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Identification of a Novel Response Element in the Rat Bone Sialoprotein (BSP) Gene Promoter that Mediates Constitutive and Fibroblast Growth Factor 2-induced Expression of BSP

2001; Elsevier BV; Volume: 276; Issue: 8 Linguagem: Inglês

10.1074/jbc.m008971200

ISSN

1083-351X

Autores

Emi Shimizu-Sasaki, Muneyoshi Yamazaki, Shunsuke Furuyama, Hiroshi Sugiya, Jaro Sodek, Yorimasa Ogata,

Tópico(s)

Connective tissue disorders research

Resumo

Bone sialoprotein (BSP) is a sulfated and phosphorylated glycoprotein, found almost exclusively in mineralized connective tissues, that may function in the nucleation of hydroxyapatite crystals. We have found that expression of BSP in osteoblastic ROS 17/2.8 cells is stimulated by fibroblast growth factor 2 (FGF2), a potent mitogen for mesenchymal cells. Stimulation of BSP mRNA with 10 ng/ml FGF2 was first evident at 3 h (∼2.6-fold) and reached maximal levels at 6 h (∼4-fold). From transient transfection assays, a FGF response element (FRE) was identified (nucleotides −92 to −85, “GGTGAGAA”) as a target of transcriptional activation by FGF2. Ligation of two copies of the FRE 5′ to an SV40 promoter was sufficient to confer FGF-responsive transcription. A sequence-specific protein-DNA complex, formed with a double-stranded oligonucleotide encompassing the FRE and nuclear extracts from ROS 17/2.8 cells, but not from fibroblasts, was increased following FGF2 stimulation. Several point mutations within the critical FRE sequence abrogated the formation of this complex and suppressed both basal and FGF2-mediated promoter activity. These studies, therefore, have identified a novel FRE in the proximal promoter of the BSP gene that mediates both constitutive and FGF2-induced BSP transcription. Bone sialoprotein (BSP) is a sulfated and phosphorylated glycoprotein, found almost exclusively in mineralized connective tissues, that may function in the nucleation of hydroxyapatite crystals. We have found that expression of BSP in osteoblastic ROS 17/2.8 cells is stimulated by fibroblast growth factor 2 (FGF2), a potent mitogen for mesenchymal cells. Stimulation of BSP mRNA with 10 ng/ml FGF2 was first evident at 3 h (∼2.6-fold) and reached maximal levels at 6 h (∼4-fold). From transient transfection assays, a FGF response element (FRE) was identified (nucleotides −92 to −85, “GGTGAGAA”) as a target of transcriptional activation by FGF2. Ligation of two copies of the FRE 5′ to an SV40 promoter was sufficient to confer FGF-responsive transcription. A sequence-specific protein-DNA complex, formed with a double-stranded oligonucleotide encompassing the FRE and nuclear extracts from ROS 17/2.8 cells, but not from fibroblasts, was increased following FGF2 stimulation. Several point mutations within the critical FRE sequence abrogated the formation of this complex and suppressed both basal and FGF2-mediated promoter activity. These studies, therefore, have identified a novel FRE in the proximal promoter of the BSP gene that mediates both constitutive and FGF2-induced BSP transcription. bone sialoprotein fibroblast growth factor 2 FGF response element fibroblast growth factor receptors nucleotide(s) protein kinase C cAMP-dependent protein kinase mitogen-activated protein kinase MAP kinase kinase pituitary-specific transcription factor-1 luciferase activator protein-1 activator protein-2 activating transcription factor glucocorticoid response element nuclear factor κB cAMP response element cAMP response element-binding protein minimum essential medium glyceraldehyde-3-phosphate dehydrogenase Bone sialoprotein (BSP)1is a highly sulfated, phosphorylated, and glycosylated protein that is characterized by its ability to bind to hydroxyapatite, through polyglutamic acid sequences, and to mediate cell attachment, through an RGD sequence (1Oldberg Å. Franzén A. Heinegård D. J. Biol. 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Furthermore, the temporo-spatial deposition of BSP into the extracellular matrix (6Chen J. Shapiro H.S. Sodek J. J. Bone Miner. Res. 1992; 7: 987-997Crossref PubMed Scopus (351) Google Scholar, 7Kasugai S. Nagata T. Sodek J. J. Cell. Physiol. 1992; 152: 467-477Crossref PubMed Scopus (126) Google Scholar, 8Bianco P. Fisher L.W. Young M.F. Termine J.D. Robey P.G. Calcif. Tissue Int. 1991; 49: 421-426Crossref PubMed Scopus (338) Google Scholar) and the ability of BSP to nucleate hydroxyapatite crystal formation (9Hunter G.K. Goldberg H.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8562-8565Crossref PubMed Scopus (576) Google Scholar) indicate a role for this protein in the initial mineralization of bone, dentin, and cementum (4Ganss B. Kim R.H. Sodek J. Crit. Rev. Oral. Biol. Med. 1999; 10: 79-98Crossref PubMed Scopus (457) Google Scholar). Recent studies have shown that BSP is also expressed in osteotropic cancers, suggesting BSP might play a role in the pathogenesis of bone metastases (10Waltregny D. Bellahcène A. Leval X.D. Florkin B. Weidle U. Castronovo V. J. Bone Miner. Res. 2000; 15: 834-843Crossref PubMed Scopus (112) Google Scholar). Thus, regulation of the BSP gene is important in the differentiation of osteoblasts, in bone matrix mineralization and in tumor metastasis. The human (11Kim R.H. Shapiro H.S. Li J.J. Wrana J.L. Sodek J. Matrix Biol. 1994; 14: 31-40Crossref PubMed Scopus (79) Google Scholar, 12Kerr J.M. Fisher L.W. Termine J.D. Wang M.G. McBride O.W. Young M.F. Genomics. 1993; 17: 408-415Crossref PubMed Scopus (65) Google Scholar), mouse (13Benson M.D. Aubin J.E. Xiao G. Thomas P.E. Franceschi R.T. J. Bone Miner. Res. 1999; 14: 396-405Crossref PubMed Scopus (68) Google Scholar), and rat (14Li J.J. Sodek J. Biochem. J. 1993; 289: 625-629Crossref PubMed Scopus (87) Google Scholar) BSP genes have been cloned and partially characterized. These promoters include a highly conserved region (BSP box) that extends upstream from the transcription start site to nt −370 (15Sodek J. Li J.J. Kim R.H. Ogata Y. Yamauchi M. Connect. Tissue Res. 1996; 35: 23-31Crossref PubMed Scopus (17) Google Scholar). This region includes a functional, inverted TATA element (nts −24 to −19) (16Li J.J. Kim R.H. Sodek J. Biochem. J. 1995; 310: 33-40Crossref PubMed Scopus (54) Google Scholar), which overlaps a vitamin d response element (17Kim R.H. Li J.J. Ogata Y. Yamauchi M. Freedman L. Sodek J. Biochem. J. 1996; 318: 219-226Crossref PubMed Scopus (71) Google Scholar). In addition, putative sites of regulation through an inverted CCAAT box (−50 to −46) (18Kim R.H. Sodek J. Cancer Res. 1999; 59: 565-571PubMed Google Scholar) and an AP-2 site (−447 to −440), which overlaps a transforming growth factor-β activation element, have been identified in the proximal promoter (19Ogata Y. Niisato N. Furuyama S. Cheifetz S. Kim R.H. Sugiya H. Sodek J. J. Cell. Biochem. 1997; 65: 501-512Crossref PubMed Scopus (76) Google Scholar). Further upstream, a glucocorticoid response element (GRE) overlapping an AP-1 site has been characterized (20Ogata Y. Yamauchi M. Kim R.H. Li J.J. Freedman L.P. Sodek J. Eur. J. Biochem. 1995; 230: 183-192Crossref PubMed Scopus (94) Google Scholar, 21Yamauchi M. Ogata Y. Kim R.H. Li J.J. Freedman L.P. Sodek J. Matrix Biol. 1996; 15: 119-130Crossref PubMed Scopus (49) Google Scholar). Recently, we have identified a pituitary-specific transcription factor-1 (Pit-1) motif through which the stimulatory effects of parathyroid hormone on BSP transcription are mediated (22Ogata Y. Nakao S. Kim R.H. Li J.J. Furuyama S. Sugiya H. Sodek J. Matrix Biol. 2000; 19: 395-407Crossref PubMed Scopus (47) Google Scholar), while studies by Benson et al. (23Benson M.D. Bargeon J.L. Xiao G. Thomas P.E. Kim A. Cui Y. Franceschi R.T. J. Biol. Chem. 2000; 275: 13907-13917Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) have indicated that a homeodomain binding element in the BSP promoter is required for osteoblast-specific expression. Fibroblast growth factor-2 (FGF2 or basic FGF), a member of the heparin-binding growth factor family of mitogens, has been implicated in a range of normal physiological processes from embryonic mesoderm induction and pattern formation to angiogenesis and wound repair (24Mason I.J. Cell. 1994; 78: 547-552Abstract Full Text PDF PubMed Scopus (525) Google Scholar,25Yamasaki M. Miyake A. Tagashira S. Itho N. J. Biol. Chem. 1996; 271: 15918-15921Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). FGF2 is also synthesized by osteoblasts and is stored in a bioactive form in the extracellular matrix (26Hurley M.M. Abreu C. Harrison J.R. Lichtler A.C. Raisz L.G. Kream B. J. Biol. Chem. 1993; 268: 5588-5593Abstract Full Text PDF PubMed Google Scholar, 27Rodan S.B. Wesolowski G. Yoon K. Rodan G.A. J. Biol. Chem. 1989; 264: 19934-19941Abstract Full Text PDF PubMed Google Scholar, 28Nakamura T. Hanada K. Tamura M. Shibanushi T. Nigi H. Tagawa M. Fukumoto S. Matsumoto T. Endocrinology. 1995; 136: 1276-1284Crossref PubMed Google Scholar, 29Noda M. Vogel R. J. Cell Biol. 1989; 109: 2529-2535Crossref PubMed Scopus (126) Google Scholar), where it acts as a local regulator of bone formation. The FGF family of molecules transduce signals to the cytoplasm via a family of transmembrane receptors with tyrosine kinase activity (30Debiais F. Hott M. Graulet A.M. Marie P.J. J. Bone Miner. Res. 1998; 13: 645-654Crossref PubMed Scopus (150) Google Scholar, 31Muenke M. Schell U. Hehr A. Robin N.H. 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Immunoglobulin-like domains II and III are sufficient for FGF binding and determine affinity (30Debiais F. Hott M. Graulet A.M. Marie P.J. J. Bone Miner. Res. 1998; 13: 645-654Crossref PubMed Scopus (150) Google Scholar, 31Muenke M. Schell U. Hehr A. Robin N.H. Losken H.W. Schinzel A. Pulleyn L.J. Rutland P. Reardon W. Malcolm S. Winter R.M. Nat. Genet. 1994; 8: 269-274Crossref PubMed Scopus (538) Google Scholar, 32Jabs E.W. Li X. Scott A.F. Meyers G. Chen W. Eccles M. Mao J. Charnas L.R. Jackson C.E. Jaye M. Nat. Genet. 1994; 8: 275-279Crossref PubMed Scopus (406) Google Scholar, 33Reardon W. Winter R.M. Rutland P. Pulleyn L.J. Jones B.M. Malcalm S. Nat. Genet. 1994; 8: 98-103Crossref PubMed Scopus (611) Google Scholar, 34Tavormina P.L. Shiang R. Thompson L.M. Zhu Y.Z. Wilkin D.J. Lachman R.S. Wilcox W.R. Rimoin D.L. Cohn D.H. Wasmuth J.J. Nat. Genet. 1995; 9: 321-328Crossref PubMed Scopus (520) Google Scholar). Mutations in the FGFR1 gene are associated with Pfeiffer syndrome, which is one of the classic autosomal dominant craniosynostosis syndromes (31Muenke M. Schell U. Hehr A. Robin N.H. Losken H.W. Schinzel A. Pulleyn L.J. Rutland P. Reardon W. Malcolm S. Winter R.M. Nat. Genet. 1994; 8: 269-274Crossref PubMed Scopus (538) Google Scholar), while mutations in FGFR2 and FGFR3 produce genetic disorders involving bone development. Jackson-Weiss and Crouzon syndromes are allelic with mutations in FGFR2 (32Jabs E.W. Li X. Scott A.F. Meyers G. Chen W. Eccles M. Mao J. Charnas L.R. Jackson C.E. Jaye M. Nat. Genet. 1994; 8: 275-279Crossref PubMed Scopus (406) Google Scholar, 33Reardon W. Winter R.M. Rutland P. Pulleyn L.J. Jones B.M. Malcalm S. Nat. Genet. 1994; 8: 98-103Crossref PubMed Scopus (611) Google Scholar). Thanatophoric dysplasia, the most common neonatal lethal skeletal dysplasia, and achondroplasia are caused by mutations in FGFR3 (34Tavormina P.L. Shiang R. Thompson L.M. Zhu Y.Z. Wilkin D.J. Lachman R.S. Wilcox W.R. Rimoin D.L. Cohn D.H. Wasmuth J.J. Nat. Genet. 1995; 9: 321-328Crossref PubMed Scopus (520) Google Scholar). Analysis of FGFR3-deficient mice has revealed prolonged bone growth, showing that FGFR3 is a negative regulator of bone growth (35Den C. Wynshaw-Boris A. Zhou F. Kuo A. Leder P. Cell. 1996; 84: 911-921Abstract Full Text Full Text PDF PubMed Scopus (912) Google Scholar). Collectively these studies, and the observation that intravenous FGF2 stimulates bone formation and mineralization (28Nakamura T. Hanada K. Tamura M. Shibanushi T. Nigi H. Tagawa M. Fukumoto S. Matsumoto T. Endocrinology. 1995; 136: 1276-1284Crossref PubMed Google Scholar, 36Liang H. Pun S. Wronski T.J. Endocrinology. 1999; 140: 5780-5788Crossref PubMed Google Scholar), indicate that FGF is an important regulator of bone formation. FGF2 inhibits alkaline phosphatase activity in ROS 17/2.8 cells (27Rodan S.B. Wesolowski G. Yoon K. Rodan G.A. J. Biol. Chem. 1989; 264: 19934-19941Abstract Full Text PDF PubMed Google Scholar) and the calcification of hypertrophic chondrocytes (37Kato Y. Iwamoto M. J. Biol. Chem. 1990; 265: 5903-5909Abstract Full Text PDF PubMed Google Scholar). FGF2 also inhibits type I collagen and osteocalcin transcription in ROS 17/2.8 cells and MC3T3-E1 cells (26Hurley M.M. Abreu C. Harrison J.R. Lichtler A.C. Raisz L.G. Kream B. J. Biol. Chem. 1993; 268: 5588-5593Abstract Full Text PDF PubMed Google Scholar, 27Rodan S.B. Wesolowski G. Yoon K. Rodan G.A. J. Biol. Chem. 1989; 264: 19934-19941Abstract Full Text PDF PubMed Google Scholar, 38Boudreaux J.M. Towler D.A. J. Biol. Chem. 1996; 271: 7508-7515Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). However, the combination of FGF2 and forskolin markedly up-regulates osteocalcin mRNA accumulation in MC3T3-E1 cells (38Boudreaux J.M. Towler D.A. J. Biol. Chem. 1996; 271: 7508-7515Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). An osteocalcin FGF2 response element (GCAGTCA motif) has been identified in the proximal promoter of the rat osteocalcin gene as a target of FGF2 and cAMP stimulation (38Boudreaux J.M. Towler D.A. J. Biol. Chem. 1996; 271: 7508-7515Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The induction of human osteocalcin transcription by FGF2 requires the interaction of CCAAT motif that overlaps with three tandem repeats of a nuclear factor-1 half-site (TTGGC) (39Schedlich L.J. Flanagan J.L. Crofts L.A. Gillies S.A. Goldberg D. Morrison N.A. Eisman J.A. J. Bone Miner. Res. 1994; 9: 143-152Crossref PubMed Scopus (35) Google Scholar). To determine the molecular mechanism of FGF2 regulation of the BSP gene, we have analyzed the effects of the FGF2 on the expression of BSP in ROS 17/2.8 cells. These studies have revealed a novel FRE that mediates both the constitutive and FGF2-induced expression of BSP in osteoblastic cells. Cell culture media, fetal bovine serum, LipofectACE, penicillin, streptomycin, and trypsin were obtained from Life Technologies, Inc., Tokyo, Japan. The pGL2-promoter vector, pSV-β-galactosidase control vector, and MEK inhibitor U0126 were purchased from Promega Co., Madison, WI. 5,6-Dichloro-1-β-d-ribofuranosyl benzimidazole was from Sigma-Aldrich Japan, Tokyo, Japan, the protein kinase inhibitors H89 and H7 were from Seikagaku Corp., Tokyo, Japan, and herbimycin A and guanidium thiocyanate were purchased from Wako Pure Chemical Industries, Ltd., Tokyo, Japan. PP1 was from Biomol Research Laboratories, Inc., Plymouth Meeting, PA, and recombinant human FGF2 was from Genzyme, Techne, Minneapolis, MN. The rat clonal cell lines, ROS 17/2.8 (generously provided by Dr. G. A. Rodan, Merck-Sharpe and Frosst, West Point, PA) was used in these studies as an osteoblastic cell line that synthesizes BSP (20Ogata Y. Yamauchi M. Kim R.H. Li J.J. Freedman L.P. Sodek J. Eur. J. Biochem. 1995; 230: 183-192Crossref PubMed Scopus (94) Google Scholar). Cells grown to confluence in 60-mm tissue culture dishes in α-MEM medium containing 10% fetal bovine serum were changed to α-MEM without serum and incubated with or without 10 ng/ml FGF2 for time periods extending over 3–24 h. To determine the effect of FGF2 on the stability of BSP mRNA, cells were first incubated for 6 h in the presence or absence of 10 ng/ml FGF2 and the incubation continued for up to 24 h in the presence of 60 μm concentration of the transcription inhibitor, 5,6-dichloro-1-β-d-ribofuranosyl benzimidazole. RNA was isolated from triplicate cultures at various time intervals and analyzed for the expression of BSP mRNA by Northern hybridization as described below. Total RNA from the culture cells was extracted with guanidium thiocyanate and, following purification, 20-μg aliquots of RNA were fractionated on a 1.2% agarose gel and transferred onto a Hybond N membrane, as described previously (20Ogata Y. Yamauchi M. Kim R.H. Li J.J. Freedman L.P. Sodek J. Eur. J. Biochem. 1995; 230: 183-192Crossref PubMed Scopus (94) Google Scholar). Hybridizations were performed at 42 °C with either a32P-labeled rat BSP or rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH), DNA probe. Following hybridization, membranes were washed four times for 5 min each at 21 °C in 300 mmsodium chloride, 30 mm trisodium citrate, pH 7.0, containing 0.1% SDS. This was followed by two, 20-min washes at 55 °C in 15 mm sodium chloride, 1.5 mmtrisodium citrate, pH 7.0, 0.1% SDS. The hybridized bands, representing the two polyadenylated forms (1.6 and 2.0 kilobases) of rat BSP mRNA, were scanned in a Bio-imaging analyzer (Fuji BAS 2000, Tokyo, Japan) and normalized to the expression of GAPDH. Exponentially growing ROS 17/2.8 cells were used for transfection assays. Twenty-four hours after plating, cells at 50-70% confluence were transfected using a LipofectACE reagent. The transfection mixture included 1 μg of a luciferase (LUC) construct (20Ogata Y. Yamauchi M. Kim R.H. Li J.J. Freedman L.P. Sodek J. Eur. J. Biochem. 1995; 230: 183-192Crossref PubMed Scopus (94) Google Scholar) and 2 μg of pSV-β-galactosidase vector as an internal control. Two days post-transfection, cells were deprived of serum for 12 h, and 10 ng/ml FGF2 was added for 6 h prior to harvesting. The luciferase assay was performed according to the supplier's protocol (picaGene, Toyo Inki, Tokyo, Japan) using a Luminescence reader BLR20 (Aloka) to measure the luciferase activity. The protein kinase inhibitor H89 (5 μm) and H7 (5 μm) were used to inhibit protein kinases A and C. Herbimycin A (1 μm) and PP1 (10 μm) were used for tyrosine kinase and Src tyrosine kinase inhibition, respectively (40Hanke J.H. Gardner J.P. Dow R.L. Changelian P.S. Brissette W.H. Weringer E.J. Pollok B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Abstract Full Text Full Text PDF PubMed Scopus (1784) Google Scholar). U0126 (5 μm) was used for MAP kinase kinase (MEK) inhibitor (41Favata M.F. Horiuchi K.Y. Manos E.J. Daulerio A.J. Stradley D.A. Feeser W.S. Van Dyk D.E. Pitts W.J. Earl R.A. Hobbs F. Copeland R.A. Magolda R.L. Scherle P.A. Trzaskos J.M. J. Biol. Chem. 1998; 273: 18623-18632Abstract Full Text Full Text PDF PubMed Scopus (2747) Google Scholar). The FRE sequence, identified from the transient transfection studies, was cloned in the BglII site of pGL2-promoter vector, immediately upstream of the enhancerless SV40 promoter. This construct was used to identify the sequence within the BSP promoter that is required for transcriptional induction by FGF2. Oligonucleotide-directed mutagenesis by PCR was utilized to introduce the dinucleotide substitutions using the Quikchange Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA). All constructs were sequenced as described previously (20Ogata Y. Yamauchi M. Kim R.H. Li J.J. Freedman L.P. Sodek J. Eur. J. Biochem. 1995; 230: 183-192Crossref PubMed Scopus (94) Google Scholar) to verify the fidelity of the mutagenesis. The pCG-ATF2 and pCG-ATF3 expression plasmids were kindly provided by Dr. T. Hai (42Chen B.P.C. Liang G. Whelan J. Hai T. J. Biol. Chem. 1994; 269: 15819-15826Abstract Full Text PDF PubMed Google Scholar). Confluent ROS 17/2.8 cells in T-75 flasks incubated for 6 and 12 h with 10 ng/ml FGF2 in α-MEM without serum were used to prepare nuclear extracts as we have described previously (19Ogata Y. Niisato N. Furuyama S. Cheifetz S. Kim R.H. Sugiya H. Sodek J. J. Cell. Biochem. 1997; 65: 501-512Crossref PubMed Scopus (76) Google Scholar, 20Ogata Y. Yamauchi M. Kim R.H. Li J.J. Freedman L.P. Sodek J. Eur. J. Biochem. 1995; 230: 183-192Crossref PubMed Scopus (94) Google Scholar, 21Yamauchi M. Ogata Y. Kim R.H. Li J.J. Freedman L.P. Sodek J. Matrix Biol. 1996; 15: 119-130Crossref PubMed Scopus (49) Google Scholar, 22Ogata Y. Nakao S. Kim R.H. Li J.J. Furuyama S. Sugiya H. Sodek J. Matrix Biol. 2000; 19: 395-407Crossref PubMed Scopus (47) Google Scholar), with the addition of extra proteinase inhibitors (the extraction buffer was 0.42 m NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 1 mm dithiothreitol, 25% (v/v) glycerol, 0.5 mmphenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml pepstatin A, 1 μg/ml aprotinin, pH 7.9). Double-stranded oligonucleotides encompassing the inverted CCAAT (nts −61 to −37, 5′-CCGTGACCGTGATTGGCTGCTGAGA) and the FRE (nts −98 to −79, 5′-TTTTCTGGTGAGAACCCACA) in the BSP promoter, together with FRE mutation 1 (FREm1; 5′-TTTTCTaaTGAGAACCCACA); FRE mutation 2 (FREm2; 5′-TTTTCTGGcaAGAACCCAC), FRE mutation 3 (FREm3; 5′-TTTTCTGGTGcaAACCCAC), FRE mutation 4 (FREm4; 5′-TTTTCTGGTGAGctCCCAC), 3′-FRE (nts −95 to −73, 5′-TCTGGTGAGAACCCACAGCCTGA), BSP-NFκB (nts −112 to −93, 5′-GTTGTAGTTACGGATTTTCT), and NFκB binding site identified in mouse Igκ enhancer (43Kurokouchi K. Kambe F. Yasukawa K. Izum R. Ishiguro N. Iwata H. Seo H. J. Bone Miner. Res. 1998; 13: 1290-1299Crossref PubMed Scopus (111) Google Scholar) (Igκ-NFκB; 5′-AGAGGGGACTTTCCGAGA), were prepared by Bio-Synthesis, Inc., Lewisville, TX; while consensus AP-1 (5′-CGCTTGATGAGTCAGCCGGAA) and GRE (5′-TCGACTGTACAGGATGTTCTAGCTACT) were purchased from Promega. For gel shift analysis the double-stranded-oligonucleotides were end-labeled with [γ-32P]ATP and T4 polynucleotide kinase. Nuclear protein extracts (3 μg) were incubated for 20 min at room temperature (room temperature = 21 °C) with 0.1 pmradiolabeled double-stranded oligonucleotide in buffer containing 50 mm KCl, 0.5 mm EDTA, 10 mmTris-HCl, pH 7.9, 1 mm dithiothreitol, 0.04% Nonidet P-40, 5% glycerol, and 1 μg of poly(dI-dC). For competition experiments unlabeled oligonucleotides for the inverted CCAAT, FRE, FREm1, 2,3,4,3′-FRE, BSP-NFκB, Igκ-NFκB, and consensus AP1 and GRE (Promega) were used at 20-, 40-, and 100-fold molar excess. Following incubation, the protein-DNA complexes were resolved by electrophoresis on 5% nondenaturing acrylamide gels (38:2 acrylamide/bis acrylamide) run at 150 V at room temperature. Following electrophoresis, the gels were dried and autoradiograms prepared and analyzed using an image analyzer. Triplicate samples were analyzed for each experiment and experiments replicated to ensure consistency of the responses to FGF2. Significant differences between control and FGF2 treatment were determined using Student's t test. To study the regulation of BSP expression by FGF2, we used ROS 17/2.8 cells, which have been shown to have osteoblastic characteristics (44Majeska R.J. Rodan S.B. Rodan G.A. Endocrinology. 1980; 107: 1494-1503Crossref PubMed Scopus (418) Google Scholar,45Majeska R.J. Rodan G.A. Calcif. Tissue Int. 1982; 34: 59-66Crossref PubMed Scopus (153) Google Scholar) and to express BSP mRNA constitutively (20Ogata Y. Yamauchi M. Kim R.H. Li J.J. Freedman L.P. Sodek J. Eur. J. Biochem. 1995; 230: 183-192Crossref PubMed Scopus (94) Google Scholar). First, a dose-response relation for FGF2 induction of BSP was established by treating the ROS 17/2.8 cells with different concentrations of FGF2 for 6 h and measuring the BSP mRNA levels by Northern blot analysis. At 1–50 ng/ml, FGF2 increased BSP mRNA with a maximal effect at 10 ng/ml (Fig. 1 A). This optimal level of FGF2 (10 ng/ml) was used to determine a time course of BSP mRNA expression (Fig. 1 B). FGF2 up-regulated BSP mRNA accumulation markedly in ROS 17/2.8 cells. A stimulation of 2.6-fold was evident 3 h after the addition of FGF2, with maximal levels (4.0-fold) of BSP mRNA obtained at 6 h. In comparison, osteopontin mRNA, which has been shown previously to be stimulated by FGF2 (27Rodan S.B. Wesolowski G. Yoon K. Rodan G.A. J. Biol. Chem. 1989; 264: 19934-19941Abstract Full Text PDF PubMed Google Scholar), was increased at 3 h and returned to base line at 12 h, whereas no effect on GAPDH mRNA was observed. To determine whether the increase in BSP mRNA was due to an increased stability of the BSP mRNA in response to FGF2 treatment, ROS 17/2.8 cells were incubated in the presence of the transcription inhibitor 5,6-dichloro-1-β-d-ribofuranosyl benzimidazole and the BSP mRNA levels determined over a 24-h period. From regression analysis at 12 of ∼16 h was determined for BSP mRNA in the ROS 17/2.8 cells with no significant change observed in the presence of FGF2, indicating that the increase in mRNA was due to increased gene transcription (data not shown). To determine the site of FGF2-regulated transcription in the 5′-flanking region of the BSP gene, various sized promoter constructs ligated to a luciferase reporter gene were transiently transfected into ROS 17/2.8 cells and their transcriptional activity determined in the presence of FGF2. The constructs used, pLUC1-pLUC5, encompassing nucleotides −116 to +60, gave a 2.9-fold increase in transcription after 6 h treatment with 10 ng/ml FGF2 (Fig.2). FGF2 also increased transcription of pLUC4 (−425 to +60) and pLUC5 (−801 to +60). Within the DNA sequence that is unique to pLUC3 (between nts −116 to −43), an inverted CCAAT box (ATTGG; between nts −50 and −46), a possible cAMP response element (CRE; between nts −75 and −68), and a pituitary-specific transcription factor-1 (Pit-1) motif (between nts −111 and −105), which is the target of parathyroid hormone stimulation, are present (Fig.3 A).Figure 3The nucleotide sequences of the rat BSP gene proximal promoter is shown from nts −116 to −43. A,the inverted CCAAT box, CRE, NFκB, Pit-1, and FRE are present.B, comparison of rat, mouse, and human DNA sequences from nucleotide −111 to −68. The rat sequence is shown on the top line and the mouse and human sequence below. DNA sequences that are identical between the species are shown by a dot.View Large Image Figure ViewerDownload Hi-res image Download (PPT) By using a series of 5′ deletion constructs between nts −116 and −43, we found that the FGF2 response was mediated by a region between nts −108 and −84 of the promoter sequence (Fig.4). A series of 2-base pair mutations were made between nts −92 and −85 within pLUC3 construct (Fig.5). All four constructs (mutations 1–4; pLUC3m1–4) had lower basal activities than pLUC3 and resulted in near abolition of the FGF2 effects on the promoter. In particular, the mutation in pLUC3m2 drastically reduced basal expression and completely abolished the FGF2 effect (Fig. 5). Thus, the GGTGAGAA motif (FRE, FGF response element) in the region nts −92 to −85 is important for basal expression as well as being necessary for the FGF2 induction of BSP promoter activity. To examine whether the FRE of the rat BSP promoter confers FGF2-dependent inducibility in the context of a promoter that is not stimulated by FGF2, the DNA segment between nts −98 and −79 in the rat BSP gene was inserted 5′ to the SV40 in theBglII site of the pGL2-promoter. While the insertion of a single FRE sequence in the same orientation as in the BSP promoter did not influence basal activity of the SV40 promoter, and only a modest increase in FGF-mediated transcription was found, two copies of FRE increased basal activity and significantly increased FGF-mediated transcription (Fig. 6). Notably, the FRE sequences identified in the rat BSP promoter are conserved in the mouse and human BSP promoters (Fig. 3 B;underlined).Figure 5GGTGAGAA motif at nts −92 to −85 in the rat BSP promoter is necessary for induction by FGF2. A series of dinucleotide substitutions were made within context of the homologous −116 to +60 BSP promoter fragment (pLUC3). The constructs were analyzed for relative promoter activity after transfection into ROS17/2.8 cells and examined for induction in the presence of FGF2 (10 ng/ml). The results of transcriptional activity obtained from three separate transfections with constructs pLUCB, pLUC3, and pLUC3 mutations 1–4 (pLUC3m1 to pLUC3m4) were combined and the values expressed with S.E. Significant differences from relative luciferase activity of pLUC3: *, p < 0.1; **, p < 0.05; ***, p < 0.02.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig

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