title: Developmental regulation of osteocalcin expression in MC3T3-E1 osteoblasts: minimal role of the proximal E-box cis-acting promoter elements.
abstract: Osteoblasts undergo a temporal sequence of development characterized by transcriptional upregulation of osteoblast-specific genes. Basic helix-loop-helix (bHLH) transcription factors may control this developmental process through binding to E-box cis-acting elements in developmentally regulated genes. To investigate the role of bHLH proteins in MC3T3-E1 osteoblasts, which undergo a developmental sequence in vitro, we analyzed the transcriptional control of osteocalcin gene expression by stable transfection of an osteocalcin promoter-luciferase chimeric gene (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 binding experiments in Ros 17/2.8 osteoblasts. We found that the activity of 637-OC luciferase promoter was low in undifferentiated 5-day-old cultures but increased in parallel with endogenous osteocalcin message expression 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 -149. 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 probe was not specific for the core CAGCTG element, whereas the CACATG site in E-box 1 oligonucleotide was required for specific binding of these nuclear factors. Stable transfection of p637OC-luc containing a mutant E1 site (p637OC-luc E1m), however, did not alter the developmental upregulation of osteocalcin promoter activity in MC3T3-E1 osteoblasts. Moreover, the E-box 1 mutation had no effect on either basal or vitamin D-stimulated activity of the osteocalcin promoter in Ros 17/2.8 osteoblasts in transient transfection experiments. These data suggest that osteoblasts contain underfined factors that bind to the E-box 1 CACATG site in the proximal osteocalcin promoter; however, this E-box element does not play a significant role in the developmental stage-specific regulation of the osteocalcin gene in MC3T3-E1 osteoblasts.
authors.affiliation: Department of Medicine, Duke University, Durham, North Carolina 27710, USA.
authors.forename: L D
authors.initials: LD
authors.lastname: Quarles
authors.forename: S R
authors.initials: SR
authors.lastname: Siddhanti
authors.forename: S
authors.initials: S
authors.lastname: Medda
mesh_terms: D016475:3T3 Cells; D000818:Animals; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology; D002117:Calcitriol / Q000494:pharmacology; D002454:Cell Differentiation; D004268:DNA-Binding Proteins / Q000235:genetics; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D018257:Helix-Loop-Helix Motifs; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism; D010006:Osteoblasts / Q000378:metabolism*; D015675:Osteocalcin / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic / Q000187:drug effects; D016212:Transforming Growth Factor beta; D015854:Up-Regulation
chemical_list: C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D016212:Transforming Growth Factor beta; D015675:Osteocalcin; D002117:Calcitriol