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Female Reproductive Tract |
a Department of Pathology, Hyogo College of Medicine, Nishinomiya, Hyogo 663-8501, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, estradiol, female reproductive tract, signal transduction, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent studies on intracellular apoptosis signaling cascades revealed that mitochondria play a pivotal role in the regulation of apoptosis [9–15]. Several apoptogenic factors such as cytochrome c, apoptosis-inducing factor (AIF), a protein called Diablo or Smac, and certain procaspases exist in the intermembrane space of mitochondria between the outer and inner membranes, and release of these factors from mitochondria into the cytosol initiates apoptosis cascades. Cytochrome c triggers the assembly of Apaf-1 and procaspase-9 into an apoptosome, thereby activating caspase-9, which in turn activates caspase-3. Diablo/Smac neutralizes caspase inhibitors in the cytosol [16, 17], and AIF moves into the nucleus, where it stimulates large-scale fragmentation of DNA and condensation of chromatin [18, 19]. The release of these apoptogenic factors has been shown to be controlled by Bcl-2 family members that include antiapoptotic and proapoptotic proteins [9–11, 13]. However, the mechanisms involved remain controversial [9–11, 13–15]. In one model, the voltage-dependent anion channel (VDAC) of the permeability transition pore (PTP) has been proposed to be a channel for apoptogenic factors [10, 11, 15, 20]. PTP is an oligo-protein channel consisting of the VDAC on the outer membrane, the adenine nucleotide translocator on the inner membrane, and additional components including matrix protein cyclophilin D. This model assumes that functional interaction of proapoptotic members such as Bak and Bax with the VDAC or another PTP component opens the VDAC, thereby allowing cytochrome c release, whereas the functional interaction of antiapoptotic members such as Bcl-2 and Bcl-X closes this channel. In other models, apoptogenic factors are believed to be released through an outer membrane channel formed by multimerization of proapoptotic Bcl-2 members themselves or as a result of nonspecific outer membrane rupture [10, 14].
Identification of genes, the expression of which increases in the uterus after estrogen deprivation, may provide some clues as to the mechanism of uterine epithelial apoptosis. Our attempts to determine such genes by the subtraction method showed that expression of VDAC genes increased in the mouse uterus after estrogen deprivation, suggesting the significance of the mitochondrial apoptosis signaling pathway in uterine epithelial cells. In this study we analyzed the expression of VDAC and Bcl-2 family members, the release of cytochrome c from mitochondria, and the activation of caspase-3 in uterine epithelial cells following estrogen deprivation in order to investigate the role of the mitochondrial apoptosis signaling pathway in uterine epithelial apoptosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The female BALB/c mice used in this study were bred in our laboratory, maintained in accordance with the Guide for Care and Use of Laboratory Animals published by the National Institutes of Health, and treated with humane care. They were kept at 25°C under controlled light conditions (12L:12D), and allowed free access to water and a pellet diet. All surgical procedures were performed after anesthesia with an i.m. injection of 0.1 ml of saline containing xylazine (0.2 mg) and ketamine hydrochloride (1 mg). All experiments involving mice were approved by the Animal Care Committee of Hyogo College of Medicine.
Treatment of Mice
Mice were ovariectomized at the age of 8 wk, and 2 wk later, a 10 mg cholesterol pellet containing 500 µg of estradiol-17ß (hereafter called the E2 pellet) was implanted s.c. into the interscapular space. Four days later, the E2 pellets were removed (designated as Day 0), and mice were killed at various times (i.e., on Day 0, 1, 2, 4, or 8) for analysis of uteri.
Apoptotic Index (Percentage of Apoptotic Cells)
Uteri were fixed in Zamboni fixative and embedded in paraffin wax. Transverse sections (5 µm in thickness) of the midportion of uteri were prepared and stained with hematoxylin and eosin. About 1000 cells in the luminal or glandular epithelium were examined to identify apoptotic cells with the following characteristics: shrinkage with eosinophilic-condensed cytoplasm and condensed chromatin, and fragmentation with eosinophilic-condensed cytoplasm and condensed chromatin [21, 22]. Our previous study [8] showed that such cells are positively stained with the in situ DNA 3'-end labeling method [23].
Preparation of Total RNA
Uteri were rapidly frozen in liquid nitrogen and pounded in a mortar with a pestle. Total RNA was isolated by the guanidium isothiocyanate-cesium chloride centrifugation method as follows. The uterine powder was homogenized gently in a Teflon homogenizer of the Potter type with a guanidium solution (50 mM Tris-HCl pH 7.5, containing 5 M guanidium isothiocyanate, 10 mM EDTA, and 5% 2-mercaptoethanol). The homogenate was centrifuged at 5980 x g at 4°C for 10 min, and the supernatant was mixed with a 1/10 volume of a 20% N-lauroylsarcosine aqueous solution and incubated at 65°C for 2 min. The sample was placed on a 5 M cesium chloride aqueous solution in a polyallomer tube (Beckman, Palo Alto, CA), and centrifuged at 113 000 x g at 4°C for 12 h in a swing rotor. The pellet that formed was dissolved in 5 mM EDTA pH 8.0, 0.5% N-lauroylsarcosine, and 5% 2-mercaptoethanol, and total RNA was extracted three times with phenol, chloroform, and isoamylalcohol (25:24:1).
Northern Blot Analysis
Total RNA (20 µg) was fractionated by 1.2% agarose-formamide gel electrophoresis, transferred to a Hybond N+ membrane (Amersham Pharmacia Biotech, Buckinghamshire, U.K.), and hybridized with 32P-labeled probes described below in an Express Hyb hybridization solution (Clontech, Palo Alto, CA) at 65°C overnight. To prepare probes, a 765 base pair (bp) fragment of DNA corresponding to nucleotides 236–1000 of mouse ß-actin cDNA; a 319 bp fragment corresponding to nucleotides 959–1277 of mouse VDAC1 cDNA; and 337, 459, and 245 bp fragments corresponding to, respectively, nucleotides 1514–1850 of mouse VDAC1 cDNA, 2122–2580 of mouse VDAC2 cDNA, and 1530–1786 of mouse VDAC3 3' untranslated region cDNA [24, 25] were labeled with [
-32P]-deoxycytidine triphosphate (specific activity, 3000 Ci/mmol) (Amersham Pharmacia Biotech) with a BcaBest labeling kit (Takara Shuzo, Kyoto, Japan) using random oligonucleotide primers. After hybridization, the blot was washed with 2x SSC (0.15 M NaCl, 0.015 M trisodium citrate) containing 0.05% SDS at room temperature for 30 min, and then with 0.1x SSC containing 0.1% SDS at 50°C for 40 min. Radio-image quantitation was carried out using a BAS 2000-P computerized image display system (Fuji Film, Tokyo, Japan). Signals were normalized to ß-actin signals, which were correlated with the amount of ribosomal RNA.
Isolation of Epithelial Cells
Uterine epithelial cells were isolated as described previously [8]. The uterus was opened longitudinally and then cut transversely into small segments. Segments were incubated in Hanks balanced salt solution containing 1% trypsin at 4°C for 90 min and then the uterine epithelium was separated from underlying tissue by gentle pipetting. The underling tissue was subjected to further vigorous pipetting to remove adherent epithelial cells. The collected epithelium and underlying tissue were used for subsequent analyses.
RNase Protection Assay
RNase protection assays of Bax, Bak, Bcl-2, Bcl-X, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were carried out using 10 µg of total RNA and a RiboQuant RNase protection assay system with an mApo-2 RiboQant mouse apoptosis multiprobe template set (Pharmingen, San Diego, CA) according to the manufacturer's instructions. Hybridized products were quantified with a BAS 2000-P computerized image display system (Fuji Film) and normalized to GAPDH signals.
Immunohistochemistry
Frozen transverse sections (6 µm in thickness) of the midportions of uteri were prepared, dried, fixed in acetone for 5 min at 4°C, and treated with 3% H2O2 in 50% methanol for 30 min at room temperature to inactivate endogenous peroxidase. The sections were incubated with goat polyclonal anti-human VDAC1 antibody (1:200 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA) or goat polyclonal anti-human caspase-3 p10 antibody (1:200 dilution) (Santa Cruz Biotechnology) at 4°C overnight, and then with Histofine simple stain MAX-PO (G) (Nichirei, Tokyo, Japan) for 30 min at room temperature. Immunoreacted cells were visualized using a Simple Stain diaminobenzidine solution (Nichirei). The sections were lightly counterstained with hematoxylin. The negative control section was processed without the anti-VDAC1 and anti-caspase-3 antibodies.
Western Blot Analysis
For Western blot analysis of Bax or Bcl-2, uteri were homogenized in ice-cold PBS containing 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS using a Pyscotron microhomogenizer (NITI-ON; Funabashi, Chiba, Japan). The homogenate was centrifuged at 500 x g at 4°C for 10 min, and the supernatant was used for Western blot analysis.
For Western blot analysis of cytochrome c, VDAC1, AIF, or caspase-3, uteri were homogenized in ice-cold 10 mM Hepes buffer pH 7.4 containing 0.3 M mannitol, 0.2 mM EGTA, and 0.1% fatty acid-free BSA using a microhomogenizer. The homogenate was centrifuged at 500 x g at 4°C for 10 min, and the supernatant was separated and centrifuged at 5000 x g at 4°C for 10 min. The resultant supernatant and pellet were used for Western blot analysis as the cytosol and mitochondrion-enriched fractions, respectively.
Thirty-microgram samples of protein were electerophoresed on 10% SDS polyacrylamide gel (Easy Gel II; Funakoshi, Tokyo, Japan) for Bax, Bcl-2, and AIF, and on 12.5% SDS polyacrylamide gel (Easy gel II, Funakoshi) for VDAC1, and 5%–10% polyacrylamide gradient gel (Easy Gel II, Funakoshi) for cytochrome c. After electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane (Fine Trap; Nihon Eido, Tokyo, Japan) using an electroblotting apparatus (Nihon Eido). After blocking nonspecific binding by incubating the membrane in a TBST solution pH 7.4 (10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20) containing 1% BSA, the membrane was incubated at room temperature for 1 h with one of the primary antibodies: rabbit polyclonal anti-mouse Bax antibody, rabbit polyclonal anti-human Bcl-2 antibody, goat polyclonal anti-human VDAC1 antibody, rabbit polyclonal anti-human cytochrome c antibody, or rabbit polyclonal anti-human AIF antibody (all from Santa Cruz Biotechnology). Binding of the primary antibody was detected using horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (Ig) G (Santa Cruz Biotechnology) and an enhanced chemiluminescence (ECL) Western blotting detection system (Amersham Pharmacia Biotech) or using biotin-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology), a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) and the Amersham ECL Western blotting detection system. Bax, Bcl-2, and VDAC1 proteins on Western blot analysis were quantitated with a densitograph (Atto, Tokyo, Japan).
For Western blot analysis of caspase-3, 50-µg protein samples were electrophoresed on 10%–20% polyacrylamide gradient gel (MultiGel 10/20; Daiichi Pure Chemicals, Tokyo, Japan). A TBST solution containing 5% skim milk was used to block nonspecific binding, and as the primary antibody, goat polyclonal anti-human caspase-3 p10 antibody (Santa Cruz Biotechnology) or rabbit polyclonal anti-human caspase-3 p20 antibody (Cell Signaling Technology, Beverly, MA) was used. The anti-human caspase-3 p10 antibody recognizes the 10 kDa subunit and 32 kDa precursor caspase-3, and the anti-human caspase-3 p20 antibody recognizes the 17 kDa subunit and 32 kDa precursor caspase-3. The membrane was incubated with each antibody at room temperature for 3 h. The membrane was then cut into two strips that contained 47–25 kDa proteins and 25–6 kDa proteins. Binding of the anti-caspase-3 p20 antibody was detected using biotin-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology), a Vectastain ABC kit (Vector Laboratories) and an ECL Western blotting detection system (Amersham Pharmacia Biotech), and that of the anti-caspase-3 p10 antibody was detected using alkaline-phosphatase-conjugated donkey anti-rabbit IgG (Santa Cruz Biotechnology), 4-nitro blue tetrazolium chloride (Sigma, St. Louis, MO) and 5-bromo-4-chloro-3-indolyl-phosphate (Sigma). A longer time was spent to detect the 17 kDa and 10 kDa subunits than was spent to detect the 32 kDa precursor caspase-3.
Statistical Analysis
The data were analyzed by one-way ANOVA. When statistical significance was found, a Bonferroni multiple comparison test was applied. A P value of less than 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cholesterol pellets containing 500 µg of E2 were implanted into ovariectomized mice and removed 4 days later (designated as Day 0), and the luminal and glandular epithelia were analyzed for apoptosis at different times. In the luminal epithelium, the apoptotic index increased rapidly, reaching its maximal level on Day 2, and it declined rapidly thereafter (Fig. 1). The apoptotic index also increased in the glandular epithelium, but to a much smaller extent than in the luminal epithelium.
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Expression of VDAC mRNA After Estrogen Deprivation
The expression of VDAC mRNA in the uterus after estrogen deprivation was examined by Northern blot analysis with a probe for VDAC1 cDNA (Fig. 2). This probe has 79.3% and 74.3% homology with VDAC2 and VDAC3 cDNAs, respectively [25, 26]. The three forms of VDAC mRNA are similar in size (1.8 kilobases) and migrate to the same spot. Our results, which may reflect changes in the level of all three forms of VDAC mRNA, showed that VDAC mRNA levels increased 1.9-fold on Day 2 and then declined to their original level on Day 6.
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We then analyzed the epithelium, stroma, and muscle on Days 0 and 2 for the expression of VDAC1, 2, and 3 mRNAs separately using probes specific for VDAC1, 2, or 3 mRNA (Fig. 3). In the epithelium, the VDAC1, 2, and 3 mRNAs accounted for 51%, 37%, and 12% of total VDAC mRNA, respectively, on Day 0. On Day 2, mRNA levels of VDAC1, 2, and 3 increased 1.7-fold, 1.7-fold, and 1.9-fold, respectively. In the stroma and muscle, the VDAC1, 2, and 3 mRNAs also accounted for 54%, 32%, and 14% of total VDAC mRNA, respectively, on Day 0, but none of these mRNAs showed significant increases in expression on Day 2.
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Western Blot Analysis of Expression of VDAC1, Release of Cytochrome C and AIF From Mitochondria, and Activation of Caspase-3
Mitochondria and cytosol of the uterus were analyzed for expression of VDAC1, and the release of cytochrome c and AIF by Western blotting. Levels of VDAC1 protein that were detected in mitochondria but not in the cytosol increased after estrogen deprivation, peaking on Day 2 (Fig. 4A and B). Cytochrome c was present in mitochondria only on Day 0, it was detected in the cytosol on Days 1 and 2, but it again became undetectable in the cytosol on Day 4 (Fig. 4A). AIF remained in mitochondria throughout the experimental period (Fig. 4A). Release of cytochrome c from mitochondria results in activation of caspase-9, which in turn activates caspase-3 [12, 27]. Immunohistochemistry showed that caspase-3 was detected only in the luminal and glandular epithelia (Fig. 5C), whereas the VDAC1 protein was present in the muscle as well as in the luminal and glandular epithelia (Fig. 5B). Activation of caspase-3 involves cleavage of a 32 kDa inactive precursor into 10 and 20 kDa subunits, and further cleavage of the 20 kDa subunit into a 17 kDa subunit. Western blot analysis showed the presence of the 17 kDa and 10 kDa subunits in the uterine cytosol on Day 2 (Fig. 6).
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Expression of Bcl-2 Family mRNAs and Proteins After Estrogen Deprivation
Expression of the Bax, Bak, Bcl-2, and Bcl-X mRNAs in the uterus after estrogen deprivation was examined by means of RNase protection assay. Estrogen deprivation resulted in gradual increases in the expression of these mRNAs (Fig. 7A and B). When the mRNA ratios of Bax:Bcl-2, Bax:Bcl-X, Bak:Bcl-2, and Bak:Bcl-X were calculated (Fig. 7C), the mRNA ratio of Bax:Bcl-2 was found to increase on Day 1, it reached its maximal level on Day 2, and then declined below the original level on Days 4 and 6. Similar changes were observed with the mRNA ratio of Bax:Bcl-X. On the other hand, the mRNA ratios of Bak:Bcl-2 and Bak:Bcl-X decreased steadily after estrogen deprivation.
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Expression of Bax, Bak, Bcl-2, and Bcl-X mRNAs in the epithelium, stroma, and muscle were also examined on Days 0 and 2 (Fig. 8). In the epithelium, Bax mRNA levels increased 3.6-fold, Bak mRNA levels rose 2-fold, Bcl-2 mRNA levels rose 1.9-fold, and Bcl-x mRNA levels rose 1.7-fold on Day 2 (Fig. 8A and B). In contrast, levels of all these mRNAs were essentially unchanged in the stroma and muscle (Fig. 8A and B). The mRNA ratios of Bax:Bcl-2 and Bax:Bcl-X increased about 2-fold in the epithelium on Day 2, whereas these ratios showed little changes in the stroma and muscle (Fig. 8C). The mRNA ratios of Bak:Bcl-2 and Bak:Bcl-X were essentially unchanged in epithelium, stroma, and muscle on Day 2 (Fig. 8C).
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Western blot analysis showed that expression of both Bax and Bcl-2 proteins increased after estrogen deprivation, with Bax protein levels increasing more rapidly than Bcl-2 protein levels on Days 1 and 2, and then more slowly on Day 4 (Fig. 9A and B). This resulted in rapid increases in the protein ratio of Bax:Bcl-2 on Days 1 and 2, followed by rapid decreases on Day 4 (Fig. 9B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, apoptotic cells were morphologically identifiable in tissue sections stained with hematoxylin and eosin according to well-established criteria [21, 22]. The validity of this identification has been previously shown in comparison to the in situ DNA 3'-end labeling method [8, 23]. In the latter method, different sensitivities of cells to the treatment with proteinase K often affect the number of positive cells with the generation of false positives [28]. Therefore, the morphological identification of apoptotic cells seems to be more reliable in comparing apoptosis in tissue sections.
The luminal apoptosis was correlated well to the release of cytochrome c from mitochondria and the activation of procaspase-3, both peaking on Day 2. In the mitochondrial apoptosis signaling pathway, cytochrome c released into the cytosol forms a complex with Apaf-1 and procaspase-9, leading to activation of caspase-9 [9, 12]. Caspase-9 activates procaspase-3, and the activated caspase-3 in turn cleaves ICAD (inhibitor of caspase-activated DNase), which leads to migration of caspase-activated DNase to the nucleus, where it degrades chromosomal DNA [29, 30]. The correlation of uterine epithelial apoptosis with the release of cytochrome c and activation of caspase-3 suggests that the mitochondrial pathway plays a role in uterine epithelial apoptosis. In addition, the immunohistochemical localization of caspase-3 in the luminal and glandular epithelia is in good agreement with the fact that apoptosis occurs exclusively in the epithelium of the uterus [1, 3–6].
AIF is known as a protein in mitochondria, which on release from mitochondria and transportation into the nucleus, triggers large-scale fragmentation of DNA and condensation of chromatin [18, 19]. However, in this study, we did not observe the release of AIF from mitochondria, suggesting that AIF has no role in uterine epithelial apoptosis.
The release of apoptogenic factors such as cytochrome c from mitochondria into the cytosol is regulated by antiapoptotic Bcl-2 family proteins, such as Bcl-2 and Bcl-X, and proapoptotic Bcl-2 family proteins such as Bax and Bak [9–11, 13]. The heterodimerization between antiapoptotic and proapoptotic proteins is considered to result in suppression of the activity of the partner proteins [9–11, 13]. Therefore, the relative amount rather than the absolute amount of proapoptotic and antiapoptotic proteins is important for the induction of apoptosis. In the present study, we showed that the ratios of Bax:Bcl-2 and Bax:Bcl-X increased at both mRNA and protein levels after estrogen deprivation, peaking on Day 2, and decreasing thereafter in the uterus. No such changes were observed in the stroma and muscle, where no apoptosis takes place. In contrast, the mRNA ratios of Bak:Bcl-2 and Bak:Bcl-X decreased after estrogen deprivation, suggesting that Bak in combination with Bcl-2 or Bcl-X plays no role in the regulation of apoptosis in the uterine epithelium after estrogen deprivation. Perlman et al. [31] also reported elevation of the Bax:Bcl-2 ratio in correlation with epithelial apoptosis of the rat ventral prostate after castration. However, using Bax knockout mice and Bcl-2 transgenic mice, Bruckheimer et al. [32] showed that Bax deficiency and overexpression of Bcl-2 had only a little influence on epithelial apoptosis of the mouse ventral prostate after castration. Further investigation with Bax knockout mice is desired to determine the significance of elevation of Bax:Bcl-2 or Bcl-X in the regulation of uterine epithelial apoptosis.
Northern blot analysis showed that the expression of mRNAs of VDACs 1, 2, and 3 increased in the uterine epithelial cells after estrogen deprivation in correlation with apoptosis. None of these mRNAs increased in expression in the stroma and muscle. We detected VDAC1 protein, the product of the most abundant mRNA species, immunohistochemically in the epithelium. These results are consistent with a role for VDAC in the release of cytochrome c from mitochondria. It has been proposed that mitochondrial apoptogenic factors are released through the VDAC of the PTP [10, 11, 15, 20]. This model assumes that the functional interaction of proapoptotic members such as Bak and Bax with the VDAC or another PTP component opens the VDAC channel, thereby allowing cytochrome c release, whereas that of antiapoptotic members such as Bcl-2 and Bcl-X, closes this channel. If VDAC is a channel for cytochrome c, an increase in VDAC levels may be associated with the vulnerability of epithelial cells to apoptosis.
Several groups have reported extracellular signals that induce uterine epithelial apoptosis after deprivation of steroid hormones. Kurita et al. [33] reported that the antiapoptotic action of progesterone on mouse uterine epithelial cells is mediated by stromal paracrine influences through stromal progesterone receptors, but not by direct action of progesterone on epithelial cells through their progesterone receptors. Suzuki et al. [34, 35] reported that in ovariectomy-induced apoptosis of mouse vaginal cells, Fas plays an important role, with tumor necrosis factor-, not Fas-ligand, serving as a ligand. However, they also reported that Fas plays only a little role in ovariectomy-induced apoptosis of uterine epithelial cells [34]. In contrast to extracellular signals, little is known about the intracellular apoptotic pathway in uterine epithelial cells after deprivation of steroid hormones. The present study suggests for the first time that the mitochondrial apoptosis signaling pathway plays a role in uterine epithelial apoptosis after estrogen deprivation, and that this pathway is regulated by Bcl-2 family members. In addition, the present study suggests the involvement of VDAC in the mitochondrial apoptosis signaling pathway in uterine epithelial cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Naoko Yamada, Department of Pathology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan. FAX: 81 798 45 6431; ynaoko{at}hyo-med.ac.jp
Received: 6 June 2002.
First decision: 27 June 2002.
Accepted: 14 October 2002.
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acts as a ligand for fas in vaginal cells. Cell Death Differ 1999 6:638-643[CrossRef][Medline]This article has been cited by other articles:
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