Stimulation of osteoprotegerin (OPG) gene expression by transforming growth factor-beta (TGF-beta). Mapping of the OPG promoter region that mediates TGF-beta 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 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-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.
Thirunavukkarasu
K
K
Gene Regulation, Bone and Inflammation Research, Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, Indiana 46285, USA.
Miles
R R
RR
Halladay
D L
DL
Yang
X
X
Galvin
R J
RJ
Chandrasekhar
S
S
Martin
T J
TJ
Onyia
J E
JE
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018920:Coculture Techniques; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D051379:Mice; D009154:Mutation; D010010:Osteoclasts / Q000166:cytology; D053244:Osteoprotegerin; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis* / Q000235:genetics; D018124:Receptors, Tumor Necrosis Factor; D015398:Signal Transduction; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism
D006023:Glycoproteins; D053244:Osteoprotegerin; D020033:Protein Isoforms; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D018124:Receptors, Tumor Necrosis Factor; C506030:TNFRSF11B protein, human; C506031:Tnfrsf11b protein, mouse; D016212:Transforming Growth Factor beta; D001616:beta-Galactosidase