Effects of Inorganic Polyphosphates on the Expression of BSP Gene

Objective: Inorganic polyphosphates (Poly (P)) are linear polymers of many tens or hundreds of orthophosphate residues linked by high-energy phosphoanhydride bonds. Poly (P) modulated mitogenic activity of fibroblast growth factors and induced calcification of osteoblast-like cells. Bone sialoprotein (BSP) is a mineralized connective tissue specific protein. Developmental expressions of BSP have shown that BSP mRNA is expressed at high levels at the onset of bone formation. The purpose of this study is to investigate the effect of poly (P) on the expression of BSP in osteoblasts. Methods: We used two kinds of Poly (P), sodium phosphate glass type 25 (SPG25) and 65 (SPG65) at the concentration of 12.5 µM. To determine the molecular basis of the transcriptional regulation of BSP gene by Poly (P), we conducted real-time PCR, transient transfection analyses and gel mobility shift assays using ROS 17/2.8 osteoblast-like cells. Results: Results of real-time PCR showed that the treatments of SPG25 and 65 (12.5 µM) increased BSP and Runx2 mRNA levels at 3 and 6 h. In transient transfection analyses, using various sized rat BSP gene promoter ligated to a luciferase reporter gene, SPG25 and 65 (12.5 µM, 12 h) stimulated luciferase activities of the constructs pLUC3 (-116 to +60) and pLUC4 (-425 to +60). Effects of SPG25 and 65 were abrogated by 2bp mutations in the FGF2 response element (FRE) in pLUC3. 2bp mutations in the homeodomain protein-binding site (HOX) in pLUC4 reduced the effect of SPG25. When FRE and HOX were used for gel shift assays, the formations of FRE- and HOX-protein complexes increased at 6 h. Conclusion: These studies indicated that two kinds of Poly (P) increased BSP transcription in ROS17/2.8 cells. SPG25 increased BSP expression mediate through FRE and HOX sites, and SPG65 regulated BSP transcription via FRE in the proximal rat BSP gene promoter.