The Promoter of Murine Follicle-Stimulating Hormone Receptor: Functional Characterization and Regulation by Transcription Factor Steroidogenic Factor 1
Jérôme Levallet,
Pasi Koskimies,
Nafis Rahman and
Ilpo Huhtaniemi
Department of Physiology University of Turku 20520 Turku,
Finland
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ABSTRACT
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The promoter of the FSH receptor (R) gene has been
cloned from several species. Although some of its regulatory elements
have been identified, its function still remains poorly characterized.
Using transient transfections of luciferase reporter constructs, driven
by various fragments of the murine (m) FSHR promoter, we identified a
cell-specific promoter region. This domain is located in the distal
part of the mFSHR promoter, -1,110 to -1,548 bp upstream of the
translation initiation site, and it contains two steroidogenic factor 1
(SF-1) like binding sites (SLBS). The cellular levels of SF-1 mRNA and
protein closely correlated in various steroidogenic cell lines with
activity of the transfected mFSHR promoter/luciferase reporter
construct carrying the distal activator domain. A dose-dependent
increase in FSHR promoter activity was shown in nonsteroidogenic HEK
293 cells transiently transfected with SF-1 cDNA. SF-1 was found to
bind to a nonconsensus 5'-CAAGGACT-3' SLBS-3 motif in the distal part
of the promoter; formation of the SF-1/SLBS-3 complex could be
reversed by addition of SF-1 antibody. Mutation in the SLBS-3 domain
abolished the SF-1/SLBS-3 complex in gel-shift assays and led to a
significant loss of SF-1-mediated mFSHR promoter activity. The second
SLBS appeared to have minor role in SF-1-regulated mFSHR expression. In
conclusion, we have identified a regulatory domain in the mFSHR
promoter participating in the cell-specific regulation of FSHR
expression. We demonstrated for the first time that the mFSHR promoter
possesses functional SF-1 binding sites and thus belongs to the group
of SF-1-regulated genes. These findings provide further evidence for
the key role of SF-1 in the regulation of genes involved in gonadal
differentiation and endocrine functions.
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INTRODUCTION
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Gonadal function is critically dependent on regulatory impulses
from the pituitary-gonadal axis. The two gonadotropins, LH and FSH,
play a key role in this regulation. FSH acts via a specific G
protein-coupled cell surface receptor consisting of a long
extracellular domain, the characteristic seven transmembrane-spanning
domains, and a short carboxy-terminal intracellular part (1). FSH
stimulation results in activation of the Gs protein and subsequent cAMP
production. In the male, it supports indirectly spermatogenesis through
action on Sertoli cells, together with the Leydig cell product
testosterone. In mice with targeted disruption of the FSH ß-subunit
or FSH receptor (FSHR) genes, males are fertile despite decreased
testis size and epididymal sperm count; however, all female homozygotes
are sterile (2, 3, 4). In the female, FSH is therefore indispensable for
recruitment and maturation of Graafian follicles through its
growth-promoting action and stimulation of granulosa cell estrogen
production.
Expression of the FSHR is strictly limited to Sertoli cells in the
testis and granulosa cells in the ovary, which may implicate the
presence of cell-specific cis-regulatory elements in the
FSHR promoter. The FSHR gene has been cloned from various species,
including the human, rat, mouse, bovine, ovine, and chicken (5, 6, 7, 8, 9, 10).
Various regulatory elements have been identified in the 5'-flanking
region of the FSHR gene, including an E box upstream-activating
sequence (CANNTG) and an initiator region conserved in the rat, human,
and mouse (11). In a murine Sertoli cell line, MSC-1, Heckert et
al. (12) identified two upstream stimulatory factors, 1 and 2, as
primary components of the complexes binding the E box. In the rat, an
activator protein 1 (AP-1) binding site in the FSHR (6) and a
cAMP-responsive element (CRE)-like sequence have also been
described (13). The FSHR expression is up- regulated by FSH-induced
cAMP production in cultured rat granulosa cells and inhibited by some
growth factors [epidermal growth factor (EGF), basic fibroblast growth
factor (bFGF)] (14). FSHR expression is posttranscriptionally
down-regulated by a cAMP-dependent mechanism in rat Sertoli cells
(15), and a biphasic effect of gonadotropins on FSHR regulation has
also been demonstrated in vivo (16). However, the mechanisms
of cell specificity of FSHR expression have not yet been studied,
partly because of the lack of permanent cell lines of gonadal somatic
cells. The expression of FSHR is extremely sensitive to in
vitro conditions and is rapidly lost in tissue culture conditions
and upon cell transformation. Immortalized lines of steroidogenic
granulosa (KK-1) (17) and Sertoli (MSC-1) (18) cells have been
generated by genetically targeted tumorigenesis in transgenic mice.
However, these cell lines do not respond to FSH stimulation: the former
lost its response to FSH after prolonged culture, and the latter only
expressed the functional catalytic subunit of adenylate
cyclase.
Recent studies have shown that an orphan nuclear receptor,
steroidogenic factor 1 (SF-1), plays an important role in the
regulation of gene expression of steroidogenic cells. SF-1 was first
recognized to regulate steroidogenic enzymes in the adrenal gland and
gonads (19). SF-1 shares common structural organization with other
members of the steroid receptor superfamily, but, in contrast to the
others, it recognizes specific hexameric response elements in target
genes and binds as a monomer (20). SF-1 expression has been detected in
steroid hormone-producing organs, including testicular Leydig and
Sertoli cells, ovarian theca and granulosa cells, and adrenocortical
cells (21, 22) as well as in the ventral medial nucleus of the
hypothalamus and in pituitary gonadotrope cells (23, 24). Targeted
disruption of the SF-1 gene resulted in pleiotropic impairment of
function of the hypothalamic-pituitary gonadal axis and adrenal cortex,
supporting the role of SF-1 as a key regulator of endocrine development
and function (25).
Herein, we have identified a cell-specific regulatory region of the
murine (m) FSHR promoter containing functional noncanonical recognition
sites for SF-1. Our aim was to characterize the SF-1 binding sites and
to study the regulation of FSHR promoter activity by SF-1 in
steroidogenic and nonsteroidogenic cell lines. Two steroidogenic cell
lines, derived from FSHR-expressing cells, were selected despite their
inability to express the endogenous FSHR in culture, i.e.
immortalized murine granulosa cells (KK-1) (17) and Sertoli cells
(MSC-1) (18). Although lacking FSHR, these cells nevertheless express
other markers specific to FSH- responsive cells. Additionally, a
Leydig cell-derived steroidogenic cell line lacking FSHR (mLTC-1) (26),
as well as a nonsteroidogenic embryonic kidney cell line (HEK 293) were
used.
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RESULTS
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Structure of the 5'-Flanking Sequence of the mFSHR Gene
Analysis of the 1,548-bp mFSHR 5'-flanking sequence revealed a
number of putative binding elements for various transcription factors
(Fig. 1
). These include two CRE-like
sequences at positions -227 and -655 bp (in relation to translation
initiation codon) and two putative estrogen response element (ERE)
half-sites at position -394 bp and -679 bp. The presence of the CREs
is consistent with the known transcriptional regulation of FSHR
expression by cAMP (14, 15). Additionally, several putative PEA 3
binding sites at positions -521, -758, and -819 bp, reported to
function as phorbol ester response elements (27) and AP-1 involved in
the protein kinase C pathway (28), were identified. These are in
agreement with findings of involvement of additional signaling
pathways, in addition to cAMP, in target cell responses to FSH
stimulation (29, 30). A consensus Sp 1 binding site (-1,313 bp) was
also identified. In addition to its general role in the transcription
of housekeeping genes, Sp 1 has regulatory functions in the mouse
differentiation process (31). The E box and the initiator region (In
R), described previously in the rat FSHR promoter (11), were located at
positions -118 bp, and -81 to -107 bp, respectively. The mouse E box
shares total homology with those of the rat and ovine, while a 1-bp
change was noticed in the human E box. The marked 94% homology between
rat and mouse promoters (32) was also evident in the initiator region.
Putative GATA (-534 bp) and transforming growth factor-ß (TGFß)
(-734 bp) binding sites were also observed. Further analysis of the
mFSHR promoter revealed additional potential binding elements. These
were three SF-1-like binding sites (SLBS), containing the core sequence
CAAGG, but differing at their 3'-ends compared with the consensus
sequence. One of these SLBS was located downstream of the main
transcription initiation site between nucleotides -289 to -281 bp
(5'-TCAAGGAAT-3', SLBS-1), and two were located in the
distal region of the promoter at positions -1,171 to -1,163 bp
(5'-AACCCTTGG-3' reverse orientation, SLBS-2) and -1,369
to -1,361 (5'-CCAAGGACT-3', SLBS-3) (Fig. 1
).

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Figure 1. DNA Sequence of the 5'-Flanking Region of Mouse
FSHR Gene
The nucleotides are numbered assigning position -1 for the first
nucleotide 5' of the translation initiation codon. A deduced sequence
for the first amino acids translated is shown below the
DNA sequence. The main transcription start site of the FSHR gene (7 ) at
-534 is indicated by an arrow. Potential binding sites
as well as the putative E box and In R regions are
underlined. The restriction endonuclease recognition
sites used for generation of deletion mutants are boxed.
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Characterization of a Steroidogenic Cell-Specific Domain in the
Distal Region of the mFSHR Promoter
We prepared chimeric constructs of the mouse FSHR promoter, in
which the immediate -1,548-bp sequence of the 5'-flanking region, and
various deletion mutants thereof, were used to drive expression of the
luciferase reporter gene. The promoter activity was investigated in
murine granulosa tumor (KK-1) and Sertoli tumor (MSC-1) cells that had
lost their endogenous FSHR expression (17, 18), as well as in a Leydig
cell line (mLTC-1) that did not express this receptor in
vivo. Additionally, a nonsteroidogenic cell line (HEK 293) was
used as a control. As shown in Fig. 2
, the promoter fragments of differential lengths displayed highly
variable transcriptional activities in the mLTC-1 and KK-1 cells.
Surprisingly, the overall luciferase/ß-galactosidase activity was
highest in Leydig mLTC-1 cells (P < 0.05), displaying
a maximum of 105-fold increase over the promoterless control plasmid
(pBL-0Luc) with the longest promoter sequence (pBL-1548Luc), as
compared with a respective 55-fold stimulation in KK-1 granulosa cells.
Deletion of region -1,548/-1,110 bp caused a dramatic 85% reduction
of promoter activity in mLTC-1 cells and 69% in KK-1 cells. Using the
same construct, a significantly less pronounced decrease of luciferase
activity was found in HEK 293 (48%) and MSC-1 (31%). A further
deletion of the promoter sequence down to -867 bp partially restored
the transcriptional activity in mLTC-1 and KK-1 cells, and totally in
MSC-1 and HEK 293 cells, whereas removal of the sequence between -867
and -555 bp did not significantly affect the promoter activity in any
of the cell types studied.

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Figure 2. Deletion Analysis of the Murine FSHR Promoter
Function
Different promoter/luciferase constructs were transiently transfected
into immortalized murine granulosa (KK-1), Leydig (mLTC-1), and Sertoli
(MSC-1) cell lines, and in human embryonic kidney cell line (HEK 293).
Luciferase/ß-galactosidase activity is presented as fold-increase
over a promoterless luciferase construct (pBL-0Luc), used as negative
control. The left side of the figure shows schematically
the different deletion mutants and the restriction sites used for their
generation. The approximate position of the main transcription
initiation site according to Huhtaniemi et al. (7 ) is
indicated by arrows. The results shown on the
right represent the mean ± SEM of
luciferase/ß-galactosidase activities (fold over promoterless
construct pBL-0Luc) of three independent experiments measured in
triplicate.
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The minimum promoter length capable of driving luciferase gene
expression in the steroidogenic and nonsteroidogenic cells resided
between nucleotides -555 to -99. The orientation of this regulatory
element was critical for transcriptional activity of the FSHR promoter,
since in reverse orientation it was nearly totally devoid of activity.
The inability of region -1,548/-555 to promote luciferase activity in
the absence of the -555/-99 region was confirmed using the
pBL-1548
-555,-99Luc construct. This construct induced only marginal
increases of luciferase activity in all cell types tested. The activity
was comparable to that evoked by pBL-99Luc, and it was negligible in
comparison to that measured with constructs carrying the minimal
promoter sequence. Conspicuously, the residual low promoter activity
observed with the short promoter fragments was roughly similar in each
cell line, indicating that the steroidogenic cell-specific
cis elements reside in the distal region of the FSHR
promoter. In the mFSHR promoter, a major transcription initiation site
was found at position -534 bp (7). The pBL-555Luc construct displays
high promoter activity in KK-1 and mLTC-1 cells, although it contains
only 21 bp upstream of the main transcription initiation site. In this
study, the positions of transcription initiation sites of the different
constructs were not assessed. Multiple transcription start sites have
been demonstrated for the rat (6) and human (5) FSHR genes. In this
regard, the presence of alternative, quantitatively less marked
transcription start sites is also possible with the mFSHR promoter
as has been previously suggested (7).
The Cell-Specific mFSHR Promoter Activity Is Correlated with the
SF-1 mRNA and Protein Contents
As SF-1 is a likely candidate for transactivation of the FSHR gene
in steroidogenic cells, the endogenous SF-1 mRNA expression and SF-1
protein contents were investigated (Fig. 3
). The endogenous 2.7-kb transcript of
SF-1 mRNA (panel A, upper part) and a 53-kDa protein (panel
B, upper) were demonstrated in KK-1, MSC-1, and at the
highest level in mLTC-1 cells. The size of the protein (53 kDa) is
consistent with the reported molecular mass of SF-1 (19, 21). Mouse
testicular and ovarian tissues, used as positive controls, also showed
SF-1 mRNA expression, at higher levels in the ovary. The
nonsteroidogenic embryonic kidney cells (HEK 293) did not express
endogenous SF-1 mRNA or protein; however, an abundant 53-kDa SF-1
protein band was detected in these cells after transient transfection
of the SF-1 expression plasmid. The endogenous SF-1 mRNA expression was
1:3 and 1:10 in KK-1 and MSC-1 cells, respectively, as compared with
mLTC-1 cells (Fig. 3A
, lower panel), and similar differences
were observed at the level of SF-1 protein (Fig. 3B
, lower
panel).

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Figure 3. Northern Blot and Immunoblot Analysis of Endogenous
SF-1 mRNA and Protein Expression
A, Twenty micrograms of total RNA were prepared from KK-1, mLTC-1,
MSC-1, and HEK 293 cells, as well as from normal mouse testicular and
ovarian tissues (upper panel). Hybridization was
performed with a [ -32P]dCTP-labeled
EcoRI/PstI fragment of the SF-1 cDNA
probe generated from pCMV119+-SF-1 plasmid (see
Materials and Methods). The ethidium bromide staining of
28S rRNA is shown below as proof of equal RNA loading. B, Ten
micrograms of nuclear extract were resolved by SDS-PAGE and blotted
onto nitrocellulose filter. The membrane was probed with a polyclonal
anti-SF-1 antibody raised in rabbit (1:5,000), and protein bands were
visualized by chemiluminescence. Lower panels, The
arbitrary densitometric unit (ADU) values of the 2.7-kb SF-1 mRNA
transcript (panel A) and of the immunoreactive 53-kDa protein (panel B)
as quantified by densitometric scanning. Values represent the mean
± SEM of three separate experiments. Bars bearing
asterisks are statistically significantly different
(P < 0.05) compared with the level of KK-1 cell
SF-1 expression.
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To obtain evidence for a cell-specific region in the mFSHR promoter,
luciferase activity of constructs pBL-1,548, -1,110, and -867Luc
(see Fig. 2
) was compared with activity of the construct carrying the
minimum mFSHR promoter (pBL-555Luc) (Fig. 4A
). The activator domain between
-1,548/-1,110 bp increased the promoter activity especially in the
steroidogenic cell lines, but with significant effect only noticed in
mLTC-1 cells, where a 2-fold increase occurred as compared with
pBL-1548Luc activity of HEK 293 cells. However, the repressor domain
that encompasses region -1,110/-867 bp provoked a decrease of
promoter activity that was similar in steroidogenic and
nonsteroidogenic cells. It is of interest that the cell-specific
increase of promoter activity with constructs containing the
-1,548/-1,110 bp region correlated significantly (P
< 0.05) with the levels of endogenous SF-1 mRNA and protein (Fig. 4B
).
In accordance, this distal promoter region possesses two nonconsensus
SF-1 recognition sites at positions -1,163 bp (SLBS-2) and -1,361 bp
(SLBS-3) (Fig. 1
). Taken together, these data are in favor of SF-1-
dependent regulation of the mFSHR promoter activity through the
distal -1,548/-1,110 bp activator domain. To study further the
SF-1-dependent mFSHR promoter activity, we concentrated on
investigating the mLTC-1 cell line, which has the dual advantage of
expressing high levels of endogenous SF-1 and no FSHR. HEK 293 cells
were used as a negative control free of endogenous SF-1 expression.

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Figure 4. Cell-Specific mFSHR Promoter Activity in Relation
to Endogenous SF-1 Expression
Panel A represents the luciferase/ß-galactosidase activities
(mean ± SEM) of pBL-1548Luc, pBL-1,110Luc, and
pBL-867Luc promoter constructs compared with activity of the construct
carrying the minimal promoter (pBL-555Luc) for each cell line, assigned
a value of 100%. The bar bearing an
asterisk is statistically significantly different
(P < 0.05) from the data obtained on HEK 293
cells. Panel B presents the correlation between pBL-1548Luc activity
and the level of endogenous SF-1 mRNA (circle) or
protein (triangle) level in the four cell lines studied.
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A Functional SF-1 Binding Site Is Present in the mFSHR Promoter
Sequence
To reveal putative interactions between SF-1 protein and the
SF-1-like binding sites (SLBS-1, -2, or -3), electrophoretic mobility
shift assays (EMSAs) were carried out. Purified nuclear extracts of
KK-1, MSC-1, mLTC-1, and HEK 293 cells, transfected or untransfected
with the SF-1 plasmid, were incubated with specific radiolabeled
oligonucleotides containing the different SLBS sites. In the
experimental conditions used, we were unable to detect specific
SF-1-shifted complex using oligonucleotides SLBS-1 and SLBS-2, either
with nuclear extracts from mLTC-1 or SF-1-transfected HEK 293 cells
(data not shown). With oligonucleotide carrying the SLBS-3 site,
purified nuclear extract from mLTC-1 cells gave rise to two slowly
migrating DNA-protein complexes (Fig. 5
, lane 3). The intensity of the lower shifted complex was markedly
decreased using KK-1, and absent using MSC-1 nuclear extract. However,
the intensity of the higher molecular mass complex was not dependent on
the cell line (lanes 25). With HEK 293 cell nuclear extract, three
shifted complexes were found (lane 5), whereas with HEK 293-SF-1 cell
nuclear extract, a new shifted complex appeared to the detriment of the
other bands (lane 6). Interestingly, this SF-1-specific complex
comigrated with the lower binding complex detected with the mLTC-1 and
KK-1 cell extracts. To verify the identity of the protein(s), gel-shift
assays were performed in the presence of a rabbit polyclonal antibody
directed against the full-length bovine SF-1 protein (33) (Fig. 6
). The addition of a 1:100 dilution of
the SF-1 antiserum reduced the intensity whereas a 1:10 dilution
totally abolished the SF-1/SLBS-3 complex generated by the HEK 293-SF-1
and mLTC-1 nuclear extracts (panel A and B, respectively). The same
dilutions of nonimmunized rabbit serum were not able to reverse the
SF-1/SLBS-3 complexes in either cell lines (data not shown). This type
of competition, termed gel-shift abrogation, has already been described
and relies on ability of the anti-SF-1 antibody to effectively block
accessibility of the DNA to the SF-1 DNA-binding domain (34, 35).
Collectively, these data support the notion that SF-1 is capable of
binding to the SLBS-3 sequence of mFSHR promoter.

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Figure 5. Intensity of a Shifted Complex in EMSA Correlates
with Cell-Specific SF-1 Protein Content
Oligonucleotides SLBS-3S and SLBS-3AS (see Table 1 ) were annealed,
end-labeled, and incubated without (lane 1) or with 10
µg of nuclear extract of KK-1, mLTC-1, MSC-1, and HEK
293 cells (lanes 25). The nuclear extract used in lane 6 was from HEK
293 cells transiently transfected with 1 µg of the
pCMV119+-SF-1 expression plasmid. The DNA-protein complexes
were resolved on a 4% polyacrylamide gel after 1 h incubation at
4 C.
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Figure 6. Inhibition of SF-1 Binding to SLBS-3 by Rabbit SF-1
Antisera
Nuclear cell extracts (10 µg) from HEK 293 cells (panel A, lane 2)
and HEK 293 transfected with SF-1 expression plasmid (panel A, lanes
35) or mLTC-1 (panel B, lanes 24) were incubated with a rabbit
polyclonal SF-1 antibody (1:10 and 1:100) (33 ) for 45 min at 4 C before
the addition of 32P-radiolabeled double-stranded SLBS-3
oligonucleotide. Protein-DNA complexes were resolved on 4%
nondenaturing polyacrylamide gel.
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The direct effect of SF-1 on mFSHR promoter activity was investigated
in HEK 293 cells devoid of endogenous SF-1 expression (Fig. 7
). Coexpression of SF-1 cDNA increased,
in a dose-dependent manner, the FSHR promoter-driven luciferase
activity, reaching 2.3-fold elevation with 1 µg of the
pCMV119+-SF-1 sense plasmid. Cotransfection of
pCMV119--SF-1 (reverse orientation) failed to
stimulate promoter activity. Hence, it can be concluded that SF-1 is
able physically to interact with the SLBS-3 domain present in the
distal region of the mFSHR promoter. Moreover, the binding of SF-1 to
the mFSHR promoter sequence enhances its effect on reporter gene
expression.

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Figure 7. Dose-Dependent Increases of mFSHR Promoter Activity
after Transient Transfection of SF-1 Expression Plasmid into Cultured
HEK 293 Cells
The cells were transiently transfected with an FSHR promoter/luciferase
reporter gene construct (pBL-1548Luc) and increasing amounts (0.1 to 1
µg) of pCMV119+-SF-1 sense (lanes 25) or 1 µg
pCMV119--SF-1 antisense (lane 6) expression plasmids. The
pCMV-ß-galactosidase plasmid was cotransfected to control for
transfection efficiency. The results shown are
luciferase/ß-galactosidase ratios in relation to the pBL-1548Luc
activity (mean 100%). The molar amount of transfected DNA was
equalized using empty pMT2 vector. Each bar represents
the mean ± SEM of three separate experiments, each
run in triplicate. Different letters above the bars
indicate that the difference between them is statistically significant
(P < 0.05).
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Integrity of the SF-1/SLBS-3 Sequence Is Needed for Full
Binding Capacity and Transcriptional Activity
To further confirm the specificity of the SF-1/SLBS-3 binding
complex, mutations were introduced into SLBS-3. As shown in Fig. 8A
(lanes 2 and 3), using mLTC-1 cell
nuclear extract, the shifted lower molecular mass DNA-protein complex
could be competed for by increasing the amount of unlabeled SLBS-3. The
same molar excess of competitor, mutated in the SF-1 core sequence,
failed to displace SF-1 binding (lanes 4 and 5). The lower molecular
mass SF-1 binding complex also disappeared totally after addition of
50-fold molar excess of competitor containing the SF-1 consensus
sequence (comparing lanes 2 and 6). An identical experiment was
performed with HEK 293-SF-1 extract, showing the same pattern of
competition (Fig. 8B
, lanes 14). In lanes 5 and 6, SLSB-2 and SLBS-1
oligonucleotides were used in high excess (x400) as competitors, and
decreases in SF-1/SLBS-3 complex intensity were observed in both cases,
SLBS-2 behaving as a stronger competitor. The latter finding suggests
that SLBS-2 and, to a lesser extent, SLBS-1 could also bind SF-1
protein, although with lesser avidity. A nonspecific AP-1 competitor
used at 200- and 400-fold excess failed to affect the SF-1/SLBS-3
binding complexes either using HEK 293-SF-1 or mLTC-1 nuclear extract
(data not shown).

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Figure 8. Characterization of the Distal FSHR SF-1 Binding
Site by Competition Studies
A, EMSA performed using nuclear extract from mLTC-1 cells preincubated
in the presence of 50- or 200-fold molar excess of unlabeled
double-strand SLBS-3 (lanes 2 and 3), SLBS-3-mut (lanes 4 and 5), and
SLBS-3-Con (lanes 6 and 7). A 32P-radiolabeled SLBS-3 probe
was added for a further 1-h incubation. The DNA protein complexes were
resolved on a polyacrylamide gel. B, EMSA performed as described above
using SF-1-transfected HEK 293 nuclear extract. Competitors were use at
200-fold excess in lanes 24, and at 400-fold excess in lanes 5
(SLBS-2) and 6 (SLBS-1). The SLBS-3-Mut was prepared using
oligonucleotides MUT-3S and MUT-3AS, to change the SF-1 core sequence
from CAAGG to CAATT. For SLBS-3-Con, CON-3S and CON-3AS
oligonucleotides were used to create a consensus SF-1 site CAAGGTCA.
All the oligonucleotides used as double-stranded competitors are listed
in Table 1 .
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The mutation that abolished SF-1 binding to SLBS-3 (CAAGG changed to
CAATT) was introduced to the SLBS-3, SLBS-2, and SLBS-1 sequences (Fig. 9
). The effects were investigated in HEK
293 cells transfected or untransfected with SF-1 expression plasmid, as
well as in mLTC-1 cells. No significant changes in promoter activity
were observed when using HEK 293 cells, regardless of the site mutated.
However, destruction of the SF-1 binding site of SLBS-3 resulted in a
3840% decrease (P < 0.05) of promoter activity in
both HEK 293-SF-1 and mLTC-1 cells as compared with nonmutated
pBL-1548Luc. Additionally, a mutation in SLBS-2 slightly but
significantly suppressed the luciferase activity (2025%) while no
effect was shown with mutated SLBS-1. Reduction of FSHR promoter
activity in mLTC-1 cells reached 50% when both SLBS-3 and SLBS-2 were
mutated (data not shown). The mutation introduced in the SLBS sites
created an E box-like element (CANNTG). To eliminate this potential
experimental artifact, a second set of mutations (CAAGG to CCGGG) in
SLBS-3 and SLBS-2 were assayed. The promoter activity of constructs
carrying the newly mutated SLBS-3 site was similarly decreased to 50%
and 37% in HEK 293-SF-1 and mLTC-1 cells, respectively. With the
second mutated SLBS-2 construct, a reduced promoter activity was also
observed in both cell lines (data not shown). The two sets of
SLBS-3-mutated primers were also used as oligoprobe in EMSA assay, and
no SF-1-specific shifted bands could be detected with either HEK
293-SF-1 or mLTC-1 nuclear extract (data not shown).

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Figure 9. Mutation in SF-1 Sites Affects the FSHR Promoter
Activity in mLTC-1 and HEK 293-SF-1 Cells
PCR-based mutagenesis was used to generate mutations in the putative
SF-1-like site of the FSHR promoter. pBL-1548Luc was used as the
template and site-directed mutagenesis was carried out as described in
Materials and Methods. The mutated constructs were
transiently transfected into mLTC-1, HEK 293, and SF-1-transfected HEK
293 cells, 24 h before luciferase and ß-galactosidase
measurements. The results shown are luciferase/ß-galactosidase
activity ratios in relation to the nonmutated pBL-1548Luc construct
(mean 100%). Each bar represents the mean ±
SEM of three separated experiments, each done in
triplicate. Bars bearing asterisks are
statistically significantly different (P < 0.05)
from nonmutated constructs in the same cell line. The left
panel depicts schematically the positions of the mutated
SF-1-like sites.
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The transformation of the SLBS-3 sequence to a consensus SF-1 site
(CAAGGTCA) had a positive effect (52% increase, P <
0.05) on SF-1-induced promoter expression in mLTC-1 cells, and a 22%
increase was observed in HEK 293-SF-1-transfected cells (Fig. 9
). These
results confirm the role of SLBS-3 and, to a lesser extent, of SLBS-2
in the SF-1-regulated mFSHR promoter activity. Furthermore, the binding
capacity and transcriptional ability are closely related and dependent
on the SF-1 DNA recognition sequence.
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DISCUSSION
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The FSHR promoter belongs to the promoter types that lack the
conventional TATA and CCAAT box elements, a characteristic shared by
ubiquitously and constitutively expressed genes. However, in contrast
to housekeeping genes, that of FSHR is tightly regulated during
development and expressed in a cell-specific manner. This subclass of
TATA-less promoters usually contains an initiator region with one or
several transcription start sites close to the ATG codon. The positive
regulatory elements needed for full activity of the rat and human FSHR
promoters have been exclusively sought and located in the proximal
region of the 5'-untranslated region, close to their respective
transcription start sites (5, 11, 13). The majority of the positive
regulatory elements described in rat and human minimal promoters were
also found in the mouse and sheep proximal promoter region (Fig. 10
). However, these putative
cis-acting domains were located downstream from the main
transcription initiation site in the mouse (-534 bp). The present
study demonstrated the active participation of the sequence between
nucleotides -99 to -555 bp in transactivation of the promoter. Thus,
the cis-acting elements located downstream of the
transcriptional start site are apparently functional in the mFSHR
promoter, as has been reported for several other genes (36, 37).
Several regions 3' of the transcription start sites were also important
for promoter function in the rat FSHR (12). The molecular mechanisms by
which these elements enhance transcription are largely unknown, but
they may act in a manner similar to 5'-promoter elements, to help
recruitment of components of the general transcription machinery, or
they may be important for start site selection or elongation. Further
studies are needed to define the proximal sequences of the promoter
regions that interact with cellular transcription factors to modulate
the expression of gonadotropin receptors and augment in selection of
transcription initiation start sites.

View larger version (21K):
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Figure 10. Alignment of Putative DNA-Responsive Elements of
the FSHR Proximal Promoter Regions
The murine (7 ), rat (6 ), human (5 ), and chicken (9 ) nucleotide
sequences are numbered assigning -1 to the first nucleotide above the
translation initiation codon (ATG). Capital letters
represent identical nucleotides.
|
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In this study, we demonstrated that additional regions far upstream of
the transcription initiation sites are also involved in the
cell-specific mFSHR expression. Deletion of a distal region between bp
-1,548 to -1,110, containing two putative SF-1 binding sites, showed
a decrease in promoter activity in highly SF-1-expressing mLTC-1 cells.
We demonstrated herein that overexpression of SF-1 induced a
dose-dependent increase of the mFSHR promoter activity in transiently
transfected HEK 293 cells. The SF-1 action was mostly mediated by a
nonconsensus SF-1 site at position -1,369 bp (5'-CCAAGGACT-3'). SF-1
was able to specifically bind to this SLBS-3 sequence and transactivate
FSHR promoter expression. A mutation of the functional SLBS-3 motif,
which abolished its binding ability, significantly, although not
totally, suppressed the promoter activity. The luciferase activity and
the intensity of the SF-1/SLBS-3 binding complex were closely related
to the endogenous SF-1 mRNA expression and SF-1 protein levels in
steroidogenic cell lines.
SF-1, alternatively known as adrenal 4-binding protein, is an orphan
nuclear receptor that is a key regulator of steroidogenic enzymes (19, 21). In mammals, SF-1 plays a key role in the development and
differentiated function of the adrenal glands and gonads. In addition
to its role during development, and consistent with its distribution,
SF-1 functions as a potent transcription factor for many genes involved
in hypothalamic-pituitary-steroidogenic function. In addition to
steroidogenic enzymes, SF-1 regulates in mice the expression of ACTH
and GnRH receptors, the LH ß-subunit, steroidogenic acute regulatory
protein (StAR), anti-Müllerian hormone, and DAX-1 (for
references, see Ref. 38). SF-1 possesses multiple putative functional
domains, including a characteristic zinc finger DNA-binding domain at
the N-terminal region, a hinge region, a dimerization domain, a
ligand-binding domain, and a conserved ligand-dependent activation
function 2 (AF-2) in the distal C terminus essential for receptor
transactivation (39).
Ueda et al. (40) demonstrated that SF-1 binds as a monomer
to its recognition element, the hexameric AGGTCA motif, which is
recognized by the two zinc fingers, leading to high-affinity binding.
In this work, the binding of SF-1 to SLBS-3 (AGGaCt) appeared less
effective than to a consensus SF-1 site. In addition, SLBS-3-Con
(carrying consensus sequence) was a more potent competitor than SLBS-3.
These data suggest a sequence- dependent binding ability of the
putative SF-1 site to SF-1 transcription factor. The differences in
SF-1 binding motifs observed in SLBS-1 (AGGaat) and SLBS-2 (TCCcaT)
lead to near-totally or severely suppressed binding ability. However,
mutation in SLBS-2 significantly affected the FSHR promoter activity.
In the rat StAR promoter, Sandhoff et al. (41) described
high- and low-affinity SF-1 binding sites, all possessing the core
CAAGG domain but with differences at their 3'-ends. All the three SLBSs
described in the FSHR promoter contain the core CAAGG sequence
essential for SF-1 binding (42). However, the differences in function
of SLBS-1, -2, and -3 suggest that the presence of this core sequence
is not sufficient for full SF-1-mediated promoter activation. Thus,
participation of additional nucleotides in the binding site and/or
spatial arrangement of the SF-1 site may contribute to full regulatory
function. In the present work, gel shift assays confirmed that the GG
dinucleotide in the core sequence was critical for SF-1 binding ability
and functional activity. Furthermore, we showed that cytosine at the
3'-end of the recognition sequence was also crucial for binding, while
the TCA motif supports the ability for high-affinity binding,
consistent with the canonical SF-1 sequence, 5'-YCAAGGYCR-3' (34, 42).
Moreover, SF-1 binding and transcriptional activity have been
demonstrated through other nonconsensus sequences (43, 44). Hence, the
involvement of SLBS-2 or even SLBS-1 sites in the SF-1-induced FSHR
promoter activation, as well as through other noncharacterized
sequences, cannot be totally excluded. Additional sequences resembling
the SF-1 recognition site have been reported in the 2.1-kb 5'-upstream
region of the ovine FSHR gene (9), but their functions have not been
explored.
Tissue-specific gene expression requires the combined action of tissue-
and promoter-specific activators and repressors. Mutation or deletion
of the functional SF-1-like motif (SLBS-3) significantly
decreased, but did not abolish, the mFSHR promoter activity. The
positive element located in the -1,548/-1,110 region of the mFSHR
promoter was not able to evoke promoter activity without cooperation
with the proximal minimal promoter, suggesting the presence of multiple
response elements for cell-specific factors in addition to the
ubiquitous regulatory sequences. We characterized various putative
binding sites for ubiquitous and cell-specific transcription factors in
the mFSHR promoter that could participate in the restricted FSHR
expression specific for FSH target cells. Interestingly, functional
interaction or direct heterodimerization has been shown between SF-1
and a number of these transcription factors, including Sp1 (45),
cAMP-responsive element binding protein (44), estrogen receptor (46),
and GATA-4 (47). These transcription factors act in synergy with SF-1
to potentiate the SF-1 regulatory function. Another regulatory factor,
DAX-1, has been extensively described as a potent repressor of SF-1
action (48, 49, 50). Moreover, coactivators and corepressors, with distinct
tissue-specific expression patterns and hormonal regulation, are also
involved in the modulation of SF-1 action (51, 52).
Targeted disruption of the murine SF-1 gene resulted in adrenal and
gonadal aplasia, male-to-female sex reversal of the internal and
external genitalia, malformations of the ventromedial hypothalamus, and
selective deficiency of GnRHR, LHß, and FSHß mRNA in the pituitary
gland (22, 25, 53). Analysis of SF-1 expression in embryonic gonads did
not only support a role for SF-1 in steroidogenesis, but also indicated
that this transcription factor may play additional roles in development
(54). The pattern of SF-1 expression correlates with the ontogeny of
FSHR expression during fetal life, as SF-1 mRNA level was detected
before the onset of FSHR expression in both sexes (25, 55). After
birth, SF-1 is mainly expressed in seminiferous tubules, and high
expression was observed in rat Sertoli cells during a limited period of
pubertal maturation before the first cycle of spermatogenesis (56),
coinciding with a peak in FSHR mRNA expression (57). SF-1 is a negative
regulator of granulosa cell mitosis; thus, enhanced SF-1 expression is
part of the molecular mechanism associated with granulosa cell
differentiation. In addition, SF-1 knockout females fail to develop
ovaries, suggesting that SF-1 is essential for ovarian organogenesis
(58).
In conclusion, the SF-1-dependent regulation of FSHR expression, as
demonstrated in the present study, is in line with functions
demonstrated earlier for this transcription factor. The concomitant
changes in FSHR promoter activities with the expression levels of SF-1
further supported SF-1 as an in vivo regulator of FSHR gene
expression.
 |
MATERIALS AND METHODS
|
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Plasmid Constructions
An XbaI/XhoI genomic DNA fragment,
containing a 1.5-kb fragment of the murine FSHR 5'-flanking region, was
obtained by PCR methods using the pFSHRI plasmid (7) carrying 7.5 kb of
the 5'-end and exon I of the mouse FSHR gene. Two endonuclease
restriction sites were introduced in pFSHRI using primers FSHR1 and
FSHR2 (Table 1
), XbaI site at position
-1,548 into the 5'-untranslated region, and XhoI site to
substitute the ATG translation start site. The -1,548/-1 promoter
fragment was subcloned into pBL-Luci plasmid in front of the luciferase
reporter gene-coding sequence using XbaI and XhoI
restriction endonucleases to obtain pBL-1548Luc. All mouse FSHR
promoter deletion mutants were prepared using endonuclease restriction
sites of the promoter region present in pBL-1548Luc. pBL-1110Luc and
pBL-867Luc mutants were constructed by digestion with PstI
and HindIII, respectively. With BamHI and
BglII we obtained two fragments, -1,548/-555 and
-555/-99, which we used to construct pBL-555Luc, pBL-99Luc,
pBL-99555Luc, and pBL-1548
-55599Luc after random ligations. The
identities of all FSHR promoter constructs were verified by restriction
mapping and sequencing.
Cell Culture
The mouse Leydig tumor cell line (mLTC-1) (26) was cultured in
HEPES (20 mM)-buffered Waymouths medium
(Sigma, St Louis, MO), supplemented with 9%
heat-inactivated horse serum (Life Technologies, Inc.,
Paisley, Scotland), and 4.5% heat-inactivated FCS (iFCS) (Bioclear,
Wilts, UK) containing 0.1 ng/ml gentamycin (Life Technologies, Inc., Gaithersburg, MD). The lines of granulosa cells (KK-1)
(17), mouse Sertoli cells (MSC-1) (18), and human embryonic kidney
cells (HEK 293) were maintained in DMEM/Hams F-12 1:1 medium
(Sigma), supplemented with 10% iFCS, containing 50 mIU/ml
ampicillin and 0.5 µg/ml streptomycin
(Sigma). The cells were allowed to grow on 10-cm diameter
plates to 7080% confluency under a humidified atmosphere of 95% and
5% CO2 at 37 C.
Transient Transfection of Cell Lines
The transient transfection method was optimized for each cell
lines using pCMV-ß-galactosidase as the marker of transfection
efficiency. Cells were seeded on six-well plates at a density of
0.5 x 106 cells per well, 1620 h
preceding transfection to achieve 6070% confluence. Cells were
transfected with 1.5 µg of one of the FSHR promoter
constructs and 0.3 µg of pCMV-ß-galactosidase as
control. The mLTC-1 cells were transiently transfected by the FuGENE 6
transfection reagent (Roche Molecular Biochemicals,
Mannheim, Germany) as described in the manufacturers protocol. After
15 min incubation of the FuGENE 6-DNA complex, it was distributed
dropwise on cells grown overnight and cultured for a further 24 h.
For HEK 293, KK-1, and MSC-1 cells, LipofectAMINE transfection reagents
(Life Technologies, Inc.) were used, according to
instructions of the manufacturer. The transfection solution was removed
after 5 h incubation at 37 C and replaced by 2 ml of complete
DMEM/Hams F-12 for a further 24-h culture.
For cotransfection experiments, we used plasmids expressing SF-1 cDNA
in correct or reverse orientation, pCMV119+-SF-1
and pCMV119--SF-1, respectively (obtained from
Dr. K. L. Parker, Durham, NC). The amount of transfected DNA was
equalized using the empty pMT2 vector. The volume of both LipofectAMINE
and FuGENE 6 reagents was increased and adjusted to the DNA amount
according to the manufacturers instructions.
After 24 h, transfection media were replaced by 100
µl of cell lysis buffer [12.5 mM
Tris-HCl, pH 7.8, 10 mM NaCl, 0.4
mM EDTA, 0.2 mM
MgSO4, 1 mM dithiothreitol
(DTT), and 0.2% Triton X-100] for 5 min. The cells were scraped off
and centrifuged for 1 min at room temperature. Luciferase activity was
measured from 10 µl of the lysate in a Victor multilabel
counter (Wallac, Inc., Turku, Finland) by adding 100
µl of luciferase assay buffer (40 mM
Tris-HCl, pH 7.8, 0.5 mM ATP, 10
mM MgSO4, 0.5
mM EDTA, 10 mM DTT, 0.5
mM coenzyme A, 0.5 mM
luciferin). The ß-galactosidase activity was assessed from the same
lysate in 100-mm phosphate buffer, pH 7.0, supplemented with 10
mM KCl, 1 mM
MgSO4, and 50 mM
ß-mercaptoethanol and incubated for 30 min at 37 C in the presence of
O-nitrophenyl-ß-D-galactopyranoside (ONPG) at
0.8 mg/ml final concentration. Luciferase activity was normalized for
transfection efficiency by dividing the luciferase activity by
ß-galactosidase activity.
Northern Hybridization Analysis
Total RNA was isolated from cells using the single-step TRIZOL
(Life Technologies, Inc.) method, according to
instructions of the supplier. Twenty micrograms of RNA per lane were
resolved on 1.2% denaturing agarose gel and transferred onto Hybond-XL
nylon membranes (Amersham International , Aylesbury,
Bucks, UK). Membranes were prehybridized overnight at 42 C in a
solution containing 5 x SSC, 5 x Denhardts, 0.5% SDS,
50% formamide, and 5 mg/ml of denatured calf thymus DNA. An
EcoRI/PstI fragment of SF-1 cDNA probe was cut
out from pCMV119+-SF-1, labeled with Prime-a-Gene
kit (Pharmacia Biotech, Uppsala, Sweden) using
[
-32P]dCTP during 4 h at 37 C and
purified with NickColumn (Pharmacia Biotech).
Hybridization was carried out at 42 C for 20 h in the same
prehybridization solution after addition of radioactively labeled
probe. After hybridization, the membranes were washed twice in 2
x SSC and 0.1% SDS at room temperature for 10 min, followed by two
washes in 0.1 x SSC and 0.1% SDS at 42 C to remove most of the
background. Membranes were exposed to x-ray film (XAR-5; Eastman Kodak Co., Rochester, NY) at -70 C for 47 days or to
phosphorimager (BAS-5000 film I
I, Fuji Photo Film Co., Ltd., Tokyo, Japan) for 424 h. The intensities of specific
bands were quantified using Tina software (Raytest, Stranbenhardt,
Germany) and related to those of the 28S rRNA, in the gel stained with
ethidium bromide. The molecular sizes of the mRNA species were
estimated by comparison with mobilities of the 18S and 28S rRNAs.
Immunoblotting
Nuclear extracts from various cell lines were resolved by 7.5%
SDS-PAGE, transferred to nitrocellulose membrane (Hybond ECL,
Amersham Pharmacia Biotech, Arlington Heights, IL) by
electroblotting, preincubated for 2 h in TBS, 1% Tween-20, and
5% nonfat dry milk, and washed three times for 10 min in the same
buffer without milk. Incubation with rabbit anti-SF-1 polyclonal
antibody (33) (1:5,000) (obtained from Dr. K. Morohashi, Fukuoka,
Japan) was performed overnight at 4 C. The filter was washed and
incubated for 1 h with 1:1,000 rabbit Ig, horseradish
peroxidase-linked antibody. After washing three times, the membrane was
subjected to chemiluminescent detection using ECL Western blotting
detection kit (Amersham Pharmacia Biotech), and finally
the membranes were exposed for 210 min to Kodak x-ray
films. The immunospecific bands were quantified by Tina software.
Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared from confluent cell cultures as
described previously (59). Complementary oligonucleotides, containing
the respective SF-1 like sequences, and competitors (Table 1
) were
annealed in 10 mM Tris HCl, pH 7.5, 1 mM EDTA,
25 mM NaCl, 10 mM MgCl2,
1 mM DTT. 5'-GG overhangs present in the double-stranded
oligonucleotide were filled with [
-32P]dCTP
for 2 h at 30 C using the Klenow DNA polymerase. The labeled
oligonucleotide probes were purified in Nick Column. Nuclear extracts
(10 µg) were incubated with 6 fmol (
40,000 cpm)
radiolabeled, double-stranded oligonucleotides for 1 h at 4 C in a
reaction buffer containing 12 mM HEPES, pH 7.9, 4
mM Tris HCl, 12% glycerol, 1
mM EDTA, 60 mM KCl, 1
mM DTT, and 300 µg/ml BSA in the
presence of 2 µg poly (dI:dC). For competition
experiments, the competitor was first incubated with nuclear extract at
4 C for 1 h in reaction buffer before addition of labeled probe.
In antibody-abrogation gel shifts, nuclear extracts were incubated with
a rabbit polyclonal antibody directed against the full-length bovine
SF-1 protein (33) or an equal dilution (1:10 and 1:100) of nonimmunized
rabbit serum for 45 min at 4 C before the addition of radiolabeled
probe (34). Protein-DNA complexes were resolved on 4% nondenaturing
polyacrylamide gels using 0.25x Tris-borate-EDTA buffer, and the gel
was dried and exposed to Kodak x-ray film at 50 C for
13 days.
Site-Directed Mutagenesis
Site-directed mutants of the FSHR promoter sequence were
prepared using QuickChange Site-Directed Mutagenesis kit
(Stratagene) according to the manufacturers protocol.
The same complementary oligonucleotides were used as competitors in gel
shift experiments and as primers for mutagenesis (Table 1
). One hundred
nanograms of the plasmid DNA template were incubated with 125 ng of
appropriate primers, 25 mM deoxynucleoside
triphosphates, and 50 µl of 1x reaction buffer in
the presence of 2.5 IU of Pfu DNA polymerase. The PCR conditions
included 16 cycles with denaturing step at 95 C for 30 sec, annealing
at 55 C for 1 min, and extension at 68 C for 13 min. The parental DNA
template was digested by adding 10 IU of DpnI restriction
endonuclease for 1 h at 37 C. One to 5 µl of PCR
reaction were used to transform XL-1 Blue Supercompetent cells. The
mutations were verified by restriction mapping using Mfe I,
Vsp I, XmaI, and/or Tsp 45I followed
by sequencing of both strands.
Statistical Analysis
All results presented are from two to six independent
experiments performed in triplicate. The data are expressed as
mean ± SEM. Statistically significant differences
between groups were determined by one-way ANOVA, followed by Duncans
test; P < 0.05 was considered statistically
significant.
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to Dr. K. L. Parker (Departments of
Medicine and Pharmacology and Howard Hughes Medical Institute, Durham,
NC) for the generous gift of pCMV119--SF-1 and
pCMV119+-SF-1 plasmids, and to Dr. K. Morohashi
(Department of Molecular Biology, Kyushu University, Fukuoka, Japan)
for providing the SF-1 antibody. We thank Dr. Matti Poutanen for the
helpful discussions during preparation of this manuscript. The
invaluable help of Dr. Pirjo Pakarinen and technical assistance of Ms.
Riikka Kytömaa are gratefully acknowledged.
 |
FOOTNOTES
|
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Address requests for reprints to: Ilpo Huhtaniemi, M.D., Ph.D., Department of Physiology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. E-mail: ilpo.huhtaniemi{at}utu.fi
This study was supported by grants from the Academy of Finland and the
Sigrid Juselius Foundation.
Received for publication March 13, 2000.
Revision received September 19, 2000.
Accepted for publication September 25, 2000.
 |
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