title: Liganded thyroid hormone receptor inhibits phorbol 12-O-tetradecanoate-13-acetate-induced enhancer activity via firefly luciferase cDNA.
abstract: Thyroid hormone receptor (TR) belongs to the nuclear hormone receptor (NHR) superfamily and regulates the transcription of its target genes in a thyroid hormone (T3)-dependent manner. While the detail of transcriptional activation by T3 (positive regulation) has been clarified, the mechanism of T3-dependent repression (negative regulation) remains to be determined. In addition to naturally occurring negative regulations typically found for the thyrotropin β gene, T3-bound TR (T3/TR) is known to cause artificial negative regulation in reporter assays with cultured cells. For example, T3/TR inhibits the transcriptional activity of the reporter plasmids harboring AP-1 site derived from pUC/pBR322-related plasmid (pUC/AP-1). Artificial negative regulation has also been suggested in the reporter assay with firefly luciferase (FFL) gene. However, identification of the DNA sequence of the FFL gene using deletion analysis was not performed because negative regulation was evaluated by measuring the enzymatic activity of FFL protein. Thus, there remains the possibility that the inhibition by T3 is mediated via a DNA sequence other than FFL cDNA, for instance, pUC/AP-1 site in plasmid backbone. To investigate the function of FFL cDNA as a transcriptional regulatory sequence, we generated pBL-FFL-CAT5 by ligating FFL cDNA in the 5' upstream region to heterologous thymidine kinase promoter in pBL-CAT5, a chloramphenicol acetyl transferase (CAT)-based reporter gene, which lacks pUC/AP-1 site. In kidney-derived CV1 and choriocarcinoma-derived JEG3 cells, pBL-FFL-CAT5, but not pBL-CAT5, was strongly activated by a protein kinase C activator, phorbol 12-O-tetradecanoate-13-acetate (TPA). TPA-induced activity of pBL-FFL-CAT5 was negatively regulated by T3/TR. Mutation of nt. 626/640 in FFL cDNA attenuated the TPA-induced activation and concomitantly abolished the T3-dependent repression. Our data demonstrate that FFL cDNA sequence mediates the TPA-induced transcriptional activity, which is inhibited by T3/TR.
authors.affiliation: Second Division, Department of Internal Medicine, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Misawa
authors.forename: Shigekazu
authors.initials: S
authors.lastname: Sasaki
authors.forename: Akio
authors.initials: A
authors.lastname: Matsushita
authors.forename: Kenji
authors.initials: K
authors.lastname: Ohba
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Iwaki
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Matsunaga
authors.forename: Shingo
authors.initials: S
authors.lastname: Suzuki
authors.forename: Keiko
authors.initials: K
authors.lastname: Ishizuka
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Oki
authors.forename: Hirotoshi
authors.initials: H
authors.lastname: Nakamura
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics*; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D008024:Ligands; D049409:Luciferases, Firefly / Q000235:genetics*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011988:Receptors, Thyroid Hormone / Q000378:metabolism*; D017384:Sequence Deletion / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives* / Q000494:pharmacology; D013937:Thymidine Kinase / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D015533:Transcriptional Activation / Q000187:drug effects; D014284:Triiodothyronine / Q000494:pharmacology
chemical_list: D018076:DNA, Complementary; D008024:Ligands; D009687:Nuclear Proteins; D011988:Receptors, Thyroid Hormone; D018808:Transcription Factor AP-1; D014284:Triiodothyronine; C016658:phorbolol myristate acetate; D049409:Luciferases, Firefly; D013937:Thymidine Kinase; D013755:Tetradecanoylphorbol Acetate