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(Received for publication, November 27, 1996)
From the Departments of Obstetrics & Gynecology and Cell Biology,
Vanderbilt University School of Medicine, Nashville, Tennessee
37232-2515, the § Department of Molecular Biology, Graduate
School of Medical Science, Kyushu University, Higashi-ku Fukuoka
812, Japan, and the ¶ Department of Clinical Biochemistry,
University of Edinburgh, Edinburgh EH3 9YW, Scotland
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
3
Volume 272, Number 12,
Issue of March 21, 1997
pp. 7960-7967
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
-Hydroxysteroid
Dehydrogenase/
5-4 Isomerase Promoter by
the Transcription Factor Steroidogenic Factor-1/Adrenal 4-binding
Protein and Phorbol Ester
-hydroxysteroid dehydrogenase (3-HSD II), the major gonadal and
adrenal isoform. Regulation of the 3-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 3-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 3-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 (TCAA
TAA to
TCAA
TAA) 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 3-HSD II promoter.
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