Transcriptional mechanisms of bone morphogenetic protein-induced osteoprotegrin gene expression

Osteoprotegerin (OPG), an osteoblast-secreted decoy receptor, specifically binds to osteoclast differentiation factor and inhibits osteoclast maturati on. Members of the transforming growth factor-beta superfamily including bo ne morphogenetic proteins (SMPs) stimulate OPG mRNA expression. In this stu dy, we have characterized the transcription mechanism of BMP-induced OPG ge ne expression. Transfection of Smadl and a constitutively active BMP type I A receptor ALK3 (Q233) stimulated the OPG promoter. Deletion analysis of th e OPG promoter identified two Hoxc-8 binding sites that respond to BMP stim ulation. Glutathione S-transferase-Hoxc-8 protein binds to these two Hox si tes specifically. Consistent with the transfection results of the native pr omoter, ALK3 or Smadl linker region, which interacts with Hoxc-8, stimulate d the activation of the reporter construct with the two Hox sites. Overexpr ession of Hoxc-8 inhibited the induced promoter activity. When the two Hox binding sites were mutated, ALK3 or Smadl linker region no longer activated the transcription. Importantly, Smadl linker region induced both OPG promo ter activity and endogenous OPG protein expression in 2T3 osteoblastic cell s. The medium from cells transfected with Smadl linker region expression pl asmid effectively inhibited osteoclastogenesis. Collectively, our data indi cate that Hox sites mediate both OPG promoter construct activity and endoge nous OPG gene expression in response to BMP stimulation.