A role for fibroblast growth factor receptor-2 in the altered osteoblast phenotype induced by Twist haploinsufficiency in the Saethre–Chotzen syndrome

Abstract

Genetic mutations of Twist, a bHLH transcription factor, induce premature fusion of cranial sutures (craniosynostosis) in the Saethre-Chotzen syndrome (SCS). The mechanisms by which Twist haploinsufficiency may alter osteoblast differentiation are poorly understood. In this study, we investigated the role of fibroblast growth factor receptor-2 (Fgfr2) in the abnormal osteoblast differentiation in SCS. Cranial osteoblasts from an SCS patient with a Y103X mutation inducing deletion of the Twist bHLH domain showed decreased Fgfr2 mRNA levels associated with decreased expression of Runx2, bone sialoprotein (BSP) and osteocalcin (OC), markers of differentiated osteoblasts, compared with wild-type osteoblasts. Transfection with Twist or Runx2 expression vectors, but not with Runx2 mutant which impairs DNA binding, restored Fgfr2, Runx2, BSP and OC expression in Twist mutant osteoblasts. EMSA analysis of mutant osteoblast nuclear extracts showed reduced Runx2 binding to a target OSE2 site in the Fgfr2 promoter. ChIP analyses showed that both Twist and Runx2 in mutant osteoblast nuclear extracts bind to a specific region in the Fgfr2 promoter. Significantly, forced expression of Fgfr2 restored Runx2 and osteoblast marker genes, whereas a dominant-negative Fgfr2 further decreased Runx2 and downstream genes in Twist mutant osteoblasts, indicating that alteration of Fgfr2 results in downregulation of osteoblast genes in Twist mutant osteoblasts. We conclude that Twist haploinsufficiency downregulates Fgfr2 mRNA expression, which in turn reduces Runx2 and downstream osteoblast-specific genes in human calvarial osteoblasts. This provides genetic and biochemical evidence for a role of Fgfr2 in the altered osteoblast phenotype induced by Twist haploinsufficiency in the SCS.

Down-Regulation of Ubiquitin Ligase Cbl Induced by Twist Haploinsufficiency in Saethre-Chotzen Syndrome Results in Increased PI3K/Akt Signaling and Osteoblast Proliferation

“…As reported previously, 9,11 Runx2 mRNA levels were decreased in Twist mutant osteoblasts compared with wildtype cells ( Figure 7A). Moreover, basal COLIA1 mRNA levels were increased in Twist mutant osteoblasts, whereas osteocalcin levels were decreased in Twist mutant osteoblasts compared with wild-type cells ( Figure Figure 5.…”

“…11 Cells were washed with phosphate-buffered saline and lysed with Extract-All (Eurobio, Courtaboeuf, France) reagent according to the manufacturer's instructions. Three g of total cellular RNA from each sample were reverse-transcribed, and the cDNA samples were then divided and amplified by PCR using specific primers.…”

“…Primers for Cbl, Runx2, COLIA1, and GAPDH were as described. 11 Autoradiographic signals were quantified using a scanner densitometer, and the signal for each gene was related to that of GAPDH.…”

Fibroblast growth factor signaling in skeletal development and disease

“…In human craniosynostosis, increased osteoblast gene expression and cranial osteogenesis induced by Apert FGFR2(S252W) mutation are related to PLCγ and PKCα activation (Lemonnier et al 2001b). The consequence is increased expression of Runx2, which is associated with premature fusion of cranial sutures in mice and humans (Zhou et al 2000;Eswarakumar et al 2002;Tanimoto et al 2004;Baroni et al 2005;Guenou et al 2005). In both mice and humans, FGFR2 levels are decreased in response to the Apert FGFR2(S252W) mutation as a result of c-CBL-mediated FGFR2 ubiquitination and degradation (Kaabeche et al 2004;Holmes et al 2009).…”

Mechanism of Transcriptional Activation by the Proto-oncogene Twist1

“…In contrast to these antagonizing functions of Twist1, a positive correlation has in several contexts been found between the expression of Twist1 and the osteogenic factor FGFR2 (55)(56)(57). Consequently, the mammalian Twist proteins most likely function as inducers in the early phases and inhibitors in later differentiation, thereby playing a dual role in osteogenesis.…”