Stimulation of Osteoprotegerin (OPG) Gene Expression by Transforming Growth Factor-β (TGF-β) MAPPING OF THE OPG PROMOTER REGION THAT MEDIATES TGF-β EFFECTS

Abstract Transforming growth factor-β (TGF-β) 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-β regulation of OPG expression, we evaluated the effects of TGF-β on osteoclast formation, OPG protein secretion, mRNA expression, and gene transcription. The marked inhibitory effect of TGF-β 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-β 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-β was mimicked by TGF-β2 and -β3 but not by BMP-4, suggesting a TGF-β signal-specific effect. Deletion analysis revealed that a 183-base pair region (−372 to −190) in the promoter was required for TGF-β responsiveness, and this region was sufficient to confer TGF-β inducibility to a heterologous (osteocalcin) minimal promoter. Substitution mutations that disrupted the Cbfa1- and/or Smad-binding elements present in the 183-base pair region resulted in a decrease in base-line expression and in the responsiveness to TGF-β and Cbfa1. Collectively, these studies indicate the involvement and possible interaction of Cbfa1 and Smad proteins in mediating the effects of TGF-β on OPG transcription.