From the Department of Biochemistry, The Cancer
Institute, Tokyo, Japanese Foundation for Cancer Research (JFCR),
1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo 170-8455, Japan and
Creative Biomolecules, Inc., Hopkinton,
Massachusetts 01748
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ABSTRACT |
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Bone morphogenetic proteins (BMPs)/osteogenic
proteins (OPs), members of the transforming growth factor-
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, termed
BMP/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-
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.
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INTRODUCTION |
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Bone morphogenetic proteins
(BMPs)1/osteogenic proteins
(OPs), members of the transforming growth factor- (TGF-
)
superfamily, were originally identified by their activity to induce
bone formation in vivo (1, 2). The BMP family includes
various proteins, which can be divided into several subgroups based on
their structural similarity; i.e. a group containing
Drosophila decapentaplegic 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
(3-5).
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 (6-8). 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 (9), and OP-1-deficient mice die shortly after birth because of poor kidney development and have eye defects and skeletal abnormalities (10, 11).
BMPs transduce their signals through binding to two different types of serine/threonine kinase receptors, type I and type II (12). 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 (13-16). Signaling by these receptors is mediated by the recently identified Smad proteins (17, 18). In the case of BMPs, phosphorylation of Smad1 (19-23) by the activated type I receptors allows association of Smad1 with Smad4 (24), and the complex moves into the nucleus, wherein Smads regulate the transcription of a subset of target genes. In Xenopus embryos, Smad5 is also able to mediate BMP signaling (25). In activin signaling, Smad2 interacts with the activin-response element of Mix.2, an immediate early activin-response gene, in concert with FAST-1, a novel member of the winged-helix family of putative transcription factor (26), 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 (27, 28).
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 (18, 29). In Xenopus, homeobox-containing genes,
Mix.1 (30), Xvent-1 (31, 32), Xvent-2
(33-35), and msx1 (36), and erythroid transcription
factors, GATA-1 (37) and GATA-2 (38), 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--inducible early gene (TIEG), a putative zinc finger protein
which is induced by BMP-2 as well as TGF-
(39).
In the present study, we report the isolation of a novel gene, termed BMP/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.
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MATERIALS AND METHODS |
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Cell Culture--
Mouse muscle myoblast C2C12 cells (40) 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-, 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.
RNA Isolation-- 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.
Differential Display--
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 (41) 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 [-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.
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 mM NaCl). 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 mM Na2HPO4·7H2O, 1 mM EDTA), 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).
RT-PCR-- 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 Taq polymerase (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 with EcoRI and EcoRV. Primers for the mouse or rat GAPDH (CLONTECH) were also used as loading controls for the RT-PCR procedure.
5'-RACE-- 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 mM MgCl2, 200 µM dNTPs, 0.2 µM B-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.
Preparation of cDNA Library and Isolation of cDNA
Clones--
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.
Isolation of BORG Genomic Clones-- 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-- 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 (42). 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 (43).
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RESULTS |
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Identification of a Novel Target Gene of BMPs-- 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 (44). 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 (41) 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. 1A) 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. 1A 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. 1B), 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.
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Expression of 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 and
3). 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.
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cDNA Cloning of BORG-- 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 polyadenylation signal, AATAAA, at the 3'-end (nucleotide 2,812) (Fig. 5).
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Sequence Analysis of BORG-- A search of sequence data bases using the BLAST program (47) revealed an unexpected feature of BORG. Although no cDNA or mRNA sequence homologous to BORG transcript was detected in the data bases, mouse genomic sequence of origin region repeat-1a transposon-like element, clone origin region repeat-F (GenBank accession number: MMU17092) was highly homologous (76.7% identical) to a part of BORG cDNA (nucleotides 607-1,041), which indicates that BORG transcript contains an interspersed repetitive sequence (Fig. 5). To further investigate whether other interspersed repetitive sequences were involved in BORG cDNA sequence, we used RepeatMasker program2 that screens DNA sequences for interspersed repeats known to exist in mammalian genomes. BORG cDNA contained a cluster of homologous regions to four types of interspersed repetitive sequences in tandem; sequence of nucleotides 354-535 was homologous to type B4A of the short interspersed nucleotide element (SINE) (62.6% identical) (48), sequence of nucleotides 536-704 was homologous to the long terminal repeat region of origin region repeat-1B (70.2% identical), a member of a superfamily of mammalian apparent long terminal repeat-retrotransposons (49), sequence of nucleotides 709-1,042 was homologous to the internal sequence of origin region repeat-1A (78.7% identical), another member of mammalian apparent long terminal repeat-retrotransposons, and sequence of nucleotides 1,043-1,343 was homologous to RMER4 (62.6% identical), a long terminal repeat sequence that was not fully characterized in the RepeatMasker program or previous publications. These repetitive elements are frequently found within introns but much less in exons. Even if found in exons, they are usually found within 5'- or 3'-noncoding exons (50).
Another unexpected feature of BORG was the lack of any extensive ORFs in the cDNA sequence because of the high density of stop codons in all three reading frames (Fig. 6). The longest ATG-initiated ORF was found in nucleotides 911-1,273 (reading frame 2) which contained 363-bp long nucleotides with a favorable context for initiation of translation (51) with a purine at the
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Genomic Organization of BORG-- To negate a possibility that BORG is a processed pseudogene, we determined the genomic structure of BORG (Fig. 7). Using BORG cDNA as a probe, we isolated several overlapping genomic clones from a mouse genomic library. Detailed restriction mapping and Southern blot analyses of the clones and sequencing of their subclones revealed that BORG consisted of three exons interrupted by two introns. The sequences of the exon-intron boundaries were consistent with the donor/acceptor splicing rule, with GT at the donor site and AG at the acceptor site of the intron. Therefore, BORG transcripts, like authentic protein- coding mRNAs, are found to be both spliced and polyadenylated, excluding the possibility that BORG is a pseudogene.
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Effect of Antisense Oligonucleotides of BORG-- To investigate the roles of BORG in BMP-induced differentiation, we transfected C2C12 cells with antisense oligonucleotides of BORG and examined their effect on BORG expression and alkaline phosphatase activity induced by BMP-2. Transfection of antisense oligonucleotides of BORG decreased the extent of BMP-2-induced expression of BORG as determined by RT-PCR (Fig. 8), indicating the effectiveness of the antisense oligonucleotides on preventing the expression of BORG. As compared with sense oligonucleotides, transfection of antisense oligonucleotides partially inhibited alkaline phosphatase activity induced by BMP-2 (Fig. 8), suggesting the possible roles of BORG in BMP-induced osteoblastic differentiation in C2C12 cells.
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DISCUSSION |
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In the present study, we identified a novel gene, termed BORG, in a C2C12 mouse myoblast cell line by using an mRNA differential display method. C2C12 cells have been shown to differentiate into myotubes under reduced concentration of serum, but trans-differentiate into osteoblastic cells in the presence of BMP-2 (44). We found that OP-1 also induced the osteoblastic phenotype in C2C12 cells including the induction of osteocalcin mRNA (Fig. 2) and alkaline phosphatase activity (data not shown).
We demonstrated that expression of BORG was strongly induced by either
BMP-2 or OP-1 in C2C12 cells, whereas TGF-, another member of
TGF-
superfamily, had a minimal effect. The induction of BORG was
first detected 2-3 h after the treatment with BMP-2 or OP-1 and peaked
after 12-24 h by Northern blot analysis in C2C12 cells (Figs.
1B, 2 and 3). Although the kinetics of BORG induction by
BMP-2 were essentially identical to that by OP-1, BMP-2 had a slightly
stronger activity in BORG induction as compared with OP-1, which
correlated well with alkaline phosphatase and osteocalcin inducing
activity by BMP-2 and OP-1 (data not shown). In contrast, TGF-
,
which does not induce an osteoblastic phenotype in C2C12 cells, induced
BORG expression very weakly (Fig. 3). Moreover, the kinetics of BORG
induction by TGF-
, which peaked after 3 h and rapidly decreased
after this, was clearly different from that by BMP-2 or OP-1. Supposing
that BORG is a specific target gene for OP-1 and BMP-2 but not TGF-
,
BORG might also be sensitive to certain nonspecific stimulations such
as TGF-
, but only specific stimulations for osteoblastic
differentiation such as BMP-2 and OP-1 may strongly increase and
maintain its expression level for a longer period.
BMP-2-dependent expression of BORG was also detected in two
other cell types, ST2 and C3H10T1/2, both of which are known to respond
to BMPs (45, 46), suggesting that BORG is a common target gene of
BMPs.
In Xenopus, homeobox-containing genes, Mix.1
(30), Xvent-1 (31, 32), Xvent-2 (33-35), and
msx1 (36), and erythroid transcription factors, GATA-1 (37)
and GATA-2 (38), have been shown to be immediate early response genes
of BMPs. The counterparts of these genes are likely candidates for
immediate early response genes of BMPs in mammalian cells. Because of
the rapid responsiveness of BORG to BMPs together with the fact that
cycloheximide failed to inhibit the induction of BORG by OP-1 (data not
shown), BORG may be classified as an immediate early response gene of
BMPs. No such genes have been reported in mammalian cells to date,
except for a putative zinc finger protein, called TGF--inducible
early gene (TIEG), which is induced by BMP-2 as well as TGF-
(39). Therefore, detailed analysis of the regulatory mechanism of BORG expression will allow us to elucidate the precise molecular pathways involved in BMP signaling.
Sequencing analysis of BORG cDNA revealed several unexpected features of BORG cDNA. One of them is the existence of a cluster of multiple interspersed repetitive sequences in the middle part of the cDNA (Fig. 5). A large fraction of the mammalian genome is composed of interspersed repetitive sequences. Most numerous such sequences are the short and long interspersed nucleotide elements represented in the human genome by Alu and L1 sequences, respectively. Mammalian apparent long terminal repeat-retrotransposons form a class of repetitive elements distinct from SINEs and long interspersed nucleotides elements (49). The cluster of multiple interspersed repetitive sequences of BORG cDNA includes one region related to SINE and three regions related to mammalian apparent long terminal repeat-retrotransposons. The significance of the cluster of multiple interspersed repetitive sequences in BORG is unknown. The lack of any extensive ORFs is another feature of BORG. Because of the high density of stop codons in all three reading frames (Fig. 6), even the longest ATG-initiated ORF in BORG cDNA contained only 363 bp with poor contexts for coding a peptide. CTG or ACG also serves naturally as a start codon (51); however, the longest ORF was still the 399-bp CTG-initiated ORF started at nucleotide 875 with the upstream strong ATG codons, again suggesting that it is unlikely to encode a long peptide. These unusual structural features of the cDNA suggest that BORG encodes a noncoding RNA.
Recently, considerable attention has been attracted to two mammalian
genes, H19 (52-54) and Xist (55-57), both of
which function as untranslated RNAs. The structural features of the two
genes resemble that of BORG in that they are spliced, polyadenylated, but have no extensive ORFs. H19 is implicated in imprinting
of the insulin-2 and insulin-like growth factor 2 gene (53), and is
demonstrated to have tumor-suppressor activity (54). Xist RNA acts in the nucleus and is essential for inactivation of most genes
along the X chromosome in female (57). Thus, the characterization of
these two RNA molecules supported a notion that untranslated RNAs can
play important biological roles. In addition, although less
characterized than H19 and Xist, three other
noncoding RNAs have been reported to date. His-1, cloned
from a common retroviral insertion site in murine leukemia
virus-induced myeloid leukemia (58), and bic, cloned from a
common retroviral insertion site in avian leukosis virus-induced B-cell
lymphoma (59), may also encode noncoding RNAs, both implicated in
growth control and oncogenesis. A novel synapse-associated noncoding
RNA, 7H4, has been cloned as a candidate synaptic regulatory molecule
from rat (60). The function of 7H4 in synaptic nuclei remains unknown,
but interestingly, 7H4 cDNA has a short region containing
homologous sequence to B1 SINE in the middle part of the cDNA,
which is a structural feature in common with BORG. Therefore, BORG
might be a new member of a growing unique class of noncoding RNAs.
However, we should also consider a possibility that BORG may encode a
small peptide. The gene product of a Drosophila heat shock
gene, hsr (61), which was suggested to act as a noncoding
RNA molecule, was recently re-evaluated to encode a small peptide of
23-27 amino acids depending on the Drosophila species. In
this case, it was suggested that the translation itself, rather than
the generation of a functional protein product, may be important in
order to monitor the state of translation activity of the cells. In
either case, it is important to identify the functional region in the
transcript of BORG. We are currently cloning the orthologs of mouse
BORG in other species to identify conserved regions that may contribute
to the function of BORG.
Although biological function of BORG remains largely unknown, observations that the induction of BORG by the treatment with BMPs preceded BMP-induced osteoblastic phenotype and that transfection of antisense oligonucleotides of BORG partially inhibited BMP-induced expression of alkaline phosphatase activity (Fig. 8) strongly reminds us of possible key roles of BORG in osteoblast differentiation. However, since we cannot exclude a possibility that the antisense oligonucleotides of BORG also inhibited other genes important for BMP-induced alkaline phosphatase activity, further experiments will be needed to disclose the functional roles of BORG in BMP-induced osteoblastic differentiation.
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ACKNOWLEDGEMENTS |
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We are grateful to M. Asashima, S. Takahashi, H. Okabayashi, and S. Noji for valuable discussion. We thank S. Oida for cDNAs for osteocalcin and GAPDH.
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FOOTNOTES |
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* This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB010885.
§ Present address: Dept. of Biomaterials Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan.
¶ To whom correspondence should be addressed: Dept. of Biomaterials Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. Tel.: 81-3-5803-5471; Fax: 81-3-5803-0192; E-mail: ichijo.det2{at}dent.tmd.ac.jp.
1
The 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-, transforming growth factor-
; bp, base pair(s);
ORF, open reading frame; PCR, polymerase chain reaction; RT, reverse
transcriptase.
2 Smit, A. F. A., and Green, P., RepeatMasker, http://ftp.genome.washington; edu/RM/RepeatMasker.html.
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