| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2004: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2003: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
Arylhydrocarbon receptor(AhR) is a ligand-activated transcription factor that mediates a spectrum of toxicological and biological effects of 2,3,7,8-tetrachloro-p-dioxin(TCDD) and related compounds. AhR knockout mice exhibited a lipid accumulation in the liver and the curvature of the spinal column. Therefore, we investigated the effect of TCDD treatment on the differentiation of human mesenchymal stem cells to adipocytes and osteocytes. The differentiation to adipocytes in human mesenchymal stem cells was depressed by TCDD treatment. On the other hand, the differentiation to osteocytes in the cells was accumulated by TCDD. These results suggest that the loss of adipose tissue mass (wasting syndrome), osteodysplasia and osteochondrosis are induced by the disturbed regulation of the differentiation in the human mesenchymal stem cells to multi-functional cells.
Furthermore, we examined the expression level of AhR in 3T3-L1 cells which have highly sensitivity to TCDD. AhR protein level was
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depressed in 3T3-L1 cells during adipogenesis. The treatment of proteosome inhibitors to preadipocytes and adipocytes in 3T3-L1 cells was no affected to the level of AhR protein. In the expression of AhR mRNA, the level was depleted in 3T3-L1 cells during adipogenesis as similarly the AhR protein level. Next, to understand the mechanism by which the AhR is depleted during adipogenesis, we analyzed the AhR promoter activity during adipose differentiation in 3T3-L1 cells. To identify the sequence of the AhR promoter region responsible for differentiation-dependent suppression of AhR transcription, a series of deletion constructs linked to the CAT reporter were transfected into 3T3-L1 cells. A comparison of CAT activity between preadipocytes and adipocytes revealed that the sequences of -399/-339 and -296/-217 are core contributor differentiation-dependent downregulation of AhR promoter activity. Gel shift analysis using the nuclear fraction showed the presence of the factor bound to the sequences of -399/-339 and -296/-217. The binding activity was higher in preadipocytes than in adipocytes. Consequently, the downregulation of the trans-acting factor may result in the suppression of AhR gene transcription during adipose differentiation. Less
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