| Budget Amount *help |
¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2001: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
The steroidogenic enzyme 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-ene-isomerase type II (3β-HSD II) is important for the biosynthesis of steroid hormones. This enzyme is exclusively localized to the adrenocortex, ovary, and testis. The objective of the present study was to evaluate the regulation of transcription of this enzyme, especially focusing on this enzyme suppression in adrenal reticularis zone. Two studies were performed. 1) Plasmid containing 5'-flanking of the human 3β-HSD II gene was constructed to luciferase reporter vector (PGL3). The promoter activities in transiently transfected to NCI-H295R cells demonstrated that -1182 to +34bo promoter sequence had no change under cyclic AMP (10 mM) and PMA (10nM) stimulation. In this region the sequence revealed a putative SF-1 regulatory element at -64 to -56bp that was essential for PMA stimulation. The upper region of the SF-1 regulatory element may have a suppressive role on this promoter. 2) Certain nuclear proto-oncogenes have be
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en implicated in the regulation of gene expression in many cell systems. In the second studies, we sought to determine IGF-1 regulating Fos and Jun family gene for steroidogenesis in NCI-H295R cells. P450c17 expression was increased from 60 min after IGF-1 stimulation. JunB expression was maximal 15 min and decreased after 60 min. Expression of c-fos was increased after 15 min. Significant expression of P450c17, c-fos, JunB was induced by IGF-1 (1, 10, 100 nM) in a dose fashion manner. To examine kinase activity involved in IGF-1 stimulation of steroidogenesis, we added either the phbsphatidylinositol 3-kinase (PI-3K) inhibitor LY294002 (50 μM) or MEK inhibitor PD98059 (50 μM) prior to IGF-I stimulation. LY294002, but not PD98059, reduced the induction of P450c17, c-fos, and JunB mRNA by IGF-1. These results indicate that PI-3K pathway plays a role in IGF-1 mediated induction of steroidogenesis in NCI-H295R cells, and suggest that Fos and Jun family participates in the regulation of steroidogenic gene expression. Less
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