β-cat signaling upregulates the expression of OSX in human pre-osteoblastic and bone marrow stromal cells by trans-activating the OSX gene promoter mainly through increased c-Jun binding at a putative c-Jun binding

Both β-catenin (β-cat) and osterix (OSX) are known to be essential for embryonic and postnatal osteoblast differentiation and bone growth. In the present study, we explored the crosstalk between β-cat signaling and OSX, and assessed its effect on the osteoblastogenic differentiation of human pre-osteoblastic cells (MG-63) and bone marrow stromal cells (HS-27A). In the HS-27A and MG-63 cells, the selective β-cat signaling inhibitor, CCT031374, and the stable overexpression of a constitutively active β-cat mutant respectively decreased and increased the cytoplasmic/soluble β-cat levels, and respectively decreased and increased TOPflash reporter activity, the mRNA levels of β-cat signaling target genes c-Myc and c-Jun, as well as the mRNA and protein expression levels of OSX. Mutational analyses and electrophoretic mobility shift assays revealed that the increased binding activity of c-Jun at a putative c-Jun binding site (-858/-852 relative to the translation start codon, which was designated as +1) in the human OSX gene promoter was required for teh β-cat signaling-induced expression of OSX in the HS-27A and MG-63 cells. During osteoblastogenic culture, stimulating β-cat signaling activity by the stable overexpression of the active β-cat mutant markedly increased alkaline phosphatase (ALP) activity and calcium deposition in the HS-27A and MG-63 cells, which was abolished by knocking down OSX using shRNA. On the other hand, the inhibition of β-cat signaling activity with CCT031374 decreased the ALP activity and calcium deposition, which was completely reversed by the overexpression of OSX. On the whole, the findings of our study suggest that β-cat signaling upregulates the expression of OSX in human pre-osteoblastic and bone marrow stromal cells by trans-activating the OSX gene promoter mainly through increased c-Jun binding at a putative c-Jun binding site; OSX largely mediates β-cat signaling-induced osteoblastogenic differentiation. The present study provides new insight into the molecular mechanisms underlying osteoblast differentiation.