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Molecular Endocrinology, doi:10.1210/me.2003-0223
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Molecular Endocrinology 18 (3): 558-573
Copyright © 2004 by The Endocrine Society

Assessment of the Role of Activator Protein-1 on Transcription of the Mouse Steroidogenic Acute Regulatory Protein Gene

Pulak R. Manna, Darrell W. Eubank and Douglas M. Stocco

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430

Address all correspondence and requests for reprints to: Douglas M. Stocco, Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430. E-mail: doug.stocco{at}ttmc.ttuhsc edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
cAMP-dependent mechanisms regulate the steroidogenic acute regulatory (StAR) protein even though its promoter lacks a consensus cAMP response-element (CRE, TGACGTCA). Transcriptional regulation of the StAR gene has been demonstrated to involve combinations of DNA sequences that provide recognition motifs for sequence-specific transcription factors. We recently identified and characterized three canonical 5'-CRE half-sites within the cAMP-responsive region (-151/-1 bp) of the mouse StAR gene. Among these CRE elements, the CRE2 half-site is analogous (TGACTGA) to an activator protein-1 (AP-1) sequence [TGA(C/G)TCA]; therefore, the role of the AP-1 transcription factor was explored in StAR gene transcription. Mutation in the AP-1 element demonstrated an approximately 50% decrease in StAR reporter activity. Using EMSA, oligonucleotide probes containing an AP-1 binding site were found to specifically bind to nuclear proteins obtained from mouse MA-10 Leydig and Y-1 adrenocortical tumor cells. The integrity of the sequence-specific AP-1 element in StAR gene transcription was assessed using the AP-1 family members, Fos (c-Fos, Fra-1, Fra-2, and Fos B) and Jun (c-Jun, Jun B, and Jun D), which demonstrated the involvement of Fos and Jun in StAR gene transcription to varying degrees. Disruption of the AP-1 binding site reversed the transcriptional responses seen with Fos and Jun. EMSA studies utilizing antibodies specific to Fos and Jun demonstrated the involvement of several AP-1 family proteins. Functional assessment of Fos and Jun was further demonstrated by transfecting antisense c-Fos, Fra-1, and dominant negative forms of Fos (A-Fos) and c-Jun (TAM-67) into MA-10 cells, which significantly (P < 0.01) repressed transcription of the StAR gene. Mutation of the AP-1 site in combination with mutations in other cis-elements resulted in a further decrease of StAR promoter activity, demonstrating a functional cooperation between these factors. Mammalian two-hybrid assays revealed high-affinity protein-protein interactions between c-Fos and c-Jun with steroidogenic factor 1, GATA-4, and CCAAT/enhancer binding protein-ß. These findings demonstrate that Fos and Jun can bind to the TGACTGA element in the StAR promoter and provide novel insights into the mechanisms regulating StAR gene transcription.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE STEROIDOGENIC ACUTE regulatory (StAR) protein plays a critical role in the regulation of steroid hormone biosynthesis by mobilizing cholesterol from the outer to the inner mitochondrial membrane (1, 2, 3, 4). Previous studies indicate that regulation of StAR expression and steroidogenesis occurs through cAMP-mediated mechanisms that involve transcriptional induction. Also, inhibition of transcription markedly affects the synthesis of the StAR protein and steroid biosynthesis in mouse Leydig cells (2, 5, 6). Transcriptional activation by cAMP is mediated through the interaction of the cAMP response element (CRE) binding protein (CREB) with a consensus CRE (5'-TGACGTCA-3') found in the promoter of target genes (7, 8, 9). However, the StAR gene promoter lacks a consensus CRE and as such, resembles the promoters of several steroid hydroxylase genes that are regulated by cAMP signaling (10). The StAR promoter sequences in mouse, rat, and human are highly homologous, and in the absence of a canonical CRE, multiple cis-elements have been demonstrated to play important roles in StAR gene expression (11, 12, 13, 14, 15, 16).

Transcriptional regulation of the mouse StAR gene has been shown to be mediated by combinations of several trans-acting factors present within the -151/-1 bp region relative to the transcription start site (15, 16, 17, 18). Recently, we have investigated the importance of three CRE half-sites within the -96/-67 region of the mouse StAR promoter and in those studies, a member of the CREB family [CREB/CRE modulator (CREM)] was shown to be involved in StAR gene regulation (16). Of these CRE elements, the CRE2 half-site [analogous to an activator protein 1 (AP-1) recognition motif] was found to be predominantly involved in regulating StAR gene expression. Several lines of evidence indicate that the TGACGTCA [CRE or activating transcription factor (ATF) binding site] and the TGA(C/G)TCA [AP-1 binding site or phorbol 12-O-tetradecanoate 13-acetate responsive element (TRE)] sequence motifs are two of the major regulatory elements that contribute to transcriptional regulation (19, 20, 21). The CRE/ATF and AP-1/TRE sites are recognized by proteins that share the basic region/leucine zipper (bZip) motif but have different specificity for DNA binding affinity (22, 23). The bZip proteins are broadly defined to at least three subfamilies of proteins, including CREB/CREM/ATF, AP-1, and C/EBPs, and cross-dimerization among these proteins has been demonstrated (22, 23, 24, 25, 26, 27). We have recently determined that CRE elements can bind to MA-10 nuclear proteins and that the protein-DNA binding was markedly decreased by a consensus AP-1, suggesting that the transcription factor AP-1 may influence StAR gene transcription (16). As such, the close similarities between the CRE/ATF and AP-1/TRE sequence motifs raise the possibility that these families of proteins may bind to the same target sequence and play roles in StAR transcription.

Mechanisms controlling transcription include the roles of enhancer elements, which function in switching on gene expression, and negative regulatory elements, which silence transcription (7, 8, 28). Members of the AP-1 transcription factor family are grouped into the Fos (c-Fos, Fra-1, Fra-2, and Fos B) and the Jun (c-Jun, Jun B, and Jun D) subfamilies, based on their amino acid similarities, and have been shown to interact with specific DNA sequences located in the promoters of target genes (29, 30, 31). However, Fos members can form heterodimers with Jun proteins whereas Jun members can form either homo- or heterodimers among themselves and have been reported to play central roles in regulating many biological functions, including proliferation, differentiation, and transformation (31, 32, 33, 34). Fos and Jun have been demonstrated to have opposite effects on transcription of the phosphoenolpyruvate carboxykinase (PEPCK) (35), GnRH (36), and myogenic helix-loop-helix (37) genes. Studies have also shown that Fos and Jun are able to interact with different proteins and mediate transcriptional responses (38, 39, 40). Recently, c-Fos has been reported to decrease StAR gene expression in Y-1 cells (41). Also, Fos and Jun were found to be involved in the epidermal growth factor-mediated attenuation of the mouse StAR promoter responsiveness in mLTC-1 mouse Leydig tumor cells (42). However, these studies lack a description of the molecular events associated with the AP-1 family proteins involved in StAR gene expression. The functional importance of the CRE2 (16), and consequently the identification of an AP-1 element (TGACTGA) in the StAR promoter resembling Fos and Jun recognition motifs, prompted us to explore the role of these factors in understanding the mechanisms involved in StAR gene transcription.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Role of AP-1 Element in StAR Gene Expression
The cAMP responsive region (-151/-1 bp) of the mouse StAR gene contains binding sites for several transcription factors that have been demonstrated to play roles in cAMP-mediated StAR gene regulation. In the present study, the involvement of an AP-1 recognition motif (that overlapped with the CRE2 element at -81/-75) in StAR gene transcription was assessed. As illustrated using EMSA, an oligonucleotide probe (-96/-67 bp) containing AP-1 and/or CRE elements binds to MA-10 nuclear extract (NE) in a dibutyryl cAMP [(Bu)2cAMP]-responsive manner (Fig. 1Go, compare lanes 2 and 4). Protein-DNA complexes (I, II, and III) were decreased after addition of its cold competitor (lanes 3 and 5), consensus CRE (Con CRE, lanes 7 and 10), and consensus AP-1 (Con AP-1, lanes 8 and 11) sequences. Importantly, mutation generated in the AP-1 core sequence (TGACTGA to TAGATCT) had no affect on protein-DNA complexes (lanes 6 and 9).



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  		Fig. 1. Binding of the -96/-67 bp Region of the StAR Promoter to MA-10 NE

Using EMSA, 10 µg of NE from control and (Bu)2cAMP (500 µM, 6 h)-stimulated cells were used to analyze protein-DNA binding with the 32P-labeled probe (-96/-67) containing CRE and AP-1 elements. A representative phosphor image illustrates formation of three protein-DNA complexes (I, II, and III) to the 32P-labeled probe. Competitors were used at 100-fold molar excess and include -96/-67 (lanes 3 and 5), consensus CRE (Con CRE, lanes 7 and 10), consensus AP-1 (Con AP-1, lanes 8 and 11), and AP-1 mutant (AP-1 Mut, lanes 6 and 9). Protein-DNA complexes were resolved on 5% polyacrylamide gels, the gels were dried, and the complexes were visualized with a phosphor imaging device. The experiments were repeated three to four times. Migration of free probes is shown on each lane.

 
The region comprising the AP-1 element in the mouse StAR promoter (-81/-75 bp) is reasonably conserved among different species (Fig. 2AGo), including rat, human, ovine, porcine, and monkey (15, 16, 17). To verify the involvement of the AP-1 element, a relatively shorter oligonucleotide probe (-83/-72 bp), specific to the AP-1 binding sequence, was examined in EMSA using (Bu)2cAMP-stimulated nuclear extract (NE) obtained either from MA-10 (lanes 2–6) or Y-1 (lanes 8–12) cells (Fig. 2BGo). Using the -83/-72 probe, protein-DNA binding occurring at complex I was nearly abolished when compared with the -96/-67 probe. However, protein-DNA complexes were clearly decreased by its cold competitor (lanes 3 and 9), Con CRE (lanes 4 and 10), and Con AP-1 (lanes 5 and 11) sequences. On the other hand, mutation in the AP-1 sequence motif did not affect protein-DNA complexes (lanes 6 and 12). The pattern of protein-DNA binding was qualitatively similar in both MA-10 and Y-1 cells. It is noted, however, that recombinant (rec) CREB protein was shown to bind to the same region of the StAR promoter (16, 18). Moreover, this region has previously been demonstrated to bind C/EBPß (43). The present results demonstrate that both CRE and AP-1 DNA binding proteins bind to the TGACTGA element and influence StAR gene expression in steroidogenic cells.



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  		Fig. 2. StAR Promoter -83/-72 bp Region in Different Species, and Binding of the -83/-72 bp Region to MA-10 and Y-1 Nes

Panel A, Sequence alignment of mouse, rat, human, ovine, porcine, and monkey StAR promoter regions (-83/-72) illustrating the AP-1 recognition motifs (underlined). Panel B, Protein-DNA binding was performed using an oligonucleotide probe specific to the AP-1 sequence motif (-83/-72), and binding reactions were carried out as described in the legend of Fig. 1Go. NE (10 µg) obtained from (Bu)2cAMP (500 µM, 6 h)-stimulated MA-10 (lanes 2–6) or Y-1 (lanes 8–12) cells was used to determine binding with the 32P-labeled probe using EMSA. Competitors were used at 100-fold molar excess, i.e. -83/-72 (lanes 3 and 9), Con CRE (lanes 4 and 10), Con AP-1 (lanes 5 and 11), and AP-1 Mut (lanes 6 and 12). Protein-DNA complexes are marked as I, II, and III. Migration of free probes is shown in both cases. A representative phosphor image depicts binding of the -83/-72 probe to MA-10 and Y-1 NEs. These experiments were repeated three to four times. Con CRE, consensus CRE; Con AP-1, consensus AP-1; AP-1 Mut, AP-1 mutant.

 
The role of the AP-1 binding motif was further investigated by determining reporter gene activity utilizing the -151/-1 bp StAR promoter segment (Fig. 3Go). This segment was chosen based on our previous observations in which the -110/-1 bp region had a decreased response in StAR promoter activity (16). MA-10 cells transfected with the -151/-1 StAR segment displayed a 3.6-fold increase in reporter activity after (Bu)2cAMP stimulation. Mutation in the AP-1 element resulted in a 52% decrease in basal reporter activity but did not significantly affect (Bu)2cAMP-stimulated fold-response, demonstrating the importance of the AP-1 motif in StAR promoter responsiveness.



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  		Fig. 3. Functional Involvement of the AP-1 Element on StAR Promoter Activity

MA-10 cells were transfected with either the -151/-1 bp StAR reporter segment or the -151/-1 containing a mutation in the AP-1 recognition site (1 µg each), in the presence of pRL-SV40 vector (renilla luciferase for determining transfection efficiency), as described in Materials and Methods. After 36 h of transfection, cells were incubated for an additional 6 h in the absence (basal) or presence of 500 µM (Bu)2cAMP (cAMP). pGL3 basic (pGL3) was used as a control. Luciferase activity in the cell lysates was determined and expressed as relative light units, RLU (luciferase/renilla). Schematic presentation illustrates wild-type and mutant AP-1 plasmids within the -151/-1 bp region. Data represent the mean ± SEM of five independent experiments.

 
Assessment of the Role of AP-1 Family Proteins in StAR Gene Transcription
To examine whether the binding of AP-1 family members to the TGACTGA element in the mouse StAR promoter was of functional significance, MA-10 and Y-1 cells were transfected in the absence or presence of Fos and Jun (Fig. 4Go). Transfection of MA-10 cells with the -151/-1 bp StAR segment resulted in a 3.4-fold increase in (Bu)2cAMP-mediated reporter activity over untreated cells. Cells transfected with the -151/-1 bp StAR segment in the presence of Fos members (c-Fos, Fra-1, Fra-2, and Fos B) demonstrated increases (150% for c-Fos and 270% for Fos B) and decreases (38% for Fra-2 and 14% for Fra-1) in basal reporter activities. However, in all cases (Bu)2cAMP-stimulated promoter responses were decreased between 28 and 45% when compared with control (pcDNA). Jun members showed a marked elevation in basal luciferase activity (570% with c-Jun, 285% with Jun B, and 350% with Jun D). After (Bu)2cAMP stimulation these responses were attenuated by 42%, 36%, and 47% with c-Jun, Jun B, and Jun D, respectively. In additional experiments, an approximately 2-fold increase in (Bu)2cAMP-stimulated reporter activity was observed when Y-1 cells were transfected with the -151/-1 bp StAR segment (Fig. 4Go). Basal luciferase responses were increased with c-Fos (136%), Fos B (195%), c-Jun (360%), Jun B (377%), and Jun D (243%) and decreased with Fra-2 (30%), respectively. In the case of Fra-1, a moderate but consistent decrease in promoter activity was observed. (Bu)2cAMP-stimulated reporter activities were decreased in all cases by 26–40% when compared with control. Moreover, the results obtained with MA-10 and Y-1 cells demonstrate that the repression of (Bu)2cAMP-stimulated StAR promoter activity seen with Fos and Jun in the absence of a canonical AP-1 element, appeared to be mediated through similar mechanisms.



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  		Fig. 4. Effects of Fos and Jun on StAR Promoter Responsiveness

Utilizing the -151/-1 StAR reporter segment containing an AP-1 binding motif, MA-10 (upper panel) and Y-1 (lower panel), cells were transfected with either empty expression vector (pcDNA) or with expression vector containing Fos and Jun (DNA ratio 1:1) in the presence of pRL-SV40 vector. After 36 h of transfection, cells were incubated for an additional 6 h without (basal) or with (Bu)2cAMP (cAMP; 500 µM), and luciferase activity in the cell lysates was determined and expressed as relative light units (RLU) (luciferase/renilla). Data represent the mean ± SEM of three to five independent experiments.

 
To obtain more insight into these mechanisms, StAR reporter activity in response to Fos and Jun was assessed using the -151/-1 bp StAR segment by generating a mutation in the AP-1 binding site. The results summarized in Fig. 5Go show that transfection of MA-10 cells with the -151/-1 bp segment resulted in a 3.5-fold increase in luciferase activity in response to (Bu)2cAMP. Mutation in the AP-1 element (as above), clearly decreased (P < 0.01) basal but did not significantly affect (Bu)2cAMP-mediated StAR reporter responses. Importantly, the inhibitory effects of Fos (c-Fos, Fra-1, and Fra-2) and Jun (c-Jun and Jun D) in (Bu)2cAMP-stimulated StAR reporter activity were apparently lost when cells were transfected with the -151/-1 bp StAR segment containing a mutation in the AP-1 element. Consequently, Fos and Jun have no significant effects on StAR gene transcription under basal conditions, demonstrating the specificity of the AP-1 binding motif in StAR promoter responsiveness.



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  		Fig. 5. Specificity of Fos and Jun binding to the AP-1 Element Involved in StAR Promoter Responsiveness

MA-10 cells were transfected either with the -151/-1 StAR reporter segment (-151/-1 StAR) or -151/-1 StAR containing a mutation in the AP-1 element (-151/-1 StAR-AP-1 Mut), without (pcDNA) or with Fos and Jun plasmids as indicated, in the presence of pRL-SV40. After 36 h of transfection, cells were incubated for a further 6 h in the absence (basal) or presence of (Bu)2cAMP (cAMP; 500 µM). Luciferase activity in the cell lysates was determined and expressed as relative light units (RLU) (luciferase/renilla). pGL3 basic (pGL3) was used as a control. These experiments were repeated three to four times, and the data (±SEM) from a representative experiment with triplicate samples are presented.

 
The involvement of Fos and Jun proteins in StAR gene expression was evaluated in EMSA by utilizing an oligonucleotide probe (-96/-67). Protein-DNA complexes (I, II, and III) observed with (Bu)2cAMP-stimulated MA-10 NE were markedly inhibited by the addition of unlabeled sequence (lane 11). Different antibodies (Abs) specific to Fos and Jun members (lanes 4–10) interfere with protein-DNA complexes to varying degrees (Fig. 6Go). Notably, c-Fos and Fra-2 Abs nearly abolished protein-DNA complexes (lanes 4 and 6). The binding was found to be decreased with Jun D Ab (lane 10). An Ab specific to CREM1 demonstrated a marked decrease in protein-DNA complexes (lane 12) apparently similar to those of c-Fos and Fra-2, an observation consistent with our previous finding (16, 18). Preimmune serum for CREM1 (lane 13) and IgG (lane 3) were also used. Qualitatively similar results were obtained when the -83/-72 probe was used in EMSA with MA-10 NE (data not shown).



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  		Fig. 6. Assessment of the Role of AP-1 Family Proteins on StAR Gene Expression

EMSA was performed with an oligonucleotide probe representing the -96/-67 bp region of the StAR promoter that contains a functional AP-1 binding motif. NE (10 µg) from (Bu)2cAMP-stimulated (500 µM, 6 h) MA-10 cells was incubated with the 32P-labeled probe in the absence or presence of different Abs to Fos and Jun (lanes 4–10) and CREM1 (lane 12). Protein-DNA complexes are marked as I, II, and III. A representative phosphor image illustrates Ab interference analysis with Fos and Jun (lanes 4–10) and CREM1 (lane 12). Cold competitor (-96/-67) was used at 100-fold molar excess (lane 11). IgG (lane 3) and preimmune serum for CREM1 (lane 13) were used in the incubation. Migration of free probes is shown in each lane. Similar results were obtained from three independent experiments.

 
In an alternative approach, the functional significance of Fos and Jun was further investigated in transcription of the StAR gene using either antisense (AS) or dominant negative forms of these proteins (Fig. 7Go). MA-10 cells transfected with AS-c-Fos, AS-Fra-1 plasmids, within the -151/-1 StAR segment, demonstrated significant decreases (P < 0.01) in basal and Fos-mediated StAR promoter responsiveness. Importantly, transfection of a dominant negative form of Fos (A-Fos) lacking a DNA binding domain markedly decreased StAR promoter activity. A-Fos has been demonstrated to inactivate the DNA binding of the Fos:Jun heterodimer and potentially inhibit Jun-dependent transactivation (44, 45). On the other hand, the introduction of a dominant negative form of c-Jun (TAM-67) showed a marked inhibition of basal and c-Jun-mediated StAR promoter responses. Fos and Jun AS and dominant negative forms were also able to decrease (Bu)2cAMP-mediated StAR promoter activity (data not shown). These findings reinforce the importance of Fos and Jun in StAR gene transcription.



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  		Fig. 7. Functional Involvement of Fos and Jun in StAR Promoter Responsiveness Using AS and Dominant Negative Fos and Jun in MA-10 Cells

Cells were transfected with empty vector (pcDNA), c-Fos, cJun, AS-c-Fos, and AS-Fra-1, and dominant negative mutants of Fos (A-Fos) and c-Jun (TAM-67) expression plasmids (1.5 µg each) or in combination as specified, in the presence of the -151/-1 StAR reporter segment (DNA ratio, 1:1) and pRL-SV40, as described in Materials and Methods. After 48 h of transfection, cells were harvested, and luciferase activity in the cell lysates was determined and expressed as relative light units (RLU) (luciferase/renilla). pGL3 basic (pGL3) was used as a control. Data represent the mean ± SEM of three independent transfections.

 
To delineate the events involved in AP-1-mediated StAR transcription, c-Fos, Fra-2, and c-Jun were further examined (Fig. 8Go). Overexpression of increasing amounts (0.5–6.0 µg) of c-Fos, Fra-2, and c-Jun, along with the -151/-1 bp StAR segment in MA-10 cells, was found to be associated with increases (c-Fos and c-Jun) and a decrease (Fra-2) in StAR reporter activity in a concentration-dependent manner (Fig. 8AGo). To determine whether the inhibitory effect of Fra-2 in StAR gene expression affects the c-Jun response, MA-10 cells were transfected with Fra-2, c-Jun, and a combination of them, utilizing the -151/-1 bp StAR segment (Fig. 8BGo). Overexpression of Fra-2 decreased basal StAR promoter activity by 38% whereas c-Jun increased it by 542%. Importantly, coexpression of Fra-2 and c-Jun resulted in a 57% decrease in reporter activity, indicating that Fra-2 is able to block the stimulatory effect of c-Jun. The addition of increasing amounts of c-Jun with a fixed concentration of Fra-2 did not alter the inhibitory effect elicited by Fra-2. In contrast, higher amounts of Fra-2 decreased the c-Jun induction of StAR promoter activity by approximately 65%, demonstrating that Fra-2 inhibits c-Jun responsiveness.



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  		Fig. 8. Alterations in Transcription of the StAR Promoter by Expression of Fos and Jun

Utilizing the -151/-1 StAR reporter segment containing a functional AP-1 element, MA-10 cells were transfected with or without increasing amounts (0.5–6 µg) of c-Fos, Fra-2, and c-Jun plasmids, in the presence of pRL-SV40 (panel A). After 36 h of transfection, cells were harvested, and luciferase activity in the cell lysates was determined and expressed as percent control. In panel B, cells were also transfected with or without Fra-2, c-Jun (2–6 µg), and a combination of them as indicated, in the presence of the -151/-1 StAR reporter segment. After 36 h of transfection, luciferase activity in the cell lysates was determined and expressed as relative light units (RLU) (luciferase/renilla). Values are the mean ± SEM of three to four independent experiments in both cases. Different letters above the bars (panel B) indicate that these groups differ significantly from each other at P < 0.05.

 
Elements Involved in StAR Gene Transcription Cooperate/Interact with AP-1
The roles of cis-acting elements within the -151/-1 bp region of the mouse StAR promoter were assessed by determining StAR reporter activity after generating mutations in the putative binding sites, either alone or in combination with AP-1 (Fig. 9Go). These mutations include the recognition motifs for Sp1, steroidogenic factor 1 (SF-1), CCAAT/enhancer binding protein (C/EBP), and GATA, and their positions within the -151/-1 StAR promoter are illustrated (Fig. 9AGo). MA-10 cells transfected with the -151/-1 StAR segment demonstrated a 3.3-fold increase in luciferase activity in response to (Bu)2cAMP. Alteration of bases in the AP-1 element demonstrated a significant decrease (P < 0.01) in StAR promoter activity. Mutations generated in Sp1, C/EBP, SF-1, and GATA binding sites attenuated basal responses by 37%, 44%, 58%, and 42% respectively, but did not affect (Bu)2cAMP-mediated fold-induction (Fig. 9BGo). Transfection of any of these mutants in combination with the AP-1 mutant further decreased StAR reporter activity when compared individually, suggesting the functional cooperation of these elements in StAR promoter responsiveness.



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  		Fig. 9. Mutations in the AP-1 and Other cis-Acting Elements, Either Alone or in Combination, Affect StAR Promoter Responsiveness

The schematic diagram illustrates the positions of the cis-elements within the -151/-1 StAR promoter region (panel A). MA-10 cells were transfected with either the -151/-1 StAR reporter segment or the -151/-1 StAR containing mutations in the AP-1 sequence either alone or in combination with Sp1, C/EBP, SF-1, and GATA elements (DNA ratio, 1:1), in the presence of pRL-SV40 (panel B). After 36 h of transfection, cells were incubated for a further 6 h without (Basal) and with 500 µM (Bu)2cAMP (cAMP), and luciferase activity in the cell lysates was determined and expressed as RLU (luciferase/renilla). The approximate positions of AP-1, Sp1, C/EBP, SF-1, and GATA elements within the -151/-1 bp StAR promoter region and a combination of them together with their mutations are illustrated. pGL3 basic (pGL3) was used as a control. These experiments were repeated three to four times, and data (±SEM) from a representative experiment with quadruplicate samples are presented.

 
Further insight into these mechanisms was obtained by determining protein-protein interactions between c-Fos and c-Jun with SF-1, GATA-4, Sp1, and C/EBPß using the mammalian two-hybrid assays. HeLa cells transfected with either c-Fos-pACT, c-Jun-pBIND, or SF-1, GATA-4, Sp1, and C/EBPß (SF-1, GATA-4, Sp1, and C/EBPß either in pACT or pBIND) plasmids resulted in limited effects on luciferase activity (Fig. 10Go). Coexpression of c-Fos-pACT with SF-1pBIND, GATA-4-pBIND, and C/EBPß-pBIND increased luciferase responses approximately 8.8-, 5.7-, 4.7-, 3.8-, 9.2-, and 6.4-fold when compared with c-Fos-pACT, SF-1-pBIND, GATA-4-pBIND, and C/EBPß-pBIND, respectively. On the other hand, cells transfected with c-Jun-pBIND in the presence of SF-1-pACT, GATA-4-pACT, and C/EBPß-pACT resulted in 18.2-, 19.5-, 12.4-, 14.2-, 16.5-, and 19.7-fold increases in luciferase activity when compared with c-Jun-pBIND, SF-1-pACT, GATA-4-pACT, and C/EBPß-pACT, respectively. Coexpression of either c-Fos-pACT and c-Jun-pBIND with Sp1-pACT or Sp1-pBIND had no significant effects in luciferase responsiveness. These results demonstrate that c-Fos and c-Jun proteins are either capable of a direct association with SF-1, GATA-4, and C/EBPß or interact in vivo through a common adapter protein.



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  		Fig. 10. Protein-Protein Interactions between c-Fos and c-Jun with SF-1, GATA-4, Sp1, and C/EBPß in the Mammalian Two-Hybrid Assay

HeLa cells were transfected with c-Fos-pACT, c-Jun-pBIND, or SF-1, GATA-4, Sp1, and C/EBPß (either in pACT or pBIND) plasmids (2 µg each), or their combination as indicated, in the presence of the pG5-luciferase vector. After 48 h of transfection, cells were harvested, and luciferase activity in the cell lysates was determined (relative light units). These experiments were repeated two to four times, and data (±SEM) from a representative experiment with triplicate samples are presented.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The CRE/ATF and AP-1/TRE sequence motifs are two of the major classes of regulatory elements that contribute to transcriptional regulation of eukaryotic gene expression. Transcriptional regulation of one such gene, the steroidogenic regulatory protein (StAR), is mediated by cAMP-dependent mechanisms even though it lacks a canonical CRE in its promoter region. Nevertheless, the mouse StAR gene has been demonstrated to be regulated by the combinatorial action of multiple DNA elements that consist of recognition motifs for sequence-specific transcription factors located within the -151/-1 bp region of the StAR promoter (6, 15, 16, 17, 42, 46). Within this region, three well-conserved 5'-CRE half-sites (-96/-67 bp) have recently been characterized, and their involvement in StAR gene expression has been demonstrated using the CRE binding proteins CREB and CREM (16, 18). One of the CRE elements, the CRE2 half-site is analogous to an AP-1 sequence motif and is highly conserved among different species. The experimental approaches used in the present study indicate the importance of the AP-1 recognition motif and provide evidence that the AP-1 family proteins are involved in controlling transcription of the StAR gene.

The AP-1 transcription factor family (Fos and Jun) are members of the bZip family, share many properties, and have been demonstrated to regulate many biological functions (22, 25, 29, 30). A variety of external stimuli, such as growth factors, tumor promoters, hormones, and analogs of the cAMP second messenger, have been shown to acutely induce the levels of Fos and Jun (36, 47, 48, 49, 50). Fos and Jun have been reported to be involved in transcription by binding to a DNA sequence, known as an AP-1/TRE element [TGA(C/G)TCA], present in the promoter of a number of genes (31, 32, 33). Homologous to the AP-1/TRE sequence, a highly conserved element (TGACTGA) was found in the mouse StAR promoter. The present data demonstrate that oligonucleotide probes containing the AP-1 binding motif can bind to MA-10 and Y-1 NE in EMSA, and that the protein-DNA binding was affected by consensus AP-1 and by mutation in the AP-1 sequence motif. In addition, disruption of the AP-1 binding site exerted an approximately 50% decrease in StAR reporter activity, demonstrating the importance of the AP-1 element in StAR gene expression. Previously, this region (the CRE2 element) was found to bind rec CREB protein and appeared to be the most important among the three CRE half-sites. Also, a mutated CRE2 probe displayed virtually no binding with MA-10 NE in comparison with other CRE mutants (18). However, it is interesting to note that the AP-1 element in the StAR promoter more closely corresponds to its consensus sequence than does the CRE2 element correspond to its consensus sequence.

The identification of an AP-1 recognition motif in the cAMP-responsive region of the mouse StAR promoter provided the opportunity to characterize the roles of Fos and Jun in StAR gene transcription in more depth. Evidence is accumulating that Fos and Jun are involved in mediating transcription of several genes, including those for PEPCK (35), atrial natriuretic factor (51), proopiomelanocortin (52), neurotensin/neuromedin N (53), GnRH (54), bone morphogenic protein-2 (49), and the early growth factor-inducible gene 1 (Erg-1) (39). Also, Fos has been demonstrated to decrease cAMP responsiveness and adversely affect the inducible effect of Jun in PEPCK gene transcription (35). Our current data demonstrate the functional involvement of Fos and Jun in controlling transcription of the StAR gene. In particular, although they have varying effects on basal StAR gene transcription, Fos and Jun are capable of repressing (Bu)2cAMP-mediated responses. Importantly, overexpression of Fra-2 was found to inhibit the induction of c-Jun-mediated transcription of StAR promoter activity. The effects of Fos and Jun in StAR gene transcription, in the absence of a canonical AP-1/TRE element in the StAR promoter, were found to be similar in steroidogenic cells. Mutation in the AP-1 sequence motif reversed the transcriptional responses elicited by Fos and Jun and demonstrated that their effects are not due to nonspecific protein-protein interactions. Utilizing the p-1862 rat StAR segment, which possesses three AP-1 binding motifs at -85, -187, and -1567 bp, c-Fos was recently reported to decrease basal and cAMP- and c-Jun-induced promoter responses in Y-1 cells (41). The contradictory effect of c-Fos on basal reporter activity (i.e. inhibitory vs. stimulatory in the present finding), could be due to the use of different StAR promoter segments (rat, p-1862 containing three AP-1 sites vs. mouse, p-151 containing one AP-1 site). The mechanism accounting for differences in Fos- and Jun-mediated StAR gene transcription may be due to the lack of sequence similarity outside the DNA-binding and dimerization domains and will be the subject of future investigations.

The participation of Fos and Jun in StAR gene expression was also investigated by determining protein binding to the AP-1 DNA element. An oligonucleotide probe containing three CRE half-sites (including the overlapping AP-1 element), demonstrated strong inhibition of protein-DNA complexes by c-Fos, Fra-2, and CREM1 Abs, supporting the notion that AP-1 and CREM proteins can form heterodimers. This event is not entirely surprising because the ability of AP-1 and CRE binding proteins to heterodimerize has been demonstrated in other systems (22, 55, 56). Studies have also shown that AP-1 complexes are not limited to Fos and Jun dimers, and these proteins have been shown to dimerize with other bZip proteins (22, 57). Also, it has been demonstrated that repression of the PEPCK gene by Fos does not require a consensus phorbol 12-O-tetradecanoate 13-acetate responsive element (TRE) but it does require an intact DNA binding region and dimerization domain, and that it binds to the C/EBP protein family (35, 58). The Fos protein has also been shown to repress its own promoter by interacting with the dyad symmetry element or serum response element, an action that is independent of a TRE (59, 60).

Notably, among the Fos and Jun family members assessed, c-Jun was found to be most potent in transactivation of the StAR gene. Therefore, the transcriptional activation potential of c-Jun might be expected to correlate with its ability to affect protein-DNA binding. However, EMSA experiments using an Ab specific to c-Jun did not indicate such a correlation, suggesting the involvement of an alternate mechanism. Studies have demonstrated that c-Jun containing tethered Fos dimers have distinct promoter specificity and biological responses. In accordance with this, the tethered c-Jun~c-Fos has been demonstrated to be the strongest activator followed by c-Jun~c-Fra-1, c-Jun~c-Fra-2, and c-Jun~ATF2 in the human collagenase gene promoter (matrix metalloproteinase 1), which possesses a consensus TRE element (61, 62, 63). Within the human cyclin D1 (CCND1) promoter, two elements containing TRE (CCND1–1) and CRE (CCND1–2) have been reported to be the targets of c-Jun-containing dimers (64, 65). Nonetheless, the tethered Jun~Fos proteins could efficiently bind to consensus TRE, be recognized by appropriate Abs with a mobility shift similar to that of a mixture of Jun and Fos, and show nuclear localization by transfection assays (63). Also, these findings have documented that c-Jun~c-Fos dimers had an exclusive binding to TRE (matrix metalloproteinase 1 and CCND1–1) as compared with CRE (CCND1–2) elements, suggesting c-Jun~Fos dimers prefer TRE-like motifs whereas Jun~ATF2 prefer CRE-like motifs. Because the AP-1/CRE2 region in the StAR promoter corresponds more closely to a consensus TRE in comparison to a CRE, it is more likely that, rather than a direct effect of c-Jun alone, the tether c-Jun~c-Fos and/or c-Jun~Fos (Fra-2) dimers play important roles in transcription of the StAR gene. We also used an additional strategy that employed AS and/or dominant negative forms of Fos and Jun. It has previously been demonstrated that the use of AS and/or dominant negative forms of these proteins resulted in inhibition of Fos and Jun expression (45, 52, 66, 67). In the present studies, AS and/or dominant negative forms of Fos and Jun were demonstrated in the repression of StAR promoter responsiveness, reinforcing the involvement of AP-1 family proteins in StAR gene transcription. Moreover, it was observed by RT-PCR analysis that MA-10 cells express c-Fos and c-Jun, and that cAMP increased levels of the former about 3-fold and marginally increased the latter (data not shown).

The 5'-flanking region of the StAR gene possesses recognition motifs for several trans-acting factors, including SF-1, GATA, Sp1, and C/EBP, that have been demonstrated to be instrumental in StAR gene expression (14, 15, 42, 43, 46, 68). SF-1, either alone or in concert with other factors, plays an important role in many cAMP-regulated genes, including StAR, which lack CREs and are involved in steroidogenesis (15, 17, 69, 70, 71). The current data using sequence- and site-specific mutational analyses indicate that SF-1, GATA, Sp1, and C/EBP elements cooperate with AP-1 in mediating StAR reporter responsiveness. Previous studies have demonstrated that mutation in the GATA binding site, either alone or in combination with SF-1 and/or C/EBPß-AP-1-nuclear receptor half-site, significantly decreases basal and cAMP-stimulated mouse StAR promoter responses (15). Therefore, it is conceivable that more than one transcription factor can bind to most, if not all, of the cis elements involved in cAMP-mediated regulation of the StAR gene, a phenomenon consistent with the PECPK gene promoter in which different transcription factor binding arrays modulate cAMP responsiveness (72, 73).

An intriguing aspect of the present results is that a functional protein-protein interaction exists between c-Fos and c-Jun with SF-1, GATA-4, and C/EBPß as determined using the mammalian two-hybrid assay. c-Fos has recently been demonstrated to interact with SF-1 and decrease SF-1-mediated rat StAR gene expression (41). Also, an interaction between AP-1 and SF-1 has been reported in cAMP-dependent activation of the ACTH receptor promoter (40). Fos has been shown to interact with the CArg box sequence and decrease Egr-1 gene transcription (39). Studies have also demonstrated that SF-1 cooperates and interacts with CREB/CREM, C/EBPß, and Sp1 in the transcriptional regulation of the StAR gene (17, 18, 71). Several lines of evidence suggest that CBP/p300 participates in the activities of many transcription factors including CREB, SF-1, c-Fos, and c-Jun by functioning as bridging proteins (74, 75). However, the interaction of Fos and Jun with other proteins, in addition to SF-1, GATA-4, and C/EBPß, which act through sequences not involving AP-1, cannot be ruled out and may play roles in StAR gene expression.

From the results of the present studies it is apparent that the TGACTGA element is the binding site for Fos and Jun. We have recently demonstrated that rec CREB protein specifically binds to this sequence (18). It is noted, however, that this region has also been demonstrated to bind C/EBPß (43). Therefore, it appears that three subfamilies of bZip proteins can bind to the same target sequence. Indeed, these families possess high degrees of homology in their binding sequences and share bZip motifs, although they have a higher binding specificity for their respective consensus sequences (22, 23, 25). Cross-talk in the form of binding between AP-1 and CRE family proteins has also been reported (25, 76, 77, 78). Studies have demonstrated that CRE binding proteins can bind to the AP-1 target sequence and suppress AP-1 reporter activity in differentiating keratinocytes (23, 25). Consistent with this, it was also observed that coexpression of Fos/Jun and CREB in the presence of the -151/-1 bp StAR promoter segment resulted in a decrease in reporter activity (Manna, P. R., and D. M. Stocco, unpublished observations). Considering the similarities in the AP-1 and CRE binding sequences, it is not surprising that both Fos/Jun and CREB compete with each other for the TGACTGA motif and result in an attenuation of StAR promoter activity. Further studies to elucidate the molecular interactions between AP-1 and CREB/CREM that should lead to a better understanding of the involvement of these factors in regulating StAR gene transcription are underway. Taken together, the present results point to an important role of Fos and Jun in regulating the transcriptional machinery of the StAR gene, leading us to propose a model presented in Fig. 11Go.



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  		Fig. 11. Proposed Model for Mouse StAR Gene Transcription

Initially, ligand-receptor interaction at the cell surface results in an activation of coupled G proteins (G), which in turn stimulates membrane-associated adenylyl cyclase (AC). This, in turn, generates cAMP and results in the dissociation of the inactive tetrameric protein kinase A (PKA) complex into the active catalytic and the regulatory subunits. The catalytic subunits migrate into the nucleus, phosphorylate transcription factors, and thereby activate StAR gene transcription. Shown are the different transcription factor binding motifs that may interact with the AP-1 element during StAR gene transcription. The concerted action of multiple transcription factors, and their interaction with most, if not all, of the cis-regulatory elements, appears to be involved in regulating StAR gene expression. It is noteworthy that Fos and Jun can specifically bind to the AP-1/CRE2 recognition motif in the StAR promoter and play important roles in transcription of the StAR gene. Based on previously available information together with our own preliminary data, it is highly likely that CBP/p300, a factor known to interact with many transcription factors including CREB, c-Fos and c-Jun, may participate as a bridging protein and influence StAR gene transactivation.

 
In summary, these findings provide compelling evidence that Fos and Jun can specifically bind to the TGACTGA element in the StAR gene promoter and are involved in controlling its transcription. Functional cooperation, interaction, alteration, or competition between transcription factors may represent cross-talk between different signaling pathways and may be important for cAMP responsiveness. A balance between the positive and negative effects of different transcription factors presumably allows for the fine tuning of the regulatory events associated with StAR gene expression. Regardless of the trans-regulatory mechanisms involved, the 5'-flanking region (-151/-1 bp) of the StAR promoter that contains three CRE half-sites and an AP-1 site, in addition to several transcription factor recognition motifs, appears to be the most important region and might function as a cAMP response unit in the transcriptional regulation of the StAR gene.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Generation of StAR Promoters and Site-Directed Mutagenesis
The proximal -151/-1 bp region of the mouse StAR promoter was demonstrated to have full cAMP responsiveness when compared with the entire promoter region (15, 16, 18, 42, 46). Therefore, this region was constructed and subcloned into the pGL3 basic vector (Promega Corp., Madison, WI) utilizing a XhoI and HindIII fragment that contains the firefly luciferase gene as a reporter (16). Using this fragment, mutations in the AP-1 and other cis-acting elements were generated using the QuikChange Site-directed mutagenesis kit (Stratagene, La Jolla, CA). The following sense strands of the oligonucleotide sequences used for mutagenesis (mutated bases in lowercase bold) were: AP-1 mutant (Mut), 5'-CCTTGACCCTCTGCACAATagaTctTGACTTTTTTATCTC-3'; Sp1 Mut, 5'-ACACAGTCTGCgaatTCCCACCTTGGCCAG-3'; SF-1 Mut, 5'-CAATCATTCCAgCtgTGACCCTCTGCAC-3'; C/EBP Mut, 5'-GCACTGCAGGATGgtcgAcTCATTCCATCCTTG-3'; GATA Mut, 5'-GACTGATGACTTTTTccggaCAAGTGATGATGCACAG-3'.

The specific SF-1 and C/EBP binding sites within the cAMP responsive region chosen for analysis were based on their predominant involvement in StAR gene expression (17, 18). Specific mutations in the target sites were assessed by restriction mapping using Bgl II (AP-1 Mut), EcoRI (Sp1 Mut), PvuII (SF-1 Mut), SalI (C/EBP Mut), and BspEI (GATA Mut), and confirmed by automated sequencing on a PE Biosystem 310 Genetic Analyzer (ABI PRISM 310, PerkinElmer Corp., Norwalk, CT) at the Texas Tech University Biotechnology Core Facility.

Construction of Fos/Jun Plasmids
The full-length mouse c-Fos, Fra-1, Fra-2, and Fos B open reading frames were synthesized using a PCR-based cloning strategy. Primer pairs employed for amplifying the different Fos members were the following: c-Fos forward, 5'-CCCCGCTCGAGATGATGTTCTCGGGTT-3'; c-Fos reverse, 5'-CCCGGGATCCCACAGGGCCAGCAGCG-3'; Fos B forward, 5'-CCCCGCTCGAGATGTTTCAAGCTTTTCC-3'; Fos B reverse, 5'-CCCGGGATCCAGAGCAAGAAGGGAGG-3'; Fra-1 forward, 5'-CCCCGCTCGAGATGTACCGAGACTACG-3'; Fra-1 reverse, 5'-CCCGGGATCCCACAAAGCCAGGAGTGTAGG-3'; Fra-2 forward, 5'-CCCCGCTCGAGATGTACCAGGATTATCC-3'; Fra-2 forward, 5'-CCCGGGATCCAGGGCTAGAAGTGTGG-3'. The 5'-primer for all Fos members contains a XhoI site whereas the 3'-primers possess a BamHI site at the 5'-end (underlined). The PCR products amplified were subcloned into the pcDNA 3.1(-)Myc-HisB (pcDNA3.1-MHB) expression vector (Invitrogen Life Technologies, Carlsbad, CA) after digestion with XhoI-BamHI restriction enzymes. The mouse Jun members (open reading frame) were similarly constructed and inserted into the pcDNA3.1-MHB expression vector (obtained from Dr. Curt M. Pfarr, Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX), using EcoR1-HindIII sites (79, 80). The identity of the inserted fragments were verified by restriction digestion and confirmed by automated sequencing (ABI PRISM 310, Perkin-Elmer Corp.).

EMSAs
Preparation of nuclear extracts (NEs) from mouse MA-10 Leydig (81) and Y-1 adrenocortical tumor cells was carried out as described previously (16, 18, 82). The oligonucleotide probes were synthesized by heating sense and antisense primers to 65 C for 5 min in annealing buffer (10 mM Tris-Cl, 100 mM NaCl, 1 mM EDTA, pH 7.5), followed by cooling at room temperature. The sense strands of the oligonucleotide sequences used in EMSA were the following: -96/-67 (CRE half-sites), 5'-GGTGACCCTCTGCACAATGACTGATGACTTTT-3'; -83/-72 (AP-1), 5'-GGAATGACTGATGA-3'; -83/-72 (AP-1 Mut), 5'-GGAATAGATCTTGA-3'; consensus AP-1 (23), 5'-GGCGCTTGATGAGTCAGCCGGAA-3'; consensus CRE (19), 5'-GGAGAGATTGCCTGACGTCAGAGAGCTAG-3'.

The doubled-stranded oligonucleotides (4 pM each) were end labeled with [{alpha}32P]dCTP (PerkinElmer Life Sciences, Inc., Boston, MA) using Klenow (Promega Corp.) fill-in reactions and protein-DNA binding assays were carried out under optimized conditions (16, 83). Briefly, 10–15 µg NE were incubated in 20 µl of reaction buffer (10 mM HEPES, 1 mM EDTA, 4% Ficoll, 10 mM dithiothreitol, 1 µg poly dIdC, 40 mg/ml BSA, and 2 µM ZnSO4; pH 7.9) for 15 min at room temperature. The 32P-labeled DNA probe (0.5 pM, ~100,000 cpm) was added alone or in the presence of 50 pM (100-fold molar excess) of unlabeled probe, and incubation was continued for an additional 15 min. When antiserum was used, binding reactions were incubated on ice for 45 min before the addition of the labeled DNA. Different Abs to the AP-1 family members used in EMSA reactions were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). These include mouse monoclonal c-Fos (sc-8047) and Jun B (sc-8051), and rabbit polyclonal Fra-1 (sc-183), Fra-2 (sc-171), Fos B (sc-48), c-Jun (sc-1694), and Jun D (sc-74). The CREM1 Ab is specific for CREM protein and cross-reacts with CREB at very high concentrations (16, 84). Mouse IgG was purchased from Santa Cruz Biotechnology, Inc. Protein-DNA complexes were then subjected to electrophoresis on 5% polyacrylamide gels for about 1.5 h at 200 V in 0.5x TBE buffer (90 mM Tris-borate, 2 mM EDTA; pH 8.3). The gels were dried, exposed to phosphor screens, and quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Mammalian Two-Hybrid Assay
Protein-protein interactions between c-Fos and c-Jun with SF-1, GATA-4, Sp1, and C/EBPß were investigated using the mammalian two-hybrid assay (Promega Corp.), as described previously (18). Briefly, c-Fos-pACT and c-Jun-pBIND plasmids were constructed using c-Fos and c-Jun cDNAs inserted into either SalI and KpnI sites of the pACT vector or BamHI and NotI sites of the pBIND vector, respectively. The full-length SF-1, GATA-4, Sp1, and C/EBPß cDNAs were inserted into either BamHI and NotI sites of the pBIND vector or SalI and KpnI sites of the pACT vector, respectively. HeLa cells were transfected with c-Fos-pACT, c-Jun-pBIND, SF-1-pACT or SF-1-pBIND, GATA-4-pACT or GATA-4-pBIND, Sp1-pACT or Sp1-pBIND, C/EBPß-pACT or C/EBPß-pBIND plasmids (2 µg each), or their combination as indicated in the figure legend, in the presence of the pG5-luciferase vector under optimized conditions (18), using FuGENE 6 reagent (Roche Diagnostics Corp., Indianapolis, IN). After 48 h of transfection, cells were harvested and luciferase activity in the cell lysates was determined using a TD 20/20 Luminometer (Turner Designs, Sunnyvale, CA).

Cell Culture, Transient Transfections, and Luciferase Assays
MA-10 (81) and Y-1 (ATCC, Manassas, VA) cells were maintained, respectively, in HEPES-buffered Waymouth’s MB/752 and Y-12K media containing antibiotics (16). HeLa cells were cultured in DMEM as described previously (18).

Transfection studies were carried out using FuGENE 6-transfection reagent (Roche Diagnostics Corp.) under optimized conditions (16, 18). In brief, wild-type and mutant StAR promoters (-151/-1 StAR/luc) were used (1 or 1.5 µg) in the absence or presence of Fos and Jun plasmids either in equal or at varied concentrations (0.5–6 µg), as specified in the figure legends. The functional specificity of Fos and Jun on StAR promoter responsiveness was also assessed using the AS c-Fos (52), AS Fra-1 (45), and dominant negative Fos [A-Fos, (45)] and c-Jun [TAM-67, (67)] plasmids. The amount of DNA used in transfections was equalized with empty vector. Transfection efficiency was normalized by cotransfecting 15 ng pRL-SV40 vector (a plasmid that constitutively expresses renilla luciferase).

Luciferase activity in the cell lysates was determined by the Dual-Luciferase reporter assay system (Promega Corp.), using a TD 20/20 Luminometer (Turner Designs) as described previously (16, 18).

Statistical Analysis
The experiments presented were repeated three to five times. Statistical analysis was performed by ANOVA using Statview (Abacus Concepts Inc., Berkeley, CA) followed by Fisher’s protected least significant differences test. Data represent the mean ± SEM, and P < 0.05 was considered significant.


   ACKNOWLEDGMENTS
 
The authors are grateful to Dr. C. M. Pfarr (Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX) for the generous gifts of Jun plasmids. We are grateful to Drs. E. Tulchinsky (University of Leicester, Division of Urology, Leicester, UK) for providing us with the dominant negative Fos; J. P. Loeffler (Institut de Physiologie et de Chimie Biologique, Strasbourg, France) for the AS c-Fos; and G. T. Bowden (Department of Pharmacology and Toxicology, The University of Arizona, Tucson, AZ) for the dominant negative c-Jun constructs. We also thank Dr. P. Sassone-Corsi (Institute de Génétique et de Biologie et Moléculaire and Cellulaire, Strasbourg, France) for generously providing the CREM1 Ab. We also acknowledge the help of Mathew T. Dyson, John D. Short, and Deborah Alberts during the course of these studies.


   FOOTNOTES
 
This work was supported by NIH Grant HD-17481 and with funds from the Robert A. Welch Foundation.

Abbreviations: Ab, Antibody; AP-1, activator protein 1; AS, antisense; ATF, activating transcription factor; (Bu)2cAMP, dibutyryl cAMP; CCND1, cyclin D1; CRE, cAMP response element; C/EBPß, CCAAT/enhancer binding protein; CREB, cAMP response-element binding protein; CREM, CRE modulator; NE, nuclear extract; PEPCK, phosphoenolpyruvate carboxykinase; rec, recombinant; SF-1, steroidogenic factor 1; StAR, steroidogenic acute regulatory protein; TRE, phorbol 12-O-tetradecanoate 13-acetate responsive element.

Received for publication June 10, 2003. Accepted for publication December 3, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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