JBC Ideal method for primary cell transfection

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J Biol Chem, Vol. 273, Issue 45, 30024-30032, November 6, 1998

Retinoic Acid Mediates Down-regulation of the alpha -Fetoprotein Gene through Decreased Expression of Hepatocyte Nuclear Factors

Thomas R. MageeDagger , Yan CaiDagger , Motawa E. El-Houseini§, Joseph Locker§, and Yu-Jui Yvonne WanDagger

From the Dagger  Department of Pathology, Harbor-UCLA Medical Center, Torrance, California 90509 and the § Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

alpha -Fetoprotein (AFP), a protein highly induced during fetal liver development, is down-regulated by retinoids in the human hepatoma cell line Hep3B, in contrast to up-regulation observed in other cell types. Previously, we have documented that such up-regulation involves direct effects through cis-retinoid X receptor-binding sites in the AFP enhancer. In this report, we show a distinctive effect of all-trans-retinoic acid (RA) in Hep3B cells. RA caused a marked decrease in AFP transcripts. Deletion analysis of the upstream regulatory region of the AFP gene revealed that cis-acting sites required for down-regulation resided near the promoter. Gel mobility shift assays for factors binding to key elements in the AFP promoter region demonstrated that hepatocyte nuclear factor (HNF) 1 binding was diminished in nuclear extracts from RA-treated cells. In addition, HNF4, which is not known to bind to the AFP promoter but does regulate HNF1, was also diminished. The levels of HNF1 and HNF4 mRNA were also decreased following RA treatment. AFP promoter-chloramphenicol acetyltransferase transient transfection assays demonstrated that the level of HNF1 had a direct impact on basal transcription as well as RA-mediated down-regulation of the AFP gene, and that co-transfection of HNF1 and HNF4, but not transfection of either factor alone, reversed the RA-mediated inhibition. Taken together these data point to an interaction among the RA, HNF1, and HNF4 signals, which is reflected in decreased expression of AFP.


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.

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