©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Epidermal Growth Factor and c-Jun Act via a Common DNA Regulatory Element to Stimulate Transcription of the Ovine P-450 Cholesterol Side Chain Cleavage (CYP11A1) Promoter (*)

(Received for publication, April 12, 1995)

Richard G. Pestell Chris Albanese Genichi Watanabe Janet Johnson Nathan Eklund Przemyslaw Lastowiecki J. Larry Jameson (§)

From the Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University Medical School, Chicago, Illinois, 60611

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The P-450 side chain cleavage (CYP11A1) gene encodes the enzyme that catalyzes the initial step in steroid biosynthesis, resulting in the conversion of cholesterol to pregnenolone. Expression of the CYP11A1 gene is increased by hormones, such as adrenocorticotropin and luteinizing hormone, as well as by a number of growth factors, suggesting that its promoter may contain regulatory elements that respond to multiple signal transduction pathways. Using transient expression assays of the ovine CYP11A1 promoter in JEG-3 placental cells, distinct regulatory elements were found to mediate transcriptional stimulation by cAMP and epidermal growth factor (EGF). The cAMP response was mediated through a GC-rich sequence localized between -117 and -92. In contrast, EGF induced CYP11A1 transcription through an adjacent but distinct sequence (-92 to -77 base pairs) that was shown previously to bind nuclear proteins in DNase I footprinting reactions. This EGF-responsive element (EGF-RE) resembles an activator protein-1 (AP-1) site and was also required for transactivation by co-transfected c-Jun. A point mutation within the EGF-RE impaired stimulation by both EGF and c-Jun, suggesting that these pathways converge on a common regulatory element. Transfer of single or multiple copies of the EGF-RE upstream of an heterologous promoter conferred EGF and c-Jun responses, providing further evidence that this element is sufficient for both responses. Transfection studies employing mutant c-Jun proteins confirmed a requirement for its DNA binding, leucine zipper and amino-terminal domains, each of which are required for activation of a classical AP-1 reporter. Gel shift studies demonstrated that protein binding to the CYP11A1 EGF-RE was competed specifically by a canonical AP-1 site, and the addition of an anti-JUN antibody confirmed the presence of AP-1 proteins. Consistent with the possibility that EGF may act in part via c-Jun, EGF stimulated the activity of a chimeric GAL4 c-Jun protein, indicating that JUN can serve as a potential target of EGF in JEG-3 cells. EGF also induced mitogen-activated protein kinase activity, and a dominant negative mutant of mitogen-activated protein kinase partially blocked EGF stimulation of GAL4 c-Jun activity. We conclude that EGF stimulates the CYP11A1 promoter through an AP-1 like element and that c-Jun is one of the targets of EGF action.


INTRODUCTION

The cholesterol side chain cleavage (P-450 SCC or CYP11A1) enzyme is highly expressed in steroidogenic tissues such as the adrenal gland, gonads, and placenta, where it catalyzes the initial step in steroid biosynthesis, converting cholesterol to pregnenolone. A number of different hormones and growth factors have been shown to regulate the expression and biosynthesis of this enzyme(1) . In the adrenal gland, adrenocorticotropin increases CYP11A1 gene expression. In the gonads, its expression is controlled in part by follicle-stimulating hormone and luteinizing hormone. Signaling via these G-protein-coupled receptors likely involves activation of the cAMP-stimulated protein kinase A pathway. CYP11A1 gene expression is also stimulated by growth factors such as epidermal growth factor (EGF) (^1)and insulin-like growth factor-1, which are produced locally in the adrenal cortex, corpus luteum, and placenta(2) , providing paracrine mechanisms for regulation.

Given the multiple hormonal and growth factor inputs that regulate CYP11A1 gene expression, it is likely that a number of distinct DNA regulatory elements are required for normal physiologic responses that alter promoter activity. Transient gene expression studies have delineated several cAMP regulatory sequences in the promoters of CYP11A1 genes(7, 8, 9, 10, 11, 12, 13) . The locations of the cAMP regulatory sequences vary among species(8, 12) , and distinguishable promoter regions have been shown to convey cAMP responsiveness of the human CYP11A1 promoter in different steroidogenic cell types(9, 10, 11) . Some of these cAMP regulatory elements differ from the canonical sequences that bind members of the B-Zip family such as CREB, activation transcription factors, c-Jun, and c-Fos and are comprised of GC-rich sequences that interact with proteins that remain to be fully characterized(8, 13) .

Although the cAMP pathway is an important regulator of both basal and hormone-induced expression, basal promoter activity is also maintained independently of the cAMP pathway, perhaps by paracrine growth factors or other mechanisms(13) . The regions of the CYP11A1 gene mediating growth factor responsiveness have not been investigated previously. In this study, we sought to identify the CYP11A1 promoter regulatory elements that mediate EGF stimulation in JEG-3 choriocarcinoma cells. EGF acts via a tyrosine kinase receptor that elicits sequential activation of p21(14) and protein kinases including the mitogen-activated protein kinases (MAPKs) and stress-activated protein kinases(15, 16, 17) . Several nuclear proteins, including c-Jun(18, 19, 20, 21) , are phosphorylated and transcriptionally activated by these signaling pathways. In conjunction with other members of the activator protein-1 (AP-1) complex, c-Jun couples extracellular signals to alterations in gene transcription(22, 23) . Therefore, we also examined a potential role for c-Jun in EGF-mediated stimulation of CYP11A1 gene expression.


MATERIALS AND METHODS

Reporter Genes and Expression Vectors

The reporter -2700 CYPLUC consists of a 2700-bp ovine CYP11A1 promoter fragment derived as an EcoRI/Pst fragment from -2700 SCC/CAT (7) and inserted into the luciferase vector pA(3)LUC (30) . The pA(3)LUC vector includes a trimerized SV40 poly(A) termination site, which reduces transcriptional read-through(33) , and does not contain AP-1-responsive vector sequences(34) . The -183 CYPLUC reporter construct, consisting of the ovine CYP11A1 promoter from -183 to +50, was cloned using polymerase chain reaction amplification of ovine genomic DNA (31) using oligonucleotide primers directed against the published sequence(7) . -117 CYPLUC consists of a 140-bp Rsa fragment, and -77 CYPLUC was created using a 115-bp PVUII fragment derived from -183 CYPLUC. The reporter, -92 CYPLUC, was created by polymerase chain reaction with amplification using specific oligonucleotide primers. Mutation of the footprinted CYP11A1 AP-1 like sequences from 5` GCT GGA GTC AGC TGG 3` to 5` GCT GGC GTT AGC TGG 3` (mutations underlined) was performed using polymerase chain reaction in the context of the -117 CYPLUC reporter to create -117 CYP AP-1 mutLUC. The glycoprotein hormone alpha-subunit (GPHalpha) reporter GPHalphaCRE TKLUC consists of the two GPHalpha CREs linked to the TK promoter(32) .

To create constructs containing the CYP AP-1 like element upstream of a heterologous promoter, double-stranded CYP AP-1-like element oligonucleotides (-92 to -77 bp) were cloned as single or multimeric sites into the TKpA(3)LUC reporter and are referred to as p(CYP AP-1)TKLUC where n is the number of CYP11A1 AP-1 sites. The constructs were made with the CYP11A1 AP-1 site in both the sense and antisense orientations. The reporter p(3)TPLUX contains trimeric wild type collagenase AP-1-responsive reporter elements(32) . An element (-114 5` GTT TGG GAG GAG CTG TGT GGG CTG 3`) previously referred to as ovine footprint 5 (OF5)(7) , which contains a putative cAMP-responsive sequence, was also cloned into the TKLUC reporter. The integrity of all new constructs was determined by restriction enzyme analysis and dideoxy DNA sequencing (35) using an Applied Biosystems automated sequencer.

The expression vectors for wild type and mutant Rous sarcoma virus c-Jun proteins have been described previously(32) . The construction of the plasmid encoding the wild type and mutant catalytic subunits of protein kinase A(39) , pCMV-p41, which contains the full-length human p41 cDNA, and p41 mut, a mutant of the ATP binding site pCMV-p41(Ala Ala)(40) , and the expression vectors GAL4 c-Jun 5-253(38) , GAL4 CREBA, and GAL4 DNA binding domain(41) , were described previously.

Cell Culture, DNA Transfection, and Luciferase Assays

JEG-3 choriocarcinoma cells (American Type Culture Collection, Rockville, MD) were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum, 1% penicillin, and 1% streptomycin. Cells were transfected by calcium phosphate precipitation, the media was changed after 6 h, and luciferase activity was determined after a further 24 h as described previously(32) . At least three different plasmid preparations of each construct were used. In co-transfection experiments a dose response was determined in each experiment with 40, 60, 80, 100, or 200 ng of c-Jun expression vector and the CYP11A1 reporter plasmids (1.6 µg). EGF treatment was performed for 6-24 h at doses from 2 to 20 ng/ml to determine maximal responses. Subsequent experiments were conducted using EGF at 20 ng/ml for 24 h. In co-transfection experiments, comparison was made between the effect of transfecting 100 ng of active expression vector with the effect of an equal amount of the parental empty expression vector. Luciferase assays were performed at room temperature using an Autolumat LB 953 (EG& Berthold). Luciferase content was measured by calculating the light emitted during the initial 30 s of the reaction, and the values are expressed in arbitrary light units(32) . Background activity from cell extracts was typically <150 arbitrary light units/30 s.

Electrophoretic Mobility Gel Shift Assays

The oligodeoxyribonucleotides used in electrophoretic mobility gel shift assays correspond to the CYP11A1 promoter AP-1-like region at -92 (5` GCT GGA GTC AGC TGG 3`), previously shown to include footprinted sequences referred to as ovine footprint 4 (OF4) (7) . The wild type AP-1 site (TCC ATT CTG ACT CAT TTT TTT TAA) and a mutant AP-1 site (TCC ATT CTG CCG CAT TTT TTT TAA) were used as controls. Nuclear extracts (7) were used in electrophoretic mobility gel shift assays as described previously(32) . The protein-DNA complexes were analyzed by electrophoresis through a 5% polyacrylamide gel in a 0.5 TBE buffer (1 TBE: 0.045 M Tris borate, 0.001 M EDTA) and 2.5% glycerol. Autoradiography was performed at -70 °C using Kodak XAR5 film with an intensifying screen.

MAPK Assays

Cell extracts were prepared from JEG-3 cells treated with EGF (10 ng/ml), tumor necrosis factor alpha (50 ng/ml), or vehicle for 20 min and used for MAPK assays as recently described(15, 43) . Staphylococcal protein A-Sepharose beads were incubated with anti-MAPK antibody (C16) (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at 4 °C. The antibody and beads were washed once with RIPA buffer and then incubated with cell lysates for 2 h at 4 °C. The immunoprecipitates were washed with RIPA buffer once, with LiCl, 0.1 M Tris base, pH 8.0, twice, and once in kinase buffer. The kinase reactions were performed at room temperature for 20 min in 30 µl of kinase buffer with 10 µCi of [P]ATP (3000 Ci/mmol; 1 Ci = 37 GBq) and 4 µg of myelin basic protein. The samples were analyzed by SDS-polyacrylamide gel electrophoresis upon termination of the reaction with Laemmli buffer and boiling. The phosphorylation of myelin basic protein was quantified by densitometry using a Fuji Bio Imaging Analyzer BAS 2000.

Western Blots

For the detection of c-Jun protein, cell fractions from the nuclear and cytoplasmic fractions were prepared as described(45) . In brief, cells were lysed in 3-5 volumes of a buffer (50 mM Tris-HCl, pH 7.4, 0.25 M NaCl, 0.1% Triton X-100, 1 mM EDTA, 50 mM NaF, 1 mM dithiothreitol, 0.1 mM Na(3)VO(4)) containing 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml of leupeptin, 10 µg/ml of soybean trypsin inhibitor, 10 µg/ml of 1-chloro-3-tosylamido-7-amino-2-heptanone, and 1 µg/ml of aprotonin. After incubation on ice, the samples were centrifuged at 14,000 rpm for 5 min at 4 °C to recover a Triton-soluble fraction. The pellet was resuspended in the original volume of the same buffer containing 1% SDS. After incubation on ice for 30 min, samples were centrifuged for 5 min at 4 °C to recover a Triton-insoluble fraction (supernatant). Separated proteins were transferred to nitrocellulose as described previously (46) and probed with a c-Jun antibody (c-jun Ab2, Oncogene Sciences, Uniondale, NY) at a 1:500 dilution. Reactive proteins were visualized using an anti-rabbit horseradish peroxidase second antibody, and reactive proteins were visualized by the enhanced chemiluminescence system (Amersham Corp.).


RESULTS

EGF Stimulates CYP11A1 Transcription-The structure of the ovine CYP11A1 promoter and several of its regulatory DNA elements is depicted in Fig. 1A. The full-length -2700 CYPLUC reporter was transfected into JEG-3 choriocarcinoma cells and examined for responsiveness to EGF. A dose-responsive increase in CYPLUC activity was observed with maximal stimulation (5-fold) at 20 ng/ml (data not shown). When cells were treated for 3, 6, 12, and 24 h, the maximal EGF effect was observed within 3 h and persisted at 24 h. A series of 5` deletions of the CYP11A1 promoter was examined to delineate the minimal region responsive to EGF. Basal activity decreased progressively with deletions from -2700 to -92 bp (Fig. 1B). Although the basal activity of the -92 construct was relatively low (3000 arbitrary light units), it was well above background luciferase activity. These same constructs were analyzed for induction by EGF (Fig. 1C). EGF responsiveness was similar (3.4-4.8-fold) for the -2700, -183, -117, and -92 bp constructs but decreased to less than 1.5-fold upon deletion from -92 to -77 bp (Fig. 1C).


Figure 1: EGF activates transcription of the ovine CYP11A1 promoter. A, schematic representation of the ovine CYP11A1 promoter. The footprinted regions between -117 and -92 (OF5) (7) (grayoval), and between -92 and -77 (OF-4) (stripedoval) are shown. Three additional footprinted regions are shown as openovals. B, basal activity of 5` deletions of the CYP11A1 promoter in JEG-3 cells. C, EGF stimulation of 5` deletions of the CYP11A1 promoter. Transfected JEG-3 cells were treated with EGF (20 ng/ml) for 24 h before measuring luciferase activity. The -fold stimulation by EGF is relative to the basal activity of the same construct in untreated cells. The data are the mean ± S.E. of at least 14 separate transfections. ALU, arbitrary light units.



The deleted region that reduced EGF responsiveness corresponds to a protein binding region detected by DNase I footprinting(7) . Site-directed mutagenesis was used to alter two nucleotides within a putative AP-1-like binding site. The mutation was studied within the context of the -117 bp promoter fragment because this region retains high basal activity and EGF responsiveness (Fig. 2A). Mutation of the putative AP-1 site decreased basal activity by greater than 80% and reduced EGF responsiveness to the level seen with the minimal promoter (-77 bp). These results indicate that the AP-1-like sequence contributes to basal expression and is necessary for EGF responsiveness.


Figure 2: Localization of an EGF response element in the CYP11A1 promoter. A, the effect of EGF was examined using wild type and mutant -117 CYPLUC constructs. The mutation was introduced into the AP-1-like sequence between -92 and -77 bp. The data are the mean ± S.E. of nine separate transfections. B, functional properties of the region between -92 and -77 (CYP AP-1) when linked to the TK promoter as single or multiple sites (p(CYP AP-1) where n is the number of copies of the element). Cells were stimulated with EGF as described in the legend to Fig. 1, and the data represent the mean ± S.E. of at least seven separate transfections. ALU, arbitrary light units.



In order to assess whether the region between -92 and -77 was sufficient for activation by EGF, this sequence was linked to a heterologous promoter as single or multimeric sites (Fig. 2B). A single copy of the CYP11A1 element (p(1)(CYP AP-1)TKLUC) conveyed 5-6-fold activation by EGF, and there was greater induction after multimerization (p(2)(CYP AP-1)TKLUC, 7-fold; p(3)(CYP AP-1)TKLUC, 9-fold; p(5)(CYP AP-1)TKLUC, 14-fold). The previously characterized AP-1 reporter p(3)TPLUX(32) , which contains a multimeric AP-1 site from the collagenase gene, was induced to a similar degree (6-fold) by EGF, whereas the control reporters, TKLUC and the plasmid vector pA(3)LUC, were not stimulated by EGF (Fig. 2B).

EGF Responsiveness Requires Distinct Sequences from Those Involved in cAMP Stimulation of the Ovine CYP11A1 Promoter

Previous studies demonstrated that the DNA sequences responsible for cAMP induction of the ovine CYP11A1 promoter were located within 183 bp of the transcriptional start site(7) . A series of CYP11A1 5` promoter deletion constructs were examined to assess whether activation of the protein kinase A pathway occurred through similar or distinct DNA sequences from those involved in EGF responsiveness. Cotransfection with the protein kinase A catalytic subunit (PKA(c)) caused 8-fold stimulation, whereas no stimulation was seen with a mutant catalytic subunit of protein kinase A (PKA) (Fig. 3A). Similar results were seen when 8-bromo-cAMP was used rather than PKA(c) (data not shown). There was a 50% reduction in PKA(c)-stimulated activity upon deletion from -117 to -92, which includes a GC-rich sequence highly homologous to the region involved in cAMP-induced transcription of the bovine CYP11A1 promoter(47) . When this (OF5) element (-117 to -92) was linked to a heterologous promoter and co-transfected into JEG-3 cells with the PKA(c) expression vector, it was sufficient to convey 6-7-fold activation (Fig. 3B). In contrast, the CYP11A1 sequences (-92 to -77 bp) sufficient for EGF responsiveness were not induced by the PKA(c) expression vector (Fig. 3B), and there was no significant additional activation by EGF in the presence of the PKA(c) expression vector (data not shown). These results indicate that the cAMP (-117 to -92 bp) and EGF (-92 to -77 bp) responsive regulatory elements are distinguishable.


Figure 3: Regions of the CYP11A1 promoter mediating cAMP activation are distinct from the EGF-RE. A, different 5` deletions of the CYP11A1 promoter were examined for stimulation by the co-expressed catalytic subunit of protein kinase A (PKA(c)). B, the -117 and -92 (CYP OF5) element was linked in the sense (s) or antisense (as) orientation to the TK promoter and examined for responsiveness to co-transfected PKA(c). A comparison was made to p(1)(CYP AP-1), p(3)(CYP AP-1), GPHalphaCRETKLUC (which contains a known CRE), and TKLUC or pA(3)LUC plasmids. -Fold stimulation was determined relative to cells transfected with a mutant PKA(c) and represents the mean ± S.E. of at least five separate transfections.



c-Jun Transactivates the CYP11A1 Promoter through the EGF-responsive Sequence

Because the EGF-RE contained an AP-1-like element, c-Jun was co-transfected to determine whether it was capable of simulating EGF-mediated activation of CYP11A1. The -2700 bp CYP11A1 promoter fragment was induced 6-fold by c-Jun (Fig. 4A), with somewhat less activation by co-transfection of Jun D (3.2-fold) or c-Fos (2.5-fold) (data not shown). Upon deletion from -92 to -77 bp, the induction by c-Jun was reduced from 4.4- to 1.4-fold (Fig. 4A), suggesting that it may act through the region required for EGF responsiveness. This issue was addressed further using the site-directed mutant that alters 2 bp within the putative AP-1 recognition site (Fig. 4B). As seen previously for EGF stimulation, this mutation reduced c-Jun-mediated transactivation by 70%.


Figure 4: The CYP11A1 -92 to -77 sequences are sufficient for c-Jun-induced reporter activity. A, the regulation of different 5` deletion mutants of the CYP11A1 promoter by transfected c-Jun was determined in JEG-3 cells. -Fold induction by c-Jun was derived by comparison with cells transfected with the parental plasmid without the cDNA insert (n = 11-33 separate transfections). B, the effect of the mutation of the AP-1-like sequence (-117 AP-1 mut CYPLUC) on c-Jun activation (n = 9 separate transfections). C, the effect of c-Jun on the region between -92 and -77 (CYP AP-1) linked to the TK promoter (n = 8 separate transfections). Control constructs are described in the legends to Fig. 2and Fig. 3.



The effect of c-Jun was also analyzed using constructs in which the -92 to -77 region was linked to the minimal TK promoter as single or multimeric sites (Fig. 4C). This sequence was sufficient to convey 6-fold stimulation by c-Jun with progressively greater activation using multimerized elements (p(2)(CYP AP-1)TKLUC, 7-fold; p(3)(CYP AP-1)TKLUC, 8-fold; and p(5)(CYP AP-1)TKLUC, 13-fold). By comparison, the collagenase AP-1 reporter, p(3)TPLUX, was stimulated 7-fold by c-Jun, whereas TKLUC and the promoterless vector, pA(3)LUC, were not stimulated by c-Jun (Fig. 4C). The CYP OF5 TKLUC, which contains the cAMP-responsive sequences, was repressed 3-fold by c-Jun as was GPHalphaCRETKLUC (5-fold repression), which contains canonical CREs that mediate repression by c-Jun(32) . The activation of the CYP11A1 AP-1-like sequences by c-Jun was therefore comparable with that of the canonical AP-1 site and was mediated by sequences that correspond to the EGF-RE.

A series of mutant c-Jun cDNAs were used to characterize the functional domains of the protein that are required for transcriptional induction of the CYP11A1 promoter (Fig. 5). These plasmids have been shown previously to express comparable amounts of protein in transfected cells(32, 38) . Compared with the effect of the wild type c-Jun expression vector, mutations in the dimerization domain (Dimer), the DNA-binding domain (DNA), the amino terminus (N22 and N51), and the A2 activation domain mutant (A2) reduced activation of -183 CYPLUC to less than 30% of wild type c-Jun. Dose response curves performed using up to 4-fold greater amounts of the mutants than wild type vector had little alteration in their effect on CYP11A1 transcription (data not shown). Similar effects of the c-Jun mutants were seen with the multimerized CYP AP-1 site (p(3)(CYP AP-1LUC)) and with p(3)TPLUX, which is analogous to chloramphenicol acetyltransferase constructs used in previous studies of these mutants(38) . These results indicate a requirement for the DNA binding and transactivation domains of c-Jun for stimulation of the CYP11A1 promoter.


Figure 5: The domains of c-Jun required for activation of CYP11A1 reporter activity. A series of c-Jun expression vectors were transfected with the -183 CYPLUC, the p(3)(CYP AP-1)TKLUC, or p(3)TPLUX, which contains three canonical AP-1 sites, in JEG-3 cells. The c-Jun mutant defective in dimerization (Dimer mutant) has amino acid substitutions at positions 303 and 317. c-Jun DNA has double amino acid substitutions at positions 277 and 278 within the c-Jun DNA binding domain(37) . The c-Jun mutant (Del 197-248), contains a deletion of amino acids 197-248 corresponding to the A2 activation domain(38) . The c-Jun mutants N22 and N51 contain 22 and 51 amino acid deletions from the amino terminus. The -fold induction by the expression plasmid was derived by comparison with cells transfected with the parental plasmid without the cDNA insert. The data are the mean ± S.E. of at least six separate transfections.



AP-1 Proteins Bind to the CYP11A1 EGF-responsive Sequence

Electrophoretic gel mobility shift assays were performed using the CYP11A1 EGF-responsive sequence (-92 to -77 bp) and nuclear extracts from JEG-3 cells (Fig. 6A). Several protein complexes bound to the radiolabeled EGF-RE. Protein binding was competed specifically by excess unlabeled CYP11A1 AP-1 site or by the canonical AP-1 site from the collagenase gene but not by a mutant AP-1 sequence. A polyclonal antibody that recognizes several JUN isoforms (37) was used to confirm the presence of AP-1 proteins among these protein complexes (Fig. 6B). The anti-JUN antibody greatly decreased the intensity of the uppermost band but had less effect on the binding of the other complexes. These results are consistent with the binding of JUN to the CYP11A1 AP-1-like sequence.


Figure 6: Binding of JEG-3 nuclear proteins to the CYP11A1 AP-1-like sequences. A, specific binding of AP-1 complexes to the CYP11A1 AP-1-like sequence. The -P-labeled CYP11A1 AP-1 probe was incubated with JEG-3 nuclear extracts in the presence of the indicated competitor oligonucleotides (lane1, no competitor; lanes2 and 3, excess homologous CYP11A1 AP-1 competitor; lanes3 and 4, excess wild type AP-1 sequence; lane5, excess mutant AP-1 site). B, effect of an anti-JUN antibody on protein binding to the CYP11A1 AP-1-like sequence. Gel shifts were performed as in panelA except for the addition of the JUN Ab (59) (which detects a variety of JUN family proteins) (Dr. V. Baichwal, personal communication).



EGF and MAPK Enhance Transactivation by c-Jun

Growth factors such as EGF can induce JUN protein levels as well as activating post-translational modifications(22, 23, 24) . Western blot analyses were used to assess the effect of EGF on c-Jun protein levels (Fig. 7). After a 2-h treatment with EGF, there was a 2-fold increase in c-Jun protein in the nuclear fraction of JEG-3 cells.


Figure 7: EGF stimulation of c-Jun protein levels in JEG-3 cells. Western blot analyses were performed on JEG-3 cells treated with EGF (10 ng/ml) for the indicated time points. JEG-3 nuclear extracts (50 µg) were immunoblotted using a c-Jun antibody (jun Ab 2, Oncogene Science, 1:500 dilution). c-Jun protein is indicated by an arrow.



EGF has been shown to induce phosphorylation of the MAPKs, p42 and p44(16) , which are capable of activating several nuclear transcription factors including c-Jun (18, 19, 20) . The effect of EGF on MAPK activity in JEG-3 cells was assessed using myelin basic protein as a substrate (Fig. 8). MAPK activity was induced 4-fold by EGF (10 ng/ml) at 20 min but was not induced by vehicle alone. Treatment of cells with tumor necrosis factor (50 ng/ml) for 20 min induced MAPK activity 2-fold.


Figure 8: EGF stimulation of p42 activity in JEG-3 cells. JEG-3 cells were treated with EGF (10 ng/ml), tumor necrosis factor alpha (TNFalpha) (50 ng/ml), or vehicle for 20 min. Cell extracts (300 µg) were immunoprecipitated using a polyclonal anti-MAPK antibody (C16), and kinase assays were performed as described under ``Materials and Methods'' using treated or untreated cell extracts. The results of a representative experiment that was repeated on three separate occasions is shown.



A chimeric GAL4 c-Jun construct was used to examine the effect of EGF upon c-Jun transcriptional activity (Fig. 9). The GAL4 DNA binding domain was linked to amino acids 5-253 of c-Jun, which encode the amino-terminal and A2 activation domains(38) . GAL4 c-Jun activity was assayed using a reporter gene containing GAL4 DNA binding sites (UASTKLUC). Transfection of GAL4 c-Jun induced UASTKLUC reporter activity 5-fold (n = 20) compared with the activity of the GAL4 DNA binding domain alone (Fig. 9). EGF increased transactivation by GAL4 c-Jun activity an additional 3-4-fold (n = 10), but did not increase the effect of GAL4 CREB (data not shown). Co-transfection of the wild type p41 expression vector with GAL4 c-Jun increased transactivation by 2.2-fold (n = 10). Overexpression of the dominant negative p41 expression vector (MAPKi) reduced basal GAL4 c-Jun activity by 20%. In the presence of MAPKi, EGF induction of GAL4 c-Jun was reduced by approximately 60%. These results demonstrate that EGF augments transactivation by c-Jun in JEG-3 cells and that a dominant negative inhibitor of the MAPK pathway impairs the effects of EGF.


Figure 9: c-Jun mediated transactivation of a heterologous reporter in JEG-3 cells. A, schematic diagram of GAL4 c-Jun and GAL4 DNA binding domain. B, the GAL4-responsive luciferase reporter gene, UASTKLUC. The effect of EGF (n = 20) and the effect of expression vectors encoding wild type or mutant p41 (n = 8) on GAL4 c-Jun was determined using the reporter UASTKLUC (4.8 µg). EGF (20 ng/ml) was used to treat the cells for 24 h (n = 10). The -fold induction of GAL4 c-Jun by EGF was corrected for a 1.4-fold effect on the reporter plasmid, UASTKLUC, in the absence of GAL4 c-Jun. GAL4 CREB was not induced by EGF (data not shown).




DISCUSSION

In this study, we have shown that the ovine P450 SCC (CYP11A1) gene is a target of transcriptional induction by EGF. The EGF-RE was localized between -92 and -77 bp, a region that includes an AP-1-like element. The EGF-responsive sequence was distinguishable from an adjacent cAMP-responsive region (-117 to -92 bp). c-Jun also stimulated the CYP11A1 promoter and required sequences that co-localized with the EGF-RE. Taken together with evidence that JUN binds to the AP-1-like sequence in the EGF-RE and that EGF stimulates transactivation by GAL4 c-jun, these findings are consistent with a role for AP-1 proteins in EGF activation of the CYP11A1 promoter.

In previous studies, cAMP regulatory elements were identified in the CYP11A1 promoters of other species. The human CYP11A1 promoter contains cell type-specific cAMP regulatory regions, which are located between -1733 and -1621 in Y1 cells(9) , between -1676 and -1620 in MA10 cells(10) , and between -108 and -89 in JEG-3 cells(11) . The -108 region in the human CYP11A1 promoter may contain AP-1 and AP-2 sites, both of which have been shown to confer regulation by cAMP in other genes(23, 53, 54) . The region required for cAMP stimulation of the rat Cyp11a1 promoter in granulosa cells lies between -73 and -38 and includes the sequence AAGTCA(12) . Together these findings suggest cAMP-induced transcription of the CYP11A1 involves several distinct DNA sequences. In the case of the ovine CYP11A1 promoter, cAMP stimulation in JEG-3 cells was conveyed primarily through a region between -117 and -92 bp. This region shares sequence homology with the site conveying cAMP responsiveness in the bovine CYP11A1 gene(8) . As with the homologous bovine sequences, the ovine -117 to -92 sequences also bind Sp-1 in electrophoretic mobility gel shift assays. (^2)

Although the cAMP-dependent pathway is an important regulator of CYP11A1 expression, additional cAMP-independent factors are also involved in the regulation of this promoter(13) . Ad4BP (also called steroidogenic factor-1, or SF-1) increases expression of the CYP11A1 promoter in steroidogenic cells(52) , and the Ad4BP site in the human CYP11A1 promoter is an important basal enhancer element(52) . Recent studies have demonstrated that insulin-like growth factor-1 stimulated the porcine CYP11A1 promoter through a GC-rich region(55) , and the identification of the EGF-RE represents yet another distinct regulatory element. Thus, the CYP11A1 promoter contains numerous distinct regulatory elements, a finding that is consistent with its complex regulation by different hormones and growth factors.

Several lines of evidence suggest that members of the AP-1 family are involved in the regulation of growth factor responsiveness(6, 22, 24, 25, 26, 54) . The JUN and FOS protein families are induced by growth factors, including EGF and fibroblast growth factor(6, 22, 25, 26) . In addition, inhibition of c-Jun expression with antisense RNA (27) or microinjection of AP-1 antibodies (28, 29) inhibits growth factor-dependent cellular proliferation and cell cycle progression. The EGF-RE in the ovine CYP11A1 promoter (GGAGTCAGCTGGAGG) resembles a subset of AP-1 sites (underlined)(56) . This AP-1-like motif is one of the few regions of the CYP11A1 promoter that is highly conserved among species(7) . A related AP-1-like sequence is found in the human CYP11A1 promoter at -605(3) , in the bovine promoter at -110 (4) and in the murine promoter at -319(5) . Of note, the bovine CYP11A1 AP-1-like site is not involved in PMA-regulated expression in cultured ovarian luteal cells and did not bind ovarian AP-1-related proteins(47) . However, the bovine CYP11A1 AP-1-like sequence (TGAGTCT) differs from the ovine motif in the boldface flanking sequences and in its contextual bases. The role of the sequences resembling the AP-1 site in the CYP11A1 promoters of other species in growth factor responsiveness will be of considerable interest.

The co-localization of the c-Jun-responsive region of the ovine CYP11A1 to the -92 to -78 region is consistent with a role for c-Jun and potentially other AP-1 proteins in the regulation of EGF-induced CYP11A1 transcription. The role of c-Jun in the activation of the CYP11A1 promoter was investigated using several complementary techniques. Expression vectors encoding mutant c-Jun proteins (38) were used to evaluate the domains of c-Jun required for transactivation of the CYP11A1 promoter. These experiments show that the leucine zipper and DNA binding domain of c-Jun are required for activation of the CYP11A1 promoter, implying that dimerization of c-Jun is required to form a functional transcriptional complex. c-Jun is known to interact with several different types of transcription factors, including members of the CREB/activation transcription factor family, NF-kappaB(36) , the helix-loop helix proteins(42) , a cell type-specific inhibitor of c-Jun (38) , the Maf proteins(57) , and several nuclear receptors (reviewed in (23) ). Further studies will be required to evaluate the potential heterodimeric partners of c-Jun that exist in the context of the CYP11A1 promoter AP-1 site. The c-Jun mutant Delta197-248 and the amino-terminal deletion mutants had less than 10% wild type activation of the CYP11A1 promoter. The 197-248 region is required for dephosphorylation-dependent activation of c-Jun by MAPK(s)(19, 24, 50, 51) , and the amino terminus of c-Jun is the target of stress-activated protein kinase activation (JNK1)(44) . Thus, several mutants that are known to alter key functional domains of c-Jun impaired or eliminated its ability to stimulate the CYP11A1 promoter.

The binding of AP-1 proteins to the CYP11A1 AP-1-like sequence in the EGF-RE provides further evidence for a direct role in the regulation of the EGF response. The gel shift assays revealed that several different specific complexes bind to the AP-1-like sequence. Each of these complexes was competed by a consensus AP-1 site, consistent with the presence of AP-1-related proteins. Although an anti-JUN antibody reduced the binding of the uppermost of these complexes, several others were minimally altered by this antibody. The nature of the different protein complexes is presently unknown. The AP-1 family of proteins includes Jun D and Jun B as well as c-Jun. As noted above, proteins in this family can bind as homodimers or as heterodimers with c-Fos or Fos-related proteins (e.g. Fra-1 and Fra-2) as well as interacting with numerous other transcription factors. In addition, phosphorylation could give rise to complexes with different mobilities. In this regard, it is notable that the CYP11A1 AP-1 site is structurally similar to a site in the Krox 24 gene (GCAGTCA)(56) . The Krox 24 gene sequences bind predominantly jun D, c-Jun, and Fos B(56) . Further studies will be necessary to characterize the different proteins that bind to the CYP11A1 EGF-RE.

EGF doubled c-Jun protein, providing one explanation for how it might enhance AP-1 activity. However, post-translational modification by phosphorylation is also critical for transcriptional activation by c-Jun(24) . c-Jun transcriptional activity can be stimulated by the MAPK, stress-activated protein kinase (JNK1) pathways in a cell type-specific manner(15, 17, 44) . We demonstrated that EGF activated MAPK activity in JEG-3 cells. Whether the stress-activated protein kinase and JNK pathways are also activated remains to be investigated. The GAL4 c-Jun chimeric protein was used to assay transcriptional activation of c-Jun since it circumvents some of the issues concerning dimerization partners and proteins that may compete at the level of the target DNA sequence. EGF treatment or overexpression of p41 induced GAL4 c-Jun transcriptional activity, indicating that c-Jun is a target of each of these pathways. The ability of a dominant negative mutant of MAPK to inhibit EGF stimulation of GAL4 c-Jun is consistent with the involvement of MAPK in this pathway. Although MAPK activates other nuclear transcription factors, including c-Myc and ETS-related proteins(48, 49, 58) , c-Myc and ETS sites are not found within the EGF-RE (-92 and -77 bp). An ETS consensus at -76 is located within a region of the promoter that is not activated by EGF. Unlike the -92 to -77 region, which is footprinted by JEG-3 cells, primary adrenal nuclear extracts, and primary placental nuclear extracts, the ETS-like sequences at -76 are not footprinted(7) . Thus, c-Jun, and not ETS proteins, appears to be the likely mediator of EGF-induced CYP11A1 promoter activity.

In conclusion, these studies demonstrate that cAMP and EGF activate the ovine CYP11A1 promoter in JEG-3 cells through distinguishable regions. EGF induced c-Jun protein levels, increased MAPK activity, and augmented transactivation by c-Jun in JEG-3 cells. The same CYP11A1 promoter sequences were required for activation by EGF and c-Jun in the context of the native or a heterologous promoter. These data are consistent with a model in which c-Jun may convey an important component of EGF-dependent activation of CYP11A1 transcription.


FOOTNOTES

*
This work was supported in part by U.S. Public Health Service Grant HD 23519 (to J. L. J.), by the NCI (National Institutes of Health) Clinical Investigator Award KO8 CA 620008 01, and by the American Cancer Society (Illinois Division, Inc.) Grant 94-27 (to R. G. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
A recipient of the Neil Hamilton Fairley Postdoctoral Fellowship from the Australian Medical Research Council and the Royal Australian College of Physicians Winthrop Travelling Fellowship. To whom correspondence should be addressed: Division of Endocrinology, Metabolism and Molecular Medicine, Tarry 15, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611. Tel.: 312 503 0644; Fax: 312 908 9032; pestell{at}merle.acns.nwu.edu.

^1
The abbreviations used are: EGF, epidermal growth factor; EGF-RE, EGF-responsive element; MAPK, mitogen-activated protein kinase; bp, base pair(s); PCR, polymerase chain reaction; GPHalpha, glycoprotein hormone alpha-subunit; CRE, cAMP-responsive element; CREB, CRE binding protein; TK, thymidine kinase; LUC, luciferase; OF4 and OF5, ovine footprint 4 and 5, respectively; RIPA buffer, radioimmune precipitation buffer.

^2
R. G. Pestell, G. Watanabe, and P. Lastowiecki, unpublished data.


ACKNOWLEDGEMENTS

We are grateful to Dr. A. Schneyer for the sheep adrenal used as a source of DNA for polymerase chain reaction of the ovine CYP11A1 promoter; Dr. T. Curran, Dr. V. R. Baichwal, Dr. R. Tjian, Dr. M. Gilman, Dr. J. Massague, Dr. J. P. Coghlan, Dr. R. Maurer, and Dr. R. Davis for plasmids; and J. Burrows for technical assistance. We thank Vidya Sundaresan for advice on MAPK assays and Dr. W. Lowe, Jr., for reading the manuscript.


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