| Budget Amount *help |
¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 1993: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1992: ¥6,100,000 (Direct Cost: ¥6,100,000)
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| Research Abstract |
We investigated the effect of TGFbeta on the expression of osteopontin gene. To do this, about 1,000bp of the promoter region of the mouse osteopontin gene was cloned and this fragment was used to construct a vector containing a reporter gene, chloramphenicol acetyl transferase (CAT). Three deletion constructs containing -777/+79, -545/+79, -253/+79, promoter fragments were also constructed. These reporter constructs were then transfected into osteoblastic osteosarcoma ROS17/2.8 cells. As osteoblastic cells, MC3T3E1, another type of cell line was also used. The latter cells were representing non-transformed class of cells with capability to induce bone formation in vitro and to form calcified nodules. For transfection, either DEAE-transfection or electroporation was found to be effective to introduce these gene constructs into the two types osteoblasts. The expression of CAT and its activity was measured to estimate the activity of the promoter and its fragments. The difference of the promoter activiy varied several fold among the constructs with varying length of the promoter. After the tansfection was completed, the cells were subsequently treated with TGF-beta1 for up tp 72 hours. The effect of the TGFbeta1 was found to be stimulatory in all the constructs examined in this study. We concluded that TGFbeta1 acts on the relatively short fragment downstream of the nucleotide position -253. In this region several consensus sites for known transcription regulators were located. Among those, E-box sequences (CANNTG) would be a candidate for the osteoblastic expression and regulation of osteopontin gene, since expression of transcription regulators with helix-loop-helix motif was found to be under the control of calcitropic regulators such as bone morphegenetic protein, which is a member of TGFbeta family, as well as calcitriol and dexamethasone. We would like to further define elements responsible for TGFbeta regulation of osteopontin gene expression.
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