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Endocrinology Vol. 145, No. 10 4685-4692
Copyright © 2004 by The Endocrine Society

Spaciotemporal Association and Bone Morphogenetic Protein Regulation of Sclerostin and Osterix Expression during Embryonic Osteogenesis

Yoshio Ohyama, Akira Nifuji, Yukiko Maeda, Teruo Amagasa and Masaki Noda

Department of Molecular Pharmacology (Y.O., A.N., Y.M., M.N.), Medical Research Institute, Integrated Action Initiative in Japan Society for Promotion of Science Core to Core Program (M.N.); Department of Maxillofacial Surgery (T.A.), Tokyo Medical and Dental University; and Center of Excellence (COE) Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone (Y.O., A.N., Y.M., M.N.), Tokyo 101-0062, Japan

Address all correspondence and requests for reprints to: Masaki Noda, Department of Molecular Pharmacology, Tokyo Medical and Dental University, 3-10 Kanda-Surugadai 2-Chome, Chiyoda-ku, Tokyo, Japan; or Akira Nifuji, Department of Molecular Pharmacology, Tokyo Medical and Dental University, 3-10 Kanda-Surugadai 2-Chome, Chiyoda-ku, Tokyo, Japan. E-mail noda.mph{at}mri.tmd.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Sclerostin (SOST), a member of the cystine-knot superfamily, is essential for proper skeletogenesis because a loss-of-function mutation in the SOST gene results in sclerosteosis featured with massive bone growth in humans. To understand the function of SOST in developmental skeletal tissue formation, we examined SOST gene expression in embryonic osteogenesis in vitro and in vivo. During osteoblastic differentiation in primary calvarial cells, the levels of SOST expression were increased along with those of alkaline phosphatase activity and nodule formation. In situ hybridization study revealed that SOST mRNA expression was observed in the digits in embryonic 13-d limb buds, and SOST expression was observed in osteogenic front in embryonic 16.5-d postcoitus embryonic calvariae, and this expression persisted in the peripheral area of cranial bone in the later developmental stage (embryonic 18.5-d post coitum). These temporal and spacial expression patterns in vivo and in vitro were in parallel to those of osterix (Osx), which is a critical transcriptional factor for bone formation. Similar coexpression of SOST and Osx mRNA was observed when the primary osteoblastic calvarial cells were cultured in the presence of bone morphogenetic protein (BMP)2 in vitro. Moreover, endogenous expression of SOST and Osx mRNA was inhibited by infection of noggin-expression adenovirus into the primary osteoblastic calvarial cells, suggesting that endogenous BMPs are required for these cells to express SOST and Osx mRNA. Thus, expression and regulation of SOST under the control of BMP were closely associated with those of Osx in vivo and in vitro.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SKELETAL DEVELOPMENT IS under the concerted control by multiple factors, cytokines, and hormones. During embryonic skeletogenesis, precursor cells form the cartilage anlage, which then leads to the formation of bone primordium. In this primordium, osteoblastic cells differentiate from the precursor cells and then express a variety of signaling molecules to stimulate themselves as well as the surrounding cells. Osteoblastic cells then deposit bone matrix proteins to form bone (1). One of such important signaling molecules during development bone is bone morphogenetic protein (BMP). Recently in addition to BMPs as ligands, BMP inhibitors have been shown to be expressed during bone development. The importance of these ligands and inhibitors has been shown by the fact that loss-of-function mutations in either BMP ligands or such BMP inhibitors resulted in severe skeletal defects. For instance, a point mutation in cartilage-derived morphogenetic proteins 1 (or human BMP14) causes chondrodysplasia Grebe type, this disease is characterized by severe limb shortening and dysmorphogenesis of the skeleton (2). Mutation in another BMP inhibitor, noggin, causes proximal symphalangism and multiple synostosis syndromes (3).

Downstream to such cytokines including BMP osteoblastic differentiation is under the control of critical transcriptional factors such as runt-related gene (Runx2) and osterix (Osx). Runx2 deficiency results in complete lack of bone (4). Osx has been considered to be downstream to Runx2, and this zinc finger transcription factor is also essential for bone formation because null mutation for Osx gene causes the disappearance of osteoblast (5).

Sclerostin (SOST) is a recently identified BMP antagonist, and mutation in this gene causes bone dysplasia called sclerosteosis (6, 7). This is an autosomal recessive disorder characterized by gigantism, widening of skull, and distortion of the face. In sclerosteosis patients each of the bone elements are severely sclerotic and x-ray indicates high bone density due to excessive mineral deposition (7). Such high activity of bone formation is reflected by the elevated alkaline phosphatase activity levels in serum.

SOST is a member of the novel cystine knot protein superfamily (8) and shares its characteristic motif with other BMP inhibitors such as dan, gremlin, and cerberus. Recently SOST has been reported to be expressed in the surface of the intramembranous as well as the endochondral bone and has been shown to be expressed in osteoclast (9). SOST binds to BMP6 and BMP7 with high affinity but not so much to BMP2 and BMP4 and decreases alkaline phosphatase expression levels induced by BMPs in MC3T3E1 cells (9).

In this study we examined SOST expressions during skeletal development in terms of its relationship to Osx. We found that in response to BMP, these two molecules are coexpressed during embryogenesis in vivo and in calvarial cells in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
Embryos were collected from mating ICR outbred mice. All animal experimental designs and procedures were approved by the Animal Ethic Committee of Tokyo Medical and Dental University.

Cell isolation and primary cultures of osteoblastic cells
Calvarial cells (osteoblast-enriched cells) were isolated by sequential enzymatic digestions from calvariae of 15.5-d post conception (E) embryos and 10-wk-old adult mouse using 1 mg/ml collagenase and 250 U/ml dispase. Digestion was conducted at 37 C, and sequential six digestions were conducted for each of 20 min. Digestion was carried out, and their fractions of the cells obtained from the third trough six digestions were collected. The cells were then plated at 10,000 cells/cm2 in {alpha}MEM medium supplemented with 10% fetal bovine serum. To induce osteoblastic differentiation, the medium was changed to the fresh medium, and the cells were cultured in the presence of 50 µg/ml ascorbic acid (AA) and 10 mM ß-glycelophosphate (ßGP) after cells reached confluency (d 0). Subsequently the cells were incubated for the indicated period of time with the change of the medium every 3 d. For the set of the cells treated with BMP, the cells were cultured in the presence of 100 ng or 400ng/ml recombinant human BMP2. BMP2 treatment was conducted for 24 h using the cells at 70% confluency.

Cytokines
Human recombinant BMP2 was obtained from Yamanouchi Pharmaceuticals (Tokyo, Japan). Recombinant human BMP2 was dissolved in buffer, including 2.5% glycine, 0.5% sucrose, 5 mM sodium chloride, and 0.01% polysorbate 80 (pH4.5).

Alkaline phosphatase activity assay
The calvarial cells were cultured as indicated above and indicated time points, and the cells were rinsed twice with 0.9% NaCl and scraped into a buffer containing 10 mM Tris-HCl, 2 mM MgCl2, and 0.05% Triton X-100 (pH 8.2). The cell lysates were briefly sonicated on ice after two cycles of freezing and thawing. The cell lysates were then mixed with the aliquot of assay mixture containing 2.2 mM p-nitrophenyl phosphate in 0.1 M 2-amino-2-metyl-1-propanol and 2.2 mM MgCl2 (pH 10.5). The mixtures were incubated at 37 C for 7 min, and the reaction was stopped by the addition of 1 M NaOH. After the incubation the amount of p-nitrophenol irrigated by the reaction was measured by a spectrophotometer at 415 nm. The lysates were also subjected to protein determination by Coomassie blue G staining according to the method described elsewhere (10). The protein levels were determined after measurement of the light absorption at 595 nm (11).

In vitro nodule formation assay
Calvaria-derived cells were cultured for 14 d in the presence of ascorbic acid and ß-glycelophosphate (osteoblastic differentiate medium). After the culture the wells were stained for mineralization used based on Von Kossa or alizarin red methods. The cell cultures were rinsed with PBS and then fixed with 0.2% glutaraldehyde for 15 min followed by subsequently rinsing with water. For the cultures subjected to Von Kossa staining, the cultures were dehydrated after 100% ethanol and then rehydrated to water, and then the cells were stained by incubating in 2% AgNO3 (silver nitrate) for 10 min and exposed to sunlight. The reaction was stopped with a 5% sodium thiosulfate solution. For alizarin red staining, the cells were stained with 1% alizarin red solution for 20 min followed by several rinsings with water.

RNA isolation
Total RNA was isolated from mouse calvarial cells according to acid guanidinium-phenol-chloroform methods (12). The RNA was isolated from mouse kidney. Briefly, the cells or tissues were homogenized in solution D containing 4 M guanidinium isothiocyanate, 25 mM sodium citrate (pH7.5), 0.5% sodium, 10% N-lauroyl-sarcosine (Sarcosyl), and 0.1 M 2-mercaptoethanol. The homogenates were mixed with 1 volume of water-saturated phenol, 0.2 volume chlorofolm-iso-amyl alcohol mixtures (49:1), and 0.1 volume 2 M sodium acetate (pH 4.0). After mixing and centrifugation, the aqueous phase was transferred to a new tube and precipitated after mixing with 1 volume of isopropanol. After centrifugation the pellet was resuspended in solution D followed by precipitation in the mixture with the isopropanol. The final pellet was resuspended in the solution containing 10 mM Tris and 0.1 mM EDTA.

Reverse transcription (RT) and RT-PCR
Aliquots of RNA 1 µg were incubated with oligo dT primer (Roche, Stockholm, Sweden) at 70 C for 10 min. Then annealing was conducted by leaving the mixture at room temperature for 5 min. The annealed product was then subjected to reveres transcription by using 200 U SuperScript II RNase H (Life Technologies Inc., Grand Island, NY) according to the manufacturer’s instruction. The RT was conducted in the solution, containing 5x first-strand synthesis buffer [0.1 M DDT, 10 mM nucleotide triphosphate mixture, and 40 U RNase inhibitor (Promega, Madison, WI)]. The reaction mixture was incubated at 50 C for 50 min to proceed RT reaction, followed by incubation at 70 C for 15 min to stop the reaction.

PCR was conducted by using 25 ng cDNA that was amplified by following cyclic treatment: 94 C for 30 sec, 62 C for 30 sec, and 72 C for 90 sec. LA Taq DNA polymerase (TAKARA, Shiga, Japan) was used for SOST amplification and TAKARA Taq (TAKARA) was used for others. The numbers of PCR cycles were 22 cycles for glyceraldehyde-3-phosphate dehydrogenase (GAPDH); 28 cycles for osteocalcin (OC); 30 cycles for noggin, chordin, and Osx; and 32 cycles for SOST, respectively.

The primers used were as follows: GAPDH, 5'-ACCACAGTCCATGCCATCAC-3' (550–569) (sense), 5'-TCCACCACCCTGTTGCTGTA-3' (982–1001) (antisense), amplified fragment length, 452 bp; OC, 5'-CTCTGTCTCTCTGACCTCACAG-3' (43–64) (sense), 5'-CAGGTCCTAAATAGTGATACCG-3' (251–272) (antisense), amplified fragment length, 230 bp; SOST, 5'-GACTGGAGCCTGTGCTACCGAGTGC-3' (–43 to –19) (sense), 5'-CTAGTAGGCGTTCTCCAGCTCCGCCT-3' (610–636) (antisense), amplified fragment length, 679 bp; alkaline phosphatase (ALP), 5'-ATTGCCCTGAAACTCCAAAACC-3' (279–290) (sense), 5'-CCTCTGGTGGCATCTCGTTATC-3' (717–738) (antisense), amplified fragment length, 460 bp; Osx, 5'-CTGGGGAAAGGAGGCACAAAGAAG-3' (214–238) (sense), 5'-GGGTTAAGGGGAGCAAAGTCAGAT-3' (663–687) (antisense), amplified fragment length, 474 bp; Runx2, 5'-GAACCAAGAAGGCACAGACA-3' (901–920) (sense), 5'-AACTGCCTGGGGTCTGAAAA-3' (1333–1352) (antisense), amplified fragment length, 452 bp; and chordin, 5'-CAAGCCTCAGCGGAAGAACCAG-3' (1380–1401) (sense), 5'-TCTTTTACCACGCCCTGAGCCT-3' (1826–1847) (antisense), amplified fragment length, 468 bp.

Real-time PCR
PCR contained 100 ng template (cDNA), 2.5 µM each forward and reverse primer, and 2x SybrGreen PCR Master Mix (Applied Biosystems, Foster City, CA) in 25 µl. Samples were amplified for 50 cycles in an ABI Prism 7700 sequence detection system (Applied Biosystems) with an initial melt at 95 C for 10 min followed by 50 cycles of 95 C for 15 sec, 60 C for 30 sec, and 78 C for 40 sec. PCR product accumulation was monitored at multiple points during each cycle by measuring the increase in fluorescence caused by the binding of SybrGreen I to double-stranded DNA. Postamplification melting curves were performed to confirm that a single PCR product was produced in each reaction. The relative amount of gene transcript present at different time points was calculated and normalized by dividing the calculated value for the gene of interest by GAPDH value at that particular time point. At least two independent experiments from cell culture to PCR were conducted, and each PCR was performed three times.

For Real time PCR, the primers used were as follows: GAPDH, 5'-GCCAAACGGGTCATCATCTC-3' (368–387) (sense), 5'-GTCATGAGCCCTTCCACAAT-3' (548–67) (antisense), amplified fragment length, 200 bp; SOST, 5'-AAGCCGGTCACCGAGTTGGT-3' (286–305) (sense), 5'-GTGAGGCGCTTGCACTTGCA-3' (468–487) (antisense), amplified fragment length, 202 bp; Osx, 5'-CTACCCAGCTCCCTTCTCAA-3' (180–199) (sense), 5'-CTTGTACCACGAGCCATAGG-3' (373–392) (antisense), amplified fragment length, 213 bp; and OC, 5'-CAAGCAGGAGGGCAATAAGG-3' (442–461) (sense), 5'-CCGTAGATGCGTTTGTAGGC-3' (796–815) (antisense) amplified fragment length, 374 bp.

Statistical analysis
The data were expressed as means ± SEM, and evaluated according to Fisher’s protected least significant difference analysis.

Isolation of mouse SOST, Osx, and chordin cDNA
To isolate cDNA fragments of SOST, Osx, and chordin, we recovered amplification products and subcloned them into pCR-Script SK (+) (Stratagene, La Jolla, CA). For SOST the cDNAs were synthesized using total cellular RNA isolated from mouse kidney. For Osx and chordin, total RNA isolated from primary osteoblastic cells was used. The primers used for Osx cDNA cloning were the sense primer 5'-TTCTCCCATTCTCCCTCCCTCTCCCTTCTC-3' (–98 to –69), the antisense primer 5'-GCTCTCTCCTATTGCATGCTATACTCTGGG-3' (1309–1338) nucleotide sequences. The primers for SOST and chordin cDNA were the same as described in Materials and Methods (RT and RT-PCR). After subcloning PCR products into the PCR vector, their nucleotide sequences were confirmed by sequencing.

Whole-mount in situ hybridization
The whole-mount in situ hybridization (WISH) was conducted as described previously (13) with a minor modification. Briefly, embryos were fixed overnight in 4% paraformaldehyde in PBS at 4 C. The embryos were dehydrated through a series of methanol/PBS containing 0.1% Tween 20 (PBT) at 4 C (25, 50, 75, and twice with 100% methanol). The embryos were rehydrated through a graded series of methanol/PBT (75, 50, and 25% methanol, twice with PBT) and bleached in 6% H2O2 in PBT for 1 h. Proteinase K treatment was performed in a reaction buffer containing 10 µg/ml proteinase K at 37 C for 25 min for E13 and twice for 30 min treatment for E16.5 and E18.5 embryos. The embryos were subjected to hybridization using probes for SOST, Osx, chordin, and BMP7 (14). Osx mRNA probe was 1436 bp including full cording sequence (1287 bp). After overnight hybridization, embryos were washed twice at 70 C for 30 min each with solution I [50% formamide, 5x saline sodium citrate (pH 4.5), 1% sodium dodecyl sulfate] and three times at 70 C for 30 min each with solution III [50% formamide, 2x saline sodium citrate (pH 4.5)] and once 1 h at 70 C.

Construction and infection of recombinant adenovirus expressing noggin
Construction and infection of recombinant adenovirus expressing noggin as follows: briefly, xenopus noggin cDNA was inserted in a cassette in a cosmid pAxCAwt. The predigested adenovirus genome tagged with a 55-kDa terminal protein was mixed with the noggin expressing cosmid cassette and transfected into human embryonic kidney 293 cells. The supernatant that contained recombinant adenovirus was collected. For infection, embryonic osteoblasts were plated and incubated with adenovirus expression vectors for noggin (Ad/noggin) or ß-galactosidase (Ad/lacZ) at multiplicity of infection 30 for 1 h.

RNA interference
Small interfering RNAs (siRNAs) were generated by using Dicer siRNA generation kit (Gene Therapy Systems, San Diego, CA) (15). Briefly, cDNA templates were amplifying by using 40 base primers containing T7 promoter as primer for sense 5'-GCGTAATACGACTCACATAGGGAGAATGCTCAAGCACCAATGGACTCC-3' and 5'-GCGTA-ATACGACTCACTATAGGGAGATAATTGCAGACGAAAGGCCTCTC-3'. Double-strand RNAs were transcribing from the PCR products as templates by T7 RNA polymerase. Then the double-strand RNAs were cleaved by Dicer enzyme to generate 22 siRNAs. Each siRNA was transfected into embryonic osteoblasts. Ten days later, the cells were recovered and subjected to real-time PCR and semiquantitative PCR.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Primary calvarial cells isolated from E15.5 embryos have characteristics of osteoblasts
Osteoblast-enriched cells enzymatically isolated from E15.5 calvariae were cultured in the presence of AA and ßGP after the cells reached confluency (d 0). ALP activity level was low on d 0 and increased along with time to peak on d 7–10 (Fig. 1AGo). Phase-contrast light microscopy of the primary osteoblastic cells showed that nodule-like morphology was apparent on d 14 (Fig. 1Go, B and C). Von Kossa staining and alizarin red staining revealed mineralized matrix deposition in the cells after 14 d of culture (Fig. 1DGo). Thus, the cells derived from E15.5 calvariae exhibited osteoblastic characteristics.



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  		FIG. 1. The primary calvarial cells isolated from E15.5 embryos exhibited characteristics of osteoblasts. The primary calvarial cells isolated from E15.5 embryos were plated at a density of 10,000/cm2 in {alpha}MEM medium with 10% fetal bovine serum. To induce osteoblastic differentiation, 50 µg/ml AA and 10 mM ßGP were added after the cells reached confluency (d 0). We measured ALP activity in these cells on d 0, 3, 7, 10, and 14 (A). The quantification was represented as means ± SD. Phase-contrast light microscopy of primary osteogenic cell cultures on d 3 (B) and d 14 (C) showed nodule-like cell aggregation. Von Kossa staining (D, upper half) and alizarin red staining (D, lower half) showed that mineralized matrix was abundant in these cells on d 14. Bar, 100 µm.

 
SOST and OSX mRNA was expressed at low levels in the early stage and increased with time during differentiation in the primary osteoblastic cells
We examined expression of SOST during in vitro differentiation of E15.5 calvaria-derived primary osteoblastic cells. Semiquantitative PCR experiment revealed that expression levels of SOST mRNA were low on d 0, and they increased by treatment with AA and ßGP in a time-dependent manner up to d 14 (Fig. 2AGo). During this time course, OC and ALP expression peaked on d 7 and maintained or slightly decreased on d 14. Interestingly, similarly to SOST, we found that mRNA expression of Osx, a critical transcriptional factor for bone formation, was low on d 0 and was increased with time (Fig. 2AGo). Another transcriptional factor for bone formation, Runx2, was increased with the expression of SOST. Likewise, mRNA expression of chordin, inhibitor of BMP, was increased in a time-dependent manner. We also performed real-time PCR as more quantitative methods to compare temporal change of SOST and OSX mRNA expression. We observed significant enhancement of both SOST and OSX expression on d 7, confirming the result of semiquantitative PCR (Fig. 2BGo). We further examined SOST expression in 10-wk-old calvariae-derived primary osteoblastic cells (Fig. 2CGo). The expression of SOST was high at d 2 and declined at d 7–14. Similarly to SOST, the expression of Osx was also high at d 2 and decreased with time. This temporal change of SOST and Osx expression was confirmed by real-time PCR (Fig. 2DGo). Thus, in in vitro primary osteoblastic cell isolated from embryonic, SOST, and Osx expression increased with time, whereas in adult calvaria-derived osteoblastic cells, peaks of temporal expression shifted to an earlier time point during the progression of osteoblastic differentiation.



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  		FIG. 2. SOST mRNA expression increased along with cell differentiation in the primary osteoblastic cells derived from E15.5 calvariae and 10-wk-old mouse calvariae. Calvarial cells (osteoblast-enriched cells) were isolated by sequential enzymatic digestions from E15.5 calvariae and 10-wk-old (see Materials and Methods). Semiquantitative (A) and real-time PCR (B) experiments were performed at confluency (d 0) or on d 7 and 14 after the cultures in the primary osteoblastic cells derived from E15.5 calvariae in the presence of 50 µg/ml AA and 10 mM ßGP. Relative expression was calculated by dividing each value by that of GAPDH (see Materials and Methods) Likewise semiquantitative PCR (C) and real-time PCR (D) were performed on d 2, 7, and 14 after the cultures in the primary osteoblastic cells derived from 10-wk-old mouse calvariae. Quantification of gene expression was represented as means ± SEM. *, Statistically significant difference was observed between d 0 and d 7 (*, P < 0.05) (B) and d 2 and d 14 (D) for Osx and SOST expression. White bars indicate d 0 for embryonic osteoblasts and d 2 for adult calvaria-derived osteoblasts, and black bars indicate d 7 for embryonic osteoblasts and d 14 for adult calvaria-derived osteoblasts.

 
In the primary osteoblastic cells, BMP treatment enhanced both SOST and Osx mRNA expression in a dose-dependent manner
Previously, it was shown that BMP treatment up-regulated Osx expression in osteoblasts and chondrocytes (5, 16). Because AA and ßGP treatment increased both SOST and Osx mRNA expression in a similar manner in the osteoblast-enriched cell cultures, we examined whether SOST expression was also regulated by BMP. After calvariae-derived osteoblastic cells reached confluency, the culture medium was changed to new one containing 100 or 400 ng/ml BMP. Twenty-four hours later, total cellular RNA was extracted and subjected to semiquantitative RT-PCR. Treatment with recombinant BMP2 enhanced SOST and Osx mRNA expression in the calvaria-derived cells (Fig. 3Go).



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  		FIG. 3. BMP2-induced expression of SOST and Osx mRNA in the primary osteoblastic cells. After the osteoblastic cells searched subconfluency, we changed the medium to new medium alone or the medium containing 5 µg/ml AA and 10 mM ßGP (AA+ßGP), 100 ng/ml or 400 ng/ml BMP2. Twenty-four hours later, the total cellular RNA was extracted and subjected to RT-PCR. BMP2 induced expression of SOST mRNA as well as Osx mRNA. CTL, Control.

 
Blocking of endogenous BMP signal by noggin-expressing adenovirus resulted in suppression of SOST and Osx mRNA expression
Because treatment with exogenous BMP up-regulated both SOST and Osx mRNA expression in primary osteoblastic cells, it was possible that the increase in the expression levels of both genes during in vitro osteogenesis would be caused by endogenous BMP action. To address this possibility, we blocked BMP signals by infecting Ad/noggin into the primary osteoblastic cells. As shown in Fig. 4Go, in Ad/lacZ-infected cells, expression of SOST and Osx mRNA increased along with osteoblastic differentiation in the calvariae-derived cell cultured in the presence of AA and ßGP. In contrast, expression of both mRNAs was hardly detectable in Ad/noggin-expressing cells. These data suggested that endogenous BMP signals would be required for the increase in both SOST and Osx gene expression in the cultures of the primary osteoblastic cells.



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  		FIG. 4. Noggin inhibited expression of SOST and Osx mRNA in the primary osteoblastic cells. Ad/noggin or Ad/lacZ was infected into the primary osteoblastic cells. After infected cells reached confluency, the medium was changed to the differentiation medium. Total cellular RNA was extracted at confluency (d 0), on d 7 or 14 and subjected to RT-PCR to examine mRNA expression of SOST and Osx.

 
SOST mRNA was expressed in embryonic bone and colocalized with Osx mRNA in vivo
In vitro data on SOST expression using the primary osteoblastic cells showed that SOST and Osx expression increased in a time-dependent manner during the osteoblastic differentiation, and the increase in their expression levels was caused by endogenous BMP action. To examine whether SOST and Osx mRNA were coexpressed in vivo, we examined SOST and Osx mRNA expression in E13 embryos and E16.5 and E18.5 calvaria by WISH.

In E13 embryos, SOST mRNA was expressed in the peridigital or interdigital regions of limb bud (Fig. 5AGo). At this stage Osx mRNA was also expressed in limb bud, but its expression was more diffuse (Fig. 5CGo). In E16.5 calvariae, SOST was expressed within the edge of frontal bone, nasal bone, and parietal bone (Fig. 6AGo). In frontal bone, SOST expression was localized in anterior-lateral edge (Fig. 6CGo). In E18.5 embryos, SOST expression was within the anterior edges (Fig. 6EGo, arrowhead) and medial edge (Fig. 6EGo, arrow). Osx mRNA was expressed in E16.5 and E18.5 calvaria bones (Fig. 6Go, D and F). In frontal bone, Osx expression was observed in the anterolateral (Fig. 6FGo, arrowhead) and medial edge (Fig. 6FGo, arrow). In E18.5 embryos, BMP7 mRNA expression was also observed in the anterior edge (Fig. 6GGo). In E18.5 embryos, BMP inhibitor chordin mRNA was expressed in an area with a diamond shape in the central region of frontal bone (Fig. 6HGo), whereas SOST and Osx mRNAs were not expressed. Likewise, both SOST and Osx mRNAs were expressed in the mandible bone primordia (Fig. 6Go, J and K), whereas chordin mRNA expression was not observed (Fig. 6LGo).



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  		FIG. 5. SOST mRNA was expressed in limb bud, and its expression domain was overlapped with that of Osx. WISH was performed in E13 embryos using cRNA antisense probes for SOST (A) and Osx (C) and sense probe for SOST (B) and OSX (D). SOST mRNA was expressed in the peripheral region of limb digit primordia (A, arrowhead). At this stage, Osx mRNA was observed in the similar region in limb bud (C, arrowhead), and its expression domain was overlapped with that of SOST.

 


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  		FIG. 6. Expression domain of SOST mRNA was overlapped with that of Osx in calvariae and mandible. We investigated SOST (A–C and E), Osx (D and F), BMP7 (G), and chordin (H) expression in cranium of E16.5 (A, C, and D) and E18.5 (B, E–H) by WISH. SOST mRNA expressed at all cranial bone primordia such as frontal bone, nasal bone, and parietal bone (A and B, arrowhead, f, n, and p, respectively in). SOST mRNA was observed in anterior-lateral border of frontal bone (C, arrowhead) in E16.5 calvaria. At E18.5 expression domain of SOST mRNA extended to anterior border (E, arrowhead) and medial border (E, arrow) in frontal bone. Osx expression was observed in broader region (D, arrowhead) in frontal bone at E16.5. On E18.5 Osx mRNA expression was localized in anterior (F, arrowhead), posterior, and medial border (F, arrow), in which SOST mRNA expression was observed. BMP7 expressed in anterior border near the place in which SOST and Osx mRNA was expressed (G, arrowhead). Another BMP inhibitor, chordin, expressed only in the center of frontal bone (H, arrowhead). The boxed area in A with dotted line corresponds to the area in which the photo in C and D was taken, and the boxed area in B with dotted line corresponds to the area in which photos in E–H were taken. Long dashed dotted line in C–H indicates frontal bone border. Caudal view of mandible (shaded yellow), mylohyoid muscle (sky blue), hyoid bone (yellow) was shown in I. The enclosed area with long dashed dotted line in I corresponds to the enclosed area in J–L. Both SOST and Osx mRNAs were expressed in mandible bone primordia (J, K), whereas chordin expression was not observed in the mandible (L). Long dashed dotted line in J–L indicated mandible border.

 
Suppression of Osx mRNA by siRNA decreased SOST mRNA expression.
Because both Osx and SOST expression was regulated in a similar manner in osteogenetic differentiation by BMP, we wanted to address mechanistic relationship between SOST and Osx. We performed siRNA experiments to suppress Osx expression in primary osteoblastic cells. We transfected Osx siRNA generated by using Dicer. OSX siRNA inhibited endogenous Osx mRNA expression to approximately half-level, compared with control green fluorescent protein (GFP) siRNA. Both control GFP siRNA and Osx siRNA did not affect GAPDH expression. In contrast, SOST as well as OC mRNAs were significantly down-regulated by Osx siRNA transfection. Thus, suppression of Osx mRNA by siRNA resulted in down-regulation of SOST mRNA expression (Fig. 7Go).



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  		FIG. 7. Suppression of Osx mRNA by siRNA decreased SOST mRNA expression. To examine mechanistic relationship between SOST and Osx expression, we performed siRNA experiments to suppress Osx mRNA expression. The primary osteoblastic cells derived from E15.5 calvariae were transfected with Osx siRNA. Ten days later, the transfected cells were recovered and subjected to real-time PCR. Suppression of Osx expression resulted in inhibition SOST expression as well as OC expression. G (white bars), Transfection of GFP siRNA. O (black bars), Transfection of Osx siRNA. The relative amount of gene transcript present was calculated and normalized by dividing the calculated value for the gene of interest by GAPDH. Quantification of gene expression was represented as means ± SEM. *, Statistically significant difference was observed between transfection of GFP and OSX siRNA (*, P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we found that SOST mRNA expression was increased along with osteoblastic differentiation in vitro and that SOST mRNA was expressed in calvariae and appendicular skeletal primordia in vivo. SOST expression was observed in the region in which skeletogenesis is ongoing, such as the osteogenic front of calvarial bone and periostea of mandibulae and limbs. These regions correspond to the place in which skeletal phenotypes were reported in sclerosteosis patients (17, 18, 19). These data provided embryological support for the observations that mutations in the SOST gene lead to the sclerosteosis in human. Furthermore, expression of SOST in limbs observed in this study may explain the pathogenesis of syndactyly that is seen in most sclerosteosis patients (17).

Spacial and temporal SOST expression patterns were associated with those of Osx. In the primary osteoblastic cells, increase in Osx expression was observed earlier than that of SOST (Fig. 2Go). Furthermore, BMP induced expression of SOST mRNA as well as that of OSX in vitro. Noggin overexpression inhibited expression of these two genes, suggesting the role of endogenous BMP signaling. In frontal bone in E18.5 calvaria, the region of BMP7 expression was similar to those of SOST and Osx. Thus, BMP induces Osx as well as negative regulator of osteogenesis SOST simultaneously to keep the balance of osteoblastic differentiation.

Most sclerosteosis patients exhibit syndactyly in their fingers (19). By WISH experiment, we observed SOST expression in interdigital regions of E13.0 limb buds. Therefore, syndactyly in sclerosteosis patients may be caused by the defects of limb patterning. Moreover, BMP signaling has been also implicated in finger patterning. BMP4 and BMP7 are expressed in the interdigital regions of limb buds (20), and implantation of recombinant BMP induces supernumerary digits in chick limb bud in vivo (21). Thus, SOST expression evoked by BMP may be required for proper limb patterning during embryogenesis.

In adult calvaria-derived osteoblasts, SOST expression was high at d 2 of culture and declined with time, whereas in embryonic-derived cells, such a peak was at d 7. The differences in temporal peak of gene expression during embryonic and adult osteoblastic differentiation may reflect on the differentiation status of the cells. Perturbation of maintenance of SOST expression in osteoblasts may cause the phenotype in sclerosteosis.

Several BMP inhibitors, including gremlin (22), cerberus (23), and dan (22, 24, 25) have been reported to be expressed during embryonic skeletogenesis. The expression of gremlin was observed in the proximal dorsal and interdigital regions of the limb buds in E11.5 embryos (22). Dan was expressed in cranial mesenchyme and somites in early headfold stage, later in limb and facial mesenchyme in E10.5 embryos (22). Chordin was expressed similarly to SOST in the primary cultures of osteoblastic cells in our experimental system. However, spacial localization of chordin expression is different from that of SOST in calvariae. Chordin mRNA was expressed in an area with a diamond shape in the central region of frontal bone in which SOST mRNA was not expressed in vivo. Chordin null mice do not show any phenotype in skeletogenesis (26), suggesting that localized expression of chordin may not be relevant to osteoblastic differentiation. In contrast, spacial localization of SOST is directly related to bone phenotypes of the gene mutation and thus could be potentially relevant to osteoblastic function. It was reported that SOST treatment suppressed ALP activity in mouse preosteoblastic cell cultures (27). However, whether SOST protein regulates osteoblastic function by attenuating BMP signaling in vivo remains to be elucidated. Elucidation of SOST function in osteoblastic differentiation will provide a clue to understand the molecular mechanism of physiological state as well as pathological state of bone formation.


   Acknowledgments
 
We thank Yamanouchi Pharmaceuticals and Drs. Enomoto and Asahina (Tokyo Medical and Dental University) for providing recombinant BMP2 protein and Dr. Richard Harlord (University of California, Berkeley, CA) for recombinant noggin protein and cDNA.


   Footnotes
 
This work was supported by grants from the Japanese Ministry of Education, Science, and Culture of Japan (14207056, 16390521, 16659405, 16027215, 16022221), Japan Space Forum, National Space Development Agency of Japan, Core Research for Evolutional Science and Technology, and Japan Society for Promotion of Science (Integrated Action Initiative in Core to Core Program) and grants-in-aid from the Japanese Ministry of Education [21st Century Center of Excellence (COE) Program, Molecular Destruction and Reconstitution of Tooth and Bone].

Abbreviations: AA, Ascorbic acid; Ad/noggin, adenovirus expressing noggin; Ad/lacZ, adenovirus expressing ß-galactosidase; ALP, alkaline phosphatase; BMP, bone morphogenetic protein; E, days post conception; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; ßGP, ß-glycelophosphate; OC, osteocalcin; Osx, osterix; PBT, PBS containing Tween 20; RT, reverse transcription; Runx2, runt-related gene; siRNA, small interfering RNA; SOST, sclerostin; WISH, whole-mount in situ hybridization.

Received November 4, 2003.

Accepted for publication June 16, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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