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Human Molecular Genetics Advance Access originally published online on April 13, 2005
Human Molecular Genetics 2005 14(11):1429-1439; doi:10.1093/hmg/ddi152
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

A role for fibroblast growth factor receptor-2 in the altered osteoblast phenotype induced by Twist haploinsufficiency in the Saethre–Chotzen syndrome

Hind Guenou, Karim Kaabeche, Sandrine Le Mée and Pierre J. Marie*

Laboratory of Osteoblast Biology and Pathology, INSERM U606, Paris, University Paris 7, Hôpital Lariboisière, Paris, France

* To whom correspondence should be addressed at: INSERM U606, Laboratory of Osteoblast Biology and Pathology, Lariboisière Hospital, 2 rue Ambroise Paré, 75475 Paris Cedex 10, France. Tel: +33 149956389; Fax: +33 149958452; Email: pierre.marie{at}larib.inserm.fr

Received February 1, 2005; Accepted April 6, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Genetic mutations of Twist, a bHLH transcription factor, induce premature fusion of cranial sutures (craniosynostosis) in the Saethre–Chotzen syndrome (SCS). The mechanisms by which Twist haploinsufficiency may alter osteoblast differentiation are poorly understood. In this study, we investigated the role of fibroblast growth factor receptor-2 (Fgfr2) in the abnormal osteoblast differentiation in SCS. Cranial osteoblasts from an SCS patient with a Y103X mutation inducing deletion of the Twist bHLH domain showed decreased Fgfr2 mRNA levels associated with decreased expression of Runx2, bone sialoprotein (BSP) and osteocalcin (OC), markers of differentiated osteoblasts, compared with wild-type osteoblasts. Transfection with Twist or Runx2 expression vectors, but not with Runx2 mutant which impairs DNA binding, restored Fgfr2, Runx2, BSP and OC expression in Twist mutant osteoblasts. EMSA analysis of mutant osteoblast nuclear extracts showed reduced Runx2 binding to a target OSE2 site in the Fgfr2 promoter. ChIP analyses showed that both Twist and Runx2 in mutant osteoblast nuclear extracts bind to a specific region in the Fgfr2 promoter. Significantly, forced expression of Fgfr2 restored Runx2 and osteoblast marker genes, whereas a dominant-negative Fgfr2 further decreased Runx2 and downstream genes in Twist mutant osteoblasts, indicating that alteration of Fgfr2 results in downregulation of osteoblast genes in Twist mutant osteoblasts. We conclude that Twist haploinsufficiency downregulates Fgfr2 mRNA expression, which in turn reduces Runx2 and downstream osteoblast-specific genes in human calvarial osteoblasts. This provides genetic and biochemical evidence for a role of Fgfr2 in the altered osteoblast phenotype induced by Twist haploinsufficiency in the SCS.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Twist is a basic helix–loop–helix (bHLH) factor involved in mesodormal, myoblast and osteoblast differentiation (1Go–4Go). Mutations in the Twist gene in humans are associated with Saethre–Chotzen syndrome (SCS), also known as acrocephalysyndactyly III (ACS III), an autosomal dominant hereditary disorder characterized by facial dysmorphism, digit defects and premature fusion of coronal sutures (5Go–8Go). Multiple mutations have been identified in the Twist gene, most of them being stop codons or missense mutations in the highly conserved bHLH domain (7Go–10Go). The phenotype in SCS patients with heterozygous deletion of Twist and Twist-null heterozygous mice suggests that Twist haploinsufficiency is the causal mechanism of the disease (9Go–11Go). Several mechanisms have been proposed to account for Twist haploinsufficiency in the SCS syndrome. Twist is known to heterodimerize with the broadly expressed bHLH E proteins, which bind DNA canonical sequences called E-boxes (CANNTG), which are consensus binding sites for bHLH proteins present in the promoter of target genes. In SCS, Twist mutations cause Twist protein degradation and loss of dimerization with E proteins, which abolish Twist binding activity to DNA (12Go,13Go). Twist target genes include the cell cycle regulator p21 (WAF1/CIP1) (14Go,15Go), cytokines involved in apoptosis (16Go,17Go) and the histone acetyltransferase P300/CBP (18Go), indicating that Twist targets several molecular mechanisms to induce its positive or negative control of cell growth, differentiation and survival.

Although craniosynostosis is a prominent feature of SCS, the mode of action of Twist in the control of human osteoblast differentiation genes is not fully understood. The expansion of cranial osteogenesis at the suture level is controlled by the balance between osteoprogenitor cell proliferation, differentiation and apoptosis (19Go,20Go). In mouse osteoblastic cells, Twist expression occurs in early progenitors and decreases with osteogenesis (21Go), suggesting that Twist is a negative regulator of osteoblast differentiation. In SCS, we previously showed that Twist haploinsufficiency results in increased collagen expression and osteogenic capability in postnatal human calvarial osteoblasts (22Go). In addition, deletion of bHLH in the Twist gene in postnatal human calvarial osteoblasts is associated with reduced expression of the Runt-related factor Runx2, a master gene involved in osteoblast maturation and osteogenesis (23Go–25Go), and with reduced expression of Runx2-regulated genes such as osteopontin, bone sialoprotein (BSP) and osteocalcin (OC) (26Go). It is intriguing to note that in the developing mouse, Twist can inhibit the functional activity of Runx2 through a Twist box identified in the C-terminal domain of Twist (27Go), suggesting that Twist may interact with Runx2 independent of the bHLH domain during early stages of mouse skeletal development. These observations suggest that Twist may control the function of osteoblasts through distinct pathways depending on the physiological or developmental context.

One mechanism by which Twist may control osteoblasts is through interaction with fibroblast growth factor (Fgf) and Fgf receptors (Fgfrs). Fgf/Fgfr signalling is an important mechanism controlling cranial bone formation (20Go,28Go,29Go). Notably, activating mutations in Fgfr2 induce premature cranial suture ossification through alterations in osteoblast differentiation and survival in murine (30Go) and human calvarial osteoblasts (31Go–33Go). Humans with features of the SCS were found to carry genetic mutations in the Twist and Fgfr2 genes (6Go), suggesting links between Twist and Fgfr signalling in the development of premature cranial ossification. In Drosophila, the Fgfr homologue DFR1 was found to depend on Twist, which acts upstream of Fgfr1 (34Go), suggesting that Twist may regulate Fgfr during development. In the mouse developing limb bud, Twist is required for the positive regulation of Fgfr and Fgf signalling (35Go) and loss of Twist function alters the expression pattern of genes associated with Fgf signalling (36Go). In the mouse developing cranial suture, Twist mRNA expression domains show some overlap with that of Fgfr2, raising the possibility that this transcription factor may control the cell proliferation–differentiation balance through regulation of Fgfr2 transcription (37Go). In addition, FGF was found to control Twist expression in mouse calvarial cells and during murine limb development (19Go,38Go), suggesting that Twist may act upstream of Fgf/Fgfr signalling, although it is unknown whether this is a stimulatory or an inhibitory effect (19Go). These observations raise the question of whether Twist haploinsufficiency in the SCS may alter the osteoblast phenotype by controlling Fgfr expression. Up to now, however, no direct functional interaction between the Twist and Fgfr genes has been established in the SCS.

In this study, we investigated the role of Fgfr2 in the osteoblast phenotype induced by Twist haploinsufficiency in SCS. We show here that the reduced Twist dosage in the SCS induces reduction in Fgfr2 mRNA expression, resulting in alteration of Runx2 and Runx2-dependent gene expression in osteoblasts. This provides novel genetic and biochemical evidence for a role of Fgfr2 in the altered osteoblast phenotype induced by Twist haploinsufficiency in the SCS.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Twist haploinsufficiency alters FGFR2 mRNA expression in mutant osteoblasts
Taking advantage of the Y103X mutation in Twist, which introduces a stop codon inducing deletion of the bHLH domain (16Go,22Go), we determined the effect of Twist haploinsufficiency on the expression of Fgfr2 transcripts in human calvarial mutant osteoblasts when compared with control (wild-type) osteoblasts. To achieve this goal, three sets of primers were designed to amplify N-terminal, intramembrane and C-terminal domains of Fgfr2. As shown in Figure 1, N-terminal Fgfr2 mRNA levels were markedly decreased in M-Tw cells compared with Nl cells. This was confirmed by the analysis of the intramembrane and C-terminal domains of Fgfr2 (Fig. 1A and B). In contrast, Fgfr1 and Fgfr3 mRNA levels were not lower in M-Tw compared with Nl cells (data not shown). These results show that Twist haploinsufficiency induced by the Y103X Twist mutation generating loss of bHLH causes a marked decrease in the expression of Fgfr2 transcripts in human mutant calvarial osteoblasts.



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  		Figure 1. Twist haploinsufficiency reduces Fgfr2 mRNA expression in osteoblasts. (A) Total RNA from calvarial osteoblasts from an SCS patient with the Y103X Twist mutation (M-Tw) and normal (wild-type) human calvarial osteoblasts (Nl) was subjected to RT–PCR analysis to detect N-terminal, C-terminal and membrane domains of Fgfr2. (B) Densitometric analysis of transcripts was determined after Southern hybridization and values were related to GAPDH (mean±SEM). Asterisks indicate significant differences with wild-type (Nl) cells (P<0.05).

 
Transfection with Twist restores the expression of Fgfr2, Runx2 and Runx2-dependent genes in Twist mutant osteoblasts
To determine the implication of Fgfr2 in the osteoblast phenotype induced by the Y103X Twist mutation, we transfected M-Tw osteoblasts with a Twist expression vector and determined the changes in Fgfr2 mRNA expression. As expected, transfection with Twist expression vector increased Twist mRNA levels in mutant osteoblasts (Fig. 2A). Significantly, transfection with Twist expression vector increased Fgfr2 mRNA levels (N-terminal domain) in Twist mutant cells (Fig. 2B). This was confirmed by the analysis of C-terminal and membrane Fgfr2 domains (data not shown). Furthermore, transfection with Twist corrected Runx2, BSP and OC mRNA expression in M-Tw cells (Fig. 2). These results indicate that correction of Twist dosage in Twist mutant osteoblasts can rescue most of Fgfr2 expression, which is associated with correction of Runx2 and Runx2-dependent gene expression.



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  		Figure 2. Transfection with Twist expression vector restores Fgfr2 and osteoblast gene expression in Twist mutant osteoblasts. (A) M-Tw calvarial osteoblasts were transfected with Twist expression vector or control vector (pcDNA3), total cellular RNA was subjected to RT–PCR analysis to detect Twist, Fgfr2, Runx2, BSP and OC and the data were compared with levels in wild-type (Nl) human calvarial osteoblasts (Nl). (B) Densitometric analysis of transcripts was determined and related to GAPDH (mean±SEM). a indicates a significant difference with Nl cells and b indicates a significant difference with pcDNA3-transfected M-Tw cells (P<0.05).

 
Twist binding to the Fgfr2 promoter
To examine whether Twist may bind to the Fgfr2 promoter in vivo, we carried out chromatin immunoprecipitation (ChIP) assay, which tests whether a protein binds to a candidate promoter in living cells. The presence of E-box sequences in immunoprecipitates was revealed by PCR amplification using S1 and S2 primers specific for two regions of the Fgfr2 promoter, which exhibit CANNTG sequences (15Go,39Go). Significantly, we found that Twist protein in nuclear extracts from M-Tw cells binds in vivo to the FGFR2 promoter region, which was amplified using S1 primers (Fig. 3A). In contrast, Twist protein in M-Tw nuclear extracts did not bind in vivo to the FGFR2 promoter region amplified using S2 primers (Fig. 3B). Purified total genomic DNA used as a PCR positive control showed a PCR product, whereas M-Tw nuclear extracts incubated without Twist antibody used as a negative control showed no PCR product (Fig. 3A and B). Similar results were obtained with nuclear extracts from wild-type (Nl) osteoblasts (data not shown). These data indicate that Twist interacts in vivo with the Fgfr2 promoter in the specific region studied, suggesting that Fgfr2 is a potential target for Twist in human calvarial osteoblasts.



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  		Figure 3. Binding of Twist to a specific Fgfr2 promoter region. ChIP assay was conducted with a Twist specific antibody and nuclear extracts from Twist mutant (M-Tw) osteoblasts. (A, B) Ethidium bromide staining of PCR products (arrows) corresponding to 301 bp S1 and 482 bp S2 fragments, respectively, of the Fgfr2 promoter. Genomic DNA was used as a PCR positive control (+) and samples without antibody was used as a negative control (–). The lower band represents free primers.

 
Transfection with Runx2 restores Fgfr2 expression in Twist mutant osteoblasts
Because we found that Twist transfection can rescue both Fgfr2 and Runx2 expression in Twist mutant osteoblasts (Fig. 2), we hypothesized that the altered Runx2 expression associated with Twist haploinsufficiency may in turn result in alteration of Fgfr2 expression in mutant cells. We then investigated whether correction of Runx2 may rescue Fgfr2 levels in M-Tw cells. As shown in Figure 4, Runx2 mRNA levels were markedly decreased in Twist mutant osteoblasts when compared with control (wild-type) cells. Significantly, transfection with Runx2 expression vector corrected Runx2 mRNA levels and increased the expression of Runx2 downstream genes BSP and OC. In addition, transfection with Runx2 corrected Fgfr2 mRNA levels (N-terminal domain) in M-Tw cells (Fig. 4A and B). Similar effects were observed with primers designed to amplify the C-terminal and membrane domains of Fgfr2 (data not shown). The finding that transfection with Runx2 restores Fgfr2 expression in Twist mutant osteoblasts indicates that Fgfr2 is a potential target for Runx2 and that the decreased Runx2 expression may be involved in the altered Fgfr2 expression in Twist mutant osteoblasts. To further analyse the role of Runx2 and investigate whether the effect of Runx2 on FGFR2 transcription is specific, we repeated these experiments with a Runx2 mutant bearing a Ser191Asn mutation in the runt domain that was shown to impair DNA binding (40Go). The levels of Runx2 products amplified using primers designed at distance from the Ser191Asn mutation were increased in M-Tw cells transfected with the Runx mutant, validating the transfection. In contrast to the Runx2 vector (Fig. 4A and B), the Runx2 mutant did not restore the expression of BSP, OC and Fgfr2 (Fig. 4C and D). This indicates that the effect of Runx2 on Fgfr2 transcription involves the DNA binding domain of Runx2.




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  		Figure 4. Transfection with Runx2 augments the expression of Fgfr2 and osteoblast genes in Twist mutant osteoblasts. M-Tw calvarial osteoblasts were transfected with Runx2 expression vector or control vector (pCMV5) (A, B), or a Runx2 mutant vector bearing a Ser191Asn mutation in the Runt domain that impairs DNA binding (C, D). Total cellular RNA was subjected to RT–PCR analysis to detect Runx2, BSP, OC and Fgfr2 (N-terminal domain) and the data were compared with normal (wild-type) human calvarial osteoblasts (Nl). Densitometric analysis of transcripts was determined and related to GAPDH (mean±SEM). a indicates a significant difference with Nl cells and b indicates a significant difference with M-Tw cells transfected with the control vector (P<0.05).

 
Twist haploinsufficiency reduces Runx2 binding to the Fgfr2 promoter
To further determine whether the reduced Fgfr2 mRNA expression in Twist mutant osteoblasts may be related to the alteration in Runx2 expression, we investigated whether Twist haploinsufficiency reduces Runx2 binding to the Fgfr2 gene using electrophoresis mobility shift assay (EMSA). As shown in Figure 5A, Runx2 in nuclear extracts from Twist mutant and wild-type osteoblasts was able to bind to an OSE2 site in the OC promoter, used here as a positive control for Runx2 binding. Notably, Runx2 binding to the OC promoter was reduced in M-Tw cells compared with wild-type cells, as expected from the reduced Runx2 protein expression in M-Tw cells (26Go). Supershift analysis using a specific Cbfa1/Runx2 antibody confirmed the specificity of the Runx2–OSE2 binding in the promoter (Fig. 5A). Similarly, we found that Runx2 binding to the BSP promoter was reduced in M-Tw cells compared with wild-type osteoblasts (data not shown). We then investigated whether Runx2 present in nuclear extracts from Twist mutant osteoblasts can bind to an OSE2 site in the Fgfr2 promoter. As shown in Figure 5B, Runx2 in nuclear extracts from M-Tw cells bound to the OSE2 site in the Fgfr2 promoter. Runx2 binding to the OSE2 site in the Fgfr2 promoter was displaced by excess of appropriate competitor (Fig. 5B). Despite repeated experiments, supershift analyses were not successful, possibly because the available antibody is not appropriate for this purpose. Because of the reduced Runx2 expression in M-Tw cells compared with Nl cells (26Go), Runx2 binding to the Fgfr2 promoter was specifically reduced in mutant osteoblasts when compared with Nl cells (Fig. 5B). These results indicate that Runx2 binds to the Fgfr2 promoter, suggesting that Fgfr2 is a target for Runx2 in osteoblasts. To further prove that Runx2 can interact with the Fgfr2 promoter, we performed a ChIP analysis in the S1 and S2 regions of the Fgfr2 promoter. As shown in Figure 5C, Runx2 protein in nuclear extracts from M-Tw cells bound in vivo to the FGFR2 promoter region, which was amplified using S1 primers. In contrast, Runx2 protein in M-Tw nuclear extracts did not bind in vivo to the FGFR2 promoter region amplified using S2 primers (data not shown). Purified total genomic DNA used as a PCR positive control showed a PCR product, whereas M-Tw nuclear extracts incubated without Runx2 antibody used as a negative control showed no PCR product (Fig. 5C). Similar results were obtained with nuclear extracts from wild-type (Nl) osteoblasts (data not shown). These results further indicate that Runx2 binds to the Fgfr2 promoter element in osteoblasts.




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  		Figure 5. Twist haploinsufficiency reduces Runx2 binding to OC and Fgfr2 genes. (A, B) EMSAs were performed using nuclear extracts from M-Tw and Nl cells and regions of the human OC (A) and Fgfr2 promoters (B). Radiolabelled double-stranded oligonucleotides containing the OSE2 site were used as a probe. The OSE2 core binding sites are underlined. Competition experiments were performed using a 100-fold excess of unlabelled oligonucleotides. Complexes were resolved on a non-denaturing electrophoresis and displayed by autoradiography. Relative positions of the bound probe are indicated (arrows). SC indicates supershift complex. (C) ChIP assay conducted with a Runx2 specific antibody and nuclear extracts from Twist mutant (M-Tw) osteoblasts, showing ethidium bromide staining of PCR products (arrow) corresponding to the 301 bp S1 fragment of the Fgfr2 promoter. Genomic DNA was used as a PCR positive control (+) and samples without antibody was used as a negative control (–). The lower band represents free primers.

 
Dominant-negative Fgfr2 further decreases Runx2 expression in Twist mutant osteoblasts
The previously mentioned results indicate that Twist haploinsufficiency induces reduced Runx2 expression and binding to Fgfr2 associated with abnormal expression of osteoblast genes. A number of osteoblast genes were found to be upregulated by Fgfr2 signalling, including Runx2, BSP and OC (20Go,28Go). We therefore hypothesized that the reduced Fgfr2 mRNA expression induced by Twist haploinsufficiency may account, at least in part, for the observed alteration in osteoblast gene expression in Twist mutant osteoblasts. To determine the role of Fgfr2 in the osteoblast phenotype induced by Twist haploinsufficiency, M-Tw cells were transfected with a dominant-negative (DN) Fgfr2 vector and changes in osteoblast gene expression was determined by reverse transcription–polymerase chain reaction (RT–PCR) analysis. We found that transfection with DN-Fgfr2 decreased Runx2 mRNA expression compared with the empty vector (Fig. 6A). This decrease in Runx2 expression in M-Tw cells was associated with a decrease in BSP and OC mRNA levels (Fig. 6A and B). Thus, reduction in Fgfr2 mRNA levels using DN-Fgfr2 further downregulates Runx2 and the downstream genes BSP and OC, which supports our finding that Fgfr2 mRNA downregulation can account, in part, for the decreased expression of Runx2 and downstream genes in Twist mutant cells.



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  		Figure 6. DN-Fgfr2 decreases osteoblast gene expression in Twist mutant osteoblasts. (A) Twist mutant osteoblasts (M-Tw) were transfected with a DN-FGFR2 expression vector or control vector (pRK5) and total cellular RNA was subjected to RT–PCR analysis to detect Runx2, BSP and OC mRNA in M-Tw osteoblasts. (B) Densitometric analysis of transcripts was determined and related to GAPDH (mean±SEM). Asterisks indicate significant differences with pRK5-transfected M-Tw cells (P<0.05).

 
Forced expression of Fgfr2 restores Runx2 and Runx2-dependent gene expression in Twist mutant osteoblasts
To further analyse the implication of Fgfr2 in the alteration of osteoblast phenotypic genes induced by Twist haploinsufficiency, we rescued Fgfr2 expression in M-Tw cells by forced expression of Fgfr2 and determined the changes in osteoblast gene expression. As shown in Figure 7A, transfection with Fgfr2 expression vector restored Fgfr2 mRNA levels in M-Tw cells. Significantly, this effect was associated with increased Runx2 mRNA levels. Consistently, forced expression of Fgfr2 increased BSP and OC mRNA levels in M-Tw cells (Fig. 7B). These results indicate that correction of Fgfr2 levels in M-Tw osteoblasts can rescue the expression of Runx2 and Runx2-dependent genes, confirming the implication of Fgfr2 in the abnormal osteoblast phenotype induced by Twist haploinsufficiency in SCS.



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  		Figure 7. Forced expression of Fgfr2 increases Runx2 and dowstream genes in Twist mutant osteoblasts. (A) Twist mutant osteoblasts (M-Tw) were transfected with Fgfr2 expression vector or control vector (pRK5) and total cellular RNA was subjected to RT–PCR analysis to detect Fgfr2, Runx2, BSP and OC mRNA. (B) Densitometric analysis of transcripts was determined and related to GAPDH (mean±SEM). Asterisks indicate significant differences with pRK5-transfected M-Tw cells (P<0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In the SCS, most Twist mutations are loss-of-function mutations occurring in the highly conserved bHLH domain and resulting in Twist haploinsufficiency (9Go,10Go). It is therefore expected that the decreased Twist ratio in the SCS may directly or indirectly induce alterations of phenotypic genes that are regulated by Twist. Previous studies in mice have suggested that Twist may interact with Fgfr signalling in cranial bone (19Go). However, whether Twist may up- or downregulate Fgfr expression in SCS has not been investigated, and interactions between Twist and Fgfr2 have not been reported in SCS up to now. The present study establishes a link between Twist and Fgfr2 in human calvarial osteoblasts in the SCS. We show here that the reduced Twist dosage induced by the Y103X mutation in Twist results specifically in a marked decrease in Fgfr2 mRNA levels in postnatal human calvarial mutant osteoblasts. Because Twist transfection restored Fgfr2 mRNA in M-Tw cells, it appears that the altered expression of Fgfr2 in M-Tw cells results from the reduced Twist dosage. Up to now, the precise molecular interactions between Twist and Fgfr2 genes are not fully understood. Although data base records indicate that DNA canonical sequences (CANNTG) are present in the human Fgfr2 promoter, it remains unknown whether Twist may bind to the Fgfr2 promoter to regulate this gene. Using ChIP analysis, we found that Twist binds to one region of the Fgfr2 promoter that contains a CANNTG sequence, revealing a putative mechanism by which Twist may regulate Fgfr2 expression. In support of a direct regulation of Fgfr2 by Twist is the observation that Ce-Twist directly interacts with the promoter of egl-15, a homologue of Fgfr in Caenorhabditis elegans (41Go). It must be noted, however, that Twist may regulate Fgfr2 by indirect mechanisms. One reported indirect mechanism is the interaction of Twist with E12 and the histone acetyl transferase p300/CBP (18Go). Thus, direct and indirect mechanisms may be involved in the altered Fgfr2 mRNA expression resulting from Twist haploinsufficiency in human calvarial osteoblasts.

The most important question was to determine the role of the altered Fgfr2 mRNA expression in the abnormal osteoblast phenotype in the SCS. We previously showed that constitutive activation of Fgfr2 signalling induced by genetic mutations promotes the expression of osteoblast-specific genes in human osteoblasts (31Go–33Go). We, therefore, hypothesized that the reduced Fgfr2 mRNA expression may be responsible for the observed alteration of osteoblast phenotypic markers in Twist mutant cells. Our finding that transfection with the Twist expression vector not only restored Fgfr2 mRNA levels, but also rescued Runx2, BSP and OC mRNA levels in M-Tw cells indicates that the alteration in Fgfr2 mRNA mediates in part the alteration of phenotypic osteoblast markers in M-Tw cells. This is consistent with the observation that activation of Fgfr signalling increases Runx2 expression in mouse calvarial osteoblastic cells (42Go,43Go) and with the finding that constitutive activation of FGFR2 in Apert syndrome upregulates OC expression in human calvarial osteoblasts (31Go,32Go). To confirm the role of Fgfr2 in the altered Runx2 expression, we transfected M-Tw cells with DN-Fgfr2 with the goal of further altering Fgfr2 expression. Our finding that DN-Fgfr2 inhibited Runx2 and downstream genes further supports the hypothesis that the reduced Fgfr2 mRNA expression is involved in the altered Runx2 expression in M-Tw osteoblasts. This was further demonstrated by our finding that forced expression of Fgfr2 rescued Runx2, BSP and OC expression in M-Tw cells. These data indicate that the altered Runx2 expression in Twist mutant osteoblasts results, in part, from the altered Fgfr2 expression. Interestingly, Runx2 was recently found to be a cell growth inhibitor in osteoblastic cells (44Go). One can thus hypothesize that the downregulation of Runx2 in M-Tw osteoblasts may be one mechanism by which cell growth is upregulated in Twist mutant human osteoblasts (22Go).

Another important finding in this study is that Runx2 may directly control Fgfr2 expression in osteoblasts. Strikingly, we found that forced expression of Runx2 not only corrected Runx2 and downstream genes BSP and OC, but also restored Fgfr2 mRNA expression in Twist mutant cells. Consistently, Runx2 binds to at least one OSE2 site in the Fgfr2 promoter, which suggests a specific interaction between Runx2 and Fgfr2. Both Twist and Runx2 can bind to the same Fgfr2 promoter region amplified by S1 primers, suggesting possible interactions between Twist and Runx2 to regulate the Fgfr2 promoter. Thus, in addition to be an essential transcription factor that regulates osteoblast differentiation genes (23Go–25Go), Runx2 may modulate the expression of Fgfr2, a receptor that is involved in the control of osteoblast differentiation (20Go). Our data also indicate that the reduced Fgfr2 expression in M-Tw cells results from Twist haploinsufficiency and from Runx2 depletion, which provides a dual molecular mechanism controlling Fgfr2 mRNA expression in Twist mutant osteoblasts (Fig. 8).



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  		Figure 8. Schematic representation of the proposed role of Fgfr2 in the altered osteoblast phenotype induced by Twist haploinsufficiency in SCS. Twist haploinsufficiency induced by bHLH deletion in Twist reduces Fgfr2 mRNA expression resulting in reduced Runx2, BSP and OC expression in Twist mutant osteoblasts. Consistent with a role in Fgfr2 in the SCS phenotype, Runx2, BSP and OC are reduced by DN-Fgfr2 and are restored by Fgfr2 overexpression. Transfection with Twist or Runx2 expression vectors restored Fgfr2 as well as Runx2, BSP and OC expression, which supports a role for Fgfr2 acting upstream of Runx2 in the altered osteoblast phenotype induced by Twist haploinsufficiency in the SCS.

 
Our finding that Twist acts through the bHLH domain to control Fgfr2 and Runx2 arises from our experiments using human postnatal osteoblasts directly derived from affected tissues and are therefore close to the in vivo situation in SCS. It is, however, possible that Twist may differently control Runx2 at earlier stages of skeletal development. Indeed, in the developing mouse, Twist was recently found to interact transiently with Runx2 through a Twist box located at the C-terminal end of the protein, thereby inhibiting Runx2 binding to the OC promoter (27Go). Because SCS can occur despite intact bHLH in the Twist gene, and there are several Twist mutations in SCS that do not affect the Twist C-terminal domain (10Go), it is likely that Twist may control osteoblast differentiation gene expression by distinct molecular mechanisms throughout skeletal development. Because the activation of FGFR2 promotes osteoblast differentiation in human craniosynostosis (31Go–33Go), it can be speculated that Twist haploinsufficiency keeps osteoprogenitor cells in a premature stage (characterized by low FGFR2 and Runx2 expression) and that the premature cranial suture closure in this syndrome may result from an increased number of pre-osteoblasts rather than from an increased differentiation rate of osteoblasts.

The present data support a model by which deletion of the bHLH domain causing Twist haploinsufficiency in SCS acts upstream of Fgfr2 to reduce Fgfr2 mRNA, which in turn affects Runx2 expression and downstream phenotypic markers in postnatal human cranial osteoblasts. Our data also indicate that the decreased Runx2 expression contributes to downregulate Fgfr2 mRNA expression in Twist mutant osteoblasts (Fig. 8). This provides genetic and biochemical evidence for a role of Fgfr2 acting upstream of Runx2 in the altered osteoblast phenotype induced by Twist haploinsufficiency in the SCS.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell cultures
Human osteoblasts from patients with genetic mutations inducing craniosynostosis are useful to characterize the molecular alterations involved in these genetic syndromes, as they are representative of the in vivo phenotype (16Go,31Go). Calvarial cells were obtained by collagenase digestion from coronal sutures in a 3.5 month SCS subject with the Y103X mutation that leads to deletion of the functional bHLH domain in the Twist gene (no mutation was found in Fgfr2 or Fgfr3 in this patient) and calvarial cells obtained from an age-matched normal subject (16Go,22Go). The resulting mutant (M-Tw) and normal (Nl) immortalized calvarial cell populations express an osteoblast phenotype which is similar to the phenotype in primary human calvarial cells in vitro and in osteoblasts in vivo (22Go). M-Tw cells show reduced Twist protein levels compared with Nl cells, confirming Twist haploinsufficiency (16Go,22Go). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with L-glutamine (292 mg/l), 10% heat inactivated fetal calf serum (FCS) and antibiotics (100 IU/ml penicillin, 100 µg/ml streptomycin).

Expression vectors and transfection analyses
For expression of DN-Fgfr2, M-Tw cells were transfected with the pRK5 expression vector containing human-DN-Fgfr2 cDNA (tyrosine kinase deleted, 3 immunoglobulin-like loop form, IIIc splicing variant; kindly provided by Dr J. Schlessinger) (45Go). For FGFR2 overexpression, we used the full-length Fgfr2 human cDNA or Fgfr2-3c immunoglobulin-like loop form cloned into the pMirb vector (kindly provided by Dr D. Ornitz). For forced expression of Twist, M-Tw cells were transfected with a pcDNA3 expression vector containing the human Twist cDNA (kindly provided by Dr V. El Ghouzzi). The Runx2 and Ser191Asn Runx2 mutant cDNAs (kindly provided by Dr G. Karsenty) (40Go) were cloned into the pCMV5 expression vector. Transfections were performed using Exgen500 (Euromedex, Mundolsheim, France). All cells were plated at 5000 cells/cm2 the day before transfection in DMEM supplemented with 10% FCS, rinsed with DMEM and 1 ml of DMEM containing 7 equiv. of Exgen500 (Promega) and 2.5 µg of different constructs with 50 ng of pCMV ß-galactosidase were added. The cells were incubated for 1 h at 37°C, then 1 ml of DMEM supplemented with 20% FCS was added to the medium. Cells were lysed 48 h after transfection. Efficiency of transfection was controlled by determination of ß-galactosidase activity.

RT–PCR analysis
The expression of transcripts in M-Tw and Nl osteoblasts was analysed by RT–PCR as described (16Go). Confluent Nl and M-Tw cells cultured in serum deprived medium (1% FCS) for 48 h were washed with PBS and lysed with Extract-All (Eurobio) reagent according to the manufacturer's instructions. Three micrograms of total cellular RNA from each sample were reverse transcribed and the cDNA samples were then divided and amplified using the following specific primers for Fgfr2 N-terminal domain: sense 5'-TTAGAGCCAGAAGAGCCACC-3', antisense 5'-AGTACAAGCATAGAGGCCGG-3', internal 5'-TCTCAACCAGAAGTGTACGTGG-3'; for Fgfr2 C-terminal domain: sense 5'-TGATGATGAGGGACTGTTGG-3', antisense 5'-TGTTTTAACACTGCCGTTTATGTGTG-3', internal 5'-CCTAGTTACCCTGACACAA-3'; for Fgfr2 intramembrane domain: sense 5'-GCGCCTGGAAGAGAAAAGGAGATTACA-3', antisense 5'-AGAGAGGCGTGTTGTTATCC-3', internal 5'-AACACGACCAAGAAGCCAGA-3'. Primers used for Twist were sense 5'-TCTTACGAGGAGCTGCAGAC-3' and antisense 5'-ATAGCTGCAGCTTGCCATC-3'. Primers for the phenotypic markers Runx2, BSP, OC and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as previously reported (16Go,22Go,26Go). Optimization of RT–PCR results was carried out by generating saturation curves of RT–PCR products of each gene and of GAPDH against cycle number (0–31 cycles). Hybridization signals were quantified using a scanner densitometer. The signal for each gene was corrected using GAPDH as internal control.

Electrophoresis mobility shift assay
Equimolar amounts of single-stranded oligonucleotides containing OSE2 binding sites (24Go,26Go) (Sigma Genosys, Cambridgeshire, UK) were annealed, purified on 6% acrylamide gel and radiolabelled using T4 polynucleotide kinase (Invitrogen, Cergy Pontoise, France) and [{gamma}-P32]dATP. The radiolabelled probes were then separated from free nucleotides on MicroSpin G-50 columns (Amersham Pharmacia Biotech Inc., les Ulis, France). Double-stranded oligonucleotides were end-labelled with {gamma}-P32 by T4 polynucleotide kinase (Invitrogen). The probe (1x105 c.p.m.) was incubated with 5 µg of nuclear extracts prepared as described (26Go) in 25 µl volume of binding reaction [20 mM HEPES, pH 7.9, 20% glycerol, 100 mM KCl, 1 mM DTT, 5 mM MgCl2, 0.1 mM EDTA, 0.082 mg/ml poly(dI·dC)] at room temperature for 20  min. In competition experiments, 100-fold excess of unlabelled double-stranded oligonucleotides was added to the binding mixture. For supershift analysis, nuclear extracts were pre-incubated with Runx2 polyclonal antibody (2 µg/µl) on ice for 30 min before the addition of labelled double-stranded oligonucleotides followed by incubation at room temperature for an additional 20 min. Following incubation, the protein–DNA complexes were resolved by electrophoresis on 5% non-denaturing acrylamide gels, the gels were dried and autoradiograms analysed using an image analyser.

Chromatin immunoprecipitation analyses
ChIP assays were used to test whether Twist or Runx2 bind to the Fgfr2 promoter in M-Tw cells in vivo. Putative transcription factor binding sites within the human Fgfr2 promoter were identified on data bases (GenBank accession no. AF233344). For ChIP analysis, chromatin from pre-confluent M-Tw cells was fixed with formaldehyde (1% final). Nuclear extracts were isolated and sonicated, and Twist or Runx2 present in nuclear extracts and bound to the chromatin were immunoprecipitated with specific anti-Twist antibody (Santa Cruz Biotechnology, Inc.) or anti-Runx2 antibody (provided by Dr G. Karsenty), protein–DNA crosslink was reversed and the isolated genomic DNA was amplified by PCR, using specific primers encompassing S1 (301 bp) or S2 (482 bp) fragments from the human Fgfr2 promoter. The primers used for S1 were sense 5'-TGAATGTGTCCTTGTCTGGC-3' and antisense 5'-CCTCAGTGGTGTGAAGGTT-3', and for S2 sense 5'-TTCCTGTTGTGGTCTTGTGG-3' and antisense 5'-GCTGAGGTGTCCGTTAATGT-3'. PCR products were separated on a 2% agarose gel and visualized by ethidium bromide staining. Total genomic DNA was used as a positive control and samples without Twist or Runx2 antibody were used as negative controls.

Statistical analysis
The data were expressed as the mean±SEM of three independent experiments. Differences between the mean values were analysed using the statistical package super-ANOVA (Macintosh, Abacus concepts, Inc., Berkeley, CA, USA) with a minimal significance of P<0.05.


   ACKNOWLEDGEMENTS
 
The authors thank Drs V. El Ghouzzi, J. Bonaventure, E. Renier and A. Munnich (INSERM U 393 and the Department of Neurosurgery, Hopital Necker-Enfants Malades, Paris, France) for bone samples, mutation analysis and Twist expression vector, Drs G. Karsenty and M. Starbuck (Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA) for the gift of Runx2 antibody and plasmids, Dr D. Ornitz (Department of Molecular Biology and Pharmacology, Washington University Medical School, St Louis, USA) for the Fgfr2 expression vector and Dr J. Schlessinger (Department of Pharmacology, Yale University School of Medicine, Yale, CT, USA) for the DN-Fgfr2 expression vector. We thank Dr M. Morgan (Institut Curie-CNRS UMR144, Paris, France) and Dr V. Geoffroy for their advice. H.G. is a recipient of a scholarship from the Association Rhumatisme et Travail (Centre Viggo-Petersen, Hôpital Lariboisière, Paris, France). K.K. is a recipient of a scholarship from the Ministère de la Recherche et de la Technologie (France).

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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