Identification of an osteocalcin gene promoter sequence that binds AP1

Osteoblasts are differentiated cells that produce bone matrix components including the bone-specific protein osteocalcin. The osteocalcin gene promoter has become a model for understanding how genes are regulated, specifically in osteoblasts. One model for cell-specific regulation suggests that osteoblast-expressed genes are regulated through common promoter sequences which bind osteoblast-specific transcriptional activators. The phenotype suppression model suggests osteoblast-specific promoters are switched off through the action of the common transcriptional activator AP1. We previously demonstrated that a short sequence element (OSCARE-2) in the osteocalcin promoter was homologous to a repressive element in the collagen type 1 (alpha 1) promoters. In this paper we use electrophoretic mobility shift (EMS) assays to examine DNA-protein interactions in the OSCARE-2 sequence. In EMS assays, OSCARE-2 binds a complex of proteins, including AP1. This supports the role of AP1 sites in contributing to the regulation of the osteocalcin promoter. Exogenous c-JUN protein bound to OSCARE-2 and increasing c-JUN incubated with nuclear extract amounts caused a progressive increase in a higher-molecular-weight complex, consistent with c-JUN involvement in protein-protein as well as DNA-protein interactions. Anti-c-FOS antibody was capable of supershifting OSCARE-2 DNA-protein complexes produced using osteoblast-like cell nuclear extracts. In addition, EMS assays of nuclear proteins from osteoblast-like cells indicated that 1,25 (OH)2D3-inducible proteins are bound to OSCARE-2. Osteocalcin promoter constructs showed that OSCARE-2 contributed to the 1,25 (OH)2D3 response, albeit in a minor way. These data support the role of AP1 protein as a regulator of osteoblast-specific gene expression during osteoblast development.