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Members of the Nuclear Factor 1 Transcription Factor Family Regulate Rat 3-Hydroxysteroid/Dihydrodiol Dehydrogenase (3-HSD/DD AKR1C9) Gene Expression: A Member of the Aldo-keto Reductase Superfamily

Department of Pharmacology University of Pennsylvania School of Medicine Philadelphia, Pennsylvania 19104-6084

Rat 3-hydroxysteroid/dihydrodiol dehydrogenase (3-HSD/DD; AKR1C9), a member of the aldo-keto reductase (AKR) superfamily, inactivates nearly all steroid hormones by converting 5- and 5ß-dihydrosteroids to their respective 3,5- and 3,5ß-tetrahydrosteroids and protects against circulating steroid hormone excess. It is highly expressed in rat liver comprising 0.5–1.0% of the soluble protein. Previously, we identified a powerful distal enhancer resident at about -4.0 kb to -2.0 kb in the 5'-flanking region of the 3-HSD/DD gene. We now report the functional dissection of this enhancer. Transfection of nested deletions of the 5'-end of the gene promoter linked to chloramphenicol acetyltransferase (CAT) into HepG2 cells located the enhancer activity between (-4673 to -4179 bp). Further internal and 5'-end deletion mutants revealed that a 73-bp fragment (from -4351 to -4279 bp) contained a major enhancer element. This fragment spanned two imperfect direct repeats GTGGAAAAACCCAGGAA and GTGGA-AAAAACCCAGGAA and contained three direct repeats of GGAAAAA. This fragment also contained three potential half-nuclear factor 1 (NF1) sites (TGGA-NNNNNGCCA) and a putative CCA-AT-enhancer binding protein (C/EBP) binding site. The 73-bp fragment enhanced CAT activity from the basal 3-HSD/DD gene promoter. Recombinant C/EBP and C/EBPß did not bind to this fragment. Electrophoretic mobility shift assays showed that HepG2 and rat liver nuclear extracts bound to this 73-bp fragment. The 73-bp protein complex was competed out by a NF1 oligonucleotide and was supershifted by an NF1 antibody. When the 73-bp fragment was fused to an 1-globin promoter-CAT construct and cotransfected with CCAAT transcription factor 1 (CTF1)/NF1 into Drosophila Schneider SL2 insect cells (which lack NF1-like proteins) trans-activation of CAT activity was observed. These results indicate that members of the NF1 transcription factor family regulate high constitutive expression of the rat 3-HSD/DD gene that is responsible for steroid hormone inactivation. The potential role of NF1 in regulating other AKR genes that have protective roles is discussed.

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