FOXC1 negatively regulates BMP-SMAD activity and Id1 expression during osteoblast differentiation.

Bone morphogenetic proteins regulate a diverse range of biological processes through their activation of SMAD1, SMAD5, or SMAD8 proteins that, in turn, regulate gene expression. These SMAD transcription factors achieve a layer of functional specificity in different cell types largely through actions with additional transcriptional regulatory molecules. In this study, we demonstrate that the forkhead box C1 (FOXC1) transcription factor can modulate bone morphogenetic protein (BMP) signaling to impair the expression of BMP4-responsive genes and prevent the efficient osteoblast differentiation. We demonstrate that repression occurs downstream of BMP signaling and impacts the ability SMAD1 or SMAD5 to activate gene expression. Repression of SMAD activity requires FOXC1 DNA-binding capacity and the transcriptional inhibitory domain of FOXC1. We report that FOXC1 inhibits BMP4 induction of Id1 expression and identify a motif in the regulatory region of mouse Id1 gene that FOXC1 binds. We determine that this inhibition by FOXC1 binding does not affect SMAD1, SMAD5, or SMAD8 binding to its target sequence in the Id1 gene. Finally, we determine that the elevated expression of FOXC1 can reduces expression osteogenic differentiation genes in mouse embryonic stems directed to the osteoblast lineage through BMP4 treatment. Together, these findings indicate that FOXC1 can negatively regulate certain aspects of BMP4 signaling required for osteoblast differentiation. We propose that FOXC1 acts to attenuate the initial BMP-activated pathways that establish osteoblast differentiation and allow for terminal osteoblast differentiation to conclude.