Synergistic activation of the human type II 3beta-hydroxysteroid dehydrogenase/delta5-delta4 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 orphan nuclear receptor/transcription factor known to regulate the P450 steroid hydroxylases; however, mechanisms that regulate the activity of SF-1/Ad4BP are not well defined. In addition, little is known about the mechanisms that regulate the human steroidogenic enzyme, type II 3beta-hydroxysteroid dehydrogenase (3beta-HSD II), the major gonadal and adrenal isoform. Regulation of the 3beta-HSD II promoter was examined using human adrenal cortical (H295R; steroidogenic) and cervical (HeLa; non-steroidogenic) carcinoma cells. H295R cells were transfected with a series of 5' deletions of 1251 base pairs (bp) of the 3beta-HSD II 5'-flanking region fused to a chloramphenicol acetyltransferase (CAT) reporter gene followed by treatment with or without phorbol ester (phorbol 12-myristate 13-acetate; PMA). CAT assay data indicated that the region from -101 to -52 bp of the promoter was required for PMA-induced expression. A putative SF-1/Ad4BP regulatory element, TCAAGGTAA, was identified by sequence homology at -64 to -56 bp of the promoter. Cotransfection of HeLa cells with the -101 3beta-HSD-CAT construct and an expression vector for SF-1/Ad4BP increased CAT activity 49-fold. Subsequent treatment with PMA induced an unexpected synergistic increase in transcriptional activity 540-fold over basal. Mutation of the putative response element (TCAAGGTAA to TCAATTTAA) abolished SF-1-induced CAT activity and the synergistic response to PMA. Gel mobility shift assays confirmed that SF-1/Ad4BP interacts with the putative element and transcripts for SF-1/Ad4BP were detected in H295R cells by Northern 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 transcription, and 3) the importance of the SF-1/Ad4BP regulatory element in the regulation of the 3beta-HSD II promoter.
Leers-Sucheta
S
S
Department of Obstetrics & Gynecology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2515, USA.
Morohashi
K
K
Mason
J I
JI
Melner
M H
MH
D000302:Adrenal Cortex / Q000166:cytology / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D051717:Fushi Tarazu Transcription Factors; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006367:HeLa Cells; D018398:Homeodomain Proteins; D006801:Humans; D009097:Multienzyme Complexes / Q000235:genetics*; D011375:Progesterone Reductase / Q000235:genetics*; D011401:Promoter Regions, Genetic; D018160:Receptors, Cytoplasmic and Nuclear; D013251:Steroid Isomerases / Q000235:genetics*; D054339:Steroidogenic Factor 1; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
C021425:3 beta-hydroxysteroid oxidoreductase-delta(5) 3-ketosteroid isomerase; D004268:DNA-Binding Proteins; D051717:Fushi Tarazu Transcription Factors; D018398:Homeodomain Proteins; D009097:Multienzyme Complexes; C515057:NR5A1 protein, human; D018160:Receptors, Cytoplasmic and Nuclear; D054339:Steroidogenic Factor 1; D014157:Transcription Factors; D011375:Progesterone Reductase; D015500:Chloramphenicol O-Acetyltransferase; D013251:Steroid Isomerases; D013755:Tetradecanoylphorbol Acetate