Department of Cell Biology (I.J.G.R., T.A., J.S.R.) Baylor
College of Medicine Houston, Texas 77030
Department of
Molecular and Cell Biology (P.B., G.L.F.) University of California
at Berkeley Berkeley, California 94720
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ABSTRACT |
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INTRODUCTION |
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The A-kinase pathway itself is regulated by many factors that control
its cellular location and state of activation. For example, the
catalytic (C) subunit of A-kinase is inhibited by the regulatory (R)
subunit of which there are several isoforms (RI/ß, RII
/ß)
encoded by distinct genes (Refs. 3, 4 and references therein). R
subunits have recently been shown to bind cellular scaffolding
proteins, called A-kinase anchor proteins (AKAPs) (5, 6, 7, 8, 9, 10). Because R
binds these scaffolding sites, they are presumed to regulate the site
of localization of the holoenzyme and provide convenient, local target
sites for the activated enzyme. However, once A-kinase is activated and
C is released from the holoenzyme, the C subunit moves rapidly to the
nucleus (11) where it phosphorylates transcription factors such as the
cAMP-regulatory element binding protein, CREB (12, 13, 14). It has been
well documented that phosphorylation of serine 133 CREB (phospho-CREB)
is obligatory for transcriptional activation of CREB (15, 16) and its
association with CREB binding protein, CBP/p300 (17, 18). This paradigm
has now been established in many cells indicating that this pathway is
a major route by which A-kinase regulates transcription.
The A-kinase pathway is negatively regulated by numerous inhibitory
mechanisms. Phosphodiesterases (PDEs) rapidly catabolize cAMP, thereby
rapidly reducing intracellular levels of cAMP and favoring the
reestablishment of the holoenzyme (19). Specific inhibitors of the PDE
isoforms present in the ovary can sustain elevated intracellular levels
of cAMP in granulosa cells vs. ooctyes (19, 20). Protein
kinase inhibitor (PKI) exists in multiple isoforms (PKI, PKIß, and
PKIß subtypes), all of which bind and inactivate the A-kinase C
subunit (21, 22, 23, 24). Although the physiological function of PKIs is not
yet clear, PKI
has been shown to enhance the export of C from the
nucleus providing one function for its binding and the inactivation of
C activity in the nucleus (25, 26, 27). By restricting residence time of
the C subunit in the nucleus, PKI
would regulate the transactivation
of A-kinase-regulated genes. Lastly, serine protein phosphatases (PPs)
are known to control the phosphorylation state of proteins (28, 29, 30),
and calcineurin (PP2B), in particular, is known to be translocated to
the nucleus in response to certain signals (31).
In the ovary, FSH and LH stimulate the A-kinase pathway and thereby
control the growth and differentiation of the ovarian follicle (1). In
response to tonic secretion of FSH, granulosa cells of small follicles
produce low levels of cAMP, proliferate, and differentiate. Genes such
as serum- and glucocorticoid-induced kinase (Sgk) (32, 33) and
serum-induced kinase (Snk) (1), as well as the cell cycle-regulatory
molecule, cyclin D2 (34, 35, 36), are induced in these cells in an
immediate-early expression pattern. In contrast, other genes exhibit a
more delayed response to hormone stimulation and do not peak until
2448 h after exposure to FSH when granulosa cell function has reached
the PO stage. Genes induced at this time include aromatase (Refs. 37, 38 and data herein), inhibin (39), LH receptor (4, 40),
and the secondary rise of Sgk (Ref. 32 and data herein). In response to
the LH surge, granulosa cells generate high levels of intracellular
cAMP and rapidly initiate a program of terminal differentiation
(luteinization) in which proliferation ceases (4, 35, 36). LH
dramatically down-regulates genes associated with follicular function
such as aromatase (4, 38) and cyclin D2 (35, 36) and rapidly but
transiently induces genes required for ovulation (41, 42, 43, 44, 45, 46, 47, 48, 49, 50): progesterone
receptor (PR) (41, 42, 43, 44, 45), prostaglandin synthase-2 (PGS-2) (46, 47, 48, 49) and
CAAT enhancer-binding protein (C/EBPß) (47, 50). During the process
of luteinization, granulosa cells appear to become refractory to
further cAMP stimulation. Genes such as cholesterol side-chain cleavage
cytochrome P450 (P450scc) (51, 52) are constitutively expressed at
elevated levels. Neither forskolin, which increases cAMP, nor H89,
which blocks A-kinase activation, alters expression of P450scc in the
luteinized cells (52). Thus, the A-kinase pathway controls the
expression of numerous genes in granulosa cells at distinct stages of
differentiation and by specific molecular events.
The diverse patterns of gene expression that are regulated by FSH in granulosa cells are dramatically altered by the LH surge-induced transition to luteal cells. Notably, luteal cells exhibit altered/impaired responses to cAMP. Therefore, these studies were designed to focus on the functional and subcellular changes in A-kinase pathway components that might regulate the rapid and reversible responses to cAMP that occur in proliferating granulosa cells compared with the irreversible and cAMP nonresponsive state in terminally differentiated luteal cells. In particular, the localization of the A-kinase C subunit has been related to the phosphorylation state of CREB as well as to the expression of two A-kinase-regulated genes, aromatase and Sgk. Because the latter is also a kinase with presumed functions related to proliferation as well as differentiation (32, 33, 53), the subcellular localization of this protein in granulosa cells and luteal cells was also examined. Inhibitors of the A-kinase pathway were used to determine the extent to which they control the pattern of A-kinase activation, its subcellular localization, and the expression patterns of aromatase and Sgk.
Two well characterized culture systems were used to compare the agonist-induced responses of immature, proliferating granulosa cells to those of terminally differentiated, nondividing luteinized granulosa cells. Specifically, the culture of granulosa cells isolated from preantral follicles of immature rats in serum-free medium permits analysis of dynamic, short-term responses to hormones (cAMP) (32, 38). In contrast, the culture of granulosa cells from PO follicles that have been exposed in vivo to surge concentrations of hCG (PO/hCG) permits analyses of responses in cells that undergo irreversible, long-term changes associated with the process of luteinization (4, 51, 52). Results indicate that granulosa cell differentiation is associated with dynamic changes in the subcellular localization of A-kinase pathway components and provide evidence for the intriguing possibility that A-kinase C subunit, phospho-CREB, and Sgk have distinct nuclear import/export controls, docking sites, and functions (?) in proliferating granulosa cells that differ from those in terminally differentiated luteal cells.
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RESULTS |
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Antibodies against peptides of either the amino terminus (NT) or
carboxy terminus (CT) of the A-kinase C subunit (NT-C and CT-
C,
respectively) recognize a 41- to 39-kDa immunoreactive band that
remained constant in granulosa cells cultured with FSH/T for 048 h
(Fig. 1A
). Likewise,
the cellular content of CREB (43 kDa) remained unchanged in response to
hormone stimulation. An antibody that specifically recognizes
activated, phosphorylated (serine 133) CREB showed that immunoreactive
phospho-CREB was low in cells cultured overnight in the absence of
hormone (0 h) but was markedly increased 6.5 ± 0.3-fold by FSH
within 1 h. This increase was transient; levels of phospho-CREB
declined to basal levels by 6 h. However, a secondary 2.1 ±
0.3-fold increase in phospho-CREB was observed at 24 h (Fig. 1A
).
Since each antibody recognized only a specific immunopositive band,
these same antibodies were used for immunolocalization of these
proteins within granulosa cells during hormone stimulation.
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Antibodies to either the N terminus (NT-C) or the C terminus
(CT-
C) of the A-kinase C subunit also exhibited specific patterns of
immunolocalization (Fig. 1B
, middle and lower panels,
respectively). NT-
C, preferentially recognized C subunit present in
the nucleus with only weak recognition of the kinase complexed to
cytoplasm structures (Fig. 1B
; middle panels, 0 h).
Specifically, immunostaining with NT-
C was low and diffuse in the
untreated granulosa cells but increased markedly in nuclei of
essentially all cells in response to FSH/T (1 h). This intense nuclear
staining of C subunit with NT-
C was transient and returned to lower
levels by 6 h. However, at 2448 h, C subunit staining in nuclei
with NT-
C appeared to increase slightly in 80% of the cells (Fig. 1B
, middle panels). This is consistent with the observation
that addition of H89, an A-kinase-selective inhibitor, to granulosa
cells cultured with FSH/T for 41 h completely blocks the
expression of aromatase and reduces expression of Sgk at 48 h
(data not shown). In contrast, CT-
C intensely stained a discrete,
small structure [possibly Golgi-related (9)] exterior to the nucleus
of untreated granulosa cells (Fig. 1B
; lower panel, 0
h). CT-
C staining was undetectable or low in the nucleus and other
regions of the cells, respectively (Fig. 1B
; lower panel,
0 h). After the addition of FSH for 1 h, immunostaining with
CT-
C was more diffuse and spread throughout the cells with punctate
staining clearly visible in granulosa cell nuclei. These changes in the
distribution of immunoreactive C subunit were rapid but transient and
occurred in all cells. At 6 h, C subunit (as detected by CT-
C)
had returned to the basket-like structure in the cytoplasm. At 24
h and 48 h of culture with FSH/T, C subunit remained in the
basket-like region of the cytoplasm but was also detected in a punctate
pattern in nuclei, especially at 24 h (Fig. 1B
, middle and
lower panels). Thus, although the amount of immunoreactive C that
is localized to nuclei at 2448 h is less than that at 1 h, it
appears to be functional and required at this stage of granulosa cell
differentiation since inhibitors of A-kinase activity (i.e.
H89) block aromatase gene expression.
These results indicate further that each C subunit antibody recognizes
a specific epitope within the protein and that in the denatured protein
both epitopes are clearly detected by each antibody (i.e.
Western blot; Fig. 1A) but are selectively detected by the antibodies
in tissue sections where proteins have been cross-linked by fixation
procedures (i.e. as in immunofluorescent analyses). The
N-terminal antigenic site of the C subunit appears to be masked
(NT-
C) when C is bound to R subunits (and other proteins?) tethered
to specific cytoplasm structures. The C-terminal antigenic site,
however, is exposed when the C subunit is present in the cytoplasmic
structures as well as when the protein is imported to the nucleus
(CT-
C). In contrast to the transient, agonist-induced release and
transport of the C subunit to the nucleus, immunostaining of the
A-kinase RII(
/ß) subunits in untreated (Fig. 1C
) and
hormone-stimulated (not shown) granulosa cells was localized to the
basket-like region within the cytoplasm, most likely related to the
Golgi (9). Changes in the intensity/pattern of RII
/ß subcellular
distribution were not observed at the time intervals examined.
Effects of PDE Inhibitors on Activation of the A-Kinase
Pathway
To determine whether the transient, FSH-induced increase in
phospho-CREB between 0 to 6 h was related to changes in
intracellular concentrations of cAMP and the residence time of C
subunit in the nucleus, two inhibitors of PDEs were added to the
cultures to maintain elevated concentrations of intracellular cAMP.
Isobutylmethylxanthine (IBMX) is a PDE inhibitor with broad specificity
whereas rolipram is a more selective inhibitor of the PDE4 isoform
known to be selectively present in granulosa cells (19). As above,
granulosa cells were cultured overnight in defined medium. At that time
either rolipram (10 µM), IBMX (50 µM-1
mM) or vehicle was added to the cells for 30 min
(pretreatment) after which forskolin (10 µM) was added (0
h). Western blots show that phospho-CREB was low in untreated cells as
well as in those pretreated with rolipram (Fig. 2A). Forskolin alone or forskolin and
rolipram stimulated similar increases in phospho-CREB at 1 h
(Fig. 2A
). However, at 6 h, phospho-CREB remained higher (7-fold)
in the forskolin- and rolipram-treated cells compared with those
exposed to forskolin alone. In addition, the decline in phospho-CREB at
24 and 48 h appeared to be more gradual, and phospho-CREB remained
higher in the rolipram-treated cells compared with those stimulated by
forskolin alone. Levels of phospho-CREB were also enhanced in granulosa
cells cultured in the presence of forskolin and high levels of IBMX (1
mM) (Fig. 2A
) whereas cells cultured with lower
concentrations of IBMX (0.050.10 mM) exhibited a pattern
of phospho-CREB similar to that of cells cultured with forskolin alone
(data not shown).
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Immunostaining of phospho-CREB and the A-kinase C subunit in granulosa
cells of these same cultures showed that forskolin alone (data not
shown) stimulated the same pattern as observed in response to FSH/T in
Fig. 1. In contrast, the PDE inhibitors promoted the retention of the C
subunit in the nucleus at 648 h (Fig. 2B
). Although immunoreactive
phospho-CREB remained present in granulosa cell nuclei at
6 h, the levels declined markedly at 24 and 48 h, and a
subset of cells (20%) exhibited cytoplasmic staining (Fig. 2B
; 48
h). Thus, despite the longer retention of C subunit in the nucleus and
the higher levels of nuclear phospho-CREB at 6 h, the pattern of
gene expression was not dramatically altered. In addition, the
heterogeneous localization of phospho-CREB in cells at 48 h
indicated that some of these cells might be acquiring characteristics
of luteal cells.
Effect of Phosphatase Inhibitors on the A-Kinase
Pathway
To determine whether the rapid changes in levels of
nuclear phospho-CREB were related to phosphatase activity, two
inhibitors of serine protein phosphatases were used. Okadaic acid (OA)
preferentially inhibits PP1 and PP2A, whereas cyclosporin A (CsA)
preferentially inhibits calcineurin (PP2B). OA (10 nM) and
CsA (100 nM) were added to granulosa cell cultures 30 min
before the addition of forskolin (0 h). OA alone increased the
levels of phospho-CREB 2-fold in the untreated cells (Fig. 3, 0 h). Cells treated with
forskolin and OA also had higher levels of phospho-CREB at 1, 6, and
24 h (1.5-, 3.0- and 3.5-fold, respectively) compared with cells
treated with forskolin alone. In these same cells, the level of CREB
remained constant (Fig. 3
). Surprisingly, the expression of Sgk protein
was completely abolished by exposing the cells to OA. The FSH/T-induced
peaks of Sgk at 1 h and 24 h were completely absent if OA was
also present in the cultures (Fig. 3
). In contrast, CsA had no apparent
effect on the levels of phospho-CREB or the expression of Sgk; the
relative increases in reponse to forskolin were not changed (Fig. 3
).
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Immunofluorescent analyses showed that low levels of phospho-CREB were
present in nuclei of untreated granulosa cells (Fig. 5; upper
panels, 0 h). Forskolin (or FSH/T; not shown) stimulated
marked increases in nuclear phospho-CREB within 45 min. As shown in
Fig. 1B
, this response was transient. After 6 h of culture with
FSH/T alone, only a few (<5%) cells exhibited intense levels of
phospho-CREB; most cells exhibited low levels of phospho-CREB (Fig. 5
;
upper panels, -Fo). At 24 h of culture with FSH/T
alone, essentially all cells showed a low level of nuclear
phospho-CREB. As in previous experiments, the staining of phospho-CREB
was more heterogeneous at 48 h. Some cells exhibited low levels of
phospho-CREB in the nucleus, whereas other (larger?) cells showed
phospho-CREB localized to a perinuclear region in the cytoplasm (Fig. 5
; two panels, 48 h; -Fo). The addition of forskolin at
6 h and 24 h increased immunoreactive phospho-CREB in nuclei
of nearly all cells within 45 min (Fig. 5
; upper panels,
+Fo). At 48 h many cells (80 ± 0.6%) exhibited nuclear
staining but a subset of cells (
20%) immunoreactive for
phospho-CREB remained localized to the cytoplasm (Fig. 5
; two
panels, 48 h; +Fo).
These dynamic changes in the CREB phosphorylation were associated with
the activation of A-kinase and its nuclear import/export (Fig. 5, middle and lower panels). Specifically, in untreated granulosa
cells, very low levels of C-subunit were detected within the nucleus
(NT-
C), whereas intense staining was observed in a small basket-like
structure within the cytoplasm (CT-
C) (Fig. 5
; -Fo; middle
and lower panels, respectively). In response to FSH/T or
forskolin, rapid and marked changes in the subcellular distribution of
immunoreactive C subunit were observed (Fig. 5
). Immunostaining to the
basket-like structure rapidly decreased as a more diffuse staining
pattern of C subunit appeared throughout the cytoplasm (CT-
C; Fig. 5
, lower panels; +Fo). Increased immunostaining of C-subunit
in the nucleus was also apparent (CT-
C) and was even more pronounced
when the N-terminal antibody was used to detect the kinase (NT-
C;
Fig. 5
, middle panels; +Fo). These changes were transient
and by 6 h the cellular distribution of C subunit was similar to
that observed in the untreated cells. Note, however, that the
size/intensity of staining to the basket-like structure increased in
cells cultured with FSH/T for 24 and 48 h (Fig. 5
; CT-
C; -Fo).
Cells stimulated with forskolin at 6, 24, and 48 h responded in a
fashion similar to that of untreated cells: rapid but transient
increases in nuclear C subunit were associated with increased
phospho-CREB. The cellular levels of C subunit and CREB remain
unchanged as indicated by Western blotting. Therefore, the
immunolocalization data indicate that there are agonist-induced and
time-dependent changes that control the rapid and transient subcellular
trafficking (nuclear import/export) of the C subunit as well as CREB
phosphorylation in granulosa cells. Although this culture system
provides an ideal model for characterizing the functional changes in
granulosa cells as they differentiate in response to FSH/T, the
heterogeneous staining pattern observed in cells at 48 h indicated
that a more precise model was needed to analyze the cellular
localization of A-kinase pathway components and Sgk in luteal cells.
Thus, we used another culture model in which granulosa cells luteinize
and exhibit altered responses to exogenous hormones and increased
intracellular cAMP (4, 51, 52).
Effect of Luteinization on the Content and Cellular Localization of
A-Kinase Pathway Components and A-Kinase-Regulated Gene Expression
The LH surge stimulates an acute elevation of intracellular cAMP
in granulosa cells of PO follicles. Within 4 h, the granulosa
cells of these LH-stimulated follicles cease dividing and differentiate
to a stable luteal cell phenotype (4, 35, 36, 51, 52). This
reprogramming or terminal differentiation of granulosa cells to luteal
cells occurs rapidly, is irreversible, and is characterized by a
regulated, genetic switch. Several genes induced by cAMP in granulosa
cells are expressed in a constitutive, non-cAMP-inducible manner in
luteal cells. Specifically, neither the addition of forskolin, which
stimulates cAMP, nor H89, which blocks A-kinase activation by cAMP,
alters expression of P450 scc (52), aromatase (37, 55), or as shown
herein, sgk. To analyze the content and cellular
localization of components of the A-kinase pathway in luteinized
granulosa cells, a second culture system (Refs. 51, 52 ; see
Materials and Methods) was used. In the second model system,
immature female rats were injected subcutaneously with a low dose (0.15
IU) of hCG twice daily for 2 days to stimulate the growth of PO
follicles (51, 52). The PO follicles were dissected manually from the
ovaries isolated from rats either before or 6 h after an
intraperitoneal (IP) injection of an ovulatory dose (10 IU) hCG
(PO/hCG). Granulosa cells were harvested from the PO and PO/hCG-treated
follicles by needle puncture and cultured in DMEM-F12 containing 1%
FBS as previously described (4, 51, 52). Granulosa cells harvested from
PO/hCG follicles luteinize, attain a stable luteal phenotype, and
thereby permit analysis of responses in cells that undergo
irreversible, long-term changes associated with the transition of
granulosa cells to luteal cells. In contrast, PO granulosa cells fail
to luteinize and require forskolin/cAMP to stimulate the maintenance of
a differentiated phenotype and expression of P450scc and
aromatase (52) and as shown herein, Sgk.
When A-kinase pathway components were analyzed in this second model,
both the A-kinase C subunit and CREB were present and expressed at
similar levels in PO granulosa cells and the luteinized PO/hCG
granulosa cells cultured for 6 days (0 h) in medium containing 1% FBS
(Fig. 6). The addition of forskolin did
not alter the levels of C subunit or CREB. Immunoreactive phospho-CREB
was detected at low levels in the PO cells before the addition of
forskolin, likely reflecting a response to some serum factor(s).
Forskolin did not stimulate a rapid increase in phospho-CREB between
45120 min in the PO granulosa cells (Fig. 6
; 2-h data are shown).
However, at 48 h the levels of phospho-CREB were increased
6.1 ± 0.5-fold. At this time (but not at 02 h), Sgk protein and
aromatase mRNA were also expressed, indicating that forskolin (cAMP) is
required in these cells to induce/maintain the differentiated state. In
the luteinized PO/hCG granulosa cells, the A-kinase C subunit and CREB
were present at levels similar to the PO granulosa cells (Fig. 6
).
However, immunoreactive phospho-CREB was elevated (5.6 ± 0.4
fold) in luteinized cells (0 h) compared with that observed in the PO
granulosa cells (0 h) (Fig. 6
). Furthermore, in the luteinized cells
phospho-CREB was not increased by the addition of forskolin at either
2 h or 48 h (Fig. 6
). The amount of Sgk protein in the
luteinized cells was even higher than that induced by forskolin in the
PO granulosa cells; no increases in Sgk protein were observed 48 after
forskolin in the luteal cells (Fig. 6
). In a similar manner, aromatase
mRNA was constitutively expressed and nonresponsive to forskolin
treatment in the luteinized cells (Fig. 6
).
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To determine whether the changes in the subcellular localization of the
C subunit and phospho-CREB in luteal cells was specific, we examined
time-dependent changes in the cellular localization of another kinase,
Sgk, in each culture system. When granulosa cells from
estradiol-treated, immature rats were cultured in a defined medium
overnight, immunoreactive Sgk protein was low and appeared diffuse
(Fig. 9A). After stimulation with FSH, immunoreactive Sgk increased at
2 h, a temporal pattern similar to that observed by Western blot
(Fig. 4
) and Northern blot (32). Notably, immunoreactive Sgk was
localized to granulosa cell nuclei in a distinct punctate pattern of
staining. Immunoreactive Sgk remained nuclear for 624 h, after which
it was preferentially localized to the cytoplasm (Fig. 9A
). In PO
granulosa cells, immunoreactive Sgk exhibited a low level of distinct
punctate staining in nuclei (Fig. 9B
). By 48 h of culture with
forskolin, the intensity of Sgk staining increased. Although this is
harder to capture by immunofluorescence than by Western blotting (Fig. 6
), Sgk remained nuclear in most of the forskolin-treated PO granulosa
cells but was also observed in the cytoplasm of some cells (50%) where
staining was more intense (Fig. 9B
; two panels, 48 h). In
the luteinized PO+hCG granulosa cells, intense immunostaining of Sgk
was preferentially localized in a punctate pattern within the cytoplasm
and remained cytoplasmic even after the addition of forskolin for
2 h (data not shown) or 48 h (Fig. 9B
). Thus, whereas Sgk is
nuclear in immature (Fig. 9A
) and PO granulosa cells (Fig. 9B
), it
exhibits marked cytoplasmic staining as granulosa cells differentiate
to luteal cells (Fig. 9
), a pattern similar to that of phospho-CREB
(Fig. 8B
).
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DISCUSSION |
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The activation of the A-kinase pathway in granulosa cells exhibits
dynamic short-term changes and critical long-term induced changes that
appear irreversible. During the acute response of undifferentiated
granulosa cells to FSH (or forskolin), the A-kinase C subunit is
rapidly but transiently imported to the nucleus (Fig. 1; Ref. 11), a
response pattern observed in other cell types and first documented by
the studies of Adams et al. (11). The acute phase of FSH
action and nuclear import of the A-kinase C subunit are associated with
specific changes in downstream targets in the A-kinase pathway, most
notable of which is the phosphorylation of CREB (present report
and Refs. 54, 56), leading to the transactivation of several ovarian
expressed genes that contain functional CRE promoter regions: aromatase
(shown herein and Refs. 4, 54), CREM (cAMP regulatory element
modulator; Ref. 57), and inhibin
(39). In addition, we have
recently shown that expression of Sgk is induced in a biphasic pattern
by A-kinase with the first peak in Sgk mRNA (32) and protein (Ref. 32
and present data) occurring between 12 h after FSH/forskolin
stimulation and dependent on the transcription factors Sp1/Sp3 (32).
The newly expressed Sgk protein is localized to the nucleus, but in
contrast to the A-kinase C subunit, remains nuclear from 224 h.
Increased nuclear C subunit in granulosa cells at 12 h after FSH is
also associated with the rapid induction of mRNA encoding the cell
cycle-regulatory molecule cyclin D2 (34, 35, 36). Thus, the early
FSH-stimulated events that are associated with proliferation as well as
the onset of differentiation appear to be dependent on the nuclear
localization of C subunit and the phosphorylation of nuclear
transcription factors, such as CREB (13, 14, 15, 54) and Sp1/Sp3
(32).
After the rapid decline in nuclear levels of C subunit and phospho-CREB
at 6 h there is a transient secondary increase that occurs at
about 24 h. These secondary changes in C subunit localization and
phospho-CREB are associated with maximal induction of aromatase mRNA
(Fig. 3 and Ref. 38) and activity (38) and the secondary increase in
Sgk mRNA and protein (present data and Ref. 32). As differentiation
proceeds to the PO phenotype, most (80%) granulosa cells retain
responsiveness to cAMP, indicating that C subunit rapidly shuttles
between the nucleus and cytoplasm. However, as granulosa cells
luteinize, their responsiveness is altered. Thus, when immature
granulosa cells were cultured with FSH/T for 24 and 48 h and then
stimulated by forskolin to reactivate additional bursts in cAMP
production, immunoreactive C subunit appears in the nucleus of most
cells (80%) but the magnitude of the increase (based on the intensity
of nuclear immunostaining) and the number of cells responding were less
than observed in response to the initial FSH stimulus, indicating that
some of these cells have begun to luteinize. The decrease in the
cAMP-induced localization of C subunit to the nucleus as granulosa
cells differentiate was associated with a subset of cells in which
nuclear phospho-CREB had decreased, indicating that CREB is one key
substrate that the A-kinase C subunit regulates during granulosa cell
differentiation.
Interestingly, the inhibitors of PDE activity delayed but did not prevent the eventual, progressive decline in nuclear phospho-CREB and C subunit 48 h after FSH/forskolin stimulation. Equally notable, the PDE inhibitors had little or no effect on the downstream targets of A-kinase: neither the magnitude nor the temporal pattern of induction of aromatase mRNA or Sgk protein was markedly altered between 624 h. These results indicate that once the program of differentiation is set in motion by the rapid increases in cAMP and nuclear C subunit at 12 h, the program cannot be acutely changed by the presence of more cAMP, nuclear C subunit, or phosphoproteins, such as CREB, suggesting that low levels of A-kinase activation and phospho-CREB are sufficient. That A-kinase remains critically important at 2448 h is emphasized by the fact that the addition of H89 to granulosa cells at either 24 h or 42 h of culture with FSH/T completely abolished (or markedly reduced) the expression of aromatase and Sgk at 48 h (our unpublished data). Thus, A-kinase, in concert with other factors and pathways, appears to control the cellular levels of aromatase mRNA, the expression of Sgk, and the transition to the differentiated phenotype that occurs between 648 h after agonist stimulation. The complexity of events and multiplicity of factors associated with differentiation are emphasized by the observation that the phosphatase inhibitor OA completely blocked the early and secondary induction of Sgk expression by forskolin in granulosa cells but did not alter other cell functions, such as the induction of aromatase. Thus, a delicate balance of phosphorylation-dephosphorylation of key regulatory protein(s) (Sp1/Sp3?; Refs. 32, 58, 59) controlling the expression of Sgk may exert potent positive or negative regulatory functions. Equally striking is the distinct transition of Sgk protein from the nucleus in immature, proliferative granulosa cells to punctate sites within the cytoplasm of differentiated, nonproliferative granulosa cells at 48 h of culture. Thus, differentiation proceeds by a progressive, irreversible change in the induced expression of specific genes and the subcellular localization of specific proteins.
The dynamics and factors controlling the localization and activation of
the A-kinase pathway and its target genes is even more dramatic in
fully luteinized PO/hCG granulosa cells. Although the PO and luteinized
PO/hCG granulosa cells contained the same cellular levels of the
A-kinase C subunit (as determined by Western blotting) as untreated
immature granulosa cells, the subcellular distribution of the C-subunit
in the luteinized PO/hCG cells was distinct, the movement of the C
subunit in response to forskolin was impaired, and genes regulated by
cAMP in granulosa cells no longer responded to acute stimulation by
forskolin. Specifically, in the luteinized PO/hCG granulosa cells,
immunoreactive C was localized to highly punctate regions within the
nucleus and the cytoplasm (NT-C) as well as to the basket-like
cytoplasm structure (CT-
C). This distribution pattern was not
altered by exposing these luteinized cells to forskolin for 2 h or
48 h, indicating that factors other than, or in addition to, cAMP
might also be regulating the distribution of C subunit in these cells.
Since nuclear localization of C subunit was also observed in PO
granulosa cells, factors in 1% serum may stimulate low levels of cAMP,
allowing C subunit to shuttle between the cytoplasm and nucleus.
However, in the PO cells (unlike the luteinized cells), the addition of
forskolin did increase nuclear uptake of C subunit, indicating that
these cells retained responsiveness to cAMP. This contrasts with the
absence of nuclear immunostaining of C subunit within undifferentiated,
immature granulosa cells (cultured in serum-free medium) in which
activation of adenylyl cyclase is dependent on addition of
hormone/agonist and obligatory for nuclear localization of C subunit.
Thus, the immature granulosa cells and the luteal cells represent two
extremes with the PO granulosa cells exhibiting an intermediate
phenotype.
The localization of Sgk further indicates that immature cells differ markedly from luteal cells. Sgk, which is mostly nuclear in immature and PO granulosa cells, was localized almost exclusively to cytoplasmic sites in the luteinized PO/hCG granulosa cells, even after treatment with forskolin. In addition, Sgk, which is induced by cAMP in immature, proliferative granulosa cells, was constitutively expressed at elevated levels in the nonproliferating, terminally differentiated PO/hCG cells even in the absence of forskolin. In a similar manner, Sgk was observed in mammary epithelial tumor cells to be nuclear in S and G2/M phases of the cell cycle but was in the cytoplasm of cells during the G1 transition or in cells arrested in G1 as a consequence of glucocorticoid-induced differentiation (33, 53). Collectively, these results add Sgk to the list of kinases that are not only regulated by A-kinase but exhibit changes in their subcellular localization during cell differentiation.
The mechanisms controlling nuclear-cytoplasmic shuttling (nuclear
import/export) and the cellular localization (by specific docking
sites?) of the A-kinase C subunit, as well as Sgk, at each stage of
granulosa cell differentiation appear complex. Nuclear transport of
molecules is controlled by many factors including nuclear localization
signals (NLS), nuclear export signals (NES), ATP and specific GTPases,
phosphorylation, and in some cases, chaperones (60, 61, 62, 63, 64, 65, 66, 67). The A-kinase C
subunit does not have a specific NLS or NES (25, 26, 27). Rather, studies
by Taylor and colleagues (25) have shown that simple diffusion accounts
for import and export in the cell types studied (25) whereas PKI and
PKIß can facilitate nuclear export of the A-kinase C subunit, an
effect explained by the ability of PKI to bind C and to the presence of
a NES within the PKI
and PKIß peptide (26, 27). The cellular
localization of A-kinase C subunit is also determined by specific
docking proteins, such as the R subunit/AKAP complexes (5, 6, 7, 8, 9, 10) as well
as I
B (67). Since the subcellular distribution pattern of the
A-kinase C subunit differs in granulosa cells and luteal cells and does
not strictly mimic that of RII
/ß and since the response to cAMP
appears impaired in luteinized PO/hCG cells, we hypothesized that
either the antigenic sites of C subunit are masked when it binds to R
subunit/AKAP complexes or the C subunit binds different tethering sites
as granulosa cell differentiation proceeds. Using two different
antibodies, we have determined that the N-terminal antigenic sites on C
subunit (recognized by NT-
C antibody) appear masked when C is
complexed with the R subunit/AKAP complex. However, it remains possible
that there are specific AKAPs present in luteal cells that alter the
nuclear import/export behavior of the C subunit. Alternatively, some C
subunit may be bound by I
B (or other docking proteins). If
associated with I
B or some other protein, the C subunit would not
released by increased intracellular cAMP (67), providing one
explanation for the apparent lack of C-subunit redistribution by
forskolin/cAMP in the luteal cells. Interestingly, neither cAMP not C
subunit have been shown to increase the phosphorylation of luteal cell
proteins, adding further evidence for the apparent diminished effect of
A-kinase in these terminally differentiated cells (68, 69). In contrast
to the A-kinase C subunit, Sgk has putative NLS and NES sequences in
its structure. Therefore, changes in the subcellular trafficking of Sgk
from the nucleus in granulosa cells and to a cytoplasmic site in luteal
cells may involve modifications (by phosphorylation or protein
binding?) of either the NLS or NES to enhance or restrict nuclear
import in relation to cell cycle progression (53) or other cell
functions.
The amount and subcellular distribution of phospho-CREB were also dramatically altered in the luteinized granulosa cells. First, the cellular levels of phospho-CREB (Western blot analysis) were elevated in luteinized granulosa cells in the absence or presence of forskolin. This is consistent with our previous observations that the addition of the A-kinase inhibitor, H89, to these cells does not alter expression of P450scc (52) or aromatase (37, 55). Furthermore, immunoreactive phospho-CREB was exclusively localized to a cytoplasmic region of the luteal cells. The cytoplasmic localization of phospho-CREB is difficult to explain. In luteal cells, immunodetection of nuclear phospho-CREB may be masked by the binding of specific nuclear proteins (coregulators) to this transcription factor. Alternatively, since immunoreactive CREB is nuclear in luteal cells, it is possible that a set of nuclear kinases are selectively active in luteal cells (but not granulosa cells) and phosphorylate CREB not only at Ser133 (as detected by the Western blot) but also at other amino acid residues to target it for nuclear export. Kinases other than A-kinase that phosphorylate CREB include CaM kinase II and CaM kinase IV, protein kinase C, RSK-2, and ERK-2 (70, 71, 72, 73, 74). Some of these have been shown to alter the nuclear movement of specific proteins, but the identity of the kinase(s) acting in luteal cells that might alter the localization of phospho-CREB remains to be determined.
In summary, the transition of proliferating granulosa cells to terminally differentiated luteal cells is characterized by a dramatic reprogramming of cellular and molecular events. The data provided herein illustrate two diverse patterns in the activation of the A-kinase pathway and the distribution of A-kinase components within the cells. Immature cells respond in a rapid and robust manner to FSH/forskolin/cAMP: C subunit is mobilized to the nucleus and phosphorylates CREB. This response is transient, reversible, and associated with maximal induction of aromatase and Sgk at 2448 h. In luteal cells forskolin/cAMP do not alter the cellular distribution of C subunit, phosphorylation of CREB, or expression of aromatase and Sgk, which are constitutively elevated. Quite unexpectedly, phospho-CREB resides in a cytoplasmic region, not in the nucleus, of luteal cells. The dramatic switch in the subcellular localization of phospho-CREB, combined with the lack of mobilization of A-kinase C subunit in luteal cells, provides biochemical and cellular evidence to help account for the shift from cAMP-regulated gene expression in granulosa cells to cAMP-independent regulation in luteal cells. Rivaling the changes in A-kinase and phospho-CREB is the dramatic egress of Sgk from the nucleus in granulosa cells to cytoplasmic sites in luteal cells. Although the mechanisms that control the subcellular trafficking of C subunit and Sgk, as well as the cytoplasmic localization of phospho-CREB, are not yet entirely clear, these data provide the novel observation that the cellular distribution patterns of specific kinases are differentiation dependent and may be functionally related to cell cycle progression.
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MATERIALS AND METHODS |
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Electrophoresis and molecular biology grade reagents were purchased
from Sigma Chemical Co., Bio-Rad Laboratories, Inc. (Richmond, CA), and Roche Molecular Biochemicals (Indianapolis, IN). Oligonucleotides were purchased
from Genosys (The Woodlands, TX). All reverse transcriptase-PCR
reagents were from Promega Corp. (Madison, WI) except for
deoxyribonucleotides (dNTPs; Roche Molecular Biochemicals). -32P[dCTP] was from ICN
Radiochemicals (Costa Mesa, CA). Hyperfilm was purchased from
Amersham Pharmacia Biotech (Arlington Heights, IL).
Animals
Intact immature (day 25 of age) Holtzman Sprague Dawley female
rats (Harlan, Indianapolis, IN) were housed under a 16-h light, 8-h
dark schedule in the Center for Comparative Medicine at Baylor College
of Medicine and provided food and water ad libitum. Animals
were treated in accordance with the NIH Guide for the Care and Use of
Laboratory Animals, as approved by the Animal Care and Use Committee at
Baylor College of Medicine (Houston, TX).
Granulosa Cell Cultures
For these studies we have used two well characterized cell
culture models in which the effects of cAMP on gene expression have
been clearly defined. The first system has been used to characterize
the immediate-early and delayed effects of FSH on granulosa cell
differentiation (32, 38). Immature rats were primed with estradiol (E;
1.5 mg/0.2 ml propylene glycol once daily for 3 days) as previously
described (32, 38). After treatment (day 27), granulosa cells were
harvested and cultured at a density of 1 x 106 cells
per 3 ml serum-free medium (DMEM-F12 containing penicillin and
streptomycin) in multiwell (35-mm) dishes that were serum coated. In
the presence of FSH/T or forskolin, these cells differentiate to a PO
phenotype in which aromatase (38), LH receptor (40), and Sgk (32) are
expressed.
In the second model system, immature female rats were injected subcutaneously with a low dose (0.15 IU) of hCG twice daily for 2 days to stimulate the growth of PO follicles (51, 52). The PO follicles were dissected manually from the ovaries either before or 6 h after an ip injection of an ovulatory dose (10 IU) hCG (PO/hCG). Granulosa cells were harvested from the PO and PO/hCG-treated follicles by needle puncture, collected by centrifugation, and cultured in 35-mm multiwell dishes in DMEM-F12 containing 1% FBS as previously described (4, 51, 52). Granulosa cells harvested from PO/hCG follicles luteinize and attain a stable luteal phenotype, whereas PO granulosa cells fail to luteinize and require forskolin/cAMP to stimulate the maintenance of a differentiated phenotype.
The culture of immature granulosa cells permits analysis of dynamic, short-term responses to hormone (cAMP), whereas the PO/hCG granulosa cells permit analysis of responses in cells that undergo irreversible, long-term changes associated with the transition of granulosa cells to luteal cells. In each culture system, hormones, agonists (FSH, forskolin), and inhibitors (IBMX, rolipram, OA, CsA) were added at the doses and times indicated in the figure legends. All experiments were repeated at least three times.
RNA Isolation, Northern Blots, and RT-PCR Assays
Cytoplasmic RNA was isolated from cultured cells with a buffer
containing 1% NP-40 (32, 38). Each RNA sample was pooled from three
replicate wells. The RNA was purified by sequential phenol,
phenol-chloroform, and chloroform extraction, followed by ethanol
precipitation. The RNA was resuspended in 0.1%
diethylpyrocarbonate-treated water and its concentration determined by
absorbance at 260 nm.
RT-PCR reactions were performed as previously described (39, 75) using specific primer pairs for rat aromatase (forward, 5'-TGCACAGGCTCGAGTATTTCC-3' and reverse 5'-ATTTCCACAATGGGGCTGTCC-3') (74) and the ribosomal protein L19 (39). The amplified cDNA products were resolved by acrylamide gel electrophoresis, and radioactivity/PCR product band was quantified on a Betascope 603 Blot Analyzer (Betagen Corp., Mt. View, CA). Data are presented as the ratio of radioactivity in the aromatase and L19 bands.
Cell Extracts and Western Blot Analyses
Total cell extracts were prepared according to a method
described by Ginty et al. (70) by adding to each well hot
(100 C) Tris-buffer containing 10% SDS and ß-mercaptoethanol. The
cells were rapidly scraped with a rubber policeman, and the extract
transferred to an Eppendorf tube at 100 C for 5 min.
Extracts were stored at 4 C until analyzed by SDS-PAGE. After SDS-PAGE,
samples were electrophretically transferred to nylon filter, washed
briefly in PBS, and blotted with either 3% BSA, or 5% Carnation milk
at room temperature for 1 h. Antibodies were added in the same
blocking solutions at the dilutions indicated in the figure legends.
Immunoreactive proteins were visualized with either
[I125]Protein A or with enhanced chemiluminescence (ECL)
according to the specification of the supplier (Pierce Chemical Co., Rockford, IL). Immunoreactive bands were quantified by
counting the radioactive bands ([I125]Protein A) or by
quantitative image analysis of autoradiograms (ECL) using AlphaImager
2000 (3.3) from Alpha Innotech Corporation (San Leandro, CA).
Immunocytochemistry
Granulosa cells in each model system were also cultured (as
above) on glass coverslips for various times in the presence or absence
of FSH and T or forskolin. Cells were fixed in fresh 4%
paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA) in
PBS for 30 min at room temperature, blocked with PBS containing 10
mM glycine, and then washed three times with PBS as
described previously (54). The fixed cells were either stored at 4 C or
the cells were immediately permeabilized with 0.5% NP-40 in PBS for 10
min and then blocked with 4% BSA in PBS for 1 h at room
temperature. The cells were incubated at 4 C for 18 h with
specific antibodies diluted in buffers according to the specifications
of the companies from which they were obtained. After several PBS
washes, cells were incubated with fluorescein-labeled goat antirabbit
IgG (1:20, Pierce Chemical Co.) in 4% BSA in PBS for
1 h at room temperature. phospho-CREB, CREB, A-kinase catalytic
subunit (C subunit), CBP, and RII/ß were visualized on a Axiophot
microscope (Carl Zeiss, Thornwood, NY).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported, in part, by NIH Grants HD-16272 (J.S.R.) and CA-71514 (G.L.F.).
Received for publication December 15, 1998. Revision received May 7, 1999. Accepted for publication May 20, 1999.
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REFERENCES |
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