GnRH Regulates Early Growth Response Protein 1 Transcription Through Multiple Promoter Elements
W. Rachel Duan,
Masafumi Ito,
Youngkyu Park,
Evelyn T. Maizels,
Mary Hunzicker-Dunn and
J. Larry Jameson
Division of Endocrinology, Metabolism, and Molecular Medicine
(W.R.D., M.I., Y.P., J.L.J.), Cell and Molecular Biology (E.T.M.,
M.H.D.), Northwestern University Medical School, Chicago, Illinois
60611
Address all correspondence and requests for reprints to: J. Larry Jameson, M.D., Ph.D., Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Tarry 15-709, 303 East Chicago Avenue, Chicago, Illinois 60611-3008. E-mail:
ljameson{at}northwestern.edu
 |
ABSTRACT
|
---|
Pulsatile secretion of GnRH is the major regulator of
gonadotropin (LH, FSH) gene expression and secretion. Recently, GnRH
has been shown to rapidly stimulate the expression of early growth
response protein-1 (Egr-1), a transcription factor that is essential
for LHß gene expression in the pituitary. In this study, we examined
the regulatory elements and signal transduction pathways by which GnRH
regulates Egr-1 transcription. Deletion analysis of the murine Egr-1
promoter identified two regions (-370 to -342 and -116 to -73) that
are critical for GnRH responsiveness in
T3 pituitary gonadotrope
cells. The first region, which contains two serum response elements
(SREs), contributed about 7080% of GnRH inducibility, whereas the
second region, which contains two SREs and one Ets binding site,
conferred an additional 2030% of activity. Mutations that
abolish protein binding to these SREs and Ets binding sites completely
eliminated GnRH-mediated transcriptional activation of the Egr-1
promoter. Mutation of cAMP response element reduced promoter activity
by 40%. Using specific protein kinase inhibitors, GnRH stimulation of
Egr-1 expression was found to be dependent on PKC/ERK pathways. In
addition, GnRH activated p90 ribosomal S6 kinase, which has the
potential to phosphorylate serum response factor and cAMP response
element binding protein. We conclude that GnRH stimulation of Egr-1
gene expression requires several distinct SREs/Ets elements and a cAMP
response element and is mediated via activation of PKC/ERK
signaling pathways.
 |
INTRODUCTION
|
---|
GnRH PLAYS AN essential role in the
regulation of gonadotropin gene expression and secretion
(1, 2, 3). The gonadotropins (LH and FSH) consist of a common
-subunit and specific ß-subunits. The
- and ß-subunits are
each stimulated by GnRH at the transcriptional level (4, 5). A pulsatile GnRH stimulus is required to stimulate
gonadotropin subunit transcription and the
, LHß, and FSHß genes
are differentially regulated by pulse frequency (4, 6).
For instance, LHß gene expression is maintained by relatively
high-frequency GnRH pulses, whereas FSHß expression is favored by
lower pulse frequencies (4).
Studies of the
-subunit gene promoter have identified several
transcription factors and regulatory elements that provide basal,
tissue-specific, and hormonally mediated regulation of gene expression
(7, 8, 9, 10). Three transcription factorsthe orphan nuclear
receptor steroidogenic factor 1 (Sf-1), the
bicoid-related homeoprotein Ptx1, and the early growth
response protein-1 (Egr-1)are essential for LHß
transcriptional activation (11, 12, 13, 14, 15, 16). Among these factors,
Egr-1 appears to be a dynamic effector of GnRH action. GnRH stimulates
Egr-1 expression but has little or no effect on the levels of Ptx1 or
Sf-1 (13, 15).
The Egr-1 gene (also referred as krox24, NGFI-A, TIS8, or zif268)
belongs to a family of immediate early response genes (17, 18). The Egr family of proteins contain a conserved zinc finger
DNA-binding domain and bind to a GC-rich sequence in the promoter
region of target genes (17). Egr-1 expression is rapidly
and transiently activated in many different cell types during
development. In adult tissues, a variety of signals, including serum,
growth factors, cytokines, and hormones, stimulate Egr-1 expression
(17). Egr-1 is also induced by changes in the local
cellular environment, such as variation in osmotic pressure, heat
shock, hypoxia, and DNA-damaging agents (19, 20, 21, 22, 23). Egr-1 is
involved in diverse cellular functions including cell proliferation,
differentiation, and apoptosis (17). A role for Egr-1 in
reproduction was demonstrated in two independent Egr-1 gene deletion
experiments (11, 12). Egr-1-deficient mice are sterile due
to defects in the anterior pituitary of both sexes and in the ovaries
of females (12). In the pituitary, the absence of Egr-1
causes a selective loss of LHß synthesis and secretion whereas
production of the
-subunit and FSHß remain intact
(12). Sequence analysis of the rat LHß gene promoter
identified two Egr-1 binding sites (11, 14). Egr-1 binds
to both sites and activates the LHß promoter by acting in combination
with Sf-1 (11, 14).
The 5'-flanking sequence of the Egr-1 gene has been cloned from several
species (24, 25, 26, 27, 28), revealing the presence of multiple serum
response elements (SREs). Additional putative regulatory elements
include an Sp1 binding site, a cAMP response element (CRE), and an Ets
binding site (17, 26, 29). The majority of studies have
mapped Egr-1 promoter inducibility to regions encompassing the SRE
clusters, although preferential utilization of specific SREs may occur
in response to specific stimulatory pathways (30, 31, 32, 33, 34). The
SRE consensus sequence contains a CC(A/T)6GG
motif, and activation of the SRE is dependent on binding of the
transcription factor, SRF (serum response factor) (27, 35, 36). Ets binding sites are located adjacent to the SREs of the
Egr-1 promoter and thereby permit the interaction of ternary complex
factors (TCFs) with the SRE-SRF complex (37, 38). Studies
showed that SRF forms a ternary complex with a TCF protein, Elk-1, at
the c-fos SRE, and phosphorylation of Elk-1 is required for
in vitro ternary complex formation with SRF (39, 40). Like the c-fos SRE, the Egr-1 SRE/Ets sites are
occupied by a multiprotein complex containing a dimer of SRF and Ets
family transcription factors (30). In addition to the SREs
and Ets motif, a proximal CRE is required for stimulation by
granulocyte-macrophage colony-stimulating factor (GM-CSF) (32, 41), whereas a distal CRE is necessary for activation by
p38/stress-activated protein kinases (SAPKs) in response to cellular
stress (20). The CRE plays a positive role in Egr-1
expression in response to depolarization in pancreatic islet ß-cells
(42) but serves as a negative regulator in synovial
fibroblasts from rheumatoid arthritis patients (43).
Activation of the Egr-1 promoter is mediated by several kinase
pathways. A member of the MAPK family, p38/SAPK2 (or p38MAPK), is
involved in stress-induced stimulation of Egr-1 (20),
whereas PKC, and ERK-1 and -2, are responsible for the Elk
phosphorylation, which ultimately mediates Egr-1 induction (19, 44, 45). Activation of the serine/threonine kinase, p90RSK (p90
ribosomal S6 kinase), which phosphorylates cAMP response element
binding protein (CREB), is critical for Egr-1 stimulation in response
to GM-CSF (34, 46).
Although GnRH is known to transiently induce Egr-1 expression in
gonadotrope cells (13, 15, 16, 47, 48), the mechanisms and
intracellular signaling pathways by which GnRH regulates Egr-1
expression remain incompletely defined. In the present study, we
investigated the 5'-regulatory elements of the murine Egr-1 gene in
cultured gonadotrope cells to identify GnRH-mediated signaling events
and transcription factors that stimulate the Egr-1 promoter.
 |
RESULTS
|
---|
GnRH Activates the Egr-1 Promoter Through SREs and Adjacent Ets
Binding Sites
To define the regulatory elements responsible for GnRH
stimulation, a 1.3-kb fragment of the murine Egr-1 promoter was
sequentially deleted. As shown in Fig. 1
, the -1,381 reporter construct displayed a 6- to 7-fold increase in
response to GnRH when transiently transfected into
T3 cells. The
fold induction by GnRH was relatively constant until deletions reached
-370 bp. Further deletion resulted in a significant loss of activity.
Approximate 2-fold stimulation by GnRH was maintained up to -116 bp.
Based on these findings, key elements that confer GnRH responsiveness
appear to be located in two regions (-370 to -342 and -116 to -73).
The distal region (region A) includes two SREs, and the proximal region
(region B) contains two SREs on both sides of an Ets binding site (Fig. 2A
).

View larger version (19K):
[in this window]
[in a new window]
|
Figure 1. Localization of Regions Involved in GnRH
Stimulation of the Murine Egr-1 Promoter
A deletion series of the Egr-1 promoter-reporter constructs was
transiently transfected into T3 cells (0.5 µg/well), and cells
were treated with or without 10 nM GnRH for 46 h before
luciferase assays. Results are the mean ± SEM of fold
induction. Data represent at least three independent experiments, with
each transfection performed in triplicate. Two critical regions
conferring GnRH responsiveness are shown in shaded
areas. *, P < 0.05 for construct -342
vs. -370 and construct -73 vs. -116.
|
|

View larger version (29K):
[in this window]
[in a new window]
|
Figure 2. Protein Binding to Wild-Type or Mutant SREs and Ets
Motifs in the Egr-1 Promoter
A, Schematic representation of the putative regulatory elements within
the Egr-1 promoter. Region A contains two SREs, and region B contains
two SREs and an Ets binding site. Mutations introduced to the EMSA
probes are also shown. B, Nuclear extracts were prepared from T3
cells. EMSA was performed with wild-type or mutant radiolabeled
oligonucleotides.
|
|
SRF Binds to Both Distal and Proximal SRE Sites in the Egr-1
Promoter
EMSAs were performed to document specific transcription factor
binding to the SRE and Ets sites. Nuclear extracts prepared from
T3
cells were incubated with radiolabeled oligonucleotide probes
corresponding to regions A and B of the Egr-1 promoter (Fig. 2A
). A
single complex was observed with the region A probe (Fig. 2B
, lane 1).
This complex was eliminated by mutations in the two SREs within region
A (Fig. 2B
, lane 2) or by a 100-fold excess of unlabeled
oligonucleotides (Fig. 2B
, lane 3). An anti-SRF antibody supershifted
the complex completely (Fig. 2B
, lane 4), suggesting that SRF is the
primary gonadotrope-derived transcription factor that binds to the
distal SREs. Two complexes were detected using the region B probe,
which contains two SREs and an Ets binding site (Fig. 2B
, lane 5). Both
DNA-protein complexes were eliminated by mutations of the SRE/Ets
binding site (Fig. 2B
, lane 6) or by addition of excess unlabeled
oligonucleotides (Fig. 2B
, lane 7). Incubation with an anti-Ets
antibody did not supershift either band (Fig. 2B
, lane 8). However,
anti-SRF antibody completely supershifted the upper complex (Fig. 2B
, lane 9), indicating that SRF is also bound to the proximal SRE. The
binding activity of SRF and Ets was not affected by treatment of the
cells with GnRH (data not shown).
Mutations in the Egr-1 SREs Impair Transcriptional Activation by
GnRH
Mutations that eliminated protein binding to regions A and B (Fig. 2A
) were introduced into the -370 construct (-370 m) to assess the
functional roles of these binding sites. The native -370 construct was
stimulated 8-fold by GnRH treatment, whereas the -370 m construct
failed to respond to GnRH (Fig. 3
),
indicating an essential contribution of the distal and proximal SREs to
GnRH stimulation of the Egr-1 promoter.

View larger version (15K):
[in this window]
[in a new window]
|
Figure 3. Effect of Mutations of the SREs and Ets Sites on
GnRH Inducibility of the Egr-1 Promoter
The wild-type -370 reporter construct and constructs containing
mutations at SREs and Ets binding sites within regions A and B (-370
m) were transiently transfected into T3 cells (0.5 µg/well). After
treatment with or without 10 nM GnRH for 46 h, cells were
harvested and luciferase activity assays were performed. Values shown
are the mean ± SEM. Data represent three experiments,
with each transfection performed in triplicate. +,
P > 0.05 vs. basal activity.
|
|
CREB Is Associated with the CRE Egr-1 Promoter
Because the Egr-1 promoter CRE has been shown to mediate induction
in response to certain cytokines (32) and stress
(20), it is possible that the CRE is also involved in GnRH
regulation. Oligonucleotide probes containing the CRE (TCACGTCA) from
the Egr-1 promoter were examined in EMSA using human embryonic kidney
TSA 201 cell nuclear extracts (Fig. 4A
).
Two binding complexes were observed with the Egr-1 promoter CRE
(Fig. 4B
, lane 1). The intensity of the upper band slightly decreased
when an anti-CREB antibody was added compared with the control with
normal serum (Fig. 4B
, lanes 2 and 3). When TSA cells were transfected
with the CREB expression vector, the intensity of the upper band
increased dramatically (lanes 46), indicating that CREB is a
component of this complex. Incubation with an anti-CREB antibody
resulted in a supershifted band (Fig. 4B
, lane 5). Similar results were
obtained with an oligonucleotide derived from the CRE sequence of the
inhibin
promoter (49) (Fig. 4B
, lanes 712), although
the lower band was not detected with the inhibin
promoter CRE. To
examine the effect of GnRH on CREB binding to the Egr-1 promoter CRE,
T3 cell were treated with or without GnRH. Similar to TSA cells, two
DNA-protein complexes bound to the wild-type CRE (Fig. 4B
, lane 13),
but they were not observed with the mutant CRE
(TCTCATCA) probe and were eliminated by excess
unlabeled oligonucleotides (Fig. 4B
, lanes 14 and 15). The intensity of
the faster-migrating band was not affected by an anti-CREB antibody.
This band was present in both TSA and
T3 cells and appeared to be
Egr-1 promoter specific, as it was not detected with the inhibin
promoter CRE. Addition of the anti-CREB antibody decreased the signal
of the slower-migrating band slightly and generated faint supershifted
bands, but the intensity of these bands was not altered by GnRH
treatment (Fig. 4B
, lanes 16 and 17). EMSA was also performed using an
antiphospho-CREB antibody that specifically recognizes protein
phosphorylated on Ser 133. In the absence of GnRH, a distinct
supershifted band was observed, and GnRH treatment significantly
increased the intensity of this band (Fig. 4B
, lanes 18 and 19).

View larger version (52K):
[in this window]
[in a new window]
|
Figure 4. CREB Bound to the CRE in the Egr-1 Promoter
A, Schematic representation of the CRE within the Egr-1 promoter. The
CRE is located between regions A and B. Mutations introduced into the
EMSA probe are also shown. B, Nuclear extracts were prepared from TSA
cells transfected with a control or CREB expression vector (TF CREB)
and T3 cells treated with or without 10 nM GnRH for 15
min, and EMSA was performed using wild-type or mutant Egr-1 CRE probes
and the inhibin CRE. The arrowhead indicates the
supershifted band.
|
|
The Egr-1 CRE Contributes to GnRH Induction of the Egr-1
Promoter
The CRE in the -692 reporter construct was mutated (-692 m) to
examine the functional role of the CRE. The native -692 construct was
stimulated 6- to 7-fold after GnRH treatment (Fig. 5
). Introduction of the CRE mutation
resulted in a 40% loss of activity in response to GnRH. Basal reporter
activity was not affected by the mutation.

View larger version (12K):
[in this window]
[in a new window]
|
Figure 5. Effect of CRE Mutations on GnRH Stimulation of the
Egr-1 Promoter
A mutation was introduced into the CRE of the -692 reporter gene
(-692 m). Wild-type or mutant reporter constructs were transiently
transfected into T3 cells (0.5 µg/well). Cells were treated with
or without 10 nM GnRH for 46 h, followed by luciferase
assays. Values shown are the mean ± SEM. Data
represent three independent experiments, with each transfection
performed in triplicate. *, P < 0.05 for -692 m
vs. -692 in GnRH-treated cells.
|
|
PKC and ERK Pathways Are Involved in GnRH Stimulation of the Egr-1
Promoter
Multiple intracellular pathways contribute to Egr-1
expression (19, 20, 44, 46, 50, 51). To elucidate the
individual contribution of intracellular signaling pathways to GnRH
regulation of Egr-1 gene expression,
T3 cells were transfected with
the -442 reporter construct and treated with or without GnRH in the
presence or absence of specific protein kinase inhibitors. As shown in
Fig. 6
, GF109203X completely blocked
transcriptional activation of the Egr-1 promoter by GnRH but had no
effect on basal activity. Pretreatment with 12-myristate 13-acetate
(PMA) to deplete PKC abolished both GnRH and PMA-induced Egr-1 promoter
activity (data not shown). Likewise, the ERK inhibitor, PD98059,
totally blocked GnRH stimulation of the Egr-1 promoter and exhibited a
slight inhibitory effect on basal activity (Fig. 6
). KN62, a
Ca2+/calmodulin-dependent kinase inhibitor,
caused a slight decrease in the promoter activity by GnRH. A PKA
inhibitor, H89, showed a partial inhibition of the Egr-1 promoter by
GnRH.

View larger version (15K):
[in this window]
[in a new window]
|
Figure 6. Effects of Protein Kinase Inhibitors on the Egr-1
Promoter Activity
A, T3 cells were transiently transfected with the -442-bp
Egr-1 promoter construct (0.5 µg/well). Forty hours after the
transfection, cells were preincubated with dimethylsulfoxide [DMSO
(control)], GF109203X (5 µM), PD98059 (50
µM), KN-62 (5 µM), or H-89 (10
µM) for 1 h. Cells were then treated with or without
10 nM GnRH for 46 h (+,
P > 0.05 vs. basal activity. *,
P < 0.05 vs. GnRH-treated cells of control
group). Cell extracts were prepared and assayed for luciferase
activity. Values shown are the mean ± SEM.
Data represent four independent experiments, with each transfection
performed in triplicate.
|
|
Western blot analyses showed that Egr-1 protein is strongly induced by
1-h treatment of
T3 cells with GnRH (Fig. 7A
). In agreement with the transfection
data, treatment with GF109203X or PD98059 substantially blocked GnRH
stimulation of Egr-1 protein expression. KN62 and H89 exhibited weak
inhibitory effects (Fig. 7
, B and C).

View larger version (31K):
[in this window]
[in a new window]
|
Figure 7. Effects of Protein Kinase Inhibitors on
GnRH-Induced Egr-1 Protein Expression
A, T3 cells were treated with 10 nM GnRH for the time
indicated. Nuclear extracts were prepared and subjected to Western blot
analysis using an anti-Egr-1 antibody. B, T3 cells were preincubated
with DMSO, GF109203X (5 µM), PD98059 (50
µM), KN-62 (5 µM), or H-89 (10
µM) for 1 h. Cells were then treated with or without
10 nM GnRH for 1 h. Nuclear extracts were prepared and
subjected to Western blot analysis using an anti-Egr-1 antibody. C,
Protein levels in panel B were quantitated using a densitometer.
|
|
Dose-response experiments were performed to further evaluate the
effects of GF109203X on GnRH-induced Egr-1 promoter activity. PMA and
forskolin were included as positive and negative controls,
respectively. PMA stimulation of Egr-1 promoter activity was reduced by
50% using 0.1 µM GF109203X. The stimulatory effect of
GnRH was less affected (17% inhibition) at this GF109203X
concentration (Fig. 8
). Using 0.5
µM or greater (1, 5, and 10 µM) GF109203X
concentrations, both GnRH and PMA-induced activation of the Egr-1
promoter were completely blocked (Fig. 8
). As expected, forskolin did
not alter Egr-1 promoter activity.

View larger version (17K):
[in this window]
[in a new window]
|
Figure 8. Effect of Different Concentrations of GF109203X on
GnRH-Induced Egr-1 Promoter Activity
T3 cells were transiently transfected with the -442-bp Egr-1
promoter construct (0.5 µg/well). Forty hours after the transfection,
cells were preincubated with different concentrations of GF109203X as
indicated for 1 h. Cells were then treated with DMSO, GnRH (10
nM), PMA (100 nM), or forskolin (10
µM) for 46 h. Cell extracts were prepared and assayed
for luciferase activity. Values shown are the mean ±
SEM of triplicate transfection. The experiment was repeated
three times, and similar data were obtained. *, P
< 0.05 vs. DMSO-treated cells.
|
|
GnRH Induces Activation of p90RSK
p90RSK, an immediate downstream kinase of MAPK pathway, is
activated in pokeweed mitogen-treated B cells (52) and in
differentiated monocytes (34) in association with Egr-1
induction. Recent evidence indicates that p90RSK can directly
phosphorylate SRF on Ser-103 in vitro (53) and
is responsible for CREB phosphorylation at Ser-133 in response to
growth factor stimulation (46, 54, 55, 56). We next examined
whether GnRH stimulates p90RSK activation using an antibody that
recognizes phosphorylated p90RSK.
T3 cells were treated with GnRH in
the absence or presence of protein kinase inhibitors. Immunoblot
analysis revealed a 3- to 4-fold increase in p90RSK phosphorylation
after GnRH stimulation (Fig. 9A
). As a
positive control, PMA also stimulated p90RSK phosphorylation. The
activation of p90RSK by GnRH and PMA was partially blocked by 1
µM GF109203X (Fig. 9A
). Higher concentrations
of GF109203X (10 µM) inhibited GnRH activation
of p90RSK by 90% (data not shown). PD98059 totally prevented the
p90RSK phosphorylation by GnRH (Fig. 9A
). Forskolin had no effect on
p90RSK activation.

View larger version (42K):
[in this window]
[in a new window]
|
Figure 9. GnRH Stimulates p90RSK and Elk-1 Phosphorylation in
T3 Cells
A, GnRH stimulation of p90RSK. T3 cells were preincubated
with DMSO, GF109203X (1 µM), or PD98059 (50
µM) for 1 h. Cells were then treated without or with
GnRH (10 nM), PMA (100 nM), or forskolin (10
µM) for 1 h. Cell extracts were prepared and
subjected to Western blot analysis using an antiphospho-p90RSK
antibody. An antibody against RSK2 was used as a control (upper
panel). Protein levels were quantitated using densitometry and
normalized to the control (RSK2) value (lower panel). B,
GnRH stimulation of Elk-1. T3 cells were preincubated with DMSO,
GF109203X (1 µM), or PD98059 (50
µM) for 1 h. Cells were then treated
without or with GnRH (10 nM), PMA (100
nM), or forskolin (10 µM)
for 1 h. Cell extracts were prepared and subjected to Western blot
analysis using antiphospho-Elk-1 antibody (upper panel).
Protein levels were quantitated using densitometry and normalized to
the control (RSK2) value (lower panel).
|
|
GnRH Stimulates Elk-1 Phosphorylation Through a PKC-Independent ERK
Pathway
To explore a link between MAPK and the SRE-associated
transcription factors in response to GnRH stimulation, we determined
whether GnRH stimulates Elk-1 activation and which signaling pathways
are involved. Studies of the c-fos SRE showed that SRF forms
a ternary complex with an accessory protein, Elk-1, which is essential
for c-fos stimulation (37, 57). Elk-1 is a well
documented major nuclear substrate of MAPK cascades (38, 40, 58), and Elk-1 phosphorylation contributes to SRE-mediated
transcriptional activation (45, 57, 59, 60, 61, 62). As shown in
Fig. 9B
, GnRH increased Elk-1 phosphorylation in
T3 cells. This
stimulatory effect of GnRH was not affected by 1
µM GF109203X or by higher concentrations of
GF109203X (5 and 10 µM, data not shown). In
contrast, PMA stimulation of Elk-1 was partially blocked by 1
µM GF109203X. PD98059 completely blocked
GnRH-induced Elk-1 phosphorylation (Fig. 9B
). Forskolin did not inhibit
GnRH-induced Elk-1 phosphorylation.
 |
DISCUSSION
|
---|
Egr-1 is a ubiquitous transcription factor that participates in a
wide range of physiological and pathophysiological processes
(17). The generation of Egr-1 knockout mice revealed an
unanticipated critical role for Egr-1 in the regulation of LHß gene
expression (11, 12). Several studies have documented that
GnRH up-regulates Egr-1 gene expression (13, 15, 16, 47).
The finding of a GnRH-responsive transcription factor offers the
opportunity to better understand how GnRH regulates LH biosynthesis,
and it also provides a proximate target of GnRH action to help unravel
signaling pathways that mediate transcriptional events.
The Egr-1 promoter contains multiple putative regulatory elements,
including two Sp1 sites, five SREs with adjacent Ets-like motifs, two
CREs, and Egr-1 binding sites (26, 29). The SREs in the
Egr-1 promoter have been extensively studied. The distal SREs are
critical for induction of Egr-1 by GH (63),
platelet-derived growth factor (64), GM-CSF
(65), urea (66), and stress
(44). The proximal SREs are necessary for maximal
induction by GM-CSF (32). By deletion analysis of the
murine Egr-1 promoter in
T3 cells, two regions that are responsible
for transcriptional activation by GnRH were localized. The first region
(A: -370 to -342) contains two functional SREs. The second region (B:
-116 to -73) spans a putative Ets motif with two adjacent SREs. The
distal SREs are predominantly responsible for GnRH action (7080%
inducibility), but the proximal SREs and Ets binding site also confer
GnRH stimulation (2030% inducibility), suggesting a degree of
redundancy or additivity in their actions. The SRF binds to the SREs in
region A as evidenced by a supershift using the anti-SRF antibody.
Binding of SRF to the CArG box is thought to recruit TCF family
members, such as Fli-1, Elk-1, and Sap-1a, to the Ets motif sequence
(35). Two complexes were observed when a proximal SRE- and
Ets-containing oligonucleotide was used. The upper band is an
SRF-containing complex. The lower band may represent TCF proteins. It
is established that Fli-1 bound the Ets sites in the Egr-1 promoter in
GM-CSF-stimulated extracts (65, 67), whereas Elk and
Sap-1a were found in DNA-protein complexes of Egr-1 promoter in
GH-stimulated 3T3-F442A cells (63). Although there is no
direct evidence of Elk-1 binding to Ets motif, we showed that Elk-1
could be phosphorylated through the ERK pathway upon GnRH
stimulation in a pituitary cell line. Activated Elk-1 has been
recognized as a transcription factor that cooperatively interacts with
the SRF and binds to the SRE/Ets motifs in the promoter region of
various genes (40, 68, 69). Phosphorylation of Elk-1 has
been reported to contribute to Egr-1 induction by urea
(60), stress (61), and a calmodulin
antagonist (62). In addition, GnRH has been shown to
stimulate Elk-1 activation as a downstream event of MAPK cascade
(70) in human granulosa-luteal cells (71),
and c-fos was proposed as a target gene.
It is notable that SRF bound to the SREs independent of GnRH
stimulation. This finding agrees with other studies in which SRF
constitutively binds SREs with no change after stimulation by growth
factors or serum (65, 72, 73, 74). It is possible that the
primary function of SRF in GnRH stimulation is to act as a docking
element and associate with other transcription factors or to stabilize
DNA binding of CREB or Ets TCF proteins, as suggested in GH induction
of Egr-1 (63). Alternatively, SRF is a phosphoprotein that
can be phosphorylated by p90RSK, calmodulin II and IV, and
MAPK-activated protein kinase in response to growth factors,
stress, or other agents (53, 75, 76). SRF phosphorylation
is thought to facilitate binding of SRF to the SRE (53, 77). Therefore, SRF is also likely a direct intracellular target
of signal transduction cascades.
CREB binding to the CRE is involved in the regulation of Egr-1 gene
expression in response to certain stimuli (20, 41, 42). We
have previously shown that GnRH-stimulated CREB phosphorylation
contributes to transcriptional activation of the
-subunit gene in
the pituitary (78). In support of the hypothesis that CREB
may mediate GnRH regulation of Egr-1 expression, EMSA revealed that
CREB constitutively binds to the CRE of the Egr-1 promoter, regardless
of the presence or absence of GnRH. After GnRH stimulation, however,
the binding of phospho-CREB to CRE substantially increased.
Phospho-CREB binding was reduced in the presence of the PKC inhibitor,
GF109203X (data not shown), suggesting that the PKC pathway is involved
in GnRH-stimulated CREB phosphorylation. In addition, we show that
p90RSK is also stimulated by GnRH, providing another pathway for CREB
phosphorylation. Although CREB recognizes the CRE of the Egr-1
promoter, it appears that other factors also bind to this element, as a
major fast-migrating band also associates with the CRE probe (Fig. 4B
).
The identity and function of this band remain to be determined.
An Egr-1 reporter gene containing a CRE mutation exhibited 3540%
reduction of GnRH-induced activation. Of note, this reduction was
observed only with reporter constructs containing the distal SREs,
implicating functional interactions between the SREs and the CRE. CREB
binding protein (CBP) has been shown to interact with the C-terminal
transactivation domains of the TCFs, Elk-1 and Sap-1a, and with
full-length SRF (79, 80, 81). CBP is also recruited to the
c-fos SRE through interactions between the bromodomain and
Elk-1 (82). Phosphorylation of Elk-1, SRF, CREB, as well
as CBP itself, may induce a conformational change that permits the
transactivation domains of CBP to contact the basal transcription
machinery and thus potentiate transcriptional initiation
(82).
Multiple intracellular pathways, including p38/SAPK2 (20),
ERK/pp90RSK (19, 24, 34, 44, 45, 46), PKC (19, 44, 45, 50), and tyrosine kinase have been shown to mediate Egr-1
stimulation (51). The PKC pathway appears to be necessary
for GnRH stimulation of the Egr-1 gene (13, 15, 47, 48).
Consistent with these studies, we found that pharmacological activation
of PKC by PMA induces Egr-1 promoter activity, and a PKC inhibitor is
sufficient to abolish Egr-1 stimulation by GnRH as well as PMA.
Although experiments using pharmacological agents must be interpreted
with caution, these findings suggest a pivotal role for the PKC pathway
in Egr-1 induction and are reminiscent of similar findings with the
-subunit promoter (7, 83, 84). Transfection of PKC
with the Egr-1 promoter appears to increase basal and GnRH-stimulated
Egr-1 promoter activity (data not shown), raising the possibility that
it may mediate PKC action. In addition to PKC, the ERK pathway is
activated by GnRH and contributes to
-promoter stimulation
(70, 85, 86). Our finding that an ERK inhibitor prevents
GnRH stimulation of Egr-1 promoter activity and protein expression
confirms the role of the ERK pathway in GnRH activation of Egr-1,
consistent with another report that PD98059 blocks GnRH stimulation of
Egr-1 mRNA (48).
Although activation of the ERK cascade by GnRH is well documented, the
downstream mediators have not been well characterized. Several lines of
evidence suggest that p90RSK activity is necessary for growth
factor-induced immediate-early gene expression (46, 52, 56). RSK family members are thought to influence gene expression
through phosphorylation of transcription factors, such as SRF and CREB
(53, 54, 87). All three members of RSK have been shown to
catalyze CREB phosphorylation in vitro and in
vivo (56). Similar to growth factors, GnRH was found
to stimulate p90RSK activation in our study. Pretreatment with PD98059
and GF109203X suppressed GnRH-induced p90RSK phosphorylation,
suggesting that PKC-Raf-MEK-ERK and p90RSK are involved in GnRH
regulation of Egr-1 gene expression. Therefore, SRF and CREB may serve
as direct intracellular targets of the ERK pathway in GnRH regulation
of the Egr-1 gene. It will therefore be interesting to determine
whether p90RSK directly phosphorylates SRF and CREB after GnRH
stimulation. In addition, it is unclear whether RSK2 and RSK3 can be
activated and participate in GnRH regulation of the Egr-1 gene. It
should be noted that a PKC-independent ERK pathway might also
contribute to GnRH stimulation of the Egr-1 gene, as Elk-1
phosphorylation was blocked by PD98059 but not by GF109203X. A recent
study indicated that activation of ERK by GnRH involved two distinct
signaling pathways: one is mediated by PKC and the other involves Ras
activation by Src and dynamin (25, 71).
Other MAPK family members, such as p38MAPK, are activated by GnRH and
contribute to GnRH induction of the c-fos promoter
(88). Jun N-terminal kinase (JNK/SAPK1) is activated by
GnRH in a PKC-independent pathway in gonadotrope cells (89, 90), and a JNK cascade appears to be necessary to elicit an
LHß promoter induction (90). Future studies may yield
insights into whether p38MAPK and JNK are involved in GnRH regulation
of Egr-1. Although PKA and Ca+2 kinase inhibitors
appear to weakly inhibit Egr-1 promoter activity and protein expression
induced by GnRH, these effects were inconsistent. Given the complex
regulatory elements in the Egr-1 promoter, it is likely that more than
one signaling pathway is involved in GnRH regulation of Egr-1. Our data
suggest that a complicated array of signaling cascades interact with
regulatory elements and transcription factors to regulate the Egr-1
promoter. In future studies, it should be informative to investigate
how pulsatile GnRH is coupled to these signaling pathways and
regulatory elements to dynamically control the level of Egr-1, which in
turn modulates LHß gene expression.
 |
MATERIALS AND METHODS
|
---|
Plasmids
The murine Egr-1 promoter (-1,381 to +79) was amplified by PCR
and subcloned into the pGL3 basic luciferase reporter construct vector
(Promega Corp., Madison, WI). A series of deletion
constructs (-1,072, -841, -692, -542, -482, -442, -392, -370,
-342, -292, -245, -192, -142, -130, -116, and -73) was made by
either restriction digestion or PCR. Reporter genes containing
mutations at the SREs, Ets binding site (-370 m), and CRE (-692 m)
were constructed by overlapping PCR. The DNA sequence of the promoter
region was confirmed using a dRhodamine terminator cycle sequencing kit
(Perkin-Elmer Corp., Norwalk, CT) and an ABI377 automated
sequencer (PE Applied Biosystems, Foster City, CA).
Cell Culture, Transfection, and Luciferase Assays
T3 cells were grown to approximately 5070% confluency in
DMEM/F12 with 10% FBS, 100 U/ml penicillin, and 100 µg/ml
streptomycin in 12-well plates. Cells were washed with serum-free media
and transfected with Egr-1 promoter-reporter constructs (0.5 µg
DNA/well) using Transfectam (Promega Corp.), according to
manufacturers instructions. Cells were incubated with the
transfection mixture at 37 C for 2.5 h, and complete media were
added to the cells. Forty hours after transfection, cells were treated
with or without 10 nM GnRH analog
(Des-Gly10,
[D-Ala6]-GnRH ethylamide;
Sigma, St. Louis, MO) for 46 h. Cell extracts were then
harvested and analyzed for luciferase activity as described previously
(78). In experiments involving protein kinase inhibitors,
cells were preincubated with an inhibitor for 1 h and treated with
GnRH or other reagents. All experiments were performed in triplicate
and repeated at least three times. Data were presented as the mean
± SEM. Statistical analyses were performed using the
t test, and P < 0.05 was considered
statistically significant.
EMSAs
Nuclear extracts were prepared from
T3 cells treated with or
without GnRH for 1 h by the method of Shapiro et al.
(91) in the presence of a protease inhibitor mixture
(Complete, Roche Molecular Biochemicals, Indianapolis, IN)
and 25 mM NaF. Nuclear extracts were also
obtained from human embryonic kidney TSA 201 cells grown in DMEM
supplemented with 10% FBS and transfected with an empty or CREB
expression vector. EMSA was performed as described previously
(92). Briefly, nuclear extracts (10 µg) were incubated
with 20 fmol of 32P-labeled oligonucleotides
(Table 1
) for 30 min on ice. For antibody
supershift experiments, nuclear extracts were incubated with antibodies
for 30 min on ice before radiolabeled probes were added. Anti-SRF,
-Ets, and -CREB antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antiphospho-CREB antibody
was purchased from Upstate Biotechnology, Inc. (Lake
Placid, NY). DNA-protein complexes were resolved on 4% native
polyacrylamide gels in 0.5x Tris-borate-EDTA buffer and subjected to
autoradiography.
Western Blot Analyses
T3 cells at about 80% confluency were preincubated
with or without a kinase inhibitor at 37 C for 1 h and then
treated with GnRH or other reagents for an additional 1 h. The
same concentration of an inhibitor was maintained during GnRH
stimulation. Cells were washed with ice-cold PBS and harvested. Nuclear
extracts were prepared as described above. In experiments involving
CREB, p90RSK, and Elk-1, cell extracts were prepared. Equal amounts of
protein (nuclear or cell extracts) were resolved by 10% SDS-PAGE and
transferred onto nitrocellulose filters. The membranes were blocked
with 3% nonfat milk for 1.5 h and then incubated overnight at 4 C
with anti-Egr-1 (Santa Cruz Biotechnology, Inc.),
phospho-CREB, phospho-p90RSK, or phospho-Elk-1 antibody (Cell Signaling
Technology, Beverly, MA). Immunoreactive proteins were detected using
an antirabbit horseradish peroxidase-conjugated antibody and the
enhanced chemiluminescence system (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Bands were quantitated using a
Personal Densitometer (Molecular Dynamics, Inc.,
Sunnyvale, CA).
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Jeffrey Weiss for helpful suggestions and Tom
Kotlar and Leah Sabacan for assistance with DNA sequencing.
 |
FOOTNOTES
|
---|
This work was conducted as a part of the National Cooperative Program
on Infertility Research and was supported by NIH Grant U54-HD-29164.
R.D. is a recipient of NIH Fellowship Award HD-08311 and a Training
Program in Signal Transduction and Cancer, T32CA-70085. Y.P. is a
recipient of NIH fellowship HD-08516.
Abbreviations: CBP, CREB-binding protein; CREB, cAMP response
element-binding protein; DMSO, dimethylsulfoxide; Egr-1, early growth
response protein 1; GM-CSF, granulocyte macrophage colony-stimulating
factor; JNK, Jun N-terminal kinase; p90RSK, p90 ribosomal S6
kinase; PMA, 12-myristate 13-acetate; SAPK, stress-activated protein
kinase; Sf-1, steroidogenic factor 1; SRE, serum response element; SRF,
serum response factor; TCF, ternary complex factor.
Received for publication November 6, 2000.
Accepted for publication October 31, 2001.
 |
REFERENCES
|
---|
-
Schally AV, Arimura A, Kastin AJ 1973 Hypothalamic
regulatory hormones. Science 179:341350[Medline]
-
Crowley Jr WF, Filicori M, Spratt DI, Santoro NF 1985 The
physiology of gonadotropin-releasing hormone (GnRH) secretion in men
and women. Recent Prog Horm Res 41:473531[Medline]
-
Clarke IJ, Cummins JT 1982 The temporal relationship between
gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH)
secretion in ovariectomized ewes. Endocrinology 111:17371739[Medline]
-
Haisenleder DJ, Dalkin AC, Marshall JC 1994 Regulation of
gonadotropin gene expression. In: Knobil E, Neill JD, eds. The
physiology of reproduction. New York: Raven Press; 17931813
-
Weiss J, Jameson JL, Burrin JM, Crowley Jr WF 1990 Divergent
responses of gonadotropin subunit messenger RNAs to continuous
vs. pulsatile gonadotropin-releasing hormone in
vitro. Mol Endocrinol 4:557564[Abstract]
-
Haisenleder DJ, Dalkin AC, Ortolano GA, Marshall JC, Shupnik
MA 1991 A pulsatile gonadotropin-releasing hormone stimulus is required
to increase transcription of the gonadotropin subunit genes: evidence
for differential regulation of transcription by pulse frequency
in vivo. Endocrinology 128:509517[Abstract]
-
Albanese C, Colin IM, Crowley WF, Ito M, Pestell RG, Weiss J,
Jameson JL 1996 The gonadotropin genes: evolution of distinct
mechanisms for hormonal control. Recent Prog Horm Res 51:2358[Medline]
-
Barnhart KM, Mellon PL 1994 The orphan nuclear receptor,
steroidogenic factor-1, regulates the glycoprotein hormone
-subunit
gene in pituitary gonadotropes. Mol Endocrinol 8:878885[Abstract]
-
Horn F, Windle JJ, Barnhart KM, Mellon PL 1992 Tissue-specific gene expression in the pituitary: the glycoprotein
hormone
-subunit gene is regulated by a gonadotrope-specific
protein. Mol Cell Biol 12:21432153[Abstract]
-
Silver BJ, Bokar JA, Virgin JB, Vallen EA, Milsted A, Nilson
JH 1987 Cyclic AMP regulation of the human glycoprotein hormone
-subunit gene is mediated by an 18-base-pair element. Proc Natl Acad
Sci USA 84:21982202[Abstract]
-
Lee SL, Sadovsky Y, Swirnoff AH, Polish JA, Goda P, Gavrilina
G, Milbrandt J 1996 Luteinizing hormone deficiency and female
infertility in mice lacking the transcription factor NGFI-A (Egr-1).
Science 273:12191221[Abstract]
-
Topilko P, Schneider-Maunoury S, Levi G, Trembleau A, Gourdji
D, Driancourt MA, Rao CV, Charnay P 1998 Multiple pituitary and ovarian
defects in Krox-24 (NGFI-A, Egr-1)-targeted mice. Mol Endocrinol 12:107122[Abstract/Free Full Text]
-
Wolfe MW, Call GB 1999 Early growth response protein 1 binds
to the luteinizing hormone-ß promoter and mediates
gonadotropin-releasing hormone-stimulated gene expression. Mol
Endocrinol 13:752763[Abstract/Free Full Text]
-
Halvorson LM, Ito M, Jameson JL, Chin WW 1998 Steroidogenic
factor-1 and early growth response protein 1 act through two composite
DNA binding sites to regulate luteinizing hormone ß-subunit gene
expression. J Biol Chem 273:1471214720[Abstract/Free Full Text]
-
Tremblay JJ, Drouin J 1999 Egr-1 is a downstream effector of
GnRH and synergizes by direct interaction with Ptx1 and SF-1 to enhance
luteinizing hormone ß gene transcription. Mol Cell Biol 19:25672576[Abstract/Free Full Text]
-
Dorn C, Ou Q, Svaren J, Crawford PA, Sadovsky Y 1999 Activation of luteinizing hormone ß gene by gonadotropin-releasing
hormone requires the synergy of early growth response-1 and
steroidogenic factor-1. J Biol Chem 274:1387013876[Abstract/Free Full Text]
-
Gashler A, Sukhatme VP 1995 Early growth response protein 1
(Egr-1): prototype of a zinc-finger family of transcription factors.
Prog Nucleic Acid Res Mol Biol 50:191224[Medline]
-
Yan SF, Pinsky DJ, Mackman N, Stern DM 2000 Egr-1: is it
always immediate and early? J Clin Invest 105:553554[Free Full Text]
-
Yan SF, Lu J, Zou YS, Soh-Won J, Cohen DM, Buttrick PM, Cooper
DR, Steinberg SF, Mackman N, Pinsky DJ, Stern DM 1999 Hypoxia-associated induction of early growth response-1 gene
expression. J Biol Chem 274:1503015040[Abstract/Free Full Text]
-
Rolli M, Kotlyarov A, Sakamoto KM, Gaestel M, Neininger A 1999 Stress-induced stimulation of early growth response gene-1 by
p38/stress-activated protein kinase 2 is mediated by a cAMP-responsive
promoter element in a MAPKAP kinase 2-independent manner. J Biol
Chem 274:1955919564[Abstract/Free Full Text]
-
Lim CP, Jain N, Cao X 1998 Stress-induced immediate-early
gene, egr-1, involves activation of p38/JNK1. Oncogene 16:29152926[CrossRef][Medline]
-
Huang RP, Adamson ED 1995 A biological role for Egr-1 in cell
survival following ultra-violet irradiation. Oncogene 10:467475[Medline]
-
Datta R, Taneja N, Sukhatme VP, Qureshi SA, Weichselbaum R,
Kufe DW 1993 Reactive oxygen intermediates target CC(A/T)6GG sequences
to mediate activation of the early growth response 1 transcription
factor gene by ionizing radiation. Proc Natl Acad Sci USA 90:24192422[Abstract]
-
Changelian PS, Feng P, King TC, Milbrandt J 1989 Structure of
the NGFI-A gene and detection of upstream sequences responsible for its
transcriptional induction by nerve growth factor. Proc Natl Acad Sci
USA 86:377381[Abstract]
-
Janssen-Timmen U, Lemaire P, Mattei MG, Revelant O, Charnay P 1989 Structure, chromosome mapping and regulation of the mouse
zinc-finger gene Krox-24; evidence for a common regulatory pathway for
immediate-early serum-response genes. Gene 80:325336[CrossRef][Medline]
-
Tsai-Morris CH, Cao XM, Sukhatme VP 1988 5' Flanking sequence
and genomic structure of Egr-1, a murine mitogen inducible zinc finger
encoding gene. Nucleic Acids Res 16:88358846[Abstract]
-
Christy B, Nathans D 1989 Functional serum response elements
upstream of the growth factor-inducible gene zif268. Mol Cell Biol 9:48894895[Medline]
-
Sakamoto KM, Bardeleben C, Yates KE, Raines MA, Golde DW,
Gasson JC 1991 5' Upstream sequence and genomic structure of the human
primary response gene, EGR-1/TIS8. Oncogene 6:867871[Medline]
-
Schwachtgen JL, Campbell CJ, Braddock M 2000 Full promoter
sequence of human early growth response factor-1 (Egr-1): demonstration
of a fifth functional serum response element. DNA Seq 10:429432[Medline]
-
McMahon SB, Monroe JG 1995 A ternary complex factor-dependent
mechanism mediates induction of egr-1 through selective serum response
elements following antigen receptor cross-linking in B lymphocytes. Mol
Cell Biol 15:10861093[Abstract]
-
DeFranco C, Damon DH, Endoh M, Wagner JA 1993 Nerve growth
factor induces transcription of NGFIA through complex regulatory
elements that are also sensitive to serum and phorbol 12-myristate
13-acetate. Mol Endocrinol 7:365379[Abstract]
-
Sakamoto KM, Fraser JK, Lee HJ, Lehman E, Gasson JC 1994 Granulocyte-macrophage colony-stimulating factor and interleukin-3
signaling pathways converge on the CREB-binding site in the human egr-1
promoter. Mol Cell Biol 14:59755985[Abstract]
-
Kharbanda S, Rubin E, Datta R, Hass R, Sukhatme V, Kufe D 1993 Transcriptional regulation of the early growth response 1 gene in human
myeloid leukemia cells by okadaic acid. Cell Growth Differ 4:1723[Abstract]
-
Kharbanda S, Saleem A, Hirano M, Emoto Y, Sukhatme V, Blenis
J, Kufe D 1994 Activation of early growth response 1 gene transcription
and pp90rsk during induction of monocytic differentiation. Cell Growth
Differ 5:259265[Abstract]
-
Treisman R 1992 The serum response element. Trends Biochem Sci 17:423426[CrossRef][Medline]
-
Norman C, Runswick M, Pollock R, Treisman R 1988 Isolation and
properties of cDNA clones encoding SRF, a transcription factor that
binds to the c-fos serum response element. Cell 55:9891003[Medline]
-
Shaw PE, Schroter H, Nordheim A 1989 The ability of a ternary
complex to form over the serum response element correlates with serum
inducibility of the human c-fos promoter. Cell 56:563572[Medline]
-
Marais R, Wynne J, Treisman R 1993 The SRF accessory protein
Elk-1 contains a growth factor-regulated transcriptional activation
domain. Cell 73:381393[Medline]
-
Papavassiliou AG 1994 The role of regulated phosphorylation in
the biological activity of transcription factors SRF and Elk-1/SAP-1.
Anticancer Res 14:19231926[Medline]
-
Gille H, Kortenjann M, Thomae O, Moomaw C, Slaughter C, Cobb
MH, Shaw P 1995 ERK phosphorylation potentiates Elk-1-mediated ternary
complex formation and transactivation. EMBO J 14:951962[Abstract]
-
Lee HJ, Mignacca RC, Sakamoto KM 1995 Transcriptional
activation of egr-1 by granulocyte-macrophage colony-stimulating factor
but not interleukin 3 requires phosphorylation of cAMP response
element-binding protein (CREB) on serine 133. J Biol Chem 270:1597915983[Abstract/Free Full Text]
-
Bernal-Mizrachi E, Wice B, Inoue H, Permutt MA 2000 Activation
of serum response factor in the depolarization induction of Egr-1
transcription in pancreatic islet ß-cells. J Biol Chem 275:2568125689[Abstract/Free Full Text]
-
Aicher WK, Dinkel A, Grimbacher B, Haas C, Seydlitz-Kurzbach
EV, Peter HH, Eibel H 1999 Serum response elements activate and cAMP
responsive elements inhibit expression of transcription factor Egr-1 in
synovial fibroblasts of rheumatoid arthritis patients. Int Immunol 11:4761[Abstract/Free Full Text]
-
Schwachtgen JL, Houston P, Campbell C, Sukhatme V, Braddock M 1998 Fluid shear stress activation of egr-1 transcription in cultured
human endothelial and epithelial cells is mediated via the
extracellular signal-related kinase 1/2 mitogen-activated protein
kinase pathway. J Clin Invest 101:25402549[Abstract/Free Full Text]
-
Hodge C, Liao J, Stofega M, Guan K, Carter-Su C, Schwartz J 1998 Growth hormone stimulates phosphorylation and activation of elk-1
and expression of c-fos, egr-1, and junB through activation
of extracellular signal-regulated kinases 1 and 2. J Biol Chem 273:3132731336[Abstract/Free Full Text]
-
Kwon EM, Raines MA, Blenis J, Sakamoto KM 2000 Granulocyte-macrophage colony-stimulating factor stimulation results in
phosphorylation of cAMP response element-binding protein through
activation of pp90RSK. Blood 95:25522558[Abstract/Free Full Text]
-
Halvorson LM, Kaiser UB, Chin WW 1999 The protein kinase C
system acts through the early growth response protein 1 to increase
LHß gene expression in synergy with steroidogenic factor-1. Mol
Endocrinol 13:106116[Abstract/Free Full Text]
-
Call GB, Wolfe MW 1999 Gonadotropin-releasing hormone
activates the equine luteinizing hormone ß promoter through a protein
kinase C/mitogen-activated protein kinase pathway. Biol Reprod 61:715723[Abstract/Free Full Text]
-
Ito M, Park Y, Weck J, Mayo KE, Jameson JL 2000 Synergistic
activation of the inhibin
-promoter by steroidogenic factor-1 and
cyclic adenosine 3',5'-monophosphate. Mol Endocrinol 14:6681[Abstract/Free Full Text]
-
Rupprecht HD, Sukhatme VP, Rupprecht AP, Sterzel RB, Coleman
DL 1994 Serum response elements mediate protein kinase C dependent
transcriptional induction of early growth response gene-1 by arginine
vasopressin in rat mesangial cells. J Cell Physiol 159:311323[Medline]
-
Qureshi SA, Cao XM, Sukhatme VP, Foster DA 1991 v-Src
activates mitogen-responsive transcription factor Egr-1 via serum
response elements. J Biol Chem 266:1080210806[Abstract/Free Full Text]
-
Chauhan D, Kharbanda SM, Uchiyama H, Urashima M, Anderson KC 1995 Activation of pp90rsk and early growth response-1 gene expression
by pokeweed mitogen in human B cells. Leuk Res 19:337344[CrossRef][Medline]
-
Rivera VM, Miranti CK, Misra RP, Ginty DD, Chen RH, Blenis J,
Greenberg ME 1993 A growth factor-induced kinase phosphorylates the
serum response factor at a site that regulates its DNA-binding
activity. Mol Cell Biol 13:62606273[Abstract]
-
Bohm M, Moellmann G, Cheng E, Alvarez-Franco M, Wagner S,
Sassone-Corsi P, Halaban R 1995 Identification of p90RSK as the
probable CREB-Ser133 kinase in human melanocytes. Cell Growth Differ 6:291302[Abstract]
-
De Cesare D, Jacquot S, Hanauer A, Sassone-Corsi P 1998 Rsk-2
activity is necessary for epidermal growth factor-induced
phosphorylation of CREB protein and transcription of c-fos
gene. Proc Natl Acad Sci USA 95:1220212207[Abstract/Free Full Text]
-
Xing J, Kornhauser JM, Xia Z, Thiele EA, Greenberg ME 1998 Nerve growth factor activates extracellular signal-regulated kinase and
p38 mitogen-activated protein kinase pathways to stimulate CREB serine
133 phosphorylation. Mol Cell Biol 18:19461955[Abstract/Free Full Text]
-
Liao J, Hodge C, Meyer D, Ho PS, Rosenspire K, Schwartz J 1997 Growth hormone regulates ternary complex factors and serum response
factor associated with the c-fos serum response element.
J Biol Chem 272:2595125958[Abstract/Free Full Text]
-
Gille H, Sharrocks AD, Shaw PE 1992 Phosphorylation of
transcription factor p62TCF by MAP kinase stimulates ternary complex
formation at c-fos promoter. Nature 358:414417[CrossRef][Medline]
-
Janknecht R, Cahill MA, Nordheim A 1995 Signal integration at
the c-fos promoter. Carcinogenesis 16:443450[Medline]
-
Cohen DM 1996 Urea-inducible Egr-1 transcription in renal
inner medullary collecting duct (mIMCD3) cells is mediated by
extracellular signal-regulated kinase activation. Proc Natl Acad Sci
USA 93:1124211247[Abstract/Free Full Text]
-
Chiu JJ, Wung BS, Hsieh HJ, Lo LW, Wang DL 1999 Nitric oxide
regulates shear stress-induced early growth response-1. Expression via
the extracellular signal-regulated kinase pathway in endothelial
cells. Circ Res 85:238246[Abstract/Free Full Text]
-
Shin SY, Kim SY, Kim JH, Min DS, Ko J, Kang UG, Kim YS, Kwon
TK, Han MY, Kim YH, Lee YH 2001 Induction of early growth response-1
gene expression by calmodulin antagonist trifluoperazine through the
activation of Elk-1 in human fibrosarcoma HT1080 cells. J Biol
Chem 276:77977805[Abstract/Free Full Text]
-
Clarkson RW, Shang CA, Levitt LK, Howard T, Waters MJ 1999 Ternary complex factors Elk-1 and Sap-1a mediate growth hormone-induced
transcription of egr-1 (early growth response factor-1) in 3T3F442A
preadipocytes. Mol Endocrinol 13:619631[Abstract/Free Full Text]
-
Rupprecht HD, Sukhatme VP, Lacy J, Sterzel RB, Coleman DL 1993 PDGF-induced Egr-1 expression in rat mesangial cells is mediated
through upstream serum response elements. Am J Physiol
265:F351F360
-
Mora-Garcia P, Sakamoto KM 2000 Granulocyte colony-stimulating
factor induces Egr-1 up-regulation through interaction of serum
response element-binding proteins. J Biol Chem 275:2241822426[Abstract/Free Full Text]
-
Cohen DM, Gullans SR, Chin WW 1996 Urea inducibility of egr-1
in murine inner medullary collecting duct cells is mediated by the
serum response element and adjacent Ets motifs. J Biol Chem 271:1290312908[Abstract/Free Full Text]
-
Watson DK, Robinson L, Hodge DR, Kola I, Papas TS, Seth A 1997 FLI1 and EWS-FLI1 function as ternary complex factors and ELK1 and
SAP1a function as ternary and quaternary complex factors on the Egr1
promoter serum response elements. Oncogene 14:213221[CrossRef][Medline]
-
Treisman R 1994 Ternary complex factors: growth factor
regulated transcriptional activators. Curr Opin Genet Dev 4:96101[Medline]
-
Gille H, Kortenjann M, Strahl T, Shaw PE 1996 Phosphorylation-dependent formation of a quaternary complex at the
c-fos SRE. Mol Cell Biol 16:10941102[Abstract]
-
Roberson MS, Misra-Press A, Laurance ME, Stork PJ, Maurer RA 1995 A role for mitogen-activated protein kinase in mediating
activation of the glycoprotein hormone
-subunit promoter by
gonadotropin-releasing hormone. Mol Cell Biol 15:35313539[Abstract]
-
Kang SK, Tai CJ, Nathwani PS, Choi KC, Leung PC 2001 Stimulation of mitogen-activated protein kinase by
gonadotropin-releasing hormone in human granulosa-luteal cells.
Endocrinology 142:671679[Abstract/Free Full Text]
-
Herrera RE, Shaw PE, Nordheim A 1989 Occupation of the
c-fos serum response element in vivo by a
multi-protein complex is unaltered by growth factor induction. Nature 340:6870[CrossRef][Medline]
-
Sheng M, Dougan ST, McFadden G, Greenberg ME 1988 Calcium and
growth factor pathways of c-fos transcriptional activation
require distinct upstream regulatory sequences. Mol Cell Biol 8:27872796[Medline]
-
Fisch TM, Prywes R, Roeder RG 1987 c-fos Sequence
necessary for basal expression and induction by epidermal growth
factor, 12-O-tetradecanoyl phorbol-13-acetate and the
calcium ionophore. Mol Cell Biol 7:34903502[Medline]
-
Miranti CK, Ginty DD, Huang G, Chatila T, Greenberg ME 1995 Calcium activates serum response factor-dependent transcription by a
Ras- and Elk-1-independent mechanism that involves a
Ca2+/calmodulin-dependent kinase. Mol Cell Biol 15:36723684[Abstract]
-
Heidenreich O, Neininger A, Schratt G, Zinck R, Cahill MA,
Engel K, Kotlyarov A, Kraft R, Kostka S, Gaestel M, Nordheim A 1999 MAPKAP kinase 2 phosphorylates serum response factor in
vitro and in vivo. J Biol Chem 274:44344443
-
Marais RM, Hsuan JJ, McGuigan C, Wynne J, Treisman R 1992 Casein kinase II phosphorylation increases the rate of serum response
factor-binding site exchange. EMBO J 11:97105[Abstract]
-
Duan WR, Shin JL, Jameson JL 1999 Estradiol suppresses
phosphorylation of cyclic adenosine 3',5'-monophosphate response
element binding protein (CREB) in the pituitary: evidence for indirect
action via gonadotropin-releasing hormone. Mol Endocrinol 13:13381352[Abstract/Free Full Text]
-
Janknecht R, Nordheim A 1996 MAP kinase-dependent
transcriptional coactivation by Elk-1 and its cofactor CBP. Biochem
Biophys Res Commun 228:831837[CrossRef][Medline]
-
Janknecht R, Nordheim A 1996 Regulation of the
c-fos promoter by the ternary complex factor Sap-1a and its
coactivator CBP. Oncogene 12:19611969[Medline]
-
Ramirez S, Ait-Si-Ali S, Robin P, Trouche D, Harel-Bellan A 1997 The CREB-binding protein (CBP) cooperates with the serum response
factor for transactivation of the c-fos serum response
element [published erratum appears in J Biol Chem 1999 Jun
18;274(25):18140]. J Biol Chem 272:3101630121[Abstract/Free Full Text]
-
Nissen LJ, Gelly JC, Hipskind RA 2000 Induction-independent
recruitment of CREB binding protein to the c-fos serum
response element through interactions between the bromo domain and
Elk-1. J Biol Chem 276:52135221[Abstract/Free Full Text]
-
Kay TW, Chedrese PJ, Jameson JL 1994 Gonadotropin-releasing
hormone causes transcriptional stimulation followed by desensitization
of the glycoprotein hormone
promoter in transfected
T3
gonadotrope cells. Endocrinology 134:568573[Abstract]
-
Weck J, Fallest PC, Pitt LK, Shupnik MA 1998 Differential
gonadotropin-releasing hormone stimulation of rat luteinizing hormone
subunit gene transcription by calcium influx and mitogen-activated
protein kinase-signaling pathways. Mol Endocrinol 12:451457[Abstract/Free Full Text]
-
Sundaresan S, Colin IM, Pestell RG, Jameson JL 1996 Stimulation of mitogen-activated protein kinase by
gonadotropin-releasing hormone: evidence for the involvement of protein
kinase C. Endocrinology 137:304311[Abstract]
-
Reiss N, Llevi LN, Shacham S, Harris D, Seger R, Naor Z 1997 Mechanism of mitogen-activated protein kinase activation by
gonadotropin-releasing hormone in the pituitary of
T3-1 cell line:
differential roles of calcium and protein kinase C. Endocrinology 138:16731682[Abstract/Free Full Text]
-
Xing J, Ginty DD, Greenberg ME 1996 Coupling of the RAS-MAPK
pathway to gene activation by RSK2, a growth factor-regulated CREB
kinase. Science 273:959963[Abstract]
-
Roberson MS, Zhang T, Li HL, Mulvaney JM 1999 Activation of
the p38 mitogen-activated protein kinase pathway by
gonadotropin-releasing hormone. Endocrinology 140:13101318[Abstract/Free Full Text]
-
Mulvaney JM, Roberson MS 2000 Divergent signaling pathways
requiring discrete calcium signals mediate concurrent activation of two
mitogen-activated protein kinases by gonadotropin-releasing hormone.
J Biol Chem 275:1418214189[Abstract/Free Full Text]
-
Yokoi T, Ohmichi M, Tasaka K, Kimura A, Kanda Y,
Hayakawa J, Tahara M, Hisamoto K, Kurachi H, Murata Y 2000 Activation
of the luteinizing hormone ß promoter by gonadotropin-releasing
hormone requires c-Jun NH2-terminal protein kinase. J Biol Chem 275:2163921647[Abstract/Free Full Text]
-
Shapiro DJ, Sharp PA, Wahli WW, Keller MJ 1988 A
high-efficiency HeLa cell nuclear transcription extract. DNA 7:4755[Medline]
-
Yu RN, Ito M, Jameson JL 1998 The murine Dax-1 promoter
is stimulated by SF-1 (steroidogenic factor-1) and inhibited by COUP-TF
(chicken ovalbumin upstream promoter-transcription factor) via a
composite nuclear receptor-regulatory element. Mol Endocrinol 12:10101022[Abstract/Free Full Text]