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Molecular Endocrinology 18(3):558–573
Copyright © 2004 by The Endocrine Society
doi: 10.1210/me.2003-0223
Assessment of the Role of Activator Protein-1 on
Transcription of the Mouse Steroidogenic Acute
Regulatory Protein Gene
PULAK R. MANNA, DARRELL W. EUBANK,
AND
DOUGLAS M. STOCCO
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center,
Lubbock, Texas 79430
cAMP-dependent mechanisms regulate the steroidogenic acute regulatory (StAR) protein even
though its promoter lacks a consensus cAMP
response-element (CRE, TGACGTCA). Transcriptional regulation of the StAR gene has been
demonstrated to involve combinations of DNA
sequences that provide recognition motifs for
sequence-specific transcription factors. We recently identified and characterized three canonical
5ⴕ-CRE half-sites within the cAMP-responsive region (ⴚ151/ⴚ1 bp) of the mouse StAR gene. Among
these CRE elements, the CRE2 half-site is analogous (TGACTGA) to an activator protein-1 (AP-1)
sequence [TGA(C/G)TCA]; therefore, the role of the
AP-1 transcription factor was explored in StAR
gene transcription. Mutation in the AP-1 element
demonstrated an approximately 50% decrease in
StAR reporter activity. Using EMSA, oligonucleotide probes containing an AP-1 binding site were
found to specifically bind to nuclear proteins obtained from mouse MA-10 Leydig and Y-1 adrenocortical tumor cells. The integrity of the sequencespecific AP-1 element in StAR gene transcription
was assessed using the AP-1 family members, Fos
(c-Fos, Fra-1, Fra-2, and Fos B) and Jun (c-Jun, Jun
B, and Jun D), which demonstrated the involvement of Fos and Jun in StAR gene transcription to
varying degrees. Disruption of the AP-1 binding
site reversed the transcriptional responses seen
with Fos and Jun. EMSA studies utilizing antibodies specific to Fos and Jun demonstrated the involvement of several AP-1 family proteins. Functional assessment of Fos and Jun was further
demonstrated by transfecting antisense c-Fos,
Fra-1, and dominant negative forms of Fos (A-Fos)
and c-Jun (TAM-67) into MA-10 cells, which significantly (P < 0.01) repressed transcription of the
StAR gene. Mutation of the AP-1 site in combination with mutations in other cis-elements resulted
in a further decrease of StAR promoter activity,
demonstrating a functional cooperation between
these factors. Mammalian two-hybrid assays revealed high-affinity protein-protein interactions
between c-Fos and c-Jun with steroidogenic factor
1, GATA-4, and CCAAT/enhancer binding protein-␤. These findings demonstrate that Fos and
Jun can bind to the TGACTGA element in the StAR
promoter and provide novel insights into the mechanisms regulating StAR gene transcription. (Molecular Endocrinology 18: 558–573, 2004)
T
tion markedly affects the synthesis of the StAR protein
and steroid biosynthesis in mouse Leydig cells (2, 5,
6). Transcriptional activation by cAMP is mediated
through the interaction of the cAMP response element
(CRE) binding protein (CREB) with a consensus CRE
(5⬘-TGACGTCA-3⬘) found in the promoter of target
genes (7–9). However, the StAR gene promoter lacks
a consensus CRE and as such, resembles the promoters of several steroid hydroxylase genes that are regulated by cAMP signaling (10). The StAR promoter
sequences in mouse, rat, and human are highly homologous, and in the absence of a canonical CRE,
multiple cis-elements have been demonstrated to play
important roles in StAR gene expression (11–16).
Transcriptional regulation of the mouse StAR gene
has been shown to be mediated by combinations
of several trans-acting factors present within the
⫺151/⫺1 bp region relative to the transcription start
site (15–18). Recently, we have investigated the importance of three CRE half-sites within the ⫺96/⫺67
region of the mouse StAR promoter and in those stud-
HE STEROIDOGENIC ACUTE regulatory (StAR)
protein plays a critical role in the regulation of
steroid hormone biosynthesis by mobilizing cholesterol from the outer to the inner mitochondrial membrane (1–4). Previous studies indicate that regulation
of StAR expression and steroidogenesis occurs
through cAMP-mediated mechanisms that involve
transcriptional induction. Also, inhibition of transcrip-
Abbreviations: Ab, Antibody; AP-1, activator protein 1; AS,
antisense; ATF, activating transcription factor; (Bu)2cAMP,
dibutyryl cAMP; CCND1, cyclin D1; CRE, cAMP response
element; C/EBP␤, CCAAT/enhancer binding protein; CREB,
cAMP response-element binding protein; CREM, CRE modulator; NE, nuclear extract; PEPCK, phosphoenolpyruvate
carboxykinase; rec, recombinant; SF-1, steroidogenic factor
1; StAR, steroidogenic acute regulatory protein; TRE, phorbol
12-O-tetradecanoate 13-acetate responsive element.
Molecular Endocrinology is published monthly by The
Endocrine Society (http://www.endo-society.org), the
foremost professional society serving the endocrine
community.
558
Manna et al. • AP-1 and StAR Gene Transcription
ies, a member of the CREB family [CREB/CRE modulator (CREM)] was shown to be involved in StAR gene
regulation (16). Of these CRE elements, the CRE2
half-site [analogous to an activator protein 1 (AP-1)
recognition motif] was found to be predominantly involved in regulating StAR gene expression. Several
lines of evidence indicate that the TGACGTCA [CRE or
activating transcription factor (ATF) binding site] and
the TGA(C/G)TCA [AP-1 binding site or phorbol 12-Otetradecanoate 13-acetate responsive element (TRE)]
sequence motifs are two of the major regulatory elements that contribute to transcriptional regulation (19–
21). The CRE/ATF and AP-1/TRE sites are recognized
by proteins that share the basic region/leucine zipper
(bZip) motif but have different specificity for DNA binding affinity (22, 23). The bZip proteins are broadly
defined to at least three subfamilies of proteins, including CREB/CREM/ATF, AP-1, and C/EBPs, and
cross-dimerization among these proteins has been
demonstrated (22–27). We have recently determined
that CRE elements can bind to MA-10 nuclear proteins
and that the protein-DNA binding was markedly decreased by a consensus AP-1, suggesting that the
transcription factor AP-1 may influence StAR gene
transcription (16). As such, the close similarities between the CRE/ATF and AP-1/TRE sequence motifs
raise the possibility that these families of proteins may
bind to the same target sequence and play roles in
StAR transcription.
Mechanisms controlling transcription include the
roles of enhancer elements, which function in switching on gene expression, and negative regulatory elements, which silence transcription (7, 8, 28). Members
of the AP-1 transcription factor family are grouped into
the Fos (c-Fos, Fra-1, Fra-2, and Fos B) and the Jun
(c-Jun, Jun B, and Jun D) subfamilies, based on their
amino acid similarities, and have been shown to interact with specific DNA sequences located in the promoters of target genes (29–31). However, Fos members can form heterodimers with Jun proteins whereas
Jun members can form either homo- or heterodimers
among themselves and have been reported to play
central roles in regulating many biological functions,
including proliferation, differentiation, and transformation (31–34). Fos and Jun have been demonstrated to
have opposite effects on transcription of the phosphoenolpyruvate carboxykinase (PEPCK) (35), GnRH
(36), and myogenic helix-loop-helix (37) genes. Studies have also shown that Fos and Jun are able to
interact with different proteins and mediate transcriptional responses (38–40). Recently, c-Fos has been
reported to decrease StAR gene expression in Y-1
cells (41). Also, Fos and Jun were found to be involved
in the epidermal growth factor-mediated attenuation
of the mouse StAR promoter responsiveness in
mLTC-1 mouse Leydig tumor cells (42). However,
these studies lack a description of the molecular
events associated with the AP-1 family proteins involved in StAR gene expression. The functional importance of the CRE2 (16), and consequently the identi-
Mol Endocrinol, March 2004, 18(3):558–573
559
fication of an AP-1 element (TGACTGA) in the StAR
promoter resembling Fos and Jun recognition motifs,
prompted us to explore the role of these factors in
understanding the mechanisms involved in StAR gene
transcription.
RESULTS
Role of AP-1 Element in StAR Gene Expression
The cAMP responsive region (⫺151/⫺1 bp) of the
mouse StAR gene contains binding sites for several
transcription factors that have been demonstrated to
play roles in cAMP-mediated StAR gene regulation. In
the present study, the involvement of an AP-1 recognition motif (that overlapped with the CRE2 element at
⫺81/⫺75) in StAR gene transcription was assessed.
As illustrated using EMSA, an oligonucleotide probe
(⫺96/⫺67 bp) containing AP-1 and/or CRE elements
binds to MA-10 nuclear extract (NE) in a dibutyryl
cAMP [(Bu)2cAMP]-responsive manner (Fig. 1, compare lanes 2 and 4). Protein-DNA complexes (I, II, and
III) were decreased after addition of its cold competitor
(lanes 3 and 5), consensus CRE (Con CRE, lanes 7 and
10), and consensus AP-1 (Con AP-1, lanes 8 and 11)
sequences. Importantly, mutation generated in the
AP-1 core sequence (TGACTGA to TAGATCT) had no
affect on protein-DNA complexes (lanes 6 and 9).
The region comprising the AP-1 element in the
mouse StAR promoter (⫺81/⫺75 bp) is reasonably
conserved among different species (Fig. 2A), including
rat, human, ovine, porcine, and monkey (15–17). To
verify the involvement of the AP-1 element, a relatively
shorter oligonucleotide probe (⫺83/⫺72 bp), specific
to the AP-1 binding sequence, was examined in EMSA
using (Bu)2cAMP-stimulated nuclear extract (NE) obtained either from MA-10 (lanes 2–6) or Y-1 (lanes
8–12) cells (Fig. 2B). Using the ⫺83/⫺72 probe, protein-DNA binding occurring at complex I was nearly
abolished when compared with the ⫺96/⫺67 probe.
However, protein-DNA complexes were clearly decreased by its cold competitor (lanes 3 and 9), Con
CRE (lanes 4 and 10), and Con AP-1 (lanes 5 and 11)
sequences. On the other hand, mutation in the AP-1
sequence motif did not affect protein-DNA complexes
(lanes 6 and 12). The pattern of protein-DNA binding
was qualitatively similar in both MA-10 and Y-1 cells.
It is noted, however, that recombinant (rec) CREB
protein was shown to bind to the same region of the
StAR promoter (16, 18). Moreover, this region has
previously been demonstrated to bind C/EBP␤ (43).
The present results demonstrate that both CRE and
AP-1 DNA binding proteins bind to the TGACTGA
element and influence StAR gene expression in steroidogenic cells.
The role of the AP-1 binding motif was further investigated by determining reporter gene activity utilizing the ⫺151/⫺1 bp StAR promoter segment (Fig. 3).
This segment was chosen based on our previous
560
Mol Endocrinol, March 2004, 18(3):558–573
Fig. 1. Binding of the ⫺96/⫺67 bp Region of the StAR Promoter to MA-10 NE
Using EMSA, 10 ␮g of NE from control and (Bu)2cAMP
(500 ␮M, 6 h)-stimulated cells were used to analyze proteinDNA binding with the 32P-labeled probe (⫺96/⫺67) containing CRE and AP-1 elements. A representative phosphor image illustrates formation of three protein-DNA complexes (I,
II, and III) to the 32P-labeled probe. Competitors were used at
100-fold molar excess and include ⫺96/⫺67 (lanes 3 and 5),
consensus CRE (Con CRE, lanes 7 and 10), consensus AP-1
(Con AP-1, lanes 8 and 11), and AP-1 mutant (AP-1 Mut,
lanes 6 and 9). Protein-DNA complexes were resolved on 5%
polyacrylamide gels, the gels were dried, and the complexes
were visualized with a phosphor imaging device. The experiments were repeated three to four times. Migration of free
probes is shown on each lane.
observations in which the ⫺110/⫺1 bp region had a
decreased response in StAR promoter activity (16).
MA-10 cells transfected with the ⫺151/⫺1 StAR segment displayed a 3.6-fold increase in reporter activity
after (Bu)2cAMP stimulation. Mutation in the AP-1 element resulted in a 52% decrease in basal reporter
activity but did not significantly affect (Bu)2cAMPstimulated fold-response, demonstrating the importance of the AP-1 motif in StAR promoter
responsiveness.
Assessment of the Role of AP-1 Family Proteins
in StAR Gene Transcription
To examine whether the binding of AP-1 family members to the TGACTGA element in the mouse StAR
promoter was of functional significance, MA-10 and
Y-1 cells were transfected in the absence or presence
Manna et al. • AP-1 and StAR Gene Transcription
of Fos and Jun (Fig. 4). Transfection of MA-10 cells
with the ⫺151/⫺1 bp StAR segment resulted in a
3.4-fold increase in (Bu)2cAMP-mediated reporter activity over untreated cells. Cells transfected with the
⫺151/⫺1 bp StAR segment in the presence of Fos
members (c-Fos, Fra-1, Fra-2, and Fos B) demonstrated increases (150% for c-Fos and 270% for Fos
B) and decreases (38% for Fra-2 and 14% for Fra-1)
in basal reporter activities. However, in all cases
(Bu)2cAMP-stimulated promoter responses were decreased between 28 and 45% when compared with
control (pcDNA). Jun members showed a marked elevation in basal luciferase activity (570% with c-Jun,
285% with Jun B, and 350% with Jun D). After
(Bu)2cAMP stimulation these responses were attenuated by 42%, 36%, and 47% with c-Jun, Jun B, and
Jun D, respectively. In additional experiments, an approximately 2-fold increase in (Bu)2cAMP-stimulated
reporter activity was observed when Y-1 cells were
transfected with the ⫺151/⫺1 bp StAR segment (Fig.
4). Basal luciferase responses were increased with
c-Fos (136%), Fos B (195%), c-Jun (360%), Jun B
(377%), and Jun D (243%) and decreased with Fra-2
(30%), respectively. In the case of Fra-1, a moderate
but consistent decrease in promoter activity was observed. (Bu)2cAMP-stimulated reporter activities were
decreased in all cases by 26–40% when compared
with control. Moreover, the results obtained with
MA-10 and Y-1 cells demonstrate that the repression
of (Bu)2cAMP-stimulated StAR promoter activity seen
with Fos and Jun in the absence of a canonical AP-1
element, appeared to be mediated through similar
mechanisms.
To obtain more insight into these mechanisms, StAR
reporter activity in response to Fos and Jun was assessed using the ⫺151/⫺1 bp StAR segment by generating a mutation in the AP-1 binding site. The results
summarized in Fig. 5 show that transfection of MA-10
cells with the ⫺151/⫺1 bp segment resulted in a 3.5fold increase in luciferase activity in response to
(Bu)2cAMP. Mutation in the AP-1 element (as above),
clearly decreased (P ⬍ 0.01) basal but did not significantly affect (Bu)2cAMP-mediated StAR reporter responses. Importantly, the inhibitory effects of Fos (cFos, Fra-1, and Fra-2) and Jun (c-Jun and Jun D) in
(Bu)2cAMP-stimulated StAR reporter activity were apparently lost when cells were transfected with the
⫺151/⫺1 bp StAR segment containing a mutation in
the AP-1 element. Consequently, Fos and Jun have no
significant effects on StAR gene transcription under
basal conditions, demonstrating the specificity of the
AP-1 binding motif in StAR promoter responsiveness.
The involvement of Fos and Jun proteins in StAR
gene expression was evaluated in EMSA by utilizing an
oligonucleotide probe (⫺96/⫺67). Protein-DNA complexes (I, II, and III) observed with (Bu)2cAMPstimulated MA-10 NE were markedly inhibited by the
addition of unlabeled sequence (lane 11). Different
antibodies (Abs) specific to Fos and Jun members
(lanes 4–10) interfere with protein-DNA complexes to
Manna et al. • AP-1 and StAR Gene Transcription
Mol Endocrinol, March 2004, 18(3):558–573
561
Fig. 2. StAR Promoter ⫺83/⫺72 bp Region in Different Species, and Binding of the ⫺83/⫺72 bp Region to MA-10 and Y-1 Nes
Panel A, Sequence alignment of mouse, rat, human, ovine, porcine, and monkey StAR promoter regions (⫺83/⫺72) illustrating
the AP-1 recognition motifs (underlined). Panel B, Protein-DNA binding was performed using an oligonucleotide probe specific
to the AP-1 sequence motif (⫺83/⫺72), and binding reactions were carried out as described in the legend of Fig. 1. NE (10 ␮g)
obtained from (Bu)2cAMP (500 ␮M, 6 h)-stimulated MA-10 (lanes 2–6) or Y-1 (lanes 8–12) cells was used to determine binding with
the 32P-labeled probe using EMSA. Competitors were used at 100-fold molar excess, i.e. ⫺83/⫺72 (lanes 3 and 9), Con CRE
(lanes 4 and 10), Con AP-1 (lanes 5 and 11), and AP-1 Mut (lanes 6 and 12). Protein-DNA complexes are marked as I, II, and III.
Migration of free probes is shown in both cases. A representative phosphor image depicts binding of the ⫺83/⫺72 probe to
MA-10 and Y-1 NEs. These experiments were repeated three to four times. Con CRE, consensus CRE; Con AP-1, consensus
AP-1; AP-1 Mut, AP-1 mutant.
varying degrees (Fig. 6). Notably, c-Fos and Fra-2 Abs
nearly abolished protein-DNA complexes (lanes 4 and
6). The binding was found to be decreased with Jun D
Ab (lane 10). An Ab specific to CREM1 demonstrated
a marked decrease in protein-DNA complexes (lane
12) apparently similar to those of c-Fos and Fra-2, an
observation consistent with our previous finding (16,
18). Preimmune serum for CREM1 (lane 13) and IgG
(lane 3) were also used. Qualitatively similar results
were obtained when the ⫺83/⫺72 probe was used in
EMSA with MA-10 NE (data not shown).
In an alternative approach, the functional significance of Fos and Jun was further investigated in transcription of the StAR gene using either antisense (AS)
or dominant negative forms of these proteins (Fig. 7).
MA-10 cells transfected with AS-c-Fos, AS-Fra-1
plasmids, within the ⫺151/⫺1 StAR segment, demonstrated significant decreases (P ⬍ 0.01) in basal and
Fos-mediated StAR promoter responsiveness. Importantly, transfection of a dominant negative form of Fos
(A-Fos) lacking a DNA binding domain markedly decreased StAR promoter activity. A-Fos has been dem-
562 Mol Endocrinol, March 2004, 18(3):558–573
Manna et al. • AP-1 and StAR Gene Transcription
Fig. 3. Functional Involvement of the AP-1 Element on StAR Promoter Activity
MA-10 cells were transfected with either the ⫺151/⫺1 bp StAR reporter segment or the ⫺151/⫺1 containing a mutation in the
AP-1 recognition site (1 ␮g each), in the presence of pRL-SV40 vector (renilla luciferase for determining transfection efficiency),
as described in Materials and Methods. After 36 h of transfection, cells were incubated for an additional 6 h in the absence (basal)
or presence of 500 ␮M (Bu)2cAMP (cAMP). pGL3 basic (pGL3) was used as a control. Luciferase activity in the cell lysates was
determined and expressed as relative light units, RLU (luciferase/renilla). Schematic presentation illustrates wild-type and mutant
AP-1 plasmids within the ⫺151/⫺1 bp region. Data represent the mean ⫾ SEM of five independent experiments.
onstrated to inactivate the DNA binding of the Fos:Jun
heterodimer and potentially inhibit Jun-dependent
transactivation (44, 45). On the other hand, the introduction of a dominant negative form of c-Jun (TAM67) showed a marked inhibition of basal and c-Junmediated StAR promoter responses. Fos and Jun AS
and dominant negative forms were also able to decrease (Bu)2cAMP-mediated StAR promoter activity
(data not shown). These findings reinforce the importance of Fos and Jun in StAR gene transcription.
To delineate the events involved in AP-1-mediated
StAR transcription, c-Fos, Fra-2, and c-Jun were further examined (Fig. 8). Overexpression of increasing
amounts (0.5–6.0 ␮g) of c-Fos, Fra-2, and c-Jun, along
with the ⫺151/⫺1 bp StAR segment in MA-10 cells,
was found to be associated with increases (c-Fos and
c-Jun) and a decrease (Fra-2) in StAR reporter activity
in a concentration-dependent manner (Fig. 8A). To
determine whether the inhibitory effect of Fra-2 in
StAR gene expression affects the c-Jun response,
MA-10 cells were transfected with Fra-2, c-Jun, and a
combination of them, utilizing the ⫺151/⫺1 bp StAR
segment (Fig. 8B). Overexpression of Fra-2 decreased
basal StAR promoter activity by 38% whereas c-Jun
increased it by 542%. Importantly, coexpression of
Fra-2 and c-Jun resulted in a 57% decrease in reporter
activity, indicating that Fra-2 is able to block the stimulatory effect of c-Jun. The addition of increasing
amounts of c-Jun with a fixed concentration of Fra-2
did not alter the inhibitory effect elicited by Fra-2. In
contrast, higher amounts of Fra-2 decreased the cJun induction of StAR promoter activity by approximately 65%, demonstrating that Fra-2 inhibits c-Jun
responsiveness.
Elements Involved in StAR Gene Transcription
Cooperate/Interact with AP-1
The roles of cis-acting elements within the ⫺151/⫺1
bp region of the mouse StAR promoter were assessed
by determining StAR reporter activity after generating
mutations in the putative binding sites, either alone or
in combination with AP-1 (Fig. 9). These mutations
include the recognition motifs for Sp1, steroidogenic
factor 1 (SF-1), CCAAT/enhancer binding protein
(C/EBP), and GATA, and their positions within the
⫺151/⫺1 StAR promoter are illustrated (Fig. 9A).
MA-10 cells transfected with the ⫺151/⫺1 StAR segment demonstrated a 3.3-fold increase in luciferase
activity in response to (Bu)2cAMP. Alteration of bases
in the AP-1 element demonstrated a significant decrease (P ⬍ 0.01) in StAR promoter activity. Mutations
generated in Sp1, C/EBP, SF-1, and GATA binding
sites attenuated basal responses by 37%, 44%, 58%,
and 42% respectively, but did not affect (Bu)2cAMPmediated fold-induction (Fig. 9B). Transfection of any
of these mutants in combination with the AP-1 mutant further decreased StAR reporter activity when
compared individually, suggesting the functional cooperation of these elements in StAR promoter
responsiveness.
Further insight into these mechanisms was obtained
by determining protein-protein interactions between
c-Fos and c-Jun with SF-1, GATA-4, Sp1, and
C/EBP␤ using the mammalian two-hybrid assays.
HeLa cells transfected with either c-Fos-pACT, c-JunpBIND, or SF-1, GATA-4, Sp1, and C/EBP␤ (SF-1,
GATA-4, Sp1, and C/EBP␤ either in pACT or pBIND)
plasmids resulted in limited effects on luciferase
Manna et al. • AP-1 and StAR Gene Transcription
Mol Endocrinol, March 2004, 18(3):558–573
563
Fig. 5. Specificity of Fos and Jun binding to the AP-1 Element Involved in StAR Promoter Responsiveness
MA-10 cells were transfected either with the ⫺151/⫺1
StAR reporter segment (⫺151/⫺1 StAR) or ⫺151/⫺1 StAR
containing a mutation in the AP-1 element (⫺151/⫺1 StARAP-1 Mut), without (pcDNA) or with Fos and Jun plasmids as
indicated, in the presence of pRL-SV40. After 36 h of transfection, cells were incubated for a further 6 h in the absence
(basal) or presence of (Bu)2cAMP (cAMP; 500 ␮M). Luciferase
activity in the cell lysates was determined and expressed as
relative light units (RLU) (luciferase/renilla). pGL3 basic
(pGL3) was used as a control. These experiments were repeated three to four times, and the data (⫾SEM) from a representative experiment with triplicate samples are presented.
Fig. 4. Effects of Fos and Jun on StAR Promoter Responsiveness
Utilizing the ⫺151/⫺1 StAR reporter segment containing
an AP-1 binding motif, MA-10 (upper panel) and Y-1 (lower
panel), cells were transfected with either empty expression
vector (pcDNA) or with expression vector containing Fos and
Jun (DNA ratio 1:1) in the presence of pRL-SV40 vector. After
36 h of transfection, cells were incubated for an additional 6 h
without (basal) or with (Bu)2cAMP (cAMP; 500 ␮M), and luciferase activity in the cell lysates was determined and expressed as relative light units (RLU) (luciferase/renilla). Data
represent the mean ⫾ SEM of three to five independent experiments.
activity (Fig. 10). Coexpression of c-Fos-pACT with
SF-1pBIND, GATA-4-pBIND, and C/EBP␤-pBIND increased luciferase responses approximately 8.8-,
5.7-, 4.7-, 3.8-, 9.2-, and 6.4-fold when compared
with c-Fos-pACT, SF-1-pBIND, GATA-4-pBIND, and
C/EBP␤-pBIND, respectively. On the other hand,
cells transfected with c-Jun-pBIND in the presence of
SF-1-pACT, GATA-4-pACT, and C/EBP␤-pACT resulted in 18.2-, 19.5-, 12.4-, 14.2-, 16.5-, and 19.7-
fold increases in luciferase activity when compared
with c-Jun-pBIND, SF-1-pACT, GATA-4-pACT, and
C/EBP␤-pACT, respectively. Coexpression of either
c-Fos-pACT and c-Jun-pBIND with Sp1-pACT or
Sp1-pBIND had no significant effects in luciferase responsiveness. These results demonstrate that c-Fos
and c-Jun proteins are either capable of a direct association with SF-1, GATA-4, and C/EBP␤ or interact
in vivo through a common adapter protein.
DISCUSSION
The CRE/ATF and AP-1/TRE sequence motifs are two
of the major classes of regulatory elements that contribute to transcriptional regulation of eukaryotic gene
expression. Transcriptional regulation of one such
gene, the steroidogenic regulatory protein (StAR), is
mediated by cAMP-dependent mechanisms even
though it lacks a canonical CRE in its promoter region.
Nevertheless, the mouse StAR gene has been demonstrated to be regulated by the combinatorial action
of multiple DNA elements that consist of recognition
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Mol Endocrinol, March 2004, 18(3):558–573
Manna et al. • AP-1 and StAR Gene Transcription
Fig. 6. Assessment of the Role of AP-1 Family Proteins on StAR Gene Expression
EMSA was performed with an oligonucleotide probe representing the ⫺96/⫺67 bp region of the StAR promoter that contains
a functional AP-1 binding motif. NE (10 ␮g) from (Bu)2cAMP-stimulated (500 ␮M, 6 h) MA-10 cells was incubated with the
32
P-labeled probe in the absence or presence of different Abs to Fos and Jun (lanes 4–10) and CREM1 (lane 12). Protein-DNA
complexes are marked as I, II, and III. A representative phosphor image illustrates Ab interference analysis with Fos and Jun (lanes
4–10) and CREM1 (lane 12). Cold competitor (⫺96/⫺67) was used at 100-fold molar excess (lane 11). IgG (lane 3) and preimmune
serum for CREM1 (lane 13) were used in the incubation. Migration of free probes is shown in each lane. Similar results were
obtained from three independent experiments.
motifs for sequence-specific transcription factors located within the ⫺151/⫺1 bp region of the StAR promoter (6, 15–17, 42, 46). Within this region, three wellconserved 5⬘-CRE half-sites (⫺96/⫺67 bp) have
recently been characterized, and their involvement in
StAR gene expression has been demonstrated using
the CRE binding proteins CREB and CREM (16, 18).
One of the CRE elements, the CRE2 half-site is analogous to an AP-1 sequence motif and is highly conserved among different species. The experimental approaches used in the present study indicate the
importance of the AP-1 recognition motif and provide
evidence that the AP-1 family proteins are involved in
controlling transcription of the StAR gene.
The AP-1 transcription factor family (Fos and Jun)
are members of the bZip family, share many properties, and have been demonstrated to regulate many
biological functions (22, 25, 29, 30). A variety of external stimuli, such as growth factors, tumor promoters,
hormones, and analogs of the cAMP second messenger, have been shown to acutely induce the levels of
Fos and Jun (36, 47–50). Fos and Jun have been
reported to be involved in transcription by binding to a
DNA sequence, known as an AP-1/TRE element
[TGA(C/G)TCA], present in the promoter of a number
of genes (31–33). Homologous to the AP-1/TRE sequence, a highly conserved element (TGACTGA) was
found in the mouse StAR promoter. The present data
demonstrate that oligonucleotide probes containing
the AP-1 binding motif can bind to MA-10 and Y-1 NE
in EMSA, and that the protein-DNA binding was affected by consensus AP-1 and by mutation in the AP-1
sequence motif. In addition, disruption of the AP-1
binding site exerted an approximately 50% decrease
in StAR reporter activity, demonstrating the importance of the AP-1 element in StAR gene expression.
Previously, this region (the CRE2 element) was found
to bind rec CREB protein and appeared to be the most
important among the three CRE half-sites. Also, a
mutated CRE2 probe displayed virtually no binding
with MA-10 NE in comparison with other CRE mutants
(18). However, it is interesting to note that the AP-1
Manna et al. • AP-1 and StAR Gene Transcription
Mol Endocrinol, March 2004, 18(3):558–573
565
Fig. 7. Functional Involvement of Fos and Jun in StAR Promoter Responsiveness Using AS and Dominant Negative Fos
and Jun in MA-10 Cells
Cells were transfected with empty vector (pcDNA), c-Fos,
cJun, AS-c-Fos, and AS-Fra-1, and dominant negative mutants of Fos (A-Fos) and c-Jun (TAM-67) expression plasmids
(1.5 ␮g each) or in combination as specified, in the presence
of the ⫺151/⫺1 StAR reporter segment (DNA ratio, 1:1) and
pRL-SV40, as described in Materials and Methods. After 48 h
of transfection, cells were harvested, and luciferase activity in
the cell lysates was determined and expressed as relative
light units (RLU) (luciferase/renilla). pGL3 basic (pGL3) was
used as a control. Data represent the mean ⫾ SEM of three
independent transfections.
element in the StAR promoter more closely corresponds to its consensus sequence than does the
CRE2 element correspond to its consensus sequence.
The identification of an AP-1 recognition motif in the
cAMP-responsive region of the mouse StAR promoter
provided the opportunity to characterize the roles of
Fos and Jun in StAR gene transcription in more depth.
Evidence is accumulating that Fos and Jun are involved in mediating transcription of several genes,
including those for PEPCK (35), atrial natriuretic factor
(51), proopiomelanocortin (52), neurotensin/neuromedin N (53), GnRH (54), bone morphogenic protein-2
(49), and the early growth factor-inducible gene 1
(Erg-1) (39). Also, Fos has been demonstrated to decrease cAMP responsiveness and adversely affect the
inducible effect of Jun in PEPCK gene transcription
(35). Our current data demonstrate the functional involvement of Fos and Jun in controlling transcription
of the StAR gene. In particular, although they have
varying effects on basal StAR gene transcription, Fos
and Jun are capable of repressing (Bu)2cAMP-mediated responses. Importantly, overexpression of Fra-2
Fig. 8. Alterations in Transcription of the StAR Promoter by
Expression of Fos and Jun
Utilizing the ⫺151/⫺1 StAR reporter segment containing a
functional AP-1 element, MA-10 cells were transfected with
or without increasing amounts (0.5–6 ␮g) of c-Fos, Fra-2, and
c-Jun plasmids, in the presence of pRL-SV40 (panel A). After
36 h of transfection, cells were harvested, and luciferase
activity in the cell lysates was determined and expressed as
percent control. In panel B, cells were also transfected with or
without Fra-2, c-Jun (2–6 ␮g), and a combination of them as
indicated, in the presence of the ⫺151/⫺1 StAR reporter
segment. After 36 h of transfection, luciferase activity in the
cell lysates was determined and expressed as relative light
units (RLU) (luciferase/renilla). Values are the mean ⫾ SEM of
three to four independent experiments in both cases. Different letters above the bars (panel B) indicate that these groups
differ significantly from each other at P ⬍ 0.05.
was found to inhibit the induction of c-Jun-mediated
transcription of StAR promoter activity. The effects of
Fos and Jun in StAR gene transcription, in the absence
of a canonical AP-1/TRE element in the StAR pro-
566 Mol Endocrinol, March 2004, 18(3):558–573
Manna et al. • AP-1 and StAR Gene Transcription
Fig. 9. Mutations in the AP-1 and Other cis-Acting Elements, Either Alone or in Combination, Affect StAR Promoter Responsiveness
The schematic diagram illustrates the positions of the cis-elements within the ⫺151/⫺1 StAR promoter region (panel A). MA-10
cells were transfected with either the ⫺151/⫺1 StAR reporter segment or the ⫺151/⫺1 StAR containing mutations in the AP-1
sequence either alone or in combination with Sp1, C/EBP, SF-1, and GATA elements (DNA ratio, 1:1), in the presence of pRL-SV40
(panel B). After 36 h of transfection, cells were incubated for a further 6 h without (Basal) and with 500 ␮M (Bu)2cAMP (cAMP),
and luciferase activity in the cell lysates was determined and expressed as RLU (luciferase/renilla). The approximate positions of
AP-1, Sp1, C/EBP, SF-1, and GATA elements within the ⫺151/⫺1 bp StAR promoter region and a combination of them together
with their mutations are illustrated. pGL3 basic (pGL3) was used as a control. These experiments were repeated three to four
times, and data (⫾SEM) from a representative experiment with quadruplicate samples are presented.
moter, were found to be similar in steroidogenic cells.
Mutation in the AP-1 sequence motif reversed the
transcriptional responses elicited by Fos and Jun and
demonstrated that their effects are not due to nonspecific protein-protein interactions. Utilizing the p-1862
rat StAR segment, which possesses three AP-1 binding motifs at ⫺85, ⫺187, and ⫺1567 bp, c-Fos was
recently reported to decrease basal and cAMP- and
c-Jun-induced promoter responses in Y-1 cells (41).
The contradictory effect of c-Fos on basal reporter
activity (i.e. inhibitory vs. stimulatory in the present
finding), could be due to the use of different StAR
promoter segments (rat, p-1862 containing three AP-1
sites vs. mouse, p-151 containing one AP-1 site). The
mechanism accounting for differences in Fos- and
Jun-mediated StAR gene transcription may be due to
the lack of sequence similarity outside the DNA-binding and dimerization domains and will be the subject of
future investigations.
The participation of Fos and Jun in StAR gene expression was also investigated by determining protein
binding to the AP-1 DNA element. An oligonucleotide
probe containing three CRE half-sites (including the
overlapping AP-1 element), demonstrated strong inhibition of protein-DNA complexes by c-Fos, Fra-2, and
CREM1 Abs, supporting the notion that AP-1 and
Manna et al. • AP-1 and StAR Gene Transcription
Mol Endocrinol, March 2004, 18(3):558–573
567
Fig. 10. Protein-Protein Interactions between c-Fos and c-Jun with SF-1, GATA-4, Sp1, and C/EBP␤ in the Mammalian
Two-Hybrid Assay
HeLa cells were transfected with c-Fos-pACT, c-Jun-pBIND, or SF-1, GATA-4, Sp1, and C/EBP␤ (either in pACT or pBIND)
plasmids (2 ␮g each), or their combination as indicated, in the presence of the pG5-luciferase vector. After 48 h of transfection,
cells were harvested, and luciferase activity in the cell lysates was determined (relative light units). These experiments were
repeated two to four times, and data (⫾SEM) from a representative experiment with triplicate samples are presented.
CREM proteins can form heterodimers. This event is
not entirely surprising because the ability of AP-1 and
CRE binding proteins to heterodimerize has been
demonstrated in other systems (22, 55, 56). Studies
have also shown that AP-1 complexes are not limited
to Fos and Jun dimers, and these proteins have been
shown to dimerize with other bZip proteins (22, 57).
Also, it has been demonstrated that repression of the
PEPCK gene by Fos does not require a consensus
phorbol 12-O-tetradecanoate 13-acetate responsive
element (TRE) but it does require an intact DNA binding region and dimerization domain, and that it binds
to the C/EBP protein family (35, 58). The Fos protein
has also been shown to repress its own promoter by
interacting with the dyad symmetry element or serum
response element, an action that is independent of a
TRE (59, 60).
Notably, among the Fos and Jun family members
assessed, c-Jun was found to be most potent in transactivation of the StAR gene. Therefore, the transcriptional activation potential of c-Jun might be expected
to correlate with its ability to affect protein-DNA binding. However, EMSA experiments using an Ab specific
to c-Jun did not indicate such a correlation, suggesting the involvement of an alternate mechanism. Studies have demonstrated that c-Jun containing tethered
Fos dimers have distinct promoter specificity and biological responses. In accordance with this, the tethered c-Jun⬃c-Fos has been demonstrated to be
the strongest activator followed by c-Jun⬃c-Fra-1,
c-Jun⬃c-Fra-2, and c-Jun⬃ATF2 in the human collagenase gene promoter (matrix metalloproteinase 1),
which possesses a consensus TRE element (61–63).
Within the human cyclin D1 (CCND1) promoter, two
elements containing TRE (CCND1–1) and CRE
(CCND1–2) have been reported to be the targets of
c-Jun-containing dimers (64, 65). Nonetheless, the
tethered Jun⬃Fos proteins could efficiently bind to
consensus TRE, be recognized by appropriate Abs
with a mobility shift similar to that of a mixture of Jun
and Fos, and show nuclear localization by transfection
assays (63). Also, these findings have documented
that c-Jun⬃c-Fos dimers had an exclusive binding to
TRE (matrix metalloproteinase 1 and CCND1–1) as
compared with CRE (CCND1–2) elements, suggesting
c-Jun⬃Fos dimers prefer TRE-like motifs whereas
568
Mol Endocrinol, March 2004, 18(3):558–573
Jun⬃ATF2 prefer CRE-like motifs. Because the AP-1/
CRE2 region in the StAR promoter corresponds more
closely to a consensus TRE in comparison to a CRE, it
is more likely that, rather than a direct effect of c-Jun
alone, the tether c-Jun⬃c-Fos and/or c-Jun⬃Fos
(Fra-2) dimers play important roles in transcription of
the StAR gene. We also used an additional strategy
that employed AS and/or dominant negative forms of
Fos and Jun. It has previously been demonstrated that
the use of AS and/or dominant negative forms of these
proteins resulted in inhibition of Fos and Jun expression (45, 52, 66, 67). In the present studies, AS and/or
dominant negative forms of Fos and Jun were demonstrated in the repression of StAR promoter responsiveness, reinforcing the involvement of AP-1 family
proteins in StAR gene transcription. Moreover, it was
observed by RT-PCR analysis that MA-10 cells express c-Fos and c-Jun, and that cAMP increased levels of the former about 3-fold and marginally increased
the latter (data not shown).
The 5⬘-flanking region of the StAR gene possesses
recognition motifs for several trans-acting factors, including SF-1, GATA, Sp1, and C/EBP, that have been
demonstrated to be instrumental in StAR gene expression (14, 15, 42, 43, 46, 68). SF-1, either alone or in
concert with other factors, plays an important role in
many cAMP-regulated genes, including StAR, which
lack CREs and are involved in steroidogenesis (15, 17,
69–71). The current data using sequence- and sitespecific mutational analyses indicate that SF-1, GATA,
Sp1, and C/EBP elements cooperate with AP-1 in
mediating StAR reporter responsiveness. Previous
studies have demonstrated that mutation in the GATA
binding site, either alone or in combination with SF-1
and/or C/EBP␤-AP-1-nuclear receptor half-site,
significantly decreases basal and cAMP-stimulated
mouse StAR promoter responses (15). Therefore, it is
conceivable that more than one transcription factor
can bind to most, if not all, of the cis elements involved
in cAMP-mediated regulation of the StAR gene, a phenomenon consistent with the PECPK gene promoter in
which different transcription factor binding arrays
modulate cAMP responsiveness (72, 73).
An intriguing aspect of the present results is that a
functional protein-protein interaction exists between
c-Fos and c-Jun with SF-1, GATA-4, and C/EBP␤ as
determined using the mammalian two-hybrid assay.
c-Fos has recently been demonstrated to interact with
SF-1 and decrease SF-1-mediated rat StAR gene expression (41). Also, an interaction between AP-1 and
SF-1 has been reported in cAMP-dependent activation of the ACTH receptor promoter (40). Fos has been
shown to interact with the CArg box sequence and
decrease Egr-1 gene transcription (39). Studies have
also demonstrated that SF-1 cooperates and interacts
with CREB/CREM, C/EBP␤, and Sp1 in the transcriptional regulation of the StAR gene (17, 18, 71). Several
lines of evidence suggest that CBP/p300 participates
in the activities of many transcription factors including
CREB, SF-1, c-Fos, and c-Jun by functioning as bridg-
Manna et al. • AP-1 and StAR Gene Transcription
ing proteins (74, 75). However, the interaction of Fos
and Jun with other proteins, in addition to SF-1,
GATA-4, and C/EBP␤, which act through sequences
not involving AP-1, cannot be ruled out and may play
roles in StAR gene expression.
From the results of the present studies it is apparent
that the TGACTGA element is the binding site for Fos
and Jun. We have recently demonstrated that rec
CREB protein specifically binds to this sequence (18).
It is noted, however, that this region has also been
demonstrated to bind C/EBP␤ (43). Therefore, it appears that three subfamilies of bZip proteins can bind
to the same target sequence. Indeed, these families
possess high degrees of homology in their binding
sequences and share bZip motifs, although they have
a higher binding specificity for their respective consensus sequences (22, 23, 25). Cross-talk in the form
of binding between AP-1 and CRE family proteins has
also been reported (25, 76–78). Studies have demonstrated that CRE binding proteins can bind to the AP-1
target sequence and suppress AP-1 reporter activity in
differentiating keratinocytes (23, 25). Consistent with
this, it was also observed that coexpression of Fos/
Jun and CREB in the presence of the ⫺151/⫺1 bp
StAR promoter segment resulted in a decrease in reporter activity (Manna, P. R., and D. M. Stocco, unpublished observations). Considering the similarities in
the AP-1 and CRE binding sequences, it is not surprising that both Fos/Jun and CREB compete with
each other for the TGACTGA motif and result in an
attenuation of StAR promoter activity. Further studies
to elucidate the molecular interactions between AP-1
and CREB/CREM that should lead to a better understanding of the involvement of these factors in regulating StAR gene transcription are underway. Taken
together, the present results point to an important role
of Fos and Jun in regulating the transcriptional machinery of the StAR gene, leading us to propose a
model presented in Fig. 11.
In summary, these findings provide compelling evidence that Fos and Jun can specifically bind to the
TGACTGA element in the StAR gene promoter and
are involved in controlling its transcription. Functional
cooperation, interaction, alteration, or competition between transcription factors may represent cross-talk
between different signaling pathways and may be important for cAMP responsiveness. A balance between
the positive and negative effects of different transcription factors presumably allows for the fine tuning of
the regulatory events associated with StAR gene
expression. Regardless of the trans-regulatory mechanisms involved, the 5⬘-flanking region (⫺151/⫺1 bp)
of the StAR promoter that contains three CRE halfsites and an AP-1 site, in addition to several transcription factor recognition motifs, appears to be the most
important region and might function as a cAMP response unit in the transcriptional regulation of the
StAR gene.
Manna et al. • AP-1 and StAR Gene Transcription
Mol Endocrinol, March 2004, 18(3):558–573
569
Fig. 11. Proposed Model for Mouse StAR Gene Transcription
Initially, ligand-receptor interaction at the cell surface results in an activation of coupled G proteins (G), which in turn stimulates
membrane-associated adenylyl cyclase (AC). This, in turn, generates cAMP and results in the dissociation of the inactive
tetrameric protein kinase A (PKA) complex into the active catalytic and the regulatory subunits. The catalytic subunits migrate into
the nucleus, phosphorylate transcription factors, and thereby activate StAR gene transcription. Shown are the different transcription factor binding motifs that may interact with the AP-1 element during StAR gene transcription. The concerted action of
multiple transcription factors, and their interaction with most, if not all, of the cis-regulatory elements, appears to be involved in
regulating StAR gene expression. It is noteworthy that Fos and Jun can specifically bind to the AP-1/CRE2 recognition motif in
the StAR promoter and play important roles in transcription of the StAR gene. Based on previously available information together
with our own preliminary data, it is highly likely that CBP/p300, a factor known to interact with many transcription factors including
CREB, c-Fos and c-Jun, may participate as a bridging protein and influence StAR gene transactivation.
MATERIALS AND METHODS
Generation of StAR Promoters and Site-Directed
Mutagenesis
The proximal ⫺151/⫺1 bp region of the mouse StAR promoter was demonstrated to have full cAMP responsiveness
when compared with the entire promoter region (15, 16, 18,
42, 46). Therefore, this region was constructed and subcloned into the pGL3 basic vector (Promega Corp., Madison,
WI) utilizing a XhoI and HindIII fragment that contains the
firefly luciferase gene as a reporter (16). Using this fragment,
mutations in the AP-1 and other cis-acting elements were
generated using the QuikChange Site-directed mutagenesis
kit (Stratagene, La Jolla, CA). The following sense strands of
the oligonucleotide sequences used for mutagenesis (mutated bases in lowercase bold) were: AP-1 mutant (Mut),
5⬘-CCTTGACCCTCTGCACAATagaTctTGACTTTTTTATCTC-3⬘; Sp1 Mut, 5⬘-ACACAGTCTGCgaatTCCCACCTTGGCCAG-3⬘; SF-1 Mut, 5⬘-CAATCATTCCAgCtgTGACCCTCTGCAC-3⬘; C/EBP Mut, 5⬘-GCACTGCAGGATGgtcgAcTCATTCCATCCTTG-3⬘; GATA Mut, 5⬘-GACTGATGACTTTTTccggaCAAGTGATGATGCACAG-3⬘.
The specific SF-1 and C/EBP binding sites within the
cAMP responsive region chosen for analysis were based on
their predominant involvement in StAR gene expression (17,
18). Specific mutations in the target sites were assessed by
restriction mapping using Bgl II (AP-1 Mut), EcoRI (Sp1 Mut),
PvuII (SF-1 Mut), SalI (C/EBP Mut), and BspEI (GATA Mut),
and confirmed by automated sequencing on a PE Biosystem
310 Genetic Analyzer (ABI PRISM 310, PerkinElmer Corp.,
Norwalk, CT) at the Texas Tech University Biotechnology
Core Facility.
Construction of Fos/Jun Plasmids
The full-length mouse c-Fos, Fra-1, Fra-2, and Fos B open
reading frames were synthesized using a PCR-based cloning
strategy. Primer pairs employed for amplifying the different
Fos members were the following: c-Fos forward, 5⬘CCCCGCTCGAGATGATGTTCTCGGGTT-3⬘; c-Fos reverse,
5⬘-CCCGGGATCCCACAGGGCCAGCAGCG-3⬘; Fos B forward, 5⬘-CCCCGCTCGAGATGTTTCAAGCTTTTCC-3⬘; Fos
B reverse, 5⬘-CCCGGGATCCAGAGCAAGAAGGGAGG-3⬘;
Fra-1 forward, 5⬘-CCCCGCTCGAGATGTACCGAGACTACG3⬘; Fra-1 reverse, 5⬘-CCCGGGATCCCACAAAGCCAGGAGTGTAGG-3⬘; Fra-2 forward, 5⬘-CCCCGCTCGAGATGTACCAGGATTATCC-3⬘; Fra-2 forward, 5⬘-CCCGGGATCCAGGGCTAGAAGTGTGG-3⬘. The 5⬘-primer for all Fos members contains a XhoI site whereas the 3⬘-primers possess a
BamHI site at the 5⬘-end (underlined). The PCR products
amplified were subcloned into the pcDNA 3.1(⫺)Myc-HisB
(pcDNA3.1-MHB) expression vector (Invitrogen Life Technologies, Carlsbad, CA) after digestion with XhoI-BamHI restriction enzymes. The mouse Jun members (open reading frame)
were similarly constructed and inserted into the pcDNA3.1MHB expression vector (obtained from Dr. Curt M. Pfarr,
Department of Cell Biology and Biochemistry, Texas Tech
570 Mol Endocrinol, March 2004, 18(3):558–573
University Health Sciences Center, Lubbock, TX), using
EcoR1-HindIII sites (79, 80). The identity of the inserted fragments were verified by restriction digestion and confirmed by
automated sequencing (ABI PRISM 310, Perkin-Elmer Corp.).
EMSAs
Preparation of nuclear extracts (NEs) from mouse MA-10
Leydig (81) and Y-1 adrenocortical tumor cells was carried
out as described previously (16, 18, 82). The oligonucleotide
probes were synthesized by heating sense and antisense
primers to 65 C for 5 min in annealing buffer (10 mM Tris-Cl,
100 mM NaCl, 1 mM EDTA, pH 7.5), followed by cooling at
room temperature. The sense strands of the oligonucleotide
sequences used in EMSA were the following: ⫺96/⫺67 (CRE
half-sites), 5⬘-GGTGACCCTCTGCACAATGACTGATGACTTTT-3⬘; ⫺83/⫺72 (AP-1), 5⬘-GGAATGACTGATGA-3⬘; ⫺83/⫺72
(AP-1 Mut), 5⬘-GGAATAGATCTTGA-3⬘; consensus AP-1 (23),
5⬘-GGCGCTTGATGAGTCAGCCGGAA-3⬘; consensus CRE
(19), 5⬘-GGAGAGATTGCCTGACGTCAGAGAGCTAG-3⬘.
The doubled-stranded oligonucleotides (4 pM each) were
end labeled with [␣32P]dCTP (PerkinElmer Life Sciences, Inc.,
Boston, MA) using Klenow (Promega Corp.) fill-in reactions
and protein-DNA binding assays were carried out under optimized conditions (16, 83). Briefly, 10–15 ␮g NE were incubated in 20 ␮l of reaction buffer (10 mM HEPES, 1 mM EDTA,
4% Ficoll, 10 mM dithiothreitol, 1 ␮g poly dIdC, 40 mg/ml
BSA, and 2 ␮M ZnSO4; pH 7.9) for 15 min at room temperature. The 32P-labeled DNA probe (0.5 pM, ⬃100,000 cpm)
was added alone or in the presence of 50 pM (100-fold molar
excess) of unlabeled probe, and incubation was continued for
an additional 15 min. When antiserum was used, binding
reactions were incubated on ice for 45 min before the addition of the labeled DNA. Different Abs to the AP-1 family
members used in EMSA reactions were obtained from Santa
Cruz Biotechnology, Inc. (Santa Cruz, CA). These include
mouse monoclonal c-Fos (sc-8047) and Jun B (sc-8051), and
rabbit polyclonal Fra-1 (sc-183), Fra-2 (sc-171), Fos B (sc48), c-Jun (sc-1694), and Jun D (sc-74). The CREM1 Ab is
specific for CREM protein and cross-reacts with CREB at
very high concentrations (16, 84). Mouse IgG was purchased
from Santa Cruz Biotechnology, Inc. Protein-DNA complexes
were then subjected to electrophoresis on 5% polyacrylamide gels for about 1.5 h at 200 V in 0.5⫻ TBE buffer (90 mM
Tris-borate, 2 mM EDTA; pH 8.3). The gels were dried, exposed to phosphor screens, and quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Mammalian Two-Hybrid Assay
Protein-protein interactions between c-Fos and c-Jun with
SF-1, GATA-4, Sp1, and C/EBP␤ were investigated using the
mammalian two-hybrid assay (Promega Corp.), as described
previously (18). Briefly, c-Fos-pACT and c-Jun-pBIND plasmids were constructed using c-Fos and c-Jun cDNAs inserted into either SalI and KpnI sites of the pACT vector or
BamHI and NotI sites of the pBIND vector, respectively. The
full-length SF-1, GATA-4, Sp1, and C/EBP␤ cDNAs were
inserted into either BamHI and NotI sites of the pBIND vector
or SalI and KpnI sites of the pACT vector, respectively. HeLa
cells were transfected with c-Fos-pACT, c-Jun-pBIND, SF1-pACT or SF-1-pBIND, GATA-4-pACT or GATA-4-pBIND,
Sp1-pACT or Sp1-pBIND, C/EBP␤-pACT or C/EBP␤-pBIND
plasmids (2 ␮g each), or their combination as indicated in the
figure legend, in the presence of the pG5-luciferase vector
under optimized conditions (18), using FuGENE 6 reagent
(Roche Diagnostics Corp., Indianapolis, IN). After 48 h of
transfection, cells were harvested and luciferase activity in
the cell lysates was determined using a TD 20/20 Luminometer (Turner Designs, Sunnyvale, CA).
Manna et al. • AP-1 and StAR Gene Transcription
Cell Culture, Transient Transfections, and
Luciferase Assays
MA-10 (81) and Y-1 (ATCC, Manassas, VA) cells were maintained, respectively, in HEPES-buffered Waymouth’s MB/752
and Y-12K media containing antibiotics (16). HeLa cells were
cultured in DMEM as described previously (18).
Transfection studies were carried out using FuGENE
6-transfection reagent (Roche Diagnostics Corp.) under optimized conditions (16, 18). In brief, wild-type and mutant
StAR promoters (⫺151/⫺1 StAR/luc) were used (1 or 1.5 ␮g)
in the absence or presence of Fos and Jun plasmids either in
equal or at varied concentrations (0.5–6 ␮g), as specified in
the figure legends. The functional specificity of Fos and Jun
on StAR promoter responsiveness was also assessed using
the AS c-Fos (52), AS Fra-1 (45), and dominant negative Fos
[A-Fos, (45)] and c-Jun [TAM-67, (67)] plasmids. The amount
of DNA used in transfections was equalized with empty vector. Transfection efficiency was normalized by cotransfecting
15 ng pRL-SV40 vector (a plasmid that constitutively expresses renilla luciferase).
Luciferase activity in the cell lysates was determined by the
Dual-Luciferase reporter assay system (Promega Corp.), using a TD 20/20 Luminometer (Turner Designs) as described
previously (16, 18).
Statistical Analysis
The experiments presented were repeated three to five times.
Statistical analysis was performed by ANOVA using Statview
(Abacus Concepts Inc., Berkeley, CA) followed by Fisher’s
protected least significant differences test. Data represent
the mean ⫾ SEM, and P ⬍ 0.05 was considered significant.
Acknowledgments
The authors are grateful to Dr. C. M. Pfarr (Department of
Cell Biology and Biochemistry, Texas Tech University Health
Sciences Center, Lubbock, TX) for the generous gifts of Jun
plasmids. We are grateful to Drs. E. Tulchinsky (University of
Leicester, Division of Urology, Leicester, UK) for providing us
with the dominant negative Fos; J. P. Loeffler (Institut de
Physiologie et de Chimie Biologique, Strasbourg, France) for
the AS c-Fos; and G. T. Bowden (Department of Pharmacology and Toxicology, The University of Arizona, Tucson, AZ)
for the dominant negative c-Jun constructs. We also thank
Dr. P. Sassone-Corsi (Institute de Génétique et de Biologie et
Moléculaire and Cellulaire, Strasbourg, France) for generously providing the CREM1 Ab. We also acknowledge the
help of Mathew T. Dyson, John D. Short, and Deborah Alberts
during the course of these studies.
Received June 10, 2003. Accepted December 3, 2003.
Address all correspondence and requests for reprints to:
Douglas M. Stocco, Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center,
Lubbock, Texas 79430. E-mail: [email protected].
edu.
This work was supported by NIH Grant HD-17481 and with
funds from the Robert A. Welch Foundation.
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