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.

Essential Role of TEA Domain Transcription Factors in the Negative Regulation of the MYH 7 Gene by Thyroid Hormone and Its Receptors

“…It may be reasonable that T3-dependent repression is detectable only when T3/TR interferes with the activity of DNA-binding transcription factors, which maintain overall transcription levels [16], [18]. Based on this hypothesis, we explored the molecular mechanism by which T3/TR regulates TEAD-dependent activity of the MYH7 promoter.…”

Essential Role of GATA2 in the Negative Regulation of Type 2 Deiodinase Gene by Liganded Thyroid Hormone Receptor β2 in Thyrotroph

“…The firefly luciferase-based reporter gene may be artificially suppressed by T3/TR [ 1 , 33 ]. This possibility was recently reported by Zhang et al [ 34 ].…”

“…−128/+37) in TSHβ-CAT was replaced by this PCR product by digesting with restriction enzymes (MluI and XhoI) to generate hD2-CAT. In this construct, the pUC-derived AP-1-like sequence was deleted because it may also mediate artifactual T3-dependent inhibition [ 1 , 33 ]. hD2-CAT was amplified by PCR with the fixed reverse primer hDIO2-DSX and the various forward primers hDIO2-U1ME (5′-ggggacgcgtctagaattcttttgcattttcttaaataagataat-3′), hDIO2-U2ME (5′-ggggacgcgtctagaattcataatgataatcagaagagagagtgttggccat-3′), hDIO-U2ME (5′-ggggacgcgtctagaattctttcctgaaggctgtcaagggt-3′), hDIO-D4UME (5′-ggggacgcgtctagaattcaaagccctctttctcaatgacg-3′), and hDIO-D5UME (5′-ggggacgcgtctagaattccacttctctattgcagcaattagc-3′) to generate PCR products for Δ1 to Δ5, respectively.…”

Liganded T3 receptor β2 inhibits the positive feedback autoregulation of the gene for GATA2, a transcription factor critical for thyrotropin production

“…Expression plasmids for rat TRβ2 (pCMX-rTRβ2) and mouse GATA2 (pcDNA3-mGATA2) have been described previously [8,17,18]. Because the firefly luciferase-based reporter gene may be artificially suppressed by liganded TR [ [13,46] and references therein], we employed a CAT-based reporter gene. Three luciferase-based reporter plasmids (generous gifts from Drs.…”

“…The human D2 promoter in hD2-CAT [47] was replaced by these PCR products after digestion with restriction enzymes (EcoRI and Bgl-II), generating 1S-CAT, (-3.9)1S-CAT and (+9.5)1S-CAT, respectively ( Fig 1B). In these constructs, the pUC-derived AP-1-like sequence was deleted because it might also mediate artifactual T3-dependent inhibition [ [13,46] and references therein]. All subcloning sites were confirmed by sequencing.…”