Correspondence to M. Amling: amling{at}uke.uni-hamburg.de
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Abbreviations used in this paper: Bsp, bone sialoprotein; Lrp5, low density lipoprotein receptor-related protein 5; Osc, osteocalcin; Osx, osterix; Phex, phosphate-regulating gene with homologies to endopeptidases located on the X-chromosome; Runx2, runt-related transcription factor 2; Tnsalp; tissue-nonspecific alkaline phosphatase.
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Introduction |
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Once the vertebrate skeleton has reached its final size and shape, bone is constantly remodeled, thereby providing the possibility to adapt to changes in daily activities and to contribute to mineral homeostasis. This remodeling process is coordinated on several levels to achieve a balance between bone formation by osteoblasts and bone resorption by osteoclasts, thereby maintaining a sufficient bone mass (Amling et al., 2000). The disturbance of this balance can lead to bone loss and osteoporosis, one of the most prevalent degenerative diseases. Therefore, the identification of mechanisms regulating the differentiation and activity of bone-forming osteoblasts is an important step toward the identification of novel therapeutic options to regain bone mass in osteoporotic patients.
Osteoblasts differentiate from mesenchymal precursor cells in a process requiring the coordinated activity of transcription factors and other signaling proteins (Harada and Rodan, 2003). Among these molecules, the runt-related transcription factor 2 (Runx2) is generally considered to be a master regulator of osteoblast differentiation and bone formation based on overwhelming genetic evidence (Karsenty, 1999; Komori, 2002). First, Runx2-deficient mice fail to form bone, which is explained by a complete absence of osteoblasts (Komori et al., 1997; Otto et al., 1997). Second, mice lacking one allele of Runx2 display a defect in intramembranous ossification that is reminiscent of cleidocranial dysplasia, an autosomal dominant disorder caused by mutations of the human Runx2 gene (Lee et al., 1997; Mundlos et al., 1997, Otto et al., 1997). Third, transgenic mice expressing a dominant-negative variant of Runx2 in differentiated osteoblasts display an osteopenic phenotype, thereby demonstrating that Runx2 is also required for the functional activity of osteoblasts (Ducy et al., 1999). This action of Runx2 is, at least in part, mediated through osterix (Osx), another transcription factor required for osteoblast differentiation, acting downstream of Runx2 (Nakashima et al., 2002).
Although Runx2 has an additional function in the regulation of chondrocyte hypertrophy (Takeda et al., 2001), the most important transcription factor in terms of chondrogenesis is Sox9, because it is required for determination, proliferation, and differentiation of mesenchymal progenitors into chondrocytes (De Crombrugghe et al., 2001). Accordingly, endochondral bone formation is inhibited at an early phase and does not take place in the absence of Sox9 (Akiyama et al., 2002). Similarly, human patients with heterozygous SOX9 mutations suffer from campomelic dysplasia, a severe skeletal malformation syndrome (Foster et al., 1994; Wagner et al., 1994). Sox9 belongs to a family of high mobility group transcription factors with close resemblance to the male sex-determining factor Sry in their DNA-binding domain and many different functions during mammalian development (Wegner, 1999; Bowles et al., 2000). Among these Sox proteins, Sox9 exhibits closest similarity to Sox8 and Sox10, and together these three proteins form the group E of the Sox family. Sox10 has been studied extensively and is required in derivatives of the neural crest and glial cells of the central nervous system at various phases of development (Herbarth et al., 1998; Southard-Smith et al., 1998; Britsch et al., 2001; Stolt et al., 2002; J. Kim et al., 2003). In contrast, much less is known about the physiologic role of Sox8.
Despite the widespread expression of Sox8 (Pfeifer et al., 2000; Schepers et al., 2000), Sox8-deficient mice appeared remarkably normal, exhibiting mainly a significant overall weight reduction (Sock et al., 2001). The lack of a more severe phenotype may be explained by the fact that in many tissues Sox8 is coexpressed with Sox9 and Sox10. Because Sox8 has similar biochemical properties as Sox9 and Sox10 in vitro, the Sox8 deficiency is possibly compensated by the remaining production of either of these two Sox proteins. Previously, we had however noticed, that ossification of tarsal bones in the hindfeet of Sox8-deficient mice was disturbed (Sock et al., 2001), arguing that Sox8 might have a previously uncharacterized role in skeletal biology. This finding prompted us to analyze the skeletal phenotype of Sox8-deficient mice.
Here, we show that Sox8-deficient mice display an osteopenic phenotype that is caused by an intrinsic osteoblast differentiation defect. Primary osteoblasts derived from these mice show an accelerated differentiation and mineralization compared with wild-type cells, where Sox8 expression ceases upon differentiation. The fact that transgenic mice that express Sox8 under the control of an osteoblast-specific Col1a1 promoter fragment display severe defects in bone formation demonstrates that the down-regulation of Sox8 is required for osteoblast differentiation and an essential step in bone remodeling.
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Results |
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Therefore, it was likely that the size reduction of the Sox8-deficient mice is caused by defects in postnatal bone formation. This possibility is underscored by the finding that bones formed by intramembranous ossification were also affected in the absence of Sox8. Besides the reduction of the clavicle size (Fig. 1 G) we observed a hypomineralization of the calvariae using µCT analysis (Fig. 1 H). The quantification of the calvarial thickness revealed that the absence of Sox8 leads to a 50% reduction at the age of 6 and 20 wk. This suggested that Sox8-deficient mice display an osteopenic phenotype that is caused by an impaired bone formation.
We next performed a complete histomorphometric analysis of bone remodeling parameters in Sox8-deficient mice and wild-type littermates at 6 and 20 wk old. Von Kossa staining of undecalcified sections confirmed the osteopenia in Sox8-deficient mice at both ages (Fig. 2, A and B). The histomorphometric analysis revealed that the trabecular bone volume in vertebral bodies and tibiae of Sox8-deficient mice was decreased by >30% compared with wild-type littermates. Further analysis demonstrated that trabecular number and trabecular thickness were significantly decreased in Sox8-deficient mice at both ages (Fig. 2 C).
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Accelerated osteoblast differentiation in the absence of Sox8
To determine whether the decreased bone formation of Sox8-deficient mice is caused by an intrinsic osteoblast differentiation defect, we next studied the behavior of wild-type and Sox8-deficient primary calvarial osteoblasts ex vivo. We first analyzed Sox8 expression in these cells by Northern blotting and found a strong expression in nondifferentiated wild-type cultures. Importantly, this expression was completely abolished 5 and 10 d after the addition of ascorbic acid and ß-glycerophosphate, two agents leading to terminal osteoblast differentiation and mineralization ex vivo (Fig. 3 A). We next determined the proliferation of wild-type and Sox8-deficient primary calvarial osteoblasts. Using a BrdU incorporation assay we found that the proliferation rate of Sox8-deficient cells was strongly reduced compared with wild-type cells, even before osteoblast differentiation was induced (Fig. 3 B). To analyze osteoblast differentiation we performed Von Kossa staining of the mineralized matrix from wild-type and Sox8-deficient cells formed before (d0) as well as 5 and 10 d after the addition of ascorbic acid and ß-glycerophosphate. Unexpectedly, we observed an accelerated mineralization of Sox8-deficient cells. In contrast to wild-type cultures, mineralized nodules were already detectable after 5 d of differentiation. After 10 d the mineralization was still much more pronounced (Fig. 3 C).
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We next analyzed the expression of osteoblast differentiation markers in wild-type and Sox8-deficient primary osteoblasts by RT-PCR. In wild-type cultures expression of tissue-nonspecific alkaline phosphatase (Tnsalp), osteocalcin (Osc), bone sialoprotein (Bsp), phosphate-regulating gene with homologies to endopeptidases located on the X-chromosome (Phex), Runx2 and Osx was observed only 5 and 10 d after the addition of ascorbic acid and ß-glycerophosphate (Fig. 4 A). In Sox8-deficient cells however, we observed a different expression pattern for all of these genes except Osc. Whereas Tnsalp was prematurely down-regulated in the absence of Sox8, Bsp, and Phex, two genes associated with ECM mineralization, were expressed in Sox8-deficient cells even without the addition of ascorbic acid and ß-glycerophosphate (Fig. 4 A). A premature expression in Sox8-deficient cells was also observed for Runx2 and Osx, two genes encoding transcription factors required for osteoblast differentiation. In contrast, we did not observe changes in the expression pattern of low density lipoprotein receptor-related protein 5 (Lrp5), a gene that was recently identified to play a major role in bone formation in a Runx2-independent manner (Kato et al., 2002).
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To further confirm the premature differentiation of Sox8-deficient osteoblasts in vivo, we performed a Northern blot expression analysis using RNA from calvaria and femur of wild-type and Sox8-deficient mice at 6 wk old (Fig. 4 C). Thereby, we found an increased expression of Bsp, Phex, and Runx2, which is consistent with the RT-PCR expression analysis described above. Together, these data suggested that Sox8 is a negative regulator of osteoblast differentiation, potentially acting through Runx2.
Impaired bone formation in Col1a1-Sox8 transgenic mice
The fact that Sox8 expression is down-regulated upon differentiation of wild-type osteoblasts raised the hypothesis that this down-regulation is a prerequisite for osteoblast differentiation. To interfere with Sox8 down-regulation in osteoblasts we generated transgenic mice that express Sox8 under the control of an osteoblast-specific Col1a1 promoter fragment that is active in osteoblast precursor cells and maintains its activity in differentiated osteoblasts (Rossert et al., 1995, 1996). The transgene consisted of the complete Sox8 ORF placed under the control of the 2.3-kb osteoblast-specific Col1a1 promoter fragment (Fig. 5 A). Four transgenic founders with similar phenotype and transgene expression were obtained. Three of them were used to generate transgenic mouse lines (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200408013/DC1). The fourth had to be killed at 10 d old because of spontaneous fracture. Expression analysis of the transgene using RT-PCR confirmed its bone-specific expression, as well as the expected lack of down-regulation during osteoblast differentiation (Fig. 5 B).
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Because this phenotype did not lead to postnatal lethality, we next analyzed wild-type and transgenic mice at 2 wk old. Using contact radiography we observed an increased radiolucency in transgenic mice demonstrating a low bone mass phenotype (Fig. 6 A). Additionally, we found a size reduction of all skeletal elements compared with wild-type littermates, including the clavicles. The severe hypomineralization of calvariae that was still prominent in the transgenic mice at 2 wk old was further visualized by µCT imaging (Fig. 6 B). Histomorphometric analysis of vertebral sections demonstrated that the trabecular bone volume in transgenic mice was reduced by 40% at 2 wk old and by 30% at 10 wk old compared with wild-type littermates (Fig. 6 C).
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We next analyzed whether the disturbed cortical bone formation in the Col1a1-Sox8 transgenic mice leads to a decreased biomechanical stability. Cross-sectional µCT scans of the femora confirmed the cortical porosity in transgenic mice at 2 wk old. When the stability of these femora was analyzed in three-pointbending assays, the force required to cause bone failure was significantly decreased in transgenic mice (Fig. 7 E). This reduced biomechanical stability is also relevant in vivo, because one founder animal displayed a spontaneous fracture of the humerus. This was observed by contact radiography and histologically confirmed by Mallory staining to verify callus formation (Fig. 7 F). Together, these data demonstrate that Col1a1-Sox8 transgenic mice display a severe osteoporosis that is caused by an impaired bone formation.
Reduced expression of Runx2 in osteoblasts from Col1a1-Sox8 transgenic mice
To confirm that the phenotype of the Col1a1-Sox8 transgenic mice is caused by an intrinsic osteoblast differentiation defect we analyzed the behavior of primary calvarial osteoblasts from transgenic mice and wild-type littermates. In contrast to wild-type cultures, no mineralized bone nodules were observed after 10 d of differentiation in transgenic cultures. After 20 d only few areas were mineralized in transgenic cultures, whereas wild-type cultures displayed many mineralized nodules (Fig. 8 A). We next performed an RT-PCR expression analysis for osteoblast differentiation markers. In contrast to the Sox8-deficient cultures, we observed a delayed and reduced expression of Osc, Bsp, Phex, Runx2, and Col1a1 compared with wild-type cultures, whereas Lrp5 expression was not significantly changed (Fig. 8 B). Again, these results were confirmed in vivo by Northern blot expression analysis using RNA from calvaria and femur of Col1a1-Sox8 transgenic mice and wild-type littermates at 2 wk old (Fig. 8 C).
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Discussion |
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Bone formation through osteoblasts is controlled on different levels (Harada and Rodan, 2003). Whereas endocrine and neuronal mechanisms are important to regulate the functional activity of osteoblasts, at least two transcription factors, Runx2 and Osx, are required for the differentiation of mesenchymal precursor cells into osteoblasts (Ducy et al., 2000; Komori, 2002; Nakashima et al., 2002). Because Osx is not expressed in the absence of Runx2, the latter one is generally considered to be a master regulator of osteoblast differentiation. Runx2 activates the expression of most genes associated with osteoblast differentiation and function, and its deficiency in mice results in a complete lack of bone formation (Ducy et al., 1997; Komori et al., 1997). Haploinsufficiency of Runx2 leads to cleidocranial dysplasia, a disorder characterized by defects in endochondral and intramembranous bone formation including short stature, open fontanelles, and hypoplastic clavicles (Lee et al., 1997; Mundlos et al., 1997; Otto et al., 1997). Given the important role of Runx2 in osteoblast differentiation, it is not surprising that its expression needs to be tightly regulated, which is underscored by the phenotype of transgenic mice that overexpress Runx2 under the control of the Col1a1 promoter. Unexpectedly, these mice displayed an osteopenia associated with spontaneous fractures (Liu et al., 2001; Geoffroy et al., 2002), thereby demonstrating that the expression level of Runx2 needs to be limited to allow a coordinated osteoblast differentiation process.
The data presented in this manuscript provide evidence that Sox8 regulates osteoblast differentiation in a Runx2-dependent manner. In fact, the accelerated differentiation and mineralization of Sox8-deficient primary osteoblasts may be explained by the premature expression of Runx2 in these cells. Likewise, the reduced proliferation of the Sox8-deficient cells can be explained by the same mechanism, because Runx2 has recently been demonstrated to trigger the exit of preosteoblasts from the cell cycle (Pratap et al., 2003). Furthermore, the severe phenotype of the Col1a1-Sox8 transgenic mice is reminiscent of cleidocranial dysplasia, because we observed a size reduction of all skeletal elements including the clavicles as well as the characteristic failure of fontanelle closure. Again, this phenotype can be well explained by the strong down-regulation of Runx2 in these mice.
At least two other transcription factors, Twist and Stat1, have been demonstrated to attenuate Runx2-action in vivo (S. Kim et al., 2003; Bialek et al., 2004). Whereas Twist is required to prevent a premature Runx2-dependent osteogenesis during skeletal development, the function of Stat1 is more important in postnatal bone remodeling. In both cases the mechanism of action is posttranscriptional. Whereas the physical interaction of Twist and Runx2 decreases the DNA-binding activity of the latter one, the interaction with Stat-1 prevents the nuclear localization of Runx2 (S. Kim et al., 2003; Bialek et al., 2004). In this study, we have identified Sox8 as another molecule that exerts its inhibitory influence on osteoblast differentiation in a Runx2-dependent manner. As is the case for Stat1, this function is more important postnatally where Sox8 is required to assure a coordinated osteoblast differentiation process during bone remodeling. In contrast to Twist and Stat1 however, Sox8 acts on the transcriptional level, because the expression of Runx2 is elevated in nondifferentiated primary osteoblasts lacking Sox8. Therefore, it is possible that Sox8 is a direct regulator of Runx2 promoter activity, although such a mechanism needs to be established by future experiments. The same is the case for the identification of molecular mechanisms regulating Sox8 expression during osteoblast differentiation. One possibility is that Sox8 is under the control of BMP signaling, because all group E Sox family members have been shown to be induced after implantation of a BMP-7 bead at the tip of the digits of chicken embryos (Chimal-Monroy et al., 2003).
Regardless of these open questions, the analyses of both mouse models described in this manuscript demonstrate that Sox8 is an important transcriptional regulator of osteoblast differentiation. Through its expression in osteoblast precursor cells Sox8 inhibits their terminal differentiation and keeps them in the proliferative stage. Upon differentiation, Sox8 expression is down-regulated, which is required for an elevation of Runx2 expression, consequently leading to the initiation of an osteogenic cascade ensuring bone matrix deposition and mineralization by the differentiated osteoblasts.
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Materials and methods |
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Skeletal analysis
For skeletal preparations newborn mice were dissected and fixed overnight in 95% ethanol. Staining with alcian blue and alizarin red was performed using standard protocols (McLeod, 1980). For radiographic and histologic analysis mice were killed, internal organs were removed and the whole skeletons were fixed in 3.7% PBS-buffered formaldehyde for 18 h at 4°C. The skeletons were analyzed by contact radiography using a Faxitron Xray cabinet (Faxitron Xray Corp.). For three-dimensional visualization the calvariae were scanned (40 kV/114 µA) in a µCT 40 (Scanco Medical) at a resolution of 12 µm. For the assessment of the cortical porosity, femora were scanned at the midshaft at a resolution of 10 µm. The raw data were manually segmented and analyzed with the µCT Evaluation Program V4.4A (Scanco Medical). For visualization, the segmented data were imported and displayed in µCT Ray V3.0 (Scanco Medical). After fixation the skeletons were incubated in 70% ethanol for 24 h. The lumbar vertebral bodies (L3L5) and one tibia of each mouse were thereafter dehydrated in ascending alcohol concentrations and embedded in methylmetacrylate as described previously (Amling et al., 1999). For details on bone histology and histomorphometry, see Online supplemental material.
Image acquisition
Images were acquired using a Axioskop2 microscope (Carl Zeiss MicroImaging Inc.) with the Zeiss Plan Neofluar objective lenses: 1.25x/0.035; 2.5x/0.075; 10x/0.3; 20x/0.5; 40x/0.75. No immersion fluid was used. Pictures were taken at RT with a Zeiss Axiocam and Zeiss Axiovision Software V3.1. For white balancing and adjustment of brightness and contrast, Adobe Photoshop 7.0 was used.
In situ hybridization, BrdU labeling, X-Gal staining, and immunohistochemistry
In situ hybridization was performed according to standard procedures on 14-µm-thick sections using digoxigenin-labeled antisense riboprobes corresponding to the 3'-untranslated region of the Col2a1-cDNA and the third exon of the ColXa1 gene. For BrdU labeling, 1-wk-old mice were injected with 100 µg BrdU per gram of body weight 2 h before dissection. Incorporated BrdU was detected by immunohistochemistry using a monoclonal anti-BrdU antibody obtained from DakoCytomation according to the manufacturer's instructions. Detection of ß-galactosidase activity was performed following standard protocols (Hogan et al., 1994). In brief, tissue specimens were fixed in 1% PBS-buffered PFA for 2 d. For detection of ß-galactosidase activity, sections were covered with X-Gal staining solution and incubated at 37°C until blue precipitates were detectable. Immunohistochemistry was performed on 14-µm cryotome sections using specific antibodies against type I collagen (Novacostra) and Runx2 (Santa Cruz Biotechnology, Inc.) as recommended by the manufacturer. Secondary antibodies conjugated to Cy3 immunofluorescent dye (Dianova) were used for detection.
Analysis of primary osteoblast differentiation
Primary osteoblasts were obtained by sequential collagenase digestion of calvariae from 3-d-old mice as described previously (Ducy et al., 2000). Osteoblast differentiation was induced at 80% confluency in -MEM containing 10% FBS, 50 µg/ml ascorbic acid, and 10 mM ß-glycerophosphate. Total RNA was extracted using the TRIzol reagent (Invitrogen) immediately before differentiation (d0), as well as 5 and 10 or 20 d thereafter (d5, d10, d20). Northern blot analysis was performed according to standard protocols with the cDNAs encoding Sox8 and Gapdh as probes. BrdU incorporation assays were performed using the Cell Proliferation ELISA Biotrak system obtained from Amersham Biosciences according to the manufacturer's instructions. Mineralization of the cultures was analyzed using Von Kossa staining as described previously (Ducy et al., 2000). The percentage of the mineralized area was quantified using computer aided image analysis (Image tool V2.0; University of Texas Health Science Center, San Antonio, TX). For details on the RT-PCR expression analysis and the electrophoretic mobility shift assays, see Online supplemental material.
Online supplemental material
Fig. S1 shows the histologic analysis of the growth plates from wild-type and Sox8-deficient mice at 1 and 6 wk old. Fig. S2 shows a comparison of transgene expression and phenotypes for the offspring derived from the three different founders carrying the Col1a1-Sox8 transgene. Fig. S3 shows a Northern blot expression analysis for Sox9 and Sox10 in wild-type and Sox8-deficient osteoblasts and bones. Further comments on the data can be found in the legends. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200408013/DC1.
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Acknowledgments |
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This work was sponsored by grants from the Deutsche Forschungsgemeinschaft to M. Wegner (SFB473) and to M. Amling (AM103/8-3).
Submitted: 3 August 2004
Accepted: 24 January 2005
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References |
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