Developmental regulation of osteocalcin expression in MC3T3-E1 osteoblasts

Minimal role of the proximal E-box cis-acting promoter elements

Leigh Quarles, Suresh R. Siddhanti, Sukumar Medda

Research output: Contribution to journalArticle

18 Citations (Scopus)

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 hound 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 Elm), 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 undefined factors that hind 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.

Original languageEnglish (US)
Pages (from-to)11-24
Number of pages14
JournalJournal of Cellular Biochemistry
Volume65
Issue number1
DOIs
StatePublished - Jan 1 1997
Externally publishedYes

Fingerprint

Osteocalcin
Osteoblasts
E-Box Elements
Genes
Transfection
Up-Regulation
Luciferases
Assays
DNA
Basic Helix-Loop-Helix Transcription Factors
Oligonucleotide Probes
Electrophoretic Mobility Shift Assay
Gene expression
Vitamin D
Oligonucleotides
Gels
Experiments
Gene Expression

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Developmental regulation of osteocalcin expression in MC3T3-E1 osteoblasts : Minimal role of the proximal E-box cis-acting promoter elements. / Quarles, Leigh; Siddhanti, Suresh R.; Medda, Sukumar.

In: Journal of Cellular Biochemistry, Vol. 65, No. 1, 01.01.1997, p. 11-24.

Research output: Contribution to journalArticle

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