Characterization of the Promoter Region of the Mouse Gene Encoding the Steroidogenic Acute Regulatory Protein
Kathleen M. Caron,
Yayoi Ikeda,
Shiu-Ching Soo,
Douglas M. Stocco,
Keith L. Parker and
Barbara J. Clark
Departments of Medicine and Pharmacology Duke University
Medical Center (K.M.C., Y.I., S.-C.S., K.L.P.) Durham, North
Carolina 27710
Department of Cell Biology and
Biochemistry Texas Tech University Health Science Center
(D.M.S.) Lubbock, Texas 79430
Department of
Biochemistry University of Louisville School of Medicine
(B.J.C.) Louisville, Kentucky 40292
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ABSTRACT
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Steroidogenic acute regulatory protein (StAR)
delivers cholesterol to the inner mitochondrial membrane, where the
cholesterol side-chain cleavage enzyme carries out the first committed
step in steroid hormone biosynthesis. StAR expression is restricted to
steroidogenic cells and is rapidly induced by treatment with trophic
hormones or cAMP. We analyzed the 5'-flanking region of the mouse
StAR gene to elucidate the mechanisms that regulate its
cell-specific and hormone-induced expression. In transient transfection
assays, a luciferase reporter gene driven by the StAR
5'-flanking region was preferentially expressed by steroidogenic Y1
adrenocortical and MA-10 Leydig cells in a cAMP-responsive manner.
5'-Deletion and site-directed mutagenesis studies identified a region
between -254 and -113 that is essential for full levels of promoter
activity. This region contains a binding site for the orphan nuclear
receptor steroidogenic factor-1 (SF-1) that, although not required for
hormone induction, is critical for basal promoter activity, thus
implicating SF-1 in StAR expression. Analyses of knockout mice
deficient in SF-1 further supported an important role for SF-1 in
StAR gene expression. These studies provide novel insights
into the mechanisms that regulate StAR gene expression and
extend our understanding of SF-1s global roles within
steroidogenic cells. Molecular Endocrinology 11: 138147, 1997)
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INTRODUCTION
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A critical component of the regulated production of steroid
hormones is the induction of their synthesis by pituitary trophic
hormones. This hormonal induction is mediated predominantly by
increases in intracellular cAMP and can be divided temporally into two
phases: acute effects, which occur within seconds to minutes, and
chronic effects, which require hours before they are manifest. The
acute induction largely reflects increased mobilization and delivery of
cholesterol, the precursor for all physiological steroid hormones, to
the mitochondrial inner membrane, where it is metabolized to
pregnenolone by the cytochrome P450 cholesterol side-chain cleavage
enzyme (reviewed in Ref.1). In contrast, chronic effects of trophic
hormones largely result from increased transcription of the genes that
encode the steroidogenic enzymes, thereby maintaining optimal capacity
for steroid production (reviewed in Ref.2).
Recent studies have implicated the steroidogenic acute regulatory
protein (StAR) as an essential component of the acute response of
steroidogenic cells to trophic hormone. StAR is a 30-kDa mitochondrial
phosphoprotein whose expression is restricted to steroidogenic cells,
where it is rapidly induced by trophic hormone in a manner that
correlates with the acute stimulation of steroidogenesis (3, 4). In
transient transfection experiments in both steroidogenic and
nonsteroidogenic cells, StAR expression directly stimulated
pregnenolone production (4, 5, 6). The onset of StAR expression during
mouse embryonic development correlated closely with the onset of
steroid hydroxylase expression and with the beginning of steroid
hormone biosynthesis (5). Definitive proof for an essential role of
StAR in regulated steroidogenesis came from studies of patients with
lipoid congenital adrenal hyperplasia, a congenital disorder
characterized by global defects in steroidogenesis (7). Analyses of
patients with this disorder revealed mutations in the StAR
gene that preclude the expression of functional StAR protein (8). These
findings provided compelling biochemical and genetic evidence for the
essential role of StAR in regulated steroidogenesis and strongly
suggested that StAR mediates the acute regulation of steroid hormone
biosynthesis.
To further our understanding of the mechanisms that regulate
StAR gene expression, we recently isolated the mouse
StAR gene and determined its structural organization and
sequence (5). We further showed that StAR messenger RNA (mRNA) levels
within steroidogenic cells were markedly increased by cAMP, suggesting
that cAMP may induce StAR transcription. To extend these studies, we
now characterize the mechanisms that regulate StAR expression in
steroidogenic cells. Our results show that the 5'-flanking sequences of
StAR can direct cell-specific and hormone-induced
expression. They further implicate steroidogenic factor-1 (SF-1; also
called adrenal-4-binding protein), an orphan nuclear receptor that
plays key roles at multiple levels of the
hypothalamic-pituitary-steroidogenic organ axis. Collectively, these
results provide novel insights into the mechanisms that control the
expression of this essential component of regulated
steroidogenesis.
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RESULTS
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The 5'-Flanking Region of the StAR Gene Directs Cell-Selective and
Hormone-Induced Expression
We previously reported the isolation and structural
characterization of the mouse StAR gene (5). From these
genomic clones, we isolated and sequenced
3.6 kilobases of the
StAR 5'-flanking region (Fig. 1
). These
sequences are numbered relative to the transcription initiation site,
which was determined by primer extension analysis (Fig. 2
). Unlike many tissue-specific and hormonally
responsive genes, neither a canonical TATA box nor a consensus
cAMP-responsive element (CRE) can be identified within this region. We
previously noted a sequence at -135 (CCAAGGTGG, bottom strand) that
resembles the consensus binding motif for the orphan nuclear receptor
SF-1 (5). Further examination of the 5'-flanking sequence revealed two
other potential binding sites for nuclear hormone receptors: the
inverted repeat (AGGTCA GGACCT) located at -890 and a
region at -42 (AGGCTG, bottom strand) that somewhat resembles the SF-1
consensus motif.

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Figure 1. Nucleotide Sequence of the 5'-Flanking Region of
the Mouse StAR Gene
The sequence of 3646 bp of the mouse StAR 5'-flanking
region is shown. The underlined sequence at -890
indicates an inverted repeat of the AGGTCA recognition motif for
nuclear receptors. Two potential SF-1-binding sites at -135 and -42
are boxed. Dashed underlined sequences
indicate repetitive motifs that resemble somewhat repetitive motifs in
the human StAR promoter. The transcription initiation site, as
determined by primer extension analysis, is designated +1 and is shown
by the arrow.
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Figure 2. Primer Extension Analysis Maps the Mouse StAR
Transcription Initiation Site
A primer complementary to positions 110130 of the StAR cDNA was used
in primer extension analysis with total RNA from mouse Y1
adrenocortical cells. As determined by comparison with end-labeled,
HaeIII-digested X DNA markers, the size of the
extended product was 130 nucleotides.
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To determine whether the StAR 5'-flanking region can direct
cell-specific expression, the proximal 966 bp were placed upstream of
the luciferase reporter gene, and the resulting plasmid (p-966StAR/luc)
was transiently transfected into cultured mouse cell lines. As shown in
Fig. 3
, the StAR 5'-flanking region directed
high levels of luciferase expression in two steroidogenic cell lines
(mouse Y1 adrenocortical and MA-10 Leydig cells) and low levels of
expression in two nonsteroidogenic cell lines (mouse L-TK-
fibroblast and
T3 gonadotrope cells). The finding that StAR promoter
activity is restricted to steroidogenic cells demonstrates that the 966
bp of the StAR 5'-flanking region are sufficient to direct
appropriate cell-selective expression in transient transfection
analyses.

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Figure 3. The StAR Promoter Is Preferentially Active in
Steroidogenic Cell Lines
Y1 adrenocortical, MA-10 Leydig, T3 gonadotrope, and
L-TK- fibroblast cells were transiently transfected with
2.5 µg p-966StAR/luc as described in Materials and
Methods. Where indicated, 8-Br-cAMP (1 mM) was
added to the culture medium 36 h after transfection. Luciferase
activities in cell lysates prepared 48 h after transfection were
determined and normalized to those of the positive control plasmid
pRSV2 luciferase, which contains the Rous sarcoma virus
promoter/enhancer driving luciferase expression. Error bars indicate
the SEMs.
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As StAR expression is hormonally induced, we also analyzed the effect
of cAMP treatment on StAR promoter activity in the same cell lines. As
shown in Fig. 3
(cross-hatched bars), treatment with cAMP
significantly induced promoter activity in the steroidogenic Y1
adrenocortical and MA-10 Leydig cells. Significant increases were also
observed in the very low levels of expression in L cells. These results
demonstrate that the proximal 966 bp of the StAR promoter are
sufficient for hormone-induced expression in steroidogenic and
nonsteroidogenic cells. Studies with a reporter construct
containing 3.6 kilobases of StAR 5'-flanking region showed
that these additional sequences did not significantly increase basal or
hormone-induced promoter activity over that seen with the proximal 966
bp (data not shown). We, therefore, concentrated our analyses of
StAR gene regulation on the proximal 966 bp of the
5'-flanking region shown to be capable of directing both cell-selective
and hormone-induced expression.
Elements Required for StAR Promoter Activity Are Located within 253
bp of the Transcription Initiation Site
We next performed 5'-deletion analyses to identify specific
sequences within the StAR 5'-flanking region that regulate
constitutive and hormone-induced promoter activity. Plasmids containing
progressively decreasing amounts of StAR 5'-flanking region
upstream of the human GH (hGH) reporter gene were transiently
transfected into MA-10 Leydig and Y1 adrenocortical cells, with or
without cAMP treatment, and promoter activity was measured by RIA for
hGH (MA-10 cells) or by Northern blotting analyses of hGH transcripts
(Y1 cells). As shown in Fig. 4A
, progressive deletion of
sequences from -966 to -426 and from -426 to -254 did not
significantly impair basal or cAMP-induced promoter activity. In fact,
there was a progressive increase in both basal and cAMP-induced
activity, suggesting that a negative regulatory element may lie between
-966 and -254. These results demonstrate that sequences within this
region do not positively regulate StAR expression in MA-10 cells.
Further deletion of the promoter from -254 to -113 considerably
diminished basal StAR promoter activity to levels that did not differ
significantly from the those of the promoterless negative control.
Moreover, there was no response to cAMP treatment in cells transfected
with the -113 construct. Similar results were obtained when the same
plasmids were transiently transfected into Y1 adrenocortical cells
(Fig. 4B
). In addition, hGH transcripts expressed from the 5'-deletion
plasmids were of the predicted size, strongly suggesting that they
arose from the authentic initiation site. These results strongly
suggest that the region between -254 and -113 contains an important
positive regulator of StAR expression in both MA-10 Leydig cells and Y1
adrenocortical cells. Interestingly, this region contains the putative
SF-1 binding site at -135.

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Figure 4. 5'-Deletion Analysis Reveals a Region Important for
StAR Promoter Activity in Both MA-10 Leydig Cells and Y1 Adrenocortical
Cells
A, MA-10 Leydig cells were transiently transfected in triplicate by the
lipofectamine method with the indicated StAR 5'-deletion plasmids and
pCMVß-gal. Twenty-four hours after transfection, the medium was
replaced, and where indicated, the cells were treated with 1
mM Bt2cAMP. Levels of promoter activity were
measured by RIA for hGH. Levels of hGH activities were normalized to
ß-galactosidase activity and are expressed as a percentage of the
basal activity of p-966StAR/hGH. Error bars depict the
SEMs from three independent experiments. B, Y1
adrenocortical cells were transiently transfected in triplicate by the
calcium phosphate precipitation method with 2.5 µg of the indicated
StAR 5'-deletion plasmids. For reference, cells were also transfected
with the positive control plasmid pXGH5, which contains the mouse
metallothionine promoter driving hGH expression, and the promoterless
plasmid pBSGH. Cells were treated with 1 mM 8-Br-cAMP
36 h after transfection, where indicated (+). Total RNA was
harvested 48 h after transfection and used in Northern blot
analysis with a hGH cDNA probe. A probe for -tubulin was used to
verify the amount and quality of the RNA.
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Induction of StAR Transcripts by cAMP Is Blocked by
Actinomycin D, but Does not Require de Novo Protein Synthesis
Previous analyses have implicated transcriptional induction as the
predominant basis for trophic hormone induction of genes required for
steroidogenesis (reviewed in Ref.2). However, the possible role of
changes in mRNA stability and the requirement for de novo
protein synthesis in hormone-induced gene expression appear to be gene
dependent (9, 10, 11). Treatment of p-966StAR/luc with cAMP significantly
increased StAR promoter activity (Fig. 3
), suggesting that hormonal
effects reflect increased StAR transcription. Consistent with this
model, trophic hormone induction of StAR mRNA levels was abolished when
cells were treated with actinomycin D to prevent the synthesis of new
transcripts (Fig. 5
). However, treatment of the cells
with cycloheximide did not prevent the cAMP-related increases in StAR
mRNA, indicating that cAMP induction of StAR mRNA is direct and does
not require de novo protein synthesis.

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Figure 5. cAMP Induction of StAR mRNA Levels Requires
Transcription, but not de Novo Protein Synthesis
MA-10 Leydig cells were treated for 2 h with 1 mM
Bt2cAMP, Bt2cAMP plus 10 µg/ml actinomycin D
(ACTD), or Bt2cAMP plus 7 µg/ml cycloheximide (CHX).
Total RNA was isolated, and StAR and actin transcripts were quantitated
by reverse transcription-PCR as described in Materials and
Methods. Radiolabeled PCR products were quantitated using a
PhosphoImager (Molecular Dynamics).
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The Potential SF-1-Binding Site at -135 Interacts Specifically
with SF-1
Our 5'-deletion studies implicated a region that includes a
potential SF-1 site in basal and cAMP-induced StAR promoter activity.
To determine whether SF-1 can bind this motif, we performed gel
mobility shift assays with oligonucleotides containing this sequence
and nuclear extracts from Y1 adrenocortical cells. As shown in Fig. 6
, a probe containing the SF-1 motif located at -135
formed a prominent shifted complex that was competed in a
dose-dependent manner by unlabeled oligonucleotides containing the
homologous sequence (CCAAGGTGG), but not by unlabeled oligonucleotides
containing a mutated SF-1-binding site
(CCATATTAT). Moreover, preincubation of the
nuclear extract with a polyclonal antiserum specific for the
DNA-binding domain of SF-1 completely abolished complex formation (Fig. 6
). Even though it was not identified as an important regulator of StAR
expression in our 5'-deletion studies, the potential SF-1 motif at -42
also competed with the SF-1-related complex. Given that the region
between -254 and -113 is important for StAR promoter activity, the
finding that SF-1 interacts specifically with the -135 element is
consistent with the model in which SF-1 regulates StAR gene
expression.

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Figure 6. SF-1 Binds the Potential SF-1-Responsive Element at
-135 in the StAR 5'-Flanking Region
An oligonucleotide corresponding to the potential SF-1-binding site at
-135 of the mouse StAR 5'-flanking region (SF-1-1) was
used in gel mobility shift assays with 10 µg nuclear extract from
mouse Y1 adrenocortical cells. Where indicated, unlabeled
oligonucleotides corresponding to the SF-1 site at -135 (SF-1-1), a
mutated variant (mSF-1-1), or the proximal SF-1 binding site at -42
(SF-1-2) were included in the reactions at the indicated molar ratios
to labeled probe. Where indicated, antiserum specific for SF-1
( -SF-1) or preimmune serum were included in the binding reaction as
described in Materials and Methods.
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Site-Directed Mutagenesis Defines the Role of the -135
SF-1-Binding Site in StAR Promoter Activity
Although the 5'-deletion and gel shift assays suggested that SF-1
regulates StAR promoter activity, they did not directly address the
roles of the SF-1 sites in StAR expression. We, therefore, used
site-directed mutagenesis to mutate the SF-1 site at -135, either
alone or in combination with the -42 site, within the context of
p-966StAR/luc. Consistent with the 5'-deletion analyses shown in Fig. 4
, mutation of the -135 SF-1-binding site decreased basal promoter
activity in Y1 adrenocortical cells to
50% of the level of the
wild-type promoter (Fig. 7
), revealing a role for this
element in StAR promoter activity. No further impairment was seen when
the -42 SF-1 site was also mutated. However, both mutated plasmids
exhibited full levels of induction in response to cAMP, suggesting that
hormone induction and regulation by SF-1 occur through different
promoter elements. Similar qualitative results were obtained when
mutated StAR promoter/hGH plasmids were transiently transfected in
MA-10 Leydig cells (data not shown). Collectively, the 5'-deletion and
site-directed mutagenesis studies thus define an important role for the
SF-1 binding motif at -135 in StAR gene expression.

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Figure 7. Mutation of the SF-1 Binding Motif at -135 Impairs
StAR Promoter Activity
Y1 adrenocortical cells were transiently transfected in triplicate with
0.5 µg of the indicated StAR promoter/luciferase plasmids. Thirty-six
hours after transfection, the cells were treated with 1 mM
8-Br-cAMP (cross-hatched bars). Luciferase activities in
cell lysates prepared 48 h after transfection were determined and
normalized to those of the positive control plasmid pRSV2 luciferase.
Error bars indicate the SEMs.
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SF-1 Is Essential for StAR Gene Expression in Vivo
Our analyses of StAR expression in transfected cell lines strongly
suggested that SF-1 positively regulates StAR expression. To address
the role of SF-1 in StAR expression in vivo, we took
advantage of knockout mice that are homozygously disrupted in the
Ftz-F1 gene that encodes SF-1 (12). As a consequence of this
gene knockout, the Ftz-F1-disrupted mice undergo
degeneration of the developing gonads and adrenal primordia on
approximately embryonic day 12 (E12). As StAR gene
expression normally commences on E10.0 to E10.5, before the gonads have
significantly degenerated, we were able to use these mice to examine
the possible role of SF-1 in StAR expression in vivo. As
shown in Fig. 8
, StAR transcripts were readily detected
in the genital ridges of wild-type embryos on both E10.5 and E11.5, but
could not be detected in the urogenital ridges of Ftz-F1
knockout mice. To the extent that the developing gonads were still
intact at these relatively early stages of gonadogenesis, these results
implicate SF-1 as an essential regulator of StAR expression in
vivo.

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Figure 8. StAR Transcripts Are not Detected in Knockout Mice
Lacking SF-1
Mouse embryos were harvested and genotyped by Southern blotting
analysis. Serial sagittal sections were prepared from wild-type (WT;
left panels) and Ftz-F1-disrupted
(right panels, -/-) embryos and analyzed by in
situ hybridization with an antisense complementary RNA probe
specific for StAR as described in Materials and Methods.
Darkfield views from E10.5 (top panels) and E11.5
(middle panels) are shown above, with
brightfield views of the E11.5 sections shown below. The
arrow points to the developing gonad. G, Genital
ridge.
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DISCUSSION
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In this study, we explore the molecular mechanisms that
regulate the expression of the mouse StAR gene. StAR has
been implicated in the acute phase of regulated steroidogenesis,
delivering cholesterol to the inner mitochondrial membrane where the
cholesterol side-chain cleavage enzyme carries out the first steps in
steroidogenesis. In keeping with its proposed function, StAR expression
is restricted to the steroidogenic cells of the adrenal cortex and
gonads (5). We now show that 966 bp of the StAR 5'-flanking
region can direct cell-selective and hormone-induced expression in
transient transfection experiments, suggesting that this region is
important in both key components of StAR gene expression. We
also demonstrate that the hormone-induced expression of StAR reflects
increased transcription of StAR mRNA, but does not require de
novo protein synthesis. Within the StAR 5'-flanking
sequences, we identify a region from -254 to -113 that is essential
for full levels of basal and hormone-induced promoter activity. This
region contains a promoter element that is bound by the orphan nuclear
receptor SF-1 and is essential for full levels of promoter activity,
implicating SF-1 in the regulation of StAR expression.
Finally, we show that StAR expression is markedly impaired in knockout
mice deficient in SF-1, providing strong evidence that SF-1 positively
regulates StAR gene expression in the intact mouse.
Our analyses of StAR promoter activity raise several interesting
points. Perhaps most significantly, both transfection studies and
analyses of knockout mice implicate the orphan nuclear receptor SF-1 in
StAR gene regulation. Although cotransfection of Y1 cells
with an SF-1 expression plasmid only slightly increased the promoter
activity of the mouse StAR 5'-flanking sequences (data not
shown), this modest induction probably reflects the fact that Y1 cells
endogenously express high levels of SF-1. Consistent with this model, a
similar role for SF-1 in the promoter activity of the human
StAR gene has recently been proposed based on SF-1 induction
of promoter activity of the human 5'-flanking sequences in transient
transfection experiments in nonsteroidogenic cells (13).
Initially identified as an essential regulator of the cytochrome P450
steroid hydroxylases (14, 15), analyses of knockout mice established
that SF-1 plays essential roles at multiple levels of the
hypothalamic-pituitary-steroidogenic organ axis, establishing it as a
key factor in reproduction (12, 1619; reviewed in ref. 20). Although
it is not entirely unexpected that SF-1 regulates StAR gene
expression in view of its global roles in steroidogenesis and
reproduction, the link between SF-1 and StAR identifies yet another
target gene by which SF-1 determines steroidogenic competence.
Another important finding is that the StAR 5'-flanking
region directs hormone-induced expression. Unlike many genes that are
induced by cAMP, increases in StAR promoter activity apparently do not
result from interactions between the transcriptional regulator
cAMP-response element binding protein and its cognate CRE (21), as no
sequence resembling the consensus CRE is found in the 5'-flanking
region of the mouse (this report) or human (13) StAR genes.
Nonetheless, our promoter analyses strongly suggest that StAR induction
by cAMP at least partly involves transcriptional activation. In this
respect, the StAR gene resembles those of several of the
steroid hydroxylases, which also are induced by cAMP in the absence of
a classical CRE (21). Although SF-1-binding sites have been implicated
in the hormonal regulation of several steroid hydroxylases (reviewed in
Ref.22), our site-directed mutagenesis studies demonstrate that the
SF-1 sites in the StAR promoter are dispensable for hormone induction.
Thus, an important goal for future studies will be to identify the
precise region(s) of the StAR gene that underlies increased
transcription in response to hormone induction.
Another key component of StAR expression is its tissue specificity,
with expression in steroidogenic cells of the adrenal cortex and
gonads, but not in those of the placenta. Our own studies indicate that
the orphan nuclear receptor SF-1 is a key regulator of cell
specificity. It is, nonetheless, clear that other mechanisms also must
limit StAR expression to the steroidogenic cells of the adrenal cortex
and gonads, as SF-1 is also expressed by pituitary gonadotropes and the
ventromedial hypothalamic nucleus, regions that do not express StAR
(5). Of interest in this regard, mutation of the SF-1 binding element
does not impair StAR promoter activity as drastically as does deletion
of the region from -254 to -113. It thus appears that other important
regulatory elements reside within this part of the StAR
5'-flanking region. An important goal for future studies will be to
identify and characterize these other regulatory elements, perhaps
leading to a better understanding of the precise mechanisms that
regulate this essential component of regulated steroidogenesis.
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MATERIALS AND METHODS
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Materials
Radionucleotides were purchased from New England
Nu-clear-DuPont (Boston, MA). Restriction and modification
enzymes were purchased from Boehringer Mannheim (Indianapolis, IN).
Reagents for sequencing were purchased from Amersham Life Science
(Arlington Heights, IL). Reagents for cell culture, including
lipofectamine reagent, were purchased from Life Technologies
(Gaithersburg, MD) and Mediatech (Washington DC). 8-Bromo-cAMP
(8-Br-cAMP) and (Bu)2cAMP were purchased from Sigma
Chemical Co. (St. Louis, MO). PCR reagents were purchased from
Perkin-Elmer (Norwalk, CT). The Double-Stranded Site Directed
Mutagenesis Kit was obtained from Stratagene (La Jolla, CA). Reagents
for luciferase assays and ß-galactosidase assays were purchased from
Promega (Madison, WI). RIA kits for hGH were purchased from Nichols
Institute (San Juan Capistrano, CA). Reagents for in
situ hybridization were purchased from Novagen (Madison, WI).
Plasmids
Plasmids were made using previously isolated sequences of the
StAR 5'-flanking region (5). Full-length and 5'-deletion
plasmids with the indicated amounts of 5'-flanking sequences were
cloned into a promoterless plasmid containing the hGH structural gene
using PCR-based strategies and convenient restriction sites. Other
plasmids used the same 5'-flanking sequences upstream of a luciferase
reporter gene in the pGL2 basic plasmid (Promega). Mutated plasmids
were generated by PCR-based strategies or double stranded site-directed
mutagenesis. The SF-1 site at -135 was mutated from wild-type
CCAAGGTGG (bottom strand) to TACGTAGTT (bottom strand). The
SF-1 site at -42 was mutated from wild-type AGGCTG (bottom strand) to
TACGTA (bottom strand).
Primer Extension Analysis
The transcription initiation site of StAR was mapped with the
Drosophila Embryo Nuclear Extract In Vitro
Transcription System (Promega) according to the manufacturers
protocol. Total RNA was isolated from Y1 adrenocortical cells by the
guanidinium isothiocyanate method using a standard protocol (23). A
primer complementary to 110130 bp of the complementary DNA (cDNA) was
used to extend a
130-nucleotide fragment, whose size was estimated
relative to the migration of end-labeled, HaeIII-digested
X DNA.
Cell Culture
Mouse Y1 adrenocortical, MA-10 Leydig, L-TK-
fibroblast, and
T3 gonadotrope cells were cultured and transfected
in triplicate by the CaPO4 precipitation technique (23) or
by lipofectamine (4), using 0.52.5 µg of the reporter gene. Levels
of hGH were assayed by RIA or Northern analysis 48 h after
transfection. In experiments with cAMP stimulation, cells were treated
with either 8-Br-cAMP for 12 h (Y1 studies) or
(Bu)2cAMP for 224 h (MA-10 studies). Total RNA was
isolated by the guanidinium isothiocyanate method using a standard
protocol and subjected to Northern blot analyses using standard methods
(23).
Reverse Transcription-PCR
MA-10 Leydig cells were either treated or untreated for 2 h
with 1 mM (Bu)2cAMP alone plus 10 µg/ml
actinomycin D or plus 7 µg/ml cycloheximide. Total RNA was extracted
using the Trizol reagent (Life Technologies, Gaithersburg, MA) and
semiquantitative reverse transcription-PCR was used to measure StAR
mRNA levels. Random hexamer oligonucleotides (Pharmacia-LKB,
Piscataway, NJ) were used for the RT reaction, which contained
deoxy-NTPs, RNAsin, 2.5 mM MgCl2, 200 U Moloney
murine leukemia virus-reverse transcriptase (Life Technologies) and 1
µg total RNA. For amplification of the cDNA, the reaction was
"spiked" with [32P]deoxy-CTP to radiolabel the
amplification products. After 25 cycles of amplification, the PCR
products were separated on a 2% agarose gel, the gel was dried and
exposed to a phosphoscreen, and the products were visualized and
quantitated using a Molecular Dynamics PSF PhosphoImager (Sunnyvale,
CA). StAR mRNA levels were normalized to ß-actin mRNA levels to
determine the effect of treatment on StAR expression.
Gel Mobility Shift Assays
Complementary oligonucleotides corresponding to each of the
putative SF-1 sequences were synthesized, annealed, and end labeled by
Klenow fill-in reaction; these duplex oligonucleotides included SF-1-1
(5'-CCTCCCACCTTGGCCA-3', positions -142 to -126) and
SF-1-2 (5'-TGCACAGCCTTCCACG-3', positions -49 to -34). Gel
mobility shift assays were performed essentially as previously
described (14). Briefly, labeled probes were incubated with 10 µg Y1
adrenocortical cell nuclear extract and 4 µg
poly[d(I-C)]-poly-[d(I-C)] as nonspecific competitor. Where
indicated, either unlabeled oligonucleotide competitors or antiserum
specific for SF-1 were preincubated with the nuclear extract before
probe addition. Samples were analyzed by electrophoresis on a 4%
nondenaturing polyacrylamide gel. Gels were dried and exposed to x-ray
film overnight.
In Situ Hybridization
Serial sagittal sections (6 µm) were deparaffinized and
hybridized overnight at 5055 C using an in situ
hybridization kit according to recommended protocol.
35S-Labeled complementary RNA probes were prepared using T3
and T7 polymerases according to the protocol supplied with a kit
purchased from Novagen (Madison, WI). After washes at high stringency,
the slides were dipped in Kodak NTB-2 emulsion (Eastman Kodak,
Rochester, NY) diluted 1:1. Exposures were carried out for 34 weeks.
After exposure, slides were developed in Kodak D-19, fixed, and
counterstained with methyl green. Both sense and antisense probes
derived from the mouse StAR cDNA (5) were used, and all hybridizations
included a control section of adult mouse adrenal gland.
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ACKNOWLEDGMENTS
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We thank Dr. Mario Ascoli for the MA-10 cells; Dr. Pamela Mellon
for the
T3 cells; Drs. Deepak Lala, Cameron Scarlett, Jerry Strauss,
and Walter Miller for helpful discussions; and Jeana Meade, LeeAnn
Baity, and Rebecca Combs for superb technical assistance.
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FOOTNOTES
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Address requests for reprints to: Dr. Keith L. Parker, 323 CARL Building, Duke University Medical Center, Durham, North Carolina 27710.
This work was supported by the Howard Hughes Medical Institute; NIH
Grants HL-48460 (to K.L.P.), HD-17481 (to D.M.S.), and DK-51656-01 (to
B.J.C.); and NIEHS Grant ES06832-03 (to B.J.C.).
Received for publication February 19, 1996.
Revision received October 17, 1996.
Accepted for publication November 5, 1996.
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