Regulation of Steroidogenesis and the Steroidogenic Acute Regulatory Protein by a Member of the cAMP Response-Element Binding Protein Family
Pulak R. Manna,
Matthew T. Dyson,
Darrell W. Eubank,
Barbara J. Clark,
Enzo Lalli,
Paolo Sassone-Corsi,
Anthony J. Zeleznik and
Douglas M. Stocco
Department of Cell Biology and Biochemistry (P.R.M., M.T.D.,
D.W.E., D.M.S.), Texas Tech University Health Sciences Center, Lubbock,
Texas 79430; Department of Biochemistry (B.J.C.), University of
Louisville School of Medicine, Louisville, Kentucky 40292; Institute de
Génétique et de Biologie et Moléculaire et cellulaire
(E.L., P.S.-C.), CNRS-INSERM, 67085 Strasbourg, France; and Department
of Cell Biology and Physiology (A.J.Z.), University of Pittsburgh
School of Medicine, Pittsburgh, Pennsylvania 15261
Address all correspondence and requests for reprints to: Douglas M. Stocco, Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430. E-mail:
doug.stocco{at}ttmc.ttuhsc.edu
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ABSTRACT
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The mitochondrial phosphoprotein, the steroidogenic acute
regulatory (StAR) protein, is an essential component in the regulation
of steroid biosynthesis in adrenal and gonadal cells through
cAMP-dependent pathways. In many cases transcriptional induction by
cAMP is mediated through the interaction of a cAMP response-element
binding protein (CREB) family member with a consensus cAMP response
element (CRE; 5'-TGACGTCA-3') found in the promoter of target genes.
The present investigation was carried out to determine whether a
CRE-binding protein (CREB) family member [CREB/CRE modulator (CREM)
family] was involved in the regulation of steroidogenesis and StAR
protein expression. Transient expression of wild- type CREB in
MA-10 mouse Leydig tumor cells further increased the levels of
(Bu)2cAMP-induced progesterone synthesis, StAR promoter
activity, StAR mRNA, and StAR protein. These responses were
significantly inhibited by transfection with a dominant-negative CREB
(A-CREB), or with a CREB mutant that cannot be phosphorylated
(CREB-M1), the latter observation indicating the importance of
phosphorylation of a CREB/CREM family member in steroidogenesis and
StAR expression. The CREB/CREM-responsive region in the mouse StAR gene
was located between -110 and -67 bp upstream of the transcriptional
start site. An oligonucleotide probe (-96/-67 bp) containing three
putative half-sites for 5'-canonical CRE sequences (TGAC) demonstrated
the formation of protein-DNA complexes in EMSAs with recombinant CREB
protein as well as with nuclear extracts from MA-10 or Y-1 mouse
adrenal tumor cells. The predominant binding factor observed with EMSA
was found to be the CREM protein as demonstrated using specific
antibodies and RT-PCR analyses. The CRE elements identified within the
-96/-67 bp region were tested for cAMP responsiveness by generating
mutations in each of the CRE half-sites either alone or in combination.
Although each of the CRE sites contribute in part to the CREM response,
the CRE2 appears to be the most important site as determined by EMSA
and by reporter gene analyses. Binding specificity was further assessed
using specific antibodies to CREB/CREM family members, cold
competitors, and mutations in the target sites that resulted in either
supershift and/or inhibition of these complexes. We also demonstrate
that the inducible cAMP early repressor markedly diminished the
endogenous effects of CREM on cAMP-induced StAR promoter activity and
on StAR mRNA expression. These are the first observations to provide
evidence for the functional involvement of a CREB/CREM family member in
the acute regulation of trophic hormone-stimulated steroidogenesis and
StAR gene expression.
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INTRODUCTION
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STEROID BIOSYNTHESIS IN response to trophic
hormone stimulation is a de novo protein synthesis requiring
a process that is regulated by the delivery of cholesterol from the
outer to the inner mitochondrial membrane where it is cleaved by the
cholesterol side chain cleavage cytochrome P450 enzyme (1, 2). Among the candidate regulatory proteins involved in this
transfer, the steroidogenic acute regulatory protein (StAR) has
essentially all of the required characteristics (2, 3).
The strongest evidence for the critical role of StAR in regulating
steroidogenesis has been demonstrated in patients suffering from
congenital lipoid adrenal hyperplasia, in which both adrenal and
gonadal steroid biosyntheses are markedly impaired due to mutations in
the StAR gene (4). An essentially identical phenotype is
observed in StAR null mice (5, 6).
Transduction of signals by a number of hormones and neurotransmitters
is known to occur via the activation of adenylate cyclase, which
increases intracellular cAMP levels and activates PKA. The expression
of the StAR protein in the adrenals and gonads is stimulated through
the cAMP signaling pathway and is closely correlated with the acute
steroidogenic response of these cells to tropic hormone stimulation
(7, 8). Several transcription factors and processes are
involved in regulating both StAR expression and steroidogenesis, but
none so far can fully account for their rapid stimulation in response
to increases in cAMP.
Response to cAMP at the gene level is typically mediated through a
palindromic conserved sequence (5'-TGACGTCA-3') referred to as the cAMP
response element (CRE) (9, 10, 11). However, the StAR gene
lacks a consensus CRE, a situation also found in several steroid
hydroxylase genes that are regulated by cAMP (12). CRE
sequences investigated to date demonstrate that the 5'-TGACG is highly
conserved in comparison to the 3'-TCA (13). A large family
of basic-leucine zipper (bZip) CRE-binding factors, including
CRE-binding protein (CREB), CRE modulator protein (CREM), and
activating transcription factor (ATF-1), have been shown to interact
with this sequence. Both activators and repressors of transcription can
be found within the CRE-binding factor family, which can homo- and
heterodimerize using a specific interaction code (14, 15, 16).
Three genes, CREB, CREM, and ATF-1, share extensive homology,
constitute the CREB/CREM subfamily (CREB/CREM), and mediate
transcriptional activation (17).
CREB/CREM plays a major role in the growth and developmental
processes of many organs, is required for survival, and mediates a
variety of biological functions (10, 17, 18). CREB/CREM is
activated after phosphorylation by PKA as well as other kinases, and
its transcriptional activities are regulated by multiple signaling
pathways (9, 14, 16). Transcriptional increases in
response to cAMP are observed after the phosphorylation of CREB at
serine-133 (19, 20) or CREM at serine-117
(21), and its binding to the nuclear protein CBP (CREB
binding protein). Recent studies have demonstrated that neither
a mutant form of CREB, unable to be phosphorylated by PKA (CREB-M1),
nor a dominant-negative form of CREB (A-CREB) is capable of activating
transcription (22, 23, 24). Also, transcriptional activation
by CREB/CREM is greatly affected by expression of CREM isoforms that
include DNA-binding and dimerization domains but lack transactivation
domains. In particular, the CREM inducible cAMP early repressor (ICER)
acts as a powerful repressor of cAMP-induced transcription (25, 26). Transcriptional repression by ICER has been shown to
occur via its binding to the CRE sites of target gene promoters
or by the formation of inactive heterodimers with CREB or other
associated transactivators (13, 25, 26).
Considerable progress has been made regarding the regulation of
hormone-stimulated steroidogenesis and StAR expression. However, the
mechanism for the acute regulation of StAR transcription by cAMP is
still not completely understood. Based on these considerations, the
present investigation was undertaken to evaluate the potential
involvement of CREB/CREM family members in the regulation of
steroidogenesis and StAR expression. Our findings provide evidence for
the first time that a CREB/CREM family member, most probably CREM, is
involved in increases in the levels of steroid synthesis, StAR promoter
activity, StAR mRNA, and StAR protein expression in steroidogenic
cells.
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RESULTS
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Involvement of CREB/CREM in the Regulation of
Steroidogenesis
The potential role of CREB/CREM in the regulation of
(Bu)2cAMP-induced progesterone (P) production was
determined. These findings demonstrated that nonstimulated MA-10 cells
transiently expressing WT-CREB significantly increased
(P < 0.05) basal P production, while
(Bu)2cAMP-stimulated P levels observed were
increased by 3040% when compared with mock-transfected cells
(expression vector not containing CREB) (Table 1
). Expression of either A-CREB or
CREB-M1 resulted in an approximately 30% decrease in
(Bu)2cAMP-stimulated P levels when compared with
that seen in control cells. However, neither A-CREB nor CREB-M1
significantly altered basal P levels in the absence of cAMP
stimulation. These results demonstrate that a CREB/CREM family member
is involved in the regulation of steroid hormone biosynthesis in mouse
Leydig cells.
The Role of CREB/CREM in StAR Expression
To determine whether StAR was involved in the observed stimulation
of P synthesis in response to CREB/CREM expression, StAR mRNA and
protein levels were analyzed in CREB transfected cells. As illustrated
in Fig. 1A
, MA-10 cells expressing
wild-type CREB (WT-CREB) modestly, but consistently, increased basal
StAR mRNA expression (P < 0.05), an observation in
keeping with the observed increase in basal P production.
(Bu)2cAMP-induced StAR mRNA expression in WT-CREB
transfected cells was significantly elevated to approximately 3-fold of
that seen in mock-transfected cells. Similar to the observation with P
synthesis, when MA-10 cells were transfected with A-CREB or CREB-M1,
(Bu)2cAMP-stimulated levels of StAR mRNA were
decreased to a level approximately 3040% below that seen in control
cells. Using the same experimental paradigms it was also determined
that the mitochondrial 30-kDa StAR protein content was slightly but
consistently increased in nonstimulated MA-10 cells after transfection
with WT-CREB and was increased approximately 2-fold by expression of
WT-CREB in (Bu)2cAMP-stimulated cells. After
expression of A-CREB or CREB-M1 there was a 40% decrease in StAR
protein levels in (Bu)2cAMP-stimulated cells when
compared with controls (Fig. 1B
). These results demonstrate the
potential involvement of a CREB/CREM family member in StAR
expression and demonstrate a direct relationship between StAR
expression and steroidogenesis.

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Figure 1. Effect of Expression of WT-CREB, A-CREB, and
CREB-M1 on (Bu)2cAMP-Stimulated StAR mRNA and StAR Protein
Expression in MA-10 Cells
Cells were transiently transfected with different CREB expression
plasmids (2 µg each) using Fugene-6 transfection reagent as described
in Materials and Methods. After 36 h of
transfection, cells were stimulated for 6 h in the absence (Basal)
or presence of 500 µmol/liter of (Bu)2cAMP (cAMP). Total
RNA and mitochondrial protein were isolated from each group separately
and subjected to RT-PCR and Western blotting to determine StAR
expression. Shown is a representative autoradiogram of cAMP-induced
StAR mRNA (panel A) and a representative Western blot of 30-kDa StAR
protein (panel B) expression in mock (MT)-, WT-CREB-, A-CREB-, and
CREB-M1-transfected cells. The integrated optical density (IOD) of StAR
expression was quantitated in both cases and normalized in the case of
RT-PCR to L19 expression. The IOD values represent the mean ±
SEM of three to five independent experiments.
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Time-Dependent Increase in cAMP-Stimulated StAR mRNA and P Levels
in Relation to CREB/CREM
The temporal relationship between StAR expression and
steroidogenesis in response to CREB/CREM was investigated. Figure 2
shows the time-dependent increases in
the steady-state levels of StAR mRNA in control and CREB-expressing
cells in response to (Bu)2cAMP stimulation.
Expression of WT-CREB marginally increased (P < 0.05)
basal StAR mRNA but significantly elevated
(Bu)2cAMP-induced StAR mRNA levels, reaching
a maximum between 6 and 8 h and being 3 times higher than
mock-transfected cells. Thereafter, the levels of StAR mRNA decreased.
The magnitude of response was evident within 1 h of cAMP induction
displaying a 70% increase in StAR mRNA levels in CREB-transfected
cells compared with mock-transfected cells.

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Figure 2. Temporal Pattern of (Bu)2cAMP-Induced
StAR mRNA Expression in Mock- and WT-CREB-Transfected MA-10 Cells
Cells were transfected with either empty vector (mock; MT) or WT-CREB
(2 µg each) as described in Fig. 1 . After 36 h of transfection,
cells were washed and stimulated for 012 h in the presence of 500
µmol/liter (Bu)2cAMP. Total RNA was extracted at the
indicated times and subjected to RT-PCR analysis of StAR mRNA
expression as described in the legend of Fig. 1 and Materials
and Methods. Panel A illustrates a representative autoradiogram
of cAMP-induced StAR mRNA expression at different times. Panel B
depicts IOD values of StAR mRNA expression from three independent
experiments (± SEM) after normalization to the L19
bands.
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The accumulation of P in the media at each time point was also
determined in mock- and WT-CREB-transfected cells after
(Bu)2cAMP stimulation and paralleled the
increases in StAR mRNA expression (Fig. 3
). The levels of P significantly
increased (P < 0.01) after 1 h of
(Bu)2cAMP stimulation, while WT-CREB increased P
levels by approximately 35% over mock-transfected cells by 6 and
8 h. The P production began to decrease beyond 8 h in both
cases as did that of StAR mRNA, further demonstrating a close
correlation between cAMP-stimulated StAR expression and
steroidogenesis.

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Figure 3. Temporal Pattern of (Bu)2cAMP-Induced P
Levels in Mock- and WT-CREB-Transfected MA-10 Cells
Cells were transiently transfected with empty vector (MT) or WT-CREB (2
µg each) as described in Fig. 2 . Thirty-six hours after transfection,
cells were washed and stimulated with 500 µmol/liter
(Bu)2cAMP for 012 h. P levels in the media at each time
point were assayed by RIA. The results are the mean ±
SEM of five independent experiments.
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Determination of CREB/CREM-Responsive Region(s) in the 5'-Flanking
Region of the Mouse StAR Gene
To identify the functional region involved in the CREB/CREM
increase in StAR mRNA levels, different mouse StAR promoter/luciferase
constructs (-966, -254, -110, and -68 StAR/luc) were generated and
assessed for (Bu)2cAMP-induced luciferase
activity in MA-10 cells. As shown in Fig. 4
, using the -966 bp fragment,
(Bu)2cAMP-stimulated luciferase activity was
increased approximately 4-fold in the WT-CREB-transfected cells when
compared with the mock-transfected control group. Further deletion of
the 5'-flanking region to -254 bp yielded a similar 4-fold
(Bu)2cAMP-induced luciferase response with
WT-CREB transfection, indicating that the regulatory elements remained
present within this region. Transfection with the -110 bp fragment
caused both a decreased basal (not illustrated) and
(Bu)2cAMP-induced promoter activity in
mock-transfected cells; however, the fold stimulation in
WT-CREB-transfected cells was similar to that of longer StAR
reporters. Conversely, even though there was an increase in
WT-CREB-mediated cAMP-induced luciferase activity when the -68 bp
fragment was used, its activity was markedly decreased to about 15% of
that seen with the longer constructs, demonstrating the presence of
element(s) responsive to CREB/CREM family members in the -110 to -68
bp region of the mouse StAR promoter.

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Figure 4. 5'-Flanking Deletion of the Mouse StAR Gene for
Determination of the CREB-Responsive Region(s)
MA-10 cells were transiently transfected with one of the
5'-deleted StAR promoter/luciferase constructs (-966 StAR/luc, -254
StAR/luc, -110 StAR/luc, and -68 StAR/luc) either alone
(StAR/luc+cAMP) or in combination with WT-CREB (StAR/luc+CREB+cAMP) in
the presence of pRL-SV40 (renilla luciferase for determining
transfection efficiency) as described in Materials and
Methods. After 36 h of transfection, cells were incubated
for a further 6 h without or with (Bu)2cAMP (500
µmol/liter). Luciferase activity in the cell lysates was determined
and expressed as relative light units (RLU, fold activation) where fold
increases after (Bu)2cAMP stimulation over respective
controls are presented. The data represent the mean ±
SEM of five independent experiments.
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Role of CREB/CREM in cAMP-Dependent StAR Promoter Activity in
MA-10, Y-1, and COS-1 Cells
The potential involvement of CREB/CREM in StAR promoter/luciferase
activity was further studied in MA-10, Y-1, and COS-1 cells using the
-254 StAR/luc segment of the StAR promoter. The -254 bp fragment was
chosen since, unlike the -110 StAR/luc fragment, there was no decrease
in the basal StAR promoter/luciferase response (Fig. 4
). The data
presented in Fig. 5A
show that
(Bu)2cAMP treatment of mock-transfected MA-10
cells resulted in a 6.6-fold stimulation of StAR promoter activity over
untreated cells. Transient expression of WT-CREB in these cells
resulted in a 2-fold increase in basal promoter activity but a highly
significant 4-fold increase in the cAMP-stimulated luciferase response
as compared with the mock-transfected group. Transfection of Y-1 cells
with WT-CREB resulted in a modest increase in basal StAR promoter
activity but in an approximately 2.3-fold increase in
(Bu)2cAMP-stimulated promoter activity compared
with mock transfected cells (Fig. 5B
). Cotransfection of the cells with
A-CREB modestly decreased basal but strongly impaired cAMP-induced
luciferase activity in both MA-10 and Y-1 cells. Similarly, CREB-M1
essentially abolished the cAMP-stimulated StAR promoter/luciferase
activity in both cell types. Nonsteroidogenic COS-1 cells transfected
with different CREB expression plasmids did not show any significant
cAMP-induced activation of the StAR promoter/luciferase response. It
was noted, however, that WT-CREB markedly increased basal StAR promoter
activity, suggesting that a CREB/CREM family member modulates StAR
promoter in COS-1 cells in a cAMP-independent manner (Fig. 5C
).

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Figure 5. Expression of WT-CREB, A-CREB, and CREB-M1 on
cAMP-Induced StAR Promoter Activity
MA-10 (panel A), Y-1 (panel B), and COS-1 (panel C) cells were
transiently transfected for 36 h with -254 StAR/luc in the
presence or absence of different CREB constructs as described in
Materials and Methods. To control the variation in
transfection efficiency, pRL-SV40 was included in each assay. Cells
were then stimulated for 6 h in the absence (Basal) or presence
(cAMP) of 500 µmol/liter (Bu)2cAMP. Luciferase activity
in the cell lysates was determined and expressed as RLU
(luciferase/renilla). The data represent the mean ±
SEM of two to five independent experiments.
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Identification of CREB/CREM-Responsive Element(s) Within the
-110/-67 bp Region of the StAR Promoter
The -110/-67 bp region is reasonably conserved among the StAR
promoters of different species (Fig. 6A
),
including mouse (27), rat (28), ovine
(GenBank accession no. AF086814), human (29), monkey
(GenBank accession no. AY007224), and pig (30) and
revealed three 5'-canonical CRE half-site sequences that could be
potential binding sites for CREB/CREM.

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Figure 6. Identification of CREB/CREM Responsive Element(s)
Between -110 and -67 bp of the Mouse StAR Gene
A, Sequence alignment of mouse, rat, ovine, human, monkey, and pig StAR
promoters depicting three putative CRE half-sites CRE1, CRE2, and CRE3
(bold), and transcription factor-binding sites for SF-1
and AP-1 (highlighted in gray) and C/EBP
(boxed). The asterisks represent the
conserved bases in the StAR promoters of different species. B,
Representative phosphorimage of an EMSA using -96 to -67 bp of the
mouse StAR promoter as a radiolabeled oligonucleotide probe as
described in Materials and Methods. The reactions
include: rec CREB protein (lanes 1 and 2), rec CREB plus ATF-1 Ab (lane
2), NE from nonstimulated MA-10 cells (lane 3) or stimulated with 0.5
mmol/liter (Bu)2cAMP (cAMP; lane 4), 0.5 mmol/liter cAMP
plus ATF-1 Ab (lane 5); transfected with WT-CREB plus cAMP (lane 6);
transfected with WT-CREB plus cAMP plus ATF-1 Ab (lane 7); and in Y-1
NE (lanes 811). Formation of specific protein-DNA complexes with rec
CREB protein (a) or with NE is marked as I, II, and III, respectively.
Arrows indicate the supershifted position (SS) of
protein-DNA complexes. The migration of free probe is shown. The
experiments were repeated three to five times.
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The CRE half-sites in the -110 and -67 bp region were tested for
CREB/CREM protein-DNA binding using EMSA. As illustrated in Fig. 6B
, a
radioactively labeled oligonucleotide probe (-96 to -67 bp)
containing the three CRE half-sites demonstrated the presence of a
specific protein-DNA complex with recombinant CREB (rec CREB) (a, lane
1). Furthermore, nuclear extracts (NE) prepared from MA-10 (lanes
37) and Y-1 (lanes 811) cells also showed formation of three
protein-DNA complexes (I, II, and III) with the -96 to -67 bp region.
As demonstrated previously (31), treatment of MA-10 cells
with (Bu)2cAMP resulted in increased formation of
protein-DNA complex I (lane 4) while only modestly increasing complexes
II and III. Complex I apparently corresponded to that observed with rec
CREB protein (a, lane 1) and WT-CREB-transfected, cAMP-stimulated NE
from MA-10 (lane 6) or Y-1 (lane 10) cells. ATF-1, an antibody (Ab)
that recognizes CREB/CREM-1/ATF-1 in immunoblots supershifted the rec
CREB protein-DNA complex (lane 2), as well as complexes formed with NE
from WT-CREB-transfected, cAMP-stimulated cells (lanes 7 and 11).
However, ATF-1 Ab did not supershift these complexes when
cAMP-stimulated NE from untransfected MA-10 cells was used; rather, a
modest inhibition was observed (compare lanes 4 and 5). These data
demonstrate that rec CREB can bind to the StAR promoter, presumably at
one or more of the canonical 5'-CRE half-site(s), and also suggest that
a CREB/CREM family member present in NE from MA-10 and Y-1 cells binds
to the -96/-67 bp region of the mouse StAR promoter.
Identification of the CREB/CREM Family Member Interacting with the
StAR Promoter
To determine the identity of the endogenous protein(s) that bind
to the -96/-67 bp region of the mouse StAR promoter,
(Bu)2cAMP-stimulated MA-10 NE binding was
assessed in EMSA using antisera specific to the CREB/CREM/ATF-1 family
members (Fig. 7A
). A specific antiserum
to CREM family members (CREM1) markedly decreased protein-DNA complexes
(lane 3) when compared with its preimmune serum (lane 2) or a nonimmune
IgG (lane 10). In contrast, different antibodies to CREB/CREM family
members CREB and ATF-1 had either modest or no effects on protein-DNA
complexes (lanes 48). Also, CCAAT/enhancer binding protein
ß Ab neither supershifted nor showed significant inhibition of
protein-DNA complexes (lane 9). Therefore, these data suggest that the
MA-10 NE species that bind to the -96/-67 region are predominantly
CREM proteins.

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Figure 7. Determination of the Binding and Relative
Expression Levels of Endogenous CREB/CREM Family Involved in StAR Gene
Expression
A, EMSA reactions with (Bu)2cAMP-stimulated MA-10 NE (lanes
19) with the 32P-labeled oligonucleotide probe (-96
to-67) in the absence or presence of Abs to different CREB family
members. Protein-DNA complexes in MA-10 NE are marked as I, II, and
III. B, Formation of protein-DNA complexes with rec CREB protein (a,
lanes 12) or MA-10 NE (I, II, and III, lanes 36) in the absence or
presence of Abs to different CREB family members using -83/-67 bp as
the probe. The arrow indicates supershift (SS) of the
retarded complexes. These experiments were repeated three to five
times, and a representative phosphorimage from each group is
illustrated. C, RT-PCR analysis of CREM and CREB mRNAs in MA-10,
Y-1, and COS-1 cells. Total RNA was extracted from these cells and
subjected to RT-PCR analysis of CREM and CREB gene expression as
described in Materials and Methods. A representative
experiment from four different experiments is shown.
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The involvement of CREM protein in protein-DNA binding as shown
with EMSA was further assessed using a shorter oligonucleotide probe
(-83 to -67) containing the CRE2 and CRE3 sites (Fig. 7B
). The
results obtained with this shorter probe were similar to those observed
with -96/-67 bp probe and demonstrated qualitatively similar
protein-DNA binding with either rec CREB protein (lanes 1 and 2) or NE
prepared from (Bu)2cAMP-stimulated MA-10 cells
(lanes 36). However, the binding observed at complex I was decreased
using this probe (-83/-67 bp), suggesting that CRE1 may play a role
in the formation of protein-DNA complexes. Importantly, CREM1-specific
Ab abolished protein-DNA complexes (lane 5) while CREB-1 Ab had no
effect on them (lane 6). These data further confirm the presence of
CREM proteins in MA-10 NE that bind to the CRE half-sites.
Since CREM proteins appeared to be the major species binding to the CRE
sites, the relative abundance of CREB and CREM mRNAs in MA-10, Y-1, and
COS-1 cells were determined using RT-PCR analysis (Fig. 7C
). Two
conclusions can be drawn from these results. 1) Both MA-10 and Y-1
cells contain several different sized CREM transcripts while there
appears to be only one CREB transcript in these cells. 2) There is a
significant increase in the total amount of CREM transcripts when
compared with the CREB transcript. Similar results were also obtained
in the nonsteroidogenic COS-1 cell line (Fig. 7C
). These results
further support our EMSA studies and are in agreement with previous
data showing CREM to be the predominant transcript in the testis
(32, 33).
Relative Importance of CRE Half-Sites
The importance of the three CRE half-sites was further assessed by
generating mutations in each CRE half-site either alone or in
combination, followed by competition analysis using EMSA. The data
presented in Fig. 8
demonstrate that
protein-DNA complexes observed with the -96/-67 bp oligonucleotide
probe using (Bu)2cAMP-stimulated MA-10 NE (lanes
115) were effectively inhibited by 100-fold molar excess addition of
its unlabeled sequences (-96/-67; lane 2). CREM protein-DNA binding
specificity was also assessed by competition with a consensus CRE
sequence (lane 3). The protein-DNA complexes were competed with
100-fold molar excess oligonucleotides bearing mutations in CRE1
(TGACCC to Tccgga; lane 4), CRE1 affecting steroidogenic factor 1
(SF-1) binding (CRE1/SF-1) (TGACCC to gaAttC; lane 5), and CRE3
(lane 7), indicating these sites did not affect CREM binding. However,
mutations in CRE1&3 sites (lane 11) demonstrated relatively weaker
competition, suggesting that both sites had a modest effect on these
complexes. On the other hand, mutation of the CRE2 site either alone
(lane 6) or in combination with other CRE sites, i.e. CRE1&2
(lane 10), CRE2&3 (lanes 12 and 13), and CRE1,2,3 (lanes 14 and 15),
were not able to compete with these complexes, suggesting the greater
importance of the CRE2 site in CREM protein-DNA binding. In fact,
mutations in the CRE2 and CRE3, or the CRE1,2,3 sites comprised of
different mutated bases (see Materials and Methods)
demonstrated qualitatively similar results (lanes 12 and 14). In
addition, radioactively labeled -96/-67 StAR probes containing
mutations in CRE1 or CRE3 alone showed formation of protein-DNA
complexes with MA-10 NE similar to -96/-67, while the formation of
these complexes was markedly affected in a probe containing the CRE2
mutation (data not shown). It is noteworthy that the CRE2 site is
analogous to an AP-1 binding motif (TGACTGA) previously identified
(31). Hence, a consensus AP-1 sequence was used in
competition experiments (lane 9) and showed strong competition with the
protein-DNA complexes. Since CREM family members can also bind to AP-1
sites (34, 35), a point mutation was introduced in the
AP-1 site (TGACgGA; lane 8), and the resulting sequence demonstrated
similar competition with the protein-DNA complexes. These data are
consistent with the hypothesis that complex formation on the CRE2/AP-1
binding site was predominantly CREM proteins. Furthermore, we have
observed that the Ap-1 family members, cfos and Fra-2 markedly decrease
CREM protein-DNA binding with the -97/-67 StAR probe while jun
members have little or no effects on these complexes (data not
shown).

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Figure 8. The Importance of the CRE Half-Sites Within -96 to
-67 bp Region of the StAR Gene
(Bu)2cAMP (0.5 mmol/liter) induced MA-10 NE was used in
EMSA as described in Materials and Methods. The
following unlabeled oligonucleotide competitors were used at 100-fold
molar excess: self (-96/-67 StAR, lane 2), consensus CRE (Cons CRE,
lane 3), and consensus AP-1 (Cons AP-1, lane 9). Also the following
unlabeled probes consisting of mutations in CRE1 (lane 4), CRE1 and
SF-1 (CRE1/SF-1; lane 5), CRE2 (lane 6), CRE3 (lane 7), AP-1 point
mutation (AP-1 pt Mut; lane 8), or double and triple mutations in CRE
sites as indicated (lanes 1015) were used. MA-10 protein-DNA
complexes are marked as I, II, and III. These experiments were repeated
three times, and the data from one representative experiment are shown.
The migration of free probe is shown in each lane.
|
|
To obtain even more insight into these mechanisms, specific mutations
were generated in each of the CRE half-sites, either alone or in
combination, in the -151/-1 bp fragment, and StAR promoter/luciferase
activity was determined. This construct was used since the -110 bp
StAR fragment demonstrated decreased basal promoter activity. MA-10
cells transfected with -151 StAR/luc demonstrated an approximately
4-fold increase in the (Bu)2cAMP-stimulated
luciferase response over nonstimulated cells (Fig. 9
). Mutation of the CRE1 site (TGACCC to
Tccgga) did not significantly affect cAMP-stimulated reporter promoter
activity, although there was an approximately 35% decrease in basal
activity when compared with the wild-type StAR promoter. On the other
hand, mutation of the CRE2 site resulted in an approximately 50%
decrease in both basal and cAMP-induced promoter activity as compared
with controls. Mutation of the CRE3 site had essentially no effect on
basal or cAMP-stimulated StAR promoter/luciferase responses.
Interestingly, mutation in the bases of CRE1 involved in SF-1 binding
(CRE1/SF-1) caused approximately 80% decreases in basal and
cAMP-stimulated activities. Attenuation of basal and cAMP-stimulated
promoter responses was consistently observed when mutations generated
in the CRE1/SF-1 site were assessed in combination with mutations in
CRE2 and/or CRE3, with the CRE1/SF-1 and CRE2 double mutation and the
triple mutation having an equally significant effect (Fig. 9
).
Consistent with these observations, transfection of WT-CREB with the
-151 CRE2 mutant significantly diminished the CREM-dependent cAMP
induction of StAR promoter activity while there was little or no effect
of mutating the CRE1 or CRE3 sites, respectively (data not shown).
These data further confirm the importance of CRE2 in CREM-induced StAR
promoter activity and demonstrate the site specificity of these CRE
elements in cAMP responsiveness.

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|
Figure 9. Functional Assessment of the CRE Half-Sites in StAR
Promoter/Luciferase Activity
MA-10 cells were transiently transfected with different -151 StAR
constructs containing mutations in one or more CRE sites (as
indicated), in the presence of pRL-SV40 as described in
Materials and Methods. pGL3-Basic (pGL3) was used as a
control. After 36 h of transfection, cells were incubated for a
further 6 h without or with (Bu)2cAMP (500
µmol/liter). Luciferase activity in the cell lysates was determined
and expressed as relative light units (RLU, luciferase/renilla). These
experiments were repeated three to five times, and data
(±SEM) from one representative experiment in triplicate
are presented.
|
|
Effect of ICER on Endogenous CREM-Mediated cAMP-Stimulated StAR
Promoter Activity and StAR mRNA Expression
The functional involvement of CREM on StAR promoter activity and
StAR expression was further assessed in MA-10 cells after
cotransfection with ICER. As before, expression of WT-CREB with the
-254 StAR/luc promoter significantly (P < 0.01)
increased (Bu)2cAMP-induced StAR promoter
activity (Fig. 10A
) and StAR gene
expression (Fig. 10B
) as compared with mock-transfected cells. Cells
transiently transfected with ICER alone or in combination with WT-CREB
marginally affected basal responses. However, the cAMP-stimulated level
of StAR promoter/luciferase activity was inhibited by approximately
90% (Fig. 10A
), and StAR mRNA expression by 80% (Fig. 10B
), in
WT-CREB+ICER-transfected cells. P levels in the media of the
corresponding incubations demonstrated qualitatively similar results
(data not shown). These data are again consistent with the involvement
of endogenous CREM in cAMP-induced StAR expression and steroid
biosynthesis in MA-10 cells.

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|
Figure 10. Effects of WT-CREB, ICER, and Their Combination on
cAMP-Induced StAR Promoter Activity and StAR mRNA Expression in MA-10
Cells
Cells were transiently transfected with -254 StAR/luc and either empty
expression vector (MT), ICER, WT-CREB (CREB) or both CREB+ICER
expression plasmids. Panel A illustrates the StAR promoter/luciferase
activity in different incubations. The variation in transfection
efficiency was monitored by a pRL-SV40 plasmid cotransfected in each
assay as described in Materials and Methods. After
36 h of transfection, cells were incubated for 6 h without
(Basal) and with (Bu)2cAMP (500 µmol/liter), and
luciferase activity in the cell lysates was determined and expressed as
RLU (luciferase/renilla). Data represent the mean ±
SEM of four experiments. To determine StAR mRNA expression,
MA-10 cells were transfected either with WT-CREB, ICER alone, or in
combination as above. After 36 h, cells were stimulated without
(Basal) or with 500 µmol/liter (Bu)2cAMP. Panel B, Total
RNA was extracted from different treatment groups and subjected to
RT-PCR analysis for StAR mRNA expression as described in the legend of
Fig. 1 and in Materials and Methods. A representative
autoradiogram from three independent experiments with similar results
is shown. The IOD values represent the mean ± SEM of
three experiments.
|
|
 |
DISCUSSION
|
---|
The StAR protein plays a critical role in steroidogenesis by
mediating the intramitochondrial transport of cholesterol to the
cholesterol side chain cleavage cytochrome P450 enzyme (1, 2, 36). Rapid expression of StAR in response to trophic hormone is
required to initiate and maintain ongoing steroid hormone biosynthesis,
and this expression is regulated via the cAMP second messenger
signaling pathway. Given the importance of StAR expression and the
purported role of cAMP in this process, we explored the possible
involvement of one or more of the members of the CREB/CREM family of
transcription factors in steroidogenesis and StAR expression. The
CREB/CREM family is distinguished by their DNA-binding bZip domains and
mediate part of the transcriptional response to cAMP signaling
(11, 37, 38, 39). To more fully understand the role of
CREB/CREM in this regulation, the present investigation used three
different inhibitors of CREB/CREM function: a dominant-negative A-CREB
that blocks the DNA binding activity in a dimerization-dependent
fashion (23), CREB-M1 (Ser 133Ala
substitution) that is unable to be phosphorylated by PKA (20, 40), and the transcription repressor, ICER, which forms inactive
heterodimers (13, 25, 26).
Our results demonstrate that cells expressing WT-CREB significantly
increased cAMP-dependent steroid production, StAR promoter activity,
StAR mRNA, and StAR protein, thus showing a potential role for a
CREB/CREM family member. In addition, 5'-deletion analysis of the mouse
StAR gene identified the presence of CREB/CREM responsive element(s)
between -110 and -68 bp of the transcription start site, a region
containing three half-sites for 5'-canonical CRE sequences. An
oligonucleotide probe (-96/-67 bp) containing these CRE half-sites
showed protein-DNA binding with rec CREB protein or NE prepared from
MA-10 cells. Utilizing a series of antibodies for various members of
the CREB/CREM family, it was determined that the CREB/CREM family
member, CREM, was the predominant form binding to the CRE sites. The
quantities of CREM and CREB transcripts were established by RT-PCR, and
it was found that CREM transcripts were present in greater abundance
than was the CREB transcript in both MA-10 and Y-1 cells. The role of
CREM was further strengthened by the observation that ICER inhibited
the ability of endogenous CREM proteins to induce steroid hormone
biosynthesis and StAR mRNA expression in response to cAMP. These
results clearly indicate an important role of CREB/CREM family members,
most notably CREM, in the cAMP-mediated regulation of steroidogenesis
and StAR expression and thus provide a potential new mechanism for the
rapid stimulation of StAR by cAMP.
The observations obtained with CREB-M1 indicates the importance of
phosphorylation and provides a mechanism for the rapid increase in StAR
expression by PKA activation (19, 20). Our findings are
consistent with previous data demonstrating that CREB-M1 inhibited
somatostatin gene transcription and adversely affected survival in
transfected F9 teratocarcinoma, rat granulosa, and Sertoli cells,
respectively (19, 22, 41). Similarly, our observation that
endogenous CREM-dependent, cAMP-induced steroid production and StAR
expression were markedly decreased by ICER is consistent with previous
studies demonstrating that ICER down-regulated the CRE-mediated
transcription of the CREB gene in Sertoli cells (42) and
suppressed both basal and cAMP-induced expression of the inhibin
-subunit in granulosa cells (43).
As discussed above, while StAR expression is regulated through a
PKA-dependent pathway, the StAR promoter lacks a consensus CRE. In such
cases it is possible that alternate elements are involved in
CREB/CREM function, a phenomenon that has previously been observed for
cAMP-regulated genes in which nonconsensus CREs mediate hormonal
induction (12). Our data on the 5'-flanking region of the
mouse StAR gene indicates that the majority of the acute cAMP
responsive region was present between -110 and -68 bp upstream of the
transcriptional start site. This is the same region required for
CREB/CREM responsiveness and supports the observation made in a
previous study (31). We identified three 5'-canonical CRE
half-sites within this region that are well conserved among different
species. Indeed, our data clearly show that an oligonucleotide probe
(-96/-67 bp) containing these CRE sites resulted in the formation of
specific protein-DNA complexes with rec CREB or with NE from MA-10 and
Y-1 cells. Specific mutations in the CRE half-sites within the
-96/-67 bp region demonstrated the predominant involvement of the
CRE2 site in CREM binding and in cAMP responsiveness. However, CREB
activation of a promoter can require the binding of multiple CRE
binding factors to several CREs rather than the binding of a single
dimer (44, 45, 46). Thus, it is possible that the CRE1 and
CRE3 sites may also play roles, albeit lesser ones, in the expression
of the StAR gene.
The CRE2 site maps to the CREs identified in two previous studies and
referred to as C/EBPß-3 (47) or the
C/EBPß/nonconsensus activating protein (AP-1)/nuclear receptor
half-site (CAN) (31). In those studies, a mutation
of the AP-1 site (the same mutation as the CRE2 mutation in the current
studies), or a different mutation in the C/EBPß-3 site both abolished
protein-DNA binding and reduced basal and cAMP/FSH-responsive StAR
promoter activity by approximately 50% (31, 47). Members
of the AP-1, CREB/CREM/ATF-1, and C/EBP families are bZip proteins that
recognize similar DNA sequences, and certain ATF/CREB factors can
heterodimerize with AP-1 (34, 35) and C/EBP
(48) family members depending on the binding site. Further
studies must be performed to determine whether bZip family heterodimers
can form in a tissue-specific fashion on the CRE2 site and whether
these heterodimers are important for the cAMP induction of StAR
expression.
Previous EMSA studies demonstrated C/EBPß binding to -87 to -70 of
the StAR promoter in ovarian extracts (47); however, in
subsequent studies, Abs to C/EBPß and AP-1 family members did not
affect complex formation in MA-10 NE bound to a similar region
(-87/-64, see Ref. 31). Moreover, as there was a cAMP-dependent
increase in MA-10 NE binding (our data and Ref. 31) and
since we found rec CREB bound to -96/-67 of the StAR promoter, Ab
supershift experiments coupled with the use of specific Abs were
performed to determine whether this region bound endogenous CREB/CREM
protein(s) present in MA-10 extracts. Indeed, the protein-DNA complexes
were essentially abolished in the presence of CREM1-specific Ab, while
antisera to CREB and ATF-1 family members demonstrated only a modest or
no inhibition of these complexes. We also corroborated previous results
(31) indicating that C/EBPß does not appear to bind to
this region.
While a role for AP-1 or other bZip family members cannot be
eliminated, the present observations suggest that CREM proteins are the
predominant binding species in MA-10 cells. Consistent with our EMSA
data, RT-PCR analyses demonstrated that the total amount of CREM mRNA
is much higher than CREB mRNA in MA-10 and Y-1 cells. These findings
are in agreement with previous studies using Northern and in
situ analyses which demonstrated that the number of CREM
transcripts in the testis are markedly higher than those of CREB
(32, 33). In the adult testis CREM transcripts exclusively
encode for the activator form of CREM, CREM
(17). Thus,
it would appear that CREM is the predominant endogenous protein in
MA-10 NE binding to the CRE-2 site. Moreover, our EMSA experiments
using MA-10 cells transfected with WT-CREB suggest that the protein-DNA
complex changes (from complex I, II, and III) to resemble that of rec
CREB (complex I only) are presumably due to titration effects. In
contrast, there was no apparent change in complex formation in Y-1
cells either in response to cAMP or when transfected with WT-CREB.
Hence, our data corroborate previous work (31, 47)
suggesting that there are cell type-specific binding differences in
-96/-67 of the StAR promoter.
Several studies have identified three elements that bind the
transcription factors SF-1 (our work and Ref. 31), C/EBP
(47, 49, 50), and GATA-4 (31, 47) within the
-110 to -64 bp region of the StAR promoter, and each site influenced
basal and/or cAMP induction. However, interaction of these factors with
CREB/CREM family members needs further exploration. Most likely, in the
absence of CREB/CREM binding, the cAMP-dependent response remains
partially intact due to coactivator recruitment by other
transcription factors bound to this specified region of the StAR
promoter. We propose that the SF-1, CRE2/AP-1/C/EBPß-3, and GATA
sites might function as a complex cAMP response unit. In support of
this, studies on the phosphoenol-pyruvate carboxykinase,
aromatase, and inhibin-
genes demonstrated that CREB synergizes
with other transcription factors to mediate cAMP responsiveness
(40, 46, 51, 52). In addition, there is extensive
documentation of the cooperation between SF-1 and C/EBPß, GATA-4, or
Sp1 in the regulation of StAR gene transcription and
steroidogenesis (31, 49, 53, 54). The interaction of
CREB/CREM with other transcription factor(s) and/or coactivators that
may bind within this region of the StAR promoter and be required for
full promoter activity will be the subject of future investigations.
Moreover, as aromatase and other steroidogenic enzymes previously shown
to be regulated by cAMP contain elements for SF-1 and/or GATA, these
genes may also be regulated by a complex cAMP response unit containing
a nonconsensus CRE site similar to the StAR promoter.
In conclusion, we present evidence the CREB/CREM family member, CREM,
is functionally important in MA-10 and Y-1 cells for maximal
cAMP-dependent steroidogenesis and StAR gene expression. The mechanism
was found to be predominantly mediated through the binding of CREM to
the CRE2 site; however, all three CRE elements identified and
characterized within the -96/-67 bp region of the mouse StAR gene
mediate at least part of the response. We also propose that trophic
hormone-stimulated steroid biosynthesis in other steroidogenic cells
may be mediated by similar mechanisms.
 |
MATERIALS AND METHODS
|
---|
Construction of StAR Promoters and CRE Mutants
Various truncations in the 5'-flanking region of the mouse StAR
gene (-966 bp, -254 bp, -110 bp, and -68 bp) were cloned upstream
of a luciferase reporter gene into the pGL2 basic vector (Promega Corp., Madison, WI), as described previously
(27).
The -151/-1 bp region of the StAR promoter was synthesized
using a PCR-based cloning strategy using -254-StAR-pGL2 as the
template. The 5' primer
5'-TAGCTCGAGTCTGCTCCCTCCCACCTTGGCCAGC-3' and the
3'-primer 5'-CTAAAGCTTGGCGCAGATCAAGTGCGCTGCCT-3',
contain a XhoI and HindIII site
(underlined) at the 5' end, respectively. The amplicon is
then subcloned into PCR2.1-TOPO vector following the TOPO-TA cloning
method of the manufacturer (CLONTECH Laboratories, Inc.,
Palo Alto, CA). The XhoI and HindIII fragments
were cleaved from the StAR-PCR2.1 vector, purified, and subcloned
into the XhoI and HindIII cloning sites of the
pGL3 basic vector (Promega Corp.), which contains firefly
luciferase as a reporter gene. The identity of the inserted
XhoI and HindIII fragments was confirmed by
sequencing using the pGL3 clockwise 5'-CTAGCAAAATAGGCTGTCCC-3' and
counterclockwise 5'-TGGAAGACGCCAAAAACATAAAG-3' primers,
corresponding to a region 3' of the multiple cloning site and the
beginning of the region encoding luciferase.
Plasmids containing a single mutation in the CRE half-sites were
generated using the Quikchange site-directed mutagenesis kit
(Stratagene, La Jolla, CA) using -151/-1 StAR-pGL3 as
the template. Double or triple mutations in the CRE sites were
generated using appropriate CRE single/double mutation vectors as the
PCR template. The sense strand of the oligonucleotide sequences used
were [mutated (Mut) bases in boldface lowercase letters]:
CRE1 Mut, 5'-GGCAATCATTCCATCCTTccggaTCTGCACAATGAC-3';
CRE1/SF1 Mut,
5'-GCAATCATTCCATCCTgaAttCTCTGCACAATGAC-3';
CRE2 Mut
5'-CCTTGACCCTCTGCACAATagaTcttGACTTTTTTATCTC-3';
CRE3 Mut 5'-CCCTCTGCACAATGACTGAgatCTTTTTTATCTC-3'; CRE2&3
Mut 5'-CCTTGACCCTCTGCACAAgcggccgcGACTTTTTTATCTCAAG-3'.
Specific mutations were tested by restriction endonuclease digestion
using Sau 3A I (CRE1 Mut), EcoRI (CRE1/SF-1 Mut),
BglII (CRE2 Mut and CRE3 Mut), or NotI (CRE2&3
Mut). Finally, a XhoI and HindIII fragment
containing the mutations was religated into the pGL3 vector. Assessment
of these mutations was confirmed by sequencing on a PE Biosystems 310 Genetic Analyzer (Perkin Elmer) at
the Texas Tech University Biotechnology Core Facility.
RNA Extraction and Quantitative RT-PCR
Total RNA was extracted from different treatment groups using
Trizol reagent (Life Technologies, Inc., Gaithersburg,
MD). The isolation and amplification of MA-10 StAR cDNA were carried
out utilizing the mouse StAR cDNA sequence (3), as
previously described (55). Briefly, the following primer
pairs were used for StAR amplification: the forward primer,
5'-GACCTTGAAAGGCTCAGGAAGAAC-3', and the reverse primer,
5'-TAGCTGAAGATGGACAGACTTGC-3', spanning bases -51 to -27 and
931908, respectively. The variation in RT-PCR efficiency was
corrected for using an L19 ribosomal protein gene, which was amplified
using the following primer pairs: the forward
5'-GAAATCGCCAATGCCAACTC-3' and the reverse 5'-TCTTAGACCTGCGAGCCTCA-3'
(56). RT and PCR of StAR and L19 were run sequentially in
the same assay tube using 2 µg of total RNA, and the parameters
including the number of cycles used were optimized to be in the
exponential phase as previously described (55, 57).
CREB and CREM cDNAs were generated with 2 µg of total RNA using the
specific primer pairs: CREB forward, 5'-GCAGTGACGGAGGAGCTTGTAC-3'
(bases 101122) and CREB reverse, 5'-TCTGATTTGTGGCAGTAAAG-3' (bases
1,1381,157); CREM forward, 5'-ACTGGGCAAATTTCAATCCCTGC-3' (bases
88110), and CREM reverse, 5'-CAAACTTCCGGGCGATGCAGCCATC-3' (bases
765789), respectively (58, 59, 60). cDNAs were amplified by
PCR for 32 cycles (94 C for 75 min, 58 C for 2 min, and 72 C for 3
min). The identity of all RT-PCR products was confirmed by automated
sequencing (as above) as described previously (55), and
the sequences obtained corresponded to previously published findings
(3, 56, 58, 59, 60).
The molecular sizes of the PCR products (StAR, CREB, CREM, and L19)
were determined in 1.2% agarose gels. The gels were stained with
ethidium bromide (CREB and CREM). For StAR and L19, the gels were
vacuum dried and exposed to Hyperfilm (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK) at 4 C
between 1 and 3 h, and relative levels of different signals were
quantitated by densitometry.
Preparation of Mitochondria and Immunodetection of StAR
Protein
Isolation of mitochondria was carried out as follows. In brief,
cells were homogenized in TSE buffer (10 mmol/liter Tris, 250
mmol/liter sucrose, 100 mmol/liter EDTA, pH 7.4) containing protease
inhibitors (1 mmol/liter dithiothreitol, 1 mmol/liter
phenylmethylsulfonyl fluoride, 2 mg/liter leupeptin, 2 mg/liter
aprotinin) at 1,200 rpm for 30 passes with a Potter-Elvehjem
homogenizer. Mitochondrial isolation was carried out by differential
centrifugation, as previously described (3, 57).
Mitochondrial proteins (1520 µg) were solubilized in sample buffer
(25 mmol/liter Tris-Cl, pH 6.8, 1% SDS, 5% ß-mercaptoethanol,
10% glycerol, and 0.01% bromophenol blue) and loaded onto 12%
SDS-polyacrylamide gels (Mini Protean II System, Bio-Rad Laboratories, Inc., Hercules, CA), as described by Laemmli
(61). Electrophoresis was performed at 200 V for 1 h,
and the proteins were electrophoretically transferred onto Immuno-blot
polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc.). The membranes were incubated in a blocking buffer
(Tris-buffered saline, containing 0.1% Tween-20 and 5% nonfat dry
milk) for 2 h at room temperature, followed by incubation with
anti-StAR peptide antibodies (3, 57). Immunodetection of
StAR protein was performed using the ECL Western blotting detection kit
(Amersham Pharmacia Biotech, Piscataway, NJ) and after
exposure of the membranes to Hyperfilm (Amersham Pharmacia Biotech) for the appropriate times, bands were quantitated using
computer- assisted image analysis (Visage 2000, BioImage, Ann Arbor,
MI).
Preparation of NE and EMSAs
The NE from different treatment groups were prepared as
described previously (62, 63), slightly modified to
achieve higher purity. Briefly, cells were washed and collected with
0.01 mol/liter PBS and centrifuged at 1,000 x g for 5
min. The pellet was resuspended in buffer A containing protease
inhibitors (10 mmol/liter HEPES, 1.5 mmol/liter
MgCl2, 10 mmol/liter KCl, 0.5 mmol/liter
dithiothreitol, 0.1% Nonidet P-40, 1 mmol/liter phenylmethylsulfonyl
fluoride, 2 mg/liter leupeptin, 2 mg/liter aprotinin, pH 7.9). After
centrifugation, the crude nuclear pellet was resuspended and allowed to
swell for 2030 min in a rotator at 4 C in buffer C (buffer A
containing 10 mmol/liter HEPES, 0.42 mmol/liter NaCl, 0.2 mmol/liter
EDTA, 25% glycerol, pH 7.9). After removing the debris by
centrifugation at 12,000 x g for 3 min, the NE was
assayed directly or stored at -80 C.
The different mouse StAR oligonucleotide probes were annealed by
heating sense and antisense primers to 65 C for 5 min in annealing
buffer (10 mmol/liter Tris-Cl, 100 mmol/liter NaCl, 1 mmol/liter EDTA,
pH 7.5) and then slowly cooled over 2 h to room temperature. The
following sense primers of the oligonucleotides and mutated (Mut)
sequences (lowercase bold) were used:
-96/-67 StAR
5'-GGTGACCCTCTGCACAATGACTGATGACTTTT-3'
-96/-67 StAR CRE1 Mut
5'-GGTccggaTCTGCACAATGACTGATGACTTTT-3'
-96/-67 StAR CRE1/SF-1 Mut
5'-GGgaAttCTCTGCACAATGACTGATGACTTTT-3'
-96/-67 StAR CRE2/AP-1 Mut
5'-GGTGACCCTCTGCACAATagaTctTGACTTTT-3'
-96/-67 StAR CRE3 Mut
5'-GGTGACCCTCTGCACAATGACTGAgatCTTTT-3'
-96/-67 StAR AP-1 point (pt) Mut
5'-GGTGACCCTCTGCACAATGACgGATGACTTTT-3'
-96/-67 StAR CRE1&2 Mut:
5'-GGTccggaTCTGCACAATagaTctTGACTTTT-3'
-96/-67 StAR CRE1&3 Mut:
5'-GGTccggaTCTGCACAATGACTGAgatCTTTT-3'
-96/-67 StAR CRE2&3 Mut:
5'-GGTGACCCTCTGCACAATagaTctgatCTTTT-3'
-96/-67 StAR CRE1,2,3 Mut:
5'-GGTccggaTCTGCACAATagaTctgatCTTTT-3'
-96/-67 StAR CRE2&3 Mut no. 2:
5'-GGTGACCCTCTGCACAAgcggccgcGACTTTT-3'
-96/-67 StAR CRE1,2,3 Mut no. 2:
5'-GGgaAttCTCTGCACAAgcggccgcGACTTTT-3'
-83/-67 StAR: 5'-GGAATGACTGATGACTTTT-3'
Consensus AP-1 (Ref. 34):
5'-GGCGCTTGATGAGTCAGCCGGAA-3'
Consensus CRE (Ref. 9):
5'-GGAGAGATTGCCTGACGTCAGAGAGCTAG-3'
The 5'-GG overhangs present in the
doubled-stranded oligonucleotides (4 pmol/liter each) were end-labeled
with [
32P]-dCTP (DuPont Easytides, 6000
Ci/mmol, Amersham Pharmacia Biotech) using Klenow
(Promega Corp.) at 25 C for 15 min. The probes were
purified using Probe Quant spin columns (Amersham Pharmacia Biotech). DNA-protein binding assays were carried out under
optimized conditions as described previously (49, 63, 64).
Briefly, 10 µg NE or 1.5 µg rec CREB protein were incubated in 1x
reaction buffer (10 mM HEPES, 1 mmol/liter EDTA, 4%
Ficoll, 10 mmol/liter dithiothreitol, 1 µg poly dI·dC, 40 ng/µl
BSA, pH 7.9) for 15 min at room temperature before the addition of
32P-labeled double-stranded DNA probe (0.5
pmol/liter,
100,000 cpm). When antiserum was used, binding reactions
were incubated an additional 45 min on ice before the addition of the
labeled DNA. The following Abs were used in EMSA reactions: monoclonal
mouse anti-human ATF-1 Ab (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA), which maps to the DNA binding and dimerization
domain and is reactive with ATF-1 p35, CREB-1 p43, and CREM1; CREM1 Ab,
which is specific for CREM and cross-reacts with CREB only at very high
protein concentrations (32, 65); CREB-1 Ab, which is
specific for CREB (65), and C/EBPß Ab, which is specific
for C/EBPß (49, 66). In addition, polyclonal
rabbit antihuman ATF-2 (specific for ATF-2), polyclonal rabbit
antihuman ATF-3 (specific for ATF-3), polyclonal goat antihuman CREB-2
(specific for CREB-2 and designated ATF-4), and mouse IgG were
purchased from Santa Cruz Biotechnology, Inc. When
competitors were used, 50 pmol/liter (100-fold molar excess) of
unlabeled oligonucleotide competitors were added at the same time
as the radiolabeled probe and incubated for 15 min. The entire
reaction was then subjected to electrophoresis at 200 V for about
1.5 h through a 5% polyacrylamide gel in 0.5x TBE buffer
(90 mmol/liter Tris-borate, 2 mmol/liter EDTA, pH 8.0). The gels were
dried and then exposed to phosphor screens. Detection and quantitation
of the protein-DNA complexes were performed using a phosphorimaging
device (Molecular Dynamics, Inc., Sunnyvale, CA).
Cell Culture, Transfections, and Luciferase Assays
The MA-10 mouse Leydig tumor cells (67) were a
generous gift from Dr. Mario Ascoli (Department of Pharmacology,
University of Iowa College of Medicine, Iowa City, IA). These cells
were maintained in HEPES-buffered Weymouths MB/752 medium
supplemented with 15% heat-inactivated horse serum (HS; Life Technologies, Inc., Gaithersburg, MD) containing 40 mg/liter of
gentamicin sulfate, in a humidified atmosphere of 95% air and 5%
CO2. The Y-1 (mouse adrenocortical tumor) and
COS-1 (African green monkey kidney) cells were obtained from
American Type Culture Collection (Manassas, VA) and grown,
respectively, in Y-12K medium supplemented with 15% HS and 2.5% FBS
and DMEM with high glucose plus 10% (FBS), containing 50,000 U/liter
penicillin and 50 mg/liter of streptomycin (Life Technologies, Inc.
Transfections were carried out when the cells were 6575% confluent
using FuGENE 6-transfection reagent (Roche Diagnostics
Corp., Indianapolis, IN) according to the manufacturers
instructions under optimized conditions (55). Briefly,
MA-10 cells were transfected with 2 µg of a particular CREB
expression construct or WT-CREB (CREB341) plus ICER II
(ICER)
expression constructs (2 µg each) where indicated (see Ref.
25) for studying both StAR expression and steroidogenesis.
The levels of P in the media were measured by RIA as described
previously (68).
For promoter studies, cells were transfected with CREB expression
plasmids together with equal amounts (1:1) of various truncations in
the 5'-flanking region of the StAR gene (-966 StAR/luc, -254
StAR/luc, -110 StAR/luc, and -68 StAR/luc) as specified elsewhere, in
the presence of 10 ng pRL-SV40 vector (a plasmid that constitutively
expresses renilla luciferase) to normalize for transfection efficiency.
Reporter assays were also carried out in combination with WT-CREB and
ICER. The effects of mutating the three CRE half-sites either alone or
in combination on StAR promoter/luciferase activity were performed in
MA-10 cells transfected with 1.0 µg of different -151 StAR-pGL3
plasmids.
Luciferase activity in the cell lysates was determined by the
Dual-luciferase reporter assay system (Promega Corp.).
Briefly, after washing, 300 µl of the reporter lysis buffer were
added to the cells. The cellular debris was pelleted by centrifugation
at 14,000 x g at 4 C, and the supernatant was measured
for relative light units in a Monolight 2010 luminometer
(Analytical Luminescence Laboratory, San Diego, CA)
following the manufacturers instructions.
Statistical Analysis
The data presented are the mean ± SEM.
Statistical analysis was performed by ANOVA using Statview (Abacus
Concepts Inc., Berkeley, CA) followed by Fishers protected least
significant differences test (Fishers PLSD). P <
0.05 was considered statistically significant.
 |
ACKNOWLEDGMENTS
|
---|
The authors would like to thank Dr. M. Montminy (Department of
Cell Biology, Harvard Medical School, Boston, MA) and Dr. R. H.
Goodman (Oregon Health Sciences University, Portland, OR) for the
generous gifts of CREB-M1 and WT-CREB, respectively. We thank Dr. S.
Williams (Department of Cell Biology and Biochemistry, Texas Tech
University Health Sciences Center, Lubbock, TX) for providing us with
C/EBPß antiserum, and Dr. M. R. Waterman (Department of
Biochemistry, Vanderbilt University School of Medicine, Nashville, TN)
for the CREB-1 antiserum for this study. The -151/-1 pGL3-StAR
plasmid was a generous gift from Dr. X. J. Wang (Texas Tech
Health Sciences Center). The technical assistance of Deborah Alberts is
gratefully acknowledged.
 |
FOOTNOTES
|
---|
This work was supported in part by NIH Grant HD-17481 and by the Robert
A. Welch Foundation.
Abbreviations: Ab, Antibody; ATF-1, activating
transcription factor 1; bZip, basic-leucine zipper; CRE, cAMP response
element; CREB, CRE binding protein; CREM, CRE modulator; ICER,
inducible cAMP early repressor; Mut, mutant; NE, nuclear extracts; P,
progesterone; rec CREB, recombinant CREB; SF-1, steroidogenic factor 1;
StAR, steroidogenic acute regulatory protein; WT-CREB, wild-type
CREB.
Received for publication January 19, 2001.
Accepted for publication September 25, 2001.
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