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• W2017230803 abstract "Bone morphogenetic proteins (BMPs)/osteogenic proteins (OPs), members of the transforming growth factor-β superfamily, have a wide variety of effects on many cell types including osteoblasts and chondroblasts, and play critical roles in embryonic development. BMPs transduce their effects through binding to two different types of serine/threonine kinase receptors, type I and type II. Signaling by these receptors is mediated by the recently identified Smad proteins. Despite the rapid progress in understanding of the signaling mechanism downstream of BMP receptors, the target genes of BMPs are poorly understood in mammals. Here we identified a novel gene, termedBMP/OP-responsive gene (BORG), in C2C12 mouse myoblast cell line which trans-differentiates into osteoblastic cells in response to BMPs. Expression of BORG was dramatically induced in C2C12 cells by the treatment with BMP-2 or OP-1 within 2 h and peaked at 12–24 h, whereas transforming growth factor-β had a minimal effect. BMP-dependent expression of BORG was also detected in other cell types which are known to respond to BMPs, suggesting that BORG is a common target gene of BMPs. Cloning and sequence analysis of BORG cDNA and the genomic clones revealed that, unexpectedly, the transcript of BORG lacks any extensive open reading frames and contains a cluster of multiple interspersed repetitive sequences in its middle part. The unusual structural features suggested that BORG may function as a noncoding RNA, although it is spliced and polyadenylated as authentic protein-coding mRNAs. Together with the observation that transfection of antisense oligonucleotides of BORG partially inhibited BMP-induced differentiation in C2C12 cells, it is possible that a new class of RNA molecules may have certain roles in the differentiation process induced by BMPs. Bone morphogenetic proteins (BMPs)/osteogenic proteins (OPs), members of the transforming growth factor-β superfamily, have a wide variety of effects on many cell types including osteoblasts and chondroblasts, and play critical roles in embryonic development. BMPs transduce their effects through binding to two different types of serine/threonine kinase receptors, type I and type II. Signaling by these receptors is mediated by the recently identified Smad proteins. Despite the rapid progress in understanding of the signaling mechanism downstream of BMP receptors, the target genes of BMPs are poorly understood in mammals. Here we identified a novel gene, termedBMP/OP-responsive gene (BORG), in C2C12 mouse myoblast cell line which trans-differentiates into osteoblastic cells in response to BMPs. Expression of BORG was dramatically induced in C2C12 cells by the treatment with BMP-2 or OP-1 within 2 h and peaked at 12–24 h, whereas transforming growth factor-β had a minimal effect. BMP-dependent expression of BORG was also detected in other cell types which are known to respond to BMPs, suggesting that BORG is a common target gene of BMPs. Cloning and sequence analysis of BORG cDNA and the genomic clones revealed that, unexpectedly, the transcript of BORG lacks any extensive open reading frames and contains a cluster of multiple interspersed repetitive sequences in its middle part. The unusual structural features suggested that BORG may function as a noncoding RNA, although it is spliced and polyadenylated as authentic protein-coding mRNAs. Together with the observation that transfection of antisense oligonucleotides of BORG partially inhibited BMP-induced differentiation in C2C12 cells, it is possible that a new class of RNA molecules may have certain roles in the differentiation process induced by BMPs. Bone morphogenetic proteins (BMPs) 1The abbreviations used are: BMP, bone morphogenetic protein; OP, osteogenic protein; BORG, BMP/OP-responsive gene; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; GAPDH, glyceraldehydephosphate dehydrogenase; RACE, rapid amplification of cDNA ends; SINE, short interspersed nucleotide element; TGF-β, transforming growth factor-β; bp, base pair(s); ORF, open reading frame; PCR, polymerase chain reaction; RT, reverse transcriptase. 1The abbreviations used are: BMP, bone morphogenetic protein; OP, osteogenic protein; BORG, BMP/OP-responsive gene; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; GAPDH, glyceraldehydephosphate dehydrogenase; RACE, rapid amplification of cDNA ends; SINE, short interspersed nucleotide element; TGF-β, transforming growth factor-β; bp, base pair(s); ORF, open reading frame; PCR, polymerase chain reaction; RT, reverse transcriptase./osteogenic proteins (OPs), members of the transforming growth factor-β (TGF-β) superfamily, were originally identified by their activity to induce bone formation in vivo (1Wozney J.M. Rosen V. Celeste A.J. Mitsock L.M. Whitters M.J. Kriz R.W. Hewick R.M. Wang E.A. Science. 1988; 242: 1528-1534Crossref PubMed Scopus (3305) Google Scholar, 2Özkaynak E. Rueger D.C. Drier E.A. Corbett C. Ridge R.J. Sampath T.K. Oppermann H. EMBO J. 1990; 9: 2085-2093Crossref PubMed Scopus (514) Google Scholar). The BMP family includes various proteins, which can be divided into several subgroups based on their structural similarity; i.e. a group containingDrosophiladecapentaplegic gene product, BMP-2, and BMP-4, a group containing Drosophila 60A gene product, OP-1/BMP-7, OP-2/BMP-8, BMP-5, and BMP-6/Vgr1, a group containing growth/differentiation factor-5, -6, and -7, and other members (3Kingsley D.M. Genes Dev. 1994; 8: 133-146Crossref PubMed Scopus (1719) Google Scholar, 4Reddi A.H. Curr. Opin. Genet. Dev. 1994; 4: 737-744Crossref PubMed Scopus (327) Google Scholar, 5Hogan B.L.M. Genes Dev. 1996; 10: 1580-1594Crossref PubMed Scopus (1708) Google Scholar). In vitro studies have revealed that BMPs have various biological effects on osteoblasts and chondroblasts, e.g.stimulation of proteoglycan synthesis in chondroblasts, and induction of collagen, alkaline phosphatase, and osteocalcin during chondrogenic and osteogenic differentiation (6Wozney J.M. Mol. Reprod. Dev. 1992; 32: 160-167Crossref PubMed Scopus (606) Google Scholar, 7Yamaguchi A. Katagiri T. Ikeda T. Wozney J.M. Rosen V. Wang E.A. Kahn A.J. Suda T. Yoshiki S. J. Cell Biol. 1991; 113: 681-687Crossref PubMed Scopus (650) Google Scholar, 8Asahina I. Sampath T.K. Hauschka P.V. Exp. Cell Res. 1996; 222: 38-47Crossref PubMed Scopus (225) Google Scholar). BMPs appear to exert various effects on many other cell types and play critical roles in embryonic development. For instance, null mutation in the BMP-2 gene leads to defects in amnion/chorion and cardiac development (9Zhang H. Bradley A. Development. 1996; 122: 2977-2986Crossref PubMed Google Scholar), and OP-1-deficient mice die shortly after birth because of poor kidney development and have eye defects and skeletal abnormalities (10Dudley A.T. Lyons K.M. Robertson E.J. Genes Dev. 1995; 9: 2795-2807Crossref PubMed Scopus (946) Google Scholar,11Luo G. Hofmann C. Bronckers A.L.J.J. Sohocki M. Bradley A. Karsenty G. Genes Dev. 1995; 9: 2808-2820Crossref PubMed Scopus (864) Google Scholar). BMPs transduce their signals through binding to two different types of serine/threonine kinase receptors, type I and type II (12Yamashita H. ten Dijke P. Heldin C.-H. Miyazono K. Bone. 1996; 19: 569-574Crossref PubMed Scopus (191) Google Scholar). Upon ligand binding followed by the formation of heteromeric receptor complexes, type I receptors are phosphorylated by type II receptors, and subsequent activation of the catalytic activity of type I receptor kinase is essential for signaling (13ten Dijke P. Yamashita H. Sampath T.K. Reddi A.H. Estevez M. Riddle D.L. Ichijo H. Heldin C.-H. Miyazono K. J. Biol. Chem. 1994; 269: 16985-16988Abstract Full Text PDF PubMed Google Scholar, 14Koenig B.B. Cook J.S. Wolsing D.H. Ting J. Tiesman J.P. Correa P.E. Olson C.A. Pecquet A.L. Ventura F. Grant R.A. Chen G.-X. Wrana J.L. Massagué J. Rosenbaum J.S. Mol. Cell. Biol. 1994; 14: 5961-5974Crossref PubMed Scopus (308) Google Scholar, 15Wrana J.L. Attisano L. Wieser R. Ventura F. Massagué J. Nature. 1994; 370: 341-347Crossref PubMed Scopus (2089) Google Scholar, 16Attisano L. Wrana J.L. Montalvo E. Massagué J. Mol. Cell. Biol. 1996; 16: 1066-1073Crossref PubMed Scopus (283) Google Scholar). Signaling by these receptors is mediated by the recently identified Smad proteins (17Massagué J. Hata A. Liu F. Trends Cell Biol. 1997; 7: 187-192Abstract Full Text PDF PubMed Scopus (269) Google Scholar,18Heldin C.-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3301) Google Scholar). In the case of BMPs, phosphorylation of Smad1 (19Graff J.M. Bansal A. Melton D.A. Cell. 1996; 85: 479-487Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 20Hoodless P.A. Haerry T. Abdollah S. Stapleton M. O'Connor M.B. Attisano L. Wrana J.L. Cell. 1996; 85: 489-500Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar, 21Thomsen G.H. Development. 1996; 122: 2359-2366PubMed Google Scholar, 22Liu F. Hata A. Baker J.C. Doody J. Càrcamo J. Harland R.M. Massagué J. Nature. 1996; 381: 620-623Crossref PubMed Scopus (587) Google Scholar, 23Kretzschmar M. Liu F. Hata A. Doody J. Massagué J. Genes Dev. 1997; 11: 984-995Crossref PubMed Scopus (474) Google Scholar) by the activated type I receptors allows association of Smad1 with Smad4 (24Lagna G. Hata A. Hemmati-Brivanlou A. Massagué J. Nature. 1996; 383: 832-836Crossref PubMed Scopus (805) Google Scholar), and the complex moves into the nucleus, wherein Smads regulate the transcription of a subset of target genes. In Xenopusembryos, Smad5 is also able to mediate BMP signaling (25Suzuki A. Chang C. Yingling J.M. Wang X.-F. Hemmati- Brivanlou A. Dev. Biol. 1997; 184: 402-405Crossref PubMed Scopus (141) Google Scholar). In activin signaling, Smad2 interacts with the activin-response element ofMix.2, an immediate early activin-response gene, in concert with FAST-1, a novel member of the winged-helix family of putative transcription factor (26Chen X. Rubock M.J. Whitman M. Nature. 1996; 383: 691-696Crossref PubMed Scopus (623) Google Scholar), whereas DNA-binding partners of Smad1 or Smad5 have yet been unknown in BMP signaling. Certain Smads have been shown to directly bind to DNAs (27Yingling J.M. Datto M.B. Wong C. Frederick J.P. Liberati N.T. Wang X.-F. Mol. Cell. Biol. 1997; 17: 7019-7028Crossref PubMed Google Scholar, 28Kim J. Johnson K. Chen H.J. Carroll S. Laughon A. Nature. 1997; 388: 304-308Crossref PubMed Scopus (446) Google Scholar). In order to elucidate how the Smad proteins and other transcription factors function in mediating BMP signals, and whether the signaling pathways not using the Smad proteins are also involved in BMP signaling, it is necessary to identify and analyze the genes directly induced by BMPs. A number of target genes of decapentaplegic gene product, the Drosophila counterpart of BMP-2, have been reported (18Heldin C.-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3301) Google Scholar, 29Padgett R.W. Savage C. Das P. Cytokine Growth Factor Rev. 1997; 8: 1-9Crossref PubMed Scopus (25) Google Scholar). In Xenopus, homeobox-containing genes,Mix.1 (30Mead P.E. Brivanlou I.H. Kelley C.M. Zon L.I. Nature. 1996; 382: 357-360Crossref PubMed Scopus (131) Google Scholar), Xvent-1 (31Gawantka V. Delius H. Hirschfeld K. Blumenstock C. Niehrs C. EMBO J. 1995; 14: 6268-6279Crossref PubMed Scopus (302) Google Scholar, 32Ault K.T. Dirksen M.-L. Jamrich M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6415-6420Crossref PubMed Scopus (85) Google Scholar), Xvent-2(33Onichtchouk D. Gawantka V. Dosch R. Delius H. Hirschfeld K. Blumenstock C. Niehrs C. Development. 1996; 122: 3045-3053Crossref PubMed Google Scholar, 34Schmidt J.E. von Dassow G. Kimelman D. Development. 1996; 122: 1711-1721Crossref PubMed Google Scholar, 35Ladher R. Mohun T.J. Smith J.C. Snape A.M. Development. 1996; 122: 2385-2394Crossref PubMed Google Scholar), and msx1 (36Suzuki A. Ueno N. Hemmati-Brivanlou A. Development. 1997; 124: 3037-3044Crossref PubMed Google Scholar), and erythroid transcription factors, GATA-1 (37Xu R.-H. Kim J. Taira M. Lin J.-J. Zhang C.-H. Sredni D. Evans T. Kung H.-F. Mol. Cell. Biol. 1997; 17: 436-443Crossref PubMed Scopus (50) Google Scholar) and GATA-2 (38Maéno M. Mead P.E. Kelley C. Xu R.H. Kung H.F. Suzuki A. Ueno N. Zon L.I. Blood. 1996; 88: 1965-1972Crossref PubMed Google Scholar), have been shown to have immediate early response to BMPs. The mammalian counterparts of these genes may function as direct target genes of BMPs; however, little is known to date in mammals about the BMP-responsive genes except for TGF-β-inducible early gene (TIEG), a putative zinc finger protein which is induced by BMP-2 as well as TGF-β (39Subramaniam M. Harris S.A. Oursler M.J. Rasmussen K. Riggs B.L. Spelsberg T.C. Nucleic Acids Res. 1995; 23: 4907-4912Crossref PubMed Scopus (220) Google Scholar). In the present study, we report the isolation of a novel gene, termedBMP/OP-responsive gene (BORG), of which expression was regulated by either BMP-2 or OP-1 in BMP-responsive cells. Interestingly, it lacks any extensive open reading frames (ORFs) and contains a cluster of multiple interspersed repetitive sequences in its middle part. A possibility that BORG may function as a noncoding RNA in the BMP-induced differentiation process is discussed. Mouse muscle myoblast C2C12 cells (40Blau H.M. Chiu C.-P. Webster C. Cell. 1983; 32: 1171-1180Abstract Full Text PDF PubMed Scopus (600) Google Scholar) and mouse embryo fibroblast C3H10T1/2 clone 8 were obtained from the American Type Culture Collection. ST2 mouse bone marrow stromal cells were obtained from the RIKEN Cell Bank (Tsukuba, Japan). C2C12 cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Nissui) containing 15% fetal bovine serum (FBS) and antibiotics (100 units/ml penicillin). When the C2C12 cells were treated with BMP-2, OP-1, or TGF-β, medium was replaced by DMEM containing 5% FBS and antibiotics. C3H10T1/2 cells and ST2 cells were maintained in basal medium Eagle's with Earle's salts (Life Technologies, Inc.) and RPMI 1640 (Nissui), respectively, in the presence of 10% FBS and antibiotics. Total RNA was isolated from the cells by using Isogen (Wako), and poly(A)+ RNA was purified by binding to Oligotex-dT30 Super (Takara Biomedicals) as described by the manufacturer's instructions. C2C12 cells were cultured in DMEM containing 15% FBS to reach confluency; the serum was reduced to 5%, and the cells were allowed to grow in the presence or absence of 300 ng/ml OP-1 for additional 2 h. Poly(A)+ RNA was extracted from the cells, and subjected to digestion with DNase I (MessageClean kit, GenHunter) for 30 min at 37 °C to remove residual contaminated DNA fragments. Poly(A)+ RNA was further purified by phenol/chloroform extraction and precipitated with ethanol. The differential display method (41Liang P. Pardee A.B. Science. 1992; 257: 967-971Crossref PubMed Scopus (4675) Google Scholar) was performed by using an RNAimage kit (GenHunter). Briefly, 0.2 μg of poly(A)+ RNA purified as above was reverse transcribed in a 20-μl reaction containing 25 mm Tris-HCl, pH 8.3, 37.6 mm KCl, 1.5 mm MgCl2, 5 mm dithiothreitol, 20 μm dNTPs, and 20 μm anchored oligo-dT primer, H-T11M (5′-AAGC(T11)(G/A/C)-3′), for 5 min at 65 °C and for 60 min at 37 °C, followed by 5 min at 75 °C. Murine Moloney leukemia virus reverse transcriptase (100 units) was added after 10 min incubation at 37 °C. PCR reaction was performed in a 20-μl reaction containing 10 mm Tris-HCl, pH 8.4, 50 mm KCl, 1.5 mm MgCl2, 0.001% gelatin, 2 μm dNTPs, 0.2 μm arbitrary 13-mer (H-AP-1 to -80), 0.2 μm H-T11M, 2 μl of reverse transcription mixture, 1 μl of [α-35S]dATP (1200 Ci/mmol, Amersham), and 1 unit of Taq polymerase (Boehringer Mannheim). PCR conditions were 40 cycles of 94 °C (15 s), 40 °C (2 min), and 72 °C (30 s), followed by 5 min at 72 °C using a Perkin-Elmer 9600 thermocycler. The amplified products were separated on a 6% denaturing polyacrylamide gel. The gel was dried and exposed to an autoradiography film (Hyperfilm MP, Amersham). Differentially expressed products were cut out from the gel, extracted with H2O, and reamplified by the same set of primers under the same condition except for the use of 20 μm dNTPs. The secondary amplified products were separated side by side with the primary PCR products on a 6% denaturing polyacrylamide gel. The gel was dried and exposed to a film; the secondary amplified product was cut out from the gel, extracted with H2O, subcloned into pGEM-T vector (Promega), sequenced, and used as a probe for Northern blot analysis. Two μg of poly(A)+ RNA was denatured, separated on a 1.2% agarose-formaldehyde gel, and blotted onto a nylon filter (Hybond-N, Amersham) with 20 × SSC (1 × SSC is 15 mm sodium citrate, 150 mmNaCl). Subcloned cDNA fragments from differential display and cDNAs for rat osteocalcin and glyceraldehydephosphate dehydrogenase (GAPDH) (gifts of Dr. S. Oida) were labeled with 32P by a Ready-To-Go DNA Labeling Kit (Pharmacia). Hybridization was performed at 43 °C overnight with labeled probe in 5 × SSPE (1 × SSPE is 180 mm NaCl, 10 mmNa2HPO4·7H2O, 1 mmEDTA), 50% formamide, 5 × Denhardt's solution, 0.5% SDS, 20 μg/ml salmon sperm DNA. The filters were washed twice in 2 × SSPE, 0.1% SDS at 43 °C for 15 min, once in 1 × SSPE, 0.1% SDS at 43 °C for 30 min, and once in 0.1 × SSPE, 0.1% SDS at room temperature for 15 min, followed by the analysis using a Fuji BAS 2000 Bio-Imaging Analyzer (Fuji Photo Film). One μg of total RNA was reverse transcribed into single strand cDNA using Superscript Preamplification System (Life Technologies, Inc.) as described by the manufacturer's instructions. PCR was performed in a 50-μl reaction containing 1 × PCR reaction buffer (Boehringer Mannheim), 200 μm dNTPs, 0.2 μm B-S4 primer (5′-TAATGGGACAGCCTAGTAGG-3′), 0.2 μm B-AS4 primer (5′-TCCGTGTAAGAAAGCTGGCC-3′), 1 μl of single strand cDNA solution, and 2.5 units of Taqpolymerase (Boehringer Mannheim). PCR conditions were 94 °C (5 min), 25 cycles of 94 °C (30 s), 55 °C (30 s), and 72 °C (30 s), followed by 10 min at 72 °C. A second round of PCR was performed with 2 μl of the first reaction as a template in the same reaction mixture except for the use of the internal primers, B-SN (5′-CAGCGGCCGCTAACTTGAGTATGTGG) and B-ASX1 (5′-CACTCGAGCTGACTATGATTTGTC-3′) instead of B-S4 and B-AS4. The second PCR conditions were 94 °C (1 min), 15 cycles of 94 °C (30 s), 55 °C (30 s), and 72 °C (30 s), followed by 10 min at 72 °C. Specificity of the PCR products was confirmed by digestion withEcoRI and EcoRV. Primers for the mouse or rat GAPDH (CLONTECH) were also used as loading controls for the RT-PCR procedure. Rapid amplification of cDNA ends (5′-RACE) was performed using a Marathon cDNA Amplification Kit (CLONTECH). Using 1 μg of poly(A)+RNA isolated from OP-1-treated C2C12 cells, a library of adapter-ligated double strand cDNA was constructed as described by the manufacturer's instruction. For the initial attempt to obtain a full-length cDNA of BORG, two sequential antisense primers, B-AS1 (5′-ATCCAAGGTGAGGCCTAGTTCAC-3′) and B-AS2 (5′-CAAGGTGGCCTCAGTGTGGATGC-3′), were designed from the sequence of the cDNA fragment obtained by the differential display. To isolate the 5′-end of BORG cDNA, B-AS6 (5′-ACGGCTGCTGGGATTTAAAC-3′) and B-ASPE (5′-GTGGTAGCTGATCTTGATTGTCAAGCTTGTTGCCC-3′) were designed from the sequence of the 5′-region of the mouse C2C12 cDNA library clone, clone P69 (see below). PCR reaction was performed in a 50-μl reaction containing 50 mm Tris-HCl, pH 9.2, 14 mm(NH4)2SO4, 1.75 mmMgCl2, 200 μm dNTPs, 0.2 μmB-AS1 primer, 0.2 μm adapter primer 1 (AP1,CLONTECH), 0.5 μl of adapter-ligated double strand cDNA solution, 2.5 units of Taq/Pwo DNA polymerase mixture (Expand Long Template PCR system, Boehringer Mannheim), and 0.3 μg of TaqStart Antibody (CLONTECH). PCR conditions were 94 °C (1 min), followed by 30 cycles of 94 °C (30 s) and 68 °C (4 min). A second round of PCR was performed with 0.5 μl of the first reaction as a template in the same reaction mixture except for the use of B-AS2 primer and nested adapter primer 2 (AP2, CLONTECH) instead of B-AS1 and AP1. The second PCR conditions were 94 °C (1 min), followed by 20 cycles of 94 °C (30 s) and 68 °C (4 min). The PCR product was subcloned into pGEM-T vector (Promega), sequenced, and used as a probe for cDNA library screening. Using poly(A)+ RNA isolated from OP-1-treated C2C12 cells, an oligo(dT)-primed cDNA library with 1 × 106 independent clones was prepared by Uni-ZAP XR/Gigapack II Gold Cloning kit (Stratagene). The unamplified cDNA library was plated and lifted onto nylon filters (Hybond-N, Amersham), and immobilized by UV cross-linking. The duplicate filters were probed with the 32P-labeled 5′-RACE product at 65 °C overnight in the hybridization buffer containing 5 × SSPE, 5 × Denhardt's solution, 0.5% SDS, 20 μg/ml salmon sperm DNA. The filters were washed at 65 °C twice in 2 × SSPE, 0.1% SDS for 15 min, once in 1 × SSPE, 0.1% SDS for 30 min, and once in 0.1 × SSPE, 0.1% SDS for 15 min, followed by autoradiography. The positive clones were isolated and rescued into pBluescript SK(−). Nucleotide sequencing was performed on both strands. One million clones of a 129SV mouse genomic library (Stratagene) were screened with the full-length BORG cDNA as a probe as described above. Phage DNA was isolated from the positive clones and subjected to digestion with appropriate restriction enzymes to generate a physical map. The digested DNA was also probed with various portions of BORG cDNA to determine their location in the genome of BORG. Fragments that hybridized with the probes were subcloned into pBluescript SK(+) for nucleotide sequencing. Antisense oligonucleotides at nucleotide positions from 902 to 921 of BORG cDNA (Fig. 5) were designed to hybridize to BORG RNA. Nucleotide sequences are: antisense, 5′-CCAGGCCACATACTCAAGTT-3′; sense, 5′-AACTTGAGTATGTGGCCTGG-3′. Both were synthesized as phosphorothionate oligonucleotides and high pressure liquid chromatography-purified by Greiner Japan (42Wagner R.W. Nature. 1994; 372: 333-335Crossref PubMed Scopus (801) Google Scholar). For transfection, 5 μm of each oligonucleotide was mixed with 2 μl/ml Tfx-50 (Promega) in DMEM containing 5% FBS, and added to C2C12 cells in the presence or absence of 300 ng/ml BMP-2. Total RNA extraction followed by RT-PCR for detecting the expression of BORG was done 6 h after the transfection as described above. Alkaline phosphatase activity was measured 36 h after the transfection as described previously (43Nishitoh H. Ichijo H. Kimura M. Matsumoto T. Makishima F. Yamaguchi A. Yamashita H. Enomoto S. Miyazono K. J. Biol. Chem. 1996; 271: 21345-21352Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). To identify a novel target gene of BMPs, we first examined the OP-1 responsiveness in a mouse myoblast cell line C2C12, which was reported to trans-differentiate into osteoblastic cells in response to BMP-2 (44Katagiri T. Yamaguchi A. Komaki M. Abe E. Takahashi N. Ikeda T. Rosen V. Wozney J.M. Fujisawa-Sehara A. Suda T. J. Cell Biol. 1994; 127: 1755-1766Crossref PubMed Scopus (1275) Google Scholar). C2C12 cells were found to start to express osteocalcin mRNA (see below), as well as alkaline phosphatase activity (data not shown), representative markers for osteoblastic phenotype, by 24 h after the treatment with 300 ng/ml OP-1. In contrast, C2C12 cells not treated with OP-1 did not undergo such osteoblastic changes (data not shown), suggesting that C2C12 cells provide a useful system for the differential screening of OP-1-induced gene expression. We applied an mRNA differential display method (41Liang P. Pardee A.B. Science. 1992; 257: 967-971Crossref PubMed Scopus (4675) Google Scholar) by using poly(A)+RNA obtained from OP-1-treated or -untreated C2C12 cells. The C2C12 cells were maintained in DMEM containing 15% FBS. When the cells reached confluency, the serum was reduced, and the cells were allowed to grow in the presence or absence of 300 ng/ml OP-1 for an additional 2 h. Thereafter, poly(A)+ RNA was extracted, reverse transcribed into first strand cDNA and applied to the PCR-based differential screening using various combinations of arbitrary primers. Differentially expressed products (exemplified in Fig. 1 A) which were observed only in the OP-1-treated material were cut out from the gel, reamplified by the same sets of the primers, and subcloned into plasmid vectors. DNA sequencing of the two differentially displayed clones as shown in Fig. 1 A revealed that they encoded the same gene product with overlapping sequences. When one of the clones, named DD-10, was used as a probe for Northern blot analysis, a major transcript of approximately 3 kilobases was found to be increased in the OP-1-treated material (Fig. 1 B), which confirmed that DD-10 corresponded to an OP-1-induced transcript in C2C12 cells. Together with the following induction data by BMP-2 (see below), the gene for this transcript was denoted BORG. A time course experiment by Northern blot analysis revealed that the expression of BORG was induced as early as 3 h after the addition of OP-1, peaked at 12–24 h, and decreased after 48 h (Figs. 2 and3). While very weak expression of BORG was observed 3–12 h after the reduction of serum even without OP-1 (Fig. 2, OP-1(−) and Fig. 3, control), the extent of expression was much less compared with that observed in OP-1-treated cells. These results strongly suggested that OP-1 specifically induced BORG expression in C2C12 cells. When the same filter was reprobed with osteocalcin cDNA, osteocalcin mRNA was found to be induced 24–48 h after the treatment with OP-1 (Fig. 2), indicating that the expression of BORG precedes osteocalcin induction by OP-1 in C2C12 cells.Figure 3Effects of OP-1, BMP-2, and TGF-β on expression of BORG RNA in C2C12 cells. Confluent C2C12 cells were incubated with DMEM containing 5% FBS in the absence (control) or presence of 300 ng/ml OP-1, 300 ng/ml BMP-2, or 25 ng/ml TGF-β for the indicated time. Poly(A)+ RNA (2 μg) extracted from the cells was applied to Northern blot analysis using BORG PCR fragment clone DD-10 as a probe. The amount of mRNAs was verified by rehybridizing the filters with GAPDH probe (data not shown). Radioactive signals were quantitated on a Fuji BAS2000 Bio-Imaging Analyzer and plotted to give a graphical representation of the results. The amount of each transcript was calculated as relative activity against the background signal and normalized by RNA loading on the gels. Relative changes were calculated by standardizing the 0-h time point as a basal level.View Large Image Figure ViewerDownload (PPT) To investigate the ability of other members of TGF-β superfamily to induce BORG expression, we treated C2C12 cells with BMP-2 or TGF-β. BORG was strongly induced by BMP-2 after 12–24 h and decreased after 48 h with a time course similar to OP-1 (Fig. 3). BORG was weakly induced in response to TGF-β; however, TGF-β-induced expression of BORG peaked at 3 h after the treatment and decreased thereafter. Taking the fact into account that TGF-β-induced expression of BORG was weak and transient, it is likely that BORG is a relatively specific target gene for OP-1 and BMP-2 but not TGF-β. To examine whether BMP-induced expression of BORG was a cell-type specific event in C2C12 cells, we tested the expression of BORG by RT-PCR in other cell lines that are known to respond to BMPs. In ST2 mouse bone marrow stromal cells (45Yamaguchi A. Ishizuya T. Kintou N. Wada Y. Katagiri T. Wozney J.M. Rosen V. Yoshiki S. Biochem. Biophys. Res. Commun. 1996; 220: 366-371Crossref PubMed Scopus (301) Google Scholar) and C3H10T1/2 mouse embryo fibroblast (46Katagiri T. Yamaguchi A. Ikeda T. Yoshiki S. Wozney J.M. Rosen V. Wang E.A. Tanaka H. Omura S. Suda T. Biochem. Biophys. Res. Commun. 1990; 172: 295-299Crossref PubMed Scopus (451) Google Scholar), BORG was found to be induced within 1 h after the treatment with BMP-2 (Fig. 4), suggesting that BORG is a common target gene of BMPs in BMP-responsive cells. To obtain a full-length cDNA for BORG, we applied 5′-RACE to poly(A)+ RNA isolated from OP-1-treated C2C12 cells by using two nested antisense primers designed from the sequence of the original PCR clone, DD-10. Specifically amplified products were subcloned into plasmid vectors, and two independent clones were sequenced. These clones encoded overlapping PCR products of 2,528 and 2,455 bp long, but a few nucleotides of these clones were different from each other in the overlapping region probably due to misincorporation of deoxynucleotides during the PCR procedure (data not shown). Next, a cDNA library was constructed using poly(A)+ RNA isolated from OP-1-treated C2C12 cells and probed with the longer 5′-RACE product (2,528 bp). Several overlapping clones were obtained, and a clone, termed P69, yielded a 2,840-bp nucleotide sequence with a polyadenylation signal, AATAAA, followed by a poly(A) tail. The majority of the other clones were found to encode the partial sequence of P69. A few clones, whose inserts were longer than that of P69, appeared to encode premature transcripts since they contained additional intron-like sequences (data not shown). To determine the 5′-end sequence of BORG RNA, we again applied 5′-RACE using two sequential antisense primers designed in the 5′-region of P69 and the cDNA templates used in the initial 5′-RACE. Sequencing of the specifically amplified products yielded an additional six nucleotides at the 5′-end of the cDNA. Thus, the combined nucleotide sequence of the 5′-RACE product and P69 was a putative full-length cDNA for BORG, containing a pol" @default.
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• W2017230803 title "Identification of a Novel Bone Morphogenetic Protein-responsive Gene That May Function as a Noncoding RNA" @default.
• W2017230803 cites W1494055834 @default.
• W2017230803 cites W1496740624 @default.
• W2017230803 cites W1529816420 @default.
• W2017230803 cites W1530364143 @default.
• W2017230803 cites W1591850953 @default.
• W2017230803 cites W1607031549 @default.
• W2017230803 cites W1639188211 @default.
• W2017230803 cites W1663371441 @default.
• W2017230803 cites W1816605246 @default.
• W2017230803 cites W1858153082 @default.
• W2017230803 cites W1935346479 @default.
• W2017230803 cites W1937940610 @default.
• W2017230803 cites W1964269746 @default.
• W2017230803 cites W1967147992 @default.
• W2017230803 cites W1967951907 @default.
• W2017230803 cites W1969356288 @default.
• W2017230803 cites W1970823660 @default.
• W2017230803 cites W1978368163 @default.
• W2017230803 cites W1980473444 @default.
• W2017230803 cites W1986469118 @default.
• W2017230803 cites W1988942273 @default.
• W2017230803 cites W1989020935 @default.
• W2017230803 cites W1989722096 @default.
• W2017230803 cites W1995574948 @default.
• W2017230803 cites W2002156733 @default.
• W2017230803 cites W2006239014 @default.
• W2017230803 cites W2019303375 @default.
• W2017230803 cites W2036734848 @default.
• W2017230803 cites W2043420357 @default.
• W2017230803 cites W2047333953 @default.
• W2017230803 cites W2048492425 @default.
• W2017230803 cites W2050434777 @default.
• W2017230803 cites W2052127095 @default.
• W2017230803 cites W2055110202 @default.
• W2017230803 cites W2056984915 @default.
• W2017230803 cites W2059267677 @default.
• W2017230803 cites W2062313490 @default.
• W2017230803 cites W2070539534 @default.
• W2017230803 cites W2074952408 @default.
• W2017230803 cites W2080546144 @default.
• W2017230803 cites W2082554124 @default.
• W2017230803 cites W2093417867 @default.
• W2017230803 cites W2093654042 @default.
• W2017230803 cites W2099681945 @default.
• W2017230803 cites W2103042473 @default.
• W2017230803 cites W2107022932 @default.
• W2017230803 cites W2114245705 @default.
• W2017230803 cites W2119282152 @default.
• W2017230803 cites W2121848695 @default.
• W2017230803 cites W2124415162 @default.
• W2017230803 cites W2131211068 @default.
• W2017230803 cites W2140970244 @default.
• W2017230803 cites W2144973569 @default.
• W2017230803 cites W2158714788 @default.
• W2017230803 cites W2185475290 @default.
• W2017230803 cites W2188247991 @default.
• W2017230803 cites W2412339092 @default.
• W2017230803 cites W4248706725 @default.
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