E2F1 effects on osteoblast differentiation and mineralization are mediated through up-regulation of frizzled-1

Abstract

Frizzled homolog 1 (FZD1) is a transmembrane receptor that mediates Wnt signaling. The transcriptional regulation of FZD1 and the role of FZD1 in osteoblast biology are not well understood. We examined the role of E2F1 in FZD1 promoter activation and osteoblast differentiation and mineralization. A putative E2F1 binding site in the FZD1 promoter region was initially identified in silico and characterized further in Saos2 cells in vitro by chromatin immunoprecipitation (ChIP), electrophoretic mobility shift (EMSA) and promoter reporter assays. Over-expression of E2F1 transactivated the FZD1 promoter and increased endogenous FZD1 mRNA and protein levels in Saos2 cells. Over-expression of E2F1 in Saos2 cells up-regulated osteoblast differentiation markers alkaline phosphatase (ALP), type I collagen α (COL1A), and osteocalcin (OCN). Furthermore, E2F1 over-expression enhanced mineralization of differentiated Saos2 cells, whereas siRNA knockdown of FZD1 diminished the effects of E2F1 on osteoblast mineralization. The effects of E2F1 on FZD1 expression and osteoblast mineralization were further confirmed in normal human FOB osteoblasts. Taken together, our experiments demonstrate a role of E2F1 in osteoblast differentiation and mineralization and suggest that FZD1 is required, in part, for E2F1 regulation of osteoblast mineralization.