Synergistic Activation of the Human Type II 3β-Hydroxysteroid Dehydrogenase/Δ5-Δ4 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 3β-hydroxysteroid dehydrogenase (3β-HSD II), the major gonadal and adrenal isoform. Regulation of the 3β-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 3β-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 3β-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 (TCAATAA to TCAATAA) 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 3β-HSD II promoter.

The steroidogenic enzyme 3␤-hydroxysteroid dehydrogenase/ ⌬ 5 -⌬ 4 -ene-isomerase (3␤-HSD) 1 is essential for the biosynthesis of all classes of steroid hormones and catalyzes the dehydrogenation and isomerization of ⌬ 5 -3␤-hydroxysteroids including pregnenolone, 17␣-hydroxypregnenolone, dehydroepiandrosterone, and 5-androstene-3␤,17␤-diol to the ⌬ 4 -3-ketosteroids progesterone, 17␣-hydroxyprogesterone, androstenedione, and testosterone, respectively. Subsequent tissuespecific metabolism of ⌬ 4 -3-ketosteroids by various cytochrome P450 enzymes results in the production of glucocorticoids, mineralocorticoids, estrogens and androgens. This crucial enzyme is present in classical steroidogenic tissues such as the adrenal cortex, ovary, testis, and placenta, and was more recently localized to peripheral tissues such as prostate, mammary gland, and skin (1). In the human, 3␤-HSD exists as two isoforms (type I and II) derived from the tissue-specific expression of two highly related but distinct genes (2,3). Human type I 3␤-HSD is predominantly expressed in placenta and skin and is the major form found in breast tissue (2). In contrast, type II 3␤-HSD expression is almost exclusively localized to the adrenal, ovary, and testis (2).
The essentiality of this enzyme in adrenal and gonadal steroidogenesis is underscored by the severe physiological consequences that arise in cases of 3␤-HSD deficiency. Congenital adrenal hyperplasia, which can be fatal if not detected and treated early, occurs in response to deficiencies in any one of the steroidogenic enzymes involved in the biosynthesis of cortisol, including 3␤-HSD (4). However, because 3␤-HSD is also involved in gonadal steroidogenesis, insufficient levels of 3␤-HSD may impair sexual differentiation, resulting in pseudohermaphroditism with incomplete masculinization of the external genitalia in males and mild virilization in females (5,6). Furthermore, ovarian synthesis of progesterone is required for the establishment and maintenance of early pregnancy (7) and a reduction in the duration or concentration of systemic progesterone, luteal phase insufficiency, is associated with impaired fertility and repeated first trimester abortion (8,9).
Numerous studies have demonstrated the role of the tran-scription factor steroidogenic factor-1 (SF-1; also called adrenal 4-binding protein; Ad4BP) in the cAMP-mediated transactivation of cytochrome P450 steroid hydroxylase genes (10 -15) in adrenal and gonadal tissues. This transcription factor is a member of the steroid hormone receptor superfamily (11,16) and is classified as an orphan nuclear receptor because an endogenous ligand has not been identified. SF-1/Ad4BP and a closely related isoform, embryonal long terminal repeat-binding protein (ELP), are transcribed from the same gene (17,18) and are homologs of fushi tarazu factor 1 (Ftz-F1), an orphan nuclear receptor that controls the fushi tarazu homeobox gene in Drosophila (19). In addition to its regulatory actions on steroid hydroxylase gene expression, targeted disruption of the Ftz-F1 gene in mice proved this transcription factor to be essential for sexual differentiation and development of the adrenal gland and gonads (20) and critical for normal development of the ventromedial hypothalamus and pituitary gonadotrophs (21). Interestingly, this nuclear receptor mediates transcriptional control over a number of genes that are involved in various aspects of reproductive function including the ␣-subunit of pituitary glycoprotein hormones (22), Mü llerian inhibiting substance (23), and oxytocin (24) genes. Additionally, pituitaries of Ftz-F1-disrupted mice lack transcripts for gonadotropin-releasing hormone receptor, as well as ␤-subunits of luteinizing hormone and follicle-stimulating hormone (25), all of which are requisite for reproductive competence.
Although it is well established that 3␤-HSD is vital for the synthesis of essential adrenal glucocorticoid and mineralocorticoid hormones, as well as gonadal production of progesterone, estrogens, and androgens (26), research aimed at elucidating the factors that regulate expression of the type II 3␤-HSD gene has lagged behind that of the cytochrome P-450 steroid hydroxylase enzymes. Human adrenocortical carcinoma (H295R) cells have provided a good physiological model in which to study 3␤-HSD-II gene regulation, in that they express the type II 3␤-HSD isozyme and treatment of the cells with either angiotensin II or phorbol ester results in enhanced expression of 3␤-HSD mRNA and synthesis of aldosterone, presumably through activation of protein kinase C (PKC; Refs. 27 and 28). The objective of the present study was to identify the specific regions of the type II 3␤-HSD promoter that confer basal and phorbol ester-induced regulation of transcription in adrenal cortical cells as a first step toward elucidating the response elements and transcription factors involved. Plasmids and Reporter Plasmid Construction-Plasmid (BlueScript KS II ϩ vector; Stratagene, San Diego, CA) containing 1251 bp of 5Јflanking and 820 bp of downstream sequence (Ϫ1251 to ϩ820 bp; relative to the transcriptional start site) of the h3␤HSD-II gene (3) subcloned into the HindIII site was a kind gift of Dr. Van Luu-The, Laval University, Quebec, Canada. Initially, sequence from Ϫ1251 to ϩ45 bp was amplified by the polymerase chain reaction (PCR) using Taq polymerase (Promega, Madison, WI), a 5Ј oligonucleotide primer identical to the published T7 promoter sequence in the BlueScript vector and a 3Ј oligonucleotide primer that spanned from ϩ27 to ϩ45 bp of untranslated exon I and was designed to contain a penultimate HindIII site. Following agarose gel purification, the Ϫ1251 to ϩ45 bp fragment was subcloned into the PCR II vector (InVitrogen, San Diego, CA), subjected to HindIII digestion, and ultimately fused to the chloramphenicol acetyltransferase (CAT) gene in the pCAT-Basic vector (Ϫ1251 CAT; Promega).

Cell
Progressive 5Ј deletion mutants of the Ϫ1251 to ϩ45 bp fragment of the h3␤HSD-II gene were generated using PCR. Five 5Ј oligonucleotide primers were used in separate reactions with the previously described 3Ј oligonucleotide primer to generate five constructs with sequence progressively deleted from the 5Ј end of the promoter. The five primer sequences were identical to Ϫ1051 to Ϫ1028 (Ϫ1051 CAT), Ϫ701 to Ϫ678 (Ϫ701 CAT), Ϫ301 to Ϫ278 (Ϫ301 CAT), Ϫ101 to Ϫ78 (Ϫ101 CAT), and Ϫ52 to Ϫ32 (Ϫ52 CAT) bp of the promoter. All oligonucleotides contained a penultimate HindIII restriction endonuclease site to facilitate subcloning. Following PCR, expected sizes of the promoter fragments were confirmed by agarose gel electrophoresis, extracted from the gel, and subcloned into the PCR II vector. Clones containing the five promoter fragments were digested with HindIII and the resulting fragments subjected to agarose gel purification prior to ligation into the pCAT-basic vector. All constructs were confirmed by sequencing through the ligation sites using the dideoxy chain-termination method (29) and Sequenase version 2.0 DNA sequencing kit (United States Biochemical Corp.).
The Ϫ101 mutant CAT promoter construct (Ϫ101M CAT) was generated by synthesizing the top and complementary DNA strands (Ϫ101 to ϩ1 bp) in three sections that, when annealed, yielded 3 doublestranded oligonucleotides containing 6-bp overhangs to facilitate ligation. The putative SF-1 response element was mutated by substituting two thymidine residues for two guanine residues (underlined) in the sequence so that TCAAGGTAA became TCAATTTAA. Both 5Ј and 3Ј ends of the construct were designed to contain HindIII restriction sites to facilitate subcloning into the pCAT-basic vector. The entire Ϫ101 mutant promoter CAT construct was sequenced in both directions to confirm mutation of the putative SF-1/Ad4BP response element.
Transient Transfections-H295R and HeLa cells were transiently transfected using a modification of the calcium phosphate co-precipitation method (31). Briefly, adherent H295R and HeLa cells were cultured to 55-65% confluency in 100-mm tissue culture dishes (Corning Scientific Products, Corning, NY) in 10 ml of the appropriate medium. Calcium phosphate-DNA co-precipitates were formed by dropwise addition of equal volumes (0.5 ml) of solution A (0.24 M CaCl 2 containing 10 g of promoter-CAT construct, 10 g of control pGEM-3Z plasmid (Promega), plus 2 g of pSV2-luc plasmid DNA for H295R cells and 10 g of promoter-CAT construct, 10 g of RSV-SF-1/Ad4BP, and 2 g of pSV2-luc plasmid DNA for HeLa cells) to Solution B (2 ϫ Hepesbuffered saline; 50 mM Hepes, 1.4 mM Na 2 HPO 4 , 0.28 M NaCl (pH 7.1)). Calcium phosphate:DNA precipitates were incubated at 23°C for at least 20 min and added to single 100-mm dishes of cells containing 9 ml of fresh medium. H295R and HeLa cells were incubated with precipitate for 4 h at 37°C (5% CO 2 and 95% air), shocked for 3 min or 30 s, respectively, with 15% (v/v) glycerol in Dulbecco's phosphate-buffered saline (D-PBS; 0.137 M NaCl, 0.137 M NaCl, 0.5 mM MgCl 2 , 6.45 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 ), washed with D-PBS, and incubated at 37°C for 24 or 36 h, respectively. During the final 24 h of incubation, cells were cultured in the presence or absence of PMA or carrier as described previously. Cells were harvested using trypsin/EDTA (Life Technologies, Inc.), pelleted, resuspended in 0.25 M Tris-HCl (pH 7.4), and stored at Ϫ70°C until assayed for CAT activity. Transfections were performed in triplicate with mock (no plasmid) serving as negative controls. Experiments were repeated identically at least twice and at least three times with modifications.
CAT and Luciferase Assays-Frozen cell pellets were thawed on ice and lysed by sonication. Soluble extract was separated from cell debris by centrifugation, divided into aliquots for CAT and luciferase assays, and stored at Ϫ70°C prior to use. Prior to CAT assay, extracts were heated to 60°C for 5 min to denature any endogenous acetylase/ deacetylase enzymes. Fluorescent CAT assays were performed as described (32) with some modification using the FLASH CAT assay kit (Stratagene). Acetyl coenzyme A (CoA) was synthesized by reaction of CoA (Pharmacia Biotech Inc.) with acetic anhydride (Sigma) as described elsewhere (33) and stored at Ϫ70°C until use. H295R cell extracts (45 l) and HeLa cell extracts (1 l of a 1:10 dilution) were incubated in 0.25 M Tris-HCl (pH 7.4) in a total reaction volume of 125 l with acetyl-CoA (8.2 mM) and fluorescent borondipyrromethene difluoride (BODIPY) chloramphenicol (CAM) substrate (1:12.5 dilution) at 37°C for 8 and 1 h, respectively. Reactions were terminated by addition of cold ethyl acetate (850 l), followed by vigorous vortexing. An aliquot (800 l) of extracted substrate and acetylated products was removed (organic phase), dried under vacuum, and resuspended in ethyl acetate (20 l) prior to separation on thin-layer chromatography plates (LK6, Whatman, Clifton, NJ) with chloroform:methanol (9:1) for 30 min. Substrate and products were visualized under long-wave UV light (366 nm) and photographed (Type 55 positive/negative film, Polaroid, Cambridge, MA). Substrate and combined product bands were scraped from the plates, extracted and diluted 1:10 in methanol prior to quantification by fluorescence spectrophotometry at excitation and emission wavelengths of 490 nm and 512 nm, respectively, using a fluorometer. Percent conversion of BODIPY CAM substrate to 1-, 3-, and 1,3-acetylated BODIPY CAM products was computed after correcting samples for background activity.
Differences in transfection efficiency between samples were monitored using a portion of the cell extracts for luciferase assays (34) with minor modification. Briefly, 10 l of cell extract was added to 350 l of reaction buffer (25 mM glycylglycine, 5 mM ATP, and 15 mM MgSO 4 (pH 7.6)). Luciferin (1 mM; Boehringer Mannheim) was injected into the reaction and relative light output determined using a Monolight 2010 luminometer (Analytical Luminescence Labs, San Diego, CA). Transfection efficiency did not vary significantly between triplicate culture dishes within any single treatment group or between treatment groups.
Preparation of Nuclear Extracts-Several 225-cm 2 flasks of H295R cells were maintained as described previously. At 55-65% confluency, cells were cultured in the presence or absence of 200 nM PMA for 24 h at which time cells were harvested using trypsin/EDTA, pelleted, washed with D-PBS, pelleted and resuspended in D-PBS (1.0 ml), and maintained on ice. Crude nuclear extracts were prepared according to the method of Dignam and co-workers (35) as modified by Andrews and Faller (36). Briefly, after pelleting and removal of D-PBS, cells were allowed to swell for 10 min on ice in a hypotonic buffer (10 mM HEPES-KOH (pH 7.9) at 4°C, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride) followed by vortexing and centrifugation. The supernatant was discarded and the pellet resuspended in a high salt buffer (20 mM HEPES-KOH (pH 7.9), 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride) for 20 min to extract nuclear proteins. The suspension was centrifuged, and the supernatant containing nuclear proteins was aliquoted and stored in liquid nitrogen. Protein concentrations were determined using the BCA method (Pierce) as modified for the presence of sulfhydryl reagents (37).
Messenger RNA Isolation and Northern Analysis-Prior to isolation of mRNA, four 225-cm 2 flasks of H295R were cultured to 65% confluency, at which time cells were treated with media containing 200 nM PMA or an equivalent amount of Me 2 SO for 24 h. In addition, one 75-cm 2 flask of untreated HeLa and two 75-cm 2 flasks of mouse F9 embryonic teratocarcinoma cells were cultured until cells were confluent. Cells were harvested using trypsin/EDTA, and the resulting pellets were stored at Ϫ70°C.
Isolation of enriched polyadenylated (poly(A) ϩ ) mRNA was performed using a messenger RNA isolation kit (Stratagene) following the protocol, including the poly(A) ϩ enrichment procedure, provided by the manufacturer. After elution from oligo(dT)-cellulose push columns, the poly(A) ϩ -enriched mRNA was ethanol/sodium acetate-precipitated overnight at Ϫ20°C using glycogen as a carrier. Following centrifugation, mRNA samples were resuspended in a small volume of elution buffer, aliquoted, and stored at Ϫ20°C in an excess of absolute ethanol.
Digital Imaging-Autoradiographs and Polaroid negatives were scanned using an Agfa Arcus II flatbed scanner and Adobe Photoshop software. Digitized images were saved as TIFF files, and all cropping and text enhancements were carried out using Aldus Pagemaker or Freehand programs.

RESULTS
To determine the region(s) of the promoter that confer phorbol ester-mediated transcriptional regulation of the type II 3␤-HSD gene, a series of 5Ј deletions of the promoter fused to a CAT reporter gene were used to transiently transfect human adrenocortical carcinoma (H295R) cells followed by treatment for 24 h in the absence (basal) or presence of PMA. As shown in Fig. 1, deletion of the promoter sequence from Ϫ101 to Ϫ52 bp resulted in a nearly 4-fold and 500-fold reduction in basal and PMA-stimulated CAT activity, respectively, suggesting that this region is important for both basal and PMA-induced regulation of type II 3␤-HSD expression. Additionally, the region from Ϫ1251 to Ϫ301 bp may contain a negative regulatory element that inhibits PMA-induced transcription since an approximately 5-fold increase in CAT activity was observed fol-lowing the deletion of Ϫ1251 to Ϫ301 bp of the promoter.
A subsequent search of the Ϫ101 to Ϫ52 bp promoter sequence revealed a putative SF-1/Ad4BP regulatory element, TCAAGGTAA, at Ϫ64 to Ϫ56 bp that differed from the reported SF-1 (PyCAAGGPyCPu; Ref. 13) and Ad4BP (C/TCAAGGT/ CC/T; Ref. 10) elements by a single nucleotide (underlined). To determine if the putative SF-1/Ad4BP element was functional, non-steroidogenic HeLa cells, which do not express SF-1/ Ad4BP, were transiently cotransfected with the Ϫ101 CAT, Ϫ52 CAT, and Ϫ101M CAT constructs in the presence and absence of an expression vector for SF-1/Ad4BP (RSV-SF-1/ Ad4BP). In addition, transfected cells were cultured for 24 h in the presence or absence of PMA (200 nM) to investigate the ability of phorbol ester to modulate SF-1/Ad4BP-mediated activity of the 3␤-HSD promoter (Fig. 2).
Cotransfection of HeLa cells with Ϫ101 CAT and RSV-SF-1/ Ad4BP increased CAT activity 49-fold over basal, whereas only small increases were observed (6-and 2-fold, respectively) in those cells transfected with Ϫ52 CAT, which lacked the putative SF-1/Ad4BP element, or Ϫ101M CAT, in which two nucleotides in the putative response element had been mutated. PMA treatment increased CAT activity 20-fold over basal in those cells transfected with Ϫ101 CAT alone. PMA had a similar effect on cells transfected with Ϫ52 CAT (17-fold increase); however, CAT activity in response to PMA was considerably less in those cells transfected with Ϫ101M CAT (5-fold). Interestingly, an unexpectedly powerful synergistic effect of SF-1/ Ad4BP and PMA on CAT activity was observed in cells cotransfected with Ϫ101 CAT and RSV-SF-1/Ad4BP and treated with PMA. Overexpression of SF-1/Ad4BP in HeLa cells transfected with Ϫ101 CAT and subsequently treated with PMA increased promoter activity 540-fold as compared with basal. An increase was observed (16-fold) in similarly treated cells transfected with the Ϫ52 CAT construct plus RSV-SF-1/Ad4BP and was most likely due to the action of PMA alone. No similar increase was observed with the Ϫ101M CAT construct. Collectively, these data suggest that SF-1/Ad4BP can specifically regulate the promoter of the human type II 3␤-HSD gene and that SF-1/Ad4BP-induced expression of the gene in HeLa cells is greatly enhanced by phorbol ester.
Although phorbol ester activation of the human type II 3␤-HSD promoter in non-steroidogenic HeLa cells appeared to be primarily mediated by SF-1, it was also necessary to evaluate the response in a physiologically relevant human steroidogenic cell line. To address this issue, adrenocortical carcinoma (H295R) cells were transfected with the Ϫ101, Ϫ52, and Ϫ101M CAT constructs followed by treatment with or without phorbol ester. As shown in Fig. 3, PMA treatment of H295R cells transfected with the Ϫ101 CAT construct containing the putative SF-1 regulatory element increased promoter activity 18-fold over basal. In contrast, deletion (Ϫ52 CAT) or mutation (Ϫ101M CAT) of the putative SF-1 regulatory element dramatically reduced PMA responsiveness to 3-fold and 2-fold over that for basal, respectively. Collectively, these data and those from the preceding HeLa cell experiments clearly demonstrate the importance of the SF-1 regulatory element in mediating phorbol ester regulation of the human type II 3␤-HSD promoter in both steroidogenic and non-steroidogenic cells.
Interaction of SF-l with the putative regulatory element was examined by EMSA using nuclear extracts prepared from H295R cells cultured in the presence or absence of PMA and a 32 P-labeled double-stranded oligonucleotide containing the putative SF-1/Ad4BP site and 10 -11 bases of 5Ј and 3Ј flanking type II 3␤-HSD promoter sequence. As shown in Fig. 4, multiple protein-DNA complexes were formed when nuclear extracts from untreated and PMA-treated H295R cells were incubated with the oligonucleotide probe. However, when extracts were incubated in the presence of increasing concentrations of unlabeled oligonucleotide, the appearance of one complex was markedly diminished as compared with the others. Preincubation of the extracts with SF-1/Ad4BP antiserum abolished the formation of that particular complex, confirming the participation of SF-1/Ad4BP. Additionally, the intensity of the band representing the SF-1/Ad4BP specific protein-DNA complex appeared to be slightly darker for control versus PMA treatment. These data may be interpreted to suggest reduced DNA binding in those extracts derived from cells treated with PMA as compared with control. However, given that the cells were harvested at only one time point and that the kinetics of PMAinduced expression of SF-1/Ad4BP mRNA are currently unknown, the results of this comparison appear to be of limited value.
To further test the specificity of DNA binding, nuclear extracts from untreated H295R cells were incubated in the presence of a nonspecific control consisting of antiserum against a secreted epididymal protein in rats or increasing concentrations of unlabeled heterologous oligonucleotides (containing GAS or PIE response elements) or unlabeled oligonucleotide (Ϫ101M) containing a mutated form of the putative SF-1/ Ad4BP response element flanked by type II 3␤-HSD promoter sequence (Fig. 5). Incubation of nuclear extracts with nonspecific antibody or increasing concentrations of unlabeled Ϫ101M, GAS, or PIE oligonucleotides failed to diminish the appearance of the complex that was abolished by SF-1/Ad4BP antiserum, suggesting that formation of this protein-DNA complex is specific to SF-1/Ad4BP. Finally, Northern analysis was used to determine if SF-1/ Ad4BP was expressed in H295R, HeLa, and mouse F9 teratocarcinoma cells. As shown in Fig. 6, SF-1/Ad4BP transcripts were detected in H295R cells and expression appeared to increase slightly upon treatment with PMA. In addition, a slightly smaller transcript was detected in F9 cells after longer autoradiography times. As anticipated, no transcripts for SF-1/Ad4BP were detected in HeLa cells. Collectively, results of the EMSA and Northern analysis demonstrate that SF-1/ Ad4BP is expressed in adrenal cortical carcinoma cells and interacts with the putative SF-1/Ad4BP regulatory element present in the promoter of the human type II 3␤-HSD gene. DISCUSSION In the present study we sought to determine regions of the human type II 3␤-HSD promoter important for regulation us-

. H295R cell nuclear proteins form a complex with the SF-1/Ad4BP response element present in the human type II 3␤-HSD promoter that is abolished by SF-1/Ad4BP antiserum.
EMSA were performed using nuclear extracts from control or PMAtreated H295R cells (20 g) and labeled oligonucleotide containing the SF-1/Ad4BP regulatory element present in the type II 3␤-HSD promoter in the presence or absence of SF-1/Ad4BP antiserum (1 l) or increasing molar concentrations (50-or 500-fold) of unlabeled oligonucleotide, as described under "Materials and Methods." Arrowhead denotes loss of gel-shifted complex in extracts incubated with SF-1/Ad4BP antiserum or unlabeled oligonucleotide.
ing a physiologically relevant cell line, H295R adrenal cortical carcinoma cells (27,28). Using deletion mutagenesis we determined that the region from Ϫ101 to Ϫ52 bp of the promoter was essential for PMA-mediated transcription of the reporter gene and contained a putative SF-1/Ad4BP regulatory element TCAAGGTAA from Ϫ64 to Ϫ52 bp. Interestingly, the human type I 3␤-HSD gene is also regulated by PMA (45); however, it does not contain a functional SF-1/Ad4BP element (TCAAAGTGA; Ref. 46) because it differs from the consensus element by one critical core nucleotide (10). As a result, the mechanism conferring PMA responsiveness to the type I 3␤-HSD gene, which is predominantly expressed in the placenta and skin, most likely differs from that of the type II isoform. The putative SF-1/Ad4BP element found in the type II 3␤-HSD promoter is another variant of the shared motif AGGTCA, the core binding sequence of several zinc-finger DNA-binding proteins including the estrogen, thyroid hormone, retinoic acid, and vitamin D 3 receptors (47). This sequence has been reported to confer cAMP responsiveness to a number of the cytochrome P450 steroid hydroxylase genes in gonadal (13,14) as well as adrenal (15,48,49) tissues. In these tissues, steroid biosynthesis is regulated by the interaction of luteinizing and/or folliclestimulating hormone or adrenocorticotrophic hormone, respectively, with their cognate cell membrane receptors, resulting in elevated production of cAMP and enhanced tissue-specific expression of the appropriate steroidogenic enzyme.
Expression of 3␤-HSD and synthesis of aldosterone in H295R cells are regulated through the action of angiotensin II via the type I angiotensin II receptor coupled to polyphosphoinositidase-C and subsequent increases in intracellular calcium (28). Because this effect can be mimicked by phorbol ester (27), it is presumed to be mediated via protein kinase C. Our results are consistent with PKC-mediated regulation of 3␤-HSD expression in H295R cells. PMA treatment of H295R cells transfected with a series of 5Ј deletion mutants of the 3␤-HSD promoter increased reporter gene activity, for all constructs except Ϫ52 CAT, greater than that observed for untreated transfected cells. The novel aspect of this finding is that the PMA-induced increase in transcriptional activity appeared to be mediated by SF-1/Ad4BP. Deletion of promoter sequence from Ϫ101 to Ϫ52 bp, later found to contain a putative SF-1/Ad4BP regulatory element, abolished PMA-stimulated CAT activity. Additionaly, mutation of the putative SF-1/Ad4BP regulatory element also inhibited PMA-induced promoter activation. To date, no similar effect of phorbol ester has been reported for any of the genes known to be regulated by SF-1/Ad4BP.
Cotransfection of HeLa cells with the Ϫ101 CAT construct and RSV-SF-1/Ad4BP expression vector yielded a significant 49-fold increase in transcriptional activity due to SF-1/Ad4BP alone, as well as a distinct synergistic effect of PMA on SF-1/ Ad4BP-mediated transcription. The 49-fold increase in CAT activity in the absence of any other treatment is greater than that reported for isolated SF-1/Ad4BP elements from other genes similarly cotransfected into non-steroidogenic cell lines (13,15,23,24,49). SF-1/Ad4BP alone was unable to activate MIS gene expression in HeLa cells, whereas coexpression of a mutant form of SF-1/Ad4BP lacking the putative ligand binding domain slightly increased transcriptional activity (23), suggesting that ligand or a cofactor is necessary for activation of the MIS promoter by SF-1/Ad4BP. In the case of some steroid hydroxylase genes, the addition of protein kinase A activators such as cAMP and forskolin or coexpression of the catalytic subunit of protein kinase A is necessary for appreciable activation by SF-1/Ad4BP in nonsteroidogenic cells. In our case, high levels of transcriptional activity due to SF-1/Ad4BP alone may result from promoter-specific sequence that confers a greater sensitivity of the type II 3␤-HSD gene to stimulation by SF-1/Ad4BP. A number of reports have suggested that nucleotides 5Ј of the response elements for SF-1/Ad4BP and another orphan nuclear receptor, nerve growth factor-inducible factor B (NGFI-B), may influence binding affinity (10,15,23,50). Additionally, a brief survey of reported functional SF-1/Ad4BP response elements in comparison to that for type II 3␤-HSD indicates that the putative 3␤-HSD SF-1/Ad4BP element is a unique variant of the AGGTCA consensus motif in that the penultimate cytidine at the 3Ј end of the sequence has been replaced with an adenine resulting in AGGTAA, which has not been reported for other genes. This difference could be in part responsible for the enhanced SF-1/Ad4BP activation of 3␤-HSD gene expression in HeLa cells.
The mechanism underlying the stimulatory effect of PMA on 3␤-HSD promoter activity in HeLa cells in the absence of SF-1/Ad4BP is less clear and may be independent of a functional SF-1/Ad4BP response element because removal of the element, Ϫ52 CAT, failed to reduce CAT activity in response to PMA. It is possible that a yet unidentified regulatory element is present in the sequence from Ϫ52 to ϩ45 bp of the promoter and perhaps PMA induces the synthesis of a HeLa cell-specific regulatory protein(s) that is responsive to PMA in the absence of SF-1/Ad4BP. Loss of PMA-induced CAT activity after mutation, but not removal, of the putative SF-1/Ad4BP response element (Ϫ101M CAT) is seemingly more complex. However, it is possible that the PMA-responsive region of the promoter may partially overlap that for SF-1/Ad4BP binding and that the loss of several nucleotides from the 5Ј-end of the PMA responsive sequence may be well tolerated whereas mutation of several of those nucleotides severely compromises PMA responsiveness. Interestingly, HeLa cells express an orphan nuclear receptor, chicken ovalbumin upstream promoter transcription factor (COUP-TF; Ref. 51), that has been reported to bind to recognition elements that partially and totally encompass the SF-1/ Ad4BP elements in the 17␣-hydroxylase (15) and oxytocin (24) promoters, respectively.
The highly synergistic effect of PMA on SF-1/Ad4BP activation of the 3␤-HSD promoter in HeLa cells has not been reported previously, and the mechanism responsible for this cell specific response is presently unknown. Because PMA is a potent activator of PKC and SF-1/Ad4BP has 10 consensus PKC phosphorylation sites by sequence homology (16) and is a phosphoprotein, 2 it is tempting to speculate that the profound response to PMA is due, at least in part, to direct phosphorylation of the transcription factor. Alternatively, it is possible that treatment with PMA induces the production and(or) phosphorylation of a HeLa cell factor, that significantly augments SF-1/Ad4BP activation of the type II 3␤-HSD promoter. Synergistic effects of phorbol ester and cholera toxin on estradiolstimulated transcription of a synthetic estrogen-responsive reporter gene were also found to be cell-specific and appeared to result from the stabilization or facilitation of the receptor with components of the transcriptional apparatus, possibly as a result of phosphorylation of the receptor or other necessary proteins (52). It is also possible that PMA provokes synthesis of an undiscovered ligand for SF-1/Ad4BP in nonsteroidogenic HeLa cells. Recent data indicate that SF-1/Ad4BP expression and action are not limited to steroidogenic tissues and the regulation of steroidogenic enzymes (22)(23)(24)(25). Thus, it is possible that the undiscovered ligand for SF-1/Ad4BP is not a steroid, as has been hypothesized (15,17,53); instead, it may be a molecule that is present in steroidogenic as well as nonsteroidogenic tissues, with specificity of activation relying solely on the tissue-specific expression of SF-1/Ad4BP.
There is evidence to indicate that other members of the steroid/thyroid hormone receptor superfamily, normally activated by ligand binding, may elicit their actions at steroidresponsive regulatory elements through ligand-independent processes that involve cross-talk between membrane-bound receptor signaling pathways and the specific nuclear steroid receptor. Dopamine can activate progesterone, estrogen (ER), vitamin D, and thyroid hormone receptor-␤, but not glucocorticoid, receptor-mediated activation of target response elements in transfected cells in the absence of steroid ligand and in the case of progesterone receptor, activation required the presence of a specific serine phosphorylation site on the receptor (54). More recently, epidermal growth factor (EGF) was reported to activate estrogen-independent transcription of a consensus estrogen response element cotransfected with an expression vector for the mouse ER into human endometrial adenocarcinoma cells (55). In the presence of estrogen, EGF had a synergistic effect on transcription. Although not determined, ER phosphorylation and(or) activation of other regulatory proteins were hypothesized as plausible mechanisms mediating the effects of EGF.
In HeLa cells, co-transfection of SF-1/Ad4BP followed by treatment with PMA results in promoter activity levels that greatly exceed the additive effect of each treatment alone and fulfills the criteria for synergism in transcriptional activation as discussed by Herschlag and Johnson (56). Therefore, the synergism implies that the two activating mechanisms function in the same pathway. While these high levels of activation might occur without physical interaction between the two activating agents, it is also highly likely that the activation of protein kinase C isoforms by phorbol ester treatment may alter the phosphorylation state of SF-1 with a corresponding change in activity.
Our investigation of basal and phorbol ester-mediated regulation of type II 3␤-HSD gene expression in human adrenocortical carcinoma cells has resulted in several novel findings. Both basal and PMA-induced transcription of the gene in H295 cells required the presence of promoter sequence containing an SF-1/Ad4BP recognition element. This is the first demonstration of SF-1/Ad4BP-mediated regulation of a non-cytochrome P450 steroidogenic enzyme promoter. Cotransfection of the reporter construct containing the putative SF-1/Ad4BP element or a mutated version of the element and an expression vector for SF-1/Ad4BP into HeLa cells confirmed the essentiality and functionality of this response element in a non-steroidogenic cell line. We also discovered a previously unreported synergistic effect of PMA on the regulation of transcription by SF-1/Ad4BP. Additionally, results of EMSA with SF-1/Ad4BP antiserum indicated that H295R cell nuclear extracts contained SF-1/Ad4BP, which specifically interacted with the putative 3␤-HSD SF-1/Ad4BP response element. Northern analysis also confirmed the presence of SF-1/Ad4BP transcripts in H295R but not HeLa cells. Collectively, these data provide considerable evidence to support a role for SF-1/Ad4BP in the regulation of the type II 3␤-HSD gene in adrenal cortical cells.
However, further research is needed to more clearly define the mechanisms underlying phorbol ester-mediated regulation of this gene in both H295R and HeLa cells.