REPRODUCTION-DEVELOPMENT
FOXL2 Is Involved in the Synergy between Activin and
Progestins on the Follicle-Stimulating Hormone ␤Subunit Promoter
Yasmin Ghochani, Jasjit K. Saini, Pamela L. Mellon, and Varykina G. Thackray
Department of Reproductive Medicine and the Center for Reproductive Science and Medicine, University
of California, San Diego, La Jolla, California 92093
Differential regulation of gonadotropin hormone production in the pituitary is critical for fertility.
Activin and progesterone signaling in gonadotrope cells is important for Fshb gene expression.
Previously, we reported that synergy between activin and progestins required the binding of SMAD
proteins and the progesterone receptor (PR) to the murine Fshb promoter. In this study, we demonstrate that the FOXL2 transcription factor is also necessary for the full synergistic response
between activin and progestins. We show that this synergy occurs in a species-specific manner and
that multiple elements in the Fshb promoter that bind forkhead box L2 (FOXL2), SMA/mothers
against decapentaplegic homologs (SMAD), and PR are required. Furthermore, we demonstrate
that FOXL2 can physically interact with PR and SMAD3. Thus, it is likely that protein-protein interactions among FOXL2, SMAD, and PR recruited to the Fshb promoter play a key role in facilitating Fshb transcription before the secondary FSH surge in rodents. (Endocrinology 153:
2023–2033, 2012)
F
SH and LH are essential for mammalian fertility because they are required for ovarian folliculogenesis,
gametogenesis and steroidogenesis (1). Gonadotropins
are heterodimeric glycoproteins composed of a common
␣-glycoprotein subunit and unique ␤-subunits that confer
biological specificity. Transcription of the ␤-subunits is
the rate-limiting step for production of the mature hormones (2, 3) and is differentially regulated during the estrous cycle. The LH surge is proceeded by an increase in
Lhb and Fshb transcription in the afternoon of proestrus
(4 – 6), while an independent secondary FSH surge occurs,
after an increase in Fshb transcription during estrus, to
promote follicular recruitment for the following cycle (3,
7, 8).
Multiple mechanisms have been proposed for the distinct regulation of Fshb vs. Lhb transcription (recently
reviewed in Refs. 9 and 10). Pulsatile secretion of GnRH
contributes to differential regulation of FSH and LH (2,
11, 12). In addition, an increase in activin levels during
estrus, due to lower follistatin and/or inhibin, is thought to
selectively favor Fshb synthesis (13, 14). Furthermore, steroid hormones such as progesterone have been shown to
induce Fshb while inhibiting Lhb expression (15, 16).
Activin strongly induces Fshb expression in gonadotrope cells (17). Activin binding to the type II receptor
leads to heterodimerization and phosphorylation of the
type I receptor (18). Subsequently, the type I receptor
phosphorylates SMA/mothers against decapentaplegic
homologs (SMAD) 2 and SMAD3, which bind to SMAD4
and translocate into the nucleus to mediate transcription
of specific target genes (18 –20). The proximal murine
Fshb promoter contains multiple activin responsive elements (reviewed in Refs. 21 and 22). The ⫺267 SMADbinding element (SBE) in the murine Fshb promoter is a
consensus SBE comprised of a palindrome sequence
GTCTAGAC (23–25). Two other elements at ⫺153 and
⫺120 are critical for activin induction in all species examined (26). The TALE homeodomain proteins, PBX1
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2012 by The Endocrine Society
doi: 10.1210/en.2011-1763 Received September 14, 2011. Accepted December 30,
2011.
First Published Online January 31, 2012
Abbreviations: DN, Dominant-negative; FOXL2, forkhead box L2; GFP, green fluorescent
protein; GST, glutathione S-transferase; HND, Hepes/NaCl/dithiothreitol; HRE, hormone
response element; PR, progesterone receptor; SBE, SMAD-binding element; SMAD, SMA/
mothers against decapentaplegic homologs.
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Activin and Progestin Synergy Involves FOXL2
and PREP1 bind the ⫺120 site, which allows tethering of
SMAD to the promoter (26, 27), whereas the forkhead
box L2 (FOXL2) transcription factor was recently shown
to bind the ⫺153 element (28, 29). Indeed, Fshb mRNA
levels are reduced in both FOXL2- and SMAD3-null mice,
indicating these transcription factors are required for normal Fshb expression (30, 31).
Differential regulation of gonadotropin gene expression may be due to interactions among the hormone signaling pathways that regulate their synthesis. Cross talk
between activin and progesterone may be important for
the secondary FSH surge because FSH levels were reported
to increase upon activin and progesterone cotreatment in
primary rat pituitary cell cultures and a progesterone antagonist suppressed activin induction of Fshb gene expression in vivo in rats (32, 33). Another study revealed that
progesterone stimulation of FSH secretion was prevented
by follistatin treatment in rats (34). More recently, we
showed synergistic induction of the Fshb promoter with
activin and progestin cotreatment of murine primary pituitary cells as well as immortalized gonadotrope-derived
L␤T2 cells (35). We also demonstrated that SMAD can
physically interact with progesterone receptor (PR) and
that both transcription factors were required to bind to the
Fshb promoter for the synergy (35).
In this study, we further investigated mechanisms of
Fshb transcriptional regulation mediated by cross talk between activin and progestin signaling pathways using
L␤T2 cells. During the course of our investigations, we
and others discovered that FOXL2 plays a role in activin
induction of the Fshb promoter (28, 29). Therefore, we
shifted our focus to understanding the potential role of
FOXL2 in mediating activin and progestin synergy on the
Fshb promoter. We show that the FOXL2 transcription
factor is involved in activin and progestin synergy on the
murine Fshb promoter and that the synergy occurs in a
species-specific manner. We demonstrate that a ⫺355
SMAD half-site and an adjacent ⫺350 FOXL2 site are
necessary for the full synergistic response along with a
previously characterized ⫺381 hormone response element
(HRE) and ⫺267 SBE. We also show that additional activin and progesterone-responsive elements are necessary
for the maximal response. Furthermore, we demonstrate
that FOXL2 physically interacts with PR, suggesting that
interactions among FOXL2, SMAD, and PR may play a
role in elevated Fshb synthesis before the secondary FSH
surge in rodents.
Materials and Methods
Hormones
Promegestone (R5020) was purchased from NEN Life Science Products Life Sciences (Boston, MA), activin from Calbio-
Endocrinology, April 2012, 153(4):2023–2033
chem (La Jolla, CA), and GnRH from Sigma-Aldrich (St. Louis,
MO).
Plasmid constructs
The construction of the ⫺1000 murine Fshb and ⫺985 ovine
FSHB luciferase (luc) reporter plasmids were described previously (16, 36). The ⫺1028/⫹7 human FSHB-luc reporter plasmid was provided by Daniel Bernard, murine PRB by Dean Edwards, and murine FOXL2 and FOXL2 ⌬133–375 by Louise
Bilezikjian (37).
Mutagenesis
The QuikChange Site-Directed Mutagenesis Kit (Stratagene, La
Jolla, CA) was used to generate deletions or mutations in plasmids
containing the murine and human FSHB promoters. Insertion of the
⫺381 HRE and/or the ⫺267 SBE into the human FSHB promoter
was performed with the following primers: 5⬘-TTTGTTTCTTCCTTCACAGTGTTCAATATGCTCTTGGAGCAATTT-3⬘ and
5⬘-AAAGATACAAAAGAAAAGTCTAGACTCTGGAGTCAC
AATTAATT-3⬘ (the mutated bases are indicated in bold). The
sequences of the promoters were confirmed by dideoxyribonucleotide sequencing. The ⫺381, ⫺273, ⫺230, ⫺197, and
⫺139 HRE mutations in the murine Fshb-luc reporter plasmid
were described previously (16) as well as the ⫺267, ⫺153, ⫺120,
and 3xSBE mutations (35). The ⫺355 SMAD, ⫺350 FOXL2,
⫺339 SF1, ⫺355 SMAD/⫺350 FOXL2, ⫺208 (5⬘ T), ⫺153 (3⬘
T), and ⫺106 mutations were also previously described (29, 38).
Cell culture and transient transfections
Cell culture and transient transfection experiments were performed using the L␤T2 cell line (39, 40). The cells were maintained in 10-cm plates in DMEM from Mediatech Inc. (Herndon,
VA) with 10% fetal bovine serum (Omega Scientific, Inc., Tarzana, CA) and penicillin/streptomycin antibiotics (Life Technologies, Inc./Invitrogen, Grand Island, NY) at 37 C and 5% CO2.
Trypsin-EDTA (1⫻) (Sigma-Aldrich) was used for cell dissociation. Cells were split at 3 ⫻ 105 cells per well into 12-well plates
and transfected 18 h later, using Fugene 6 reagent (Roche Molecular Biochemicals, Indianapolis, IN) following the manufacturer’s instructions. For all experiments, the cells were transfected with 400 ng of the indicated reporter plasmid, 10 ng of
mouse PRB expression vector, and 200 ng of a ␤-galactosidase
reporter plasmid driven by the herpes virus thymidine kinase
promoter to control for transfection efficiency. Cells were
switched to serum-free DMEM containing 0.1% BSA, 5 mg/liter
transferrin, and 50 mM sodium selenite 6 h after transfection. The
cells were treated with various hormones after overnight starvation in serum-free media. Vehicle control for R5020 was 0.1%
ethanol; for activin and GnRH, it was 0.1% BSA. The cells were
treated with 10 ng/ml activin (unless stated otherwise), 10⫺9 M
R5020 for 24 h or 10⫺8 M GnRH for 6 h.
Luciferase and ␤-galactosidase assays
After hormone treatment, cells were washed with 1⫻ PBS and
lysed with 0.1 M K-phosphate buffer (pH 7.8) containing 0.2%
Triton X-100. Lysed cells were assayed for luciferase activity
using a buffer containing 100 mM Tris-HCl (pH 7.8), 15 mM
MgSO4, 10 mM ATP, and 65 ␮M luciferin. ␤-Galactosidase activity was assayed using the Tropix Galacto-light assay (Applied
Biosystems, Foster City, CA), according to the manufacturer’s
Endocrinology, April 2012, 153(4):2023–2033
protocol. Both assays were measured using a Veritas Microplate
Luminometer (Promega, Madison, WI).
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A
Glutathione S-transferase (GST) interaction assay
GST-FOXL2 was creating by ligating Flag-tagged murine
FOXL2 (37) into the BamHI and EcoRI sites of the pGEX-5X-1
expression vector. GST-SMAD3 was provided by Rik Derynck.
35
S-labeled proteins were produced using the TnT Coupled Reticulolysate System (Promega). Bacteria transformed with GST
plasmids were grown to OD of 0.6 and induced with isopropyl
␤-D-1-thiogalactopyranoside overnight at 30 C (41). Bacterial
pellets were sonicated in 0.1% Triton X-100, 5 mM EDTA in 1⫻
PBS and centrifuged, and the supernatant was bound to glutathione Sepharose 4B resin (Amersham Pharmacia Biotech, Piscataway, NJ). Beads were washed 4⫻ in PBS and in Hepes/NaCl/
dithiothreitol (HND) buffer [10 mg/ml BSA, 20 mM HEPES (pH
7.8), 50 mM NaCl, 5 mM dithiothreitol, and 0.1% Nonidet P-40].
For the interaction assay, 40 ␮l of 35S-labeled in vitro transcribed-translated PR or 5 ␮l of green fluorescent protein (GFP)
was added to the beads with 400 ␮l HND buffer. Beads were
incubated overnight at 4 C and washed twice with HND buffer
and twice with 0.1% Nonidet P-40 in PBS. Thirty microliters of
2⫻ Laemmli load buffer were added, and the samples were
boiled and electrophoresed on a 10% SDS-polyacrylamide gel.
One tenth of the 35S-labeled in vitro transcribed-translated product was loaded onto the gel as input.
Statistical analysis
Transient transfection experiments were repeated independently at least three times. Data were normalized for transfection
efficiency with luciferase activity relative to ␤-galactosidase.
Data were also normalized to the pGL3 plasmid (to control for
hormone effects on the vector DNA). In the figures, the error bars
represent the SEM. Data were analyzed by one-way ANOVA,
followed by post hoc comparisons with the Tukey-Kramer honestly significant difference test or two-way ANOVA to determine
synergy (as described in Ref. 42) using the statistical package
JMP version 9.0 (SAS, Cary, NC). In all analyses, the result was
considered significant if P ⱕ 0.05.
Results
Low levels of exogenous PR are sufficient for
progestin responsiveness and synergy between
activin and progestins on the Fshb promoter
One concern from our previous studies was the high
levels of exogenous PR transfected into L␤T2 cells to measure progestin responsiveness (16, 35). In the current
study, we performed a titration to determine the minimal
effective level of PR for progestin induction of the murine
Fshb promoter (Fig. 1A). Because 10 ng PR resulted in a
robust induction, we used this amount of receptor for all
of the subsequent experiments. Ten nanograms of PR resulted in an 11-fold response to R5020 (a synthetic progestin) on the Fshb promoter, whereas the synergistic response to activin and progestin was 47-fold (Fig. 1B).
B
FIG. 1. In contrast to activin with progestins or activin with GnRH,
R5020, and GnRH do not synergistically regulate Fshb gene expression.
Panel A, The ⫺1000 murine Fshb-luc reporter was transiently
transfected into L␤T2 cells along with increasing amounts of PR as
indicated. After overnight starvation in serum-free media, cells were
treated with vehicle or 10⫺9 M R5020. Panel B, The ⫺1000 murine
Fshb-luc reporter was transiently transfected into L␤T2 cells along with
10 ng PR. After overnight starvation in serum-free media, cells were
treated with vehicle (V), 10 ng/ml activin (A), 10⫺9 M R5020 (P), or 10
nM GnRH (G), individually or with the indicated combinations. The
results represent the mean ⫾ SEM of at least three experiments. *,
Significant difference from the vehicle-treated control using one-way
ANOVA followed by Tukey’s post hoc test; #, significant interaction as
defined by two-way ANOVA (42).
GnRH and progestin signaling pathways do not
synergize on the Fshb promoter
As shown previously (23, 35, 43), activin with progestins or activin with GnRH synergistically enhanced transcription of a Fshb-luc reporter (Fig. 1B). Because activin,
GnRH, and progesterone are all present during the estrous
cycle to regulate Fshb gene expression, we investigated
whether GnRH and progestin signaling pathways interact
to mediate Fshb gene expression. As shown in Fig. 1B,
GnRH induced the Fshb promoter 2.6-fold and R5020
11-fold. However, GnRH and R5020 cotreatment up-regulated Fshb gene expression 12-fold, indicating that they
do not interact to regulate the Fshb promoter.
Synergy between activin and progestins is not
conserved on the ovine and human FSHB
promoters
Activin has been shown to regulate ovine FSHB gene
expression by SMAD and TGF-␤-activated kinase-1-de-
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Activin and Progestin Synergy Involves FOXL2
pendent signaling pathways (26, 44, 45). It is less clear
whether progesterone modulates transcription of ovine
FSHB in gonadotropes. FSHB mRNA levels decreased after progestin treatment in ovine primary pituitary cells
(46). On the other hand, a reporter gene containing the
proximal ovine FSHB promoter was induced by progesterone in ovine primary pituitary cells (47). Data regarding
transcriptional regulation of the human FSHB gene by
activin and progestins are limited. The human FSHB promoter has been reported to be relatively insensitive to activin
treatment (48, 49). A polymorphism in the human FSHB
promoter was mapped to a conserved region containing a
putative HRE, suggesting that progestin regulation of FSHB
synthesis may be conserved among mammals (50).
To determine whether the ovine and human promoters
respond to progestins alone or synergistically with activin
and progestins, ⫺1000 murine, ⫺985 ovine, or ⫺1028/⫹7
human FSHB-luc reporter constructs were transiently transfected into L␤T2 cells along with PR (Fig. 2A). In contrast to
the murine promoter, which was induced 15-fold by proges-
A
B
FIG. 2. Activin and progestin synergy is not conserved on the ovine
and human FSHB promoters and addition of the ⫺381 HRE and/or
⫺267 SBE to the human FSHB promoter is not sufficient for synergy.
A, The ⫺1000 murine (m), ⫺985 ovine (o), or ⫺1028/⫹7 human (h)
FSHB-luc reporter constructs were transiently transfected into L␤T2
cells, as indicated. B, Wild-type (WT) ⫺1028/⫹7 hFSHB-luc or mutants
containing the ⫺381 HRE, ⫺267 SBE, or both were transiently
transfected into L␤T2 cells, as indicated. After overnight starvation in
serum-free media, cells were treated for 24 h with vehicle (veh), 50
ng/ml activin, 10⫺9 M R5020, or both. The results represent the
mean ⫾ SEM of at least three experiments. *, Significant difference
from the vehicle-treated control using one-way ANOVA followed by
Tukey’s post hoc test; #, significant interaction as defined by two-way
ANOVA.
Endocrinology, April 2012, 153(4):2023–2033
tin treatment and 82-fold by activin and progestins, the ovine
and the human FSHB promoters did not exhibit significant
progestin responsiveness alone or in the presence of 50 ng/ml
activin.
Addition of the ⴚ381 HRE and/or the ⴚ267 SBE to
the human FSHB promoter is not sufficient for
activin and progestin synergy
The ⫺267 SBE is present in rodent Fshb promoters but
is not conserved in other mammalian species. Addition of
the ⫺267 SBE to the human FSHB promoter increased
activin responsiveness (48). The ⫺381 HRE is necessary
for the synergistic induction of murine Fshb gene expression by activin and progestins, because mutating it reduced the synergy by 73% (35). We, therefore, investigated whether addition of the murine ⫺381 HRE, ⫺267
SBE, or both to the human FSHB promoter would be sufficient to observe synergy between activin and progestins.
With the addition of the ⫺267 SBE site, the human FSHB
promoter responded to activin 3-fold more than the wildtype human promoter, as previously demonstrated (48).
On the other hand, addition of the ⫺381 HRE did not
result in progestin responsiveness or synergy between activin and R5020, indicating that the ⫺381 HRE is not
sufficient (Fig. 2B). Furthermore, although addition of the
⫺267 SBE to the human promoter resulted in substantial
activin responsiveness, it was unable to facilitate activin
and progestin synergy.
The ⴚ273, ⴚ230, ⴚ197, and ⴚ139 HRE are
necessary for activin and progestin synergy
In addition to the ⫺381 HRE, several other HREs on
the murine Fshb promoter are necessary for the full progestin response (16) (Fig. 3A). Because the ⫺381 HRE is
not sufficient for the synergy between activin and progestins, mutations in these additional HRE sites were tested
for responsiveness to activin, progestins, and cotreatment.
The ⫺273 and ⫺139 HRE mutations resulted in a significant decrease in the activin response, perhaps due to proximity of these sites to the ⫺267 and ⫺153 activin responsive elements, respectively (Fig. 3B). The ⫺197 HRE
mutation increased the activin response by approximately
50%, probably due to enhancement of the adjacent ⫺208
activin responsive element as an unintended consequence
of the mutation. Indeed, an individual base-pair change at
⫺193 instead of the double mutation at ⫺196 and ⫺193
did not exhibit an increased activin response (see Supplemental Fig. 1, published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). All
HRE mutants showed a significant decrease in progestin
responsiveness and synergistic induction by activin and
R5020 cotreatment (Fig. 3B). The ⫺381 HRE mutation
Endocrinology, April 2012, 153(4):2023–2033
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A
treatment with activin and progestins, as previously reported (35).
B
The region between the ⴚ381 HRE and the ⴚ267
SBE contains a FOXL2 site and SMAD half-site
necessary for synergy between activin and
progestins
Because it appeared, from our studies, that additional
elements might be involved in the synergy between activin
and progestins (Fig. 2) and that the ⫺381 HRE, ⫺273
HRE, and ⫺267 SBE are important elements for this synergy (Fig. 3), we undertook a systematic analysis of the
region between these elements. Internal deletions of 10 bp
from ⫺370 to ⫺271 in the murine ⫺1000 Fshb promoter
were created because the helical periodicity of DNA is
approximately 10 bp per turn, and the orientation of the
promoter DNA would not be altered. The responsiveness
of the 10-bp deletions to activin, progestin, or cotreatment
was measured. The ⫺370/⫺361 deletion overlapped the
last 4 bp of the 3⬘ end of the ⫺381 HRE and thus led to a
significant reduction of R5020 responsiveness and synergy compared with the wild-type promoter (Fig. 4). Deletions at ⫺360/⫺351, ⫺350/⫺341, and ⫺320/⫺311 also
resulted in significant reductions in the induction of Fshb
gene expression by R5020 and cotreatment (Fig. 4). The
⫺320/⫺311 region was recently identified as playing a
role in activin induction of the Fshb promoter (29), but it
remains to be determined what transcription factors bind
to this region.
Closer examination of the 10-bp internal deletions
from ⫺360 to ⫺341 found that a putative SMAD half-site
at ⫺355 bp and a newly characterized FOXL2 site at
C
FIG. 3. Multiple response elements are necessary for the full
synergistic response between activin and progestins. A, Schematic of
the mFshb promoter showing the position of previously identified HRE,
SBE, and activin-responsive elements (ARE). B and C, The wild-type
(WT) ⫺1000 mFshb-luc reporter and mutations in specific response
elements were transiently transfected into L␤T2 cells, as indicated.
After overnight starvation in serum-free media, cells were treated for
24 h with vehicle (veh), 10 ng/ml activin, 10⫺9 M R5020, or both. The
results represent the mean ⫾ SEM of at least three experiments. *,
Significant difference from the wild-type Fshb promoter using one-way
ANOVA followed by Tukey’s post hoc test.
resulted in a complete lack of responsiveness to R5020
alone or in combination with activin.
Disruption of individual activin-responsive
elements prevents cross talk between activin and
progestins
The expression of a luciferase reporter containing three
mutated activin-responsive elements in the murine ⫺1000
Fshb proximal promoter (3xSBE) was previously studied
for its responsiveness to activin and progestins (35). Here,
we measured responsiveness to activin, R5020, and cotreatment of individual mutations in these elements. Mutations in the ⫺267, ⫺153, and ⫺120 elements showed a
significant decrease in activin responsiveness compared
with the wild-type promoter (Fig. 3C). None of the mutated promoters showed any significant difference in their
progestin response. The synergistic induction of the mutated promoters due to activin and R5020 cotreatment
was significantly reduced (Fig. 3C). The 3xSBE mutation
led to a complete lack of response to activin alone or co-
FIG. 4. The 10-bp internal deletions between the ⫺381 HRE and the
⫺267 SBE reveal several regions involved in activin and progestin
synergy on the murine Fshb promoter. The wild-type (WT) ⫺1000
mFshb-luc reporter or mutants with the indicated 10-bp deletions were
transiently transfected into L␤T2 cells. After overnight starvation in
serum-free media, cells were treated for 24 h with vehicle (veh), 10
ng/ml activin, 10⫺9 M R5020, or both. The results represent the
mean ⫾ SEM of at least three experiments. *, Significant difference
from the vehicle-treated control using one-way ANOVA followed by
Tukey’s post hoc test; #, significant interaction as defined by two-way
ANOVA.
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Activin and Progestin Synergy Involves FOXL2
⫺350 bp described by Corpuz et al. (29) (Fig. 5A) had been
deleted. The expression of specific mutations in each of the
above-mentioned sites and a double mutant was measured
to understand the effects of these sites on hormonal induction of the Fshb promoter. As expected from the deletion studies, mutation of a putative SF1 site did not significantly alter Fshb induction by activin, R5020, or
cotreatment (Fig. 5B), indicating that this element does not
play a significant role. The ⫺355 SMAD half-site and the
⫺350 FOXL2 site mutations did not significantly alter
activin induction of the Fshb promoter. On the other
hand, mutation of the ⫺355 SMAD half-site reduced the
progestin response by 53% and the synergistic induction
by 49% of the wild-type promoter induction. Similarly,
the ⫺350 FOXL2 mutation reduced the progestin response by 58% and synergistic induction by 70% of the
wild-type promoter. Interestingly, the SMAD half-site/
A
B
C
FIG. 5. The ⫺355 SMAD half-site and ⫺350 FOXL2 site on the murine
Fshb promoter are necessary for the synergy between activin and
progestins. A, Schematic illustrating the ⫺381 PR binding site, ⫺355
putative SMAD half-site, ⫺350 FOXL2 site, and ⫺339 putative SF1 site
on the murine Fshb promoter. B, The wild-type (WT) ⫺1000 mFshb-luc
reporter or mutations in specific sites were transiently transfected into
L␤T2 cells, as indicated. C, The WT ⫺1000 mFshb-luc reporter
construct or deletions in the region between the ⫺381 HRE and the
⫺267 SBE were transiently transfected into L␤T2 cells, as indicated.
After overnight starvation in serum-free media, cells were treated for
24 h with vehicle (veh), 10 ng/ml activin, 10⫺9 M R5020, or both. The
results represent the mean ⫾ SEM of at least three experiments. *,
Significant difference from the wild-type Fshb promoter using one-way
ANOVA followed by Tukey’s post hoc test.
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FOXL2 double mutation decreased the progestin response
and the synergy by approximately 80 –90% of the wildtype promoter without significantly reducing activin
induction.
Because the ⫺355 putative SMAD half-site and the
⫺350 FOXL2 site are located between the ⫺381 HRE and
⫺267 SBE, we hypothesized that these sites may help to
stabilize a ternary complex formed by PR, SMAD, and
FOXL2. If this is the case, bringing the ⫺381 HRE and
⫺267 SBE closer together on the promoter may remove
the necessity for the SMAD and FOXL2 sites. Deletions of
21 or 85 bp were created in the murine Fshb promoter
from ⫺359/⫺338 and ⫺359/⫺274, respectively. Similarly to Fig. 5B, the ⫺359/⫺338 deletion encompassing
the SMAD half-site and FOXL2 site significantly reduced
Fshb gene expression (Fig. 5C). Notably, because the
⫺381 HRE and the ⫺267 SBE were brought closer together with the 85-bp deletion, the progestin response and
the synergy were partially rescued. For the 21- and 85-bp
deletions, the progestin response was reduced by 66 and
30%, whereas the synergy was reduced by 81 and 44% of
the wild-type promoter, respectively (Fig. 5C).
The ⴚ208, ⴚ153, and ⴚ106 FOXL2 sites are
necessary for maximal synergistic response
In addition to the ⫺350 FOXL2 site, several other
FOXL2 sites have been recently characterized that play a
role in activin induction of the murine Fshb promoter (28,
29, 51). We used specific mutations in the ⫺208, ⫺153,
and ⫺106 elements to determine whether they are also
necessary for progestin responsiveness and the synergy
between activin and progestins. In the ⫺208 (5⬘ T) mutation, two thymines at ⫺204 and ⫺203 were changed to
cytosines, whereas in the ⫺153 (3⬘ T) mutation, three thymines at ⫺142/⫺140 were altered to guanines (Fig. 6A)
(29). The ⫺208 (5⬘ T) mutation reduced the response to
activin, progestins, and cotreatment by 69, 25, and 54%
of the wild-type response, respectively, whereas the ⫺153
(3⬘ T) mutation reduced the response to activin, progestins, and cotreatment by 61, 24, and 66% (Fig. 6B). The
⫺106 mutation (38) reduced the response to activin and
cotreatment by 60 and 25% of the wild-type response,
whereas the response to progestins was increased by 23%.
Overexpression of a dominant-negative (DN)
FOXL2 reduces synergy between activin and
progestins on the Fshb promoter
Because FOXL2 binding sites are necessary for activin
and progestin synergy, we asked whether the FOXL2 transcription factor is required for the synergy to occur. Transient transfection of L␤T2 cells with an expression plasmid containing murine FOXL2 resulted in only a slight
Endocrinology, April 2012, 153(4):2023–2033
endo.endojournals.org
A
A
B
B
FIG. 6. The ⫺208, ⫺153, and ⫺106 FOXL2 sites are necessary for the
full synergistic response between activin and progestins on the murine
Fshb promoter. A, Schematic of the FOXL2 sites on the mFshb
promoter with the mutated bases underlined (29, 38). B, The ⫺1000
mFshb-luc reporter or mutants were transiently transfected into L␤T2
cells, as indicated. After overnight starvation in serum-free media, cells
were treated for 24 h with vehicle (veh), 10 ng/ml activin, 10⫺9 M
R5020, or both. The results represent the mean ⫾ SEM of at least three
experiments. *, Significant difference from the wild-type Fshb
promoter using one-way ANOVA followed by Tukey’s post hoc test.
increase in Fshb gene expression compared with the empty
vector, likely due to the fact that the cells already contain
high levels of FOXL2 protein. L␤T2 cells were also transfected with an expression plasmid containing FOXL2
⌬133–375, which lacks part of the DNA-binding forkhead domain and the carboxyl-terminal alanine-rich region (Fig. 7A). Several mutations in patients with blepharophimosis-ptosis-epicanthus inversus syndrome produce
truncated proteins lacking part of the forkhead-binding
domain and/or a polyalanine tract (52, 53). Truncated
FOXL2 proteins have been shown to exert DN effects on
wild-type FOXL2 (54). Overexpression of the DN
FOXL2 ⌬133–375 resulted in 40% reduction in the synergy between activin and progestins (Fig. 7B).
FOXL2 interacts with PR and SMAD3
Because FOXL2 is necessary for the maximal synergistic response between activin and progestins on the murine
Fshb promoter, we investigated whether FOXL2 can
physically interact with PR. FOXL2 has been reported to
interact with SMAD3 (37), and we have shown that
SMAD2, -3, and -4 can interact with PR (35). We tested
whether FOXL2 interacts with PR by incubating a GSTFOXL2 fusion protein with in vitro-transcribed and
-translated PR in pull-down experiments (Fig. 7C).
FOXL2 bound to both the full-length PRB and PRA isoforms. As a positive control, we also showed that FOXL2
interacts with SMAD3, as previously reported (37). In
contrast, there was minimal interaction between the GST-
2029
C
FIG. 7. DN FOXL2 inhibits activin and progestin synergy, and FOXL2
interacts directly with PR. A, Schematic showing the structure of
FOXL2 and DN FOXL2 ⌬133–375. B, The ⫺1000 mFshb-luc reporter
was transiently transfected into L␤T2 cells along with empty vector
(EV), wild-type or DN FOXL2, as indicated. After overnight starvation in
serum-free media, cells were treated for 24 h with vehicle (veh), 10
ng/ml activin, 10⫺9 M R5020, or both. The results represent the
mean ⫾ SEM of at least three experiments. *, Significant difference
from the wild-type Fshb promoter using one-way ANOVA followed by
Tukey’s post hoc test. C, GST interaction assays were performed using
bacterially expressed GST-fusion proteins (indicated above each lane)
and 35S-labeled in vitro-transcribed and -translated PRB, PRA, and GFP
(indicated on the left). GFP was used as a negative control. The GSTfusion proteins included GST alone, GST-FOXL2, and GST-SMAD3. One
tenth of the protein used in the interaction assay was loaded in the
lane marked input. The experiment was repeated several times with
the same results, and a representative experiment is shown.
FOXL2 fusion protein and the negative control (GFP) or
with GST alone incubated with PR (Fig. 7C).
Discussion
Interactions among multiple hormone signaling pathways
are likely responsible for the differential regulation of Fshb
synthesis before the secondary FSH surge. In investigating
combinatorial interactions among activin, progestins, and
GnRH, we first confirmed that there are synergistic interactions between activin and progestins or activin and
GnRH on the murine Fshb promoter, as previously reported (23, 35, 43). In contrast, our studies demonstrate
that there is no significant interaction between GnRH and
progestins on the murine Fshb promoter in L␤T2 cells.
This is particularly interesting because cross talk with PR
2030
Ghochani et al.
Activin and Progestin Synergy Involves FOXL2
has been implicated as critical for GnRH self-priming in
pituitary gonadotropes (55, 56), and progestins inhibit
GnRH induction of Lhb gene expression (57), suggesting
that interactions between GnRH and progesterone signaling pathways may be important for LH synthesis and secretion but not for Fshb transcriptional regulation.
Several of the HREs previously characterized in the murine Fshb promoter have a degree of conservation across
multiple species, suggesting that steroid hormones such as
progesterone may regulate other mammalian FSHB promoters (16). However, we demonstrated that neither the
ovine nor human promoter exhibited a progestin response
or synergistic induction of the promoter upon cotreatment
with activin and progestins. This result suggests that progesterone does not directly regulate ovine or human FSHB
transcription in gonadotrope cells due to the lack of species-specific regulatory elements present on the rodent
promoter. Because our data differ from what was observed in ovine primary pituitary cells with an ovine
FSHB-luc reporter (47), they suggest that the progesterone
induction observed in ovine pituitary cells was due to
paracrine influences from other cells in the primary cultures. On the other hand, our results agree with experiments in sheep and ovine pituitary cells showing that progestins do not up-regulate FSHB mRNA (46, 58).
To test whether elements in the murine promoter, not
conserved in the ovine and human promoters, are sufficient to induce a progestin response and synergy with activin, we inserted the murine ⫺381 HRE, ⫺267 SBE, or
both into the human FSHB promoter. Addition of the
⫺267 SBE resulted in a substantial activin response, as
previously described (48). In contrast, addition of the
⫺381 HRE to the human FSHB promoter was not sufficient for a progestin response alone or in combination
with activin, suggesting that additional regulatory elements are required.
Because the extent of steroid hormone response conveyed by a single HRE is often weak, multiple HRE are
often found in the promoters of steroid-responsive genes
(59). We confirmed that the ⫺381 HRE is required for the
progestin response and for synergistic induction of the
promoter by activin and progestin cotreatment, in agreement with our previous studies (16, 35). We also showed
that additional HRE previously characterized on the murine Fshb promoter at ⫺273, ⫺230, ⫺197, and ⫺139 as
playing a role in the progestin response (16) are also necessary for maximal synergistic induction of the promoter
by activin and progestins. It is noteworthy that mutation
of the elements at ⫺273, ⫺197, and ⫺139 also affected
activin responsiveness, possibly due to the proximity or
overlap of these sites with the ⫺267 SBE, ⫺208, and ⫺153
FOXL2 sites, respectively. These results also indicate that
Endocrinology, April 2012, 153(4):2023–2033
there may be strong coregulation between elements that
bind PR and FOXL2/SMAD.
Our previous studies showed that SMAD binding to the
Fshb promoter was necessary for the synergistic interaction between activin and progestins (57). As previously
reported, mutation of three activin-responsive elements
(3xSBE) led to a lack of activin responsiveness and disappearance of the synergistic induction of the promoter. Individual disruption of the ⫺267 SBE substantially reduced
cross talk between activin and progestins and did not allow for maximal synergistic induction of the promoter.
However, mutation of the ⫺267 SBE did not completely
abolish activin induction or synergy on the Fshb promoter,
indicating that this hormonal regulation requires the binding of additional factors to multiple activin-responsive elements in the Fshb promoter. PBX1/PREP1 were reported
to bind a CTGTCTATCCAA element encompassing a putative SMAD half-site (underlined) at ⫺120 in the murine
Fshb promoter and recruit SMAD3/4 to the promoter
(26). Not surprisingly, mutation of the ⫺120 element not
only blocked activin induction, as previously described,
but also greatly reduced the synergy between activin and
progestins. These data confirm that activin signaling
through SMAD proteins is critical for synergy between
activin and progestins on the Fshb promoter.
In addition to transcription factors such as SMAD and
PBX1/PREP1, recent studies have characterized the binding of the FOXL2 transcription factor at several elements
in the murine Fshb promoter (28, 29). In the current experiments, mutation of the ⫺350 FOXL2 site and a nearby
⫺355 putative SMAD half-site had little effect on activin
responsiveness, in contrast to our previous study, which
showed a significant reduction with a mutation of the
⫺350 FOXL2 site (29). It remains to be determined
whether the ⫺355 element is a bona fide SBE because
SMAD4 did not supershift protein complexes bound to an
oligo encompassing this site (29). On the other hand, deletion or disruption of these two sites greatly reduced the
response to progestins, alone or in combination with activin (Figs. 4 and 5, B and C). Indeed, a double FOXL2/
SMAD mutation almost completely abolished progestin
responsiveness. These results indicate that the FOXL2 site
and nearby SMAD half-site are necessary for progestin
responsiveness and the synergy between activin and progestins on the murine Fshb promoter. They also suggest
that these elements play a role in supporting progestin
action at the nearby ⫺381 HRE via protein-protein interactions among FOXL2, SMAD, and PR. The fact that the
⫺359/⫺274 deletion (which moved the ⫺273 HRE and
⫺267 SBE much closer to the ⫺381 HRE) partially restored progestin responsiveness and synergy between activin and progestins also suggests that multiple elements
Endocrinology, April 2012, 153(4):2023–2033
are necessary to stabilize interactions among SMAD,
FOXL2, and PR on the Fshb promoter.
We also determined whether additional FOXL2 binding elements that play a role in activin responsiveness on
the murine Fshb promoter also play a role in progestin
responsiveness alone or in combination with activin. Interestingly, these FOXL2 elements are in close proximity
to other elements important for the synergy. Because the
⫺208 FOXL2 element overlaps the ⫺197 HRE by 2 bp,
we mutated the 5⬘ T to try to separate the effect of FOXL2
vs. PR. The 5⬘ T mutation resulted in a decrease in progestin responsiveness and the synergy (Fig. 6B), in addition
to the previously reported effect on activin induction (29).
The ⫺153 FOXL2 element (originally identified as a putative SMAD half-site at ⫺149) (26) overlaps the ⫺139
HRE by 2 bp. Mutation of residues in the SMAD half-site
reduced activin response and, thus, the synergy (Fig. 3C),
whereas the 3⬘ T mutation also decreased progestin induction of Fshb (Fig. 6B). Mutation of the ⫺106 FOXL2
site increased progestin response, but the synergy was still
reduced, presumably due to the role of the ⫺106 element
in activin induction (Fig. 6B). Similarly to the ⫺350
FOXL2 site, these results suggest that the ⫺208, ⫺153,
and ⫺106 FOXL2 elements are necessary for a maximal
progestin response and synergy between activin and progestins. This idea is supported by the fact that cotransfection with the DN FOXL2 ⌬133–375 reduced the synergistic induction of Fshb gene expression by activin and
progestins.
Because FOXL2 is necessary for the full synergistic response between activin and progestins, we tested whether
there was a direct interaction between FOXL2 and PR.
Using a GST interaction assay, we showed that FOXL2
can physically interact with PR as well as with SMAD3.
Our data expand the set of proteins that can directly partner with FOXL2. Moreover, these results also suggest that
FOXL2 may interact with PR directly and/or indirectly via
SMAD3 and that a tripartite complex of FOXL2, SMAD,
and PR may be important for the synergy between activin
and progestins on the murine Fshb promoter.
In summary, our experimental results provide evidence
that the FOXL2 transcription factor is involved in the
cooperative interaction between activin and progesterone
signaling pathways in pituitary gonadotrope cells. We
demonstrate that the murine Fshb promoter contains a
cluster of species-specific elements critical for activin and
progestin synergy that may play a crucial role in the rapid
elevation in Fshb mRNA before the secondary FSH surge
in rodents. These elements are conserved on the rat promoter and likely function in a similar manner. In contrast,
the ovine and human FSHB promoters are not induced by
progestins alone or in combination with activin, suggest-
endo.endojournals.org
2031
ing that other mechanisms regulate FSHB gene expression
in these species. Our data also indicate that the cooperation between activin and progesterone signaling pathways
requires the binding of FOXL2, SMAD, and PR to their
respective elements on the murine Fshb promoter and suggest that physical interactions among these transcription
factors may be important for the synergy. Future experiments characterizing these interactions will shed light on
how synergy between activin and progesterone signaling
pathways regulates reproductive fitness. Cooperation
among transcription factors such as FOXL2, SMAD, and
PR may also prove to be important for the regulation of
gene expression in other reproductive tissues such as the
ovary (60).
Acknowledgments
We thank Djurdjica Coss, Kellie Breen, and Scott Kelley for their
comments, suggestions, helpful discussions, and critical reading
of the manuscript. We thank Susan Mayo for technical assistance. We are grateful to Malcolm Low, Dan Bernard, Dean
Edwards, Louise Bilezikjian, Djurdjica Coss, Rik Derynck, and
Douglass Forbes for providing reagents. We also acknowledge
using the DNA Sequencing Shared Resource, UCSD Cancer Center, funded in part by NCI Cancer Center Support Grant P30
CA023100 for sequencing.
Address all correspondence and requests for reprints to: Varykina G. Thackray, Ph.D., Department of Reproductive Medicine, University of California at San Diego, 9500 Gilman Drive,
La Jolla, California 92093. E-mail: vthackray@ucsd.edu.
This work was supported by National Institutes of Health
(NIH) Grant K01 DK080467 and R01 HD067448 (to V.G.T.)
and Eunice Kennedy Shriver National Institute of Child Health
and Human Development/NIH through a cooperative agreement (U54 HD012303) as part of the Specialized Cooperative
Centers Program in Reproduction and Infertility Research and
R01s HD020377 and DK044838 (to P.L.M.). P.L.M. was partially supported by P30 DK063491, P30 CA023100, and P42
ES010337. J.K.S. was supported by an Endocrine Society Student Summer Research Fellowship.
Disclosure Summary: The authors have nothing to disclose.
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