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-Dependent Pathway1
Departments of Physiology (G.F., A.P., J.J.P.) and Obstetrics and Gynecology (K.L., 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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, 
, 
,
and 
. Of these PKC isoforms, only PKC has been shown to be
activated by TPA. In apoptotic SIGCs, PKC levels were depleted. When
PKC levels were reduced by pretreatment with 500 nM TPA,
neither bFGF nor 10 nM TPA suppressed apoptosis.
Collectively, these observations suggest that bFGF maintains
[Ca2+]i and thereby SIGC viability through a
PKC-dependent pathway. |
Introduction |
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Unfortunately, little is known about the signal transduction pathways through which any of these factors prevent granulosa cell apoptosis. For example, bFGF is known to bind to high affinity cell surface receptors that possess intrinsic tyrosine kinase activity (8). Ligand activation of this receptor induces receptor dimerizaiton, tyrosine kinase activity, and autophosphorylation (8). Further, studies have shown that genistein, a tyrosine kinase inhibitor, blocks bFGF’s ability to prevent granulosa cell apoptosis (5). While this is consistent with bFGF’s known mechanism of action, no studies have been conducted to elucidate the antiapoptotic signaling events downstream of the tyrosine phosphorylation of the FGF receptor. There is one report that suggests that granulosa cells undergoing apoptosis in vivo have a reduction in the activity of the Raf-1-MEK-ERK signaling pathway (9). Because bFGF can stimulate this pathway (8), it is possible that this decrease in Raf-1-MEK-ERK signaling could be due to the lack of bFGF stimulation but this issue has not been addressed.
One reason for not knowing more about the signal transduction pathways
through which bFGF prevents granulosa cell apoptosis may be related to
not having a granulosa cell line. To resolve this problem, we have
tested a spontaneously immortalized granulosa cell line
(i.e. SIGCs). These cells were developed by Dr. Burghardt
(Texas A&M University) and derived from 45-day-old Berlin Duckery (BD
IV) rats (10). These cells remain undifferentiated and do
not spontaneously luteinize in culture. Like undifferentiated granulosa
cells, they express cytochrome P450scc and
synthesize limited amounts of estradiol and progesterone
(10). In this paper, we will present data that demonstrate
that the SIGCs undergo apoptosis in the absence of bFGF. Data will also
be presented that reveals that bFGF mediates its antiapoptotic action
by maintaining calcium homeostasis through a protein kinase
C-dependent mechanism.
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Materials and Methods |
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Identification of apoptotic cells
Apoptosis was assessed by either in situ staining
using the nuclear dye, YOPRO-1, by annexin V/propidium iodide staining
or by the detection of 185 bp DNA fragments. To stain apoptotic cells,
YOPRO-1 was added directly into each culture chamber at a final
concentration of 10 µM (11). The
cells were incubated for 10 min at 37 C and then observed at a
magnification of 200x under fluorescent optics using the FITC 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 200 cells/well were counted. The percentage of apoptotic cells
was then calculated.
SIGCs were stained with annexin V and propidium iodide using reagents and protocol provided by Oncogene Research Products (Cambridge, MA). The only deviation from the manufacturer’s protocol was that the concentration of annexin V was doubled. Those cells that bound annexin V but did not stain with propidium iodide were considered to be in the early stages of apoptosis.
DNA fragmentation was assessed using the Quick Apoptotic DNA Ladder Detection protocol and reagents provide by BioVision Inc. (Palo Alto, CA). For these studies, cells were plated in 100 mm glass Petri dishes as previously described. Five hours after serum withdrawal, the dishes were placed on an orbital mixer (Thermolyne RotoMix, Fisher Scientific, Pittsburgh, PA). The dishes were shaken for 5 min at room temperature at 100 rpm. The "loosely attached" cells were then collected by aspirating the medium. The attached cells were harvested by exposing cells to a trypsin/EGTA solution for 2 min at 37 C and subsequently aspirating the medium. The media containing either the "loosely attached" or attached cells, respectively, were centrifuged at 600 x g for 10 min at room temperature. The medium was removed, and the DNA from the cell pellet was processed per the BioVision protocol. Ten to 15 microliters of DNA sample was loaded onto a 1% agarose gel containing 0.5 µg/ml of ethidium bromide. The gel was run at 5 V/cm for 2 h. The ethidium bromide-stained DNA was then visualized by transillumination with UV light. The image was then captured using an Alpha Imager 2000 system (Alpha Innotech, San Leandro, CA).
[Ca2+]i measurements
SIGCs were plated in serum-supplemented medium on round cover
glass for 24 h and then 24 h in serum-free medium with bFGF.
The cells were then loaded at room temperature with Fluo-4 AM, a
calcium dye indicator, according to the protocol provided by
Molecular Probes, Inc. in the presence of Pluronic F-127,
sulfinpyrazone and bFGF. After loading, the cover glass was placed in a
coverslip clamp culture chamber (ALA Scientific Instruments, Inc.,
Westbury, NY). The cells were incubated at room temperature in 0.5 ml
of Krebs-HEPES buffer supplemented with bFGF. A field of cells was
selected based on their phase image. Fluorescent (i.e.
Fluo-4) images were then captured at 30 sec intervals. To allow the
cells to establish a baseline level, the first 7 images
(i.e. first 3.5 min) were discarded. Fluorescent images were
collected from cells in the presence or absence of bFGF. The intensity
of the Fluo-4 fluorescence was assessed in each cell using IP Lab
Spectrum Software (Signal Analytics Corp., Vienna, VA).
[Ca2+]i levels were
expressed as a fold change compared with the 3.5 min value. Similar
experiments were conducted to assess the effect of the PKC inhibitor,
chelerythrine chloride, as described (12). In this study,
bFGF was present continuously and then either chelerythrine chloride (1
µM) or DMSO was added after 3.5 min.
[Ca2+]i levels were
determined as outlined above.
Western blot analysis
After the cells were collected, 1 ml of boiling lysate buffer
(125 nM Tris pH 6.8; 4% SDS, 10% glycerol, 0.006%
bromphenol blue and 2% mercaptoethanol) was added to each cell
preparation. The cells were then passed through a 26 g needle
several times to reduce viscosity. The samples were then boiled for 5
min and subsequently centrifuged for 5 min at 13,000 x
g at 4 C to remove insoluble material. For each experiment,
a parallel culture was extracted with a boiling lysate buffer which
contained 1% SDS, 1.0 mM sodium ortho-vanadate,
and 10 mM Tris (pH 7.4). Protein determinations
were made on these samples using the BCA method (Pierce Chemical Co., Rockford, IL). Typically, 10 µg of lysate were loaded
onto each lane and the sample electrophoresed on a 10% polyacrylamide
gel at 100 V. Proteins were then transferred to nitrocellulose and
incubated with 5% nonfat milk (Nestlé Food Company, Glendale,
CA) in Tris-buffered saline with 0.1% Tween-20 (TBS-T) for 1 h
with agitation at 4 C.
The nitrocellulose blot was then 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. For comparison, positive controls for each isotype were run with the SIGC lysates. The positive controls were also provided by Transduction Laboratories, Inc. The blots were washed four times with 1% nonfat milk in TBS-T and incubated with a 1:25,000 dilution of a peroxidase-labeled goat antimouse IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD) in 1% nonfat dry milk for 1 h with agitation at room temperature. The specific protein was detected by chemiluminescence using the SuperSignal ULTRA detection system (Pierce Chemical Co.). Specific staining is assessed by omitting the primary antibody from the Western blot protocol.
Statistical analysis
Experiments involving annexin V/propidium iodide staining, DNA
fragmentation analysis, Western blot analysis, and
[Ca2+]i measurements were
repeated two to three times with each experiment yielding essentially
identical results. The experiments in which apoptosis was assessed by
YOPRO-1 staining were done in quadruplicate with each experiment
replicated two to three times. These data were pooled and analyzed by a
one way ANOVA followed by a Student’s-Newman-Keuls test, when
appropriate. Comparisons between two groups were made by a Student’s
t test. Regardless of the test, P values of less
than 0.05 were considered to be significant.
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Results |
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). These cells were isolated by brief
agitation and aspirated from the culture dish. Approximately 90% of
the "loosely attached" cells stained with YOPRO-1. Subsequent DNA
fragmentation analysis revealed that the "loosely attached" cells
possessed a DNA ladder pattern characteristic of apoptotic cells. The
DNA was intact in those cells that maintained contact with the culture
dish after being deprived of serum for 5 h (Fig. 1). The
percentage of cells that lost contact and stained with YOPRO-1 after
serum withdrawal increased to 20–30% by 5 h (Fig. 2).
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). Cells that stained
with propidium iodide were rarely observed in either treatment group
(Fig. 3).
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).
Interestingly, bFGF had to be present within 30 min of serum withdrawal
to prevent apoptosis (Fig. 4B).
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, [Ca2+]i levels were
maintained at a relatively constant level in the presence of bFGF. Upon
the removal of bFGF,
[Ca2+]i levels rapidly
increased. This coincided with an apparent increase in calcium within
the nucleus (Fig. 5). To determine whether the observed increase in
[Ca2+]i played an
essential role in SIGC apoptosis, cells were loaded with an
intracellular calcium chelator, BAPTA-AM, and then serum was removed.
Under these conditions serum depletion resulted in an increase in
apoptosis, but this increase was attenuated by pretreatment with BAPTA
(Fig. 6).
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). Like bFGF, TPA was
required to be present within 30 min of serum withdrawal (Fig. 7B).
Further, the ability of bFGF to prevent apoptosis was attenuated by the
PKC inhibitor, bisindolylmaleimide II, but not by its inactive analog,
bisindolylamleimide V (Fig. 8).
Similarly, the PKC inhibitor, chelerythrine chloride, interfered with
the ability of bFGF to maintain calcium homeostasis (Fig. 9). Free calcium levels also appeared to
increase within the nuclei of the chelerythrine-treated cells (Fig. 9).
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, 
, , and were
expressed by SIGCs (Fig. 10) and
primary rat granulosa cells (data not shown). Of these PKC isoforms,
PKC was the only one that can be activated by TPA (16).
Serum withdrawal did not alter the PKC level in cells that remained
attached (Fig. 11). In "loosely
attached" cells, the amount of PKC was dramatically reduced (Fig. 11). Levels of PKC were not affected by serum withdrawal (Fig. 11).
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in regulating SIGC apoptosis,
cells were pretreated with a 500 nM dose of TPA and the
levels of PKC and assessed by Western blot. Under these
conditions PKC but not PKC levels were reduced to nearly
nondetectable levels (Fig. 12).
Interestingly, neither bFGF (Fig. 13A)
nor 10 nM TPA (Fig. 13B) prevented apoptosis in SIGCs in
which the level of PKC was reduced by pretreatment with 500
nM TPA.
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Discussion |
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Interestingly, the DNA is intact in those cells that adhere to the culture dish after 5 h of serum deprivation. Also, the morphology of these cells appears similar to those maintained in serum-supplemented medium. In spite of their normal morphology and intact DNA, those cells that remain attached to the culture dish under serum-free conditions are in the early stages of apoptosis. This statement is based on the finding that most of these attached cells bind annexin V but do not stain with propidium iodide. Annexin V binding is detected very early in the apoptotic pathway, well before major changes in membrane permeability as assessed by propidium iodide staining or the formation of apoptotic bodies (17, 18). Taken together, the present study indicates that the serum-deprived SIGCs provide a useful model to study granulosa cell apoptosis in that SIGCs in either the early or late-stages of apoptosis can be isolated and assessed. Moreover, this study shows that SIGC apoptosis is inhibited by bFGF. Thus, the SIGCs provide a useful cell line in which to elucidate the signal transduction pathways through which bFGF prevents granulosa cell apoptosis.
As has been shown for primary granulosa cells (15), [Ca2+]i levels increase in SIGCs after removal of survival factors. This increase in [Ca2+]i likely plays an essential role in regulating SIGC apoptosis given that BAPTA, an intracellular calcium chelator, prevents these cells from undergoing apoptosis in response to serum withdrawal. Apparently, SIGCs can tolerate either elevated levels of [Ca2+]i or the calcium-induced changes for up to 15 min since bFGF or TPA given by 15 min after serum withdrawal, prevents apoptosis. However by 30 min, the elevated [Ca2+]i apparently alters the cell such that it cannot be maintained by bFGF.
There are at least two mechanisms by which a prolonged increase in
[Ca2+]i could promote
apoptosis. First, the increase in
[Ca2+]i is likely to
increase calcium levels within the mitochondria (19). This
has been shown to promote the release of cytochrome c from mitochondria
(19). Cytochrome c then could act to increase caspase
activity (20). Ultimately, caspases cleave various
substrates that are essential for cell viability. One such substrate is
PKC (21, 22). The finding that PKC levels are
reduced in apoptotic SIGCs is consistent with enhanced caspase-3
activity. Furthermore, an increase in caspase-3 activity occurs during
both primary granulosa cell (2) and SIGCs apoptosis
(Peluso, unpublished observation). A second consequence of prolonged
elevations in
[Ca2+]i is the
apparent accumulation of calcium within the nucleus. This putative
increase in nuclear free calcium levels would activate endonucleases
that cut the DNA into 185 bp fragments (23). This would
account for the presence of the DNA ladders in the apoptotic SIGCs.
Collectively, these observations are consistent with the concept that
elevated [Ca2+]i levels
leads to an increase in both caspase and endonuclease activity and
thereby cause the SIGCs to undergo apoptosis.
The present studies also reveal that bFGF prevents SIGC apoptosis in part by maintaining [Ca2+]i levels. The ability to maintain calcium homeostasis is also involved the mechanism through which bFGF prevents neural cell death (24). In SIGCs, bFGF regulates calcium homeostasis in part through a PKC-dependent pathway because the PKC inhibitor, chelerythrine chloride, results in an rapid increase in [Ca2+]i even in the presence of bFGF. In chicken granulosa cells, PKC activation blocks transient increases in [Ca2+]i (25). In these cells, [Ca2+]i is released from its intracellular stores. The emptying of these stores seems to activate calcium channels within the cell membrane, thereby triggering a calcium influx. This results in a further increase in [Ca2+]i. The influx of calcium is inhibited through a PKC-dependent pathway (25). It has been proposed that PKC activation phosphorylates and subsequently inactivates these calcium channels. This stops the influx of calcium and terminates the increase in [Ca2+]i. This would allow the calcium-ATPase pump to move calcium back into its cellular stores thereby returning [Ca2+]i levels to a normal range (25). In addition, the calcium-ATPase transport pump could be directly activated by PKC (for review see Ref. 16). These mechanisms may both be involved in regulating [Ca2+]i in SIGCs because they are not mutually exclusive. However, which if either of these two mechanisms are involved in maintaining calcium homeostasis in SIGCs remains to be determined.
As previously indicted, details regarding the signal transduction
pathway through which bFGF regulates
[Ca2+]i and apoptosis in
both primary granulosa cells and SIGCs are very limited. Tilly et
al. (5) first demonstrated that bFGF prevents
granulosa cell apoptosis. Moreover, these investigators showed that
bFGF’s actions were blocked by genistein, a tyrosine kinase inhibitor
(5). Because the FGF receptor is a member of the receptor
tyrosine kinase family (8), it is likely that genistein
blocks the initial activation of the FGF receptor. The events
downstream of FGF receptor activation are largely unknown. Once ligand
activated (i.e. tyrosine phosphorylated), the FGF receptor
stimulates a number of different signal transduction pathways
(8). These include the activation of PLC
that leads to
the hydrolysis of PIP2 generating
IP3 and diacylglcerol (DAG) (8). The
IP3 induces an increase in
[Ca2+]i, whereas DAG
activates PKC. The data in the present study argue that in SIGCs the
antiapoptotic action of bFGF is mediated through its ability to
activate a PKC-dependent mechanism. This is based on the findings that
an activator of PKC, TPA, mimics whereas PKC inhibitors block the
effects of bFGF. This is consistent with the findings of Amsterdam and
associates that showed phorbol esters prevent cAMP-induced granulosa
cell apoptosis (13, 26).
Although the present data implicate PKC as the mediator of bFGF’s
antiapoptotic action, it important to appreciate that there are several
different isotypes of PKC (25, 27, 28, 29). Briefly, the PKC
isotypes can be classified into three groups: conventional, novel, and
atypical. Conventional PKCs are calcium-dependent, DAG/TPA-activated
and include isotypes 
, ß1,
ß2, and . Novel PKCs (i.e. ,
, 
, 
,) are calcium independent but DAG/TPA activated. Finally,
the atypical PKCs (, /) are calcium-dependent but not
responsive to either DAG or TPA. Our data show that PKC, ,
/ are present in SIGCs and primary granulosa cells. Although in
some cell types PKC appears to be involved in promoting apoptosis
(for review see Ref. 27), the following observations
indicate that PKC acts as the mediator of bFGF’s antiapo-ptotic
action in SIGCs. First, TPA mimics the antiapoptotic actions of bFGF.
This argues that PKC transduces the antiapototic action of TPA
because PKC is the only PKC isotype in SIGCs that is capable of
responding to TPA. Further, the effective dose of TPA is 10
nM. This is similar to the 9.6
nM Kd for TPA binding to
PKC (28). Second, the TPA-induced depletion of PKC
attenuates the ability of both bFGF and TPA to prevent SIGC apoptosis.
High doses of TPA can down-regulate the FGF receptor, and this could
explain the inability of bFGF to maintain SIGCs after PKC depletion
(30). However, this would not account for TPA’s failure
to prevent apoptosis in PKC-depleted SIGCs. All of these studies are
consistent with the hypothesis that PKC mediates bFGF’s
antiapoptotic action. However, additional studies are required to
conclusively prove this concept.
The concept that PKC promotes survival in some cell types including
SIGCs while inducing apoptosis in others is somewhat perplexing
(27). One reason for these opposite effects may be related
to the cellular location of PKC. For example, PKC is usually
associated with the membrane in viable cells (31). At this
location, PKC could regulate the phosphorylation status of the
calcium channels as previously proposed (24). In other
cells, PKC could be either localized or translocated to the nucleus
as part of the apoptotic cascade (31, 32, 33). Once in the
nucleus PKC associates with the DNA-dependent protein kinase
catalytic subunit (34). This results in the
phosphorylation of DNA-dependent protein kinase catalytic subunit,
which ultimately leads to the apoptotic death of human leukemia cells.
Interestingly, TPA maintains the membrane localization of PKC and
prevents apoptosis in human leukemia cells (34). Whether
bFGF or TPA maintains SIGC viability by regulating the localization of
PKC is now being assessed.
In summary, the present data support the hypothesis that bFGF activates
PKC, which in turn maintains
[Ca2+]i within a normal
physiological range. It is further proposed that it is the ability to
regulate calcium homeostasis that accounts in part for bFGF’s
antiapoptotic action.
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Acknowledgments |
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Footnotes |
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Received May 5, 2000.
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References |
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is activated by caspase-dependent proteolysis
during ultraviolet radiation-induced apoptosis of human keratinocytes.
J Biol Chem 273:29995–30002. Eur J
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and - to the cytosol. Implications in
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