Members of the nuclear factor 1 transcription factor family regulate rat 3alpha-hydroxysteroid/dihydrodiol dihydrodiol dehydrogenase (3 alpha-HSD/DDAKR1C9) gene expression: A member of the aldo-keto reductase superfamily

Pat 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase (3 alpha-HSD/DD; AKR1C 9), a member of the aldo-keto reductase (AKR) superfamily, inactivates near ly all steroid hormones by converting 5 alpha- and 5b-dihydrosteroids to th eir respective 3 alpha,5 alpha- and 3 alpha,5 beta-tetrahydrosteroids and p rotects against circulating steroid hormone excess. It is highly expressed in rat liver comprising 0.5-1.0% of the soluble protein. Previously, we ide ntified a powerful distal enhancer resident at about -4.0 kb to -2.0 kb in the 5'-flanking region of the 3 alpha-HSD/DD gene. We now report the functi onal dissection of this enhancer. Transfection of nested deletions of the 5 '-end of the gene promoter linked to chloramphenicol acefyitransferase (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 fragm ent (from -4351 to -4279 bp) contained a major enhancer element. This fragm ent spanned two imperfect direct repeats GTGGAAAAACCCAGGAA and GTGGAAAAAACC CAGGAA and contained three direct repeats of GGAAAAA. This fragment also co ntained three potential half-nuclear factor 1 (NF1) sites (TGGA-NNNNNGCGA) acid a putative CCAAT-enhancer binding protein (C/EBP) binding site. The 73 -bp fragment enhanced CAT activity from the basal 3 alpha-HSD/DD gene promo ter. Recombinant C/EBP alpha and C/EBPb did not bind to this fragment. Elec trophoretic mobility shift assays showed that HepG2 and rat liver nuclear e xtracts bound to this 73-bp fragment. The 73-bp protein complex was compete d out by a NF1 oligonucleotide and was supershifted by an NF1 antibody. Whe n the 73-bp fragment was fused to an alpha 1-globin promoter-CAT construct and cotransfected with CCAAT transcription factor 1 (CTF1)/NF1 into Drosoph ila Schneider SL2 insect cells (which lack NF1-like proteins) trans-activat ion of CAT activity was observed. These results indicate that members of th e NF1 transcription factor family regulate high constitutive expression of the rat 3a-HSD/DD gene that is responsible for steroid hormone inactivation . The potential role of NF1 in regulating other AKR genes that have protect ive roles is discussed.