Inhibition of β-catenin signaling causes defects in postnatal cartilage development

Endochondral bone formation is a complex process that is initiated by mesenchymal cell condensation at specific sites and differentiation into chondrocyte precursors. Immature chondrocytes develop in the center of mesenchymal condensation and undergo a highly regulated maturation process that sequentially involves chondrocyte proliferation, hypertrophy and matrix calcification. Calcified cartilage is subsequently invaded by blood vessels that deliver osteoblasts and osteoclasts precursors, in order to begin the primary bone formation and remodeling. Since each step is precisely regulated, mutations of genes involved in these processes frequently result in skeletal malformation or abnormal bone formation (Kronenberg, 2003). Several growth factors and signaling proteins have been reported to be involved in the regulation of chondrocyte differentiation and maturation, including FGF, BMP, Ihh, PTHrP, Runx2, Runx3 and Wnt (Kolpakova and Olsen, 2005; Komori, 2003; Kronenberg, 2003; Ornitz, 2005; Yoon and Lyons, 2004).

β-catenin is a key component of the canonical Wnt signaling pathway and plays a crucial role in multiple steps during chondrogenesis and chondrocyte maturation. Constitutional gene deletion of β-catenin results in lethality at stage E7.5 (Haegel et al., 1995), prior to formation of the skeletal elements. Conditional deletion of the β-catenin gene in early mesenchymal precursors results in enhanced chondrogenesis (Day et al., 2005; Hill et al., 2005), suggesting that β-catenin inhibits early mesenchymal cell differentiation into cartilage. However, conditional deletion of the β-catenin gene in chondrocytes using the Col2a1-Cre transgene causes decreased chondrocyte proliferation and delayed chondrocyte maturation (Akiyama et al., 2004). Thus, whereas β-catenin inhibits chondrogenesis, once cartilage has formed, it promotes the maturation of growth plate chondrocytes. Whereas conditional gene deletions targeting cartilage have provided insight into the role of β-catenin in the regulation of cartilage, these mutants perish before or shortly after birth, and have skeletal dysplasia as well as altered cartilage and limb morphologies that limit the understanding of the role of β-catenin as a regulator of endochondral bone formation (Akiyama et al., 2004). Thus, the role of β-catenin in postnatal development remains unknown.

Inhibitor of β-catenin and TCF (ICAT) is an 82-amino-acid peptide that was first identified using the yeast two-hybrid assay (Tago et al., 2000). Its crystal structure reveals that ICAT binds to the armadillo repeats of β-catenin and disrupts the ability of β-catenin and TCF/LEF to form a complex (Daniels and Weis, 2002; Graham et al., 2002). In vitro studies show that ICAT inhibits β-catenin signaling but not cell adhesion (Daniels and Weis, 2002). ICAT knockout (KO) mice exhibit malformation of the forebrain and craniofacial bones, and lack of kidney formation (Satoh et al., 2004). These abnormalities are due to the activation of the canonical Wnt/β-catenin signaling in specific tissues where ICAT is typically highly expressed. The role of ICAT in chondrocyte function has not been reported.

In order to specifically inhibit β-catenin signaling without disturbing cell adhesion in chondrocytes, we generated Col2a1-ICAT transgenic mice. Mice are viable after birth, which allowed us to investigate the functional role of Wnt/β-catenin signaling in chondrocytes during postnatal growth and development. We found that chondrocyte proliferation and differentiation were inhibited in Col2a1-ICAT transgenic mice. Defects in chondrocyte maturation and formation of the secondary ossification center were observed. VegfA (Vegf) is a downstream target gene of the β-catenin signaling and β-catenin directly activates Vegf gene transcription with BMP2 in chondrocytes. These findings provide new insights into the mechanism of β-catenin signaling in chondrocyte maturation.

To investigate whether β-catenin signaling is active in chondrocytes during cartilage development, we examined the X-Gal staining pattern in chondrocytes in tissue sections obtained from TOP-gal transgenic embryos (DasGupta and Fuchs, 1999) in which the lacZ reporter gene is under the control of 3×TCF responsive elements. We performed X-Gal staining using E14.5 and E16.5 mouse embryos. Extensive X-Gal staining was noted throughout the entire cartilage elements, including the skull, vertebral column, ribs and long bones in E14.5 TOP-gal embryos (Fig. 1A-D). In E16.5 embryos, X-Gal staining was present in proliferating (Fig. 1E), and hypertrophic chondrocytes (Fig. 1F), and was also observed in osteoblasts (Fig. 1F). These results demonstrate that β-catenin signaling is active in chondrocytes during embryonic cartilage development.

To investigate the function of β-catenin signaling in cartilage, we generated Col2a1-ICAT transgenic mice in which expression of the ICAT transgene was targeted to chondrocytes using the 1.0 kb type II collagen promoter (Col2a1) followed by the col2 enhancer (Fig. 2A) (Krebsbach et al., 1996; Metsaranta et al., 1991). Col2a1-ICAT transgenic mice are viable and fertile. We established three independent lines of Col2a1-ICAT transgenic mice, all of which displayed similar phenotypes. The transgenic mice were initially identified by Southern blot analysis and then genotyped using PCR of the DNA extracted from the tail tissues after mice were weaned (Fig. 2B). Body-weight measurements demonstrated that the transgenic mice exhibit an ∼30%-40% reduction in body weight compared with wild-type (WT) littermates (Fig. 2C). X-ray radiographic analysis showed that bone growth is delayed in 1-week-old, 2-week-old and 4-week-old Col2a1-ICAT transgenic mice (Fig. 2D). The size of newborn Col2a1-ICAT transgenic mice is similar to their WT littermates (Fig. 2E), suggesting that embryonic development is relatively normal in Col2a1-ICAT transgenic mice. However, Col2a1-ICAT transgenic mice are significantly smaller than WT littermates at 2 weeks of age (Fig. 2F), suggesting that the postnatal cartilage development is impaired in Col2a1-ICAT transgenic mice.

Expression of the Flag-ICAT protein was analyzed by western blotting and immunostaining using an anti-Flag antibody in primary sternal chondrocytes derived from WT and Col2a1-ICAT transgenic mice. Expression of the Flag-ICAT protein was detected by western blotting (Fig. 3A) and immunostaining (Fig. 3B) in chondrocytes isolated from Col2a1-ICAT transgenic mice but not in those from WT mice. Immunostaining was also performed using histological sections from 2-week-old mice. Expression of Flag-ICAT was detected in proliferating chondrocytes in growth plate and in articular chondrocytes in Col2a1-ICAT transgenic mice (Fig. 3C). To determine whether β-catenin signaling is blocked in Col2a1-ICAT transgenic mice, we transfected the TOP-flash reporter construct into primary chondrocytes derived from Col2a1-ICAT transgenic mice and WT littermates. The activity of the TOP-flash reporter is about 30% lower in chondrocytes derived from Col2a1-ICAT transgenic mice than in those derived from WT mice. Wnt3a-induced reporter activity was completely blocked in chondrocytes derived from Col2a1-ICAT transgenic mice (Fig. 3D). These results suggest that β-catenin signaling is inhibited in Col2a1-ICAT transgenic chondrocytes. To further determine the specificity of the ICAT transgene expression, total RNA was extracted from multiple tissues and the expression of Flag-ICAT mRNA was examined. We found that strong expression of Flag-ICAT was detected from ribs, weak expression of Flag-ICAT was detected from the brain and the transgene was not detected from other tissues, including spleen, kidney, lung and liver (Fig. 3E). Because β-catenin is a bi-functional molecule that also regulates cell adhesion through interaction with the membrane protein, cadherin adhesion assays were performed using primary chondrocytes isolated from Col2a1-ICAT transgenic mice and WT littermates (McCrea et al., 1991; Peifer et al., 1992). Cadherin-mediated cell adhesion is protected by Ca²⁺ (Xue et al., 2005). Therefore, chondrocytes were treated with trypsin-CaCl₂ (TC) or trypsin-EDTA (TE) and the relative cell aggregation was determined by the TC:TE ratio (Xue et al., 2005). Protein levels of cadherin and β-catenin were unchanged in Col2a1-ICAT transgenic mice (data not shown). Overexpression of ICAT did not alter chondrocyte cell adhesion (Fig. 3F). However, as a positive control, deletion of the β-catenin gene – by using Ad-Cre infection – in primary chondrocytes isolated from the β-catenin^fx/fx mice (Brault et al., 2001) reduced chondrocyte cell adhesion (Fig. 3F). These results establish that ICAT specifically inhibits β-catenin signaling without affecting cell adhesion in Col2a1-ICAT transgenic chondrocytes.

Analysis of histological sections showed a dramatic delay in the formation of the secondary ossification center (SOC) in Col2a1-ICAT transgenic mice compared with their WT littermates, especially in 2-week-old transgenic mice (Fig. 4A). In WT mice, chondrocyte hypertrophy and vascular invasion were observed in the epiphyseal area in 1-week-old mice. By contrast, vascular invasion was delayed until the age of 2 weeks in Col2a1-ICAT transgenic mice (Fig. 4A), indicating defects in chondrocyte maturation and endochondral bone formation. Histomorphometric analyses showed that the width of both the proliferating and the hypertrophic zones in the growth plate of the tibia and the femur were reduced in 2-week-old Col2a1-ICAT transgenic mice (Fig. 4B-F), suggesting alterations in both chondrocyte proliferation and maturation in chondrocytes with inhibition of β-catenin signaling.

To evaluate chondrocyte proliferation, we performed Ki-67 and DAPI double immunostaining and counted percentage Ki-67-positive cells by normalizing to DAPI-positive cells. We found that cell proliferation was reduced 24% in growth-plate chondrocytes derived from Col2a1-ICAT transgenic mice (Fig. 5A,B). Consistent with this finding, Western blot analysis showed reductions in the expression of cyclin D1, D2 and PCNA proteins in primary chondrocytes isolated from Col2a1-ICAT transgenic mice (Fig. 5C). In contrast, the expression of cyclin A was not significantly changed (Fig. 5C). In addition, no significant changes in total and phosphorylated β-catenin and phosphorylated Rb proteins were observed (Fig. 5C). The expression of chondrocyte differentiation marker genes, including collagen type X (Col10a1, hereafter referred to as colX), alkaline phosphatase liver/bone/kidney (hereafter referred to as Alp), vascular endothelial growth factor A (hereafter referred to as Vegf) and matrix metalloproteinase 13 (Mmp13) were examined by real-time reverse transcriptase (RT)-PCR in primary chondrocytes isolated from sterna of 3-day-old neonatal Col2a1-ICAT transgenic mice and their WT littermates. The expression of all of the chondrocyte marker genes was significantly decreased in the Col2a1-ICAT transgenic mice (Fig. 5D). Apoptosis, as determined by TUNEL staining showed that chondrocyte apoptosis was significantly increased in hypertrophic zone of Col2a1-ICAT transgenic mice (Fig. 5E). These results suggest that the reduced width of the hypertrophic zone could be owing to less chondrocytes entering the hypertrophic zone (decreased chondrocyte proliferation) and more cells leaving the hypertrophic zone (increased apoptosis in hypertrophic chondrocytes). Altogether, the findings show a reduction in chondrocyte proliferation and differentiation and an increase in chondrocyte apoptosis in Col2a1-ICAT transgenic mice, leading to reduced widths of the proliferating and hypertrophic zones, delayed formation of secondary ossification center, and a reduced rate of skeletal growth.

The observations in the Col2a1-ICAT transgenic mice confirm a role of β-catenin in the promotion of chondrocyte maturation in the growth plate. Although the BMP signaling pathway has also been shown to have a role in promoting chondrocyte maturation, interactions between β-catenin and BMP signaling in chondrocytes have not been described previously. Since prior work in our laboratory established that TGFβ stimulates β-catenin signaling (Li et al., 2006a), initial experiments were performed to examine whether BMP2 activates β-catenin signaling in chondrocytes. Primary chondrocytes were isolated from TOP-gal transgenic mice and treated with BMP2 (100 ng/ml) for 24 hours. We found that BMP2 did not stimulate β-catenin signaling in β-Gal assay (data not shown). However, treatment with Wnt3a (100 ng/ml) stimulated Bmp2 and Bmp4 mRNA expression in primary chondrocytes (Fig. 6A) and upregulated the BMP signaling reporter (12×SBE) activity (Fig. 6B), providing evidence of cross-talk between these pathways. When Wnt3a was cultured with primary chondrocytes for 4 and 6 days, it induced colX mRNA expression. Addition of noggin (400 ng/ml), an antagonist of BMP2 and BMP4 ligands, completely blocked this effect (Fig. 6C). In Col2a1-ICAT transgenic chondrocytes, the expression of Bmp2 and Bmp4 is reduced (Fig. 6D). These results suggest that BMP signaling is downstream of β-catenin signaling in chondrocytes and that β-catenin stimulates chondrocyte maturation at least in part through activation of BMP signaling.

To determine whether there is a cooperative role between BMP2 and β-catenin in the induction of chondrocyte maturational markers, primary chondrocytes isolated from WT and Col2a1-ICAT transgenic mice were treated with BMP2. Treatment with BMP2 restored colX and Alp mRNA expression in chondrocytes isolated from Col2a1-ICAT transgenic mice and compensated for the loss of β-catenin signaling (Fig. 7A,B). By contrast, BMP2 failed to restore Vegf and Mmp13 expression in Col2a1-ICAT transgenic chondrocytes, despite the BMP2 responsiveness of these genes in WT chondrocytes (Fig. 7C,D). These results suggest that BMP2 regulates Vegf and Mmp13 expression in a β-catenin-signaling-dependent manner.

β-catenin has been reported to regulate VEGF gene transcription in cancer cells (Easwaran et al., 2003). In the growth plate, VEGF is secreted by mature chondrocytes (Zelzer et al., 2004) and induces the invasion of blood vessels. In Col2a1-ICAT transgenic mice, the formation of the secondary ossification center in the epiphysis of the growth plate was substantially delayed. These results suggest that Vegf expression is directly controlled by β-catenin in chondrocytes. To further determine the regulatory mechanism of Vegf expression by β-catenin and BMP signaling, cells of the the chondrogenic cell line RCJ3.1C5.18 (RCJ3.1) were treated with BMP2 (100 ng/ml) and the GSK-3β inhibitor (2′Z,3′E)-6-bromoindirubin-3′-oxime (BIO; 1 μg/ml) in the presence or absence of noggin (400 ng/ml) or Dkk1 (1 μg/ml), which inhibit BMP or Wnt signaling, respectively. BMP2 significantly induced Vegf expression within 2 hours. Dkk1 completely inhibited BMP2-induced Vegf expression (Fig. 7E). Similar to BMP2, BIO also induced Vegf expression at the same time frame in these cells. Addition of noggin only partially inhibited BIO-induced Vegf expression (Fig. 7F). These results suggest that Vegf is the direct downstream target gene for Wnt/β-catenin signaling in chondrocytes and that induction by either signaling pathway requires cooperative interactions with the other pathway. To further confirm the regulatory role of β-catenin signaling in the expression of VEGF and MMP13, we performed immunostaining assays to examine changes in the expression of VEGF and MMP13 in Col2a1-ICAT transgenic mice. The results demonstrated that the expression of VEGF and MMP13 proteins was significantly reduced in Col2a1-ICAT transgenic mice (Fig. 7G,H).

To determine whether β-catenin/TCF directly regulates VegF gene transcription, the mouse VEGFa promoter (since Vegfa is the Vegf isoform expressed in chondrocytes) was cloned and the effect of β-catenin on Vegf promoter activity was examined in RCJ3.1 chondrogenic cells. Transfection of constitutively active β-catenin (β-catenin^S33Y) stimulated Vegf promoter activity more than threefold (Fig. 8A). Subsequent experiments using serial deletion constructs of the Vegf promoter show a marked reduction in promoter activity in the –140/+87 reporter (Fig. 8B), while a threefold induction of the promoter is maintained in the –1341/+87 and –940/+87 reporter constructs. Sequence analysis shows multiple TCF-response elements located in the 1-kb region of the Vegf proximal promoter and there are five putative TCF/LEF-binding sites within the 1-kb Vegf proximal promoter (Fig. 8C). To determine whether β-catenin binds to the Vegf promoter, RCJ3.1 cells were treated with BIO, and ChIP assay was performed. Binding of β-catenin to one of these TCF-binding regions located in the proximal promoter was detected by ChIP assay and, as expected, β-catenin addition of BIO significantly enhanced binding of β-catenin to the Vegf promoter (Fig. 8D). These results demonstrate that β-catenin directly activates Vegf gene transcription in chondrocytes.

Delayed formation of secondary ossification center might be related to the decreased expression of Vegf and Mmp13. To determine whether there is a defect in the vascularization in Col2a1-ICAT transgenic mice, we examined the expression of endothelial cell marker platelet endothelial cell adhesion molecule precursor (PECA1, hereafter referred PECAM-1) in WT and Col2a1-ICAT transgenic mice and found that expression of PECAM-1 was significantly decreased in Col2a1-ICAT transgenic mice (Fig. 9A). The vascularization was then further examined in 2-week-old WT and Col2a1-ICAT transgenic mice by using microtomography (microCT) (Zhang et al., 2005). The results showed that the Col2a1-ICAT transgenic mice had less vascular structure than the WT controls (Fig. 9B), demonstrating defects of angiogenesis in cartilage of Col2a1-ICAT transgenic mice. To determine the specificity of this defect, we also performed the lead perfusion experiments in livers using WT and transgenic mice of the same age. Compared with the defects in cartilage angiogenesis, only a small difference between WT and Col2a1-ICAT transgenic mice was found in the vascularization of liver (Fig. 9C). These results indicate the specific defects in vascularization in the cartilage tissues of Col2a1-ICAT transgenic mice.

Limb development is a complicated process and is regulated by bone-growth factors, signaling molecules and key transcription factors. β-catenin is a central molecule in canonical Wnt signaling pathway and has a crucial role in the various steps involved in endochondral bone formation. β-catenin activity is regulated by several proteins including ICAT, which inhibits the interaction of β-catenin with TCF/LEF. Col2a1-ICAT transgenic mice with chondrocyte-specific transgene expression were generated in order to determine the role of β-catenin signaling in chondrocyte development. A marked effect was observed on postnatal chondrocyte maturation in Col2a1-ICAT transgenic mice, establishing for the first time that Wnt/β-catenin signaling is required for postnatal chondrocyte development. In the present study, we generated seven Col2a1-ICAT founder mice. We could not establish transgenic lines with two founder mice because their offspring is very small and mice usually died within 1 month after birth. We have established and characterized three lines of the Col2a1-ICAT transgenic mice that survive into adulthood and show significant defects in postnatal cartilage development. Other transgenic mice show only a moderate phenotype. These observations suggest a dosage effect for the transgene expression and severity of the phenotype.

The crucial role of β-catenin in endochondral bone formation has been demonstrated in animal models using tissue-specific deletion or conditional activation of the β-catenin gene in chondrocytes (Akiyama et al., 2004). However, the prenatal or perinatal lethality due to the severe developmental defects restricts the usage of these models to further analyze the role of β-catenin signaling in postnatal chondrocyte development. Since ICAT only inhibits canonical Wnt signaling without interruption of cell adhesion and because ICAT might be a reversible inhibitor of β-catenin/TCF interaction, use of the Col2a1-ICAT transgenic mouse model provides us with an opportunity to investigate the role of β-catenin signaling in postnatal cartilage development. Our findings clearly demonstrate that β-catenin has an essential role in postnatal chondrocyte maturation and endochondral bone formation within long bones.

In Col2a1-ICAT transgenic mice, chondrocyte maturation is delayed and the width of the hypertrophic zone is reduced, especially in 1-week-old and 2-week-old Col2a1-ICAT transgenic mice. Consistent with a previous report (Akiyama et al., 2004), chondrocyte proliferation was reduced in Col2a1-ICAT transgenic mice, demonstrated by the reduction of Ki-67-positive chondrocytes and the decreased width of the proliferating zone in 1-week-old and 2-week-old Col2a1-ICAT transgenic mice. Since cell apoptosis is increased in the hypertrophic zone of Col2a1-ICAT transgenic mice, the reduced hypertrophic zone observed in Col2a1-ICAT transgenic mice might be owing to the combined effect of decreased chondrocytes entering into the hypertrophic zone and increased chondrocyte apoptosis.

In this study, we described that β-catenin signaling is necessary for normal vascularization of cartilage. Evidence in support include findings that: (1) Vegf and Mmp13 expression was reduced in Col2a1-ICAT transgenic mice and both genes have an important role in vascularization and endochondral conversion; (2) PECAM-1 is a vascular endothelial protein whose expression in the growth plate is decreased in Col2a1-ICAT transgenic mice; (3) vascular invasion is a crucial initial step for the formation of the secondary ossification center, which was significantly delayed in Col2a1-ICAT transgenic mice and; (4) lead perfusion and microCT vascularization analysis demonstrated that blood-vessel formation is dramatically impaired in Col2a1-ICAT transgenic mice. These results indicate that vascularization is crucial for postnatal cartilage development and β-catenin signaling plays an important role in this process. The mouse Vegfa gene has three alternative splicing products, VEGF120, VEGF164 and VEGF188. Mice expressing only VEGF120 die right after birth (Maes et al., 2002; Maes et al., 2004). Mice expressing only VEGF164 show normal bone development (Maes et al., 2002; Maes et al., 2004). However, mice expressing only VEGF188 display a phenotype similar to that observed by us in Col2a1-ICAT transgenic mice, including delayed formation of the secondary ossification center, defects in epiphysis angiogenesis and increased cell apoptosis (Maes et al., 2004).

Using the primary sternal chondrocytes and chondrogenic cell lines, we demonstrated that Vegf is the direct downstream target gene for β-catenin/TCF signaling in chondrocytes. Activation of BMP2 and Wnt signaling pathways resulted in stimulation of Vegf gene expression. We demonstrated the following: (1) Wnt3a and BMP2 stimulate expression of Vegf mRNA within 2 hours. (2) In Col2a1-ICAT transgenic chondrocytes both basal and BMP2-induced Vegf expression is significantly reduced. Although our experiments were performed in isolated primary chondrocytes, β-catenin might have similar effects on Vegf expression in mesenchymal cells and neighboring cells in vivo. (3) Wnt3a-induced Vegf expression is completely blocked by addition of noggin, a BMP-ligand antagonist. (4) The Vegf promoter region responsive to β-catenin is located in the 1-kb region of the Vegf proximal promoter. (5) Five potential TCF-binding sites and one potential Smad1/Smad5-binding site have been identified in this region by sequence analysis and – demonstrated by ChIP assay – β-catenin binds to this region. Although further analysis is still required, current findings strongly suggest that BMP2 and Wnt signaling molecules activate Vegf gene transcription in a coordinated fashion.

Previously, interactions between Wnt/β-catenin and BMP signaling pathways have been reported; in osteoblasts, BMP2 and β-catenin stimulate osteoblast differentiation synergistically (Mbalaviele et al., 2005; Rawadi et al., 2003). Here, we demonstrated that (1) Wnt3a and β-catenin stimulate Bmp2 and Bmp4 mRNA expression; (2) in Col2a1-ICAT transgenic mice, Bmp2 and Bmp4 expression was reduced; and (3) Wnt3a-induced colX expression was completely inhibited by the addition of the BMP antagonist noggin. Whereas these results suggest that BMP signaling is downstream of the Wnt/β-catenin signaling in chondrocytes during the process of chondrocyte maturation, our findings also show that Wnt/β-catenin and BMP signaling molecules act coordinately during the activation of Vegf gene transcription. Thus, in chondrocytes, the relationship between these two signaling pathways is complex and, in some cases, gene specific. Our findings establish that these two signaling pathways are highly inter-related and have a crucial role in postnatal chondrocyte development.

β-galactosidase (β-gal) staining was performed in embryos. In brief, specimens were dissected in PBS and prefixed in PBS containing 1% formaldehyde, 0.2% glutaraldehyde, 2 mM MgCl₂, 5 mM EGTA, and 0.02% Nonidet P-40 at 4°C for 30-90 minutes. Samples were washed three times in PBS containing 0.02% Nonidet P-40 at room temperature for 30 minutes before they were stained in PBS containing 1 mg/ml X-Gal, 5 mM potassium ferricyanide, 2 mM potassium ferrocyanide, 2 mM MgCl₂, 0.01% sodium deoxycholate, and 0.02% Nonidet P-40 at 30°C for 6-16 hours. The specimens were subsequently fixed in formaldehyde and processed for paraffin or frozen sections.

TOP-gal mice were obtained from The Jackson Laboratory (Bar Harbor, ME). To generate the Col2a1-ICAT transgenic mice, DNA fragments encoding ICAT were cloned into the NotI site of a collagen 2 α1 (Col2a1)-based expression vector PKN185 (Tanaka et al., 2000; Tsuda et al., 2003). The resulting vector contains the Flag-ICAT expression unit and includes the 5′ NdeI site of the Col2a1 promoter (nucleotides 1940-2971, GenBank accession number: M65161), the β-globin intron cassette, Flag-ICAT, SV40 poly (A) and the Col2a1 enhancer (nucleotide 4930–5571, GenBank accession number: M65161). The expression unit of Flag-ICAT was excised by NdeI and HindIII digestion. The ICAT transgene was then purified and injected into pronuclei of fertilized eggs (C57BL/6J). Positive transgenic founder mice were identified by PCR and confirmed by Southern blot analysis.

Primary mouse sternal chondrocytes were isolated as described (Li et al., 2006b). Briefly, 3-day-old neonatal mice were sacrificed and genotyped using tail tissues obtained at sacrifice. The anterior rib cage and sternum were harvested en-bloc, washed with PBS and then digested with Pronase (Roche Applied Science, Indianapolis, IN) dissolved in PBS (2 mg/ml) in a 37°C water bath with continuous shaking for 60 minutes. This was followed by incubation in a solution of collagenase D (3 mg/ml dissolved in serum-free Dulbecco's modified Eagle's medium (DMEM; Roche Applied Science) for 90 minutes at 37°C. The soft tissue debris was removed and the remaining sterna and costosternal junctions were further digested in fresh collagenase D solution in Petri dishes in a 37°C incubator for 5 hours with intermittent shaking. This step allows remnant fibroblasts to attach to the Petri dish while the chondrocytes remain afloat in the medium. The digestion solution was filtered through Swinex to remove all residual bone fragments. The solution was centrifuged, and the cells were resuspended in complete medium [DMEM with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 100 mM L-glutamine, and 50 μg/ml ascorbic acid, pH 7.1]. Cells were then counted and plated at the appropriate density. To remove any remaining fibroblasts, 24-hour cultures were treated with 0.05% trypsin for 1 minute to lift the fibroblasts from the culture dish while allowing the chondrocytes to remain attached.

The following antibodies were used in this study: anti-β-catenin monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-cyclin D1 monoclonal antibody (1:1000 dilution, Upstate, Charlottesville, VA), anti-Flag monoclonal antibody (1:200 dilution for immuno-fluorescence assay and 1:1000 dilution for western blotting, Sigma), rabbit anti-Ki67 monoclonal antibody (1:200 dilution for immunostaining, Lab Vision, Fremont, CA), and anti-PECAM-1 antibody (1:100 for immunostaining, Santa Cruz Technology). BIO was purchased from Calbiochem (San Diego, CA), and dissolved in DMSO. BMP2, Wnt3a, noggin and Dkk1 were obtained from R&D Systems (Minneapolis, MN).

Cells were lysed on ice for 30 minutes in a buffer containing 50 mM Tris-HCL pH 7.4, 150 mM NaCl, 1% Nonidet P-40, and 0.1% SDS supplemented with protease inhibitors (10 μg/ml leupeptin, 10 μg/ml pepstatin A, and 10 μg/ml asprotinin) and phosphatase inhibitors (1 mM NaF and 1 mM Na₃VO₄). Proteins were fractionated by SDS-PAGE, transferred to nitrocellulose membrane, and detected using the respective antibodies. Bound primary antibodies were detected with horseradish-peroxidase-conjugated secondary antibodies followed by ECL-mediated visualization (Amersham, Piscataway, NJ).

Mouse tissues were dissected in PBS, fixed in 10% buffered formalin, and paraffin embedded. Tissue sections were subject to immune complex staining using an avidin–biotinylated-enzyme complex according to the manufacturer's protocol (Vector Laboratories, Burlingame, CA). Rabbit monoclonal anti-Ki-67 was used in the analysis. Bound primary antibodies were detected with fluorescein-conjugated secondary antibodies (Amersham, Piscataway, NJ). Immunostaining of cultured cells was performed using indirect fluorescent staining technique. Briefly, cells were fixed with 4% paraformaldehyde for 10 minutes and treated with 0.5% Triton X-100 for 15 minutes and 50 mM glycine for 10 minutes followed by blocking with the PBS-Triton buffer containing 3% BSA for 30 minutes. Samples were then incubated with primary antibody for 1 hour and fluorescent conjugated secondary antibody for 45 minutes and mounted with vectashield (Lab Vision, Fremont, CA). The anti-Flag mouse monoclonal antibody was used as primary antibody.

Primary sternal chondrocytes isolated from wild-type (WT) or Col2a1-ICAT transgenic mice were plated into 12-well culture plates and grown to 60% confluence. The cells were treated with the growth factors as indicated. 48 hours after incubation, cells were washed twice with PBS, and cell lysates extracted with passive lysis buffer (Promega, Madison, WI). ALP activity in cell lysates was measured using a Sigma ALP assay kit (Sigma, St Louis, MO) and normalized by the protein content.

Total cellular and tissue RNA was prepared by Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. One microgram total RNA was used to synthesize cDNA using iScripts cDNA Synthesis Kit (Bio-Rad, Hercules, CA). PCR primers were: VEGF Fw: 5-TTACTGCTGTACCTCCACC-3, Rev: 5′-ACAGGACGGCTTGAAGATG-3′; MMP13 Fw: 5′-TTTGAGAACACGGGGAAGA-3′, Rev: 5-ACTTTGTTGCCAATTCCAGG-3′; Col-X Fw: 5′-ACCCCAAGGACCTAAAGGAA-3′, Rev: 5′-CCCCAGGATACCCTGTTTTT-3′; BMP2 Fw: 5′-GCTTTTCTCGTTTGTGGAGC-3′, Rev: 5′-TGGAAGTGGCCCATTTAGAG-3′; BMP-4 Fw: 5′-GAGGAGGAGGAAGAGCAGAG-3′, Rev: 5′-TGGGATGTTCTCCAGATGTT-3′; ALP Fw: 5′-TGACCTTCTCTCCTCCATCC-3′, Rev: 5′-CTTCCTGGGAGTCTCATCCT-3′.

A TUNEL staining kit (DeadEnd Fluorometric TUNEL System, Promega, Madison, WI) was used to assess cell death by catalytically incorporating fluorescein-12-dUTP at 3′-OH DNA ends using the terminal deoxynucleotidyl transferase and recombinant enzyme (rTDT). After paraffin removal, the tissue sections were placed in equilibration buffer and then in a solution containing the equilibration buffer, nucleotide mix, and rTDT enzyme and incubated at 37°C for 1 hour. The reaction was stopped with 2× saline sodium citrate (SSC). Hoechst dye 33342 was used to stain the nuclei. Fluorescence microscopy (Zeiss, Axiovert 40 CFL, Chester, VA) was used to identify apoptotic cells.

We used the 2.7 kb mouse BMP2 promoter and 2.0 kb mouse BMP-4 promoter (Feng et al., 1995; Feng et al., 2003; Garrett et al., 2003), constitutively active β-catenin, β-catenin^S33Y (gift from Kenneth Kinzler) (Morin et al., 1997), and the BMP signaling reporter, 12×SBE-luc (Zhao et al., 2003).

Cells were transfected by Lipofectamine 2000 (Invitrogen) with various combinations of plasmids. 0.5 μg of reporter plasmids (TOP-flash, 12×SBE-luc) and 0.01 μg of internal control SV-40 Renilla plasmid were using in the reporter assays. The luciferase activity was measured using the Promega dual system kit.

Chromatin IP was performed as described in the manual of the ChIP Assay kit (Upstate, Charlottesville, VA). Briefly, RCJ3.1C518 chondrocytes were cultured in 4×15 cm culture dishes to 70-80% confluence. Chondrocytes were then treated with 1 μM BIO for 4 hours, followed by crosslinking using formaldehyde at 37°C for 10 minutes. The crosslink reaction was stopped using glycine buffer, cells were washed with a protease inhibitor cocktail (Roche Applied Science) and harvested. Chondrocytes were then incubated with lysis buffer on ice for 30 minutes and the cell lysate was sonicated to shear the genomic DNA to 200-bp to 500-bp fragments. After centrifugation, the supernatant was incubated with protein G beads saturated with salmon sperm DNA for 30 minutes to pre-clean the cell lysate. A total of 50 μl anti-β-catenin antibody (Santa Cruz Technology) was added to the cell lysate and incubated for overnight at 4°C. Lysates were then incubated with 100 μl of proteinG beads for 2 hours. Beads were pelleted by centrifugation for 2 minutes at 4000 rpm and washed according to the manufacturer's protocol. The protein-DNA complex was eluted with ChIP elution buffer containing 0.01M NaHCO₃ and 0.5% SDS. After centrifugation, 5M NaCl was added to the eluted solution to a final concentration of 200 mM and then incubated at 65°C overnight. After reversing the crosslink, the samples were further digested by proteinase K. The DNA was extracted by phenol-chloroform, precipitated with ethanol and prepared for PCR. Primer sequences were: TRE Fw: 5′-ACTCTAGTTGTCCCTATCCTCA-3′, TRE Rev: 5′-TCTGCGCTTCTCACCGGTAACA-3′; Cont Fw: 5′-AGAGCTTGCCCGAGGAATGT-3′, Cont Rev: 5′-CTCCGATACCTGTGGGAAGA-3′; GAPDH Fw: 5′-AACGACCCCTTCATTGAC-3′, GAPDH Rev: 5′-TCCACGACATACTCAGCAC-3′.

The bone vascular network was examined on tissue sections of animals following perfusion of a lead-chromate-based contrast agent using microCT analysis. Microfil MV-122 contrast medium, a radiopaque silicone rubber compound containing lead chromate, was perfused through the heart together with 4% paraformaldehyde. After perfusion, the hind limbs were decalcified using 10% EDTA solution. After complete decalcification, the samples were scanned again to image vascularization.

We thank Kenneth Kinzler (The Johns Hopkins University Medical Institutions, Baltimore, Maryland) for providing us the β-catenin^S33Y plasmid and Tetsu Akiyama (University of Tokyo, Japan) for the ICAT plasmid. This work was supported by grants AR038945 (to R.J.O.), AR053717 (to R.J.O.) and AR054465 (to D.C.) from the National Institute of Health.