Inhibición del receptor FGFR3 por ARNs de interferencia para la acondroplasia

Achondroplasia is a short-limbed dwarfism resulting from mutation and gain-of-function in fibroblast growth factor receptor 3 (FGFR3). Effective therapy for this condition has not as yet been established. We have tested the efficiency of three different small interference RNAs (siRNAs) to abrogate the FGFR3 expression in human immortalized chondrocytes carrying the achondroplasia mutation (G380R). Two siRNA sequences induced markedly decrease of FGFR3 mRNA (up to 75% reduction) and protein levels (up to 61% reduction). Furthemore, siRNA-mediated knockdown of FGFR3 blocked the activation of the downstream signal transduction ERK pathway.La acondroplasia es un tipo de enanismo caracterizado por extremidades cortas resultante de una mutación en el receptor de crecimiento de fibroblastos de tipo 3 (FGFR3). Aún no se ha establecido una terapia efectiva para esta enfermedad. Nosotros hemos testado la eficiencia de tres diferentes small interference RNAs (siRNAs) para bloquear la expresión del receptor FGFR3 en condrocitos humanos inmortalizados portadores de la mutación acondroplásica (G380R). Dos secuencias de siRNAs indujeron un marcado descenso de la expresión de ARN mensajero del receptor FGFR3 (hasta un 75%) así como de los niveles de proteína (hasta un 61%). Además, el bloqueo de la expresión del receptor FGFR3 mediado por los siRNAs redujo la activación de la cascada de transducción de las ERK

Human immortalized chondrocytes carrying heterozygous FGFR3 mutations: An in vitro model to study chondrodysplasias

AbstractAchondroplasia and thanatophoric dysplasia are human chondrodysplasias caused by mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. We have developed an immortalized human chondrocyte culture model to study the regulation of chondrocyte functions. One control and eight mutant chondrocytic lines expressing different FGFR3 heterozygous mutations were obtained. FGFR3 signaling pathways were modified in the mutant lines as revealed by the constitutive activation of the STAT pathway and an increased level of P21WAF1/CIP1 protein. This model will be useful for the study of FGFR3 function in cartilage studies and future therapeutic approaches in chondrodysplasias

The impact of polyphenols on chondrocyte growth and survival: a preliminary report

Background: Imbalances in the functional binding of fibroblast growth factors (FGFs) to their receptors (FGFRs) have consequences for cell proliferation and differentiation that in chondrocytes may lead to degraded cartilage. The toxic, proinflammatory, and oxidative response of cytokines and FGFs can be mitigated by dietary polyphenols. Objective: We explored the possible effects of polyphenols in the management of osteoarticular diseases using a model based on the transduction of a mutated human FGFR3 (G380R) in murine chondrocytes. This mutation is present in most cases of skeletal dysplasia and is responsible for the overexpression of FGFR3 that, in the presence of its ligand, FGF9, results in toxic effects leading to altered cellular growth. Design: Different combinations of dietary polyphenols derived from plant extracts were assayed in FGFR3 (G380R) mutated murine chondrocytes, exploring cell survival, chloride efflux, extracellular matrix (ECM) generation, and grade of activation of mitogen-activated protein kinases. Results: Bioactive compounds from Hibiscus sabdariffa reversed the toxic effects of FGF9 and restored normal growth, suggesting a probable translation to clinical requests in humans. Indeed, these compounds activated the intracellular chloride efflux, increased ECM generation, and stimulated cell proliferation. The inhibition of mitogen-activated protein kinase phosphorylation was interpreted as the main mechanism governing these beneficial effects. Conclusions: These findings support the rationale behind the encouragement of the development of drugs that repress the overexpression of FGFRs and suggest the dietary incorporation of supplementary nutrients in the management of degraded cartilage.The authors are grateful for the constant support provided by the Hospital Universitari de Sant Joan and the Universitat Rovira i Virgili. Salvador Fernández-Arroyo is the recipient of a Sara Borrell grant (CD12/00672) from the Instituto de Salud Carlos III, Madrid, Spain. The authors also thank the Andalusian Regional Government Council of Innovation and Science for the Excellence Project P11-CTS-7625 and Generalitat Valenciana for the project PROMETEO/2012/007. This work was also supported by projects of the Fundación Areces and the Fundación MAGAR

Impact de l'activation constitutive de FGFR3 sur l'ossification endochondrale

PARIS5-BU Méd.Cochin (751142101) / SudocSudocFranceF

Un nouveau modèle pour étudier la physiopathologie des chondrodysplasies liées à la FGFR3

FGFR3 (Fibroblast Growth Factor Receptor 3) est responsable d'une famille de chondrodysplasies de sévérité variable regroupant l'hypochondroplasie, forme modérée, l'achondroplasie, nanisme le plus fréquent et le nanisme thanatophore, forme sévère. Afin de comprendre les conséquences des mutations activatrices sur le développement squelettique, un nouveau modèle murin a été généré exprimant une mutation de nanisme thanatophore. Les souris mutantes présentent un nanisme sévère évolutif avec des os longs courts et trapus, une plaque de croissance désorganisée et un retard d'ossification épiphysaire. De plus, des anomalies de l'épithélium sensoriel de la cochlée ont été mises en évidence et sont responsables d'une surdité chez la souris mutante. Parallèlement, une étude a été réalisée chez l'humain montrant également un retard d'âge osseux et une surdité neurosensorielle dans l'achondroplasie. Ces résultats confirment le rôle primordial de FGFR3 dans l'ossification enchondrale et l'audition.FGFR3 (Fibroblast Growth Factor Receptor 3) cause several chondrodysplasias, including hypochondroplasia, mild phenotype, achondroplasia, most common form of human dwarfism, and thanatophoric dysplasia, severe dwarfism. To investigate the role of activating FGFR3 mutation in skeletal development, we introduced fgfrS mutation in mouse genome corresponding to the thanatophoric dysplasia. The mutant mice displayed severe dwarfism with shortened long bones, growth plate disturbed and secondary ossification center delayed. In addition, the mutant mice exhibited mild deafness with defect in epithelial sensory cells of the inner ear. At the same time, a human study was performed: a bone age delay and a sensorineural hearing loss were also observed in achondroplasia patients. Our results demonstrate the crucial role of FGFRS in endochondral ossification and auditory system.PARIS5-BU Méd.Cochin (751142101) / SudocSudocFranceF

Novel therapeutic approaches for the treatment of achondroplasia

International audienceAchondroplasia is the most common form of human dwarfism. The molecular basis of achondroplasia was elucidated in 1994 with the identification of the fibroblast growth factor receptor 3 (FGFR3) as the causative gene. Missense mutations causing achondroplasia result in activation of FGFR3 and its downstream signaling pathways, disturbing chondrogenesis, osteogenesis, and long bone elongation. A more accurate understanding of the clinical and molecular aspects of achondroplasia has allowed new therapeutic approaches to be developed. These are based on: clear understanding of the natural history of the disease; proof-of-concept preclinical studies in mouse models; and the current state of knowledge regarding FGFR3 and related growth plate homeostatic pathways. This review provides a brief overview of the preclinical mouse models of achondroplasia that have led to new, non-surgical therapeutic strategies being assessed and applied to children with achondroplasia through pioneering clinical trials

An Fgfr3-activating mutation in immature osteoblasts affects the appendicular and craniofacial skeleton

International audienceAchondroplasia (ACH), the most common form of dwarfism is caused by a missense mutation in the gene coding for fibroblast growth factor receptor 3 (FGFR3). The resulting increase in FGFR3 signaling perturbs the proliferation and differentiation of chondrocytes (CCs), alters the process of endochondral ossification and thus reduces bone elongation. Increased FGFR3 signaling in osteoblasts (OBs) might also contribute to bone anomalies in ACH. In the present study of a mouse model of ACH, we sought to determine whether or not FGFR3 overactivation in OBs leads to bone modifications. The model carries an Fgfr3 activating mutation (Fgfr3 Y367C/+) that accurately mimics ACH; we targeted the mutation to either immature OBs and hypertrophic CCs or to mature OBs by using the Osx-cre and collagen 1α1 (2.3kb-Col1α1)-cre mouse strains, respectively. We observed that Fgfr3 activation in immature OBs and hypertrophic CCs (Osx-Fgfr3) not only perturbed the hypertrophic cells of the growth plate (thus affecting long bone growth) but also led to osteopenia and low cortical thickness in long bones in adult (3-month-old) mice but not in growing (3-week-old) mice. Importantly, craniofacial membranous bone defects were present in the adult mice. In contrast, activation of Fgfr3 in mature OBs (Col1-Fgfr3) had very limited effects on skeletal shape, size and micro-architecture. In vitro, we observed that Fgfr3 activation in immature OBs was associated with low mineralization activity. In conclusion, immature OBs appears to be affected by Fgfr3 overactivation, which might contribute to the bone modifications observed in ACH independently of CCs

Activating Fgfr3 Y367C mutation causes hearing loss and inner ear defect in a mouse model of chondrodysplasia.

International audienceFibroblast growth factor receptor 3 (FGFR3) is a key regulator of skeletal development and activating mutations in FGFR3 cause skeletal dysplasias, including hypochondroplasia, achondroplasia and thanatophoric dysplasia. The introduction of the Y367C mutation corresponding to the human Y373C thanatophoric dysplasia type I (TDI) mutation into the mouse genome, resulted in dwarfism with a skeletal phenotype remarkably similar to that of human chondrodysplasia. To investigate the role of the activating Fgfr3 Y367C mutation in auditory function, the middle and inner ear of the heterozygous mutant Fgfr3(Y367C/+) mice were examined. The mutant Fgfr3(Y367C/+) mice exhibit fully penetrant deafness with a significantly elevated auditory brainstem response threshold for all frequencies tested. The inner ear defect is mainly associated with an increased number of pillar cells or modified supporting cells in the organ of Corti. Hearing loss in the Fgfr3(Y367C/+) mouse model demonstrates the crucial role of Fgfr3 in the development of the inner ear and provides novel insight on the biological consequences of FGFR3 mutations in chondrodysplasia