From the Departments of Biological Regulation and
¶ Molecular Cell Biology, the Weizmann Institute of Science,
Rehovot 71600, Israel,
Biochemie-Zentrum Heidelberg, University
of Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany, and
Center for research on Reproduction and
Women's Health, University of Pennsylvania,
Philadelphia, Pennsylvania 19104
Received for publication, July 31, 2000, and in revised form, January 10, 2001
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ABSTRACT |
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The response of granulosa cells to
luteinizing hormone (LH) and follicle-stimulating hormone (FSH)
is mediated mainly by cAMP/protein kinase A (PKA) signaling. Notably,
the activity of the extracellular signal-regulated kinase (ERK)
signaling cascade is elevated in response to these stimuli as well. We
studied the involvement of the ERK cascade in LH- and FSH-induced
steroidogenesis in two granulosa-derived cell lines, rLHR-4 and
rFSHR-17, respectively. We found that stimulation of these cells with
the appropriate gonadotropin induced ERK activation as well as
progesterone production downstream of PKA. Inhibition of ERK activity
enhanced gonadotropin-stimulated progesterone production, which was
correlated with increased expression of the steroidogenic acute
regulatory protein (StAR), a key regulator of progesterone synthesis.
Therefore, it is likely that gonadotropin-stimulated progesterone
formation is regulated by a pathway that includes PKA and StAR, and
this process is down-regulated by ERK, due to attenuation of StAR
expression. Our results suggest that activation of PKA signaling by
gonadotropins not only induces steroidogenesis but also activates
down-regulation machinery involving the ERK cascade. The activation of
ERK by gonadotropins as well as by other agents may be a key mechanism
for the modulation of gonadotropin-induced steroidogenesis.
Gonadotropic hormones, follicle-stimulating hormone
(FSH)1 and luteinizing
hormone (LH), which are released from the pituitary, play a crucial
role in controlling reproductive function in males and females. The
pleotropic effects of gonadotropins are manifested in various cells of
the reproductive system including LH and FSH in ovarian granulosa
cells, LH in theca interna cells, FSH in testicular Sertoli cells, and
LH in Leydig cells (1-3). One of the main effects of both LH and FSH
on the ovary is the stimulation of the production of estradiol and
progesterone, which play important roles in ovarian function and
control of the reproductive cycle (reviewed in Ref. 4). The mechanisms
involved in the regulation of progesterone production by ovarian
granulosa cells have been characterized in detail. Gonadotropins exert
their stimulatory activity via interaction with specific
seven-transmembrane receptors, the LH receptor and FSH receptor. Upon
binding of the gonadotropins, both receptors stimulate the
Gs protein, which activates the membrane-associated adenylyl cyclase, causing an elevation of intracellular cAMP (5). This
cyclic nucleotide serves as a second messenger for the up-regulation of
the steroidogenic acute regulatory protein (StAR) and the cytochrome P450 (P450scc) enzyme system (reviewed in Refs. 6 and 7).
Activation of alternative signaling pathways by the gonadotropin
receptors was described in the last decade, including calcium ion
mobilization, activation of the phosphoinositol pathway, and stimulation of chloride ion influx (reviewed in Ref. 8). However, these
gonadotropin-induced signaling processes were not previously implicated
in the modulation of steroidogenesis (5). Another process that plays an
important role in inhibiting gonadotropin-induced steroidogenesis is
the desensitization of the gonadotropin receptor (3). G-protein-coupled
receptor kinase phosphorylation of the gonadotropin receptors, the
adaptor protein arrestin, and massive internalization of the receptors
are thought to play a role in the down-regulation of gonadotropin
signaling. However, since desensitization precedes the internalization
of the gonadotropin receptor (9), additional mechanisms are likely to
participate in the rapid attenuation of gonadotropin signals downstream
of the receptors.
The extracellular signal-regulated kinases (ERKs) include three kinases
(p42ERK2, p44ERK1, p46ERK1b) that belong to the family of the signaling
mitogen-activated protein kinases (MAPKs). Upon extracellular
stimulation, the ERKs are activated by a network of interacting
proteins, which funnel the signals into a multitier kinase cascade
(reviewed in Refs. 10 and 11). The activated ERKs in turn regulate
additional signaling kinases (e.g. RSK) or can by themselves
phosphorylate and activate target regulatory proteins (e.g.
Elk1) that govern various cellular processes. Although the ERKs were
first implicated in the regulation of proliferation and
differentiation, it is presently known that these kinases participate
also in the control of cellular morphology, learning and memory in the
central nervous system, apoptosis, and carcinogenesis (11).
It has previously been shown that ovarian granulosa cell ERK is
activated (2-5-fold) in response to LH and FSH (12, 13). These effects
were mimicked by elevation of intracellular cAMP, and the FSH effect
was inhibited by inhibitors of PKA, indicating that ERK transduces
signals downstream of PKA in gonadotropin-induced granulosa cells. In
the present work, we show that gonadotropins induce ERK activation and
progesterone production via cAMP in immortalized granulosa cell lines.
These cell lines are homogeneous populations, unlike follicular
granulosa cells, which represent a heterogeneous population with
respect to LH receptor content and the degree of maturation (14).
Interestingly, inhibition of ERK activation causes an elevation in
gonadotropin-cAMP-induced progesterone production, while activation of
ERK inhibits this process. Moreover, the addition of a MEK inhibitor
elevated the intracellular content of StAR, which operates downstream
of cAMP, suggesting that the inhibitory effect of the ERK on
steroidogenesis may be mediated by the reduction in the expression of
StAR. Therefore, it is likely that gonadotropin-induced progesterone
formation is regulated by PKA, which induces not only the expression of StAR but also a counteracting down-regulating mechanism. These two
mechanisms are simultaneously brought into play by the activation of
ERK, which reduces StAR expression.
Stimulants, Inhibitors, Antibodies, and Other
Reagents--
Human FSH, human LH, and human chorionic
gonadotropin (hCG) were kindly provided by the National Institutes of
Health and Dr. Parlow. Deglycosylated hCG was enzymatically prepared as
previously described (15). Mouse monoclonal anti-diphospho-ERK
(anti-active ERK/MAPK) antibodies (DP-ERK Ab) and anti-general ERK
antibody were obtained from Sigma, Israel (Rehovot, Israel). Anti
C-terminal ERK1 antibody (C16) was purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Polyclonal antibodies to human
StAR were raised in rabbits (16). Alkaline phosphatase-,
horseradish peroxidase-, and flourescein-conjugated secondary
antibodies were purchased from Jackson ImmunoResearch Laboratories Inc.
(West Grove, PA). PD98059 and U0126 were purchased from Calbiochem.
H89, forskolin, and 8-Br-cAMP were obtained from Sigma.
Cell Lines--
rLHR-4 cell line was established by
cotransfection of rat preovulatory granulosa cells with mutated p53
(Val135-p53), Ha-ras genes and plasmid
expressing the rat LH/CG receptor (17). The rFSHR-17 cell line was
established by immortalization of preovulatory rat granulosa cells via
cotransfection of primary cells with SV40 DNA and an HA-ras
gene. Cells were transfected with plasmid expressing the rat FSH
receptor (18). The cells were maintained in F-12/DMEM medium (1:1)
containing 5% fetal calf serum.
Stimulation and Harvesting of Cells--
Subconfluent cultures
were serum-starved for 16 h and subsequently incubated for
selected time intervals with the indicated agents in the presence or
absence of various inhibitors. Following stimulation, cells were washed
twice with ice-cold phosphate-buffered saline and once with buffer A
(50 mM Transfection of PKI and ERK Plasmids into Cells--
The rLHR-4
and rFSHR-17 cells were grown in DMEM supplemented with 10% fetal calf
serum up to 70% confluency. The plasmids used were RSV-PKI and
RSV-PKImutant (20) (a generous gift from Dr. R. A. Maurer; Oregon Health Sciences University, Portland, OR) and pGFP-ERK2
(21). The plasmids were introduced into the two cell types using
LipofectAMINE (Life Technologies, Inc.) according to the
manufacturer's instruction. About 15-20% transfection was observed
in the two cell lines using a Zeiss fluorescent microscope. After
transfection, the rLHR-4 cells were grown in DMEM plus 10% fetal calf
serum for 6 h and then starved in DMEM plus 0.1% fetal calf serum
for an additional 14 h. The rFSHR-17 cells were grown in DMEM plus
10% fetal calf serum for 20 h. The transfected cells were then
stimulated and harvested as above.
Western Blot Analysis--
Cell supernatants, which contained
cytosolic proteins were collected, and aliquots from each sample (30 µg) were separated on 10% SDS-polyacrylamide gel electrophoresis
followed by Western blotting with the appropriate antibodies.
Alternatively, immunoprecipitated proteins were boiled in sample buffer
and subjected to SDS-polyacrylamide gel electrophoresis and Western
blotting. The blots were developed with alkaline phosphatase or
horseradish peroxidase-conjugated anti-mouse or anti-rabbit Fab antibodies.
Determination of ERK Activity by Phosphorylation--
Cell
supernatants (200 µg of proteins) were subjected to
immunoprecipitation with monoclonal anti-ERK C-terminal antibodies (C16; Santa Cruz Biotechnology) as described above. During the final
step of immunoprecipitation, pellets were washed with buffer A,
resuspended in 15 µl of buffer A, and incubated (20 min, 30 °C)
with 5 µl of 2 mg/ml myelin basic protein (MBP) and 10 µl of 3×
reaction mix (30 mM MgCl2, 4.5 mM
dithiothreitol, 75 mM Progesterone Assay--
Progesterone secreted into the culture
medium was assayed by radioimmunoassay as previously described
(22).
Localization of StAR by Immunofluorescence--
Cells were
cultured on 24 × 24-mm cover glasses placed in 35-mm plastic
tissue culture dishes. Cells were fixed with 3% paraformaldehyde subsequent to 24-h incubation at 37 °C with the appropriate
stimulants and visualized in a Zeiss fluorescent microscope following
incubation with a 1:200 dilution of antiserum to human StAR and goat
anti-rabbit antibodies conjugated to fluorescein. For negative
controls, cells were incubated with nonimmune rabbit serum followed by
the second antibodies.
Stimulation of granulosa cells with the gonadotropin LH or FSH
induces several cellular processes, including de novo
synthesis of steroid hormones. To study the signaling pathways that
couple gonadotropin receptors to the regulation of progesterone
production, we used two distinct granulosa cell lines expressing either
LH/CG or FSH receptors: rLHR-4 and rFSHR-17. The addition of the
appropriate gonadotropins to these cells has previously been shown to
stimulate cAMP production, activation of PKA, and induction of
steroidogenesis (Ref. 18 and data not shown). Since the ERK cascade was
implicated in the signaling of G protein-coupled receptors (23), we
first examined whether the ERK cascade is also activated in the rLHR-4 and rFSHR-17 cell lines.
Activation of ERK by hCG, Deglycosylated hCG (dghCG), and cAMP in
rLHR-4 Cells--
Serum-starved rLHR-4 cells were stimulated with hCG,
which signals via the LH receptor (3), and phosphorylation of the activation TEY motif of ERK was then assessed using a Western blot
analysis with DP-ERK Ab (24). Considerable staining of three bands at
42, 44, and 46 kDa (ERK2, ERK1, and ERK1b respectively (19)) was
detected in the resting, nonstimulated cells. The intensity of staining
of ERK2 and ERK1 was enhanced (~5-fold) 5-20 min after the addition
of hCG and remained high (~3-fold) up to 60 min after stimulation.
The appearance of p46 ERK1b is of particular interest, because although
ERK1b has been reported to exist in rat and human (19), its abundance
and relative activity as compared with that of ERK1 and ERK2 are
usually small. Interestingly, the basal activity of ERK1b in rLHR-4
cells was as high as that of ERK1, only modestly increased 5-20 min
after stimulation (~2-fold), and it declined back to basal level 40 min later. The kinetics of activation, which are different from those
of ERK1 and ERK2, suggests a differential mode of ERK1b regulation as
recently demonstrated in EJ cells (19).
We next examined LH, which, like hCG, specifically acts via the LH/CG
receptors. The effect of LH on ERK activity was essentially the same as
that of hCG under all conditions examined (data not shown).
dghCG, which has previously been reported to maintain the same
affinity for binding to the LH receptor as the intact hormone but
retains only a residual activity for stimulation of steroidogenesis
(25), also caused activation of ERK. However, this activation was
significantly lower than that achieved by the intact hormone
(2.5-fold activation 20 min after dghCG treatment as compared
with 4.5-fold 20 min after hCG treatment (Fig.
1)). Since LH and hCG have previously
shown to transmit their signal via Gs and cAMP (25), we
examined the role of cAMP-elevating agents on the ERK activity. Indeed,
both 8-Br-cAMP, and forskolin, which activate adenylyl cyclase,
significantly activated ERK phosphorylation in the rLHR-4 cells (data
not shown), indicating that the hCG-induced ERK activation may be
dependent on elevation of intracellular cAMP.
Besides ERK phosphorylation of the TEY motif, which mainly reflects MEK
activity, we also measured the activity of ERK itself. This was
performed by immunoprecipitation with anti C-terminal ERK1 antibody
followed by phosphorylation of the general substrate, MBP (19). As
expected, this method revealed that the activity of ERK correlated well
with the regulatory phosphorylation of ERK (Fig. 1, bottom
two panels), verifying that both hCG and dghCG cause a 4-5-fold activation of ERK1 activity in rLHR-4 cells. The
addition of the MEK inhibitor, PD98059, reduced both hCG-stimulated and
-nonstimulated activity of ERK to below basal levels, and a similar
reduction was observed for dghCG-, forskolin-, and 8-Br-cAMP-stimulated activity of ERK (Fig. 1 and data not shown). None of the treatments caused any significant change in the total amount of the ERKs as judged
by staining with an anti-general ERK antibody (7884), which recognizes
both ERK1 and ERK2 much better than ERK1b (Fig. 1;
G-ERK).
Activation of ERK by FSH and cAMP in rFSHR-17 Cells--
We tested
the ability of FSH to stimulate ERK activity in the rat
granulosa-derived cell line, rFSHR-17. Similarly to the rLHR-4 line,
there was considerable staining of all three ERK isoforms, ERK2, ERK1,
and ERK1b, in Western blots from extracts of serum-starved cells. This
staining was enhanced by the addition of FSH to the cells, in kinetics
that were slightly slower than the kinetics of hCG stimulation in
rLHR-4 cells (Fig. 2, upper lanes). The staining of the three ERK isoforms was enhanced
5 min after FSH stimulation, peaked (5-fold above basal level) at 20 min after stimulation, and slightly decreased at 60 min. Also in these
cells, the cAMP-stimulating agents, forskolin and 8-Br-cAMP, enhanced
the phosphorylation of the three ERK isoforms (3- and 5-fold above
basal level, respectively). None of the treatments caused any change in
the amount of the ERK isoforms as judged by the staining with a general
anti-ERK antibody, confirming that as for the hCG experiment, the
changes detected by the DP-ERK Ab are indeed due to changes in ERK
phosphorylation and not due to induction of ERK expression. In
addition, we examined ERK activity by immunoprecipitation and
phosphorylation of MBP. We found (Fig. 2, bottom) that not
only ERK phosphorylation but also ERK activity was stimulated by
FSH, forskolin and 8-Br-cAMP and was attenuated by PD98059,
confirming that both LH and FSH receptors can transmit signals to the
ERK pathway in the examined cell lines.
PD98059 Stimulates FSH and hCG-induced Steroidogenesis--
One of
the important cellular processes that is stimulated by gonadotropins in
granulosa cells is stereoidogenesis (26). Indeed, a significant
increase in progesterone production was observed 24 and 48 h after
LH stimulation of rLHR-4 cell line (Fig.
3A). hCG had a similar effect
to that of LH (data not shown), while dghCG had a very small effect,
and forskolin caused a 2-fold greater induction of progesterone
production than LH. To examine whether the activated MAPK cascade is
also involved in the induction of progesterone production, we incubated
the rLHR-4 cells with the MEK inhibitor, PD98059. This inhibitor had no
effect by itself on progesterone production by rLHR-4 cells. However,
when the cells were incubated with PD98059 for 15 min prior to LH
induction, there was a 3-fold increase in LH-induced progesterone
production (Fig. 3), under conditions where ERK activity was completely
abolished (Fig. 1). A similar stimulatory effect on progesterone
production was observed when the MEK inhibitor was added prior to
stimulation of the cells with forskolin (Fig. 3), hCG, and 8-Br-cAMP
(data not shown). Similar to the rLHR-4 cells, MEK inhibitor
significantly increased steroidogenesis in rFSHR-17 cells. Thus, in
these cells, FSH and forskolin caused a significant elevation of
progesterone production after 24 and 48 h, which was dramatically
amplified by the addition of PD98059. In contrast to the induction by
the MEK inhibitor, TPA, which is a known activator of the ERK cascade (27), had a negative effect on the forskolin-induced production of
progesterone in both cell lines after 24 and 48 h. Taken together, these results suggest that the ERK signaling cascade suppresses gonadotropin-stimulated progesterone production.
MEK Inhibitors Stimulate Expression of StAR--
StAR plays
a crucial role in the regulation of cholesterol transport from the
outer to the inner mitochondrial membrane, where cytochrome P450scc
participates as a rate-limiting enzyme in steroidogenesis, converting
cholesterol into pregnenolone (7). The induction of StAR and its
downstream effects are likely to be cAMP-dependent processes as reported for gonadotropin-induced steroidogenesis in the
gonads and ACTH-stimulated steroidogenesis in the fasciculata cells of
the adrenal (7). Moreover, since StAR is known to have a short
functional half-life (28), we studied whether down-regulation of StAR
may explain the effect of the ERK cascade on progesterone production.
Thus, rLHR-4 cells were treated with the various agents described above
and examined for the expression of StAR 24 h after stimulation. As
expected, LH, hCG, forskolin, and to a considerably lesser extent
dghCG, induced the expression of StAR under the conditions examined
(Fig. 4). PD98059 alone caused an
induction of StAR by itself, but when the cells where preincubated with this MEK inhibitor prior to the addition of forskolin, LH, and hCG,
there was a synergistic elevation in the production of StAR. Similar
results were obtained also in the rFSHR-17 cells, where PD98059
dramatically increased the forskolin- and FSH-induced expression of
StAR. Thus, the ERK cascade may negatively regulate steroidogenesis,
and this can be explained by the attenuation of StAR expression, which
may be the regulatory component that integrates the signals from both
the cAMP and the ERK pathway to regulate the rate of
steroidogenesis.
To further verify the results obtained with PD98059, we used an
additional specific MEK inhibitor, the U0126 (29). As observed with the
PD98059, the addition of this inhibitor to both rLHR-4 and rFSHR-17
cells caused an elevation in the amount of 30-kDa mature StAR (30)
within 24 h (Fig. 5). The addition
of the gonadotropins alone also elevated this expression, but when the
inhibitor was added together with the appropriate gonadotropins, the
expression of StAR was significantly higher and reached up to 10-fold
above basal expression levels. This was significantly higher compared with the amounts expected from the expression induced by U0126 and
gonadotropin alone. Interestingly, in some of the experiments, a 37-kDa
pre-StAR (30) was detected by the anti-StAR antibody (Fig.
5A). This cytosolic protein is known to be maintained in a
low steady state level, because it rapidly matures into the 30-kDa form
of StAR in the mitochondria (30). Unlike the 30-kDa StAR, the
relatively low amount of this protein did not change upon the addition
of LH, FSH, or MEK inhibitors (Fig. 5). We then studied the
effect of U0126 on steroidogenesis in the rLHR-4 and the rFSHR-17
cells. Similar to the results of PD98059, U0126 did not induce
steroidogenesis by itself but synergized with the gonadotropins to
produce high amounts of progesterone (Fig. 5).
Taken together, our results indicate that MEK inhibitors dramatically
increase gonadotropin-induced StAR expression and steroidogenesis. However, the MEK inhibitors themselves induced clear elevation of StAR
expression without corresponding elevation in progesterone production.
This is probably due to the fact that in the immortalized granulosa
cell lines no basal levels of the cytochrome p450scc, the activity of
which is obligatory for the conversion of cholesterol to pregnenolone,
can be detected (31). This notion is supported by our preliminary
findings that in primary rat granulosa cells obtained from preovulatory
follicles and containing p450scc, PD98059 by itself increased
progesterone production. On the other hand, MEK inhibitors do synergize
with gonadotropin/cAMP stimulation of stereoidogenesis because of the
de novo synthesis of the cytochrome p450scc, which is
stimulated by gonadotropin/cAMP in the granulosa cell lines (17,
31).
Subcellular Localization of the Overexpressed StAR--
To examine
whether the enhancement of StAR expression by PD98059, gonadotropins,
and cAMP-elevating agents is mainly located in mitochondria (32), we
stained rFSHR-17 cells with anti-StAR antibodies prior to or following
PD98059, FSH, and forskolin stimulation (Fig.
6). In nonstimulated cells, StAR could
not be detected in mitochondria (a). In contrast, clear
elevation in mitochondrial StAR was evident following 24 h of
treatment with PD98059 (b). LH clearly increased the StAR
content in the mitochondria (c), while PD98059 dramatically
increased mitochondrial StAR content (d). Forskolin
augmented StAR levels (e), while PD98059 further enhanced
StAR content in the mitochondria (f). Thus, the
immunocytochemical observations confirmed the data obtained by Western
blot on the elevation of StAR expression by PD98059.
Gonadotropin-induced ERK Activation and StAR Production Are
Mediated by PKA--
Although we showed that an elevation of cAMP is
sufficient to activate ERK, it was not clear whether cAMP and PKA are
the major mediators of the gonadotropin-generated signaling to ERK. Therefore, we used H89, which is a potent and selective inhibitor of
PKA to study the involvement of cAMP/PKA in the activation of ERK in
rLHR-4 and rFSHR-17 cells. The addition of 3 µM H89 15 min prior to gonadotropin stimulation did not change the basal activity
of the three ERKs but completely abrogated the induction of ERK by hCG
in rLHR-4 cells and by FSH in rFSHR-17 cells (Fig. 7). As expected, ERK activation by
forskolin and 8-Br-cAMP in both cell lines was also inhibited by H89
(data not shown), indicating that ERK activation is mediated mainly by
PKA and probably not via cAMP-dependent guanine nucleotide exchange
factor (33).
To further verify the involvement of PKA in the activation of ERK by
gonadotropins, we coexpressed GFP-ERK2 (21) together with the potent
PKA inhibitor PKI or its inactive mutant (PKImutant) (20).
Specific activation of ERK in the transfected cells was measured by the
incorporation of phosphate into the activation loop of the GFP-ERK2
with anti-diphosphorylated ERK antibodies. As observed with H89,
inhibition of PKA with PKI significantly inhibited ERK activation by
gonadotropins and by forskolin (Fig. 8).
Taken together, these results clearly indicate that the activation of
ERK by gonadotropin in the cell lines examined is mostly
PKA-dependent.
We then examined whether StAR activation is mediated by PKA alone.
Indeed, when the PKA inhibitor H89 was added to rLHR-4 and rFSHR-17
cells, it significantly inhibited hCG- and FSH- stimulated StAR
expression (Fig. 9). As expected, H89
also attenuated forskolin-induced StAR expression (Fig. 9), indicating
that StAR production is regulated by PKA in the cell lines examined. As
expected, progesterone production was also significantly inhibited by
the H89 inhibitor (data not shown), indicating that the processes
examined may function mainly downstream of PKA. However, progesterone
production most probably lies downstream of PKA and of StAR, whereas
ERK, although activated by PKA, serves as a negative regulator of this
pathway due to its suppression of StAR (Fig.
10).
In this paper, we demonstrate a mechanism for cross-talk between
two signaling pathways, the cAMP/PKA and the ERK cascade in a
Gs-induced system. The interaction between these two
cascades has been extensively studied in several cellular systems over the past few years (34). In many systems, such as in epidermal growth
factor-stimulated Rat1 fibroblasts (35) or platelet-derived growth
factor-stimulated human arterial smooth muscle cells (36), it was shown
that cAMP inhibits the activation of the ERK cascade. This inhibition
seems to occur by either inhibitory phosphorylation of Raf-1 (35) or by
activation of the small GTPase, Rap-1, which competes with Ras for the
activation of Raf-1 (37). In other cell systems such as nerve growth
factor-stimulated PC12 cells, cAMP not only does not inhibit the ERK
cascade but in fact activates it to induce various mitogenic or
differentiation processes. One mechanism that activates the ERK cascade
by PKA includes the activation of the cAMP-responsive guanine
nucleotide exchange factors for the small GTPase Rap1, Epac1 and Epac2.
Upon binding of cAMP, these components rapidly activate Rap1, which
then promotes the activation of B-Raf (but not Raf-1) and the rest of
the ERK cascade (33). However, in the rLHR-4 and rFSHR-17 cells used in
our study, the activation of ERK seems to be downstream of PKA,
indicating that the Epac factors are probably not involved in
the ERK activation. This pathway may then involve an activation of the
Rap-1 GTPase by PKA, which causes the tight association with B-Raf and
induction of the ERK cascade. Recently, it was shown that activation of ERK by cAMP in the brain might occur via a cAMP-responsive STE-20-like kinase, MST3b (38). Although this specific isoform does not seem to be
expressed in granulosa cells, it is possible that another MAP4K or
MAP3K is involved in the transmission of PKA signals to ERK in the
rLHR-4 and rFSHR-17 cells.
The involvement of PKA in gonadotropin-dependent ERK
activation is demonstrated in the present work both by pharmacological means using PKA inhibitor H89 and by genetic means, i.e.
transfection of cells with plasmid encoding for PKI. The data using
both methods are in good agreement that PKA plays a major role in
transducing gonadotropin signaling toward ERK. Nevertheless, it should
be noted that although PKI completely suppressed forskolin-induced ERK
activation, it did not completely inhibit the gonadotropin-induced ERK
activation. Therefore, it is quite possible that the gonadotropin receptors are using other G proteins or the G Cooperation between the cAMP/PKA and the ERK pathways has been
demonstrated in several cells. For example, it was shown that cAMP
causes sustained activation of the ERK cascade, which is important for
neurite outgrowth in PC-12 cells (40). In human cyst epithelial cells,
elevation of cAMP causes a mitogenic response that is mediated
primarily by the ERK cascade (41). However, it was also shown that
cAMP-induced processes might contribute to a late down-regulation of
ERK-mediated processes. An example of this interaction of the
PKA-induced CPG16 kinase, which seems to partially inhibit the activity
of the transcription factor CREB (42), suggests its involvement in the
down-regulation of cAMP- and ERK cascade-induced transcription. In
contrast to this type of interaction, we show here that the activation
of processes downstream of PKA may also be inhibited by an ERK-mediated mechanism.
The inhibition of cAMP-induced progesterone production could occur at
the level of phosphorylation-dephosphorylation of proteins that play a
role in the steroidogenic pathway. In the present study, we examined
the expression of StAR, which is known to be phosphorylated on serine
or threonine residues (43). Although StAR phosphorylation may play a
role under distinct circumstances, it did not seem to correlate with
the induction of PKA or ERK cascades, and we could not detect any
direct phosphorylation of StAR by ERK (data not shown). However, we did
observe an inverse correlation between ERK activity and StAR expression
in the mitochondria. The blockade of ERK activity caused an elevation
in the amount of StAR, while activation of ERK by TPA reduced StAR
expression in granulosa cells. Therefore, it is probable that the two
cascades interact to regulate StAR gene transcription, the primary
mechanism for regulating StAR expression in granulosa cells (44).
Several transcription factors including steroidogenic factor-1,
CCAAT/enhancer-binding protein, and the negative regulator DAX-1
(45-47) participate in the transcriptional regulation of this gene.
StAR gene transcription is probably driven by the steroidogenic factor-1 and CCAAT/enhancer-binding protein downstream of PKA, but it
is unlikely that these components participate in the down-regulation of
StAR expression via the ERK cascade, because both have been shown to be
stimulated by ERK (48, 49). Therefore, it is possible that the negative
regulation of StAR expression occurs at the level DAX-1 or some yet to
be identified transcription factor. Alternatively, StAR expression
could be controlled by induction of potent phosphatases that abolish
both the PKA and ERK phosphorylation of steroidogenic factor-1 and
CCAAT/enhancer-binding protein or induce a proteolytic system that
reduces the half-life of the StAR.
Another explanation for the mechanism by which ERK can inhibit
steroidogenesis could be its involvement in desensitization of the
gonadotropin receptors. Prolonged incubation of granulosa cells with
gonadotropic hormones has previously been shown to cause
desensitization of the cells to further stimulation, which is
characterized by down-regulation of cAMP formation as well as of
steroidogenesis (9). Moreover, it has previously been demonstrated that
the ERK cascade could activate G-protein-coupled receptor kinase 2 (50), which in turn induces down-regulation of seven transmembrane
receptors. However, it is unlikely that this is the mechanism in our
case, because the inhibitory effects of ERK were demonstrated when
cells were stimulated by cAMP. Since this activator can bypass the
receptor to directly activate PKA, most of the inhibitory signals are
probably receptor-independent. Nevertheless, under physiological
conditions, the gonadotropins play a key role in modulation of ERK
activity. Moreover, activation of ERK can explain the mitogenic
signals exerted by FSH during folliculogenesis.
It is known that unlike the initiation of steroidogenesis, which is
proportional to the duration and extent of cAMP production, full
activation of ERK can be achieved as a consequence of even modest
increases in intracellular cAMP. This amplification occurs due to a
switch-like mechanism of the ERK cascade, which allows a strong
signaling output even by weak extracellular signals (51). The stronger
activity of ERK, which functions downstream of cAMP, may explain the
suppression of steroidogenesis upon weak gonadotropic signals, which
lead to steroidogenesis. Therefore, such a situation may explain the
low levels of steroidogenesis induced by dghCG, which is able to induce
only weak signals by the gonadotropic receptors.
In summary, the present study shows that activation of cAMP/PKA
signaling by gonadotropins not only induces steroidogenesis but also
activates down-regulation machinery that involves the ERK cascade. This
potent down-regulation machinery inhibits the gonadotropin-induced
steroidogenic pathway by mechanisms that are different from the well
characterized receptor desensitization mechanisms. Activation of the
ERK cascade downstream of PKA in turn regulates the level of StAR
expression, which is probably the key participant in these
down-regulation processes. Thus, PKA not only mediates
gonadotropin-induced steroidogenesis, it also activates the
down-regulation mechanism that can silence steroidogenesis under
certain conditions. Moreover, our findings raise the possibility that
activation or inhibition of ERK by other pathways could be an important
mechanism for diminution or amplification of gonadotropin-stimulated
steroidogenesis. This could contribute to functional luteolysis, a
process in which luteinized granulosa cells show reduced
sensitivity to LH despite maintenance of LH receptor or to
up-regulation of the steroidogenic machinery during luteinization of
granulosa cells (reviewed in Ref. 52).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerophosphate, pH 7.3, 1.5 mM
EGTA, 1 mM EDTA, 1 mM dithiothreitol, and 0.1 mM sodium vanadate (19)) and were subsequently harvested in
ice-cold buffer A plus proteinase inhibitors (19). Cell lysates were
centrifuged at 20,000 × g, for 20 min. The supernatant
was assayed for protein content and subjected to a Western blot
analysis or to immunoprecipitation as below. For the detection of
StAR, cells were lysed in radioimmune precipitation buffer (19)
and subjected to Western blot analysis.
-glycerophosphate, pH 7.3, 0.15 mM Na3VO4, 3.75 mM
EGTA, 30 µM calmidazolium, 2.5 mg/ml bovine serum
albumin, and 100 µM [
-32P]ATP (2 cpm/fmol)). The phosphorylation reactions were terminated by the
addition of sample buffer and boiling (5 min), and the samples were
analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography
as previously described (19).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (44K):
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Fig. 1.
Activation of ERK/MAPK by hCG and dghCG in
rLHR-4 cells. rLHR-4 cells were serum-starved for 16 h and
then stimulated with hCG (3 IU/ml) with or without PD98059
(PD; 15-min prestimulation, 25 µM), with
PD98059 (25 µM) alone, or with dghCG (3 IU/ml) for the
indicated times. Cytosolic extracts (50 µg) were subjected to
immunoblotting with DP-ERK Ab (upper panel) or
with anti-general ERK antibody (G-ERK; second
panel). Alternatively, the cytosolic extracts were subjected
to immunoprecipitation with anti-C-terminal ERK1 antibody (C16)
followed by in vitro phosphorylation of MBP as described
under "Experimental Procedures" (third panel,
Phospho.). The amount of immunoprecipitated ERK for the
phosphorylation reaction was determined by Western blotting with the
anti-general ERK antibody (bottom panel). The
positions of ERK2, ERK1, ERK1b, MBP, and IgG are indicated. Each of
these experiments was reproduced at least three times.
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Fig. 2.
Activation of ERK/MAPK by FSH/cAMP in
rFSHR-17 cells. rFSHR-17 cells were serum-starved for 16 h
and then stimulated with FSH (3 IU/ml) with forskolin (FK;
50 µM), with 8-Br-cAMP (Br-cAMP; 50 µM), with or without PD98059 (PD, 15-min
prestimulation, 25 µM), or with PD98059 (25 µM) alone for the indicated times. Cytosolic extracts (50 µg) were subjected to immunoblotting with DP-ERK Ab (upper
panel) or with anti-general ERK antibody (G-ERK,
second panel). Alternatively, the cytosolic
extracts were subjected to immunoprecipitation with anti C-terminal
ERK1 antibody (C16) followed by in vitro phosphorylation of
MBP as described under "Experimental Procedures"
(Phospho., third panel). The amount of
immunoprecipitated ERK for the phosphorylation reaction was determined
by Western blot with the anti-general ERK antibody (bottom
panel). The positions of ERK2, ERK1, ERK1b, MBP, and IgG are
indicated. Each of these experiments was reproduced at least three
times.
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Fig. 3.
Enhancement of progesterone production by MEK
inhibitor in gonadotropins, and cAMP-stimulated rLHR-4 and rFSHR-17
cells. Subconfluent cultures were treated with PD98059 alone
(PD; 25 µM), hCG (3 IU/ml), human FSH (3 IU/ml), dghCG (3 IU/ml), forskolin (FK; 50 µM), TPA (100 nM), or the same reagent with
PD98059 for 24 or 48 h, after which progesterone production was
determined as described under "Experimental Procedures." Data are
means of triplicate determinations ± S.E. These experiments
were repeated four times.
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Fig. 4.
Expression of StAR in rLHR-4 and rFSHR-17
cells. Subconfluent cultures were stimulated with forskolin
(FK; 50 µM), PD98059 (PD; 25 µM), hCG (3 IU/ml), human FSH (3 IU/ml), dghCG (3 IU/ml),
human LH (3 IU/ml), or a combination of them for 24 h. Then the
cells were extracted as described under "Experimental Procedures,"
and the extracts were subjected to SDS-polyacrylamide gel
electrophoresis and Western blotting using anti-StAR antibodies. The
arrow indicates mature StAR at 30 kDa. These experiments
were repeated three times.
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Fig. 5.
Effect of U0126 on StAR expression and
progesterone production. Subconfluent cultures of either rLHR-4
(A) or rFSHR-17 (B) cells were stimulated for
24 h with LH (3 IU/ml; A) human FSH (3 IU/ml;
B), U0126 (10 µM), or a combination of the
gonadotropins with U0126 in the same concentrations. Expression of StAR
(upper panel) and progesterone production
(lower panel) were detected as described
above.
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Fig. 6.
Subcellular localization of StAR upon
induction with FSH and PD98059. Immunofluorescence of cells
stained with anti-StAR antibodies followed by goat anti-rabbit IgG
conjugated to fluorescein is shown. Subconfluent rFSHR-17 cells
were stained with anti-StAR antibodies prior to or following
PD98059, FSH, and forskolin stimulation. a, no treatment;
b, 24-h incubation with PD98059 (25 µM);
c, 24-h incubation with LH (3 IU/ml); d, 24-h
incubation with PD98059 (25 µM) and LH (3 IU/ml);
e, 24-h incubation with forskolin (50 µM);
f, 24-h incubation with PD98059 (25 µM) and
forskolin (50 µM) (fluorescence microscopy, × 1000). The
arrow indicates StAR staining in the mitochondria.
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Fig. 7.
Effect of H89 on activation of ERK in
gonadotropin-treated rLHR-4 and rFSHR-17 cells. rLHR-4 or rFSHR-17
cells were serum-starved for 16 h and then stimulated with the
appropriate gonadotropins (3 IU/ml, 10 min) with or without the PKA
inhibitor, H89 (15-min prestimulation, 3 µM). Cytosolic
extracts (50 µg) were subjected to immunoblotting with DP-ERK Ab
(upper panel), or with anti-general ERK antibody
(G-ERK, second panel). The ERK2, ERK1, and ERK1b
are indicated.
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Fig. 8.
Effect of PKI on ERK-activation by
gonadotropins and by forskolin. rLHR-4 (A) and rFSHR-17
(B) cells were transfected with pGFP-ERK2 alone (no plasmid)
or cotransfected with pGFP-ERK2 together with RSV-PKI (PKI) and
RSV-PKImutant (PKI-M, which is inactive PKI). After
transfection, the cells were treated as described under "Experimental
Procedures" for 18 h and then stimulated with LH(3 IU/ml), FSH(3
IU/ml) or forskolin (FK; 50 µM) for 10 min or
left untreated (B). The cells were then harvested, and
cytosolic extracts were subjected to Western blot analysis with the
anti-DP-ERK Ab and anti-C16 antibodies (G-ERK); the 70-kDa
band which represents GFP-ERK2 is shown in the upper
panels. Densitometric scanning of the DP-ERK Ab
lanes (arbitrary units) were used as a measure for ERK
activity (bar graphs, bottom
panels). The results in the bar graphs
are average and S.E. of three experiments.
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Fig. 9.
Effect of H89 on the expression of StAR in
gonadotropin-treated rLHR-4 and rFSHR-17 cells. rLHR-4 or rFSHR-17
cells were serum-starved for 16 h and then stimulated with the
appropriate gonadotropins (3 IU/ml, 10 min) with or without the PKA
inhibitor, H89 (15-min prestimulation, 3 µM). Cytosolic
extracts (50 µg) were subjected to immunoblotting with anti-StAR
antibody. The arrow indicates mature StAR at 30 kDa.
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[in a new window]
Fig. 10.
Schematic representation of the signaling
pathways controlling gonadotropin-induced steroidogenesis.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits of
Gs protein to activate the ERK cascade as was observed for
other receptors and cell types (reviewed in Refs. 23 and 39).
Interestingly, we recently found that basic fibroblast growth factor
suppresses progesterone production in the granulosa cell lines (data
not shown), which would suggest that there may be
gonadotropin/cAMP-independent pathways in these cells that suppress
steroidogenesis via the ERK cascade.
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FOOTNOTES |
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* This work was supported by grants from the Benozyio Institute for Molecular Medicine at the Weizmann Institute of Science and from the Estate of Siegmund Landau (to R. S.) and the Israel Academy of Science (to A. A.) and National Institutes of Health Grant HD-06224 (to J. F. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence may be addressed. Tel.: 972-8-9343602; Fax: 972-8-9344116; E-mail: rony.seger@weizmann.ac.il.
§§ The incumbent of the Joyce and Ben. B. Eisenberg professorial chair in molecular endocrinology and cancer research. To whom correspondence may be addressed. Tel: 972-8-9343713; Fax: 972-8-9344125; E-mail: abraham.amsterdam@weizmann.ac.il.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M006852200
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ABBREVIATIONS |
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The abbreviations used are: FSH, follicle-stimulating hormone; DP-ERK Ab, anti diphospho-ERK antibody; dghCG, deglycosylated hCG; ERK, extracellular signal-regulated kinase; hCG, human chorionic gonadotropin; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; LH, luteinizing hormone; PKA, protein kinase A; StAR, steroidogenic acute regulatory protein; TPA, 12-O-tetradecanoylphorbol-13-acetate; CG, chorionic gonadotropin; hCG, human CG; DMEM, Dulbecco's modified Eagle's medium; PKI, protein kinase inhibitor; 8-Br-cAMP, 8-bromo-cyclic AMP.
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