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KMID : 0617320000090010192
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Journal of Pharmacetical Sceiences Ewha Womans University
2000 Volume.9 No. 1 p.192 ~ p.199
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Inhibition of 2,3,7,8,-Tetrachlorodibenzo-p-dioxin(TCDD)-Stimulated Cyplal Promoter Activity by Hypoxic Agents
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Kim Ji-E.
SHEEN Yhun-Y.
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Abstract
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Since hypoxia-inducible factor-la (HIF-1α) and the arylhydrocarbon receptor (AhR) shared the AhR nuclear translocator (Amt) for hypoxia- and AhR-mediated signaling, respectively, it was possible to establish the hypothesis that hypoxia could regulate cytochrome P450 lal (Cyplal) expression. In order to test this hypothesis, we undertook to examine the effect of hypoxia on Cyplal transcription in Hepa-I cells. Mouse Cyplal 5´-flanking DNA, 1.6 kb was cloned into pGL3 expression vector in order to construct pmCyplal Luc. Hepa-I cells were transfected with pmCyplal-Luc and treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the presence or absence of various hypoxic agents such as 1-100 μM cobalt chloride. 1-100 μM picolinic acid, and 1-100 μM desferrioxamine. Luciferase activity of the reporter gene was measured from pmCyplal-Luc-transfected Hepa-I cell lysate which contains 2 ㎍ total protein using luciferin as a substrate. Hypoxic agents such as cobalt chloride, picolinic acid, and desferrioxamine showed inhibition of luciferase activity that was induced by 1-nM TCDD treatment in a dose-and time-dependent manner. Concomitant treatment of 150 μM ferrous sulfate with 1-100 μM desferrioxamine or 1-100 μM picolinic acid recovered luciferase activity from that inhibited by hypoxic agents or induced by TCDD. These data demonstrated that iron-chelating and hypoxic agents inhibited dioxin-induced Cyplal transcription in Hepa-I cells. Thus, we might suggest that hypoxia inhibits TCDD-induced Cyplal expression due to the competition between HIF-1α and the AhR for the Amt in Hepa-I cells.
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