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Reproductive Biology Unit and Division of Reproductive Medicine, Departments of Obstetrics and Gynecology and Cellular and Molecular Medicine, University of Ottawa; and Ottawa Health Research Institute, The Ottawa Hospital (Civic Campus), Ottawa, Ontario, Canada K1Y 4E9
Address all correspondence and requests for reprints to: Benjamin K. Tsang, Ph.D., Ottawa Health Research Institute, The Ottawa Hospital (Civic Campus), 725 Parkdale Avenue, Ottawa, Ontario, Canada K1Y 4E9. E-mail: btsang{at}lri.ca
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PI 3-K is composed of a catalytic (p110) and a regulatory (p85) subunit (7). The p110 sequence includes a p85-binding region, a Ras-binding domain, and a catalytic core (8, 9, 10). Similarly, the p85 sequence includes an N-terminal Src homology 3 (SH3) domain, a Bcr homologous region flanked by two proline-rich regions, and two SH2 domains separated by an inter-SH2 region (8, 11, 12). It has been shown that the association of p110 with the GTP-binding protein Ras following ligand-receptor binding results in PI 3-K activation (13, 14). PI 3-K activation is also facilitated by the association of p85 with p110 (15) and the recruitment of the p85:p110 dimer by activated protein tyrosine kinase (16).
The inhibitor of apoptosis proteins (IAPs) is a family of intracellular antiapoptotic proteins that were first identified in baculovirus. They include X-linked IAP (XIAP and cIAP-3), human IAP-1 (HIAP-1 and cIAP-2), human IAP-2 (HIAP-2 and cIAP-1), neuronal IAP (NAIP), and survivin (17, 18). IAPs are characterized by the presence of a caspase recruitment domain and an N-terminal baculovirus inhibitor of apoptosis repeat motif, which are necessary for biological activity. With the exception of NIAP and survivin, the IAPs also contain a C-terminal ring-zinc finger domain believed to be required for protein-protein interactions. Only a few reports to date have addressed the mechanisms of action of these antiapoptotic proteins. XIAP has been shown to be a direct inhibitor of caspase-3 and caspase-7 (19) and also to modulate the Bax/cytochrome c pathway by inhibiting caspase-9 (20). Overexpression of XIAP has been shown to protect Chinese hamster ovary (CHO) and Rat-1 cells from menadione or growth factor withdrawal-mediated apoptosis (18) and from apoptosis in HeLa cells induced by transient transfection with interleukin-1ß-converting enzyme (21) as well as suppress apoptosis induced by Sindbis virus (22).
The presence of gonadotropin during follicular development is critical for the selection and survival of the growing follicles (see Ref. 23 for a review). We have previously shown that IAP contents in granulosa cells are increased during follicular growth in response to gonadotropin stimulation in vivo and that gonadotropin withdrawal resulted in decreased granulosa cell IAP expression and apoptosis (24). However, the mechanism(s) by which gonadotropin increased granulosa cell XIAP level and suppressed apoptosis is not known. Moreover, although both PI 3-K and IAPs are well established cell survival intermediates, if and how they interact to determine the fate of the granulosa cells (survival vs. apoptosis) has not been investigated. The objective of the present study was to address this question and to determine how XIAP block programmed cell death during follicular development.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animal preparation
Immature (23–24 days of age) female Sprague Dawley rats (50–60
g; Charles River Laboratories, Inc. Canada,
Montréal, Canada) were injected with eCG (15 IU, ip) or saline
(0.9% NaCl) and 24 h later, with 100 µl of either NRS (saline
and eCG groups) or anti-eCG antiserum (anti-eCG groups). Animals were
killed 24 h after NRS or antiserum injection. Ovaries were excised
and fixed in 10% formalin for XIAP immunohistochemistry and in
situ terminal deoxynucleotidyl transferase-mediated
deoxy-UTP-biotin end labeling (TUNEL) of apoptotic cells. In addition,
granulosa cells from each group of animals were harvested by follicle
puncture as previously described (25). The animals were
fed Prolab RMH4018 (Agway, Inc., Syracuse, NY) and water ad
libitum. A 14-h light, 10-h dark cycle was maintained, with light
cycle initiated at 0600 h.
Cell culture
Granulosa cells (1 x 106), isolated
24 h after eCG injection (15 IU, sc), were cultured in RPMI 1640
containing 10% FBS, 1% nonessential amino acids, 0.5%
streptomycin-penicillin, and 0.25% fungizone for 48 at 37 C in an
atmosphere of 5% CO2. For XIAP sense and
antisense adenoviral infections, the spent medium was discarded, and
0.5 ml fresh 10% FBS medium containing sense XIAP adenovirus
[multiplicity of infection (MOI), 0, 1, 2.5, and 5] or antisense XIAP
(MOI, 0, 10, 20, and 40). lacZ adenoviral vector was used as
the control and was also used in combination with XIAP sense and
antisense DNA adenovirals to attain a final viral concentration with
MOI of 5 and 40, respectively. The cells were incubated with adenoviral
vectors for 1 h with frequent shaking to ensure maximal infection
and then cultured in 2 ml FBS-free medium for 48 h. At the end of
the culture period, both floating cells and attached cells [recovered
by trypsin treatment (0.05% trypsin and 0.53 mM
EDTA, 37 C for 3–5 min)] were pooled and centrifuged (1900 x
g, 5 min). For protein extraction, cell pellets were
resuspended in a lysis buffer (PBS, pH 7.4) containing NaCl (150
mM), SDS (0.1%), sodium deoxycholate (0.5%),
Nonidet P-40 (1%), and the protease inhibitor
phenylmethylsulfonylfluoride (1 mM).
Immunohistochemistry
Ovarian tissues sections were deparaffinized in xylene and
rehydrated through a graded series of alcohol. Immunohistochemistry was
performed using a Dakko IHC kit (Dakko Diagnostics Canada, Inc.,
Mississauga, Canada) with slight modification to the manufacturer’s
procedure. Briefly, sections were blocked for 5 min with blocking
solution, washed with PBS, and incubated with polyclonal antihuman XIAP
antibody (1:50 dilution, room temperature, 1 h). Sections were
washed briefly with PBS and incubated for 30 min with a secondary
fluorescent antirabbit IgG-FITC antibody, washed again with PBS, and
mounted. For phospho-Akt immunohistochemistry, antigens were retrieved
by heating the sections with microwave in citrate buffer (0.01
M, pH 6.0) for 20 min (four times, 5 min each time). The
sections were then blocked with 1.5% normal serum containing avidin
(30 min, room temperature), incubated with sheep anti-phospho-AKT1
antibody (1:250, 1 h at room temperature or overnight at 4 C),
washed with PBS (three times for 5 min each time), and incubated with a
biotinylated secondary antibody (1:100 in PBS containing 1.5% normal
serum; 30 min at room temperature). They were again washed with PBS
(three times, 5 min each time), incubated with fluorescein-avidin D (25
µg/ml; 30 min), and mounted for fluorescence imaging.
In situ TUNEL
In situ TUNEL was carried out on ovarian sections and
cultured granulosa cells using a TUNEL kit (Roche, Laval,
Canada) in accordance with manufacturer’s instructions. At the end of
granulosa cell culture period, floating cells were collected by
aspiration, and cells attached to the growth surface were subjected to
trypsin treatment (0.05% trypsin and 0.53 mM
EDTA; 3–5 min, 37 C). The two cell fractions (floating and attached
cells) were combined, and an aliquot of this cell mixture was fixed on
a microscope slide. At least a total of 200 cells in a randomly
selected area were counted for each experimental group. The counter was
blinded and was not aware of treatment, so as to avoid experimental
bias.
Protein extraction and Western analysis
Cells were sonicated in the lysis buffer (10 sec), and the
sonicates were centrifuged (12,000 x g, 20 min, 4 C)
to remove insoluble material. Supernatant was recovered and stored at
-20 C until further processing. Protein content was determined with a
DC protein assay (Bio-Rad Laboratories, Inc., Richmond,
CA). Protein extracts were heated (3 min, 94 C), resolved by 10%
SDS-PAGE, and electrotransferred to nitrocellulose membranes (15 V, 30
min). The membranes were first stained with SYPRO Ruby protein gel
stain (Molecular Probes, Inc., Cedarlane, CA) to test
whether the protein loading was constant among samples. Total protein
stains were quantitated densitometrically. The membranes were then
blocked in PBS containing 5% milk powder (room temperature, 2 h)
and incubated (room temperature, 2 h) with antibody for XIAP
(1:2500), Akt (1:1000), or Phospho-Akt (1:1000) and subsequently with
horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000,
room temperature, 45 min). Peroxidase activity was visualized with the
enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Arlington Heights, IL) according to the manufacturer’s
instructions. The content of the protein of interest was likewise
scanned and determined by dividing its signal intensity by that of the
corresponding total protein to correct for any loading differences
between lanes.
Statistical analysis
Results are expressed as the mean ± SEM of
four experiments. Statistical analysis were carried out by one-way
ANOVA (PRISM software version 2.0, GraphPad Software, Inc., San Diego, CA). Significant differences between treatment
groups were determined by Tukey’s test. Statistical significance was
inferred at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 2). Immunohistochemical examination of
ovarian sections from eCG-treated rats revealed high Xiap and
phospho-Akt immunoreactivity as well as low TUNEL positivity in
granulosa cells whereas the opposite was true in sections from the eCG-
plus anti-eCG-treated group (Fig. 1). A similar pattern of Xiap and
phospho-Akt immunosignals was observed in the thecal layer, although
their intensities were considerably higher. Western blot analysis of
granulosa cell extracts indicates that whereas gonadotropin treatment
in vivo increased granulosa cell XIAP (P <
0.05) and phospho-Akt (P < 0.05) contents, withdrawal
of gonadotropin support with anti-eCG antibody administration (eCG and
anti-eCG) resulted in decreases in these parameters to levels observed
in the saline group and induced apoptosis (P < 0.05;
Fig. 2). Likewise, ovarian weight was significantly increased in the
eCG group (P < 0.05), a phenomenon effectively
attenuated by gonadotropin withdrawal. Total Akt protein content,
however, was not significantly affected by any of the treatments (Fig. 2).
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). Down-regulation of XIAP by antisense
expression reduced phospho-Akt content (P < 0.05) and
increased apoptosis (P < 0.05) in a concentration-
dependent manner. At a MOI of 40, adenoviral XIAP antisense
significantly decreased the levels of both XIAP (P <
0.05) and phospho-Akt (P < 0.05) by 38%. Total
Akt content remained constant throughout the experiment (Fig. 3).
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). Infection of
granulosa cells with increasing concentrations of adenoviral XIAP sense
cDNA resulted in concentration-dependent increases not only in XIAP
content but also in phospho-Akt levels. Under these conditions, no
differences in total Akt protein levels were detected between
experimental groups, and the incidence of apoptosis remained low (Fig. 4).
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). The presence of LY294002
significantly decreased Akt phosphorylation and increased apoptosis in
a dose-dependent manner (P < 0.05), but had no effect
on total Akt content (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Other evidence for the importance of the PI 3-K/Akt pathway in granulosa cell regulation came with the finding that high levels of phosphorylated Akt were present in granulosa cells in vitro under nonstimulated conditions. It is well established that phospho-Akt is the active form of this protein and suppresses cell death activity (see Ref. 27 for a review). The serine-threonine kinase Akt has been shown to be directly phosphorylated and activated by PI 3-K. Activation of Akt, in turn, phosphorylates the proapoptotic protein BAD, a member of the Bcl-2 family (5, 6). The phosphorylated BAD then associates with 14–3-3 protein, preventing BAD from binding with Bcl-xL and thereby promoting cell survival (28). Whereas overexpression of BAD induced rat granulosa cell apoptosis (29), exposure of immature rats to eCG decreased granulosa cell phospho-BAD content (30). Thus, the high levels of phospho-Akt in the eCG-primed granulosa cells observed in the present study may be important in preventing these cells from undergoing apoptosis. This idea is supported by the observation that treatment of granulosa cells with the specific PI 3-K inhibitor LY294002 not only significantly decreased phospho-Akt content, but also increased apoptosis in a concentration-dependent manner. Thus, it is possible that inhibition of the PI 3-K pathway may be a key event in the induction of follicular atresia in vivo, whereas activation of PI 3-K is essential for granulosa cell survival and follicular development.
In the present study we demonstrated that XIAP is involved in the regulation of granulosa cell Akt activity. Whereas eCG treatment in vivo increased granulosa cell XIAP and phospho-Akt contents, these responses were attenuated with gonadotropin withdrawal. Moreover, adenoviral XIAP sense cDNA expression increased granulosa cell phospho-Akt contents, whereas XIAP down-regulation by adenoviral antisense suppressed the phosphorylation of Akt and induced apoptosis. Total Akt levels were not affected by these treatments. Moreover, the relative abundance of XIAP may be a key element in cell fate determination, as it is a potent endogenous inhibitor of caspase-3, -7 (31), and -9 (20, 32). Caspases are a family of aspartate-specific proteases that play an essential role in maintaining cell viability because they are activated in response to a cell death signal. We have previously demonstrated the presence of caspase-3 in granulosa cells of atretic, but not healthy, follicles (33). In the present study it is possible that granulosa cell caspase activation and activity, which were otherwise suppressed by high levels of XIAP, were reactivated when this cell survival protein was down-regulated after antisense expression, thus leading to increased apoptosis. Furthermore, it has been reported that activation of Akt results in procaspase-9 phosphorylation and blockade of the caspase cascade (34), although others have demonstrated that the Akt phosphorylation site found in human caspase-9 was not present in mouse caspase-9 (35). The role of Akt activation in the regulation of granulosa cell survival remains to be elucidated.
The precise mechanism(s) involved in the control of PI 3-K activity by XIAP is not known, and different candidates may be involved. Phosphoinositide-dependent kinase has been shown to be responsible for Akt phosphorylation at the threonine 308 position (36), although this phosphorylation alone was not sufficient for maximal AKT activation, which also required serine 473 phosphorylation in a PtdIns(3, 4, 5)P3 (the product of PI 3-K)-dependent manner. Recently, it has been shown that integrin-linked kinase (ILK; a serine and threonine protein kinase) may directly phosphorylate Akt on Ser473 in vitro (26). On the other hand, as these cellular processes are regulated by both protein tyrosine kinases and protein tyrosine phosphatases, it is also possible that XIAP may regulate phosphatases known to decrease PI 3-K activity. Recombinant PTEN (a tumor suppressor protein and a phospholipid phosphatase) is capable of dephosphorylating both phosphotyrosine and phosphothreonine as well as the product of PI 3-K [PtdIns (3, 4, 5)P3] (37, 38). A recent study demonstrated high ILK activity and Akt phosphorylation in PTEN-mutant prostate cancer cells and that PTEN wild-type transfection suppressed ILK activity and Akt activation (39). As ILK is sensitive to and activated by high levels of PtdIns(3, 4, 5)P3 (26), mutation in the PTEN gene led to PtdIns(3, 4, 5)P3 accumulation and ILK activation. Another possible regulatory protein of PI 3-K is the Src homology 2-containing protein tyrosine phosphatase 1, which functions by direct association with its p85 subunit (40). However, which of these kinases and/or phosphatases are indeed putative candidates involved in the regulation of the phosphorylated (activated) status of Akt and survival in granulosa cells by XIAP remains to be determined.
In conclusion, the results of the present study demonstrate that the PI 3-K/Akt pathway has an important regulatory function in granulosa cells during follicular development. The activation of this survival pathway in granulosa cells is dependent on gonadotropin support and may involve an XIAP-mediated increase in phospho-Akt content.
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Acknowledgments |
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Footnotes |
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2 Current address: Département de Chimie-Biologie, Section
Biologie Médicale, Université du Québec à
Trois-Rivières, 3351 boulevard des Forges, C.P. 500,
Trois-Rivières, Québec, Canada G9A 5H7.
Received October 19, 2000.
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References |
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and the complexed heterodimeric p110.p85 forms of the mammalian
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