Abstract
The current study demonstrates that T3-activated transcription of the NADPH:cytochrome P450 oxidoreductase (P450R) gene is dependent on the thyroid hormonal status of the animal, with both transcriptional and post-transcriptional pathways being important in regulating the cellular P450R mRNA level. The region required for transcriptional activation of the P450R gene by T3 has been identified. Nuclear run-on experiments demonstrated that the effects of T3 on P450R transcription are dependent on thyroid status, with a transcriptional enhancement obtained in T3-treated hypothyroid rat liver (1.8-fold increase) but not in T3-treated euthyroid animals. Transient cotransfection of P450R promoter/chloramphenicol acetyl transferase (CAT) constructs and the thyroid hormone receptor β1 (TRβ1) expression plasmid into rat hepatoma H4IIE cells resulted in a 2.4-fold induction of promoter activity that was both T3 and TRβ1 dependent. Analysis of promoter deletion constructs identified a P450R-thyroid response region (P450R-TRE; bases, −564 to −536) containing three imperfect direct repeats of the thyroid response motif, AGGTCA. Mutational analysis further established that T3 induction was dependent only on the upstream direct repeat, having the sequence AGGTGAgctgAGGCCA. Footprint analysis showed that all three motifs were protected by proteins present in rat liver nuclear extracts, and a direct interaction between P450R-TRE and T3 receptors TRα1 and TRβ1 was demonstrated by gel-shift analysis. In vitrobinding studies with P450R-TRE revealed the formation of heterodimeric complexes when TRα1 was coincubated with either the retinoic X receptor α or nuclear extract from rat liver, COS, or H4IIE cells. In addition, placement of the P450R-TRE upstream of the T3-nonresponsive heterologous thymidine kinase promoter resulted in a 2.7-fold transcriptional enhancement that was both T3 and TRβ1 dependent. Previous studies have demonstrated that T3 augments P450R mRNA levels ∼20–30-fold and ∼12-fold, respectively, in hypothyroid and euthyroid rats. Hence, for the hypothyroid state, transcriptional and post-transcriptional events contribute to the T3-induced mRNA increases; however, the marked increase in message level in T3-treated euthyroid animals depends primarily on post-transcriptional pathways.
Footnotes
- Received November 25, 1996.
- Accepted April 7, 1997.
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Send reprint requests to: Dr. Charles B. Kasper, University of Wisconsin, McArdle Laboratory, 1400 University Avenue, Madison, WI 53706. or Dr. David J. Waxman, Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215. E-mail:djw{at}bio.bu.edu
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This research was supported by Grants CA22484 and CA0930 (C. B. K.) from the National Institutes of Health and ACS Grant CN-14 (D. J. W.).
- The American Society for Pharmacology and Experimental Therapeutics
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Thyroid Regulation of NADPH:Cytochrome P450 Oxidoreductase: Identification of a Thyroid-Responsive Element in the 5′-Flank of the Oxidoreductase Gene
