SYNERGISTIC ACTIVATION OF THE HUMAN TYPE-II 3-BETA-HYDROXYSTEROID DEHYDROGENASE DELTA(5)-DELTA(4) ISOMERASE PROMOTER BY THE TRANSCRIPTION FACTOR STEROIDOGENIC FACTOR-1/ADRENAL 4-BINDING PROTEIN AND PHORBOL ESTER/

Steroidogenic factor-1/adrenal 4-binding protein (SF-1/Ad4BP) is an or phan nuclear receptor/transcription factor known to regulate the P450 steroid hydroxylases; however, mechanisms that regulate the activity o f SF-1/Ad4BP are not well defined, In addition, little is known about the mechanisms that regulate the human steroidogenic enzyme, type II 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD II), the major gonadal and adrenal isoform, Regulation of the 3 beta-HSD II promoter was exam ined using human adrenal cortical (H295R; steroidogenic) and cervical (HeLa; non-steroidogenic) carcinoma cells, H295R cells were transfecte d with a series of 5' deletions of 1251 base pairs (bp) of the 3 beta- HSD II 5'-flanking region fused to a chloramphenicol acetyltransferase (CAT) reporter gene followed by treatment with or without phorbol est er (phorbol 12-myristate 13-acetate; PMA). CAT assay data indicated th at the region from -101 to -52 bp of the promoter was required for PMA -induced expression, A putative SF-1/Ad4BP regulatory element, TCAAGGT AA, was identified by sequence homology at -64 to -56 bp of the promot er, Cotransfection of HeLa cells with the -101 3 beta-HSD-CAT construc t and an expression vector for SF-1/Ad4BP increased CAT activity 49-fo ld, Subsequent treatment with PMA induced an unexpected synergistic in crease in transcriptional activity 540-fold over basal, Mutation of th e putative response element (TCAAGGTAA to TCAATTTAA) abolished SF-1-in duced CAT activity and the synergistic response to PMA. Gel mobility s hift assays confirmed that SF-II Ad4BP interacts with the putative ele ment and transcripts for SF-1/Ad4BP were detected in H295R cells by No rthern analysis, These data are the first to demonstrate 1) regulation of a non-cytochrome P450 steroidogenic enzyme promoter by SF-1/Ad4BP, 2) a powerful synergistic effect of PMA on SF-1/Ad4BP-induced transcr iption, and 3) the importance of the SF-1/Ad4BP regulatory element in the regulation of the 3 beta-HSD II promoter.