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

Missense FGFR3 mutations create cysteine residues in thanatophoric dwarfism type I (TD1

Thanatophoric dwarfism (TD) is a sporadic lethal skeletal dysplasia with micromelic shortening of the limbs, macrocephaly, platyspondyly and reduced thoracic cavity. In the most common subtype (TD1), femurs are curved, while in TD2, straight femurs are associated with cloverleaf skull. Mutations in the fibroblast growth factor receptor 3 (FGFR3) gene were identified in both subtypes. While TD2 was accounted for by a single recurrent mutation in the tyrosine kinase 2 domain, TD1 resulted from either stop codon mutations or missense mutations in the extracellular domain of the gene. Here, we report the identification of FGFR3 mutations in 25/26 TD cases. Two novel missense mutations (Y373C and G370C) were detected in 8/26 and 1/26 TD1 cases respectively. Both mutations created cysteine residues in the juxta extramembrane domain of the receptor. Sixteen cases carried the previously reported R248C (9/26 cases), S249C (2/26 cases) or stop codon FGFR3 mutations (5/26 cases). Our results suggest that TD1 is a genetically homogeneous condition and give additional support to the view that newly created cysteine residues in the extracellular domain of the protein play a key role in the severity of the disease

Tyrosine kinases regulate chondrocyte hypertrophy: promising drug targets for Osteoarthritis

Osteoarthritis (OA) is a major health problem worldwide that affects the joints and causes severe disability. It is characterized by pain and low-grade inflammation. However, the exact pathogenesis remains unknown and the therapeutic options are limited. In OA articular chondrocytes undergo a phenotypic transition becoming hypertrophic, which leads to cartilage damage, aggravating the disease. Therefore, a therapeutic agent inhibiting hypertrophy would be a promising disease-modifying drug. The therapeutic use of tyrosine kinase inhibitors has been mainly focused on oncology, but the Food and Drug Administration (FDA) approval of the Janus kinase inhibitor Tofacitinib in Rheumatoid Arthritis has broadened the applicability of these compounds to other diseases. Interestingly, tyrosine kinases have been associated with chondrocyte hypertrophy. In this review, we discuss the experimental evidence that implicates specific tyrosine kinases in signaling pathways promoting chondrocyte hypertrophy, highlighting their potential as therapeutic targets for OA.Biomaterials & Tissue Biomechanic