RNA interference and inhibition of MEK-ERK signaling prevent abnormal skeletal phenotypes in a mouse model of craniosynostosis

Abstract

Premature fusion of one or more of the cranial sutures (craniosynostosis) in humans causes over 100 skeletal diseases, which occur in 1 of approximately 2,500 live births. Among them is Apert syndrome, one of the most severe forms of craniosynostosis, primarily caused by missense mutations leading to amino acid changes S252W or P253R in fibroblast growth factor receptor 2 (FGFR2). Here we show that a small hairpin RNA targeting the dominant mutant form of Fgfr2 (Fgfr2(S252W)) completely prevents Apert-like syndrome in mice. Restoration of normal FGFR2 signaling is manifested by an alteration of the activity of extracellular signal-regulated kinases 1 and 2 (ERK1/2), implicating the gene encoding ERK and the genes downstream of it in disease expressivity. Furthermore, treatment of the mutant mice with U0126, an inhibitor of mitogen-activated protein (MAP) kinase kinase 1 and 2 (MEK1/2) that blocks phosphorylation and activation of ERK1/2, significantly inhibits craniosynostosis. These results illustrate a pathogenic role for ERK activation in craniosynostosis resulting from FGFR2 with the S252W substitution and introduce a new concept of small-molecule inhibitor-mediated prevention and therapy for diseases caused by gain-of-function mutations in the human genome.

Fibroblast Growth Factor Receptor 2 Promotes Osteogenic Differentiation in Mesenchymal Cells via ERK1/2 and Protein Kinase C Signaling

“…Recent studies suggest that ERK1/2 and PI3K signaling pathways may play an important role in osteogenic differentiation (16 -20). Moreover, a pathogenic role for ERK activation has been established in murine craniosynostosis resulting from activated FGFR2 due to a S252W substitution (48), although the target cells and cellular phenotype remain to be determined. In the present study, we showed that both WT and MT FGFR2 activated ERK1/2 phosphorylation, whereas JNK, PI3K, and AKT pathways were not significantly affected, suggesting a central role of ERK1/2 signaling in the phenotype induced by FGFR2 in these cells.…”

“…These results suggest that ERK1/2 activation mediates the increased C3H10T1/2 cell proliferation induced by WT or MT FGFR2. Activation of ERK1/2 has been recently proposed to be involved in the skeletal phenotype induced by the activating S252W FGFR2 mutation in a mouse model of Apert syndrome (48). To determine the role of ERK1/2 on osteoblast differentiation induced by FGFR2 in C3H10T1/2 cells, the cells were transfected with DN-ERK (43), and the expression of osteoblast differentiation markers was determined.…”

Fibroblast growth factor signaling in skeletal development and disease

“…In several mouse models, Apert FGFR2(S252W) mutations activate ERK1/2, p38 MAPK, AKT, β-catenin, and PLCγ in osteogenic cells, which may contribute to premature cranial ossification (Chen et al 2003;Kim et al 2003;Shukla et al 2007;Yin et al 2008;Wang et al 2010;Suzuki et al 2012). Interestingly, reduced dosage of ERF, an inhibitory ETS transcription factor directly bound by ERK1/2 signaling, was found to cause craniosynostosis in humans and mice, implying a role of ERF downstream from ERK signaling in cranial suture ossification (Twigg et al 2013).…”

“…In vivo, selective uncoupling of the docking protein FRS2α and the Crouzon-like activated FGFR2c mutant was found to attenuate FGFR signaling and prevent premature suture fusion in mice (Eswarakumar et al 2006). In mice, treatments with a shRNA targeting the Apert FGFR2(S252W) mutation or the use of a ERK1/2 MAPK inhibitor (Shukla et al 2007) or a soluble mutant form of FGFR2 Yokota et al 2014) were also found to prevent craniosynostosis. The recent finding that FGFR signaling interacts with PDGFRα signaling to induce premature suture fusion in mice and humans may offer alternative therapeutic strategies to indirectly attenuate signals induced by activating FGFR mutations (Moenning et al 2009;Miraoui et al 2010a).…”

The molecular and cellular basis of Apert syndrome

“…Another study also verified that the FGFR2 S252W AS mice treated with U1206, a pharmacological inhibitor of MEK1/2 that blocks phosphorylation and activation of ERK1/2 during pregnancy and early postnatal stages, significantly repressed craniosynostosis and improved skeletal abnormalities. It also showed that a small hairpin RNA targeting the dominant mutant form of FGFR2 (FGFR2S252W) without affecting wild-type mRNA levels completely prevents the Apert-like syndrome in mice, during which the alterations of ERK1/2 activity and FGF-FGFR modulators and downstream genes were observed (41). These data clearly demonstrate that ERK1/2 plays a vital role in osteoblast differentiation and AS occurrence.…”