Molecular silencing of Twist1 enhances osteogenic differentiation of murine mesenchymal stem cells: Implication of FGFR2 signaling

Abstract

The capacity of mesenchymal stem cells (MSCs) to differentiate into functional osteoblasts is tightly controlled by transcription factors that trigger osteoblast commitment and differentiation. The role of Twist1, a basic helix-loop-helix (bHLH) transcription factor, in osteogenic differentiation of MSCs remains unclear. Here we investigated the role of Twist1 in the osteogenic differentiation program of murine C3H10T1/2 mesenchymal cells. We showed that molecular silencing of Twist1 using short hairpin RNA (shRNA) expression moderately increased C3H10T1/2 cell proliferation and had no effect on cell survival. In contrast, Twist1 silencing enhanced osteoblast gene expression and matrix mineralization in vitro. Biochemical analyses revealed that Twist1 silencing increased the expression of FGFR2 protein level, which was reduced by a mutant Runx2. Consistent with this finding, Twist1 silencing increased ERK1/2 and PI3K signaling. Moreover, molecular or pharmacological inhibition of FGFR2 or of ERK1/2 and PI3K signaling partly abolished the increased osteoblast gene expression induced by Twist1 silencing in C3H10T1/2 cells. These results reveal that Twist1 silencing upregulates osteoblast differentiation of murine mesenchymal cells in part via activation of FGFR2 expression and downstream signaling pathways, which provides novel insights into the molecular signals by which this transcription factor regulates the osteogenic differentiation program in MSCs.

Pivotal role of Twist in skeletal biology and pathology

“…In cultured murine mesenchymal stem cells, we showed that Twist1 silencing reduces adipocyte gene expression and differentiation, although the mechanisms involved appear to be indirect. We found that Twist1 silencing increases FGF Receptor 2 (FGFR2) signaling (Miraoui et al, 2010). This results in reduction in adipocyte differentiation (Miraoui et al, 2010) because FGFR2 negatively controls adipocyte differentiation in murine mesenchymal stem cells (Miraoui et al, 2009).…”

Fibroblast growth factor signaling in skeletal development and disease

“…FGFR2 also plays an important role in osteoblastogenesis. In mesenchymal cells, constitutive Fgfr2 activation results in increased osteoblast differentiation and decreased adipocyte differentiation through ERK1/2 and PKCα signaling, resulting in increased Runx2 expression (Miraoui et al 2009(Miraoui et al , 2010b. Consistently, mice conditionally lacking Fgfr2 or harboring a mutation in Fgfr2c, the mesenchymal splice variant of Fgfr2, show decreased Runx2 expression, retarded ossification, and decreased bone mineral density, suggesting that Fgfr2 is a positive regulator of osteoblast maturation (Eswarakumar et al 2002;Yu et al 2003).…”

Transcriptional Regulatory Cascades in Runx2-Dependent Bone Development

“…Conversely, Twist-1 overexpression inhibits osteogenesis without affecting Runx2 expression, 42 whereas Twist-1 silencing enhances osteogenesis of MSCs. 43 Further molecular analysis shows that Twist directly binds to the Runt DNA-binding domain of Runx2 to decrease binding of Runx2 to DNA. 38 These data show that Twist determines the onset of osteogenesis as an antagonist of Runx2.…”