The authors have no conflict of interest.
Identification of Novel Regulators Associated With Early-Phase Osteoblast Differentiation†
Version of Record online: 20 FEB 2004
Copyright © 2004 ASBMR
Journal of Bone and Mineral Research
Volume 19, Issue 6, pages 947–958, June 2004
How to Cite
de Jong, D. S., Vaes, B. L., Dechering, K. J., Feijen, A., Hendriks, J. M., Wehrens, R., Mummery, C. L., van Zoelen, E. J., Olijve, W. and Steegenga, W. T. (2004), Identification of Novel Regulators Associated With Early-Phase Osteoblast Differentiation. J Bone Miner Res, 19: 947–958. doi: 10.1359/JBMR.040216
This article contains supplementary material (Tables 1 and 2) that is only available in the online version of the manuscript
- Issue online: 2 DEC 2009
- Version of Record online: 20 FEB 2004
- Manuscript Accepted: 20 JAN 2004
- Manuscript Revised: 27 NOV 2003
- Manuscript Received: 28 MAY 2003
- bone morphogenetic protein 2;
- transcription factors
Key regulatory components of the BMP-induced osteoblast differentiation cascade remain to be established. Microarray and subsequent expression analyses in mice identified two transcription factors, Hey1 and Tcf7, with in vitro and in vivo expression characteristics very similar to Cbfa1. Transfection studies suggest that Tcf7 modulates BMP2-induced osteoblast differentiation. This study contributes to a better definition of the onset of BMP-induced osteoblast differentiation.
Introduction: Elucidation of the genetic cascade guiding mesenchymal stem cells to become osteoblasts is of extreme importance for improving the treatment of bone-related diseases such as osteoporosis. The aim of this study was to identify regulators of the early phases of bone morphogenetic protein (BMP)2-induced osteoblast differentiation.
Materials and Methods: Osteoblast differentiation of mouse C2C12 cells was induced by treatment with BMP2, and regulation of gene expression was studied during the subsequent 24 h using high-density microarrays. The regulated genes were grouped by means of model-based clustering, and protein functions were assigned. Real-time quantitative RT-PCR analysis was used to validate BMP2-induced gene expression patterns in C2C12 cells. Osteoblast specificity was studied by comparing these expression patterns with those in C3H10T1/2 and NIH3T3 cells under similar conditions. In situ hybridization of mRNA in embryos at embryonic day (E)14.5 and E16.5 of gestation and on newborn mouse tails were used to study in vivo expression patterns. Cells constitutively expressing the regulated gene Tcf7 were used to investigate its influence on BMP-induced osteoblast differentiation.
Results and Conclusions: A total of 184 genes and expressed sequence tags (ESTs) were differentially expressed in the first 24 h after BMP2 treatment and grouped in subsets of immediate early, intermediate early, and late early response genes. Signal transduction regulatory factors mainly represented the subset of immediate early genes. Regulation of expression of these genes was direct, independent of de novo protein synthesis and independent of the cell type studied. The intermediate early and late early genes consisted primarily of genes related to processes that modulate morphology, basement membrane formation, and synthesis of extracellular calcified matrix. The late early genes require de novo protein synthesis and show osteoblast specificity. In vivo and in vitro experiments showed that the transcription factors Hey1 and Tcf7 exhibited expression characteristics and cell type specificity very similar to those of the osteoblast specific transcription factor Cbfa1, and constitutive expression of Tcf7 in C2C12 cells differentially regulated osteoblast differentiation marker genes.
MESENCHYMAL STEM CELLS have the potential to differentiate into various cell types that include osteoblasts, chondrocytes, adipocytes, myoblasts, and fibroblasts,(1,2) depending on the signaling cascades activated during the initial phase of differentiation. Each of these signaling mechanisms is likely to regulate a specific set of early response genes that ultimately determine the final fate of the differentiation process. Bone is a highly specialized tissue, but osteoblasts are nevertheless very similar to fibroblasts in terms of gene expression, and only a very limited number of osteoblast-specific genes have been identified.(3)
Among the few established osteoblast specific genes are the transcription factors Core-binding factor 1 (Cbfa1) and Osterix (Osx). Cbfa1 is a key regulator of osteoblast differentiation,(4–6) and its regulation and function have been studied extensively over the last decade. Several isoforms of Cbfa1 have now been identified, of which the type II isoform is bone-specific.(7,8) The activity of the Cbfa1 protein is modulated through post-translational modifications as well as through protein-protein interactions.(9–12) Most of the well-established bone markers, such as alkaline phosphatase, collagen I, bone sialoprotein, osteopontin, and osteocalcin, are known target genes of at least one of the Cbfa1 isoforms.(4,13–15) Moreover, the transcription factor Osx acts downstream of Cbfa1, and both Cbfa1 and Osx knockout mice exhibit a complete absence of skeletal ossification.(6,16)
Osteoblast differentiation can be induced by various stimuli, but the most potent inducers are the bone morphogenetic proteins (BMPs). Although the BMPs were first identified for their ability to induce ectopic bone formation in vivo,(17) these proteins seem to be multifunctional regulators of morphogenesis during embryonal development.(18) In addition to the BMP-induced Smad signal transduction pathway, several major signal transduction pathways, including the Wnt and Notch pathways, have been implicated in osteoblast differentiation. Nevertheless, only parts of the complete gene regulatory program guiding mesenchymal stem cells to become mature osteoblasts are currently known, and many gaps still need filling.
Recently, we have shown that gene expression microarray analysis is a powerful approach to identifying late phase bone marker genes(19) and to examine differential responses induced by BMP2, transforming growth factor (TGF)β1, and activin A.(20) The aim of this study was to identify novel potential regulators of the osteoblast differentiation process that are regulated during the early phases (within 24 h) of BMP-induced osteoblast differentiation using high-density microarrays. Model-based clustering and functional analysis identified two osteoblast-related transcription factors, Hey1 and Tcf7, which exhibited very similar expression characteristics to the osteoblast-specific type II Cbfa1 isoform. In addition, Tcf7 was shown to differentially regulate osteoblast differentiation marker genes.
MATERIALS AND METHODS
Tissue culture and growth factors
The murine mesenchymal progenitor cell lines C2C12 and C3H10T1/2 and the fibroblast cell line NIH3T3 (obtained from the American Type Culture Collection) were maintained in DMEM supplemented with 10% newborn calf serum (NCS), penicillin (100 U/ml), and streptomycin (100 μg/ml) as described previously.(20) For the various experiments, cells were plated at a density of 2.0 × 104 cells/cm2 and grown for 24 h in DMEM supplemented with 10% NCS. Subsequently, medium was replaced by DMEM containing 5% NCS either in the presence or in the absence of 300 ng/ml recombinant human BMP2 with or without 50 μM cyclohexamide (CHX). Recombinant human BMP2 was kindly provided by Wyeth (Andover). Cyclohexamide was purchased from Sigma.
Total RNA was extracted according to an acid guanidium thiocyanate-phenol-chloroform method (TriPure Isolation Reagent; Roche Diagnostics), and RNA concentrations were determined by measuring the absorbance at 260 nm. Poly A+ RNA was prepared from total RNA using the Oligotex method (Qiagen). Quantification of poly A+ RNA was carried out according to the Ribogreen RNA quantitation assay (Molecular Probes).
Microarray and statistical analysis
Microarray analysis was performed as described before.(20) For each time point, the BMP2-treated samples were hybridized versus control samples that were incubated for the same period in DMEM supplemented with 5% NCS. For the hybridization experiments, the mouse Unigene 1 array from Incyte Genomics (Palo Alto, CA, USA) was used, and the experiments were performed in duplicate with a fluor reversal to minimize possible bias caused by the molecular structure of the Cy3 and Cy5 dyes. Background-subtracted element signals were used to calculate Cy3/Cy5 ratios. Genes were selected for further analysis if the signal to background ratio for a particular gene was >2.5 and at least 40% of the area on chip could be used for signal computation in at least one of the two channels. To account for systematic errors caused by nonlinear differences in signal intensities of the Cy3 and Cy5 channel, statistical analysis was performed as described before.(20) Genes with significant (p < 0.01) differential expression regulation for at least one of the time points were selected for further analysis. Before clustering, microarray data were log-transformed (base 10) as follows: logI = log (ICy3/ICy5), if ICy3 > ICy5, and logI = −log(ICy5/ICy3), if ICy5 > ICy3, in which I is the intensity of each spot on microarray. Log values were averaged over duplicate experiments and scaled to unit vector length (normalization). Subsequently, normalized log values were clustered using model-based clustering.(21) Briefly, in this method, clusters are considered to be groups displaying multivariate normal distributions. Various models were fitted to the data, and an estimation was made of the accuracy of the model describing the data by assuming that underlying probability distributions occur for each model. Subsequently, a selection was made based on maximizing Bayesian Information Criterion (BIC) values for the specific model and number of clusters that represented the data best. Optimal clustering was obtained using a clustering method in which the distribution of the expression ratios within a cluster were ellipsoidal, the cluster volumes were variable, the cluster shapes were equal, and the cluster orientations were variable (VEV-model). Protein functions of the genes were identified according to Incytes Protein Function Hierarchy.
For real-time RT-PCR, the ABI Prism Sequence Detection System 5700 and Primer Express Software (PE Biosystems) were used. For each gene, a set of primers was designed using sequences obtained from Genbank, and amplicons of 50-100 bp with TM between 58°C and 60°C were selected. Before cDNA synthesis, 2 μg of total RNA were DNase (Invitrogen) treated for 15 minutes at 37°C, after which RNA was reverse-transcribed using random hexamer primers (Amersham Pharmacia) in a total volume of 50 μl using the SUPERSCRIPT II reverse transcriptase kit (Invitrogen). Subsequently, aliquots of first-strand cDNA (2 μl) were amplified using SYBR Green PCR Master mix (Applied Biosystems) under the following conditions: initial denaturation for 10 minutes at 95°C followed by 40 cycles consisting of 15 s at 94°C and 1 minute at 60°C. Fold inductions and expression ratios were calculated from differences in threshold cycles at which an increase in reporter fluorescence above a baseline signal could first be detected (CT) between two samples and averaged for duplicate experiments.
In situ hybridization
Mouse embryos were collected from F1 females (Cba × C57B16) at embryonic days 14.5 and 16.5 (E14.5 and E16.5), dissected free from the uterus and membranes, fixed overnight at 4°C in 4% paraformaldehyde, dehydrated, embedded in paraffin wax, and sectioned (6 μm) onto coated slides (Klinipath, Duiven, The Netherlands). Neonatal mouse tails that were kindly provided by Dr Marcel Karperien (Leiden University Medical Center, Leiden, The Netherlands) were collected from Swiss albino mice on day 5 after birth and fixed in 4% paraformaldehyde, decalcified by incubation in 12.5% sodium EDTA (pH 8.0) for 1 week at 4°C, embedded in paraffin wax, and sectioned (5 μm) as described previously.(22) The slides were dewaxed, rehydrated, and pretreated with Proteinase K and acetic anhydride and hybridized overnight at 55°C essentially as described previously.(23) Constructs were cloned into pSKII− in the antisense orientation in the T7 promoter. RNA probes were generated by transcription of the T7 RNA polymerase in the presence of [α-35S]-uridine triphosphate (UTP). After hybridization, the slides were washed at high stringency for 30 minutes at 62°C, treated with RNase A for 30 minutes at 37°C, washed again at high stringency, dehydrated, and allowed to dry. Autoradiography was performed using Ilford G5 photo emulsion. The slides were exposed for 2 weeks at 4°C. Photographs were made from combined bright-field (blue filter) and dark-field images (red filter).
Plasmids and transfections
The mouse Tcf7 cDNA cloned in pcDNA1 was kindly provided by Dr Hans Clevers (Hubrecht Laboratory, Utrecht, The Netherlands), whereas the mouse Cbfa1 cDNA cloned in pBluescript and the p6OSE2 luciferase reporter construct were kindly provided by Dr Patricia Ducy (Baylor College of Medicine, Houston, TX, USA). The renilla reporter construct pRL-SV40 was purchased from Promega. The Tcf7 coding region was excised from the plasmid by HindIII/XbaI digestion and ligated into HindIII/XbaI-digested pcDNA3.1. The Cbfa1 coding region was excised from the plasmid by EcoRV/XbaI digestion and ligated into EcoRV/XbaI-digested pcDNA3.1. The integrity of the resulting pTcf7-3.1 and pCbfa1-3.1 plasmids was verified by DNA sequence analysis. For stable transfections, pTcf7-3.1 was introduced into C2C12 cells using the Lipofectamin method according to the manufacturer's specifications (Gibco/BRL). Monoclonal stable transfectants were selected on G418 (400 μg/ml), and Tcf7 expression levels were analyzed by real-time RT-PCR. For transient transfections, pCbfa1-3.1, pTcf7-3.1, p6OSE2, and pRL-SV40 (Promega) were introduced into C2C12 cells using the Lipofectamin method according to the manufacturer's specifications (Gibco/BRL). Twenty-four hours after transfection, luciferase levels were measured.
BMP2-induced expression profiles
On BMP2 treatment, C2C12 mesenchymal progenitor cells differentiate into osteoblastic cells, whereas they form myotubes in response to low serum conditions.(24) Previously, we have shown that, under identical experimental conditions as used here, C2C12 cells differentiate into osteoblasts that strongly express bone markers such as alkaline phosphatase (ALP) and osteocalcin (OCN) 2 days after BMP2 treatment.(19) To identify novel potential regulators of BMP2-induced differentiation, we studied the onset of the differentiation process by microarray analysis (Fig. 1). Of the 9330 genes spotted on the array, 184 sequences were found to exhibit significant (p < 0.01) differential expression in the first 24 h after BMP2 treatment.
Model-based clustering, a method recently described for efficient handling of microarray data,(25,26) was used to group the 184 genes by similarities in their expression profiles, resulting in the identification of 10 distinct clusters. When classified into functional groups according to the function of their cognate protein (Table 1, supplementary materials), the majority (94%) of the genes in our data set fell in the groups “Signal Transduction,” “Localized and Structural Proteins,” or “Expressed Sequence Tags” (ESTs). Figure 1 shows an overview of the clustering results and the distribution of the genes over the functional categories is indicated per cluster. Figure 1A shows the six clusters with genes for which the most notable differential expression is positive, and Fig. 1B shows the four clusters with genes for which this is negative. Globally, genes from clusters 2, 3, 4, 7, and 10 can be categorized as immediate early genes that display distinct regulation 2 h after BMP2 treatment. Clusters 5, 6, and 9 represent intermediate early genes that were regulated from 6 h onward, and clusters 1 and 8 represent late early genes for which no expression regulation was observed until 24 h after BMP2 treatment.
Of the immediate early genes, 90% (clusters 2, 3, 4, and 7) showed increased expression, whereas only 10% (cluster 10) exhibited decreased expression after BMP2 treatment. In contrast, the intermediate early responses showed a balance in up- and downregulated genes (clusters 5 and 6 and cluster 9, respectively). This indicates that the initial response to BMP2 is a transcriptional activation response. Furthermore, a time-dependent shift could be observed in the distribution of gene functions. Specifically, 65% (34 of 52) of the immediate early genes could be classified as Signal Transduction proteins, while only 12% (6 of 52) of these genes represented Structural Proteins. During proceeding differentiation, less Signal Transduction-related genes were regulated (17% and 28% for intermediate early and late early genes, respectively), whereas an increasing number of Structural Proteins were regulated (19% and 38%, respectively). These Structural Proteins include cell morphology-related genes such as the Troponins C and T and keratin complex genes, and matrix-related genes such as procollagen IV α1 (Col4a1), matrix Gla protein, nidogen1, and the laminins α5 and β3. Using model-based clustering, we have thus been able to distinguish different time-dependent expression profiles of the regulated genes that coincide with specific functions of the genes present in each cluster.
Validation of the microarray data
We and others have shown previously that microarray data are highly comparable with results obtained using other methods such as real-time RT-PCR and Northern blotting, but that the ratio of expression regulation measured with these other methods vary, probably because of the distinct hybridization conditions of the various methodologies.(20,27) To validate the expression profiles measured in this microarray experiment, 52 genes were tested as representatives of all 10 clusters, both in original RNA material used for the microarray and in RNA material obtained from an identical but independent experiment by real-time RT-PCR. The expression profiles of 92% (48 of 52) of the genes tested was confirmed (Table 2, supplementary materials). Only the expression pattern of genes present in cluster 4, which were relatively weakly regulated and reached a maximum of 1.4-fold expression regulation, could not be confirmed. In conclusion, using real-time RT-PCR, we have been able to confirm both the pattern of the expression profiles as well as the level of differential expression regulation for the majority of the genes in this microarray experiment.
Transcriptional regulators of osteoblast differentiation
To identify novel potential regulators of osteoblast differentiation, we primarily focused on the transcription factors with altered expression during differentiation. In the complete set of 184 regulated sequences, 17 transcription factors were identified (Table 3). The real-time RT-PCR analysis described above confirmed the BMP2-induced differential expression for 15 of the 17 transcription factors. It should be noted that on the microarray, sequences uniquely representing the bone-specific Cbfa1 isoform (type II) were not present. Hence, type II Cbfa1 was not included in our microarray data set.
Transcription factors that are specifically expressed in osteoblasts but not in other cell types are likely to be regulators of osteoblast differentiation. We analyzed the cell type specificity for 13 transcription factors that showed a robust response to BMP2 treatment compared with control medium (Table 3). To this end, gene expression regulation by BMP2 was assessed in mouse C2C12 and C3H10T1/2 mesenchymal progenitor cell lines, which both can differentiate into osteoblasts, and for comparison, in NIH3T3 fibroblasts, which cannot differentiate into osteoblasts (Fig. 2). To test the validity of this approach, expression regulation of the bone-specific isoform of Cbfa1 (Cbfa1, type II) was analyzed by real-time RT-PCR in all three cell types and compared with expression regulation of sequences correlating with all isoforms (Cbfa1, bipartite sequences). Figure 2A shows a 5-fold BMP-specific induction for Cbfa1 type II in the osteoblastic cell types after 24 h, whereas no expression regulation was detected in the NIH3T3 fibroblasts. In contrast, the Cbfa1 bipartite sequences displayed low levels of BMP2-induced expression in the C2C12 cells, whereas no differential expression was observed in the C3H10T1/2 or NIH3T3 cells. These data show that this approach enables us to discriminate between osteoblast-restricted and common BMP responses.
Subsequently, the expression regulation of the 13 transcription factors was tested for cell type specificity (Fig. 2B; Table 3). Nine of these transcription factors were regulated in response to BMP2 treatment in all three cell lines to approximately the same extent and will further be referred to as “general BMP-response transcription factors.” The genes L-myc and MyoD were only expressed in C2C12 cells. C2C12 cells differentiate into myoblasts under low serum conditions, whereas C3H10T1/2 cells do not; therefore, these transcription factors could be characteristic for the pre-myoblastic phenotype of C2C12 cells. Finally, the transcription factors Hey1 and Tcf7 showed specific BMP2-induced upregulation in both C2C12 and C3H10T1/2 cells, similar to the osteoblast specific transcription factor Cbfa1 type II, but were not or were very weakly regulated in NIH3T3 cells. These results suggest that the BMP2-induced regulation of Hey1 and Tcf7 is osteoblast-specific.
To be able to distinguish between direct and indirect effects in response to BMP2, we analyzed gene expression in the presence or absence of cyclohexamide (Chx), an inhibitor of de novo protein synthesis. The data summarized in Table 3 shows that the immediate early general BMP-response transcription factors were not dependent on protein synthesis for their BMP2-induced gene expression, at least at the 2 and 6 h time points. mRNA of some of these genes was actually superinduced in the presence of BMP2 and Chx, probably as a result of an increase in mRNA stability or loss of transcriptional repressors by Chx as previously reported for Smad7.(28) Furthermore, the late early genes Tcf7 and Cbfa1 type II were fully impaired in their BMP2-induced expression in the presence of cyclohexamide. Induction of Hey1 was severely repressed because of Chx treatment, but its expression was still induced >13-fold at 24 h in the presence of the protein synthesis inhibitor, which suggests that regulation of Hey1 might be both direct and indirect. These results indicate that the classification of the genes based on our clustering method correlates with de novo protein synthesis-dependent transcription regulation.
Expression of Hey1 and Tcf7 during mouse development
Results obtained from the above-described experiments indicate that Hey1 and Tcf7 could be potential novel regulators of osteoblast differentiation. To study the involvement of both genes in embryonic bone development, expression of these genes was analyzed in mouse embryos at E14.5 and E16.5 of gestation and in neonatal mouse tails by the use of in situ hybridization. Neonatal mouse tails were used because their primary centers of endochondral ossification are still developing in the most distal vertebrae. These vertebrae consist of ALP− cartilage, whereas the most proximal vertebrae are ALP+ and mineralized.(22) The expression patterns of Tcf7 and Hey1 were compared with that of Cbfa1, which has previously been shown to be expressed during early skeletal development, in cells of mesenchymal condensations, and in the osteoblast lineage. In later development, it is expressed in ossification centers of all bones, independent of their embryonic origin and mechanism of ossification (intramembraneous or endochondral), but not in hypertrophic chondrocytes.(4) We first confirmed the expression of Cbfa1 at E14.5 in bone in transverse sections of the head at the level of the jaws (Fig. 3A), at E16.5 in sagittal sections of the head at the level of the nasal cavities (Fig. 3D), and in proximal vertebrae of the mouse neonatal tail (Fig. 3G). The ossification centers of the developing mandibular and maxillary bones, nasal bone, clavicle, humerus, and scapula, as well as proliferating chondroblasts, periosteal osteoblasts, and trabecular osteoblasts, all showed clear expression of Cbfa1, as expected.(4) In agreement with previous data, high levels of Cbfa1 expression were also observed in the mandibular region/dental mesenchyme surrounding the developing tooth.(19)
The Tcf7 gene showed an expression pattern remarkably similar to that of Cbfa1 in embryos at E14.5 and E16.5 of gestation (Figs. 3B and 3E). Except for the C1 vertebra, expression of Tcf7 was observed in all bones analyzed, although restricted to the periosteum/compacted mesenchyme of these bones. In the mouse neonatal tail vertebrae, both the periosteal and trabecular osteoblasts showed high levels of Tcf7 expression similar to Cbfa1, but no expression of Tcf7 was observed in proliferating chondroblasts (Fig. 3H). With respect to the Hey1 gene, no expression could be detected in the clavicle, humerus, scapula, or C1 vertebra. However, low but significant levels of expression were observed in the mandibular and maxillary bones (Figs. 3C and 3F). Specifically, Hey1 expression was restricted to the central parts of the bones (trabecular bone) and was absent in the periosteum. In the newborn tail vertebrae, the Hey1 gene showed specific expression in trabecular osteoblasts, albeit somewhat weaker than the Cbfa1 and Tcf7 genes (Fig. 3I). These data show that the two osteoblastic transcription factors identified in this study are expressed during in vivo bone differentiation, with individual expression patterns similar but not equal to Cbfa1.
Tcf7 differentially affects BMP2-induced osteoblast differentiation
To further investigate the role of Tcf7 and Hey1 expression during osteoblast development, we introduced expression vectors for their cognate genes into C2C12. We identified cells expressing high levels of Tcf7 (Fig. 4A) but were unable to isolate clones overexpressing Hey1 (data not shown). The differentiation capacity of Tcf7-overexpressing cells (C2C12-Tcf7) was assessed by measuring ALP activity, as well as the expression of the myogenic differentiation markers myogenin and MyoD and of the osteogenic differentiation markers ALP, Wnt inhibitory factor 1 (Wif1),(19)Cbfa1, and OCN after BMP2 treatment. The data represented in Figs. 4B and 4C show that the C2C12-Tcf7 cells were still able to differentiate to the osteoblast lineage, as evidenced by the induction of ALP activity and transcripts and the induction of the late-phase differentiation marker Wif1. The levels of these markers were slightly elevated in the C2C12-Tcf7 cells compared with the control cells. In contrast, the expression of Cbfa1 was slightly lower in C2C12-Tcf7 cells, whereas the expression of OCN was completely abrogated. These data suggest that Tcf7 differentially affects osteoblast differentiation. In control C2C12 cells, the expression of the myogenic differentiation markers myogenin and MyoD was induced during conditions that favor myoblast differentiation and repressed on BMP2 treatment. In contrast, the C2C12-Tcf7 cells failed to induce myogenin and MyoD transcription under myogenic differentiation conditions, indicating that differentiation was impaired. The abrogation of OCN expression in the C2C12-Tcf7 cells could be a result of the slightly reduced Cbfa1 levels in these cells or from a direct action of Tcf7 on OCN transcription. The latter notion is in line with published data that show that lymphoid enhancer-binding factor 1 (Lef1), a functional homolog of Tcf7, represses Cbfa1-dependent transcription from the OCN gene promoter.(29) The repression of Lef1 is mediated through direct interaction with Cbfa1 and could be visualized on p6OSE2, which contains an artificial promoter consisting of 6 Cbfa1 recognition sites upstream of the basal osteocalcin promoter (−34/+13), and a luciferase reporter gene. To investigate whether a similar mechanism could play a role in our system, we co-transfected combinations of Tcf7, Cbfa1, and p6OSE2 into C2C12 cells. As expected, Cbfa1 but not Tcf7 induced activity of the luciferase reporter (Fig. 4D). Interestingly, co-expression of Tcf7 significantly reduced the Cbfa1-induced activity of the reporter. The SV40 promoter driven expression of the renilla-reporter gene that was co-transfected to monitor differences in transfection efficiency was not affected by Tcf7 (data not shown), indicating that the observed effect is specific for Cbfa1-driven transcription.
Genetic cascades regulated during the initial phase of differentiation guide mesenchymal stem cells to become osteoblasts, and activating and repressing transcription factors are very important in directing this differentiation process. In our data set of 184 regulated genes and ESTs, we observed an immediate upregulation of transcription factors and other Signal Transduction proteins (2 h), but a slight delay in the repression of genes such as those related to myoblast differentiation (6 h). The near absence of repression of gene expression 2 h after BMP treatment observed in this study might be a reflection of the observation that Smads are transcriptional activators that first need to activate transcriptional repressors to inhibit gene expression.(30) In accordance with this, a number of transcription factors have been identified among the immediate early genes, such as the inhibitor of differentiation (Id) genes (Id1, Id2, and Id3) and the transforming growth factorβ1-induced transcript 4 (TSC-22) gene, which can function as transcriptional repressors.(31,32) The immediate early transcription factors may subsequently regulate the expression of genes involved in remodeling of cell architecture (between 6 and 24 h). However, commitment to the osteogenic lineage was not apparent until 24 h, when the osteoblast-specific transcription factor Cbfa1 and the novel osteoblast-related genes Hey1 and Tcf7 were found to be regulated. Previously, we have shown that the onset of regulation of bone markers such as ALP and OCN starts around 48 h of BMP treatment.(19) These results are slightly different from data reported recently by Balint et al.,(33) who showed both activation and repression of differentiation-related genes within the first hours of osteoblast differentiation, followed by commitment and establishment of the bone phenotype in C2C12 cells within 24 h of BMP treatment. It should be noted here that there are substantial differences in the experimental design, such as the cell density at the onset of BMP treatment and the absence of serum in their experiments that might explain the observed differences.
Our study identified 15 transcription factors that are induced within the first 24 h of BMP2-induced osteoblast differentiation. We investigated whether additional osteoblast-related transcription factors might be identified among the three immediate early ESTs identified in this study. However, in silico sequence analysis did not reveal specific DNA binding domains or characteristic transcription-activating domains, suggesting that these ESTs are not novel transcription factors (data not shown). Of the 15 known transcription factors in our data set, 9 immediate early general BMP-response transcription factors did not display osteoblast-specific expression regulation, but were specifically induced by BMP2 and therefore might have a function in the regulation of osteoblast differentiation. Indeed, eight of these nine transcription factors have previously been reported to be involved in osteoblast differentiation or function. Old astrocyte specifically induced substance (OASIS) has been described to be expressed in pre-osteoblasts in vivo, and its expression pattern coincides with that of osteopontin.(34)Prx2 cooperates with Prx1 in regulating skeletogenesis in the craniofacial region, inner ear, and limbs, as was shown in knockout experiments.(35) Transient induction of the Id genes by BMP has previously been described in osteoblastic cells,(24,36–38) and Id1 was found to be able to inhibit OCN gene expression.(39) Furthermore, Smad6 and Smad7 are negative regulators of the BMP signal transduction pathway that are capable of repressing BMP-induced osteoblast differentiation.(40,41)Tieg was first identified in human osteoblastic cells(42) and is able to induce ALP expression and inhibit OCN expression in human osteoblasts similar to TGFβ.(43) Until now, direct involvement of SnaI in osteoblast differentiation has not been reported. However, SnaI has been described to be an inhibitor of E-cadherin expression in different cell types,(44–46) and E-cadherin itself has been reported to be essential for optimal BMP-induced osteoblast differentiation,(47) suggesting that SnaI might be indirectly involved in osteoblast differentiation.
Although bone is a highly specialized tissue, osteoblasts have previously been described as sophisticated fibroblasts, exhibiting only a few osteoblast-specific transcription factors and marker genes, such as Cbfa1, Osx, and OCN.(3) It could be postulated that, to establish a highly specialized tissue such as bone, the action of several specific key regulatory transcription factors might not be required. Instead, a limited set of osteoblast-specific transcription factors in combination with general BMP-responsive transcription factors might be sufficient for guiding a mesenchymal stem cell to become a mature osteoblast. In our experiments, BMP2-induced transcription of Hey1 and Tcf7 was restricted to osteoblastic cells, suggesting that these factors may contribute to further specifying osteoblast fate delineated by bone-specific transcription factors like Cbfa1 and Osx. This notion is further substantiated by our observation that both Hey1 and Tcf7 are expressed in developing bone tissues in vivo.
Hey1 belongs to the Hairy and enhancer of split (Hes1) family of transcription factors and is a primary target of Notch,(48) which has been reported to inhibit osteoblast differentiation.(49,50)Hey1 functions as an inhibitor of myoblast differentiation in C2C12 cells(51) and is able to associate to its family member Hes1 and form stable heterodimers with Hes1 on DNA binding.(48)Hes1 has been reported to be a negative regulator of osteoblast differentiation,(52) but it can also stimulate the transactivating function of the osteoblast specific transcription factor Cbfa1.(9) To the best of our knowledge, Hey1 knockout mice have not been reported, and Hey1 has not previously been reported to be involved in osteoblast differentiation. Our in vitro data indicate that Hey1 expression by BMP2 is restricted to osteoblasts and not observed in fibroblasts. Furthermore, Hey1 is expressed during in vivo bone formation with an expression pattern that partially overlaps with that of Cbfa1 and Tcf7. We speculate that Hey1 might affect osteoblast differentiation in a comparable manner to or in association with Hes1. This putative mechanism is the subject of our current studies.
Tcf7 is a member of the T-lymphocyte-specific enhancer/lymphoid enhancer binding factor (Tcf/Lef) family and an effector of Wnt signaling.(53) The canonical Wnt pathway has been implicated in the control of bone mass and osteoblast differentiation. Inactivating mutations in the Wnt co-receptor LRP5 decrease bone accrual during growth, whereas activating mutations lead to a high bone mass phenotype,(54) and stabilized β-catenin in osteoblasts enhances their differentiation capacity.(55,56) However, the downstream effectors of Wnt/β-catenin in osteoblasts remain to be established. Our data indicate that Tcf7 is a BMP2 target gene in osteoblasts but not in fibroblasts. In vivo, expression of Tcf7 has been reported during embryonic bone development up to E14.5.(57) Our data confirm and extend these results and show that Tcf7 expression at E14.5 and E16.5 of gestation is highly but not completely similar to Cbfa1 expression. In the developing mouse tail, Tcf7 expression is limited to osteoblasts, and unlike Cbfa1, is not seen in hypertrophic chondrocytes. It is tempting to speculate that Tcf7 plays a role as a downstream effector of BMP2/Wnt signaling and provides a molecular access point where these two signaling pathways integrate. In C2C12 cells that overexpress Tcf7, we observed a downregulation of myogenin and MyoD similar to the downregulation mediated by Cbfa1,(58) suggesting a role of Tcf7 in suppression of myogenic differentiation. In addition, we observed a BMP2-dependent differential effect on expression of genes that mark osteoblast differentiation. Our observation that Tcf7 suppresses Cbfa1-induced transcriptional activity of the p6OSE2 reporter is in perfect agreement with published work that showed that the Tcf7 family member Lef1 exerts an identical effect on Cbfa1-mediated transcription.(29) The exact mechanisms by which Tcf7 modulates osteoblast and/or myogenic differentiation are not clear and are the subject of our current investigations.
In conclusion, our results show that BMP-induced osteoblast differentiation employs a cascade in which the initial phase of differentiation is based on general BMP-response genes. During the progression of osteoblast differentiation, osteoblast commitment genes and osteoblast-specific genes are regulated in concert with general BMP-response genes, which ultimately leads to a functional osteoblast. In addition, Hey1 and Tcf7 have been identified with similar expression characteristics as the osteoblast specific transcription factor Cbfa1, and Tcf7 was shown to modulate BMP2-induced osteoblast differentiation. Further elucidation of the function of these transcription factors in osteoblast differentiation may contribute to drug target research to treat bone diseases such as osteoporosis.
We thank Wyeth (Andover) for kindly providing recombinant human BMP2. We thank Dr H Clevers (Hubrecht Laboratory, Utrecht, The Netherlands) for providing the Tcf7 pCDNA1 construct, Dr HBJ Karperien (Department of Endocrinology, Leiden University Medical Center, The Netherlands) for providing the mouse neonatal tails, and Dr Patricia Ducy (Baylor College of Medicine, Houston, TX, USA) for providing the Cbfa1 cDNA and p6OSE2 luciferase promoter constructs. Furthermore, we thank L van der Meer for technical assistance and Dr Ester Piek for support and fruitful discussion on this manuscript. This work was supported by the Netherlands Institute for Earth and Life Sciences Grants 809.67.023 (to DSJ) and 809.67.024 (to AF).
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