Foxc1 Expression in Early Osteogenic Differentiation Is Regulated by BMP4-SMAD Activity.

FOXC1 is an important regulator of the initial steps in intramembranous and endochondral ossification processes. As BMP signalling is a key initiator of these processes, we sought to determine whether Foxc1 expression is regulated by such signalling factors. BMP4 treatment of C2C12 cells resulted in an induction in Foxc1 mRNA levels. Chromatin immunoprecipitation assays demonstrated that SMAD proteins interacted with the mouse Foxc1 promoter approximately 300 bp upstream of the transcription start site. This ChIP positive region was cloned into a luciferase reporter and demonstrated to be responsive to BMP4 stimulation. Reduction of Foxc1 levels in C2C12 cells though siRNA impaired BMP4 osteogenic differentiation. In contrast, BMP4 treatment repressed Foxc1 expression in 10T1/2 or D1-ORL mesenchymal cells and MC3T3 preosteoblasts. Finally, siRNA knock-down of Foxc1 in MC3T3 cells resulted in an induction of markers of osteoblast differentiation and an accelerated mineralization. These data indicate that Foxc1 expression is regulated by BMP4 and FOXC1 functions in the commitment of progenitor cells to the osteoblast fate and its expression is reduced when differentiation proceeds. J. Cell. Biochem. 117: 1707-1717, 2016.