Regulation of osteocalcin gene expression by a novel Ku antigen transcription factor complex

J Biol Chem. 2002 Oct 4;277(40):37280-91. doi: 10.1074/jbc.M206482200. Epub 2002 Jul 26.

Abstract

We previously described an osteocalcin (OC) fibroblast growth factor (FGF) response element (FRE) DNA binding activity as a target of Msx2 transcriptional regulation. We now identify Ku70, Ku80, and Tbdn100, a variant of Tubedown-1, as constituents of the purified OCFRE-binding complex. Northern and Western blot analyses demonstrate expression of Ku and Tbdn100 in MC3T3E1 osteoblasts. FGF2 treatment regulates Ku, but not Tbdn100, protein accumulation. Gel supershift studies confirm sequence-specific DNA binding of Ku in the OCFRE complex; chromatin immunoprecipitation assays confirm association of Ku and Tbdn100 with the endogenous OC promoter. In the promoter region -154 to -113, the OCFRE is juxtaposed to OSE2, an osteoblast-specific element that binds Runx2 (Osf2, Cbfa1). Expression of the Ku.Tbdn100 complex up-regulates both the basal and Runx2-dependent transcription driven by this 42-bp OC promoter element, reconstituted in CV-1 cells. Synergistic transactivation occurs in the presence of activated FGF receptor 2 signaling. Msx2 suppresses Ku- and Runx2-dependent transcription; suppression is dependent upon the Msx2 homeodomain NH(2)-terminal arm and extension. Pull-down assays confirm physical interactions between Ku and these co-regulatory transcription factors, consistent with the functional interactions identified. Finally, cultured Ku70 -/- calvarial cells exhibit a profound, selective deficiency in OC expression as compared with wild-type calvarial cells, confirming the biochemical data showing a role for Ku in OC transcription. In toto, these data indicate that a novel Ku antigen complex assembles on the OC promoter, functioning in concert with Msx2 and Runx2 to regulate OC gene expression.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3T3 Cells
  • Amino Acid Sequence
  • Animals
  • Antigens, Nuclear*
  • Base Sequence
  • Cell Line
  • Cell Nucleus / metabolism
  • Cells, Cultured
  • Colforsin / pharmacology
  • DNA Helicases*
  • DNA, Complementary
  • DNA-Binding Proteins / metabolism*
  • Fibroblast Growth Factor 2 / pharmacology
  • Gene Expression Regulation*
  • Humans
  • Ku Autoantigen
  • Mice
  • Molecular Sequence Data
  • Nuclear Proteins / metabolism*
  • Osteoblasts / metabolism*
  • Osteocalcin / genetics*
  • Transcription Factors / metabolism*

Substances

  • Antigens, Nuclear
  • DNA, Complementary
  • DNA-Binding Proteins
  • Nuclear Proteins
  • Transcription Factors
  • Fibroblast Growth Factor 2
  • Osteocalcin
  • Colforsin
  • DNA Helicases
  • XRCC5 protein, human
  • Xrcc6 protein, human
  • Xrcc6 protein, mouse
  • Ku Autoantigen

Associated data

  • GENBANK/AF112670