The Mouse Adrenocorticotropin Receptor Gene: Cloning and Characterization of Its Promoter and Evidence for a Role for the Orphan Nuclear Receptor Steroidogenic Factor 1
Florence M. Cammas1,
Gill D. Pullinger,
Stewart Barker and
Adrian J. L. Clark
Molecular Endocrinology Section, Department of Chemical
Endocrinology, St Bartholomews and the Royal London School of
Medicine, and Dentistry, London EC1A 7BE, United Kingdom
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ABSTRACT
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To elucidate the mechanism underlying the
tissue-specific expression of the ACTH receptor/MC2 receptor (ACTH-R)
in the adrenal cortex, we have cloned the mouse ACTH-R promoter. The
analysis of the cDNA 5'-end showed an untranslated region of 321 bp,
and the ACTH-R gene was demonstrated to be composed of two exons of 113
and 112 bp lying upstream of the single coding exon. S1 nuclease
protection assay showed two major transcription start sites separated
by 4 bp; 1.8 kb of the 5'-flanking region inserted in a luciferase
reporter vector had cell-specific promoter activity because it was
functional only in mouse adrenocortical Y1 cells but not in mouse
Leydig TM3 cells or fibroblast L cells. There was no dramatic change in
the promoter activity in Y1 cells for all the deletions tested up to
-113 bp upstream of the transcription start site. In contrast,
expression in TM3 cells was switched on from deletion -908 bp, while
remaining undetectable in L cells. Site-directed mutagenesis of a
steroidogenic factor 1 (SF1)-like site at position -25 bp resulted in
a significant reduction in luciferase expression by the promoter in Y1
cells. Gel shift analysis of this site indicated specific binding of a
protein in extracts of Y1 and TM3 cells. Moreover, expression of SF1 in
L cells induced promoter activity of the construct p(908). In
conclusion, cell-specific expression of the mouse ACTH-R appears to be
controlled by at least two factors. One of them, most probably SF1, is
responsible for steroidogenic cell-specific expression. The other as
yet unknown factor binding between position -1236 bp and -908 bp acts
as a strong inhibitory factor in nonadrenal steroidogenic cells,
resulting in the adrenal-specific expression of ACTH-R.
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INTRODUCTION
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The production of glucocorticoids by the adrenal cortex is
predominantly regulated by pituitary ACTH (for review see Refs. 1 and
2). This hormone acts via a seven-transmembrane domain receptor
belonging to the G protein-coupled receptor superfamily (ACTH-R), also
known as the MC2 receptor (3). The identity of this receptor as the
ACTH-R was supported by association of mutations in this receptor and
adrenal unresponsiveness to ACTH in familial glucocorticoid deficiency
(for review see 4 . Although it is generally accepted that the
ACTH-R is expressed in the adrenal cortex, a number of groups have
suggested that the ACTH-R is also expressed in peripheral blood
mononuclear leukocytes (5, 6), adipocytes (7, 8, 9), and skin (10) on the
basis of binding studies and/or mRNA detection. There is very little
evidence of ACTH-R expression in other tissues, implying that an
effective mechanism exists to restrict expression of this gene.
The most probable explanation for this phenomenon could be the
existence of tissue-specific regulatory elements in the ACTH-R
promoter. Of particular relevance to ACTH-R expression are several
factors that have been shown to be involved in steroidogenic or
adrenal-specific expression. The orphan nuclear receptor steroidogenic
factor 1 (SF1) is probably the best characterized (11, 12) and has been
shown to be involved in steroidogenic cell-specific expression of
several genes (13, 14, 15, 16, 17, 18, 19) and in the development of the adrenal and gonad
(20, 21, 22). An adrenal-specific nuclear protein (ASP) has also been shown
to be involved in the human CYP21B gene expression (23, 24).
Disruption of the orphan nuclear receptor DAX-1 has been implicated in
X-linked adrenal hypoplasia congenita, suggesting a specific role in
adrenal and gonadal development (25). Furthermore, some ubiquitous
factors such as NGFI-B (26) and AP1 (27) have been shown to be involved
in the regulation of adrenal-specific gene expression.
We have previously reported the cloning and expression of the mouse
ACTH-R (28, 29). In this report we describe the characterization of the
mouse ACTH-R gene structure and the cloning of its promoter. This
promoter has been functionally analyzed in the well characterized Y1
mouse adrenocortical cell line, which has been shown to express the
ACTH-R and respond to ACTH (30, 31). Our data indicate the existence of
two important regions that regulate tissue-specific expression of the
gene. One of these regions suggests a role for SF1, or a closely
related protein, in the determination of steroidogenic cell specificity
of ACTH-R expression.
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RESULTS
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Characterization of the Mouse ACTH-R cDNA 5'-End
To identify the 5'-end of the ACTH-R cDNA and therefore to
characterize the structure of the gene, we used the rapid amplification
of cDNA ends (RACE) protocol. PCR with a sense primer complementary to
the ligated adapter at the cDNA 5'-end and an antisense primer
complementary to the ACTH-R coding sequence produced a single 1.1-kb
fragment (Fig. 1A
). This was subcloned, sequenced, and
shown to comprise the ACTH-R coding sequence and a 5'-untranslated
sequence of 241 bp (Fig. 1B
). Comparison between this cDNA fragment and
the ACTH-R genomic sequence indicated that the mouse ACTH-R gene
consisted of at least two exons with an intron/exon junction 96 bp
upstream of the translation start codon (Fig. 1B
). The exonic nature of
the sequence was confirmed using S1 nuclease protection assay (32) with
mouse adrenal mRNA and a genomic probe spanning the putative
intron/exon junction in which only the predicted exonic sequence was
protected against S1 nuclease digestion. Another probe derived from the
5'-RACE cDNA product was fully protected against digestion (data not
shown).

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Figure 1. Cloning of the Mouse ACTH-R cDNA 5'-Ends
A, Agarose gel analysis of the RACE product performed on 1 mg mouse
adrenal mRNA with an antisense primer in the mouse ACTH-R coding
sequence. Different dilutions of the final ligation mixture: 0, 1, 1:5,
1:10, 1:25, 1:50 were used as template for PCR. A predominant 1.1-kb
band in all samples is indicated. B, 241-bp sequence of the
5'-untranslated region of the mouse ACTH-R gene. The
underlined sequence represents the 96-bp untranslated
part of the coding exon, and the translation initiation codon is
indicated in bold.
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Tissue-Specific Distribution of ACTH-R mRNA
Taking advantage of the multiexonic nature of the ACTH-R gene, we
used RT-PCR with intron-skipping primers to assess ACTH-R expression in
mouse adrenal gland, liver, lung, spleen, abdominal adipose tissue,
whole brain, heart, testis, and kidney. The PCR products were analyzed
by Southern blotting with the RACE cDNA fragment described above as the
probe to obtain the maximum sensitivity. RNA quality and the efficiency
of the reverse transcription step were controlled by carrying out
parallel RT-PCR with ß-actin primers. As shown in Fig. 2
, the ACTH-R mRNA was relatively abundantly expressed
in adrenal tissue. However, low expression of this gene was also
apparent in abdominal adipose tissue. Expression could not be detected
in spleen, testis, liver, lung, heart, whole brain, or kidney.

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Figure 2. Tissue Distribution of the Mouse ACTH-R mRNA
A, Ethidium bromide-stained agarose gel analysis of RT-PCR performed
with ß-actin exon-skipping primers and 10 µg total RNA obtained
from the mouse tissues indicated, or a water control (-) to check the
RNA quality and the reverse transcription efficiency. B, RT-PCR was
performed with the mouse ACTH-R intron-skipping primers and the same
RNA samples, and the product was analyzed by Southern blotting after
agarose gel electrophoresis using the mouse ACTH-R 5'-RACE cDNA
fragment as the probe.
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Cloning of the Mouse ACTH-R Gene Promoter
A probe corresponding to the first 144 bp of the 5'-end of the
RACE product was used to screen 106 pfu of a mouse genomic
library under high stringency conditions. Five positive clones were
isolated that were found to have distinct restriction patterns after
digestion with Bgl2, NcoI, and PvuII and Southern
blot analysis using the same probe as for the screening (Fig. 3
). The restriction fragments marked with
arrows in the figure were subcloned and sequenced. In this
way the 5'-untranslated region was shown to consist of two separate
exons. Exon 1 overlapped the 5'-end of the RACE clone by 33 bp. Exon 2
consisted of 112 bp, which together with the 96 bp at the 5'-end of
exon 3, encode the 5'-untranslated region of this gene. The genomic
clones 4 and 5 contained exon 2 while clones 1, 2, and 3 contained exon
1. The NcoI-positive fragment from clone 1, containing exon
1, was subcloned, sequenced, and shown to contain 1.8 kb upstream
sequence expected to be the mouse ACTH-R promoter. This fragment was
therefore analyzed further.

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Figure 3. Isolation of Genomic Clones Containing the Mouse
ACTH-R 5'-End Exons
A, Agarose gel showing restriction enzyme analysis (using
NcoI, PvuII, or Bgl2) of
the five positive genomic clones (15) obtained after screening with
the most 5' 144 bp of the RACE cDNA product. M represents DNA size
markers of 21.6 kb, 9.4 kb, 6.6 kb, and 4.4 kb. B, Southern blot
analysis of the five positive clones using the 5'-RACE cDNA product as
the probe. The positive bands of 4 kb (clone 5), 2.2 kb (clone 1), and
1 kb (clone 4) marked with arrows were subcloned and
sequenced.
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The precise size of exon 1 was assessed by S1 nuclease protection
assay with mouse adrenal mRNA and a probe spanning the expected
transcription start site. It was demonstrated that the actual size of
the first exon is 113 bp (Fig. 4
). There was a second
protected fragment corresponding to an exon of 109 bp implying that
there were two major start sites for transcription separated by 4 bp.
The intensity of these two protected fragments was similar, indicating
that both transcription start sites are used with equivalent
efficiency.

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Figure 4. ACTH-R Gene Structure and 5'-End Determination
A, Schematic representation of the ACTH-R gene, the genomic clones from
which each exon was identified, the mature mRNA product, and the site
and extent of the 5'-RACE cDNA clone. B, Determination of the
transcription start site by S1 nuclease protection assay. A probe
overlapping the putative transcription start site was obtained as
described in Materials and Methods. The protected
fragments of 104 bp and 100 bp marked with the bottom
arrows indicate two transcription start sites corresponding to
first exons of 113 bp and 109 bp, respectively. The undigested probe of
300 bp is marked with the top arrow. The (-) lane
indicates the use of 10 µg of transfer RNA as template.
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Sequencing of the 5'-untranslated region (Fig. 5
) did
not reveal any of the classic promoter features such as a TATA box,
CAAT box, or GC-rich region. However, there is an initiator-like
sequence overlapping the transcription start site (dashed
underlining in Fig. 5
), and there are a number of consensus
response elements as depicted in Fig. 5
including an OctB site, an Sp1
site, a glucocorticoid response element, an AP2 site, an AP1 site, and
two SF1-like sites at positions -897 bp and -25 bp, respectively.

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Figure 5. Sequence of Mouse ACTH-R Gene
Lowercase letters represent intronic sequences. Only the
extremities of the introns are shown separated by dots.
The light-face uppercase sequence is the promoter, and
the bold uppercase sequences are the three exons.
Putative regulatory sequences are underlined with a
solid line, and the putative initiator sequence are
underlined with a dashed line. The
arrows show the ends of the different deletion
constructs.
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Functional Characterization of the Mouse ACTH-R Promoter
To functionally characterize the promoter, the region between +104
bp and -1808 bp was subcloned into the pGL3-basic luciferase reporter
vector. This construct was then transfected by electroporation into
mouse Y1 adrenocortical cells and two other mouse cell lines derived
from tissues that do not express ACTH-R: TM3 cells, derived from Leydig
cells, and L cells, a fibroblast cell line. With this full length
promoter construct, luciferase activity was detectable in Y1 cells, but
no activity was apparent in TM3 cells. The level of this activity was
equivalent to 510% of the activity of Rous sarcoma virus
(RSV).luciferase in the same cells. L cells produced some activity,
although this was extremely low and statistically indistinguishable
from zero (Fig. 6A
), showing the expected cell-specific
activity of this promoter.

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Figure 6. Functional Characterization of the Mouse ACTH-R
Promoter
A, Luciferase activity of the mouse ACTH-R promoter deletions in Y1
cells, TM3 cells, and L cells. The deletion constructs transfected into
the three cell lines are described on the left with the
numbering refering to the transcription start site of the longest
transcript. The results are expressed as a mean (± SEM)
arbitrary light units as described earlier for the same number of
independent experiments. B, Luciferase activity obtained in three
independent experiments (mean ± SEM) using the
full-length promoter, the minimal promoter fragment p(113), and the SF1
site mutant of p(113). Results were compared using Students
t test.
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To identify the important regulatory regions for expression of the
ACTH-R gene, a series of deletion constructs of the promoter were
generated as indicated in Fig. 5
. Figure 6A
summarizes the effect of
these deletions on luciferase reporter activity in the three cell
lines. There was no significant difference between the full length
[p(1808)] construct and all the other deletions up to -113 bp in Y1
cells. No significant luciferase activity was detected for any of the
constructs in L cells. In TM3 cells no luciferase activity was detected
after transfection with the full length construct or the two constructs
p(1590) and p(1236). In contrast, when the deletion constructs p(908),
p(717), p(641), and p(113) were transfected, they all showed luciferase
activity in TM3 cells at a significantly lower level than in Y1 cells
with the exception of p(113), which appeared to induce transcription at
an equivalent level to the minimal promoter in Y1 cells.
Identification of a Binding Site for SF1
Of the transcription factor consensus binding motifs
identified, the SF1-like sequence at position -25 bp was of particular
interest in connection with ACTH-R tissue-specific expression because
it is the only recognized site in the construct p(113) that showed
promoter activity specifically in Y1 cells and TM3 cells. We therefore
used site-directed mutagenesis to alter the -25 SF1 consensus sequence
in the p(113) construct at two critical nucleotides SF1 binding site
(AAGGTT
ATTGTT). The results of an
independent series of experiments performed in Y1 cells are shown in
Fig. 6B
and indicate a highly significant loss of promoter activity in
p(113)mutant to 38% of that of p(113) itself (P <
0.01).
Further evidence for the involvement of SF1 at the -25-bp site was
sought using gel shift analysis. A 46-bp fragment surrounding the
SF1-like binding site at position -25 bp was used as the labeled probe
(see Fig. 7A
). A DNA/protein complex (CI) was found to
be formed using cellular extracts from Y1 cells. A similar complex was
apparent when a cellular extract from TM3 cells was used. However, no
such complex was observed with extracts from L cells (Fig. 7B
),
although a certain amount of very slowly migrating nonspecific material
was present. In Y1 cell extracts a second complex (CII) was also
formed. Specificity of the interaction was confirmed by displacement of
the DNA/protein complexes by a 200-fold molar excess of an unlabeled
consensus SF1- binding site derived from the rat CYP17 gene
(19), providing further evidence that the protein involved in these
complexes was SF1 (Fig. 7B
).

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Figure 7. Electrophoretic Mobility Gel Shift Assay with the
SF1-Like Site at Position -25 bp
A, Sequence of the probe (derived from the ACTH-R sequence) and the
consensus SF1 competitor (derived from the cytochrome P450
17 -hydroxylase gene) used for gel retardation assays: the
SF1-binding sites are in bold. B, Thirty micrograms of
Y1, TM3, and L cell cellular extracts were incubated with the
end-labeled SF1-like probe without or with 200 x or 500 x
molar excess of unlabeled consensus SF1 competitor. Complexes I and II
are indicated on the side of the figure as CI and CII.
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SF1 Induces ACTH-R Promoter Activity in Fibroblasts
To confirm the functional relevance of SF1 in the regulation of
ACTH-R expression, an expression vector containing the SF1-coding
sequence was cotransfected with the promoter construct p(908) into L
cells. P(908) was chosen to avoid any influence of the putative
repressor identified in TM3 cells in case it was also present in L
cells. These cells do not express SF1 endogenously, as shown above
(Fig. 7B
), and do not have any ACTH-R promoter activity (Fig. 6A
).
Heterologous expression of SF1 in these cells resulted in significant
induction of ACTH-R promoter activity (P < 0.05)
whereas no effect was observed using a promoterless construct (Fig. 8
), lending support to the hypothesis that SF1 is an
essential factor for ACTH-R expression.

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Figure 8. Expression of SF1 in Fibroblast Cells Induces
ACTH-R Promoter Activity
Five micrograms of the promoterless luciferase reporter plasmid pGL3 or
the ACTH-R promoter-luciferase construct p(908) were cotransfected with
or without the SF1 expression vector under the control of a
cytomegalovirus promoter, and luciferase activity was measured after
72 h as described earlier. The results are the mean
(±SEM) of three independent experiments. The
asterisk represents a significant increase
(P < 0.05) using one-tailed Students test.
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DISCUSSION
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We have demonstrated that the mouse ACTH-R gene consists
of at least three exons of lengths 113 bp, 112 bp and >1000 bp. The
two first exons contain the 5'-untranslated region while the third exon
contains 96 bp of 5'-untranslated region and the entire coding
sequence. The functional relevance of the two untranslated exons is not
known. Interestingly, a single exon upstream of the coding exon has
been identified in the human ACTH-R gene (33), and this is highly
homologous to the mouse exon 1, suggesting that any physiological role
of the mouse exon 2 is not required in the human gene.
The knowledge of this structure has enabled us to use RT-PCR as a
highly sensitive method of mRNA detection with intron-skipping primers
followed by Southern blotting. As shown in Fig. 3
, ACTH-R mRNA is
relatively abundantly expressed in the adrenal gland, as is well
recognized. However, there is also low expression in abdominal adipose
tissues. This site has already been shown to express ACTH-R by binding
studies, and recently this has been confirmed by Northern blot analysis
(9) and has been proposed to mediate the lipolytic action of ACTH.
Thus, our data confirm the tissue-specific nature of ACTH-R gene
expression, and it is the molecular mechanism of this regulation,
especially at the level of the adrenal cortex, that has been the focus
of our further studies. It is of interest that an abstract recently
presented by another group (34) indicated the existence of a fourth
alternatively spliced exon lying between exons 1 and 2. This is
probably an uncommon splice variant that we have been unable to
identify but may be represented by the weak higher molecular weight
band in the adrenal RT-PCR shown in Fig. 2B
.
Sequence analysis of the 5'-untranslated flanking region of the ACTH-R
gene indicated that the region immediately upstream of exon 1 does not
show any typical characteristics of promoter regions such as a TATA
box, CAAT box, or GC-rich region. However, there is an initiator-like
site overlapping the transcription start site. The initiator element
(Inr), first identified for viral transcriptional regulation (for
review see 35 , has been shown to be involved in transcription
initiation of a growing number of TATA box-containing and TATA-less
promoters. In addition to this Inr-like sequence, there are several
putative sites for transcription factors as shown in Fig. 5
. Of
particular interest is the presence of a partial consensus sequence at
position -25 bp for binding of the mouse transcription factor SF1,
which has been shown to be involved in tissue-specific expression of
several steroidogenic enzymes (11, 12, 13, 20) as well as
Mullerian-inhibiting substance (15), and the glycoprotein hormone
-subunit (16). SF1 has also been shown to be involved in the cAMP
responsiveness of the cytochrome P450c17 enzyme expression (19).
Therefore, this sequence motif represents a strong candidate for
regulation of ACTH-R gene expression. Recently, Naville et
al. (33) have reported the promoter sequence for the human ACTH-R
gene revealing that it also contains an Inr element and an SF1-like
site at position -35.
The presence of a glucocorticoid response element in the ACTH-R
promoter is also important because glucocorticoids have been shown to
induce a significant increase of ACTH-R expression in ovine
adrenocortical cells (36). The molecular mechanism of this effect is
not yet known but may occur at the transcriptional level. It is
noteworthy that the ACTH-R promoter does not contain any consensus cAMP
response element (CRE) despite the fact that ACTH is recognized to
stimulate ACTH-R expression via cAMP (31, 37). However, this lack of a
consensus CRE is a common feature for the cAMP-regulated steroidogenic
enzymes and may suggest the existence of a yet uncharacterized
cAMP-dependent regulatory factor. In the case of CYP17, this
function has been shown to be fulfilled by SF1 (19), but that does not
seem to be the case for the other steroidogenic enzymes. The functional
importance of both of these regulatory elements will be addressed in
our future work. In contrast, the human ACTH-R contains seven CRE-like
elements in the proximal 700 bp of this promoter, although these have
not yet been shown to be functional (33).
The promoter activity of the ACTH-R 5'-untranslated region was assessed
after insertion upstream of the luciferase coding sequence in the pGL3
basic vector. It has been shown that this DNA fragment is necessary and
sufficient to drive expression of a heterologous gene in a
cell-specific manner, since the signal was detectable in Y1 cells but
not in TM3 or L cells. Deletion studies allowed us to define two
important regions for cell-specific expression. Promoter activity
became detectable in TM3 cells when sequences upstream of position
-908 bp were removed, despite remaining undetectable in L cells (Fig. 6A
). These results suggest that the ACTH-R promoter is potentially
active in steroidogenic tissues other than the adrenal, but is totally
inhibited by the binding of a factor between position -1236 bp and
-908 bp that would act as a repressor of expression in nonadrenal
steroidogenic cells such as the Leydig cell. The existence of such
transcriptional repressors or silencers responsible for tissue-specific
expression of genes has been reported for several genes. However, our
results are the first evidence for a repressor restricting gene
expression in the adrenal gland.
In Y1 and TM3 cells, the minimal promoter extended to -113 bp, which
includes the putative SF1-like binding site described earlier. This
suggests that SF1 might be a strong candidate for determining
steroidogenic-specific expression of the ACTH-R as it does for several
other genes. Our data lend support to the notion that SF1 or an
SF1-like factor is involved in ACTH-R cell-specific regulation since 1)
Mutation of the SF1 site leads to significant loss of promoter
activity; 2) it binds specifically to this site as shown by gel shift
analysis; and 3) expression of SF1 in a cell line that normally does
not have any ACTH-R promoter activity leads to an induction of its
activity. However, the level of promoter activity obtained in these
conditions is low, indicating that SF1 may be essential to induce the
promoter in an "active conformation" whereas other factors are
probably necessary for full promoter activity. In keeping with this, it
is noteworthy that mutation of the SF1 site leads only to a significant
reduction of activity and not complete loss of promoter
function. The role of the second SF1-binding site at position -897 bp
has not been extensively studied. However, it seems that SF1 binding at
this site of the promoter may be more critical in TM3 cells than in Y1
cells because its absence in both constructs p(717) and p(641) resulted
in a significant decrease of promoter activity in TM3 cells whereas it
was almost without effect in Y1 cells. The significance of these
results will need further investigation.
In conclusion, the tissue-specific expression of the ACTH-R gene
appears to be mainly under the control of two factors, with SF1, or an
SF1-like factor, responsible for steroidogenic cell specificity, and
another as yet unknown factor binding between position -1236 bp and
-908 bp silencing the promoter in nonadrenal steroidogenic cells. It
might be postulated that binding of such a repressor protein upstream
of the SF1-binding site prevents the formation of an SF1-dependent
transcriptionally active complex.
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MATERIALS AND METHODS
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Chemicals
All chemicals were obtained from Sigma Chemical Co. (Poole,
Dorset, UK); FCS, horse serum, Hams F10 and F12 media were obtained
from GIBCO BRL (Renfrewshire, UK). All radionucleotides were obtained
from Amersham International plc (Amersham, Bucks. UK).
Rapid Amplification of cDNA Ends
The Marathon cDNA Amplification kit (Clontech, Cambridge, UK)
was used following the manufacturers protocol. The mouse ACTH-R gene
antisense primer was 5'-GACCTGGAAGAGAGACATGTAG3-'. RNA was extracted
from mouse adrenal tissues using RNazolB (Biogenesis, Poole, Dorset,
UK), and mRNA was purified using a Sephadex oligo-dT column (Pharmacia,
Herts, UK). One microgram of this mRNA was used per reaction. The PCR
product was subcloned directly in the pGEM-T vector (Promega,
Southampton, UK) and sequenced using sequenase 2.0 (Amersham).
RT-PCR
Ten micrograms of total RNA from the following frozen mouse
tissues were analyzed: whole brain, kidney, lung, spleen, abdominal
adipose tissue, lung, adrenal, heart, and testis. One hundred nanograms
of oligo-dT were used to prime the Superscriptase (GIBCO BRL) for
1 h at 42 C in a 20-µl reaction mixture. One microliter was used
as template for PCR with: mouse ACTH-R exon 1 sense primer:
(5'-CTTGCCGAGAAAGATCCT-3') and exon 3 antisense primer:
(5'-AGCGATGTGAAGGTGAGC-3'), or the ß-actin sense primer:
(5'-GTAACCAACTGGGACGAT-3') and antisense primer
(5'-GACCACACCCCACTATGG-3').
Reaction conditions were 30 sec at 94 C and 30 sec at 56 C for ACTH-R
or 58 C for ß-actin and 1 min at 72 C for 30 cycles.
In the case of the ACTH-R, PCR products were run on a 1% agarose gel,
transferred to nitrocellulose membrane, and hybridized in 50%
formamide, 5 x NaCl-sodium citrate, 0.05 M sodium
phosphate buffer (pH 6.8), 0.3 mg/ml herring sperm DNA, 5 x
Denharts solution, 0.05% SDS, with approximately 106cpm
-32P-labeled ACTH-R probe at 42 C for 16 h. The
filters were washed under stringent conditions (1 x NaCl-sodium
citrate, 0.1% SDS) at 65 C and exposed to Kodak XAR-5 film (Eastman
Kodak, Rochester, NY) at -80 C for 3 h or overnight for the long
exposure.
S1 Nuclease Protection Assay
The reaction conditions were as described previously (32). PCR
was performed with a biotinylated sense primer MP237S:
5'-TACAACCTTCACAATCTGC-3' designed in the putative promoter region and
a phosphorylated antisense primer at the 3'-end of exon 1:
5'-CTGAAGTAGGATCTTTCTCG-3'. Fifty micrograms of total mouse adrenal RNA
were hybridized with this probe overnight at 43 C.
Plasmid Constructs
For the full-length construct p(1808), PCR was performed using
Vent polymerase (New England Biolabs., Beverly, MA) with a sense
primer: 5'-GACCATTAACTTTGAATTAGG-3' at the 5'-end of the
NcoI fragment and an antisense primer complementary to exon
1: 5'-CTGAAGTAGGATCTTTCTCG-3' using the genomic positive clone 1 as
template. This was subcloned into the SmaI site of the
pGL3-basic luciferase reporter vector (Promega, Madison, WI), and PCR
artefacts were excluded by sequencing. For the promoter deletions,
p(1808) was digested with specific enzymes as follows: -1590 bp,
KpnI; -1236 bp, NheI; after separation of the
insert and the backbone, the plasmid was religated on itself. For the
following deletions, p(1808) was cut with KpnI and another
specific enzyme, the ends were filled using Klenow fragment, and the
plasmid was religated: -908 bp, Bsg1; -717 bp,
TthIII.1; -641 bp, ApaI; -113 bp,
BstX1. The constructs or at least their 5'- extremities were
checked by sequencing.
Cell Culture and Transfection Experiments
Mouse Y1 adrenal cells were maintained in 50% DMEM and 50%
Hams F10 supplemented with 12.5% horse serum and 2.5% FCS. Mouse
TM3 cells (kindly provided by Dr Ray Iles) were grown in 50% DMEM and
50% Hams F12 supplemented with 5% horse serum, 2.5% FCS, and 20
mM HEPES (pH 7.4). Mouse L cells (kindly given by Prof. Tom
MacDonald) were grown in RPMI containing 5% FCS. Approximately
106 cells were washed and resuspended in HEPES- buffered
saline and 500 µl were used per electroporation at 300 V and 1000
milliFarads in 2-mm wide cuvettes with 5 µg of the ACTH-R gene
construction and 10 µg of RSV-CAT plasmid. Cells were left 1 min at
room temperature and then resuspended in their respective growth medium
in 60-mm plates for 72 h. Five micrograms of SF1 expression vector
were cotransfected with 5 µg of the p(908) construct in L cells in
the conditions described above. Because the transfection efficiency was
constant, RSV-CAT was not used systematically in all experiments.
Luciferase Activity and Chloramphenicol Acetyltransferase (CAT)
Protein Measurement
The luciferase assay kit (Promega) was used for this purpose.
The cells were washed twice at room temperature with PBS, covered with
400 µl lysis reporter buffer for 20 min at room temperature, scraped,
recovered in Eppendorf tubes, and snap frozen at -70 C. The cell
debris was pelleted and 100 µl of the supernatant were used for
luciferase activity measurement with 400 µl lysis reporter buffer and
100 µl luciferase substrate solution. One hundred fifty microliters
of the supernatant were used for measurement of CAT protein level
according to the CAT ELISA protocol (Boehringer Mannheim, Lewes, UK).
Mutagenesis of the -25 bp site in the p(113) plasmid was accomplished
using the QuikChange technique (Stratagene Ltd., Cambridge, UK) with
Pfu polymerase. The identity of mutated clones was
determined by DNA sequencing.
Electrophoretic Mobility Shift Assay
To prepare the probe an antisense ACTH-R primer,
5'-GACAGCTACTTGTTATCC-3', was labeled with [
32P]ATP
using T4 polynucleotide kinase (New England Biolabs.) and used for PCR
with an unlabeled ACTH-R sense primer, 5'-CCGTTTATTTCTAGTGAC-3'
producing a 46-bp fragment surrounding the SF1-like site, which was
purified with microcentrifuge 5000 tubes (Sigma). Binding reactions of
10 µl were carried out in buffer containing 15 mM HEPES,
75 mM KCl, 9 mM MgCl2, 0.075
mM EDTA, 12.5% glycerol, 1 mM dithiothreitol,
0.1 mg/ml sperm DNA, approximately 5 x 103cpm of
radiolabeled probe, and 30 mg of cellular extracts proteins for the
three cell lines prepared as described previously (36). The reactions
were incubated for 15 min at 30 C. Competitor was added 15 min before
the probe and incubated at 30 C. Free and bound DNA were separated on a
4% nondenaturing polyacrylamide gel in 0.5 x Tris-borate-EDTA at
4 C. The gels were dried and analyzed by autoradiography.
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to Dr. Keith Parker (Duke University,
Durham, NC) for providing us with the SF1 expression vector and to Dr.
Joy Hinson (Queen Mary & Westfield College, London) for provision of
mouse adrenal tissue. This work has benefited from helpful discussion
with Dr. Paul Lavender (Wellcome CRC, Cambridge, UK) and Avtar Roopra
(University College, London). F.M.C. was supported by a Studentship
from the Joint Research Board of St Bartholomews Hospital, and G.D.P.
was supported by the Medical Research Council.
 |
FOOTNOTES
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Address requests for reprints to: Adrian J. L. Clark, Department of Chemical Endocrinology, St. Bartholomews & Royal London School of Medicine and Dentistry, West Smithfield, London ECIA 7BE, UK.
1 Present address: IGBMC, BP163, C.U. de Strasbourg, 67404 Illkirch,
France. 
Received for publication August 7, 1996.
Revision received February 17, 1997.
Accepted for publication February 28, 1997.
 |
REFERENCES
|
---|
-
Koritz SB, Bhargava G, Schwartz 1977 ACTH action on
adrenal steroidogenesis. Ann NY Acad Sci 297:329335[Medline]
-
Simpson ER, Waterman MR 1983 Regulation by ACTH of steroid
hormone biosynthesis in the adrenal cortex. Can J Biochem Cell
Biol 61:692707[Medline]
-
Mountjoy KG, Robbins LS, Mortrud MT, Cone RD 1992 The cloning
of a family of genes that encode melanocortin receptors. Science 257:12481251[Medline]
-
Clark AJL, Weber A 1994 Molecular insights into inherited
ACTH resistance syndromes. Trends Endocrinol Metab 5:209214
-
Johnson HM, Torres BA, Smith EM, Blalock JE 1982 Regulation
of the in vitro antibody response by neuroendocrine
hormones. Proc Natl Acad Sci USA 79:41714174[Abstract]
-
Clarke BL, Bost KL 1989 Differential expression of functional
adrenocorticotropic hormone by subpopulations of lymphocytes. J
Immunol 143:464469[Abstract/Free Full Text]
-
Grunfeld C, Hagman J, Sabin EA, Buckley DI, Jones DS,
Ramachandran J 1985 Characterization of adrenocorticotropin that
appears when 3T3L1 cells differentiate into adipocytes. Endocrinology 116:113117[Abstract]
-
Izawa T, Mochizuki T, Komabayashi T, Suda K, Tsuboi 1994 Increase in cytosolic free Ca2+ in corticotropin-stimulated white
adipocytes. Am J Physiol 266:E418E426
-
Boston BA, Cone RD 1996 Characterization of melanocortin
receptor subtype expression in murine adipose tissues and in 3T3L1
cell line. Endocrinology 137:20432050[Abstract]
-
Slominski A, Ermak G, Mihm M 1996 ACTH receptor, CYP11A1,
CYP17 and CYP21A2 genes are expressed in skin. J Clin Endocrinol
Metab 81:27462749[Abstract]
-
Lala DS, Rice DA, Parker KL 1992 Steroidogenic factor 1, a key
regulator of steroidogenic enzyme expression, is the mouse homolog of
fushi tarazu-factor 1. Mol Endocrinol 6:12491258[Abstract]
-
Morohashi KI, Honda SI, Inomata Y, Handa H, Omura T 1992 A
common trans-acting factor, Ad4-binding protein, to the promoters of
steroidogenic P450. J Biol Chem 267:1791317919[Abstract/Free Full Text]
-
Lynch JP, Lala DS, Peluso JJ, Luo W, Parker KL, White BA 1993 Steroidogenic factor 1, an orphan nuclear receptor, regulates the
expression of the rat aromatase gene in gonadal tissues. Mol Endocrinol 7:776786[Abstract]
-
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]
-
Shen WH, Moore CCD, Ikeda Y, Parker KL, Ingraham HA 1994 Nuclear receptor steroidogenic factor 1 regulates the Mullerian
inhibiting substance gene: a link to the sex determination cascade.
Cell 77:651661[Medline]
-
Barnhart KM, Mellon PL 1994 The orphan nuclear receptor,
steroidogenic factor-1, regulates the glycoprotein hormone a-subunit
gene in the pituitary gonadotropes. Mol Endocrinol 8:878885[Abstract]
-
Burris TP, Guo W, Le T, McCabe ERB 1995 Identification of a
putative steroidogenic factor-1 response element in the DAX-1 promoter.
Biochem Biophys Res Commun 214:576581[CrossRef][Medline]
-
Bakke M, Lund J 1995 Transcriptional regulation of the bovine
CYP17 gene: two nuclear orphan receptors determine activity of
cAMP-responsive sequence 2. Endocr Res 21:509516[Medline]
-
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. Mol Endocrinol 10:147158[Abstract]
-
Ikeda Y, Shen WH, Ingraham HA, Parker KL 1994 Developmental
expression of mouse steroidogenic factor-1, an essential regulator of
the steroid hydoxylases. Mol Endocrinol 8:654662[Abstract]
-
Luo X, Ikeda Y, Parker KL 1994 A cell-specific nuclear
receptor is essential for adrenal and gonadal development and sexual
differentiation. Cell 77:481490[Medline]
-
Sadovsky Y, Crawford PA, Woodson KG, Polish JA, Clements MA,
Tourtelotte LM, Simburger K, Milbrandt J 1995 Mice deficient in the
orphan receptor steroidogenic factor 1 lack adrenal glands and gonads
but express P450 side-chain-cleavage enzyme in the placenta and have
normal embryonic serum levels of corticosteroids. Proc Natl Acad Sci
USA 92:1093910943[Abstract]
-
Kagawa N, Waterman MR 1991 Evidence that an adrenal-specific
nuclear protein regulates the cAMP responsiveness of the human CYP21B
(P450C21) gene. J Biol Chem 266:1119912204[Abstract/Free Full Text]
-
Kagawa N, Waterman MR 1992 Purification and characterization
of a transcription factor which appears to regulate cAMP responsiveness
of the human CYP21B gene. J Biol Chem 267:2521325219[Abstract/Free Full Text]
-
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, Camerino G 1994 An unusual member of the nuclear
hormone receptor superfamilly responsible for X-linked adrenal
hypoplasia congenita. Nature 372:635641[CrossRef][Medline]
-
Wilson TE, Mouw AR, Weaver CA, Milbrandt J, Parker KL 1993 The
orphan nuclear receptor NGFI-B regulates expression of the gene
encoding steroid 21-hydroxylase. Mol Cell Biol 13:861868[Abstract]
-
Mukai K, Mitani F, Shimada H, Ishimura Y 1995 Involvement of
an AP1 complex in the zone-specific expression of the CYP11B1 gene in
the rat adrenal cortex. Mol Cell Biol 15:60036012[Abstract]
-
Cammas FM, Kapas S, Barker S, Clark AJL 1995 Cloning,
characterization and expression of a functional mouse ACTH receptor.
Biochem Biophys Res Commun 212:912918[CrossRef][Medline]
-
Kapas S, Cammas FM, Clark AJL 1996 Characterization of the
action of ACTH peptides on the cloned mouse ACTH receptor expressed in
stably transfected Hela cell line. Endocrinology 137:32913294[Abstract]
-
Schimmer BP, Schulz P 1985 The roles of c AMP and c
AMP-dependent protein kinase in forskolins actions on Y 1
adrenocortical tumor cells. Endocr Res 11:199209[Medline]
-
Mountjoy KG, Bird IM, Rainey WE, Cone RD 1994 ACTH induces
up-regulation of ACTH receptor mRNA in mouse and human adrenocortical
cell lines. Mol Cell Endocrinol 99:R17R20
-
Cammas FM, Clark AJL 1996 S1 nuclease protection assay using
streptavidin dynabeads purified single stranded DNA. Anal Biochem 236:182184[CrossRef][Medline]
-
Naville D, Jaillard C, Barjhoux L, Durand P, Begeot M 1997 Genomic structure and promoter characterization of the human ACTH
receptor gene. Biochem Biophys Res Commun 230:712[CrossRef][Medline]
-
Shimizu C, Kubo M, Saeki T, Matsumura T, Kakinuma M, Koike T,
The genomic organization of the mouse ACTH receptor. Program of the
10th International Congress of Endocrinology, San Francisco, CA, 1996 (Abstract P3188)
-
Weis L, Reinberg D 1992 Transcription by RNA polymerase II:
initiator-directed formation of transcription-competent complexes.
FASEB J 6:33003309[Abstract/Free Full Text]
-
Darbeira H, Durand P 1990 Mechanism of glucocorticoid
enhancement of the responsiveness of ovine adrenocortical cells to
adrenocorticotropin. Biochem Biophys Res Commun 166:11831191[Medline]
-
Lebrethon MC, Naville D, Begeot M, Saez JM 1994 Regulation of
corticotropin receptor number and messenger RNA in cultured human
adrenocortical cells by corticotropin and angiotensin II. J Clin
Invest 93:18281833[Medline]