FGFR3 intracellular mutations induce tyrosine phosphorylation in the Golgi and defective glycosylation.

Mutations of the Fibroblast Growth Factor Receptor 3 (FGFR3) gene have been implicated in a series of skeletal dysplasias including hypochondroplasia, achondroplasia and thanatophoric dysplasia. The severity of these diseases ranges from mild dwarfism to severe dwarfism and to perinatal lethality, respectively. Although it is considered that the mutations give rise to constitutively active receptors, it remains unclear how the different mutations are functionally linked to the severity of the different pathologies. By examining various FGFR3 mutations in a HEK cell culture model, including the uncharacterized X807R mutation, it was found that only the mutations affecting the intracellular domain, induced premature receptor phosphorylation and inhibited receptor glycosylation, suggesting that premature receptor tyrosine phosphorylation of the native receptor inhibits its glycosylation. Moreover, these mutations appeared to be associated with elevated receptor signaling in the Golgi apparatus. In conclusion, although pathological severity could not be correlated with a single factor arising from FGFR3 mutations, these results suggest that intracellular domain mutations define a distinct means by which mutated FGFR3 could disrupt bone development.

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

Achondroplasia 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 P21(WAF1/CIP1) protein. This model will be useful for the study of FGFR3 function in cartilage studies and future therapeutic approaches in chondrodysplasias.

The localization of FGFR3 mutations causing thanatophoric dysplasia type I differentially affects phosphorylation, processing and ubiquitylation of the receptor.

Recurrent missense fibroblast growth factor receptor 3 (FGFR3) mutations have been ascribed to skeletal dysplasias of variable severity including the lethal neonatal thanatophoric dysplasia types I (TDI) and II (TDII). To elucidate the role of activating mutations causing TDI on receptor trafficking and endocytosis, a series of four mutants located in different domains of the receptor were generated and transiently expressed. The putatively elongated X807R receptor was identified as three isoforms. The fully glycosylated mature isoform was constitutively but mildly phosphorylated. Similarly, mutations affecting the extracellular domain (R248C and Y373C) induced moderate constitutive receptor phosphorylation. By contrast, the K650M mutation affecting the tyrosine kinase 2 (TK2) domain produced heavy phosphorylation of the nonglycosylated and mannose-rich isoforms that impaired receptor trafficking through the Golgi network. This resulted in defective expression of the mature isoform at the cell surface. Normal processing was rescued by tyrosine kinase inhibitor treatment. Internalization of the R248C and Y373C mutant receptors, which form stable disulfide-bonded dimers at the cell surface was less efficient than the wild-type, whereas ubiquitylation was markedly increased but apparently independent of the E3 ubiquitin-ligase casitas B-lineage lymphoma (c-Cbl). Constitutive phosphorylation of c-Cbl by the K650M mutant appeared to be related to the intracellular retention of the receptor. Therefore, although mutation K650M affecting the TK2 domain induces defective targeting of the overphosphorylated receptor, a different mechanism characterized by receptor retention at the plasma membrane, excessive ubiquitylation and reduced degradation results from mutations that affect the extracellular domain and the stop codon.

Novel FGFR3 mutations creating cysteine residues in the extracellular domain of the receptor cause achondroplasia or severe forms of hypochondroplasia.

Achondroplasia (ACH) and hypochondroplasia (HCH) are two autosomal-dominant skeletal disorders caused by recurrent missense FGFR3 mutations in the transmembrane (TM) and tyrosine kinase 1 (TK1) domains of the receptor. Although 98% of ACH cases are accounted for by a single G380R substitution in the TM, a common mutation (N540K) in the TK1 region is detected in only 60-65% of HCH cases. The aim of this study was to determine whether the frequency of mutations in patients with HCH was the result of incomplete mutation screening or genetic heterogeneity. Eighteen exons of the FGFR3 gene were entirely sequenced in a cohort of 25 HCH and one ACH patients in whom common mutations had been excluded. Seven novel missense FGFR3 mutations were identified, one causing ACH and six resulting in HCH. Six of these substitutions were located in the extracellular region and four of them creating additional cysteine residues, were associated with severe phenotypes. No mutations were detected in 19 clinically diagnosed HCH patients. Our results demonstrate that the spectrum of FGFR3 mutations causing short-limb dwarfism is wider than originally recognised and emphasise the requirement for complete screening of the FGFR3 gene if appropriate genetic counselling is to be offered to patients with HCH or ACH lacking the most common mutations and their families.

Defective chondrocyte proliferation and differentiation in osteochondromas of MHE patients.

Multiple hereditary exostoses (MHE) is an autosomal dominant skeletal disorder caused by mutations in one of the two EXT genes and characterized by multiple osteochondromas that generally arise near the ends of growing long bones. Defective endochondral ossification is likely to be involved in the formation of osteochondromas. In order to investigate potential changes in chondrocyte proliferation and/or differentiation during this process, osteochondroma samples from MHE patients were obtained and used for genetic, morphological, immunohistological, and in situ hybridization studies. The expression patterns of IHH (Indian hedgehog) and FGFR3 (Fibroblast Growth Factor Receptor 3) were similar with transcripts expressed throughout osteochondromas. Expression of PTHR1 (Parathyroid Hormone Receptor 1) transcripts was restricted to a narrow zone of prehypertrophic chondrocytes. Numerous cells forming osteochondromas although resembling prehypertrophic chondrocytes, stained positively with an anti-proliferating cell nuclear antigen (PCNA) antibody. In addition, ectopic expression of collagen type I and abnormal presence of osteocalcin (OC), osteopontin (OP), and bone sialoprotein (BSP) were observed in the cartilaginous osteochondromas. These data indicate that most chondrocytes involved in the growth of osteochondromas can proliferate, and that some of them exhibit bone-forming cell characteristics. We conclude that in MHE, defective heparan sulfate biosynthesis caused by EXT mutations maintains the proliferative capacity of chondrocytes and promotes phenotypic modification to bone-forming cells.

New insight on FGFR3-related chondrodysplasias molecular physiopathology revealed by human chondrocyte gene expression profiling.

Endochondral ossification is the process by which the appendicular skeleton, facial bones, vertebrae and medial clavicles are formed and relies on the tight control of chondrocyte maturation. Fibroblast growth factor receptor (FGFR)3 plays a role in bone development and maintenance and belongs to a family of proteins which differ in their ligand affinities and tissue distribution. Activating mutations of the FGFR3 gene lead to craniosynostosis and multiple types of skeletal dysplasia with varying degrees of severity: thanatophoric dysplasia (TD), achondroplasia and hypochondroplasia. Despite progress in the characterization of FGFR3-mediated regulation of cartilage development, many aspects remain unclear. The aim and the novelty of our study was to examine whole gene expression differences occurring in primary human chondrocytes isolated from normal cartilage or pathological cartilage from TD-affected fetuses, using Affymetrix technology. The phenotype of the primary cells was confirmed by the high expression of chondrocytic markers. Altered expression of genes associated with many cellular processes was observed, including cell growth and proliferation, cell cycle, cell adhesion, cell motility, metabolic pathways, signal transduction, cell cycle process and cell signaling. Most of the cell cycle process genes were down-regulated and consisted of genes involved in cell cycle progression, DNA biosynthesis, spindle dynamics and cytokinesis. About eight percent of all modulated genes were found to impact extracellular matrix (ECM) structure and turnover, especially glycosaminoglycan (GAG) and proteoglycan biosynthesis and sulfation. Altogether, the gene expression analyses provide new insight into the consequences of FGFR3 mutations in cell cycle regulation, onset of pre-hypertrophic differentiation and concomitant metabolism changes. Moreover, impaired motility and ECM properties may also provide clues about growth plate disorganization. These results also suggest that many signaling pathways may be directly or indirectly altered by FGFR3 and confirm the crucial role of FGFR3 in the control of growth plate development.