Inhibition or activation of Apert syndrome FGFR2 (S252W) signaling by specific glycosaminoglycans

Most Apert syndrome patients harbor a single amino acid mutation (S252W) in fibroblast growth factor (FGF) receptor 2 (FGFR2), which leads to abnormal FGF/FGFR2 signaling. Here we show that specific combinations of FGFs and glycosaminoglycans activate both alternative splice forms of the mutant but not of the wild-type FGF receptors. More importantly, 2-O- and N-sulfated heparan sulfate, prepared by a combined chemical and enzymatic synthesis, antagonized the over-activated FGFR2b (S252W) to basal levels at nanomolar concentrations. These studies demonstrated that specific glycosaminoglycans could be useful in treating ligand-dependent FGFR signaling-related diseases, such as Apert syndrome and cancer.     

Inhibited Wnt Signaling Causes Age-Dependent Abnormalities in the Bone Matrix Mineralization in the Apert Syndrome FGFR2S252W/+ Mice

Apert syndrome (AS) is a type of autosomal dominant disease characterized by premature fusion of the cranial sutures, severe syndactyly, and other abnormalities in internal organs. Approximately 70% of AS cases are caused by a single mutation, S252W, in fibroblast growth factor receptor 2 (FGFR2). Two groups have generated FGFR2 knock-in mice Fgfr2^S252W/+ that exhibit features of AS. During the present study of AS using the Fgfr2^S252W/+ mouse model, an age-related phenotype of bone homeostasis was discovered. The long bone mass was lower in 2 month old mutant mice than in age-matched controls but higher in 5 month old mutant mice. This unusual phenotype suggested that bone marrow-derived mesenchymal stem cells (BMSCs), which are vital to maintain bone homeostasis, might be involved. BMSCs were isolated from Fgfr2^S252W/+ mice and found that S252W mutation could impair osteogenic differentiation BMSCs but enhance mineralization of more mature osteoblasts. A microarray analysis revealed that Wnt pathway inhibitors SRFP1/2/4 were up-regulated in mutant BMSCs. This work provides evidence to show that the Wnt/β-catenin pathway is inhibited in both mutant BMSCs and osteoblasts, and differentiation defects of these cells can be ameliorated by Wnt3a treatment. The present study suggested that the bone abnormalities caused by deregulation of Wnt pathway may underlie the symptoms of AS.     

Mesodermal expression of Fgfr2S252W is necessary and sufficient to induce craniosynostosis in a mouse model of Apert syndrome

Coordinated growth of the skull and brain are vital to normal human development. Craniosynostosis, the premature fusion of the calvarial bones of the skull, is a relatively common pediatric disease, occurring in 1 in 2500 births, and requires significant surgical management, especially in syndromic cases. Syndromic craniosynostosis is caused by a variety of genetic lesions, most commonly by activating mutations of FGFRs 1-3, and inactivating mutations of TWIST1. In a mouse model of TWIST1 haploinsufficiency, cell mixing between the neural crest-derived frontal bone and mesoderm-derived parietal bone accompanies coronal suture fusion during embryonic development. However, the relevance of lineage mixing in craniosynostosis induced by activating FGFR mutations is unknown. Here, we demonstrate a novel mechanism of suture fusion in the Apert Fgfr2(S252W) mouse model. Using Cre/lox recombination we simultaneously induce expression of Fgfr2(S252W) and β-galactosidase in either the neural crest or mesoderm of the skull. We show that mutation of the mesoderm alone is necessary and sufficient to cause craniosynostosis, while mutation of the neural crest is neither. The lineage border is not disrupted by aberrant cell migration during fusion. Instead, the suture mesenchyme itself remains intact and is induced to undergo osteogenesis. We eliminate postulated roles for dura mater or skull base changes in craniosynostosis. The viability of conditionally mutant mice also allows post-natal assessment of other aspects of Apert syndrome.     

A soluble form of fibroblast growth factor receptor 2 (FGFR2) with S252W mutation acts as an efficient inhibitor for the enhanced osteoblastic differentiation caused by FGFR2 activation in Apert syndrome

Apert syndrome is an autosomal dominant disease characterized by craniosynostosis and bony syndactyly associated with point mutations (S252W and P253R) in the fibroblast growth factor receptor (FGFR) 2 that cause FGFR2 activation. Here we investigated the role of the S252W mutation of FGFR2 on osteoblastic differentiation. Osteoblastic cells derived from digital bone in two Apert patients with the S252W mutation showed more prominent alkaline phosphatase activity, osteocalcin and osteopontin mRNA expression, and mineralized nodule formation compared with the control osteoblastic cells derived from two independent non-syndromic polydactyly patients. Stable clones of the human MG63 osteosarcoma cells (MG63-Ap and MG63-IIIc) overexpressing a splice variant form of FGFR2 with or without the S252W mutation (FGFR2IIIcS252W and FGFR2IIIc) showed a higher RUNX2 mRNA expression than parental MG63 cells. Furthermore MG63-Ap exhibited a higher osteopontin mRNA expression than did MG63-IIIc. The enhanced osteoblastic marker gene expression and mineralized nodule formation of the MG63-Ap was inhibited by the conditioned medium from the COS-1 cells overexpressing the soluble FGFR2IIIcS252W. Furthermore the FGF2-induced osteogenic response in the mouse calvarial organ culture system was blocked by the soluble FGFR2IIIcS252W. These results show that the S252W mutation in the FGFR2 gene enhances the osteoblast phenotype in human osteoblasts and that a soluble FGFR2 with the S252W mutation controls osteoblast differentiation induced by the S252W mutation through a dominant negative effect on FGFR2 signaling in Apert syndrome.     

Apert syndrome with preaxial polydactyly showing the typical mutation Ser252Trp in the FGFR2 gene

The Apert syndrome is characterized by craniosynostosis and syndactyly of hands and feet. Although most cases are sporadic, an autosomal dominant mode of inheritance is well documented. Two mutations in the FGFR2 gene (Ser252Trp and Pro253Arg) account for most of the cases. We report a patient with a rare form of Apert syndrome with polydactyly. The proposita has turribrachycephaly. complete syndactyly of 2nd to 5th digits ("mitten hands" and cutaneous fusion of all toes). The X-rays revealed craniosynostosis of the coronal suture and preaxial polydactyly of hands and feet with distal bony fusion. Molecular analysis found a C755G transversion (Ser252Trp) in the FGFR2 gene. Only eight patients with Apert syndrome and preaxial polydactyly have been reported and this is the first case in which molecular diagnosis is available. On the basis of the molecular findings in this patient, polydactyly should be considered part of the spectrum of abnormalities in the Apert syndrome. This assertion would establish the need for a new molecular classification of the acrocephalopolysyndactylies.     

Analysis of phenotypic features and FGFR2 mutations in Apert syndrome.

A phenotypic and genotypic survey was conducted on 36 Apert syndrome patients. In all but one patient, an FGFR2 mutation, either S252W or P253R, was found in exon IIIa (exon U or 7). The frequency was 71% and 26%, for the mutations S252W and P253R, respectively. These mutations occur in the linker region between immunoglobulin-like domains II and III, which are involved in activation of the receptor by ligand binding and dimerization. The fact that one patient did not have a mutation in the same exon suggests further genetic heterogeneity in Apert syndrome. The frequencies of occurrence or means for measurements of 29 different clinical features (including severity of craniofacial features, syndactyly of the hands and feet, and multisystem involvement) were determined for all patients and for the two subgroups defined by their mutations. Comparison between the subgroups for the different clinical features was performed and suggested no statistically significant differences. These results are not unexpected, because the two common mutations for Apert syndrome alter FGFR2 at adjacent amino acids that are likely to have similar biological, and therefore phenotypic, consequences.     

Therapeutic Effect of Nanogel-Based Delivery of Soluble FGFR2 with S252W Mutation on Craniosynostosis

Apert syndrome is an autosomal dominantly inherited disorder caused by missense mutations in fibroblast growth factor receptor 2 (FGFR2). Surgical procedures are frequently required to reduce morphological and functional defects in patients with Apert syndrome; therefore, the development of noninvasive procedures to treat Apert syndrome is critical. Here we aimed to clarify the etiological mechanisms of craniosynostosis in mouse models of Apert syndrome and verify the effects of purified soluble FGFR2 harboring the S252W mutation (sFGFR2IIIc^S252W) on calvarial sutures in Apert syndrome mice in vitro. We observed increased expression of Fgf10, Esrp1, and Fgfr2IIIb, which are indispensable for epidermal development, in coronal sutures in Apert syndrome mice. Purified sFGFR2IIIc^S252W exhibited binding affinity for fibroblast growth factor (Fgf) 2 but also formed heterodimers with FGFR2IIIc, FGFR2IIIc^S252W, and FGFR2IIIb^S252W. Administration of sFGFR2IIIc^S252W also inhibited Fgf2-dependent proliferation, phosphorylation of intracellular signaling molecules, and mineralization of FGFR2^S252W-overexpressing MC3T3-E1 osteoblasts. sFGFR2IIIc^S252W complexed with nanogels maintained the patency of coronal sutures, whereas synostosis was observed where the nanogel without sFGFR2^S252W was applied. Thus, based on our current data, we suggest that increased Fgf10 and Fgfr2IIIb expression may induce the onset of craniosynostosis in patients with Apert syndrome and that the appropriate delivery of purified sFGFR2IIIc^S252W could be effective for treating this disorder.     

FGF/FGFR signaling coordinates skull development by modulating magnitude of morphological integration: evidence from Apert syndrome mouse models

The fibroblast growth factor and receptor system (FGF/FGFR) mediates cell communication and pattern formation in many tissue types (e.g., osseous, nervous, vascular). In those craniosynostosis syndromes caused by FGFR1-3 mutations, alteration of signaling in the FGF/FGFR system leads to dysmorphology of the skull, brain and limbs, among other organs. Since this molecular pathway is widely expressed throughout head development, we explore whether and how two specific mutations on Fgfr2 causing Apert syndrome in humans affect the pattern and level of integration between the facial skeleton and the neurocranium using inbred Apert syndrome mouse models Fgfr2(+/S252W) and Fgfr2(+/P253R) and their non-mutant littermates at P0. Skull morphological integration (MI), which can reflect developmental interactions among traits by measuring the intensity of statistical associations among them, was assessed using data from microCT images of the skull of Apert syndrome mouse models and 3D geometric morphometric methods. Our results show that mutant Apert syndrome mice share the general pattern of MI with their non-mutant littermates, but the magnitude of integration between and within the facial skeleton and the neurocranium is increased, especially in Fgfr2(+/S252W) mice. This indicates that although Fgfr2 mutations do not disrupt skull MI, FGF/FGFR signaling is a covariance-generating process in skull development that acts as a global factor modulating the intensity of MI. As this pathway evolved early in vertebrate evolution, it may have played a significant role in establishing the patterns of skull MI and coordinating proper skull development.     

Differential effects of FGFR2 mutations on syndactyly and cleft palate in Apert syndrome.

Apert syndrome is a distinctive human malformation characterized by craniosynostosis and severe syndactyly of the hands and feet. It is caused by specific missense substitutions involving adjacent amino acids (Ser252Trp or Pro253Arg) in the linker between the second and third extracellular immunoglobulin domains of fibroblast growth factor receptor 2 (FGFR2). We have developed a simple PCR assay for these mutations in genomic DNA, based on the creation of novel (SfiI) and (BstUI) restriction sites. Analysis of DNA from 70 unrelated patients with Apert syndrome showed that 45 had the Ser252Trp mutation and 25 had the Pro253Arg mutation. Phenotypic differences between these two groups of patients were investigated. Significant differences were found for severity of syndactyly and presence of cleft palate. The syndactyly was more severe with the Pro253Arg mutation, for both the hands and the feet. In contrast, cleft palate was significantly more common in the Ser252Trp patients. No convincing differences were found in the prevalence of other malformations associated with Apert syndrome. We conclude that, although the phenotype attributable to the two mutations is very similar, there are subtle differences. The opposite trends for severity of syndactyly and cleft palate in relation to the two mutations may relate to the varying patterns of temporal and tissue-specific expression of different fibroblast growth factors, the ligands for FGFR2.     

Facial suture synostosis of newborn Fgfr1(P250R/+) and Fgfr2(S252W/+) mouse models of Pfeiffer and Apert syndromes

Apert and Pfeiffer syndromes are hereditary forms of craniosynostosis characterized by midfacial hypoplasia and malformations of the limbs and skull. A serious consequence of midfacial hypoplasia in these syndromes is respiratory compromise due to airway obstruction. In this study, we have evaluated Fgfr1(P250R/+) and Fgfr2(S252W/+) mouse models of these human conditions to study the pathogenesis of midfacial hypoplasia. Our histologic and micro-CT evaluation revealed premature synostosis of the premaxillary-maxillary, nasal-frontal, and maxillary-palatine sutures of the face and dysplasia of the premaxilla, maxilla, and palatine bones. These midfacial abnormalities were detected in the absence of premature ossification of the cranial base at postnatal day 0. Our results indicate that midfacial hypoplasia is not secondary to premature cranial base ossification but rather primary synostosis of facial sutures. Birth Defects Research (Part A), 2011.     

Apert p.Ser252Trp mutation in FGFR2 alters osteogenic potential and gene expression of cranial periosteal cells

Apert syndrome (AS), a severe form of craniosynostosis, is caused by dominant gain-of-function mutations in FGFR2. Because the periosteum contribution to AS cranial pathophysiology is unknown, we tested the osteogenic potential of AS periosteal cells (p.Ser252Trp mutation) and observed that these cells are more committed toward the osteoblast lineage. To delineate the gene expression profile involved in this abnormal behavior, we performed a global gene expression analysis of coronal suture periosteal cells from seven AS patients (p.Ser252Trp), and matched controls. We identified 263 genes with significantly altered expression in AS samples (118 upregulated, 145 downregulated; SNR >or= |0.4|, P 相似文献   

人成纤维细胞生长因子受体2IIIc及其突变型重组腺病毒的获得和在乳腺癌细胞中的表达

获得人成纤维细胞生长因子受体2Ⅲc(FGFR2Ⅲc)及其S252W突变型重组腺病毒,感染乳腺癌细胞MDA-MB-231,为下一步研究FGFR2Ⅲc基因的功能和作用机制奠定基础。以本实验室保存的含FGFR2Ⅲc基因的质粒为模板,PCR扩增得到FGFR2Ⅲc基因,重叠延伸法PCR获得FGFR2ⅢcS252W突变型基因;分别将上述野生型和突变型基因克隆至腺病毒穿梭质粒pAdTrack-CMV上,得到重组穿梭质粒pAdTrack-FGFR2Ⅲc和pAdTrack-FGFR2ⅢcS252W,DNA测序证实。Pme I酶切后分别与腺病毒骨架质粒pAdEasy-1共转化BJ-5183感受态细菌同源重组,得到的重组表达质粒Ad-FGFR2Ⅲc和Ad-FGFR2ⅢcS252W Pac I酶切线性化后转染HEK293A细胞进行重组腺病毒的包装和扩增,通过GFP报告基因观察病毒表达情况。收集重组病毒颗粒并测定滴度,进一步感染乳腺癌细胞MDA-MB-231,RT-PCR和Western blotting方法检测目的基因的表达,3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)法和流式细胞术分析细胞增殖情况。结果表明,成功构建了人FGFR2Ⅲc及其S252W突变型基因的重组腺病毒表达载体,获得的重组腺病毒颗粒能高效感染MDA-MB-231细胞,并表达目的基因。MTT结果显示FGFR2Ⅲc和S252W均能抑制MDA-MB-231细胞增殖,S252W抑制效果更加明显。流式细胞术表明FGFR2Ⅲc和S252W均能使MDA-MB-231细胞周期停滞于G0/G1期,抑制细胞增殖。     

Apert syndrome mutant FGFR2 and its soluble form reciprocally alter osteogenesis of primary calvarial osteoblasts

Apert syndrome is characterized by craniosynostosis and syndactyly, and is predominantly caused by mutation of either S252W or P253W in the fibroblast growth factor receptor (FGFR) 2 gene. In this study, we characterized the effects of one of the mutations (S252W) using primary calvarial osteoblasts derived from transgenic mice, Ap-Tg and sAp-Tg, that expressed an Apert-type mutant FGFR2 (FGFR2IIIc-S252W; FGFR2IIIc-Ap), and the soluble form (extracellular domain only) of the mutant FGFR2 (sFGFR2IIIc-Ap), respectively. Compared to WT-derived osteoblasts, osteoblasts from Ap-Tg mouse showed a higher proliferative activity and enhanced differentiation, while those from sAp-Tg mouse exhibited reduced potential for proliferation and osteogenic differentiation. When transplanted with β-tricalcium phosphate (β-TCP) granules into immunodeficient mice, Ap-Tg-derived osteoblasts showed a higher bone forming capacity, whereas sAp-Tg-derived osteoblasts were completely deficient for this phenotype. Phosphorylation of extracellular signal-regulated kinase (ERK), MEK, PLCγ, and p38 was increased in Ap-Tg-derived osteoblasts, whereas phosphorylation of these signaling molecules was reduced in sAp-Tg-derived osteoblasts. Interestingly, when these experiments were carried out using osteoblasts from the mice generated by crossing Ap-Tg and sAp-Tg (Ap/sAp-Tg), which co-expressed FGFR2IIIc-Ap and sFGFR2IIIc-Ap, the results were comparable to those obtained from WT-derived osteoblasts. Taken together, these results indicate that osteoblasts expressing FGFR2IIIc-Ap proliferate and differentiate via highly activated MEK, ERK, and p38 pathways, while these pathways are suppressed in osteoblasts expressing sFGFR2IIIc-Ap. Our findings also suggest that altered FGFR2IIIc signaling in osteoblasts is mostly responsible for the phenotypes seen in Apert syndrome, therefore these osteoblast cell lines are useful tools for investigating the pathogenesis of Apert syndrome.     

De novo alu-element insertions in FGFR2 identify a distinct pathological basis for Apert syndrome.

Apert syndrome, one of five craniosynostosis syndromes caused by allelic mutations of fibroblast growth-factor receptor 2 (FGFR2), is characterized by symmetrical bony syndactyly of the hands and feet. We have analyzed 260 unrelated patients, all but 2 of whom have missense mutations in exon 7, which affect a dipeptide in the linker region between the second and third immunoglobulin-like domains. Hence, the molecular mechanism of Apert syndrome is exquisitely specific. FGFR2 mutations in the remaining two patients are distinct in position and nature. Surprisingly, each patient harbors an Alu-element insertion of approximately 360 bp, in one case just upstream of exon 9 and in the other case within exon 9 itself. The insertions are likely to be pathological, because they have arisen de novo; in both cases this occurred on the paternal chromosome. FGFR2 is present in alternatively spliced isoforms characterized by either the IIIb (exon 8) or IIIc (exon 9) domains (keratinocyte growth-factor receptor [KGFR] and bacterially expressed kinase, respectively), which are differentially expressed in mouse limbs on embryonic day 13. Splicing of exon 9 was examined in RNA extracted from fibroblasts and keratinocytes from one patient with an Alu insertion and two patients with Pfeiffer syndrome who had nucleotide substitutions of the exon 9 acceptor splice site. Ectopic expression of KGFR in the fibroblast lines correlated with the severity of limb abnormalities. This provides the first genetic evidence that signaling through KGFR causes syndactyly in Apert syndrome.     

A Ser252Trp Mutation in Fibroblast Growth Factor Receptor 2 (FGFR2) Mimicking Human Apert Syndrome Reveals an Essential Role for FGF Signaling in the Regulation of Endochondral Bone Formation

A S252W mutation of fibroblast growth factor receptor 2 (FGFR2), which is responsible for nearly two-thirds of Apert syndrome (AS) cases, causes retarded development of the skeleton and skull malformation resulting from premature fusion of the craniofacial sutures. We utilized a Fgfr2^+/S252W mouse (a knock-in mouse model mimicking human AS) to demonstrate decreased bone mass due to reduced trabecular bone volume, reduced bone mineral density, and shortened growth plates in the long bones. In vitro bone mesenchymal stem cells (BMSCs) culture studies revealed that the mutant mice showed reduced BMSC proliferation, a reduction in chondrogenic differentiation, and reduced mineralization. Our results suggest that these phenomena are caused by up-regulation of p38 and Erk1/2 phosphorylation. Treatment of cultured mutant bone rudiments with SB203580 or PD98059 resulted in partial rescue of the bone growth retardation. The p38 signaling pathway especially was found to be responsible for the retarded long bone development. Our data indicate that the S252W mutation in FGFR2 directly affects endochondral ossification, resulting in growth retardation of the long bone. We also show that the p38 and Erk1/2 signaling pathways partially mediate the effects of the S252W mutation of FGFR2 on long bone development.     

Expression of the c-fos proto-oncogene in bone, cartilage and tooth forming tissues during mouse development

The c-fos proto-oncogene is the cellular homologue of v-fos identified as the bone transforming gene of the FBJ and the FBR murine osteosarcoma viruses. We show here, using a sensitive in situ hybridization method, that the c-fos proto-oncogene is expressed in the cartilage, bone and tooth forming tissues during mouse development. This result suggests that the tumors observed after infection by the FBJ viral complex and c-fos overexpression in transgenic mice occur in those tissues in which c-fos is expressed during development.