Page 1 of 41 Articles in PresS. Am J Physiol Cell Physiol (November 8, 2006). doi:10.1152/ajpcell.00181.2006
Cell Cycle Dependent Calcium Oscillations in Mouse Embryonic Stem Cells
Nidhi Kapur, Gregory A. Mignery and Kathrin Banach Department of Physiology, Stritch School of Medicine, Loyola University Chicago
Ca-Oscillations Accompany Cell Cycle Progression in ES Cells
Corresponding Author: Kathrin Banach, Ph.D. Department of Physiology Stritch School of Medicine Loyola University Chicago 2160 South First Ave. Maywood IL 60153 Phone : 708-216 9113 Fax : 708-216 6308 e-mail:
[email protected]
Copyright © 2006 by the American Physiological Society.
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1 Abstract During cell cycle progression, somatic cells exhibit different patterns of intracellular calcium signals during the G0 phase, the transition from G1 to S and from G2 to M. Because pluripotent embryonic stem (ES) cells progress through cell cycle without the gap-phases G1 and G2 we aimed to determine if mES cells still exhibit characteristic changes of [Ca2+]i during cell cycle progression. With confocal imaging of the Ca-sensitive dye Fluo4-AM we identified that undifferentiated mES-cells exhibit spontaneous Ca-oscillations. In control cultures where 50.4 % of the cells reside in the S-phase of the cell cycle, oscillations appeared in 36 % of the cells within a colony. Oscillations were not initiated by Ca-influx but depended on IP3 mediated Ca-release and the refilling of intracellular stores by a store operated Ca-influx (SOC) mechanism. Using cell cycle synchronization, we determined that Ca-oscillations were confined to the G1/S phase (~70% oscillating cells vs. G2/M: ~15 % oscillating cells) of the cell cycle. ATP induced Ca-oscillations, and activation of SOC could be induced in G1/S and G2/M synchronized cells. Intracellular Ca-stores were not depleted and all three IP3 receptor isoforms were present throughout the cell-cycle. Cell cycle analysis after EGTA, BAPTA/AM, 2-APB, thapsigargin or U-73122 treatment, underlined that IP3 mediated Ca release is necessary for cell cycle progression through G1/S. Because the IP3 receptor sensitizer thimerosal induced Caoscillations only in G1/S we propose that changes in IP3R sensitivity or basal levels of IP3 could be the basis for the G1/S confined Ca oscillations. Keywords: Ca-oscillation, embryonic stem cell, cell cycle, pluripotent
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2 Introduction Pluripotent mouse embryonic stem cells have a high rate of proliferation (6, 23, 40) that does not depend on the addition of serum to the culture media (41). mES cells further remain uninfluenced by contact inhibition. Their cell cycle is characterized by a virtual lack of the G1 and G2 gap phases that in somatic cells accompany and control cell cycle progression. As a consequence, about 50% of the ES cells sojourn in the S-phase at any given point of culture (11, 23, 41). In ES cells and cells of the inner cell mass of the blastocyst, an active phosphatidylinositol signaling system is described (9, 13, 23, 34). Thereby the phosphatidylinositol-3 kinase (PI3K) appears as a key regulator for cell survival as well as cell cycle progression. Blocking PI3K activity results in increased DNA fragmentation and induces a reversible cell cycle arrest during G1/S transition (23). Apoptosis as well as proliferation seem to involve the Akt pathway whereas the MAP kinase-ERK pathway played no significant role in either process (23). The other important branch of the phosphatidylinositol (PtdIns) signal transduction cascade involves the activation of phospholipase C (PLC) where the hydrolysis of phosphatidylinositol (4,5) bisphospate (PIP2) results in the production of the second messengers inositol(1,4,5) trisphosphate (IP3) and diacylglycerol (DAG). IP3 and DAG mediate the release of Ca from intracellular stores and the activation of PKC, respectively. In mouse, inhibition or knockout of PLC prevents blastocyst formation as well as proliferation of ES cells in vitro (34, 42). Although experimental results point to a relevance of the intracellular Ca-
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3 release in proliferation, the mechanisms of Ca signaling during cell cycle progression are not yet described. In mammalian somatic cells the importance of intracellular Ca-signaling during cell cycle progression is well established (3, 37). Spontaneous Caoscillations can be observed in the G0/G1 phase where they correlate with the activation of immediate early genes that initiate reentry into the cell cycle. After a quiescent phase, oscillations resume in late G1 prior to the S phase initiation, and promote DNA synthesis. During G2/M transition, the increase in [Ca2+]i which was shown to depend on an increase in IP3 production, is further related to the breakdown of the nuclear envelope (7, 37). The cell cycle-dependent Ca signaling relies on the presence of Ca in the extracellular solution as well as on the presence of intact intracellular Ca stores. Removal of Ca from the solution or inhibition of Ca reuptake into the endoplasmic reticulum results in most cells in cell cycle arrest in the G1/S phase (19, 30). The store-operated Ca (SOC) entry as well as T-type Ca channels have been described as Ca entry mechanisms relevant for cell proliferation (12, 30, 32, 35, 44). The Ca signals that can be recorded during the cell cycle vary from continuous increases in the basal Ca level to individual transient increases and continuous Ca-oscillations (30). Recent studies showed mouse embryonic stem cells comparable to mesenchymal stem cells, have functional IP3R regulated intracellular Ca-stores. They are sensitive to stimulation with ATP, histamine and platelet derived growth factor (PDGF) and depend on SOC entry for the refilling of their intracellular Ca stores. However, in
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4 contrast to mesenchymal stem cells, no Ca-oscillations were described (26, 36, 47). In the present study, we tested the hypothesis that in mouse ES cells, IP3 mediated Ca oscillations are present in the different phases of the cell cycle and that they are a key element for cell cycle progression. Our results demonstrate that ES cells exhibit spontaneous Ca oscillations that depend on IP3 mediated Ca release. Although the principal mechanisms for Ca signaling are maintained throughout the cell cycle, oscillations are confined to the transition from the G1 to the S phase of the cell cycle. The data reveal new information about the regulation of Ca signaling in mES cells and its contribution to cell cycle progression. Materials and Methods Cell culture Mouse ES cells of the cell line CRL (Specialty Media, Chemicon Intl.) were propagated in culture as previously described (1, 18). The cells were maintained on a layer of mitomycin inactivated primary mouse fibroblasts (Specialty Media, Chemicon Intl.) in DMEM media supplemented with 15 % Fetal Calf Serum (FCS; Invitrogen) and Leukemia inhibiting factor (LIF: ESGRO; 1000 U/ml) to prevent differentiation. For the experiments, ES cells were trypsinized and plated onto gelatin-coated cover slips (Ø 25 mm) in a density of 0.1*106 cells/ml. No specific measures were taken to remove the fibroblasts from the culture; on average an individual coverslip therefore contained 7*104 fibroblasts/ml. The proliferation of fibroblasts was inhibited by mitomycin treatment prior to co-culture therefore they
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5 were outnumbered within 24h of culture. Their distinct morphology allowed their identification and Ca measurements were only obtained within ES cell colonies. All cultures were maintained in LIF. Experiments were performed on control cultures the first day after plating. Pharmacological synchronization was started 24h after plating and cells were incubated for a minimum of 12 h before experimental use. Cell cycle synchronization and FACS sorting: Cell cycle synchronization of mES cells was achieved by 12 h incubation of the cells in hydroxyurea (HU: 2 mM), aphidicoline (APC: 20 mg/ml) or mimosine (MIM: 500 µM) for synchronization in the transition from G1 to S phase and in Nocodazole (NOC: 100 ng/ml) or Demecolcine (DC: 20 ng/ml) for synchronization in the transition from G2 to M phase (21). After 12 h the cells were washed with tyrode and used for Ca-imaging experiments within 2 h. Cell cycle distribution of control and synchronized cultures were determined by FACS sorting (Becton Dickinson FACStar Plus). Cells were washed with PBS, fixed 10 min incubation in ice cold ethanol (70%) and after washing, treated with RNase (100 mg/million cells in PBS; 30 min at 37oC). After washing, the cells were stained with propidiumiodide (50 µg/ml) for 1hr at 4oC.
Propidiumiodide intercalates into the cells double
stranded nucleic acid and results in fluorescence that can be detected around 600 nm when excited at 488 nm. The FACS analysis then allows cell sorting for fluorescent intensity and therefore DNA content. Data were acquired with Cell Quest software and the percentage of G1, S and G2 phase cells was calculated with MODFIT software.
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6 Microsomes: Cells were harvested and microsomes were prepared as described previously (30, 31). Briefly, cells were harvested and transferred into 50 mM Tris-HCl, pH 8.3, 1 mM EDTA, 1 mM J-mercaptoethanol, 1 mM PMSF, 10 KM leupeptin, 10 KM pepstatin, 100 Kg/ml trypsin inhibitor; and lysed by 40 passages through a 27-gauge needle. Membranes were pelleted by a 20-min centrifugation
(289,000 × gav),
resuspended in
buffer,
and
either
used
immediately or frozen at -80 °C. SDS-PAGE and Immunoblotting: Microsomes were analyzed by 5% SDSpolyacrylamide gel electrophoresis (SDS-PAGE) followed by electro-transfer to nitrocellulose membranes and subjected to immuno-detection with InsP3R isoform
specific
antibodies
described
previously
(38,
39)
using
chemiluminescence reagents (Amersham Pharmacia Biotech). Fluorescence measurements: Spatially averaged measurements of the change of [Ca2+]i were performed on individual cells within an ES cell colony using the Ca2+-sensitive dye Fluo-4/AM. Fluo-4/AM (50 µg) was dissolved in DMSO (50 µl), Pluronic (50 µl of 20% stock in DMSO) and FCS (87.5 µl). We achieved a final dye concentration of 2.5 µM in standard extracellular solution. The cover-slips with ES cells were incubated with the dye solution for 15 min and de-esterification of the dye was allowed for another 15 min. Solutions: During the experiment, the cells were superfused with a standard extracellular tyrode solution containing (in mM): NaCl 140; KCl 5.0; MgCl2 1.0; Glucose 5.5; HEPES 10; CaCl2 2; pH 7.4. To analyze the contribution of Ca-influx to the spontaneous oscillations, in some experiments Ca was omitted
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7 (Ca-free) from the extracellular solution. Further pharmacological tools used were 2-aminoethoxydiphenyl borate (2-APB: 10 µM), ATP (10 µM), BAPTA/AM (1µM), caffeine (10 mM), lanthanum (0.1 mM), ryanodine (10 µM), thapsigargin (1µM), thimerosal (10 µM) and U-73122 (10 µM). All chemicals were purchased from Sigma. Confocal Imaging: The intracellular Ca concentration ([Ca2+]i) was monitored with 2-dimensional confocal imaging (Zeiss LSM-410 microscope). Fluo4-AM was excited at a wavelength of 488 nm and emission was detected at >515 nm. Changes of [Ca2+]i are expressed as R = F/F0, where R is the fluorescence (F) normalized to the resting fluorescence (F0). Due to the slow time course of spontaneous Ca-oscillations two-dimensional confocal images were taken at a time interval of 5 s. Intracellular Ca was analyzed in defined regions of interest (ROI) that included one entire cell, over the entire time series. Results Spontaneous Ca-oscillations in ES cells Undifferentiated ES cells in culture form multi-cellular colonies which exhibit a distinct round boundary without signs of outgrowth (8, 41). After one day in culture in the presence of LIF the observed colony size ranged from 19.8 µm to 73 µm (n = 46, mean = 45.9 ± 15 µm) with a mean cell diameter of 10.3 µm. Following the loading of the Ca-sensitive dye Fluo-4/AM we monitored the timedependent change of [Ca2+]i in individual cells within a colony. Figure 1AB shows a representative example of an ES cell colony where individual cells exhibited
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8 spontaneous Ca-oscillations. The average amplitude of spontaneous oscillations was 1.57 ± 0.7 ([Ca2+]o = 2 mM) They occurred with an inter-transient interval (ITI) of 76 ± 24 s and a mean duration of 38 ± 10 s (at 50% amplitude; n = 32 experiments). The rise in [Ca2+]i was not strongly synchronized among neighboring cells although intercellular coupling of cell colonies could be confirmed by intercellular diffusion of the dye lucifer yellow (data now shown). Overall, in control colonies spontaneous oscillations occurred in 36% of the cells within the colony. The results demonstrate that undifferentiated ES cells exhibit spontaneous Ca oscillations; however, in control cultures the cells are heterogeneous in their spontaneous Ca signaling. Ca-oscillations do not depend on Ca-influx but on intracellular Ca-release Spontaneous Ca oscillations can be driven either by Ca influx (L-, T-type or SOC entry) or by Ca release from intracellular Ca stores like the endoplasmic reticulum (3, 4, 25). We tested if Ca influx induces spontaneous Ca oscillations in ES cells by superfusing ES cell colonies with a Ca-free extracellular solution. As shown in Fig. 2A, spontaneous changes in [Ca2+]i continued even in the absence of Ca from the extracellular solution. This result was confirmed in 5 independent experiments including 22 oscillating cells. However, as shown in Fig. 2C (n = 5), in most cells Ca transient amplitudes decreased with time in Ca-free solution. The data underline that extracellular Ca is not required for the induction of Caoscillations however it is required to maintain the filling of the intracellular stores. Ca- oscillations depend on IP3 mediated Ca release
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9 In most electrically excitable cells Ca is released from the intracellular stores through the ryanodine receptor (RyR; (10)). The signal is mediated by a Ca-induced Ca-release mechanism (CICR). In electrically unexcitable cells like endothelial cells and the mouse oocyte, Ca is mostly released through IP3Rs . To determine if the spontaneous oscillations in ES cells are mediated by CICR we superfused the cultures with ryanodine (Ry: 10 µM), an antagonist of RyR which at this concentration, blocks the RyR in its open configuration (10, 29). Figure 2B shows a representative recording from an ES-cell exhibiting spontaneous Caoscillations. In all spontaneously oscillating ES cells (n = 5), superfusion with Ry did not block spontaneous oscillations or change the ITI (Fig. 2D) or the amplitude of the oscillations (Fig. 2E). To confirm the result, we superfused cells with the RyR agonist caffeine (10 mM; not shown); however, no caffeine induced Ca-release could be observed. Caffeine exposure was reduced to less than 5 s. The results indicate that spontaneous oscillations of Ca do not depend on RyR mediated Ca release or CICR. To determine if spontaneous oscillations of [Ca2+]i depend on the presence of IP3 sensitive intracellular-stores we superfused ES cell colonies with the IP3R antagonist 2-APB (10 µM) (46). Application of 2-APB resulted in a reversible inhibition of Ca oscillations indicating the contribution of Ca release from IP3 sensitive stores (Fig. 3A; n=5; 15 oscillating cells). 2-APB is a rather unspecific inhibitor of the IP3R and has further inhibitory action on gap junction channels and store operated Ca entry (16, 33). In an alternative approach we tested the aminosteroid U-73122 (1-(6-[{(17 )-3-methoxyestra-1,3,5[10]-trien-17-
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10 yl}amino]hexyl)-1H-pyrrole-2,5-dione; 10 µM) an inhibitor of PLC which was previously used to examine the signaling mechanisms in ES-cells (15, 34). Also in this case, as shown in Fig. 3B spontaneous oscillations were suppressed in 4 independent experiments. Our data indicate that spontaneous oscillations in undifferentiated ES cells depend on the release of Ca from IP3R regulated intracellular stores and are in accordance with previous reports that demonstrate the presence of all three IP3R isoforms in mouse ES cells (45) (see also Fig. 6D). Store operated Ca2+ entry in ES cells Recent data demonstrated the presence of a store operated Ca entry mechanism in mesenchymal as well as mouse ES cells (26, 47). To determine if this mechanism is present in our cell system and it contributes to the refilling of the intracellular Ca stores, we superfused mouse ES cells with the Ca-ATPase inhibitor thapsigargin (1 µM) in a Ca-free solution (47). In the experiment shown in Fig. 4A thapsigargin induced a transient increase in Ca due to the uncompensated release of Ca from the intracellular stores. Re-addition of Ca (4 mM) to the culture resulted in a reversible increase of Ca. 75 % of this Ca-influx could be blocked by lanthanum (0.1 mM) an inhibitor of SOC (Fig. 4B; n = 9 cells). The data indicate that refilling of intracellular Ca stores in ES cells is mediated by a store operated Ca (SOC) influx mechanism. ES cells exhibit heterogeneity in their spontaneous Ca-oscillations mES cells are derived from the inner cell mass of the mouse blastocyst, they are pluripotent and can be propagated indefinitely (8). Although they should
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11 represent a uniform population of cells, spontaneous Ca-oscillations could only be observed in 36 % of the cells within individual colonies. To determine if the heterogeneity depends on the ES cells cell cycle progression, we synchronized ES-cell cultures at different stages of the cell cycle before examining their Caoscillations. To evaluate if the Ca-oscillations coincide with the cells transition from G1 to S phase we synchronized the cell cultures 24 h after plating by 12 h incubation in mimosine (MIM: 500 µM) (21, 31, 43). For synchronization of the cells in the transition from G2 to M phase cells were incubated for 12 h with demecolcine (DC: 20 ng/ml). FACS analysis of PI stained MIM and DC treated cultures revealed a shift of the cell cycle distribution from control conditions. Whereas in control 50.4 ± 1.3% of the cells reside in the S phase, the number decreased to 38.6 ± 0.7 % and 14.8 ± 1.9 % in MIM and DC treated cultures respectively (see Fig. 5AB). In MIM cultures the majority of the cells resided in the G1 phase (Ctrl: 19.6 ± 4.1%, MIM: 50.1 ± 11.1%) whereas in DC treated cultures the cells accumulated in G2/M (Ctrl: 32.1 ± 7.4 %, DC: 72.8 ± 11.9%) demonstrating a successful shift in cell cycle distribution. Analysis of the spontaneous Ca-oscillations in the synchronized cultures revealed that incubation of the cells with MIM resulted in an increase in the number of cells exhibiting Ca-oscillations (Ctrl: 36 ± 23%, MIM: 58 ± 23%; Fig.6A). This increase correlates well with the accumulation of the cells in the G1 phase of the cell cycle (Ctrl: 19.6%, MIM: 50.1%; Fig. 5A). In contrast, in DC treated cultures only 8 ± 8 % oscillating cells were found within individual ES cell colonies. Also in this case, the significant decrease in oscillating cells correlates
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12 with the low number of cells residing in the G1 phase of the cell cycle after DC synchronization (4.0 ± 3%). To rule out that the change in the number of oscillating cells was due to the cell cycle drugs themselves rather than due to cell cycle synchronization, we repeated the experiment with other cell cycle inhibitors. To block the transition from the G1 to the S phase we additionally used either aphidicoline (APC: 20 µg/ml) or hydroxyurea (HU: 2 mM), to block the transition from G2 to M we further used nocodazole (NOC: 100 ng/ml) (21, 43). Analysis of the Ca oscillations in the APC, HU or NOC treated cells revealed comparable results to those obtained with MIM and DC respectively. The number of cells with Ca oscillations increased to 72 ± 13 % and 76 ± 22 % in APC and HU treated colonies respectively, whereas Ca oscillations decreased to 15 ± 11 % of the cells in NOC treated cultures (Fig. 6A). The data show that Ca oscillations within ES cell colonies occur during the cells progression through the cell cycle and in synchronized colonies they correlate with the percentage of cells residing in the G1 phase. We measured and compared the frequency and the duration of the spontaneous oscillations of [Ca2+]i that we recorded in the synchronized cultures. The analysis revealed that neither ITI nor the transient duration (TD) were significantly different in control cultures and cultures treated with DC, NOC, MIM, APC or HU. Mean ITIs ranged from 70 s to 99 s whereas mean TDs ranged from 38 s to 47 s (Fig. 6 BC). The comparability of the Ca transients observed in the control and synchronized cultures further underlines that we are looking at a
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13 defined Ca oscillation pattern that marks the transition from the G1 to the S phase of the cell cycle. Ca-signaling mechanisms during the different phases of the cell cycle To understand the mechanism of the time restricted presence of Caoscillations in the transition from G1 to S we determined the presence of cell cycle dependent changes in the Ca-handling mechanisms e.g. IP3R expression, IP3 mediated Ca-release, SOC and the basal activity of PLC. In Fig. 6D immunoblots are shown that were obtained from control cultures, cultures maintained in 0% FCS for 12 h and cultures synchronized with either HU in the G1/S transition or NOC in the G2/M transition. In all cultures examined, we identified the IP3R isoforms 1, 2 and 3. The data indicate that the confinement of the Ca-oscillations to the G1/S phase most likely does not depend on a change in IP3R isoform expression. To exclude that the reduction of Ca-oscillations in the G2/M phase of the cell cycle is due to a depletion of the intracellular Ca-stores and a reduction of the store operated Ca-entry mechanism we
superfused
HU
and
DC
synchronized
cultures
with
thapsigargin.
Representative experiments for a HU (Fig. 7A, n = 4) and a DC (Fig. 7B, n = 3) treated culture are shown. In both cultures, thapsigargin induced a transient increase in [Ca2+]i due to the leak of Ca from the intracellular stores. Re-addition of Ca (2 mM) to the extracellular solution resulted in the previously described Cainflux mediated by SOC (see also Fig. 4A). The data point out that the lack of Caoscillations in G2/M synchronized ES cells is not based on a depletion of the intracellular Ca stores or the lack of Ca-influx through SOC.
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14 It was reported that ATP induces a transient elevation of [Ca2+]i in undifferentiated ES cells (47). The ATP induced Ca-release is mediated by PLC dependent IP3 production, stimulated by a G-protein coupled purinergic receptor . Cells that were blocked in their cell cycle progression by DC or NOC between the G2 and M phase, or by MIM, APC or HU between the G1 and S phase, all responded with a transient increase in [Ca2+]i upon stimulation with ATP (G1/S: n = 4; G2/M: n = 5). Representative recordings of a culture synchronized with HU (Aa) and DC (Ba) are shown in Fig. 8Aa, Ba. The data underline that receptor mediated IP3 production and IP3 mediated Ca-release is not impaired in either phase of the cell cycle. Spontaneous changes in IP3 production as they occur in sea urchin eggs (7, 20, 29) have been discussed to induce spontaneous Ca-oscillations during cell cycle progression. We tested for potential differences in IP3 levels or IP3R sensitivity in cultures synchronized either by DC or NOC (block between G2 and M) or by MIM, APC or HU (block between G1 and S). We superfused the synchronized cultures with thimerosal, a thiol reagent described to increase the sensitivity of IP3R for its agonist (5). In control, APC and HU synchronized cells thimerosal (10 µM) rapidly increased the frequency of oscillations which ultimately resulted in an increase of the basal Ca-levels (n = 4). However, in DC or NOC treated cultures thimerosal remained without effect (n = 3). Since IP3 mediated Ca-release is present at all stages of the cell cycle, the transition from the G1 to the S phase seems to coincide with changes of IP3R open probability or IP3 production.
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15 Relevance of Ca-oscillations for cell cycle progression To determine the relevance of the Ca-oscillations observed for cell cycle progression, we determined the growth rate of undifferentiated ES cells in control medium and medium that was supplemented with either the PLC inhibitor U73122 (10 µM) or EGTA (3 mM). A semi-logarithmic plot of the cell numbers counted in one experiment is shown in Fig. 8D. The reduced slope in the cultures treated with U-73122 and EGTA indicate a decreased rate of proliferation. The mean values of 5 independent experiments reveal that under control conditions the cells had an average doubling time of 10.2 ± 0.16 h, whereas in the presence of U-73122 or EGTA cultures exhibited an increased doubling time of 15.3 ± 2.3 h and 20.9 ± 1.27 h, respectively (n=5). A similar effect was obtained after incubation of proliferating ES cell cultures with 2-APB (10 µM), thapsigargin (1 µM), or the membrane permeable Ca buffer BAPTA/AM (1 µM) to block an increase in [Ca]i. In all cases a significant slowing in cell cycle progression could be detected in comparison to control cultures (Fig. 9A). FACS analysis of U73122, 2-APB, thapsigargin and BATA/AM treated cultures after 24h of incubation revealed in all cases a reduced number of cells residing in the G2/M phase whereas the number of cells in the G1 phase of the cell cycle was increased (Fig. 9B). U73122, 2-APB, and BAPTA/AM treated cultures in contrast to thapsigargin also increased the number of cells in the S phase. The data underline the relevance of PLC mediated Ca-oscillations for cell cycle progression through the G1/S phase in mES-cells.
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16 Discussion In the present paper, we demonstrate that cell cycle progression of mouse ES cells through the G1/S phase is accompanied by spontaneous oscillations of [Ca2+]i. Because intracellular Ca-signaling mechanisms like the presence of IP3R isoforms, loading of Ca-stores, PLC mediated IP3 production and SOC seem to remain unchanged during the cell cycle we propose that these Ca-oscillations depend on an increased sensitivity of IP3R or increased levels of IP3 synthesis. Ca-oscillations in mouse ES cells In mammalian cells Ca is used as an ubiquitous second messenger (4) and in mES cells that were differentiated into endodermal cells Ca-oscillations mediate exo- and endo-cytotic vesicle shutteling. There is substantial evidence that also in cell cycle progression Ca oscillations plays an important role (38). In fibroblasts, HeLa and smooth muscle cells Ca oscillations accompany the transition from the G0 to the G1 phase (2) and in the transition from G1 to S a basal elevation of [Ca2+]i is described. The ES cell cycle is characterized by a virtual lack of G1 and G2 gap phases and the majority of the cells (50%; see Fig. 4AB) sojourn in the S phase at any given point of culture (11, 23, 29, 41). We identified Ca-oscillations in 36% of the cells within an ES-cell colony under control conditions. The lack of observation in previous studies might be related to the low incidence of oscillating cells (26, 47). Ca oscillations in ES cell preparations are described in primitive endodermal cells after induction of differentiation (absence of LIF, culture in an embryoid body)(39). Although the oscillatory pattern is comparable, the progressed differentiation might explain
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17 their dependence on extracellular Ca and sensitivity to nickel (39). We determined that the oscillations in mES-cells coincide with the G1 phase of the cell cycle by pharmacologically blocking cell cycle progression. The substances used MIM, APC and HU induce arrest at the transition from the G1 to the S phase by inhibiting the formation of replication forks, and by DNA polymerase or ribonucleotide diphosphate reductase inhibition respectively (21, 31, 43). Although the effect of the drugs we used is different, all resulted in an increased number of oscillating cells (58%, 72% and 76% respectively for MIM, APC and HU, respectively). The consistent outcome excludes that the increase in oscillations was due to an unspecific effect of the individual substances. We confirmed the change in cell cycle distribution by FACS analysis of MIM treated cultures. The number of cells residing in G1 increased from 19±4% to 50±11% (n=2) after 12 h of MIM treatment. The data underlined that in MIM treated cultures the G1 phase is the dominating cell cycle stage. The effectiveness of cell cycle synchronization is in good agreement with previously published data (11, 43). The percentage of oscillating cells correlated well with the number of cells in G1. Nevertheless, since we observe 8% and 15% oscillating cells in mES-cell cultures synchronized with either DC or NOC, we can not rule out that oscillations occur in other stages of the cell cycle. In other cell types, Caoscillations are described in the G2 as well as the M phase of the cell cycle (29, 38). However, transient duration and frequency of the Ca oscillations in DC or NOC treated cultures were comparable to those observed in G1/S synchronized cultures. Because FACS analysis of DC treated cultures reveals that still 5% of
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18 the cells ranked in the G1 and 14% in the S phase of the cell cycle we hypothesize that the observed Ca-oscillations in ES-cells are characteristic to the transition from the G1 to the S phase of the cell cycle. IP3R mediated Ca-release The regulation of intracellular Ca-signals depends on the interplay of intracellular and extracellular Ca-sources (4). Entry of Ca into the cell occurs through voltage dependent, receptor operated and store operated Ca-channels whereas the endoplasmic reticulum (ER) functions as an intracellular Ca-source. Ca-release from the ER is controlled by IP3- and/or Ry-receptors (4). To maintain Ca-oscillations, cells need a source for the immediate increase of [Ca2+]i. Our experimental data indicate that not Ca influx (Fig. 2A) but the release of Ca from intracellular stores is the primary source for the oscillatory rise in [Ca2+]i. The identification of all three IP3R isoforms (Fig. 6D;(47)), the sensitivity of the oscillations to 2-APB (Fig. 3A) and their insensitivity to Ry (Fig. 3B) support the hypothesis that IP3R operated intracellular stores contribute to the oscillatory rise in [Ca2+]i. Ca-influx and extrusion mechanisms After the release of Ca from the intracellular stores, Ca-removal from the cytoplasm can either occur through reuptake of Ca into the intracellular stores or extrusion over the plasma membrane by the Na/Ca exchange (NCX) mechanism or the plasma membrane Ca-ATPase (PMCA). NCX as well as PMCA are expressed in undifferentiated ES cells (47) however, the continuation of Caoscillations for up to 15 min (Fig. 2A) indicates reuptake of Ca into intracellular
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19 stores. However, average data show a decrease of the transient amplitude in the presence of Ca-free solution (Fig. 2C) supporting the hypothesis that Ca influx is necessary to maintain the amplitude of the oscillations. Overall our data support the hypothesis that SOC entry is a major Ca influx mechanism in mouse ES cells. It therefore can maintain the filling of the intracellular Ca stores after depletion (Fig. 4A; 7AB). This is also supported by previous reports that demonstrate the presence of SOC but they failed to identify other influx mechanisms via voltage dependent T- or L-type Ca channels (47, 48). Maintenance of intracellular Ca-oscillations Different paradigms exist to explain sustained intracellular Ca-oscillations. In undifferentiated ES cells the increase of Ca depends on its release from IP3R operated stores. That this increase also coincides with the generation of IP3 by a PLC dependent mechanism is supported by the block of the oscillations by the PLC inhibitor U-73122 (Fig. 3B) and their amplification in the presence of thimerosal (Fig. 8A). The presence of IP3 mediated Ca-signaling mechanisms is further supported by the induction of Ca transients after receptor mediated stimulation of the PLC signaling pathways by either ATP (Fig. 8Aa,Ba) or histamine (47). The critical question is why Ca oscillations dominate in the G1/S phase of the cell cycle. Our experimental results indicate that the SOC entry is present in G1/S as well as in G2/M synchronized cells (Fig. 7) and as a result, Ca stores are also filled in both phases of the cell cycle (Fig. 7). These data exclude depletion of the stores as a reason for the lack of Ca oscillations in G2/M. We observed Ca-transients as a result of receptor stimulated IP3 production (Fig.
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20 8Aa,Ba) and detected all three IP3R isoforms in G1/S and G2/M synchronized cells (Fig.6D). Therefore, lack of IP3Rs or IP3 generating second messenger pathways cannot account for the lack of Ca-oscillations. In sea urchin eggs, changes in the basal concentration of IP3 during the cell cycle were described as the basis for changes in [Ca2+]i (7). A similar mechanism could be present in undifferentiated ES cells. The lack of thimerosal induced Ca oscillations in G2/M synchronized cells could indicate that the basal second messenger concentration is not high enough to induce IP3 mediated Ca release or that the sensitivity of IP3R is reduced (Fig. 8Ab, Bb) at this stage. These changes could be brought about by subtle changes in the basal level of [Ca2+]i and the activity of CaM or CamKII (45). Other regulators of Ca homoeostasis have been related to cell cycle regulation and the expression levels of proteins such as PMCAs (22), CaM or CaMK have yet to be determined (38) in ES cells. IP3R mediated Ca-release and cell cycle progression A role of the phosphatidyl inositol signaling pathway in mES cell cycle progression has previously been proposed, but its relation to IP3 mediated Carelease was not yet demonstrated (9, 14, 24). Our data show ES cell proliferation significantly decreases in the presence of drugs that prevent a rise in [Ca]i or more specifically PLC and IP3 mediated Ca release (Fig. 8D; 9A). They are in good agreement with a recent report that EGF stimulates proliferation of mES cells via PLC dependent changes in [Ca2+]i (17). Although changes in [Ca]i might also be important for other phases of the cell cycle, a clear shift of the cell cycle distribution to the G1/S phase could be observed, further supporting the
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21 relevance of this signaling mechanism in this phase of the cell cycle. The significant decrease of cells in the S phase after thapsigargin treatment could be based on the apoptotic action of thapsigargin (28), however it remains to be determined if prolonged suppression of IP3 mediated signaling could promote a change in the cellular phenotype by induction of differentiation. The IP3 mediated Ca release might not be the only mechanism involved in the ES cells cell cycle regulation. Other potential Ca-entry pathways like SOC or the voltage operated L- and T-type Ca-channels have been attributed to regulate cell proliferation in pulmonary smooth muscle, rat neonatal cardiomyocytes as well as tumor cells (27, 32, 35). Although we have not excluded their influence individually, analysis of mRNA does no indicate the presence of voltage dependent Ca-channels in these cells (47, 48). Conclusion In the present study we demonstrate that mES cells exhibit IP3 mediated oscillations of [Ca2+]i that are confined to the transition from the G1 phase to S phase of the cell cycle and that play a role in cell cycle progression. We propose that changes in IP3R sensitivity or changes of the basal level of IP3 could be the basis for the oscillations and we plan to address this in future experiments. The results reveal further insight into ES cells cell cycle progression and describe a signaling mechanism that could promote the cells rapid transition out of G1 and therefore support the preservation of their pluripotent state. Acknowledgements
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22 This work was supported by grants from the AHA (0330393Z) to KB, the Potts Foundation Loyola University Chicago (RFC 11086) to KB and the NIH (MH533367) to GAM.
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28 Figure Legends Figure 1: Mouse ES cells exhibit spontaneous Ca oscillations. Twodimensional confocal images were taken at 5 s intervals from an mES cell colony loaded with the Ca sensitive dye Fluo-4/AM. An image sequence (A) visualizes the oscillatory increase of [Ca2+]i within individual cells of the colony. The sequence of images is displayed from left to right and top to bottom at 25 s interval. (B) The fluorescence of individual cells plotted as a function of time displays oscillatory changes in [Ca2+]i that are not synchronized among neighboring cells. The numbers of individual traces correspond to the cells numbered in the first image of (A); * mark the points of time that correspond to the images in (A). Oscillations exhibited a mean interval of 76 ± 24 s, a duration of 38 ± 10 s
(at 50% amplitude) and an amplitude of 1.57 ±0.7 (n=32
experiments).
Figure 2: Modulation of mES cell Ca oscillations by [Ca2+]O or RyR. A: Representative trace of [Ca2+]i from one cell within an mES cell colony, during superfusion with control ([Ca2+]o = 2 mM) and Ca-free solution. Ca transient amplitude (TA) decreased slowly during continuous superfusion with Ca-free solution (C) (mean ± SE). TAs were normalized to the last transient prior to application of Ca-free solution (n = 5 experiments). B: Characteristic recording of Ca oscillations before and during superfusion with ryanodine (10µM). ITI (D) and amplitude (E) of Ca oscillations remained unchanged in the presence of
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29 ryanodine (% of control; n = 4). Data in D and E are presented as mean ± SD; in D and E changes were not significant (Student t-test: P > 0.05).
Figure 3: Ca oscillations depend on IP3R mediated Ca release. Representative recordings of [Ca2+]i within a single cell during the superfusion with A. the IP3R antagonist 2-APB and B: the PLC inhibitor U-73122. 2-APB and U73122 both inhibit spontaneous oscillations of [Ca2+]i in undifferentiated ES cells.
Figure 4: Store operated Ca entry in mES cells. A: Changes of [Ca2+]i in a single mES cell during store depletion with the SERCA inhibitor thapsigargin in the absence and presence of [Ca2+]o and during superfusion with La3+. B: La3+ reversibly blocks 75% of the Ca entry induced by depletion of the intracellular Ca stores. Data are presented as mean ± SD.
Figure
5:
Pharmacologically
induced
changes
of
cell
cycle
distribution. A: Histogram of cell cycle distribution of individual experiments analyzed by FACS sorting. Histograms of control (V), MIM (• V X) and DC (… …) synchronized cultures are superimposed. B: Bar graph allows the direct comparison of the percentage of cells that reside in the G0/G1, S or G2/M phase of the cell cycle in control conditions and after MIM or DC treatment. Data represent the mean ± SD obtained from 3 independent experiments.
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30 Figure 6: Oscillations of [Ca2+]i in synchronized mES cells. Bar graphs present (A) the percentage of oscillating cells within individual mES cell colonies, (B) the interval of the Ca transients and (C) their transient duration in control cultures and cultures that were synchronized with DC, NOC, MIM, APC or HU. (D) Immunoblots demonstrate the presence of the IP3R isoforms T1, T2 and T3 in control cultures and cultures that were serum starved (0%) or synchronized by HU or NOC respectively. Data are expressed as mean ± SD (Student t-test: * indicates P > 0.05; † indicates P> 0.05).
Figure 7: Presence of SOC in HU and DC synchronized mES cells. Representative recordings of changes in [Ca2+]i in individual mES cells from cultures that were synchronized by treatment with either (A) HU or (B) DC. In both stages of the cell cycle thapsigargin induced depletion of the intracellular Ca stores that resulted in the activation of SOC.
Figure 8: IP3 mediated Ca oscillations in synchronized mES cells. Representative recordings of ATP or thimerosal induced changes in [Ca2+]i . The response from individual mES cells is shown that were synchronized by treatment with either (A) HU or (B) DC. (C) Comparison of the mean change of ATP induced Ca transients in control cultures and cultures synchronized either at G1/S (APC, HU) or G2/M (NOC, DC). Data are expressed as mean ± SD. (D) Cell cycle progression of control cultures and cultures that were maintained in the presence of either U-73122 (Z[10 µM) or EGTA (3 mM).
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31
Figure 9: Delay of cell cycle progression by inhibitors of IP3 mediated Ca release. Cell cycle progression of control, 2-APB (n=6), BAPTA/AM (n=4), U73122 (n=2), and thapsigargin (n=6) treated cultures normalized to the number of control cells (A). FACS analysis of the same treated cultures after 12 h incubation reveals a shift of the cell cycle distribution toward the G1/S phase of the cell cycle (B). Data are represented as normalized change from control cultures. (The significance of the change in cell cycle distribution was confirmed by chi2 test; P
