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Endocrinology, doi:10.1210/en.2003-0685
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Endocrinology Vol. 145, No. 7 3434-3442
Copyright © 2004 by The Endocrine Society

Noggin Inhibits Chondrogenic But Not Osteogenic Differentiation in Mesodermal Stem Cell Line C1 and Skeletal Cells

Akira Nifuji, Odile Kellermann and Masaki Noda

Department of Molecular Pharmacology, Medical Research Institute (A.N., M.N.), 21st Century Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone (M.N.), Integrated Action Initiative in JSPS Core to Core Program (M.N.), Tokyo Medical and Dental University, Tokyo 101-0062, Japan; and Laboratoire de Differenciation Cellulaire et Prions, Centre National de la Recherche Scientifique Unité Propre de Recherche 1983 (O.K.), Institut Pasteur, 94801 Villejuif, France

Address all correspondence and requests for reprints to: Masaki Noda, Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10, Kanda-surugadai, Chiyoda-ku, Tokyo 101-0062, Japan. E-mail: noda.mph{at}mri.tmd.ac.jp; or Akira Nifuji, Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10, Kanda-surugadai, Chiyoda-ku, Tokyo 101-0062, Japan. E-mail: akiki.mph{at}mri.tmd.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Osteoblasts and chondroblasts are derived from common mesenchymal progenitors. Although bone morphogenetic protein induces mesenchymal differentiation into both osteogenic and chodrogenic lineage cells in vitro, its inhibitor, Noggin, is expressed exclusively during chondrogenic but not osteogenic differentiation in an embryonal carcinoma-derived mesodermal cell line, C1. We hypothesized that Noggin may regulate cell differentiation in a lineage-specific manner. To test this hypothesis, Noggin was overexpressed using recombinant adenovirus (Ad/Noggin) in mesodermal C1 cells to examine whether Noggin specifically inhibits chondrogenic differentiation. Noggin overexpression by recombinant adenovirus infection reduced Sox9, patched, Ihh, and type II, X, and XI collagen mRNA expression levels in C1 cell aggregates that were induced to differentiate into chondrocyte lineage by culturing in differentiation medium. In contrast, Noggin overexpression did not affect osteogenic differentiation in C1 cells because osteoblast phenotypic markers such as osteocalcin and alkaline phosphatase mRNA levels were not altered. We further examined whether Noggin also differentially affects chondrogenesis and osteogenesis in limb development by using organ cultures of long bone. Ad/Noggin infection into 15.5 d post conception limb skeletal rudiments that were cultured on filter membrane in vitro or on the chorioallantoic membranes in ovo inhibited the levels of chondrogenesis, which were evaluated based on alcian blue staining. These results suggest that Noggin specifically blocks chondrogenic differentiation, rather than osteogenic differentiation, in mesodermal stem cell line C1 and skeletal cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CHONDROCYTES AND OSTEOBLASTS are derived from mesenchymal stem cells, and such stem cells may exist in adult tissues as well as in embryonic tissues. One of these examples is the limb mesenchyme, which gives rise to both chondrocytes and osteoblasts of appendicular skeleton. Before the appearance of skeletal cell aggregation, cells in the limb express early mesenchymal markers such as twist and Foxc2 (mesenchymal forkhead 1) (1, 2, 3). Concomitant with cell aggregate formation, early chondroblast markers such as Sox9 and type II collagen as well as an osteoblast/chondrocyte marker, Runx2, are expressed in the primordia of skeleton (4, 5, 6). One of the mesodermal stem cell lines, C1 cells, exhibits characteristics of mesenchymal stem cells and is capable of differentiating into skeletal cells (7). The C1 cells express many mesodermal markers such as Id, twist, and FoxC2 (Mfh1) (3, 7). On the onset of differentiation by forming cell aggregates, C1 cells express early phenotypic markers for both differentiation lineages such as type II collagen mRNA for chondrocytes and type I collagen mRNA for osteoblasts (8). When the cells are cultured in the differentiation medium for chondrogenesis, these cells start to express selectively mature chondrogenic markers, whereas expression levels of osteogenic markers decline after their differentiation into chondrocytes. Thus, an alternative switching mechanism may exist in mesenchymal cells in vivo as well as in vitro.

Bone morphogenetic protein (BMP) is a potent regulator of mesenchymal differentiation. In vitro, the addition of recombinant BMP2 and BMP7 proteins or overexpression of BMP cDNAs in a mesenchymal cell line, C3H10T1/2, induces cell differentiation into three lineages: osteoblasts, chondroblasts, and adipoblasts (9, 10, 11, 12). BMP2 enhances mRNA levels for chondroblast markers such as type II collagen and Noggin in another mesenchymal cell line, C1 (13). In developmental skeletogenesis, BMP ligands and receptors are expressed in various sites and stages. BMP4 and BMP7 are expressed in the perichondrium of the skeletal blastemas and in the precursors for muscles and ligaments (14, 15). BMP5 and BMP14 (GDF5) are expressed in the joint forming mesenchymal tissues (16). BMP receptors, such as BMPRIA, BMPRIB, and BMRII, are expressed in the mesenchyme as well as in the later skeletal primordia (17, 18). A loss-of-function study in mice revealed that BMP5 and BMP14 are essential for proper development of skeletal elements (16). These in vitro and in vivo data indicate that BMPs play pivotal roles in mesenchymal cell differentiation; however, it is not yet clear at this moment whether BMPs function as cell lineage-specific determinants for osteogenic or chondrogenic differentiation pathways.

In addition to BMP ligands, BMP inhibitors are also expressed in the mesenchymal cells and skeletal cells in vivo as well as in vitro. For example, Chordin is expressed in the resting chondrocytes in the embryonic growth plate as well as in mesenchymal tissue surrounding skeletal blastemas (19). Twisted gastrulation mRNA is localized in articular chondrocytes (20, 21), and Dan is expressed in mesenchymal cells and perichondrium (22, 23). Another BMP inhibitor, Noggin, is expressed in cartilage primordia in most skeletal tissue. In the embryonic calvaria, where membranous bone formation occurs, Noggin expression is not detected by in situ hybridization (14). In in vitro differentiation of the C1 mesodermal cell, we previously reported that Noggin is expressed during the chondrogenic differentiation but is not expressed during the osteogenic differentiation (13). These specific expression patterns of Noggin lead us to hypothesize that Noggin has lineage-specific roles in chondrocyte differentiation rather than osteoblast differentiation. To test this hypothesis, we constructed a recombinant adenovirus to express Noggin (Ad/Noggin). We infected Ad/Noggin into C1 cells before induction of differentiation and then cultured them to differentiate into chondrogenic or osteogenic lineage. We also examined how Noggin affects differentiation of limb skeletal cells in the organ culture system. Noggin blocks chondrogenic differentiation but not osteogenic differentiation in a mesodermal stem cell line, C1, and skeletal cells in limb bud cultures.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of recombinant adenovirus expressing Noggin
Xenopus Noggin cDNA was inserted in the Swa I site in cassette cosmid pAxCAwt (Takara, Osaka, Japan). 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. After collecting the medium supernatant that contains recombinant adenovirus, multiplicity of infection (MOI) for the recombinant adenovirus was determined according to the standard protocol using 293 cells (24).

For infection, C1 cells were collected at the time of replating and incubated with Noggin (Ad/Noggin)- or ß-galactosidase (Ad/LacZ)-expressing adenovirus at MOI 30 for 1 h. For adenovirus infection into long bone, a pair of humeri or femora was incubated in BGJb medium containing either Ad/Noggin or Ad/LacZ adenovirus in 2.5 x 107 colony forming units during the first 3 d and then cultured in the fresh medium without adenovirus (see below). For long bones cultured on chorioallantoic membranes (CAMs), long bone rudiments were incubated with medium containing the 2.5 x 107 colony forming unit adenovirus for 2.5 h and were placed on the CAMs of chick embryos (see below).

Cell cultures
C1 cells were routinely cultured in DMEM supplemented with 10% fetal calf serum at 37 C in a humidified atmosphere of 5% CO2 (7). After adenovirus infection, C1 cells were seeded at 5 x 105 cells in 10 ml DMEM supplemented with 10% fetal bovine serum onto bacteriological dishes to form cell aggregates. After 10 d, cells were refed with DMEM containing 1% fetal bovine serum and induced to differentiate along the chondrogenic pathway by the addition of 10–6 M dexamethasone (Dex) into medium or along the osteogenic pathway by the addition of 10 mM ß-glycerophosphate (bGP) and 50 µg/ml ascorbic acid (AA). At appropriate time points, the cells were recovered and subjected to RNA extraction. For the experiment of BMP treatment, infected C1 cells were seeded at 1 x 104 cells/well in a 24-well dish and BMP was added 1 d later. Three days later, the cells were recovered and alkaline phosphatase (ALP) activities were measured.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
The infected cells were plated at a density of 1000 cells/well in a 96-well plate. Three days after infection, the medium was removed and 50 µl of 1 mg/ml MTT (Wako Chemicals, Osaka, Japan) solution were added to each well containing cells. Then, the plate was incubated in a CO2 incubator at 37 C for 3 h. At the end of incubation, MTT solution was removed and 50 µl propanol were added to each well to dissolve the blue crystals. After transfer to a plate reader, absorbance was measured at 570 nm.

ALP assay
The cells were lysed in a lysis buffer containing 10 mM Tris HCl, 0.5 mM MgCl2, and 0.1% Triton X-100. The ALP activities in the samples were measured using sodium p-nitrophenyl phosphate as a substrate (25) in 0.1 M 2-amino-2-methhyl-1-propanol (pH 10.5). Protein contents were determined according to the Coomassie Blue G methods.

RNA preparation and Northern blot analysis
RNA preparation and Northern blot analysis were performed as described previously (13). Briefly, total cellular RNA was extracted according to the single step of the acid-phenol-chloroform method. Ten micrograms of total RNA were fractionated by electrophoresis on 1% agarose containing 0.22 M formaldehyde and transferred onto nylon filters by electroblotting. Hybridization was performed at 42 C in a hybridization solution containing 50% formamide, 5x SSC, 5x Denhardt’s, 0.1% SDS, 100 µg/ml salmon sperm DNA, and radiolabeled probes at a concentration of 106 disintegrations per minute per milliliter.

Probes
The probes used for hybridization were 1.4-kb mouse ALP cDNA insert (26), 0.5-kb rat osteocalcin (OC) cDNA insert (27), and 1.2-kb human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA insert (28). The cDNA fragments were radiolabeled using 32P dCTP and Oligolabeling kit (Amersham Biosciences, Piscataway, NJ) according to random priming methods as directed by the manufacturer’s protocol.

Semiquantitative RT-PCR experiments
One microgram of total cellular RNA was primed with oligo d(T) and used to synthesize the first-strand cDNA with Moloney murine leukemia virus reverse transcriptase (Invitogen, Carlsbad, CA). We subsequently performed PCR reaction from 50 ng cDNA as a template, under the following conditions: initial denaturation for 3 min at 94 C, followed by cycles consisting of denaturation for 30 sec at 94 C, annealing for 20 sec at 60 C, and extension for 1 min at 72 C. We used TaqDNA polymerase (Takara) for PCR reaction. GAPDH was used as an internal control of RT-PCR experiments, and a PCR condition was established to detect logarithmic amplification of PCR products. The accession numbers, numbers of PCR cycles, and primer sequences used for each PCR experiment are summarized in Table 1Go.


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  		TABLE 1. Primers for semiquantitative PCR experiments

 
Real-time PCR experiment
After cDNA synthesis, PCR reactions were performed using 3 or 12 ng of cDNA template, 625 nM of each forward and reverse primer, and 1x 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 denaturation 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 dissociation curves were performed to confirm that a single PCR product was produced in each reaction. The relative amount of gene transcripts were calculated and normalized by dividing the calculated value for the gene by that for GAPDH gene. At least two independent experiments from cell culture to PCR were performed, and each PCR was performed three times. The accession numbers and primer sequences used for each PCR experiment are summarized in Table 2Go.


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  		TABLE 2. Primers for real-time PCR experiments

 
Organ culture study of skeletal primordia
The experimental design and procedures for the animals were approved by the Animal Ethics Committee of Tokyo Medical and Dental University.

Bone rudiments for humeri or femora were dissected out from 15.5 d post conception mouse embryos. For the first series of experiments (n = 9 pairs), we placed limb bone rudiments on membrane filters supported by metal grids according to the Trowel technique (14) and cultured them in BGJB medium (Invitrogen) with recombinant adenovirus for 3 d. Then, the BGJB medium was changed to a new one without adenovirus for 4 d. For the second series of experiments (n = 7 pairs), we incubated bone rudiments with virus in 100 µl DMEM for 2.5 h in a 96-well dish. Then, we placed bone rudiments onto a nylon filter, which was transferred onto CAMs of embryonic d 8 chick embryos. The bone rudiments incubated on the CAM were recovered 7 d later.

Alizarin red and alcian blue for limb bone rudiments
Alizarin red stains mineralized bone and alcian blue-stained proteoglycans indicating cartilage. We followed the procedure for the both staining embryos in toto as described previously (29).

Histological examination
The limb bone rudiments were fixed in 4% paraformaldehyde and embedded in paraffin. The sections were prepared in 6-µm thickness. After hydration, the sections were stained with 1% alcian blue in 0.1N HCl or 1% alizarin red.

5-Bromo-4-chloro-3-indolyl-D-galactoside (X-gal) staining for limb bone rudiments
For X-gal staining in toto, bone rudiments were fixed in 0.2% glutaraldehyde and 100 mM MgCl2 in PBS at room temperature for 2 h. The rudiments were washed in PBS and then placed in X-gal staining solution containing 1 mg/ml X-gal (Takara), 2 mM MgCl2, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 0.01% sodium deoxycholate, and 0.02% Nonidet-P40 at room temperature overnight. For sections, the stained bone rudiments were fixed in paraformaldehyde overnight, embedded in paraffin, and cut into 5-µm sections.

Bromodeoxyuridine (BrdU) labeling experiment for limb bone rudiments
Ad/LacZ-infected or Ad/Nog-infected (n = 3) humeral bone rudiments were cultured in BGJB medium for 2 d and then labeled with 10 µM BrdU (Sigma, St. Louis, MO) for an additional 3 h. Infected long bone were then fixed in paraformaldehyde at 4 C overnight, embedded in paraffin, and cut into 5-µm sections. Immunohistochemistry to detect BrdU-positive cells on sections was performed by using a BrdU detection kit (PharMingen, San Diego, CA). BrdU-labeled cells from sections of three separate long bone rudiments were counted. Sagittal sections from near the center of the bone were selected for counting. Labeled cells in the fixed area at epiphysis were counted for growing chondrocytes. For cells in the bone collar, BrdU-labeled cells that reside in the outer layer at diaphysis were counted. The effect of Ad/Noggin on cell proliferation in long bone was evaluated as a ratio of the BrdU-labeled cell number in Ad/Noggin-infected long bone against the control (Ad/LacZ-infected contralateral long bone).

Statistical evaluations
The results were presented as averages ± SD. Statistical analysis was performed by Mann-Whitney U test. A value of P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We constructed an adenovirus vector to express a Xenopus Noggin cDNA (a gift from R. Harland, Berkeley, CA), using COS-TCP methods (24). We infected a LacZ-expressing adenovirus (Ad/Lac Z) at MOI 30 or a Noggin-expressing adenovirus (Ad/Noggin) into C1 cells at MOI 30, respectively. After infection of adenovirus, we cultured the C1 cells to form aggregates and performed RT-PCR to detect exogenous expression of Noggin. C1 cells expressed low levels of endogenous Noggin, which was hardly detectable by Northern blot analysis (13) but detectable by 33 rounds of PCR amplification. In contrast, 25 rounds of PCR amplification could detect expression of Xenopus Noggin mRNA in the C1 cells infected with Ad/Noggin but not in Ad/lacZ-infected control C1 cells (Fig. 1AGo). We also confirmed that Ad/Noggin blocks the effects of BMP on C1 cells. Ad/Noggin at MOI 30 suppressed elevation of ALP activity that was caused by exogenously delivered BMP in C1 cells (Fig. 1BGo).



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  		FIG. 1. Adenovirus expression of Noggin (Ad/Noggin) blocked BMP effects on C1 cells. After C1 cells were infected with adenovirus expressing Noggin (Ad/Nog) or LacZ (Ad/LacZ), expression of Xenopus Noggin and mouse (endogenous) Noggin mRNAs were examined by RT-PCR (A). Then, we examined whether Ad/Nog inhibits the effects of BMP on C1 cells. C1 cells were infected with Ad/LacZ or Ad/Nog at MOI 30 at the time of replating. After C1 cells reached confluency, BMP2 at various doses was added to the culture medium. Two days later, ALP activity was measured. ALP activity in Ad/LacZ-infected C1 cells was increased by BMP in a dose-dependent manner. In contrast, infection of Ad/Nog suppressed ALP activity to the basal level, even when C1 cells were cultured in the presence of 100 ng/ml BMP2 (B).

 
BMP is known to regulate proliferation of certain types of cells, including osteoblastic cells (12, 30). Therefore, blocking of BMP signals may affect cell proliferation, resulting in secondly influence on differentiation phenotype. To examine whether exogenously expressed Noggin affects proliferation in C1 cells, we performed the MTT assay 3 d after infection (Fig. 2Go). Ad/ Noggin infection at MOI 30 did not alter the level of MTT activity in C1 cells, when compared with control C1 cells. We also compared the cell numbers between Ad/LacZ- and Ad/Noggin-infected C1 cells cultured in the presence of Dex in monolayer culture. We found that Ad/Noggin infection did not affect cell numbers (data not shown). These results suggest that Noggin infection did not affect cell proliferation in C1 cells.



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  		FIG. 2. Adenovirus expression of Noggin did not alter proliferation in C1 cells. To examine whether exogenously expressed Noggin affects proliferation in C1 cells, we performed the MTT assay. The infected cells were plated at a density of 1000 cells/well in a 96-well plate, and the levels of MTT activity in C1 cells were measured 3 d after infection (n = 4).

 
C1 cells expressed early phenotypic markers of osteoblasts, chondroblasts, and adipocytes at their confluence (7). We asked whether intrinsic BMP ligands and receptors are expressed to be ready to transmit the BMP signal in the autocrine manner at this stage. Real-time PCR experiments revealed that C1 cells expressed BMP4, BMP7, and GDF5 as ligands (Fig. 3Go), however, BMP2 expression was not detected at their confluence (data not shown). C1 cells expressed ALK1, ALK2, BMPR IA (ALK3), and BMPRII mRNA as receptors (Fig. 3AGo and data not shown). The expression of those ligands and receptors was also observed in the C1 cells cultured under osteogenic and chondrogenic differentiation conditions as well (Fig. 3Go). The expression level of BMP4 and BMP7 was relatively higher in osteoblastic differentiation than in chondrogenic differentiation, whereas GDF5 expression was higher in chondrogenic differentiation (Fig. 3Go, A–C). Thus, C1 cells expressed BMP ligands and receptors both at their immature and more differentiated stages, suggesting that endogenous BMP signaling may exist during differentiation of mesodermal C1 cells.



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  		FIG. 3. C1 cells express BMP ligands and receptors. After C1 cells were infected with adenovirus expressing Noggin (Ad/Nog) or lacZ (Ad/LacZ), C1 cells were formed in cell aggregates and further differentiated into the chondrogenic pathway by the addition of Dex or into the osteoblastic pathways by the addition of bGP and AA for 15 d. Total RNA was extracted at the time when cell aggregates were formed (Aggregates) and when cells were differentiated into the chondrogenic pathway for 15 d (Chondrogenesis) or the osteoblastic pathways for 15 d (Osteogenesis). Each RNA sample was subjected to real-time PCR using specific probes for BMP4 (A), BMP7 (B), GDF5 (C), and ALK2 (D). *, Significant difference (P < 0.05).

 
We then asked whether Noggin regulates C1 cell differentiation. To examine whether overexpression of Noggin affects chondrogenesis in C1 cells, we infected the recombinant adenovirus into C1 cells at the time of plating and cultured them to form aggregates. Then, we changed to differentiation medium containing Dex to induce chondrogenic differentiation. We examined mRNA expression of cartilage phenotypic genes by conducting real-time PCR (Fig. 4AGo.) and semiquantitative PCR (Fig. 4BGo). On d 15 of Dex treatment, C1 cells infected by the Ad/LacZ adenovirus (control) expressed transcripts for type II, X, and XI collagen, Sox9, Indian hedgehog, and Gli, all of which are phenotypic markers of chondrogenesis (Fig. 4Go, A and B). In contrast, expression of type XI collagen, Indian hedgehog, and Gli mRNAs were suppressed in Ad/Noggin-infected C1 cells (Fig. 4Go, A and B). Likewise, type II and X collagen and Sox9 expression levels were lowered when compared with the control cells (Fig. 4Go, A and B), suggesting that Noggin inhibits chondrogenic differentiation of C1 cells.



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  		FIG. 4. Noggin blocks chondrogenic differentiation in C1 cells. To examine whether Noggin functions to regulate chondrogenesis in C1 cells, we infected the recombinant adenovirus into C1 cells at the time of replating and cultured them to form aggregates, followed by the addition of Dex in the medium to induce chondrogenic differentiation. After 15 d of Dex treatment, total cellular RNA was extracted and was subjected to real-time PCR (A) and semiquantitative RT-PCR (B). *, Significant difference (P < 0.05).

 
Because C1 cells are able to differentiate into the osteoblastic pathways, we next examined whether overexpression of Noggin influences osteoblastic differentiation in C1 cells. Cell aggregates of C1 were formed after adenovirus infection and were cultured in the presence of bGP and AA to differentiate into the osteoblastic pathway. In contrast to chondrogenic differentiation, expression levels of osteoblast markers, such as OC and ALP, did not differ between the Ad/Noggin-infected cells and the control cells both at d 7 and 15 of treatment (Fig. 5Go).



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  		FIG. 5. Noggin does not affect expression of the osteoblastic phenotype in C1 cells. To examine whether Noggin overexpression affects osteoblastic differentiation of C1 cells, we infected the recombinant adenovirus into C1 cells at the time of replating and cultured them to form aggregates, followed by the addition of bGP and AA to induce osteogenic differentiation. Seven and 15 d later, total cellular RNA was extracted and was subjected to Northern blot hybridization. Mouse ALP and rat OC cDNAs were used as probes for the osteoblastic phenotype, and GAPDH probe was used as a reference. We also performed real-time PCR in two other independent experiments and obtained similar data (data not shown).

 
During C1 cell differentiation, Noggin suppressed chondrogenesis, whereas Noggin did not affect osteoblastic differentiation. To examine further whether the selective effects of Noggin would be observed in embryonic skeletogenesis, we infected the adenovirus into long bone rudiments. We isolated pairs of humerus or femur primordia from 15.5 d post conception embryos (Fig. 6AGo). We infected Ad/Noggin into one of limb bone pair and Ad/LacZ into the other side that served as a control. To confirm that the genes delivered by adenovirus are expressed in the isolated long bone, we stained the lacZ-infected long bone with X-gal as a substrate. As shown in Fig. 6Go, B and C, infected cells were observed in peripheral cells of the bone rudiments.



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  		FIG. 6. Noggin inhibits chondrogenic growth of long bone rudiments. We isolated one pair of humerus or femoral bone rudiments from 15.5 d post conception embryos (A). We infected Ad/Noggin into one of limb bone pair and Ad/LacZ into the other bone that served as a control. To confirm that genes delivered by the adenovirus construct were expressed in the isolated long bone rudiments, we stained the Ad/LacZ-infected long bone by using X-gal as a substrate in toto (B) and in section (C). Ad/LacZ-infected cells were observed in the peripheral cells of bone rudiments but not inside, as were shown in the section (C). In the first series of experiments (experiment 1), we cultured the infected bone rudiments in the culture dish (D). In the second series of experiments (experiment 2), we cultured the infected bone rudiments on the CAM of the chick embryos (E). The infected embryos were fixed 7 d later, followed by alizarin red and alcian blue staining in toto. Then, we measured the longitudinal length of the alcian blue staining area and alizarin red staining area (see Fig. 7Go). N, Ad/Noggin-infected limb; C, Ad/LacZ-infected limb. Bars: A, D, and E, 500 µm.

 
In the first series of experiments, we cultured infected bone rudiments in the culture dish according to the Trowel culture technique (13). After 7 d of culture, the limb bone rudiments were fixed and stained with alcian blue and alizarin red. The blue-stained area by alcian blue shows proteoglycan deposition and reflects chondrogenic growth (Fig. 6DGo). We measured the longitudinal length of zone stained with alcian blue (Ch1 plus Ch2) and that stained with alizarin red (Os). We determined the value of the chondrogenic growth as a ratio of Ch1 plus Ch2 in Ad/Noggin-infected limb bone to that in control limb bone (Fig. 7Go). Similarly, we determined the value of the osteogenic growth as a ratio of Os in the Ad/Noggin-infected limb bone to the control. The ratio of chondrogenic growth after a 7-d incubation was significantly lower than the value 1, showing that Noggin infection suppressed cartilage growth by 14% (Fig. 7Go). The ratio of osteogenic growth is approximately 1, suggesting that osteogenic growth was not altered between LacZ- and Noggin-infected bone (Fig. 7Go). Although infected cells were observed in peripheral cells but not inside of long bone rudiments (Fig. 6CGo), the effects of Ad/Noggin on chondrogenesis were evident.



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  		FIG. 7. Chondrogenic growth but not osteogenic growth was suppressed by Noggin. Chondrogenic growth was determined as a ratio of Ch1 plus Ch2 of Ad/Noggin against the control (Ad/LacZ-infected contralateral long bone). Likewise, osteogenic growth was determined as a ratio of Os in Ad/Noggin-infected bone against the control. The ratios of the chondrogenic growth were significantly less than 1.0 (baseline) in both experiments 1 (Exp 1) and experiment 2 (Exp 2), showing the inhibition of chondrogenesis by Ad/Noggin. In contrast, the ratios of the osteogenic growth were approximately 1.0 in both experiments, suggesting that osteogenic growth in long bone was not altered by Ad/Noggin expression. *, Significantly different from the baseline value, 1.0 (P < 0.05).

 
Because the Trowel culture condition does not favor osteogenesis that requires vascularization, the effects of adenovirus infection on osteogenic growth may not be properly evaluated. We, therefore, cultured long bones on a CAM of the 8-d chick embryo, which provides vascular tissue that migrates into the isolated bone and supports osteogenic growth during the cultivation. Clearly, progression of limb bone ostegenesis, judged by alizarin red staining, was faster in the CAM culture condition compared with the in vitro culture condition (Fig. 6EGo). Overall length of the long bone on CAM was longer than the bone that was cultured in the dish. The ratio of chondrogenic growth was 0.773, showing that Noggin inhibited condrogenesis similarly to the results of the experiments conducted in the Towel culture (Fig. 7Go). Histological examination revealed that the hypertrophic cartilage zone of Ad/Noggin-infected long bone was shorter than that of ad/lacZ-infected long bone (Fig. 8Go, A and B). In contrast, longitudinal growth of the mineralized bone collar was not altered in Ad/Noggin-infected long bone compared with Ad/LacZ-infected long bone, which was stained in toto (Fig. 6EGo) or in sections (Fig. 8Go, C and D).



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  		FIG. 8. Histological examination of the adenovirus-infected long bone. Several pairs of the long bones were sectioned and subjected to histological examination 7 d after infection (A–D). The alcian blue staining of the sections revealed that the hypertrophic cartilage (HC) zone in Ad/Noggin-infected long bone (B) was shorter than Ad/LacZ-infected long bone (A). In contrast, alizarin red staining of the sections showed the longitudinal growth of the mineralized bone collar was not altered in Ad/Noggin-infected long bone (D) compared with Ad/LacZ-infected long bones (C). The dotted lines showed the ends of the HC zone (A and B), and a bracket-like line indicates the area of mineralized bone collar (C and D). Bar: C, 200 µm.

 
Because the hypertrophic cartilage zone was shorter in Ad/Noggin-infected long bone, we further examined whether cell proliferation in chondrocytes is affected by Noggin expression. In Ad/LacZ-infected long bone, BrdU-labeled cells were observed in the resting and proliferating chondrocytes as well as in the bone collar (Fig. 9Go, A–C). The effect of Ad/Noggin on cell proliferation of chondrocytes in long bone was evaluated as a ratio of the BrdU-labeled cell number in the resting and proliferating chondrocytes in Ad/Noggin-infected long bone against the control (Ad/lacZ-infected contralateral long bone). Likewise, the effect of Ad/ Noggin on cell proliferation of cells in the bone collar (perichondrium/periosteum) was determined as a ratio of the labeled cell number in Ad/Noggin-infected bone against the control (Fig. 9Go, D–F). The ratio of chondrocyte proliferation was 0.49 ± 0.22 (n = 3), showing the inhibition of proliferation by Ad/Noggin. In contrast, the ratio of cell proliferation in the bone collar (perichondrium/periosteum) was 0.95 ± 0.2, suggesting that cell proliferation was not affected. Thus, in these organ culture experiments, Noggin significantly inhibited chondrogenic growth, whereas it did not block osteogenic growth.



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  		FIG. 9. BrdU labeling of the adenovirus-infected long bone. Ad/LacZ-infected (A–C) or Ad/Nog-infected (D–F) (n = 3) humerus rudiments were cultured in BGJB medium for 2 d and then labeled with 10 µM BrdU for additional 3 h. Infected bone rudiments were then fixed in paraformaldehyde and subjected to immunohistochemistry to detect BrdU-positive cells on sections by using a BrdU detection kit (PharMingen). B and E are higher magnifications of A and D, respectively, in growing chondrocytes. C and F are higher magnifications of A and D, respectively, in the bone collar. The arrows indicate BrdU-positive cells that reside in the bone collar. Bar: A, 200 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We previously showed that Noggin mRNA is expressed specifically in chondrogenesis during cell differentiation of C1 cells and in the embryonic skeletogenesis, whereas Noggin expression was not observed in osteoblastic differentiation in vitro and in embryonic bone (13, 14). The present study showed that blocking of the BMP signal by Noggin inhibits expression of the cartilage marker but not that of osteoblastic markers in mesenchymal C1 cell differentiation. In addition to the C1 experiments, in organ culture studies using long bone primordia, Noggin inhibited chondrogenesis in the limb, whereas growth of the osteogenic zone was not affected by Noggin over-expression. These results suggest that intrinsic signals affected by Noggin in cartilage differentiation are not the same as those of osteoblastic differentiation. These selective effects of Noggin may imply that Noggin is a specific inhibitor of chondrogenesis rather than that of osteogenesis during skeletal cell differentiation.

Noggin is known to bind BMP2, BMP4, BMP7, and GDF5 and block their action by tethering ligands in extracellular space (31, 32). C1 cells express BMP4, BMP7, and GDF5 as ligands and express ALK2, ALK3 (BMP type IA receptor), and BMPRII as receptors. Ligands to receptor matching are known for BMP4 to ALK3 and ALK6, BMP7 to ALK2, and GDF5 to ALK6. Noggin blocked many of the cartilage-specific phenotypic markers, raising one possibility that autocrine BMP signaling that uses possibly BMP4, BMP7, and/or GDF5 is indispensable for C1 mesodermal cells to differentiate into the chondrogenic pathway. This is supported by the results of organ culture studies using embryonic long bones, where BMP4, BMP7, and GDF5 as ligands and type I and II BMP receptors are expressed as well. Indeed, previous study showed that dominant-negative BMP receptors inhibited chondrogenic differentiation and clearly indicated that proper BMP signals are required to differentiate into mature chondrocytes (33).

Ad/Noggin infection significantly inhibits the effects of exogenous BMP on C1 cells, suggesting that Ad/Noggin is effective to block the BMP signal. Regarding the effects of Noggin on endogenous BMP signaling, we used an antibody against phosphoSmad1 to examine the levels of phosphorylation of Smad1 without any BMP treatment. Because it takes more than 2 wk for C1 cells to differentiate into chondrocyte, it is hard to show change of endogenous phosphoSmad1 and to know when endogenous BMP signaling is activated and how Ad/Noggin affects it (data not shown). Although Ad/Noggin blocks the effects of exogenous BMP on C1 cells, we could not rule out other possibilities that Noggin may regulate chondrogenic differentiation independent of BMP signals.

In contrast to the effects on chondrogenic differentiation, Noggin did not block osteoblastic differentiation in C1 cells. In our protocol, the addition of bGP and AA induced ALP and OC expression in C1 cells, however Ad/Noggin infection at MOI 30 in C1 cells did not affect those expression levels. Xiao et al. (34) reported that AA treatment induces OC expression in MC3T3E1 cells, and these effects are blocked by Noggin recombinant protein at 50 ng/ml. One explanation for this discrepancy could be that MC3T3E1 cells are committed osteoblasts, whereas C1 cells are mesodermal cells that have not yet committed to either osteoblasts or chondroblasts. Therefore, Noggin may block BMP signals to promote maturation of osteoblastic cells but not block the BMP action to induce cells at a competent stage into osteoblastic pathways. Another possibility could be that the effective dose of BMP signaling to require chondrogenesis was not the same as that for osteoblastic differentiation. This possibility may be related to the differential BMP ligand repertoire during chondrogenic vs. osteoblastic differentiation in C1 cells. Noggin at MOI 30 does not inhibit osteogenesis, however, much higher doses of Noggin may block osteoblastic differentiation. A recent report on Noggin function in vivo showed that Noggin(–/–) mutant mice have calvarial defects, and this observation suggests that Noggin may positively function in membranous bone formation (35). At least a certain amount of BMP signaling is required for C1 cell differentiation because overexpression of inhibitory Smads by adenovirus in C1 cells did suppress endogenous ALP activity during osteogenic differentiation (Nifujif, A., unpublished observation).

Noggin did not affect proliferation in C1 cells, whereas it reduced the cell number of BrdU-positive cells in resting and proliferating chondrocytes in long bone rudiments. The discrepancy could be explained by differences in experimental condition for adenovirus infection. C1 cells were infected in the condition in which cells were cultured in a monolayer dish, and almost all the cells were infected. In long bone rudiments, infected cells resided in peripheral areas of long bone. The resting and proliferating chondrocytes, which were not directly infected by Ad/Noggin, showed a reduced number of BrdU-positive cells. It is possible that Noggin may indirectly affect proliferation, initially by altering expression levels of other molecules, then subsequently by regulating growth signals. Notably, Minina and co-workers (36, 37) showed that BMPs in limb pericodrium affect Indian hedgehog expression in cartilage, which in turn regulates chondrocyte proliferation and differentiation.

Several other soluble BMP inhibitors are described, including Dan, Cerberus, Gremlin, Chordin, and Twisted gastrulation (20, 22, 23, 38, 39, 40, 41). Each BMP inhibitor is expressed in certain aspects of skeletogenesis and is shown to bind BMP ligands at extracellular space. Noggin and Chordin are shown to have differential effects in cell commitment in embryonic stem cells (42). Mutant mice null for DAN, Cerberus, and Chordin do not reveal any phenotype in skeletal development, suggesting that functional redundancy may exist between these inhibitors (39, 40, 43). In contrast, Noggin(–/–) mutant mice show severe defects in skeletogenesis, including hyperdysplasia of cartilage, joint defects, and aberrant craniofacial bone growth (44). Clearly, a proper level of Noggin expression is indispensable for skeletal development. The present study further demonstrated the possibility that Noggin may function as a lineage-restricted regulator. It is likely that BMP signaling that is required for a cartilage-specific pathway differs from that for an osteogenic-specific pathway and Noggin may block the former but not the latter. It is still yet to be determined how the signaling pathways through which Noggin exerts its selective effects on skeletal differentiation.


   Acknowledgments
 
We are grateful to Drs. I. Saito and K. Takeda for technical advice on adenovirus vector construction. We thank to Dr. R. Harland for providing Xenopus Noggin cDNA.


   Footnotes
 
This work was supported by grants from the Ministry of Education, Science and Culture of Japan (13557153 and 13671895), Core Research for Engineering and Science Technology, and Japan Society for Promotion of Science.

Abbreviations: AA, Ascorbic acid; ALP, alkaline phophatase; bGP, ß-glycerophophate; BMP, bone morphogenetic protein; BrdU, bromodeoxyuridine; CAM, chorioallantoic membrane; Dex, dexamethasone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MOI, multiplicity of infection; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OC, osteocalcin; X-gal, 5-bromo-4-chloro-3-indolyl- D-galactoside.

Received June 2, 2003.

Accepted for publication March 10, 2004.


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 Results
 Discussion
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