Effects of Kaempferol on Bone Sialoprotein Gene Transcription

Objectives: Bone sialoprotein (BSP) is a major noncollagenous protein in the mineralized connective tissues that has been implicated in the nucleation of hydroxyapatite. Regulation of the BSP gene is important in the differentiation of osteoblasts, in bone matrix mineralization and in tumor metastasis. Kaempferol is a typical flavonol-type flavonoid and is present in a variety of vegetables, and its antioxidant effect implies its possible role in the prevention of oxidative stress related chronic diseases. We have previously reported that isoflavone induced BSP gene transcription via inverted CCAAT box in the rat BSP gene promoter. Methods: To determine the molecular basis of the transcriptional regulation of BSP gene by kaempferol, we conducted Northern hybridization, real-time PCR, transient transfection analyses with chimeric constructs of the rat BSP gene promoter linked to a luciferase reporter gene and gel shift assays. Results: Using rat osteoblast-like UMR106 cells, we revealed that BSP mRNA levels increased by 5 µM kaempferol at 12 h and Runx2 and Osterix mRNA expressions at 6 h. In transient transfection analyses, kaempferol (5 µM, 12 h) increased luciferase activity of the construct, pLUC3, which encompasses nucleotides -116 to +60, as well as longer constructs. Effects of kaempferol abrogated in constructs included 2 bp mutations in the inverted CCAAT, cAMP response element (CRE) and FGF2 response element (FRE). Gel shift analyses showed that kaempferol increased binding of CRE and FRE elements. Whereas the CCAAT-protein complex did not change after stimulation by kaempferol. Conclusion: These data suggest that kaempferol stimulates BSP gene transcription by targeting the inverted CCAAT, CRE and FRE elements in the rat BSP gene promoter.