(Received for publication, April 12, 1995)
From the
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.
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) ( 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
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 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
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
(
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
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
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
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 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
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
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
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
Figure 8:
EGF stimulation of
p42
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
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
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. ( 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 ( 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- 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 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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)and insulin-like growth factor-1,
which are produced locally in the adrenal cortex, corpus luteum, and
placenta(2) , providing paracrine mechanisms for regulation.
(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.
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 pALUC (30) . The
pA
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
-subunit (GPH
) reporter GPH
CRE TKLUC consists of the two
GPH
CREs linked to the TK promoter(32) .
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
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.
, 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 (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
NaVO
) 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.).
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).
(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.
(CYP AP-1)TKLUC) conveyed
5-6-fold activation by EGF, and there was greater induction after
multimerization (p
(CYP AP-1)TKLUC, 7-fold;
p
(CYP AP-1)TKLUC, 9-fold; p
(CYP AP-1)TKLUC,
14-fold). The previously characterized AP-1 reporter
p
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
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) 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
(data not shown). There was a 50%
reduction in PKA
-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
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
expression vector (Fig. 3B), and there was no significant additional
activation by EGF in the presence of the PKA
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.
). 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
. A comparison was made to
p
(CYP AP-1), p
(CYP AP-1), GPH
CRETKLUC
(which contains a known CRE), and TKLUC or pA
LUC plasmids.
-Fold stimulation was determined relative to cells transfected with a
mutant PKA
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%.
(CYP
AP-1)TKLUC, 7-fold; p
(CYP AP-1)TKLUC, 8-fold; and
p
(CYP AP-1)TKLUC, 13-fold). By comparison, the collagenase
AP-1 reporter, p
TPLUX, was stimulated 7-fold by c-Jun,
whereas TKLUC and the promoterless vector, pA
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 GPH
CRETKLUC (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.
), 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
(CYP AP-1LUC)) and with p
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.
(CYP AP-1)TKLUC, or p
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.
-
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.
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.
activity in JEG-3 cells. JEG-3 cells were
treated with EGF (10 ng/ml), tumor necrosis factor
(TNF
) (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.
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.
(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).
)
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.
B(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
197-248 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.
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.
, glycoprotein hormone
-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.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.