Induction of Relaxin Messenger RNA Expression in Response to Prolactin Receptor Activation Requires Protein Kinase C
Signaling
Carl A. Peters,
Evelyn T. Maizels,
May C. Robertson,
Robert P.C. Shiu,
Melvin S. Soloff and
Mary Hunzicker-Dunn
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
 |
ABSTRACT
|
---|
The ability of PRL or rat placental lactogen
(rPL)-1 to induce relaxin mRNA expression was analyzed in a luteinized
rat granulosa cell culture model. PRL receptor activation induced
relaxin mRNA expression in a concentration- and time-dependent manner.
High concentrations of PRL receptor agonist, equivalent to those of the
second half of pregnancy in rats, were required to elicit relaxin mRNA
expression. A 40-fold induction of relaxin mRNA was observed in cells
treated 24 h with 1 µg/ml of rPL-1. Estrogen enhanced relaxin
expression induced by PRL but did not affect relaxin expression on its
own. PRL/rPL-1 induction of relaxin expression was independent of the
extracellular regulated kinase (ERK) members of the mitogen-activated
protein kinase (MAPK) pathway, based on the inability of the ERK kinase
inhibitor PD98059 to block induction of relaxin expression. PRL/rPL-1
induction of relaxin expression required protein kinase C (PKC)
,
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
|
---|
Relaxin is a 6000-Da polypeptide hormone that has been implicated
in the successful delivery and survival of pups in the rat (1, 2). The
corpus luteum is the primary site of relaxin expression (3). Relaxin
mRNA is initially detected in luteal samples on day 11 of pregnancy;
expression increases until day 19 or 20 of pregnancy and declines
before parturition (3, 4). Translated relaxin is stored in secretory
granules in the corpus luteum before secretion (5, 6). Secreted relaxin
in the serum of pregnant rats is increasingly detected during the
second half of pregnancy and peaks on the day before parturition (7).
Both relaxin mRNA expression in corpora lutea and serum levels of
relaxin during pregnancy in rats have been shown to depend upon a
placental factor and to be enhanced by estrogen
(E2) (8, 9, 10, 11, 12). Coincident with the increase in
relaxin mRNA expression, the corpus luteum switches from reliance on
pituitary PRL or decidual luteotropin to reliance on rat placental
lactogen (rPL)-1 for maintenance of luteal function, as assessed by
progesterone production (13, 14). In contrast to the pulsatile release
of PRL in the first half of pregnancy, in which serum PRL levels never
exceed 100 ng/ml, rPL-1 and rPL-2 release, although of limited
duration, is continuous, and serum levels reach 3 µg/ml in the second
half of pregnancy (13, 15). PRL, rPL-1, and rPL-2 all bind to the two
forms of the PRL receptor, designated as long and short forms, detected
in the ovary of the rat (16, 17).
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.
 |
RESULTS
|
---|
PRL Induces Relaxin Expression
Although relaxin expression is known to require a
placental-derived factor (8, 9, 10, 11), this factor has not been identified.
We analyzed whether this factor might be rPL-1, which is detected at
high levels in the serum of pregnant rats immediately before the
increase in relaxin mRNA expression (3, 4, 14). Since rPL-1 employs the
same receptor as PRL (16), we initially analyzed the ability of PRL to
induce relaxin mRNA expression. Luteinized granulosa cells were
cultured in the presence of increasing concentrations of PRL for 9
days. Little or no relaxin mRNA could be detected when cells were
cultured in the absence of PRL or in the presence of 20 ng/ml of PRL
(Fig. 1
). However, treatment with 500 or
3000 ng/ml PRL induced relaxin mRNA expression 20-fold and greater than
30-fold, respectively.

View larger version (46K):
[in this window]
[in a new window]
|
Figure 1. PRL Induces Relaxin Expression in a
Concentration-Dependent Manner
Luteinized granulosa cells were cultured for 9 days in the presence of
the indicated concentrations of PRL and mRNA isolated. A, Relaxin mRNA
expression was assessed by Northern blot analysis followed by reprobing
of the same membrane to detect L19 to assess loading. B, Fold induction
of relaxin mRNA expression ([relaxin probe auto-radiographic density/L
19 autoradiographic density for indicated PRL
concentrations]/[relaxin probe auto-radiographic density/L 19
autoradiographic density for control]) is shown; 0 PRL = 1. For
rest of details see Materials and Methods. Result is
representative of two experiments.
|
|
Treatment of luteinized granulosa cells with 1 µg/ml of PRL induced
detectable relaxin mRNA expression in as little as 4 h (Fig. 2A
). 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.

View larger version (49K):
[in this window]
[in a new window]
|
Figure 2. PRL Induces Relaxin Expression in a Time-Dependent
Manner
Luteinized granulosa cells were cultured 448 h (A) or 5 or 9 days (B)
as indicated in the absence (-) or presence (+) of 1 µg/ml PRL, and
mRNA was isolated. Relaxin mRNA expression was assessed by Northern
blot analysis followed by reprobing of the same membrane to detect L19
for assessment of loading. For remainder of details see
Materials and Methods. Each result is representative of
two experiments.
|
|
PRL-Dependent Induction of Relaxin mRNA Expression is Enhanced by
E2
E2 has been shown to enhance both the
expression of relaxin mRNA in the corpus luteum and the production of
relaxin protein, as detected in the serum of pregnant rats (9, 12). We therefore analyzed whether E2 could
induce relaxin mRNA expression either in the absence or presence of
rPL-1. Results presented in Fig. 3
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.

View larger version (57K):
[in this window]
[in a new window]
|
Figure 3. PRL-Dependent Induction of Relaxin mRNA Expression
Is Enhanced by E2
Luteinized granulosa cells were cultured for 12 days in the absence
(-) or presence (+) of 10 nM E2. For the final
4 days of culture, cells were also treated with the indicated
concentration of rPL-1. After mRNA isolation, relaxin mRNA expression
was assessed by Northern blot analysis followed by reprobing of the
same membrane to detect L19 for assessment of loading. Results are
representative of five experiments.
|
|
PKC Signaling Is Necessary but Not Sufficient for
PRL-Dependent Induction of Relaxin mRNA Expression
We have previously shown that PKC
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 1012 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.

View larger version (42K):
[in this window]
[in a new window]
|
Figure 4. PKC Signaling Is Necessary but Not Sufficient for
PRL-Dependent Induction of Relaxin mRNA Expression
Luteinized granulosa cells were cultured for 8 days in the presence of
E2. On day 8 cells were pretreated 30 min with 10
µM rottlerin or 50 µM PD98059 as indicated
(A) or overnight with 10 nM PMA (B). Cells were
subsequently treated overnight with the rPL-1 vehicle (control) or with
3 µg/ml rPL-1 or 10 nM PMA, as indicated, and RNA
collected. Relaxin mRNA expression was assessed by Northern blot
analysis followed by reprobing of the same membrane to detect L19 for
assessment of loading. Results are representative of four experiments.
C, Average (± SEM) fold induction from four experiments of
relaxin mRNA expression ([relaxin probe autoradiographic density/L19
autoradiographic density for the indicated treatment]/[relaxin probe
autoradiographic density/L19 autoradiographic density for control),
control = 1. D, Cells were pretreated for 30 min with or without
50 µM PD98059 (PD) and then treated for 10 min with rPL-1
vehicle (control), 3 µg/ml rPL-1, or 10 nM PMA. Protein
from the cells was then collected in a membrane extracting buffer and
50 µg of total protein/lane were analyzed for ERK/MAPK activation
using an antibody that detects dually phosphorylated p44 MAPK
(upper panel). Blots were subsequently reprobed with a
MAPK antibody (lower panel). For remainder of details
see Materials and Methods. Results are representative of
three experiments.
|
|
ERK/MAPK Signaling is Not Necessary for PRL-Dependent Induction
of Relaxin mRNA Expression
The ERK members of the MAPK family have also been reported to be
activated downstream of the PRL receptor (25, 41, 42); therefore, we
analyzed whether or not the ERK/MAPK kinase (MEK) inhibitor PD98059
(54) affected the ability of rPL-1 to induce relaxin mRNA expression.
As shown in Fig. 4
, 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.

View larger version (87K):
[in this window]
[in a new window]
|
Figure 5. Effect of Rottlerin on Stat 3 DNA Binding after
rPL-1 Treatment
Luteinized granulosa cells were cultured 8 days in the presence of
E2. On day 8, cells were pretreated with 10
µM rottlerin (30 min) as indicated, and cells were
subsequently treated with rPL-1 vehicle (control) or 3 µg/ml rPL-1
for 10 min. Nuclear extracts were prepared and employed in EMSAs. For
details see Materials and Methods. Oligonucleotides
containing Stat 1(A), Stat 3 (B and C), or Stat 5 (DF) consensus
DNA-binding elements were end-labeled with [32P] and
mixed with nuclear extracts either alone (A, B, and D) or in the
presence of Stat 3 (C), Stat 5a (E), or 5b (F) polyclonal antibodies
(Ab). Results are representative of three separate experiments.
|
|
Effect of Rottlerin on Stat 3 Phosphorylation and
Coimmunoprecipitation with PKC
after rPL-1 Treatment
We next sought to identify the PKC
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).

View larger version (63K):
[in this window]
[in a new window]
|
Figure 6. Effect of Rottlerin on Stat 3 Phosphorylation after
rPL-1 Treatment
Luteinized granulosa cells were cultured for 8 days in the presence of
E2. On day 8, cells were pretreated with 10
µM rottlerin (30 min) as indicated, and cells were
subsequently treated with rPL-1 vehicle (control) or 3 µg/ml rPL-1
for 10 min. Cell lysates were prepared in a membrane extracting buffer
and analyzed for Stat 3 and Stat 1 phosphorylation by Western blotting.
For details see Materials and Methods. A, Stat 3
phosphorylation on tyrosine-705 (upper panel) and
serine-727 (second panel) was detected by Western
blotting with phospho-epitope-specific antibodies, and the presence of
Stat 3 (bottom panel) was detected by Western blotting
with a Stat 3 monoclonal antibody. B, Phosphorylation of Stat 1 on
tyrosine-701 (upper panel) was detected by Western
blotting with phospho-epitope-specific antibody, and the presence of
Stat 1 (lower panel) was detected by Western blotting
with a Stat 1 monoclonal antibody. Results are representative of two
experiments.
|
|
To assess whether a Stat 3/PKC
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.
Phosphorylation and Coimmunoprecipitation of Stat 3 with PKC
during Pregnancy
Finally, we wanted to determine whether the association between
PKC
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).

View larger version (26K):
[in this window]
[in a new window]
|
Figure 8. Phosphorylation of Luteal Stat 3 on Serine-727 and
Tyrosine-705 during Pregnancy
Lysates were prepared from rat corpora lutea, obtained on indicated
days of pregnancy, in a membrane extracting buffer, as described in
Materials and Methods. Fifty micrograms of total
protein/lane were analyzed for phosphorylation of Stat 3 on
tyrosine-705 (A) or serine-727 (B) by Western blotting with
phospho-epitope specific antibodies (upper panels), and
the presence of Stat 3 was detected by Western blotting with a Stat 3
monoclonal antibody (lower panels). Phosphorylation of
Stat 1 on tyrosine-701 (C) was detected by Western blotting with
phospho-epitope-specific antibody (upper panel), and the
presence of Stat 1 was detected by Western blotting with a Stat 1
monoclonal antibody (lower panel). Results are
representative of three experiments.
|
|
We also determined whether Stat 3 coimmunoprecipitated with PKC
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).

View larger version (22K):
[in this window]
[in a new window]
|
Figure 9. Coimmunoprecipitation of Luteal Stat 3 and JAK-2
with PKC during Pregnancy
A, Lysates were prepared in membrane extracting buffer from corpora
lutea obtained on the indicated days of pregnancy, PKC
immunoprecipitations from 500 µg of total protein were performed as
described in Materials and Methods. Right
panels display Stat 3, Stat 5, Stat 1, and PKC Western
blots performed on PKC immunoprecipitates from the indicated days
of pregnancy. For comparison, left panels display Stat
3, Stat 5, Stat 1, and PKC Western blots performed on 50 µg of
total protein extract from the indicated days of pregnancy. B, JAK-2
immunoprecipitation from 500 µg of total luteal protein extract. Blot
was probed for PKC and reprobed with a JAK-2 antibody. Results are
representative of two experiments.
|
|
 |
DISCUSSION
|
---|
The results presented herein demonstrate that rPL-1/PRL, at
concentrations equivalent to those of rPL-1 present in rat serum in the
second half of pregnancy (14), strongly induces relaxin mRNA expression
in luteinized rat granulosa cells. Concentrations of PRL receptor
agonist equivalent to those present in the first half of pregnancy do
not elicit relaxin expression in luteinized granulosa cells, consistent
with the absence of relaxin expression during the first half of
pregnancy. E2 synergized with PRL receptor
agonist in the luteinized granulosa cell model to promote increased
relaxin expression, consistent with the presence in the second half of
pregnancy not only of rPL-1 and, subsequently rPL-2, but also of
increasing concentrations of serum testosterone, aromatized to
E2 in the corpus luteum (51). Although relaxin
expression could be induced rapidly (in as little as 4 h), PRL
receptor agonist continued over time (up to 9 days) to promote relaxin
expression in the luteinized granulosa cell model, also consistent with
the sustained expression of relaxin by the corpus luteum between days
12 and 20 of pregnancy (3, 7). Thus, the temporal pattern of relaxin
expression during pregnancy appears to be mimicked in the luteinized
granulosa cell model by the luteotrophic complex of hormones known to
be required to sustain progesterone production by the corpus luteum in
the second half of pregnancy (56). This is the first demonstration, to
our knowledge, of direct evidence for the regulation of relaxin
expression by PRL receptor agonists in concert with
E2. These results are consistent with earlier
reports showing that relaxin expression depended on a signal or signals
derived from the placental unit, which was lost by removal of
conceptuses (fetus and placenta) or hysterectomy of pregnant rats and
temporarily rescued by E2 (11, 12, 57). Our
results thus support the hypothesis that the placental derived signal
consists of a combination of rPL-1 (and later, rPL-2) and androgen,
which is aromatized to estrogen by the corpus luteum (14, 51, 58).
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.
 |
MATERIALS AND METHODS
|
---|
Materials
The following materials were purchased: [
-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/Hams 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 Denhardts 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
|
---|
Address requests for reprints to: Mary Hunzicker-Dunn, Department of Cell and Molecular Biology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago Illinois 60611.
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.
 |
REFERENCES
|
---|
-
Lao Guico-Lamm M, Sherwood OD 1988 Monoclonal antibodies
specific for rat relaxin. II. Passive immunization with monoclonal
antibodies throughout the second half of pregnancy disrupts birth in
intact rats. Endocrinology 123:24792485[Abstract]
-
Hwang JJ, Sherwood OD 1988 Monoclonal antibodies specific for
rat relaxin. III. Passive immunization with monoclonal antibodies
throughout the second half of pregnancy reduces cervical growth and
extensibility in intact rats. Endocrinology 123:24862490[Abstract]
-
Crish JF, Soloff MS, Shaw AR 1986 Changes in relaxin
precursor mRNA levels in the rat ovary during pregnancy. J Biol
Chem 261:19091913[Abstract/Free Full Text]
-
Gunnersen JM, Crawford RJ, Tregear GW 1995 Expression of the
relaxin gene in rat tissues. Mol Cell Endocrinol 110:5564[CrossRef][Medline]
-
Anderson MB, Sherwood OD 1984 Ultrastructural localization of
relaxin immunoreactivity in corpora lutea of pregnant rats.
Endocrinology 114:11241127[Abstract]
-
Fields PA 1984 Intracellular localization of relaxin in
membrane-bound granules in the pregnant rat luteal cell. Biol Reprod 30:753762[Abstract]
-
Sherwood OD, Crnekovic VE, Gordon WL, Rutherford JE 1980 Radioimmunoassay of relaxin throughout pregnancy and during parturition
in the rat. Endocrinology 107:691698[Abstract]
-
Goldsmith LT, Crob HS, Scherer KJ, Surve A, Steinetz BG,
Weiss G 1981 Placental control of ovarian immunoreactive relaxin
secretion in the pregnant rat. Endocrinology 109:584552
-
Goldsmith LT, De La Cruz JL, Weiss G, Castracane VD 1982 Steroid effects on relaxin secretion in the rat. Biol Reprod 27:886890[Abstract]
-
Sherwood OD, Golos TG, Key RH 1986 Influence of the
conceptuses and the material pituitary on the distribution of multiple
components of serum relaxin immunoreactivity during pregnancy in the
rat. Endocrinology 119:21432147[Abstract]
-
Crish JF, Soloff MS, Shaw AR 1986 Changes in relaxin precursor
messenger ribonucleic acid levels in ovaries of rats after hysterectomy
and removal of conceptuses, and during the estrous cycle. Endocrinology 119:12221228[Abstract]
-
Lao Guico MS, Sherwood OD 1985 Effect of oestradiol-17 beta on
ovarian and serum concentrations of relaxin during the second half of
pregnancy in the rat. J Reprod Fertil 74:6570[Abstract]
-
Smith MS, Neill JD 1976 Termination at midpregnancy of the two
daily surges of plasma prolactin initiated by mating in the rat.
Endocrinology 98:696701[Abstract]
-
Robertson MC, Gillespie B, Friesen HG 1982 Characterization of
the two forms of rat placental lactogen (rPL): rPL-I and rPL-II.
Endocrinology 111:18621886[Medline]
-
Shiu RPC, Kelly PA, Friesen HG 1973 Radioreceptor assay for
prolactin and other lactogenic hormones. Science 180:968971[Medline]
-
Sakal E, Robertson MC, Chapnik-Coohen N, Tchelet A, Gertler A 1996 Interaction of recombinant rat placental lactogen-1 with
extracellular domains of prolactin receptors from three species. Recept
Sig Trans 6:3542
-
Shirota M, Banville D, Ali S, Jolicoeur C, Boutin JM, Edery M,
Djiane J, Kelly PA 1990 Expression of two forms of prolactin receptor
in rat ovary and liver. Mol Endocrinol 4:11361143[Abstract]
-
Dusanter-Fourt I, Muller O, Ziemiecki A, Mayeux P, Drucker B,
Djiane J, Wilks A, Harpur A, G, Fischer S, Gisselbrecht S 1994 Identification of JAK protein tyrosine kinases as signaling molecules
for prolactin. Functional analysis of prolactin receptor and
prolactin-erythropoietin receptor chimera expressed in lymphoid cells.
EMBO J 13:25832591[Abstract]
-
Rui H, Kirken RA, Farrar WL 1994 Activation of
receptor-associated tyrosine kinase JAK2 by prolactin. J Biol Chem 269:53645368[Abstract/Free Full Text]
-
DaSilva L, Howard OMZ, Rui H, Kirken RA, Farrar WL 1994 Growth
signaling and JAK2 association mediated by membrane-proximal
cytoplasmic regions of prolactin receptors. J Biol Chem 269:1826718270[Abstract/Free Full Text]
-
Rui H, Lebrun J-J, Kirken RA, Kelly PA, Farrar WL 1994 JAK2
activation and cell proliferation induced by antibody-mediated
prolactin receptor dimerization. Endocrinology 135:12991306[Abstract]
-
Pezet A, Ferrag F, Kelly PA, Edery M 1997 Tyrosine docking
sites of the rat prolactin receptor required for association and
activation of stat5. J Biol Chem 272:2504325050[Abstract/Free Full Text]
-
Kirken RA, Malabarba MG, Xu J, Liu X, Farrar WL, Hennignausen
L, Larner AC, Grimley PM, Rui H 1997 Prolactin stimulates
serine/tyrosine phosphorylation and formation of heterocomplexes of
multiple STAT5 isoforms in Nb2 lymphocytes. J Biol Chem 272:1409814103[Abstract/Free Full Text]
-
DaSilva L, Rui H, Erwin RA, Howard OM, Kirken RA, Malabarba
MG, Hackett RH, Larner AC, Farrar WL 1996 Prolactin recruits STAT1,
STAT3 and STAT5 independent of conserved receptor tyrosines TYR402,
TYR479, TYR515 and TYR580. Mol Cell Endocrinol 117:131140[CrossRef][Medline]
-
Wartmann M, Cella N, Hofer P, Groner B, Liu X, Hennighausen L,
Hynes NE 1996 Lactogenic hormone activation of Stat5 and transcription
of the ß-casein gene in mammary epithelial cells is independent of
p42 ERK2 mitogen-activated protein kinase activity. J Biol Chem 271:3186331868[Abstract/Free Full Text]
-
Chida D, Wakao H, Yoshimura A, Miyajima A 1998 Transcriptional
regulation of the beta-casein gene by cytokines: cross-talk between
STAT5 and other signaling molecules. Mol Endocrinol 12:17921806[Abstract/Free Full Text]
-
Ruff SJ, Leers-Sucheta S, Melner MH, Cohen S 1996 Induction
and activation of Stat 5 in the ovaries of pseudopregnant rats.
Endocrinology 137:40954099[Abstract]
-
Feltus FA, Groner B, Melner MH 1999 Stat5-mediated regulation
of the human type II 3 beta-hydroxysteroid dehydrogenase/
5-
4
isomerase gene: activation by prolactin. Mol Endocrinol 13:10841093[Abstract/Free Full Text]
-
Russell DL, Norman RL, Dajee M, Liu X, Henninghausen L,
Richards JS 1996 Prolactin-induced activation and binding of stat
proteins to the IL-6RE of the
2-macroglobulin (
2M) promoter:
relation to the expression of
2M in the rat ovary. Biol Reprod 55:10291038[Abstract]
-
Dajee M, Fey GH, Richards JS 1998 Stat5b and the orphan
nuclear receptors regulate expression of the
2-macroglobulin (
2M)
gene in rat ovarian granulosa cells. Mol Endocrinol 12:13931409[Abstract/Free Full Text]
-
Udy GB, Towers RP, Snell RG, Wilkins RJ, Park S-H, Ram PA,
Waxman DJ, Davey HW 1997 Requirement of STAT5b for sexual dimorphism of
body growth rates and liver gene expression. Proc Natl Acad Sci USA 94:72397244[Abstract/Free Full Text]
-
Yamashita H, Xu J, Erwin RA, Farrar WL, Kirken RA, Rui H 1998 Differential control of the phosphorylation state of proline-juxtaposed
serine residues ser725 of Stat5a and
Ser730 of Stat5b in prolactin-sensitive cells.
J Biol Chem 273:3021830224[Abstract/Free Full Text]
-
Berlanga JJ, Gualillo O, Buteau H, Applanat M, Kelly PA, Edery
M 1997 Prolactin activates tyrosyl phosphorylation of insulin receptor
substrate 1 and phosphatidylinositol-3-OH kinase. J Biol Chem 272:20502052[Abstract/Free Full Text]
-
Wen A, Zhong Z, Darnell Jr JE 1995 Maximal activation of
transcription by Stat1 and Stat3 requires both tyrosine and serine
phosphorylation. Cell 82:241250[Medline]
-
Zhang X, Blenis J, Li HC, Schindler C, Chen-Kiang S 1995 Requirement of serine phosphorylation for formation of STAT-promoter
complexes. Science 267:19901994[Medline]
-
Ng J, Cantrell D 1997 STAT3 is a serine kinase target in T
lymphocytes. J Biol Chem 272:2454224549[Abstract/Free Full Text]
-
Wen Z, Darnell Jr JE 1997 Mapping of Stat3 serine
phosphorylation to a single residue (727) and evidence that serine
phosphorylation has no influence on DNA binding of Stat1 and Stat3.
Nucleic Acids Res 25:20622067[Abstract/Free Full Text]
-
Chung J, Uchida E, Grammer TC, Blenis J 1997 STAT3 serine
phosphorylation by ERK-dependent and -independent pathways negatively
modulates its tyrosine phosphorylation. Mol Cell Biol 17:65086516[Abstract]
-
David M, Petricoin III E, Benjamin C, Pine R, Weber MJ, Larner
AC 1995 Requirement for MAP kinase (ERK2) activity in interferon
-
and interferon ß-stimulated gene expression through STAT proteins.
Science 269:17211723[Medline]
-
Jain N, Zhang T, Kee WH, Li W, Cao X 1999 Protein kinase C
associates with and phosphorylates Stat3 in an interleukin-6-dependent
manner. J Biol Chem 274:2439224400[Abstract/Free Full Text]
-
Das R, Vonderhaar BK 1996 Involvement of SHC, GRB2, SOS and
RAS in prolactin signal transduction in mammary epithelial cells.
Oncogene 13:11391145[Medline]
-
Carey GB, Liberti JP 1995 Stimulation of receptor-associated
kinase, tyrosine kinase, and MAP kinase is required for
prolactin-mediated macromolecular biosynthesis and mitogenesis in Nb2
lymphoma. Arch Biochem Biophys 316:179189[CrossRef][Medline]
-
Rillema JA, Waters SB, Tarrant TM 1989 Studies of the possible
role of protein kinase C in the prolactin regulation of cell
replication in Nb2 node lymphoma cells. Proc Soc Exp Biol Med 192:140144[Abstract]
-
Saruo MD, Zorn NE 1991 Prolactin induces proliferation of
vascular smooth muscle cells through a protein kinase C-dependent
mechanism. J Cell Physiol 148:133138[Medline]
-
DeVito WJ, Avakian C, Stone S, Okulicz WC 1993 Prolactin-stimulated mitogenesis of cultured astrocytes is mediated by
a protein kinase-C dependent mechanism. J Neurochem 60:832842[Medline]
-
Mellor H, Parker PJ 1998 The extended protein kinase C
superfamily. Biochem J 332:281292[Medline]
-
Cutler Jr RE, Maizels ET, Brooks EJ, Mizuno K, Ohno S,
Hunzicker-Dunn M 1993 Regulation of
protein kinase C during rat
ovarian differentiation. Biochim Biophys Acta 1179:260270[Medline]
-
Peters CA, Cutler Jr RE, Maizels ET, Robertson MC, Shiu RP,
Fields PA, Hunzicker-Dunn M, Regulation of PKC delta expression by
estrogen, rat placental lactogen-1 in luteinized rat ovarian granulosa
cells. Mol Cell Endocrinol, in press
-
Cutler Jr RE, Maizels ET, Hunzicker-Dunn M 1994 Delta protein
kinase-C in the rat ovary: estrogen regulation and localization.
Endocrinology 135:16691678[Abstract]
-
Peters CA, Maizels ET, Hunzicker-Dunn M 1999 Activation of PKC
delta in the rat corpus luteum during pregnancy: potential role of
prolactin signaling. J Biol Chem 274:3749937505[Abstract/Free Full Text]
-
Gibori G, Chatterton RT, Chien JL 1979 Ovarian and serum
concentrations of androgen throughout pregnancy in the rat. Biol Reprod 21:5356[Medline]
-
Gschwendt M, Muller H-J, Keilbassa K, Zang R, Kittstein W,
Rincke G, Marks F 1994 Rottlerin, a novel protein kinase inhibitor.
Biochem Biophys Res Commun 199:9398[CrossRef][Medline]
-
Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T,
Ajakane M, Baudett V, Boissin P, Boursier E, Loriolle F, Duhamel L,
Charon D, Kirilovsky J 1991 The bisindolylmalemide GF 109203X is a
potent and selective inhibitor of protein kinase C. J Biol Chem 266:1577115781[Abstract/Free Full Text]
-
Dudley DT, Pang L, Decker SJ, Bridges Aj, Saltiel AR 1995 A
synthetic inhibitor of the mitogen-activated protein kinase cascade.
Proc Natl Acad Sci USA 92:76867689[Abstract]
-
Schonwasser DC, Marais RM, Marshall CJ, Parker PJ 1998 Activation of the mitogen-activated protein kinase/extracellular
signal-regulated kinase pathway by conventional, novel, and atypical
protein kinase C isotypes. Mol Cell Biol 18:790798[Abstract/Free Full Text]
-
Gibori G, Khan I, Warshaw ML, McLean MP, Puryear TK, Nelson S,
Durkee TJ, Azhar S, Steinschneider A, Rao MC 1988 Placental-derived
regulators and the complex control of luteal cell function. Recent Prog
Horm Res 44:377425[Medline]
-
Golos TG, Sherwood OD 1984 Evidence that the maternal
pituitary suppresses the secretion of relaxin in the pregnant rat.
Endocrinology 115:10041010[Abstract]
-
Kalison B, Warshaw ML, Gibori G 1985 Contrasting effects of
prolactin on luteal and follicular steroidogenesis. J Endocrinol 104:241250[Abstract]
-
Pasqualini C, Guilbert B, Frain O, Leviel V 1994 Evidence for
protein kinase C involvement in the short-term activation of prolactin
of tyrosine hydroxylase in tuberoinfundibular dopaminergic neurons.
J Neurochem 62:967977[Medline]
-
Buckley AR, Buckley DJ 1991 Prolactin-stimulated ornithine
decarboxylase induction in rat hepatocytes: coupling to diacylglycerol
generation and protein kinase C. Life Sci 48:237243[CrossRef][Medline]
-
Fanjul LF, Marrero I, Gonzalez J, Quintana J, Santana P,
Estevez F, Mato JM, Ruiz de Galarreta CM 1993 Does
oligosaccharide-phosphatidylinositol (glycosyl-phosphatidylinositol)
hydrolysis mediate prolactin signal transduction in granulosa cells?
Eur J Biochem 216:747755[Abstract]
-
Ratovondrahona D, Fournier B, Odessa MF, Dufy B 1998 Prolactin
stimulation of phosphoinositide metabolism in CHO cells stably
expressing the PRL receptor. Biochem Biophys Res Commun 243:127130[CrossRef][Medline]
-
Hunter S, Koch BL, Anderson SM 1997 Phosphorylation of cbl
after stimulation of Nb2 cells with prolactin and its association with
phosphatidylinositol 3-kinase. Mol Endocrinol 11:12131222[Abstract/Free Full Text]
-
Toker A, Meyer M, Reddy KK, Falck JR, Aneja R, Aneja S, Parra
A, Burns DJ, Ballas LM, Cantley LC 1994 Activation of protein kinase C
family members by the novel polyphosphoinositides PtdIns-3,4-P2 and
PtdIns-3,4,5-P3. J Biol Chem 269:3235832367[Abstract/Free Full Text]
-
LeGood JA, Ziegler WH, Parekh DB, Alessi DR, Cohen P, Parker
PJ 1998 Protein kinase C isotypes controlled by phosphoinositide
3-kinase through the protein kinase PDK1. Science 281:20422045[Abstract/Free Full Text]
-
Horvath CM, Darnell Jr JE 1997 The state of STATs: recent
developments in the study of signal transduction to the nucleus. Curr
Opin Cell Biol 9:233239[CrossRef][Medline]
-
Lebrun JJ, Ali S, Ullrich A, Kelly PA 1995 Proline-rich
sequence-mediated Jak2 association to the prolactin receptor is
required but not sufficient for signal transduction. J Biol Chem 270:1066410670[Abstract/Free Full Text]
-
Berchtold S, Volarevic S, Moriggl R, Mercep M, Groner B 1998 Dominant negative variants of the SHP-2 tyrosine phosphatase inhibit
prolactin activation of JAK2 (Janus kinase 2) and induction of Stat5
(signal transducer of activator of transcription 5)-dependent
transcription. Mol Endocrinol 12:556567[Abstract/Free Full Text]
-
Yin T, Shen R, Feng GS, Yang YC 1997 Molecular
characterization of specific interactions between SHP-2 phosphatase and
JAK tyrosine kinases. J Biol Chem 272:10321037[Abstract/Free Full Text]
-
Berlanga JJ, Vara JAF, Martin-Perez J, Garcia-Ruiz J 1995 Prolactin receptor is associated with c-src kinase in rat liver. Mol
Endocrinol 9:14611467[Abstract]
-
Turkson J, Bowman T, Garcia R, Caldenhoven E, DeGroot RP, Jove
R 1998 Stat3 activation by Src induces specific gene regulation and is
required for cell transformation. Mol Cell Biol 18:25452552[Abstract/Free Full Text]
-
Saharinen P, Ekman N, Sarvas K, Parker P, Alitalo K,
Silvennoinen O 1997 The Bmx tyrosine kinase induces activation of the
Stat signaling pathway, which is specifically inhibited by protein
kinase C delta. Blood 90:43414354[Abstract/Free Full Text]
-
Hata A, Akita Y, Suzuki K, Ohno S 1993 Functional divergence
of protein kinase C (PKC) family members. J Biol Chem 268:91229129[Abstract/Free Full Text]
-
Hickey GJ, Krasnow JS, Beattie WG, Richards JS 1990 Aromatase
cytochrome P450 in rat ovarian granulosa cells before and after
luteinization: adenosine 3',5'-monophosphate-dependent and independent
regulation. Cloning and sequencing of rat aromatase cDNA and 5' genomic
DNA. Mol Endocrinol 4:312[Abstract]
-
Carr DW, DeManno DA, Atwood A, Hunzicker-Dunn M, Scott JD 1993 Follicle-stimulating hormone regulation of A-kinase anchoring proteins
in granulosa cells. J Biol Chem 268:2072920732[Abstract/Free Full Text]
-
Jackiw V, Hunzicker-Dunn M 1992 Luteinization-associated
changes in protein stability of the regulatory subunit of the type I
cAMP-dependent protein kinase. J Biol Chem 267:1433514344[Abstract/Free Full Text]
-
Heller DL, Gianola KM, Leinwand LA 1988 A highly conserved
mouse gene with a propensity to form pseudogenes in mammals. Mol Cell
Biol 8:27972803[Medline]
-
Harpold MM, Evans RM, Salditt-Georgieff M, Darnell JE 1979 Production of mRNA in Chinese hamster cells: relationship of the rate
of synthesis to the cytoplasmic concentration of nine specific mRNA
sequences. Cell 17:1021035
-
Lowry OW, Rosebrough NJ, Farr AL, Randall RJ 1951 Protein
measurement with the Folin phenol reagent. J Biol Chem 193:265275[Free Full Text]