ASCORBIC ACID-DEPENDENT ACTIVATION OF THE OSTEOCALCIN PROMOTER IN MC3T3-E1 PREOSTEOBLASTS - REQUIREMENT FOR COLLAGEN MATRIX SYNTHESIS AND THE PRESENCE OF AN INTACT OSE2 SEQUENCE

Osteocalcin is a hormonally regulated calcium-binding protein made alm ost exclusively by osteoblasts. In normal cells, osteocalcin expressio n requires ascorbic acid (AA), an essential cofactor for osteoblast di fferentiation both in vivo and in vitro. To determine the mechanism of this regulation, subclones of MC3T3-E1 preosteoblasts were transientl y transfected with 1.3 kb of the mouse osteocalcin gene 2 promoter dri ving expression of firefly luciferase. AA stimulated luciferase activi ty 20-fold after 4-5 days. This response was stereospecific to L-ascor bic acid and was only detected in MC3T3-E1 subclones showing strong AA induction of the endogenous osteocalcin gene. Similar results were al so obtained in MC3T3-E1 cells stably transfected with the osteocalcin promoter. A specific inhibitor of collagen synthesis, 3,4-dehydroproli ne, blocked AA-dependent induction of promoter activity, indicating th at regulation of the osteocalcin gene requires collagen matrix synthes is. Deletion analysis of the mOG2 promoter identified an essential reg ion for AA responsiveness between -147 and -116 bp. This region contai ns a single copy of the previously described osteoblast-specific eleme nt, OSE2. Deletion and mutation of OSE2 in DNA transfection assays est ablished the requirement for this element in the AA response. Furtherm ore, DNA-binding assays revealed that MC3T3-E1 cells contain OSF2, the nuclear factor binding to OSE2, and that binding of OSF2 to OSE2 is u p-regulated by AA treatment. Taken collectively, our results indicate that an intact OSE2 sequence is required for the induction of osteocal cin expression by AA.