FGF and TGFbeta signalling in an in-vitro model of craniosynostosis.
Doctoral thesis, UCL (University College London).
Fibroblast Growth Factor (FGF) and Transforming Growth Factor beta (TGFbeta) are key regulators of bone development. Constitutively activating mutations of FGF Receptors (FGFR) 1-3 result in craniosynostosis, premature fusion of cranial sutures. The aim of this thesis was to determine how FGF signalling is impaired in osteoblasts with the mutation FGFR2-C278F, known to induce craniosnostosis and investigate possible interactions with TGFbeta signalling. To this purpose MC3T3-E1 osteoblasts (derived from newborn mouse calvaria) that had been stably transfected with human FGFR2 (wild type FGFR2-WT or mutated FGFR2-C278F) were used as an in-vitro model and these cell lines were named R2-WT and R2-C278F. These cell lines were characterised at the cellular and molecular level to define the craniosynostotic phenotype. Gene expression was assessed with real time PCR, proliferation using both fluorescence activated cell sorting (FACS) and the methylene blue assay and protein expression by FACS, immunocytochemistry and Western blotting. Cell proliferation was reduced and apoptosis increased in the R2-C278F mutant and differentiation increased, as shown by reduced expression of differentiation marker osteopontin and an increase in osteocalcin. The effect of FGF signalling on cell growth was demonstrated by using FGFR inhibitor SU5402. This study suggested that FGFR2-C278F decreases the level of FGFR signalling. FGFR2-C278F impairs TGFbeta signalling as shown by: i) reduced Tgfbeta1 and -3 expression in R2-C278F cells; ii) maximal reduction of cell growth only in R2-C278F cells following TGFbeta inhibition using SB431542 (1μM); iii) the inability of exogenous TGFbeta1 to induce proliferation in R2-C278F cells. This suggests that exogenous TGFbeta1 cannot rescue the impaired TGFbeta signalling caused by FGFR2-C278F mutation. Fgf and Tgfbeta signalling may converge to affect osteoblast proliferation via extracellular related kinase 1/2 (Erk1/2). Analysis of the Erk1/2 protein expression in R2-C278F cells showed that Erk1 isoform had increased relative to Erk2. This change has been associated with growth arrest in osteoblasts, fibroblasts and hepatocytes and therefore it is likely to underlie the defect in proliferative response to Fgf and Tgfbeta signalling in R2-C278F cells. In summary FGFR2-C278F in MC3T3 cells impairs Fgf and Tgfbeta signalling, resulting in a proliferation defect for which increased differentiation is implicated as a secondary effect. A key convergence between FGFR2-C278F and Tgfbeta appears to be via impaired Erk1/2 signal transduction. These findings provide a valuable basis for future investigations of other Erk1/2 upstream pathways and their contribution to the craniosynostotic osteoblast phenotype.
|Title:||FGF and TGFbeta signalling in an in-vitro model of craniosynostosis|
|Open access status:||An open access version is available from UCL Discovery|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Population Health Sciences > Institute of Child Health > Department of Infection and Immunity > ICH - Infectious Diseases and Microbiology|
Archive Staff Only