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Endocrinology Vol. 148, No. 4 1804-1812
Copyright © 2007 by The Endocrine Society

Activating Protein-1 Cooperates with Steroidogenic Factor-1 to Regulate 3',5'-Cyclic Adenosine 5'-Monophosphate-Dependent Human CYP11A1 Transcription in Vitro and in Vivo

Ing-Cherng Guo, Chiung-Yi Huang, Chi-Kuang Leo Wang and Bon-chu Chung

Institute of Molecular Biology (I.-C.G., C.-Y.H., C.-K.L.W., B.-c.C.), Academia Sinica, Nankang, Taipei, 115 Taiwan; and Department of Veterinary Medicine (I.-C.G., C.-Y.H.), College of Bio-Resources and Agriculture, National Taiwan University, Taipei, 10617 Taiwan

Address all correspondence and requests for reprints to: Dr. Bon-chu Chung, Institute of Molecular Biology, 48, Academia Sinica, Nankang, Taipei, 115 Taiwan. E-mail: mbchung{at}sinica.edu.tw; or Dr. Ing-Cherng Guo, Department of Veterinary Medicine, National Taiwan University, Taipei, 10617 Taiwan. E-mail: iguo{at}ntu.edu.tw.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The CYP11A1 encodes cytochrome P450scc, catalyzing the first step of steroidogenesis in adrenals and gonads under the control of cAMP-mediated hormonal signals. The cAMP-induced activation of human CYP11A1 has been suggested to depend on the transcription factor cAMP-responsive element-binding protein (CREB), but the CREB action cannot explain the chronic cAMP effect on CYP11A1 activation. To further understand the mechanism of human CYP11A1 activation, we dissected the functions of the upstream cAMP responsive sequences (U-CRS) containing a core sequence, U identical to the steroidogenic factor-1 (SF-1)-binding site, and two flanking TPA-responsive element/cAMP-responsive element-like elements, C1 and C2. The EMSA assays showed that the binding activities of U with SF-1 as well as C1 or C2 with activating protein-1 (AP-1)/CREB-like proteins are induced by cAMP. The results from the site-directed mutagenesis analyses revealed that all three elements are required for the U-CRS function and any mutation of C1, C2, or U impairs the response to cAMP stimulation. In transgenic mice, the single or double mutations of C1 and C2 resulted in the reduction of reporter gene expression accompanied with poor hormonal response. The cAMP induction on the U-CRS activity was mimicked and enhanced by the overexpressed c-Jun in the presence of SF-1, but was abolished by the overexpression of an AP-1 dominant-negative mutant, FosB2. Furthermore, we have observed the interdependent transactivation between SF-1 and c-Jun on the U-CRS function. These results collectively demonstrate that SF-1 and AP-1 cooperate to activate CYP11A1 transcription in vitro and in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STEROID HORMONES CONTROLLING the balance of minerals, glucose, and sexual characteristics are essential for the maintenance of life and for the continuation of animal species (1). CYP11A1 encodes cytochrome P450scc enzyme that catalyzes the conversion of cholesterol into pregnenolone in the first step of steroid biosynthesis (2). P450scc is mainly expressed in all steroidogenic tissues such as adrenal glands and gonads (3), and regulated by pituitary hormones, including ACTH (4), LH, and FSH (5), through a common intracellular second messenger cAMP (6). The expression profiles of CYP11A1 at both levels of protein and mRNA reveal that CYP11A1 expression responds to a chronic cAMP stimulation (7, 8). Investigations of the promoter function of human CYP11A1 reveal that the 5'-flanking region contains sequences conferring cAMP responsiveness (1, 9, 10). Transcription factor steroidogenic factor-1 (SF-1)/Ad4-binding protein (also named NR5A1) (11) plays a central role to mediate the cAMP signal (12). Previous studies suggested that the expression of CYP11A1 was controlled by the interaction of SF-1 with adjacent elements like specific protein 1 (Sp1)-binding site (13, 14, 15) and cAMP-responsive element (CRE) (16, 17). Analyses of the human CYP11A1 promoter activity in adrenal Y1 cells (18), adrenal NCI-H295 cells (19, 20), testis MA10 cells (21), and placental JEG-3 cells (22) indicated that the cAMP stimulation of gene expression depended on the cell type.

We had identified upstream cAMP-responsive sequences (U-CRS) spanning –1640 to –1500 from the transcription start site of human CYP11A1 gene (7, 13). The U-CRS contains an SF-1-binding site U (13, 23) and two sequences (C1 and C2) highly homologous to both CRE (24) and phorbol ester TPA-responsive element (TRE) (25). C1 was found to bind the cAMP-responsive element-binding protein (CREB) very weakly (16, 17). CREB mediates the cAMP-dependent protein kinase A (PKA) signal to modulate the transcriptional activities (26). TRE is known to bind with activating protein-1 (AP-1), which consists of the Jun and Fos protein dimer to activate transcription (25) and is originally known as a mediator of protein kinase C (PKC) signal (27). The involvement of CREB, AP-1, and SF-1 activated by cAMP/PKA, PKC, and the other pathways in the regulation of steroidogenic acute regulatory protein (StAR) and the StAR-modulated steroidogenesis have been investigated (28). The CREB and AP-1 bound to the same noncanonical AP-1/CREB motif (–79/–73, TGACTGA), the sequence of which is actually identical to C2 site of CYP11A1, and interacted with SF-1 to regulate mouse StAR gene transcription (29, 30, 31, 32).

TRE has been shown to participate in the tissue-specific and cAMP-dependent expression of CYP11B1 (33), which is a close family member of CYP11A1. The presence of TRE or its variants in the 5'-flanking region of both CYP11A1 and CYP11B1 indicates that TRE may be an important regulatory element for the transcription of this gene family. However, the functions of TRE/CRE sequence in the CYP11A1 transcription have not been carefully studied, although some initial reports suggest that CREB may play a role (17, 34). The roles of SF-1 and AP-1/CREB-like proteins and their interactions in the cAMP signaling also need further examination.

In the present paper, we use the transfected cells and the transgenic mice to demonstrate that the interactions of TRE/CRE and SF-1 elements mediate the cAMP-dependent CYP11A1 transcription. In addition, we also demonstrate that c-Jun cooperates with SF-1 to activate the CYP11A1 promoter via TRE/CRE and SF-1 elements in U-CRS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
All animal experimentation was conducted in accordance with the accepted standards of humane animal care and the rules established by the Animal Committee at the Institute of Molecular Biology, Academia Sinica (Protocol No. MMiIMBCB20000097).

Cell culture and transfection
Mouse adrenocortical Y1 (35) and human choriocarcinoma JEG-3 (36) tumor cells were grown in F-10 medium supplemented with 10% heat-inactivated horse serum and 2.5% heat-inactivated fetal calf serum at 37 C in 5% CO2. Transfection with linear or circular DNA was carried out by Lipofectamine (Life Technologies, Bethesda, MD) or calcium phosphate method (37). The RSV-ßgal was used as the internal control for the tested promoter connected with chloramphenicol acetyltransferase (CAT) reporter gene; and the rous sarcoma virus-CAT (RSVCAT) was used as the internal control when the tested promoter was connected to the LacZ reporter gene. The amounts of DNA used in each transfection are described in the figures. For cAMP treatment, each dish of transfected cells was split into two dishes on the second day after transfection; one was kept in the original medium and the other was treated with 1 mM 8-Br-cAMP for 24 h. Forty-eight hours posttransfection, the lysate of transfected cells was harvested and subjected to ß-galactosidase activity assay using the chemiluminescent detection assay kit (Galacto-Light Plus; TROPIX, Bedford, MA) previously described (38, 39), and CAT activity assay (40). To normalize the reporter activities, the reporter activities from the test promoter were divided by those from the internal control.

EMSA
The procedure was described earlier (13). Briefly, the 32P-labeled probe was incubated with 5 µg nuclear proteins in the presence or absence of competitor oligonucleotide, or in the presence of anti-SF-1 serum or normal serum. Sequences and description of the sense strands of oligonucleotides used in EMSA are listed in Table 1Go.


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  		TABLE 1. Sequences of the sense strands of the oligonucleotides synthesized for EMSA and site-directed mutagenesis

 
Construction of plasmids
Plasmids pSCC#9 (7, 13), RSV-ßgal (41), RSVCAT (42), and pCREtkCAT (43), as well as expression plasmids for Jun/Fos (24, 44, 45) and SF-1 (46) were described before. The C1, U, and C2 sequences of phSCC2.3LacZ (47) were separately mutated using the Sculptor in vivo Mutagenesis System (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer’s instructions. The sequences of mutant oligonucleotides, including C1mt, Umt, and C2mt used for generating site-directed mutants are listed in Table 1Go. All mutant clones were confirmed by sequencing.

Generation and genotyping of transgenic mouse lines
The DNA fragments injected into mouse embryo at the one-cell stage are composed of the wild-type or mutated CYP11A1 promoter joined to LacZ and SV40 polyA. Mice were housed under the standard specific pathogen-free laboratory conditions. The generation of transgenic mice follows the standard protocol used previously (39). The transgene copy numbers in the genome of transgenic mice, as determined by Southern blotting, vary between 1–22, but mostly less than 4 (data not shown). All transgenic founders and their offsprings are of the FVB strain. Genomic DNA was prepared from mouse tails. Genotyping was performed by the PCR amplification of LacZ with primers 5'-Gal (AGG CAT TGG TCT GGA CAC CAG CAA) and 3'-Gal (GAT GAA ACG CCG AGT TAA CGC CAT) to produce a 476-bp fragment.

Hormonal stimulation test
For the adrenal function test, five to eight female transgenic mice were sc injected with 1 IU ACTH (Cortrosyn, Organon, Oss, The Netherlands) once a day for 7 d. Mice were anesthetized with ether 2 h after the last injection. In total, three sets of experiments had been done. For each set of experiments, eight mice of the wild-type and five mice of each mutant were injected with 1 IU ACTH or saline once a day. For the testicular function test, male transgenic mice were ip injected with 10 IU human chorionic gonadotropin (hCG; Sigma Chemical Co., St. Louis, MO) twice a day for 7 d. The levels of serum corticosterone and testosterone were measured using the 125I-labeled RIA kit (ICN Biomedicals, Costa Mesa, CA, and DSLabs, Webster, TX). The adrenal and testicular lysates were assayed for ß-galactosidase activity using the chemiluminescent detection assay kit (Galacto-Light Plus; TROPIX) counted in a luminometer as previously described (38, 39), and normalized with the amounts of proteins in the adrenal or testicular extracts.

Statistical analysis
Results are presented as mean ± SD. Statistical comparison between the wild-type and mutant or between control group and treated group was performed using Student’s t test. Statistical difference is indicated as *, P < 0.05, or **, P < 0.01.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TRE/CRE-like and SF-1-binding elements in U-CRS
The U-CRS spanning –1640 to –1500 from the transcription start site of human CYP11A1 gene contains an SF-1-binding site (U) and two TRE/CRE-like sequences, termed C1 and C2. The C1 site at –1633/–1626, TGATGTCA, homologous to the consensus TRE (TGAC/GTCA) or CRE (TGACGTCA) except the middle T, was found to bind CREB very weakly (16, 17). The C2 site, TGACTGA, with only 1 bp mismatch to the consensus TRE/CRE, is located at –1559/–1553. The U site at –1617/–1609, TCAAGGTCA, is located between C1 and C2 (Fig. 1AGo).


Figure 1
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  		FIG. 1. The protein-binding activities of TRE/CRE-like and SF-1-binding elements in CYP11A1 upstream region are enhanced by cAMP. A, The locations of C1, C2, and U elements in the U-CRS and the basal regulatory elements (B) of CYP11A1 are indicated. Numbers delineate the boundaries of each motif. B and C, The EMSA analyses show the cAMP-induced protein-binding activities of U (B), and C1 and C2 (C). Radioactive probes (P) were incubated with 5 µg of Y1 nuclear extracts in the presence or absence (–) of 500-fold molar excess of cold competitors listed on the top of each lane. Nuclear extracts treated with (+) or without (–) 1 mM 8-Br-cAMP for 24 h are indicated. The specific DNA-protein complexes are marked with arrowheads. These results reveal that the interactions of U with SF-1, and C1 and C2 with the AP-1/CREB-like proteins are increased by the cAMP treatment.

 
To identify the roles these elements play in the cAMP-dependent function of U-CRS, we first examined their binding properties in the EMSA. Three oligonucleotide probes corresponding to C1, C2, and U, respectively, were incubated with Y1 nuclear extracts and subjected to the EMSA analyses. A specific DNA-protein complex formed by the interaction of U probe with Y1 nuclear extracts was completely erased by cold oligonucleotides of U and –55/–29 fragment of human CYP11A1 promoter containing a SF-1-binding sequence (15, 48), and the antiserum specifically recognizing the DNA-binding domain of SF-1 (Fig. 1BGo). The –117/–94 fragment of human CYP11A1 promoter containing a imperfect Sp1-binding sequence (13) and normal serum had no influence on the formation of DNA-protein complex. This shows that the U-binding protein is SF-1, whose binding activity was increased by treatment with 8-Br-cAMP (Fig. 1BGo). Similarly, cAMP induced the binding intensities of Y1 nuclear extracts with C1 or C2 probe (Fig. 1CGo). These DNA-protein complexes were competed away by consensus TRE or CRE, and by C1 and C2 themselves (Fig. 1CGo). These results suggest that C1 and C2 interact with AP-1/CREB-like proteins in Y1 cells and their binding activities are stimulated by cAMP signaling.

C1, C2, and U are required for cAMP stimulation in cell culture
After examining proteins binding to U-CRS, we further verified the functions of U-CRS. At first, we generated a LacZ reporter construct driven by a 2.3-kb human CYP11A1 promoter (38), and then specifically mutated the sites of C1, C2, or/and U by the site-directed mutagenesis. The mutated sequences, C1mt, Umt, and C2mt (Table 1Go), could not bind to specific proteins in the EMSA assays (Fig. 2AGo). Seven mutant constructs in total were produced, including three single mutants (C1mt, Umt, and C2mt), three double mutants (C1/Umt, U/C2mt, and C1/C2mt), and one triple mutant (triple mt) (Fig. 2BGo). The basal activities of all mutant constructs were similar to their wild-type parent in transfection of Y1 cells (data not shown), but some mutants became refractory to cAMP stimulation (Fig. 2CGo). The responses of all three single mutants and the C1/C2mt double mutant to cAMP were less impaired, whereas the cAMP responses of C1/Umt and U/C2mt double mutants and C1/U/C2mt triple mutant were significantly reduced in comparison with the wild-type (Fig. 2CGo). These results imply that all three elements contribute to the cAMP responsiveness of human CYP11A1 promoter, although U may play a greater role than C1 and C2.


Figure 2
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  		FIG. 2. Combined mutations of C1, C2, and U elements result in the reduction of cAMP-induced CYP11A1 promoter activities in Y1 cells. A, Mutated C1mt, C2mt, and Umt do not compete for binding in EMSA. Nuclear extracts (5 µg) from Y1 cells treated with 1 mM 8-Br-cAMP for 24 h were incubated with the 32P-end-labeled C1, C2, or U. Except the control groups (–), the reactions were preincubated with 100- to 500-fold excess of the wild-type, C1mt, C2mt, or Umt. Only protein-DNA complexes are shown. B, The mutated sites of seven mutant plasmids (C1mt, Umt, C2mt, C1/Umt, U/C2mt, C1/C2mt, Triple mt) in the context of 2.3-kb CYP11A1 promoter (the wild-type, WT) connected with LacZ reporter gene are diagramed. The names and mutated elements are indicated. C, All three elements contribute to full cAMP responsiveness of the CYP11A1 promoter. Two million Y1 cells were transfected with 4 µg of linear DNA wild-type or mutant DNA together with 0.8 µg of the internal control RSVCAT. The ß-galactosidase activity was divided by CAT activity for normalization. The fold of cAMP induction was calculated by dividing cAMP-induced reporter activities by the basal activities. These values are shown after normalization with that of the wild-type, which is set at 100%. Statistical analyses were calculated from four independent experiments using Student’s t test, and the significant difference between mutant and the wild-type is indicated as * (P < 0.05) or ** (P < 0.01).

 
C1, C2, and U cooperate to control CYP11A1 promoter in transgenic mice
To look at the physiological functions of C1, C2, and U in vivo, we generated transgenic mice harboring the wild-type or mutant constructs. About 10–25 founder transgenic mice were obtained for each construct and bred to establish the transgenic mouse lines. The adrenal homogenates of mouse offspring were used for the reporter ß-galactosidase activity assay to monitor the tested promoter activity (Fig. 3Go). Higher than 1000 U of ß-galactosidase activity was considered as positive expression (represented by filled circles). There are variations of reporter gene activities due to the difference in the site of transgene integration into the mouse chromosome in each line, as shown before (39). Nevertheless, we observed a high frequency of strong transgene expression driven by the wild-type CYP11A1 promoter, as shown by the higher mean values in the ß-galactosidase activity (shown by the horizontal line). The reduced expression rates of adrenal ß-galactosidase activities were observed in the single U, C1, and C2 mutant lines, and they are more severely impaired in the double C1/C2 and U/C2 mutants. These transgenic results suggest that each of C1, C2, and U elements is required for intact CYP11A1 promoter activity in vivo.


Figure 3
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  		FIG. 3. Mutations of C1, C2, and U elements result in the reduction of CYP11A1 promoter activities in transgenic mice. The female adrenal homogenates of transgenic mouse lines carrying the human 2.3-kb CYP11A1 promoter linked to the LacZ gene without (WT) or with mutation in U (Umt), C1 (C1mt), C2 (C2mt), C1/C2 (C1/C2mt), or U/C2 (U/C2mt) were collected. Each circle represents an individual transgenic line. Open circles represent lines that express ß-galactosidase activity at lower than the cutoff value of 1000, which is shown as the dashed line. Filled circles represent positive expression above 1000 U. The mean expression levels of mouse lines from the same construct are shown by horizontal lines. All mutants have lower expression levels than the wild-type, and the double mutants are worse than the single mutants, suggesting that C1, C2, and U elements all are needed for full CYP11A1 promoter activity in vivo.

 
Combination of C1 and C2 are essential for hormonal regulation in transgenic mice
We previously showed that the U mutation resulted in loss of the hormonal response in the transgenic mice (12). We would like to test whether C1 and C2 also play a similar role in mice. Most of the mutant transgenic mice already lost their ability to express ß-galactosidase, but few of them still have reasonable activities. To test whether the residual promoter activity could respond to the hormonal regulation, we injected hormones into these best expressors and measured their adrenal ß-galactosidase activities. Comparing to the wild-type, the ACTH-stimulated adrenal ß-galactosidase activities of all tested mutants, including C1mt, C2mt, and C1/C2mt, were reduced (Fig. 4AGo). The C1mt still responded to ACTH somewhat, but C2mt retained little response. Moreover, the C1/C2mt line was completely resistant to the ACTH stimulation. The ACTH-stimulated serum corticosterone levels from all tested mutant lines, comparable to that of the wild-type, approved the efficacy of injected ACTH in the mutant mice (Fig. 4BGo).


Figure 4
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  		FIG. 4. Lack of ACTH induction from the mutated CYP11A1 promoter. A, Adrenal expression of mutated CYP11A1 promoter-driven reporter gene in response to ACTH. The female transgenic mouse lines carrying the wild-type or mutant human 2.3-kb CYP11A1 promoter linked to the LacZ gene were injected with ACTH and the reporter activities from adrenal were measured 7 d later. Eight wild-type mice and five mice from each mutant genotype were used. B, Normal induction of serum corticosterone levels indicates normal ACTH function in these mice. Despite a normal ACTH-regulated hormone profile, the combined mutations of C1 and C2 completely abolish the ACTH responsiveness of the tested CYP11A1 promoter in adrenals.

 
The effect of hCG injection on the reporter gene expression in testes was also assayed. Unlike the wild-type, the testicular ß-galactosidase activities of these tested mutant lines were silent to hCG stimulation (Fig. 5AGo). Similar to that of the wild-type, the serum testosterone levels of the tested mutants were dramatically increased by hCG (Fig. 5BGo). These results document that either one of C1 and C2 elements is essential for the full response of CYP11A1 to ACTH and hCG in the adrenals and testes, respectively, yet C2 seems more important with respect to adrenal expression.


Figure 5
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  		FIG. 5. Lack of hCG induction from the mutated CYP11A1 promoter. A, Testicular expression of mutated CYP11A1 promoter-driven reporter gene in response to hCG. The male transgenic lines carrying the wild-type or mutant human 2.3-kb CYP11A1 promoter linked to the LacZ gene were injected with hCG, and the reporter activities from testis were measured 7 d later. Eight wild-type mice and five mice from each mutant genotype were used. B, Normal induction of serum testosterone levels indicates normal hCG function in these mice. All single and double mutants of C1 and C2 completely lose CYP11A1 promoter activities in testes.

 
Involvement of AP-1 in U-CRS function
To test whether AP-1 is involved in the cAMP-stimulated transcription, a dominant-negative mutant of AP-1, FosB2 (44), was used. As the concentrations of cotransfected FosB2 expression plasmid were increased in Y1 cells, the cAMP-induced reporter CAT activities of pSCC#9, which contains U-CRS linked to the CAT reporter gene driven by a minimal promoter, were gradually diminished (Fig. 6AGo). This inhibitory effect of FosB2 is specific for U-CRS because the increasing FosB2 did not affect the cAMP induction of classical CRE-controlled transactivation (Fig. 6BGo). These results indicate that AP-1 proteins could mediate the cAMP-dependent transcription through interacting with U-CRS.


Figure 6
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  		FIG. 6. Effect of FosB2 on U-CRS function. Plasmids pSCC#9 containing U-CRS of human CYP11A1 linked to tkCAT (A) or control pCREtkCAT (B) (10 µg) was cotransfected with 0, 5, 10 or 20 µg FosB2 expression plasmids plus 2 µg of the internal control RSV-ßgal into Y1 cells, and the cAMP-dependent CAT reporter activities are shown in the top gel. The substrate chloramphenicol and acetylated products are indicated by arrow and arrowhead, respectively. The bottom graph shows the reporter activity after normalization with the internal control ß-galactosidase activity. The data from pSCC#9 are calculated from five repeated experiments and graphed with the mean ± SD value. Similar results from two independent experiments for pCREtkCAT were seen, and one of them is shown.

 
The AP-1 protein family contains many members including Jun, Fos, Maf, and activating transcription factor subfamilies (49). To test which members of the AP-1 family interact with U-CRS, the expression plasmids for different Jun/Fos members were separately cotransfected with pSCC#9 into Y1 cells. As shown in Fig. 7AGo, the U-CRS activity was increased with the overexpression of c-Jun, and further elevated by concomitant overexpression of c-Jun and c-Fos. JunB and JunD had no effect in the presence or absence of Fos proteins. The transactivation by c-Jun was completely blocked by the cotransfection of FosB (Fig. 7AGo). Besides, the addition of cAMP enhanced c-Jun or c-Jun/c-Fos transactivation by 2-fold (Fig. 7BGo). These results reveal that the overexpression of AP-1, c-Jun homodimer, or c-Jun/c-Fos heterodimer, mimics part of cAMP effect, and retains response to cAMP induction.


Figure 7
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  		FIG. 7. Function of AP-1 for U-CRS function in Y1 cells. A, Relative reporter activities from plasmid pSCC#9 cotransfected with (+) or without (–) the expression plasmids for c-Jun, JunB, or JunD, in combination with c-Fos or FosB. B, Relative reporter activities from plasmid 10 µg pSCC#9 cotransfected with (+) or without (–) the expression plasmids for 5 µg c-Jun, c-Fos, or FosB in the presence or absence of 8-Br-cAMP. The data are calculated from four repeated experiments and graphed with the mean ± SD value. Error bars do not show up if they are too small.

 
U and C1/C2 sites mediate synergism of SF-1 and AP-1 regulating CYP11A1 promoter activity
To further clarify the interaction of SF-1 with AP-1 members, we selected JEG-3 as a cell model because of its lack of SF-1. As shown in Fig. 8AGo, SF-1 is required for the reporter activity of pSCC#9 and exogenous c-Jun enhanced SF-1-activated reporter activities. The synergistic effect between SF-1 and c-Jun was further elevated by c-Fos, but abolished by FosB. These results demonstrate the interaction between SF-1 and AP-1 in regulating U-CRS activity and confirm the dominant-negative role of FosB in AP-1 function.


Figure 8
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  		FIG. 8. Synergistic effect between AP-1 and SF-1 on U-CRS activity is mediated by C1, C2, and U sites. A, AP-1 promotes the SF-1-enhanced activity of U-CRS. Relative reporter activities from JEG-3 cells cotransfected with 10 µg pSCC#9 or ptkCAT with (+) or without (–) the expression plasmids for 5 µg SF-1, c-Jun, c-Fos, or FosB are shown. The data are calculated from four repeated experiments and graphed with the mean ± SD value. B, Side-directed mutations of U, C1, and C2 abolish the transactivation of the CYP11A1 promoter by SF-1 and AP-1. Reporter activities from JEG-3 cells transfected with 200 ng wild-type or mutant linear plasmids (C1mt, Umt, C2mt, C1/Umt, U/C2mt, C1/C2mt, or Triple mt), 40 ng RSVCAT, 200 ng of SF-1 expression plasmid, or/and 40 ng c-Jun expression plasmids are shown. The data are calculated from three repeated experiments and graphed with the mean ± SD value. Some error bars are too small to be presented. These results indicate that U mediates SF-1 transactivation, and C1 and C2 are required for synergistic effect of AP-1 with SF-1.

 
Finally, we examine whether U and C1/C2 sites are required for the synergistic action of SF-1 and AP-1. The 2.3-kb CYP11A1 promoter with mutations at these sites was cotransfected with SF-1 and/or c-Jun (Fig. 8BGo). For the wild-type promoter, SF-1 greatly enhanced its promoter activity, which was further enhanced by cotransfection with c-Jun. However, SF-1 failed to activate any one of U-mutated constructs such as Umt, C1/Umt, U/C2mt, and triple mt; even though all constructs with normal U site like the wild-type, C1mt, C2mt, and C1/C2mt was activated by SF-1. These results demonstrate that the U site mediates SF-1 functions. Furthermore, the synergistic effect of c-Jun and SF-1 disappeared in C1mt, C2mt, C1/C2mt, and triple mt. It indicates that both C1 and C2 sites are required for the synergistic action of c-Jun and SF-1.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have characterized the functional roles of two TRE/CRE-like elements and one SF-1 element in the U-CRS of the human CYP11A1 promoter in detail. These sites bind to AP-1 and SF-1, respectively, in a cAMP-dependent manner. Furthermore, SF-1 and c-Jun act synergistically to increase CYP11A1 transcription. Transgenic mouse studies also showed that these sites are important for basal and hormone-regulated reporter gene expression in vivo. Taken together, our evidence suggests that AP-1 cooperates with SF-1 to regulate cAMP-dependent CYP11A1 transcription.

For some genes, CRE alone is sufficient to mediate cAMP response by interacting with CREB (50). Given that C1 and C2 are similar to CRE with only 1-bp mismatch, they might be expected to mediate cAMP response like CRE. Yet our results do not support this notion. We found that C1 and C2 alone are not sufficient to mediate cAMP response of U-CRS from a heterologous tk promoter (data not shown). Besides, the nature of U-CRS is distinct from that of a typical CRE because the dominant-negative mutant of AP-1 blocked U-CRS but not CRE activity (Fig. 6Go). This may be due to a weaker interaction between the imperfect TRE/CRE and AP-1/CREB proteins. The cooperation among transcription factors to form a more stable DNA-protein complex is probably required. This is consistent with the result that c-Jun alone does not activate CYP11A1 promoter; it only functions when SF-1 is present (Fig. 8Go). Similar situation was observed in the proximal StAR promoter. Multiple elements within the cAMP-response region (–105/–60) of mouse StAR gene formed DNA-protein complexes with SF-1, AP-1/CREB, CCAAT/enhancer binding protein-ß, and GATA-4 to stabilize the StAR promoter activity (29, 30, 31, 32). Furthermore, proteins that are bound to U-CRS of the CYP11A1 promoter are increased by cAMP (Fig. 1Go); this result agrees with earlier observations that cAMP increases DNA-binding activity of SF-1 (17, 51, 52).

Typically, AP-1 mediates the PKC signaling (27). The cross talks between cAMP-PKA and PKC pathways have been observed. For example, the TPA-induced TRE activation is increased by the enhanced cAMP levels (53), and PKA activates Jun (54); reciprocally, Jun/AP-1 activates CRE through a direct binding (55). Similar cross talks between the cAMP signaling and AP-1 also control CYP11A1 transcription.

cAMP stimulates gene expression in both early and delayed fashions. Genes like phosphoenolpyruvate carboxykinase are stimulated by cAMP within minutes of induction (56), yet the stimulation of CYP11A1 by cAMP signaling is slow and usually takes many hours (7, 13). Therefore, the regulatory mechanism for this delayed gene stimulation must be different from that of the immediate early genes. c-Fos expression is rapidly but transiently induced by cAMP in both Y1 (57) and the rat adrenal cells (58). This transient expression of c-Fos can be involved in the rapid but not the delayed action of cAMP. Although c-Fos can activate CYP11A1 promoter when supplemented exogenously (Figs. 7Go and 8AGo), the lack of its continued presence from the adrenal cells precludes c-Fos from playing a role in activating CYP11A1 in vivo. c-Jun, on the contrary, is present in the adrenals even many hours after cAMP stimulation (58). Thus c-Jun could be the important factor for hormonal stimulation of CYP11A1 expression.

Our in vitro studies of site-specific promoter mutations indicate that the U, C1, and C2 elements contribute to cAMP induction of CYP11A1 promoter activity (Fig. 2BGo). In transgenic mouse studies, the combination of both C1 and C2 were required to fully mediate hormonal signals (Figs. 4Go and 5Go). These data complement our previous observations in transgenic mice that indicate the requirement of U in hormonal regulation (47). Altogether, our transgenic results demonstrate that each of the C1, C2, and SF-1 elements is essential for hormonal stimulation of CYP11A1 transcription. These are the first in vivo evidence to reveal the individual and coordinated roles of C1, C2, and U elements in the cAMP-dependent transcriptional regulation of CYP11A1.

In summary, the cAMP-regulated CYP11A1 expression does not totally depend on any single element. We in this paper verify the roles of U, C1, and C2 elements in CYP11A1 promoter, and provide evidence that cAMP regulates CYP11A1 transcription in vitro and in vivo through the possible interactions between SF-1 and c-Jun that bind to the U, C1, and C2 elements of U-CRS.


   Acknowledgments
 
We thank Ya-Hui Tsai and Shu-Jan Chou for excellent technical assistance. Transgenic mice were generated by Transgenic Core Facility at Academia Sinica.


   Footnotes
 
This work was supported by the Academia Sinica and the National Taiwan University, and by Grants NSC 89-2311-B-001-114-B25 (to B.-c.C.) and NSC 95-2313-B-002-061-MY3 (to I.-C.G.) from the National Science Council, and DOH95-TD-HF-111-003(1/3) (to I.-C.G.) from the Department of Health, Executive Yuan, Republic of China.

Disclosure Summary: I.-C.G., C.-Y.H., C.-K.L.W., and B.-c.C. have nothing to declare.

First Published Online January 11, 2007

Abbreviations: AP-1, Activating protein-1; CAT, chloramphenicol acetyltransferase; CRE, cAMP-responsive element; CREB, cAMP-responsive element-binding protein; hCG, human chorionic gonadotropin; PKA, protein kinase A; PKC, protein kinase C; RSVCAT, rous sarcoma virus-CAT; SF-1, steroidogenic factor-1; Sp1, specific protein 1; StAR, steroidogenic acute regulatory protein; TRE, TPA-responsive element; U-CRS, upstream cAMP-responsive sequences.

Received July 13, 2006.

Accepted for publication January 2, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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