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

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.