REPRODUCTION-DEVELOPMENT
Progesterone Inhibits Basal and GonadotropinReleasing Hormone Induction of Luteinizing
Hormone ␤-Subunit Gene Expression
Varykina G. Thackray, Jennifer L. Hunnicutt, Aisha K. Memon, Yasmin Ghochani,
and Pamela L. Mellon
Departments of Reproductive Medicine and Neurosciences and the Center for Reproductive Science and Medicine,
University of California, San Diego, La Jolla, California 92093-0674
LH and FSH play critical roles in mammalian reproduction by mediating steroidogenesis and gametogenesis in the gonad. Gonadal steroid hormone feedback to the hypothalamus and pituitary
influences production of the gonadotropins. We previously demonstrated that progesterone differentially regulates the expression of the LH and FSH ␤-subunits at the level of the gonadotrope:
FSH␤ transcription is induced, whereas LH␤ is repressed. In this study, we investigated the mechanism of progesterone repression of LH␤ gene expression using immortalized gonadotrope-derived L␤T2 cells. The progesterone suppression of both basal and GnRH-induced LH␤ gene expression occurs in a hormone- and receptor-dependent manner. Chromatin immunoprecipitation
demonstrates that the hormone-bound progesterone receptor (PR) is recruited to the endogenous
mouse LH␤ promoter. In addition, suppression requires both the amino-terminal and DNA-binding
regions of PR. Furthermore, progesterone suppression does not require direct PR binding to the
promoter, and, thus, PR is likely recruited to the promoter via indirect binding through other
transcription factors. These data demonstrate that the molecular mechanism for progesterone
action on the LH␤ promoter is distinct from FSH␤, which involves direct PR binding to the promoter
to produce activation. It also differs from androgen repression of LH␤ gene expression in that,
rather than Sp1 or steroidogenic factor-1 elements, it requires elements within ⫺300/⫺250 and
⫺200/⫺150 that also contribute to basal expression of the LH␤ promoter. Altogether, our data
indicate that progesterone feedback at the level of the pituitary gonadotrope is likely to play a key
role in differential production of the gonadotropin genes. (Endocrinology 150: 2395–2403, 2009)
he mammalian hypothalamic-pituitary-gonadal axis controls reproduction, including sexual development, puberty,
the menstrual cycle, pregnancy, and menopause. GnRH is secreted in a pulsatile manner directly into the hypophyseal portal
system by neurons in the hypothalamus (1). GnRH then activates
its receptor on the surface of gonadotrope cells within the anterior pituitary. Activation of the GnRH receptor leads to synthesis
and secretion of LH and FSH, consisting of a common ␣-subunit
and a unique ␤-subunit (2). These glycoprotein hormones are
secreted into the bloodstream to regulate gametogenesis; folliculogenesis; and production of testosterone, estrogen, and progesterone in the gonads (3–5). Subsequently these steroids feed back
T
to regulate expression and secretion of GnRH, LH, and FSH in
the hypothalamus and the anterior pituitary.
Because synthesis of LH␤ and FSH␤ is the rate-limiting step
in gonadotropin production (6, 7), transcriptional regulation is
a key focus. The conserved proximal promoter of LH␤ binds
several transcriptional regulators that modulate cell-specific expression including steroidogenic factor (SF)-1 (8, 9), early
growth response protein (Egr)-1, and pituitary homeobox
1/orthodenticle homeobox 1 (10 –12). These proteins, as well as
those binding to more distal elements, interact to elicit basal
promoter activity. GnRH induction of the rat LH␤ promoter has
been shown to involve a tripartite GnRH response element, com-
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2009 by The Endocrine Society
doi: 10.1210/en.2008-1027 Received July 23, 2008. Accepted December 17, 2008.
First Published Online December 23, 2008
Abbreviations: AR, Androgen receptor; ChIP, chromatin Immunoprecipitation; DBD, DNAbinding domain; Egr, early growth response proteín; PR, progesterone receptor; PRE,
progesterone response element; SF, steroidogenic factor; TK, thymidine kinase.
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PR Suppresses LH␤ Gene Expression
posed of two Sp1 sites in the distal promoter, and proximal pairs
of SF-1 and Egr-1 binding elements (10).
In addition to GnRH, steroid hormones play a pivotal role in
LH synthesis. Many studies have shown that LH␤ mRNA levels
increase after gonadectomy and subsequently decrease with reintroduction of gonadal steroid hormones (13, 14). Steroid hormone receptors are expressed in gonadotrope cells (15–17), indicating that regulation by gonadal steroids likely occurs directly
at the level of the pituitary, in addition to the hypothalamus.
Estrogens suppress LH␤ synthesis due to negative feedback at the
hypothalamus (18, 19), but they also exert a direct effect on the
pituitary by inducing LH␤ gene expression in gonadotropes
(20, 21). In addition, many studies indicate that androgens
repress LH␤ mRNA directly in the pituitary. Androgens decrease LH␤ mRNA levels in castrated, GnRH antagonisttreated rats (22) and primary pituitary cells (23). Furthermore,
androgens suppress LH␤ gene expression in immortalized gonadotrope cells (24, 25).
Despite evidence for progesterone enhancement of GnRHinduced LH secretion (26), it is unclear whether progesterone
regulates LH␤ gene expression directly in the pituitary gonadotrope. Because levels of LH␤ mRNA are lowest during the luteal
phase of the ovulatory cycle when circulating progesterone levels
are at their highest, progesterone is an excellent candidate for
countering the stimulatory effects of GnRH on LH␤ gene expression. Many studies have shown that, in the presence of estrogen, progesterone can suppress LH␤ mRNA levels in rodents
(27–29), although these experiments did not differentiate between hypothalamic and pituitary sites of action. Contrary to
other studies that did not report an effect of progesterone on LH␤
(30, 31), we recently demonstrated that progesterone can suppress LH␤ gene expression in gonadotrope cells, in contrast to its
stimulatory effect on the FSH␤ promoter (32), indicating that
progesterone may serve as a differential regulatory influence on
the two gonadotropins during the menstrual cycle.
In mammals, gonadotropes comprise approximately 10% of
the anterior pituitary cell population, making it difficult to conduct mechanistic studies of their function (33). Development of
the immortalized L␤T2 cells provided the opportunity to analyze
mechanisms of steroid hormonal regulation of the gonadotropin
genes in the context of a pure population of gonadotrope cells
(34). The immortalized L␤T2 cell line expresses many markers of
a mature gonadotrope including FSH␤, LH␤, activin, follistatin,
and inhibin as well as activin, inhibin, and steroid receptors (32,
35–38). In this study, we used transient transfection experiments
in L␤T2 cells to demonstrate that progesterone suppresses both
basal transcription and GnRH-induced LH␤ gene expression in
gonadotrope cells. Because estrogen is necessary for the induction of progesterone receptor (PR) but may mask the repressive
effects of progesterone due to its stimulatory effect on LH␤ gene
expression, we enhanced PR levels directly by overexpressing the
receptor in L␤T2 cells. We defined and characterized a progesterone-responsive region in the proximal LH␤ promoter necessary and sufficient for the suppressive effect. We determined that
progesterone suppression is dependent on the presence of PR and
that PR is recruited to the endogenous LH␤ promoter after hormone treatment in L␤T2 cells. Interestingly, direct binding of PR
Endocrinology, May 2009, 150(5):2395–2403
to the LH␤ promoter does not appear to be necessary for the
suppression, although the DNA-binding domain (DBD) of PR is
required. These results suggest that the progesterone suppression
of LH␤ gene expression is through indirect binding of PR to the
DNA via other transcription factors.
Materials and Methods
Cell culture and transient transfection
Cell culture and transient transfections were performed as described
previously (32). L␤T2 cells were plated in 12-well plates at 3 ⫻ 105
cells/well. Wells were transfected with 400 ng of reporter plasmid and
100 ng of a PR expression vector, unless otherwise noted. Cells were also
transfected with 200 ng of a thymidine kinase (TK)-␤-gal reporter as a
control for transfection efficiency. Six hours after transfection, cells were
switched to serum-free DMEM. Eighteen hours later, cells were treated
for 6 h (unless otherwise noted) with one of the following treatments:
0.1% ethanol and 0.1% BSA (vehicle control); 10⫺7 M promegestone
(R5020) and 0.1% BSA; 0.1% ethanol and 10⫺8 M GnRH; or both 10⫺7
⫺8
M R5020 and 10
M GnRH. R5020 was purchased from NEN Life
Science Products Life Sciences (Boston, MA) and progesterone, mifepristone (RU486) and GnRH from Sigma (St. Louis, MO). Luciferase
and ␤-galactosidase activity were measured as previously described (32).
Transfection data were analyzed by one-way ANOVA, followed by post
hoc comparisons with the Tukey-Kramer honestly significant difference
test or two-way ANOVA using the statistical package JMP 7.0 (SAS,
Cary, NC).
Subcloning and mutagenesis
PCR was performed using the appropriate primers (supplemental
Table 1, published as supplemental data on The Endocrine Society’s
Journals Online web site at http://endo.endojournals.org) to create 5⬘
truncations of the ⫺1800-bp LH␤ promoter at ⫺500, ⫺300, ⫺150, and
⫺87. Fragments were inserted between KpnI and HindIII in pGL3. A
similar strategy was used to insert ⫺300/⫺150 of the LH␤ promoter into
a ⫺81-TK-luc reporter in forward or reverse orientation.
The PRB C577A DBD mutant unable to bind DNA was described
previously (32). The QuikChange kit (Stratagene, La Jolla, CA) was used
to generate the 5⬘ Sp1/CArG, 3⬘ Sp1, triple Sp1/CArG, 5⬘ SF-1, 3⬘ SF-1,
5⬘ Egr-1, and 3⬘ Egr-1 mutations in the ⫺1800-bp LH␤ promoter (oligonucleotides in supplemental Table 1). The progesterone response element (PRE) mutation was made in the ⫺500 LH␤ promoter. The ⫺300/⫺150,
⫺300/⫺250, ⫺250/⫺200, and ⫺200/⫺150 deletions were made in the
⫺1800- or ⫺500-bp LH␤ promoters. Dideoxynucleotide sequencing
confirmed mutagenesis.
EMSA
Full-length human PRB was overexpressed in Sf9 cells via a baculovirus expression system. The cell lysate was centrifuged at 40,000 rpm for
30 min, and the supernatant was taken as a soluble whole-cell extract or
purified as described previously (39) and used in EMSA as described (32).
The 1294 PR mouse monoclonal antibody was used to supershift PR and
mouse IgG was used as a control. Oligonucleotides used for EMSA are
shown in supplemental Table 1.
Chromatin immunoprecipitation (ChIP)
Confluent L␤T2 cells in 15-cm plates were treated with vehicle or
10⫺7 M R5020 and cross-linked with 1% formaldehyde. The nuclear
fraction was obtained and chromatin was sonicated. Protein-DNA complexes were incubated overnight with 1294 PR antibody or IgG control
and precipitated with protein A/G beads. Beads were washed, the protein-DNA complexes eluted, the cross-links reversed, and the DNA precipitated, as described (32). PCR primers for the LH␤ promoter spanned
Endocrinology, May 2009, 150(5):2395–2403
the 220-bp sequence in the mouse LH␤ gene from ⫺180 to ⫹40 (supplemental Table 1).
Results
LH␤ gene expression is suppressed in immortalized
gonadotropes by progesterone
Recently we demonstrated that both testosterone and progesterone can differentially modulate gonadotropin synthesis in
pituitary gonadotrope cells by enhancing FSH␤ and repressing
LH␤ gene expression (32). Because testosterone has been shown
to suppress GnRH induction of LH␤ gene transcription (24, 25),
we hypothesized that progesterone might have a similar effect.
Progesterone treatment suppressed both basal promoter activity
and GnRH-induced LH␤ gene transcription (Fig. 1A). Progesterone repressed basal gene expression by 10%. Treatment of the
L␤T2 cells with GnRH alone resulted in a 4.8-fold induction,
which was reduced 65% by progesterone. This suppression was
maintained for over 24 h (data not shown). Treatment of the cells
with the synthetic progestin, R5020, elicited a similar suppression on basal and GnRH-induced LH␤ gene expression
(Fig. 1A), indicating that R5020 acts in an analogous manner
to progesterone.
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To test whether suppression of GnRH induction was dosedependent, cells were treated with vehicle, 10⫺7 M R5020, 10⫺8 M
GnRH, or 10⫺8 M GnRH and increasing concentrations of
R5020 from 10⫺12 to 10⫺7 M (Fig. 1B). In these experiments, the
progestin alone repressed basal LH␤ gene expression by 30%
(shown on an expanded scale as an inset in Fig. 1B). GnRH
treatment resulted in a 4.2-fold induction. As the R5020 concentration was increased, the suppression of GnRH induction
increased in concert until saturation at 10⫺9 M (approximately
70% reduction by 10⫺9 to 10⫺7 M). All subsequent experiments
used 10⫺7 M R5020 to ensure saturation of the receptor. Together these experiments demonstrate that the progesterone regulation of LH␤ occurs in a saturable, dose-dependent manner.
Ligand-bound PR is necessary for maximal suppression
of LH␤ gene expression
We next examined whether the suppression of LH␤ by progesterone required the presence of its classical nuclear receptor.
L␤T2 cells were transfected with increasing concentrations of the
PRB expression vector to mimic estrogen induction of PR levels
without the complication of estrogen action on LH␤ gene expression. Basal LH␤ gene expression was not suppressed by
R5020 unless PR was transfected into the cells (Fig. 1C). With
100 and 400 ng of PR expression vector, R5020 repressed basal
gene expression by 25 and 33%, respectively.
As increasing amounts of PR were transB
A
fected into the cells, GnRH induction (in
the absence of R5020) decreased from 3.8fold (when no receptor was added) to 3.5-fold
with 100 ng PR and then down to 2.5-fold with
400 ng PR, indicating that the unliganded PR
may slightly repress GnRH-induced LH␤ gene
expression (Fig. 1C), although it had no effect
on basal LH␤ levels (data not shown). Whereas
D
C
GnRH induction decreased due to higher levels
of exogenous receptor, the suppression of the
GnRH induction by PR liganded with R5020
remained relatively constant. Thus, although
low levels of PR are expressed endogenously
(32, 40), transfection of exogenous receptor
was necessary to observe significant suppression of both basal transcription and GnRH induction. Because suppression did not increase
FIG. 1. Progesterone suppresses basal and GnRH-induced LH␤ gene expression in immortalized
with greater amounts of exogenous receptor,
gonadotrope cells. A, The ⫺1800-bp LH␤-luc reporter plasmid was transiently transfected into L␤T2 cells
all subsequent experiments used 100 ng of PR
with 100 ng of rat PRB expression vector. After overnight starvation in serum-free media, the cells were
treated for 6 h with vehicle, 100 nM progesterone, 100 nM R5020, 10 nM GnRH, or both GnRH and the
expression vector.
relevant progestin as indicated. B, The ⫺1800-bp LH␤-luc reporter plasmid was transiently transfected
Once we demonstrated that the ligandinto L␤T2 cells with 100 ng of rat PRB expression vector. After overnight starvation in serum-free media,
bound PR was required for suppression of
cells were treated with vehicle, 100 nM R5020, 10 nM GnRH, or GnRH with increasing amounts of R5020
(10⫺12 to 10⫺7 M). Inset illustrates the decrease in basal due to R5020 alone on an expanded scale. C,
LH␤ gene expression by progesterone, we
The ⫺1800-bp LH␤-luc reporter plasmid was transiently transfected into L␤T2 cells along with 0, 100, or
tested whether this effect could be blocked by
400 ng of PRB expression vector. After overnight starvation in serum-free media, the cells were treated
the PR antagonist, RU486. Treatment with
for 6 h with vehicle, 100 nM R5020, 10 nM GnRH, or both GnRH and R5020 as indicated. D, The ⫺1800bp LH␤-luc reporter plasmid was transiently transfected into L␤T2 cells with 100 ng of PRB expression
100-fold higher levels of RU486 abrogated
vector. After overnight starvation in serum-free media, the cells were treated for 6 h with vehicle, 100 nM
the suppressive effects of R5020 on basal and
R5020, 10 nM GnRH, or both GnRH and R5020 as well as either vehicle or 1 ␮M RU486. The data were
GnRH-induced LH␤ gene expression (Fig.
normalized for transfection efficiency by expressing luciferase (luc) activity relative to ␤-galactosidase
activity and relative to the empty pGL3 plasmid to control for hormone effects on the vector DNA. Results
1D). Interestingly, the GnRH induction was
represent the mean ⫾ SEM of at least three independent experiments performed in triplicate and are
also suppressed 30% by the RU486 antagopresented as fold induction of hormone treatment relative to the vehicle control. *, Significant differences
nist in the absence of the R5020 agonist,
from the vehicle-treated control; †, interaction as defined by a two-way ANOVA (P ⬍ 0.05) (45).
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PR DNA-binding domain is essential for progesterone
suppression of LH␤ gene expression
To determine whether PR can specifically associate with the
endogenous mouse LH␤ promoter in L␤T2 cells, ChIP analysis
with an antibody specific to PR was used. Figure 2A demonstrates
that PR localizes to the endogenous mouse LH␤ promoter; weakly
in the absence of hormone and more strongly in the presence of
R5020 (Fig. 2A, lanes 4 and 6). In contrast, there was no precipitation of LH␤ promoter DNA with a nonspecific IgG control (lanes
3 and 5). The LH␤ promoter was amplified from input chromatin
(lanes 1 and 2) as a positive control for genomic DNA preparation
and PCR conditions. This experiment demonstrated that PR is recruited to the endogenous proximal LH␤ promoter, suggesting that
the mechanism of progesterone suppression involves a direct action
of the agonist-bound receptor on the LH␤ promoter.
PR has two isoforms: PRB and PRA. The PRA isoform lacks
the amino-terminal transactivation domain of PRB. Both iso-
forms are expressed in pituitary gonadotropes from the same
gene and modulation of their ratio is thought to contribute to the
degree of progesterone augmentation of GnRH-induced secretion (40). To determine which regions of PR play a critical role
in the suppression of LH␤ gene expression, transient transfection
assays were performed using the PRA isoform and a PRB C557A
DBD mutant receptor unable to bind to DNA. As observed previously, PRB suppressed basal LH␤ promoter activity by 46%
and the GnRH induction by 56%. The PRB-DBD mutant resulted in a complete disruption of R5020 repression in the absence or presence of GnRH induction (Fig. 2B). Similarly, when
an expression vector for the mouse PRA isoform was transfected
into L␤T2 cells, there was no significant suppression of basal
gene expression by progesterone (Fig. 2C). In the presence of
PRA, suppression of GnRH induction was reduced by 19%,
but the trend did not approach statistical significance. These
data suggest that the PR DBD plays a critical role and that the
unique amino-terminal region of PRB is involved in suppression of basal transcription and GnRH induction of the LH␤
subunit.
FIG. 2. PR binds to the endogenous LH␤ promoter in L␤T2 cells, and the DNAbinding domain is necessary for progesterone suppression of LH␤ gene
expression. A, ChIP was performed using cross-linked protein/chromatin from
L␤T2 cells treated with vehicle (Veh) or R5020 and antibodies directed against PR
or nonspecific IgG as a negative control. PCR primers encompassing the proximal
promoter of LH␤ were used to detect precipitation of genomic DNA. PCR
amplification was performed on 0.2% chromatin input (lanes 1 and 2), and
chromatin was precipitated with either mouse IgG (lanes 3 and 5) or PR antibody
(lanes 4 and 6). B, The ⫺1800-bp LH␤-luc reporter plasmid was cotransfected
with wild-type PRB or PRB DBD mutant (PRC577A). C, The ⫺1800-bp LH␤-luc
reporter plasmid was cotransfected with mouse PRB or PRA. After overnight
starvation in serum-free media, the cells were treated with vehicle, 100 nM
R5020, 10 nM GnRH, or both hormones as indicated. *, Significant differences
from the vehicle-treated control; †, interaction as defined by a two-way ANOVA
(P ⬍ 0.05). luc, Luciferase.
Mutation of Sp1, SF-1, or Egr-1 elements in the distal or
proximal LH␤ promoter does not alleviate progesterone
suppression of LH␤ transcription
One potential mechanism for progesterone suppression of
LH␤ transcription is through an interaction between ligandbound PR and the transcription factor Sp1 similar to the interaction
reported between androgen receptor (AR) and Sp1 on the rat LH␤
promoter (24). Alternatively, ligand-bound PR may interact with
proximal promoter binding transcription factors, like the AR/SF-1
interaction observed on the bovine LH␤ promoter (25).
To investigate the hypothesis that Sp1 binding sites are critical
for PR suppression of LH␤ transcription, specific mutations in
Sp1 elements were created within the ⫺1800-bp rat LH␤ promoter. Figure 3A illustrates the location of the transcription factor binding sites on the LH␤ promoter. The 5⬘ Sp1/CArG double
mutation and the 3⬘ Sp1 mutation increased basal transcription
compared with wild type (1.5- and 1.8-fold, respectively),
whereas the triple mutation decreased basal activity by 93%
(data not shown). In contrast, GnRH induction was not significantly altered by these mutations (Fig. 3B). There was no discernible difference in basal gene expression or GnRH induction
of LH␤ among the wild-type LH␤ or these mutants after R5020
treatment. These data suggest that the Sp1-binding elements do
not play a critical role in suppression of GnRH induction by
progesterone.
Site-specific mutations were also generated in the 5⬘ SF-1, 3⬘
SF-1, 5⬘ Egr-1, and 3⬘ Egr-1 binding elements within the
⫺1800-bp rat LH␤ promoter. Each of these four mutations resulted in a reduction of basal promoter activity by approximately
40% (data not shown). GnRH treatment produced a 3.7-fold
induction of the wild-type promoter (⫺1800 LH␤, Fig. 3C). Mutation of the 5⬘ SF-1 site led to a greater stimulation by GnRH,
whereas mutation of the other proximal promoter binding sites
resulted in a diminished response to GnRH: 3-fold with the 3⬘
SF-1 mutant, 1.6-fold with the 5⬘ Egr-1 mutant, and 1.5-fold
with the 3⬘ Egr-1 mutant, similar to previous reports (8, 9, 43).
probably due to its known actions as a partial agonist when
bound to PR (41, 42).
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FIG. 3. Mutation of Sp1, SF-1, and Egr-1 binding elements on the LH␤ promoter
does not alleviate progesterone suppression of LH␤ transcription. A, A schematic
of the rat LH␤ promoter illustrating the known transcription factor binding sites
involved in basal and GnRH-induction of LH␤ gene expression. The rat LH␤
promoter contains two Sp1 sites at ⫺451/⫺442 and ⫺398/⫺386. The most
distal Sp1 element overlaps with a CArG box (⫺443/⫺434). The proximal
promoter includes two SF-1 binding elements at ⫺127/⫺119 and ⫺58/⫺51 and
two Egr-1 binding sites at ⫺112/⫺104 and ⫺49/⫺41. B and C, L␤T2 cells were
transiently transfected with either the ⫺1800-bp LH␤-luc reporter plasmid or
reporter plasmids containing mutations in the Sp1, SF-1, or Egr-1 binding
elements as indicated. After overnight starvation in serum-free media, the cells
were treated with vehicle, 100 nM R5020, 10 nM GnRH, or both hormones as
indicated. *, Significant differences from the vehicle-treated control; †, interaction as
defined by a two-way ANOVA (P ⬍ 0.05). luc, Luciferase.
Regardless of the mutation, R5020 still repressed basal gene expression and suppressed the remaining GnRH induction, indicating
that these proximal promoter elements are not essential for the
suppression of LH␤ gene expression by progestins.
Progesterone suppression of LH␤ transcription maps to
a region located ⴚ300/ⴚ150 upstream of the
transcription start site of the LH␤ promoter
Because the suppression of LH␤ gene expression by progesterone did not appear to require the known transcription factor
binding elements, we used truncation analysis to identify the
necessary regions. Truncation to ⫺500, ⫺150, or ⫺87 resulted
in significant reductions in basal activity in the absence of any
hormones compared with the ⫺1800-bp LH␤-luc reporter,
whereas the ⫺300 truncation increased basal gene expression
(Fig. 4A). These results suggest that transcription factors important for basal gene expression also bind the ⫺300/⫺150 region,
in addition to the more distal and proximal regions of the LH␤
promoter.
GnRH induction of the ⫺500 LH␤ truncation was comparable with that seen with the ⫺1800-bp LH␤-luc reporter (Fig.
4B). However, the ⫺500 LH␤ truncation exhibited only a partial
reduction by progesterone of basal promoter activity and GnRH
FIG. 4. Progesterone suppression of basal and GnRH-induced LH␤ gene
expression maps to the ⫺300 to ⫺150 region of the LH␤ promoter. A, L␤T2 cells
were transiently transfected with either the ⫺1800-bp LH␤-luc reporter plasmid
or 5⬘ promoter truncations to compare basal transcriptional activity in the
absence of hormone treatment. luc, Luciferase. B, L␤T2 cells were transiently
transfected with either the ⫺1800-bp LH␤-luc reporter plasmid or 5⬘ promoter
truncations to compare the effects of hormone treatments. C, L␤T2 cells were
transiently transfected with either ⫺1800-bp or ⫺500 LH␤-luc reporter plasmids
or ⫺1800-bp or ⫺500 LH␤-luc reporter plasmids containing a 150-bp deletion of
the region between ⫺300/⫺150 of the LH␤ promoter. D, Cells were transfected
with a TK-luc reporter plasmid containing the ⫺300/⫺150 repressive element in
the forward or reverse orientation. After overnight starvation in serum-free
media, the cells were treated with vehicle, 100 nM R5020, 10 nM GnRH, or both
hormones as indicated (B–D). Luciferase activity was normalized to ␤galactosidase activity and set relative to the empty reporter vector (A–C, pGL3;
and D, TK-luc). Results represent the mean ⫾ SEM of at least three independent
experiments performed in triplicate and are presented as luc/␤-gal for basal gene
expression (A) or fold induction of hormone treatment relative to the vehicle
control (B–D). *, Significant differences from the vehicle-treated control;
†, interaction as defined by a two-way ANOVA (P ⬍ 0.05).
induction compared with the ⫺1800-bp LH␤-luc reporter, suggesting the presence of an upstream region involved in the suppression of GnRH induction of LH␤ by progesterone. The ⫺300
LH␤-luc reporter showed a significant repression of basal activity and GnRH induction after progesterone treatment (Fig. 4B).
In contrast, the ⫺150 LH␤ truncation lost the suppression of
basal gene expression and GnRH induction by progesterone,
suggesting that the ⫺300/⫺150 region of the LH␤ promoter is
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critical for the progesterone suppression. Interestingly, when the
⫺87 LH␤ promoter was tested, the effects were reversed. R5020
treatment resulted in a significant increase in ⫺87 LH␤ transcription (23.5%), and cells treated with both GnRH and R5020 showed
a synergistic induction compared with the individual hormones.
The repressive element at ⴚ300/ⴚ150 of the LH␤
promoter is necessary and sufficient for progesterone
suppression
Because the suppression mapped to the ⫺300/⫺150 region
using 5⬘ truncations of the LH␤ promoter, we tested whether this
region was necessary for progesterone repression of both basal
LH␤ gene expression and GnRH induction. Deletion of this region within the context of the ⫺1800-bp LH␤ promoter still
resulted in progesterone suppression (Fig. 4C). Given the results
from the truncation analysis, we hypothesized that an upstream
region may compensate for the repressive element at ⫺300/
⫺150, so we created a 150-bp deletion within the ⫺500 LH␤
promoter. Basal gene expression was reduced by 40% (data not
shown), indicating that this region has a role in LH␤ basal transcriptional regulation. Interestingly, there was no suppression of
basal LH␤ gene expression or the GnRH induction by R5020
with the repressive element deleted in the context of the ⫺500
LH␤ promoter (Fig. 4C), indicating that this 150-bp region
(⫺300/⫺150) is necessary for the progesterone suppression in
the context of the proximal promoter.
To assess whether the ⫺300/⫺150 repressive element in the
LH␤ promoter is sufficient for suppression by progesterone, we
inserted the 150-bp element upstream of a heterologous TK-luc
reporter in the forward or reverse orientation. Basal TK promoter activity was suppressed more than 50% by the addition of
this element (data not shown). Progesterone further suppressed
promoter activity by 30% when the ⫺300/⫺150 repressive element was in the forward or reverse orientation (Fig. 4D), indicating that this 150-bp element from the proximal LH␤ promoter is sufficient to elicit the suppression by progesterone.
PR binding to the repressive element at ⴚ300/ⴚ150 is
not critical for LH␤ suppression
Given that the ⫺300/⫺150 region of the LH␤ promoter was
essential for progesterone suppression, EMSA was performed to
assess whether PR can bind to the proximal rat LH␤ promoter in
vitro. Six 35-mer oligonucleotide probes were designed to span
the ⫺300/⫺150 region. Purified PRB bound the ⫺225/⫺191
oligonucleotide probe (Fig. 5A, lane 1), whereas no binding to
the other probes in this region was observed (data not shown). To
further demonstrate that PR bound specifically to the ⫺225/
⫺191 probe, the resulting complex was supershifted by a PRspecific antibody (lane 3) but not by IgG (lane 4). This complex
also showed evidence of competition with a consensus PRE (data
not shown). To further define where PR binds to the proximal
LH␤ promoter, EMSA with the wild-type ⫺225/⫺191 probe
and 12 3-bp scanning mutations were used as probes. The mutated probes that could not bind PR (Fig. 5B, lanes 2, 3, 5, and
6) encompassed a putative PRE at ⫺221/⫺207.
Once we had ascertained that PR could bind a putative PRE
at ⫺221/⫺207, transient transfection assays were used to deter-
FIG. 5. PR binds to a putative PRE at ⫺221/⫺207, but mutation of the PRE does
not alter progesterone suppression. A, Purified PRB was incubated with a wildtype (lane 1) or PRE mutant (lane 2) ⫺225/⫺191 probe (supplemental Table 1)
and tested for complex formation in EMSA. The addition of a PR antibody (PR
Ab, lane 3) or nonspecific IgG control antibody (IgG, lane 4) to the binding
reaction, are indicated. PR binding and the antibody supershift are also indicated.
B, EMSA was performed with PR whole-cell extract using either wild-type (WT)
⫺225/⫺191-labeled probe or oligonucleotides containing 3-bp scanning
mutations as indicated, each with three adjacent A substitutions in the 3-bp
sequence shown above each of the lanes. C, L␤T2 cells were transfected with
either the ⫺500 LH␤-luc reporter plasmid or a reporter containing a PRE cis
mutation in which the G and C residues important for high-affinity DNA binding
were mutated (supplemental Table 1). Luciferase (Luc) activity was normalized to
␤-galactosidase activity and set relative to the empty reporter vector. Results
represent the mean ⫾ SEM of at least three independent experiments performed
in triplicate and are presented as fold induction of hormone treatment relative to
the vehicle control. *, Significant differences from the vehicle-treated control;
†, interaction as defined by a two-way ANOVA (P ⬍ 0.05).
mine whether direct PR binding to the PRE played a functional
role in the progesterone suppression of LH␤ gene expression. For
this experiment, L␤T2 cells were transiently transfected with the
⫺500 LH␤-luc reporter or a PRE cis mutation in which the G and
C residues important for high-affinity DNA binding were mutated (supplemental Table 1). This PRE mutant does not bind PR
in EMSA (Fig. 5A, lane 2). R5020 suppressed the basal promoter
activity and the GnRH-induction of the wild-type LH␤ promoter
and the PRE mutant (Fig. 5C), indicating that direct PR binding
Endocrinology, May 2009, 150(5):2395–2403
to the putative ⫺221/⫺207 PRE is not necessary for the suppressive effect of progesterone.
Two 50-bp regions of the repressive element are critical
for LH␤ suppression
Because direct binding of PR to the repressive region did not
appear to be required for progesterone suppression, we created
three sequential 50-bp deletions within the ⫺500 LH␤ promoter.
Each of these three deletions resulted in greater than 50% reduction in basal gene expression (Fig. 6A), indicating that these
regions must contribute to basal gene expression, most likely by
binding specific transcriptional activators. The promoter containing the ⫺250/⫺200 deletion was suppressed by progesterone
for both basal LH␤ expression and GnRH induction (Fig. 6B). In
contrast, there was no significant progesterone suppression after
either the ⫺300/⫺250 or ⫺200/⫺150 regions were deleted, indicating that more than one region of the 300-bp element is necessary
for suppression by progesterone. In silico analysis of these 50-bp
regions revealed a number of putative transcription factor binding
elements. However, 10-bp scanning deletions through the two important regions did not prevent progesterone suppression of GnRH
induction of the LH␤ promoter (data not shown), suggesting that
multiple elements within each region may be important.
FIG. 6. Multiple elements are required for suppression. A, L␤T2 cells were
transiently transfected with either the ⫺500 bp LH␤-luc reporter plasmid or
⫺500 LH␤-luc reporter plasmids containing specific 50-bp deletions, as indicated,
to compare basal transcriptional activity in the absence of hormone treatment. B,
L␤T2 cells were transfected with either the ⫺500 LH␤-luc reporter plasmid or
reporter plasmids containing specific 50-bp deletions as indicated. After
overnight starvation in serum-free media, the cells were treated with vehicle, 100
nM R5020, 10 nM GnRH, or both hormones as indicated. Luciferase (Luc) activity
was normalized to ␤-galactosidase activity and set relative to the empty reporter
vector. Results represent the mean ⫾ SEM of at least three independent
experiments performed in triplicate and are presented as luc/␤-gal for basal gene
expression (A) or fold induction of hormone treatment relative to the vehicle
control (B). *, Significant differences from the vehicle-treated control; †, interaction
as defined by a two-way ANOVA P ⬍ 0.05).
endo.endojournals.org
2401
Discussion
Our study demonstrates that progestins can suppress both basal
transcription and GnRH induction of the LH␤ gene in gonadotrope cells. Similar to androgens, progesterone suppression occurs in a hormone- and receptor-dependent manner, indicating
that the actions of progesterone are through the classical PR.
Moreover, the fact that PR is recruited to the endogenous LH␤
promoter after hormone treatment and that the suppression occurred in as little as 6 h, indicate that progesterone mediates
suppression of LH␤ gene expression through a mechanism directly involving the LH␤ promoter.
Maximal suppression of LH␤ transcription by progesterone
requires the hormone-bound PRB complex containing an intact
DBD. Disruption of the PR DBD abrogated the repression of
basal transcription as well as the suppression of GnRH induction
by progesterone. One possible role for the PR DBD is for direct
binding of PR to the LH␤ promoter. To investigate this possibility, we searched for PREs within the ⫺300/⫺150 repressive
element necessary for progesterone suppression. We identified a
putative PRE at ⫺225/⫺191 that bound PR in EMSA. However,
mutation of this site or deletion of a 50-bp region from ⫺250/
⫺200 that encompassed this element did not relieve progesterone suppression of LH␤ transcription, suggesting that direct
DNA binding by PR is not necessary for this effect, in contrast to
progesterone induction of the FSH␤ gene that requires direct
binding of PR to the proximal promoter. Another possibility for
the role of the PR DBD in the suppression of LH␤ transcription
by progesterone is through a protein-protein interaction with
another transcription factor involved in LH␤ expression, rather
than direct DNA binding. For instance, Curtin et al. (24) demonstrated that the AR DBD interacts directly with Sp1 and as a
result reduces Sp1 binding to the rat LH␤ promoter, thereby
decreasing the GnRH response. However, it is unlikely that Sp1
is involved in progesterone suppression because mutation of
both of the Sp1 elements in the distal LH␤ promoter had no effect
and overexpression of Sp1 did not relieve repression (data not
shown).
In addition to the PR DBD, we also demonstrated that the
unique amino-terminal region of the PRB isoform is necessary
for the full suppression of LH␤ by progesterone. This region has
been shown to contain a transactivation function that is thought
to be the reason that PRB is generally a stronger activator than
PRA. As a result of this additional region, PRB likely forms a
more stable secondary and/or tertiary structure than PRA [recently reviewed by Bain et al. (44)] that favors the interaction of
factor(s) necessary for progesterone suppression of LH␤ gene
expression.
Initially, we had hypothesized that the mechanism of action
for progesterone suppression would be similar to the reported
androgen suppression on either the rat (24) or bovine (25) promoter. Similarities between the two mechanisms include the necessity for liganded receptor and an intact DBD. The most significant difference is that the site of action for PR on the LH␤
promoter is distinct from that of AR. Mutations in the distal Sp1
sites failed to relieve progesterone suppression of both basal and
GnRH-induced LH␤ gene expression, indicating that these ele-
2402
Thackray et al.
PR Suppresses LH␤ Gene Expression
ments are not critical for the suppression by progesterone. Mutation of the SF-1 or Egr-1 binding sites in the proximal promoter
also did not relieve suppression by progesterone, suggesting that
these elements do not play a critical role in progesterone suppression of LH␤ mRNA levels. Our experiments with the 5⬘
truncations of the LH␤ promoter provided supporting evidence
for these conclusions. Specifically, a ⫺300 LH␤-luc reporter
lacking the distal Sp1 elements was sufficient for suppression by
progesterone, and conversely, a ⫺150 LH␤ truncation (containing both Egr-1 and SF-1 binding sites) was not suppressed by
progesterone.
In addition to highlighting the different mechanisms of repression by progestins vs. androgens, our analysis also revealed
that several regions in the LH␤ promoter contribute to the suppression by progesterone. Truncation analysis identified a repressive element between ⫺300/⫺150 that was both necessary
and sufficient to elicit progesterone suppression. Because this
region is less conserved among mammalian species than the
proximal LH␤ promoter, it remains to be determined whether
progesterone suppression of LH␤ transcription occurs in a species-specific manner. The fact that deletion of the repressive element resulted in loss of suppression in the context of the ⫺500
LH␤ promoter but not the ⫺1800-bp promoter suggests that a
region upstream of ⫺500 also contributes to progesterone suppression of both basal and GnRH-induced LH␤ gene expression
by progesterone.
To further characterize the critical region necessary for suppression of LH␤ transcription in the proximal promoter, we
created and tested three 50-bp deletions in the ⫺300/⫺150 region. All three deletions reduced basal activity of the LH␤ promoter by about 50%, indicating that they likely bind factors
important for LH␤ gene expression. Deletion of either the region
from ⫺300/⫺250 or ⫺200/⫺150 resulted in a lack of suppression by progesterone, indicating that multiple elements may be
required but that spacing is less critical because the middle 50 bp
can be deleted without effects on progesterone regulation. Also
supporting the idea that multiple elements in the proximal promoter may be responsible for the progesterone suppression is the
fact that 10-bp scanning deletions through these two regions did
not prevent progesterone suppression of GnRH induction. This
situation is reminiscent of the mechanism of androgen suppression on the bovine LH␤ promoter, in which cis mutations
or block replacements affecting Egr-1, SF-1, or pituitary homeobox 1 binding elements had no effect, but the proximal
promoter clearly mediated the androgen suppression (25).
Furthermore, the data thus far support the conclusion that the
repression of basal activity and GnRH induction of the LH␤
gene by PR occur through modulation of factor(s) in common
between the two processes.
In summary, our results demonstrate that progesterone can
suppress both basal transcription and GnRH induction of LH␤
gene expression in a hormone- and receptor-dependent manner
in gonadotrope cells. We determined that the full suppressive
effect of progesterone on LH␤ gene expression requires the
unique amino-terminal region of the PRB isoform and an intact
DBD. However, we did not find any evidence that the progesterone suppression involves direct binding of PR to the LH␤
Endocrinology, May 2009, 150(5):2395–2403
promoter, although it is recruited to the endogenous promoter in
live cells. Rather, our data suggest that these domains are necessary for tethering or binding to other transcription factor(s).
Furthermore, we identified a repressive element in the proximal
LH␤ promoter that is both necessary and sufficient to elicit suppression by progesterone. Multiple regions at ⫺300/⫺200 and
⫺200/⫺150 appear to be involved in both basal transcription of
the LH␤ gene and suppression by progesterone, further supporting the concept that PR may act through other transcription
factors bound to these regions. We also demonstrated that there
is a region upstream of ⫺500 in the rat LH␤ promoter that may
also be involved in the suppression of LH␤ transcription by progesterone. Additional experiments will be necessary to define the
cis-regulatory elements and transcription factors that play a role
in the regulation of LH␤ gene expression by ligand-bound PR.
Altogether this work has revealed new insights into the pituitary
action of progesterone. In particular, it has highlighted the role
that progesterone may play in limiting preovulatory GnRH-induced LH secretion via progesterone suppression of GnRH-induced LH␤ transcription.
Acknowledgments
The authors thank Djurdjica Coss and other members of the Mellon lab
for helpful discussions and comments. We also thank Scott Kelley for his
suggestions and critical reading of the manuscript. We are grateful to
Mark Lawson, Benita Katzenellenbogen, and Dean Edwards for providing reagents. The ⫺1800-bp rat LH␤ luciferase-reporter plasmid (in
pGL3) was donated by Mark Lawson. Rat PRB (in pCMV5) was provided by Benita Katzenellenbogen; the 1294 PR antibody and mouse PRB
and PRA plasmids (in pcDNA-I) were donated by Dean Edwards. We
also acknowledge the University of California, San Diego, Cancer Center
DNA Sequencing Shared Resource for sequencing and the University of
Colorado Cancer Center Tissue Culture Core Facility for baculovirus
production.
Address all correspondence and requests for reprints to: Pamela L.
Mellon, Ph.D., Department of Reproductive Medicine, University of
California, San Diego, 9500 Gilman Drive, La Jolla, California 92093.
E-mail: pmellon@ucsd.edu.
This work was supported by National Institute of Child Health and
Human Development/National Institutes of Health (NIH) through a
cooperative agreement (U54 HD12303) as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research (to
P.L.M.). This work was also supported by NIH Grant R01 HD20377 (to
P.L.M.). V.G.T. was supported by NIH Grants F32 DK065437 and T32
HD07203.
Disclosure Summary: The authors have nothing to disclose.
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