Hypochondroplasia gain-of-function mutation in FGFR3 causes defective bone mineralization in mice

Hypochondroplasia (HCH) is a mild dwarfism caused by missense mutations in fibroblast growth factor receptor 3 (FGFR3), with the majority of cases resulting from a heterozygous p.Asn540Lys gain-of-function mutation. Here, we report the generation and characterization of the first mouse model (Fgfr3Asn534Lys/+) of HCH to our knowledge. Fgfr3Asn534Lys/+ mice exhibited progressive dwarfism and impairment of the synchondroses of the cranial base, resulting in defective formation of the foramen magnum. The appendicular and axial skeletons were both severely affected and we demonstrated an important role of FGFR3 in regulation of cortical and trabecular bone structure. Trabecular bone mineral density (BMD) of long bones and vertebral bodies was decreased, but cortical BMD increased with age in both tibiae and femurs. These results demonstrate that bones in Fgfr3Asn534Lys/+ mice, due to FGFR3 activation, exhibit some characteristics of osteoporosis. The present findings emphasize the detrimental effect of gain-of-function mutations in the Fgfr3 gene on long bone modeling during both developmental and aging processes, with potential implications for the management of elderly patients with hypochondroplasia and osteoporosis

Growth disorders analysis in hypochondroplasia mouse model and its therapeutic approaches

L'hypochondroplasie (HCH) est un nanisme rhizomélique caractérisé par un raccourcissement des membres, une lordose lombaire et des troubles cognitifs avec des anomalies de l'hippocampe et une dysgénésie des lobes frontaux. Cette chondrodysplasie est associée à des mutations localisées dans le gène Fibroblast Growth Factor Receptor 3 (FGFR3), la mutation gain de fonction la plus fréquente (p.Asn540Lys) est située dans le domaine tyrosine kinase I de la protéine. FGFR3 fait partie d'une famille de 4 récepteurs à activité tyrosine kinase (FGFR1-4). Les FGFRs forment un complexe avec les ligands FGFs (Fibroblast Growth Factor) et activent des voies de signalisation en aval des FGFRs comme les MapKinases, PLCg, STATs et AKT. Ces voies de signalisation vont réguler les processus de survie cellulaire, de différenciation et de prolifération à la fois dans le squelette et le cerveau. La génération du premier modèle murin d'HCH a permis de mieux définir les caractéristiques cliniques de l'HCH. L'étude des souris (Fgfr3Asn534Lys/+) Hch a montré un retard de croissance progressif avec une réduction de la taille des os longs. Les crânes des souris Hch montre une macrocéphalie, un prognathisme, une fusion prématurée des synchondroses de la base du crâne ainsi qu'une diminution de l'aire du foramen magnum. L'étude du squelette axial des souris Hch a montré une modification de la forme du disque intervertébral dès le jour 1. Les analyses de la microarchitecture de la vertèbre lombaire L5 ont montré une diminution de la densité osseuse de l'os trabéculaire. Les tests de compressions des vertèbres Hch ont confirmé la fragilité osseuse des vertèbres De façon surprenante au sein de la plaque de croissance à J14, la prolifération chondrocytaire n'est pas affectée chez les mutants Hch. En revanche, la différenciation chondrocytaire est diminuée. L'analyse de la microarchitecture des fémurs et tibias montrent une diminution de la densité osseuse de l'os trabéculaire chez la souris Hch adulte, alors que la densité osseuse était fortement augmentée au niveau de l'os cortical. Malgré l'augmentation de la densité osseuse de l'os cortical, l'os est plus fragile résistant moins aux fractures. Dans un second temps, nous nous sommes intéressés à l'effet de la mutation HCH dans le cerveau. L'analyse des IRM du cerveau n'ont pas montré de différence de volume mais des modifications de forme du cerveau des souris Hch. Ensuite, nous avons réalisé des tests de comportement afin de comprendre l'effet de la mutation sur les processus mémoriels. Les souris Hch présentent des anomalies des processus de mémoire épisodique et spatiale. Nous avons pu restaurer ce phénotype cognitif en traitant les souris avec un inhibiteur des tyrosines kinases confirmant ainsi le rôle de FGFR3 dans les processus mémoriels. En outre des tests de comportement évaluant la dépression ont permis d'observer que la mutation N540K a un effet antidépresseur. Enfin, j'ai participé à deux études visant à étudier de nouvelles cibles thérapeutiques pour le traitement de l'achondroplasie et l'hypochondroplasie. La molécule (-)-epicatechine isolée du cacao, agit sur la croissance de fémurs des souris Fgfr3Y367C/+ ex vivo et in vivo et normalise l'expression de SOX9 et ERK1/2 dans le cartilage. (-)-epicatechine agit sur la taille du cil primaire des chondrocytes et améliore l'architecture de la plaque de croissance des fémurs. La deuxième molécule étudiée est un inhibiteur de phosphatase (LB100), qui combiné au BMN111, un analogue du CNP, accroit l'effet de BMN111 sur la croissance des fémurs ex vivo dans un modèle murin Fgfr3Y367C/+. L'analyse du premier modèle murin Hch a permis de mieux définir le phénotype squelettique de l'HCH, de montrer que FGFR3 contrôlait la formation de l'os cortical et trabéculaire et de mettre en évidence des troubles de comportement dans le modèle Hch. Toutes ces données permettront à l'avenir une meilleure prise en charge des patients atteints d'hypochondroplasie.Hypochondroplasia (HCH) is a rhizomelic dwarfism characterized by limb shortening and lumbar lordosis and cognitive impairment with hippocampus abnormalities and frontal lobes dysgenesis. This pathology is associated with mutations in the Fibroblast Growth Factor Receptor 3 (FGFR3) gene, the most common mutation p.Asn540Lys is located in the tyrosine kinase I domain of the protein. FGFR3 belong to a family of four receptor with tyrosine kinase activity (FGFR1 to 4). FGFRs form a complex with FGFs (fibroblast growth factor receptor) ligands and their co-factors and activate downstream signaling pathways (Map kinase, PLC', STATs and AKT pathway). These signaling pathways will regulate the processes of cell survival, differentiation and proliferation in skeleton and brain. The generation of the first mouse model of hypochondroplasia helps us to define the clinical characteristics of hypochondroplasia. The study of Hch mice showed progressive growth retardation with a reduction of long bones size. Micro computed tomography of Hch mice skull revealed macrocephaly and prognathism. But also, we observed a premature fusion of the skull base synchondroses and a decrease of foramen magnum area. Axial skeleton analyses of Hch mice showed a change of the intervertebral disc shape starting at P1. Microarchitectural analyses of the L5 lumbar vertebrae showed decreased bone density and trabecular size. In addition, L5 vertebrae compression analyzes in the mouse model confirmed the decrease of the axial skeleton mineralization. Study of the appendicular skeleton from childhood to adulthood of Hch mice show no alteration in cell proliferation, but a decrease in chondrocyte differentiation. In addition, the trabecular bone parameters of the femurs and tibias are altered over time. However, this mutation increased mineralization in the cortical bone in femurs and tibias. Despite, this increased cortical bone density, the bone is less resistant to fracture. In a second part, we looked at the effect of the Hch mutation on brain. We performed MRIs on Hch mice, there is no difference in volume, but the structure of the brain is abnormal. After, we performed behavioral tests on the Hch mouse model to understand the effect of this gain-of-function mutation on memory processes. The Hch mouse model present anomalies in episodic and spatial memory processes. Also, behavior test for depression show that FGFR3 gain-of-function N540K mutation has an antidepressant effect. Finally, I participated in two publications testing new therapeutic approaches for Achondroplasia and Hypochondroplasia treatment. A molecule isolated from cocoa, (-)- epicatechin acts on the growth of Fgfr3Y367C/+ ex vivo femur mice. But also, this molecule increases the growth of Fgfr3Y367C/+ mice in vivo and normalizes the expression of FGFR3, SOX9 and ERK1 / 2. It also acts on the primary cilium by restoring its size in the femurs growth plate and allows the chondrocytes to regain a plate stack conformation. Another study is the combination of a phosphatase inhibitor LB100 and BMN111, a CNP analogue. This combination enhances the effect of BMN111 on ex vivo femur growth of an Fgfr3Y367C/+ mouse model. Study of the first hypochondroplasia mouse model allowed us to better understand the skeletal phenotype of HCH and to show that FGFR3 is implicated in the formation of cortical and trabecular bone and to highlight behavioral disorders in the Hch model. All of these data will allow better management of hypochondroplasia patient in the future

Elevated secretion of pro-collagen I-alpha and vascular endothelial growth factor as biomarkers of acetabular labrum degeneration and calcification in hip osteoarthritis: An explant study

Background: Hip osteoarthritis (OA) involves structural degeneration of different joint compartments, including femoral head cartilage, periarticular ligaments and the acetabular labrum. However, the molecular mechanisms underlying labrum degeneration in hip OA remain poorly understood. Aim: To assess secretion of putative biomarkers for OA from explanted human labrum tissues under basal and inflammatory conditions and to determine whether these could differentiate between OA and calcification status compared to fracture controls. Methods: Intact labrum specimens were collected from patients undergoing joint arthroplasty for primary hip OA (n ​= ​15, mean age 70) or non-OA femoral neck fracture (n ​= ​5, mean age 64). Tissues were dissected in equal-sized samples and explanted for one week. To mimic activation of inflammatory signaling by endogenous damage-associated molecular patterns (DAMP) tissue were stimulated with a toll-like receptor 4 (TLR4) agonist (1 ​μg/mL LPS). The involvement of transforming growth factor-beta (TGF-beta) signaling was evaluated by treatment with a TGF-beta type 1 receptor inhibitor (10 ​μM SB-505124). Secretion of aggrecan (ACAN), pro-collagen-I alpha (Pro-Col-Iα), cartilage oligomeric matrix protein (COMP), interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) was assessed by enzyme-linked immunosorbent assay (ELISA). Labrum calcification was evaluated by 3D whole mount fluorescent microscopy of ethyl cinnamate-based optically cleared tissues stained with Alcian blue/Alizarin red. Results: Whole mount microscopy revealed non-OA fracture controls were non-calcified, whereas six OA labra (40%) were partially calcified or ossified. Basal secretion of Pro-Col-Iα and VEGF was increased four-fold in OA versus non-OA labra. Pro-Col-Iα levels were correlated with those of VEGF (r ​= ​0.65) and COMP (r ​= ​0.54). Stimulation of DAMP signaling through TLR4 affected secretion of IL-6, VEGF, COMP and Pro-Col-Iα, with distinct responses between non-OA and OA tissues. Inhibition of TGF-beta signaling specifically reduced elevated secretion of Pro-Col- Iα and VEGF in calcified OA labrum. Conclusions: Secretion of the putative OA biomarkers Pro-Col-Iα and VEGF is elevated in degenerated human acetabular labrum and may serve as indicators of OA and calcification status. Secretion of both factors was partially regulated by TGF-beta signaling in calcified OA labrum tissues.The Translational potential of this article:Our findings suggest that a biomarker panel consisting of Pro-Col-Iα/VEGF/COMP may be valuable for assessing subradiographic labrum degeneration and calcification in hip OA. Targeting TGF-beta signaling may offer a means to reduce vascular invasion and fibrosis in acetabular labrum tissue

Phosphatase inhibition by LB-100 enhances BMN-111 stimulation of bone growth

Activating mutations in fibroblast growth factor receptor 3 (FGFR3) and inactivating mutations in the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase both result in decreased production of cyclic GMP in chondrocytes and severe short stature, causing achondroplasia (ACH) and acromesomelic dysplasia, type Maroteaux, respectively. Previously, we showed that an NPR2 agonist BMN-111 (vosoritide) increases bone growth in mice mimicking ACH (Fgfr3Y367C/+). Here, because FGFR3 signaling decreases NPR2 activity by dephosphorylating the NPR2 protein, we tested whether a phosphatase inhibitor (LB-100) could enhance BMN-111–stimulated bone growth in ACH. Measurements of cGMP production in chondrocytes of living tibias, and of NPR2 phosphorylation in primary chondrocytes, showed that LB-100 counteracted FGF-induced dephosphorylation and inactivation of NPR2. In ex vivo experiments with Fgfr3Y367C/+ mice, the combination of BMN-111 and LB-100 increased bone length and cartilage area, restored chondrocyte terminal differentiation, and increased the proliferative growth plate area, more than BMN-111 alone. The combination treatment also reduced the abnormal elevation of MAP kinase activity in the growth plate of Fgfr3Y367C/+ mice and improved the skull base anomalies. Our results provide a proof of concept that a phosphatase inhibitor could be used together with an NPR2 agonist to enhance cGMP production as a therapy for ACH

Theobroma cacao improves bone growth by modulating defective ciliogenesis in a mouse model of achondroplasia

International audienceAbstract A gain-of-function mutation in the fibroblast growth factor receptor 3 gene ( FGFR3 ) results in achondroplasia (ACH), the most frequent form of dwarfism. Constitutive activation of FGFR3 impairs bone formation and elongation and many signal transduction pathways. Identification of new and relevant compounds targeting the FGFR3 signaling pathway is of broad importance for the treatment of ACH, and natural plant compounds are prime drug candidate sources. Here, we found that the phenolic compound (-)-epicatechin, isolated from Theobroma cacao , effectively inhibited FGFR3’s downstream signaling pathways. Transcriptomic analysis in an Fgfr3 mouse model showed that ciliary mRNA expression was modified and influenced significantly by the Indian hedgehog and PKA pathways. (-)-Epicatechin is able to rescue mRNA expression impairments that control both the structural organization of the primary cilium and ciliogenesis-related genes. In femurs isolated from a mouse model ( Fgfr3 Y367C/+ ) of ACH, we showed that (-)-epicatechin eliminated bone growth impairment during 6 days of ex vivo culture. In vivo, we confirmed that daily subcutaneous injections of (-)-epicatechin to Fgfr3 Y367C/+ mice increased bone elongation and rescued the primary cilium defects observed in chondrocytes. This modification to the primary cilia promoted the typical columnar arrangement of flat proliferative chondrocytes and thus enhanced bone elongation. The results of the present proof-of-principle study support (-)-epicatechin as a potential drug for the treatment of ACH

FGFR3 overactivation in the brain is responsible for memory impairments in Crouzon syndrome mouse model

International audienceCrouzon syndrome with acanthosis nigricans (CAN, a rare type of craniosynostosis characterized by premature suture fusion and neurological impairments) has been linked to a gain-of-function mutation (p.Ala391Glu) in fibroblast growth factor receptor 3 (FGFR3). To characterize the CAN mutation's impact on the skull and on brain functions, we developed the first mouse model (Fgfr3 A385E/+) of this syndrome. Surprisingly, Fgfr3 A385E/+ mice did not exhibit craniosynostosis but did show severe memory impairments, a structurally abnormal hippocampus, low activity-dependent synaptic plasticity, and overactivation of MAPK/ERK and Akt signaling pathways in the hippocampus. Systemic or brain-specific pharmacological inhibition of FGFR3 overactivation by BGJ398 injections rescued the memory impairments observed in Fgfr3 A385E/+ mice. The present study is the first to have demonstrated cognitive impairments associated with brain FGFR3 overactivation, independently of skull abnormalities. Our results provide a better understanding of FGFR3's functional role and the impact of its gain-of-function mutation on brain functions. The modulation of FGFR3 signaling might be of value for treating the neurological disorders associated with craniosynostosis