| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Departments of Reproductive Medicine (F.P., V.V.V., S.B.R., J.S.B., P.L.M.) and Neuroscience (V.V.V., P.L.M.), University of California-San Diego, La Jolla, California 92093-0674; and Department of Biochemistry, North Carolina State University (H.-J.H., W.L.M.), Raleigh, North Carolina 27695
Address all correspondence and requests for reprints to: Pamela L. Mellon, Ph.D., Department of Reproductive Medicine 0674, University of California-San Diego, 9500 Gilman Drive, La Jolla, California 92093-0674. E-mail: pmellon{at}ucsd.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
- and ß-subunits of
LH, but was thought not to express FSH. Using RT-PCR, we show that
these cells synthesize FSH ß- subunit messenger RNA, which is
induced by activin and inhibited by follistatin. Furthermore, in
transient transfections an ovine FSHß 5'-regulatory region (5.5 kb)
confers LßT2 cell-specific expression to a reporter gene compared
with other pituitary and nonpituitary cell lines. This FSHß
regulatory region responds to activin specifically in LßT2 cells, an
effect that is blocked by follistatin. The LHß, -subunit, and GnRH
receptor regulatory regions are induced by activin and blocked by
follistatin. Furthermore, LßT2 cells express the components of the
activin system, and addition of follistatin alone reduces FSHß gene
expression, demonstrating that an endogenous activin autocrine loop
regulates FSH in these cells. In addition, GnRH stimulates both the
FSHß and LHß regulatory regions, specifically in LßT2 cells.
Surprisingly, GnRH induction is reduced by follistatin, suggesting its
dependence on endogenous activin. As the mouse GnRH receptor promoter
is inhibited by follistatin, reduction of GnRH receptor levels might be
one mechanism by which follistatin interferes with GnRH induction of
gonadotropin genes. In summary, LßT2 cells exhibit the
characteristics of fully differentiated gonadotropes, including the
expression of LH, FSH, GnRH receptor, and components of the
activin/follistatin system, as well as display the appropriate
responses to activin and GnRH. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-subunit, but also contains a unique ß-subunit that confers
physiological specificity. Pituitary gonadotropin messenger RNA (mRNA) expression and protein secretion are regulated by pulses of GnRH from the hypothalamus (2). Differential control of LH vs. FSH production is determined by the frequency and amplitude of the GnRH pulses (3). Another important point of control at the pituitary level is the synthesis and activity of the seven-transmembrane G protein-coupled GnRH receptor (GnRH-R), which, along with the gonadotropins, is regulated by both steroid and peptide hormones (4, 5).
Activin and inhibin, members of the transforming growth factor-ß
family of growth factors, also play important roles in the modulation
of hormone secretion from pituitary gonadotropes. Activin stimulates
the synthesis of FSH (6), and inhibin represses the
synthesis of FSH in the pituitary (7, 8). Activin is
secreted by the gonads and within the pituitary gland, where it acts as
an autocrine and/or paracrine regulator of the reproductive axis.
Activin occurs as a homo- or heterodimer of two highly related
ß-subunits (ßA and
ßB) (9). Inhibin is an activin
antagonist composed of an activin ß- subunit
(ßA or ßB) and a unique
-subunit (8) secreted by the pituitary, gonads, adrenal
gland, and brain (10). Follistatin, a potent
activin-binding protein, is primarily secreted by gonadotrope and
folliculostellate cells within the pituitary (11). It
inhibits binding of activin to its receptors and thereby specifically
inhibits the synthesis and secretion of FSH by the pituitary
(12). This remarkable confluence of interacting hormones,
growth factors, and receptors within a single cell type indicates a
complex set of autocrine loops and endocrine responses that can be
differentially modulated to regulate the transcription and
secretion of the gonadotropins, LH and FSH.
Currently, very little is known about FSHß regulation at the transcriptional level, largely due to the lack of a differentiated gonadotrope cell line that produces endogenous FSHß in which to perform these studies. Primary cell culture, transfections into heterologous cells, and transgenic mice have been employed in the past to study transcriptional regulation of the FSHß gene. Each of these approaches has inherent disadvantages. A problem encountered when using primary pituitary cell culture preparations has been the multiplicity of endocrine cell types in this tissue. Gonadotropes comprise only 5–10% of pituitary cells, making it difficult to distinguish cell-specific responses over the background of activities in multiple other cell types. Moreover, the magnitude of a given response may be small due to the limited proportion of each cell type in the culture, making the study of modestly expressed genes difficult. The use of heterologous cells (13, 14) does not accurately reflect cell-specific responses, because distinct sets of transcription factors, kinases, G proteins, receptors, and other classes of molecules are expressed in different cell types. Finally, the use of transgenic mice has proven to be a very laborious and time-consuming approach (15) that does not allow analysis of signaling pathways or DNA-protein interactions.
Targeted expression of oncogenes in transgenic mice has been used to
immortalize specific cell types that can then serve as valuable
cultured model systems. Our laboratory has used 1.8 kb of the rat LHß
promoter to direct expression of the oncogene simian virus 40 T antigen
to the pituitary in mice (16). One of the cell lines
isolated from the tumors induced by this transgene, LßT2, has many
characteristics of a mature gonadotrope cell, including expression of
GnRH-R, steroidogenic factor 1 (SF-1), estrogen, and
progesterone receptors (17) and both the - and
ß-subunits of LH (16).
In the present study we show that the LßT2 cell line also expresses
FSH ß-subunit mRNA and that it is induced by activin and repressed by
follistatin. To investigate the potential of LßT2 cells as a model
for the study of FSH gene regulation, we performed transient
transfections with the 5.5-kb flanking region of the ovine FSHß
(oFSHß) gene, a region that successfully targets expression to the
pituitary of transgenic mice (18). We found that this
5.5-kb region is specifically expressed in the LßT2 gonadotrope cells
compared with other pituitary and nonpituitary cell lines. It also
responds to activin, an effect that is inhibited by follistatin.
Surprisingly, activin regulation is LßT2 cell specific, failing to
occur in T3–1 cells despite the fact that they are known to be
activin responsive (19, 20). Furthermore, LßT2 cells
express all four known activin receptors as well as activin
ßB-subunit, follistatin, and inhibin
-subunit. In addition, GnRH stimulates transcription from FSHß and
LHß, and this response is inhibited by follistatin, suggesting that
the GnRH response depends on endogenously produced activin. In concert
with this observation, the GnRH-R promoter is repressed by follistatin
in LßT2 cells, indicating that the activin dependence of the FSHß
response to GnRH may be due at least partially to activin regulation of
the GnRH-R gene. In summary, the 5'-flanking region of the ovine FSHß
gene is specifically expressed in LßT2 cells and responds
appropriately to its primary regulators, activin and GnRH. Thus, these
cells constitute an excellent model system to study the mechanisms
involved in FSHß gene regulation.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Construction of plasmids for transfection analysis
A 5.5-kb region of the ovine FSHß gene (oFSHß) encompassing
4741 bp of the promoter and 759 bp downstream from the +1 transcription
start site, including the first intron of the oFSHß gene (the oFSHß
translation start site was mutated to permit translation to initiate at
the reporter gene AUG), was subcloned into the KpnI and
XbaI restriction sites of the pGL3-Basic luciferase reporter
plasmid (Promega Corp., Madison, WI). The resulting
plasmid was named oFSHß-Luc.
The mouse GnRH-R promoter sequence was amplified by PCR. The 1227-bp band was verified by DNA sequencing and cloned in the BamHI/XhoI sites of the pUC18 backbone and named pGRBX. This plasmid was then digested with SphI (5'-end), filled with Klenow polymerase to generate a blunt end, and subsequently digested with XhoI. The isolated fragment was then cloned into the SmaI/XhoI sites of pGL3-Basic (Promega Corp.) and named GnRH-R-Luc.
The GSU-Luc plasmid contains -507 to +46 bp of the mouse
glycoprotein -subunit promoter and was provided by Dr. Richard
Maurer (21). The LHß-Luc plasmid contains the region
between -1800 and +5 bp of the rat LHß promoter (provided by Dr.
James Roberts) cloned upstream of a luciferase reporter gene in the
pUC18 plasmid backbone.
The thymidine kinase (TK)-Luc plasmid contains the -81 bp region of the herpes simplex I thymidine kinase promoter cloned in pXP2, described by Dr. Steve Nordeen (22), provided by Dr. Sylvia Evans. This region of the TK promoter is missing both the CCAAT box and the distal GC box present in the 109-bp fragment of the promoter.
Plasmid DNA was prepared from overnight bacterial cultures using plasmid columns according to the supplier’s protocol (QIAGEN, Chatsworth, CA) or a cesium chloride protocol adapted from Sambrook et al. (23).
Cell culture and transient transfections
Cells were grown in 60-mm diameter dishes to 60–70% confluence
in DMEM (Cellgro, Mediatech, Inc., Herndon, VA) supplemented with 10%
FBS (Omega Scientific, Inc., Tarzana, CA) at 37 C with 5%
CO2. Transient transfections were performed using
FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN), following the manufacturer’s
protocol. For the activin/follistatin experiments, cells were
pretreated with follistatin (250 ng/ml) at the time of transfection.
After overnight incubation (16 h), cells were washed twice with 1
x PBS, and each independent treatment was then added in DMEM with 1%
FBS: activin (100 ng/ml), follistatin (250 ng/ml), or activin plus
follistatin. Cells were then incubated for an additional 24 h.
GnRH treatments were performed at 1 nM for 6 h before
harvest. The cells were then harvested, and luciferase and
ß-galactosidase assays performed. All experiments were performed
using 0.44 pmol of the reporter plasmids, unless noted differently.
Rous sarcoma virus (RSV)-ß-galactosidase (0.5 µg) was used as an
internal control unless noted differently. All transfection experiments
were performed at least three times.
Luciferase and ß-galactosidase assays
Cells were washed twice in 1 x PBS and then 1 ml
harvesting buffer (0.15 M NaCl, 1 mM EDTA, and
40 mM Tris-HCl, pH 7.4) was added to each dish. Cells were
then scraped, transferred to microcentrifuge tubes, and collected by
centrifugation at 3000 rpm for 5 min. The supernatant was discarded,
and the cells were resuspended in 50–100 µl lysis solution
(Galacto-light assay system, Tropix, Bedford, MA).
Luciferase activity was measured using an E.G.&G. Berthold Microplate Luminometer by injecting 100 µl of a buffer containing 100 mM Tris-HCl (pH 7.8), 15 mM MgSO4, 10 mM ATP, and 65 µM luciferin/well. ß-Galactosidase assays were performed using the Galacto-light assay system following the manufacturer’s protocol. Before each ß-galactosidase assay, cell extracts were heat-inactivated at 48 C for 50 min.
Reverse transcriptase, PCR, and Southern blotting
Total RNA was isolated by the method of Chomczynski and
Sacchi (24). Polyadenylated
[poly(A)+] mRNA was extracted using the
PolyATract mRNA Isolation System IV (Promega Corp.)
according to the manufacturer’s protocol. The concentration of each
RNA sample was determined by UV absorbance measurement. Twenty
nanograms of each RNA sample were annealed to 300 pmol of random
hexamers (pdN6, Pharmacia Biotech, Piscataway, NJ) by
heating at 70 C for 10 min and then chilling on ice. The RT reaction
buffer, 7.5 mM dithiothreitol, and 0.37 mM
deoxy-NTP mix were added to the annealed sample, and the mix was heated
for 10 min at 25 C, and then incubated for 2 min at 42 C, after which
200 U Superscript TM II RT (Life Technologies, Inc.,
Gaithersburg, MD) were added. The reaction was then incubated for an
additional 50 min at 42 C, after which the enzyme was inactivated by
incubation at 70 C for 10 min. A 1-µl aliquot of the RT reaction was
used as a template for the PCR reaction. Mouse FSHß primers (sense,
5'-GGACGTAGCTGTTTACTTCCCAG-3'; antisense, 5'-AGGGAGTCTGAGTGGCGGGC-3')
were designed to span intron 2. The expected FSHß DNA products were
235 bp (spanning 97 bp of the 3'-end of exon 2 plus 138 bp of the
5'-end of exon 3) and 512 bp (235 bp of exons 2 and 3 sequences as
described above plus 227 bp corresponding to intron 2) for
complementary DNA (cDNA) and genomic products (25),
respectively. The PCR reaction was performed in a final volume of 30
µl with 1.25 U Taq DNA polymerase (Amersham Pharmacia Biotech), 3 µl 10 x Taq buffer, 0.5
mM deoxy-NTP mix, and 10 pmol of each primer. We
then performed 35 PCR cycles: 1 min at 94 C, 1 min at 63 C, and 1 min
at 68 C, followed by an elongation step for 10 min at 68 C. PCR
products were electrophoresed on 2% agarose and transferred to a nylon
membrane (Hybond N+, Amersham Pharmacia Biotech, Arlington Heights, IL) following the Southern blot
protocol described by Sambrook et al. (23). The
rat FSHß cDNA (26) probe fragment was labeled by random
priming (27).
Northern blotting
Total RNA was isolated using the TRIzol reagent, (Life Technologies, Inc.), following the manufacturer’s protocol and
poly(A)+ RNA was extracted using PolyATract mRNA
Isolation System IV (Promega Corp., Madison, WI) according
to the manufacturer’s protocol. Two µg of each
polyA+ RNA sample were electrophoresed in a
denaturing 1% agarose gel containing formaldehyde and transferred to
Hybond N+ nylon membrane (Amersham Pharmacia Biotech). The RNA was fixed by UV cross-linking using
a UV chamber (Bio-Rad Laboratories, Inc., Richmond, CA).
Membranes were hybridized overnight with
32P-labeled cDNA probe at 55 C in a 25%
formamide solution. DNA probes were labeled by random priming
(27). Excess probe was removed by washing at 65 C with
0.2 x SSPE (36 mM NaCl, 2 mM sodium
phosphate, and 0.2 mM EDTA) and 0.1% SDS.
Statistics
In the transfection experiments, luciferase to ß-galactosidase
ratios were compared for each independent condition by the ANOVA
factorial test, followed by the Fisher’s protected least significant
difference post-hoc test. In all analyses, P
< 0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-subunit, LH
ß-subunit, and other gonadotrope-specific markers, such as GnRH-R and
SF-1; however, FSHß was not detected by this method
(28). The FSHß gene is regulated differently from the
LHß gene via mechanisms including activin stimulation and low
pulsatile GnRH frequency. We hypothesized that FSHß might be
expressed at low levels in LßT2 cells in the absence of these
physiological stimulators. We then performed RT-PCR, a more sensitive
method, to detect even low levels of FSHß RNA in LßT2 cells. LßT2
cell poly(A)+ mRNA was extracted from cells
pretreated for 16 h with follistatin to block endogenously
produced activin. The pretreatment was followed by a 48-h treatment
with activin, follistatin, or a combination of both.
Poly(A)+ mRNA from T3–1 cells treated under
the same conditions was used as a negative control, as these cells
represent a precursor gonadotrope that expresses GnRH-R, SF-1, and
-subunit, but neither of the ß-subunits (29).
Multiple RT-PCR products were observed on an agarose gel, including the
expected size for the amplified mouse FSHß cDNA fragment (235 bp;
data not shown). A Southern blot of the RT-PCR gel, probed with a rat
FSHß cDNA fragment (26), detected the 235-bp
FSHß-specific band (Fig. 1
) in
pretreated (-) LßT2 cell RNA. Treatment with activin increased the
expression of FSHß in LßT2 cells, as indicated by the stronger
intensity of the hybridization signal of the 235-bp PCR-amplified band.
Follistatin blocks the synthesis of FSHß RNA in LßT2 cells alone or
in combination with activin, as shown by the absence of detectable
FSHß signal under these conditions. Furthermore, no FSHß signal was
present in T3–1 cell samples regardless of activin and/or
follistatin treatments.
|
), we performed transient transfection
experiments using a plasmid encompassing 5.5 kb of the ovine FSHß
5'-regulatory region driving expression of the luciferase reporter gene
(oFSHß-Luc). An RSV long-terminal repeat promoter driving expression
of a ß-galactosidase gene (RSV-ßgal) was cotransfected as an
internal control. A RSV luciferase plasmid (RSV-Luc) was transfected in
parallel with the oFSHß-Luc reporter gene to control for differences
in transfection efficiency, transcription rates, reporter mRNA
half-life, luciferase and ß-galactosidase protein stability, and
metabolic factors between distinct cell types. The ratio of the RSV-Luc
value (parallel control) to the RSV-ßgal value (internal control) was
set at 100 for each cell line, and the oFSHß-Luc values from each
cell line were normalized to the RSV-Luc/RSV-ßgal value. To validate
this approach, we tested the activities of the promoterless luciferase
expression vector pGL3 and a TK-Luc plasmid and found each to be
expressed at the same level in T3–1 and LßT2 cells relative to
RSV-Luc (data not shown). Basal expression of the oFSHß-Luc plasmid
is approximately 4-fold higher in LßT2 cells than in the
nongonadotrope cell lines tested (P < 0.001). The
luciferase activity in the gonadotrope progenitor cells (T3–1) is
greater than in other cells tested, but is still 2.2-fold lower than in
LßT2 cells. The corticotrope-derived (AtT20), hypothalamic
neuroendocrine (GT1–7) (30), fibroblast-derived
(NIH-3T3), and kidney-derived (CV1) cells show equivalently low levels
of expression of the reporter gene, indicating that the 5.5-kb oFSHß
regulatory region confers LßT2 cell specificity.
|
). It has been demonstrated that follistatin decreases
steady state FSHß mRNA levels in primary pituitary cell cultures by
destabilizing FSHß RNA (31). To analyze the
transcriptional effects of activin and/or follistatin treatments on the
oFSHß regulatory region, we transiently transfected the oFSHß-Luc
reporter plasmid into LßT2, T3–1, and NIH-3T3 cells. RSV-ßgal
was used as an internal control, and RSV-Luc was transfected into each
cell line in parallel to allow for normalization and comparison between
distinct cell lines. At the time of transfection, cells were subjected
to follistatin pretreatment to block endogenous activin. One set of
plates was transfected and not pretreated to allow observation of
autocrine activin effects. Sixteen hours after transfection, additional
treatments with activin and/or follistatin were performed for 24
h, after which the cells were harvested. As shown in Fig. 3, activin stimulation of transcription
from the oFSHß 5'-flanking region was only observed in LßT2 cells.
In these cells reporter gene expression was approximately 3- to 4-fold
higher in activin-treated cells compared with cells pretreated with
follistatin (P < 0.001). As expected, follistatin
treatment alone did not have a statistically significant effect on
transcription from the 5'-flanking region of the oFSHß gene compared
with cells that only received follistatin pretreatment. When activin
and follistatin were combined, follistatin completely blocked the
stimulatory effect of activin. In untreated cells, oFSHß activity was
approximately 2-fold higher than in the pretreated cells, indicating
that LßT2 cells synthesize endogenous activin, which is blocked by
pretreatment with follistatin (P < 0.001). The oFSHß
5.5-kb region did not respond to activin in either NIH-3T3 or T3–1
cells despite the fact that T3–1 cells respond to activin by
regulating both the GnRH-R gene (19) and the -subunit
gene (20). This suggests that members of the activin
signal transduction pathway involved in FSHß gene stimulation might
be present only in the more differentiated gonadotrope LßT2 cells.
Alternatively, activin induction of FSHß gene expression might be
dependent upon additional cell type- specific transcription factors
present only in cells that naturally express FSH.
|
-subunit, LHß, and
GnRH-R promoters in LßT2 cells-subunit (-GSU) promoter is repressed by
activin in transient transfections in the gonadotrope precursor
T3–1 cells (19) whereas the GnRH-R promoter is
stimulated by activin in these cells, effects that are blocked by
follistatin (20). Transient transfections with luciferase
reporter plasmids containing the oFSHß regulatory region, -507 to
+46 region of the mouse -GSU promoter (GSU-Luc), the 1.2-kb mouse
GnRH-R promoter (GnRH-R-Luc), or 1.8 kb of the rat LHß promoter
(rLH-Luc), were performed in LßT2 cells. TK-Luc and a promoterless
luciferase vector (pGL3) were used as negative controls (Fig. 4). Cells were pretreated with
follistatin for 16 h, followed by a 24-h treatment with activin,
follistatin, or a combination of both. To facilitate interpretation of
the data, the values for the follistatin-pretreated condition were set
at 1 for each plasmid tested. The oFSHß-Luc plasmid reproduced the
results shown in Fig. 3, in which activin treatment stimulated the
promoter 4-fold, and treatment with both follistatin and activin
prevented the induction of reporter gene expression. The GnRH-R
promoter was stimulated 6-fold by activin, compared with the pretreated
condition, and this effect was completely abolished by follistatin
(Fig. 4). Follistatin alone decreased GnRH-R (-) expression by 65%,
indicating that endogenous activin plays a role in the expression of
the GnRH-R promoter. Surprisingly, the LHß promoter showed a
statistically significant 60% increase (P < 0.0001)
upon activin treatment, and this stimulatory effect of activin was
completely blocked upon cotreatment with follistatin. However, the
LHß promoter was not affected by pretreatment with follistatin,
indicating that LßT2 endogenous activin is not sufficient to increase
the transcription of this promoter. In the case of the -GSU
promoter, an approximately 60% decrease in the activity of the
promoter in pretreated cells (P < 0.001) was observed
compared with that in the untreated group, demonstrating that the
expression of this promoter is maintained by endogenous activin in
LßT2 cells. Moreover, activin treatment led to a 40% increase in
transcription of the -GSU promoter (P < 0.01), an
effect that was abolished when activin was combined with follistatin.
As expected, the TK promoter does not respond to any of the
activin/follistatin treatments, nor does pGL3, demonstrating that the
activin/follistatin responses observed in the LßT2 cells are promoter
specific.
|
, 3, and 4), indicate that a functional
activin/follistatin signaling system is present in these cells. To
investigate whether activin, inhibin, and follistatin mRNAs are
expressed in LßT2 cells, we performed Northern blots with
poly(A)+ RNA extracted from LßT2, T3–1, and
NIH-3T3 cells and probed them with rat cDNA fragments coding for
activin ßB-subunit, follistatin, and inhibin
-subunit. Activin ßB- subunit
transcripts (4.2 and 3.4 kb) were observed (Fig. 5) in both LßT2 and T3–1 cells,
whereas NIH-3T3 cells did not express any of the
ßB-subunit RNA forms. These results indicate
that an activin autocrine loop is probably present in the LßT2 and
T3–1 cell cultures. As a positive control, we used ovary RNA, in
which we observed only the 4.2 kb ßB-subunit
form, in agreement with previous observations (32). As
pituitary gonadotropes have been shown to synthesize activin
ßB-subunit RNA (33), the presence
of endogenous activin in LßT2 cells models mature gonadotropes
in vivo and validates the use of this cell for the study of
activin regulation of gonadotropin gene expression. Two follistatin
mRNA species (2.5 and 1.6 kb) were observed in all three cell lines
tested (Fig. 5). It is not surprising that the NIH-3T3
fibroblast-derived cells also synthesize follistatin mRNA, as it has
been described in a wide variety of tissues, including pituitary
(34). Inhibin -subunit mRNA (1.6 kb) is observed only
in LßT2 cells (Fig. 5). Surprisingly, inhibin -subunit RNA was not
detectable in the less mature gonadotrope cell line, T3–1,
suggesting that inhibin is either expressed at very low levels or is
absent from T3–1 cells. In support of this idea, in rat embryos,
inhibin -subunit is detected by in situ hybridization
only later in development, between embryonic days 16 and 20
(35).
|
T3–1, and NIH-3T3
cells and probed with mouse cDNA fragments from each of the activin
receptors: Act RI, Act RIB, Act RII, and Act RIIB (Fig. 6). The expected 3.5-kb mRNA coding for
mouse Act RI (38) was observed in all three cell lines
tested, as was the 4.4-kb RNA transcript coding for activin receptor
type RIB (38). LßT2, T3–1, and NIH-3T3 cells also
expressed both the 6.9- and 2.1-kb RNA species corresponding to Act RII
and the 14-, 11-, 3.8-, and 2.4-kb RNA forms described for Act RIIB
(39). Thus, the pituitary-derived cells, LßT2 and
T3–1, and the fibroblast-derived NIH-3T3 cells synthesize RNA
transcripts corresponding to all four types of activin receptors,
indicating that these cell lines are capable of responding to activin
stimulation. Swiss 3T3 fibroblast cells have been shown to undergo
mitogenesis upon activin treatment (40); therefore it is
not surprising that activin receptor RNAs are present in NIH-3T3
fibroblasts.
|
T3–1 cells (41).
Furthermore, the rat LHß 5'-flanking region responds to GnRH after
transfection into LßT2 cells (42). To investigate the
responsiveness of the FSHß regulatory region in LßT2 cells, we
transfected the oFSHß-Luc expression vector and treated cells with 1
nM GnRH for 6 h, the optimal time and concentration
for induction of this gene (Vasilyev, V., F. Pernasetti, and P. L.
Mellon, in preparation). We compared the response of the oFSHß
regulatory region to those of the LHß and TK promoters in both LßT2
and T3–1 cells (Fig. 7). Both
oFSHß-Luc and rLHß-Luc were induced by GnRH in LßT2 cells, by 2-
and 1.6-fold, respectively. The minimal TK promoter did not respond to
GnRH in either of the cells tested. Remarkably, neither of the
ß-subunit promoters responded to GnRH treatment in the gonadotrope
precursor T3–1 cells despite the known GnRH responsiveness of this
cell line (21, 41), indicating that under these
conditions, the GnRH response of these regulatory regions is LßT2
cell specific.
|
), and this promoter is induced by GnRH in LßT2 cells (Fig. 7), we also tested whether the response of the LHß promoter to GnRH
in these cells is affected by follistatin. All transfected cells were
pretreated with follistatin to eliminate the effects of endogenously
secreted activin and then tested for GnRH response with or without
continued follistatin treatment. GnRH alone stimulates the oFSHß
reporter approximately 2-fold, whereas follistatin completely inhibits
the stimulatory effect of GnRH (Fig. 8),
bringing the activity of the oFSHß promoter to the same level as that
with follistatin alone. These data strongly suggest that endogenously
secreted activin acts within an autocrine loop that is essential for
the GnRH response of the FSHß promoter in LßT2 cells.
|
), which is largely blocked (by
70%) by follistatin (Fig. 8). This inhibition of GnRH induction is
only partial, as the value for the GnRH plus FS is statistically
different from the pretreated only condition and the pretreated plus
follistatin condition, indicating that follistatin is capable of
interfering with the GnRH response of the LHß promoter as well, but
to a lesser extent than the oFSHß promoter. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
T3–1 cells display functional GnRH-R and
express both SF-1 and glycoprotein hormone -subunit RNAs, indicating
that they reside in the gonadotrope lineage (29). As they
do not express the FSHß or LHß subunits, we proposed that this cell
line represents a fetal gonadotrope progenitor arrested between
embryonic days 13.5 and 16.5 of development within the period when GnRH
receptors are detectable (43), but before the expression
of the LHß gene (44). Our previous observation that
LßT2 cells express the LH ß- subunit gene in addition to the
gonadotrope markers expressed by the T3–1 cells had led us to
conclude that LßT2 represented gonadotrope cells arrested between
embryonic days 16.5 and 17.5 of mouse development (16),
before the expression of the FSH ß-subunit gene
(44). Here we have demonstrated that the LßT2 cells do
express FSHß mRNA, using a sensitive detection method [RT-PCR on
poly(A)+ mRNA], and that the transfected FSHß
regulatory region recapitulates gonadotrope specificity. Furthermore,
LßT2 cells express the components of the activin and follistatin
system as well as responding to activin and GnRH by regulating the
gonadotropin ß-subunit genes. Thus, we now conclude that the LßT2
cell represents a gonadotrope with the major hallmarks of the fully
differentiated state in the adult mouse pituitary. FSH ß-subunit mRNA is detectable in untreated LßT2 cells by RT-PCR. It is strongly induced by activin and rendered undetectable by follistatin. In a previous study FSHß mRNA was not observed in untreated LßT2 cells by ribonuclease protection (45), although it increased in a dose-dependent manner in response to activin (45). Our detection of FSHß mRNA in the absence of exogenously added activin is probably due to the acute sensitivity of the RT-PCR assay. Alternatively, different cell culture conditions could account for the different observations by affecting the endogenous production of activin or steroid levels. For example, Graham et al. (45) cultured the cells in 10% charcoal-stripped serum supplemented with dexamethasone (20 nM) in DMEM/Ham’s F-12 (1:1), whereas we used 1% nonstripped serum in DMEM.
Transcription from the ovine FSHß regulatory region is significantly
higher in the LßT2 gonadotrope cell line than in other cell types.
Expression levels are intermediate in the less differentiated
gonadotrope cell line, T3–1. Although very little information is
available regarding the transcription factors involved in the
expression of FSHß, SF-1 is known to play a key role in the
expression of the LHß gene, interacting with pituitary homeobox 1
(Ptx1) and early growth response protein 1 (Egr-1) (46).
Despite the importance of these proteins in the expression of the LHß
gene, SF-1, Ptx-1, and Egr-1 cannot entirely account for the
gonadotrope-specific expression of either the LH or FSH ß-subunit
genes because they are all present in T3–1 cells, which do not
express either of the ß-subunit mRNAs (46). Targeted
disruption of the Egr-1 gene in mice results in specific loss of LHß
gene expression, yet FSHß gene expression is maintained
(47). In contrast, a null mutation in the SF-1 gene
selectively reduces expression of both gonadotropins in transgenic mice
(48), whereas Ptx-1 null mice have decreased numbers of
gonadotrope cells and lower levels of LHß and FSHß
(49). These observations suggest that both common and
distinct proteins are likely to regulate the expression of the LHß
and FSHß genes in LßT2 cells.
Expression of activin ßB-subunit and all four
activin receptor RNA forms together with the ability to respond
appropriately to activin signaling indicate that LßT2 cells
accurately recapitulate activin regulation of the FSHß gene in the
natural gonadotrope. Our transient transfection experiments with
activin show that the oFSHß regulatory region is induced by activin
treatment in LßT2 cells. This result is in agreement with experiments
performed in primary pituitary cells in which activin causes an
increase in FSHß primary transcripts as detected by RT-PCR
(50). Cotreatment with the transcriptional inhibitor
actinomycin D blocked activin stimulation in primary cells, indicating
that the activin response involves transcriptional activation of the
FSHß gene (50). Interestingly, the oFSHß regulatory
region is not regulated by activin in the gonadotrope precursor cell,
T3–1. This lack of response does not appear to be due to the
absence of activin receptors in T3–1 cells, as we have demonstrated
that these cells synthesize mRNA for all four activin receptor types.
Furthermore, the GnRH-R (19) and -subunit
(20) genes are regulated by activin in T3–1 cells.
Thus, the lack of responsiveness of FSHß to activin in T3–1 cells
implies the involvement of LßT2 cell-specific partners for the
downstream activin-signaling molecules that are not present or active
in T3–1 cells. Smad 2 and Smad 3, members of the novel Smad family
of cytoplasmic proteins (51), are downstream targets of
activin. Scanning of the oFSHß 5.5-kb regulatory region of the
oFSHß gene for Smad protein consensus DNA-binding sites
(5'-GTCTAGAC-3') (52) revealed at least 23 sites with 6 or
7 of 8 bases conserved, suggesting a possible role for these proteins
in the activin regulation of oFSHß gene expression. The cell
specificity of activin regulation of the oFSHß gene may involve Smad
proteins present only in more mature gonadotropes, or perhaps
interaction of Smad proteins with specific transcription factors that
may be present in LßT2 cells, but not in the less mature T3–1
cell type. Alternatively, there could be repressor molecules in
T3–1 cells that are not present in LßT2 cells.
Our experiments comparing the activin responsiveness of several
gonadotrope-specific promoters in LßT2 cells demonstrate that these
responses are not only cell specific, but also promoter specific. The
GnRH-R promoter is strongly stimulated by activin in these cells
(6-fold), similar to the approximately 3-fold induction found in
T3–1 cells (19). Interestingly, activin also induces
the -GSU promoter by 40% in LßT2, and this induction is blocked
by the addition of follistatin. These data are in agreement with
observations in primary rat pituitary cultures, where activin had a
small, but significant (10–20%), stimulatory effect on -subunit
synthesis and secretion after 48–72 h (53). However, this
is in contrast to previous observations in T3–1 cells, where
activin was reported to decrease transcription of the -GSU promoter
by approximately 50% (20). These differences in
responsiveness of the -GSU promoter may be due to the presence of
distinct activin response mechanisms in each cell line, suggesting
distinct activin transcriptional regulation of the -subunit gene at
different stages of gonadotrope development. Attardi et al.
(20) suggested that activin might suppress -subunit
gene expression in the embryonic pituitary until the specific
ß-subunit genes are expressed. Our observation that the -subunit
promoter is stimulated by activin in LßT2 cells, which express both
ß-subunits, supports this hypothesis. Activin regulation of
-subunit gene expression may recapitulate a developmental program in
the pituitary gonadotropes and deserves further exploration. Another
possibility is that these different observations result from distinct
experimental conditions. We performed our experiments in DMEM with 1%
FBS, pretreating the LßT2 cells with 250 ng/ml follistatin and
treating with 100 ng/ml activin, whereas the T3–1 cells
(20) were grown in Eagle’s MEM with 10% dextran-coated
charcoal-stripped FBS, no pretreatment was performed, and only 10 ng/ml
activin was added. The different results obtained in stripped
vs. whole serum might indicate a role for steroid hormones
or other growth factors in this regulatory process.
We also observed that the LHß promoter is significantly stimulated by
exogenous activin in LßT2 cells, and this effect is blocked by
follistatin (Fig. 4). The negative controls, TK-Luc and pGL3, did not
respond to any of the treatments, supporting the specificity of the
LHß promoter responses under these experimental conditions.
Interestingly, the pretreatment with follistatin to remove endogenous
activin does not have a significant repressive effect on the LHß
promoter, indicating that higher concentrations of activin might be
necessary to observe regulation of LHß transcription. To our
knowledge, these data are the first to demonstrate an effect of activin
on transcription of the LHß gene. Several reports have failed to
detect an effect of activin on LH secretion (54, 55).
However, Stouffer et al. (56) have shown acute
and sustained increases in LH secretion in response to activin in
female rhesus monkeys. These researchers observed a 2-fold increase in
LH within 8 h, and the levels remained 10-fold higher than control
values after 4–5 days. McLachlan and co-workers (57)
reported that 2-day activin infusion in adult male monkeys
significantly increased LH as well as FSH release in response to GnRH
injection. It has also been previously demonstrated that treatment of
primary pituitary cell cultures with activin for 24 h results in
small, but significant, increases in LH secretion and cell content as
well as in steady state LHß mRNA levels (53). These data
indicate that activin might be a physiological regulator of LHß
transcription and that the LßT2 cells can serve as a model in which
to further investigate the regulatory pathways involved in this
response.
Our transient transfection experiments show that continuous GnRH treatment regulates oFSHß in LßT2 cells, with a maximal 2-fold increase in reporter activity at 1 nM GnRH after a 6-h incubation. This is in agreement with transient transfection experiments in heterologous HeLa and COS-7 cells cotransfected with expression vectors for the GnRH-R, in which the 5.5-kb regulatory region of oFSHß is stimulated 3.1- and 2.7-fold, respectively, by treatment with 100 nM GnRH for 12 h (14).
Surprisingly, we observed that FSHß regulation by GnRH is LßT2 cell
specific (Fig. 7) compared with the less differentiated gonadotrope,
T3–1. We have previously shown that T3–1 cells synthesize
GnRH-R and are capable of responding to GnRH by inducing -GSU
transcription (41). The LßT2 cell specificity of the
GnRH response suggests that this effect most likely involves factors
present only in this highly differentiated gonadotrope cell line. We
observed the same cell specificity in the activin response of oFSHß
(Fig 3), reinforcing the hypothesis that the LßT2 cell line
represents a highly differentiated stage in gonadotrope development,
and that these cells synthesize specific sets of transcription factors
involved in the molecular pathways of both activin and GnRH
responses.
Interestingly, the response of the LHß promoter to GnRH is also
LßT2 cell specific under our experimental conditions. Previous work
in T3–1 cells by Weck and co-workers (58) showed
transcriptional regulation by GnRH of a transiently transfected
fragment of the rat LHß promoter, containing only the -617 to -245
bp region fused to a minimal TK promoter. This region was induced
approximately 2-fold by a 6-h treatment with 10 nM GnRH,
whereas the TK promoter did not respond under the same conditions.
These observations suggest that the full LHß promoter might contain
additional GnRH regulatory regions, including possible repressive
elements, that are not present in the small promoter region from -617
to -245 bp. Thus, we hypothesize that in the context of the precursor
gonadotrope T3–1 cells, which do not synthesize gonadotropin
ß-subunits, the intact rat LHß promoter is not induced by GnRH,
perhaps due to inhibitory factors expressed in these cells that are not
present or active in LßT2 cells. Alternatively, T3–1 cells could
lack key factors that allow the LHß and FSHß regulatory regions to
respond to GnRH or, for that matter, activin.
The observation that follistatin blocks GnRH stimulation of the regulatory region of the ß-subunit genes in transient transfections in LßT2 cells suggests that activity of the activin autocrine loop is required for GnRH stimulation of these genes. These data are in agreement with the observation that GnRH stimulation of FSHß endogenous mRNA is dependent on activin and blocked by follistatin both in perifusion of primary pituitary cells and in vivo (59). However, follistatin has also been shown to bind to inhibin (60), bone morphogenic protein-4 (BMP4), and BMP7 (61), albeit with much lower affinity than that for activin. We have previously described the presence of BMP7 mRNA transcripts in LßT2 cells, by RT-PCR, whereas BMP4 and BMP2 transcripts were not detected (62). However, although BMP7 is capable of binding to Act RI, Act RII, and Act RIIB, with affinities 3-fold lower than activin, it did not stimulate FSH secretion from primary rat pituitary cells (63). These findings do not exclude the possibility that BMPs and transforming growth factor-ß family members play a role in FSHß gene transcription, but strongly suggest an important role for the interaction between activin and follistatin in this process.
The follistatin blockade of GnRH responsiveness of both the FSHß and
LHß promoters (Fig. 8) suggests two possible mechanisms. The first
involves a direct effect of the activin/follistatin system on the
signaling pathways employed by GnRH to exert its activity, and the
second mechanism involves regulation of the GnRH-R itself, and hence
GnRH sensitivity, by the activin/follistatin system. Taken together,
our data provide evidence that both of these mechanisms may play roles
in follistatin blockade of the GnRH response. The 6-fold stimulation by
activin of the GnRH-R regulatory region is completely blocked by
follistatin. In addition, this regulatory region is inhibited 2-fold by
follistatin treatment alone, indicating that levels of GnRH-R may be
reduced after follistatin treatment alone, probably resulting in a
reduction of the responsiveness to GnRH. These data tend to support the
second mechanism. On the other hand, although the GnRH response of the
FSHß promoter is completely blocked by follistatin, the LHß
promoter response to GnRH is only reduced by 70% in the presence of
follistatin. This suggests that the inhibition of GnRH-R synthesis in
these cells might not be the only pathway through which follistatin is
interfering with the GnRH response. Thus, at least in the case of LHß
gene regulation, cross-talk between the activin and GnRH signal
transduction pathways might play a role in the dependence of GnRH
action on the activin/follistatin system.
In conclusion, LßT2 cells are a unique gonadotrope cell model in
which to study regulation of the LHß and FSHß genes. Our studies
place these cells in the category of a fully differentiated
gonadotrope, expressing the full spectrum of characteristic markers for
these cells: -subunit, SF-1, activin
ßB-subunit, follistatin, inhibin, activin
receptors, GnRH-R, and LHß and FSHß subunits. The oFSHß
regulatory region is specifically active in LßT2 cells, compared with
other pituitary and nonpituitary cell lines, and its response to FSH’s
main physiological regulators, activin and GnRH, is LßT2 cell
specific. We have also revealed a requirement for an endogenous activin
autocrine loop for GnRH regulation of the gonadotropin ß-subunit
genes, indicating interaction between GnRH action and the
activin/follistatin system in LßT2 cells.
Identification of the transcriptional elements and proteins involved in the regulation of both FSHß and LHß genes is now feasible using the homologous gonadotrope LßT2 cell model system. Such studies will shed further light on the mechanisms of action and likely interactions between activin and GnRH signaling pathways in the gonadotrope. Understanding the regulation of LH and FSH gene expression in gonadotrope cells will contribute critical knowledge for clinical applications in a variety of endocrine and reproductive conditions.
|
Acknowledgments |
|---|
-subunit promoter plasmid. Probes for the activin
receptors, follistatin, and activin/inhibin subunits were kindly
provided by W. Vale, S. Shimasaki, and V. Roberts. |
Footnotes |
|---|
2 Supported by an American Heart Association postdoctoral fellowship
and a fellowship from the Lalor Foundation.
3 Supported by the NIH Training Grant T32-DK-07541.
4 V.V.V. and S.B.R. contributed equally to this work.
5 Supported by the NIH Training Grant T32-GM-08666.
Received October 27, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, ßA, and ßB subunits in
various tissues predicts diverse functions. Proc Natl Acad Sci USA 85:247–51-subunit gene expression and secretion by
activin in T3–1 cells. Mol Endocrinol 9:1737–1749-subunit gene. Mol Endocrinol 6:893–903This article has been cited by other articles:
 |
|
 |
|
  J. Chen, B.-S. An, L. Cheng, G. L. Hammond, and P. C. K. Leung Gonadotropin-Releasing Hormone-Mediated Phosphorylation of Estrogen Receptor-{alpha} Contributes to fosB Expression in Mouse Gonadotrophs Endocrinology, October 1, 2009; 150(10): 4583 - 4593. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Wang, P. K Nicholls, P. G Stanton, C. A Harrison, M. Sarraj, R. B Gilchrist, J. K Findlay, and P. G Farnworth Extra-ovarian expression and activity of growth differentiation factor 9 J. Endocrinol., September 1, 2009; 202(3): 419 - 430. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Wiater, K. A. Lewis, C. Donaldson, J. Vaughan, L. Bilezikjian, and W. Vale Endogenous Betaglycan Is Essential for High-Potency Inhibin Antagonism in Gonadotropes Mol. Endocrinol., July 1, 2009; 23(7): 1033 - 1042. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. G. Thackray, J. L. Hunnicutt, A. K. Memon, Y. Ghochani, and P. L. Mellon Progesterone Inhibits Basal and Gonadotropin-Releasing Hormone Induction of Luteinizing Hormone {beta}-Subunit Gene Expression Endocrinology, May 1, 2009; 150(5): 2395 - 2403. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Bak, L. Carpio, J. L Kipp, P. Lamba, Y. Wang, R.-S. Ge, M. P Hardy, K. E Mayo, and D. J Bernard Activins regulate 17{beta}-hydroxysteroid dehydrogenase type I transcription in murine gonadotrope cells J. Endocrinol., April 1, 2009; 201(1): 89 - 104. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Wang, J. Fortin, P. Lamba, M. Bonomi, L. Persani, M. S. Roberson, and D. J. Bernard Activator Protein-1 and Smad Proteins Synergistically Regulate Human Follicle-Stimulating Hormone {beta}-Promoter Activity Endocrinology, November 1, 2008; 149(11): 5577 - 5591. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Feng, M. A. Lawson, and P. Melamed A Proteomic Comparison of Immature and Mature Mouse Gonadotrophs Reveals Novel Differentially Expressed Nuclear Proteins that Regulate Gonadotropin Gene Transcription and RNA Splicing Biol Reprod, September 1, 2008; 79(3): 546 - 561. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. A. Ciccone, C. T. Lacza, M. Y. Hou, S. J. Gregory, K.-Y. Kam, S. Xu, and U. B. Kaiser A Composite Element that Binds Basic Helix Loop Helix and Basic Leucine Zipper Transcription Factors Is Important for Gonadotropin-Releasing Hormone Regulation of the Follicle-Stimulating Hormone {beta} Gene Mol. Endocrinol., August 1, 2008; 22(8): 1908 - 1923. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Sasson, S. H. Luu, V. G. Thackray, and P. L. Mellon Glucocorticoids Induce Human Glycoprotein Hormone {alpha}-Subunit Gene Expression in the Gonadotrope Endocrinology, July 1, 2008; 149(7): 3643 - 3655. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Lamba, V. Khivansara, A. C. D'Alessio, M. M. Santos, and D. J. Bernard Paired-Like Homeodomain Transcription Factors 1 and 2 Regulate Follicle-Stimulating Hormone {beta}-Subunit Transcription through a Conserved cis-Element Endocrinology, June 1, 2008; 149(6): 3095 - 3108. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L Nicol, M-O Faure, J R McNeilly, J Fontaine, C Taragnat, and A S McNeilly Bone morphogenetic protein-4 interacts with activin and GnRH to modulate gonadotrophin secretion in L{beta}T2 gonadotrophs J. Endocrinol., March 1, 2008; 196(3): 497 - 507. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. G. Thackray and P. L. Mellon Synergistic Induction of Follicle-Stimulating Hormone {beta}-Subunit Gene Expression by Gonadal Steroid Hormone Receptors and Smad Proteins Endocrinology, March 1, 2008; 149(3): 1091 - 1102. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Gardner, S. Maudsley, R. P. Millar, and A. J. Pawson Nuclear Stabilization of {beta}-Catenin and Inactivation of Glycogen Synthase Kinase-3{beta} by Gonadotropin-Releasing Hormone: Targeting Wnt Signaling in the Pituitary Gonadotrope Mol. Endocrinol., December 1, 2007; 21(12): 3028 - 3038. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Coss, C. M. Hand, K. K. J. Yaphockun, H. A. Ely, and P. L. Mellon p38 Mitogen-Activated Protein Kinase Is Critical for Synergistic Induction of the FSH{beta} Gene by Gonadotropin-Releasing Hormone and Activin through Augmentation of c-Fos Induction and Smad Phosphorylation Mol. Endocrinol., December 1, 2007; 21(12): 3071 - 3086. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. L. Chand, G. T. Ooi, C. A. Harrison, A. N. Shelling, and D. M. Robertson Functional analysis of the human inhibin {alpha} subunit variant A257T and its potential role in premature ovarian failure Hum. Reprod., December 1, 2007; 22(12): 3241 - 3248. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. A Ferris, H. E Walsh, J. Stevens, P. C Fallest, and M. A Shupnik Luteinizing Hormone Beta Promoter Stimulation by Adenylyl Cyclase and Cooperation with Gonadotropin-Releasing Hormone 1 in Transgenic Mice and LBetaT2 Cells Biol Reprod, December 1, 2007; 77(6): 1073 - 1080. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Takeda, F. Otsuka, H. Otani, K. Inagaki, T. Miyoshi, J. Suzuki, Y. Mimura, T. Ogura, and H. Makino Effects of peroxisome proliferator-activated receptor activation on gonadotropin transcription and cell mitosis induced by bone morphogenetic proteins in mouse gonadotrope L{beta}T2 cells J. Endocrinol., July 1, 2007; 194(1): 87 - 99. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Lim, M. Luo, M. Koh, M. Yang, M. N. bin Abdul Kadir, J. H. Tan, Z. Ye, W. Wang, and P. Melamed Distinct Mechanisms Involving Diverse Histone Deacetylases Repress Expression of the Two Gonadotropin {beta}-Subunit Genes in Immature Gonadotropes, and Their Actions Are Overcome by Gonadotropin-Releasing Hormone Mol. Cell. Biol., June 1, 2007; 27(11): 4105 - 4120. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Lawson, R. Tsutsumi, H. Zhang, I. Talukdar, B. K. Butler, S. J. Santos, P. L. Mellon, and N. J. G. Webster Pulse Sensitivity of the Luteinizing Hormone {beta} Promoter Is Determined by a Negative Feedback Loop Involving Early Growth Response-1 and Ngfi-A Binding Protein 1 and 2 Mol. Endocrinol., May 1, 2007; 21(5): 1175 - 1191. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. B Lee, V. Khivansara, M. M Santos, P. Lamba, T. Yuen, S. C Sealfon, and D. J Bernard Bone morphogenetic protein 2 and activin A synergistically stimulate follicle-stimulating hormone {beta} subunit transcription J. Mol. Endocrinol., February 1, 2007; 38(2): 315 - 330. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. McGillivray, V. G. Thackray, D. Coss, and P. L. Mellon Activin and Glucocorticoids Synergistically Activate Follicle-Stimulating Hormone {beta}-Subunit Gene Expression in the Immortalized L{beta}T2 Gonadotrope Cell Line Endocrinology, February 1, 2007; 148(2): 762 - 773. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Larder, D. Karali, N. Nelson, and P. Brown Fanconi Anemia a Is a Nucleocytoplasmic Shuttling Molecule Required for Gonadotropin-Releasing Hormone (GnRH) Transduction of the GnRH Receptor Endocrinology, December 1, 2006; 147(12): 5676 - 5689. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Zhang, J. S. Bailey, D. Coss, B. Lin, R. Tsutsumi, M. A. Lawson, P. L. Mellon, and N. J. G. Webster Activin Modulates the Transcriptional Response of LssT2 Cells to Gonadotropin-Releasing Hormone and Alters Cellular Proliferation Mol. Endocrinol., November 1, 2006; 20(11): 2909 - 2930. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. G. Thackray, S. M. McGillivray, and P. L. Mellon Androgens, Progestins, and Glucocorticoids Induce Follicle-Stimulating Hormone {beta}-Subunit Gene Expression at the Level of the Gonadotrope Mol. Endocrinol., September 1, 2006; 20(9): 2062 - 2079. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. M Bilezikjian, A. L Blount, C. J Donaldson, and W. W Vale Pituitary actions of ligands of the TGF-{beta} family: activins and inhibins. Reproduction, August 1, 2006; 132(2): 207 - 215. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Z. Altuntas, J. M. Johnson, and V. K. Tuohy Autoimmune Targeted Disruption of the Pituitary-Ovarian Axis Causes Premature Ovarian Failure J. Immunol., August 1, 2006; 177(3): 1988 - 1996. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Lamba, M. M Santos, D. P Philips, and D. J Bernard Acute regulation of murine follicle-stimulating hormone {beta} subunit transcription by activin A J. Mol. Endocrinol., February 1, 2006; 36(1): 201 - 220. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Kanasaki, G. Y. Bedecarrats, K.-Y. Kam, S. Xu, and U. B. Kaiser Gonadotropin-Releasing Hormone Pulse Frequency-Dependent Activation of Extracellular Signal-Regulated Kinase Pathways in Perifused L{beta}T2 Cells Endocrinology, December 1, 2005; 146(12): 5503 - 5513. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Safwat, J. Ninomiya-Tsuji, A. J. Gore, and W. L. Miller Transforming Growth Factor {beta}-Activated Kinase 1 Is a Key Mediator of Ovine Follicle-Stimulating Hormone {beta}-Subunit Expression Endocrinology, November 1, 2005; 146(11): 4814 - 4824. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Coss, V. G. Thackray, C.-X. Deng, and P. L. Mellon Activin Regulates Luteinizing Hormone {beta}-Subunit Gene Expression through Smad-Binding and Homeobox Elements Mol. Endocrinol., October 1, 2005; 19(10): 2610 - 2623. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Su, J. C. Wu, J. R. Sommer, A. J. Gore, R. M. Petters, and W. L. Miller Conditional Induction of Ovulation in Mice Biol Reprod, October 1, 2005; 73(4): 681 - 687. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. I. Suszko, D. M. Balkin, Y. Chen, and T. K. Woodruff Smad3 Mediates Activin-Induced Transcription of Follicle-Stimulating Hormone {beta}-Subunit Gene Mol. Endocrinol., July 1, 2005; 19(7): 1849 - 1858. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L. Givens, N. Rave-Harel, V. D. Goonewardena, R. Kurotani, S. E. Berdy, C. H. Swan, J. L. R. Rubenstein, B. Robert, and P. L. Mellon Developmental Regulation of Gonadotropin-releasing Hormone Gene Expression by the MSX and DLX Homeodomain Protein Families J. Biol. Chem., May 13, 2005; 280(19): 19156 - 19165. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Zhao, R. Teng, K. Asanuma, Y. Okouchi, K. Johkura, N. Ogiwara, and K. Sasaki Differentiation of Mouse Embryonic Stem Cells Into Gonadotrope-like Cells In Vitro Reproductive Sciences, May 1, 2005; 12(4): 257 - 262. [Abstract] [PDF]
|
|
|
|
 |
|
 C. K. Cheng and P. C. K. Leung Molecular Biology of Gonadotropin-Releasing Hormone (GnRH)-I, GnRH-II, and Their Receptors in Humans Endocr. Rev., April 1, 2005; 26(2): 283 - 306. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. Gregory, C. T. Lacza, A. A. Detz, S. Xu, L. A. Petrillo, and U. B. Kaiser Synergy between Activin A and Gonadotropin-Releasing Hormone in Transcriptional Activation of the Rat Follicle-Stimulating Hormone-{beta} Gene Mol. Endocrinol., January 1, 2005; 19(1): 237 - 254. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L L Burger, D J Haisenleder, A C Dalkin, and J C Marshall Regulation of gonadotropin subunit gene transcription J. Mol. Endocrinol., December 1, 2004; 33(3): 559 - 584. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. L. Givens, R. Kurotani, N. Rave-Harel, N. L. G. Miller, and P. L. Mellon Phylogenetic Footprinting Reveals Evolutionarily Conserved Regions of the Gonadotropin-Releasing Hormone Gene that Enhance Cell-Specific Expression Mol. Endocrinol., December 1, 2004; 18(12): 2950 - 2966. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Wu, P. Su, N. W. Safwat, J. Sebastian, and W. L. Miller Rapid, Efficient Isolation of Murine Gonadotropes and Their Use in Revealing Control of Follicle-Stimulating Hormone by Paracrine Pituitary Factors Endocrinology, December 1, 2004; 145(12): 5832 - 5839. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. E. West, G. E. Parker, J. J. Savage, P. Kiratipranon, K. S. Toomey, L. R. Beach, S. C. Colvin, K. W. Sloop, and S. J. Rhodes Regulation of the Follicle-Stimulating Hormone {beta} Gene by the LHX3 LIM-Homeodomain Transcription Factor Endocrinology, November 1, 2004; 145(11): 4866 - 4879. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Larder, L. Chang, M. Clinton, and P. Brown Gonadotropin-Releasing Hormone Regulates Expression of the DNA Damage Repair Gene, Fanconi anemia A, in Pituitary Gonadotroph Cells Biol Reprod, September 1, 2004; 71(3): 828 - 836. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Harrison, P. C. Gray, W. H. Fischer, C. Donaldson, S. Choe, and W. Vale An Activin Mutant with Disrupted ALK4 Binding Blocks Signaling via Type II Receptors J. Biol. Chem., July 2, 2004; 279(27): 28036 - 28044. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J Winters and J. P Moore Intra-pituitary regulation of gonadotrophs in male rodents and primates Reproduction, July 1, 2004; 128(1): 13 - 23. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Bailey, N. Rave-Harel, S. M. McGillivray, D. Coss, and P. L. Mellon Activin Regulation of the Follicle-Stimulating Hormone {beta}-Subunit Gene Involves Smads and the TALE Homeodomain Proteins Pbx1 and Prep1 Mol. Endocrinol., May 1, 2004; 18(5): 1158 - 1170. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Bonfil, D. Chuderland, S. Kraus, D. Shahbazian, I. Friedberg, R. Seger, and Z. Naor Extracellular Signal-Regulated Kinase, Jun N-Terminal Kinase, p38, and c-Src Are Involved in Gonadotropin-Releasing Hormone-Stimulated Activity of the Glycoprotein Hormone Follicle-Stimulating Hormone {beta}-Subunit Promoter Endocrinology, May 1, 2004; 145(5): 2228 - 2244. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. J. Spady, R. Shayya, V. G. Thackray, L. Ehrensberger, J. S. Bailey, and P. L. Mellon Androgen Regulates Follicle-Stimulating Hormone {beta} Gene Expression in an Activin-Dependent Manner in Immortalized Gonadotropes Mol. Endocrinol., April 1, 2004; 18(4): 925 - 940. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Bernard Both SMAD2 and SMAD3 Mediate Activin-Stimulated Expression of the Follicle-Stimulating Hormone {beta} Subunit in Mouse Gonadotrope Cells Mol. Endocrinol., March 1, 2004; 18(3): 606 - 623. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Coss, S. B. R. Jacobs, C. E. Bender, and P. L. Mellon A Novel AP-1 Site Is Critical for Maximal Induction of the Follicle-stimulating Hormone {beta} Gene by Gonadotropin-releasing Hormone J. Biol. Chem., January 2, 2004; 279(1): 152 - 162. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. L. Burger, D. J. Haisenleder, K. W. Aylor, A. C. Dalkin, K. A Prendergast, and J. C. Marshall Regulation of Luteinizing Hormone-{beta} and Follicle-Stimulating Hormone (FSH)-{beta} Gene Transcription by Androgens: Testosterone Directly Stimulates FSH-{beta} Transcription Independent from Its Role on Follistatin Gene Expression Endocrinology, January 1, 2004; 145(1): 71 - 78. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Yamada, H. Yamamoto, T. Yonehara, H. Kanasaki, H. Nakanishi, E. Miyamoto, and K. Miyazaki Differential Activation of the Luteinizing Hormone {beta}-Subunit Promoter by Activin and Gonadotropin-Releasing Hormone: A Role for the Mitogen-Activated Protein Kinase Signaling Pathway in L{beta}T2 Gonadotrophs Biol Reprod, January 1, 2004; 70(1): 236 - 243. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Liu, D. A. Austin, and N. J. G. Webster Gonadotropin-Releasing Hormone-Desensitized L{beta}T2 Gonadotrope Cells Are Refractory to Acute Protein Kinase C, Cyclic AMP, and Calcium-Dependent Signaling Endocrinology, October 1, 2003; 144(10): 4354 - 4365. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Mellor, E. M. A. Ball, A. E. O'Connor, J.-F. Ethier, M. Cranfield, J. F. Schmitt, D. J. Phillips, N. P. Groome, and G. P. Risbridger Activin {beta}C-Subunit Heterodimers Provide a New Mechanism of Regulating Activin Levels in the Prostate Endocrinology, October 1, 2003; 144(10): 4410 - 4419. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Y. Bedecarrats, F. H. O'Neill, E. R. Norwitz, U. B. Kaiser, and J. Teixeira Regulation of gonadotropin gene expression by Mullerian inhibiting substance PNAS, August 5, 2003; 100(16): 9348 - 9353. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. B. R. Jacobs, D. Coss, S. M. McGillivray, and P. L. Mellon Nuclear Factor Y and Steroidogenic Factor 1 Physically and Functionally Interact to Contribute to Cell-Specific Expression of the Mouse Follicle-Stimulating Hormone-{beta} Gene Mol. Endocrinol., August 1, 2003; 17(8): 1470 - 1483. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Y. Bedecarrats and U. B. Kaiser Differential Regulation of Gonadotropin Subunit Gene Promoter Activity by Pulsatile Gonadotropin-Releasing Hormone (GnRH) in Perifused L{beta}T2 Cells: Role of GnRH Receptor Concentration Endocrinology, May 1, 2003; 144(5): 1802 - 1811. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Dupont, J. McNeilly, A. Vaiman, S. Canepa, Y. Combarnous, and C. Taragnat Activin Signaling Pathways in Ovine Pituitary and L{beta}T2 Gonadotrope Cells Biol Reprod, May 1, 2003; 68(5): 1877 - 1887. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. S. Ellsworth, B. R. White, A. T. Burns, B. D. Cherrington, A. M. Otis, and C. M. Clay c-Jun N-Terminal Kinase Activation of Activator Protein-1 Underlies Homologous Regulation of the Gonadotropin-Releasing Hormone Receptor Gene in {alpha}T3-1 Cells Endocrinology, March 1, 2003; 144(3): 839 - 849. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. I. Suszko, D. J. Lo, H. Suh, S. A. Camper, and T. K. Woodruff Regulation of the Rat Follicle-Stimulating Hormone {beta}-Subunit Promoter by Activin Mol. Endocrinol., March 1, 2003; 17(3): 318 - 332. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-F. Ethier, P. G. Farnworth, J. K. Findlay, and G. T. Ooi Transforming Growth Factor-{beta} Modulates Inhibin A Bioactivity in the L{beta}T2 Gonadotrope Cell Line by Competing for Binding to Betaglycan Mol. Endocrinol., December 1, 2002; 16(12): 2754 - 2763. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Welt, Y. Sidis, H. Keutmann, and A. Schneyer Activins, Inhibins, and Follistatins: From Endocrinology to Signaling. A Paradigm for the New Millennium Experimental Biology and Medicine, October 1, 2002; 227(9): 724 - 752. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. R. Norwitz, S. Xu, J. Xu, L. B. Spiryda, J. S. Park, K.-H. Jeong, E. A. McGee, and U. B. Kaiser Direct Binding of AP-1 (Fos/Jun) Proteins to a SMAD Binding Element Facilitates Both Gonadotropin-releasing Hormone (GnRH)- and Activin-mediated Transcriptional Activation of the Mouse GnRH Receptor Gene J. Biol. Chem., September 27, 2002; 277(40): 37469 - 37478. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. L. Burger, A. C. Dalkin, K. W. Aylor, D. J. Haisenleder, and J. C. Marshall GnRH Pulse Frequency Modulation of Gonadotropin Subunit Gene Transcription in Normal Gonadotropes--Assessment by Primary Transcript Assay Provides Evidence for Roles of GnRH and Follistatin Endocrinology, September 1, 2002; 143(9): 3243 - 3249. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. V. Vasilyev, M. A. Lawson, D. Dipaolo, N. J. G. Webster, and P. L. Mellon Different Signaling Pathways Control Acute Induction versus Long-Term Repression of LH{beta} Transcription by GnRH Endocrinology, September 1, 2002; 143(9): 3414 - 3426. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. C. Fowkes, P. King, and J. M. Burrin Regulation of Human Glycoprotein Hormone {alpha}-Subunit Gene Transcription in L{beta}T2 Gonadotropes by Protein Kinase C and Extracellular Signal-Regulated Kinase 1/2 Biol Reprod, September 1, 2002; 67(3): 725 - 734. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. M. Zakaria, K.-H. Jeong, C. Lacza, and U. B. Kaiser Pituitary Homeobox 1 Activates the Rat FSH{beta} (rFSH{beta}) Gene through Both Direct and Indirect Interactions with the rFSH{beta} Gene Promoter Mol. Endocrinol., August 1, 2002; 16(8): 1840 - 1852. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. B. Rosenberg and P. L. Mellon An Otx-Related Homeodomain Protein Binds an LH{beta} Promoter Element Important for Activation During Gonadotrope Maturation Mol. Endocrinol., June 1, 2002; 16(6): 1280 - 1298. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Sidis, D. V. Tortoriello, W. E. Holmes, Y. Pan, H. T. Keutmann, and A. L. Schneyer Follistatin-Related Protein and Follistatin Differentially Neutralize Endogenous vs. Exogenous Activin Endocrinology, May 1, 2002; 143(5): 1613 - 1624. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. V. Vasilyev, F. Pernasetti, S. B. Rosenberg, M. J. Barsoum, D. A. Austin, N. J. G. Webster, and P. L. Mellon Transcriptional Activation of the Ovine Follicle-Stimulating Hormone-{beta} Gene by Gonadotropin- Releasing Hormone Involves Multiple Signal Transduction Pathways Endocrinology, May 1, 2002; 143(5): 1651 - 1659. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Liu, D. A. Austin, P. L. Mellon, J. M. Olefsky, and N. J. G. Webster GnRH Activates ERK1/2 Leading to the Induction of c-fos and LH{beta} Protein Expression in L{beta}T2 Cells Mol. Endocrinol., March 1, 2002; 16(3): 419 - 434. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. R. Norwitz, S. Xu, K.-H. Jeong, G. Y. Bedecarrats, L. D. Winebrenner, W. W. Chin, and U. B. Kaiser Activin A Augments GnRH-Mediated Transcriptional Activation of the Mouse GnRH Receptor Gene Endocrinology, March 1, 2002; 143(3): 985 - 997. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
