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J Bone Miner Metab (2001) 19:345351

Springer-Verlag 2001Original articles

Expression of mouse osteocalcin transcripts, OG1 and OG2,

is differently regulated in bone tissues and osteoblast culturesTakeshi Yanai1,2, Takenobu Katagiri1, Shuichi Akiyama1, Mana Imada1, Takeyoshi Yamashita3,Hiroshige Chiba2, Naoyuki Takahashi1, and Tatsuo Suda1

1 Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan2 Department of Oral and Maxillofacial Surgery, Tokyo Medical University, Tokyo, Japan3 Pharmaceutical Research Laboratory, Kirin Brewery, Gunma, Japan

Introduction

Bone essentially consists of hydroxyapatite crystals and

matrix proteins secreted by osteoblasts. Type I collagenand osteocalcin are the most abundant collagen andnoncollagenous protein, respectively, in bone matrixproteins [1,2]. Osteocalcin is synthesized and secretedby mature osteoblasts and odontoblasts. Three glutamicacid residues of the secreted osteocalcin have beenfound to be -carboxylated by vitamin K-dependent-carboxylase. These -carboxyglutamic acid residues(Gla) function in binding to hydroxyapatite crystalswith a high affinity [3]. Osteoid, which is a nonmine-ralized bone matrix, gradually mineralizes with hy-droxyapatite crystals in developing animals. Circulatingosteocalcin is found in both fetal and adult animals, but

it accumulates in bone only after matrix mineralization[4]. It is known that the administration of warfarin, anantagonist of vitamin K, inhibits the activity of vitaminK-dependent -carboxylase, leading to a loss of Glaresidues in osteocalcin in vivo and in vitro. In warfarin-administered rats, osteocalcin is continuously circulatedin serum, but not accumulated in bone matrix [5]. Dueto its binding activity to hydroxyapatite crystals, osteo-calcin has been thought for a long time to be a regulatorof mineralization of bone matrix.

Recently, three copies of osteocalcin genes, calledOG1/OC-A, OG2/OC-B, and ORG/OC-X, were identi-

fied as a gene cluster within a 23-kb span of the mousegenome [6,7], and their genomic and deduced amino-acid sequences showed a high similarity. Recently, micedeficient for both the OG1 and OG2 alleles were gener-ated by gene targeting in embryonic stem cells [8].These OG1/OG2-deficient mice showed increased boneformation without abnormalities in bone mineraliza-tion. The phenotype of OG1/OG2-deficient mice sug-gests that osteocalcin functions as an inhibitor of boneformation, rather than as a regulator of bone mineral-ization in vivo.

Offprint requests to: T. KatagiriReceived: February 2, 2001 / Accepted: June 4, 2001

Abstract Osteocalcin is a noncollagenous protein that isabundant in mineralized bone matrix. Mice have a gene clus-ter of osteocalcin that consists ofOG1, OG2, and ORG. Weestablished a new method to directly analyze the expression

levels of OG1, OG2, and ORG mRNAs relative to totalosteocalcin mRNA. They were amplified as 371-bp fragmentsby reverse transcription-polymerase chain reaction (RT-PCR)at the same time using common primers, digested withApaLI,and separated in a polyacrylamide gel. ApaLI digestion didnot affect the mobility of the OG1-derived 371-bp fragment,whereas both 371-bp fragments, derived from OG2 and ORG,were digested into 350bp. Total RNA prepared from mousebone was then subjected to RT-PCR followed by ApaLI di-gestion. OG1 and OG2 mRNAs were found to be expressed atratios of 80%86% and 14%20%, respectively, to the totalosteocalcin mRNA in mouse bone. The ratios were almostconstant in various bones in vivo, independent of the animals

genetic background, age, or gender, or different parts of bone.RT-PCR using specific primers revealed that mouse bone tis-sues strongly expressed osteocalcin mRNA derived from OG1and OG2, but not ORG. In contrast, cells cultured in vitroshowed different expression ratios of osteocalcin mRNA:53%65% for OG1 and 35%47% for OG2 to the totalosteocalcin mRNA in the osteoblast cell line and primaryosteoblasts in culture even though they formed many mineral-ized bone nodules. Similar results were obtained in bothKS483 osteoblasts and C2C12 myoblasts, when they were cul-tured with bone morphogenetic protein-2 (BMP-2) to induceosteocalcin mRNA. Taken together, these findings indicatethat OG1 is the predominant transcript among the threeosteocalcin genes in mouse bone in vivo. It is also suggested

that the expression of OG1 and OG2 is regulated differentlyin bone tissues and osteoblast cultures.

Key words osteocalcin gene expression osteoblasts mice

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346 T. Yanai et al.: Expression of osteocalcin transcripts in mice

Because osteocalcin is secreted specifically by bone-forming mature osteoblasts, the expression of osteo-calcin is thought to be a marker for bone formationand osteoblast differentiation. Of the three osteocalcingenes in mice, OG1 and OG2 transcripts were specifi-cally expressed in bone [7]. In contrast, the ORG tran-script was mainly expressed in gonadal tissues, and at

lower levels in several other tissues, in mice [7,9]. Todate, however, the expression levels of the threeosteocalcin genes, especially OG1 and OG2, in bone arenot clear. In the present study, we established a newmethod, using a reverse transcription-polymerase chainreaction (RT-PCR), followed by restriction enzyme di-gestion, to directly analyze the amounts of OG1, OG2,and ORG transcripts relative to the total amount ofosteocalcin mRNA. Using this method, we found thatOG1 was the major transcript, with expression levelshigher than 80% of the total osteocalcin mRNA inmouse bone tissues in vivo. However, OG1 and OG2

were expressed at roughly equal levels in osteoblasticcells tested in culture. These findings suggest that theregulation of the expression of mouse osteocalcinmRNA is different in bone tissues in vivo and in osteo-blast cultures in vitro.

Materials and methods

RNA preparation from animals, and cultured cells

Mice were obtained from Sankyo Laboratories AnimalCenter (Tokyo, Japan). Tibia were obtained from 7-week-old male mice of different genetic backgrounds

(ddY, C57BL/6, BALB/c, and C3H/He). Seven-week-old female ddY mice were also used as a sourceof tibia. Calvaria were obtained from newborn ddYmice. Kidney, muscle, and liver were obtained from 7-week-old male ddY mice. The tissues obtained werecleaned, washed and minced in ice-cold phosphate-buffered saline, and then homogenized in Trizol(GIBCO BRL, Grand Island, NY, USA) to extract totalRNA.

A mouse osteoblastic cell line, KS483 [10], and amouse myoblast cell line, C2C12, were cultured in -minimal essential medium (-MEM) containing 10%

fetal bovine serum (FBS; Iwaki, Chiba, Japan) andDulbeccos modified Eagles medium (DMEM) con-taining 15% FBS, respectively. To examine the effect ofbone morphogenetic protein-2 (BMP-2) on the expres-sion of osteocalcin mRNA, these cell lines were cul-tured with or without 300ng/ml of recombinant humanBMP-2 (kindly supplied by Yamanouchi Pharmaceuti-cals, Tokyo, Japan). Primary osteoblasts were preparedfrom newborn mouse calvaria by sequential collage-nase-dispase digestion, as described previously [11].The cells released as fractions IIIV were pooled and

inoculated with -MEM containing 10% FBS withoutsubcultures. Total cellular RNA was extracted fromthese cultured cells, using Trizol (GIBCO BRL).

Quantification of the expression levels of OG1,

OG2, and ORG relative to the total amount of

osteocalcin mRNA

Twenty micrograms of total RNA was treated withRNase-free DNase I (Takara Biochemicals, Shiga, Ja-pan), then extracted with Trizol again. First-strandcDNA was synthesized from 10g of DNase I-treatedRNA, using Superscript II (GIBCO BRL) and randomprimers in a final volume of 20l. One microliter of thereaction mixture was subjected to PCR amplificationwith EX Taq DNA polymerase (Takara Biochemicals)in the presence of [-33P]dCTP, in a final volume of50l. Primer sets used in PCR for total osteocalcin,ORG, and glyceraldehyde-3-phosphate dehydrogenase

(GAPDH) were: total osteocalcin, 5-CAAGTCCCACACAGCAGCTT-3 (primer A) and 5-AAAGCCGAGCTGCCAGAGTT-3 (primer B) [7];ORG, 5-AGCATTCTGGCCTTCTGGTG-3 (primerC) and primer B; and GAPDH, 5-TGAAGGTCGGTGTGAACGGATTTGGC-3 and 5-CATGTAGGCCATGAGGTCCACCAC-3, respectively. PCR con-ditions were 1min at 94C, 1min at 60C, and 1min at72C for 25 cycles (total osteocalcin and GAPDH), andthe same conditions for 30 cycles for ORG. The prod-ucts amplified in PCR were resolved by electrophoresisin a 5% polyacrylamide gel. The PCR products for

total osteocalcin were precipitated in ethanol withEthachinmate (Nippon Gene, Tokyo, Japan), and dis-solved in a digestion buffer for ApaLI. The mixturewas separated into two tubes and then incubated forat least 2h at 37 C with and without ApaLI (TakaraBiochemicals) before electrophoresis. The gels weredried and exposed to X-ray films at 80C. The radio-activity of each band on the membrane was quantifiedwith a Bioimage Analyzer BAS2000 (Fuji Film, Tokyo,Japan).

To analyze the DNA sequence of the PCR products,the 371-bp fragment was amplified from bone andkidney cDNAs of 7-week-old ddY mice by RT-PCR,

as described above, and subcloned in pGEM-T Easy(Promega, Madison, WI, USA). The sequence of theinsert DNA was analyzed with a GeneRapid DNAsequencer (Amersham Pharmacia Biotech, Buckingh-amshire, UK). Plasmids carrying OG1, OG2, and ORGcDNAs (pGEM-T/OG1, pGEM-T/OG2, and pGEM-T/ORG, respectively) were used as positive controls forthe total osteocalcin in PCR, as described above.

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T. Yanai et al.: Expression of osteocalcin transcripts in mice 347

Northern blot analysis

Northern blot analysis was performed as described pre-viously [12]. In brief, 20g of total RNA was resolvedby electrophoresis in a 1.2% agarose-formaldehydegel, and transferred onto a Hybond-N membrane(Amersham International, Amersham, UK). The mem-brane was sequentially hybridized with [32P]-labeledcDNA probes for mouse OG1 as osteocalcin andGAPDH. In some experiments, additional OG2and ORG probes were used. Probes for OG1, OG2, andORG were obtained by EcoRI digestion from pGEM-T/OG1, pGEM-T/OG2, and pGEM-T/ORG, re-spectively, as described above.

Results

Cloning of mouse OG1, OG2 and ORG cDNAs

First, we obtained cDNA clones encoding OG1, OG2,and ORG by RT-PCR from tibia and kidney of ddYmice, then we subcloned and sequenced them in plas-mid vectors (Fig. 1). The DNA sequence showed that,of 35 clones obtained from tibia, 27 were for OG1, 8were for OG2, and none for ORG. In contrast, 5 of 5clones obtained from the kidney were for ORG. Therewere one or two nucleotide substitutions in osteocalcincDNAs: OG1 (314C to T), OG2 (266C to G and 297Gto A), and ORG (118C to G) from the sequencesderived from 129Sv mice, which have been depositedon a database. The substitutions in OG2 and ORG

caused amino-acid changes, from Asp75 to Glu andAla86 to Thr, and Thr26 to Ser, respectively. Thenucleotide substitution in ORG destroyed a Cfr10 Irestriction site.

Establishment of a method to analyze the expression

levels of OG1, OG2, and ORG in the total amount of

osteocalcin mRNA

Next, we examined the expression levels of OG1, OG2,and ORG in mouse tissues by Northern blotting. Asshown in Fig. 2A, all of the probes for OG1, OG2, andORG hybridized with ~0.6kb mRNA in bone, but nosignals were detected in the kidney, skeletal muscle, andliver.

We then attempted PCR for total osteocalcin, usingcommon primers A and B, which amplify OG1, OG2,and ORG equally, followed by ApaLI digestion (Fig.2B). Plasmids carrying OG1, OG2 and ORG cDNAswere used as the templates. We obtained a single 371-bpband from each of the plasmids (Fig. 2C). No fragmentswere observed from an empty vector (data not shown).When the PCR products were treated with ApaLI, the371-bp fragments derived from OG2 and ORG werecompletely digested into a 350-bp length (Fig. 2C). In

contrast, ApaLI digestion did not alter the 371-bpfragment amplified from OG1. These findings indicatedthat an OG1-derived product can be separated from themixture of OG1, OG2, and ORG-derived products bydetecting the 371-bp fragment afterApaLI digestion.

This method was employed for measuring the expres-sion level of OG1 relative to total osteocalcin mRNA inmouse tissues. As shown in Fig. 2D, the 371-bp frag-ment was amplified from bone RNA on RT-PCR, usingcommon primers A and B. A portion of this fragmentwas digested into a 350-bp fragment by ApaLI diges-tion. Quantification of the radioactivity in these two

fragments showed that the ratios of the 371-bp and 350-bp fragments to the total were approximately 80% and20%, respectively. A longer period ofApaLI digestion(4h) did not affect the relative amounts of the two

Fig. 1. Alignment of OG1, OG2, and ORG cDNAs. TotalRNA was prepared from tibia and kidney of 7-week-old maleddY mice, and subjected to reverse transcription-polymerasechain reaction (RT-PCR), using primers A and B. A 371-bpfragment was subcloned then sequenced in plasmid vectors.

OG1 and OG2 cDNAs were obtained from tibia and ORGcDNA was obtained from the kidney. Identical bases in OG1,OG2, and ORG are boxed. Primers A and B are shown byarrows.ApaLI sites (gtgcac) in OG2 and ORG are underlined

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fragments (data not shown). We specifically amplifiedORG on RT-PCR, using a specific primer C, and acommon primer B (Fig. 2B). A very faint band, 546bp

in length, was observed in bone and kidney, but not inskeletal muscle and liver under the present PCR condi-tions (Fig. 2D). In agreement with the findings in aprevious study [7], the expression level of the 546-bpfragment in bone was lower than that in the kidney.

OG1 is the major transcript in total osteocalcin

mRNA in mouse bone in vivo

We further examined the expression levels of OG1,OG2, and ORG in tibia prepared from 7-week-old male

mice of various genetic backgrounds, including C57BL/6, BALB/c, C3H/He, and ddY. Osteocalcin mRNA wasdetected in all strains examined by Northern blotting

and the amplification of the 371-bp fragment on RT-PCR (Fig. 3A). The 350-bp fragment appeared afterApaLI digestion in all samples. In these mice, the ratiosof the 371-bp, and 350-bp fragments to the total amountof osteocalcin mRNA were 80.0%86.2% and 13.8%20.0%, respectively (Fig. 3B). Similar findings wereobserved not only in tibia prepared from 7-week-oldfemale ddY mice but also in calvaria prepared fromnewborn ddY mice (Fig. 3A,B). ORG-specific PCRamplified an extremely faint 546-bp fragment in all ofthe samples.

Fig. 2. Establishment of a method to directly compare theexpression ratios of OG1, OG2, and ORG transcripts to thetotal amount of osteocalcin mRNA. A Northern blot analysisfor OG1, OG2, and ORG. Total RNA was prepared fromtibia (T), kidney (K), skeletal muscle (M), and liver (L) of 7-week-old male ddY mice. Twenty micrograms of total RNAwas resolved by electrophoresis in a 1.2% agarose gel, and

transferred onto a membrane. The membrane was hybridizedwith cDNA probes for OG1, OG2, ORG, and GAPDH.B Schematic structure of OG1, OG2, and ORG cDNAs, 371-bp in length, amplified on RT-PCR. Coding regions are shownby solid boxes. Primers A, B, and C used for RT-PCR areshown by arrows. Positions of the ApaLI site in OG2 andORG are shown by open triangles. C Characterization of a371-bp fragment amplified separately from OG1, OG2, and

ORG cDNAs. PCR was performed with primers A and B,using pGEM-T/OG1, pGEM-T/OG2, and pGEM-T/ORG astemplates. The PCR products were digested with (w/) andwithout (w/o)ApaLI before electrophoresis in a 5% polyacry-lamide gel. Note that both OG2 and ORG were digested into350-bp fragments byApaLI, but OG1 was not. D Analysis ofthe expression levels of osteocalcin transcripts in total

osteocalcin mRNA. Total RNA was prepared from tibia (T),kidney (K), skeletal muscle (M), and liver (L) of 7-week-oldmale ddY mice. DNase I-treated total RNA was transcribedinto cDNA, using random primers, and subjected to PCR,using primers A and B for osteocalcin, primers C and B forORG, and specific primers for GAPDH. The PCR productsfor osteocalcin were digested with (w/) and without (w/o)ApaLI before electrophoresis in a 5% polyacrylamide gel

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OG1 and OG2 are expressed equally in osteoblast

cultures in vitro

Finally, we examined the expression levels of OG1,OG2, and ORG in osteoblast cultures. Primary osteo-blasts were prepared from the calvaria of newborn ddYmice, which expressed predominantly OG1 mRNA invivo (Fig. 3). The expression of osteocalcin mRNA byprimary osteoblasts began before day 4, reached a peak

at day 6, and gradually decreased thereafter (Fig. 4A).RT-PCR followed byApaLI digestion showed that theratios of the 371-bp and 350-bp fragments to the totalamount osteocalcin of mRNA were 54.2%66.5% and34.5%45.8%, respectively (Fig. 4A,B). We also usedthe osteoblastic cell line, KS483 cells, which expressesosteocalcin mRNA and forms many mineralized bonenodules in vitro [10]. In KS483 cells, the ratio of the 371-bp fragment to the total osteocalcin mRNA was 53.2%62.4% and that of the 350-bp fragment was 37.6%46.8% (Fig. 4A,B). Treatment with BMP-2 for 3 days

induced the expression of amount of osteocalcinmRNA in both KS483 osteoblasts and C2C12 myoblasts(Fig. 4A). In the presence of BMP-2, the ratios of the371-bp fragment to the total amount of osteocalcinmRNA were 46.5% in KS483 cells and 57.5% in C2C12cells (Fig. 4A,B).

Discussion

In the present study, we established a new method todirectly examine the expression levels of OG1, OG2,and ORG mRNAs in mice. We were able to amplifythese cDNAs as 371-bp fragments on RT-PCR, usingcommon primers A and B [7]. When we treated the371-bp fragments with ApaLI, OG2 and ORG weredigested into 350-bp fragments, but OG1 was not. Thismethod allowed us to separate OG1 and OG2/ORG inthe 371-bp fragments mixture. The 350-bp fragment

Fig. 3. OG1 is the major transcript in the total amount ofosteocalcin mRNA in mouse bone in vivo. A Tibias (T) wereobtained from 7-week-old male (M) and female (F) mice ofvarious genetic backgrounds, including C57BL/6, BALB/c,C3H/He, and ddY. Newborn ddY mice (NB) were examinedfor the preparation calvaria (C). Twenty micrograms of totalRNA extracted from bones was studied in Northern blot

analysis (two upper panels). DNase I-treated total RNAwas subjected to RT-PCR for total osteocalcin, ORG, and

GAPDH (four lower panels). The PCR products for totalosteocalcin were digested with (w/) and without (w/o)ApaLIbefore electrophoresis in a 5% polyacrylamide gel. B Quanti-fication of two fragments, of 371bp and 350bp, in A. Theradioactivity of the two fragments, of 371bp (open bars) and350bp (closed bars) after ApaLI digestion was determinedwith a Bioimage Analyzer (BAS2000; Fuji Film, Tokyo,

Japan)

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must be derived mainly from OG2 in bone, because theexpression level of ORG was essentially zero whenevaluated by the ORG-specific RT-PCR. This was fur-

ther confirmed by the direct DNA sequencing of the371-bp fragments. Taken together, these findings sug-gest that the new method established in this study isuseful for analyzing, simply and directly, the ratios ofOG1 and OG2/ORG transcripts relative to the totalamount of osteocalcin mRNA.

Using this method, we examined the relative expres-sion levels of OG1 and OG2 in mice in vivo and in vitro.In bone tissues, the OG1 transcript was always detectedat a level of greater than 80% of the total osteocalcinmRNA. Similar findings were observed in all mice ex-amined regardless of the various genetic backgrounds,

age, and gender, and regardless of different parts ofbone. These findings indicated that OG1 was the majortranscript among the three osteocalcin genes in mousebone tissues in vivo. There are three amino acid substi-tutions in the signal peptides of OG1 and OG2. Becausethe signal peptide is essential for protein secretion, it ispossible that the difference affects the efficiency of OG1and OG2 secretion by osteoblasts. The development ofantibodies that can recognize the difference betweenOG1 and OG2 proteins would help to clarify thisquestion.

Fig. 4. OG1 and OG2 are expressed equally in osteoblasticcell cultures in vitro. A Primary osteoblasts (POB), KS483osteoblasts, and C2C12 myoblasts were cultured with or with-out 300 ng/ml of bone morphogenetic protein (BMP)-2 for theindicated periods. Total RNA was extracted from these cellsat each time point. Twenty micrograms of total RNA wasexamined in Northern blot analysis (two upper panels). DNaseI-treated total RNA was subjected to RT-PCR for total

osteocalcin, ORG, and GAPDH (four lower panels). ThePCR products for total osteocalcin were digested with (w/)and without (w/o)ApaLI before electrophoresis in a 5% poly-acrylamide gel. B Quantification of two fragments, of 371bpand 350bp, in A. Radioactivity of the two fragments, of 371bp(open bars) and 350bp (closed bars) afterApaLI digestion wasdetermined using the Bioimage Analyzer (BAS2000; FujiFilm). N.D., Not determined

In contrast to the in vivo situation, osteoblastic cellsexpressed roughly equal amounts of OG1 and OG2transcripts in culture. These findings were observed not

only in a clonal osteoblastic cell line (KS483 cells) butalso in primary osteoblasts, which predominantly ex-pressed the OG1 transcript in vivo. We have reportedthat BMP-2 induces osteoblast differentiation of themouse myoblast cell line, C2C12 [12]. In C2C12, as wellas KS483 cells, osteocalcin mRNA induced by BMP-2consisted of roughly equal amounts of OG1 and OG2mRNAs. The reason for the different expression levelsof OG1 and OG2 in vivo and in vitro is not clear atpresent, but there are several possible explanations forthis. There may be two types of osteocalcin-expressingcells in bone in vivo. One type of cell expresses almost

equal amounts of OG1 and OG2 mRNA, as seen incultured cells. Another type of cell predominantly ex-presses OG1 mRNA in vivo. The latter may have poorproliferating capacity in vitro. Thiede et al. [13] re-ported that megakaryocytes in bone marrow and plate-lets in peripheral blood expressed osteocalcin mRNA,in rats and humans. These cells present in mouse bonetissues may affect the expression levels of OG1 andOG2 in vivo. It is necessary to examine whether thesecells also express osteocalcin mRNA in mice in vivo andin vitro. It is also possible that a factor(s) that stimulates

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the expression of OG1 in vivo may be deficient in cul-ture conditions. Conversely, the levels of an inhibitor(s)of OG1 expression may be different in in vivo and invitro conditions. Further studies are necessary to clarifythese issues.

In bone cells, Runx 2/Cbfa-1/Osf-2 has been identi-fied as a transcription factor required for the bone-

specific expression of osteocalcin [1416]. The OSE2,which is a Runx 2 consensus sequence in the osteocalcinpromoter, is present at the same regions in both OG1and OG2 promoters [17]. Furthermore, these two pro-moters have very similar DNA sequences, at least up to1.0kb [17]. The present findings, however, showedthat the expression of OG1 was predominant in mousebone in vivo. These findings suggest that the OG1 pro-moter has additional regulatory elements, which arerecognized by some unidentified transcriptional factors,rather than by Runx 2. All these factors may be re-quired for the full expression of osteocalcin mRNA

in mice in vivo. It was reported that BMP-2 inducedosteocalcin expression in Runx 2 (/) cells in vitro[18]. It would, therefore, be interesting to examine theratio of OG1 and OG2 mRNAs in Runx 2 (/) cellsstimulated by BMP-2. Further studies are necessary toclarify the molecular mechanisms of osteocalcin mRNAexpression.

In conclusion, we have developed a new method todetermine directly the expression levels of mouse OG1,OG2, and ORG transcripts relative to the total amountof osteocalcin mRNA. Using this method, we foundthat OG1 was the major transcript among the threeosteocalcin mRNAs in mouse bone in vivo. However,

OG1 and OG2 were almost equally expressed in osteo-blasts in vitro. These findings suggest that there is anovel mechanism regulating the full expression ofosteocalcin mRNA in vivo.

Acknowledgments. We thank Yamanouchi Pharmaceu-tical, Japan for their kind supply of BMP-2. This workwas supported in part by a Grant-in-Aid from theMinistry of Science, Education, and Culture of Japan(11771146) and a Grant-in-Aid for young researchersfrom the Kitasato University Alumni Association (toT. K.).

References

1. Hauschka PV, Lian JB, Cole DEC, Gundberg CM (1989)Osteocalcin and matrix Gla protein: vitamin K-dependent pro-teins in bone. Physiol Rev 69:9901047

2. Desbois C, Karsenty G (1995) Osteocalcin cluster: implicationsfor functional studies. J Cell Biochem 57:379383

3. Poser JW, Price PA (1979) A method for decarboxylation of-carboxyglutamic acid in proteins. J Biol Chem 254:431436

4. Price PA, Lothringer JW, Nishimoto SK (1980) Absence of thevitamin K-dependent bone protein in fetal rat mineral: evidencefor another -carboxyglutamic acid containing component inbone. J Biol Chem 255:29382942

5. Price PA, Williamson MK (1981) Effects of warfarin on bone. J

Biol Chem 256:12754127596. Rahman S, Oberdorf A, Montecino M, Tanhauser SM, Lian JB,

Stein GS, Laipis PJ, Stein JL (1993) Multiple copies of thebone-specific osteocalcin gene in mouse and rat. Endocrinology133:30503053

7. Desbois C, Hogue DA, Karsenty G (1994) The mouse osteocalcingene cluster contains three genes with two separate spatial andtemporal patterns of expression. J Biol Chem 269:11831190

8. Ducy P, Desbois C, Boyce B, Pinero G, Story B, Dunstan C,Smith E, Bonadio J, Goldstein S, Gundberg C, Bradley A,Karsenty G (1996) Increased bone formation in osteocalcin-deficient mice. Nature 382:448452

9. Sato M, Tada N (1995) Preferential expression of osteocalcin-related protein mRNA in gonadal tissues of male mice. BiochemBiophys Res Commun 215:412421

10. Yamashita T, Ishii H, Shimoda K, Sampath TK, Katagiri T, WadaM, Osawa T, Suda T (1996) Subcloning of three osteoblastic celllines with distinct differentiation phenotypes from the mouseosteoblastic cell line KS-4. Bone 19:429436

11. Komaki M, Katagiri T, Suda T (1996) Bone morphogeneticprotein-2 does not alter the differentiation pathway of committedprogenitors of osteoblasts and chondroblasts. Cell Tissue Res284:917

12. Katagiri T, Yamaguchi A, Komaki M, Abe E, Takahashi N, IkedaT, Rosen V, Wozney JM, Fujisawa-Sehara A, Suda T (1994) Bonemorphogenetic protein-2 converts the differentiation pathway ofC2C12 myoblasts into the osteoblast lineage. J Cell Biol 127:17551766

13. Thiede MA, Smock SL, Petersen DN, Grasser WA, ThompsonDD, Nishimoto SK (1994) Presence of messenger ribonucleic acidencoding osteocalcin, a marker of bone turnover, in bone marrow

megakaryocytes and peripheral blood platelets. Endocrinology135:92993714. Geoffroy V, Ducy P, Karsenty G (1995) A PEBP2/AML-1 related

factor increases osteocalcin promoter activity through its bindingto an osteoblast-specific cis-acting element. J Biol Chem270:3097330979

15. Banerjee C, Hiebert SW, Stein JL, Lian JB, Stein GS (1996) AnAML-1 consensus sequence binds an osteoblast-specific complexand transcriptionally activates the osteocalcin gene. Proc NatlAcad Sci USA 93:49684973

16. Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G (1997)Osf2/Cbfa1: a transcriptional activator of osteoblast differentia-tion. Cell 89:747754

17. Ducy P, Karsenty G (1995) Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene.Mol Cell Biol 15:18581869

18. Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, DeguchiK, Shimizu Y, Bronson RT, Gao YH, Inada M, Sato M, OkamotoR, Kitamura Y, Yoshiki S, Kishimoto T (1997) Targeted disrup-tion of Cbfa1 results in a complete lack of bone formation owingto maturational arrest of osteoblasts. Cell 89:755764

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