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 oste oblasts and odontoblasts undergoing mineralization. OC expression is u p-regulated in part by signals initiated by basic fibroblast growth fa ctor (FGF2), cyclic AMP or forskolin (FSK), and calcitriol via defined elements and DNA-protein interactions in the OC promoter, We identifi ed the OC gene as a target for transcriptional suppression by Msx2, a homeodomain transcription factor that controls ossification in the dev eloping skull, in this study, we examine the effects of Msx2 expressio n on OC promoter activation (luciferase reporter) by FGF2/FSK and calc itriol in MC3T3-E1 osteoblasts, Expression of Msx2 decreases basal act ivity of the 1-kilobase (-1050 to +32) rat OC promoter by 80%; however , the promoter is still inducible 3-fold by calcitriol, By contrast, O C promoter induction by FGF2/FSK is completely abrogated by Msx2, Beca use intrinsic Msx2 DNA binding activity is not required for the Msx2 s uppressor function, we assessed whether Msx2 represses OC activation b y regulating DNA-protein interactions at the FGF2 response element (OC FRE) and compared these interactions with those occurring at the calci triol response element (VDRE), Treatment of MC3T3-E1 cells with FGF2/F SK or calcitriol up-regulates specific DNA-protein interactions at the OCFRE or VDRE, respectively, as detected by gel shift assay, Preincub ation of crude nuclear extracts with recombinant glutathione S-transfe rase (GST)-Msx2 dose-dependently inhibits OCFRE DNA binding activity, whereas GST has no effect, Msx2 itself does not bind the OCFRE. Residu es 132-148 required for Msx2 core suppressor function in transfection assays are also required to inhibit OCFRE DNA binding activity, By con trast, GST-Msx2 has no effect on calcitriol-regulated DNA-protein inte ractions at the VDRE, Using gel shift as an assay, the OCFRE DNA-bindi ng protein OCFREB was purified to about 50% homogeneity from MG63 oste osarcoma cells, Recombinant Msx2 inhibits purified OCFREB DNA binding activity, whereas the Msx2 variant lacking residues 132-148 is inactiv e, Thus, Msx2 abrogates up-regulation of the OC promoter by FGF2/FSK i n part by inhibiting OCFREB binding to the OCFRE.