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Signaling
Departments of Cell and Molecular Biology (C.A.P., E.T.M.,
M.H.-D.) Northwestern University Medical School Chicago,
Illinois 60611
Department of Physiology (M.C.R.,
R.P.C.S.) University of Manitoba Winnipeg, Manitoba, Canada
R3E0W3
Department of Obstetrics and Gynecology
(M.S.S.) University of Texas Medical Branch Galveston, Texas
77555
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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,
based on the ability of the preferential PKC inhibitor rottlerin to
abolish induction of relaxin expression. Direct activation of PKC by
phorbol myristate acetate, however, was not sufficient to promote
induction of relaxin mRNA expression. Stats (signal transducers and
activators of transcription) 3 and 5 DNA binding activities were
induced by PRL/rPL-1 treatment of luteinized granulosa cells but only
Stat 3 DNA binding was reduced by rottlerin. PRL/rPL-1 treatment of
luteinized granulosa cells resulted in increased phosphorylation on
tyrosine-705 and serine-727 of Stat 3, and these responses were reduced
and blocked, respectively, by rottlerin. Tyrosine and serine
phosphorylations of Stat 3 in the corpus luteum were also increased in
the second half of pregnancy when PL levels are highest. Stat 3, but
not Stat 1 or 5, coimmunoprecipitated with luteal PKC during
pregnancy; Stat 3 transiently coimmunoprecipitated with PKC from
luteinized granulosa cells in response to PRL receptor activation; and
Stat 3/PKC complex formation required PKC kinase activity.
Taken together, these results show that PKC is obligatory for
PRL/rPL-1-dependent relaxin expression, that PKC complexes with
Stat 3 in response to PRL receptor activation, and that PKC is
involved in the regulation of Stat 3 phosphorylation downstream of the
PRL receptor. These results demonstrate that PRL/rPL-1 promotes relaxin
expression in luteal cells and that this event is mediated, at least in
part, via PKC . |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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PRL receptors do not have intrinsic kinase activity. They instead rely
on janus kinase (JAK)-2 which is constitutively bound to the membrane
proximal domain of the PRL receptor (18, 19, 20). Upon agonist binding, PRL
receptors dimerize and JAK-2 tyrosine kinase activity is induced
causing the phosphorylation of JAK-2 and the PRL receptor (19, 21).
Tyrosine-phosphorylated receptor and JAK-2 likely serve as docking
sites for additional signal transduction proteins (22). Signal
transducers and activators of transcription (Stat)-family members 1, 3,
5a, and 5b have been shown in various cellular models to be tyrosine
phosphorylated after PRL receptor activation, presumably by JAK-2 (23, 24). Tyrosine phosphorylation of the Stat proteins is required for
their dimerization and subsequent translocation to the nucleus where
they bind to response elements and induce gene transcription. Stats 5a
and 5b are well characterized for their role in induction of
PRL-responsive genes such as ß-casein in breast tissue (22, 25, 26).
In the ovary, Stat 5 expression is induced concomitant with luteal
formation (27) and Stat 5 has recently been shown to mediate the
PRL-dependent expression of the steroidogenic enzyme responsible for
conversion of pregnenolone to progesterone (28). Stat 5b has been shown
to be vital for PRL-dependent induction of 
2-macroglobulin (M)
expression in rat corpora lutea (29, 30). Consistent with the evidence
that Stat 5 plays a major role in transducing PRL actions in luteal
cells, mice deficient in Stat 5b abort in the second half of pregnancy
(31).
In addition to PRL-stimulated tyrosine phosphorylation of the Stats,
Stats 1, 3, 5a, and 5b are each phosphorylated on at least one serine
residue. However, the functional significance of the serine
phosphorylation of the Stats is still controversial. Serine-725 of Stat
5a is constitutively phosphorylated as is a second unidentified serine
residue of Stat 5a, while phosphorylation of serine-730 of Stat 5b is
induced in response to PRL in COS-7 and Nb2 cells (32). However,
phosphorylation of serine-725 or -730 on Stats 5a or 5b, respectively,
is not essential for DNA binding or transcriptional activation of a
ß-casein reporter gene in COS-7 cells (29). In contrast to Stats 5a
and 5b, ligand-dependent phosphorylation of serine-727 of Stat 1 is
reported to enhance its transcriptional activity but not its DNA
binding activity (33, 34, 35). Like Stat 1, ligand-dependent
phosphorylation of serine-727 of Stat 3 does not affect Stat 3 DNA
binding but does enhance transcriptional activity (34, 36, 37).
Although the serine kinases responsible for the phosphorylation of
Stats 1 and 5 have yet to be identified, experimental evidence in some
cellular models supports a role for the extracellular regulated kinase
(ERK) members of the mitogen-activated protein kinase (MAPK) family
(36, 38, 39) and, in other cells, for protein kinase C (PKC) as
Stat 3 kinases (40).
Consistent with these reports, PRL is reported in mammary and lymphoma cells to activate the ERKs (25, 41, 42). Signaling through the PRL receptor has also been shown to activate the serine/threonine kinases of the PKC family in Nb2 cells, astrocytes, and vascular smooth muscle cells, based on the ability of PRL to induce translocation of PKC to the Triton-soluble fraction in these cells (43, 44, 45).
The PKC family is a group of 11 related but separate isoforms. These
isoforms exhibit distinct responsiveness to physiological stimuli and
have unique tissue distributions, subcellular localizations, and
substrate specificities, indicating isoform-specific functions (46).
Ovarian tissues in the rat express the , ßI, ßII, , 
, and
isoforms of PKC (47, 48). PKC is distinguished from the other
isoforms expressed in the rat ovary by the striking increase in PKC
mRNA and protein that is observed beginning on day 11 of pregnancy in
the corpus luteum (49). PKC protein and mRNA expression in corpora
lutea continue to increase up to 25-fold by day 18 of pregnancy, remain
high until day 21, and then decrease just before parturition (49). In
addition, based on its translocation to the Triton-soluble cellular
fraction, immunecomplex kinase activity, and phosphorylation, we have
shown that PKC is activated in the corpus luteum during pregnancy
and after PRL treatment in cultured luteinized granulosa cells
(50).
These results taken together led us to hypothesize that relaxin
expression in the second half of pregnancy was stimulated by PRL
receptor activation and mediated, in part, by PKC . Current studies
were therefore undertaken to begin to address the possible role of
signaling through the PRL receptor by the high concentrations of rPL-1
present in the second half of pregnancy in the induction of relaxin
mRNA expression. We employed a luteinized granulosa cell culture system
and found that treatment with high concentrations of PRL or rPL-1
strongly induced relaxin mRNA expression in a concentration- and
time-dependent manner. In addition, our results showed that induction
of relaxin mRNA expression required PKC kinase activity and
implicated Stat 3 as a PKC target leading to relaxin mRNA
expression. Our results position PKC as a vital regulator of
relaxin expression in response to PRL signaling in rat luteal
cells.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). However, treatment with 500 or
3000 ng/ml PRL induced relaxin mRNA expression 20-fold and greater than
30-fold, respectively.
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). However, relaxin mRNA expression was
more evident after 24 and 48 h of PRL treatment. PRL-dependent
relaxin mRNA expression was still readily detected after 5 and 9 days
of PRL treatment (Fig. 2B). Cells cultured in the absence of PRL
exhibited no relaxin mRNA expression regardless of the length of the
culture period.
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show
that E2 by itself did not induce relaxin mRNA
expression (lanes 1 and 2). However, E2 enhanced
relaxin mRNA expression induced by either 0.5 or 3 µg/ml rPL-1.
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is the sole PKC isoform
whose expression is increased in the second half of pregnancy,
beginning between days 10 and 12 of pregnancy (49). Days 10–12 of
pregnancy target a time of elevated serum rPL-1 and rising serum
androgen titers and of increasing relaxin expression by the corpus
luteum (3, 7, 15, 51). PKC protein expression is increased by
E2 in rat corpora lutea of pseudopregnancy (49)
and in luteinized granulosa cells (48). We have also shown that PKC
is acutely activated by PRL in luteinized granulosa cells cultured in
the presence of E2, based on the translocation of
PKC to a Triton-soluble membrane fraction, its increased immune
complex kinase activity, and its phosphorylation on serine-662 in
response to PRL (50). Therefore, we assessed whether PKC signaling was
required for PRL-mediated relaxin mRNA expression in luteinized
granulosa cells cultured in the presence of E2.
Cells were pretreated either overnight with 10 nM phorbol
myristate acetate (PMA) to down-regulate PKC isoforms or for 30 min
with the PKC preferential inhibitor rottlerin (40, 52) before
overnight treatment with rPL-1. As shown in Fig. 4, A and B, and depicted graphically in
Fig. 4C, rPL-1 strongly induced relaxin mRNA expression. Pretreatment
with the PKC preferential inhibitor rottlerin blocked the ability
of rPL-1 to induce relaxin mRNA expression (Fig. 4, A and C).
Similarly, overnight pretreatment with PMA before treatment with rPL-1
down-regulated PKC isoform expression (data not shown) and blocked
rPL-1-dependent induction of relaxin mRNA expression (Fig. 4, B and C).
Pretreatment of cells for 30 min with the general PKC inhibitor
GF109203X (53) also abated rPL-1-dependent relaxin induction (not
shown). However, PKC signaling was not sufficient to induce relaxin
mRNA expression since direct activation of PKC with PMA did not induce
relaxin mRNA expression (Fig. 4, A and C). These results suggest that
the induction of relaxin expression by rPL-1 requires PKC kinase
activity.
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, A and C, pretreatment of luteinized granulosa cells
with the MEK inhibitor had no significant effect on the rPL-1-dependent
induction of relaxin mRNA expression. Figure 4D shows that the
inability of the MEK inhibitor to block rPL-1-induced relaxin mRNA
expression was not due to a lack of activation of ERK/MAPK by rPL-1.
rPL-1 promoted the activation of p44 MAPK, as detected with an antibody
that detects dually phosphorylated MAPK, and this activation was
completely blocked when cells were pretreated with PD98059. Consistent
with reports that several PKC isoforms are capable of activating
ERK/MAPK (55), PMA treatment of luteinized granulosa cells also induced
activation of p44 MAPK (Fig. 4D), yet PMA did not promote relaxin mRNA
expression (Fig. 4A). These results suggest that rPL-1-dependent
relaxin expression is independent of the ERK/MAPK signaling
pathway.
Effect of Rottlerin on PRL-Induced Stat 3 DNA Binding
Stat 5b has been shown to be activated in corpora lutea during the
second half of pregnancy, based on the ability of Stat 5b to bind to
DNA response elements of the 2-M promoter (29, 30). Minimal Stat 5a
and Stat 3 DNA binding and no Stat 1 DNA binding to the 2-M promoter
could be identified (29). However, since PRL receptor signaling can
employ Stats 1, 3, and 5 (24) after JAK-2 activation (19), any of these
Stats could play a role in regulation of relaxin mRNA expression
downstream of the PRL receptor. As the relaxin promoter sequence has
not been published, we sought to determine whether or not Stats 1, 3,
or 5 DNA binding activity was induced by PRL and whether or not
pretreatment with rottlerin altered this DNA binding.
E2-primed luteinized granulosa cells were treated
with PRL for 10 min and nuclear extracts were prepared. Electrophoretic
mobility shift assays (EMSAs) were performed using Stat 1, 3, and 5
consensus DNA binding elements. Figure 5A
shows that Stat 1 DNA binding is not induced by PRL in luteinized
granulosa cells. In contrast, oligonucleotide probes containing the
consensus Stat 3 (panel B) and Stat 5 (panel D) DNA binding sites were
shifted in response to PRL. Bands observed with Stat 3- (panel B) and
Stat 5- (panel D) specific oligonucleotide probes were competed with
their respective unlabeled oligonucleotides (data not shown). The
specificity of the Stat 3 complex was confirmed by the ability of a
Stat 3 antibody to supershift the complex (panel C). Since the PKC
inhibitor rottlerin blocks relaxin expression, the effect of rottlerin
on PRL-stimulated DNA binding was also determined. Stat 3 DNA binding
was reduced in cells pretreated with rottlerin (panel B) while Stat 5
DNA binding was unaffected (panel D). Furthermore, we confirmed that
the complex formed using the Stat 5-specific oligonucleotide probe
indeed contained Stat 5. Incubation with antibodies to Stat 5a or 5b
both supershifted the complexes (panels E and F), confirming that PRL
activates the DNA binding of both Stat 5a and Stat 5b in luteinized
granulosa cells.
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after rPL-1 Treatment target downstream of the
PRL receptor. Activation of transcription by Stat 3 has been shown to
be regulated by both tyrosine and serine phosphorylation (34, 36, 37),
and PKC has recently been identified as a Stat 3 serine kinase
(40). In contrast, neither Stat 5a nor 5b requires serine
phosphorylation for DNA binding or activation of transcription (32).
Therefore, we considered Stat 3 the most likely PKC target. Using
the E2-primed luteinized granulosa cell model, we
assessed whether PRL receptor activation increased the tyrosine and
serine phosphorylation of Stat 3. As shown in Fig. 6A, a basal level of tyrosine-705 and
serine-727 Stat 3 phosphorylation was detected, consistent with reports
in other cells (36). Treatment of cells with rPL-1 for 10 min increased
the phosphorylation of Stat 3 both on tyrosine-705 (top
panel) and serine-727 (middle panel). We then
determined whether rPL-1-stimulated serine phosphorylation of Stat 3
was dependent on PKC activation by pretreating cells with
rottlerin. Pretreatment of cells with rottlerin nearly completely
blocked phosphorylation of Stat 3 on serine-727 and reduced the
phosphorylation of Stat 3 on tyrosine-705 to the level detected before
rPL-1 treatment. Consistent with evidence that Stat 3 DNA binding is
regulated by tyrosine but not serine phosphorylation (34, 36, 37), our
results show a basal level of Stat 3 DNA binding and tyrosine
phosphorylation (compare Figs. 5B and 6A, top panel). Stat 3
DNA binding and tyrosine phosphorylation exhibit a similar increase
with PRL receptor activation and reduction by rottlerin. In contrast to
Stat 3, Stat 1 phosphorylation of tyrosine-701 was not affected by
treatment of luteinized granulosa cells with rPL-1 (Fig. 6B, top
panel) even though Stat 1 protein is clearly present in these
cells (Fig. 6B, bottom panel) consistent with our DNA
binding results (Fig. 5A). Hela cells treated with (positive) and
without (negative) interferon served as controls for Stat 1
tyrosine phosphorylation. Despite the ability of PRL to enhance Stat 5
DNA binding activity (Fig. 5D), the tyrosine phosphorylation of Stat 5
in PRL-treated cells was not detected (not shown).
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complex is formed in response to
activation of the PRL receptor, we determined whether Stats 1, 3, or 5
coimmunoprecipitated with PKC after rPL-1 treatment of
E2-primed luteinized granulosa cells.
Treatment of cells with rPL-1 for 5 min caused both Stats 3 and 1 to
coimmunoprecipitate with PKC (Fig. 7). This effect was blocked by rottlerin
treatment, suggesting that PKC activation is obligatory for complex
formation between PKC and Stats 1 and 3. Coimmunoprecipitation of
Stats 1 and 3 with PKC in response to rPL-1 was transient since
neither Stat 1 nor Stat 3 coimmunoprecipitated with PKC in samples
from cells treated with rPL-1 for 30 min. Stat 5 did not
coimmunoprecipitate with PKC . As blots were first probed for PKC
(bottom panel) and subsequently probed for Stats 1, 3, and 5
(first, second, and third panels, respectively),
residual PKC immunoreactivity is detected in the Stat Western
blots.
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during Pregnancy and Stat 3 was reproduced in an in vivo setting in
which luteal cells are exposed to sustained and high levels of PLs. We
thus assessed Stat 3 phosphorylation on tyrosine-705 and serine-727 in
luteal extracts obtained on days 11 and 18 of pregnancy. Stat 3
tyrosine (Fig. 8A) and serine (Fig. 8B)
phosphorylations both increased as pregnancy progressed, as shown by
Western blots of luteal lysates from the indicated days of pregnancy.
In contrast to Stat 3, Stat 1 was not active during pregnancy, as
assessed by Western blot analysis to detect Stat 1 phosphorylated on
tyrosine-701 (Fig. 8C). Neither total luteal Stat 1 nor, as previously
shown (29), Stat 3 protein expression was regulated in the second half
of pregnancy (Fig. 8, lower portion of each panel).
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from luteal extracts obtained on days 11 and 18 of pregnancy. Results
(Fig. 9A, top right panel)
showed that Stat 3 indeed coimmunoprecipitated with PKC on days 11
and 18 of pregnancy and that higher levels of Stat 3 were detected in
PKC immunoprecipitates on day 18 compared with day 11, consistent
with results of Fig. 8A. Neither Stats 1 nor 5 coimmunoprecipitated
with PKC , although both were detected in luteal membrane extracts
on these two representative days of pregnancy (Fig. 9A, left
panels). Coimmunoprecipitation of PKC with JAK-2 from luteal
membrane extracts was also detected on day 18 of pregnancy (Fig. 9B).
The absence of detectable PKC in the Jak-2 immunoprecipitate from
corpora lutea obtained on day 11 of pregnancy could reflect the lower
luteal levels of both PKC and Jak-2 on this day (Fig. 9, A and B,
bottom panels).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Our results demonstrate that the induction of relaxin mRNA expression
in response to PRL receptor activation in luteinized granulosa cells
requires signaling through PKC . PKC is strongly induced in the
second half of pregnancy, and its pattern of expression temporally
coincides with the pattern of relaxin expression (3, 7, 49). Although
we do not know how PKC expression is regulated in corpora lutea
during the second half of pregnancy, we have observed that
E2 increases PKC protein expression 
2-fold in every rat ovarian system that we have analyzed to date (48, 49). It is therefore possible that the enhancing effect of
E2 on PRL/rPL-1-stimulated relaxin expression by
luteinized granulosa cells, as seen in Fig. 3, results from the
E2-stimulated increase in PKC expression.
Since rPL-1/PRL, in concentrations present in the second half of
pregnancy, also synergizes with E2 to further
increase PKC protein and mRNA expression in luteinized rat
granulosa cells (48), the effect of PRL/rPL-1 on relaxin expression in
E2-primed cells might also be due in part to
increased expression of PKC .
Although signaling through PKC is required for PRL-dependent
relaxin expression in luteinized granulosa cells, PKC signaling is not
sufficient to induce relaxin mRNA expression since treatment of cells
with PMA, a direct activator of PKC isoforms such as PKC , does not
induce relaxin mRNA expression. Therefore, relaxin expression requires
not only activation of PKC but also of additional pathways
downstream of the PRL receptor. This is the first report, to our
knowledge, that links a specific PKC isoform to PRL receptor action.
However, our results implicating PKC as a necessary mediator of PRL
induction of relaxin mRNA expression are not the first instance in
which PKC has been implicated in the actions of PRL. PRL-dependent
proliferation in Nb2 lymphoma and liver cells is either potentiated or
mimicked by the PKC activator phorbol ester and is inhibited by
inhibition of PKC activity (43, 44). PRL-dependent activation of
tyrosine hydroxylase in neurons is blocked by a PKC inhibitor but not
by protein kinase A or calmodulin inhibitors (59).
PRL could activate PKC through various routes. PRL has been
reported to induce production of diacylglycerol by the liver
both in vivo and in vitro (60), as well as by rat
granulosa cells (61), apparently independent of PI 4,5-bisphosphate
hydrolysis. Alternatively, PKC may be activated downstream of the
PRL receptor by an increase in the level of phosphatidyl inositol
(3, 4, 5)triphosphate [PI(3, 4, 5)P3] in a PI3-kinase-dependent
fashion (33, 62, 63). PI(3, 4, 5)P3 has been shown to activate several
PKC isoforms both in vitro and after activation of
PI3-kinase in vivo (64). Furthermore, a recent report by Le
Good et al. (65) showed that PKC is phosphorylated on
its activation loop by 3-phosphoinositide-dependent protein
kinase-1 (65). This activation loop phosphorylation of PKC
is enhanced by PI(3, 4, 5)P3 in vitro and is dependent on
PI3-kinase in vivo, leading to increased catalytic activity
of PKC (65). Clearly, several pathways could be employed downstream
of the PRL receptor to affect the activation state of PKC .
Our results indicate that Stat 3 is a likely target for PKC
downstream of the PRL receptor to affect relaxin expression. Tyrosine
phosphorylation of the Stats is required for their dimerization,
translocation to the nucleus, and transcriptional activity (66).
Transcriptional activation by Stat 3 additionally appears to be
optimized, at least in some cells, by phosphorylation on serine-727
(34, 36, 37). PKC is a Stat 3 serine kinase (40). We show that
rPL-1 treatment of luteinized granulosa cells promotes increased
phosphorylation of Stat 3 both on tyrosine-705 and serine-727.
Pretreatment of cells with the PKC inhibitor before rPL-1 treatment
nearly ablated serine-727 phosphorylation of Stat 3 and reduced
tyrosine-705 phosphorylation to a level similar to that seen on Stat 3
before rPL-1/PRL treatment. The rottlerin-dependent reduction of Stat 3
tyrosine-705 phosphorylation coincides with a reduction in Stat 3 DNA
binding activity. These results show that the PRL/rPL-1-dependent
phosphorylation of Stat 3 on both serine and tyrosine is dependent on
the activity of PKC. Consistent with our evidence supporting Stat 3
as a PKC target in luteinized granulosa cells, the extent of Stat 3
tyrosine-705 and serine-727 phosphorylations is higher in corpora lutea
obtained on day 18 compared with day 11 of pregnancy, consistent with
higher expression of both PKC and relaxin mRNA at this time of
pregnancy (3, 49). Moreover, Stat 3 transiently coimmunoprecipitates
with PKC after rPL-1 treatment of luteinized granulosa cells, and
this coprecipitation is blocked when cells are pretreated with
rottlerin. This result suggests that a Stat 3/PKC complex is formed
in response to PRL/rPL-1-dependent PKC activation. In support of
this hypothesis, we have observed that during pregnancy, luteal Stat 3
and JAK-2, but not Stats 1 or 5, coimmunoprecipitate with PKC ,
especially on day 18 of pregnancy when PKC levels are elevated.
Perhaps JAK-2 serves as a docking site for Stat 3 and PKC after PRL
receptor activation (24, 67). Consistent with our results, Stat 3 has
recently been reported to transiently associate with PKC in an
interleukin-6-dependent manner (40). While PKC -dependent Stat 3
phosphorylations on serine-727 and tyrosine-705 closely correlate with
PRL-dependent relaxin expression, it is not clear whether Stat 3 exerts
a positive or negative effect on relaxin expression. Stat 3 serine-727
phosphorylation has been shown both to enhance transcription (33, 34)
and to inhibit Stat 3 transcriptional activity (38, 40). Additional
studies will be necessary to determine how Stat 3 modulates PRL-
dependent relaxin expression.
There are a number of possible mechanisms by which PKC could
regulate the serine and tyrosine phosphorylations of Stat 3. These
include direct phosphorylation of Stat 3 on serine-727 by PKC , as
recently demonstrated in a PKC immune complex kinase assay using
tagged Stat 3 as substrate (40), and/or modulation via PKC
-catalyzed phosphorylation of other Stat 3 serine kinases or Stat 3
tyrosine kinases, including JAK-2 and Src, or of associated
phosphatases (19, 60, 68, 69, 70, 71). Future studies are needed to elucidate
the precise mechanism of the PKC -dependent regulation of Stat 3
phosphorylations in luteinized granulosa cells.
Our results suggest that Stat 1 but not Stat 5 may also be a PKC
target in luteinized rat granulosa cells. Stat 1 did not exhibit either
DNA binding activity or tyrosine-701 phosphorylation in response to PRL
in luteinized granulosa cells, and Stat 1 tyrosine-701 phosphorylation
was undetectable in luteal extracts of pregnancy. These results are
consistent with the previously reported lack of Stat 1 DNA binding
during pregnancy (29). However, while Stat 1 was not readily detected
in PKC immunoprecipitates from corpora lutea obtained during
pregnancy, Stat 1 did coimmunoprecipitate with PKC in response to
acute rPL-1 treatment of luteinized granulosa cells. The
coimmunoprecipitation of Stat 1 with PKC in luteinized granulosa
cells was transient, an observation that perhaps explains why Stat 1
did not readily coimmunoprecipitate with PKC from corpora lutea of
pregnancy that are chronically exposed to rPLs. However, scrutiny of
the Stat 1 blot in Fig. 9 shows that a very small amount of Stat 1 is
detectable on day 18 in the PKC immunoprecipitate, consistent with
a very transient association of Stat 1 with PKC . In luteinized
granulosa cells, the coimmunoprecipitation of Stat 1 with PKC
appeared to be dependent on PKC kinase activity, as it was
inhibited by pretreatment of cells with rottlerin, but to be
independent of Stat 1 tyrosine phosphorylation since rPL-1 did not
stimulate detectable tyrosine phosphorylation of Stat 1. PKC has
been shown to inhibit the tyrosine phosphorylation of Stat 1 catalyzed
by Bmx kinase (72). Perhaps a similar mechanism might explain the
absence of the tyrosine phosphorylation of Stat 1 and therefore the
apparent absence of signaling through Stat 1 by PRL in luteal cells.
Consistent with this hypothesis, the transient association of PKC
and Stat 1 in luteinized granulosa cells might reflect an inhibitory
serine phosphorylation of Stat 1. In contrast to Stat 1, Stat 5 has
been shown to be activated during the second half of pregnancy (29, 30)
at the appropriate time to play a role in regulating relaxin expression
(3). Corpora lutea of Stat 5b null mice cease to produce progesterone
on day 12 of pregnancy, and these mice abort their fetuses (31),
consistent with recent evidence that Stat 5 regulates expression of
3ß-hydroxysteroid dehydrogenase expression in a PRL-dependent manner
(28). In luteinized granulosa cells, Stat 5 DNA binding is clearly
induced by PRL (see Fig. 5). However, unlike Stat 3, Stat 5 DNA binding
and transcriptional activities are solely regulated by tyrosine
phosphorylation (29) and, in luteinized granulosa cells, Stat 5 DNA
binding is unchanged by PKC inhibition. Moreover, Stat 5 did not
coimmunoprecipitate with PKC from either luteinized granulosa cells
or corpora lutea of pregnant rats. Therefore, although a role for Stat
5 in regulation of relaxin expression cannot be ruled out, it is
apparent that Stat 5 is not a direct target for PKC -dependent
regulation of relaxin expression.
In contrast to PKC , ERK/MAPK signaling is not necessary for relaxin
expression although PRL/rPL-1 does induce ERK/MAPK activation, as
detected by the tyrosine and threonine phosphorylations of p44 MAPK in
luteinized granulosa cells. ERK/MAPK activation is blocked by
pretreatment with the MEK inhibitor while this inhibitor does not alter
the PRL/rPL-1-dependent induction relaxin expression. Furthermore, PMA
treatment of luteinized granulosa cells induces p44 MAPK tyrosine and
threonine phosphorylations but does not induce relaxin mRNA expression.
While the lack of a direct effect of PMA on relaxin expression
indicates that typical PMA-regulated transcription involving activating
protein-1 (AP-1) and non-AP-1 transacting factors through PMA and serum
response elements (73) are not sufficient to induce relaxin expression,
synergy between these transcription factors and Stat transcription
factors has not been ruled out.
In conclusion, we have found that signaling through the PRL receptor is
capable of strongly inducing relaxin mRNA expression in luteinized
granulosa cells. PKC is necessary but not sufficient for induction
of relaxin mRNA expression. PKC associates with Stat 3 during
pregnancy and after PRL/rPL-1 treatment in luteinized granulosa cells.
PKC /Stat 3 association in luteinized granulosa cells after
PRL/rPL-1 treatment is transient and blocked by the PKC inhibitor
rottlerin. Similarly, Stat 3 serine phosphorylation is induced after
PRL treatment and blocked by pretreatment with rottlerin. These results
support a role for PKC in the induction of relaxin mRNA expression
during pregnancy in response to activation of the PRL receptor by
saturating agonist concentrations at a time when PKC is highly
expressed and active.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-32P]
deoxy-CTP (specific activity, 3000 Ci/mmol), [
-32P]
ATP (specific activity, 3000 Ci/mmol) from NEN Life Science Products (Boston, MA); SDS/PAGE reagents from Bio-Rad Laboratories, Inc. (Hercules, CA); protein standards from
Diversified Biotech; Nytran nylon membranes from Schleicher & Schuell, Inc. (Keene, NH); Hybond C-extra nitrocellulose and ECL
reagents from Amersham Pharmacia Biotech (Arlington
Heights, IL); TRIzol reagent from Life Technologies, Inc.
(Gaithersburg, MD); PKC specific monoclonal antibody (directed to
the N terminus) and Stats 1, 3, and 5 monoclonal antibodies from
Transduction Laboratories, Inc. (Lexington, KY); 4G-10
(antiphosphotyrosine) monoclonal antibody and antibodies for JAK-2 and
PI3 kinase from Upstate Biotechnology, Inc. (Lake Placid,
NY); active-ERK/MAPK antibody and T4 polynucleotide kinase from
Promega Corp. (Madison, WI); ERK/MAPK antibody from
Zymed Laboratories, Inc. (South San Francisco, CA);
phospho-epitope antibodies that detect Stat 3 phosphorylated on
serine-727 or tyrosine-705 or Stat 1 phosphorylated on tyrosine-710,
and HeLa cell extracts prepared with and without interferon
treatment from New England Biolabs, Inc. (Beverly, MA);
Stats 1 p84/91, 3, and 5 gel shift oligonucleotides, and Stats 3, 5a,
and 5b polyclonal antibodies from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA); rottlerin from Alexis; PD98059 from Calbiochem
(La Jolla, CA). All other biochemical reagents were purchased from
Sigma (St. Louis, MO). Final concentrations are indicated
throughout.
Granulosa Cell Culture
Rats (Sasco strains) were obtained at 21 days of age from
Charles River Laboratories, Inc. (Wilmington, MA) and
maintained in accordance with "Guidelines for the Care and Use of
Experimental Animals" by protocols approved by the Northwestern
University Animal Care and Use Committee. Follicles were
collected from 30-day-old rats that had been administered a low dose of
hCG (0.15 IU) given subcutaneously twice daily for 2 days. On the
following day a high dose of hCG (10 IU) was given to rats via tail
vein injection, and ovaries were isolated 7 h later (74). Cells
were harvested by mechanical dispersion and put into culture (75). The
medium used for all procedures was DMEM/Ham’s F-12 (DMEM/F-12, 1:1)
without phenol red and with 15 mM HEPES, 3.15 g/liter
glucose, 1% charcoal-stripped FBS, 100 IU penicillin G, and 100
µg/ml streptomycin. After sequential incubations at 37 C in 6
mM EGTA in DMEM/F-12 and 0.5 M sucrose in
DMEM/F-12, ovaries were returned to DMEM/F-12. Granulosa cells were
released into the medium from all follicles using 30-gauge needles and
gentle pressure. Cells were pelleted at 100 x g for 15
min, counted using trypan blue, and plated at a density of
approximately 1 x 106 cells/ml on plastic
dishes (Falcon, Becton Dickinson and Co, Lincoln Park,
NJ). Cells were cultured in humidified atmosphere at 37 C, 5%
CO2 with or without 10 nM
E2 (in ethanol, final concentration,
0.5%) for up to 12 days as indicated. Alternatively cells were
cultured in the presence of the indicated concentration of PRL (ovine
PRL-20, NIDDK) for up to 9 days. Media was changed every 3 days.
Pregnant Rats
Pregnant rats were Sasco strains obtained from Charles River Laboratories, Inc. and maintained as described above. On
the appropriate day of pregnancy rats were killed, ovaries removed,
corpora lutea dissected, and luteal lysates prepared as described
below. Day 1 of pregnancy is sperm positive day.
RNA Preparation and Northern Blot Analysis
Equivalent results were obtained when total RNA was isolated by
a one-step isolation procedure according to Life Technologies, Inc. specifications for use of TRIzol reagent or isolated in a
buffer containing 3 M LiCl and 6 M urea (76).
Ten micrograms of RNA were separated by electrophoresis in a 1%
agarose-formaldehyde gel. RNA was transferred to nylon membrane,
covalently attached using UV cross-linking, and the membrane hybridized
with relaxin cDNA that had been labeled with
[-32P] deoxy-CTP using random hexamer
primers and the Klenow fragment of Escherichia coli DNA
polymerase. Northern blots were reprobed with L19 (77), a probe that
detects the LLrep3 gene family (78), to assess the amount of mRNA
present in each lane. Hybridizations were carried out in 50%
formamide, 5 x SSPE, 2 x Denhardt’s reagent, 10% dextran
sulfate, 0.1% SDS, and 100 µg/ml salmon sperm DNA at 42 C. Membranes
were washed in 2 x SSC at room temperature for 15 min, at 50 C
for 30 min, and in 1 x SSC at 50 C for 30 min and then exposed to
X-AR film Eastman Kodak Co. (Rochester, NY) at -80 C.
Electrophoretic Mobility Shift Assay
Cells were solubilized in 100 µl/60-mm culture dish of EMSA
lysis buffer [20 mM HEPES, pH 7.0, 10 mM KCl,
1 mM MgCl2, 20% glycerol, 0.2%
Nonidet P-40, 1 mM orthovanadate, 25 mM NaF,
200 µM phenylmethylsulfonyl fluoride (PMSF), 5 µg/ml
aprotinin, 1 µg/ml pepstatin A, and 2 µg/ml leupeptin]. Lysates
were incubated on ice for 20 min and then clarified by centrifugation
at 20,000 x g for 20 min at 4 C. Oligonucleotides were
labeled with [-32P] ATP, using T4
polynucleotide kinase according to manufacturers specifications.
Binding reactions (20 µl) were incubated at room temperature for 20
min and contained 0.5 ng DNA probe, ± 10 µg extract in 10
mM Tris (pH 7.5), 50 mM
NaCl, 1 mM dithiothreitol, 1
mM EDTA, 5% glycerol, and 1 µg poly (dI-dC).
Polyacrylamide gels (5%) containing 2.5% glycerol and 0.5 x TBE
were prerun in 0.5 x TBE for 30 min at 350 V and run at room
temperature at 350 V after samples were loaded. Gels were wrapped in
plastic wrap and exposed to film with intensifying screen at -70 C.
The oligonucleotide probes used for gel mobility shift studies were as
follows: Stat 1, 5'-CAT-GTT-ATG-CAT-ATT-CCT-GTA-AGT-G-3'; Stat 3,
5'-GAT-CCT-TCT-GGG-AAT-TCC-TAG-ATC-3'; Stat 5, 5'-AGA-TTT-CTA-
GGA-ATT-CAA-TCC.
Lysate Preparation and Western Immunoblot Analysis
Clarified lysates were prepared by homogenization of cells in
100 µl/60 mm culture dish of a lysis buffer (10 mM
potassium phosphate, pH 7.0, 1 mM EDTA, 5 mM
EGTA, 10 mM MgCl2, 2 mM
dithiothreitol, 1 mM sodium vanadate, 50 mM
ß-glycerophosphate, 1 mM PMSF, 0.5% NP-40, 0.1%
deoxycholic acid) followed by centrifugation of homogenates at
15,000 x g for 10 min. Samples were denatured by
addition of 3 x stop (3% SDS, 150 mM
Tris-HCl, 2.4 mM EDTA, 3% ß-mercaptoethanol,
30% glycerol, and 0.5% bromphenol blue). A similar procedure was
employed for pregnant rat corpora lutea tissue samples. Protein
concentrations were determined (79) using BSA as a standard. Protein
samples were separated by SDS-PAGE and transferred to membranes for
Western blot analysis. Western blot analysis was performed using the
Amersham Pharmacia Biotech ECL detection system following
the provided protocol. Where appropriate, membranes were stripped of
antibodies according to the protocol provided with the ECL detection
system. Densitometric quantitation was performed by image analysis
using Molecular Analyst software from Bio-Rad Laboratories
(Hercules, CA).
Immunoprecipitation
Immunoprecipitations were performed on lysates containing 500
µg of total protein using the indicated antibodies. Antibody-antigen
complexes were precipitated by further incubation with an antimouse Ig
antibody, where applicable, and protein A-conjugated Sepharose or with
protein A/G-conjugated agarose alone. After washing the pelleted
proteins with low-salt (10 mM Tris-HCl, pH 7.2, 150
mM NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% SDS, 1
mM sodium vanadate, and 40 µg/ml PMSF) and high-salt (10
mM Tris-HCl, pH 7.2, 1 M NaCl, 0.1% NP-40, 1
mM sodium vanadate, and 40 µg/ml PMSF)
radioimmunoprecipitation assay (RIPA) buffer, precipitated proteins
were stopped in a 1x stop solution and denatured in a boiling water
bath.
|
FOOTNOTES |
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Supported by NIH Grant P01 HD-21921 (M.H.D.) and the P30 Center for Research on Fertility and Infertility, Northwestern University (NIH Grant P30 HD-28048).
Received for publication February 8, 1999. Revision received January 11, 2000. Accepted for publication January 18, 2000.
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