Stimulus-selective inhibition of rat osteocalcin promoter induction and protein-DNA interactions by the homeodomain repressor Msx2.
Osteocalcin (OC) is a matrix calcium-binding protein expressed in osteoblasts and odontoblasts undergoing mineralization. OC expression is up-regulated in part by signals initiated by basic fibroblast growth factor (FGF2), cyclic AMP or forskolin (FSK), and calcitriol via defined elements and DNA-protein interactions in the OC promoter. We identified the OC gene as a target for transcriptional suppression by Msx2, a homeodomain transcription factor that controls ossification in the developing skull. In this study, we examine the effects of Msx2 expression on OC promoter activation (luciferase reporter) by FGF2/FSK and calcitriol in MC3T3-E1 osteoblasts. Expression of Msx2 decreases basal activity of the 1-kilobase (-1050 to +32) rat OC promoter by 80%; however, the promoter is still inducible 3-fold by calcitriol. By contrast, OC promoter induction by FGF2/FSK is completely abrogated by Msx2. Because intrinsic Msx2 DNA binding activity is not required for the Msx2 suppressor function, we assessed whether Msx2 represses OC activation by regulating DNA-protein interactions at the FGF2 response element (OCFRE) and compared these interactions with those occurring at the calcitriol response element (VDRE). Treatment of MC3T3-E1 cells with FGF2/FSK or calcitriol up-regulates specific DNA-protein interactions at the OCFRE or VDRE, respectively, as detected by gel shift assay. Preincubation of crude nuclear extracts with recombinant glutathione S-transferase (GST)-Msx2 dose-dependently inhibits OCFRE DNA binding activity, whereas GST has no effect. Msx2 itself does not bind the OCFRE. Residues 132-148 required for Msx2 core suppressor function in transfection assays are also required to inhibit OCFRE DNA binding activity. By contrast, GST-Msx2 has no effect on calcitriol-regulated DNA-protein interactions at the VDRE. Using gel shift as an assay, the OCFRE DNA-binding protein OCFREB was purified to about 50% homogeneity from MG63 osteosarcoma cells. Recombinant Msx2 inhibits purified OCFREB DNA binding activity, whereas the Msx2 variant lacking residues 132-148 is inactive. Thus, Msx2 abrogates up-regulation of the OC promoter by FGF2/FSK in part by inhibiting OCFREB binding to the OCFRE.
Newberry
E P
EP
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Boudreaux
J M
JM
Towler
D A
DA
D000818:Animals; D002117:Calcitriol / Q000494:pharmacology; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D018398:Homeodomain Proteins / Q000378:metabolism*; D051379:Mice; D015675:Osteocalcin / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C084119:MSX2 protein; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D015675:Osteocalcin; D005576:Colforsin; D004247:DNA; D002117:Calcitriol