Human Molecular Genetics, 2002, Vol. 11, No. 4 359-369
© 2002 Oxford University Press
Twist haploinsufficiency in Saethre–Chotzen syndrome induces calvarial osteoblast apoptosis due to increased TNF
expression and caspase-2 activation
Laboratory of Osteoblast Biology and Pathology, INSERM U349 affiliated CNRS, Hopital Lariboisiere, 2 rue Ambroise Pare 75475 Paris cedex 10, France and 1INSERM U393, Hopital Necker-Enfants Malades, 75015 Paris, France
Received August 9, 2001; Revised and Accepted December 6, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Saethre–Chotzen syndrome (SCS) is a human autosomal dominant disorder characterized by premature fusion of cranial sutures caused by mutations of the Twist gene encoding a basic helix–loop–helix (bHLH) transcription factor. We previously showed that Twist haploinsufficiency caused by a Y103X nonsense mutation in SCS alters both proliferation and osteoblast gene expression in human calvarial osteoblasts, indicating that Twist is an important regulator of osteoblast differentiation. Here we show that Twist haploinsufficiency alters osteoblast apoptosis in SCS. Analysis of terminal deoxynucleotidyl transferase-mediated nick-end labelling (TUNEL) demonstrated increased osteoblast and osteocyte apoptosis in coronal sutures from two SCS patients with nonsense mutations (Y103X and Q109X) that result in the synthesis of bHLH-truncated proteins, and one patient with a missense mutation in the basic domain (R118C) that abolishes Twist DNA binding. To assess the mechanisms involved, we studied osteoblast apoptosis in mutant (M-Tw) calvarial cells bearing the Y103X mutation resulting in decreased Twist mRNA and protein levels. M-Tw cells cultured in low serum conditions showed enhanced DNA fragmentation compared to normal (Nl) age-matched calvarial cells. Biochemical analysis showed increased activity of initiator caspases-2 and -8 and downstream effector caspases-3, -6 and -7 in mutant osteoblasts. Caspase-2 was upstream of caspase-8 and effector caspases-3, -6 and -7 because their activities were suppressed by a specific caspase-2 inhibitor. M-Tw osteoblasts also showed increased cytochrome c release from the mitochondria. However, the activity of the downstream effector caspase-9 was not increased due to overexpression of the antagonist protein Hsp70. Detection of differentially expressed genes using cDNA expression array revealed increased Bax and TNF
mRNA levels in M-Tw compared to Nl cells, a finding confirmed by RT–PCR and western blot analyses. Neutralization of TNF overexpression using anti-TNF or anti-TNF receptor 1 antibodies abolished the increased activity of caspase-2, caspase-8 and caspases-3, -6 and -7 in M-Tw osteoblasts. These studies provide novel evidence that Twist haploinsufficiency in SCS promotes osteoblast apoptosis by a TNF-caspase-2-caspase-8-caspases-3, -6, -7 cascade, and uncover a molecular mechanism in which Twist plays an anti-apoptotic role in human calvarial osteoblasts. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Saethre–Chotzen syndrome (SCS), also known as acrocephaly-syndactyly III (ACS III), is an autosomal dominant hereditary disorder (1,2) characterized by limb abnormalities, facial dysmorphisms and premature fusion of cranial sutures (craniosynostosis) (3). Multiple mutations in the coding region of the Twist gene were found to account for this syndrome (4–7). Twist-null heterozygous mouse skeletal phenotype resembles those of human SCS, which implicates Twist in the pathophysiology of craniosynostosis in SCS (4,8). The Twist protein is an evolutionarily highly conserved transcription factor that belongs to the family of basic helix–loop–helix (bHLH) proteins which recognize and bind to a specific DNA consensus sequence CANNTG (E-box) present in the promoter region of responsive genes. Because of the need for the protein to dimerize prior to DNA binding, Twist mutations in SCS result from haploinsufficiency (9) due to loss of the Twist protein function induced by protein instability or loss of either dimerization with the ubiquitous E12 protein or ability of the dimerized complex to bind DNA (10,11).
Twist is expressed specifically in mesodermal and cranial neural crest cells during embryogenesis and regulates mesoderm differentiation and myogenesis (12–14). The prominent feature of craniosynostosis in SCS suggests that the Twist gene also plays a crucial role in cranial osteogenesis. Calvaria bone formation occurs by membranous ossification, characterized by direct formation of bone matrix synthesized by osteoblasts. Sutures present at the junction of ossification plaques are composed of two adjacent membranous bone ends formed by osteoblasts arising from the differentiation of pre-osteoblasts originating from mesenchymal cells (15). During membranous ossification, Twist is expressed early during osteoblast differentiation, suggesting that it is involved in osteoblast maturation (16–19). Consistently, we recently showed that Twist haploinsufficiency in SCS results in increased growth, type I collagen expression and osteogenic capability of human calvarial osteoblasts (20), indicating that Twist plays a role in osteoblast differentiation. Apert syndrome, another severe craniosynostosis condition resulting from fibroblast growth factor receptor-2 (FGFR-2) activating mutations (21) is also associated with accelerated differentiation of calvarial osteoblasts (22,23), indicating that, in both Apert sydrome and SCS, abnormal maturation of osteoblasts results in coronal synostosis (20,24).
Twist may also be involved in the control of cell survival. Twist-null mice show a massive wave of apoptosis in the developing somites, in particular sclerotome, and in cranial ganglia arising from cranial crests where Twist is expressed (25). Moreover, in vitro data indicate that ectopic Twist overexpression reduces apoptosis (26). The role of Twist in the control of human calvarial osteoblast survival is however unknown. Apoptosis is a natural component of cellular differentiation and development, functioning as an essential mechanism of normal tissue homeostasis. In the mouse coronal suture, apoptotic cell death occurs at the same time and place as suture initiation (27,28), suggesting a role in suture development. The characteristics of apoptotic cells include chromatin condensation, nuclear fragmentation and cell shrinkage (29). A central component of apoptosis is the proteolytic system that involves the family of intracellular cysteine proteases, named caspases (30). Cysteine proteases play a key role in apoptosis either as upstream initiators (caspases-1, -2, -8, -9 and -10) or as downstream effectors (caspases-3, -6 and -7) that cleave intracellular substrates during the execution phase of apoptosis. One upstream pathway is triggered by activation of cell-surface death receptors by Fas ligand or tumor necrosis factor (TNF) leading to caspase-8 activation (31), which in turn cleaves and activates effector caspases-3, -6 and -7. Another pathway is triggered by the pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins, whose dimerization regulate cytochrome c flow (32). Cytochrome c release from mitochondria promotes activation of caspase-9, mediated by its binding to Apaf-1, leading to further activation of effector caspases and DNA fragmentation (33,34).
In this study, we investigated the effect of Twist haploinsufficiency on osteoblast apoptosis in SCS. We show here that Twist haploinsufficiency in this syndrome promotes apoptosis in human calvarial osteoblasts through a TNF-caspase-2-caspase-8-caspases-3, -6, -7 cascade.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Twist haploinsufficiency induces osteoblast and osteocyte apoptosis in vivo
We first determined the effect of Twist haploinsufficiency on apoptosis in vivo. Coronal suture samples from SCS patients with various heterozygous Twist mutations were obtained. Two patients showed nonsense mutations (Y103X and Q109X) that result in the synthesis of bHLH-truncated proteins that are rapidly degraded (10,20), and one patient had a missense mutation in the basic domain (R118C) that abolishes Twist DNA binding capacities (11). Histological sections of coronal sutures in SCS patients and age-matched controls were processed using a modified terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) assay. Our histological analysis of the coronal sutures of patients with the Y103X, Q109X and R118C (Fig. 1) Twist mutations showed increased number of TUNEL-positive osteoblasts and osteocytes compared to age-matched control subjects (Fig. 1). Quantification of the number of TUNEL labelled-positive cells showed that all mutations causing Twist haploinsufficiency markedly increased osteoblast and osteocyte apoptosis compared to age-matched control samples (Fig. 2). Indeed, Twist mutations induced a 2–3-fold increase in apoptotic osteoblasts and a 5-fold increase in the number of osteocytes undergoing apoptosis in vivo. These results show that Twist haploinsufficiency caused by various Twist mutations induces apoptosis in osteoblasts and osteocytes in the coronal sutures in SCS.
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Twist haploinsufficiency increases osteoblast apoptosis and DNA fragmentation in vitro
To determine the mechanisms involved in osteoblast apoptosis induced by Twist insufficiency, we investigated the effect of the Y103X Twist mutation, which induces deletion of the functional bHLH domain, on calvarial cell behaviour, compared to age-matched control calvarial cells. In these cells, Twist mRNA and protein levels are decreased by 50% compared to control cells (20). We previously showed that these cells express the osteoblast phenotype in culture (20). Nl and M-Tw cells were cultured in serum-deprived (1%) medium for 48 h to induce apoptosis and cells undergoing apoptosis were detected by TUNEL-labelling. As shown in Figure 3B, the number of apoptotic cells was higher in cultured M-Tw cells compared to Nl cells, confirming the apoptotic effect of Twist haploinsufficiency in calvarial osteoblasts. To confirm this finding, Nl and M-Tw cells were cultured in the same conditions described above and DNA fragmentation was analysed by DNA laddering. DNA extracted from M-Tw cells revealed a specific endonuclease-mediated fragmentation characteristic of apoptotic cells, resulting in a diffuse smear on electrophoretic gels, compared to Nl cells (Fig. 3A). This confirms that Twist haploinsufficiency enhances apoptosis induced by serum deprivation in calvarial osteoblasts.
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Twist haploinsufficiency increases expression of apoptotic-related genes in mutant osteoblasts
We then used DNA microarray technology to identify the differential apoptotic-related genes expression profile in M-Tw and Nl cells. The Atlas array is composed of 588 cDNAs corresponding to genes that are known to play a role in various processes, including apoptosis. Differences in the levels of hybridization signals of specific genes on the array correlate with the abundance of these genes (35). Several housekeeping genes [ubiquitin, phospholipase A2, G3PDH,
-tubulin, ß-actin, highly basic protein (HBP)] were used as internal controls. Screening of membranes hybridized with cDNA from confluent NI and M-Tw cells identified apoptosis-related genes whose expression was constitutively increased in mutant osteoblasts. These were MDM2, TNF, DAD1 (defenser against cell death), c-myc and Bax (Fig. 4). The three last left columns of each membrane shown in Figure 4 correspond to DNA synthesis, repair and recombination-related genes. Expression of NRF1, a transcription factor that controls TNF expression, was also up-regulated (Fig. 4).
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We confirmed the overexpression of some of the apoptosis-related genes by determining their expression by RT–PCR or western blot analysis. As shown in Figure 5, the mRNA levels of TNF
and MDM2 were higher in M-Tw cells compared to Nl cells, which is consistent with the gene array analysis. Because the tumor suppressor p53, which plays a critical role in regulating cell death, is controlled by MDM2 (36), we determined the expression of p53 in M-Tw osteoblasts. We found that p53 mRNA levels were similar in M-Tw cells compared to Nl cells (Fig. 5). Moreover, p53 protein levels as well as phosphorylated p53 at Ser20 levels did not differ in M-Tw and Nl cells (Fig. 6), indicating that the Twist mutation did not affect p53 levels nor its phosphorylation at position Ser20 in mutant calvarial osteoblasts. We also found that mRNA levels for Hsp 70, a heat shock protein which inhibits pro-caspase-9 activity, were increased in M-Tw cells compared to Nl cells (Fig. 5). As shown in Figure 6, the protein levels of TNF and Bax were increased in M-Tw cells compared to Nl cells. In contrast, Bcl-2 protein levels were not markedly altered in mutant cells (Fig. 6). These results confirm the gene array analysis and show that Twist haploinsufficiency induced up-regulation of apoptosis-related genes in calvarial osteoblasts.
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Twist haploinsufficiency induces cytochrome c release
Cytochrome c normally located in the mitochondrial intermembrane space is released into the cytosol following an increase in the Bax/Bcl-2 ratio (29,32). We therefore investigated whether the observed increase in Bax levels in mutant osteoblasts was associated with cytochrome c release. By immunoblotting, we found a decreased mitochondrial cytochrome c content associated with cytochrome c release in the cytosol in M-Tw cells compared to Nl cells (Fig. 7). Cox-4, a mitochondrial membrane protein which remains in the mitochondria, was not found in the cytosol but was present in the mitochondrial extracts, validating the purity of the preparation. These results indicate that Twist haploinsufficiency induces mitochondrial release of cytochrome c in calvarial osteoblasts.
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Twist haploinsufficiency increases initiator and effector caspases activity
We then determined the role of initiator caspases-2, -8 and -9, that induce effector caspases-3, -6 and -7 activity (37) in the apoptotic effect induced by Twist haploinsufficiency. As shown in Figure 8A, Twist mutant osteoblasts cultured in serum-deprived medium showed increased caspase-8 activity compared to Nl cells cultured in the same conditions. Moreover, caspase-2 activity was increased in M-Tw cells compared to Nl cells. In contrast, caspase-9 activity did not differ in Nl and M-Tw cells. Consistent with the TUNEL analysis, the activity of effector caspases-3, -6, -7 was increased in M-Tw osteoblasts compared to Nl cells (Fig. 8A). To determine the role of initiator caspases-2 and -8 in effector caspases-3, -6, -7 activity, M-Tw cells were treated with caspase-2 or caspase-8-specific inhibitors and the activity of effector caspases and caspases-2 and -8 was tested. Treatment of M-Tw osteoblasts with the caspase-8 inhibitor suppressed caspases-3, -6, -7 activity, but not caspase-2 activity. In contrast, treatment with caspase-2 inhibitor inhibited the activity of both caspase-8 and caspases-3, -6, -7 in mutant osteoblasts (Fig. 7B), indicating that caspase-8 is downstream of caspase-2 activation in M-Tw cells. These results indicate that Twist haploinsufficiency induces activation of caspase-2, but not caspase-9, leading to subsequent activation of caspase-8 and effector caspases-3, -6, -7, last step before induction of DNA fragmentation.
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Inhibition of TNF
pathway inhibits apoptosis in Twist mutant osteoblastsBecause TNF
is a known activator of apoptosis in various cell types (31), we hypothesized that the observed increase in TNF expression may play a role in the induction of apoptosis induced by Twist insufficiency. To test this hypothesis, we used specific neutralizing anti-TNF and anti-TNFR1 antibodies which inhibit TNF activity (38). Treatment of Twist mutant cells with anti-TNF neutralizing antibodies (1 µg/ml) decreased caspases-3, -6, -7 activity compared to mutant control cells treated with a non-specific IgG (Fig. 8B and C). Similarly, anti-TNF neutralizing antibodies decreased caspase-2 and caspase-8 activity in mutant osteoblasts compared to mutant control cells treated with a non-specific IgG (Fig. 9A). To confirm this finding, M-Tw cells were treated with anti-TNFR1 neutralizing antibodies (1 µg/ml) and the activity of caspases was determined. As shown in Figure 9A, treatment of Twist mutant cells with anti-TNFR1 neutralizing antibodies reduced caspases-3, -6, -7 activity compared to IgG-treated M-Tw cells. Moreover, anti-TNFR1 neutralizing antibodies reduced caspase-2 and caspase-8 activity in mutant cells compared to IgG-treated M-Tw cells. Treatments with the two neutralizing antibodies were able to restore normal caspase levels in mutant cells (Fig. 9B and C), indicating that suppression of TNF activity abolished the increased caspase activity induced by the Twist mutation in osteoblasts. Overall, the results show that Twist haploinsufficiency induces calvarial osteoblast apoptosis in vivo and in vitro by a mechanism involving TNF overexpression, causing increased caspase-2 activity resulting in increased caspase-8 and caspases-3, -6, -7 activity and DNA fragmentation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We previously showed that Twist haploinsufficiency in SCS alters both proliferation and osteoblast gene expression in human calvarial osteoblasts, indicating that Twist is an important regulator of the osteoblast phenotype (20). In the present study, we show that mutations causing Twist haploinsufficiency decrease osteoblast survival in SCS both in vivo and in vitro. Several Twist mutations account for SCS (3–7,10). In this study, we studied the effects of Twist mutations causing Twist haploinsufficiency either by inducing truncated Twist proteins (Y103X, Q109X) or by causing loss of Twist ability to bind to its target E-Box (R118C). We showed that these Twist mutations caused an increase in apoptosis of calvarial osteoblasts in coronal sutures. Increased apoptosis was also found in osteocytes, which is consistent with the occurrence of apoptosis in the two cell types in various human bone pathologies (39) including craniosynostosis (40). The finding that the three mutations caused both Twist haploinsufficiency and an increased osteoblast apoptosis suggests that this phenotype is caused by reduced Twist activity. To confirm this finding, we performed in vitro analysis using immortalized calvarial osteoblasts bearing the Y103X Twist mutation, a mutation that induces deletion of the functional bHLH domain (5,7,10) and reduces Twist mRNA and protein levels (20). Using TUNEL analysis and DNA fragmentation, we confirmed the increased apoptosis in M-Tw calvarial osteoblasts, further demonstrating that Twist haploinsufficiency in SCS enhances osteoblast apoptosis in vitro. Although immortalization per se may alter the properties of the cells, we previously demonstrated that the osteoblast phenotype (both differentiation and apoptosis) in immortalized human calvaria cells is consistent with the phenotype obtained in primary human calvaria cells in vitro and in vivo (22,23,40,41). Thus, it is likely that apoptosis in immortalized calvarial osteoblasts reflected the phenotype in primary cultures. Our finding that Twist haploinsufficiency enhances apoptosis in human calvarial osteoblasts in vitro and in vivo supports the observation that Twist-null mice show a massive wave of apoptosis during development (25). This is also consistent with the previous finding that Twist overexpression can protect from serum starvation-induced apoptosis (26) and with the recent observation that Twist is involved in the anti-apoptotic actions of the insulin-like growth factor-I receptor in vitro (42).
We found several mechanisms by which Twist haploinsufficiency increased apoptosis in calvarial osteoblasts. Using gene microarray analysis, we identified apoptotic-related genes that are constitutively overexpressed in Twist mutant cells. Among them, two important pro-apoptotic genes were increased, namely TNF, a cytokine that induces cell death via a cellular cascade of proteases and caspases (31,43), and Bax, a pro-apoptotic member of the Bcl-2 family (32). Analysis of gene and protein expression confirmed TNF and Bax overexpression in mutant cells, further indicating that these genes are involved in apoptosis induced by the mutation. The pro-apoptotic protein Bax plays a central role in controlling cell death. This involves homodimerization or heterodimerization with the anti-apoptotic protein Bcl-2, leading to activation of caspases according to the Bax/Bcl2 protein ratio (32,44). Our finding that Twist haploinsufficiency in M-Tw osteoblasts increased Bax protein level whereas Bcl-2 level was unchanged, suggests that the pro-apoptotic protein Bax, but not Bcl-2, is involved in the increased apoptosis in M-Tw cells. These results in Twist mutant human osteoblasts are consistent with the down-regulation of Bax mRNA and the unchanged expression of Bcl-2 mRNA observed in Twist overexpressing cells (26). Transcription of Bax has been reported to be directly regulated by the p53 protein (45). However, overexpression of Bax in M-Tw cells was not associated with alteration in p53 mRNA or protein expression, consistent with the reported lack of effect of Twist ectopic expression on p53 mRNA or protein expression in vitro (26). Moreover, phosphorylation of p53 on Ser20, which prevents p53 inhibition by the MDM2 protein (46) was not changed in mutant cells, indicating that osteoblast apoptosis induced by Twist haploinsufficiency does not involve change in p53 expression or activation through phosphorylation at Ser20. However, other activating post-translational modifications of p53, such as acetylation by p300 (47), a known target of Twist protein (48), cannot be excluded. Interestingly, we found increased MDM2 mRNA expression in M-Tw cells, which is consistent with the down-regulation of MDM2 mRNA in Twist overexpressing cells (26). Although MDM2 is known to increase p53 degradation (36) and to inhibit the apoptotic function of p53 (49), this protein also has p53-independent activities (50). The role of MDM2 on osteoblast growth and apoptosis induced by Twist haploinsufficiency remains, therefore, to be determined.
We also investigated the role of Bax in apoptosis induced by Twist haploinsufficiency in osteoblasts. Translocation of Bax to mitochondrial membranes induces cytochrome c release by the mitochondria (51). In the presence of dATP or ATP, cytochrome c forms a complex with Apaf-1, termed apoptosome, enabling it to recruit the initiator pro-caspase-9 (33). Once activated, caspase-9 mediates activation of the downstream effector caspases-3, -6, -7, which is essential for dismantling cell components. Despite increased release of cytochrome c into the cytosol, the activity of caspase-9 was not altered in M-Tw cells. This lack of caspase-9 activation may be related to overexpression of Hsp70, a heat shock protein that protects cells from apoptosis. Hsp70 exerts anti-apoptotic effects through its association with the caspase-recruitment domain (CARD) of Apaf-1, preventing apoptosome formation and thereby inhibiting activation of pro-caspase-9 (52–54). Our finding that Hsp70 is overexpressed in M-Tw cells provides a mechanism by which caspase-9 activation cannot be induced by cytochrome c release in mutant cells. It remains possible, however, that the release of cytochrome c may contribute to the TNF-mediated apoptosis in M-Tw cells by amplifying the effects of caspase-8 on activation of downstream caspases, as recently found in the Xenopus cell free system (55).
The other major apoptotic gene that we found to be overexpressed in M-Tw cells is TNF. TNF activates apoptosis in various cell types and its known downstream apoptotic pathway implicates TNF receptor 1 (TNFR1), a member of the tumor necrosis factor receptor family characterized by a death domain in the cytoplasmic region (43). Activation of TNFR1 by TNF results in receptor aggregation and triggers recruitment of Fas-associated death domain (FADD), allowing recruitment of caspase-8 pro-enzyme, activation of caspase-8 and downstream caspases-3, -6, -7 (29,43). Consistent with the increased TNF expression, we found that caspase-8 and downstream caspases-3, -6, -7 activity was increased in M-Tw mutant osteoblasts. The effect mirrors the reduced caspase-3 activity previously found in Twist overexpressing cells (26). Our finding that inhibition of caspase-2 abolished both caspase-8 and caspases-3, -6, -7 activity in M-Tw cells demonstrates the implication of caspase-2 upstream of caspase-8 and effector caspases. Thus, Twist haploinsufficiency induces apoptosis in human osteoblasts by mechanisms implicating activation of caspase-2, caspase-8 and effector caspases, leading to DNA degradation and cell death. Our finding that neutralizing anti-TNF antibodies suppressed the increased caspases-2 and -8 as well as caspases-3, -6, -7 activity indicates that TNF overexpression is involved in the pro-apoptotic effect induced by Twist haploinsufficiency. Further blockade of TNFR1 by a specific antibody, which neutralizes both soluble and membrane TNF receptor I, also led to abolish the increased caspases-2, -8 and -3, -6, -7 activity in M-Tw cells, which confirms that apoptosis is mediated by the TNF signaling pathway in Twist mutant osteoblasts.
There are several potential molecular mechanisms by which Twist haploinsufficiency may induce TNF overexpression in mutant osteoblasts. One palindromic E-box motif is present in the TNF promoter, suggesting that Twist may directly regulate TNF expression. The increased expression of NRF1, a transcription factor that binds to the TNF promoter and activates TNF transcription (56,57) may provide another mechanism for TNF overexpression in M-Tw cells. Interestingly, our finding that the R118C mutation which abolishes DNA binding of Twist, produced the same phenotype on osteoblast apoptosis in vivo as other mutations resulting in truncated proteins, suggest a direct control of Twist on the expression of apoptotic genes in osteoblasts. Alternatively, Twist haploinsufficiency may induce osteoblast apoptosis by altering FGFR signaling. Drosophila Twist was found to regulate the fly FGF receptor homologue DFR1 (58). The expression patterns of Twist and FGFRs overlap in the fetal mouse coronal suture and Twist was suggested to inhibit FGFR2 expression (18). Although we found that Twist haploinsufficiency does not alter FGFR1 expression in M-Tw calvarial osteoblasts (20), our recent data indicate that FGFR2 expression is increased in Y103X Twist mutant osteoblasts (M.Yousfi, F.Lasmoles and P.J.Marie, manuscript in preparation). We recently showed that activating FGFR2 mutations promote osteoblast and osteocyte apoptosis in Apert craniosynostosis (40). It is thus possible that Twist haploinsufficiency results in increased FGFR2 expression, leading to activation of apoptosis in calvarial osteoblasts in SCS.
In summary, the present study provides genetic and biochemical evidence that Twist haploinsufficiency in SCS promotes calvarial osteoblast apoptosis in vivo and in vitro by a TNF-caspase-2-caspase-8-caspases-3, -6, -7 cascade. The anti-apoptotic role of Twist in human calvarial osteoblasts may, together with alterations in osteoblast proliferation and differentiation (20), play a role in the premature suture ossification in SCS.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Bone samples and calvaria cell cultures
Calvarial bone samples at the coronal suture level from three infants (aged 3.5–7 months) with clinical evidence of SCS associated with genetic Twist mutations were obtained from Dr V.El Ghouzzi. Coronal suture samples in SCS patients were obtained by surgical operation and normal calvarial bone samples at equivalent areas were obtained from three normal age-matched infants who underwent local reconstruction of the skull unrelated to bone diseases (Prof. D.Renier, Hopital Necker-Enfants Malades, Paris). Mutations were determined as previously described (4,6,10,11). The mutations studied cause Twist haploinsufficiency by inducing either degradation of truncated (Twist) proteins (Y103X, Q109X) (10,20) or loss of Twist DNA binding capacities (R118C) (11). Coronal sutures from patients and control subjects were fixed in 10% formaldehyde and embedded in paraffin for detection of apoptosis in vivo, as described previously (40,59). All processes were done according to the French ethical committee recommendations.
Our previous studies showed that the use of human mutant osteoblasts constitute a valuable model allowing characterization of the cellular phenotype and molecular alterations induced by genetic mutations (20,22,23,35,40). To further determine the cellular and molecular phenotype induced by Twist haploinsufficiency, we used calvarial cell populations obtained by collagenase digestion from coronal sutures in a subject with the Y103X mutation that leads to deletion of the functional bHLH domain, and in an age-matched normal subject. The lack of sufficient postnatal tissue samples did not allow us to derive sufficient primary cultures to perform in vitro analyses. Therefore, the two different cell populations were immortalized, giving rise to mutant (M-Tw) and normal (Nl) immortalized calvarial cell populations (20). Because any individual cell line may behave atypically, no attempt has been made to develop and analyse several cell lines from the whole population. M-Tw and Nl immortalized calvarial cells express characteristics of the osteoblast phenotype in culture, such as alkaline phosphatase activity, type I collagen expression and expression of the osteoblast specific gene osteocalcin (20). We previously showed that the osteoblast phenotype (differentiation and apoptosis) found in immortalized human calvaria cells is consistent with the phenotype obtained in primary human calvaria cells in vitro and in vivo (22,23,40,41).
Determination of apoptosis in vivo and in vitro
Detection of apoptotic nuclei in vivo and in vitro was assessed by the TUNEL assay (Boehringer Mannheim) as described previously (40,41). To examine apoptosis in situ, paraffin-embedded coronal samples obtained from the three SCS and three age-matched control subjects were deparaffinized in xylene and rehydrated through a graded series of ethanol. Sections were digested with 20 µg/ml proteinase K for 15 min at 37°C. Endogenous peroxidase was quenched with 0.3% H2O2 for 1 h (59). Sections were permeabilized with 0.1% Triton X-100, at 4°C for 2 min, incubated for 1 h at 37°C with the TUNEL reaction mixture containing the terminal deoxynucleotidyl transferase. TUNEL signal was revealed with diaminobenzidine and mounted. TUNEL-positive cells were revealed by brown nuclei and apoptotic morphology, reflecting the specific dNTP transfer to 3'-hydroxy ends of DNA. Positive controls consisted of sections treated with 50 U/ml DNase I for 15 min at 37°C. Negative controls were obtained by omitting the transferase from the reaction. The number of total and TUNEL-positive osteoblasts along the bone surface in the sample was counted, and the percentage of apoptotic osteoblasts was expressed as a percentage (± SEM) of total osteoblasts. The total number of osteocytes and the number of TUNEL-positive osteocytes in the bone matrix was also recorded and the percentage of apoptotic osteocytes was expressed as a percentage (± SEM) of total osteocytes in the sample. Five different sections per subject were counted. The average total number of osteoblasts and osteocytes assayed per sample was 500 and 1000, respectively.
To assess the effect of Twist haploinsufficiency on osteoblast apoptosis in vitro, M-Tw and Nl cells were cultured on Labtek chambers in serum deprived conditions (1% FCS) for 48 h. Cells were washed with PBS, fixed with paraformaldehyde (4% in PBS) and endogenous peroxidase was quenched with 0.3% H2O2. Then TUNEL assay was performed as described above. Positive and negative controls of cell apoptosis were also obtained as described above.
Apoptosis was also determined by DNA fragmentation. Confluent M-Tw and Nl cells were cultured in 1% FCS for 48 h and analysis of DNA fragmentation was carried out. Briefly, the cells were washed with PBS and lysed with 10 mM Tris, 1 mM EDTA, 0.2% Triton X-100. Lysates were treated with 100 µg of DNase-free RNase A (Boehringer Mannheim) for 30 min at 37°C and incubated with 1 mg of proteinase K (Boehringer Mannheim) for 30 min at 37°C. After precipitation in 5 M NaCl (0.1 vol), isopropanol (v/v) and centrifugation (12 000 g, 10 min), DNA pellet was dissolved in 10 mM Tris, 1 mM EDTA. Electrophoresis was performed by running aliquots of DNA on 1% agarose gel containing ethidium bromide and DNA laddering was visualized with UV light.
Atlas cDNA expression analysis
The signalling molecules involved in apoptosis induced by Twist haploinsufficiency were identified using microarray analysis. Total cellular RNA was extracted from confluent M-Tw and Nl osteoblasts using the Extract-All solution (Eurobio). Residual genomic DNA in RNA preparations was removed by incubating total RNA in the presence of 30 U RNase-free DNase I (Ambion Inc.) for 15 h. After ammonium acetate precipitation, the RNA pellet was dissolved in deionized H2O and was used to prepare labelled cDNA probes according to the Atlas Human Expression Array Kit (Clontech). The gene array consisted of 588 PCR-amplified cDNA fragments (200–500 bp) divided into six groups of genes. In parallel, 5 µg of total RNA from M-Tw and Nl cells were converted into 32P-labelled or 33P-labelled first-strand cDNA at 50°C for 20 min. Labelled cDNAs were purified using Chroma Spin-200 DEPC-H2O column (Boehringer) and 2 x 106 c.p.m. were hybridized overnight on the Atlas membranes with continuous agitation at 68°C. The hybridized Atlas arrays were washed four times in 2x SSC/0.5% SDS for 20 min at 68°C followed by two additional 20 min washes in 0.1x SSC/0.5% SDS at 68°C, wrapped and exposed to Kodak BioMax MS X-ray film with intensifying screen at –70°C. Housekeeping genes were used as internal controls for Nl and M-Tw cells.
RT–PCR analysis
The differential expression of transcripts detected by the microarray analysis in M-Tw and Nl osteoblasts was confirmed by RT–PCR. Confluent Nl and M-Tw cells were cultured in serum deprived medium (1% FCS) for 48 h, 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 TNF: sense, 5'-GCAGTCAGATCATCTTCTCG-3'; antisense, 5'-GCCAGCAATCACTGTGCAG-3'. For MDM2: sense, 5'-CTCAGGTACATCTGTTGAGTG-5'; antisense, 5'-CCTCAACACATGACTCTCTG-3'. For Hsp70: sense, 5'-AAGGTGCAGGTGAGCTACAA-3'; antisense, 5'-CTTGTTCTGGCTGATGTCCT-3'. For p53: sense, 5'-ACCCAGGTCCAGATGAAGCT-3'; antisense, 5'-GCTATCTGAGCAGCGCTCAT-3'. Optimization of RT–PCR results was carried out by generating saturation curves of RT–PCR products of each gene and of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) against cycle number (0–35 cycles) (20). We chose the same cycle number (24 cycles) for all genes except for TNF (30 cycles) in which the amplification was linear. Southern blots were performed by running aliquots of amplified cDNA on 1.2% agarose gel followed by transfer onto nylon membrane (Q Biogen). Hybridization of blots was carried out overnight at 50°C with [
-32P]ATP-labelled internal primers. Membranes were washed twice in 2x SSC/0.1% SDS at room temperature for 10 min, once in 0.1x SSC/0.1% SDS at 50°C for 10 min, then exposed to X-ray films. Autoradiographic signals were quantified using a scanner densitometer. The signal for each gene was corrected using GAPDH as internal control.
Western blot analysis
The expression of proteins encoded by pro-apoptotic and anti-apoptotic genes differentially expressed in Nl and M-Tw cells was confirmed by western blot analysis. Confluent M-Tw and Nl cells were cultured in 1% FCS for 48 h, washed twice with cold PBS and scrapped in 1 ml of ice-cold lysis buffer (10 mM Tris–HCl, 5 mM EDTA, 150 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 1 mM Na3VO4 and 0.5% Triton X-100) containing 10% glycerol and protease inhibitors (Boehringer Mannheim). Protein samples were solubilized in SDS loading buffer and boiled at 95°C for 5 min, then subjected to 4–15% SDS–PAGE (Bio-Rad) using ß-actin (Sigma) as internal control for protein loading. Proteins were transferred onto PVDF membranes (Hybond-P, Amersham) in buffer containing 20% methanol. The membranes were incubated overnight with 1% blocking buffer (Roche) in TBS (50 mM Tris–HCl, 150 mM NaCl) containing 0.1% Tween-20, and then incubated with primary antibodies: monoclonal anti-Bax (B-9, 1/500, Santa Cruz Biotechnology), monoclonal anti-Bcl-2 (sc-7382, 1/500, Santa Cruz Biotechnology), polyclonal anti-TNF (N-19, 1/250, Santa Cruz Biotechnology), monoclonal anti-p53 (Pab1801, 1/1000, Santa Cruz Biotechnology), polyclonal anti-phospho-p53 (Ser20, 1/500, Cell Signalling Technology), or polyclonal anti-ß-actin (Sigma). After 1 h at room temperature, the membranes were washed twice with TBS/0.1% Tween-20 and 0.5% blocking buffer, then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Following incubation with appropriate secondary antibodies, the membranes were washed and the signals were visualized with ECL Plus blotting substrate (Amersham Pharmacia).
Cytochrome c measurement
To determine the involvement of cytochrome c in apoptosis induced by Twist haploinsufficiency in osteoblasts, mitochondrial and cytosolic fractions from confluent M-Tw and Nl cells, cultured in serum deprived medium (1% FCS) for 48 h, were prepared by differential centrifugation using the Apoalert Cell Fractionation Kit (Clontech). Briefly, after trypsinization, the cells were centrifugated (600 g for 5 min at 4°C), resuspended in 1 ml ice-cold wash buffer, centrifugated (600 g for 5 min at 4°C), resuspended in fractionation buffer, lysed on ice with a glass Dounce homogenizer (pestle A, 5 strokes; pestle B, 60 strokes) and centrifugated (700 g for 10 min at 4°C). The supernatant was centrifugated at 10 000 g for 25 min at 4°C. The mitochondrial fraction consisted of the pellet resuspended in 0.1 ml of fractionation buffer and the cytosolic fraction was found in the supernatant. Protein samples (150 µg for the cytosolic fraction and 100 µg for the mitochondrial fraction) were loaded on SDS–18% polyacrylamide gels, subjected to electrophoresis, and then transferred to nitrocellulose membranes. Western blots were probed with either a primary rabbit anti-cytochrome c antibody (1/100, Clontech), a mouse antibody recognizing Cox-4 (1/500, Clontech), a component of the mitochondrial membrane, or a polyclonal anti-ß-actin antibody, then probed with appropriate secondary horseradish peroxidase-conjugated antibodies and developed with ECL Plus blotting substrate (Amersham Pharmacia).
Determination of caspase activity
To identify the caspases induced by the Twist haploinsufficiency in osteoblasts, confluent M-Tw and Nl cells were cultured in serum deprived medium (1% FCS) for 48 h and lysed in 400 µl lysis buffer (10 mM Tris pH 7.4, 200 mM NaCl, 5 mM EDTA, 10% glycerol, 1% NP-40) for 30 min on ice. Lysates were centrifugated (12 000 g for 10 min at 4°C) and the cytosolic fraction was collected. The activity of initiator caspases (caspase-2, caspase-8, caspase-9) and effector caspases (caspases-3, -6, -7) was determined by the cleavage of synthetic fluorogenic substrates containing the amino acid sequence recognized by specific caspases. The substrates were as follows: DEVD (Asp-Glu-Val-Asp) for caspase-3-like (caspases-3, -6, -7); DEHD (Asp-Glu-His-Asp) for caspase-2; IETD (Ile-Glu-Thr-Asp) for caspase-8 and LEHD (Leu-Glu-His-Asp) for caspase-9, all combined to a fluorophore (7-amino-4-methylcoumarin, AMC) (Biosource International). Upon cleavage of the substrate by caspases, free AMC fluorescence emission was detected using a spectrofluorimeter. For the assay, aliquots of 100 µl were incubated for 2 h at 37°C with 200 µl reaction buffer (0.1 mM PMSF, 10 mM DTT, 10 mM HEPES/NaOH pH 7.4) containing 10 µl specific substrate (200 µM). The fluorescence released in samples was measured by excitation at 367 nm and lecture was made at 440 nm. The negative control was the buffer mix and the positive control was free AMC (10 µM in PBS). Results were expressed as arbitrary units and corrected for protein content. To further identify the caspases involved in osteoblast apoptosis induced by Twist haploinsufficiency, confluent M-Tw and Nl cells were treated for 24 h in serum deprived medium (1% FCS) containing the specific caspase-2 inhibitor (Z-VDVAD-FMK) or caspase-8 inhibitor (Z-IETD-FMK) (20 µM) (R&D System), and the activity of caspases was determined as described above.
Inhibition of TNF and TNFR1 activity
To investigate the role of TNF in the pathogenesis of apoptosis induced by Twist haploinsufficiency in osteoblasts, confluent M-Tw osteoblasts were treated for 24 h in serum deprived medium (1% FCS) either with neutralizing anti-TNF antibody (MAB210, mouse IgG class, 1 µg/ml), neutralizing anti-soluble TNFR1 antibody (MAB225, mouse IgG class, 1 µg/ml) which also neutralizes the cell surface TNFR1 (R&D System), or with a non-specific mouse IgG1 (1 µg/ml) (DAKO). The cells were washed with PBS, and caspases-3, -6, -7, caspase-2 and caspase-8 activities were determined as described above.
Statistical analysis
The data were expressed as the mean ± SEM of independent experiments. Differences between the mean values were analysed using the statistical package super-ANOVA (Macintosh, Abacus Concepts Inc., Berkeley, CA) with a minimal significance of P < 0.05.
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ACKNOWLEDGEMENTS |
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The authors thank Dr A.-L.Delezoide (Hopital R. Debre, Paris, France), Dr J.Bonaventure, Dr E.Lajeunie, Prof. A.Munnich (INSERM U393, Hopital Necker-Enfants Malades, Paris, France) and Prof. D.Renier (Department of Neurosurgery, Hopital Necker-Enfants Malades, Paris, France) for providing human calvaria samples and for the mutation analyses. We also thank Dr A.Lomri (INSERM U349) for immortalization of the cells. M.Y. is a recipient of a grant from the Ministere de la Recherche et de la Technologie.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +33 1 49 95 63 58; Fax: +33 1 49 95 84 52; Email: pierre.marie@inserm.lrb.ap-hop-paris.fr
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REFERENCES |
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