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Department of Biological Sciences, The University of Notre Dame, Notre Dame, Indiana 46556
Address all correspondence and requests for reprints to: A. L. Johnson, Ph.D., Department of Biological Sciences, P.O. Box 369, The University of Notre Dame, Notre Dame, Indiana 46556. E-mail: johnson.128{at}nd.edu
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Abstract |
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, a factor previously found
to attenuate apoptosis and apoptosis-inducing agents
(e.g. paclitaxel, C8-ceramide, daunorubicin, UV
irradiation) failed to phosphorylate Bcl-XLONG. From these
studies, it is concluded that both the phosphorylation of
Bcl-XLONG (a short-term response) and increased levels of
Bcl-XLONG (a comparatively slower response) in hen
granulosa cells are promoted by gonadotropins via the adenylyl
cyclase/cAMP signaling pathway. Moreover, elevated levels of chicken
Bcl-XLONG protein expression and its phosphorylated state
are correlated with resistance to apoptotic cell death in granulosa
cells in vitro and ultimately a resistance to ovarian
follicle atresia in vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Both Bcl-2 and Bcl-XLONG proteins can be phosphorylated in vivo, and this posttranslational modification has been proposed to determine biological function. For example, in a variety of transformed cell lines, it has been concluded that phosphorylation of Bcl-2 blocks its antiapoptotic activity, possibly by inhibiting the formation of Bcl-2/Bax heterodimers and increasing intracellular levels of Bax monomers and homodimers (18, 19, 20, 21). Alternatively, the effects of Bcl-2 phosphorylation have been reported to occur independent of Bax or Bad heterodimerization and to be associated with activation of Raf-1 kinase and Raf-1 phosphorylation (22). This latter mechanism is proposed to mediate apoptotic cell death induced by chemotherapeutics that disrupt microtubule architecture and prevent cell division (e.g. paclitaxel, vincristine; Refs. 21, 23, 24). Recently, the phosphorylation of Bcl-XLONG has similarly been associated with proapoptotic cell death, but to date, these studies have been conducted only in transformed cell lines (18).
On the other hand, phosphorylation of Bcl-2 at serine/threonine residues is also found to correlate with increased antiapoptotic activity (25, 26). For example, Ras-induced apoptosis is blocked by Bcl-2 expression but not when Bcl-2 phosphorylation is prevented by serine/threonine kinase inhibitors (26). Although reasons for the apparent functional differences (anti- vs. proapoptotic actions) based upon phosphorylated state are currently not clear, it is possible that the effect(s) of phosphorylation is variable, depending on the location within the molecule and extent of phosphorylation, cell type (e.g. transformed vs. primary culture, and cell origin), and/or death-inducing signal. An alternative, although as yet unsubstantiated, possibility is that phosphorylation of Bcl-2 and/or Bcl-XLONG is not directly related to either cell death or survival but is instead a reflection of unrelated cellular processes (27).
We have previously reported that only the long, alternatively-spliced
(death-suppressing) form of the bcl-x messenger RNA (mRNA)
transcript (bcl-XLONG) is detected in hen
granulosa cells, and that levels of bcl-XLONG
mRNA expression in granulosa are positively correlated with resistance
to apoptosis in vitro and follicle viability in
vivo (8). Specifically, hen prehierarchal follicles (
8 mm) are
highly susceptible to undergoing atresia, and it has been estimated
that fewer than 10% will survive to be recruited into the preovulatory
hierarchy for final differentiation before ovulation. A significant
percentage of granulosa cells from prehierarchal follicles undergo
apoptosis after incubation in defined medium (estimated at 60–70%
after only 6 h of incubation; Ref. 28), though a proportion of
these cells is resistant to apoptosis, as evidenced by their ability to
survive and slowly proliferate under conventional culture conditions.
It has been speculated that cells from prehierarchal follicles that
survive in culture may constitutively express higher levels of
Bcl-XLONG protein. Moreover, follicles already recruited
into the hierarchy (preovulatory follicles) do not become atretic under
normal physiological conditions, and granulosa cells from such
follicles are resistant to apoptosis when cultured in vitro.
Preovulatory follicle granulosa cells express approximately 5-fold the
levels of Bcl-XLONG mRNA, compared with granulosa from
prehierarchal follicles (8).
Therefore, the present studies were conducted to characterize levels of Bcl-XLONG protein in hen ovarian follicles during development, to identify cellular mechanisms that regulate Bcl-XLONG expression, and to establish the relationship between the state of Bcl-XLONG phosphorylation and apoptosis in granulosa cells. Our results provide further support for a relationship between Bcl-XLONG expression and granulosa cell resistance to apoptosis, and they indicate that Bcl-XLONG phosphorylation in this nontransformed epithelial cell type may be associated with its antiapoptotic actions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant transforming growth factor- (TGF) was obtained from
Bachem California Inc. (Torrance, CA), and
8-bromo-cAMP (8-br-cAMP), phorbol 12-myristate 13-acetate (PMA),
3-isobutyl-1-methylxanthine (IBMX), and paclitaxel were acquired from
Sigma Chemical Co. (St. Louis, MO). Recombinant
human FSH (rhFSH, lot R1) and ovine LH (oLH, lot 26) were from the
National Hormone and Pituitary Program. N-octanoylsphingosine
(C8-ceramide) was from BIOMOL Research Laboratories Inc. (Plymouth Meeting, PA), and daunorubicin-HCl plus
H-89 were from Calbiochem (San Diego, CA). The Bcl-x
polyclonal antiserum was generated in a rabbit against a 101-amino acid
(AA) chicken Bcl-x fusion protein [AAs 42–142 from the chicken
Bcl-XLONG coding sequence (7); Upstate Biotechnology, Inc., Lake Placid, NY]. The fusion protein was produced by
ligating the Bcl-XLONG complementary DNA into the pCAL-n
vector, then transforming BL21(DE3)pLysS Competent Cells
(Stratagene, La Jolla, CA). The overexpressed protein was
gel-purified on a 10% SDS-polyacrylamide gel, then mixed 1:1 (vol:vol)
with Complete Freund’s adjuvant (first injection) or Incomplete
Freund’s adjuvant (booster injections) for immunization (100 µg
fusion protein per injection).
Tissue collection
Tissues collected for analysis of Bcl-XLONG protein
expression included cerebrum, oviduct, spleen, kidney, and bone marrow.
In addition, granulosa and theca tissue from the largest (F1), second
largest (F2), and third largest (F3) preovulatory follicles, follicles
recently selected into the follicle hierarchy (9- to 12-mm diameter
follicles), prehierarchal (6–8 mm and 3–5 mm) follicles, and stromal
tissue was collected and prepared as previously described (29).
Granulosa cell cultures
Granulosa cells were collected from preovulatory (F1 and F2) or
prehierarchal (6–8 mm) follicles and pooled within their respective
group. Granulosa cell layers from each follicle group were immediately
frozen at -70 C (T0 controls) or were dispersed with 0.3% collagenase
(type 2; Worthington Biochemical Corp., Freehold, NJ) and
plated in 6-well polystyrene culture plates (Falcon 3046; Fisher Scientific, Itasca, IL) at a density of approximately
1.5 x 106/well in 2 ml M199-HEPES supplemented with
Hanks’ salts (Life Technologies, Inc., Gaithersburg, MD;
M199-HEPES) and 2.5% FBS (Life Technologies, Inc.). We
have previously reported that the plating efficiency of preovulatory
follicle granulosa cells is greater than 80%, whereas that for
prehierarchal follicle granulosa is approximately 30%, with the
remainder of cells determined to undergo apoptosis (28).
In the first set of culture experiments, granulosa cells from
prehierarchal or preovulatory follicles were plated in the absence and
presence of 1 mM 8-br-cAMP, 9 nM TGF, or 167
nM PMA and cultured for 20 h. These agents and doses
were selected for use based upon their previously established actions
to affect cell viability and/or cell differentiation in granulosa cells
(8, 29, 30). The following day, viable, adherent cells were washed once
with fresh medium and collected directly into 125 µl protein lysis
buffer (30). Proteins were stored frozen at -70 C until used.
In a second set of culture experiments, granulosa cells from
prehierarchal follicles were precultured for 20 h, then treated
for 15 min in the absence (Con) or presence of 1 mM
8-br-cAMP, 100 mIU/ml rhFSH, 100 ng/ml oLH, 167 nM PMA, or
9 nM TGF. In addition, preovulatory follicle granulosa
cells were precultured for 20 h, then treated for 15 min in the
absence (Con) or presence of 1 mM 8-br-cAMP, 100 mIU/ml
rhFSH, 100 ng/ml oLH, 50 µM C8-ceramide, 200
nM daunorubicin, or 200 nM paclitaxel (31). One
additional well of cells was UV irradiated (120,000
µJ/cm2), then cultured for an additional 15 min. A 15-min
treatment time was chosen to monitor rapid phosphorylation events that
may be implicated in influencing upstream cell signaling. All cells in
this experiment were then washed once with fresh medium and collected
directly into 125 µl protein lysis buffer, as described above.
Proteins were stored frozen at -70 C until used. The ability of
C8-ceramide and daunorubicin treatments, plus UV irradiation, to induce
apoptotic cell death in cultured granulosa cells was verified by
evaluating oligonucleosome formation after 24 h of treatment.
Although paclitaxel treatment did not induce apoptosis in cultured
granulosa cells, the effectiveness of the paclitaxel preparation to
initiate apoptosis was verified in HeLa cells (data not shown).
A final set of experiments was conducted to further evaluate the role of the adenylyl cyclase/cAMP signaling pathway in BclXLONG phosphorylation. Granulosa cells from prehierarchal follicles were precultured for 20 h, then treated for 15 min in the absence (Con) or presence of IBMX (10 mM), oLH (1, 10, or 100 ng/ml), or IBMX plus 1 ng oLH/ml. Additional cells were pretreated for 15 min with the protein kinase A inhibitor, H-89 (33 or 100 µM; Ref. 32), then cultured an additional 15 min in the presence or absence of 10 ng oLH/ml. Samples were processed for protein, as described above.
Phosphatase treatment of cell lysates containing
Bcl-XLONG
Preovulatory follicle granulosa cells were prepared fresh (T0
sample) or precultured for 20 h, then treated with 1
mM 8-br-cAMP or vehicle (M199/HEPES) for 15 min. Cells were
subsequently collected and cell lysates prepared as described above.
Ten micrograms of protein was incubated in the absence or presence of
the serine/threonine/tyrosine phosphatase, lambda protein phosphatase
(2000 U; New England Biolabs, Inc., Beverly, MA) at 30 C
for 30 min. Protein samples were subsequently analyzed by Western blot,
as described below.
Western blot analysis
Granulosa and other tissues were homogenized in lysis buffer,
then centrifuged at 12,000 x g at room temperature
(RT) for 10 min, as previously described (30). The supernatant was
collected and protein quantified using the DC Protein Assay kit
(Bio-Rad Laboratories, Inc., Hercules, CA). Protein
samples were subsequently diluted with sample buffer, heated at 70 C
for 10 min (with appropriate size markers), then centrifuged at
12,000 x g for 2 min. Samples were separated on a 12%
SDS-PAGE gel before being transferred to a nitrocellulose membrane
(Nitrobind, Fisher Scientific).
Membranes were blocked 1 h at RT in Western blocking solution [5% nonfat dry milk, TBST: 10 mM Tris (pH 8.0), 100 mM sodium chloride, 0.1% Tween 20]. The Bcl-XLONG antibody was diluted 1:10,000 in blocking solution and incubated with membranes at 4 C overnight, then membranes were washed (3 x 10 min) in blocking solution. Goat antirabbit serum : horseradish peroxidase (Pierce Chemical Co., Rockford, IL) was diluted 1:10,000 in blocking solution and incubated with membranes for 1 h at RT. Membranes were successively washed in TBST (3 x 10 min). Finally, blots were incubated with ECL Western blotting detection reagent (Amersham Pharmacia Biotech) for 1 min, then were wrapped and exposed to autoradiographic film for 1–5 min. The extent of antibody binding was standardized to ß-tubulin (30) as quantitated by densitometry (UltraScan XL laser densitometer, Pharmacia LKB, Piscataway, NJ).
Immunocytochemistry
Tissues were fixed in Dietrick’s fixative (4% formalin, 30%
ethanol, 2% glacial acetic acid), dehydrated through graded ethanols,
and paraffin embedded. Sections were cut at 6 µ and mounted on Plus
slides (Fisher Scientific). Slides were deparaffinized in three changes
of xylene (10 min each) and rehydrated through graded ethanol washes
(100%, 90%, 70%, 50%, and distilled H2O). Tissue
sections were blocked for 1 h at RT in TBS [10 mM
Tris (pH 8.0), 100 mM sodium chloride] containing 1.5%
goat serum, followed by incubation in a humidified chamber with Bcl-x
antibody (diluted 1:200 in blocking solution) for 1.5 h at RT or
overnight at 4 C. Contol sections were incubated with preimmune serum.
Slides were subsequently washed 3 x 5 min in TBS, followed by
incubation in goat antirabbit IgG fluorescein isothiocyanate-conjugated
secondary antibody (diluted 1:200 in blocking solution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 30 min at
RT. Finally, all slides were washed 3 x 5 min in PBS and for 30
sec in PBS + 1% Triton-X100 (Fisher Scientific), rinsed in
H2O, and mounted in an aqueous quencher (Aqua poly mount,
Polysciences Inc., Warrington, PA). Images were collected with a
MRC1024 laser confocal microscope (Bio-Rad Laboratories, Inc.) and analyzed using the Lasersharp software package
(Bio-Rad Laboratories, Inc.).
Data analysis
Northern and Western blot experiments were repeated a minimum
total of three times. Some of the data were analyzed by paired
t test (e.g. T0 vs. T20 Con; see Figs. 5
and 8). Bcl-XLONG protein levels (total protein in
doublet) and phosphorylated Bcl-XLONG (upper
band of doublet) were also expressed as fold-difference (mean
± SEM) vs. a designated reference tissue
(e.g. analysis of nonovarian and follicle hierarchy
tissues), and cultured cells were compared with levels found in freshly
collected (T0) cells (value for reference tissue arbitrarily set at 1).
Before statistical analysis of culture experiments, it was established
that only treatment groups, compared with the control cultured group,
would be evaluated. Data were analyzed by one-way ANOVA (analysis of
fold-difference data did not include the reference value) and the
Fisher protected least-significant-difference multiple-range test
(33).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). By contrast, Bcl-XLONG
levels are not different during development within the theca layer
(Fig. 2). Immunocytochemical analysis of
preovulatory and prehierarchal follicles largely confirms the increased
expression of cytoplasmic Bcl-XLONG within the preovulatory
follicle granulosa layer, and it shows scattered regions of
Bcl-XLONG expression within the theca of both prehierarchal
and preovulatory follicles (Fig. 3).
Outside of the ovary, the highest levels of Bcl-XLONG
protein expression occur in the kidney and immune system tissues, bone
marrow, and spleen, whereas comparatively lower levels are consistently
detected in the oviduct and cerebrum (Fig. 4). Also of note was the detection of
the alternatively spliced Bcl-XSHORT in theca, kidney,
spleen, and cerebrum tissues, but not granulosa cells.
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, top). However, a further increase in protein, above already
elevated levels, compared with prehierarchal follicles (see Fig. 1), is
not observed after an overnight plating of granulosa cells from
preovulatory follicles (T0 vs. Con; Fig. 5, bottom). Granulosa cells from follicles at both stages of
development, cultured for 20 h in the presence of 8-br-cAMP, show
a significant increase in Bcl-XLONG levels, compared with
Con cells (P < 0.01), whereas TGF has no effect;
PMA treatment decreases Bcl-XLONG levels only in
preovulatory follicle granulosa cells (P < 0.05).
Phosphorylation of Bcl-XLONG
Short-term treatment of cultured granulosa cells with 8-br-cAMP
preferentially increases the proportion of a slower migrating
(upper) band of Bcl-XLONG protein (Fig. 6). Treatment of cell lysate from
8-br-cAMP-treated cells with 
protein phosphatase virtually
eliminates this upper band of Bcl-XLONG, and it
results in accumulation of protein within the faster migrating, lower
band. There is no evidence from Coomassie-stained gels (data not shown)
or from the immunoreactive Bcl-XLONG or tubulin proteins
(Fig. 6A) that the phosphatase treatment has any nonspecific
proteolytic effects. It is concluded that, as recently reported for
mammalian species (18), the upper band of the doublet represents an
inducible, phosphorylated form of Bcl-XLONG.
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) was conducted, and it
was determined that levels within theca tissue during follicle
development do not change (P > 0.10). By contrast, the
amount of phosphorylated protein in the granulosa layer, expressed as a
fold-difference vs. 3- to 5-mm follicles, is higher within
each of the three largest preovulatory follicles, compared with 9- to
12-mm or 6- to 8-mm follicles (P < 0.05; Fig. 6B).
Short-term culture (15 min) in the absence or presence of various
physiological and pharmacological agents did not alter the total amount
of total Bcl-XLONG protein quantitated (e.g. the
sum of the doublet protein) in granulosa cells from either
prehierarchal or preovulatory follicles (P > 0.01;
summarized data not shown). By contrast, 15 min of treatment with
8-br-cAMP, rhFSH, or oLH increased levels of phosphorylated
Bcl-XLONG, compared with Con cells in granulosa from
prehierarchal follicles (Fig. 7, top), whereas 8-br-cAMP and oLH (but not rhFSH) treatment
significantly increased levels of phosphorylated Bcl-XLONG
in preovulatory follicle granulosa (Fig. 7, bottom). Agents
determined to induce apoptosis in hen preovulatory follicle granulosa
cells (e.g. C8-ceramide, daunorubicin, and UV treatment;
Ref. 28) or to promote Bcl-x phosphorylation in human malignant cells
(e.g. paclitaxel; Ref. 18) did not increase levels of
phosphorylated Bcl-XLONG, compared with control cells.
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Treatment with oLH, at doses of 10 and 100 (but not 1) ng/ml,
significantly increases the level of phosphorylation, compared with
Con. Although IBMX alone fails to alter levels of phosphorylation
(compared with Con), IBMX plus 1 ng oLH/ml significantly increases
phosphorylation. Finally, pretreatment of cultured preovulatory
follicle granulosa, with the higher dose of the protein kinase A
inhibitor, H-89 (100 µM), blocks the phosphorylation
response after oLH treatment. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This is the first report to document patterns of chicken
Bcl-XLONG (28 kDa) and Bcl-XSHORT (18 kDa)
protein expression in avian cells, and the results presented generally
parallel previously published profiles of
bcl-XLONG mRNA from immune and ovarian tissues
(7, 8). Specifically, there is no evidence for the expression of the
alternatively spliced, proapoptotic Bcl-XSHORT form of the
protein in granulosa cells, although the theca and several nonovarian
tissues (kidney, spleen, and cerebrum) show varying levels of the
protein. Bcl-XSHORT expression has previously been
documented within tissues of the immune system (7), but the precise
function and mechanisms by which it putatively acts in a proapoptotic
fashion have not been fully elucidated. Expression of
Bcl-XSHORT has also been documented within the rat ovary
(11); however, these studies did not differentiate expression within
the granulosa vs. theca layers. The present results suggest
that the low levels of Bcl-XSHORT detected in the rat ovary
may be of thecal origin. The highest levels of Bcl-XLONG
were consistently detected within the granulosa layer of preovulatory
follicles, as determined by both immunoblot analysis and
immunocytochemistry (
Figs. 1–4). The pattern of granulosa cell
cytoplasmic staining, in the absence of nuclear localization, is
similar to that previously reported for a variety of mouse and human
tissues (34) and is consistent with its well-documented cytoplasmic and
mitochondrial sites of actions (5). Given the very low levels of
Bcl-XSHORT detectable by Western blot analysis (Fig. 4), it
is unlikely that this alternatively-spliced form contributes much, or
any, signal detected by immunocytochemistry.
However, unlike bcl-XLONG mRNA, protein levels were not found to be increased in granulosa from 9- to 12-mm follicles (recruited within the previous 24 h from a cohort of prehierarchal follicles), compared with 6- to 8-mm follicle granulosa. Presumably, this is attributable to a lag in time from mRNA transcription to translation of the Bcl-XLONG protein. Though much, if not all, of the increased Bcl-XLONG protein is attributed to newly synthesized protein, we cannot entirely rule out the possibility of a small decrease in protein degradation. We have previously reported that follicle selection into the preovulatory hierarchy is associated with both increased bcl-XLONG mRNA expression and the acquisition of resistance to apoptosis, in vitro, in granulosa from 9- to 12-mm follicles. We speculated that the increased bcl-XLONG mRNA levels within the 9- to 12-mm follicle granulosa layer may be prerequisite for facilitating follicle selection (8, 35). Though data provided herein do not seem to support this initial proposal, the presence of elevated Bcl-XLONG protein in granulosa from the largest preovulatory follicles is consistent with a role for this antiapoptotic protein in protection against cell death, in vitro, and follicle atresia, in vivo. It remains possible that increased expression of other putative antiapoptotic proteins, such as the chicken inhibitor of apoptosis protein, ITA, may be more critical for the process of follicle selection into the preovulatory hierarchy (35).
Similar to the human Bcl-XLONG protein (18, 36), the
approximate 28-kDa chicken homolog is commonly detected as a protein
doublet, with the slower migrating band representing a phosphorylated
form. Sequence analysis of the chicken Bcl-XLONG primary AA
sequence reveals a total of 20 serine and 12 threonine residues that
could potentially serve as phosphorylation sites throughout the protein
(7). However, it has previously been reported that agonist-induced
phosphorylation of the human Bcl-2 and Bcl-x proteins occurs on serine
and threonine residues primarily within a highly variable loop region
located between the BH4 and BH3 domains (19, 37, 38) and specifically
at serine-70 (39). Significantly, there are 7 serine (including a
conserved serine-70) and 3 threonine residues plus one cAMP consensus
phosphorylation site (Arg-Thr-Asp-Thr) within this region in the
chicken Bcl-XLONG protein. Though there generally occurs an
increase in a single predominant band of phosphorylated protein after
treatment with the gonadotropins, LH and FSH, occasionally there is
evidence of additional phosphorylated forms (hyperphosphorylation)
after treatment with the supraphysiological cAMP analog, 8-br-cAMP
(e.g. Fig. 7, bottom). It is not yet clear
whether such forms are of physiological relevance.
It is of significance to note that 8-br-cAMP and oLH, but not rhFSH, induce phosphorylation of Bcl-XLONG in preovulatory follicle granulosa. Furthermore, IBMX potentiates the effects of a noneffective dose of oLH, whereas inhibition of protein kinase A activity, using a pharmacologic blocker (H-89; Ref. 32), blocks the ability of oLH (10 ng/ml) to phosphorylate Bcl-XLONG. First, these findings indicate involvement of the adenylyl cyclase/cAMP pathway, perhaps acting at the cAMP consensus phosphorylation motif described above. Second, the differential phosphorylation in response to oLH, compared with rhFSH, is correlated to expression of the highest LH-receptor (R) mRNA levels during follicle development, and decreasing levels of FSH-R expression, in preovulatory follicle granulosa cells; moreover, rhFSH treatment fails to induce steroidogenesis in preovulatory follicle granulosa (40, 41, 42).
By comparison, both gonadotropins promote Bcl-XLONG
phosphorylation in prehierarchal follicle granulosa cells that survive,
after an overnight plating. We have previously reported that there
exist high levels of the FSH-R transcript in 6- to 8-mm follicle
granulosa, and that rhFSH treatment of such cells, in vitro,
induces cAMP accumulation (29). Similarly, oLH promotes
Bcl-XLONG phosphorylation, even though levels of LH-R mRNA
are comparatively low in cultured 6- to 8-mm granulosa cells. It is of
significance to note that 8-br-cAMP treatment attenuates the
progression of apoptosis in incubated prehierarchal follicle
granulosa cells (8). Although TGF is also known to attenuate the
progression of apoptotic cell death in prehierarchal follicle granulosa
cells, its actions are thought to be mediated via tyrosine kinase (not
serine/threonine kinase) activity and thus predicted not to induce
Bcl-XLONG phosphorylation. Finally, Bcl-XLONG
phosphorylation also occurs in primary cultures of theca, ovarian
stroma, and liver tissue after a 15-min treatment with 8-br-cAMP (data
not shown), indicating that this phosphorylation event is not limited
to cells of epithelial origin.
We have previously reported that a majority of prehierarchal follicle
granulosa cells rapidly succumbs to apoptosis when cultured, in
vitro, whereas a small proportion of these cells is resistant to
cell death and survives to form a monolayer (8). Consistent with
previously published results for bcl-XLONG mRNA
(30) was the finding that Bcl-XLONG protein levels were
similarly increased (by 2.4-fold) in the population of 6- to 8-mm
follicle granulosa cells that successfully plated after culture for
20 h (Fig. 5, top). This result suggests that the
survival of prehierarchal follicle granulosa cells, in
vitro, may be directly or indirectly associated with elevated
levels of Bcl-XLONG protein. Also consistent with
bcl-XLONG mRNA (30) and levels of phosphorylated
Bcl-XLONG (presented herein) was the finding that a 20-h
treatment with 8-br-cAMP, but not TGF or PMA, increased levels of
Bcl-XLONG protein in both prehierarchal and preovulatory
follicle granulosa cells (Fig. 5, top and
bottom). Collectively, the implication of these findings is
that both FSH and LH, acting via cAMP, function not only to promote
granulosa cell proliferation and differentiation but also cell
survival. The significance of the decrease in Bcl-XLONG
levels in preovulatory follicle granulosa cells after treatment with
PMA is less obvious, because this phorbol ester was previously found
not to alter levels of bcl-x mRNA.
There are a number of recent reports documenting the relationship
between agonist-induced phosphorylation of Bcl-2 and induction of
apoptosis in a variety of transformed mammalian cell lines
(e.g. 21, 22). Therefore, it was of interest to evaluate the
effects of several agents previously shown to induce apoptotic cell
death in primary cultures of hen preovulatory follicle granulosa cells.
Several agents used in the present experiments are proposed to initiate
cell death via different cellular mechanims, although the complete
pathway for any of these has yet to be defined. These include
daunorubicin, which acts via the release of endogenous ceramide and
activation of the stress-activated protein kinase pathway (28, 43, 44),
and UV irradiation, which may target activation of the p53 tumor
suppressor gene (28, 45). In addition, paclitaxel has previously been
shown to promote Bcl-2 phosphorylation and induce apoptosis in several
mammalian cell types, and its cell death-promoting effects are thought
to be related to the ability of the drug to induce mitotic arrest
(20, 21, 22, 31). Nevertheless, none of these agents was found to alter
levels of phosphorylated Bcl-XLONG after short-term
treatment of cultured preovulatory follicle granulosa (Fig. 7, bottom), thus providing no evidence for a relationship
between Bcl-XLONG phosphorylation and the induction of
apoptosis in this untransformed cell type. The inability of
paclitaxel to induce apoptotic cell death in preovulatory
follicle granulosa was not entirely unexpected, because the vast
majority of preovulatory follicle granulosa cells are terminally
differentiated and nonmitotic. More importantly, however, it has
recently been questioned whether there is a direct relationship between
bcl-2 phosphorylation and paclitaxel-induced cell death (27). For
instance, phosphorylated Bcl-2 was found to remain complexed with Bax
in transformed cells after paclitaxel treatment. Furthermore,
phosphorylation of Bcl-2 was proposed to be more closely associated
with the Cdc2 signaling cascade and mitotic activity than with events
signaling apoptosis.
In conclusion, results from the present studies provide evidence for an association between Bcl-XLONG protein levels (total and phosphorylated) and its putative antiapoptotic function in hen ovarian granulosa cells. Though the only other report to date describing physiological consequence of Bcl-x phosphorylation concludes that its function is proapoptotic (18), it is noted that this study, as well as most studies of bcl-2 (e.g. 20–24), involved only transformed cell lines. Accordingly, it is difficult to directly relate results from transformed cells to primary cell cultures. It is possible that multiple serine/threonine kinases, differentially expressed during cell differentiation and/or regionally localized within the cell cytoplasm, may be involved in Bcl-XLONG phosphorylation. Such differences, including the number and location of phosphorylated residues within the primary sequence, may ultimately determine whether Bcl-XLONG exhibits an antiapoptotic or proapoptotic function. Alternatively, it remains a possibility that phosphorylation of Bcl-XLONG, as recently discussed for Bcl-2 (27), may not be directly related to facilitation or inhibition of apoptotic cell death but, rather, is a reflection of kinase activity involved in the regulation of mitosis or cell differentiation. Additional studies are required to differentiate among the above possibilities.
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Acknowledgments |
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Footnotes |
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Received December 1, 1998.
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References |
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G. Riedlinger, R. Okagaki, K.-U. Wagner, E. B. Rucker III, T. Oka, K. Miyoshi, J. A. Flaws, and L. Hennighausen Bcl-x Is Not Required for Maintenance of Follicles and Corpus Luteum in the Postnatal Mouse Ovary Biol Reprod, February 1, 2002; 66(2): 438 - 444. [Abstract] [Full Text] [PDF]
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S.-Y. Chun, H.-W. Bae, W.-J. Kim, J.-H. Park, S. Y. Hsu, and A. J. W. Hsueh Expression of Messenger Ribonucleic Acid for the Antiapoptosis Gene P11 in the Rat Ovary: Gonadotropin Stimulation in Granulosa Cells of Preovulatory Follicles Endocrinology, June 1, 2001; 142(6): 2311 - 2317. [Abstract] [Full Text] [PDF]
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A.L. Johnson, J.T. Bridgham, and J.A. Swenson Activation of the Akt/Protein Kinase B Signaling Pathway Is Associated with Granulosa Cell Survival Biol Reprod, May 1, 2001; 64(5): 1566 - 1574. [Abstract] [Full Text]
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A.L. Johnson and J.T. Bridgham Caspase-3 and -6 Expression and Enzyme Activity in Hen Granulosa Cells Biol Reprod, March 1, 2000; 62(3): 589 - 598. [Abstract] [Full Text]
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A. L. Johnson Editorial: Mcl-1--Just Another Antiapoptotic Bcl-2 Homolog? Endocrinology, December 1, 1999; 140(12): 5465 - 5468. [Full Text]
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