Activation of bone sialoprotein gene transcription by flavonoids is mediated through an inverted CCAAT box in ROS 17/2.8 cells

Emi Shimizu, Yorimasa Ogata

Research output: Contribution to journalArticle

Abstract

Bone sialoprotein (BSP) is a major noncollagenous protein of the mineralized bone extracellular matrix that has been implicated in the nucleation of hydroxyapatite. Recent studies have shown that BSP is also expressed by osteotropic cancers suggesting BSP might play a role in the pathogenesis of bone metastases. The present study investigates regulation of BSP transcription in rat osteosarcoma ROS 17/2.8 cells by flavonoids: genistein (an inhibitor of protein tyrosine kinases), daidzein (an inactive compound of genistein), flavone, and flavanone. Genistein, daidzein, and flavone (50 μM) increased steady state levels of BSP mRNA about 1.7-fold at 12 h. From transient transfection assays using various sized BSP promoter-luciferase constructs, genistein increased luciferase activities within 12 h. Constructs including the promoter sequence nucleotides (nts) -116 to -43 (pLUC3) were found to enhance transcriptional activity ∼2.6-fold in ROS 17/2.8 cells treated with genistein (50 μM). Daidzein, flavone, and flavanone (50 μM) also increased luciferase activities. In contrast, the tyrosine kinase inhibitors, herbimycin A and lavendustin A, which do not have a flavonoid structure, did not stimulate BSP transcription. Transcriptional stimulation by genistein was almost completely abrogated in a construct that included 2 bp mutations in the inverted CCAAT box. A monoclonal antibody against NF-YA, a CCAAT box-binding transcription factor, inhibited formation of DNA-NF-Y protein complex in gel shift assays formed by nuclear extracts of ROS 17/2.8 cells. These data suggest that the inverted CCAAT box is required for flavonoid-induced BSP expression and that the stimulatory action is dependent on the flavone structure and does not involve an inhibitory action on protein tyrosine kinase.

Original languageEnglish (US)
Pages (from-to)35-44
Number of pages10
JournalJournal of Cellular Biochemistry
Volume86
Issue number1
DOIs
StatePublished - 2002

Fingerprint

Integrin-Binding Sialoprotein
flavone
Transcription
Flavonoids
Genistein
Genes
Chemical activation
CCAAT-Binding Factor
Luciferases
Protein-Tyrosine Kinases
Assays
Bone
Bone Neoplasms
Bone Matrix
Osteosarcoma
Durapatite
Extracellular Matrix
Transfection
Rats
Nucleation

Keywords

  • Bone sialoprotein
  • CCAAT box
  • Flavonoid
  • Gene regulation
  • Mineralized tissue
  • Transcription

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology

Cite this

Activation of bone sialoprotein gene transcription by flavonoids is mediated through an inverted CCAAT box in ROS 17/2.8 cells. / Shimizu, Emi; Ogata, Yorimasa.

In: Journal of Cellular Biochemistry, Vol. 86, No. 1, 2002, p. 35-44.

Research output: Contribution to journalArticle

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AB - Bone sialoprotein (BSP) is a major noncollagenous protein of the mineralized bone extracellular matrix that has been implicated in the nucleation of hydroxyapatite. Recent studies have shown that BSP is also expressed by osteotropic cancers suggesting BSP might play a role in the pathogenesis of bone metastases. The present study investigates regulation of BSP transcription in rat osteosarcoma ROS 17/2.8 cells by flavonoids: genistein (an inhibitor of protein tyrosine kinases), daidzein (an inactive compound of genistein), flavone, and flavanone. Genistein, daidzein, and flavone (50 μM) increased steady state levels of BSP mRNA about 1.7-fold at 12 h. From transient transfection assays using various sized BSP promoter-luciferase constructs, genistein increased luciferase activities within 12 h. Constructs including the promoter sequence nucleotides (nts) -116 to -43 (pLUC3) were found to enhance transcriptional activity ∼2.6-fold in ROS 17/2.8 cells treated with genistein (50 μM). Daidzein, flavone, and flavanone (50 μM) also increased luciferase activities. In contrast, the tyrosine kinase inhibitors, herbimycin A and lavendustin A, which do not have a flavonoid structure, did not stimulate BSP transcription. Transcriptional stimulation by genistein was almost completely abrogated in a construct that included 2 bp mutations in the inverted CCAAT box. A monoclonal antibody against NF-YA, a CCAAT box-binding transcription factor, inhibited formation of DNA-NF-Y protein complex in gel shift assays formed by nuclear extracts of ROS 17/2.8 cells. These data suggest that the inverted CCAAT box is required for flavonoid-induced BSP expression and that the stimulatory action is dependent on the flavone structure and does not involve an inhibitory action on protein tyrosine kinase.

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