ∼15-fold molar excess. Mutant oligonucleotides described in Fig. 4A (lanes 4–12) contained the probe A sequence as described above except for the single base substitutions described in the figure. The "flanking mutant" competitor (lane 3) used the following top strand sequence: 5′-GCACTGAAACCACAGAATAAG-3′. For supershift reactions, incubations were carried out as normal and then antibody (2 μg) was added and reactions were incubated 30 additional min at 4 °C. Antibodies used were the PEBP2αA/Cbfa1 (S-19) goat polyclonal IgG (Santa Cruz Biotechnology) or the Integrinβ2 K-19 goat polyclonal IgG (Santa Cruz Biotechnology). SOST Proximal Promoter Cloning and Function in SAOS-2 versus MG-63 Cells—To search for homology between noncoding sequences in the SOST genomic locus, we used the VISTA program to compare mouse and human sequences deposited in the respective Celera databases (Fig. 1A) (30Dubchak I. Brudno M. Loots G.G. Pachter L. Mayor C. Rubin E.M. Frazer K.A. Genome Res. 2000; 10: 1304-1306Google Scholar, 31Mayor C. Brudno M. Schwartz J.R. Poliakov A. Rubin E.M. Frazer K.A. Pachter L.S. Dubchak I. Bioinformatics. 2000; 16: 1046-1047Google Scholar). Human and mouse noncoding sequences showed greater than 50% homology in regions immediately upstream of the SOST open reading frame, in the gene's first intron, immediately downstream of the transcribed sequence, and in several more distant 3′-regions. Because noncoding sequences immediately upstream of genes may contain important proximal control elements, we decided to focus on the well-conserved 1.8-kb sequence directly upstream of the SOST gene. This region, referred to hereafter as the SOST promoter fragment, contained ∼780 bp of highly conserved sequence in its 5′-end and a shorter well-conserved region just upstream of the SOST open reading frame (Fig. 1A). We inserted the 1.8-kb sequence into the pGL2-basic luciferase reporter vector (Promega) and tested the ability of this promoter fragment to drive luciferase expression in the SAOS-2 and MG-63 osteosarcoma cell lines. By several criteria, SAOS-2 cells appear to represent a more differentiated osteoblast cell type than MG-63 cells. SAOS cells stain intensely positive for alkaline phosphatase and appear more rounded, whereas MG-63 cells are only weakly positive for alkaline phosphatase activity and have a spindly fibroblast-like appearance (Fig. 1B). When we examined both cell lines for SOST expression using Taqman PCR, basal SOST expression was readily detected in SAOS-2 but not MG-63 cells (Ct cutoff = 36), consistent with the previously reported expression of SOST in differentiated osteoblast cells (Fig. 1C) (6Balemans W. Ebeling M. Patel N. Van Hul E. Olson P. Dioszegi M. Lacza C. Wuyts W. Van Den Ende J. Willems P. Paes-Alves A.F. Hill S. Bueno M. Ramos F.J. Tacconi P. Dikkers F.G. Stratakis C. Lindpaintner K. Vickery B. Foernzler D. Van Hul W. Hum. Mol. Genet. 2001; 10: 537-543Google Scholar). 4B. R. Sevetson and P. Yang, unpublished data. SOST expression appeared to be up-regulated by vitamin D in SAOS-2 cells, and could be weakly induced by a combination of vitamin D and OIM (Osteogenesis Induction Medium) in MG-63 cells, but was not detected in untreated MG-63 cells. To test whether our SOST promoter fragment contained elements necessary to recapitulate the differences in SOST expression between SAOS-2 and MG-63 cells, we transfected the reporter construct into both cell types. The SOST-luc reporter consistently showed a 5-fold increase in luciferase activity compared with empty vector in SAOS-2 cells but no such increase in MG-63 cells, in agreement with the expression pattern of the SOST protein in these cell lines (Fig. 1D). This result suggested that SAOS-2 cells endogenously express a specific factor(s) that could bind this promoter element to up-regulate the expression of SOST. Deletion Analysis of the SOST Proximal Promoter Fragment—To delineate the promoter element(s) required for SAOS-specific SOST expression, we generated a series of deletion constructs using the promoter region's native restriction sites. Because we particularly wished to test the function of a large block of well-conserved (82% identity) sequence in the 5′-region of the promoter, we deleted promoter elements in a 5′ to 3′ fashion (Fig. 2A). Deletion of 5′-promoter elements encompassing the well-conserved region did not decrease luciferase activity, but instead revealed an ∼2-fold repressive effect of this region. Because this upstream element was dispensable for SAOS-specific activity of this promoter construct, we chose not to characterize it further. Further deletions to the 5′-boundary of the EcoRV restriction site, leaving only 140 nt of 3′-SOST promoter sequence, also failed to reduce SAOS-specific SOST activity, suggesting that the remaining 140 nt contained SAOS-specific regulatory positive elements. To confirm this observation, we tested a construct containing all SOST promoter sequences except those downstream (3′) of the EcoRV site. This construct had activity similar to that of empty vector alone (Fig. 2A, bottom construct), confirming that important promoter elements were contained in this 140-nt sequence. Finer scale deletion analysis of the EcoRV fragment (Fig. 2B, EcoRV: 1744-1836) suggested that its 5′-sequence encompassed important regulatory elements. Sequence examination revealed consensus binding sites for a number of transcription factors in this region, including two E-box elements and single binding sites for Cbfa1/RUNX2 and C/EBP proteins (Fig. 2C). Electrophoretic Mobility Shift Analysis of Active SOST Promoter Fragment—Because SOST is expressed in SAOS-2 but not MG-63 cells, we reasoned that nuclear extracts from these two osteosarcoma lines would bind differentially to the active element(s) of the SOST promoter, permitting isolation of the factor(s) responsible for SAOS-specific expression. Further deletion analysis within the EcoRV promoter fragment demonstrated that 5′-sequences could not be deleted without a loss of activity, but that some 3′-sequences, including the downstream E-box, could be eliminated without affecting promoter activity (Fig. 3A). Thus, a minimal region of 75 nt retained maximal transcriptional activity and was chosen as a probe in gel mobility shift assays. The lack of contribution of the downstream E-box was also confirmed by site-directed mutagenesis experiments (Fig. 6A). Nuclear extracts were prepared from untreated SAOS-2 and MG-63 cells and incubated with the 32P-labeled probe containing the minimal active SOST element, followed by gel electrophoresis. Both SAOS-2 and MG-63 nuclear extracts produced a slow-migrating band of essentially equal mobility (Fig. 3B, top arrow), but SAOS-2 extracts also produced a faster migrating band that was not detected in MG-63 cells (Fig. 3B, bottom arrow). To identify the region of the probe responsible for the faster migrating SAOS-specific band, we tested the nuclear extracts using 32P-labeled annealed oligonucleotide pairs representing the portions of the full-length probe designated A, B, C, and D (Fig. 3B). Probes B and D weakly bound factors present in both MG-63 and SAOS-2 cells, whereas probe A strongly and specifically bound to a factor present only in SAOS-2 cells (Fig. 3B, lane 2). The importance of region A, which co