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Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California 94305-5317
Address all correspondence and requests for reprints to: Aaron J. W. Hsueh, Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California 94305-5317.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, basic fibroblast growth factor, and insulin growth
factor-I), and cytokines (interleukin-1ß) can act as survival factors
to rescue follicles from apoptotic demise (1, 2, 3, 4). In contrast,
androgens, GnRH, and tumor necrosis factor-, are atretogenic factors
(1, 5, 6). Although the extracellular signals regulating cell survival
are often cell- and tissue-specific, a growing body of evidence
suggests that the intracellular death program that is activated during
apoptosis is similar in different cell types and conserved during
evolution (2, 7). Recent investigations have shown that during apoptosis the activation of caspases, cysteine proteases related to the Caenorhabditis elegans protease ced (cell death)-3, results in cell death via proteolytic cleavage of substrate proteins vital to the maintenance of cellular homeostasis (7). In addition, the Bcl-2 family of proteins that consists of different anti- and proapoptotic members is important in the decision step of apoptosis (8). Bcl-2-related proteins act upstream from caspase proteases in the cell death pathway (9), and the balance between pro- and antiapoptotic Bcl-2-related proteins determines the fate of the cell (10). Recent studies further demonstrated that another C. elegans gene, ced-4, could bind to both Bcl-2 family members and caspases thus bridging the essential components of the cell death machinery (11).
The exact mechanism by which hormonal signals regulate the intracellular cell death process is not clear. A possible link between signal transduction initiated by extracellular survival factors and the cell death pathway is Bcl-XL/Bcl-2-associated death promoter (BAD), a proapoptotic member of the Bcl-2 family (12, 13) that lacks the transmembrane domain. In addition to heterodimerizing with other Bcl-2 family proteins, BAD was recently found to bind to 14–3-3, a group of proteins involved in intracellular signaling and cell cycle progression (13, 14).
Targeted overexpression of Bcl-2 into ovarian cells leads to decreased apoptosis of granulosa cells on gonadotropin withdrawal and is associated with higher ovulation rate, increased litter size, and ovarian teratoma formation (15). These findings suggest that exogenous Bcl-2 protein can interact with the Bcl-2 system in ovarian somatic cells to modulate the cell’s susceptibility to undergo apoptosis. To characterize Bcl-2- interacting proteins in the ovary, we screened an ovarian fusion complementary DNA (cDNA) library by using the yeast two-hybrid system (16) and obtained several positive clones encoded for BAD, a proapoptotic Bcl-2 family member. To understand the potential role of BAD in the regulation of ovarian follicle atresia, we studied its messenger RNA (mRNA) expression and hormonal regulation in the rat ovarian cells and assessed its proapoptotic function in a primary culture of granulosa cells and in a gonadal cell line following the overexpression of BAD protein.
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Materials and Methods |
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To analyze interactions between BAD and different Bcl-2 mutants, truncated human Bcl-2 cDNAs were derived by using PCR amplification: the Bcl-2 homology (BH)4 domain mutant encompasses amino acids 31–239, the BH2 domain mutant amino acids 1–185, and the transmembrane domain mutant amino acids 1–218 of the human Bcl-2 (17). For the quantitation of protein-protein interactions, yeast cells were cotransformed with different pairs of fusion proteins and cultured in media deficient in tryptophan and leucine for 36 h. After counting, cells were lysed, and ß-galactosidase activity estimated by a colorimetric assay using 2-nitrophenyl-ß-D-galactopyranosid as the substrate.
Preparation of nucleic acid probes
For the generation of hybridization probes, a clone encoding for
the full-length rat BAD cDNA was excised from the pGADGH vector with
restriction enzymes and subcloned into pGEM4Z (Promega, Madison, WI)
vector for the production of antisense and sense complementary RNAs
(cRNAs) using the Riboprobe System (Promega). A glyceraldehyde
3-phosphate dehydrogenase (GAPDH) probe was generated using the
pTRI-GAPDH plasmid linearized with HindIII (Ambion, Austin,
TX) as a template to derive a 404-bp cRNA.
Northern blot hybridization
Sprague-Dawley rats were obtained from Simonsen Labs. (Gilroy,
CA). Ovaries from rats between 15–27 days of age were isolated and
dissected free of adherent tissue under a stereomicroscope. Immature
(24-day-old) rats were implanted with an estrogen [diethylstilbestrol
(DES); Sigma, St Louis, MO] capsule to stimulate development of
multiple early antral follicles. Ovaries were collected 3 days
following the estrogen implantation. To induce follicle cell apoptosis,
the DES capsules were removed at 3 days after implantation, and the
ovaries were collected 1 day after the removal of the implant.
For the extraction of total RNA, ovaries and other tissues were homogenized using a Tissue-tearor (Bio-Spec Products, Bartlesville, OK) in Tri-Reagent solution (Molecular Research Center, Cincinnati, OH). At least two pools of tissues from each treatment group were used. Following RNA extraction, two aliquots of 250 µg RNA from each treatment were used for subsequent isolation of poly(A)+ RNA using the Oligotex oligo(dT) resin (Quiagen, Chatsworth, CA). For Northern blot hybridization, 1.0 µg each of poly(A)+ RNA was denatured and fractionated in 1% agarose gels containing formaldehyde, followed by capillary blotting onto Gene Screen nylon membranes (Du Pont, NEN Research Products, Boston, MA) and covalent cross-linking using a UV cross-linker (Stratagene, La Jolla, CA). Membranes were prehybridized for 4 h at 65 C in a solution containing 50% formamide, 5 x sodium phosphate buffer (SSPE), 5 x Denhardt’s solution, 0.5% SDS, and 500 µg/ml yeast transfer RNA, followed by overnight hybridization in the same conditions with 1 x 106 cpm/ml of 32P-labeled BAD or a GAPDH cRNA probe. After hybridization, the membranes were washed twice in 2 x SSPE, 1% SDS at room temperature, followed by two washes in 1 x SSPE, 1% SDS at 65 C. Membranes were exposed to Kodak RX films (Eastman Kodak, Rochester, NY) for 3 days at -70 C under intensifying screens. Each Northern blot experiment was repeated three times and one representative blot is shown.
In situ hybridization
Ovaries from immature, 26-day-old rats were isolated and fixed
at 4 C for 4 h by immersion in 4% paraformaldehyde in PBS (pH
7.4), followed by dehydration in 0.5 M sucrose overnight.
Tissue blocks were embedded in Tissue-Tek solution (Sakura Finetek USA,
Torrence, CA) and snap frozen in liquid nitrogen. Twelve-micron thick
cryosections were mounted on charged microscopic slides (Fisher
Scientific, Pittsburgh, PA), postfixed in 4% paraformaldehyde, and
stored at -70 C up to 1 month. Hybridization and washes of
cryosections were adapted from previously described methods (18, 19).
After 2 weeks of exposure under NTB2 emulsion (Kodak, Rochester, NY),
the slides were developed, counterstained, and mounted with Permount
(Fisher Scientific, Fair Lawn, NJ) for observation and photography.
Overexpression of BAD in cultured granulosa cells and a gonadal
tumor (TT) cell line
To test the function of BAD in ovarian cell survival,
full-length rat BAD cDNA was subcloned into the mammalian expression
vector pCDNA3 (Invitrogen, San Diego, CA) under the control of the
cytomegalovirus promoter. BAD cDNA subcloned in the reverse orientation
(reverse BAD) or the same vector without an insert were used as control
plasmids. Granulosa cells were obtained from estrogen-treated immature
rats (DES implantation at 24 days of age) at 4 days after hormone
treatment. Cells were isolated by needle puncture and plated at a
density of 6 x 105 cells/35 mm dish (Nunc, Rotskilde,
Denmark) in DMEM/F12 (Gibco BRL, Grand Island, NY) supplemented with
10% BSA (Sigma), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2
mM glutamine (Bio Whittaker, Walkersville, MD). Twenty
hours later, granulosa cells were washed with the fresh medium four to
six times to remove nonattached, dead cells. TT cells were plated at a
density of 2 x 105 cells/35 mm dish. Twenty four
hours after plating, the cells were transfected using the lipofectamine
reagent (Life Technologies, Gaithersburg, MD) with the expression
plasmids, together with 1/10 of a reporter plasmid pCMV-ß-gal to
allow the identification of transfected cells. A 10-fold excess of the
expression vectors was used to ensure that most of the cells expressing
the ß-galactosidase also expressed the protein of interest (20, 21).
Cells were incubated with liposome-DNA complexes for 12 h in the
serum-free medium, followed by the addition of FBS to a final
concentration of 5%, and further incubation for 12 h. A
cell-permeable caspase inhibitor z-Val-Ala-Asp-CH2F (zVAD-fmk; Enzyme
Systems Products, Dublin, CA) was added to the culture medium at
12 h after start of the transfection. After an additional culture
in fresh medium containing 10% FBS for 12 h, the cells were fixed
by 0.3% glutaraldehyde and stained with X-gal (4 mg/ml in a
buffer containing 150 mM NaCl, 100 mM
Na2HPO4, 1 mM MgCl2, 3.3
mM K4Fe(CN)6.3H2O and 3.3 mM
K3Fe(CN)6, pH 7.0) for 12 h to detect blue coloration as an
indication of ß-galactosidase expression. The number of blue cells
was counted by microscopic examination at x100 magnification.
Transfection efficiency was 3–7% for granulosa cells and 10% for TT
cells. Data are expressed as number of blue cells/35 mm dish (mean +/-
SEM) based on counting six to nine independent samples of
three separate experiments. Statistical differences between
treatment groups were analyzed using one-way ANOVA and
Tukey’s multiple range tests.
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Results |
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In yeast cells overexpressing the BAD-GAL4-AD fusion protein and
the Bcl-2-GAL4-BD fusion protein, a strong interaction between these
proteins was detected as assessed by yeast growth in media deficient in
tryptophan, leucine, and histidine, and supplemented with 30
mM 3-aminotriazole [Fig. 1
, Bcl-2 wild-type (WT)]. In addition, yeast cells cotransformed with BAD
and truncated Bcl-2 mutants devoid of either BH domains 4 or 2, showed
retarded growth, indicating the importance of these BH domains in the
hetero-dimerization of BAD and Bcl-2. Likewise, yeast cells
coexpressing a truncated Bcl-2 protein devoid of its transmembrane
domain showed moderate growth, suggesting weaker interactions of this
Bcl-2 mutant with BAD as compared with the WT Bcl-2. In contrast, BAD
failed to dimerize with rat BAX or to homodimerize with itself as
indicated by the lack of growth of yeast cells cotransformed with these
vectors. No yeast cell growth was apparent when BAD was cotransformed
with an empty vector or an unrelated protein (lamin). The observed
protein-protein interactions were confirmed based on the analysis of
the activities of the ß-galactosidase reporter gene (OD value for
yeast cells expressing BAD and different proteins: Bcl-2: 253 ± 8;
Bcl-2 minus BH4: 28 ± 3; Bcl-2 minus BH2: 35 ± 9; Bcl-2 minus TM
(transmembrane region): 50 ± 8; BAX: 33 ± 9; BAD: 37 ± 2; pGBT9: 33
± 9; lamin: 33 ± 12; n = 3).
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, varying levels
of BAD mRNA of 1.2 and 1.7 kb in size were detected in all tissues
examined. Corrected for GAPDH mRNA levels, the highest mRNA expression
was seen in the lung, ovary, uterus, and brain (Fig. 2). Lower levels
were found in the heart, small intestine, liver, spleen, and thymus. In
addition, the lung and liver showed predominant expression of the
1.2-kb BAD mRNA, whereas the thymus showed a mRNA of 1.7 kb. For other
tissues tested, both forms of BAD messages could be found. These data
suggested that BAD message is widely expressed in diverse tissues.
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, A and B). BAD mRNA was detected
in follicles of different developmental stages ranging from the
secondary to the antral follicles. In contrast, hybridization with the
sense BAD cRNA showed only low background signals (Fig. 3, C and
D).
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, similar levels of BAD mRNA were found
in isolated granulosa cells (lane 1) and the residual ovarian tissues
(lane 2). These data verify the in situ findings of a wide
distribution of BAD mRNA expression in different ovarian cell
types.
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, BAD mRNA was evenly expressed at all
time points studied (lanes 1–5). Earlier studies also showed a major
increase in follicle cell apoptosis following estrogen withdrawal in
immature rats (23). Similarly, BAD mRNA levels were unaltered during
apoptosis induction associated with estrogen withdrawal (Fig. 5, lanes
6–7).
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and p53 knock-out mice (24).
A reporter plasmid encoding ß-galactosidase was cotransfected with
the BAD expression vector or control plasmids (BAD in reverse
orientation or the empty vector) in a ratio of 1:10 to ensure that all
cells expressing ß-galactosidase also contained BAD-encoding or
control plasmids. At 24 h posttransfection, the cells were fixed
and stained with X-gal to identify transfected (blue) cells (20, 21).
Transfection of BAD cDNA into cultured granulosa cells resulted in a
major loss of ß-galactosidase-expressing (survival) cells, indicating
that BAD exerts a proapoptotic effect on granulosa cells (Fig. 6A). In contrast, addition of a
membrane-permeable caspase inhibitor VAD-fmk completely reversed the
effect of BAD overexpression and restored the number of survival cells
to levels comparable with those transfected with control plasmids (Fig. 6A). Likewise, transfection of the BAD-encoding vector into TT cells
also showed a similar proapoptotic effect as seen in primary cultures
of granulosa cells (Fig. 6B). Treatment of TT cells with a caspase
inhibitor also reversed the proapoptotic effect of BAD, indicating that
the action of BAD occurs by apoptosis via the activation of caspase
cysteine proteases.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Homo- and hetero-dimerization of Bcl-2-related proteins mediated by the consensus BH domains are important in the decision step of apoptosis (26, 27). Consistent with these observations, the present yeast two-hybrid assay indicated that the interaction of Bcl-2 with BAD is dependent on both BH2 and BH4 domains of Bcl-2. In contrast, deletion of the transmembrane domain of Bcl-2 still allowed weaker interaction between Bcl-2 and BAD. Furthermore, BAD neither homo-dimerized with itself nor hetero-dimerized with BAX in yeast cells consistent with previous findings in mammalian cells (12).
Relatively high levels of BAD mRNA were found in the ovary and uterus. These organs are characterized by dependency on trophic endocrine stimulation and exhibit cyclic apoptosis during the reproductive cycle (28, 29). In situ and Northern blot hybridization analyses showed BAD mRNA expression in all ovarian somatic cells. Expression in granulosa cells was detected during both preantral and antral stages of ovarian follicle development. At the mRNA level, BAD expression was unaltered during the first wave of follicular cell apoptosis and remained constant during the onset of apoptosis induced by estrogen withdrawal. These data suggest that BAD is constitutively expressed as an essential part of the cell death process of the granulosa and other ovarian cells.
Although BAD mRNA levels do not vary under different physiological and experimental conditions, it is, however, possible that the function of BAD is regulated posttranslationally rather than at the transcriptional level. It was recently demonstrated that BAD can act as a molecular switch between signal transduction pathways and the cell death process, and that the proapoptotic activity of BAD is modulated by the degree of BAD phosphorylation (13). When BAD is in its hypophosphorylated form, it dimerizes preferably with Bcl-2 or Bcl-XL to prevent their antiapoptotic activity, possibly by liberating BAX, a proapoptotic Bcl-2-related protein (12). In contrast, when the cell is stimulated by survival factors, BAD is phosphorylated. BAD that is phosphorylated at specific serine residues in two 14–3-3 consensus binding sites could be bound by the constitutively active protein 14–3-3, which also binds to key components of the signal transduction cascades, including Raf-1 kinase, protein kinase C isoforms, and phosphatidyl inositol 3 kinase (30). When BAD is phosphorylated and bound to 14–3-3, it no longer binds to Bcl-2 or Bcl-XL, thus hindering its proapoptotic potential. The exact kinase(s) responsible for the phosphorylation of BAD remain(s) to be identified (13). Because of the low affinity of the currently available BAD antibodies, possible modulation of BAD protein phosphorylation in the ovarian cells could not be addressed at the present time. Future analysis of the phosphorylation status of BAD in granulosa cells and the regulation of the putative BAD kinase(s) should provide information regarding the signal transduction pathways by which survival factors regulate the Bcl-2 cell death process in the mammalian ovary.
The potential proapoptotic effect of BAD in ovarian granulosa cells was examined by using a transfection assay. BAD exerted proapoptotic effects in both primary cultures of granulosa cells and in the tumorous gonadal TT cell line. The observed proapoptotic effects of BAD indicate that granulosa cells express a set of cell death pathway proteins to which BAD can bind and, when BAD is overexpressed, shift the balance of cell death/survival effectors to favor apoptotic demise of the cell. The exact proteins to which BAD binds in the granulosa cells are not known, but recent investigations have demonstrated the expression of both Bcl-2 and Bcl-X mRNAs in the ovary (31, 32). Future studies using the present ovarian fusion cDNA library and the yeast two-hybrid screening could allow identification of the exact hetero-dimerization partner(s) of BAD in ovarian cells.
Apoptosis of granulosa and TT cells, induced following BAD overexpression, was inhibited by a cell-permeable, specific caspase inhibitor, indicating that the effect of BAD is mediated by cysteine proteases of the caspase family. It appears that the Bcl-2 family of proteins exerts actions upstream from the caspases (9, 25). Recently, one of the bridging molecules between Bcl-2 family members and the caspases was identified in mammals (33). The C. elegans gene ced-4 can simultaneously bind to Bcl-XL and to caspase 1 or 8 (11). Ultimately, the activation of caspases will lead to proteolytic damage and the activation of endonucleases that are responsible for the internucleosomal DNA cleavage observed during granulosa cell apoptosis (34, 35, 36).
In addition to BAX (31), BAD is the second proapoptotic protein of the Bcl-2 family found to be expressed in ovarian granulosa cells, the cell type undergoing apoptosis during follicle atresia (23). Expression of BAD in the ovary is of special interest because it is a cytosolic protein of the Bcl-2 family, thus enabling it to regulate other Bcl-2 survival proteins, all of which have a transmembrane region and are presumably associated with the outer mitochondrial membrane and the nuclear envelope (12, 13). The development and maturation of ovarian follicles are dependent on constant trophic support by hormonal signals (1, 2). During follicle growth, the antral transition presents a critical stage for the developing follicles (37, 38). At this stage, FSH has been shown to be the most potent survival factor (38). BAD expression in these follicles may represent a molecular sensor for mediating survival factor support of the developing follicle. If the follicle is receiving sufficient FSH support, BAD would remain in its phosphorylated, inactive form rendering the cell less likely to undergo apoptosis.
In summary, the death agonist BAD was identified as a Bcl-2- interacting protein expressed in the rat ovary. BAD mRNA is constitutively expressed in different ovarian cells suggesting its potential role as an essential part of the cell death process. BAD overexpression in the ovarian granulosa cells and in an ovarian tumor cell line leads to apoptotic cell demise that can be inhibited by a caspase inhibitor. Future investigations focusing on the posttranslational modifications of BAD protein in the ovary and its regulation by follicle survival factors could provide insight into the molecular mechanisms regarding the hormonal regulation of ovarian follicle atresia.
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Acknowledgments |
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Footnotes |
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2 Recipient of the Lalor Foundation postdoctoral fellowship.
Received June 6, 1997.
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