Impaired Steroidogenic Factor 1 (NR5A1) Activity in Mutant Y1 Mouse Adrenocortical Tumor Cells
Claudia Frigeri,
Jennivine Tsao,
Waldemar Czerwinski and
Bernard P. Schimmer
Banting and Best Department of Medical Research and Department of
Pharmacology University of Toronto Toronto, Ontario, Canada M5G
1L6
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ABSTRACT
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Mutants isolated from the Y1 mouse adrenocortical
tumor cell line (clones 10r-9 and 10r-6) are resistant to ACTH because
they fail to express the melanocortin-2 receptor (MC2R). In this study,
we show that a luciferase reporter plasmid driven by 1800 bp of the
proximal promoter region of the MC2R was expressed poorly in the mutant
cells compared with parent Y1 cells. The differential expression of the
MC2R in parent and mutant cells resulted from impaired activity of the
orphan nuclear receptor NR5A1 (SF1) on the promoter as determined by
5'-deletion analysis. Furthermore, the activity of an SF1 expression
plasmid on an SF1-dependent reporter plasmid was compromised in mutant
clones. The site-specific DNA binding properties of SF1 from parent and
mutant cells did not differ as determined in electrophoretic mobility
shift assays, and the addition of the activation domain of VP16 to the
amino terminus of SF1 restored the transcriptional activity of the
protein. In addition, the levels of SF1 and other cofactors including
WT1, CBP/p300, and steroid receptor coactivator 1 did not differ
appreciably between parent and mutant cells. Taken together, these
results suggest that ACTH resistance in the mutant clones resulted from
a defect that affected the activation properties of SF1 rather than its
DNA binding activity. Consistent with the observed impairment in SF1
function, other SF1-dependent genes, including Cyp11b1 and
steroidogenic acute regulatory protein (StAR), were poorly expressed
and global steroidogenesis, as evidenced by the metabolism of
22(R)-hydroxycholesterol to steroid products, was impaired.
Interestingly, MC2R, Cyp11a, Cyp11b1, and StAR
transcripts were not affected to the same degree, suggesting that each
of these genes may have a different absolute requirement for SF1. These
mutants thus provide an experimental paradigm to identify factors that
influence SF1 function and to evaluate the relative importance of SF1
in the expression of genes essential for adrenal steroidogenesis.
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INTRODUCTION
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The melanocortin-2 receptor (ACTH receptor, MC2R) is a member of
the melanocortin receptor subfamily of G protein-coupled receptors (1)
that is selectively expressed at high levels in cells of the adrenal
cortex. The cell-selective expression of the mouse MC2R appears to be
under the control of the proximal promoter region of the gene (bases
-1808 to +104, relative to the transcription start site), as
determined in transfection experiments using established cell lines.
Distinct promoter regions that contribute to the adrenal cell-selective
expression of the MC2R have been identified previously (2) and include:
a regulatory element at -25 under control of the nuclear receptor
NR5A1 (SF1),1 a negative
control region from -1236 to -908, and an initiator-like core
promoter that overlaps the transcription start site. The SF1 element
enhances MC2R expression in those cell types where SF1 is expressed
(3), whereas the negative control region seems to prevent MC2R
expression in SF1-containing cells other than the adrenal cortex (2).
SF1 elements also contribute to the cell-selective expression of the
human MC2R gene (4).
To gain a better understanding of the factors that govern MC2R gene
expression, we have investigated several mutants isolated from the Y1
mouse adrenocortical tumor cell line that fail to express the MC2R gene
and consequently are ACTH resistant (5, 6, 7). In the present study, we
demonstrate that the mutation leading to impaired MC2R expression also
affects the transcriptional activity of the mouse MC2R promoter at the
proximal SF1 site. Moreover, we demonstrate that the activation
function of SF1, rather than its DNA binding activity, is compromised.
The mutation leading to ACTH resistance adversely affected the
expression of Cyp11b1 and the steroidogenic acute regulatory
protein (StAR) and affected Cyp11a expression to a lesser
degree. These mutants thus provide an experimental paradigm to identify
factors that influence SF1 function and to evaluate the relative
importance of SF1 in the expression of the MC2R and other genes
essential for adrenal steroidogenesis.
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RESULTS
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MC2R Promoter Activity in Parent and Mutant Clones
The 5'-flanking region and part of the 5'-untranslated sequence of
the MC2R gene (bases -1808 to +104 relative to the transcription start
site, Ref. 2) were placed ahead of a luciferase reporter gene and
examined for activity in parental Y1 cells and in two MC2R-deficient
mutants, clones 10r-9 and 10r-6. In parental Y1 cells, the MC2R
promoter enhanced luciferase expression approximately 50-fold over
levels obtained with the promoterless reporter plasmid (8). In
contrast, the ACTH promoter was only 5% and 7% as effective in the
MC2R-deficient mutants 10r-9 and 10r-6, respectively (Fig. 1
; P < 0.05). The
activity of the MC2R promoter also was markedly reduced in two other
MC2R-deficient mutants (clones Y6 and OS3, data not shown). These
observations indicate that the mutations affecting MC2R
expression also affected the activity of the proximal promoter region
of the MC2R gene.

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Figure 1. MC2R Promoter Activity in Parent Y1 and Mutant
10r-6 and 10r-9 Cells
Parent and mutant cells were transfected with luciferase reporter
plasmids (1 µg) containing various lengths of the mouse MC2R
promoter as indicated. Cells were harvested 48 h after
transfection, and cell extracts were assayed for luciferase activity.
Results were normalized for variations in transfection efficiency as
described in Materials and Methods and expressed as
percentages of the mean activity achieved with the -1808 to +104 MC2R
promoter vector in Y1 cells ± SEM. Results obtained
with the -1808 to +104 construct were from seven independent
experiments; results obtained with the 5'-deletion constructs were
averaged from six independent experiments.
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To determine whether the differential expression of the MC2R in parent
and mutant clones resulted from altered activity at a specific site
within the promoter, we evaluated the effects of a limited set of
5'-deletions on reporter gene activity (Fig. 1
). Deletion of the
promoter to -106 resulted in a segment that retained the ability to
support luciferase expression much more effectively in parent Y1 cells
than in the MC2R-deficient mutants (P < 0.05) despite
the removal of the negative regulatory region. This promoter construct
had approximately 20% of the activity seen with the -1808 to +104
construct in Y1 cells, indicating the presence of another positive
regulatory element in the upstream region of the promoter. Truncating
the promoter to -13, so as to remove the SF1 site, resulted in a
promoter with a lower activity that was equivalent in parent and mutant
clones. These results raised the possibility that the compromised
activity of the ACTH promoter in the mutants resulted from impaired
activity at the SF1 site.
Transcriptional Activity of SF1 in Parent and Mutant Cells
To further evaluate SF1 function in parent and mutant
clones, the expression of a reporter plasmid (p-25 Luc) containing two
copies of the SF1 site from the MC2R promoter upstream of an SV40 core
promoter and luciferase gene was examined in transient transfection
assays. Adding SF1 sites before the core promoter did not enhance the
basal expression of the reporter gene; however, it rendered the
construct responsive to SF1. As shown in Fig. 2a
, luciferase expression was increased
approximately 10-fold (P < 0.05) when p-25 Luc was
transfected into Y1 cells together with an SF1 expression plasmid; SF1
had no effect on luciferase expression from the reporter plasmid
lacking SF1 sites (data not shown). In contrast, the SF1 expression
plasmid did not enhance p-25 Luc expression in the ACTH-resistant
mutants, 10r-9 and 10r-6 (Fig. 2a
), supporting the hypothesis that SF1
function was compromised in the mutant clones.

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Figure 2. Transcriptional Activities of SF1 and VP16/SF1 at
the Mouse MC2R SF1 Site
Parent and mutant cells were transfected with 1 µg of p-25 Luc in the
absence (-) or presence (+) of 1 µg of expression plasmids encoding
SF1 (panel a) or VP16/SF1 (panel b). Cells were harvested 48 h
after transfection and assayed for luciferase activity as described in
the legend to Fig. 1 . Results were compiled from six experiments in
panel a and from five experiments in panel b. In each case results were
normalized to luciferase expression from a reporter plasmid under
control of the SV40 promoter to correct for variations in transfection
efficiencies among clones. Data are presented as means ±
SEM.
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SF1 function also was evaluated in parent and mutant clones using p-65
Luc, a reporter plasmid driven by multiple copies of the SF1 site at
-65 in the Cyp21 gene (9). This SF1 site is distinct from
most other SF1 sites in that it also binds the orphan nuclear receptor,
NR4A1 (Nur77, Ref. 10) (see footnote 1), and thus permits a comparison
of SF1 and Nur77 actions, each regulated by the cytomegalovirus (CMV)
promoter and each acting on the same response element. In parent Y1
cells, the SF1 expression plasmid increased luciferase expression
56 ± 16-fold from p-65 Luc (n = 11). The SF1 expression
plasmid was significantly less effective in the 10r-9 and 10r-6 mutants
(P < 0.05), and displayed only 13% ± 2% (n =
10) and 34% ± 3% (n = 6) of the respective activities seen in
Y1 cells (Fig. 3a
). Optimally effective
concentrations of the Nur77 expression plasmid increased luciferase
expression from p-65 Luc approximately 275-fold in parent Y1 cells; in
the mutants 10r-9 and 10r-6, the Nur77 vector was 1.5- to 2.0-fold more
effective than in the parent cell line (Fig. 3b
). These latter
observations indicated that the mutations affecting MC2R expression
affected SF1 function without affecting the activity of Nur77 or the
transcriptional cofactors shared by these nuclear receptors.

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Figure 3. Transcriptional Activities of SF1, VP16/SF1, and
Nur77 at the Cyp21 SF1 Site
Parent and mutant cells were transfected with 2.5 µg of p-65 Luc in
the absence (-) or presence (+) of 0.5 µg (panel a) or 2.0 µg
(panels b and c) of expression plasmids encoding SF1, VP16/SF1, or
Nur77 as indicated. The activity of Nur77 on p-65 Luc (panel b) is to
be compared with the activity of p-65 Luc alone as shown in panel a.
Cells were harvested 48 h after transfection and assayed for
luciferase activity as described in the legend to Fig. 1 . Results were
expressed as means ± SEM after normalization to
luciferase expression from a reporter plasmid under control of the RSV
promoter to correct for variations in transfection efficiencies among
clones. The number of determinations (n) were: panel
a,Y1 (n = 11), 10r-9 (n = 10), 10r-6 (n = 6); panel b,
Y1 (n = 3), 10r-9 and 10r-6 (n = 4); panel c,Y1 + SF1 (n
= 5), all others (n = 4).
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Effects of VP16/SF1 in Parent and Mutant Cells
A VP16/SF1 chimeric protein also was evaluated for activity in
parent and mutant cells. VP16/SF1 is a construct that retains the DNA
binding specificity of SF1 and incorporates an activation domain from
VP16 at the amino terminus (11). A VP16/SF1 expression plasmid was
transfected into parent and mutant cells and evaluated for its ability
to regulate gene expression from reporter plasmids under control of the
SF1 sites of the MC2R promoter (Fig. 2b
) or theCyp21
promoter (Fig. 3c
). In parent Y1 cells, VP16/SF1 significantly enhanced
expression from both reporter plasmids (P < 0.05) but
did not affect the reporter plasmid lacking SF1 sites (data not shown).
In the mutant clones, VP16/SF1 also significantly enhanced luciferase
expression from both SF1-dependent reporter plasmids (P
< 0.05) and gave levels of activity that approached those seen in
parental Y1 cells (Figs. 2b
and 3c
). Thus, the addition of a strong
activation domain to SF1 was sufficient to overcome the differential
activity of SF1 on these reporter plasmids in parent and mutant clones.
These results suggested that the mutations in 10r-9 and 10r-6 did not
affect SF1 binding to DNA; rather, the mutation seemed to impair the
activation function of SF1.
Levels of SF1, WT1, CBP (CREB-Binding Protein), p300, and
Steroid Receptor Coactivator 1 in Parent and Mutant Clones
Despite the apparent loss of SF1 function in the mutants
(e.g., Fig. 1
), nuclear extracts from parent and mutant
clones were not deficient in SF1 protein as determined on Western blots
with an SF1-specific antiserum (Fig. 4
).
Inasmuch as SF1 interacts with a number of regulatory proteins,
including WT1 (12), CBP/p300 (13), steroid receptor coactivator 1 (14, 15), Dax-1 (11, 16), and SOX9 (17), we also screened parent and mutant
clones for the presence of several of these interacting proteins. As
determined by Western blot analysis, mutant cells also were not
deficient in WT1, p300, CBP, or steroid receptor coactivator 1 (Fig. 4
).

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Figure 4. Levels of SF1, WT1, and SF1 Coactivators in Parent
and Mutant Cells
Nuclear extracts (50 µg) from parent Y1 cells and from mutant 10r-6
and 10r-9 cells were electrophoresed on SDS-polyacrylamide gels (41 )
and electroblotted onto nitrocellulose membranes. The membranes were
immunoblotted with a goat antibody against mouse steroid receptor
coactivator 1 (SRC1), with rabbit antibodies against mouse WT1, CBP,
and p300 (purchased from Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA) or with a rabbit antibody against bovine SF1/AD4BP
generously provided by K. Morohashi (National Institute of Basic
Biology, Okazaki, Japan). Antigen-antibody interactions were detected
using horseradish peroxidase-labeled secondary antibodies and the ECL
detection system (Amersham Pharmacia Biotech).
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DNA Binding Activity of SF1 in Parent and Mutant Clones
To assess the DNA binding activity of SF-1 more directly,
electrophoretic mobility shift assays were conducted on nuclear
extracts from parent and mutant clones (Fig. 5
). Nuclear extracts from parent and
mutant cells exhibited three shifted complexes when incubated
with a labeled SF1 probe and then electrophoresed (lanes 1 and 7); one
of the complexes represented SF1-specific DNA binding activity as
indicated. The formation of this labeled complex was inhibited by an
excess of oligonucleotides corresponding to the SF1 sites from the MC2R
promoter (lanes 3 and 9) or the Cyp21 promoter (lanes 5 and
11). In contrast, the labeled complex persisted in the presence of an
excess of an unrelated oligonucleotide (lanes 2 and 8) or in the
presence of an excess of SF1-related oligonucleotides containing base
substitutions that disrupted the SF1 binding sites (lanes 4, 6, 10, and
12; Refs. 2, 18). As shown in Fig. 6
, similar concentrations of an unlabeled SF1 oligonucleotide were
required to displace a labeled probe from SF1 complexes in parent and
mutant extracts, indicating that the SF1 from parent and mutant cells
had similar apparent affinities for their binding sites. Taken
together, these results suggested that the failure of mutant clones
to express the MC2R did not result from impaired binding of SF1 to DNA.
Rather an inability of SF1 to activate transcription seemed to underlie
the loss of MC2R expression.

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Figure 5. DNA Binding Activity of SF1 in Parent and Mutant
Cells
Nuclear extracts from parent Y1 cells (lanes 16) and mutant 10r-6
cells (lanes 712) were incubated with a 32P-labeled
SF1 probe (3.6 nM) in the absence (lanes 1 and 7) or
presence of an 80-fold molar excess of unlabeled oligonucleotides as
competitors. DNA binding activity was evaluated in an electrophoretic
mobility shift assay. The competitors used corresponded to: the SF1
site from the MC2R promoter (lanes 3 and 9); the SF1 site from the
Cyp21 promoter (lanes 5 and 11), mutated forms of the
SF1 oligonucleotides (lanes 4 and 10 and lanes 6 and 12); an unrelated
oligonucleotide corresponding to the sequence at -170 in the
Cyp21 promoter (lanes 2 and 8). Shifted complexes were
not observed in the absence of nuclear extract (not shown). The
arrows identify the positions of the SF1 complex and
free probe.
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Figure 6. Apparent Affinities of SF1 from Parent and Mutant
Clones for Their DNA Sites
The DNA binding properties of SF1 from parent Y1 (filled
circles) and mutant 10r-9 (open circles) and
10r-6 (filled squares) cells were assessed by
electrophoretic mobility shift assays as described in Fig. 5 . Each
sample contained an 80-fold molar excess of the unrelated
double-stranded oligonucleotide (-170) to minimize nonspecific
interactions and varying concentrations of the MC2R SF1 oligonucleotide
as indicated. The amount of labeled SF1 complex formed was determined
by phosphorimage analysis using the Molecular Imager phosphorimager
from Bio-Rad Laboratories, Inc. (Mississauga,
Ontario, Canada) and normalized to the amount of shifted complex
obtained with each extract in the absence of competitor. The results
presented are averaged from two separate experiments.
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Steroidogenic Activity in Parent and Mutant Cells
Since SF1 has also been implicated in the expression of other
genes required for adrenal steroidogenesis (3), we examined the
consequences of impaired SF1 function in our mutants on other
components of the steroidogenic pathway. As shown in Fig. 7
, the 10r-9 mutant had a markedly
diminished capacity for steroidogenesis compared with parental Y1
cells. In Y1 cells, ACTH and other agents that raise intracellular
levels of cAMP stimulated steroidogenesis approximately 7.5-fold. Not
surprisingly, the MC2R-deficient 10r-9 mutant failed to respond to ACTH
with increased steroidogenesis. The mutant, however, also did not
respond to 8-Br-cAMP or forskolin and failed to convert
22(R)-hydroxycholesterol to steroid products, indicating a significant
loss of steroidogenic capacity. The steroidogenic capacity of mutant
10r-6 cells was similarly affected (data not shown). As determined by
Northern blot hybridization analysis, the loss of steroidogenic
capacity was accompanied by diminished levels of transcripts encoding
Cyp11b1 and StAR (Fig. 8
)two
key proteins required for corticosteroid biosynthesis.
Cyp11b1 transcripts in the mutants were reduced
approximately 95% compared with parent Y1 cells and seemed to be more
greatly affected than StAR transcripts, which were reduced
approximately 75%. The levels of Cyp11a did not show as
consistent a pattern of change as did the other transcripts. In the
10r-9 mutant, Cyp11a transcript levels were 45% of those
seen in parental Y1 cells, whereas in the 10r-6 mutant, the levels of
Cyp11a transcripts were reduced to approximately 20% (Fig. 8
).

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Figure 7. Steroid Production from Endogenous Cholesterol and
from Exogenous 22(R)-Hydroxycholesterol in Parent and Mutant Cells
Cultures of Y1 and 10r-9 cells were incubated for 6 h in the
absence (basal) or presence of ACTH (5 mU/ml), forskolin (10
µM), 8-Br-cAMP (3 mM), or
22(R)-hydroxycholesterol (20 µM) as indicated. The medium
from each sample was collected, extracted, and quantitated for
fluorescent steroids as detailed in Materials and
Methods. Results presented are means ± SEM
(n = 3).
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Figure 8. Cyp11a, Cyp11b1, and
StAR Transcripts in Parent and Mutant Cells
Total RNA (25 µg) from parent Y1 and mutant 10r-9 and 10r-6 clones
was blotted onto nylon membranes and probed for Cyp11a1,
Cyp11b1, StAR, and transketolase (Tkt) transcripts as
described in Materials and Methods. A representative
Northern blot is shown on the left. Results from three
separate experiments, presented as percentages of the signals obtained
in parent Y1 cells ± SEM, are shown on the
right. Hybridization signals were quantitated by
phosphorimage analysis using the Molecular Imager phosphorimager from
Bio-Rad Laboratories, Inc. and normalized to the levels of
transketolase transcripts to account for variations in mRNA recovery,
loading, and transfer among samples.
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DISCUSSION
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As demonstrated previously (7), ACTH resistance in mutant Y1
adrenocortical tumor cells results from a defect that markedly
diminishes the transcription of the MC2R gene. As shown here, the loss
of MC2R expression was correlated with impaired activity of SF1 at the
MC2R promoter. In support of this hypothesis, we found that the
proximal promoter region of the MC2R functioned poorly in the mutant
clones (Fig. 1
and Ref. 8); the differences in MC2R promoter activity
in parent and mutant cells were abolished upon deletion of the SF1 site
(Fig. 1
); an SF1 expression plasmid regulated SF1-dependent reporter
genes less effectively in mutant cells than in parent Y1 cells (Figs. 2b
and 3a
). The latter finding further indicated that the loss of
SF1-dependent activity resulted from a secondary event affecting SF1
function rather than from a mutation in SF1 per se.
Nevertheless, the loss of SF1 function seemed relatively specific since
the activity of another orphan nuclear receptor, Nur77, was not
compromised in the mutant clones (Fig. 3b
).
The loss of SF1 function seemed to result from defects in SF1-dependent
activation of gene expression rather than from DNA binding defects.
These conclusions were supported by the findings that the DNA binding
activities of SF1 from parent and mutant clones were indistinguishable
(Figs. 5
and 6
). Furthermore, the addition of the VP16 activation
domain to SF1 restored its ability to activate SF1-dependent reporter
genes in mutant clones (Figs. 2
and 3
). Possible mechanisms underlying
this loss of SF1 function include changes in posttranslational
modification of the protein or changes in a coregulatory protein or
activating ligand. Inspection of the SF1 sequence reveals consensus
sites for phosphorylation by cAMP-dependent protein kinase, protein
kinase C, mitogen-activated protein kinase, and casein-kinase II; only
the mitogen-activated protein kinase site has been shown to be
important for SF1 function, at least in the context of placental JEG
cells (19). We have not yet examined SF1 phosphorylation in Y1 and
mutant 10r-6 and 10r-9 cells; however, the mutant cells appear to have
mitogen-activated protein kinase and cAMP-dependent protein kinase
signaling pathways intact (B.P. Schimmer and T. Le, unpublished
observations, and Ref. 6). Oxysterols can activate SF1 (20), provided
that assays of SF1 function are carried out in appropriate cell types
(e.g. CV1 cells) maintained in charcoal-absorbed delipidated
serum. Since the assays of SF1 function described here were carried out
in the presence of complete serum, it is unlikely that changes in
oxysterol production contributed to the mutant phenotype, although
contributions of other endogenous ligands cannot be excluded. Finally,
we have shown that the levels of several regulatory proteins known to
interact with SF1, including WT1 (12), CBP and p300 (13), and steroid
receptor coactivator 1 (14, 15), are not altered in the mutants (Fig. 4
). Nevertheless, it remains to be determined whether alterations that
affect the activities of these or other transcription regulators
contribute to the mutant phenotype in the Y1 mutants.
SF1 is also thought to be important for the cell-selective expression
of other genes involved in adrenal steroid hormone biosynthesis,
including the cytochrome P450 steroid hydroxylases, 3ß-hydroxysteroid
dehydrogenase and StAR (reviewed in Ref. 3). The roles for SF1 in the
expression of these genes, however, have been inferred from analyses of
proximal promoter sequences of these genes in transient transfection
assays. The finding that the 10r-9 and 10r-6 mutants had impaired SF1
activity raised the possibility that the mutants might provide a
paradigm to examine the roles of SF1 in expression of other genes
involved in steroidogenesis. As shown here, the mutants were unable to
form fluorogenic steroid products from 22(R)-hydroxycholesterol (Fig. 7
). 22(R)-Hydroxycholesterol is the first intermediate in the
conversion of cholesterol to pregnenolone (21) and is efficiently
metabolized to steroid products bypassing the hormonally controlled
rate-limiting steps of cholesterol mobilization and metabolism (22). In
parent Y1 cells, these steroid products are 20
-progesterone and
11ß,20
-dihydroxyprogesterone (23, 24). In association with this
defect, transcripts encoding StAR, Cyp11a, and
Cyp11b1 were also reduced in the mutant clones. Thus, the
mutant clones appeared to have major defects in steroid biosynthesis,
supporting the hypothesis that SF1 is important for the expression of
genes required for steroidogenesis. Furthermore, these observations
raise the possibility that transcripts encoding other steroidogenic
enzymes also might be affected. Intriguingly, MC2R, Cyp11a,
Cyp11b1, and StAR transcripts were not affected to the same
degree. Whereas MC2R and Cyp11b1 transcripts were reduced to
nearly undetectable levels, StAR transcripts were reduced by 75%, and
Cyp11a transcripts exhibited greater variation (55% and
80% reduction) between the two clones (e.g., Fig. 8
and
Ref. 8). These observations suggest that each of these genes may have
different absolute requirements for SF1. Interestingly,
Cyp11a also is not dependent on SF1 for expression in
placenta (25), spleen (26), primitive gut (27), and brain (28).
The mutant clones described here were isolated by virtue of their
resistance to the growth-inhibiting effects of the diterpene, forskolin
(6), and arose from single mutational events as determined by
fluctuation analysis (29). As we have shown previously, the
growth-inhibiting effects of forskolin in Y1 cells are cAMP mediated,
and resistance results from a failure of forskolin to maximally
activate adenylyl cyclase rather than from a defect in cAMP action (6).
While the underlying mutations have yet to be identified, much of the
phenotype, including loss of MC2R expression, seems to have resulted
from impaired G protein ß/
activity. In functional reconstitution
assays, G protein ß/
activity from mutant cells was impaired (30),
and genes encoding wildtype ß/
, when transfected into the
mutants, restored MC2R expression (8). These results thus raised the
possibility that MC2R gene expression was impaired in the mutants
because of an underlying defect in a Gß/
-dependent signaling
process. The ß/
-dimer has been shown to regulate a number of
signaling pathways that potentially impact on gene expression. These
include both positive and negative effects on the cAMP-signaling
cascade, activation of Ca2+ signaling pathways,
and protein kinase C and activation of the mitogen-activated protein
kinase cascade (31). The results presented here, demonstrating impaired
SF1 function at the MC2R promoter, identify SF1 as a possible link
between Gß/
and MC2R expression and offer a starting point to
trace the signaling events that mediate ß/
effects on gene
expression.
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MATERIALS AND METHODS
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Oligonucleotides and Plasmids
The complementary oligonucleotides corresponding to SF1 sequence
at -25 in the mouse MC2R promoter with BamHI and
BglII overhanging ends were 5'-GATCCTTTAG
TCAAGGTTAGGATAAA-3' and 5'-GATCTTTATCCTAACCTTGAGTAAAG-3'. The mutated
MC2R SF1 oligonucleotides were blunt-end complementary sequences with
the core sequence AAGGTT mutated to ATTGTT as
described (2). The complementary oligonucleotides corresponding to the
SF1 site at -140 in the Cyp21 promoter
(5'-ACAGATTCTCCAAGGCTGAT-3'), the mutated SF1 site at -140 with the
core sequence CAAGGCTG mutated to CTCGGCTG (18),
and the SF1-independent site at -170 in Cyp21
(5'-TGATGGATGGTCCCATCTTTGATCC-3'; Ref. 32) were also described
previously.
The plasmids used in this study included: a luciferase reporter plasmid
under control of the mouse MC2R promoter (bases -1808 to +104; Ref.
2); a luciferase reporter plasmid under control of six copies of the
element at -65 in the mouse Cyp21 gene (p65 Luc; Ref. 9); a
luciferase reporter plasmid containing SV40 promoter and enhancer
sequences (pGL3-control; obtained from Promega Corp.,
Madison, WI); a luciferase reporter plasmid under control of the Rous
sarcoma virus promoter (from -142 to +36; obtained from Dr. H.
Elsholtz, University of Toronto, Toronto, Ontario, Canada). Luciferase
reporter plasmids containing 5'-deletions of the mouse MC2R promoter
were prepared using the PCR with appropriate primers. The luciferase
reporter plasmid, p-25 Luc, was prepared by ligating two copies of a
double-stranded oligonucleotide corresponding the SF1 site at -25 in
the mouse MC2R promoter into the BamHI site of the pGL3
promoter vector (a luciferase reporter plasmid containing the SV40 core
promoter; Promega Corp.). The SF- 1 expression plasmid was
prepared by subcloning mouse SF-1 cDNA (9) into the EcoRI
site of pcDNA3.1/His (Invitrogen, Carlsbad, CA). This
construct resulted in a cDNA encoding SF1 with
(His)6 - and epitope-tags at the amino terminus
under control of the CMV promoter. Nur77 (a 2.2-kb cDNA that includes
the entire coding region) was isolated by K. L. Parker (University
of Texas, Southwestern Medical Center, Dallas, TX) from a mouse adrenal
cDNA library and subcloned into the CMV promoter-based expression
plasmid pCMV5 (33).
Cells, Cell Culture, and Gene Transfer
The Y1 mouse adrenocortical tumor cell line used in this study
is a stable subclone (34) of the population originally isolated by
Yasumura et al. (35). ACTH receptor-deficient Y1 mutants,
including clones 10r-9 and 10r-6, were isolated on the basis of their
resistance to the growth-inhibiting effects of forskolin as described
(6). Cells were cultured as monolayers at 36.5 C under a humidified
atmosphere of 95% air-5% CO2 in nutrient
mixture F10 supplemented with 15% heat-inactivated horse serum, 2.5%
heat-inactivated FBS, and antibiotics. Tissue culture reagents were
obtained from Canadian Life Technologies, Inc. (Burlington, Ontario,
Canada).
For gene transfer experiments, cells (2 x
105) were replicate plated in 60-mm tissue
culture dishes and grown for 3 days. Cells were transferred to
-minimal essential medium supplemented with serum and antibiotics
and transfected with super-coiled plasmid DNA using a calcium phosphate
precipitation technique described previously (36, 37). Cells were
incubated with the DNA/calcium phosphate precipitates for 24 h,
rinsed to remove the DNA, and incubated in fresh medium for an
additional 48 h before being harvested for analysis. To correct
for variations in transfection efficiencies, separate plates of cells
were transfected with a luciferase expression vector under control of
either the SV40 promoter/enhancer or the Rous sarcoma virus
(RSV) promoter as indicated.
Electrophoretic Mobility Shift Assays
The DNA binding activity of nuclear extracts from parent and
mutant cells was assayed in electrophoretic mobility shift assays
essentially as described previously (38). Each reaction contained 5
µg nuclear protein, 3.6 nM labeled probe (50,000 cpm), 2
µg double-stranded poly(dI·dC) in a total of 20 µl of binding
buffer (10 mM Tris·HCl, pH 7.5, 50 mM NaCl, 1
mM EDTA, 1 mM dithiothreitol, and 5%
glycerol). DNA-protein complexes were resolved by electrophoresis on
4% nondenaturing polyacrylamide gels and visualized by fluorography.
The probe was prepared by annealing complementary oligonucleotides
corresponding to the SF1 sequence at -25 in the MC2R promoter and
filling in the 3'-BamHI/BglII ends using the
Klenow fragment of DNA polymerase-I (Amersham Pharmacia Biotech, Baie dUrfe, Quebec, Canada) in the presence of
[
-32P]dGTP (3,000 Ci/mmol; Mandel Scientific
Co., Guelph, Ontario, Canada).
Luciferase Activity
Cell extracts were prepared by scraping cells into a lysis
buffer containing 50 mM
Tris.2-[N-morpholino]ethanesulfonic
acid, pH 7.8, 1% Triton X-100, 4 mM EDTA, and 1
mM dithiothreitol; extracts were clarified by
centrifugation at 4 C. Cell supernatants (4 to 400 µg protein) were
assayed for luciferase activity in a reaction cocktail (250 µl)
containing 200 µl of cell extract, 45 mM
Tris.2-[N-morpholino]ethanesulfonic
acid, pH 7.8, 9 mM magnesium acetate, 3
mM disodium ATP, and 0.12
mM luciferin as described (39). Luminescence was
measured using a Berthold Lumat LB Luminometer under conditions where
signals were proportional to the amount of supernatant protein
added.
Steroid Production
Cells (5 x 104) were plated in 60-mm
tissue culture dishes, grown for 57 days, and then incubated for
6 h in 2 ml
-minimal essential medium containing serum and
antibiotics. At the end of the incubation, steroids were extracted from
the medium using methylene chloride and quantitated by fluorescence in
65% sulfuric acid-35% ethanol using corticosterone as a standard
(23).
 |
ACKNOWLEDGMENTS
|
---|
We thank Adrian Clark for the MC2R promoter-luciferase
construct, Keith L. Parker for cDNAs encoding SF1 and Nur77, Jeff
Milbrandt for the expression vector encoding the VP16/SF1 fusion
protein and for the p-65 Luc reporter plasmid, Ken Morohashi for the
SF1/AD4BP antiserum, and Harry Elsholtz for the RSV-Luc expression
plasmid. Fanny Lee and Brian Wong provided expert technical
assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Bernard P. Schimmer, Ph.D., Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, Ontario M5G 1L6 Canada.
This work was supported by research grants from the Medical Research
Council of Canada. C.F. was supported by a studentship from the Medical
Research Council of Canada and a University of Toronto Open Studentship
Award.
1 Members of the nuclear receptor family are
identified by official names as proposed under a unified nomenclature
system (40 ). 
Received for publication July 21, 1999.
Revision received January 5, 2000.
Accepted for publication January 10, 2000.
 |
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