Molecular Human Reproduction, Vol. 9, No. 12, pp. 775-783, 2003
© European Society of Human Reproduction and Embryology 2003; all rights reserved
Guanylyl cyclase inhibitors NS2028 and ODQ and protein kinase G (PKG) inhibitor KT5823 trigger apoptotic DNA fragmentation in immortalized uterine epithelial cells: anti-apoptotic effects of basal cGMP/PKG
Department of Physiology (Faculty of Medicine), Epithelial Cell Biology Research Centre and Centre for Gerontology and Geriatrics, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
1 To whom correspondence should be addressed at: Department of Physiology, BMSB Rm. 507, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, China. e-mail: ronfiscus{at}cuhk.edu.hk
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The cGMP/protein kinase G (PKG) signalling pathway, at basal levels, has anti-apoptotic/pro-survival effects in certain neural cells. The present study determined apoptosis-regulating effects of basal cGMP/PKG in an immortalized uterine epithelial cell line, HRE-H9 cells, using two soluble guanylyl cyclase (sGC) inhibitors, NS2028 and ODQ, and a PKG inhibitor, KT5823. A new quantitative, ultrasensitive technique using capillary electrophoresis with laser-induced fluorescent detector (CE-LIF), recently developed in our laboratory, was used to quantify levels of apoptotic DNA fragmentation. NS2028 and ODQ increased apoptotic DNA fragmentation by 3.5- and 9-fold respectively, suggesting that lowering basal cGMP levels causes spontaneous apoptosis. 8-Br-cGMP, a cell-permeable cGMP analogue that directly activates PKG, reduced ODQ-induced apoptosis by 81%, indicating that replacement of lowered cGMP with a direct PKG activator prevents apoptosis. Western blot analysis, using PKG type I (PKG-I)-specific antibody, indicated that HRE-H9 cells express PKG-I at moderate levels. Inhibiting basal PKG activity with KT5823 increased apoptotic DNA fragmentation by 9.8-fold. Overall, the data show that inhibitors of basal sGC and PKG activities in immortalized uterine epithelial cells cause apoptosis, suggesting that normal basal levels of cGMP and PKG activity may be necessary to prevent a spontaneous development of apoptosis in these cells.
Key words: apoptosis/endometrium/guanylyl cyclase/protein kinase G/uterine epithelial cells
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Introduction |
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Apoptosis is important to many physiological/pathological processes. Dysregulated apoptosis may contribute to the pathogenesis of a number of diseases, including cancer (Liebermann et al., 1995), autoimmune diseases (Reap et al., 1995), AIDS (acquired immunodeficiency syndrome) (Ameisen, 1992), neurodegenerative disorders (Estevez et al., 1998; Fiscus and Ming, 2000; Fiscus, 2002) and leukaemia (Meinhardt et al., 1999; Kolb, 2000). Altering the apoptotic threshold may change the natural progression of some of these diseases. For example, the effectiveness of chemotherapeutic drugs for the treatment of various cancers can depend on the ability of these agents to induce apoptosis preferentially in the cancer cells (Li et al., 2000).
In the female reproductive system, apoptosis of the uterine epithelial cells is now recognized to be an important step in the normal onset of menstruation (Tabibzadeh, 1995; Sato et al., 1997; Tao, 1997; Toki, 1998) as well as the normal implantation of an embryo into the uterine wall during pregnancy (Parr et al., 1987; Kamijo et al., 1998; Galan et al., 2000). Dysregulation of uterine epithelial apoptosis may thus lead to dysmenorrhoea and infertility. Abnormal apoptosis of uterine epithelial cells is also thought to contribute to the pathogenesis of endometriosis (Gebel et al., 1998; Garcia-Velasco et al., 2002). Therefore, uterine epithelial apoptosis is an important part of the physiological/pathological regulation of the female reproductive system.
Nitric oxide (NO) is known to be involved in the regulation of apoptosis, both as an anti-apoptotic factor and as a pro-apoptotic factor, depending on the type of cells, the concentrations of NO and the experimental conditions (for reviews, see Fiscus, 2002; Fiscus et al., 2002). Previously, NO had been shown to cause toxicity and cell death (both apoptosis and necrosis), notably in the central and peripheral nervous systems, where NO is thought to contribute to the neuronal damage of various neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) (Beckman and Koppenol, 1996), Alzheimer’s disease and Parkinson’s disease (Fiscus and Ming, 2000; Fiscus, 2002; Fiscus et al., 2002). Recently, NO has been shown to cause apoptosis in a human endometrial carcinoma cell line, RL95-2 cells, and this type of NO-induced apoptosis has been proposed to be responsible for, at least in part, the apoptosis of uterine epithelial cells that occurs during menstruation and embryonic implantation (Li et al., 2001).
NO also exerts anti-apoptotic effects in certain cells, either through mechanisms involving activation of soluble guanylate cyclase (sGC) and subsequent elevation of cGMP levels or, in some cells, through mechanisms independent of sGC (Kim et al., 1997; Kolb, 2000; Fiscus, 2002; Fiscus et al., 2002). Endogenous NO synthesis or exposure to low levels of NO donors has now been shown to inhibit apoptosis in a number of different kinds of cells, including B lymphocytes (Mannick et al., 1994; Genaro et al., 1995), eosinophils (Beauvais et al., 1995), ovarian follicles (Chun et al., 1995), endothelial cells (Dimmeler et al., 1997) and rat cerebellar granule cells and cortical cells (Pantazis et al., 1998; Fernandez-Tome et al., 1999). In some of these cells, the anti-apoptotic actions of NO were shown to be dependent on the cGMP elevations (Beauvais et al., 1995; Chun et al., 1995; Genaro et al., 1995), while in other cells, the anti-apoptotic mechanism of NO was independent of cGMP (Mannick et al., 1994).
Previously, we had shown that two natriuretic peptides, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), which activate particulate guanylyl cyclase (pGC) and cause prolonged elevations of cGMP levels, are potent and effective inhibitors of apoptotic cell death caused by serum deprivation-induced stress in PC12 cells (Fiscus et al., 2001b). This effect resulted in a significant prolongation of the survival of the PC12 cells. Furthermore, we found that 8-Br-cGMP, a cell-permeable analogue of cGMP that directly activates protein kinase G (PKG), also inhibited apoptosis in stressed PC12 cells. Thus, cGMP and PKG appear to be intimately involved in the prevention of apoptosis and the prolongation of cell survival, at least in certain types of cells, like PC12 cells. Many other neural cells have also been found to possess a similar anti-apoptotic/pro-survival pathway involving the cGMP/PKG signalling pathway (for reviews, see Fiscus, 2002; Fiscus et al., 2002). Furthermore, in some neural cells, such as N1E-115 and NG108-15 cells, even the basal levels of cGMP and basal activity of PKG appear to be sufficient to cause anti-apoptotic effects, protecting these cells against a spontaneous development of apoptosis (Yuen and Fiscus, 2001; Fiscus, 2002).
To our knowledge, no previous report has shown the involvement of cGMP and PKG in the regulation of apoptosis in uterine epithelial cells. Furthermore, no previous report has focused on the potential involvement of basal cGMP levels and basal PKG activity in protecting epithelial and/or reproductive cells against spontaneous development of apoptosis. The present study was designed to determine if basal cGMP levels and basal PKG activity are involved in the control of apoptosis in uterine epithelial cells, using an immortalized cell line, the HRE-H9 cells. We determined the levels of apoptosis in these cells by quantifying the internucleosomal fragmentation of the genomic DNA, a hallmark of apoptosis in uterine epithelial cells (Fiscus et al., 2001a). The apoptotic DNA fragmentation was measured by a new quantitative ultrasensitive technique using capillary electrophoresis with laser-induced fluorescence detector (CE-LIF), recently developed in our laboratory (Fiscus et al., 2001a,b, 2002; Fiscus, 2002).
We used two sGC inhibitors, 4H-8bromo-1,2,4-oxadiazola(3,4-d)benz(b-1,4)oxazin-1-one (NS2028) and 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), to determine if basal sGC activity and basal levels of cGMP are involved in regulating apoptosis in uterine epithelial cells. Some of the ODQ-treated cells were further treated with 8-Br-cGMP to determine if replacement of cGMP with a cGMP analogue can relieve the pro-apoptotic effects induced by ODQ. Because of the potential involvement of PKG in uterine epithelial cells, the present study also determined if HRE-H9 cells express PKG, using Western blot analysis, and if basal activity of PKG regulates apoptosis in these cells. The selective PKG inhibitor KT5823 was used to inhibit the basal intracellular activity of PKG in unstressed HRE-H9 cells. Like the sGC inhibitors NS2028 and ODQ, the PKG inhibitor KT5823 also caused significant increases in the levels of apoptotic DNA fragmentation.
Preliminary data of the present study, showing pro-apoptotic effects of ODQ and KT5823 in HRE-H9 cells, have been presented previously in abstract form (Chan and Fiscus, 2002).
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Materials and methods |
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Cell line
The HRE-H9 cells, a rabbit immortalized uterine epithelial cell line, was obtained from Dr Lih-Yuh C.Wing (National Cheng Kung University Medical College, Tainan, Taiwan). The HRE-H9 cells were originally developed by immortalizing primary cultures of endometrial epithelial cells, derived from hCG-treated pseudopregnant rabbits, with SV40 temperature-sensitive (ts) mutant virus (Li et al., 1989; Chen et al., 1991).
Equipment
Capillary electrophoresis with laser-induced fluorescence detector (CE-LIF) was from Bio-Rad (USA).
Materials
Materials were purchased as follows: Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin–streptomycin (P/S), Hanks’ balanced salt solution (HBSS, without phenol red), phosphate-buffered saline (PBS), Trypsin–EDTA (0.05%) 1x, and buffer–saturated phenol were all from Gibco BRL (Gaithersburg, USA). NS2028, Tris–HCl, Triton X-100 and trypsin were from Sigma Chemical Company (USA). Ethanol (absolute, AR) was from Merck (KGaA, Germany). Anti-Protein-Kinase-G antibody (Catalogue no. 370661, which recognizes both PKG-I
and PKG-Iß, but not PKG-II) and ODQ were from Calbiochem-Novabiochem (USA). CE-LIF-dsDNA-1000 kits were from Bio-Rad. Anti-rabbit Ig (horseradish peroxidase-linked) antibody, ECLTM (for enhanced chemiluminescence) detection kits and HyperfilmTM ECLTM were from Amersham Pharmacia Biotechology (USA). Bradford reagents were from Bio-Rad.
Cell cultures
The cells were grown in DMEM with phenol red containing 4% heat-inactivated fetal bovine serum, plus penicillin (100 IU/ml) and streptomycin (100 µg/ml), in the presence of 5% CO2 at 37°C, a slight modification of the method of Wing et al. (1998). During passaging, the cells were washed with HBSS (without phenol red), harvested by trypsinization and counted with a haemocytometer. In all experiments, the cells were used between passage 20 and 45. For preparation of all experiments, cells were seeded in medium supplemented with 4% serum in 6-well culture plates at a density of 1x106 cells per well. After 24 h, the medium was aspirated and replaced by medium of the different conditions indicated in the Results.
Assessment of apoptotic DNA fragmentation
Apoptotic DNA fragmentation in HRE-H9 cells was analysed by capillary electrophoresis with laser-induced fluorescence detector (CE-LIF), using a quantitative ultrasensitive technique developed in our laboratory (Fiscus et al., 2001a,b, 2002; Fiscus, 2002). Both floating cells and adherent cells were combined for the assessment of apoptosis. Floating cells were collected by removing the medium from each well and centrifuging at 800 g for 3 min. The adherent cells were washed with HBSS. DNA was extracted by the following procedure. Epithelial cells were incubated with lysis buffer (5 mmol/l Tris–HCl, 0.5% Triton X-100 and 20 mmol/l EDTA, pH 8.0). The pellets of the floating cells were collected after centrifugation and were added to the corresponding cell lysate. The cell lysates, together with the added floating cells, were mixed and incubated on ice for 15 min. Cell lysates were then centrifuged at 4800 g for 15 min at 4°C and the supernatants were collected. DNA in the supernatant fractions was extracted with phenol (supernatant:phenol, 1:1) and then extracted with phenol:chloroform:isoamyl alcohol (25:24:1). The fragmented DNA was precipitated in absolute ethanol (5:2) and sodium acetate (3 mol/l, pH 5.2) (10:1) at –20°C overnight. The DNA precipitate was collected by centrifugation of the sample at 16 500 g for 10 min at 4°C. The DNA pellet was washed in 70% ethanol and centrifuged at 15 500 g for 10 min at room temperature. The DNA pellet was then air-dried, dissolved in TE buffer (5 mmol/l Tris HCl, pH 8.0, 20 mmol/l EDTA) and treated with RNase (10 mg/ml) at room temperature for 1 h to remove RNA. The DNA was then applied to the CE-LIF for electrophoretic separation and analysis of the DNA fragments.
The CE-LIF technique used dsDNA1000 kits (Bio-Rad) to analyse the apoptotic DNA fragments following a procedure previously used in our laboratory for analysing apoptosis in cultured neural (PC12 and NG108-15) cells (Fiscus, 2001b; Fiscus, 2002; Fiscus et al., 2002) and uterine epithelial cells (Fiscus et al., 2001a). Briefly, the procedure used a coated capillary (24 cmx75 µmol/l), electrophoretic injection at 10 kV for 2 s, run at 2.5 kV for 40 min at 4°C and an argon laser at 488 nm. The kits included SYBR Green I as the dsDNA-specific fluorescent label. Standards of known amounts of a 550 bp DNA fragment were run in the CE-LIF using identical conditions as the samples. The resulting standard curve was then used for calculating the amount of apoptotic DNA in the 540 bp peak, based on area under the peak/migration time.
Protein extraction and western blot analysis
The cells were grown to 
80% of confluence in 6-well plates and then washed twice with phosphate-buffered saline (PBS). The lysis buffer (150 µl, Lysis Buffer 1363727; Boehringer Mannheim GmbH, Germany) was added to the cells to attain a protein concentration of 2 µg/µl (determined by Bradford reagent) and the samples were incubated on ice for 10 min. The samples were centrifuged at 12 000 g for 10 min at 4°C. The supernatant fractions were transferred to fresh tubes and boiled with an equal volume of 2xsample loading buffer for 5 min. The samples, each representing 20 µg of total protein, were applied to sodium dodecyl sulphate (SDS)–10% acrylamide gels and the proteins were resolved by SDS–polyacrylamide gel electrophoresis at a constant voltage of 100 V for 2 h. The gels were separated from the glass plates and placed on a semi-dry blotter. The semi-dry blotting was conducted at a constant voltage of 15 V for 1 h. The membrane was removed and kept wet in 1xTBS solution supplement with 0.1% of Tween-20 (TBS-T). The membranes were then blocked with TBS-T with 5% milk powder for 1 h at room temperature with constant shaking. The membranes were incubated with the primary antibody (anti-PKG-I/ß, diluted 1:1000) at 4°C for 16 h with constant shaking. The membranes were then washed in TBS-T solution for three times, each for 15 min. Next, the membranes were incubated with horse-radish peroxidase-conjugated secondary antibody (diluted 1:2000) in the blocking solution for 1 h at room temperature. The washing step was then repeated. ECLTM (enhanced chemiluminescence) detected kits and exposure to HyperfilmTM ECLTM were used to detect the presence of specific PKG-I labelling.
Statistical analysis
The data of the amounts of DNA fragmentation are presented as mean ± SEM. The n value indicates the number of individual experiments conducted. The comparisons between groups of data were made by Student’s paired t-tests, if there were only two treatment groups. If the number of treatment groups was more than two, the comparisons between groups of data were made by one-way analysis of variance, followed by Tukey’s test. All analyses were performed using the software GraphPad Prism (Version 3.0, GraphPad Software, Inc., USA; www.graphpad.com).
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Results |
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Apoptotic DNA fragmentation induced by the sGC inhibitors NS2028 and ODQ in unstressed HRE-H9 cells
To examine whether basal levels of sGC activity and basal levels of cGMP are involved in regulating the apoptosis of uterine epithelial cells, the HRE-H9 cells were incubated in the presence or absence of NS2028 or ODQ, two potent and selective inhibitors of sGC (Moro et al., 1996; Mulsch et al., 1997; Olesen et al., 1998). All experiments were conducted using unstressed cells (i.e. cells incubated in normal medium containing serum). Exposure of HRE-H9 cells to NS2028 for 24 h caused dose-dependent increases in DNA fragmentation, measured by CE-LIF (Figure 1). Figure 2 shows that 1 µmol/l NS2028 did not have a significant effect on apoptosis, but NS2028 at 10 and 100 µmol/l significantly increased apoptotic DNA fragmentation in HRE-H9 cells compared with the controls (P < 0.05; n = 3). NS2028 at 10 and 100 µmol/l caused 2.4- and 3.5-fold increases in DNA fragmentation (Figure 2).
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Previous studies from our laboratory had shown that ODQ (40 µmol/l, 24 h), by itself, lowers basal levels of cGMP to one-fifth of the normal levels and triggers the onset of apoptosis in two neural cell lines, NG108-15 (Yuen and Fiscus, 2001) and NIE-115 (Fiscus, 2002). In the present study, we found that ODQ (40 µmol/l, 24 h) increased the apoptotic DNA fragmentation in HRE-H9 cells, analysed by both agarose gels (Figure 3) and CE-LIF (Figure 4). ODQ (40 µmol/l) clearly increased the levels of apoptotic DNA fragmentation in both agarose gels and CE-LIF. Figure 5 shows the combination of data from five experiments using CE-LIF, showing that ODQ (40 µmol/l, 24 h) had significantly increased DNA fragmentation by 9.9-fold in HRE-H9 cells compared with the controls (P < 0.05; n = 5). Therefore, both NS2028 and ODQ can significantly and substantially induce apoptosis in unstressed HRE-H9 cells. The data suggest that cGMP at basal levels may be necessary to prevent spontaneous development of apoptosis in these cells.
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The cGMP analogue 8-Br-cGMP prevented the ODQ-induced apoptosis
To test whether the reduction in basal cGMP levels caused by ODQ was responsible for the induction of apoptosis, we determined if replacing the cGMP with a cell-permeable analogue of cGMP, 8-Br-cGMP, could prevent ODQ-induced apoptosis. 8-Br-cGMP at 100 µmol/l and 1 mmol/l substantially decreased the apoptotic DNA fragmentation caused by ODQ, as determined by both agarose gels (Figure 6) and CE-LIF (Figure 7). Figure 8 shows the combined data of four experiments using CE-LIF, indicating that 8-Br-cGMP (100 µmol/l and 1 mmol/l) caused 42 and 81% inhibition of the ODQ-induced DNA fragmentation respectively. 8-Br-cGMP (100 µmol/l and 1 mmol/l) by itself had no significant effect on the levels of apoptosis (Figure 8).
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Protein expression levels of PKG in HRE-H9 cells
HRE-H9 cells were analysed for the presence of PKG protein using Western blot analysis. The antibody recognized both the type I
and type Iß forms of PKG. Figure 9 shows the relative levels of PKG-I/ß expression in HRE-H9 cells, compared with expression levels in three other types of cells (PC12, N1E-115 and NG108-15 cells) that display cGMP/PKG-mediated inhibition of apoptosis (Fiscus et al., 2001b, 2002; Fiscus, 2002). Whereas PC12 cells showed barely detectable levels of PKG, NG108-15 and N1E-115 cells had high levels of PKG expression. The HRE-H9 cells had intermediate levels of PKG expression. The band of the HRE-H9 cells appeared to have a slightly higher molecular weight (estimated to be 76 kDa) compared with the bands of NG108-15 and N1E-115 cells with estimated molecular weights of 74 kDa each. This suggests that the HRE-H9 cells may selectively express the ß form of PKG-I, which has a molecular weight of 76 kDa, compared with PKG-I with a molecular weight of 74 kDa. However, further analysis using more specific antibodies will be needed in future studies to precisely determine which isoform of PKG is expressed in HRE-H9 cells.
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Because some cells, such as vascular smooth muscle cells (Lincoln and Cornwell, 1993) and PC12 cells (present study, Figure 9) tend to lose their expression of PKG during passaging, we also tested if the levels of PKG expression in HRE-H9 cells were altered during passaging. Figure 9 shows that PKG levels are expressed at moderate levels in all HRE-H9 cells at the three different passages (22, 43 and 66) tested. Thus, there was no indication that PKG levels are lost or reduced during passaging in the HRE-H9 cells.
Apoptotic DNA fragmentation induced by the PKG inhibitor KT5823 in unstressed HRE-H9 cells
We suspected that the anti-apoptotic effects of cGMP (at basal levels) were mediated by partial activation of PKG (i.e. basal PKG activity) in HRE-H9 cells. To test this, we used a potent and highly selective PKG inhibitor, KT5823. Exposure of HRE-H9 cells to KT5823 in a wide range of concentrations for 24 h caused dose-dependent increases in apoptotic DNA fragmentation, measured by CE-LIF (Figure 10). Because normal medium (with serum) was used, the HRE-H9 cells were under unstressed conditions (except for the exposure to KT5823). While KT5823 at 1 nmol/l and 10 nmol/l did not affect the levels of apoptotic DNA fragmentation, KT5823 at 100 nmol/l and 1 µmol/l (concentrations expected to selectively inhibit PKG; Ki = 234 nmol/l, Calbiochem catalogue 2003/2004) substantially increased DNA fragmentation, compared with controls (Figure 10). KT5823 at 100 nmol/l and 1 µmol/l caused 5.9- and 9.8-fold increases in the DNA fragmentation levels respectively (Figure 11). The statistical significance of these effects was high (P < 0.001), due to the large pro-apoptotic effects of this PKG inhibitor. The data suggest that the low-level activation of PKG that occurs under normal culturing conditions, presumably because of the basal cGMP levels, may play an essential role in preventing spontaneous development of apoptosis in unstressed HRE-H9 cells.
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Discussion |
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The present study demonstrates that two sGC inhbitiors, NS2028 and ODQ, and a PKG inhibitor, KT2823, all cause induction of apoptotic DNA fragmentation in the HRE-H9 immortalized uterine epithelial cells. Because the induction of apoptosis occurred at concentrations of the inhibitors that would be expected to cause specific inhibition of sGC or PKG, the data suggest that the basal levels of sGC and PKG activity may be essential to prevent spontaneous development of apoptosis in these cells. Furthermore, the present data show that addition of the cGMP analogue 8-Br-cGMP, a cell-permeable direct activator of PKG, can prevent the apoptosis induced by ODQ. Thus, the cGMP/PKG signalling pathway appears to play an important role in the regulation of apoptosis in immortalized uterine epithelial cells.
sGC is the main intracellular receptor for NO, whereas the particulate form of this enzyme (pGC) is the receptor for the natriuretic peptides ANP and BNP (Waldman and Murad, 1987; Fiscus, 2002). Elevations in cGMP levels are thought to mediate the anti-apoptotic effects of NO in eosinophis (Beauvais et al., 1995), splenocytes (Genaro et al., 1995), hepatocytes (Kim et al., 1997), rat dorsal root ganglia neurons (Thippeswamy and Morris, 1997), motor neurons (Estevez et al., 1998), pulmonary cells (Janssen et al., 1998), PC12 cells (Kim et al., 1999) and macrophages (Heinloth et al., 2002). Furthermore, our laboratory has shown that ANP and BNP cause anti-apoptotic and survival-prolonging effects in stressed PC12 cells, which are mediated by prolonged elevations of cGMP levels (Fiscus et al., 2001b). The PKG activator 8-Br-cGMP also protected these cells against stress-induced apoptosis, suggesting that activation of PKG plays an important role in mediating the anti-apoptotic/pro-survival effects of cGMP in PC12 cells. Many other neural cells show similar protection against apoptosis when cGMP levels and PKG activity are increased (Fiscus, 2002). Thus, activation of the cGMP/PKG pathway appears to promote cell survival in a number of different types of cells.
Very few reports, however, have focused on the potential involvement of basal cGMP levels or basal PKG activity in the regulation of cell survival. (Garthwaithe and Garthwaite, 1988) showed that exposure of young rat cerebellar slices to a sGC inhibitor, methylene blue, caused progressive destruction of differentiating neurons, and that co-administration of a cGMP analogue protected against this destruction, suggesting that basal cGMP may have neuroprotective effects. However, because of the known toxic effects of methylene blue (e.g. generation of superoxide) (Fiscus, 2002), it was not clear whether the destructive effects were caused by inhibition of sGC (and subsequent lowering of cGMP levels) or by the toxic effects of methylene blue. More recent experiments using a specific sGC inhibitor, ODQ, showed reduced survival of rat dorsal root ganglion neurons (Thippeswamy and Morris, 1997) and rat motor neurons (Estevez et al., 1998). Inhibition of sGC with ODQ was also shown to decrease the survival and activate a pro-apoptotic pathway in L1210 leukaemia cells (Flamigni et al., 2001). Recently, we have shown that inhibition of sGC with ODQ (40 µmol/l) lowers basal cGMP levels to 1/5 of normal levels and causes the development of apoptosis in two established neural cell lines, N1E-115 and NG108-15 cells (Yuen and Fiscus, 2001; Fiscus, 2002). These data suggest that basal sGC activity/cGMP levels are involved in protecting neural and leukaemia cells against spontaneous development of apoptosis.
The present study, showing that two sGC inhibitors, NS2028 and ODQ, both cause apoptotic DNA fragmentation in uterine epithelial cells, suggests that the basal sGC activity and basal cGMP levels protect these cells against spontaneous development of apoptosis. Unlike other so-called sGC inhibitors, such as LY83583 and methylene blue that are not potent inhibitors of sGC and have many other biological actions, ODQ has been shown to be a potent and selective inhibitor of sGC, blocking the NO-induced activation of sGC in cerebellar slices and endothelial cells (Garthwaite et al., 1995) as well as platelets and vascular smooth muscle cells (Moro et al., 1996). Furthermore, ODQ (10 µmol/l) has been shown to lower basal levels of cGMP to about one-third of normal levels in endothelial cells (Garthwaite et al., 1995). Data from our laboratory have further shown that ODQ, when used at 40 µmol/l, the same concentration as used in the present study, completely inhibits sGC activity and effectively lowers basal cellular levels of cGMP to about one-fifth of normal levels (Yuen and Fiscus, 2001; Fiscus, 2002). NS2028 has also been shown to selectively block both basal activity of sGC and NO-induced activition of sGC (Mulsch et al., 1997; Olesen et al., 1998). Thus, the data of the present study using ODQ and NS2028 in uterine epithelial cells provide additional evidence that basal sGC activity and basal cGMP levels have anti-apoptotic effects, and show, for the first time, that this cellular mechanism may be essential for protecting against spontaneous development of apoptosis in uterine epithelial cells.
Several downstream pathways have been proposed to mediate the anti-apoptotic effects of elevating cGMP levels (above the basal levels), including induction of Bcl-2 mRNA and protein (Beauvais et al., 1995; Genaro et al., 1995), suppression of the gene expression of the pro-apoptotic Bcl-2-binding protein BNIP3 gene (Zamora et al., 2001), prevention of cytochrome C release from the mitochondria (Kim et al., 1997, 1999) and activation of c-Src and subsequent induction of tyrosine phosphorylation of Bcl-2 (Tejedo et al., 2001). In astrocytes, the anti-apoptotic effects of cGMP elevations have been attributed to inhibition of the mitochondrial permeable transition pore (Takuma et al., 2001). Further experiments with uterine epithelial cells will be needed to determine which signalling pathway is involved in the anti-apoptotic effects of basal sGC activity and basal cGMP levels in these cells.
PKG has been shown to be activated by cGMP both in the test tube (for review, see Lincoln and Cornwell, 1993) and in various intact mammalian tissues exposed to either NO (Fiscus et al., 1983, 1984) or the natriuretic peptide ANP (Fiscus et al., 1985; Fiscus and Murad, 1988), suggesting that PKG is the major downstream target protein in the cGMP signal transduction pathway. Recently, the anti-apoptotic effects of both elevated and basal levels of cGMP have been proposed to be mediated by PKG activity in neural cells (for review, see Fiscus, 2002). In the present study, we found that HRE-H9 cells express moderate levels of PKG, intermediate between the levels in other cells showing anti-apoptotic effects of cGMP (i.e. NG108-15 and N1E-115 cells with high PKG levels and PC12 cells with low PKG levels). We also found that the PKG inhibitor KT5823 by itself causes apoptosis in HRE-H9 cells. Because these pro-apoptotic effects of KT5823 occurred at concentrations (i.e. 100 nmol/l and 1 µmol/l) that would be expected to selectively inhibit PKG, the data suggest that PKG activity under basal conditions (i.e. without elevation of cGMP levels) is sufficient to provide continuous anti-apoptotic effects in HRE-H9 cells.
In summary, the present study shows that selective inhibitors of sGC or PKG cause apoptosis in HRE-H9 immortalized uterine epithelial cells, suggesting that sGC and PKG activities under basal/unstimulated conditions may be essential to prevent spontaneous development of apoptosis. We found that HRE-H9 cells express PKG at moderate levels, which may be sufficient to provide effective, continuous anti-apoptotic effects (even at basal levels of cGMP), thus promoting the survival of these cells. Because of the importance of uterine epithelial apoptosis to normal menstruation and fertility and in the pathogenesis of endometriosis, the anti-apoptotic effects of the cGMP/PKG signalling pathway in uterine epithelial cells, described in the present study, may have special importance in the regulation of many female reproductive functions.
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Acknowledgements |
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The authors thank Jessie Yuen for her expert technical assistance performing the Western blot analysis. This work was supported by a Direct Grant for Research from the Research Grants Council of Hong Kong awarded to R.R.Fiscus and by departmental funding for partial research support of graduate students in the Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, to Siu Lan Chan.
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Submitted on May 21, 2003; resubmitted on July 19, 2003. accepted on July 28, 2003
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