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Department of Obstetrics and Gynecology, University of Connecticut Health Center, Farmington, Connecticut 06030
Address all correspondence and requests for reprints to: John J. Peluso, Department of Obstetrics and Gynecology, University of Connecticut Health Center, Farmington, Connecticut 06030.
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Abstract |
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The present studies also show that when ROSE cells establish contact with 3T3 cells that express N-cadherin, [Ca2+]i levels are maintained at low basal levels. In contrast, cell contact with 3T3 cells that do not express N-cadherin results in elevated [Ca2+]i levels. Similarly, a synthetic N-cadherin peptide, which inhibits homophilic N-cadherin binding, increases [Ca2+]i levels. Taken together, these data indicate that homophilic N-cadherin binding between adhering cells plays an important role in maintaining calcium homeostasis. Further, these data support the concept that HGF/SF’s ability to promote the dissociation of ROSE cells accounts in part for its ability to increase [Ca2+]i levels.
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Introduction |
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Because cell contact is important in maintaining both granulosa cell and ROSE cell viability, factors that enhance cell contact would likely promote viability, whereas factors that disrupt cell contact would likely induce apoptosis. In nonovarian tissue, Hepatocyte growth factor (HGF), also know as scatter factor (SF) often reduces cell contact (7, 8). Interestingly, HGF/SF is expressed by ovarian stromal and thecal cells (9), and its expression stimulated by estrogen (10). Further, c-met, the receptor for HGF/SF, is expressed by ovarian surface epithelial cells (11, 12). In spite of the fact that both HGF/SF and its receptor are expressed within the ovary, a role for HGF/SF in regulating ovarian function has not been clearly identified. Based on these findings, it is proposed that HGF/SF promotes ROSE cells to dissociate and subsequently become apoptotic. The present studies were conducted with a ROSE cell line as described by Hoffman et al. (13). These cells were derived from continuous passage of primary rat ovarian surface epithelial cell cultures. Although spontaneously immortalized, they are not tumorigenic (13). Specific experiments were designed to assess the effects of HGF/SF on mitosis, cell contact, and apoptosis. Intracellular free calcium ([Ca2+]i) levels were also examined, because an increase in [Ca2+]i is often associated with apoptosis (14). Finally, the ability of bFGF to modulate HGF/SF’s effects was studied, because the effects of cell contact can be mimicked by bFGF (4).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Determination of ROSE proliferation
ROSE cell proliferation was determined using an in
situ cell counting procedure as previously described with the
following modifications (5). Briefly, cells were plated in
eight-chamber lab-tek slides at 30,000 cells/400 µl and cultured in
serum-supplemented medium for 24 h. The ROSE cells were then
washed and cultured in serum-free medium (control) supplemented with
either HGF/SF or bFGF. After 0 or 24 h of serum-free culture, the
cultures were stained with hydroethidine (Polysciences, Warrington, PA;
14 µg/ml in DMEM/F-12 for 30 min) and observed through an inverted
microscope with an epifluorescence attachment (15). The number of ROSE
cells in four different grids (160 µm2) within each
lab-tek well were counted. The grids were located at quadrants formed
at the intersection of the horizontal and vertical axis of each well.
Cell proliferation was expressed as the number of cells/640
µm2. Each proliferation experiment was conducted in
duplicate and repeated at least three times.
Identification of apoptotic nuclei
After 6, 8, and 24 h of incubation with various treatments,
ROSE cells were assessed for apoptosis. The nuclear structure of ROSE
cells was revealed by staining the DNA with hydroethidine for 30 min as
previously described (15). For each treatment at least 200 cells were
examined, and those cells that possessed condensed and fragmented
nuclei were considered to be apoptotic (15). The percentage of
apoptotic cells was then calculated. These experiments were conducted
in duplicate and repeated at least three times.
Assessment of [Ca2+]i levels
After 24 h in serum-supplemented medium, ROSE cells were
loaded with Fluo-3 (Molecular Probes) as previously described (15).
Cells were incubated with Fluo-3 (Molecular Probes, Eugene, OR) for 45
min in a 5% CO2/air atmosphere at 37 C. The cells were
then washed with serum-free DMEM/F-12 and observed at 1–4 h of culture
to assess the relative change in [Ca2+]i.
The relative level of [Ca2+]i was estimated by examining cells in four preselected fields that were located in the center of each well. Phase and fluorescent images of each field were collected and analyzed using IP Gel Lab software (Signal Analytics Corp., Vienna, VA). The cell size (area) and mean fluorescent intensity (fluorescent intensity/pixel) of each cell was determined from the fluorescent images with the phase images used to verify the position of each cell. The background fluorescent intensity was also determined in five areas within each field. The mean background fluorescent intensity was then calculated for each treatment and subtracted from the mean fluorescent intensity of each cell, thereby yielding a specific measurement of Fluo-3 fluorescence. In each experiment, the specific Fluo-3 fluorescence (i.e. [Ca2+]i) for 50–100 cells/treatment was determined. Each experiment was repeated at least three times. As part of the Fluo-3 validation procedure, a standard curve was generated in which the relationship between known fluorescence of a standard fluorescent particle was plotted against the fluorescent intensity units obtained using IP Gel Lab software. Fluorescent standards were constructed by first determining the fluorescence of a known particle under maximum fluorescent illumination. The same particle was then exposed to decreasing fluorescent illumination by reducing the amount of electrical current to the fluorescent illuminator using a reostat and then capturing the fluorescent image. The mean fluorescent intensity units obtained from each captured image was plotted against the percentage of fluorescent illumination. This study demonstrated that over the range in which Fluo-3 measurements were made, the mean fluorescent intensity increased linearly with increasing fluorescence of the particle as described by the equation: y = -92.4 + 0.51 X (R2 = 0.92).
Cell contact assessed by time-lapse photography
After 24 h of incubation in serum-supplemented medium, ROSE
cells were washed with serum-free medium and then either cultured in
serum-free medium or BAPTA-supplemented medium in the presence or
absence of HGF/SF. For these studies, the lab-tek slide was sealed with
a mixture of vasoline and paraffin (20:1) (MicroVideo Instruments,
Arrow, MA) to maintain the pH of the medium. The lab-tek slide was then
placed in a 37 C-humidified plexiglass microscope culture chamber
(Nikon Corp., Japan). A field with several ROSE cell aggregates was
selected and observed under either phase or Hoffman optics. Sequential
images were collected at 0.5-h intervals over a 4-h period.
To ensure an accurate assessment of cell contact, only ROSE cells with three or less cell contacts were examined. A cell contact was considered lost when the cells were completely detached. The number of initial cell contacts that remained intact at each time interval was then counted and a percentage calculated. Experiments were done on 6 separate days, and at least 50 cell contacts were examined.
Coculture with N-cadherin-expressing 3T3 cells
Vector control 3T3 cells and N-cadherin-expressing 3T3 cells
have been previously described (16) and were generously provided by Dr.
Patrick Doherty (Guy’s Hospital, University of London, London, UK).
These cells were routinely cultured in DMEM medium that was
supplemented with 10% FBS. These cultures were maintained in a 8%
CO2/air atmosphere at 37 C. For experimental procedures,
vector control 3T3 cells, N-cadherin-expressing 3T3 cells, and ROSE
cells were cultured in eight-chamber glass lab-tek slides with
serum-supplemented medium until confluency.
A separate population of ROSE cells was collected and loaded with Fluo-3 as previously described, except that the loading and subsequent washing was done in a test tube. The confluent ROSE cells, vector control 3T3 cells, and N-cadherin-expressing 3T3 cells were then washed with serum-free medium. The cocultures were established by adding 1 x 104 Fluo-3-loaded ROSE cells in 400 µl serum-free medium. Additionally, Fluo-3-loaded ROSE cells were plated in glass lab-tek wells without an established cell layer. The cocultures were incubated at 37 C in either 8% CO2 (3T3 cultures) or 5% CO2 (ROSE cell cultures). The cultures were observed after 4 h to assess the relative change in [Ca2+]i as previously described. Fluo-3-loaded ROSE cells were easily identified in the cocultures because they were in a higher focal plane than the other cells and fluoresced when observed under epifluorescent illumination. For those cells plated on a glass substrate without a cellular monolayer, [Ca2+]i levels were assayed for single cells and aggregated ROSE cells. The coculture experiments were conducted in duplicate on 3 different days.
To further investigate N-cadherin’s role in regulating [Ca2+]i levels, Fluo-3-loaded ROSE cells were plated on a ROSE cell monolayer in the presence or absence of synthetic N-cadherin peptide. The synthetic N-cadherin peptide was synthesized at the Peptide Synthesis Facility of Yale University and used at a concentration of 1 mg/ml. The sequence of this peptide, free-LRAHAVDVNG-amide, was derived from the extracellular domain of the avian N-cadherin molecule and has been shown to block the ability of cell contact to prevent apoptosis (5).
Statistical analysis
Experiments that assessed cell proliferation were evaluated
using Student’s t tests. Changes in cell contact were
analyzed by chi-square and linear regression analysis. Data from
experiments that assessed apoptosis were analyzed by ANOVA after
determining that the percentage values were normally distributed. The
data were then analyzed by a Student-Newman-Keuls multiple range test
where appropriate. Regardless of the statistical test, only
P values < 0.05 were considered to be significant.
Experiments involving Fluo-3 fluorescence (i.e. [Ca2+]i) were conducted on at least 3 different days and analyzed by ANOVA as described above. In each of the experiments, the specific Fluo-3 fluorescence for each cell was normalized to a control in an individual study. Therefore, all values were reported as a fold increase in [Ca2+]i levels over control values. The values were presented as the means ± SEM. The graphic presentation of changes in [Ca2+]i levels are from a representative experiment, except for the HGF/SF time course experiment where the data were pooled.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The increase in apoptotic nuclei
was first detected 6 h after HGF/SF, with a maximum response
observed at 24 h (Fig. 1). Although both Act D and CHX increased
the percentage of apoptotic nuclei, HGF/SF’s apoptotic action was not
observed in the presence of either Act D or CHX (Fig. 2). The Act D- and CHX-induced increase in apoptosis was
expected, because RNA and protein synthesis are required for cell
survival.
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, A–C). Cell size (area)
in control cultures also remained constant averaging 850 ± 12
µm2. In addition, 95 ± 1% of the cell contacts
were maintained over the 4-h culture period (Fig. 4).
HGF/SF increased [Ca2+]i levels by
approximately 3-fold at 3 and 4 h (Fig. 3C). In some
HGF/SF-treated cells [Ca2+]i was distributed
in a punctate pattern within the perinuclear region of the cytoplasm
(Fig. 3B). Cell area remained at control values for up to 2 h of
culture with HGF/SF and then decreased by approximately 40% at 3 and
4 h post HGF/SF treatment (520 ± 30 µm2 for
3 h and 510 ± 20 for 4 h; P < 0.05
compared with 2 h HGF/SF treatment and 3 h and 4 h
control values). However, cell contact progressively decreased in
response to HGF/SF, with the first significant decrease observed after
1 h of culture (Fig. 4). This HGF/SF-induced decrease in cell
contact was also observed in the presence of BAPTA (Fig. 4).
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).
Similarly, BAPTA blocked HGF/SF’s apoptotic effects (Fig. 5B).
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Similar experiments were conducted in which Fluo-3-loaded ROSE cells
were cocultured with either N-cadherin-expressing 3T3 cells or vector
control 3T3 cells. Those ROSE cells that attached to the
N-cadherin-expressing 3T3 cells had a relatively low level of Fluo-3
fluorescence; whereas attachment to the vector control 3T3 cells
resulted in a 3-fold increase in Fluo-3 fluorescence (Fig. 6B).
Likewise, Fluo-3-loaded ROSE cells that established cell contact in the
presence of synthetic N-cadherin peptide had higher
[Ca2+]i levels than controls (Fig. 6C).
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3-fold in the absence
(Fig. 7A) but not in the presence of Act D (Fig. 7B).
Bay K induced an increase in [Ca2+]i even in
the presence of Act D (Fig. 7C).
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). In addition,
bFGF attenuated HGF/SF’s effects on apoptosis (Fig. 8),
[Ca2+]i levels (Fig. 8), and cell contact
(data not shown). However, bFGF only inhibited HGF/SF’s effect if
it was added at the time of or 2 h after HGF/SF. If added 4 h
after HGF/SF, bFGF did not prevent apoptosis (Fig. 9).
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) and increased ROSE cell apoptosis by 24 h of
culture (Fig. 11). Unlike HGF/SF, Thap and Bay K
mediated their apoptotic action in the presence of either Act D or CHX
(Figs. 2
and 12). bFGF attenuated both Thap- and Bay
K-induced increase in [Ca2+]i levels (Fig. 10) and apoptosis (Fig. 11). Further, bFGF inhibited Bay K-induced
apoptosis in the presence of Act D (Fig. 12).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cascade of cellular and molecular events that induce cells to become apoptotic is poorly defined (17). It has been proposed that once a cell reaches a point in the apoptotic cascade, it is committed to die (18). Although an increase in [Ca2+]i is not always observed during apoptosis (14, 18), an increase in [Ca2+]i is observed 3 h post HGF/SF and is sustained for at least 1 h. This sustained increase in [Ca2+]i appears to demark the commitment point for ROSE cells, because BAPTA prevents both the HGF/SF-induced increase in [Ca2+]i levels and apoptosis. This concept is further supported by the following observations. First, bFGF attenuates HGF/SF’s ability to induce both [Ca2+]i and apoptosis. Second, bFGF cannot attenuate HGF/SF’s apoptotic action if exposed to cells after the increase in [Ca2+]i (i.e. 4 h after HGF/SF). Third, Act D and CHX block HGF/SF’s ability to induce an increase in [Ca2+]i and apoptosis. Note that Bay K induces an increase in [Ca2+]i and apoptosis even in the presence of Act D. This indicates that Act D per se does not prevent the ROSE cells from responding to HGF/SF with an increase in [Ca2+]i. Rather it supports the concept that de novo RNA and protein synthesis are required for HGF/SF to increase [Ca2+]i. Finally, Thap and Bay K, agents that increase [Ca2+]i, induce apoptosis in an RNA/protein synthesis-independent manner. This demonstrates that the components of the apoptotic cascade distal to the increase in [Ca2+]i are present within ROSE cells. This is consistent with the concept that calcium-dependent proteases and endonucleases are already present within the cells, and that all that is necessary to induce apoptosis is for these enzymes to be activated by an increase in [Ca2+]i (19, 20. Because [Ca2+]i plays an essential role in initiating apoptosis, attention needs to be focused on how HGF/SF promotes a sustained elevation in [Ca2+]i levels.
HGF/SF decreases cell contact before the increase in [Ca2+]i levels. This temporal relationship suggests that a decrease in cell contact leads to an increase in [Ca2+]i. This hypothesis is supported by three sets of observations. First, ROSE cells that form a cell-cell contact have [Ca2+]i levels that are one-third those of single ROSE cells. Second, ROSE cells that contact N-cadherin-expressing 3T3 cells possess [Ca2+]i levels that are one-third those of ROSE cells that contact control vector 3T3 cells. Finally, a synthetic N-cadherin peptide allows ROSE cells to establish cell contact with each other, but attentuates the ability of cell contact to maintain low basal [Ca2+]i levels. Collectively, these studies demonstrate that homophilic N-cadherin binding between adjacent cells maintains calcium homeostasis. Similarly, N-cadherin plays an essential role in maintaining low basal levels of [Ca2+]i in glial cells (21). In nonovarian cells, HGF/SF decreases cadherin levels (22) and/or stimulates the tyrosine phosphorylation of catenins (23), with the end result being a loss of cell contact (24). Either or both of these actions could be responsible for HGF/SF’s ability to promote the disaggregation of ROSE cells and therefore, explain the HGF/SF-induced increase in [Ca2+]i levels.
Although the loss of cell contact between ROSE cells undoubtedly accounts in part for the increase in [Ca2+]i, HGF/SF may have additional actions that lead to the disregulation of [Ca2+]i and ultimately to cell death. Ovarian surface epithelial cells express c-met, the receptor for HGF/SF (11). Ligand activation of c-met results in its autotyrosine phosphorylation and subsequent increase in inositol 1,4,5-triphosphate (IP3) (7, 8). In hepatocytes, intracellular levels of IP3 are elevated within minutes of exposure to HGF/SF and remain elevated for up to 5 h (25). IP3 binds to IP3 receptors promoting the release of calcium from its stores (26). This results in an immediate increase in [Ca2+]i that is generally transient, lasting only a few minutes, and that is associated with mitosis (25). HGF/SF also stimulates an increase in [Ca2+]i in ROSE cells but does not induce mitosis. Rather, HGF/SF promotes ROSE cell apoptosis. Because the present studies were not designed to detect transient changes in [Ca2+]i as observed in response to mitogenic stimuli, it is possible that brief calcium transients occur immediately after HGF/SF. The present studies do detect major changes in [Ca2+]i that are associated with ROSE cell apoptosis. These changes differ from those associated with mitosis in that they 1) are delayed, increasing 2- to 3- fold over control values by 3 h of HGF/SF treatment; 2) are prolonged, lasting at least 1 h; and 3) require de novo RNA and protein synthesis. The identity of newly synthesized proteins that may act in concert with IP3 to increase [Ca2+]i are unknown. Recently, the expression of type 3 IP3 receptors has been shown to be induced during glucocorticoid-stimulated apoptosis of lymphocytes (24). Activation of the type 3 IP3 receptor results in a prolonged 4-fold increase in [Ca2+]i (24). It is possible then that type 3 IP3 receptors are one of several HGF/SF-induced proteins that deregulate [Ca2+]i. This hypothesis is currently being tested.
The present data also demonstrate that in ROSE cells bFGF is not mitogenic but rather functions as a survival factor. In other cells, protein kinase C activation mediates bFGF’s antiapoptotic action (27, 28). Ovarian epithelial cells express FGF receptors (29), and ligand binding to the FGF receptors stimulates a signal transduction pathway that leads to the generation of IP3 and the activation of protein kinase C (30). One consequence of protein kinase C activation is to stimulate the uptake of calcium into its cellular stores (26, 31). This counteracts the IP3-induced increase in [Ca2+]i, thereby maintaining calcium homeostasis. The present studies show that bFGF mediates its antiapoptotic action by blocking HGF/SF-, Thap-, and Bay K-induced increase in [Ca2+]i. Similarly, bFGF has been shown to prevent neuronal apoptosis by stabilizing calcium homeostasis (32). Based on these findings, it is possible that stimulation of protein kinase C activity could account for bFGF’s ability to block the sustained increase in [Ca2+]i levels, thus preventing ROSE cell apoptosis. This concept is consistent with the observation that the ability of bFGF to regulate [Ca2+]i and apoptosis is not dependent on de novo RNA and protein synthesis.
Finally, these in vitro studies suggest that the apoptotic action of HGF/SF could play an important role in the ovulatory process. For example, as preovulatory follicles develop, they secrete estrogen with maximum estrogen levels being obtained just before ovulation (33). Because estrogen increases the expression of HGF/SF (11), it is likely that ovarian HGF/SF levels are at their maximum at this time. Estrogen also induces an LH surge that initiates an array of physiological events that ultimately result in ovulation. One of these ovulatory events is the breakdown of the basement membrane of the ovulatory follicle (34). This would expose the surface epithelial cells to stromal/thecal cell-derived HGF/SF. HGF/SF could then stimulate the surface epithelial cells to undergo apoptosis. This hypothesis is consistent with the observed apoptosis of the surface epithelial cell layer that overlies the ovulatory follicles (2, 3). This apoptotic event is an important part of the ovulation cascade because it creates an opening through which the oocyte is released. Further, failure of the surface epithelial cells to undergo apoptosis would entrap the oocyte and potentially result in a luteinized unruptured follicle.
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Acknowledgments |
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Received October 10, 1996.
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