IDENTIFICATION OF MULTIPLE GLUCOCORTICOID RECEPTOR-BINDING SITES IN THE RAT OSTEOCALCIN GENE PROMOTER

The biosynthesis Of Osteocalcin (OC), a bone-specific, noncollagenous protein, is stringently regulated during differentation of the osteobl ast phenotype. Glucocorticoids, and also 1,25(OH)2D3, mediate the deve lopmental regulation of OC gene transcription. In this study, we estab lished that the-1097 to +23 promoter (pOCZCat) of the rat OC gene conf ers glucocorticoid responsiveness to both basal and vitamin D-induced OC expression. The presence of multiple glucocorticoid receptor (GR) b inding sites in the proximal rat OC gene promoter was determined by th e combined use of DNase I footprinting, dimethyl sulfate fingerprintin g, and gel mobility shift analysis with glucocorticoid receptor protei n. One glucocorticoid receptor binding element (GRE) resides immediate ly downstream of the TATA box (-16 to -1). In vivo activity was establ ished by cotransfection of ROS 17/2.8 osteosarcoma cells with an OC-CA T construct in the presence of cloned GRE sequences (wild type or muta nt) as competitors. A putative second, less protected GR binding site is located further upstream in the OC gene basal promoter within the r egion overlapping the TATA box. This is in direct contrast to the orga nization of GREs in the human OC proximal promoter wherein GR binding at the upstream GRE overlapping the TATA is stronger than at the downs tream GRE. In addition, we detected sequence-specific binding of GR pr otein to another basal promoter element, the OC box (-99 to -76), whic h contains a central CCAAT motif. The presence of multiple GR binding sites in the rat OC gene proximal promoter indicates that regulation o f basal and vitamin D-enhanced transcription by glucocorticoids may in volve the integrated activities of multiple, independent GREs.