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

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Abstract
Rat 3α-hydroxysteroid/dihydrodiol dehydrogenase (3α-HSD/DD; AKR1C9), a member of the aldo-keto reductase (AKR) superfamily, inactivates nearly all steroid hormones by converting 5α- and 5β-dihydrosteroids to their respective 3α,5α- and 3α,5β-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 3α-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 3α-HSD/DD gene promoter. Recombinant C/EBPα and C/EBPβ 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 α1-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 3α-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.