S14-09 SESSION 14: FACIOCRANIOSYNOSTOSIS – PART III MITIGATION OF SHP2 ACTIVATION PREVENTS FGFR2 SIGNALING INDUCED CRANIOSYNOSTOSIS

Abstract

Introduction: Activating-dominant mutations in the fibroblast growth factor receptor IIIc splice variant gene (Fgfr2c) cause premature cranial suture fusion (craniosynostosis), which can restrict growth of the craniofacial complex. Fgfr2c signaling is mediated via the docking protein Frs2a, which interacts with both Shp2, a tyrosine phosphatase, and Grb2, an adapter protein, to induce ERK-MAPK activation. The role of both Shp2 and Grb2 in craniosynostosis and craniofacial growth remains unknown; however, it is known that gain-of-function Fgfr2c mutant mice demonstrate hyperactivation of the ERK-MAPK signaling cascade and osteoblast activity. The objective of the current study is to determine the role Shp2 and Grb2 may play in Fgfr2-induced craniosynostosis and craniofacial growth. Methods: Gain-of-function Fgfr2c mice were crossed with mice deficient in two Shp2- or four Grb2-specific tyrosine phosphorylation sites within Frs2a. Skull morphology and sutures were analyzed grossly, histologically, and by micro-Computed Tomography. Coronal sutures were analyzed for ERK protein activation and whole calvaria were cultured and analyzed for alkaline phosphatase activity. Fluorescence-lifetime imaging microscopy – Fluorescence Resonance Energy Transfer (FLIM-FRET) was performed to study the interactions between FRS2a, Shp2, and Grb2 within WT or gain-of-function Fgfr2c mutant HEK293T cells. Analysis of Variance was used to compare three or more groups. An observed P value ≤ 0.05 was considered statistically significant. Results: Reduction of Shp2 activation prevents coronal suture fusion and restores normal craniofacial growth (P < 0.05), ERK-MAPK signaling (P < 0.05), and osteoblast activity (P < 0.05) in a mouse model of Fgfr2c-induced craniosynostosis via an Frs2a-dependent mechanism. Complete uncoupling of Grb2 from Frs2a resulted in only partial rescue of normal suture formation and growth. FLIM-FRET analysis revealed a complex interplay between FRS2a, Shp2, and Grb2 in the setting of dysregulated Fgfr2c signaling. Conclusion: This study provides mechanistic insight into craniofacial development and identifies Shp2 as a key regulator of craniofacial growth and development and highlights the therapeutic potential of Shp2 modulation for the treatment of Fgfr2c-related disorders.