Study on Regulatory Region on Secreted Phosphoprotein 1 gene. -A Gene Encoding a Molecule Responsible for the Attachment of Metastatic Tumor & Bone Cells.-

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This project was aimed to investigate the mechanisms of the regulation of Spp-1 gene expression. For this purpose, about 1,000bp of mouse Spp-1 gene promoter was cloned and several deletion mutants were generated by using mung bean exonuclease. These mutants include three fragments of the promoter DNAs, whose length varies between 800-200bp. The fragments of the promoter were linked to CAT (chloramphenicol acetyltransferase) in plasmid vectors. The vectors were transfected into several osteoblast-like cell lines, including ROS17/2.8 (rat osteoblastic osteosarcoma) cells and mouse osteoblastic cell line MC3T3E1. The cells were then treated with several calcitropic effectors such as FGF, TGFbeta, BMP2 endothelin and other cytokines and hormones, followed by assay for the CAT activity to estimate their regulatory effects on the promoter. FGF (fibroblast growth factor) has been known to up-regulate Spp-1 mRNA level in ROS cells. When the ROS cells which were transfected with the promoter constructs were treated with FGF, CAT activity in the cell lysates was also enhanced, indicating the mediation of the FGF effects via the Spp-1 promoter region. Using the deletion constructs, we located the response region in the middle 500-200bp region of the Spp-1 promoter. With regard to the regulation by BMP2, we observed the up-regulation of Spp-1 mRNA by the treatment with human recombinant BMP2 at 100 ng/ml. The longest promoter of the Spp-1, however, did not respond to the BMP2 treatment, suggesting that response element may be either outside the 1kb region or inhibitory elements may be located in the similar region as stimulatory elements. For transcriptional control of the other calcitropic modifiers fine mapping would be also followed after the findings described in this report.