(Received for publication, September 6, 1995; and in revised form, December 13, 1995)
From the
The orphan nuclear receptor, steroidogenic factor-1 (SF-1), is
expressed in the pituitary and in the gonadotrope precursor cell line,
T3-1, where it is believed to enhance expression of the common
gonadotropin
-subunit gene through transactivation of the
gonadotrope-specific element (GSE). Sequence analysis of the rat
luteinizing hormone
-subunit (LH
) gene promoter revealed the
presence of a consensus GSE at -127 to -119 (TGACCTTGT). We
have demonstrated the ability of SF-1 to bind specifically to this
putative GSE sequence by electrophoretic mobility shift assay,
utilizing both
T3-1 nuclear extracts and in vitro translated SF-1. In addition, mutation of the putative LH
-GSE
(TGAAATTGT) eliminated specific DNA binding. To examine the ability of
SF-1 to enhance LH
promoter activity, CV-1 cells, which lack
endogenous SF-1, were cotransfected with an SF-1-containing expression
vector and an LH
-luciferase reporter construct. When cotransfected
with -209/+5 of the LH
promoter, SF-1 increased
luciferase activity by 56-fold. SF-1 responsiveness was markedly
diminished with loss of the putative GSE region in deletion constructs
and in the presence of a two base pair mutation, analogous to the
mutation which eliminated DNA binding. Finally, the LH
-GSE was
able to confer SF-1 responsiveness on a heterologous minimal growth
hormone promoter, GH50 (57-fold). We conclude that SF-1 both binds to
and transactivates the rat LH
promoter. These data suggest that
SF-1 may participate in the expression of the LH
gene by the
gonadotrope.
The pituitary gonadotropins, luteinizing hormone and
follicle-stimulating hormone, are critical modulators of gamete
maturation and gonadal steroidogenesis. These hormones are composed of
a common -subunit linked noncovalently to unique
-subunits
which specify physiologic actions(1) .
Several DNA
regulatory elements have been defined for the -subunit gene
promoter. GnRH(
)-stimulated expression is believed to be
mediated through a regulatory element located between positions
-346 and -244 in the human
-subunit gene promoter, a
region separate from those involved in basal and cAMP-stimulated
expression(2) . Activation of a cAMP response element (CRE)
appears to be important for expression in both pituitary and placental
cell types, while placental-specific expression occurs through the
activation of a trophoblast-specific element acting in concert with the
CRE(3) . Pituitary-specific expression of the
-subunit
gene has been attributed to the presence of both a pituitary
glycoprotein basal element and a gonadotrope-specific element
(GSE)(4, 5) .
The consensus GSE sequence
(TGACCTTGT), defined in the common -subunit by Mellon and
colleagues, resembles a nuclear receptor binding
half-site(4, 6) . Variations of this sequence,
alternatively called the Ad4 response element, are also present in the
promoter regions of multiple genes which play a role in
steroidogenesis, sexual differentiation, and adult reproductive
function(7) . The GSE/Ad4 element has been shown to interact
with the transcription factor, steroidogenic factor-1 (SF-1), in a
number of genes, including the steroidogenic P450, the aromatase, and
the Müllerian inhibiting substance
genes(8, 9, 10) . SF-1 is an orphan member of
the nuclear hormone receptor superfamily. Best known for its selective
expression in adrenal and gonadal cells, it has more recently been
identified in the pituitary gland with localization to the
gonadotrope(6, 7) .
In studies of the human
-subunit gene promoter, SF-1 has been shown to bind to the GSE
region by electrophoretic gel mobility shift assay. Furthermore,
reporter constructs which contain the
-subunit GSE site are
expressed at higher levels in cell lines which contain endogenous SF-1
than in those cells which lack SF-1, consistent with a role for SF-1 in
tissue-specific transcriptional activation of the
-subunit
gene(4, 6) .
In contrast with the -subunit,
the cis-acting elements responsible for expression of either the
LH
- or FSH
-subunit mRNAs are poorly understood.
Interestingly, transgenic mice null for the gene which encodes SF-1 not
only express the
-subunit in low levels, but also fail to express
the
-subunits, suggesting a functional role for SF-1 in LH
gene expression(7) . As previous studies of the bovine LH
gene promoter have shown that the proximal 776 base pairs are
sufficient to direct pituitary-specific expression in transgenic mice (11) , we analyzed the corresponding region of the rat LH
promoter for the presence of an SF-1-binding site, or GSE.
The rat
LH gene promoter contains a consensus GSE at position -127
to -119 relative to the transcriptional start site (Fig. 1). Interestingly, this sequence is highly conserved
across species among the LH
genes, suggesting physiologic
significance(11, 14) . Inasmuch as the consensus GSE
sequence is present in the LH
gene promoter, we wished to
determine whether this putative GSE region has functional significance.
We, therefore, investigated the ability of SF-1 to bind to and
transactivate the rat LH
gene promoter.
Figure 1:
A consensus
SF-1-binding site is present in the rat LH gene promoter. The core
GSE sequence as defined in the human glycoprotein
-subunit (4) is aligned with the putative GSE sites of the LH
gene
in rat(12) , cow(13) , pig(14) , and human (15) . Also shown are regions of homology with the SF-1-binding
sites in the 21-hydroxylase(8) , aromatase(9) , and
Mullerian inhibiting substance (10) gene promoters. Variant
nucleotides are underlined.
Figure 3:
Mutation of the putative LH-GSE
sequence defines nucleotides essential for binding by
T3-1 nuclear
extract. Binding reactions included
T3-1 nuclear extracts and
either the
P-labeled wild-type LH
gene
oligonucleotide (LHSF, lanes 1-4) or the
P-labeled mutated oligonucleotide (LHSFM, lanes 5-8). Competition with 500-fold molar excess of
unlabeled LHSF, LHSFM, or Pit-1 oligonucleotide was performed as
indicated. The specific and nonspecific binding complexes are indicated
by an arrowhead and asterisk,
respectively.
Sense and antisense oligonucleotides were annealed and
end-labeled with [-
P]ATP by T4
polynucleotide kinase and purified over a NICK column (Pharmacia
Biotech Inc.).
The SF-1 expression vector contained 2.1 kilobase pairs of the mouse SF-1 cDNA driven by cytomegalovirus promoter sequences(18) . The Pit-1 expression vector was created by placing 915 base pairs of the rat Pit-1/growth hormone factor-1 cDNA sequence from pBluescript SK(-) (Stratagene, La Jolla, CA) into the pcDNAI vector (Invitrogen, San Diego, CA) using HindIII/NotI restriction enzyme sites(21) .
Figure 2:
T3-1 nuclear extracts and in
vitro translated SF-1 bind the putative LH
-GSE region with
similar specificity. Binding reactions included
P-labeled
LHSF as a probe and, as indicated, either
T3-1 nuclear extract (lanes 1-4) or 1, 3, or 5 µl of in vitro translated SF-1 (lanes 5-11). Competition with
500-fold molar excess of unlabeled LHSF is shown for both the nuclear
extract and in vitro translated SF-1 (lanes 2 and 8). Incubation with antiserum specific to SF-1 (lanes 3 and 9) or Pit-1 (lanes 4 and 10) was
also performed using both protein preparations. Note that 3 µl of in vitro translated SF-1 were used in the cold competition and
antibody studies (lanes 8-10) and therefore band
intensity should be compared against lane 6. Lane 11 contains
a probe and the unprogrammed reticulocyte lysate used for in vitro translation. The arrowhead indicates the specific binding
complex. A nonspecific band, indicated by the asterisk, is
present in unprogrammed reticulocyte
lysate.
In order to confirm that the complex identified in Fig. 2contained SF-1, we investigated the effect of a
SF-1-specific antibody on the formation of the T3-1 nuclear
extract-LHSF complex. This antibody has previously been shown to block
the ability of SF-1 to bind to the promoter element of a number of
genes, including the glycoprotein hormone
-subunit, aromatase, and
21-hydroxylase genes(6, 9, 25) . Treatment
with this SF-1-specific antiserum substantially decreased the intensity
of the protein-DNA complex while the addition of an anti-Pit-1
antiserum, used as a negative control, had no effect (Fig. 2, lanes 3 and 4). This result confirms that the GSE of
the LH
gene promoter is bound by SF-1, or an immunologically
related protein, present in
T3-1 nuclear extracts.
Parallel
EMSA was performed using the oligonucleotide LHSF as a probe in the
presence of nuclear extracts from cell lines that do not contain SF-1.
No specific protein-DNA interactions were detected with the use of
nuclear extracts from either monkey kidney fibroblast cells (CV-1) or
rat somatolactotrope cells (GH) (data not shown).
These studies were therefore
performed in the monkey kidney fibroblast cell line, CV-1, a cell line
which has previously been shown to support SF-1-induced transactivation
of the bovine P-450 CYP11B promoter. By Northern blot analysis, this
cell line lacks the mRNA which encodes the SF-1 homolog,
Ad4BP(27) . As stated previously, we have also demonstrated
that CV-1 nuclear extract fails to bind the LH-GSE region by EMSA,
consistent with the absence of endogenous SF-1 (data not shown).
Utilizing the CV-1 cell line, basal LH
gene promoter activity
exceeded expression of the promoterless reporter plasmid (pXP2) by an
average of 15-fold.
In Fig. 4A, CV-1 cells were
cotransfected with region -209 to +5 of the LH gene
promoter and cytomegalovirus-driven expression vectors containing
either the SF-1 or Pit-1 cDNA. The presence of SF-1 markedly increased
LH
promoter activity (56 ± 5-fold). In contrast, the
pituitary transcription factor Pit-1 did not alter luciferase levels,
indicating the specificity of the SF-1 response.
Figure 4:
An intact putative LH-GSE region
confers SF-1 responsiveness to both the LH
promoter and a
heterologous minimal promoter. CV-1 cells were transiently transfected
with luciferase reporter constructs which contained various regions of
the rat LH
gene promoter. Cells were cotransfected with plasmids
encoding either SF-1 or Pit-1 and with an RSV-
-galactosidase
expression vector. Luciferase activity was normalized to
-galactosidase activity. Promoter activity was then calculated as
fold change over expression in the presence of the appropriate control
expression vector. Results are shown as the mean ± S.E. of at
least nine samples in three independent experiments. A,
comparison of LH
promoter activity in response to SF-1 versus Pit-1. B, SF-1 stimulation of LH
promoter activity
with loss of the intact GSE sequence by sequential 5`-deletion or
mutagenesis. C, SF-1 responsiveness of the growth hormone
minimal promoter (GH50) (20) or GH50 preceded by two
copies of the putative LH
-GSE region (GSE2-GH50).
Of importance, we
have recently confirmed the ability of SF-1 to increase LH
promoter activity in the rat pituitary-derived somatolactotrope cell
line, GH
. Utilizing transiently transfected GH
cells and conditions similar to those in CV-1 cells, SF-1
increased LH
promoter activity in the -209/+5 construct
by 15 ± 1.5-fold.
The evaluation of
sequential 5`-deletion constructs revealed persistent SF-1 stimulation
of LH promoter activity with deletion to position -134,
followed by an abrupt loss of the SF-1 response with deletion to
position -82 (Fig. 4B). Based on these data, loss
of LH
promoter sequences across the putative GSE region (positions
-127 to -119) correlates with the loss of SF-1-stimulated
promoter activity.
Further definition of the SF-1-responsive
cis-acting element was obtained by the introduction of a two base pair
mutation into the putative GSE site of the -209LH luciferase
reporter construct to form -209LH
-MUT. This small change,
analogous to the mutation which eliminated DNA-binding by nuclear
extract (Fig. 3), substantially decreased the ability of SF-1 to
increase promoter activity (6 ± 1-fold versus 56
± 5-fold) (Fig. 4B). Thus, transactivation of
the LH
promoter by SF-1 appears to be critically dependent on the
presence of an intact GSE sequence.
Other investigators have
shown previously that the human -subunit promoter GSE (identical
to the putative rat LH
-GSE, see Fig. 1) increases thymidine
kinase minimal promoter activity in SF-1 containing cell lines, but not
in cell lines which lack SF-1(6) . However, interpretation of
this study was limited by the possibility that additional cell-specific
factors were contributing to the observed differences in
transcriptional activity. As our results were obtained in a single cell
line, stimulation of promoter activity in the presence of the GSE can
be attributed solely to SF-1-induced effects.
Our results clearly demonstrate that SF-1 binds specifically
to the putative GSE region of the rat LH gene promoter and that,
through this interaction, SF-1 substantially increases LH
gene
promoter activity. Furthermore, we have shown that the introduction of
a two base pair mutation within the LH
-GSE sequence markedly
blunts the ability of SF-1 to either bind to or transactivate this
promoter.
It is of interest to note that the SF-1 response was not
fully lost in the mutated construct, suggesting a role for additional
nucleotides within the GSE in effecting SF-1-induced transactivation.
While parallel functional assays have not been performed, it has been
clearly demonstrated that DNA binding by SF-1 is severely blunted by
mutation of nucleotide pairs at other positions within the GSE/Ad4
element(8) . Alternatively, additional SF-1 binding sites may
contribute to the regulation of LH promoter activity. Sequence
analysis of the rat LH
promoter identifies a number of regions
which resemble the consensus GSE. It will be of interest to investigate
the possible functional significance of these regions in future
studies.
SF-1 has been shown to act at multiple levels of the
reproductive axis, including the hypothalamus, pituitary, and
gonad(7) . Within the pituitary, Mellon and colleagues have
demonstrated SF-1-stimulated expression of the glycoprotein
-subunit gene(6) . In conjunction with their results, our
data regarding the regulation of LH
gene expression suggests that
SF-1 may play a critical role in the coordinated expression of both
subunit genes required for luteinizing hormone biosynthesis in the
gonadotrope.
In the functional studies reported here, we utilized a
heterologous system in which both an SF-1 expression vector and a
reporter construct containing LH promoter sequences were
transiently transfected into a fibroblast cell line, CV-1. In
preliminary studies, we have also observed SF-1-mediated increases in
LH
promoter activity using a pituitary-derived cell line, the rat
somatolactotrope cell line, GH
. Although these studies
would ideally have been performed in a gonadotrope-derived cell line,
currently available cell lines fail to express either endogenous or
exogenous gonadotropin
-subunits at appreciable
levels(28) .
As gonadotrope-derived T3-1 cells are
known to contain SF-1(7) , the lack of LH
gene expression
may seem inconsistent with a role for SF-1 in activation of the LH
promoter. However, a number of potential explanations are possible. For
example, this cell line may be arrested at a stage of development in
which both
-subunit (E12.5) and pituitary SF-1 (E13.5) expression
have been established, but LH
-subunit expression is absent
(E16.5)(4, 7, 28) . The
T3-1 cells may
therefore be appropriately expressing an inhibitory factor(s) which is
responsible for suppression of LH
promoter activity at this
developmental stage. Alternatively,
T3-1 cells may lack the SF-1
ligand (currently postulated but as yet unidentified) or an additional
cofactor required for LH
promoter activation. This explanation
seems less likely, however, in view of the magnitude of the observed
response in CV-1 cells, a cell line that does not express endogenous
SF-1 nor any of the identified SF-1-regulated genes and would therefore
be predicted to be less likely to contain the necessary cofactors.
The results presented here clearly demonstrate that the presence of
the GSE element is sufficient to direct SF-1 responsiveness in the
context of both the LH gene promoter and the heterologous minimal
growth hormone promoter, GH50. The question remains, however, as to
whether SF-1 is required for LH
gene expression. Interestingly, in
transgenic mice null for the Ftz-F1 gene which encodes SF-1,
GnRH replacement was able to restore gonadotropin expression in four
out of five animals(7, 29) . These results suggest
that cells from the gonadotrope lineage are present in these animals
and are capable of expressing the LH
gene despite the lack of
SF-1. However, it is important to note that quantitatively normal
levels of LH in the absence of SF-1 have yet to be shown. Furthermore,
as is true for all gene ``knockout'' paradigms, this
transgenic model system does not exclude possible redundancy in the
pool of potential transactivating factors for this critical
reproductive gene. Thus, while SF-1 may not be absolutely required for
LH
gene expression, it may in fact be required for normal levels
of expression in the intact animal. The magnitude of the SF-1-directed
increase in promoter activity observed in both the CV-1 and GH
cell lines strongly implies physiological significance.
Our
results do not directly address whether SF-1, a member of the nuclear
hormone receptor superfamily, functions as a monomer or whether it has
the ability, and/or requirement, to undergo dimerization(18) .
Members of this family are best known for binding to pairs of
recognition half-sites arranged as tandem or inverted repeats. More
recently, an alternative mechanism for DNA interaction, monomer binding
to a single 5`-extended half-site, has been described for both SF-1 and
another orphan nuclear receptor, NGFI-B(30) . The results
presented here are at least consistent with the ability of SF-1 to bind
to a single GSE element in the LH gene promoter. By sequence
analysis, the rat LH
-GSE at positions -127 to -119 is
present as a single site. Furthermore, as shown by EMSA, SF-1 is able
to bind to an oligonucleotide probe in which the LH
-GSE site is
flanked by fewer than 8 additional base pairs on each side. SF-1 has
also been shown to bind to similarly short GSE-containing promoter
sequences from a variety of other genes, including the glycoprotein
hormone
-subunit, aromatase, and Mullerian inhibiting substance
genes(6, 9, 10) .
The lack of a second
DNA-response element in the region of the LH-GSE does not exclude
potential SF-1 dimerization with a non-DNA-binding partner such has
been shown to occur between the orphan nuclear receptor NurrI/NGFI-B
and the 9-cis-retinoic acid receptor (31) . Intriguingly,
mutation of the DAX-1 gene in humans results in adrenal and
gonadal hypoplasia similar to that observed in SF-1 deficient mice,
implicating DAX-1 as a potential SF-1 dimerization
partner(7, 32) .
The current studies clearly define
a role for SF-1 in the regulation of basal expression of the LH
gene. Within the pituitary gland, SF-1 expression is restricted to the
gonadotrope subpopulation and may therefore be an important modulator
of cell-specific expression(7) . SF-1 cannot, however, be the
sole determinant of gonadotrope-specific expression as it is also
expressed in non-LH-producing tissues such as the ventromedial
hypothalamus, gonad, and adrenal gland(7, 29) . It
will also be of interest to determine whether SF-1-induced increases in
LH
promoter activity interact with GnRH-stimulated responses. The
potential role of SF-1 in both tissue-specific and hormonally-mediated
expression of the LH
gene awaits further exploration.