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-Dependent Pathway
Departments of Physiology (J.J.P., A.P., G.F.) and Obstetrics and Gynecology (J.J.P.), University of Connecticut Health Center, Farmington, Connecticut 06030
Address all correspondence and requests for reprints to: John J. Peluso, Ph.D., Department of Physiology, University of Connecticut Health Center, Farmington, Connecticut 06030. E-mail: peluso{at}nso2.uchc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, -
, and -
. Finally, the
PKC-specific inhibitor, rottlerin, blocked basic fibroblast growth
factor’s antiapoptotic action in granulosa cells and spontaneously
immortalized granulosa cells. These studies suggest that basic
fibroblast growth factor regulates intracellular free calcium through a
PKC-dependent mechanism and that a sustained increase in
intracellular free calcium is sufficient to induce and is required
for granulosa cell apoptosis.
Additional studies demonstrated that in spontaneously immortalized
granulosa cells, basic fibroblast growth factor increased PKC
activity by 60% within 2.5 min compared with serum-free control
levels. Rottlerin attenuated basic fibroblast growth factor’s ability
to stimulate PKC activity and to maintain intracellular free
calcium. Further, intracellular free calcium levels in spontaneously
immortalized granulosa cells transfected with a PKC antibody in the
presence of basic fibroblast growth factor were 2-fold higher than
those spontaneously immortalized granulosa cells transfected with IgG.
Similarly, transfecting spontaneously immortalized granulosa cells with
a specific PKC-substrate increased intracellular free calcium
compared with spontaneously immortalized granulosa cells transfected
with a specific substrate for PKC
. Moreover, basic fibroblast growth
factor increased and rottlerin attenuated 45Ca efflux by
50% compared with that in basic fibroblast growth factor-treated
cells. Finally, an inhibitor of the plasma membrane
calciumadenosine triphosphatase pump suppressed 45Ca
efflux, elevated intracellular free calcium, and induced apoptosis.
Collectively, these studies demonstrate that basic fibroblast
growth factor activates PKC, which, in turn, stimulates
calcium efflux, accounting in part for basic fibroblast growth
factor’s ability to maintain calcium homeostasis and, ultimately,
granulosa cell viability.
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Introduction |
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It is likely that the downstream signaling events involve both acute
and genomic actions (2, 6, 7, 8). Although the genomic
actions are important, the acute actions are critical, as bFGF
deprivation for just 30 min commits spontaneously immortalized
granulosa cells (SIGCs) to undergo apoptosis (8). These
acute actions of bFGF appear to be related to changes in intracellular
free calcium ([Ca2+]i)
(8). This is based on the observations that an increase in
[Ca2+]i occurs before
SIGCs undergo apoptosis (8), and bFGF prevents this
increase in [Ca2+]i
(8). In SIGCs the antiapoptotic effect of bFGF is mimicked
by 12-O-tetradecanoylphorbol-13-acetate (TPA) and attenuated
by general PKC inhibitors (8). Further, PKC inhibitors
abrogate the ability of bFGF to maintain normal basal
[Ca2+]i (8).
As PKC is the only PKC isotype that is expressed by SIGCs
(8) that can be activated by TPA (9, 10),
PKC has been implicated as the mediator of bFGF’s action. However,
the studies that implicate PKC as a mediator of bFGF’s
antiapoptotic action have all been conducted on SIGCs and not primary
GCs. Therefore, the present studies were undertaken to determine
whether PKC mediates bFGF’s antiapoptotic action in primary
GCs. This is likely, because in GCs bFGF increases the level of
diacylglycerol (DAG), an endogenous PKC activator, by 2- to 3-fold
within 2 min of exposure (11).
Subsequent studies were conducted to elucidate the mechanism through
which bFGF-activated PKC regulates
[Ca2+]i. These studies
will demonstrate that bFGF-activated PKC ultimately stimulates
calcium efflux. In this manner, bFGF maintains
[Ca2+]i within a
physiological range and thereby promotes GC viability.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Granulosa cell isolation and culture
Immature female Wistar rats (22 d of age) were obtained from
Charles River Laboratories, Inc. (Wilmington, MA), and
housed under controlled conditions of temperature, humidity, and
photoperiod (12 h of light, 12 h of darkness; lights on at
0700 h). On the day of the experiment, immature animals were 23 or
24 d of age. The rats were cervically dislocated between 0930 and
1000 h, the ovaries were removed, and GCs were isolated. This
protocol was approved by the animal care committee of the University of
Connecticut Health Center.
GCs were isolated according to the procedure of Lederer et al. (12). Once collected, small and large GC populations were separated by Percoll gradient centrifugation as previously described (12). The large GC population was composed of approximately 80% large GCs and 20% small GCs. Only the large GC population was used in these experiments, because they undergo apoptosis when deprived of growth factors (13). Unless otherwise stated, this cell population was washed, resuspended in RPMI 1640, and used in various protocols as outlined in subsequent sections. For the apoptosis studies the freshly isolated GCs were plated with the various treatments in 0.5 ml serum-free medium in Lab-Tek slides at a density of 1.25 x 105 cells/ml, and cultures were maintained in 5% CO2 at 37 C for 5 h.
SIGC culture
SIGCs were cultured in DMEM/F-12 supplemented with 5% FBS as
previously described (8). The SIGCs were routinely
maintained in Falcon T-flasks (Becton Dickinson and Co.,
Lincoln Park, NJ). SIGCs were plated in 100-mm glass (Kimax,
Fisher Scientific, Pittsburgh, PA) culture dishes at a
density of 4 x 105 cells/ml in 12 ml
medium. SIGCs were plated in 0.5 ml medium at 1.25 x
105 cells/ml in 8-chamber glass Lab-Tek slides
(Nunc Inc., Naperville, IL). Unless otherwise stated, the cells were
initially cultured in DMEM/F-12 supplemented with 5% FBS for 24
h. The serum-supplemented medium was removed, and the cells were
cultured in serum-free DMEM/F-12 with various reagents for up to 5
additional h.
Identification of apoptotic cells
Apoptosis was assessed by in situ staining using the
nuclear dye, YOPRO-1 (Molecular Probes, Inc.)
(8, 14). To stain apoptotic cells, YOPRO-1 was added
directly into each culture chamber at a final concentration of 10
µM (8). The cells were incubated
for 10 min at 37 C and then observed at a magnification of x200 under
fluorescent optics using the fluorescein isothiocyanate filter set. The
number of fluorescent cells (i.e. apoptotic cells) in a
field was counted. The total number of cells in that field was also
counted under phase optics. The process was continued until at least
100 cells/well were counted. The percentage of apoptotic cells was then
calculated.
[Ca2+]i measurements
Before loading, GCs were plated on cover glass in 35-mm dishes
for 2 h in serum-supplemented medium. SIGCs were plated on cover
glass in 35-mm dishes in serum-supplemented for 24 h, then for
24 h with serum-free bFGF-supplemented medium. After the culture
period, both GCs and SIGCs were then loaded at room temperature with
fluo-4/AM, a calcium dye indicator, in the presence of bFGF
(8). After loading, the cover glass was washed and placed
in a coverslip clamp culture chamber (ALA Scientific Instruments, Inc.,
Westbury, NY). The cells were incubated at room temperature in 0.5 ml
Krebs-HEPES buffer supplemented with bFGF. To allow the cells to
establish a baseline level, the images from the first 3.5 min were
discarded. Fluorescent images were collected at 30-sec intervals from
cells as indicated for each experimental design. Unless otherwise
stated, the intensity of the fluo-4 fluorescence was expressed as a
fold change compared with the 3.5-min value.
Western blot analysis of PKC isotypes
PKC expression in primary GCs was assessed immediately after
isolation. SIGCs were harvested from serum-supplemented medium. After
the cells were collected, they were processed for Western blot analysis
as previously described (8). Briefly, 10 µg lysate was
loaded onto each lane, and the sample was electrophoresed on a 10%
polyacrylamide gel at 100 V, then transferred to nitrocellulose. The
nitrocellulose blot was incubated for 2 h with agitation at room
temperature with monoclonal antibodies built against various PKC
isotypes (Transduction Laboratories, Inc., Lexington, KY)
at the dilution recommended by the manufacturer. The blot was then
process as previously described (8). The specific protein
was detected by chemiluminescence using the LumiGLO detection system
(Kirkegaard & Perry Laboratories, Gaithersburg, MD).
Specific staining was assessed by omitting the primary antibody from
the Western blot protocol.
bFGF and PKC activity
PKC activity was assessed using a phosphospecific PKC
antibody (Cell Signaling, Beverly, MA). When PKC is activated, it
undergoes autophosphorylation on Ser662.
Therefore, the amount of phosphorylated PKC is a direct assessment
of PKC activity (15). In this study SIGCs were cultured
for 24 h in serum, then for 24 h in serum-free medium with
bFGF, then either bFGF was removed or rottlerin was added to the
cultures. After 2.5 min at room temperature, the cells were harvested.
A crude membrane preparation (16) was made and used for
Western blot analysis. The blots were first probed with a
phosphospecific PKC antibody. This antibody detects PKC
, -ß, -
,
and - that are phosphorylated at Ser662. As
SIGCs express PKC and not PKC or PKCß, the amount of
phospho-PKC is a direct assessment of PKC activity in SIGCs. The
blots were then stripped and reprobed with a PKC-specific
antibody.
A quantitative estimate of PKC activity was obtained by scanning the
films and determining the density of the phospho-PKC bands using IPGel
software (Scanalytics, Inc., Vienna, VA). To ensure valid quantitation
of the phospho-PKC bands, several exposures of each Western blot were
prepared. The exposures selected for analysis possessed gray scale
values from 101–194 on a linear gray scale of 0–255. All treatments
from each experiment were run on the same gel and therefore subjected
to the same Western blot procedure (i.e. incubation times,
etc.). For analysis, the background from each film was subtracted from
each band to yield a specific density. The specific density of each
band was then divided by the specific density of the control treatment,
resulting in a fold increase from the control. This was done to correct
for the relatively small variation between experiments.
Effect of PKC antibody or PKC-specific substrate peptide on
[Ca2+]i
To establish a cause and effect relationship between
PKC and the maintenance of
[Ca2+]i, either a PKC
antibody or peptide substrate was delivered into the cells using
protein transfection. To demonstrate the feasibility and effectiveness
of this approach, SIGCs were exposed to tetramethylrhodamine B
isothiocyanate-labeled IgG in the presence or absence of the protein
transfection agent, Chariot, according to manufacturer’s instructions
(Active Motif, Carlsbad, CA). After transfection, the cells were loaded
with the calcium indicator dye, fluo-4. The percentage of cells that
incorporated tetramethylrhodamine B isothiocyanate-IgG and their
ability to load with Fluo-4 were then assessed.
Once the manufacturer’s protocol was shown to be effective for SIGCs,
it was used in the following study. First, either the PKC antibody
or IgG was mixed with transfection agent at a ratio of 1 part antibody
to 30 parts transfecting reagent. The SIGCs were then incubated with
the antibody mixture for a 30 min at 22 C and then for 3 h at 37
C. The antibody mixture was then removed, and the cells were loaded
with fluo-4 as previously described (8). Similar studies
were conducted with the substrate peptides for either PKC or PKC
(Calbiochem, San Diego, CA). Each peptide was used at a
concentration of 500 ng/transfection reaction.
After fluo-4 loading, cells were observed for a 3.5-min base line period. At 3.5 min an image of the fluo-4 fluorescent intensity was captured to determine the fluo-4 fluorescence (F). Then calcium ionophore, A23187 (50 µM; Sigma), was added to determine maximum fluorescent intensity (Fmax). EGTA (7 mM; Sigma) was then added, and the minimum fluorescent intensity was determined (Fmin). [Ca2+]i was estimated by the following equation: intracellular free calcium (nM) = 345 nM (F - Fmin)/(Fmax - F) (17). This approach to estimate [Ca2+]i levels was used to allow comparisons between treatment groups.
Calcium efflux measurements
SIGCs were loaded in the presence of bFGF and
45Ca (10 µCi/ml) for 18 h and washed with
45Ca-free medium as described by Husain and
associates (18). The bFGF-treated cultures were placed at
room temperature, and a 100-µl aliquot was taken. Then either vehicle
or test agent was added. Samples were taken at 15- or 30-sec intervals
over the next 3 min. The samples were added to liquid scintillation
cocktail and counted for 1 min in an LKB 6500 liquid
scintillation counter (Rockville, MD). The values were corrected for
the removal of medium, and the cumulative amount of
45Ca extruded was determined. Cumulative
45Ca efflux was expressed as the fold increase
from the value obtained 15 sec after the addition of vehicle or test
agent.
Statistical analysis
All experiments were repeated several times, as indicated in the
figures. The experiments in which apoptosis was assessed by YOPRO-1
staining were performed in quadruplicate, with each experiment
replicated two or three times. These data were pooled and analyzed by
one-way ANOVA, followed by Student-Newman-Keuls test when appropriate.
Comparisons between two groups were made by t test.
Regardless of the test, P < 0.05 was considered
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In addition,
treatment with thapsigargin, an agent known to increase
[Ca2+]i, induced GC
apoptosis even in the presence of bFGF (Fig. 1B).
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, both bFGF and TPA
reduced the percentage of apoptotic GCs to approximately 35%. Given
that the percentage of apoptotic before culture is about 30–35%, this
indicates that both bFGF and TPA are extremely effective in suppressing
GC apoptosis. Further, these observations confirm previous work with
SIGCs (8) that suggest a major role for PKC in regulating
GC viability. This concept was further supported by the observation
that in GCs the ability of bFGF to prevent apoptosis was attenuated by
the general PKC inhibitor, bisindolylmaleimide II, but not by its
inactive analog, bisindolylamleimide V (Fig. 2B). In addition, bFGF’s
ability to maintain
[Ca2+]i levels was
blocked by chelerythrine chloride, another general PKC inhibitor
(Fig. 3).
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, -, and - were
expressed by GCs (Fig. 4). As TPA mimics
bFGF’s action, and PKC is responsive to TPA (9, 10),
it is likely that PKC mediates bFGF’s action. To test this
hypothesis, the effect of rottlerin, a PKC-specific inhibitor, on GC
and SIGC apoptosis was determined. In this study rottlerin inhibited
the ability of bFGF to preserve GC and SIGC viability (Fig. 5, A and B).
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activity was monitored using a phospho-PKC antibody. As shown in
Fig. 6, PKC activity was 1.4- to
1.7-fold higher in bFGF-treated cells than in cells cultured for 2.5
min in serum-free medium. Further, rottlerin, a PKC-specific
inhibitor, blocked the ability of bFGF to increase PKC activity.
Although phospho-PKC levels were altered, the overall amount of
PKC was similar in all treatment groups (Fig. 6).
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in mediating bFGF’s ability to maintain
normal basal [Ca2+]i was
also examined by exposing SIGCs to rottlerin in the presence of bFGF.
Under these conditions rottlerin induced an increase in
[Ca2+]i (Fig. 7). The second approach used to determine
the relationship between bFGF, PKC and
[Ca2+]i involved
transfecting SIGCs with either IgG or a PKC antibody. To demonstrate
the feasibility of this approach, SIGCs were exposed to TRITC-labeled
IgG in the presence or absence of the protein transfection agent,
Chariot. After transfection, the cells were loaded with the calcium
indicator dye, fluo-4. TRITC-IgG was not incorporated into the SIGCs
unless the transfection agent was present (data not shown). In the
presence of the transfection agent the cells appeared healthy, as
judged by phase optics (Fig. 8A, upper panel), and approximately 90% of these cells
incorporated TRITC-IgG (Fig. 8A, middle panel). These cells
were also able to be loaded with fluo-4 (Fig. 8A, lower
panel).
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antibody or PKC-specific substrate
was transfected into SIGCs. Under these conditions, the PKC antibody
increased [Ca2+]i nearly
2-fold compared with IgG. A smaller, but significant, increase in
[Ca2+]i levels was
observed after transfecting SIGCs with a specific PKC substrate
peptide compared with a specific PKC substrate peptide (Fig. 8B
).
There are several possible mechanisms through which PKC could
regulate normal basal
[Ca2+]i. However, it is
likely that the molecular targets of PKC are within the plasma
membrane, because activated PKC is often localized to the plasma
membrane (9, 10). Given this, one possibility is that
PKC influences the activity of proteins that regulate calcium
efflux. As shown in Fig. 9, bFGF
stimulated 45Ca efflux compared with that in
serum-free controls. Both rottlerin and lanthanum, an inhibitor of the
plasma membrane calcium-adenosine triphosphatase (PMCA)
(19), suppressed bFGF-induced 45Ca
efflux to the level in serum-free controls (Fig. 9, A and B).
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). However,
[Ca2+]i levels only
increased by 25–50% after a 10-min exposure, compared with a nearly
2-fold increase associated with rottlerin treatment (compare Figs. 7 and 10A). Finally, both lanthanum chloride and eosin, another PMCA
inhibitor (20), attenuated bFGF’s ability to maintain
viable SIGCs (Fig. 10B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mediates bFGF’s
antiapoptotic action by maintaining
[Ca2+]i within a
physiological range (8). However, for these findings to be
physiologically relevant, a similar PKCdependent mechanism
would also have to be involved in the signal transduction cascade
through which bFGF maintains GCs. Specifically, it is essential to
determine whether GCs undergo calcium-dependent apoptosis and whether
bFGF regulates [Ca2+]i
through a PKC-dependent mechanism. Previous studies have shown that an increase in [Ca2+]i occurs before GCs undergo apoptosis (13). That the dysregulation of [Ca2+]i is an essential component of the apoptotic cascade is illustrated by the fact that BAPTA, an intracellular calcium chelator, prevents GCs from undergoing apoptosis in the absence of bFGF. In addition, thapsigargin, which increases [Ca2+]i by releasing calcium from its intracellular stores, induces apoptosis even in the presence of bFGF. These data demonstrate that GCs undergo calcium-induced apoptosis.
The present studies also show that bFGF maintains relatively constant
levels of [Ca2+]i, and
the PKC inhibitor, chelerythrine chloride, attenuates bFGF’s ability
to regulate [Ca2+]i in
GCs. Further, PKC is implicated in regulating
[Ca2+]i and GC viability
by two sets of observations. First, the PKC-specific inhibitor,
rottlerin, abrogates bFGF’s antiapoptotic action. Second, an activator
of PKC, the phorbol ester TPA, prevents GC apoptosis. Like SIGCs, GCs
express PKC, -, and -. Of these three PKC isotypes, only
PKC possesses a phorbol ester-binding site and is capable of being
activated by TPA (9, 10). Moreover, the fact that GCs
express PKC confirms the in situ hybridization studies of
Hunnzicker-Dunn and associates (22). These in
situ hybridization studies reveal that PKC is expressed
predominately by GCs of healthy follicles (22). The
observation that PKC is selectively expressed by GCs of healthy
follicles is consistent with our hypothesis that PKC plays an
essential role in regulating the viability of GCs. As these findings in
GCs are identical to the observations made in SIGCs, they indicate that
SIGCs accurately mimic the physiological responses of GCs to bFGF. This
validates the use of SIGCs for the more mechanistic biochemical studies
that require large numbers of cells.
In our first biochemical study with SIGCs, PKC activity was
monitored using a phosphospecific PKC antibody. When PKC is
activated, it undergoes autophosphorylation on
Ser662 (15). Therefore, the amount
of phosphorylated PKC is a direct assessment of PKC activity
(15). This study supports our hypothesis by revealing that
bFGF stimulated and rottlerin inhibited the activation of PKC.
Collectively, these data suggest that bFGF regulates GC and SIGCs
viability by maintaining
[Ca2+]i through a
PKC-dependent mechanism.
To definitively establish a cause and effect relationship between the
activation of PKC and bFGF’s ability to maintain normal basal
[Ca2+]i levels, three
separate approaches were used. First, the PKC-specific inhibitor,
rottlerin, was shown to suppress PKC activity and promote an
increase in [Ca2+]i even
in the presence of bFGF. The second approach involved transfecting
SIGCs with either IgG or a PKC antibody. This study demonstrates
that even in the presence of bFGF the PKC antibody increased
[Ca2+]i levels nearly
2-fold compared with IgG. Finally, a specific PKC substrate was
transfected into SIGCs. This substrate competes with endogenous PKC
substrates, thereby blocking the physiological responses to PKC
activation. By using this approach, a smaller, but significant,
increase in [Ca2+]i
levels was observed after transfecting SIGCs with a specific PKC
substrate peptide compared with a specific PKC substrate peptide.
Taken together, these studies clearly demonstrate that a cause and
effect relationship exists between the bFGF activation of PKC and
the maintenance of normal basal
[Ca2+]i.
How, then, might PKC regulate normal basal
[Ca2+]i levels? There are
at least five possible sites of actions (23, 24). First,
PKC could stimulate the sacroplasmic/endoplasmic reticulum
calcium-adenosine triphosphatase pump, which would lower
[Ca2+]i by pumping
intracellular calcium into its intracellular stores (23).
Second, PKC could inhibit IP3 receptors,
thereby stopping IP3-induced calcium release from
intracellular stores. Type 1 and 3 IP3 receptors
are expressed by both GCs and SIGCs, but these receptors were not
localized to the plasma membrane (Peluso, J. J., unpublished
observations). Because of their cellular localization, it is unlikely
that DAG-activated PKC, which is localized to the plasma membrane,
could regulate either the sacroplasmic/endoplasmic reticulum
calcium-adenosine triphosphatase pump or the IP3
receptors.
The third possible site is action could involve voltage-operated
calcium channels, which are present in the plasma membrane of GCs
(25). These channels can be inhibited by TPA
(i.e. an activator of PKC) (24). Similarly,
channels that regulate capacitative calcium entry represent a fourth
possibility (24). These channels are activated by the
depletion of calcium stores. These channels are independent of voltage,
stimulated by IP3, highly selective for calcium,
and inhibited by PKC (24). It is possible that
DAG-activated PKC could inhibit either of these types of
channels.
The fifth site of PKC’s action could involve the regulation of
proteins that promote calcium efflux. In vascular smooth muscle cells,
normal basal [Ca2+]i
levels are maintained in part by removing excess calcium from the
cytoplasm (23). The removal of excess cytoplasmic calcium
is mediated by two mechanisms. The first is the PMCA pump
(26). The PMCA pump has a high affinity for calcium.
However, in many cells it has a low capacity because it is expressed at
a low level. The second mechanism involves two proteins: the
sodium/potassium-adenosine triphosphatase (NKA) pump (27)
and the sodium/calcium exchanger (NCX) (28, 29). The NKA
moves sodium out of the cell. This provides the gradient that activates
the NCX, which pumps calcium out and sodium into the cell. The NCX has
a high capacity to produce calcium efflux. Both the PMCA pump and the
NCX/NKA are localized to the plasma membrane, and their activities are
influenced by PKC (26, 27, 28, 29).
Although PKC could regulate that function of voltage-operated
calcium channels and channels that regulate capacitative calcium entry,
the present data suggest the calcium efflux is regulated by bFGF in a
PKC-dependent manner. This is based on the observations that bFGF
stimulates calcium efflux compared with that in control cells, and
rottlerin inhibits bFGF-induced calcium efflux. Further, lanthanum, an
inhibitor of PMCA, not only suppresses bFGF-induced calcium efflux, but
also results in a gradual increase in
[Ca2+]i and, ultimately,
apoptosis. These findings demonstrate that inhibiting calcium efflux
results in a sustained increase in
[Ca2+]i that is
sufficient to induce apoptosis after 5 h. It is important to note
that the lanthanum-induced increase in
[Ca2+]i is considerably
less than the 2- to 3-fold increase in
[Ca2+]i observed after
PKC inhibition with rottlerin. This could indicate that another
component, possibly NCX and NKA, may be regulated by bFGF-activated
PKC.
The present data indicate that bFGF-activated PKC stimulates calcium
efflux. There is one study in swine granulosa cells that describes the
kinetics of calcium efflux. In this study TPA was shown to inhibit
calcium efflux (30). However, this study monitored calcium
efflux over the course of 4 h. Under these experimental
conditions, two different pools of intracellular calcium contribute to
the overall amount of calcium that is extruded. The first compartment
involves the rapid release of calcium and is not inhibited by TPA. The
second compartment influences the later or slow phase of calcium efflux
and is attenuated by TPA. It is difficult to interpret this finding
because exposure to TPA for just a few hours can down-regulate PKC
levels (8). If TPA depleted endogenous PKC levels in swine
GCs, then the previously published report on calcium efflux would
indicate that PKC is involved in stimulating calcium efflux, which
would be consistent with the data of the present studies.
Based on these studies, we propose that ligand activation of the FGF
receptor increases intracellular levels of DAG (11). DAG
remains associated with the plasma membrane, where it activates PKC.
Once activated, PKC functions to serine/threonine phosphorylate
various molecular targets that may include calcium efflux regulators,
such as PMCA. As has been shown in other cell types, TPA-dependent
phosphorylation of PMCA enhances the rate of calcium efflux, thereby
maintaining calcium homeostasis (26). Future studies will
be directed to test this hypothesis.
Finally, the present in vitro studies imply that by
regulating GC viability, bFGF promotes follicular development and
inhibits follicular atresia in vivo. The findings that GCs
of developing follicles synthesize bFGF and express high affinity FGF
receptors (1) are consistent with this hypothesis.
Further, PKC is selectively expressed by GCs of healthy follicles of
all sizes (22). Our in vitro studies establish
a causal relationship among bFGF, PKC, and GC viability by
demonstrating that bFGF’s antiapoptotic action is mediated by PKC.
These findings therefore provide insights into the putative mechanism
by which bFGF prevents GC apoptosis and follicular atresia in
vivo.
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Acknowledgments |
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Footnotes |
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Abbreviations: BAPTA, 1,2 bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; bFGF, Basic fibroblast growth factor; [Ca2+]i, intracellular free calcium; DAG, diacylglycerol; GC, granulosa cell; NCX, sodium/calcium exchanger; NKA, sodium/potassium-adenosine triphosphatase; PMCA, plasma membrane calcium-adenosine triphosphatase; SIGC, spontaneously immortalized granulosa cells; TPA, 12-O-tetradecanoylphorbol-13-acetate.
Received May 8, 2001.
Accepted for publication July 6, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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J.J. Peluso and A. Pappalardo Progesterone Regulates Granulosa Cell Viability Through a Protein Kinase G-Dependent Mechanism That May Involve 14-3-3{sigma} Biol Reprod, December 1, 2004; 71(6): 1870 - 1878. [Abstract] [Full Text] [PDF]
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J.J. Peluso, G. Fernandez, A. Pappalardo, and B.A. White Membrane-Initiated Events Account for Progesterone's Ability to Regulate Intracellular Free Calcium Levels and Inhibit Rat Granulosa Cell Mitosis Biol Reprod, August 1, 2002; 67(2): 379 - 385. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. |
