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MOLECULAR PHARMACOLOGY 52:46-53 (1997).

Thyroid Regulation of NADPH:Cytochrome P450 Oxidoreductase: Identification of a Thyroid-Responsive Element in the 5-Flank of the Oxidoreductase Gene

Kathleen A. O'Leary, Huan-Chen Li, Prabha A. Ram, Patricia McQuiddy, David J. Waxman, and Charles B. Kasper

McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin 53706 (K.A.O., P.M., C.B.K.), and Division of Cell and Molecular Biology, Department of Biology, Boston University, Boston, Massachusetts 02215 (H.-C.L., P.A.R., D.J.W.)

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 1 (TR1) expression plasmid into rat hepatoma H4IIE cells resulted in a 2.4-fold induction of promoter activity that was both T₃ and TR1 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 TR1 and TR1 was demonstrated by gel-shift analysis. In vitro binding studies with P450R-TRE revealed the formation of heterodimeric complexes when TR1 was coincubated with either the retinoic X receptor  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 TR1 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.