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

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

Saethre-Chotzen mutations cause TWIST protein degradation or impaired nuclear location

International audienceH-TWIST belongs to the family of basic helix-loop-helix (bHLH) transcription factors known to exert their activity through dimer formation. We have demonstrated recently that mutations in H-TWIST account for Saethre-Chotzen syndrome (SCS), an autosomal dominant craniosynostosis syndrome characterized by premature fusion of coronal sutures and limb abnormalities of variable severity. Although insertions, deletions, nonsense and missense mutations have been identified, no genotype-phenotype correlation could be found, suggesting that the gene alterations lead to a loss of protein function irrespective of the mutation. To assess this hypothesis, we studied stability, dimerization capacities and subcellular distribution of three types of TWIST mutant. Here, we show that: (i) nonsense mutations resulted in truncated protein instability; (ii) missense mutations involving the helical domains led to a complete loss of H-TWIST heterodimerization with the E12 bHLH protein in the two-hybrid system and dramatically altered the ability of the TWIST protein to localize in the nucleus of COS-transfected cells; and (iii) in-frame insertion or missense mutations within the loop significantly altered dimer formation but not the nuclear location of the protein. We conclude that at least two distinct mechanisms account for loss of TWIST protein function in SCS patients, namely protein degradation and subcellular mislocalization

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

FGFR3IIIS: a novel soluble FGFR3 spliced variant that modulates growth is frequently expressed in tumour cells

Fibroblast growth factor receptor 3 (FGFR3) is one of four high-affinity tyrosine kinase receptors for the FGF family of ligands, frequently associated with growth arrest and induction of differentiation. The extracellular immunoglobulin (IgG)-like domains II and III are responsible for ligand binding; alternative usage of exons IIIb and IIIc of the Ig-like domain III determining the ligand-binding specificity of the receptor. By reverse transcriptase polymerase chain reaction (RT–PCR) a novel FGFR3IIIc variant FGFR3IIIS, expressed in a high proportion of tumours and tumour cell lines but rarely in normal tissues, has been identified. Unlike recently described nonsense transcripts of FGFR3, the coding region of FGFR3IIIS remains in-frame producing a novel protein. The protein product is coexpressed with FGFR3IIIc in the membrane and soluble cell fractions; expression in the soluble fraction is decreased after exposure to bFGF but not aFGF. Knockout of FGFR3IIIS using antisense has a growth-inhibitory effect in vitro, suggesting a dominant-negative function for FGFR3IIIS inhibiting FGFR3-induced growth arrest. In summary, alternative splicing of the FGFR3 Ig-domain III represents a mechanism for the generation of receptor diversity. FGFR3IIIS may regulate FGF and FGFR trafficking and function, possibly contributing to the development of a malignant phenotype

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

FGFR3 – a Central Player in Bladder Cancer Pathogenesis?

The identification of mutations in FGFR3 in bladder tumors in 1999 led to major interest in this receptor and during the subsequent 20 years much has been learnt about the mutational profiles found in bladder cancer, the phenotypes associated with these and the potential of this mutated protein as a target for therapy. Based on mutational and expression data, it is estimated that >80% of non-muscle-invasive bladder cancers (NMIBC) and ∼40% of muscle-invasive bladder cancers (MIBC) have upregulated FGFR3 signalling, and these frequencies are likely to be even higher if alternative splicing of the receptor, expression of ligands and changes in regulatory mechanisms are taken into account. Major efforts by the pharmaceutical industry have led to development of a range of agents targeting FGFR3 and other FGF receptors. Several of these have entered clinical trials, and some have presented very encouraging early results in advanced bladder cancer. Recent reviews have summarised the drugs and related clinical trials in this area. This review will summarise what is known about the effects of FGFR3 and its mutant forms in normal urothelium and bladder tumors, will suggest when and how this protein contributes to urothelial cancer pathogenesis and will highlight areas that may benefit from further study

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