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 beta1 (TR beta1) expression plasmid 
into rat hepatoma H4IIE cells resulted in a 2.4-fold induction of promoter 
activity that was both T3 and TR beta1 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 TR alpha1 and TR beta1 was demonstrated by gel-shift 
analysis. In vitro binding studies with P450R-TRE revealed the formation of 
heterodimeric complexes when TR alpha1 was coincubated with either the retinoic 
X receptor alpha 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 TR beta1 dependent. Previous studies have 
demonstrated that T3 augments P450R mRNA levels approximately 20-30-fold and 
approximately 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.