WHITELAW Murry The University of Adelaide, 講師
GUSTAFSSON J カロリンスカ研究所, 教授
POELLINGER Lorenz Karolinska Institute, 教授
EMA Masatsugu Tohoku University, 特別研究員
KIKUCHI Yasuo Tohoku University, 理学研究科, 助手 (10004467)
SOGAWA Kazuhiro Tohoku University, 理学研究科, 助教授 (80175421)
GUSTAFSSON Jan-Ake Karolinska Institute
|Budget Amount *help
¥3,000,000 (Direct Cost : ¥3,000,000)
Fiscal Year 1996 : ¥1,600,000 (Direct Cost : ¥1,600,000)
Fiscal Year 1995 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Ah receptor (or dioxin receptor), a bHLH/PAS transcription factor, is present in the cytoplasm in association with HSP90. Upon binding with a ligand such as 2,3,7,8 dibenzodioxin and 3-methylcholanthrene, Ah receptor translocates to the nuclei, During nuclear translocation, Ah receptor dissociates from HSP90 and forms a complex with Arnt, another bHLH/PAS protein. This year, we revealed that CBP/p300 mediates the transactivation activity from Ah receptor/Arnt heterodimer to the basic transcriptional machnery, and that Ah receptor is actually involved in the malformtion of palate and kidney by the toxic effect of TCDD.We isolated novel cDNA clones for mSim1, mSim2, HLF,and Arnt2.
Since E1A suppressed inducible expression of CYP1A1 gene by TCDD,it was expected that CBP/p300 is involved in the induction of CYP1A1 gene expression as a coactivator. Coimmunoprecipitation and GST-Arnt pull down assays demonstrated that the transactivation domain of Arnt interacted with the CREB binding domain
of CBP/p300. The interaction between CBP/p300 and Arnt was confirmed by the yeast two hybrid method. Transfection of CBP/p300 expression plasmid further enhanced the inducible expression of CYP1A1 gene by TCDD.
In addition to mSim1 and mSim2 cDNAs, Arnt2 and HLF cDNAs were isolated by the RT-PCR and two hybrid methods. Arnt 2 shows close sequence similarity with, but a different expression pattern from Arnt. Arnt 2 mRNA is expressed specifically in the neural tube during embryogenesis. HLF structurally resembles HIF-1alpha especially in the bHLH/PAS domain. Its transcription activity was activated in a hypoxic condition, similarly to HIF-1alpha. However, the pattern of its mRNA expression was quite distinct from that of HIF-1alpha. The expression of HLF mRNA was much higher in the vascular system and lung than that of HIF-1alpha mRNA in embryogenesis. During parturition, the expression of HLF mRNA was markedly enhanced in parallel with that of VEGF mRNA,suggesting that HLF regulates the expression of VEGF.In situ hybridization analysis using VEGF and HLF cDNAs as probes supported this suggestion.
We produced mice lacking the Ah receptor gene by the homologous recombination technique. By using the Ah receptor-null mice, it was proved that Ah receptor mediated the teratogenic effects of TCDD on the formation of palate shelf and kidney. Less