Estrogen Modulation of Prolactin Gene Expression Requires an Intact Mitogen-Activated Protein Kinase Signal Transduction Pathway in Cultured Rat Pituitary Cells
Jyoti J. Watters,
Tae-Yon Chun,
Yong-Nyun Kim,
Paul J. Bertics and
Jack Gorski
Department of Biochemistry (J.J.W., T.-Y.C., J.G.) and
Department of Biomolecular Chemistry (Y.-N.K., P.J.B.) University
of Wisconsin Madison, Wisconsin 53706
 |
ABSTRACT
|
---|
Expression of the PRL gene is regulated by many
factors, including cAMP, estradiol (E2),
phorbol esters, epidermal growth factor (EGF), and TRH. The promoter
region of the rat PRL gene has been shown to contain DNA sequences that
are thought to support the direct interaction of estrogen receptors
(ERs) with DNA. It is by this direct ER/DNA interaction that estrogen
is thought to modulate expression of PRL. We report here that
estrogen-induced PRL expression requires an intact mitogen-activated
protein kinase (MAPK) signal transduction pathway in cultured rat
pituitary cells (PR1 lactotroph and GH3 somatolactotroph cell lines).
Interfering with the MAPK signaling cascade by inhibiting the activity
of MAPK kinase (MEK) ablates the ability of estrogen to induce PRL mRNA
and protein. In these cell lines, estrogen activates extracellular
regulated protein kinases ERK-1 and ERK-2 enzyme activities
maximally within 10 min of 1 nM
E2 treatment. This activity is blocked by
pretreatment of the cells with the MEK inhibitors PD98059 and UO126.
The mechanism by which ERKs-1 and -2 are activated by estrogen appears
to be independent of c-Src since the effects of estrogen on PRL gene
expression are not affected by herbimycin A or PP1 administration.
c-Raf-1 may be involved in the effects of E2
because estrogen causes the rapid and transient tyrosine
phosphorylation of c-Raf-1. The ER antagonist ICI 182,780 blocks both
ERK-1 and ERK-2 activation in addition to PRL protein and mRNA,
implying a central role for the classical ER in the activation of the
MAPK pathway resulting in PRL gene expression.
 |
INTRODUCTION
|
---|
Estrogen controls the expression of PRL in pituitary lactotrophs,
primarily by a transcription-dependent mechanism (1). Classically,
estrogen acts by binding to its nuclear receptors that, in turn,
interact with specific sequences of DNA (estrogen response elements,
EREs) present in the promoters of many genes to modulate their
expression. The PRL promoter contains potential EREs (1, 2, 3, 4, 5, 6, 7, 8, 9) although
thus far, only large regions of PRL promoter DNA have been shown to
confer estrogen responsiveness in reporter gene assays (5, 10). The
estrogen response of the PRL promoter is also complicated by the
presence of Pit-1 binding sites that have been shown to be required for
both basal PRL expression as well as estrogeninduced PRL
expression (11, 12, 13, 14, 15).
There are increasing reports of signal transduction cascades that seem
to be activated by estrogen in various tissues and cell lines. The
mitogen-activated protein kinase (MAPK) pathway is one such pathway,
although an exact biochemical mechanism and the endogenous,
physiologically relevant consequences of this activation remain
unclear. Upon expression of the human estrogen receptor (ER) in the
non-ERexpressing COS cells, Migliaccio et al. (16)
showed that estrogen activates the tyrosine kinase c-Src, and by this
means, transiently activates the MAPKs ERK-1 and -2 (extracellular
regulated protein kinases 1 and 2). They showed that estrogen also
activates MAPKs in MCF-7 cells. The physical interaction between c-Src
and the ER were performed in vitro using ER purified from
MCF-7 cells and columns to which c-Src was immobilized. Under these
conditions it was shown that the ER interacts with c-Src, and
presumably, this interaction results in the estrogen-induced activation
of the MAPKs. A physiological consequence of estrogen activation of
this pathway, cellular proliferation, has since been shown both in
breast cancer cells and in transfected cell systems (17, 18).
Similarly, other groups have described the rapid and transient
activation of ERKs 1 and 2 in various other cell lines and tissues
(cardiomyocytes, colon carcinoma cells, bone cells, and breast
carcinoma cells) (19, 20, 21).
We describe here, in a naturally estrogen-responsive, intact cell
system, the relationship between estrogen activation of ERKs -1 and -2
and PRL gene expression in pituitary cells. The PRL secreting pituitary
cell line, PR1, used in these studies was derived from a
diethylstilbestrol-treated Fischer 344 female rat. These cells have
been described previously (22, 23) and have been shown to respond to
estrogens by increased PRL synthesis and cell proliferation. We have
studied the signal transduction pathways activated by estrogen in both
PR1 cells and in the more commonly used GH3 pituitary cell line and
have used the endogenously expressed and estrogen-regulated PRL gene as
a physiological measure and pharmacological readout of the activation
of these pathways. The primary function of pituitary lactotrophs is to
synthesize and secrete PRL. Our results indicate that the rapid and
transient activation of ERKs-1 and -2 by estrogen in these cells
results in PRL gene transcription since pretreatment of the cells with
MAPK kinase (MEK) inhibitors blocks the ability of estrogen to promote
PRL transcription. Estrogen treatment of these cells also appears to
result in the rapid tyrosine phosphorylation of c-Raf-1, an event that
is thought to be important in enzymatic activation and biological
function of the c-Raf-1 enzyme (24). The mechanism of c-Raf tyrosine
phosphorylation/activation by estrogen remains unclear; however, it
appears to be performed by a herbimycin- and PP1-insensitive tyrosine
kinase, i.e. by an enzyme(s) other than those of c-Src
family of soluble tyrosine kinases.
 |
RESULTS
|
---|
Effects of Estrogen and MEK Inhibitors on PRL Protein
Production
Treatment of PR1 cells with 17ß-estradiol for 24 h results
in a dose-dependent increase in PRL protein production (Fig. 1A
, top panel). Pretreatment of the cells
with the MEK inhibitor PD98059 interferes with the ability of estrogen
to promote PRL protein accumulation, while the inhibitor alone had no
effect. Intracellular PRL protein accumulation (Fig. 1A
, lower
panel) is difficult to detect in this cell line (22); however, the
highest dose of estrogen (1 nM) increased
intracellular levels of PRL detectably within 24 h. And as seen
with secreted PRL in the top panel, administration of the
MEK inhibitor also reduced PRL accumulation in the intracellular pools,
indicating that the effects of the MEK inhibitor are not solely an
effect on secretion in these cells. Figure 1B
graphically presents the
optical densities of bands measured from Western blots of secreted PRL
from cultured PR1 cells after 24 h of treatment with the agents
used in Fig. 1A
. Data represent the mean ±
SEM graphed as fold induction over
vehicle-treated control cells (n
4). The MEK inhibitor had no
effect on cell viability as assessed by trypan blue staining,
MTT assay (OD490, vehicle, 0.218 ±
0.029; PD98059, 0.219 ± .014),
[3H]methionine incorporation into protein
(vehicle, 26,842 ± 829 cpm; PD98059, 26,457 ± 857 cpm), or
on DNA synthesis as measured by [3H]thymidine
incorporation (vehicle, 7,843 ± 940 cpm; PD98059 8519 ± 397
cpm) (mean ± SEM). To more specifically
control for the effects of the MEK inhibitor on the expression of other
genes, another estrogen-responsive gene, cyclin D3, was evaluated.
Cyclin D1 has been shown to be transcriptionally responsive to estrogen
(25, 26), most likely by virtue of the presence of an ERE in its
promoter (25). Interestingly, cyclin D1 is not expressed at detectable
levels in pituitary cells. However, a related gene, cyclin D3, is the
predominant cyclin expressed in pituitary cells (Ref. 27 and J. J.
Watters and J. Gorski, unpublished observations). We show here that
cyclin D3 is also regulated by estrogen (Fig. 1C
, third
panel), in a manner that is dependent upon the function of the ER,
since the pure antiestrogen ICI 182,780 ablates the increase in cyclin
D3 protein accumulation (Fig. 1C
, second panel). As
indicated in the lowest panel of Fig. 1C
, in the presence of
the MEK inhibitor PD98059, the ER retains the capacity to promote
cyclin D3 protein increases, suggesting that the MEK inhibitor is not
affecting all transcriptional functions of the ER in these cells but,
rather, that it is specific for the effects of estrogen on the PRL
gene.

View larger version (39K):
[in this window]
[in a new window]
|
Figure 1. Effect of Estrogen and a MEK Inhibitor on PRL
Synthesis in PR1
Cells PR1 cells were treated for 24 h with ethanol vehicle, 1
nM, 10 pM, or 10 fM
E2. Medium was removed and used for analysis of
secreted PRL (PRLs). Whole cell lysates were prepared and analyzed for
intracellular PRL (PRLi) protein content as described in
Materials and Methods. A, Western blot of PR1 cell medium
(upper panel) and whole cell lysates (lower
panel). Estrogen dose dependently increased the amount of PRL
produced by these cells. Administration of the MEK inhibitor PD98059
(50 µM) dramatically reduced the ability of
estrogen to promote PRL synthesis. B, Graphic representation of the
quantification of optical densities obtained from the immunoblots
described above (n 4). C, Cyclin D3 immunoblot of PR1 cells
grown in medium containing DCCSS and ethanol vehicle (upper
panel); medium same as control, but containing 100
nM ICI 182,780 for the times indicated
(second panel); medium same as control, but containing 1
nM E2 (third
panel); and medium same as control, containing
E2 at the indicated concentrations and 50
µM PD98059 for 24 h (fourth
panel).
|
|
Involvement of Tyrosine Kinases and MEK in Estrogen-Stimulated PRL
Protein Synthesis
GH3 cells, another rat pituitary cell line, shows the same
response to estrogen in terms of PRL production as the PR1 cells (Fig. 2A
). Also, as seen previously in PR1
cells, the effect of estrogen on induced PRL gene expression is
attenuated by administration of the MEK inhibitor PD98059. Another MEK
inhibitor, UO126, with a mechanism of action distinct from that of the
PD98059 compound, also blocks the ability of estrogen to promote PRL
gene transcription. Interestingly, another rapid estrogen response, the
down-regulation of ER
, was not affected by the MEK inhibitors (E. T.
Alarid, J. J. Watters and J. Gorski, unpublished
observations) providing further evidence that the inhibitors are not
interfering with all estrogen responses in these cells. Because it is
clear that MEK is involved in the estrogen stimulation of PRL in these
lactotroph cell lines, we undertook to determine the identity of the
kinase whose activity was induced by estrogen and which might be an
upstream activator (either directly or indirectly) of MEK. To do so, we
used the broad specificity tyrosine kinase inhibitor, herbimycin A.
Herbimycin A is known to inhibit tyrosine kinases of the Src family,
among others. When administered at 880 nM or at 4.4
µM, herbimycin A failed to interfere with the ability of
estrogen to promote PRL expression in GH3 cells, suggesting that the
tyrosine kinases whose activities are sensitive to herbimycin treatment
are not involved in the effects of estrogen investigated here (Fig. 2A
). This is unlike what has been reported previously (16, 21, 27), in
the human breast cancer cell line MCF-7 and in the human colon cancer
cell line Caco-2. These groups report that the tyrosine kinases c-Src
and c-Yes are involved in transducing the mitogenic estrogen signal to
the MAPKs. Genistein is a broad specificity tyrosine kinase inhibitor
with effects on the c-Src family of enzymes. It is also a compound with
estrogenic attributes when used at high concentrations (high µM
range). When genistein was used at concentrations normally used to
inhibit tyrosine kinase activity (low nM range), it too failed to
inhibit the increases in PRL gene expression stimulated by
E2 treatment (data not shown). These data support
the notion that the c-Src family of enzymes may not be involved in the
effects of estrogen on PRL gene expression in pituitary cells. To test
the specificity of the MEK inhibitor effects we observed, epidermal
growth factor (EGF) was used as a positive control for the activation
of the MAPK cascade. Additionally, a pharmacological inhibitor of
protein kinase A (PKA) (H89) and a negative control for the PD compound
(SB202474) were used (Fig. 2B
). H89 had no effect on estrogen- or
EGF-induced PRL synthesis, while PD98059 diminished the effects of both
agents. The negative control SB202474 (a structural analog of PD98059)
(28) had no effect on PRL expression in response to treatment with
either E2 or EGF. The pure anti-estrogen ICI
182,780 reduced PRL levels stimulated both by estrogen and EGF down to
those observed with vehicle treatment (Fig. 2B
), implying a role for
estrogen receptor
(ER
) both in the actions of estrogen and in
those of EGF. The ability of the pure antiestrogen to decrease
EGF-induced PRL gene expression is interesting and may be explained by
several possible mechanisms. The ER mediates many of the growth
responses of EGF in tissues such as the uterus, and EGF receptor
activation can induce ligand-independent transcriptional activation of
the ER. This may be one explanation for the antiestrogen blockade of
EGF-induced PRL gene expression, although this explanation cannot be
all of the answer. If it were, then some of the EGF-responsive elements
in the PRL promoter would have mapped to the estrogen-responsive sites.
But this does not appear to be the case, with what is known of the PRL
promoter at this time.

View larger version (42K):
[in this window]
[in a new window]
|
Figure 2. Effect of Estrogen and Pharmacological Inhibitors
of MEK and Tyrosine Kinases on PRL Production in GH3 Cells
A, Cells were treated for 24 h with ethanol vehicle or 1
nM E2 in the presence or absence of the MEK
inhibitors PD98059 (50 µM) or UO126 (10 µM)
or the tyrosine kinase inhibitor herbimycin A (880 nM and
4.4 µM). PRL secreted into the tissue culture medium was
immunoblotted (representative blot from at least four separate
experiments). B, Cells were treated with ethanol vehicle, 1
nM E2, or 50 ng/ml EGF in the presence or
absence of the PKA inhibitor H89 (5 µM), the MEK
inhibitor PD98059 (50 µM), the inactive structural analog
of PD98059, SB202474 (50 µM), or the pure antiestrogen
ICI 182,780 (100 nM). Secreted PRL was measured by
immunoblotting (representative blot from at least three separate
experiments).
|
|
Signaling Enzymes Involved in Estrogen Effects on PRL Gene
Expression
As shown previously, estrogen stimulation of PRL expression is
decreased by administration of the MEK inhibitors PD98059 and UO126
(Fig. 3A
). Because it had previously been
reported in MCF-7 cells that the initiating enzyme of the
estrogen-induced MAPK cascade was c-Src, and we saw no effect on
estrogen-stimulated PRL gene expression using the broad specificity
tyrosine kinase inhibitors herbimycin and genistein (Fig. 2A
), we used
a more specific inhibitor of the c-Src family of soluble protein
tyrosine kinases, PP1. As shown in Fig. 3A
, PP1 fails to interfere with
the ability of estrogen to induce PRL gene expression. PP1 was used at
several concentrations, two of which are shown in Fig. 3A
. The doses
used here were the same as those empirically determined to inhibit
whole-cell tyrosine phosphorylation in GH3 cells stimulated by EGF
(data not shown). These data suggest that MEK activity is involved in
the mechanism by which estrogen increases PRL expression, while
tyrosine kinases such as c-Src may not be. To further investigate the
role of c-Src in the actions of estrogen, we treated GH3 cells with 1
nM E2, immunoprecipitated c-Src,
electrophoresed the proteins by SDS-PAGE, and immunoblotted with
antiphosphotyrosine-specific antibodies. The c-Src tyrosine kinase
enzyme is activated by tyrosine phosphorylation on either of its two
tyrosine residues (reviewed in Refs. 29, 30). If estrogen were
stimulating Src tyrosine kinase activity, then an increase in the
overall tyrosine phosphorylation status of the c-Src protein would be
observed. As shown in Fig. 3B
, the overall tyrosine phosphorylation of
the c-Src protein was unchanged in response to estrogen treatment, but
was slightly decreased in the presence of PP1, the Src kinase
inhibitor, indicating that the PP1 compound had the capacity to affect
basal Src kinase activity in these experiments. The lower
panel shows the above blot, stripped and reprobed with c-Src
antibodies, to show that fairly equal amounts of protein were
immunoprecipitated. Since MEK activity appears important for
estrogen-induced PRL expression, and the enzyme c-Raf-1 is well known
to phosphorylate and activate MEK, we evaluated the ability of estrogen
to activate c-Raf-1. This was done utilizing Western blotting
techniques and a phosphospecific antibody directed against the dually
phosphorylated tyrosine resides 340 and 341 of the c-Raf-1
protein. Phosphorylation of these residues is believed to correlate
with enzymatic activation (24). As shown in Fig. 3C
, estrogen rapidly
and transiently results in the tyrosine phosphorylation of c-Raf-1 in
PR1 cells (Fig. 3C
, upper panel), while having no effect on
total c-Raf-1 protein (Fig. 3C
, lower panel). Investigation
of the enzymes that follow Raf and MEK in the MAPK signaling cascade,
the MAPKs ERK-1 and ERK-2, indicated that they too were activated in a
rapid and transient manner by estrogen in PR1 cells. The upper
panel of Fig. 3D
shows an immunoblot of the enzymatically active
ERK-1 and -2 enzymes, using an antibody that specifically recognizes
the dually phosphorylated and hence enzymatically active MAPKs.
Estrogen appears to activate these enzymes maximally within 10 min of
treatment. Activation of c-Raf-1 appeared maximal within 5 min of
estradiol treatment, a time frame that precedes that of ERK activation,
suggesting that Raf activation is upstream of ERK activation.
Pretreatment of cells with the MEK inhibitor PD98059 blocks the ability
of estrogen to activate the MAPKs, implying that the activation of MEK
by estrogen is involved in ERK activation. The lower panel
of Fig. 3D
shows the results of a reprobing of the blot in the
upper panel, with an antibody that recognizes total ERK-1
and -2 proteins (both active and inactive). This blot shows that while
there is not detectable activated ERK isoform activity in all lanes of
the upper panel, there are abundant and approximately equal
amounts of total ERK protein in each lane.

View larger version (42K):
[in this window]
[in a new window]
|
Figure 3. Effect of Estrogen on PRL Synthesis in the Presence
of the c-Src Inhibitor PP1 in GH3 Cells
Effect of estrogen on c-Raf-1 and ERK 1 and ERK 2 activation in PR1
cells. A, GH3 cells were treated with ethanol vehicle or 1
nM E2 for 24 h in the presence or absence
of the MEK inhibitors PD98059 (50 µM and 100
µM) and UO126 (10 µM and 30
µM) or the c-Src family of tyrosine kinase inhibitors PP1
(1 µM and 2 µM). Secreted PRL was evaluated
by immunoblotting techniques (representative blot from at least three
separate experiments). B, Phosphotyrosine immunoblot (i.b.) of c-Src
immunoprecipitation (ip) from GH3 cells treated with ethanol vehicle or
1 nM E2 for the indicated times or the c-Src
inhibitor PP1 (2 µM). C, PR1 cells were treated with 1
nM E2 for 5, 10,15, 30, 60, or 120 min. Control
cells (0) were treated with ethanol vehicle. Supernatants (100,000
x g) were collected as described in Materials
and Methods and were immunoblotted using an antibody that
specifically recognizes the phosphorylation of the c-Raf-1 protein at
tyrosine residues 340 and 341 (upper panel). The
lower panel shows the blot from the
upper, stripped and reprobed with an antibody that
recognizes total c-Raf-1 protein, illustrating that total c-Raf-1
protein is unchanged by estrogen treatment. D, PR1 cells were treated
with 1 nM E2 for 5, 10,15, 30, 60, or 120 min.
Control cells (0) were treated with ethanol vehicle. Supernatants
(100,000 x g) were immunoblotted using an antibody
to the dually phosphorylated and enzymatically active MAPKs ERK-1 and
ERK-2 (upper panel). The blot was stripped and reprobed
with an antibody that recognizes both ERK 1 and ERK 2 total protein.
(Blots shown are representative blots from at least five separate
experiments.)
|
|
Estrogen Dose-Response and Steroid Specificity of MAPK
Activation
Active ERK-1 and -2 levels in GH3 cells were determined
by immunoblotting with the antibodies described above. Optical
densities were measured by a Molecular Dynamics, Inc.
densitometer as described in Materials and Methods. Data
obtained in this way are shown graphically in Fig. 4
, expressed as the ratio between the
active (phospho-ERK) level and that of total ERK in arbitrary units.
Each graph is representative of at least three separate experiments.
Figure 4A
illustrates a dose-response curve of estrogen on MAPK
activation; 1 nM E2
activated both ERK-1 and ERK-2 activities as shown in Fig. 3
; 10
pM E2 also appeared to
stimulate the MAPKs although to a lesser degree than the higher dose of
E2. The lowest dose of estrogen tested, 10
fM, did not have measurable activity by this
method. Figure 4B
shows the results of progesterone, both at 1
nM and 10 pM. Progesterone,
1 nM 17
-estradiol, and testosterone (Fig. 3C
)
lacked the ability to induce ERK-1 and ERK-2 activation, illustrating
the specificity of this activation by estrogen. These hormones also had
little capacity to promote PRL gene expression in this cell system
(data not shown). The pure ER antagonist ICI 182,780 blocked the
ability of estrogen to promote activation of the MAPKs ERK-1 and -2.
These results are in agreement with our observations that the ICI
compound decreases the effects of E2 on PRL
protein expression.
Effect of Estrogen and MEK Inhibitors on PRL mRNA
Total RNA was isolated from PR1 cells and subjected to
standard agarose-formaldehyde electrophoresis as described in
Materials and Methods. Cells were treated for the times
indicated, and Northern blot analyses were performed. Blots were
hybridized with a radioactively labeled mouse PRL cDNA probe and imaged
on a Phosphorimager (Molecular Dynamics, Inc., Sunnyvale,
CA). Blots were stripped and reprobed with CHOB (Fig. 5
, A and B, lower panels), a
ribosomal protein RNA as a control for equal loading. There was a
measurable increase in PRL mRNA in response to E2
treatment for 12 h (Fig. 5A
, upper panel). This effect
was blocked by administration of the MEK inhibitor PD98059. The
increase in PRL mRNA elicited by estrogen treatment was also diminished
by administration of the ICI compound (Fig. 5B
, upper
panel). The PD compound was able to inhibit the effects of
estrogen at all time points tested (Fig. 5
, A and B, upper
panels). These data support the contention that the effects of the
MEK inhibitors are occurring not only at the level of the protein
itself, but also at the level of the RNA. Since it is widely believed
that the effects of estrogen on PRL gene expression are primarily
transcriptional (1, 2, 3, 4, 5, 6, 7, 8, 9, 31, 32, 33), and the MEK inhibitor can block the
increases in estrogen-induced PRL mRNA, it is likely that MEK (or its
downstream kinases) exert their effects transcriptionally on the PRL
gene.

View larger version (64K):
[in this window]
[in a new window]
|
Figure 5. Effect of Estrogen, MEK Inhibitor, and Antiestrogen
on PRL mRNA in PR1 Cells
A, Time course of 1 nM E2 on PRL mRNA
induction (upper panel) in the presence or absence of
the MEK inhibitor PD98059 (50 µM). PR1 cells were
treated as specified in Fig. 5A for 3, 6, 12, or 18 h and then
total RNA harvested and electrophoresed as indicated in
Materials and Methods. The blot was stripped and
reprobed with a cDNA to CHOB, a ribosomal RNA protein as a control for
loading. B, PRL mRNA expression in PR1 cells after 1
nM E2, 50 µM PD98059,
or 100 nM ICI 182,780 treatment for 18 or 24 h.
|
|
 |
DISCUSSION
|
---|
The data reported here strongly imply a role for the MAPK
pathway in the actions of estrogen on PRL gene expression. Estrogen has
been known since 1995 to activate the MAPK pathway in certain cell
types. We show for the first time, that the estrogen-induced activation
of PRL gene expression in pituitary lactotroph cells requires an intact
MAPK cascade. Interference with this pathway by utilizing inhibitors of
MEK ablates the ability of estrogen to increase PRL synthesis (Fig. 1
).
Additionally, we have shown that estrogen activates the MAPKs ERK-1 and
ERK-2 in pituitary lactotroph cell lines (Figs. 2
and 3
). Fischer 344
rats are a strain highly sensitive to the estrogen induction of
lactotroph tumors. When treated with diethylstilbestrol for 121 days,
these animals show an increase in MAPK activity in whole pituitary
extracts (J. J. Watters and J. Gorski, unpublished observations).
These data indicate that estrogen activation of MAPKs in lactotrophs
may also play a role in pituitary function in vivo.
The identity of the tyrosine kinase activated by estrogen in pituitary
lactotrophs that induces the tyrosine phosphorylation of c-Raf-1
remains unknown. Based on our observations using the pharmacological
inhibitors herbimycin A and PP1, the c-Src family of soluble tyrosine
kinases are likely not involved in mediating the effects of estrogen on
the PRL gene, although the c-Src family kinases have been found to be
involved in the estrogen activation of tyrosine phosphorylation and
MAPK activation in MCF-7 cells (16, 27). The involvement of p21 Ras in
this pathway in our cell system is also not yet clear. The activation
of rapid tyrosine phosphorylation and subsequent MAPK pathway
activation is generally associated with the actions of a
membrane-localized receptor related to those of the growth
factor/cytokine receptor family. There have been several reports of
membrane ERs (34, 35, 36, 37). In fact, GH3 cells have recently been shown to
express ERs on their plasma membranes (38, 39). The presence of a
receptor for estradiol on the cell surface may allow for interactions
with proteins that result in the tyrosine phosphorylation of Raf family
kinases and subsequent MAPK pathway activation.
The promoter of the PRL gene has been extensively studied in
terms of its ability to respond to various hormones and growth factors.
TRH, EGF, cAMP, phorbol esters, and estrogen can regulate expression of
the PRL gene. It is believed that the means by which estrogen modulates
the expression of this gene is by the direct binding of activated ERs
to specific DNA sequences located in the promoter of the PRL gene
(5, 6, 7, 8, 9). This promoter also contains binding sites for additional
transcription factors some of whose functions are regulated by protein
kinases activated by the MAPK and/or PKA pathways. Pit-1 protein
function has also been shown to be required for estrogen activation of
the PRL promoter (11, 13). Recently, it has been shown that ERs and
Pit-1 proteins physically interact in the GH3 and PR1 cell lines used
in our studies here, in an estrogen-dependent manner (40). However, it
is not yet known whether this interaction with the ER is dependent upon
the phosphorylation status of Pit-1 or the ER.
It is also possible that estrogen-induced activation of the MAPK
pathway results in the functional modification of a MAPK-responsive
transcription factor that controls the expression of PRL, which in
concert with the DNA sequences to which ERs bind, results in the
cooperative activation of PRL gene expression. It might be hypothesized
that independent MAPK activation of a transcription factor that
interacts with the PRL promoter would result in the estrogen-responsive
sites of the PRL promoter mapping to some elements that are common to
those which respond to growth factors and other kinases. However, the
activation of signal transduction cascades, although the enzymes
themselves are common to different stimuli, are in reality quite
specific for the agonist. Therefore, the fact that estrogen-responsive
elements in the PRL promoter may not at this time be known to coincide
with other transcription factor binding sites known to be responsive to
activation of this pathway by other agonists does not necessarily
negate the possibility that estrogen may be stimulating the direct
phosphorylation of some MAPK-responsive transcription factor.
Another possible explanation for the observed effects with the
MEK inhibitors might be related to the function of the ER itself. The
ER
is phosphorylated on ser167 by pp90rsk1 (a downstream target
enzyme of the ERKs) (41), and on ser118 by the MAPKs themselves (42).
These residues appear important in the recruitment of transcription
coactivators such as p68 (43) since the mutation of either or both of
these sites results in significant inhibition of ER transcriptional
activity (41). Therefore, another possible mechanism for the effects we
have observed may involve the estrogen-stimulated MAPK activity having
effects on the phosphorylation status of the ER, which in turn results
in a further transcriptional enhancement of ER function. Thus, estrogen
would serve a dual function: to bind the receptor and stimulate its
transcriptional activity while simultaneously interacting with the
MAPKs to further increase its function. At this time, there are several
possibilities that could explain our observations. There is likely not
one single mechanism at work at the PRL promoter, but rather a complex
interaction between estrogen, the ER, and other factors involved in the
MAPK pathway.
 |
MATERIALS AND METHODS
|
---|
Reagents
PD98059 and PP1 were obtained from Calbiochem
(La Jolla, CA). 17ß-Estradiol, 17
-estradiol, testosterone,
progesterone, and herbimycin A were acquired from Sigma
(St. Louis, MO). UO126 was purchased from Promega Corp.
(Madison, WI).
Tissue Culture
PR1 and GH3 cells were maintained in 100-mm Falcon plates
(Becton Dickinson and Co., Franklin Lakes, NJ) in phenol
red-free DMEM (MediaTech, Herndon, VA) containing 100 U/ml Penicillin G
sulfate, 100 µg/ml Streptomycin sulfate, 0.25 µg/ml
Amphotericin, and 2 mM L-Glutamine
(Life Technologies, Inc., Gaithersburg, MD) in the
presence of 10% FBS. Culture medium was changed every 2 days until
cells reached confluency. For experimentation with steroids, cells were
washed twice with HBSS and the growth medium was replaced with DMEM
containing the above supplements and 10% 3X dextran-coated
charcoal-stripped FBS (DCCSS) (44). In addition, cells were treated
with 100 nM ICI 182,780 (generously provided by
Zeneca Pharmaceuticals, Macclesfield, UK) for 23 days to
eliminate endogenous estrogenic activity present in the FBS used to
propagate the cell line. After this time, cells were washed twice with
HBSS and replaced with fresh DMEM/DCCSS for 1 day before stimulation
with E2, EGF (Upstate Biotechnology, Inc., Lake Placid, NY), or other hormones as indicated in the
figure legends.
PRL, Cyclin D3, and c-Src Protein Analyses
PR1 and GH3 cells were propagated as described above. Cells were
washed and replated in DMEM/DCCSS in six-well dishes (Falcon) at a
density of 7.5 x 105 to 1.0 x
106 in 2 ml culture medium. The next day, the
medium was replaced and the cells were treated with hormones at the
concentrations indicated in the figure legends. Twenty four hours after
treatment, equal volumes of medium were removed from the wells and
mixed with 2X SDS/PAGE sample buffer and assayed by Western blot for
the presence of secreted PRL. The cells were lysed in RIPA buffer (1X
PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 µg/ml
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, 2 µg/ml pepstatin, 1 mM sodium orthovanadate)
on ice for 30 min. DNA was sheared by passing the sample through a
22-gauge needle and centrifuged at 15,000 rpm for 20 min. Protein
content in the supernatant was assayed by Bradford Protein Assay
(Bio-Rad Laboratories, Inc. Hercules, CA), and equal
amounts of protein were used for c-Src immunoprecipitations or were
loaded per lane and separated by a 10% SDS/PAGE gel (45). Proteins
were transferred to Immobilon polyvinylidene difluoride membrane
(Millipore Corp., Bedford, MA) and blocked overnight in
10% nonfat milk in PBST (PBS with 0.5% Tween-20). Membranes were
incubated with rabbit polyclonal anti-rat PRL antibodies (provided by
NIDDK- National Hormone and Pituitary Program, Torrance, CA) or cyclin
D3 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
antibodies at a dilution of 1:1000 for 2 h at room temperature in
PBST/5% milk. After washing, horseradish peroxidase-linked secondary
antibodies were added and target protein bands were detected using the
Renaissance enhanced chemiluminescence detection system (NEN Life Science Products, Boston, MA). Relative band intensities were
quantified using a Densitometer and Image-Quant software
(Molecular Dynamics, Inc.). For c-Src
immunoprecipitations, RIPA supernatants were precleared and treated as
previously described (46). Briefly, supernatants were precleared by
incubating with Protein A-conjugated agarose beads at 4 C for 1 h,
followed by centrifugation at 4 C for 1 min at 14,000 rpm. The
supernatants were then subjected to immunoprecipitation using 2 µg of
polyclonal anti-Src antibody (SC-18) (Santa Cruz Biotechnology, Inc.), or nonspecific IgG overnight at 4 C. This was followed by
an incubation with Protein A-agarose beads (Santa Cruz Biotechnology, Inc.) for 2 h at 4 C. After washing the
beads three times with ice-cold RIPA buffer, the bound proteins were
eluted with SDS-PAGE sample buffer and subjected to electrophoresis and
immunoblot analysis using a cocktail of antiphosphotyrosine antibodies
4G10 and PY-20 (Upstate Biotechnology, Inc., Lake Placid,
NY, and Transduction Laboratories, Inc., Lexington, KY,
respectively) or a monoclonal anti-c-Src antibody (clone GD11)
(Upstate Biotechnology, Inc.).
PRL mRNA Analysis
PR1 cell total RNA was isolated using Tri-Reagent
(Molecular Research Center, Inc., Cincinnati, OH). RNA (20
µg) was electrophoresed in 1.2% agarose-formaldehyde gels in
MOPS buffer (0.04 M MOPS, 0.01 M sodium
acetate, 1 mM EDTA, pH 7.0). Gels were rinsed in
diethylpyrocarbonate-treated water to remove residual
formaldehyde and transferred to Hybond-N nylon membrane (Amersham Pharmacia Biotech, Piscataway, NJ) by capillary transfer
overnight in 20x SSC (3 M sodium chloride, 0.3
M sodium citrate). Membranes were briefly washed in 6x SSC
after transfer and UV cross-linked using the UV Stratalinker 1800
(Stratagene, La Jolla, CA) and air dried. The mouse PRL
cDNA was excised from the pGEM2 plasmid (kindly provided by Michael
Karin, UCSD) using HindIII and EcoRI, and gel
purified. PRL cDNA probe was randomly primed using the Prime-It II
Random Primer Labeling kit (Stratagene) and
32P
-dATP. Incorporated probe was separated
from unincorporated nucleotides using Quick Spin G-50 Sephadex columns
(Roche Molecular Biochemicals, Indianapolis, IN).
Membranes were hybridized in QuikHyb hybridization solution
(Stratagene) containing 1 x
106 cpm/ml radiolabeled PRL cDNA probe for 4
h at 50 C. Membranes were washed once in 2x SSC/0.5% SDS for 15 min
at room temperature, once in 1x SSC/0.1%SDS for 15 min at room
temperature, and then once in 1x SSC/0.1%SDS for 20 min at 65 C.
Washed membranes were apposed to a phosphorimage screen and read by a
Phosphorimager (Molecular Dynamics, Inc., Sunnyvale,
CA).
ERK and c-Raf Kinase Activation
PR1 and GH3 cells were grown as above in 100-mm plates. They
were serum starved (phenol red-free DMEM with supplements) for 16
h before stimulation with steroids or growth factor. Plates were washed
twice with ice-cold PBS and twice with ice- cold harvesting buffer H
(50 mM ß-glycerophosphate, 1.5 mM EGTA, 0.1
mM sodium orthovanadate, 1 mM DTT, 10 µg/ml
aprotinin, 2 µg/ml pepstatin, 10 µg/ml leupeptin, 1 mM
benzamidine, and 0.2 mM phenylmethylsulfonyl fluoride).
Cells were scraped off the plate in 500 µl harvesting buffer H,
sonicated, and subjected to centrifugation at 100,000 x
g for 20 min. Supernatants were removed and assayed for
protein content as described above. Cytosolic extract was loaded (50
µg per lane) and subjected to 10% SDS-PAGE. Transfer was done as
described above. Membranes were blocked overnight at 4 C in 1%
BSA/TBST (200 mM Tris, 1.37
M NaCl, 1% Tween-20, pH 7.6). Phosphospecific
antibodies against ERKs-1 and -2 (Promega Corp.) were
diluted 1:5000 in 0.1% BSA/TBST and incubated at room temperature for
2 h. Blots were washed and incubated in horseradish
peroxidase-linked secondary antibody and visualized as described above.
Antiphosphotyrosine (340/341)-specific c-Raf-1 antibodies
(Biosource International, Camarillo, CA) were used at
1:5000. To control for equal protein loading, blots were stripped (in
100 mM 2mercaptoethanol, 2% SDS, 62.5
mM Tris, pH 6.7) at 50 C for 30 min, blocked at
room temperature for 30 min in 10% nonfat milk/PBST, and then reprobed
with an ERK-1 antibody (Santa Cruz Biotechnology, Inc.) at
1:5000 in 5% milk/PBST or with an anti-c-Raf-1 antibody in 5%
milk/TBST.
 |
ACKNOWLEDGMENTS
|
---|
We would like to thank Ms. Maria Boardman and Mr. Cris VanHout
for their technical assistance and Dr. Elaine Alarid for her helpful
comments and suggestions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Jack Gorski, Department of Biochemistry, 433 Babcock Drive, Madison, Wisconsin 53706. E-mail:
gorski{at}biochem.wisc.edu
This work was supported by NIH Grants HD-08192 and HH-07259 (J.G.) and
National Research Service Award Grant F32CA81733 (J.J.W.).
Received for publication December 20, 1999.
Revision received July 27, 2000.
Accepted for publication August 4, 2000.
 |
REFERENCES
|
---|
-
Maurer RA 1982 Estradiol regulates the transcription of
the prolactin gene. J Biol Chem 257:21332136[Abstract/Free Full Text]
-
Maurer RA, Erwin CR, Donelson JE 1981 Analysis of 5' flanking
sequences and intron-exon boundaries of the rat prolactin gene. J
Biol Chem 256:1052410528[Free Full Text]
-
Maurer RA 1985 Selective binding of the estradiol receptor to
a region at least one kilobase upstream from the rat prolactin gene.
DNA 4:19[Medline]
-
Maurer RA, Notides AC 1987 Identification of an
estrogen-responsive element from the 5'-flanking region of the rat
prolactin gene. Mol Cell Biol 7:42474254[Medline]
-
Waterman ML, Adler S, Nelson C, Greene GL, Evans RM,
Rosenfeld MG 1988 A single domain of the estrogen receptor confers
deoxyribonucleic acid binding and transcriptional activation of the rat
prolactin gene. Mol Endocrinol 2:1421[Abstract]
-
Day RN, Walder JA, Maurer RA 1989 A protein kinase inhibitor
gene reduces both basal and multihormone-stimulated prolactin gene
transcription. J Biol Chem 264:431436[Abstract/Free Full Text]
-
Day RN, Maurer RA 1989 Thyroid hormone-responsive elements of
the prolactin gene: evidence for both positive and negative regulation.
Mol Endocrinol 3:931938[Abstract]
-
Lannigan DA, Notides AC 1989 Estrogen receptor selectively
binds the "coding strand" of an estrogen responsive element. Proc
Natl Acad Sci USA 86:863867[Abstract]
-
Murdoch FE, Meier DA, Furlow JD, Grunwald KA, Gorski J 1990 Estrogen receptor binding to a DNA response element in vitro
is not dependent upon estradiol. J Biochem 29:83778385
-
Day RN, Maurer RA 1989 The distal enhancer region of the rat
prolactin gene contains elements conferring response to multiple
hormones. Mol Endocrinol 3(1):39
-
Day RN, Koike S, Sakai M, Muramatsu M, Maurer RA 1990 Both
Pit-1 and the estrogen receptor are required for estrogen
responsiveness of the rat prolactin gene. Mol Endocrinol 4:19641971[Abstract]
-
Howard PW, Maurer RA 1995 A composite Ets/Pit-1 binding site
in the prolactin gene can mediate transcriptional responses to multiple
signal transduction pathways. J Biol Chem 270:2093020936[Abstract/Free Full Text]
-
Nowakowski BE, Maurer RA 1994 Multiple Pit-1-binding sites
facilitate estrogen responsiveness of the prolactin gene. Mol
Endocrinol 8:17421749[Abstract]
-
Tsukahara S, Kambe F, Suganuma N, Tomoda Y, Seo H 1994 Increase in Pit-1 mRNA is not required for the estrogen-induced
expression of prolactin gene and lactotroph proliferation. Endocr J 41:579584[Medline]
-
Fischberg DJ, Chen XH, Bancroft C 1994 A Pit-1 phosphorylation
mutant can mediate both basal and induced prolactin and growth hormone
promoter activity [see comments]. Mol Endocrinol 8:15661573[Abstract]
-
Migliaccio A, Di Domenico M, Castoria G, de Falco A, Bontempo
P, Nola E, Auricchio F 1996 Tyrosine kinase/p21ras/MAP-kinase pathway
activation by estradiolreceptor complex in MCF-7 cells. EMBO J 15:12921300[Abstract]
-
Migliaccio A, Piccolo D, Castoria G, Di Domenico M, Bilancio
A, Lombardi M, Gong W, Beato M, Auricchio F 1998 Activation of the
Src/p21ras/Erk pathway by progesterone receptor via cross-talk with
estrogen receptor. EMBO J 17:20082018[Free Full Text]
-
Castoria G, Barone MV, Di Domenico M, Bilancio A, Ametrano D,
Migliaccio A, Auricchio F 1999 Non-transcriptional action of oestradiol
and progestin triggers DNA synthesis. EMBO J 18:25002510[Abstract/Free Full Text]
-
Nuedling S, Kahlert S, Loebbert K, Meyer R, Vetter H, Grohe C 1999 Differential effects of 17ß-estradiol on mitogen-activated
protein kinase pathways in rat cardiomyocytes. FEBS Lett 454:271276[CrossRef][Medline]
-
Endoh H, Sasaki H, Maruyama K, Takeyama K, Waga I, Shimizu T,
Kato S, Kawashima H 1997 Rapid activation of MAP kinase by estrogen in
the bone cell line. Biochem Biophys Res Commun 235:99102[CrossRef][Medline]
-
Di Domenico M, Castoria G, Bilancio A, Migliaccio A, Auricchio
F 1996 Estradiol activation of human colon carcinoma-derived Caco-2
cell growth. Cancer Res 56:45164521[Abstract]
-
Chun TY, Gregg D, Sarkar DK, Gorski J 1998 Differential
regulation by estrogens of growth and prolactin synthesis in pituitary
cells suggests that only a small pool of estrogen receptors is required
for growth. Proc Natl Acad Sci USA 95:23252330[Abstract/Free Full Text]
-
Sarkar DK, Pastorcic M, De A, Engel M, Moses H, Ghasemzadeh MB 1998 Role of transforming growth factor (TGF)-ß type I and TGF-ß
type II receptors in the TGF-ß1-regulated gene expression in
pituitary prolactin-secreting lactotropes. Endocrinology 139:36203628[Abstract/Free Full Text]
-
Fabian JR, Daar IO, Morrison DK 1993 Critical tyrosine
residues regulate the enzymatic and biological activity of Raf-1
kinase. Mol Cell Biol 13:71707179[Abstract]
-
Altucci L, Addeo R, Cicatiello L, Dauvois S, Parker MG, Truss
M, Beato M, Sica V, Bresciani F, Weisz A 1996 17ß-Estradiol induces
cyclin D1 gene transcription, p36D1p34cdk4 complex activation, and
p105Rb phosphorylation during mitogenic stimulation of G(1)-arrested
human breast cancer cells. Oncogene 12:23152324[Medline]
-
Geum D, Sun W, Paik SK, Lee CC, Kim K 1997 Estrogen-induced
cyclin D1 and D3 gene expressions during mouse uterine cell
proliferation in vivo: differential induction mechanism of
cyclin D1 and D3. Mol Reprod Dev 46:450458[CrossRef][Medline]
-
Migliaccio A, Pagano M, Auricchio F 1993 Immediate and
transient stimulation of protein tyrosine phosphorylation by estradiol
in MCF-7 cells. Oncogene 8:21832191[Medline]
-
Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green
D, McNulty D, Blumenthal MJ, Heys JR, Landvatter SW, et al. 1994 A protein kinase involved in the regulation of inflammatory
cytokine biosynthesis. Nature 372:739746[CrossRef][Medline]
-
Biscardi JS, Tice DA, Parsons SJ 1999 c-Src, receptor tyrosine
kinases, and human cancer. Adv Cancer Res 76:61119[Medline]
-
Abram CL, Courtneidge SA 2000 Src family tyrosine kinases and
growth factor signaling. Exp Cell Res 254:113[CrossRef][Medline]
-
Shull JD, Walent JH, Gorski J 1987 Estradiol stimulates
prolactin gene transcription in primary cultures of rat anterior
pituitary cells. J Steroid Biochem 26:451456[CrossRef][Medline]
-
Shull JD, Gorski J 1985 Estrogen regulates the
transcription of the rat prolactin gene in vivo through at
least two independent mechanisms. Endocrinology 116:24562462[Abstract]
-
Shull JD, Gorski J 1984 Estrogen stimulates prolactin gene
transcription by a mechanism independent of pituitary protein
synthesis. Endocrinology 114:15501557[Abstract]
-
Berthois Y, Pourreau-Schneider N, Gandilhon P, Mittre H,
Tubiana N, Martin PM 1986 Estradiol membrane binding sites on human
breast cancer cell lines. Use of a fluorescent estradiol conjugate to
demonstrate plasma membrane binding systems. J Steroid Biochem 25:963972[CrossRef][Medline]
-
Pappas TC, Gametchu B, Watson CS 1995 Membrane estrogen
receptors identified by multiple antibody labeling and impeded-ligand
binding. FASEB J 9:404410[Abstract/Free Full Text]
-
Watters JJ, Campbell JS, Cunningham MJ, Krebs EG, Dorsa DM 1997 Rapid membrane effects of steroids in neuroblastoma cells: effects
of estrogen on mitogen activated protein kinase signalling cascade and
c-fos immediate early gene transcription. Endocrinology 138:40304033[Abstract/Free Full Text]
-
Razandi M, Pedram A, Greene GL, Levin ER 1999 Cell membrane
and nuclear estrogen receptors (ERs) originate from a single
transcript: studies of ER
and ERß expressed in Chinese hamster
ovary cells. Mol Endocrinol 13:307319[Abstract/Free Full Text]
-
Norfleet AM, Thomas ML, Gametchu B, Watson CS 1999 Estrogen
receptor-
detected on the plasma membrane of aldehyde-fixed
GH3/B6/F10 rat pituitary tumor cells by enzyme-linked
immunocytochemistry. Endocrinology 140:38053814[Abstract/Free Full Text]
-
Watson CS, Norfleet AM, Pappas TC, Gametchu B 1999 Rapid
actions of estrogens in GH3/B6 pituitary tumor cells via a plasma
membrane version of estrogen receptor-
. Steroids 64:513[CrossRef][Medline]
-
Ying C, Lin DH, Sarkar DK, Chen TT 1999 Interaction between
estrogen receptor and Pit-1 protein is influenced by estrogen in
pituitary cells. J Steroid Biochem Mol Biol 68:145152[CrossRef][Medline]
-
Joel PB, Traish AM, Lannigan DA 1998 Estradiol-induced
phosphorylation of serine 118 in the estrogen receptor is independent
of p42/p44 mitogen-activated protein kinase. J Biol Chem 273:1331713323[Abstract/Free Full Text]
-
Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H,
Masushige S, Gotoh Y, Nishida E, Kawashima H, Metzger D, Chambon
P 1995 Activation of the estrogen receptor through
phosphorylation by mitogen-activated protein kinase. Science 270:14911494[Abstract]
-
Endoh H, Maruyama K, Masuhiro Y, Kobayashi Y, Goto M, Tai H,
Yanagisawa J, Metzger D, Hashimoto S, Kato S 1999 Purification and
identification of p68 RNA helicase acting as a transcriptional
coactivator specific for the activation function 1 of human estrogen
receptor
. Mol Cell Biol 19:53635372[Abstract/Free Full Text]
-
Horwitz KB, Costlow ME, McGuire WL 1975 MCF-7: a human breast
cancer cell line with estrogen, androgen, progesterone, and
glucocorticoid receptors. Steroids 26:785795[CrossRef][Medline]
-
Laemmli UK 1970 Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227:680685[Medline]
-
Kim Y-N, Wiepz GJ, Guadarrama AG, Bertics PJ 2000 Epidermal
growth factor-stimulated tyrosine phosphorylation of caveolin-1. J
Biol Chem 275:74817491[Abstract/Free Full Text]