SF-1 (Steroidogenic Factor-1), C/EBPß (CCAAT/Enhancer Binding Protein), and Ubiquitous Transcription Factors NF1 (Nuclear Factor 1) and Sp1 (Selective Promoter Factor 1) Are Required for Regulation of the Mouse Aldose Reductase-Like Gene (AKR1B7) Expression in Adrenocortical Cells
Christelle Aigueperse,
Pierre Val,
Corinne Pacot,
Christian Darne,
Enzo Lalli,
Paolo Sassone-Corsi,
Georges Veyssiere,
Claude Jean and
Antoine Martinez
UMR Centre National de la Recherche Scientifique 6547
Physiologie Comparée et Endocrinologie Moléculaire (C.A.,
P.V., C.P., C.D., G.V., C.J., A.M.) Université Blaise Pascal
Clermont II, Complexe Universitaire des Cézeaux 63177
Aubière cedex, France
Institut de Génétique
et de Biologie Moléculaire et Cellulaire (E.L., P.S.C.),
Centre National de la Recherche Scientifique, INSERM
Université Louis Pasteur, BP 163 67404 Illkirch, Strasbourg,
France
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ABSTRACT
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The MVDP (mouse vas deferens protein)
gene encodes an aldose reductase-like protein (AKR1B7) that is
responsible for detoxifying isocaproaldehyde generated by
steroidogenesis. In adrenocortical cell cultures, hormonal regulation
of MVDP gene occurs through the cAMP pathway. We show that
in adrenals, the pituitary hormone ACTH regulates MVDP gene
expression in a coordinate fashion with steroidogenic genes. Cell
transfection and DNA-binding studies were used to investigate the
molecular mechanisms underlying MVDP gene regulation in Y1
adrenocortical cells. Progressive deletions of upstream regulatory
regions identified a -121/+41 fragment that was sufficient for basal
and cAMP-mediated transcriptional activities. Gel shift assays
showed that CTF1/nuclear factor 1 (NF1), CCAAT enhancer binding
protein-ß (C/EBPß), and selective promoter factor 1 (Sp1) factors
bound to cis-acting elements at positions -76, -61, and
-52, respectively. We report that the cell-specific steroidogenic
factor-1 (SF-1) interacts specifically with a novel regulatory element
located in the downstream half-site of the proximal androgen response
element (AREp) at position -102. Functional analysis of SF-1 and NF1
sites in the -121/+41 promoter showed that mutation of one of them
decreases both constitutive and forskolin-stimulated promoter activity
without affecting the fold induction (forskolin stimulated/basal).
Individual mutations of C/EBP and Sp1 sites resulted in a loss of more
than 50% of the cAMP-dependent induction. When both sites were mutated
simultaneously, cAMP responsiveness was nearly abolished. Thus, in
adrenocortical cells, both SF-1 and NF1 are required for high
expression of the MVDP promoter while Sp1 and C/EBPß
functionally interact in an additive manner to mediate
cAMP-dependent regulation. Furthermore, we report that
MVDP gene regulation is impaired in stably transfected Y1
clones expressing DAX-1. Taken together, our findings suggest that
detoxifying enzymes of the aldose reductase family may constitute new
potential targets for regulators of adrenal and gonadal differentiation
and function, e.g. SF-1 and DAX-1. (Molecular
Endocrinology 15: 93111, 2001)
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INTRODUCTION
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The adult adrenal cortex is divided into three morphologically and
functionally distinguishable zones characterized by different patterns
of steroidogenic enzyme gene expression (1). The zona glomerulosa
produces the mineralocorticoid aldosterone, whereas the zona
fasciculata synthesizes glucocorticoids: cortisol in the human and
bovine species, corticosterone in rodents. The zona reticularis cells
produce cortisol and, in primates, the adrenal androgen
dehydroepiandrosterone (1). It is also well established that
adrenocortical cell functions are regulated by the circulating hormones
ACTH and angiotensin II acting on the inner adrenocortical zones and
the outer zona glomerulosa, respectively (2, 3). cAMP acts as a second
intracellular messenger to transduce the effect of ACTH both acutely
and in a long-term manner (2). The acute effect, which occurs within
seconds to minutes, increases biosynthesis by making cholesterol
available to the steroidogenic enzymes (4). Biochemical and genetic
evidence have implicated the steroidogenic acute regulatory (StAR)
protein, which accelerates cholesterol transport into the inner
mitochondrial membrane, as an essential component of the acute response
(4, 5). The long-term effects of ACTH, achieved after many hours,
result from increased transcription of several key cAMP-responsive
genes that encode steroidogenic enzymes (2). Previous studies have
shown that a common factor in this pathway is the orphan nuclear
receptor steroidogenic factor 1 (SF-1) (6). SF-1 has been shown to
regulate the transcription of many genes involved in steroidogenesis,
including steroid hydroxylase genes (7, 8, 9) and the StAR protein gene
(10). SF-1 also regulates the expression of genes involved in the
steroidogenesis regulation such as the LHß subunit (11), the ACTH
receptor (12), and the GnRH receptor (13). Whether SF-1 is hormonally
regulated is still unclear. It has been shown that DAX-1
(dosage-sensitive sex reversal-adrenal hypoplasia congenita critical
region on the X-chromosome, gene 1) gene expression is up-regulated by
SF-1 in vitro as in vivo (14, 15) and that DAX-1
inhibits SF-1-mediated transactivation (16). Finally, DAX-1 has been
proposed to be involved in the regulation of steroidogenesis by
repressing StAR, P450scc, and 3ß-hydroxysteroid
dehydrogenase (3ß-HSD) expression (17). Thus, it seems that
regulation of SF-1 target genes is ensured by antagonistic pathways
that are necessary to establish proper development and functions of
steroidogenic organs.
The first step of steroidogenesis is the cleavage of the cholesterol
side chain by P450scc, resulting in the formation of
pregnenolone and isocaproaldehyde (4-methyl pentanal), which is
further metabolized to isocaproic acid and isocapryl alcohol (18).
While detailed mechanisms of enzymatic catalysis and gene regulation
have been extensively studied for steroidogenic enzymes, little is
known about those involved in steroid metabolism. Recent studies have
suggested that members of the aldo-keto reductase (AKR) family could be
related to steroid metabolism. First, it has been shown that
degradation of steroids by reduction of their aldehyde and ketone
groups is not primarily due to specific dehydrogenases but results from
the broad specificity of enzymes of the AKR family (19, 20). Second,
members of this family may be responsible for detoxifying aldehydes
generated by steroidogenesis or derived from lipid peroxidation in
mammalian adrenals (21). The AKR family consists of at least 42 members
that catalyze the reduction of a broad range of substrates including
aldoses, aliphatic and aromatic aldehydes and ketones, prostaglandins,
and xenobiotics such as chemotherapeutic drugs (see Ref. 22 for review
and nomenclature). Among the AKR family, aldose reductase has been
implicated in the etiology of diabetic complications (23). A subgroup
of the AKR family, including the mouse vas deferens protein (MVDP:
AKR1B7) (24), the mouse fibroblast growth factor 1-regulated protein
(FR-1: AKR1B8) (25), and the Chinese hamster ovary cell-derived CHO
reductase (AKR1B9) (26), is closely related to aldose reductase. The
presence of human homologs of this subgroup has recently been reported
(27, 28). Although MVDP represents a major secretory component of the
vas deferens, this expression profile is restricted to the mouse
species (29). More recently, MVDP was shown to be highly expressed in
the adrenals from various rodents under the control of the
hypothalamo-pituitary-adrenal axis (30, 31). This evolutionary
conserved expression of MVDP is restricted to the zona fasciculata and,
in human and murine adrenocortical cells, the MVDP gene is induced by
cAMP at both mRNA and protein levels (31). The involvement of MVDP in
steroidogenic functions was recently demonstrated by our group (32). In
adrenocortical cells and in adrenal glands, MVDP plays an essential
role in detoxifying isocaproaldehyde generated by the conversion of
cholesterol to pregnenolone, which is the rate-limiting step in
steroidogenesis.
In light of these data, our objective was to examine the
MVDP upstream regulatory regions for potential binding sites
for transcription factors that may be involved in the hormonal
regulation of MVDP gene transcription in adrenocortical
cells. Deletion and site- directed mutagenesis allowed us to
identify a proximal promoter region spanning positions -121 to +41
that confers basal and cAMP-induced regulation through the
combinatorial action of SF-1, nuclear factor 1 (NF1), C/EBPß, and
selective promoter factor (Sp1) factors. Furthermore, we demonstrate
that SF-1 interacts with a novel regulatory element lying within a
previously characterized androgen responsive element. Our reports also
suggest that MVDP, a gene encoding a detoxifying enzyme, may
be considered as a novel target gene for SF-1/DAX-1 antagonistic
actions.
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RESULTS
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MVDP Expression in Adrenals Is Regulated by ACTH
As previously shown, the MVDP gene is highly expressed
in adrenal glands from adult mouse (Fig. 1
). To determine whether or not the
accumulation of MVDP mRNA in adrenals was under the control
of pituitary ACTH, as has been shown for steroidogenic genes, hormonal
treatments were performed. In adult mice treated for 5 days with
dexamethasone to inhibit ACTH secretion, MVDP mRNA levels
were dramatically decreased by more than 4-fold when compared with
control animals (vehicle treated) (Fig. 1
). A 2-day ACTH administration
to dexamethasone-treated animals was sufficient to induce a 9-fold
increase in MVDP mRNA levels. As shown in Fig. 1
, the
responsiveness of MVDP gene to variations of ACTH
concentrations was similar to that of steroidogenic genes such as
P450scc and StAR the mRNA
levels of which were decreased by 3-fold and induced over 3-fold in the
same experimental conditions.

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Figure 1. Hormonal Regulation of MVDP in
Adrenal Glands
mRNA levels of MVDP, P450scc, and
StAR were determined by Northern blotting using pooled
adrenals from six adult mice treated with vehicle alone (5 days),
dexamethasone (5 days, DEX), and dexamethasone (5 days) plus ACTH for
the last 2 days. Analyses were performed using 25 µg of total RNAs.
Blots were sequentially probed with the indicated
32P-labeled cDNA probes.
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MVDP Transcription Start Site in Adrenals
In vas deferens, a major transcription start site has been located
on an adenine residue 46 nucleotides upstream from the ATG initiation
codon (33). RNAse protection experiments were performed to map the
transcription start site in adrenal glands (Fig. 2
). Total RNAs from adrenals and vas
deferens were hybridized to a radiolabeled riboprobe encompassing the
previously identified transcription start site. Nonhybridized RNAs were
digested by RNAse, and resistant complexes were resolved on a
sequencing gel. As shown by the similar profiles in the two organs, the
transcription start site is the same in both adrenals and vas deferens
and maps to the previously identified start site (33). This
result was also confirmed by 5'-RACE (rapid amplification of cDNA ends)
experiments (data not shown).

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Figure 2. Identification of MVDP Transcription
Start Site in Adrenals
Total RNA from mouse adrenals (10 µg) and vas deferens (2 µg) was
subjected to a RNase protection reaction with a probe encompassing the
previously identified transcription start site (33 ).
Digestion-resistant hybrids were resolved in a 6% polyacrylamide
denaturing gel next to a dideoxy sequencing reaction of the probe. Note
that RNA migration is slightly retarded compared with DNA in such
denaturing gels. Arrowhead indicates a major
transcription start site. Asterisks indicate minor start
sites.
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A Proximal Region (-121 to +41) of the MVDP Gene Is Sufficient for
Both Constitutive and cAMP-Induced Expression
Our previous results showed that a 3-h treatment of Y1
adrenocortical cells with forskolin is sufficient to reach maximal
levels of MVDP mRNA, suggesting that cAMP may regulate
MVDP gene expression at transcriptional levels (31). To
determine DNA regions that confer basal and cAMP-induced transcription,
a series of 5'-deletion mutants of the 1.8-kb upstream regulatory
region of the MVDP gene were fused to the chloramphenicol
acetyltransferase (CAT) reporter gene and then transiently transfected
into Y1 cells. All MVDP promoter constructs had significant
transcriptional activity. Compared with the p0.16 CAT plasmid activity
(referred to as 100%), basal CAT activity varied slightly after
5'-deletions from 35% with the p1.8 CAT plasmid to 115% with the p0.5
CAT construct (Fig. 3
). In contrast, a
drastic decrease (83%) of CAT activity was observed when the sequence
from -121 to -40 was deleted (pTATA CAT plasmid). These results
suggest that the region between -121 to-40 contains important
elements for constitutive expression of the MVDP promoter in
Y1 adrenal cells.

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Figure 3. Identification of Regulatory Regions Responsible
for Constitutive and cAMP-Induced Expression of MVDP
Promoter in Y1 Cells
Restriction fragments from MVDP gene 5'-flanking region
were cloned upstream of the CAT reporter gene. Nucleotide numbering is
according to the transcription start site, which is indicated by an
arrow. CAT activity was determined in protein extracts
of Y1 cells transfected with 1.5 µg of each MVDP-CAT construct and
incubated in the absence or presence of 10-5 M
forskolin (24 h). Results were expressed as percentages of the mean
value achieved with p0.16 CAT construct. Fold activation represents the
forskolin-stimulated reporter activity divided by the unstimulated
level. Values are means ± SEM of at least five
independent transfections.
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After transfection, cells were incubated for 24 h in the absence
or presence of forskolin. All constructs showed significant increases
in CAT activity (from 3.3 to 46.3-fold) in the presence of forskolin.
This effect was specifically mediated by regulatory regions of the
MVDP gene since the activity of the SV-40 enhancer/promoter
sequence (control pSV2-CAT vector) was unaffected by this treatment
(fold induction, 1.1). Thus, it appears that cAMP stimulation of the
MVDP gene was achieved, at least in part, at the
transcriptional level. The minimal promoter construct limited to the
TATA sequence itself (-40/+41) was able to ensure a low level of
forskolin-dependent transcription (3.3-fold). On the other hand, a
13.4-fold induction was achieved by the p0.16 CAT plasmid, indicating
that cAMP responsiveness of the MVDP minimal promoter was
markedly potentiated by the -121/-40 region. Thus, cis
elements located between positions -121 to -40 were required to
direct strong cAMP-induced transcription. However, the region between
-1,285 to +41 gave a higher induction of CAT activity, suggesting that
the sequence spanning positions -1,285 to -121 contains important
element(s) needed for full cAMP inducibility. Next, the -121 to -40
region sequence was examined to identify putative protein-binding sites
that might mediate cAMP-stimulated expression of MVDP in Y1 cells.
Surprisingly, sequence analysis of the 162 bp did not reveal any
regulatory motifs that resemble the canonical CRE (cAMP response
element). This region contains several putative binding sites for known
transcription factors including the selective promoter factor 1 (Sp1,
-52 to -44), C/EBP (-61 to -54), nuclear factor 1 (NF1, -76 to
-63), and androgen receptor (AR, -111 to -97). Gel mobility shift
assays were then performed to further examine the nuclear proteins that
bind to various regions of the proximal promoter.
Nuclear Proteins from Y1 Cells Bind Specifically to the Sp1, C/EBP,
and NF1 Binding Sites of the MVDP Promoter
As shown in Fig. 4A
, lane 1,
addition of nuclear extracts from Y1 cells to end-labeled
oligonucleotide containing the -52 Sp1 site (Table 1
) resulted in the formation of three
retarded bands. Complex 1 was the major protein/DNA interaction
detected. All three complexes were specific as judged by the ability of
an excess of unlabeled -52 Sp1 or canonical Sp1 oligonucleotides to
abolish the presence of the shifted bands (Fig. 4A
, lanes 3 and 4).
This pattern was not affected by an excess of oligonucleotides
containing a mutated Sp1 site (-52 Sp1m, Table 1
) (Fig. 4A
, lane 7).
The addition of anti-Sp1 antibodies in electrophoretic mobility shift
assay (EMSA) experiments consistently abrogated the formation of
complexes 1 and 2 (Fig. 4A
, lane 10). Sp1 is not the only protein
binding to GC boxes. Additional factors such as early growth response
protein-1 (Egr-1) and the Wilms tumor suppressor (WT1) may also
bind to these GC-rich sequences or related motifs (Table 1
). Thus,
oligonucleotides containing high-affinity binding sites for WT1 (WTE)
or for Egr-1 were designed and used as competitors. As shown in Fig. 4A
, lane 5, complex 3 could be competed by an excess of the WTE
oligonucleotide, whereas complexes 1 and 2 were unaffected. However,
none of the three complexes were affected by monoclonal or polyclonal
antibodies against WT1 (not shown). In addition, none of the three
complexes could be displaced by either an excess of unlabeled Egr-1
oligonucleotide (Fig. 4A
lane 6) or by addition of antibodies directed
against Egr-1 (not shown). Although the nature of the minor complex 3
remains unknown, these results show that in Y1 cells, proteins of the
Sp1 family generate the major binding complexes to the -52/-44
element of the MVDP gene. Because cAMP induced the
MVDP proximal promoter activity, we examined whether the
relative amount of factors bound to the -52 Sp1 probe was changed in
nuclear extracts from forskolin-treated cells. There was no apparent
effect of forskolin treatment on the binding activity of the Sp1
protein family (Fig. 4A
, compare lanes 1 and 2).

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Figure 4. Binding of Sp1, C/EBPß, and NF1, Present in Y1
Nuclear Extracts, to the -121/+41 Promoter of the MVDP
Gene
A, Identification of proteins binding to the -52 Sp1 binding site. Y1
nuclear proteins (5 µg), with or without 24 h stimulation with
forskolin, were incubated with a 32P-labeled probe
corresponding to the -52 Sp1 binding site in the absence or presence
of a 50-fold molar excess of unlabeled competitor oligonucleotides. The
competitors were either the unlabeled wild-type Sp1 site (-52 Sp1),
canonical Sp1 site (Sp1c), WT1 (WTE) and
Egr-1 binding sites (Table 1 ), mutant version of the
MVDP Sp1 site (-52 Sp1m), or an unrelated site (-76
NF-1). Arrows indicate the three specific DNA-protein
complexes. Complexes 1 and 2 were abrogated upon addition of
Sp1-specific antibody. B, Identification of proteins binding to the
-61 C/EBP binding site. Five micrograms of nuclear proteins prepared
from unstimulated or forskolin-stimulated Y1 cells or 3 µl of
in vitro translated C/EBPß were incubated with
32P-labeled probe corresponding to the -61 C/EBP binding
site. The competitors were either the wild-type C/EBP binding site
(-61 C/EBP), canonical C/EBP site (C/EBPc), mutant version of the
MVDP C/EBP site (-61 C/EBPm), or -76 NF1 binding site.
Arrow indicates the DNA-protein complex. Specific
complexes were supershifted upon addition of C/EBPß-specific
antibody. C, Identification of proteins binding to the -76 NF1 binding
site. Y1 nuclear proteins (5 µg), with or without 24 h
stimulation with forskolin, were incubated with a
32P-labeled probe corresponding to the -76 NF1 binding
site in the absence or presence of a 50-fold molar excess of unlabeled
competitor oligonucleotides. The competitors were either the wild-type
NF1 site (-76 NF1), canonical NF-1 site (NF1c), mutant version of
the MVDP NF-1 site (-76 NF1m), or -52 Sp1 binding
site. Arrow indicates the prominent DNA/protein complex.
The major part of this complex was shifted upon addition of
anti-CTF1/NF1 antibody. NS, Nonspecific complexes.
Asterisks indicate supershifted complexes.
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As shown in Fig. 4B
, lane 1, incubation of nuclear extracts from Y1
cells with the -61 C/EBP-labeled probe resulted in the formation of a
retarded complex that probably contains several proteins. The
specificity of the retarded complex was checked by competition with an
excess of unlabeled oligonucleotides: either homologous (-61 C/EBP,
lane 5), canonical (C/EBPc, lane 6), mutated (-61 C/EBPm, lane 7), or
unrelated (Egr-1, lane 8). As shown in Table 1
, the -61 C/EBP binding
site was more related to the enhancer core motif than to the GCAAT
motif. The intensity of the specific complex increased when nuclear
extracts from forskolin-treated Y1 cells were used (Fig. 4B
, compare
lanes 1 and 2). The addition of an antibody directed against C/EBPß
reduced the mobility but did not alter the whole DNA-protein complexes,
indicating that other interactions may occur (Fig. 4B
, lanes 3 and 4).
The resulting supershifted complexes resolved into two distinct bands,
and only the intensity of the upper band was increased by forskolin
treatment (Fig. 4B
, compare lanes 3 and 4). Note that the intensity of
the remaining nonsupershifted complex was also increased upon forskolin
stimulation. Antibodies against members of the ATF/CREB family, such as
ATF1, ATF2, ATF3, and ATF4, did not affect the profile of the proteins
bound to the -61 C/EBP oligonucleotide, suggesting that the main
binding activity of this MVDP promoter region is due to
C/EBP proteins (not shown). To further characterize this binding site,
EMSA were performed using in vitro translated C/EBPß
protein produced in rabbit reticulocyte lysate. A specific DNA-protein
complex was formed when the -61 C/EBP probe was incubated with
in vitro translated C/EBPß that was absent in unprogrammed
lysate (Fig. 4B
, compare lane 9 and 13). This complex was completely
supershifted by anti-C/EBPß antibody and was specifically competed by
an excess of unlabeled -61 C/EBP oligonucleotide or by a canonical
C/EBPc site. Note that the supershifted complex comigrates with the
upper band formed with nuclear extracts (Fig. 4B
, compare lanes 3, 4,
and 10). Taken together, these data demonstrate that forskolin
treatment increases expression, availability, or DNA-binding activity
of C/EBPß and other yet unidentified protein bands to the -61 C/EBP
element of MVDP promoter.
When a labeled oligonucleotide containing the putative -76 NF1 binding
site was incubated with Y1 cells nuclear extracts, one prominent
DNA-protein complex was observed (Fig. 4C
, lane 1). This interaction
was competed by a 50-fold molar excess of the unlabeled homologous
(-76 NF1) or canonical NF1 (NF1c) oligonucleotides whereas no
competition was observed with either the unrelated Sp1 or mutated NF1
oligonucleotides (Fig. 4C
, lanes 26). The prominent complex was
markedly reduced and supershifted by anti-CTF1/NF1 antibody (Fig. 4C
lane 8), indicating that CTF1 is a major component of the binding
activity. As described above for the -52 Sp1 probe, the intensity of
the -76 NF1 complex was unaffected by forskolin stimulation of Y1
cells (Fig. 4C
, compare lanes 1 and 2).
SF-1 Binds to the Androgen Response Element Sequence
When a 33-bp labeled oligonucleotide harboring the proximal
MVDP androgen response element (AREp) sequence (Table 1
) was
incubated with nuclear extracts from Y1 cells, a major DNA protein
complex was observed (Fig. 5A
, lane 1).
This could not be due to the AR because Y1 cells lack such a receptor
(31). Addition of increasing levels of the unlabeled AREp
oligonucleotide gradually diminished the DNA-protein complex but failed
to completely eliminate it (Fig. 5A
, lanes 24), suggesting a specific
but rather low-affinity interaction. As SF-1 has been shown to be an
important regulator of several genes involved in adrenal development
and function (Ref. 6 for review), we used a known SF-1 binding site as
DNA competitor and an anti-SF-1 antibody (directed against SF-1
DNA-binding domain) in mobility shift assays. As shown (Fig. 5A
, lane
5; Fig. 5B
, lane 2), the major DNA-protein complex was efficiently
competed by a 50-fold molar excess of the unlabeled probe containing a
high-affinity SF-1 binding site from the mouse 21-hydroxylase (21-OH)
gene promoter (Table 1
) and was eliminated by addition of an antibody
to SF-1 (Fig. 5A
, lane 7). As expected, no competitive effect was
observed when the unrelated -76 NF1 oligonucleotide was used (Fig. 5B
, lane 3). To further characterize this binding site, EMSAs were carried
out using SF-1 protein produced in rabbit reticulocyte lysate. We
showed that the in vitro translated SF-1 protein generated
an identical gel shift binding pattern to that observed with Y1 nuclear
extracts (Fig. 5B
, compare lanes 1 and 4). Furthermore, the complex was
either partially or totally abolished by a 50-fold molar excess of
unlabeled oligonucleotides corresponding to the MVDP AREp
and SF-1 binding site from the 21-OH gene, respectively (Fig. 5B
, lanes
5 and 6). Unprogrammed control lysate failed to form any specific
complex with MVDP AREp probe (Fig. 5B
, lane 7).
Collectively, these data show that the transcriptional activator SF-1
is able to interact in a specific manner to the MVDP AREp
probe.

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Figure 5. SF-1 Binds to an Unusual Site Contained within the
Proximal ARE (AREp)
Identification of proteins binding to the AREp. Y1 nuclear extracts (5
µg) (A) and in vitro translated SF-1 protein produced
in rabbit reticulocyte lysate (2 µl) (B) were incubated with a
32P-labeled probe corresponding to the MVDP
AREp sequence in the absence or presence of an excess of unlabeled
competitor oligonucleotides. The competitors were either the wild-type
AREp, a known SF-1 binding site from the mouse 21-OH gene, or the -76
NF1 binding site. Arrow indicates the DNA-protein
complex. The specific DNA-protein complex was eliminated upon addition
of the anti-SF-1 antibody. L, Unprogrammed lysate.
Asterisk indicates binding activity of endogenous
reticulocyte lysate proteins.
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As shown in Table 1
, the region extending from -117 to -93 lacks a
consensus SF-1 binding site (PyCAAGGPyPy or PuPuAGGTCA). A second
potential ARE (AREd) has been described in the 5'-flanking region of
the MVDP gene at position -1,186 (35). To check whether
AREd was a potential binding site for SF-1, this site was assayed for
its ability to bind Y1 cell nuclear proteins in EMSA. The DNA-protein
complex formed was not affected by a 50-fold excess of unlabeled
oligonucleotide containing a high-affinity SF-1 site of the 21-OH gene
(Fig. 6A
, lanes 1 and 2), indicating that
the protein interacting with the probe is not SF-1. These data confirm
that the binding of SF-1 to AREp is sequence specific and suggest that
nucleotides that differ between AREp and AREd sequences may be
important for SF-1 binding. The AR binds a motif consisting of an
imperfect palindromic repeat of the core sequence 5'-TGTTCT-3',
separated by three nonconserved nucleotides (Table 1
and Fig. 6D
). To
characterize this noncanonical SF-1 binding site more precisely,
oligonucleotides containing point mutations within or adjacent to the
core ARE sequence were used. The first mutant used in these studies had
the downstream half-site and 3'-flanking nucleotides of AREp changed to
that of AREd (Mut 1). As shown in Fig. 6A
, lane 4, this replacement
appeared to disrupt SF-1 binding. When compared with the
MVDP AREp core sequence TGTTCT, AREd contains a C instead of
a T at position 3 (TGCTCT). This substitution introduced in AREp (Mut
2) also prevented SF-1 binding (Fig. 6A
, lane 5). The G-to-T
substitution in the downstream half-site of AREp (Mut 3) abolished SF-1
protein binding (Fig. 6A
, lane 6), suggesting that this nucleotide is
also involved in SF-1/DNA interactions. Mutations generated in the
three nonconserved spacer base pairs (Mut 4) or in the upstream
half-site of the palindrome (Mut 5, 6, 7) did not affect SF-1 binding
significantly (Fig. 6A
, lanes 710). To confirm the data obtained with
Y1 nuclear extracts, in vitro translated SF-1 was incubated
with either wild-type or mutated AREp probes, and SF-1-containing
complexes were checked by competition with an excess of the specific
SF1 oligonucleotide from the 21-OH gene. As shown in Fig. 6B
, in
vitro translated SF-1 bound to AREp probe while no SF-1-containing
complexes were formed using Mut1, -2, or -3 probes (lanes 510). The
asterisk indicates an upper migrating band competed by 21-OH
oligonucleotide that was unrelated to SF-1 since it was also detected
with unprogrammed lysate. Finally, experiments using unlabeled DNA as
competitor (Fig. 6C
) showed that the complex bound to AREp probe (lane
1) was unaffected by a 200-fold excess of AREd or Mut 3
oligonucleotides (lanes 2 and 3) whereas a 50-fold excess of a
high-affinity binding site for SF-1 (from the 21-OH promoter, Fig. 6C
, lane 4) was sufficient to abolish it. Thus, it appears that SF-1
binding activity is retained by sequences beginning at position -102
and spanning the downstream palindromic hexanucleotide of AREp.

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Figure 6. Characterization of the SF-1 Binding Site within
AREp Oligonucleotide
A, Nuclear extracts (5 µg) from Y1 cells were incubated with
32P-labeled probes corresponding to the distal androgen
response element of the MVDP promoter (AREd, see also
Table 1 ), AREp, or mutant versions of AREp (Mut 1 to Mut 7). B,
In vitro translated SF-1 protein (2 µl) was incubated
with the indicated 32P-labeled probes in the absence or
presence of an excess of the unlabeled high-affinity SF-1 binding
oligonucleotide from 21-OH gene. L, Unprogrammed lysate. C, Nuclear
extracts (5 µg) from Y1 cells were incubated with a
32P-labeled probe corresponding to AREp in the absence or
presence of the indicated molar excess of unlabeled competitor
oligonucleotides. D, The sequences of ARE probes are shown. The two
half-sites of ARE motifs are shown in bold, and the
nucleotide substitutions are underlined. The bases
important for SF-1 binding are boxed.
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SF-1 and NF1 Binding Sites Are Required for Constitutive Expression
of the MVDP Gene
EMSA studies demonstrated the authenticity of the Sp1, C/EBP, NF1,
and SF-1 binding sites found in the proximal upstream region of the
MVDP gene. To determine whether these factors could affect
either constitutive or cAMP-induced expression of MVDP
promoter, additional reporter constructs were generated in which the
different binding sites were mutated in the context of the proximal
promoter. CAT reporter constructs used for these experiments were
driven by the wild-type -121/+41 proximal promoter or by mutant forms
carrying substitutions within one or two of these sites. All the
mutations disrupted the specific DNA-protein complexes observed in gel
shifts (see above results). As shown in Fig. 7
, mutation in the -102 SF-1 site, which
abolished SF-1 binding (0.16 SF-1m), resulted in a 77% reduction in
basal CAT activity. Mutation in the -76 NF1 binding site had similar
effects: basal CAT activity was reduced by 81%. When expressed as
fold-increase over basal level, the constructions lacking NF1 or
SF-1 binding sites were able to mediate a significant stimulation of
CAT activity in response to forskolin, and the fold-inductions of the
two reporters were relatively unchanged compared with the wild-type
promoter. However, forskolin- induced CAT activity, expressed as
percentage conversion, was significantly reduced by 82% and 86% after
mutation of SF-1 or NF1 binding sites, respectively. These data
indicate that both the -102 SF-1 and -76 NF1 binding sites are
required to ensure full transactivation of the MVDP proximal
promoter.

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Figure 7. Functional Activity of SF-1, NF1, C/EBPß, and Sp1
Binding Sites in Both Constitutive and cAMP-Induced Expression of the
MVDP Proximal Promoter
Y1 cells were transfected with 1.5 µg of the wild-type or mutated
versions of the p0.16 CAT construct, and reporter activity was measured
from forskolin-stimulated or unstimulated cells. Results were expressed
as percentages of the mean value achieved with the p0.16 CAT construct.
Fold activation represents the forskolin-stimulated reporter activity
divided by the unstimulated level. Values are means ±
SEM of at least four independent transfections. *,
Significantly different (P < 0.01) from basal
activity observed with control (p0.16 CAT).
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To determine whether the differential expression of wild-type and -102
SF-1 mutated MVDP promoters in Y1 cells resulted from the
presence of SF-1 factor, we compared their activities in steroidogenic
cells that are devoid of SF-1, the placental JEG-3 cell line (Fig. 8A
). Disruption of the -102 SF-1 site
within the p.016 promoter resulted in a 77% decrease in reporter
activity in Y1 cells whereas this mutation had virtually no effect in
JEG-3 cells. We next examined the ability of exogenous SF-1 to
transactivate MVDP promoter through the -102 site in
SF-1-deficient nonsteroidogenic CV1 cells. A SF-1 expression vector was
cotransfected with the wild-type and mutant promoter construct.
MVDP p0.16Luc proximal promoter was stimulated approximately
3.1-fold by SF-1, and this effect was lost when the -102 site was
mutated (Fig. 8B
). Taken together, these data indicate that
differences between wild-type and -102 site mutant promoters
activities were abolished in steroidogenic SF-1-deficient cells (JEG-3)
and that transactivation of the MVDP proximal promoter by
exogenous SF-1 requires intact -102 site.

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Figure 8. Transcriptional Activity of SF-1 at the AREp Motif
of MVDP Proximal Promoter
A, Y1 and SF-1-deficient JEG-3 cells were transfected with 1.5 and 3
µg of CAT reporter plasmids, respectively, driven either by the
wild-type (p0.16) or -102 site mutant (p0.16 SF-1 m) promoters.
Results were expressed as percentages of the mean activity achieved
with the p0.16 CAT construct. B, CV1 cells were cotransfected with 3
µg of the wild-type (p0.16 Luc) or mutated version at the -102 site
(p0.16 SF-1 m Luc) and 5 ng of SF-1 expression vector or empty vector.
Results were expressed as percentages of the mean activity achieved
with the p0.16 Luc plasmid cotransfected with the empty vector. Values
are means ± SEM of at least four independent
transfections.
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Cooperativity between Sp1 and C/EBP in cAMP-Induced Activation of
the MVDP Promoter
As shown in Fig. 7
, the mutation of Sp1 binding site decreased
both basal and forskolin-induced CAT activities by 80% and 95%,
respectively. The construct retained cAMP responsiveness, but the
fold-increase (6.9-fold) was lower than that determined with the
wild-type promoter (13.4-fold). The mutant promoter for C/EBP binding
was more effective in driving basal expression of the CAT reporter gene
(+119%) than the wild-type promoter (p0.16 CAT). Expressed as
fold-increase over basal values, the ability of forskolin to elicit
cAMP responsiveness was reduced by at least 50% after mutation of the
C/EBP binding site. To determine whether the Sp1 and C/EBP binding
sites could account for full cAMP stimulation of MVDP
proximal promoter, plasmids containing double mutations were generated.
As observed in Fig. 7
, the double mutation of both -52 Sp1 and -61
C/EBP binding sequences reduced the forskolin responsiveness of the
p0.16 CAT promoter to the residual induction level observed with pTATA
construct. When the SF-1 binding site was mutated in the context of
C/EBP or Sp1 mutant promoters, fold-activation by forskolin treatment
was either not affected or reduced to values observed with Sp1 mutant
promoter, respectively. These results indicate that a cooperative or
additive functional interaction between Sp1 and C/EBPß seems critical
for cAMP activation of MVDP proximal promoter in Y1
cells.
To better understand the respective roles of C/EBPß and Sp factors in
MVDP gene transcription, we performed transactivation assays
in a nonsteroidogenic cell line, CV1 cells, that lacks C/EBPß
expression (Fig. 9
). Increasing amounts
of C/EBP expression vector were cotransfected with the wild-type
promoter or mutant promoters for C/EBP and/or Sp1 sites. The p0.16 CAT
promoter was stimulated in a dose-dependent manner, and this effect was
diminished by approximately 67% and 77% when either the -61 C/EBP or
-52 Sp1 sites were mutated, respectively (Fig. 9
, 25 ng). Mutation of
both sites nearly abolished the responsiveness of MVDP
promoter to C/EBPß. These data indicate that full C/EBPß-dependent
activation of the MVDP promoter requires the integrity of
both -61 C/EBP and -52 Sp1 sites.

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Figure 9. Requirement of -61 C/EBP and -52 Sp1 Sites for
Transactivation of MVDP Proximal Promoter by C/EBPß
CV1 cells were cotransfected with 8 µg of wild-type or mutated
versions of the p0.16 CAT construct and increasing amounts (25 and 100
ng) of C/EBPß expression vector or empty vector. Results were
expressed as percentages of the mean value achieved with p0.16 CAT
plasmid cotransfected with the empty vector. Values are means ±
SEM of at least four independent transfections. *,
Significantly different (P < 0.01) between
activity of wild-type and C/EBP mutant version plasmid with 25 ng
C/EBPß expression vector. °, Significantly different
(P < 0.05) between activity of wild-type and C/EBP
mutant version plasmid with 100 ng C/EBPß expression vector.
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Forskolin-Induced MVDP Expression in Y1 cells Is Inhibited by
DAX-1
It has been shown that DAX-1 can affect steroidogenesis in Y1
cells by inhibiting the expression of genes involved in critical steps
of this process (17). To test whether DAX-1 can affect MVDP
expression, we used stably transfected Y1 clones expressing human DAX-1
(Y1/hDAX-1) and control cells containing the empty plasmid expressing
the neomycin-resistance gene (Y1/neo) (38). Constitutive
MVDP mRNA levels are very low in Y1/neo cells as well as in
Y1/hDAX-1 cells (Fig. 10A
). Forskolin
treatment led to the expected increase of MVDP mRNA levels
in Y1/neo cells, the induction being maximal 3 h after addition of
the activator. Conversely, MVDP transcripts stayed at very
low levels in Y1/hDAX-1 cells after forskolin treatment. The same
results have been obtained by Western blot analysis of cell extracts:
MVDP concentrations were up-regulated by forskolin in control cells but
not in Y1 clones expressing hDAX-1 (Fig. 10B
). These results
demonstrate that the expression of DAX-1 in Y1 cells significantly
impaired cAMP-stimulated expression of the MVDP gene at
least by controlling mRNA levels.

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Figure 10. MVDP Forskolin-Induced Expression
Is Inhibited by hDAX1 Expression in Y1 Cells
Stably transfected Y1 cells expressing hDAX-1 (Y1/hDAX-1) and control
cells only expressing the neomycin-resistance gene (Y1/neo) were
cultured in the presence of 10-5 M forskolin
(Fsk) for the indicated times. A, Northern blot analysis. Twenty five
micrograms of total RNAs were loaded per well and hybridized with
either 32P-labeled MVDP cDNA or ß-actin
cDNA probes. B, Western blot analysis of 15 µg proteins. After
SDS-PAGE the proteins were transferred to nitrocellulose and incubated
with anti-MVDP polyclonal antibody (dilution 1:3,000).
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DISCUSSION
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The level and pattern of expression of an eukaryotic gene are
mainly governed by the combinatorial action of multiple transcriptional
activators and repressors binding to distinct promoter and enhancer
elements (39). Using Y1 murine adrenocortical cells, our studies
demonstrate that basal and cAMP-induced activities of the
MVDP proximal promoter involve both cell-specific
(SF-1), more restricted (C/EBPß), and ubiquitous (NF1, Sp1)
transcriptional activators that act in combination.
Several lines of evidence indicate that the transcriptional activator
SF-1 interacts with a cis-acting regulatory element
positioned at -102 within the proximal ARE (AREp) of the
MVDP gene. First, a known pure SF-1 binding site [from
murine 21-steroid hydroxylase gene promoter, position -140 (37)]
competed efficiently for the formation of the DNA/protein complex in
gel mobility shift assays. Second, immunoreactivity in gel mobility
shift assay using an anti-SF-1 antibody provides evidence that the
protein in Y1 nuclear extracts interacting with the
cis-acting sequence is closely related to SF-1. Third, a
specific DNA-protein complex was formed when the AREp probe was
incubated with in vitro translated SF-1 that was absent in
unprogrammed lysate. Finally, expression of exogenous SF-1
transactivates MVDP promoter depending on the integrity of
the AREp site. SF-1 is an orphan member of the nuclear receptor
superfamily expressed in all major steroidogenic tissues, binding as a
monomer to consensus response elements PyCAAGGPyPy or PuPuAGGTCA in the
upstream region of a number of genes (40, 41). Surprisingly, the AREp
probe we used in our experiments does not contain a SF-1 consensus
sequence. Two recent examples of noncanonical SF-1 site usage are the
murine DAX-1 and the Inhibin
-promoters that are both stimulated via
cryptic SF-1 sites located at proximal positions -126 and -137,
respectively (42, 43). These two sites share a common sequence
TCAG/TGGCCA but seem unrelated to AREp. Although the precise sequence
involved in SF-1 binding to AREp remains to be determined, mutation
analyses demonstrate that this binding site resides in the downstream
half-site of the AREp sequence (5'-TGTTCT-3').
Within this binding motif, the GT dinucleotide (underlined)
is critical for SF-1 binding since mutation of either G or T abolished
the interaction with SF-1. However, the importance of these nucleotides
is dependent upon the sequences flanking the AREp half-site since an
identical TGTTCT motif lying within the distal ARE (AREd), but in the
opposite strand (AGAACA), is unable to bind SF-1. Interestingly, the GT
dinucleotide was highly conserved in functional AREs identified in a
number of androgen-regulated genes, and it has been shown that in
vitro, as well as in vivo, a G nucleotide at position 2
of the downstream half-site is essential for AR/DNA interaction (35, 44, 45). This SF-1 binding region has been previously shown to be a
functional ARE in human mammary carcinoma cells (35). This suggests
that in different tissues the same DNA motif may respond differently to
the binding of several nuclear receptors. Mutation within this SF-1
binding site nearly abolishes basal CAT activity, suggesting that
constitutive expression of the gene in adrenocortical Y1 cells requires
the presence of promoter sequence containing an SF-1 recognition
element. SF-1 is known to regulate a large number of genes involved in
steroidogenesis and reproduction (reviewed in Ref. 6). Within the
murine adrenocortical cells, it has been shown that SF-1 stimulated
expression of the cytochrome P450 steroid hydroxylases (6), StAR (10, 46), high density lipoprotein (HDL) receptor (47), and ACTH
receptor (12). Our data regarding the MVDP (AKR1B7) gene
expression are the first to demonstrate that SF-1 regulates a gene
involved in the detoxification of products of the steroidogenesis.
Taken together, these results suggest that SF-1 plays a critical role
in the coordinated expression of ACTH receptor, proteins involved in
delivery of cholesterol, steroidogenic enzymes responsible for the
biosynthesis of steroid hormones, and nonsteroidogenic enzymes involved
in the reduction of reactive aldehydes generated by steroidogenesis
(32).
Mutational analysis also identified a second element that is required
for basal expression of the MVDP promoter. The NF1 binding
site appears to be equally important for MVDP gene
transcription as its mutation nearly abolishes basal promoter activity
in Y1 cells. Thus, both SF-1 and NF1 binding sites must be intact for
constitutive transactivation of the MVDP promoter in
adrenocortical cells. In addition to the vas deferens expression that
is characteristic of the mouse species, MVDP is essentially expressed
in steroidogenic cells (Ref. 31 and our unpublished results),
and the NF1/DNA interaction cannot explain the restricted pattern of
MVDP. Members of the CTF/NF1 family are ubiquitous factors known to
function as positive regulators of gene transcription (48). Our results
rather suggest that SF-1 participates in the adrenocortical expression
of the MVDP gene.
In common with many cAMP-regulated steroidogenic genes, the
MVDP proximal promoter does not contain any consensus CRE,
suggesting the existence of yet uncharacterized cAMP-dependent
regulatory factors. Surprisingly, each steroidogenic gene appears to be
regulated by a different mechanism, even though the transcription of
these genes is temporally coordinated (49). Recent studies have shown
that whatever the mechanism involved, SF-1 is a common factor in this
pathway that mediates basal and/or hormone-induced expression of the
steroid hydroxylase genes (reviewed in Ref. 6). After mutation within
SF-1 or NF1 binding sites, the absolute cAMP-induced CAT activities of
the two reporters were strongly reduced. However, cAMP fold-induction
values were preserved, indicating that the SF-1 and NF1 sites are
important for full transcriptional responsiveness but dispensable for
the cAMP-dependent regulation of the MVDP gene in Y1 cells.
Thus, SF-1 may represent a tissue-specific factor rather than a
mediator of cAMP induction.
All experimental features, including EMSAs and functional analyses
using mutated constructs, showed that maximal responsiveness to cAMP
required two cis-acting components: the Sp1 and C/EBP
binding sites. Inactivation of any one component decreased the cAMP
responsiveness measured as fold-induction, suggesting that cAMP
stimulation of MVDP promoter activity is mediated by both
actions of the transcription factors bound to the -52 Sp1 and -61
C/EBP elements. Although originally associated with constitutive
transcription, it is now evident that Sp1 also regulates several
inducible genes. The promoter region of the genes encoding the steroid
hydroxylase usually contains atypical cAMP-responsive sequences
unrelated to the classical CRE/CREB system (reviewed in Ref. 49). It
has recently been shown that Sp1 is important for the regulation of the
CYP11A gene through binding to an element required for cAMP
responsiveness (50, 51, 52). The mechanism by which Sp1 confers cAMP
responsiveness to the MVDP promoter has not yet been
determined. Possible mechanisms include regulation of the level of Sp1
protein, Sp1 activation by posttranslational modification, affinity for
DNA, and interaction with other factors. In adrenocortical cells the
levels of Sp1 protein are not affected by forskolin treatment (52).
Recent data have demonstrated that PKA (protein kinase A)
phosphorylates Sp1 and up-regulates its DNA-binding and
trans-activating properties (53). On the other hand, it has
been recently reported that both Sp1 and SF-1 are required to regulate
the cAMP-dependent expression of bovine CYP11A gene (54) and that the
cooperation between these two factors is mediated by a direct
protein-protein interaction and/or by the common activator CBP (CREB
binding protein) (55). Whether or not the -52 Sp1 element of the
MVDP promoter is involved in the same way in transmitting a
cAMP response remains to be determined. We have previously shown that
exposure of T47D cells to (Bu)2cAMP had no effect
on the basal expression of CAT driven by the MVDP proximal
promoter (56). Our site-directed mutagenesis showed that the SF-1
binding site is required for high MVDP promoter activity in
the absence as well as in the presence of forskolin. We do not know yet
how cAMP responsiveness is restricted to Y1 cells, but one explanation
could be that Y1-enriched specific factors such as SF-1 may interact
with Sp1 bound to the MVDP promoter to confer cAMP
induction.
Immunoreactivity and in vitro translated protein in gel
shift assays as well as transactivation experiments demonstrated that
C/EBPß-related proteins are essential components of the binding
activity at position -61. The C/EBP family has been associated with
differentiation, lipid biosynthesis, and metabolism (Ref. 57 for
review). C/EBPß has been implicated in the immune response and
recently in female reproduction (57, 58). Mutation of the -61 C/EBP
binding site increases basal promoter activity to 119% of wild-type
levels, suggesting that C/EBPß acts as a repressor of the
MVDP gene. A few cases of repressor effects have been
described in relation to the C/EBP family. Two isoforms of C/EBPß are
generated from a single mRNA: the full-length protein (also called LAP:
liver enriched activator protein) and a truncated protein (LIP: liver
enriched inhibitor protein) that is a transcriptional repressor (59).
Thus, the ability of C/EBPß to activate or repress a promoter is
probably determined, at least in part, by the LAP/LIP ratio. Our
results also define C/EBPß as a possible cAMP-regulated activator of
MVDP gene transcription in Y1 cells. C/EBPß expressed in
other steroidogenic cells, such as Leydig (60) and granulosa cells
(58), is believed to play an important role in regulating the
expression of genes involved in steroid hormone synthesis. In support
of a role of C/EBPß in mediating the cAMP-dependent regulation of
gene expression in steroidogenic cells, recent data have revealed that
this factor is involved in transcriptional activation of the murine and
human StAR gene (61, 62). However, when the C/EBP binding sites
identified in the StAR gene were mutated, a pronounced decrease in
basal promoter activity occurred in Leydig or granulosa cells, but the
fold-stimulation by cAMP was unaffected (61, 62). As shown by
mutational analysis, the -61 C/EBP site participates in cAMP
responsiveness of the MVDP gene promoter expressed as
fold-induction. Thus, it seems that participation of C/EBP factors in
cAMP responsiveness could be achieved by a different mechanism in Y1
cells. In agreement with this possibility, we found that forskolin
treatment increased the intensity of the C/EBPß complexes bound to
the -61 C/EBP site in nuclear extracts of Y1 cells. In some
cell types, increases in intracellular cAMP levels have been shown to
up-regulate C/EBPß via different mechanisms including the level of
expression and the subcellular localization of the protein (63, 64).
Moreover, the activity of C/EBPß may also be controlled at the
posttranslational level, presumably by the PKA pathway (65). Since the
stimulation of MVDP gene expression by forskolin does not
require continuous protein synthesis (31), it seems likely that cAMP
exerts its action through subcellular localization or posttranslational
modifications of C/EBPß proteins. An alternative explanation is that
forskolin treatment increases the ratio of LAP to LIP as reported in
Sertoli cells (64).
Mutational analyses identified two critical regulatory elements for
cAMP responsiveness of the MVDP promoter. The two adjacent
-52 Sp1 and -61 C/EBP binding sites appear to be important for cAMP-
induced transactivation as their mutation similarly decreased the
fold-activation. cAMP inducibility was nearly abolished when both Sp1
and C/EBP sites were mutated. Consistent with these observations, in
CV1 cells, C/EBP fails to transactivate efficiently the p0.16 CAT
MVDP promoter when either the -52 Sp1 or -61 C/EBP sites
are mutated, implying that a cooperative functional interaction between
Sp1 and C/EBPß is required for cAMP activation of the MVDP
promoter in Y1 cells. In support of this model, it has been reported
that C/EBPß, but not C/EBP
, is able to cooperate with Sp1 at the
level of both transactivation and DNA binding to their adjacent sites,
to regulate the expression of the rat CYP2D5 gene (66, 67). However,
gel shift assays using a probe containing both -61 C/EBP and -52 Sp1
sites (-69 to -37, Table 1
) did not reveal any higher order
Sp1-C/EBP-DNA complex formation, suggesting that mechanisms other than
cooperative binding might be involved in MVDP promoter cAMP
activation (data not shown). cAMP responsiveness has been shown to be
fulfilled by SF-1 in the case of CYP17 gene (9), SF-1 plus Sp1 in the
CYP11A gene (54), SF-1 plus C/EBP in the StAR gene (61, 62), and Sp1
plus C/EBP in the MVDP gene (present results).
Inappropriate DAX-1 expression causes defect in adrenal and gonadal
development and function (Ref. 68 for review). DAX-1 is supposed to act
in these processes at least by suppressing SF-1-mediated transcription
through different mechanisms, including interaction with SF-1 (16, 69)
and/or binding to DNA stem-loop structures formed in the promoters of
target genes (17, 38). Interestingly, we found that DAX-1 is also able
to repress cAMP-induced MVDP gene expression in Y1 cells but
the mechanism involved will need further investigation. Indeed, in
addition to the -102 SF-1 site, analysis of more upstream DNA sequence
reveals the presence of four putative SF-1 binding sites at positions
-279, -458, -503, and -578 that could constitute additional
potential targets for SF-1/DAX1 actions. These data are the first
report pointing out detoxifying enzymes of the aldose reductase family
as new potential targets of SF-1/DAX-1 functional interactions in
steroidogenic organs.
In conclusion, we show that a complex array of nuclear proteins bind to
the MVDP promoter. At least four functional interactions
have been demonstrated within the -121/-40 bp fragment to be
sufficient for basal and hormonal-regulated expression. The SF-1
protein, which binds to an unusual cis-acting element
located in the proximal ARE, regulates the basal level of
MVDP gene expression and boosts the overall response to
forskolin. In addition, the adjacent NF1 binding site contributes
equally to these two processes. Our study also establishes the role of
Sp1 and C/EBPß as important transducers of the cAMP-positive signal
and points out DAX-1 as a negative actor in this pathway. Further
studies will be required to determine whether the interactions between
the regulatory proteins could be direct or through the recruitment of
coactivators/corepressors to the MVDP promoter.
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MATERIALS AND METHODS
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Animals
Mice of the Swiss strain (CD-1; Charles River Laboratories, Inc. Saint Aubin Les Elbeuf, France) were used. To
investigate the role of pituitary ACTH, adult males (8 weeks old) were
injected sc with dexamethasone acetate for 5 days (75 µg dissolved in
sesame oil twice daily, Sigma, St. Louis, MO) or with
dexamethasone acetate (5 days) plus ACTH im (12 daily, Synacthene,
Novartis Pharma S.A., Rueil-Malmaison, France) for the last 2 days.
Control animals received injection of vehicle only. Animals were killed
6 h after the last injection. Adrenals were removed, frozen in
liquid nitrogen, and stored at -70 C until Northern blot analysis.
RNase Protection Assay
A 32P-labeled antisense riboprobe
complementary to MVDP sequences from -185 to +94,
encompassing the previously identified transcription start site (33),
was synthesized using RiboProbe Combination System-T7/SP6 kit from
Promega Corp. (Madison, WI); 2.5 105
dpm of the probe were coprecipitated with each total RNA sample
(adrenals, 10 µg; vas deferens, 2 µg). The pellet was then
resuspended in 30 µl of hybridization buffer [80% formamide, 40
mM
piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), pH 6.4,
0.4 M sodium acetate, 1 mM
EDTA] and incubated at 85 C for 5 min. After an overnight incubation
at 45 C, nonhybridized RNAs were digested by RNAse ONE ribonuclease
(adrenals, 7 U; vas deferens, 5 U; Promega Corp.) for 45
min at 37 C in 300 µl of digestion buffer (10
mM Tris, pH 7.5, 5 mM EDTA,
200 mM sodium acetate). Reaction was stopped by
addition of 20 µl 10% SDS and 5 µl 10 mg/ml proteinase K, followed
by incubation at 37 C for 15 min. Proteins were then removed by
phenol-chloroform-isoamyl alcohol extraction. Digestion- resistant
RNA-RNA hybrids were precipitated, resuspended in 5 µl loading dye
(80% formamide, 10 mM EDTA, 0.1% bromophenol
blue, 0.1% xylene cyanol, 0.1% SDS) and run on a 6% polyacrylamide
denaturing sequencing gel. A molecular size marker was provided by a
dideoxy sequence of the DNA fragment used as the probe template, run
next to the RNase mapping reactions.
5'-RACE-PCR
Five'-RACE-PCR has been performed as described in Ref. 70
with the following exceptions. Ten micrograms total RNA from adrenals
and vas deferens were used as template. cDNA were purified using
QiaQuick PCR purification kit (QIAGEN, Hilden, Germany).
Oligonucelotidic single-stranded anchor:
PO3-ACTATCGATTCTGGAACCTTCAGAGG-NH2.
Anchor PCR primer: 5'-CCACCTCTGAAGGTTCCAGAATCGATAG-3'. MVDP
specific primers were 5'-GTGATACACATAGGCACAGTCAAT-3' (external primer,
complementary to MVDP cDNA sequence from position 127 to
150, where A of ATG translation start codon is 1) for the first 35
cycles and 5'-AATGGCCGCCTTCACGGCTTCCTT-3' (internal primer,
complementary to MVDP cDNA sequence from positions 85 to 108) for the
next 35 cycles. DNA was denatured for 5 min at 94 C, and amplification
was carried out using Takara rTaq (Boehringer Ingelheim
Bioproducts, Heidelberg, Germany), for 35 cycles with the
following parameters: denaturation, 94 C for 45 sec; annealing 60 C
(first 35 cycles) or 63 C (next 35 cycles) for 45 sec; then synthesis,
72 C for 2 min. A 10-min synthesis period was finally applied to
samples. One microliter of a fifth dilution of the reaction was used to
set up the second 35-cycle amplification round, using internal primer.
An aliquot of the PCR reactions was run in a 2% agarose gel in
Tris-acetate-EDTA electrophoresis buffer, blotted to a nylon membrane
(Hybond N+, Amersham Pharmacia Biotech France SA, Les
Ulis, France) and hybridized with a
5'-32P-labeled oligonucleotide probe specific to
the first exon (5'-GCATCTTGGCTTTGGTACT-3', position 22 to 40) overnight
at 37 C. Sequencing was carried out using a T7 sequencing
KIT (Pharmacia Biotech, Piscataway,
NJ) after cloning into pGEM-T Easy (Promega Corp.).
Northern Blot Analysis
Total RNAs were extracted from Y1/hDAX-1, Y1/neo cells, or
adrenals with RNAzol (Bioprobe Systems, Montreuil-sous-Bois, France),
according to the manufacturers instructions. Total RNA (25 µg) was
electrophoresed in a 1% (wt/vol) agarose, 2.2 M
formaldehyde gel and transferred to nylon filters. A 5-min UV exposure
covalently bound RNAs to the filters. Probes used in these experiments
include: MVDP, a 1.2-kb EcoRI-BamHI fragment of
MVDP cDNA; StAR, a RT-PCR-generated fragment from murine
StAR cDNA; and P450scc, a generous gift from Dr.
M. Begeot (INSERM-INRA U418, Lyon, France). The cDNAs were labeled with
32P-dCTP using the Megaprime DNA Labeling System
(Amersham Pharmacia Biotech, Les Ulis, France).
Prehybridization was carried for 1.5 h at 65 C in a solution
containing 3 x SSC (standard sodium citrate buffer),
polyvinylpyrrolidone (0.2%), Ficoll (0.2%), polyethyleneglycol (5%),
glycine (1%), SDS salt (1%), and 100 µg/ml denatured salmon sperm
DNA. Hybridization was performed overnight at 65 C in the same solution
containing 1 x 106 cpm/ml of
32P-labeled probes. Filters were washed at 65 C.
To normalize the quantities of MVDP mRNAs, Northern blots
were stripped and rehybridized in the buffer described above, at 65 C
with a 32P-labeled ß-actin cDNA probe.
Autoradiography was carried out by exposing the blots to x-ray films
(Cronex, Sterling Diagnostic, Newark, DE) for 48 h with an
intensifying screen at -80 C.
Western Blot Analysis
A fraction of cell samples was collected before RNA extraction
(RNA Plus, Quantum Bioprobe, Montreuil-sous-Bois, France). The
concentration of soluble proteins was determined by the Bradford method
(Bio-Rad Laboratories, Inc. München, Germany).
Soluble cellular extracts (15 µg proteins) were subjected to SDS-PAGE
on 10% acrylamide gels and transferred to nitrocellulose membranes
according to the manufacturers instructions (Bio-Rad Laboratories, Inc.). Nonspecific protein binding sites were
blocked by incubation of nitrocellulose for 1 h at room
temperature in TBST [50 mM Tris-HCl (pH 8), 150
mM NaCl, 0.1% (vol/vol) Tween 20] containing 5% (wt/vol)
nonfat dry milk, and incubation with the polyclonal anti-MVDP antibody
(diluted 1:10,000) was carried out for 1 h at room temperature as
previously described (71). After three washes in TBST, the sheets were
incubated for 1 h at room temperature with the second antibody
(peroxidase-conjugated antirabbit IgG) diluted 1:15,000. The blots were
washed again, and peroxidase activity was detected by autoradiography
with the ECL enhanced chemiluminescence system (Amersham Pharmacia Biotech).
Cell Culture and Transfections
Murine Y1 adrenocortical tumor cells (72) and human placental
JEG-3 were grown in DMEM-Hams F-12 medium (1:1) supplemented with
10% FCS, 2 mM glutamine, penicillin (100 U/ml), and
streptomycin (100 µg/ml) (Life Technologies Ltd,
Paisley, UK).
DAX-1-expressing Y1 cell lines were a generous gift from E. Lalli (38).
Stably transfected Y1 clones were obtained using the expression
plasmids pSG.DAX-1 (73) and pD383, which carry the neomycin-resistance
gene under the control of the phosphoglycerate kinase gene promoter.
Y1/hDAX-1 and Y1/neo cells were grown in DMEM-Hams F-12 medium (1:1)
supplemented with 15% horse serum, 2.5% FCS, 500 µg/ml geneticin
(G418) (Roche Molecular Biochemicals, Meylan, France), 2
mM glutamine, penicillin (100 U/ml), and streptomycin (100
µg/ml).
One day before transfection, Y1 or JEG-3 cells were plated at a density
of 1 x 106 cells/60-mm dish.
MVDP-CAT reporter plasmids (1.5 or 3 µg) were transfected
into cultured cells with FuGENE6 transfection reagent according to the
manufacturers instructions (Roche Molecular Biochemicals). Twenty-four hours after transfection, the
medium was replaced, and the cells were treated or not with forskolin
[10-5 M, dissolved in
dimethylsulfoxide (DMSO)] for 24 h. Cells were harvested 48
h after lipofection and subjected to CAT assays.
CV1 cells were transfected by the calcium phosphate method as
previously described (74). Twelve hours before transfection, cells were
seeded at 0.5 106 cells per 60-mm dish or at
0.25 x 106 cells per well in six-well
culture plates for CAT assays or luciferase assays, respectively. CV1
cells were transferred to DMEM supplemented with 5% FCS and
transfected with various amounts of effector and reporter plasmids
along with carrier DNA to equalize the total amount of DNA to 10 µg
for CAT assays (8 µg CAT reporter construct) and 5 µg for
luciferase assays (3 µg luciferase reporter construct). Cells were
incubated with the DNA/calcium phosphate precipitates for 12 h,
rinsed to remove DNA, and incubated in fresh medium for an additional
36 h before being harvested for analysis. All transfection
experiments were performed at least four times. To correct the
variations in transfection efficiencies, separate plates of cells were
transfected with a CAT reporter plasmid under the control of the SV40
promoter/enhancer (pSV2-CAT) as indicated.
CAT and Luciferase Assays
For CAT assays, cells were lysed by four rounds of freezing and
thawing. The cell lysates were heat treated 10 min at 65 C and
clarified by centrifugation. Under conditions where signals were
proportional to the amount of supernatant added, 340 µl of
supernatant were combined with 2 µl of
[14C]chloramphenicol (1.850 Mbq/ml, NEN Life Science Products, Boston, MA), 2.5 µl of 8 mM
acetylcoenzyme A, and 250 mM Tris-HCl, pH 7.8, to a final
volume of 100 µl. The mixtures were incubated at 37 C for 3 h,
and the reactions were stopped by extraction with 550 µl ethyl
acetate. The products were separated by TLC, acetylated forms of
[14C] chloramphenicol were excised
individually, and radioactivity was counted in a scintillation
counter.
For luciferase assays, cell extracts were prepared by scraping using
150 µl luciferase assay system reporter lysis buffer (Promega Corp., Lyon, France); extracts were then clarified by
centrifugation at 13,000 x g at 4 C. Supernatants (40
µl) were assayed for luciferase activity using a luciferase assay kit
(Genofax A, Prodemat, Anduse, France) exactly as described by the
protocol provided with the kit. Luminescence was measured using a
Berthold LB 96V Microplate Luminometer (Perkin-Elmer,
Courta Boeuf, France) under conditions in which signals were
proportional to the amount of supernatant added.
Plasmid Constructs
All plasmid constructs were prepared using standard methods
(75). MVDP-CAT reporter plasmids contain -1,804 to +41
bp (and deletions thereof) of the murine MVDP
promoter sequence linked to the coding region of bacterial CAT gene in
the pBlCAT3 vector as previously described (35). pTATA CAT was
constructed as follows: a DNA fragment encompassing regions from -40
to +41 of the MVDP gene was amplified by a standard PCR
reaction using p0.16 CAT plasmid as a template. PCR products were
cloned into a PstI restriction site of the pBlCAT3 vector.
Resulting plasmid was completely sequenced to confirm the presence and
orientation of the expected insert. Mutations of the AREp (-117/-93)
and NF1 (-84/-59) binding site contained in the p0.16 CAT construct
have been described previously (76). The Gene Editor kit (Promega Corp., Lyon, France) was used to introduce the other mutations
into p0.16 CAT. The oligonucleotides used to generate the mutations are
shown in Table 1
(-52 Sp1m, -61 C/EBPm, -76 NF1m) and Fig. 6D
(Mut
3). Resulting plasmids were partially sequenced to confirm that the
sites had been mutated as expected. Luciferase reporter plasmids, p0.16
Luc and p0.16 SF1m Luc, were constructed by introducing into the pGL3
plasmid (Promega Corp., Lyon, France), the -121/+41
promoter fragment either intact or mutated (Mut 3) within the AREp
sequence, respectively.
In vitro translation experiments were performed using
pGEM-SF-1 plasmid that was constructed as follows: a 2-kb
EcoRI insert containing the murine SF-1 cDNA was excised
from the pCMV5 plasmid (kindly provided by Dr. K.L. Parker, University
of Texas Southwestern Medical Center, Dallas, TX) and introduced into
the EcoRI site of pGEM-T Easy plasmid (Promega Corp., Lyon, France). The pCDNA1-LAP plasmid expressing C/EBPß
under the control of a cytomegalovirus (CMV) promoter was kindly
provided by Dr. U. Schibler (University of Geneva, Geneva, Switzerland)
and used for both transfections and in vitro translation
experiments.
EMSA
Nuclear extracts were prepared from confluent Y1 adrenocortical
cell cultures. Briefly, cell monolayers were rinsed once with PBS and
scraped in 500 µl of 10% glycerol PBS. The cells were pelleted by
centrifugation at 500 x g for 5 min, washed in 500
µl buffer A (20 mM HEPES, 50
mM KCl, 1 mM EDTA, 0.25
mM EGTA, 0.15 mM spermine,
0.5 mM spermidine, 0.5 mM
sucrose, 1 mM phenylmethylsulfonyl fluoride, 1
mM dithiothreitol, 1% aprotinin) and pelleted.
Pellets were resuspended in 500 µl buffer A with 0.5% Nonidet P-40
and vortexed. The cells were expanded for 15 min on ice. The
homogenates were centrifuged 10 min at 2,500 x g and
rinsed with buffer A without NP-40. After a centrifugation at
2,500 x g for 10 min to pellet the nuclei, 2550 µ
l buffer C (20 mM HEPES, 0.45
M NaCl, 1 mM EDTA, 0.25
mM EGTA, 0.15 mM spermine,
0.5 mM spermidine, 0.5 mM
sucrose, 1 mM phenylmethylsulfonyl fluoride, 1
mM dithiothreitol, 1% aprotinin) were added, and
the samples were expanded for 10 min on ice. The nuclear lysate was
then centrifuged for 20 min at 13,000 x g at 4 C, and
the supernatant was placed into a fresh microfuge tube and stored at
-80 C.
To produce in vitro translated SF-1 or CEBPß, pGEM-SF-1 or
pCDNA1-LAP plasmids were transcribed using SP6 or T7 polymerase,
respectively, and translated using a TNT kit (Promega Corp., Lyon, France).
Oligonucleotides used in this study are listed in Table 1
and Fig. 6D
.
Ten picomoles of the double-stranded oligonucleotides were labeled with
T4 polynucleotide kinase and [
-32P dATP]
(110 TBq/mmol, Amersham Pharmacia Biotech, Les Ulis,
France). All probes were purified by precipitation. Binding reactions
were performed by mixing 5 µg Y1 nuclear extract or 13 µl
in vitro translated protein with 0.05 pmol of probe, in the
presence or absence of 50- to 200-fold molar excess of unlabeled
competitor in a 1x binding buffer (20 mM HEPES,
0.2 mM EDTA, 100 mM NaCl,
100 mM KCl, 10 mM
MgCl2, 8 mM spermidine, 4 mM
dithiothreitol, 200 µg/ml albumin, 8% Ficoll) and 1 µg
poly(deoxyinosinic-deoxycytidylic)acid. Where indicated, antiserum
specific for SF-1, Sp1, C/EBPß, ATF family (ATF 14)
(Santa Cruz Biotechnology, Inc. Santa Cruz, CA) or
CTF1/NF1 (kindly provided by Dr. Naoko Tanese, NYU Medical Center, New
York, NY) were incubated 10 min after probe addition. Complexes were
resolved by electrophoresis through 6% nondenaturing polyacrylamide
gels in 0.5 x Tris-borate-EDTA (TBE) electrophoresis buffer. The
gels were dried and autoradiographed.
 |
ACKNOWLEDGMENTS
|
---|
The authors would like to thank Anne-Marie Lefrançois,
Michèle Manin, and Jean-Marc Lobaccaro for critical reading of
the manuscript. We wish to thank Alain Halère for his excellent
technical assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Antoine Martinez, UMR CNRS 6547, Physiologie Comparée et Endocrinologie Moléculaire, Université Blaise Pascal, Clermont II, Complexe Universitaire des Cézeaux, 24 avenue des Landais, 63177 Aubière cedex, France. E-mail: antoine.martinez{at}geem.univ-bpclermont.fr
Received for publication January 18, 2000.
Revision received September 14, 2000.
Accepted for publication September 20, 2000.
 |
REFERENCES
|
---|
-
Orth DN, Kovacs NJ 1998 The adrenal cortex. In: Wilson
JD, Foster DW, Kronenberg HM, Reed Larsen P (eds) Williams Textbook of
Endocrinology, ed 9. WB Saunders Co, Philadelphia, pp 517664
-
Simpson ER, Waterman MR 1988 Regulation of the synthesis of
steroidogenic enzymes in adrenocortical cells by ACTH. Annu Rev Physiol 50:427440[CrossRef][Medline]
-
Quinn SJ, Williams GH 1988 Regulation of aldosterone
secretion. Annu Rev Physiol 50:409426[CrossRef][Medline]
-
Stocco DM, Clark BJ 1996 Regulation of the acute production
of steroids in steroidogenic cells. Endocr Rev 17:221244[Medline]
-
Lin D, Sugawara T, Strauss III JF, Clark BJ, Stocco DM,
Saenger P, Rogol A, Miller WL 1995 Role of steroidogenic acute
regulatory protein in adrenal and gonadal steroidogenesis. Science 267:18281831[Medline]
-
Parker KL, Schimmer BP 1997 Steroidogenic factor 1: a key
determinant of endocrine development and function. Endocr Rev 18:361377[Abstract/Free Full Text]
-
Morohashi KI, Zanger UM, Honda SI, Hara M, Waterman MR, Omura
T 1993 Activation of CYP11A and CYP11B gene promoters by the
steroidogenic cell-specific transcription factor, Ad4BP. Mol Endocrinol 7:11961204[Abstract]
-
Chau YM, Crawford PA, Woodson KG, Polish JA, Olson LM,
Sadovsky Y 1997 Role of steroidogenic-factor 1 in basal and
3',5'-cyclic adenosine monophosphate- mediated regulation of
cytochrome P450 side-chain cleavage enzyme in the mouse. Biol Reprod 57:765771[Abstract]
-
Zhang P, Mellon SH 1996 The orphan nuclear receptor
steroidogenic factor-1 regulates the cyclic adenosine 3',
5'-monophosphate mediated transcriptional activation of rat cytochrome
P450c17 (17
-hydroxylase/C1720 lyase). Mol Endocrinol 10:147158[Abstract]
-
Sugawara T, Kiriakidou M, Mc Allister JM, Holt JA, Arakane F,
Strauss JF 1997 Regulation of expression of the steroidogenic acute
regulatory protein (StAR) gene: a central role for steroidogenic factor
1. Steroids 62:59[CrossRef][Medline]
-
Keri RA, Nilson JH 1996 A steroidogenic factor-1 binding site
is required for activity of the luteinizing hormone ß subunit
promoter in gonadotropes of transgenic mice. J Biol Chem 271:1078210785[Abstract/Free Full Text]
-
Cammas FM, Pullinger GD, Barker S, Clark AJ 1997 The mouse
adrenocorticotropin receptor gene: cloning and characterization of its
promoter and evidence for a role for the orphan nuclear receptor
steroidogenic factor-1. Mol Endocrinol 11:867876[Abstract/Free Full Text]
-
Duval DL, Nelson SE, Clay CM 1997 A binding site for
steroidogenic factor-1 is part of a complex enhancer that mediates
expression of the murine gonadotropin-releasing hormone receptor gene.
Biol Reprod 56:160168[Abstract]
-
Vilain E, Guo W, Zhang YH, Mc Cabe ERB 1997 DAX-1 gene
expression upregulated by steroidogenic factor 1 in an adrenocortical
carcinoma cell line. Biochem Mol Med 61:18[CrossRef][Medline]
-
Kawabe K, Shikayama T, Tsuboi H, Oka S, Oba K, Yanase T,
Nawata H, Morohashi K 1999 Dax-1 as one of the target genes of
Ad4BP/SF-1. Mol Endocrinol 13:12671284[Abstract/Free Full Text]
-
Ito M, Yu R, Jameson JL 1997 DAX-1 inhibits SF-1 mediated
transactivation via a carboxy-terminal domain that is deleted in
adrenal hypoplasia congenita. Mol Cell Biol 17:14761483[Abstract]
-
Lalli E, Melner MH, Stocco DM, Sassone-Corsi P 1998 DAX-1
blocks steroid production at multiple levels. Endocrinology 139:42374243[Abstract/Free Full Text]
-
Hall PF 1970 Endocrinology of the testis. In: Johnson AD Gomes
WR, Vandemark NL (eds) The Testis. Academic Press, New York, pp 172
-
Stolz A, Rahimi-Kiami M, Ameis D, Chan E, Reak M, Shively JE 1991 Molecular structure of rat hepatic 3
-hydroxy steroid
dehydrogenase, a member of the oxidoreductase gene family. J Biol
Chem 266:1525315257[Abstract/Free Full Text]
-
Warren JC, Murdock GL, Ma Y, Goodman SR, Zimmer WE 1993 Molecular cloning of testicular 20
-hydroxy-steroid
dehydrogenase: identity with aldose reductase. Biochemistry 32:14011406[Medline]
-
Matsuura K, Deyashiki Y, Bunai Y, Ohya I, Hara A 1996 Aldose
reductase is a major reductase for isocaproaldehyde, a product of
side-chain cleavage of cholesterol in human and adrenal glands. Arch
Biochem Biophys 328:265271[CrossRef][Medline]
-
Jez JM, Flynn TG, Penning TM 1997 A new nomenclature for the
aldo-keto reductase superfamily. Biochem Pharmacol 54:639647[CrossRef][Medline]
-
Kinoshita JH, Nishimura C 1988 The involvement of aldose
reductase in diabetic complications. Diabetes Metab Rev 4:323337[Medline]
-
Pailhoux EA, Martinez A, Veyssière GM, Jean CG 1990 Androgen-dependent protein from mouse vas deferens. cDNA cloning and
protein homology with the aldo-keto reductase superfamily. J Biol
Chem 265:1993219936[Abstract/Free Full Text]
-
Donohue PJ, Alberts GF, Hampton BS, Winkles JA 1994 A
delayed-early gene activated by fibroblast growth factor-1 encodes a
protein related to aldose reductase. J Biol Chem 269:86048609[Abstract/Free Full Text]
-
Hyndman DJ, Takenoshita R, Vera NL, Pang SC, Flynn TG 1997 Cloning, sequencing and enzymatic activity of an inducible aldo-keto
reductase from Chinese hamster ovary cells. J Biol Chem 272:1328613291[Abstract/Free Full Text]
-
Cao D, Fan ST, Chung SSM 1998 Identification and
characterization of a novel human aldose reductase-like gene. J
Biol Chem 273:1142911435[Abstract/Free Full Text]
-
Hyndman DJ, Flynn TG 1998 Sequence and expression levels in
human tissues of a new member of the aldo-keto reductase family.
Biochim Biophys Acta 1399:198202[Medline]
-
Taragnat C, Berger M, Jean Cl 1988 Preliminary
characterization, androgen-dependence and ontogeny of an abundant
protein from mouse vas deferens. J Reprod Fertil 83:835842[Abstract]
-
Lau ET, Cao D, Lin C, Chung SK, Chung SS 1995 Tissue-specific
expression of two aldose reductase-like genes in mice: abundant
expression of mouse vas deferens protein and fibroblast growth
factor-regulated protein in the adrenal gland. Biochem J 312:609615[Medline]
-
Aigueperse C, Martinez A, Lefrançois-Martinez AM,
Veyssière G, Jean Cl 1999 Cyclic AMP regulates expression of the
gene coding for a protein related to the aldo-keto reductase
superfamily (MVDP) in human and murine adrenocortical cells. J
Endocrinol 160:147154[Abstract/Free Full Text]
-
Lefrançois-Martinez AM, Tournaire C, Martinez A, Berger
M, Daoudal S, Tritsch D, Veyssière G, Jean Cl 1999 Product of
side-chain cleavage of cholesterol, isocaproaldehyde, is an endogenous
specific substrate of mouse vas deferens protein, an aldose
reductase-like protein in adrenocortical cells. J Biol Chem 274:3287532880[Abstract/Free Full Text]
-
Pailhoux E, Veyssière G, Fabre S, Tournaire C, Jean Cl 1992 The genomic organization and DNA sequence of the mouse vas
deferens androgen-regulated protein gene. J Steroid Biochem Mol Biol 42:561568[CrossRef][Medline]
-
Nakagama H, Heinrich G, Pelletier J, Housman DE 1995 Sequence
and structural requirements for high-affinity DNA binding by the WT1
gene product. Mol Cell Biol 15:14891498[Abstract]
-
Fabre S, Manin M, Pailhoux E, Veyssière G, Jean Cl 1994 Identification of a functional androgen response element in the
promoter of the gene for the androgen-regulated aldose reductase-like
protein specific to the mouse vas deferens. J Biol Chem 269:58575864[Abstract/Free Full Text]
-
Akira S, Isshiki H, Sugita T, Tanabe O, Kinoshita S, Nishio Y,
Nakajima T, Hirano T, Kishimoto T 1990 A nuclear factor for IL-6
expression (NF-IL6) is a member of a C/EBP family. EMBO J 9:18971906[Abstract]
-
Rice DA, Mouw AR, Bogerd AM, Parker KL 1991 A shared promoter
element regulates the expression of three steroidogenic enzymes. Mol
Endocrinol 5:15521561[Abstract]
-
Zazopoulos E, Lalli E, Stocco DM, Sassone-Corsi P 1997 DNA
binding and transcriptional repression by DAX-1 blocks steroidogenesis.
Nature 390:311315[CrossRef][Medline]
-
Tjian R, Maniatis T 1994 Transcriptional activation: a complex
puzzle with few easy pieces. Cell 77:58[Medline]
-
Morohashi K, Honda S, Inomata Y, Handa H, Omura T 1992 A
common trans-acting factor, Ad4-binding protein, to the promoters of
steroidogenic P-450s. J Biol Chem 267:1791317919[Abstract/Free Full Text]
-
Wilson TE, Fahrner TJ, Milbrandt J 1993 The orphan receptors
NGFI-B and steroidogenic factor 1 establish monomer binding as a third
paradigm of nuclear receptor-DNA interaction. Mol Cell Biol 13:45945804
-
Yu RN, Ito M, Jameson JL 1998 The murine DAX-1 promoter is
stimulated by SF1 (steroidogenic factor-1) and inhibited by COUP-TF
(chicken ovalbumin upstream promoter-transcription factor) via a
composite nuclear receptor-regulatory element. Mol Endocrinol 12:10101022[Abstract/Free Full Text]
-
Ito M, Park Y, Weck J, Mayo KE, Jameson JL 2000 Synergistic
activation of the inhibin
-promoter by steroidogenic factor-1
and cyclic adenosine 5',3'-monophosphate. Mol Endocrinol 14:6681[Abstract/Free Full Text]
-
Claessens F, Celis L, Peeters B, Heyns W, Verhoeven G,
Rombauts W 1989 Functional characterization of an androgen response
element in the first intron of the C3(1) gene of prostatic binding
protein. Biochem Biophys Res Commun 164:833840[Medline]
-
Crossley M, Ludwig M, Stowell KM, De Vos P, Olek K, Brownlee
GG 1992 Recovery from hemophilia B leyden: an androgen responsive
element in the factor IX promoter. Science 257:377379[Medline]
-
Caron KM, Ikeda Y, Soo SC, Stocco DM, Parker KL, Clark BJ 1997 Characterization of the promoter region of the mouse gene encoding the
steroidogenic acute regulatory protein (StAR). Mol Endocrinol 11:138147[Abstract/Free Full Text]
-
Cao G, Garcia CK, Wyne KL, Schultz RA, Parker KL, Hobbs HH 1997 Structure and localization of the human gene encoding SR-BI/CLA-1.
J Biol Chem 272:3306833076[Abstract/Free Full Text]
-
Jones KA, Kadonaga JT, Rosenfeld PJ, Kelly TJ, Tjian R 1987 A
cellular DNA-binding protein that activates eukaryotic transcription
and DNA replication. Cell 48:7989[Medline]
-
Waterman MR 1994 Biochemical diversity of cAMP dependent
transcription of steroid hydroxylase genes in the adrenal cortex.
J Biol Chem 269:2778327786[Free Full Text]
-
Guo IC, Tsai HM, Chung BC 1994 Actions of two different
cAMP-responsive sequences and an enhancer of the human CYP11A (P450scc)
gene in adrenal Y1 and placental JEG-3 cells. J Biol Chem 269:63626369[Abstract/Free Full Text]
-
Momoi K, Waterman MR, Simpson ER, Zanger UM 1992 3',5'-cyclic
adenosine monophosylate-dependent transcription of the CYP11A
(cholesterol side chain cleavage cytochrome P450) gene involves a DNA
response element containing a putative binding site for transcription
factor Sp1. Mol Endocrinol 6:16821690[Abstract]
-
Ahlgren R, Saske G, Waterman MR, Lund J 1999 Role of Sp1 in
cAMP-dependent transcriptional regulation of the bovine CYP11A gene.
J Biol Chem 274:1942219428[Abstract/Free Full Text]
-
Rohlff C, Ahmad S, Borellini F, Lei J, Glazer RI 1997 Modulation of transcription factor Sp1 by cAMP-dependent protein
kinase. J Biol Chem 272:2113721141[Abstract/Free Full Text]
-
Liu Z, Simpson ER 1997 Steroidogenic factor 1 (SF1) and Sp1
are required for regulation of bovine CYP11A gene expression in
steroidogenic cells. Mol Endocrinol 11:127137[Abstract/Free Full Text]
-
Liu Z, Simpson ER 1999 Molecular mechanism for cooperation
between Sp1 and steroidogenic factor-1 (SF-1) to regulate bovine CYP11A
gene expression. Mol Cell Endocrinol 153:183196[CrossRef][Medline]
-
Fabre S, Darne C, Veyssière G, Jean Cl 1996 Protein
kinase C pathway potentiates androgen-mediated gene expression of the
mouse vas deferens specific aldose reductase like protein (MVDP). Mol
Cell Endocrinol 124:7986[CrossRef][Medline]
-
Lekstrom-Himes J, Xanthopoulos KG 1998 Biological role of the
CCAAT/enhancer binding protein family of transcription factors. J
Biol Chem 273:2854528548[Abstract/Free Full Text]
-
Sterneck E, Tessarollo L, Johnson PF 1997 An essential role
for C/EBPß in female reproduction. Genes Dev 11:21532162[Abstract/Free Full Text]
-
Descombes P, Schibler U 1991 A liver-enriched transcriptional
activator protein, LAP, and a transcriptional inhibitory protein, LIP,
are translated from the same mRNA. Cell 67:569579[Medline]
-
Nalbant D, Williams SL, Stocco DM, Khan SA, 1998 Luteinizing
hormone-dependent gene regulation in Leydig cells may be mediated by
CCAAT/enhancer binding protein-ß. Endocrinology 139:272279[Abstract/Free Full Text]
-
Reinhart AJ, Williams SL, Clark BJ, Stocco DM 1999 SF-1
(Steroidogenic factor 1) and C/EBPß (CCAAT/enhancer binding
protein-ß) cooperate to regulate the murine StAR (steroidogenic acute
regulatory) promoter. Mol Endocrinol 13:729741[Abstract/Free Full Text]
-
Christenson LK, Johnson PF, Mc Allister JM, Strauss JF 1999 CCAAT/enhancer binding proteins regulate expression of the human
steroidogenic acute regulatory protein (StAR) gene. J Biol Chem 274:2659126598[Abstract/Free Full Text]
-
Metz R, Ziff E 1991 cAMP stimulates the C/EBP-related
transcription factor rNFIL-6 to translocate to the nucleus and induce
c-fos transcription. Genes Dev 5:17541766[Abstract]
-
Gronning LM, Dahle MK, Tasken KA, Enerbäck S, Hedin L,
Tasken K, Knutsen HK 1999 Isoform-specific regulation of the
CCAAT/enhancer-binding protein family of transcription factors by
3',5'-cyclic adenosine monophosphate in Sertoli cells. Endocrinology 140:835843[Abstract/Free Full Text]
-
Chinery R, Brockman JA, Dransfield DT, Coffey RJ 1997 Antioxidant-induced nuclear translocation of CCAAT/enhancer-binding
protein ß. A critical role for protein kinase A-mediated
phosphorylation of Ser 299. J Biol Chem 272:3035630361[Abstract/Free Full Text]
-
Lee YH, Masahiko Y, Liu SY, Matsunaga E, Johnson PF, Gonzales
FJ 1994 A novel cis-acting element controlling the rat CYP2D5 gene and
requiring cooperativity between C/EBPß and an Sp1 factor. Mol
Cell Biol 14:13831394[Abstract]
-
Lee YH, Williams SC, Baer M, Sterneck E, Gonzalez FJ, Johnson
PF 1997 The ability of C/EBP beta but not C/EBP alpha to synergize with
an Sp1 protein is specified by the leucine zipper and activation
domain. Mol Cell Biol 17:20382047[Abstract]
-
Goodfellow PN, Camerino G 1999 DAX-1, an "antitestis"
gene. Cell Mol Life Sci 55:857863[CrossRef][Medline]
-
Crawford PA, Dorn C, Sadovsky Y, Milbrandt J 1998 Nuclear
receptor DAX-1 recruits nuclear receptor corepressor N-CoR to
steroidogenic factor 1. Mol Cell Biol 18:29492956[Abstract/Free Full Text]
-
Apte AN, Siebert PD 1993 Anchor-ligated cDNA libraries: a
technique for generating a cDNA library for immediate cloning of the 5'
ends of mRNAs. Biotechniques 15:890893[Medline]
-
Taragnat C, Berger M, Jean Cl 1990 Tissue and species
specificity of mouse ductus deferens protein. J Androl 11:279286[Abstract/Free Full Text]
-
Yasumura Y, Buonassisi V, Sato G 1966 Clonal analysis of
differentiated function in animal cell cultures. I. Possible correlated
maintenance of differentiated function and the diploid karyotype.
Cancer Res 26:529535[Medline]
-
Zanaria E, Muscatelli F, Bardoni B, Strom TM, Guioli S, Guo W,
Lalli E, Moser C, Walker AP, McCabe ERB, Meitinger T, Monaco AP,
Sassone-Corsi P, Camerio G 1994 An unusual member of the nuclear
hormone receptor superfamily responsible for X-linked adrenal
hypoplasia congenita. Nature 372:635641[CrossRef][Medline]
-
Chen C, Okayama H 1987 High-efficiency transformation of
mammalian cells by plasmid DNA. Mol Cell Biol 7:27452752[Medline]
-
Sambrook J, Fritsch EF, Maniatis T 1989 Molecular Cloning: A
Laboratory Manual, ed 2 . Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY, vol 1:6871
-
Darne C, Morel L, Claessens F, Manin M, Fabre S,
Veyssière G, Rombauts W, Jean Cl 1997 Ubiquitous transcription
factors NF1 and Sp1 are involved in the androgen activation of the
mouse vas deferens protein promoter. Mol Cell Endocrinol 132:1323[CrossRef][Medline]