Stimulation of osteoprotegerin (OPG) gene expression by transforming growth factor-β (TGF-β) -: Mapping of the OPG promoter region that mediates TGF-β effects

Transforming growth factor-beta (TGF-beta) regulates osteoclastogenesis and osteoclast survival, in part through the induction of osteoprotegerin (OPG), a protein known to inhibit osteoclast formation and function. To explore the molecular basis of TGF-beta regulation of OPG expression, we evaluated the effects of TGF-beta on osteoclast formation, OPG protein secretion, mRNA expression, and gene transcription. The marked inhibitory effect of TGF-beta on osteoclast differentiation was confirmed in a co-culture model utilizing murine stromal/osteoblastic BALC cells and bone marrow hematopoietic precursors. This inhibition in osteoclast differentiation was preceded by a decrease in RANKL mRNA expression (5-fold) and a reciprocal increase in OPG mRNA (6.1-fold) and protein (7.1-fold) expression in BALC cells. At the promoter/transcriptional level, TGF-beta treatment resulted in a 3-10-fold increase in reporter gene activity directed by a 5.9-kilobase fragment of the human OPG promoter in transfection assays performed in UMR106 cells. The effect of TGF-beta was mimicked by TGF-beta2 and -beta3 but not by BMP-4, suggesting a TGF-beta signal-specific effect. Deletion analysis revealed that a 183-base pair region (-372 to -190) in the promoter was required for TGF-beta responsiveness, and this region was sufficient to confer TGF-beta inducibility to a heterologous (osteocalcin) minimal promoter. Substitution mutations that disrupted the Cbfal- and/or Smad-binding elements present in the 183-base pair region resulted in a decrease in baseline expression and in the responsiveness to TGF-beta and Cbfa1. Collectively, these studies indicate the involvement and possible interaction of Cbfa1 and Smad proteins in mediating the effects of TGF-beta on OPG transcription.