DEVELOPMENTAL REGULATION OF OSTEOCALCIN EXPRESSION IN MC3T3-E1 OSTEOBLASTS - MINIMAL ROLE OF THE PROXIMAL E-BOX CIS-ACTING PROMOTER ELEMENTS

Osteoblasts undergo a temporal sequence of development characterized b y transcriptional upregulation of osteoblast-specific genes. Basic hel ix-loop-helix (bHLH) transcription factors may control this developmen tal process through binding to E-box cis-acting elements in developmen tally regulated genes. To investigate the role of bHLH proteins in MC3 T3-E1 osteoblasts, which undergo a developmental sequence in vitro, we analyzed the transcriptional control of osteocalcin gene expression b y stable transfection of an osteocalcin promoter-luciferase chimeric g ene (p637OC-luc) and assessed the role of E-box cis-acting elements in osteocalcin promoter by DNA binding assays. We compared our findings in MC3T3-E1 osteoblasts with transient DNA transfections and DNA bindi ng experiments in Ros 17/2.8 osteoblasts. We found that the activity o f 637-OC luciferase promoter was tow in undifferentiated 5-day-old cul tures but increased in parallel with endogenous osteocalcin message ex pression in mature MC3T3-E1 osteoblasts, consistent with developmental stage-specific transcriptional upregulation of the osteocalcin gene. We identified two putative E-box elements in the proximal osteocalcin promoter, E-box 1 (CACATG) at -102 and E-box 2 (CAGCTG) at position -1 49. In gel mobility shift assays, factors present in nuclear extracts derived from differentiated osteoblast bound to oligonucleotide probes containing the E-box 1 and E-box 2 elements. Binding to the E-box 2 p robe was not specific for the core CACCTG element, whereas the CACATG site in E-box 1 oligonucleotide was required for specific binding of t hese nuclear factors. Stable transfection of p637OC-luc containing a m utant E1 site (p6370C-luc E1m), however, did not alter the development al upregulation of osteocalcin promoter activity in MC3T3-E1 osteoblas ts. Moreover, the E-box 1: mutation had no effect on either basal or v itamin D-stimulated activity of the osteocalcin promoter in Ros 17/2.8 osteoblasts in transient transfection experiments. These data suggest that osteoblasts contain undefined factors that bind to the E-box 1 C ACATG site in the proximal osteocalcin promoter; however, this E-box e lement does not play a significant role in the developmental stage-spe cific regulation of the osteocalcin gene in MC3T3-E1 osteoblasts. (C) 1997 Wiley-Liss, Inc.