Developmental Regulation of Mitogen-Activated Protein Kinase-Activated Kinases-2 and -3 (MAPKAPK-2/-3) in Vivo during Corpus Luteum Formation in the Rat
Evelyn T. Maizels,
Abir Mukherjee1,
Gunamani Sithanandam,
Carl A. Peters2,
Joshua Cottom3,
Kelly E. Mayo and
Mary Hunzicker-Dunn
Department of Cell and Molecular Biology (E.T.M., C.A.P., J.C.,
M.H.-D.) Northwestern University Medical School Chicago,
Illinois 60611
Department of Biochemistry, Molecular Biology
and Cell Biology (A.M., K.E.M.) Northwestern University
Evanston, Illinois 60208 Intramural Research Support Program
(G.S.) SAIC-Frederick National Cancer Institute
Frederick Cancer Research and Development Center Frederick,
Maryland 21702-1201
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ABSTRACT
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The current study investigates the activation
in vivo and regulation of the expression of components of
the p38 mitogen-activated protein kinase (MAPK) pathway during
gonadotropin-induced formation and development of the rat corpus
luteum, employing a sequential PMSG/human CG (hCG) treatment paradigm.
We postulated that the p38 MAPK pathway could serve to promote
phosphorylation of key substrates during luteal maturation, since
maturing luteal cells, thought to be cAMP-nonresponsive, nevertheless
maintain critical phosphoproteins. Both p38 MAPK and its upstream
activator MAPK kinase-6 (MKK6) were found to be chronically activated
during the luteal maturation phase, with activation detected by 24
h post hCG and maintained through 4 days post hCG. The p38 MAPK
downstream protein kinase target termed MAPK-activated protein
kinase-3 (MAPKAPK-3) was newly induced at both mRNA and protein levels
during luteal formation and maturation, while mRNA and protein
expression of the closely related MAPKAPK-2 diminished. Two
potential substrates for MAPKAPKs, the small heat shock protein
HSP-27 and the cAMP regulatory element binding protein CREB, were
monitored in vivo for phosphorylation. HSP-27
phosphorylation was not modulated during luteal maturation. In
contrast, we observed sustained luteal-phase CREB phosphorylation
in vivo, consistent with upstream MKK6/p38 MAPK
activation and MAPKAPK-3 induction. MAPKAPK-3-specific immune
complex kinase assays provided direct evidence that MAPKAPK-3 was in an
activated state during luteal maturation in vivo. Cellular
inhibitor studies indicated that an intact p38 MAPK path was required
for CREB phosphorylation in a cellular model of luteinization, as
treatment of luteinized granulosa cells with the p38 MAPK inhibitor SB
203580 strongly inhibited CREB phosphorylation. Transient transfection
studies provided direct evidence that MAPKAPK-3 was capable of
signaling to activate CREB transcriptional activity, as assessed by
means of GAL4-CREB fusion protein construct coexpressed with
GAL4-luciferase reporter construct. Introduction of wild-type, but not
kinase-dead mutant, MAPKAPK-3 cDNA, into a mouse ovarian cell line
stimulated GAL4-CREB- dependent transcriptional activity
approximately 3-fold. Thus MAPKAPK-3 is indeed uniquely poised to
support luteal maturation through the phosphorylation and
activation of the nuclear transcription factor CREB.
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INTRODUCTION
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Mitogen-activated protein kinases (MAPKs) comprise a superfamily
of kinases activated in response to mitogenic, stressful, and
differentiation-inducing stimuli (reviewed in Ref. 1). MAPKs are
recognized to regulate cellular responses both through the
phosphorylation of transcription factors, and through the
phosphorylation/activation of downstream target protein kinases. The
mammalian MAPK superfamily contains at least three subgroups, the
extracellular-regulated kinases (ERK/MAPKs), the stress-activated
protein kinases/Jun kinases (SAPK-1/Jun kinases), and the p38 MAPKs,
orthologs of the yeast osmosensitive HOG-1 kinase. Upstream
MAPK-kinases (MKKs) MKK6 and MKK3 serve to activate p38 MAPK through
phosphorylation (2, 3). Once activated, p38 MAPK then phosphorylates,
and may thereby activate, a number of substrates including two closely
related downstream target kinases called mitogen-activated protein
kinase-activated protein kinases-2 and -3 (MAPKAPK-2 and MAPKAPK-3)
(4, 5, 6, 7, 8). These MAPKAPKs, in turn, phosphorylate a set of substrates that
include both the small heat shock protein HSP-27 (9, 10) and the
nuclear transcription factor, cAMP-response element binding protein
(CREB), on the activation-related site serine 133 (11, 12, 13).
Activation of both p42/p44 ERK/MAPK and p38 MAPK has been documented
during ovarian response to hormonal stimulation. ERK/MAPK activation is
elicited by treatment of granulosa cells with epidermal growth factor
(14), FSH (15, 16, 17), or LH (16), and in luteal cells by treatment with
PGF2
(18). The consequent phosphorylation/activation of the
downstream ERK/MAPK target protein kinase p90rsk
was demonstrated in FSH-treated immature rat granulosa cells (15) (J.
Cottom, Y. Park, E. T. Maizels, L. Salvador, J. C. R. Jones,
R. V. Schillace, D. W. Carr, P. Cheung, C. D. Allis, J. L. Jameson, and
M. Hunzicker-Dunn, submitted). Additionally, FSH treatment of
immature granulosa cells elicits p38 MAPK activation (19, 20), with
resultant phosphorylation of the MAPKAPK-2/-3 substrate HSP-27 and
modulation of cell shape (19), and UV light, a stress stimulus,
activates p38 MAPK in bovine luteal cells (21).
In the rat, activation of preovulatory follicular granulosa cell LH
receptors by the proestrus LH surge, or by pharmacological treatment
with the LH receptor agonist, human CG (hCG), causes ovulation and
differentiation of the ovulated follicle into a corpus luteum.
LH/hCG-induced ovulation and luteal formation are accompanied by a
pattern of distinct biochemical changes (reviewed in Ref. 22),
including, for example, down-regulation followed by later reappearance
of the LH receptor and aromatase, the transient induction of the
progesterone receptor, the induction of PGHsynthase-2, and the
down-regulation of inhibin
expression. Protein phosphorylation
is recognized to be a key ovarian response to hormonal stimulation.
LH/hCG-induced cAMP formation and consequent protein kinase A
(PKA)-mediated phosphorylation of target proteins are required for both
ovulation and luteal formation. However, once luteal formation is
initiated, the mechanism for the LH/hCG-stimulated cAMP production
becomes desensitized (23), the requirement for continued cAMP-dependent
signaling is lost, and a state of cAMP nonresponsiveness is thought to
characterize the maturing luteal cell (24, 25). Nevertheless,
phosphorylation of luteal proteins, notably CREB, remains evident (25);
therefore, kinases other than PKA would be expected to control
phosphorylation of target proteins during luteal maturation. Based on
our evidence that the p38 MAPK pathway is required for immature
granulosa cell response to FSH (19) and that this pathway is known to
be regulated by a large number of input signals from many G
protein-coupled receptors as well as growth factor receptors in other
cellular models (1), we postulated that the p38 MAPK path would serve
to phosphorylate essential targets during the cAMP-nonresponsive luteal
maturation phase. We undertook to examine components of the p38 MAPK
pathway for evidence of regulation during hCG-induced luteal maturation
with special interest in MAPKAPKs, as those kinases could regulate the
phosphorylation of CREB (11, 12, 13, 26). We found that expression of
MAPKAPKs was inversely regulated. While MAPKAPK-2 expression diminished
with luteal development, MAPKAPK-3 was newly induced during this
developmental transition. Moreover, MAPKAPK-3 induction was accompanied
by upstream kinase activation, and by downstream substrate
phosphorylation; thus, MAPKAPK-3 is uniquely poised to subserve the
role of critical kinase during luteal maturation.
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RESULTS
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Activation of Ovarian p38 MAPK and MKK3/6 in Vivo
during Hormone-Induced Luteinization
We investigated the activation states of upstream component
kinases in the p38 MAPK cascade in vivo during
hormone-induced follicle maturation, ovulation, and luteinization in
the rat. Immature rats were subjected to the well characterized
sequential gonadotropin-induced luteinization paradigm (27), comprised
of initial subcutaneous PMSG injection to induce follicular maturation
to the preovulatory stage, followed 48 h later by subcutaneous hCG
injection to induce ovulation and luteinization.
p38 MAPK, analogous with other MAPK family members (1), is activated by
dual phosphorylation on threonine and tyrosine within the TXY motif in
the activation loop (28). For p38 MAPK, these phosphorylations are
catalyzed by upstream dual-specificity kinases, the MAPK-kinases MKK6
and MKK3 (2, 3). In turn, these MKKs are activated by phosphorylation
on homologous serine and threonine residues (29). We tracked the
phosphorylation states of these phosphor-ylation-dependent kinases
as a measure of their activation states, by means of immunoblotting
with phospho-specific antibodies.
First, immunoblots were performed on ovarian lysates prepared
from rats at various times post PMSG and hCG injections to detect
activation of MKK6 and MKK3 by using a phospho-specific antibody that
recognizes activation-specific phosphorylation sites (29),
phosphoserine 207 of MKK6 and the corresponding phosphoserine 189 of
MKK3, respectively (Fig. 1
, upper
panel). Protein levels were determined by immunoblotting with
control antibodies specific for MKK6 and MKK3 (Fig. 1
, middle and
lower panels, respectively). Both the 35-kDa MKK3 and the 37-kDa
MKK6 showed a small degree of activation by 1 h in response to
both PMSG (Fig. 1
, top panel, lane 2) and hCG (lane 6) in
follicular and periovulatory phases. However, while MKK3 failed to show
sustained activation as luteal maturation progressed (Fig. 1
, lanes
912), strong sustained activation of the 37-kDa MKK6 accompanied
luteal maturation (Fig. 1
, top panel, lanes 812).

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Figure 1. MKK3 and MKK6 Activation during Sequential PMSG-
and hCG-Induced Follicle Maturation, Ovulation, and Luteinization
Immunoblots were performed on ovarian lysate proteins obtained at
indicated times post PMSG injection and post hCG injection. Top
panel, Immunoblot probed with phosphospecific MKK6/MKK3
antibody, detecting phosphorylated active MKK6 (phos MKK6) at 37 kDa
and phosphorylated active MKK3 (phos MKK3) at 35 kDa. Densitometric
quantitation of the 37 kDa phos MKK6 and 35 kDa phos MKK3 bands,
respectively, is represented graphically below. Middle
panel, Immunoblot probed with control MKK6 antibody, detecting
MKK6 (con MKK6) at 37 kDa. Bottom panel, Immunoblot
probed with control MKK3 antibody, detecting MKK3 (con MKK3) at 35 kDa.
Brackets indicate the time periods corresponding to
follicular maturation, periovulatory, and luteal maturation phases.
Immunoblots and corresponding graphs show the results obtained from a
representative time course, from two independent time courses, each
containing a minimum of 11 time points.
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Next, immunoblots were performed on ovarian lysates prepared at various
times post PMSG and post hCG injections to detect activation of p38
MAPK using a phospho-specific antibody that recognizes the activated
form (Fig. 2A
, upper panel).
Protein levels were determined by immunoblotting with control antibody
specific for p38 MAPK (Fig. 2A
, lower panel). p38 MAPK
showed a small degree of activation in response to PMSG in the
follicular phase (Fig. 2A
, top panel, lane 2). p38 MAPK
underwent strong biphasic activation in response to hCG, with initial
acute activation 1 h post hCG (Fig. 2A
, lane 6), and a second
sustained chronic activation phase accompanying luteal maturation (Fig. 2A
, lanes 811).

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Figure 2. p38 MAPK and ERK/MAPK Activation during Sequential
PMSG- and hCG-Induced Follicle Maturation, Ovulation, and Luteinization
Immunoblots were performed on ovarian lysate proteins obtained at
indicated times post PMSG injection and post hCG injection. Panel A,
top panel: immunoblot probed with phospho-specific p38
MAPK antibody, detecting phosphorylated, active p38 MAPK (phos p38);
bottom panel: immunoblot probed with control p38 MAPK
(con p38). Panel B, top panel: immunoblot probed with
phospho-specific ERK/MAPK antibody, detecting phosphorylated active
ERK-1 at 44 kDa and phosphorylated active ERK-2 at 42 kDa;
bottom panel: immunoblot probed with control ERK
antibody, detecting ERK-1 and ERK-2 (indicated as con ERK-1 and con
ERK-2). Both phospho-specific and control immunoblots were subjected to
densitometry, and graphs show the densitometric ratio (phos/con) for
each time point. Immunoblots and corresponding graphs show the results
obtained from a representative time course, from two independent time
courses, each containing a minimum of 11 time points.
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Finally, to compare and contrast the activation pattern of p38 MAPK
with that of the well characterized ERK/MAPKs, immunoblots to detect
activation (phosphorylation-specific antibody, Fig. 2B
, upper
panel), as well as protein expression of ERK/MAPKs (control
antibody, Fig. 2B
, lower panel), were performed on ovarian
lysates prepared at various times post PMSG and post hCG injections.
ERK/MAPK displayed slight activation post PMSG in the follicular phase
(Fig. 2B
, lane 3). Strong acute ERK/MAPK activation was detected at
1 h post hCG in a manner similar to p38 MAPK; however, ERK/MAPKs
failed to undergo a second sustained activation phase as luteal
maturation progressed. The lack of a second sustained luteal ERK/MAPK
activation phase suggests that, unlike p38 MAPK, the ERK/MAPKs are not
poised to participate in phosphorylation events during luteal
maturation.
Inverse Modulation of MAPKAPK-2 and MAPKAPK-3 Expression
Accompanying Hormone-Induced Follicular Maturation and
Luteinization
We proceeded to investigate the ovarian pattern of expression of
the two related p38 MAPK target protein kinases, MAPKAPK-2 and
MAPKAPK-3, in vivo during hormone-induced follicle
maturation, ovulation, and luteinization. We studied alterations in
ovarian expression of each MAPKAPK at various time points post PMSG
injection or post hCG injection.
First, immunoblots to detect protein expression of both MAPKAPKs were
performed on ovarian lysates prepared at various times post PMSG and
post hCG injections. As seen in Fig. 3A
, MAPKAPK-2 protein, detected as two isoforms at 47 and 54 kDa (30, 31),
was abundant at early time points throughout follicular maturation
(lanes 18), and then decreased markedly at later time points post
ovulation, as luteal maturation progressed (lanes 912). In contrast
to the decreased expression of MAPKAPK-2 noted above, MAPKAPK-3 protein
expression, detected at 42 kDa, was minimal at early time points during
follicular development (Fig. 3B
, lanes 17), and was strongly induced
as luteal maturation progressed, increasing from 33 h post hCG
onward through the end of the observation period. (Fig. 3B
, lanes
811).

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Figure 3. Ovarian MAPKAPK-2 and -3 Protein Expression during
Sequential PMSG- and hCG-Induced Follicle Maturation, Ovulation, and
Luteinization
Immunoblots were performed on ovarian lysate proteins obtained at
indicated times post PMSG injection and post hCG injection. Panel A,
top section: immunoblot probed with MAPKAPK-2 antibody,
detecting MAPKAPK-2 as a doublet at 47 and 54 kDa;
bottom section: graphical representation
of densitometric quantitation of the 47- and 54-kDa immunoreactive
MAPKAPK-2 bands, respectively. Panel B, top section:
immunoblot probed with MAPKAPK-3 antibody, detecting MAPKAPK-3 at 42
kDa; bottom section: graphical representation of
densitometric quantitation of the 42-kDa immunoreactive MAPKAPK-3 band.
Immunoblots and corresponding graphs show the results obtained from a
representative time course, from two independent time courses, each
containing a minimum of 11 time points.
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Next, ovarian slices obtained from rats at various times post hCG
injection were subjected to in situ hybridization using cRNA
probes specific to each MAPKAPK, respectively, to detect mRNA
expression. MAPKAPK-2 mRNA signal (Fig. 4A
, top panel), detected in
the earliest time points post hCG (weakly at 0 and more strongly at 1
and 8 h post hCG), diminished in the luteal phase slices (24 h and
33 h post hCG), indicating that loss of MAPKAPK-2 mRNA expression
preceded the loss of MAPKAPK-2 protein expression observed during the
luteal maturation phase (Fig. 3A
). In contrast, MAPKAPK-3 mRNA signal
(Fig. 4A
, middle panel; Fig. 4B
), was minimal in the
earliest time points post hCG (Fig. 4A
, 0 and 1 h; also Fig. 4B
, 4 h), appeared in periovulatory follicles (8 h post hCG), and was
sustained in forming corpora lutea (24 and 33 h post hCG),
indicating that induction of MAPKAPK-3 mRNA paralleled the subsequent
induction of MAPKAPK-3 protein expression observed during luteal
formation and maturation (Fig. 3B
). Hybridization with a cRNA probe
specific for inhibin
was performed on the same slices to serve as a
control to verify ovarian response to hCG, since expression of inhibin
, prominent in preovulatory follicles, is recognized to decrease
during gonadotropin-induced ovulation and luteinization (32). As
expected, inhibin
mRNA expression was strongly evident in
follicular structures in 0 and 1 h post hCG slices and diminished
thereafter (Fig. 4A
, bottom panel).

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Figure 4. Ovarian MAPKAPK-2 and -3 mRNA Expression during
hCG-Induced Ovulation and Corpus Luteum Formation
In situ hybridization was performed on serial ovarian
slices obtained at the indicated times post hCG injection with
antisense [35S]riboprobes for MAPKAPK-2, MAPKAPK-3 and
inhibin , as indicated. Panel A shows film autoradiograms. Panel B
shows emulsion autoradiography. Panel B, bottom, shows
darkfield microscopy of slices hybridized with antisense MAPKAPK-3
(sections B, D, F, and H); top shows the corresponding
brightfield microscopy (sections A, C, E, and G). Arrows
indicate corpora lutea. Control hybridizations with sense riboprobes
showed no binding (not shown). Results are representative of two
independent time courses.
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Phosphorylation State of MAPKAPK Substrates in Vivo:
HSP-27
In light of the striking expression changes in ovarian
MAPKAPKs during follicular maturation and luteinization and in light of
clear evidence of in vivo upstream MKK6/p38 MAPK activation
accompanying luteal maturation, we wished to evaluate the
phosphorylation state of known MAPKAPK substrates for evidence of
modulated phosphorylation in vivo. Two well recognized
MAPKAPK substrates were considered: CREB (see section below) and
HSP-27 (6, 8, 33).
The small heat shock protein HSP-27 from rodent sources can be
phosphorylated by MAPKAPKs on two potential phosphorylation sites
corresponding to serine 15 and serine 86 (34). Phosphorylation on one
or both of these sites results in the appearance of HSP-27
phosphoisoforms displaying distinct migration positions on
two-dimensional isoelectric focusing (IEF)/SDS PAGE gels (19, 35). Two-dimensional immunoblots, to detect phosphoisoforms of HSP-27
as a measure of in vivo HSP-27 phosphorylation state, were
performed on ovarian lysates prepared at various times post PMSG and
post hCG injections. As seen in Fig. 5
(top), all two-dimensional blots displayed spots
corresponding to basic unphosphorylated HSP-27 isoforms (designated by
arrowhead a), more acidic monophosphorylated
HSP-27 phosphoisoforms (designated by arrowheads
b and b'), and diphosphorylated HSP 27
phosphoisoforms (designated by arrowhead c), with no
detectable alteration in HSP-27 phosphoisoform content. We calculated
relative HSP-27 phosphorylation levels from observed densities of spots
corresponding to mono- or diphosphorylated HSP-27 phosphoisoforms (Fig. 5
, bottom). No modulation of HSP-27 phosphorylation levels
was observed in ovarian lysates obtained during either follicular or
luteal phases of development. Thus, the phosphorylation state of HSP-27
did not correlate with the activation state of the p38 MAPK/MAPKAPK-3
axis during the luteal phase; however, HSP-27 phosphorylation could
be maintained at constant levels by alternative mechanisms,
e.g. through phosphorylation by other recognized HSP-27
kinases (35), or alternatively through the action of HSP-27
phosphatases (36, 37). We have recently observed that the
isoform
of protein kinase C (PKC-
), an efficient HSP-27 kinase (35), is
detected in ovarian extracts in a constitutively activated state during
luteinization (L. M. Salvador, E. Maizels, E. Miyamoto, H.
Yamamoto, and M. Hunzicker-Dunn, in preparation) and would
account for the constant phosphorylation of HSP-27 during this
transition.

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Figure 5. HSP-27 Phosphorylation during Hormone-Induced
Follicular and Luteal Maturation
Immunoblots of ovarian lysates collected at indicated times post PMSG
or post hCG injection, separated by two-dimensional electrophoresis and
then probed with HSP-27 antibody are shown. Cathode for IEF is
indicated by (-), and anode for IEF is indicated by (+). Positions of
immunoreactive HSP-27 phosphoisoforms are indicated by
arrowheads marked "a" (unphosphorylated HSP-27),
"b", "b'" (monophosphorylated HSP-27), and "c"
(diphosphorylated HSP-27), respectively. Graph shows HSP-27
phosphorylation index, calculated as the weighted sum of densities
(b+b'+2c). Immunoblots and corresponding graphs show the results
obtained from a representative time course, from two independent time
courses, each containing a minimum of four time points.
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Phosphorylation/Activation of CREB in Vivo, in Vitro,
and in Cellular Models
The nuclear transcription factor CREB has been reported to be a
substrate for both MAPKAPK-2 and MAPKAPK-3 (11, 12, 13), with
phosphorylation on the activation-regulating site serine 133. We
evaluated CREB as a potential target for the p38 MAPK/MAPKAPK
pathway in the ovary in vivo, in vitro in immune
complex kinase assays, by pharmacological studies in a cellular model
of luteinization, and by transfection assays using a CREB-sensitive
reporter.
We evaluated the phosphorylation state of CREB in vivo
during hormone-induced follicle maturation, ovulation, and
luteinization. Immunoblots were performed on ovarian lysates prepared
at various times post PMSG and post hCG injections to detect
phosphorylation of CREB using a phospho-specific antibody that
recognizes CREB phosphorylated on serine 133 (Fig. 6
, upper panel). Protein
levels were determined by immunoblotting with control antibody specific
for CREB (Fig. 6
, lower panel). Increased phosphorylation of
CREB on serine 133 was detected in response to PMSG treatment (Fig. 6
, lanes 2 and 3). Additionally, CREB underwent biphasic phosphorylation
in response to hCG. Phosphorylation of CREB increased acutely at 1
h post hCG (Fig. 6
, lane 6), decreased to a nadir at 8 h post hCG
(Fig. 6
, lane 7), and then began to rise again by 48 h post hCG
(Fig. 6
, lane 9). Densitometric values normalized for protein content,
shown graphically in the lower section of Fig. 6
, indicated
that CREB phosphorylation levels had risen to approximately one third
the maximal value by 48 h post hCG, a level compatible with
the extent of induction of MAPKAPK-3 that has occurred at this
time. Strong CREB phosphorylation was maintained as luteal maturation
progressed (Fig. 6
, lanes 1012). The sustained CREB phosphorylation
observed during later time points of luteal maturation is consistent
with CREB functioning as an in vivo phosphorylation target
for the newly induced MAPKAPK-3.

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Figure 6. CREB Phosphorylation during Sequential PMSG- and
hCG-Induced Follicle Maturation, Ovulation, and Luteinization
Panel A, Immunoblots were performed on ovarian lysate proteins obtained
at indicated times post PMSG injection and post hCG injection. Panel A,
top section: immunoblot probed with phospho-specific
CREB antibody, detecting phosphorylated CREB (phos CREB) as a doublet
at 43 kDa. Panel A, bottom section: immunoblot probed
with control CREB antibody detecting CREB (con CREB) as a doublet at 43
kDa. Phospho-specific and control immunoblots were subjected to
densitometry, and graph shows the densitometric ratio (phos/con) for
each time point. Immunoblots and corresponding graphs show the results
obtained from a representative time course, from two independent time
courses, each containing a minimum of 11 time points.
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Upstream kinase activation, as well as downstream substrate
phosphorylation, provided correlative evidence that MAPKAPK-3 would
be found in an activated state during luteal maturation. We sought
direct evidence for activation of MAPKAPK-3 in vivo during
luteal maturation by use of a MAPKAPK-3-specific immune complex kinase
assay protocol (8). MAPKAPK-3 is activated by p38 MAPK-catalyzed
phosphorylation on threonines 201 and 313 (8). Immunoprecipitations
with MAPKAPK-3-specific antibody were performed in the presence of
phosphatase inhibitors to maintain the phosphorylation/activation state
of MAPKAPK-3 that had been achieved in vivo. The collected
immune complexes were assayed for kinase activity by incubation with
recombinant CREB as substrate in the presence of
[
-32P]ATP, and the resulting
32P incorporation into CREB was demonstrated by
SDS-PAGE and autoradiography. Assays were performed on MAPKAPK-3 immune
complexes collected from ovarian extracts obtained at various times
post hCG injection in vivo (Fig. 7A
). Results showed that there was
minimal [32P]phosphate incorporation into CREB
by immune complexes prepared from early time points (0 and 1 day post
hCG) consistent with the low level of MAPKAPK-3 protein expression at
these times. In contrast, immune complexes collected at later time
points during the luteal maturation phase (2 and 4 days post hCG)
catalyzed increased [32P]phosphate
incorporation into CREB, indicating sustained activation of MAPKAPK-3
accompanying luteal maturation. Control immune complexes, collected
either in the absence of MAPKAPK-3 antibody or in the absence of
ovarian extract, showed no detectable phosphorylation of CREB (Fig. 7A
, right panel). These assay results provide direct evidence
that newly induced MAPKAPK-3 undergoes sustained activation in
vivo during luteal maturation, consistent with sustained
activation of upstream kinases MKK6 and p38 MAPK, and the sustained
phosphorylation of CREB. Additionally, these immune complex kinase
assays confirmed previous reports indicating that MAPKAPK-3 can
directly phosphorylate CREB in vitro (12, 26).

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Figure 7. Modulation of CREB Phosphorylation: Immune Complex
Kinase Assays and Luteinized Granulosa Cell Studies
Panel A, Immune complex kinases assays, to detect activation state of
MAPKAPK-3 immunoprecipitated from ovarian extracts collected at
indicated times post hCG injection, were performed as described in
Materials and Methods, with recombinant CREB as
substrate. Autoradiograms show [32P]phosphate
incorporation into CREB. Control immunoprecipitations, performed either
in the absence of tissue extract but in the presence of antibody, or in
the presence of extract (4 days post hCG) but in the absence of
antibody are shown as indicated. Panel B, left:
Immunoblot was performed to detect MAPKAPK-3 expression in luteinized
granulosa cells (luteinized GCs). Panel B, right:
Immunoblots were performed to detect phosphorylated CREB (ser 133) and
control CREB in luteinized granulosa cells treated with or without 10
µM SB 203580, as indicated. Panel C, Immunoblots to
detect phosphorylated CREB (ser 133) were performed on subcellular
fractions prepared from ovaries obtained at indicated times post hCG
injection in vivo (lanes 16) and on subcellular
fractions prepared from luteinized granulosa cells (lanes 7 and 8), as
described in Materials and Methods. s, Soluble fraction;
p, nuclear-enriched particulate fraction.
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In light of the correlation of MAPKAPK-3 induction and activation with
CREB phosphorylation during luteal maturation in vivo, we
wished to evaluate the extent to which p38 MAPK-dependent events might
contribute to CREB phosphorylation in a luteal context. The pyridinyl
imidazole SB 203580, a competitive p38 MAPK inhibitor that binds to the
ATP binding site and thereby inhibits catalytic activity (38, 39), is
widely used in cell studies to demonstrate the involvement of the p38
MAPK pathway in cellular events. Luteinized granulosa cells, collected
and cultured according to a protocol similar to that employed by
Gonzalez-Robayna et al. (25), provided a suitable
cellular model of luteinization for evaluating the effects of
pharmacological p38 MAPK inhibition, as luteinized granulosa cells
express MAPKAPK-3 (Fig. 7B
, left panel), and these cells
have previously been reported to show persistent CREB phosphorylation
in a cAMP-nonresponsive manner (25). Luteinized granulosa cells were
treated with or without p38 MAPK inhibitor SB 203580 for 4 h and
then harvested and analyzed for CREB phosphorylation by immunoblotting.
As seen in Fig. 7B
, right panel, CREB serine 133
phosphorylation was prominent in control cells and was markedly reduced
by treatment with SB 203580. These data are consistent with CREB
phosphorylation being a p38 MAPK pathway-dependent event in this
cellular model of luteinization.
A recent immunofluorescence study of cultured luteinized granulosa
cells found phosphorylated CREB largely in the soluble compartment, a
compartment in which transcriptional function of phosphorylated CREB
would be abrogated, rather than the expected nuclear compartment in
which phosphorylated CREB functions as a transcription factor (25).
Based on the results of that study, we wished to identify the
subcellular localization of phosphorylated CREB in our in
vivo luteinization model. Ovarian extracts obtained at various
times post hCG injection in vivo were separated by
centrifugation into soluble fractions and nuclear enriched-particulate
fractions, respectively. We additionally prepared soluble and nuclear
enriched-particulate fractions from luteinized granulosa cells to serve
as controls. Immunoblots to visualize phosphorylated CREB were
performed on both soluble and nuclear-enriched particulate fractions
(Fig. 7C
). Results showed that luteinized granulosa cells displayed
phosphorylated CREB primarily in the soluble fraction (Fig. 7C
, lane
7), confirming the report of Gonzales-Robayna et al. (25).
Notably in contrast, phosphorylated CREB was localized to the
nuclear-enriched particulate fraction at all time points examined using
the in vivo luteinization protocol (Fig. 7C
, lanes 2, 4, and
6). Thus, phosphorylated CREB was retained in a functional compartment
in vivo during luteal maturation.
In light of the temporal correlation of upstream kinase
activation, MAPKAPK-3 expression and activation, and sustained CREB
serine 133 phosphorylation during luteal maturation, we wished to
directly evaluate the ability of MAPKAPK-3 to modulate CREB
transcriptional activity in ovarian cells. We employed the sensitive
GAL4-linked CREB transcriptional activity assay system (40, 41). This
assay system measures GAL4-driven transcription activated in response
to phosphorylation of the activating serine residue (corresponding to
serine 133) of the CREB moiety within a fusion protein comprised of
full-length CREB linked to the DNA-binding region of the yeast
transcription factor GAL4. GRMO2 cells, representing a stable mature
granulosa cell line (42), were cotransfected with MAPKAPK-3 cDNAs
together with CREB-GAL4 fusion protein expression vector and GAL4
binding site-luciferase reporter gene construct (Fig. 8
). Cotransfection with 50 ng wild-type
MAPKAPK-3 cDNA increased CREB-GAL4-mediated reporter activity, to
levels approximately 3-fold over basal activity seen with empty vector,
a modest but significant increase (P < 0.05, Fig. 8
).
Notably, the modest reporter activity achieved by MAPKAPK-3 in the
absence of exogenous p38 MAPK activators likely represents partial
activation of wild-type MAPKAPK-3 supported by inclusion of serum in
GRMO2 culture medium, as serum serves as only a mild activator for
MAPKAPK-3 (8). The strong p38 MAPK activators anisomycin and arsenite
were tested as exogenous agents that might further activate MAPKAPK-3;
however, these treatments were toxic to GRMO2 cells at the
concentrations required to activate p38 MAPK and thus could not be
further evaluated. CREB-GAL4-mediated reporter activity in the presence
of wild-type MAPKAPK-3 corresponded to approximately 60% of that
achieved through activation of PKA in the presence of forskolin, and
approximately 20% of that achieved through transfection of PKA
catalytic subunit cDNA (not shown). Importantly, in contrast to
wild-type MAPKAPK-3 cDNA, 50 ng of the kinase-dead K>M MAPKAPK-3
mutant cDNA (8) failed to support increased CREB-GAL4-mediated reporter
activity, indicating that the kinase activity of MAPKAPK-3 is necessary
for the positive effect of MAPKAPK-3 on CREB transcriptional activity.
Thus MAPKAPK-3 can signal to enhance CREB transcriptional activity in
ovarian cells in a kinase-dependent manner, i.e. through
phosphorylation. Based on the results of these cotransfection
experiments, MAPKAPK-3 induced during luteal maturation in
vivo would be capable of signaling to enhance CREB transcriptional
activity through phosphorylation.

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|
Figure 8. CREB Activity in GRMO2 Cells in Response to
MAPKAPK-3
GRMO2 cells were cotransfected in triplicate with a CREB-GAL4 fusion
protein expression vector (50 ng) and a GAL4 binding site-luciferase
reporter gene construct (500 ng, indicated as GAL4BS-LUC) and 50 ng
MAPKAPK-3 expression constructs (empty vector, wild-type MAPKAPK-3 or
kinase-dead K>M mutant MAPKAPK-3) (8 ), as described in
Materials and Methods. Cells were harvested for
luciferase assay 12 h after transfection. Relative luciferase
activities per µg of cellular protein are presented as bar
graphs for each transfection (mean ± SEM,
n = 3). Results are from a single experiment and are
representative of two independent experiments. *, P
< 0.05 for wild- type MAPKAPK-3 compared with empty vector.
|
|
 |
DISCUSSION
|
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It is well established that the preovulatory surge of LH, which is
required for ovulation and luteinization of follicular cells to form
the corpus luteum, promotes the expression of a number of genes in a
time-dependent manner as well as the repression of other genes
(reviewed in Refs. 22, 43). LH, in common with FSH, is well
recognized to promote activation of adenylyl cyclase to generate cAMP,
with the resultant activation of PKA and the phosphorylation of PKA
substrates. Additionally, kinases other than PKA would carry out
critical phosphorylation during luteal maturation, a time when luteal
cells are thought to be cAMP-nonresponsive (24, 25). Based on the known
role of the p38 MAPK path in FSH signaling (19), and based on the known
function of the p38 MAPK-activated MAPKAPKs as CREB kinases, we
examined ovarian expression and activation patterns for the p38 MAPK
cascade in vivo, employing the sequential PMSG- and
hCG-induced luteinization paradigm. We found that an inverse pattern of
MAPKAPK expression accompanied luteinization, as MAPKAPK-2
expression diminished when MAPKAPK-3 appeared. MAPKAPK-3, newly induced
during luteal maturation, is positioned to support critical luteal
phosphorylation.
MAPKAPK-2 (44) and -3 (6, 7) are closely related kinases with 75%
amino acid sequence homology (7), and shared structural features that
include corresponding N-terminal SH3 binding domains, C-terminal
nuclear localization sequences, C-terminal
-helices, and analogous
activating phosphorylation sites (7). The mechanism of activation has
been defined for MAPKAPK-2: phosphorylation of activating sites by p38
MAPK (45) induces a conformational change that moves the inhibitory
C-terminal
helix-regulatory region away from the kinase catalytic
domain, resulting in activation (46). Conservation of the involved
structures in MAPKAPK-3 (7) suggests that the mechanism of activation
would be comparable. MAPKAPK-2 and -3 share common upstream activators
and overlapping (6, 8, 12), although not identical (7), substrate
specificities. Indeed, MAPKAPK-2 and MAPKAPK-3 have often been treated
as interchangeable (47, 48). However, the striking inverse pattern of
expression of MAPKAPKs during follicle maturation, ovulation, and
subsequent luteal development in the rat suggests that these enzymes
may, in fact, play unique rather than interchangeable roles in the
ovary.
Upstream components of the p38 MAPK cascade displayed discrete patterns
of activation in response to gonadotropin treatments in
vivo. p38 MAPK underwent initial activation in response to both
PMSG and hCG, as well as a sustained chronic activation phase
accompanying luteal maturation. Initial responses to PMSG and hCG were
accompanied by activation of upstream MKKs MKK3 and MKK6, while the
luteal phase response was accompanied by activation of MKK6
exclusively. The initial modest follicular phase activation of p38 MAPK
in response to PMSG is consistent with the previous finding that FSH
could stimulate p38 MAPK activation in immature rat granulosa cells
(19, 20). The initial acute activation of p38 MAPK in response to hCG
activation is interesting and is consistent with the possibility that
p38 MAPK-dependent signaling may participate in the mediation or
modulation of hCG-stimulated events critical to ovulation, such as the
induction of PG synthase-2 or progesterone receptor (43, 49, 50). The
sustained chronic activation of p38 MAPK during luteal maturation would
provide for the observed sustained activation of the newly induced
luteal kinase MAPKAPK-3, as monitored by immune complex kinase
assays.
What stimuli support extended p38 MAPK activation and thus MAPKAPK-3
activation during luteal maturation in the ovary in vivo?
While the gonadotropins FSH and hCG indeed elicit acute activation of
p38 MAPK in immature and preovulatory granulosa cells, respectively
(Refs. 19, 20 ; L. M. Salvador, E. Maizels, E. Miyamoto, H. Yamamoto,
and M. Hunzicker-Dunn, in preparation), and treatment with hCG
elicited p38 MAPK activation acutely in the periovulatory period in the
current study, hCG would be unlikely to be responsible for prolonged
luteal phase activation, as LH receptors would have undergone both
desensitization and down-regulation in response to the ovulatory
stimulus (23). In addition to gonadotropins, a number of cytokines and
growth factors are recognized to participate in control of ovarian
function (51, 52). Interleukin-1ß (IL-1ß), a cytokine known to
elicit p38 MAPK activation in target cells (53), is transiently induced
in the periovulatory period (54, 55, 56) and may participate in the
periovulatory activation of p38 MAPK. However, IL-1ß levels have
fallen by 48 h post hCG (54), and thus this cytokine would be
unlikely to represent the prolonged luteal-phase p38 MAPK stimulus. The
growth factors insulin-like growth factor-1 (IGF-I) and fibroblast
growth factor (FGF) can each elicit p38 MAPK/MAPKAPK activation leading
to CREB phosphorylation in other experimental systems (11, 13),
suggesting that these growth factors would be capable of serving as
initiating stimuli during luteal maturation. IGF-I is well recognized
to synergize with gonadotropins to elicit granulosa cell function and
differentiation (52, 57). Moreover, IGF-I receptor levels (58) and
basic FGF levels (59) are elevated during early luteal development in
the rat. Finally, the proangiogenic growth factor vascular endothelial
growth factor (VEGF), a critical factor in corpus luteum formation and
growth (60, 61, 62, 63), is appreciated to activate p38 MAPK in target cells
(64). Thus, the growth factors IGF-I, FGF, and VEGF comprise a set of
likely candidates for luteal-phase stimulus to support the prolonged
activation of p38 MAPK and MAPKAPK-3. Further experiments will focus on
delineating the contribution of each of these critical growth
factors.
CREB was evaluated as a potential ovarian target for newly induced and
activated MAPKAPK-3 during luteal maturation. The nuclear transcription
factor CREB requires phosphorylation on serine 133 to bind the
coactivator CREB-binding protein (CBP) and recruit transcriptional
machinery (65). CREB was initially described as a PKA substrate (66);
however, CREB is now recognized to serve as a phosphorylation target
for a number of distinct kinases in several diverse signaling pathways.
Several groups have demonstrated CREB serine 133 phosphorylation
catalyzed by kinases downstream of activated p38 MAPK (11, 67, 68, 69). p38
MAPK-dependent CREB phosphorylation was catalyzed by MAPKAPK-2 or a
closely related kinase such as MAPKAPK-3 in FGF-treated fibroblasts
(11), and by MAPKAPK-3 in IGF-I-treated PC12 cells (13). Moreover,
MAPKAPK-3 readily phosphorylates CREB in vitro (12, 26), a
finding we have confirmed in the current study through immune complex
kinase assays.
CREB underwent clear phosphorylation coinciding with the induction of
and activation of MAPKAPK-3. We observed three peaks of CREB
phosphorylation in vivo, the first during hormone-induced
follicular maturation, the second acute peak in response to hCG in the
periovulatory follicle, and the third chronic peak during luteal
maturation. The first and second peaks of CREB phosphorylation
correspond to previously described cAMP-responsive PKA-mediated events
(25, 70, 71). In contrast, the third luteal-phase CREB phosphorylation
represents what is thought to be a cAMP-nonresponsive event (25);
therefore, this phase of phosphorylation is expected to be catalyzed by
a CREB kinase other than PKA. Notably, we detected substantial
sustained phosphorylation of CREB accompanying the increased expression
and activation of MAPKAPK-3 as well as the activation of upstream
kinases MKK6/p38 MAPK as luteal maturation progressed.
To further delineate the role of the p38 MAPK pathway in CREB
phosphorylation during luteinization, we performed additional studies
with the p38 MAPK inhibitor SB 203580 (38). We found that SB 203580
strongly inhibited CREB phosphorylation in luteinized granulosa cells.
These studies are consistent with a requirement for p38 MAPK- mediated
signaling events in CREB phosphorylation in this luteal model, in good
agreement with in vivo luteal maturation phase profiles.
However, it has been suggested recently that p38 inhibitor SB 203580
can impact other signaling pathways in addition to p38 MAPK.
Specifically, SB 203580 can impact signaling through ERK (72) and
phosphatidylinositol-3 kinase/Akt pathways (73, 74) either through
direct interaction with other kinases (72, 73) or through inhibition of
cross-talk between p38 MAPK and other kinases (74). We can detect both
ERK and Akt activation in the follicular and periovulatory periods
in vivo (Fig. 2B
and E. T. Maizels, L. M. Salvador, J. E.
Cottom, and M. Hunzicker-Dunn, manuscript in preparation).
However, we detect neither ERK nor Akt activation during the luteal
maturation phase (48 h to 6 days post hCG); thus neither of these
kinase pathway can be implicated in CREB phosphorylation during the
luteal maturation phase in vivo (Fig. 2B
and E. T. Maizels,
L. M. Salvador, J. E. Cottom, and M. Hunzicker-Dunn, manuscript in
preparation). In light of the lack of participation of either ERK or
Akt in luteal-phase downstream signaling events in vivo, the
role of the p38 MAPK path in luteal-phase CREB phosphorylation is
unambiguous.
We investigated phosphorylation of an additional MAPKAPK substrate, the
small heat shock protein HSP-27. In contrast to CREB, we found no
modulation in vivo of the phosphorylation state of HSP-27, a
finding that would be explained by the constitutive activation of an
alternate HSP-27 kinase, PKC-
(35), accompanying ovulation and
luteinization (Salvador et al., in preparation).
Findings implicating newly induced MAPKAPK-3 as CREB kinase in
vivo and in vitro were reinforced by transfection
studies, which yielded direct evidence that MAPKAPK-3 could signal to
activate CREB transcriptional activity in a phosphorylation-dependent
manner in ovarian cells.
Previous reports indicate that luteal CREB expression and/or function
can vary depending on the choice of experimental model of luteinization
employed. For example CREB expression is maintained in luteinizing
granulosa cells in the rat (25, 70), although CREB protein expression
is completely lost upon luteinization in the primate (75), indicating
that there is species specificity in luteal CREB expression.
Additionally, a recent study detected phosphorylated CREB primarily
localized to the soluble compartment in luteinized granulosa cells in
primary cell culture (25), a compartment in which phosphorylated CREB
would be unable to fulfill its function as a transcription factor. We
therefore evaluated the subcellular localization of phosphorylated CREB
in our in vivo luteinization model. We were able to confirm
that phosphorylated CREB is indeed a soluble protein in cultured
luteinized granulosa cells. In contrast, phosphorylated CREB was
primarily localized in the nuclear-enriched particulate fraction in the
in vivo ovarian samples at all time points, including the
4-day post-hCG time point coinciding with MAPKAPK-3 expression as well
as upstream p38 MAPK/MKK6 activation. Thus, phosphorylated CREB is
retained in a subcellular compartment compatible with its function as a
transcription factor during luteal maturation in vivo.
CREB phosphorylation has been previously implicated in the
transcriptional regulation of several important ovarian target genes
(43), making it an interesting potential target for MAPKAPKs in the
ovary. It is well established that CREB participates in transcriptional
activation of the CYP19 aromatase gene (76, 77). Aromatase catalyzes
the conversion of androgen precursor to estrogen. Aromatase, initially
induced by FSH (or PMSG) in granulosa cells of maturing follicles, is
lost in response to the ovulatory LH surge (78, 79) but reappears in
the corpus luteum by early- to mid-pregnancy to allow production of
estrogen by the maturing corpus luteum (80, 81). Notably, in an
analogous in vivo hCG-stimulated rat luteinization model,
aromatase mRNA was induced by 3-day post hCG (82), correlating well
with the peak of phosphorylated CREB observed during luteal maturation
in our study. The strong temporal correlation indicates aromatase as a
potential transcriptional target for MAPKAPK-3-catalyzed CREB
phosphorylation accompanying luteal maturation.
In summary, we have described the developmental pattern of regulation
of components of the p38 MAPK cascade as ovarian follicles undergo
PMSG-induced maturation followed by hCG-induced ovulation, luteal
formation, and luteal maturation. The closely related kinases MAPKAPK-2
and MAPKAPK-3 underwent inverse changes in expression level, with
loss of MAPKAPK-2 mRNA and protein expression, and induction of
MAPKAPK-3 mRNA and protein expression accompanying these developmental
transitions. During the luteal maturation phase, MAPKAPK-3 induction
was accompanied by sustained activation of upstream activating kinases
p38 MAPK and MKK6, and by sustained phosphorylation of its substrate
CREB. MAPKAPK-3, activated during luteal maturation in vivo,
readily catalyzed CREB phosphorylation in immune complex kinase assays,
and phosphorylation of CREB was shown to depend on an intact p38 MAPK
signaling pathway in a cellular model of luteinization. Wild-type, but
not kinase-dead, MAPKAPK-3 enhanced CREB transcriptional activity in
cotransfection studies, demonstrating directly MAPKAPK-3s ability to
signal to activate CREB. Thus MAPKAPK-3 is indeed uniquely poised to
support luteal maturation through the phosphorylation and activation of
the nuclear transcription factor CREB. Further studies will attempt to
uncover the stimulus of MKK6/p38 MAPK activation, as well as define
transcriptional targets of MAPKAPK-3/CREB during luteal maturation.
 |
MATERIALS AND METHODS
|
---|
Materials
The following were purchased from indicated vendors or kindly
provided by indicated colleagues: MAPKAPK-2 antibody, StressGen
Biotechnology (Victoria, British Columbia, Canada); HSP-27 monoclonal
antibody, Dr. Michael Welsh, University of Michigan, Ann Arbor, MI
(83); phospho-specific p38 MAPK (T180,Y182) and monoclonal ERK/MAPK
(T202,Y204) antibodies, New England Biolabs, Inc. (Beverly, MA);
control p38 MAPK, MKK3 and MKK6 antibodies, and agarose-linked protein
A+G, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA);
phospho-specific MKK3 (S189)/MKK6 (S207) antibody and SB 203580,
Calbiochem (San Diego, CA); phospho-specific CREB (S133)
and control CREB antibodies, Upstate Biotechnology, Inc.
(Lake Placid, NY); control ERK/MAPK antibody, Zymed Laboratories, Inc. (South San Francisco, CA); PAGE reagents and Bradford
protein assay reagents, Bio-Rad Laboratories, Inc.
(Richmond, CA); luciferin (sodium salt), Analytical Luminescence laboratory (San Diego, CA); other chemicals, Sigma
(St. Louis, MO). Polyclonal MAPKAPK-3/3pK antibody was produced as
described (7). Wild-type MAPKAPK-3 cDNA and kinase-dead K>M mutant
MAPKAPK-3(73K>M) cDNA were prepared and
transfected as previously described (8). The expression plasmid coding
for the CREB-GAL4 fusion protein comprised of full-length CREB (1341)
linked to the DNA binding domain of GAL4 (1147) was provided by Drs.
J. Kornhauser and M. E. Greenberg, Childrens Hospital, Boston MA
(40). The GAL4-binding site-luciferase reporter gene construct,
comprised of five tandem repeats of the GAL4 binding sequence, TATA
box, and luciferase reporter gene, was obtained from
Stratagene (La Jolla, CA). Recombinant CREB was provided
by Dr. R. A. Maurer, Oregon Health Sciences University, Portland,
OR (84).
Animals
Sprague Dawley rats (Charles River Laboratories, Inc. Portage, MI) were housed at Northwestern University animal
care facilities, maintained in accordance with the "Guidelines for
the Care and Use of Laboratory Animals" by protocols approved by the
Northwestern University ACUC committee. With the exception of
luteinized granulosa cell culture experiments performed as described
below, immature female rats (2627 days old) were injected
subcutaneously with 25 IU PMSG. Indicated animals were further injected
subcutaneously with 25 IU of hCG 48 h following PMSG injection.
Ovaries were harvested at the indicated times post PMSG injection or
post hCG injection and either immediately frozen at -70 C for
subsequent in situ hybridization analysis or subjected to
tissue extract preparations as described below.
Tissue Extract Preparation
Whole ovarian extracts were prepared by homogenization in lysis
buffer (15) containing 10 mM potassium phosphate, pH 7.0, 1
mM EDTA, 5 mM EGTA, 10 mM
MgCl2, 50 mM ß-glycerophosphate, 1
mM Na orthovanadate, 2 mM dithiothreitol, 1
mM phenylmethylsulfonyl fluoride, 0.5% NP-40, and 0.1%
sodium deoxycholate. A clarified lysate, containing both soluble
proteins and detergent-solubilized membrane proteins, was obtained by
centrifuging the homogenate at 12,000 x g for 10 min
at 4 C. Alternatively, subcellular fractionation was performed by
homogenization of ovaries in protease- and
phosphatase-inhibitor-enriched homogenization buffer (PPI buffer) as
described previously (85), followed by centrifugation at 105,000
x g for 70 min. The separated soluble and nuclear-enriched
particulate fractions were prepared for SDS-PAGE by suspension in equal
volumes of SDS-containing sample buffer followed by heat denaturation
(100 C, 5 min).
Protein concentrations were measured by the method of Lowry et
al. (86) using crystalline BSA as a standard.
Luteinized Granulosa Cell Culture
Primary culture of luteinized granulosa cells was performed as
previously described (85). Briefly, immature rats were injected sc with
0.15 IU hCG for 2 days. On the third day, rats were injected sc with 10
IU hCG, and ovaries were removed 7 h post injection. Granulosa
cells from large preovulatory follicles were cultured for 9 days in the
presence of 1% FBS as described (85). For inhibitor studies, 9-day
cultured cells were removed from serum for 14 h, and then
subjected to 4-h treatments with vehicle or 10 µM SB
203580, and cell lysates were prepared in the presence of lysis buffer
as described in Tissue Extract Preparation. Alternatively,
9-day cultured cells were harvested and homogenized in the presence of
PPI buffer and subjected to subcellular fractionation as described
above.
Protein Separation
Separation of ovarian lysate proteins was by SDS-PAGE using 10%
or 12% separating gels (87). For two-dimensional gel electrophoresis,
ovarian lysate proteins were separated by isoelectric focusing using
mixed ampholines (4 parts pH range of 58 with 1 part pH range 310),
and then by SDS-PAGE (87). For immunoblots, proteins were
electrophoretically transferred to Hybond Nitrocellulose C-extra,
incubated with primary antibody overnight at 4 C, and detected by
enhanced chemiluminescence (Amersham Pharmacia Biotech).
Densitometry was analyzed with Molecular Analyst software
(Bio-Rad Laboratories, Inc.).
In Situ Hybridization
Twenty-micrometer sections of frozen ovaries were prepared using
a Reichert 820 cryostat (AO/Reichert, Buffalo, NY) and mounted
onto gelatin-coated glass slides for in situ hybridization
as described previously (88). Hybridization probes used were
[35S]UTP-labeled riboprobes derived from the
full-length rat inhibin
-subunit cDNA (89, 90), a 243-bp long
fragment of rat MAPKAPK-2 cDNA corresponding to amino acids 119199 of
mouse MAPKAPK-2 (44), and a 216-bp long fragment of rat MAPKAPK-3
isolated by RT-PCR and corresponding to amino acids 267338 of human
MAPKAPK-3 (7). Hybridization was continued for 1218 h at 47 C in a
humidified chamber. Sense riboprobes were used as controls.
Subsequently, the slides were washed to a final stringency of 0.1x SSC
at 65 C after a 1 h treatment with 20 µg/ml RNAse at 37 C.
Slides were then processed for film and emulsion autoradiography
(NTB-2, Eastman Kodak Co., Rochester, NY). Exposure time
on film was 3 days and on emulsion was 2 weeks. After development of
the slides, they were stained with hematoxylin to visualize the nuclei.
The sections were then examined and photographed using a microscope
(Nikon Optiphot, Nippon Kogaku (USA) Inc., Garden City,
NY) or the film autoradiograms were scanned using a Microtek flatbed
scanner.
Immune Complex Kinase Assays
Ovarian lysates (500 µg protein) were subjected to
immunoprecipitation (IP), as described previously (8), in the presence
of 10 µl of MAPKAPK-3-specific antibody and 30 µl agarose-linked
protein A+G in 500 µl incubation volume for 2 h at 4 C.
Additional control IPs were done in the absence of antibody (with 4 day
post hCG lysate), or with antibody but in the absence of lysate.
Complexes were collected by centrifugation, washed three times with
RIPA (10 mM Tris-HCl, pH 7.2, 150 mM NaCl,
1.0% deoxycholate, 1.0% Triton X-100, 0.1% SDS, 1 mM Na
orthovanadate, 40 µg/ml phenylmethylsulfonylfluoride) and then once
with TE (10 mM Tris, pH 7.5, 0.1 mM EGTA).
Complexes were resuspended in 50 µl TE, and assayed for kinase
activity for 7 min at 30 C in 115 µl reaction volume in the presence
of 42 mM
-glycerolphosphate, pH 7.0, 8.4 mM
MgCl2, 0.8 mM dithiothreitol, 42
mM ATP, 4.8 µCi [
-32P]ATP, and
4.8 µg purified recombinant CREB. Reactions were terminated by
addition of 50 µl of SDS stop solution and heat denaturation (100 C,
5 min).
GRMO2 Cell Culture, Transfection, and Luciferase Assays
Cationic liposomes, prepared as described (91), were used for
transient transfection (92) of GRMO2 cells (42) (provided by N.V.
Innogenetics, Ghent, Belgium) that were cultured as described (42, 93)
in HDTIS (DMEM-F12, 1:1, 10 µg/ml insulin, 5 nM sodium
selenite, 5 µg/ml transferrin, and 100 mg/liter sodium pyruvate)
supplemented with 2% FBS in a humidified incubator at 37 C and 5%
CO2. DNA for transfection was preincubated at
room temperature with lipofection reagent for 2030 min in OptiMEM and
then added to cells washed with PBS. GRMO2 cells, grown in 12-well
culture dishes, were transfected (per well) with 500 ng of a GAL4
binding site-luciferase reporter plasmid DNA and 50 ng of CREB-GAL4
fusion protein expression construct, and 50 ng of MAPKAPK-3 constructs
(empty MAPKAPK-3 vector, wild-type MAPKAPK-3, or kinase-dead K>M
mutant MAPKAPK-3), as indicated. After 6 h of transfection, the
DNA-lipid mixture was replaced with fresh HDTIS containing 2% FBS.
Cells were incubated for 12 h, washed with PBS, and then subjected
to lysis by gentle agitation on ice in the presence of cell-lysis
buffer (25 mM HEPES pH 7.8, 15 mM
MgSO4, 1 mM dithiothreitol, 0.1%
Triton X-100). Luciferase assays were performed essentially as
described previously (94). One hundred microliters of the cell lysates
were added to 400 µl of assay buffer (25 mM HEPES, pH
7.8, 15 mM MgSO4, 5 mM
ATP, 1 µg/ml BSA), and then 100 µl of 1 mM luciferin
were added and emitted luminescence was measured using a 2010
luminometer (Analytical Luminescence, San Diego, CA) for 10 sec.
Protein content of cell lysates was determined by the Bradford method
(95). Results were analyzed using Students t test
(P < 0.05) (96).
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Michael J. Welsh (University of Michigan Medical
School, Ann Arbor, MI), Dr. Richard A. Maurer (Oregon Health Sciences
University, Portland, OR), and Drs. John Kornhauser and Michael
Greenberg (Childrens Hospital, Boston, MA) for kindly providing
reagents. We thank N.V. Innogenetics, Ghent, Belgium for providing
GRMO2 cells. We thank Qiong Wang for performing the immunoblot of
MAPKAPK-3 in luteinized granulosa cells.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Mary Hunzicker-Dunn, Department of Cell and Molecular Biology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611-3008. E-mail:
mhd{at}northwestern.edu
This research was supported by the following grants: NIH PO1 HD-21921
(M.H.D., K.E.M.), P30 Center grant HD-28048 (K.E.M.) Training Programs
in Reproductive Biology T32 HD-07068 (A.M.) and Endocrinology T32
DK-07169 (C.A.P.), and National Cancer Institute contract no.
NO1-CO-56000 (G.S.).
1 Current address: Fred Hutchinson Cancer Research Center, Seattle,
Washington 98109. 
2 Current address: Cancer Research Institute, University of
California, San Francisco California 94115. 
3 Current address: Wyeth-Ayerst Laboratories, Inc.
Womens Health Research Institute, Radnor Pennsylvania 19087. 
Received for publication March 3, 2000.
Revision received February 6, 2001.
Accepted for publication February 9, 2001.
 |
REFERENCES
|
---|
-
Widmann C, Gibson S, Jarpe MB, Johnson GL 1999 Mitogen-activated protein kinase: conservation of a three-kinase module
from yeast to human. Physiol Rev 79:143180[Abstract/Free Full Text]
-
Derijard B, Raingeaud J, Barrett T, Wu IH, Han J, Ulevitch
RJ, Davis RJ 1995 Independent human MAP-kinase signal transduction
pathways defined by MEK and MKK isoforms. Science 267:682685[Medline]
-
Moriguchi T, Toyoshima F, Gotoh Y, Iwamatsu A, Irie K, Mori
E, Kuroyanagi N, Hagiwara M, Matsumoto K, Nishida E 1996 Purification
and identification of a major activator for p38 from osmotically
shocked cells. Activation of mitogen-activated protein kinase kinase 6
by osmotic shock, tumor necrosis factor-
, and
H2O2. J Biol Chem 271:2698126988[Abstract/Free Full Text]
-
Rouse J, Cohen P, Trigon S, Morange M, Alonso-Llamazares A,
Zamanillo D, Hunt T, Nebreda AR 1994 A novel kinase cascade triggered
by stress and heat shock that stimulates MAPKAP kinase-2 and
phosphorylation of the small heat shock proteins. Cell 78:10271037[Medline]
-
Freshney NW, Rawlinson L, Guesdon F, Jones E, Cowley S, Hsuan
J, Saklatvala J 1994 Interleukin-1 activates a novel protein kinase
cascade that results in the phosphorylation of Hsp27. Cell 78:10391049[Medline]
-
McLaughlin MM, Kumar S, McDonnell PC, Van Horn S, Lee JC,
Livi GP, Young PR 1996 Identification of mitogen-activated protein
(MAP) kinase-activated protein kinase-3, a novel substrate of CSBP p38
MAP kinase. J Biol Chem 271:84888492[Abstract/Free Full Text]
-
Sithanandam G, Latif F, Duh FM, Bernal R, Smola U, Li H,
Kuzmin I, Wixler V, Geil L, Shrestha S 1996 3pK, a new
mitogen-activated protein kinase-activated protein kinase located in
the small cell lung cancer tumor suppressor gene region. Mol Cell Biol 16:868876[Abstract]
-
Ludwig S, Engel K, Hoffmeyer A, Sithanandam G, Neufeld B,
Palm D, Gaestel M, Rapp UR 1996 3pK, a novel mitogen-activated protein
(MAP) kinase-activated protein kinase, is targeted by three MAP kinase
pathways. Mol Cell Biol 16:66876697[Abstract]
-
Landry J, Huot J 1995 Modulation of actin dynamics during
stress and physiological stimulation by a signaling pathway involving
p38 MAP kinase and heat-shock protein 27. Biochem Cell Biol 73:703707[Medline]
-
Welsh MJ, Gaestel M 1998 Small heat-shock protein family:
function in health and disease. Ann NY Acad Sci 851:2835[Free Full Text]
-
Tan Y, Rouse J, Zhang A, Cariati S, Cohen P, Comb MJ 1996 FGF
and stress regulate CREB and ATF-1 via a pathway involving p38 MAP
kinase and MAPKAP kinase-2. EMBO J 15:46294642[Abstract]
-
Clifton AD, Young PR, Cohen P 1996 A comparison of the
substrate specificity of MAPKAP kinase-2 and MAPKAP kinase-3 and their
activation by cytokines and cellular stress. FEBS Lett 392:209214[CrossRef][Medline]
-
Pugazhenthi S, Miller E, Sable C, Young P, Heidenreich KA,
Boxer LM, Reusch JE 1999 Insulin-like growth factor-I induces bcl-2
promoter through the transcription factor cAMP-response element-binding
protein. J Biol Chem 274:2752927535[Abstract/Free Full Text]
-
Keel BA, Hildebrandt JM, May JV, Davis JS 1995 Effects of
epidermal growth factor on the tyrosine phosphorylation of
mitogen-activated protein kinases in monolayer cultures of porcine
granulosa cells. Endocrinology 136:11971204[Abstract]
-
Das S, Maizels ET, DeManno D, St, Adam SA, Hunzicker-Dunn M 1996 A stimulatory role of cyclic adenosine 3',5'-monophosphate in
follicle-stimulating hormoneactivated mitogen-activated protein
kinase signaling pathway in rat ovarian granulosa cells. Endocrinology 137:967974[Abstract]
-
Cameron MR, Foster JS, Bukovsky A, Wimalasena J 1996 Activation of mitogen-activated protein kinases by gonadotropins and
cyclic adenosine 5'-monophosphates in porcine granulosa cells. Biol
Reprod 55:111119[Abstract]
-
Babu PS, Krishnamurthy H, Chedrese PJ, Sairam MR 2000 Activation of extracellular-regulated kinase pathways in ovarian
granulosa cells by the novel growth factor type 1 follicle-stimulating
hormone receptor. Role in hormone signaling and cell proliferation.
J Biol Chem 275:2761527626[Abstract/Free Full Text]
-
Chen DB, Westfall SD, Fong HW, Roberson MS, Davis JS 1998 Prostaglandin F2alpha stimulates the Raf/MEK1/mitogen-activated protein
kinase signaling cascade in bovine luteal cells. Endocrinology 139:38763885[Abstract/Free Full Text]
-
Maizels ET, Cottom J, Jones JC, Hunzicker-Dunn M 1998 Follicle
stimulating hormone (FSH) activates the p38 mitogen-activated protein
kinase pathway, inducing small heat shock protein phosphorylation and
cell rounding in immature rat ovarian granulosa cells. Endocrinology 139:33533356[Abstract/Free Full Text]
-
Gonzalez-Robayna IJ, Falender AE, Ochsner S, Firestone GL,
Richards JS 2000 Follicle-stimulating hormone (FSH) stimulates
phosphorylation and activation of protein kinase B (PKB/Akt) and serum
and glucocorticoid-induced kinase (Sgk): evidence for A
kinase-independent signaling by FSH in granulosa cells. Mol Endocrinol 14:12831300[Abstract/Free Full Text]
-
Rueda BR, Hendry IR, Ndjountche L, Suter J, Davis JS 2000 Stress-induced mitogen-activated protein kinase signaling in the corpus
luteum. Mol Cell Endocrinol 164:5967[CrossRef][Medline]
-
Richards JS, Fitzpatrick SL, Clemens JW, Morris JK, Alliston
T, Sirois J 1995 Ovarian cell differentiation: a cascade of multiple
hormones, cellular signals, and regulated genes. Recent Prog Horm Res 50:223254[Medline]
-
Hunzicker-Dunn M, Birnbaumer L 1976 Adenylyl cyclase
activities in ovarian tissues. III. Regulation of responsiveness to LH,
FSH, and PGE1 in the prepubertal, cycling, pregnant, and pseudopregnant
rat. Endocrinology 99:198210[Abstract]
-
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]
-
Gonzalez-Robayna IJ, Alliston TN, Buse P, Firestone GL,
Richards JS 1999 Functional and subcellular changes in the
A-kinase-signaling pathway: relation to aromatase and Sgk expression
during the transition of granulosa cells to luteal cells. Mol
Endocrinol 13:13181337[Abstract/Free Full Text]
-
Neufeld B, Grosse-Wilde A, Hoffmeyer A, Jordan BW, Chen P,
Dinev D, Ludwig S, Rapp UR 2000 Serine/Threonine kinases 3pK and
MAPK-activated protein kinase 2 interact with the basic
helix-loop-helix transcription factor E47 and repress its
transcriptional activity. J Biol Chem 275:2023920242[Abstract/Free Full Text]
-
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]
-
Raingeaud J, Gupta S, Rogers JS, Dickens M, Han J, Ulevitch
RJ, Davis RJ 1995 Pro-inflammatory cytokines and environmental stress
cause p38 mitogen-activated protein kinase activation by dual
phosphorylation on tyrosine and threonine. J Biol Chem 270:74207426[Abstract/Free Full Text]
-
Raingeaud J, Whitmarsh AJ, Barrett T, Derijard B, Davis RJ 1996 MKK3- and MKK6-regulated gene expression is mediated by the p38
mitogen-activated protein kinase signal transduction pathway. Mol Cell
Biol 16:12471255[Abstract]
-
Huot J, Lambert H, Lavoie JN, Guimond A, Houle F, Landry J 1995 Characterization of 45-kDa/54-kDa HSP27 kinase, a stress-sensitive
kinase which may activate the phosphorylation-dependent protective
function of mammalian 27-kDa heat-shock protein HSP27. Eur J
Biochem 227:416427[Abstract]
-
Cano E, Doza YN, Ben-Levy R, Cohen P, Mahadevan LC 1996 Identification of anisomycin-activated kinases p45 and p55 in murine
cells as MAPKAP kinase-2. Oncogene 12:805812[Medline]
-
Woodruff TK, Mayo KE 1990 Regulation of inhibin synthesis in
the rat ovary. Annu Rev Physiol 52:807821[CrossRef][Medline]
-
Stokoe D, Engel K, Campbell DG, Cohen P, Gaestel M 1992 Identification of MAPKAP kinase 2 as a major enzyme responsible for the
phosphorylation of the small mammalian heat shock proteins. FEBS Lett 313:307313[CrossRef][Medline]
-
Gaestel M, Schroder W, Benndorf R, Lippmann C, Buchner K,
Hucho F, Erdmann VA, Bielka H 1991 Identification of the
phosphorylation sites of the murine small heat shock protein hsp25.
J Biol Chem 266:1472114724[Abstract/Free Full Text]
-
Maizels ET, Peters CA, Kline M, Cutler Jr RE, Shanmugam M,
Hunzicker-Dunn M 1998 Heat-shock protein-25/27 phosphorylation by the
isoform of protein kinase C. Biochem J 332:703712[Medline]
-
Cairns J, Qin S, Philp R, Tan YH, Guy GR 1994 Dephosphorylation of the small heat shock protein Hsp27 in
vivo by protein phosphatase 2A. J Biol Chem 269:91769183[Abstract/Free Full Text]
-
Gaestel M, Benndorf R, Hayess K, Priemer E, Engel K 1992 Dephosphorylation of the small heat shock protein hsp25 by
calcium/calmodulin-dependent (type 2B) protein phosphatase. J Biol
Chem 267:2160721611[Abstract/Free Full Text]
-
Young PR, McLaughlin MM, Kumar S, Kassis S, Doyle ML, McNulty
D, Gallagher TF, Fisher S, McDonnell PC, Carr SA, Huddleston MJ, Seibel
G, Porter TG, Livi GP, Adams JL, Lee JC 1997 Pyridinyl imidazole
inhibitors of p38 mitogen-activated protein kinase bind in the ATP
site. J Biol Chem 272:1211612121[Abstract/Free Full Text]
-
Kumar S, Jiang MS, Adams JL, Lee JC 1999 Pyridinylimidazole compound SB 203580 inhibits the activity but
not the activation of p38 mitogen-activated protein kinase. Biochem
Biophys Res Commun 263:825831[CrossRef][Medline]
-
Sheng M, Thompson MA, Greenberg ME 1991 CREB: a
Ca(2+)-regulated transcription factor phosphorylated by
calmodulin-dependent kinases. Science 252:14271430[Medline]
-
Pugazhenthi S, Boras T, OConnor D, Meintzer MK, Heidenreich
KA, Reusch JE 1999 Insulin-like growth factor I-mediated activation of
the transcription factor cAMP response element-binding protein in PC12
cells. Involvement of p38 mitogen-activated protein kinase-mediated
pathway. J Biol Chem 274:28292837[Abstract/Free Full Text]
-
Briers TW, van de Voorde, Vanderstichele H 1993 Characterization of immortalized mouse granulosa cell lines. In Vitro
Cell Dev Biol Anim 29A:847854
-
Richards JS 1994 Hormonal control of gene expression in the
ovary. Endocr Rev 15:725751[Medline]
-
Engel K, Plath K, Gaestel M 1993 The MAP kinase-activated
protein kinase 2 contains a proline-rich SH3-binding domain. FEBS Lett 336:143147[CrossRef][Medline]
-
Ben-Levy R, Leighton IA, Doza YN, Attwood P, Morrice N,
Marshall CJ, Cohen P 1995 Identification of novel phosphorylation sites
required for activation of MAPKAP kinase-2. EMBO J 14:59205930[Abstract]
-
Engel K, Schultz H, Martin F, Kotlyarov A, Plath K, Hahn M,
Heinemann U, Gaestel M 1995 Constitutive activation of
mitogen-activated protein kinase-activated protein kinase 2 by mutation
of phosphorylation sites and an A-helix motif. J Biol Chem 270:2721327221[Abstract/Free Full Text]
-
Guay J, Lambert H, Gingras-Breton G, Lavoie JN, Huot J, Landry
J 1997 Regulation of actin filament dynamics by p38 MAP
kinase-mediated phosphorylation of heat shock protein 27. J Cell
Sci 110:357368[Abstract/Free Full Text]
-
Goebeler M, Kilian K, Gillitzer R, Kunz M, Yoshimura T,
Brocker EB, Rapp UR, Ludwig S 1999 The MKK6/p38 stress kinase cascade
is critical for tumor necrosis factor-
-induced expression of
monocyte-chemoattractant protein-1 in endothelial cells. Blood 93:857865[Abstract/Free Full Text]
-
Park-Sarge OK, Mayo KE 1994 Regulation of the progesterone
receptor gene by gonadotropins and cyclic adenosine 3',5'-monophosphate
in rat granulosa cells. Endocrinology 134:709718[Abstract]
-
Ko C, In YH, Park-Sarge OK 1999 Role of progesterone receptor
activation in pituitary adenylate cyclase activating polypeptide gene
expression in rat ovary. Endocrinology 140:51855194[Abstract/Free Full Text]
-
Brannstrom M, Norman RJ, Seamark RF, Robertson SA 1994 Rat
ovary produces cytokines during ovulation. Biol Reprod 50:8894[Abstract]
-
Adashi EY, Resnick CE, DErcole AJ, Svoboda ME, Van Wyk JJ 1985 Insulin-like growth factors as intraovarian regulators of
granulosa cell growth and function. Endocr Rev 6:400420[Abstract]
-
ONeill LA, Greene C 1998 Signal transduction pathways
activated by the IL-1 receptor family: ancient signaling machinery in
mammals, insects, and plants. J Leukoc Biol 63:650657[Abstract]
-
Hurwitz A, Ricciarelli E, Botero L, Rohan RM, Hernandez ER,
Adashi EY 1991 Endocrine- and autocrine-mediated regulation of rat
ovarian (theca-interstitial) interleukin-1 ß gene expression:
gonadotropin-dependent preovulatory acquisition. Endocrinology 129:34273429[Abstract]
-
Adashi EY 1996 Immune modulators in the context of the
ovulatory process: a role for interleukin-1. Am J Reprod Immunol 35:190194[Medline]
-
Wang LJ, Brannstrom M, Cui KH, Simula AP, Hart RP, Maddocks S,
Norman RJ 1997 Localisation of mRNA for interleukin-1 receptor and
interleukin-1 receptor antagonist in the rat ovary. J Endocrinol 152:1117[Abstract]
-
Adashi EY 1995 Insulin-like growth factors as determinants of
follicular fate. J Soc Gynecol Invest 2:721726[CrossRef][Medline]
-
Sugino N, Telleria CM, Tessier C, Gibori G 1999 Regulation and
role of the insulin-like growth factor I system in rat luteal cells. J
Reprod Fertil 115:349355[Abstract]
-
Asakai R, Tamura K, Eishi Y, Iwamoto M, Kato Y, Okamoto R 1993 Basic fibroblast growth factor (bFGF) receptors decrease with luteal
age in rat ovarian luteal cells: colocalization of bFGF receptors and
bFGF in luteal cells. Endocrinology 133:10741084[Abstract]
-
Ferrara N, Chen H, Davis-Smyth T, Gerber HP, Nguyen TN, Peers
D, Chisholm V, Hillan KJ, Schwall RH 1998 Vascular endothelial growth
factor is essential for corpus luteum angiogenesis. Nat Med 4:336340[Medline]
-
Sugino N, Kashida S, Takiguchi S, Karube A, Kato H 2000 Expression of vascular endothelial growth factor and its receptors in
the human corpus luteum during the menstrual cycle and in early
pregnancy. J Clin Endocrinol Metab 85:39193924[Abstract/Free Full Text]
-
Redmer DA, Dai Y, Li J, Charnock-Jones DS, Smith SK, Reynolds
LP, Moor RM 1996 Characterization and expression of vascular
endothelial growth factor (VEGF) in the ovine corpus luteum. J Reprod
Fertil 108:157165[Abstract]
-
Levitas E, Chamoun D, Udoff LC, Ando M, Resnick CE, Adashi EY 2000 Periovulatory and interleukin-1 ß-dependent up-regulation of
intraovarian vascular endothelial growth factor (VEGF) in the rat:
potential role for VEGF in the promotion of periovulatory angiogenesis
and vascular permeability. J Soc Gynecol Investig 7:5160[CrossRef][Medline]
-
Rousseau S, Houle F, Kotanides H, Witte L, Waltenberger J,
Landry J, Huot J 2000 Vascular endothelial growth factor (VEGF)-driven
actin-based motility is mediated by VEGFR2 and requires concerted
activation of stress-activated protein kinase 2 (SAPK2/p38) and
geldanamycin-sensitive phosphorylation of focal adhesion kinase. J
Biol Chem 275:1066110672[Abstract/Free Full Text]
-
Kwok RP, Lundblad JR, Chrivia JC, Richards JP, Bachinger HP,
Brennan RG, Roberts SG, Green MR, Goodman RH 1994 Nuclear protein CBP
is a coactivator for the transcription factor CREB. Nature 370:223226[CrossRef][Medline]
-
Gonzalez GA, Montminy MR 1989 Cyclic AMP stimulates
somatostatin gene transcription by phosphorylation of CREB at serine
133. Cell 59:675680[Medline]
-
Deak M, Clifton AD, Lucocq LM, Alessi DR 1998 Mitogen- and
stress-activated protein kinase-1 (MSK1) is directly activated by MAPK
and SAPK2/p38, and may mediate activation of CREB. EMBO J 17:44264441[Abstract/Free Full Text]
-
Xing J, Kornhauser JM, Xia Z, Thiele EA, Greenberg ME 1998 Nerve growth factor activates extracellular signal-regulated kinase and
p38 mitogen-activated protein kinase pathways to stimulate CREB serine
133 phosphorylation. Mol Cell Biol 18:19461955[Abstract/Free Full Text]
-
Rolli M, Kotlyarov A, Sakamoto KM, Gaestel M, Neininger A 1999 Stress-induced stimulation of early growth response gene-1 by
p38/stress-activated protein kinase 2 is mediated by a cAMP-responsive
promoter element in a MAPKAP kinase 2-independent manner. J Biol
Chem 274:1955919564[Abstract/Free Full Text]
-
Mukherjee A, Park-Sarge OK, Mayo KE 1996 Gonadotropins induce
rapid phosphorylation of the 3',5'-cyclic adenosine monophosphate
response element binding protein in ovarian granulosa cells.
Endocrinology 137:32343245[Abstract]
-
DeManno DA, Cottom JE, Kline MP, Peters CA, Maizels ET,
Hunzicker-Dunn M 1999 Follicle-stimulating hormone promotes histone H3
phosphorylation on serine-10. Mol Endocrinol 13:91105[Abstract/Free Full Text]
-
Birkenkamp KU, Tuyt LM, Lummen C, Wierenga AT, Kruijer W,
Vellenga E 2000 The p38 MAP kinase inhibitor SB203580 enhances nuclear
factor-
B transcriptional activity by a non-specific effect upon the
ERK pathway. Br J Pharmacol 131:99107[Abstract/Free Full Text]
-
Lali FV, Hunt AE, Turner SJ, Foxwell BM 2000 The pyridinyl
imidazole inhibitor SB203580 blocks phosphoinositide-dependent protein
kinase activity, protein kinase B phosphorylation, and
retinoblastoma hyperphosphorylation in interleukin-2-stimulated T
cells independently of p38 mitogen-activated protein kinase. J
Biol Chem 275:73957402[Abstract/Free Full Text]
-
Rane MJ, Coxon PY, Powell DW, Webster R, Klein JB, Ping P,
Pierce W, McLeish KR 2000 p38 kinase-dependent MAPKAPK-2 activation
functions as 3-phosphoinositide-dependent kinase-2 for Akt in human
neutrophils. J Biol Chem 276:35173523[Abstract/Free Full Text]
-
Somers JP, Benyo DF, Little-Ihrig L, Zeleznik AJ 1995 Luteinization in primates is accompanied by loss of a 43-kilodalton
adenosine 3',5'-monophosphate response element-binding protein isoform.
Endocrinology 136:47624768[Abstract]
-
Fitzpatrick SL, Richards JS 1994 Identification of a cyclic
adenosine 3',5'-monophosphate-response element in the rat aromatase
promoter that is required for transcriptional activation in rat
granulosa cells and R2C Leydig cells. Mol Endocrinol 8:13091319[Abstract]
-
Carlone DL, Richards JS 1997 Functional interactions,
phosphorylation, and levels of 3',5'-cyclic adenosine
monophosphate-regulatory element binding protein and steroidogenic
factor-1 mediate hormone-regulated and constitutive expression of
aromatase in gonadal cells. Mol Endocrinol 11:292304[Abstract/Free Full Text]
-
Agarwal P, Peluso JJ, White BA 1996 Steroidogenic factor-1
expression is transiently repressed and c-myc expression and
deoxyribonucleic acid synthesis are induced in rat granulosa cells
during the periovulatory period. Biol Reprod 55:12711275[Abstract]
-
Fitzpatrick SL, Carlone DL, Robker RL, Richards JS 1997 Expression of aromatase in the ovary: down-regulation of mRNA by the
ovulatory luteinizing hormone surge. Steroids 62:197206[CrossRef][Medline]
-
Yoshinaga-Hirabayashi T, Ishimura K, Fujita H, Kitawaki J,
Osawa Y 1990 Immunocytochemical localization of aromatase in immature
rat ovaries treated with PMSG and hCG, and in pregnant rat ovaries.
Histochemistry 93:223228[Medline]
-
Hickey GJ, Oonk RB, Hall PF, Richards JS 1989 Aromatase
cytochrome P450 and cholesterol side-chain cleavage cytochrome P450 in
corpora lutea of pregnant rats: diverse regulation by peptide and
steroid hormones. Endocrinology 125:16731682[Abstract]
-
Krasnow JS, Hickey GJ, Richards JS 1990 Regulation of
aromatase mRNA and estradiol biosynthesis in rat ovarian granulosa and
luteal cells by prolactin. Mol Endocrinol 4:1321[Abstract]
-
Smoyer WE, Gupta A, Mundel P, Ballew JD, Welsh MJ 1996 Altered
expression of glomerular heat shock protein 27 in experimental
nephrotic syndrome. J Clin Invest 97:26972704[Abstract/Free Full Text]
-
Pei L, Dodson R, Schoderbek WE, Maurer RA, Mayo KE 1991 Regulation of the
inhibin gene by cyclic adenosine
3',5'-monophosphate after transfection into rat granulosa cells. Mol
Endocrinol 5:521534[Abstract]
-
Peters CA, Maizels ET, Hunzicker-Dunn M 1999 Activation of PKC
in the rat corpus luteum during pregnancy. Potential role of
prolactin signaling. J Biol Chem 274:3749937505[Abstract/Free Full Text]
-
Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ 1951 Protein
measurement with the Folin phenol reagent. J Biol Chem 193:265275[Free Full Text]
-
Hunzicker-Dunn M, Cutler REJ, Maizels ET, DeManno DA,
Lamm ML, Erlichman J, Sanwal BD, LaBarbera AR 1991 Isozymes of
cAMP-dependent protein kinase present in the rat corpus luteum. J
Biol Chem 266:71667175[Abstract/Free Full Text]
-
Park OK, Mayo KE 1991 Transient expression of progesterone
receptor messenger RNA in ovarian granulosa cells after the
preovulatory luteinizing hormone surge. Mol Endocrinol 5:967978[Abstract]
-
Woodruff TK, DAgostino J, Schwartz NB, Mayo KE 1988 Dynamic
changes in inhibin messenger RNAs in rat ovarian follicles during the
reproductive cycle. Science 239:12961299[Medline]
-
Woodruff TK, Meunier H, Jones PB, Hsueh AJ, Mayo KE 1987 Rat
inhibin: molecular cloning of
- and ß-subunit complementary
deoxyribonucleic acids and expression in the ovary. Mol Endocrinol 1:561568[Abstract]
-
Campbell MJ 1995 Lipofection reagents prepared by a simple
ethanol injection technique. Biotechniques 18:10271032[Medline]
-
Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M,
Northrop JP, Ringold GM, Danielsen M 1987 Lipofection: a highly
efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad
Sci USA 84:74137417[Abstract]
-
Vanderstichele H, Delaey B, de Winter J, de Jong F, Rombauts
L, Verhoeven G, Dello C, van de Voorde, Briers T 1994 Secretion of
steroids, growth factors, and cytokines by immortalized mouse granulosa
cell lines. Biol Reprod 50:11901202[Abstract]
-
de Wet J, Wood KV, DeLuca M, Helinski DR, Subramani S 1987 Firefly luciferase gene: structure and expression in mammalian cells.
Mol Cell Biol 7:725737[Medline]
-
Bradford MM 1976 A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the principle
of protein-dye binding. Anal Biochem 72:248254[CrossRef][Medline]
-
Bender FE, Douglass LW, Kramer A 1982 Statistical Methods for
Food and Agriculture. AVI Publishing Co, Inc, Westport, CT, pp
87107