From the Department of Biochemistry, Nijmegen Center
for Molecular Life Sciences, Nijmegen NL-6500 HB and the
Department of Pathology, Academic Medical Center, Amsterdam
Zuidoost 1105 AZ, The Netherlands
Received for publication, November 4, 2002, and in revised form, January 7, 2003
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ABSTRACT |
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Evidence in the literature implicating
both Ras-like Ras (R-Ras) and intracellular Ca2+ in
programmed cell death and integrin-mediated adhesion prompted us to
investigate the possibility that R-Ras alters cellular Ca2+
handling. Chinese hamster ovary cells expressing the cholecystokinin (CCK)-A receptor were loaded with indo-1 to study the effects of
constitutively active V38R-Ras and dominant negative N43R-Ras on the
kinetics of the thapsigargin (Tg)- and CCK8-induced
Ca2+ rises using high speed confocal microscopy. In the
absence of extracellular Ca2+, both 1 µM Tg,
a potent and selective inhibitor of the Ca2+ pump of the
intracellular Ca2+ store, and 100 nM
CCK8 evoked a transient rise in Ca2+, the size
of which was decreased significantly after expression of V38R-Ras. At
0.1 nM, CCK8 evoked periodic Ca2+
rises. The frequency of these Ca2+ oscillations was reduced
significantly in V38R-Ras-expressing cells. In contrast to V38R-Ras,
N43R-Ras did not alter the kinetics of the Tg- and
CCK8-induced Ca2+ rises. The present findings
are compatible with the idea that V38R-Ras expression increases the
passive leak of Ca2+ of the store leading to a decrease in
Ca2+ content of this store, which, in turn, leads to a
decrease in frequency of the CCK8-induced cytosolic
Ca2+ oscillations. The effect of V38R-Ras on the
Ca2+ content of the intracellular Ca2+ store
closely resembles that of the antiapoptotic protein Bcl-2 observed
earlier. Together with reports on the role of dynamic Ca2+
changes in integrin-mediated adhesion, this leads us to propose that
the reduction in endoplasmic reticulum Ca2+ content may
underlie the antiapoptotic effect of R-Ras, whereas the decrease in
frequency of stimulus-induced Ca2+ oscillations may play a
role in the inhibitory effect of R-Ras on stimulus-induced cell
detachment and migration.
The Ras-related G-protein,
R-Ras,1 is a member of the
Ras subfamily of small GTP-binding proteins (1, 2). The R-Ras
protein is localized at the inner leaflet of the outer membrane and
shares 55% identity with the prototypic Ras, but is 26 amino acids
longer at its N terminus. In vitro, R-Ras interacts with
several known Ras regulatory proteins including RasGRF1,
RasGRP/CalDAG-GEFII, RasGRP3/CalDAG-GEFIII, GAP1IP4BP (3),
and the three downstream effector proteins Raf1, phosphatidylinositol 3-kinase, and RalGDS (4-6).
Several of the Ras exchange factors with which R-Ras interacts
including RasGRF1, CalDAG-GEFII (RasGRP), CalDAG-GEFIII
(RasGRP3), and CalDAG-GEFIII are sensitive to Ca2+
and/or diacylglycerol (DAG), the endogenous activator of protein kinase C, whereas the GTPase-activating protein GAP1IP4BP
with which R-Ras also interacts is activated by inositol
1,3,4,5-tetrakisphosphate (3).
R-Ras has been implicated in cell transformation, cell adhesion (7),
and cell cycle control (6, 8). These functions appear to be mediated by
few, if any, of the signaling pathways taken by Ras (4, 6, 9-11).
Several studies have shown that Ras and R-Ras have opposing effects on
apoptosis, or programmed cell death, in that R-Ras stimulates this
process under conditions where Ras is protective (1, 12, 13). Early
studies employing the yeast two hybrid system suggested a physical
interaction between R-Ras and the antiapoptotic Bcl-2 (14). Thus far,
however, this interaction could not be demonstrated in a mammalian cell
system (15). Other studies have shown that under certain experimental conditions activated mutants of R-Ras can act through the
phosphatidylinositol 3-kinase pathway to inhibit cell death (16, 17).
Finally, constitutively active V38R-Ras has been shown to keep cellular integrins in an active state thus allowing attachment to surfaces coated with integrin ligands (7).
Recent studies have implicated the
Ca2+-dependent enzyme calpain in cell
detachment during cell migration (18) and inhibition of
integrin-induced stress fiber assembly and cell spreading (19). These
findings explain previously reported effects of alterations in
intracellular Ca2+ concentration on integrin-mediated
adhesion (20). A role for Ca2+ in apoptosis became apparent
when it was shown that a modest reduction in endoplasmic
reticulum (ER) Ca2+ content prevented cell death (21,
22).
The involvement of R-Ras and intracellular Ca2+ in both
programmed cell death and integrin-mediated adhesion prompted us to investigate the possibility that R-Ras might exert its actions through
an effect on cellular Ca2+ handling. Here we show that
constitutively active V38R-Ras decreases the ER Ca2+
content in a manner similar to the proapoptotic protein Bcl-2 and slows
down the frequency of stimulus-induced periodic Ca2+ rises.
We propose that the reduction in ER Ca2+ content may
underlie the antiapoptotic effect of R-Ras described by Suzuki and
co-workers (16) and Broadway and Engel (17). Furthermore, we
propose that the decrease in frequency of the stimulus-induced
cytosolic Ca2+ rises may inhibit stimulus-induced
activation of calpain, thus causing inhibition of cell detachment and
migration and favoring integrin-mediated cell attachment and spreading.
Transient Transfection of CHO Cells with R-Ras Mutants--
The
development of a CHO cell line stably expressing the CCKA
receptor (CHO-CCKA) has been described in detail elsewhere
(23). CHO-CCKA cells were grown to confluence in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum in a humidified atmosphere of 5% CO2 at 37 °C.
For transfection, cells were trypsinized (5 × 106
cells/300 µl) and electroporated (280 V, 975 microfarads) in the
presence of 2 µg of plasmid pGFP-N1 (Clontech,
Palo Alto, CA) and 18 µg of either pMT2-HA-V38R-Ras or
pMT2-HA-N43R-Ras (24). Subsequently, cells were seeded on a glass
coverslip (15,000 cells/30 µl) and allowed to attach for 30 min.
Culture medium was added, and the cells were grown for 48 h.
Detection of R-Ras Mutants in CHO Cells--
GFP-positive
(GFP+) and GFP Single Cell Ca2+ Imaging--
Cells were loaded with
indo-1 for 30 min at 37 °C in Dulbecco's modified Eagle's medium
containing 10% fetal calf serum, 3 µM indo-1/AM, and
0.025% (w/v) pluronic F-127. Excess indo-1 was removed by washing
twice with calcium-free HEPES/Tris medium containing 133 mM
NaCl, 4.2 mM KCl, 1.0 mM MgCl2, 5.8 mM glucose, an amino acid mixture according to Eagle, 0.1%
(w/v) bovine serum albumin, and 10 mM HEPES, adjusted with
Tris to pH 7.4. 22-mm cover slips were subsequently mounted in a Leiden
chamber (25) and placed on the stage of an inverted microscope (Nikon,
Diaphot), attached to a videorate confocal microscope (Noran
instruments, Middleton, WI). A water immersion objective (×40, NA 1.2)
was used, allowing a field of view of about 15 cells. Within each field
(165 × 155 µm), cells of comparable size were selected.
GFP+ cells were identified by recording their green
emission at 525 ± 25 nm after 488 nm excitation delivered by an
argon ion laser (Omnichrome Inc., Chino, CA). Specific excitation of
indo-1 (351 nm) was provided by a high power argon ion laser (Coherent
Enterprise, Santa Clara, CA). Indo-1 fluorescence emission was
monitored at 405 ± 45 nm and 485 ± 45 nm at 30 Hz by using
a 455 nm dichroic mirror. The OZ hardware set-up and acquisition
were controlled with Intervision software (version 1.6, Noran
Instruments) running under IRIX 6.2 on an Indy work station
(Silicon Graphics Inc., Mountain View, CA) equipped with 128 Mb of RAM.
Fluorescence signals were collected in real time (30 Hz, 5-10 min
total recording time) from eight rectangular regions of interest
(including a cell-free area for background correction) drawn on
full-frame images (512 × 480 pixels). The zoom factor was 0.6, and the pixel size was 0.323 µm as calibrated with a graticule (26).
To reduce noise and to ensure that each cell was fully within the
confocal volume, no slit was applied (optical section thickness of
10.53 µm). Between recordings, hardware settings (i.e.
brightness, contrast, and laser power) were kept constant. The laser
power used (28 µW at the back of the objective lens) was minimal to
prevent cytotoxic and/or heating artifacts. The cells were incubated
for 2 min in calcium-free HEPES/Tris medium containing 0.5 mM EGTA prior to the start of the recording. Subsequently,
the cells were stimulated with either 0.1 nM or 100 nM CCK8 or 1 µM thapsigargin
(Tg). The fluorescence emission ratio at 405 and 485 nm was
monitored as a measure of [Ca2+]i
after excitation at 351 nm.
For long term recordings, cells were loaded with fura-2 in the presence
of 3 µM fura-2/AM and 0.025% (w/v) pluronic F-127 as
described above. Coverslips were mounted in a thermostatic (37 °C)
perfusion chamber placed on the stage of an inverted microscope (Nikon,
Diaphot). Dynamic video imaging was carried out as described previously
(27) using the MagiCal hardware and TARDIS software provided by Joyce
Loebl (Dukesway, Team Valley, Gateshead, UK). By using an
epifluorescent ×40 oil immersion objective we were able to monitor
simultaneously the cytosolic Ca2+ concentration in close to
50 individual cells. GFP+ cells were identified by their
green emission (525 ± 20 nm) at an excitation wavelength of 490 nm. Fura-2 emission was monitored at 492 nm during alternating
excitation at 340 and 380 nm. The fluorescence emission ratio at 492 nm
was monitored as a measure of
[Ca2+]i after excitation at 340 and 380 nm. 0.1 nM CCK8 was added by means of a
custom-made superfusion system.
Inositol 1,4,5-Trisphosphate Measurements--
At 24 h
after transfection, GFP Data Analysis--
Data were analyzed using Origin Pro 6.1 (Microcal, Northampton, MA) and Image Pro Plus 4.1 image analysis
software (Media Cybernetics, Silver Spring, MD). The results presented
are the mean ± S.E. Overall statistical significance was
determined by analysis of variance. In the case of significance,
individual groups were compared according to Fischer, and p
values < 0.05 were considered significant. For linear fits the
least squares algorithm was applied using both Pearson's r
and p values as a measure for the quality of the fit. In all
graphs, indo-1 ratio signals were normalized to the basal
(prestimulatory) level.
Materials--
CCK8 was obtained from Sigma, Tg from
LC Services (Woburn, MA), and tissue culture medium with additives from
Invitrogen. Indo-1/AM, fura-2/AM, and pluronic F-127 were purchased
from Molecular Probes Inc. (Leiden, The Netherlands).
D-myo-[3H]Inositol 1,4,5-trisphosphate (51.4 Ci/mmol) was obtained from Amersham Biosciences. All other chemicals
were of reagent grade.
Coexpression of R-Ras and GFP in CHO Cells--
CHO cells
expressing the rat CCKA receptor (CHO-CCKA
cells) were cotransfected with GFP and either constitutively active
HA-V38R-Ras or dominant negative HA-N43R-Ras. At 48 h
post-transfection, cells were loaded with the fluorescent
Ca2+ indicator indo-1 and visualized by confocal
microscopy. Fig. 1A shows a
representative example of a cluster of GFP+ cells
cotransfected with V38R-Ras. When
identical hardware settings were used
during acquisition, no significant differences in GFP intensity between
V38R-Ras- and N43R-Ras-transfected cells were observed (Table
I). The corresponding indo-1 image (Fig. 1B) shows a
second cluster of GFP
To demonstrate that GFP expression reports expression of R-Ras,
GFP+ and GFP Morphology of R-Ras-expressing Cells--
To demonstrate
alterations in cellular morphology induced by R-Ras, we compared both
the cross-sectional area and morphology between GFP V38R-Ras Alters the Kinetics of the Thapsigargin-induced
Ca2+ Rise--
To assess possible effects of R-Ras on
cellular Ca2+ handling, CHO-CCKA cells
transiently expressing either N43R-Ras or V38R-Ras were treated with
Tg, a specific inhibitor of the sarcoplasmic and ER
Ca2+-ATPase (SERCA). The cells were loaded with indo-1, and
the Tg-induced changes in cytosolic Ca2+ concentration were
monitored by means of high speed confocal microscopy. Tg increases the
cytosolic Ca2+ concentration by preventing the active
reuptake of the Ca2+ ions that continuously leak out of the
ER Ca2+ store. In the absence of extracellular
Ca2+, these Ca2+ ions are removed from the
cytosol by the action of the plasma membrane Ca2+-ATPase
(PMCA).
Fig. 2A shows the effect of 1 µM Tg on the average cytosolic Ca2+
concentration of four V38R-Ras expressing cells (filled
circles) and four GFP
The duration of the Tg-induced Ca2+ transient was shortened
significantly from 190 ± 12 s (n = 38 cells)
in GFP
The amplitude of the transient (Fig. 2A) seemed to
be decreased in V38R-Ras-expressing cells (2.63 ± 0.12;
n = 24 cells) compared with GFP
The rising phase of the Tg-induced Ca2+ transient continues
as long as the rate of Ca2+ leak from the ER exceeds that
of active cytosolic Ca2+ removal by the PMCA. The kinetics
of this phase was described adequately by a sigmoid (Boltzmann)
equation (y = A2 + (A1
The decay phase of the Tg-induced Ca2+ transient starts
when the rate of Ca2+ leak from the ER becomes smaller than
the rate of PMCA-mediated Ca2+ removal from the cytosol.
The kinetics of this phase was adequately described by a
monoexponential equation (y = y0 + A·e
Calculation of the integrated area underneath the Ca2+
transient, as a measure of the amount of Ca2+ released into
the cytosol, revealed a significant decrease from 162 ± 10 arbitrary units (n = 38) and 169 ± 15 arbitrary
units (n = 10) in GFP V38R-Ras Alters the Kinetics of the 100 nM
CCK8-induced Single Ca2+ Rise--
In
CHO-CCKA cells, CCK-induced cytosolic Ca2+
signals arise from Ins (1,4,5)P3-mediated Ca2+
release from the ER (23), paralleled by capacitative Ca2+
entry across the plasma membrane (29). To investigate whether R-Ras
affects stimulus-induced Ca2+ release from the ER , cells
were stimulated with 100 nM CCK8 in the absence
of extracellular Ca2+. At this concentration,
CCK8 evokes a single Ca2+ transient that is not
followed by repetitive Ca2+ transients (Ca2+
oscillations). Fig. 2 (D and F) shows that both
GFP+ and GFP
V38R-Ras-expressing cells displayed a Ca2+ transient that
was less wide (Fig. 2D) and rose more slowly (Fig.
2E) than that in the corresponding GFP
Analysis of the second (fast) phase of Ca2+ decay revealed
a µ value that was of the same order of magnitude for
N43R-Ras-expressing cells (27.8 ± 0.2 s;
r2 = 0.79; n = 4) and the
corresponding GFP
Finally, calculation of the integrated area underneath the
Ca2+ transient revealed a significant decrease from
187 ± 17 arbitrary units (n = 58) and 177 ± 13 arbitrary units (n = 19) in GFP CCK8-induced Ins(1,4,5)P3 Formation Is Not
Altered in R-Ras-expressing Cells--
CCK8 acts
through Ins(1,4,5)P3 to increase the cytosolic free
Ca2+ concentration in CHO-CCKA cells. Fig.
3 shows the dose-response curve for the
effect of CCK8 on the cellular Ins(1,4,5)P3
content, measured at 20 s after the onset of stimulation. Neither
V38R-Ras (Fig. 3A) nor N43R-Ras (Fig.
3B) interfered with the CCK8-induced production of Ins(1,4,5)P3.
V38R-Ras Does Not Alter the Kinetics of the 0.1 nM
CCK8-induced Oscillatory Ca2+ Rises--
To
assess possible effects of V38R-Ras expression on physiologically
relevant Ca2+ signals, we studied the kinetics of the
CCK8-induced repetitive Ca2+ rises
(Ca2+ oscillations) in CHO-CCKA cells. When
added at a 1,000-fold lower concentration (0.1 nM),
CCK8 readily induced oscillatory Ca2+ rises
(Fig. 4A; (30)). Of note, this
measurement was performed in the absence of extracellular
Ca2+. Under this condition, the amplitude of the
Ca2+ oscillations gradually decreased as a function of
time. Fig. 4B shows that the rate of Ca2+
release (dotted line) was much slower for the last
(b) than for the first (a)
Ca2+ oscillation. These findings are indicative of a
gradual decrease of the Ca2+ content of the ER because of
the action of the PMCA removing part of the released Ca2+
out of the cell during each oscillation.
Analysis of the first oscillatory Ca2+ rise revealed no
significant differences in amplitude and width between
V38R-Ras-expressing cells and GFP
Analysis of the rate of Ca2+ rise revealed
Moreover, analysis of the decay phase revealed µ values of 5.6 ± 0.8 s (r2 = 0.96; n = 3)
and 5.3 ± 0.6 s (r2 = 0.96;
n = 4) for V38R-Ras-expressing cells and corresponding GFP Reduced Frequency of Stimulus-induced Repetitive Ca2+
Rises in V38R-Ras-expressing Cells--
To demonstrate a possible
effect of V38R-Ras-expression on the temporal characteristics of the
cytosolic Ca2+ oscillations, we stimulated the cells with
0.1 nM CCK8 in the presence of 1 mM
extracellular Ca2+. The latter prevented depletion of the
ER Ca2+ store and allowed recording of Ca2+
oscillations during prolonged periods of time. Cells were loaded with
fura-2, and videoimaging microscopy was used to monitor the CCK8-induced Ca2+ changes. Fig.
5 shows that the oscillation frequency
was reduced significantly in V38R-Ras-expressing cells but not in
N43R-Ras-expressing cells (p < 0.01; n = 307, 31, and 157 cells for GFP Evidence in the literature has implicated both R-Ras and
intracellular Ca2+ in programmed cell death (1, 12, 13, 16,
17, 21, 22) and integrin-mediated cell adhesion (7, 8, 19, 20). This prompted us to investigate the possibility that R-Ras might exert
its effects by altering the activities of proteins and/or organelles
involved in cellular Ca2+ handling. To study the effects of
R-Ras, CHO cells stably expressing the CCK-A receptor were
cotransfected with GFP and either constitutively active V38R-Ras or
dominant negative N43R-Ras. Separation of GFP+ and
GFP V38R-Ras Causes Cell Rounding and Enlargement--
For
fluorescence measurements, cells were seeded on a glass coverslip
immediately after transfection and grown for 48 h. This procedure
provided us with the unique opportunity to monitor simultaneously the
cytosolic Ca2+ changes in R-Ras-expressing
(GFP+) cells and the corresponding sham-transfected
(GFP V38R-Ras Expression Decreases Both the ER Ca2+ Content
and the Frequency of the CCK8-induced Cytosolic
Ca2+ Rises--
Cells transiently expressing either
V38R-Ras or N43R-Ras were loaded with indo-1 or fura-2, and the changes
in cytosolic free Ca2+ concentration were monitored by
means of high speed confocal or conventional videoimaging microscopy,
respectively. To start with, the cells were treated with Tg, a potent
and selective inhibitor of the Ca2+ pump of the ER
Ca2+ store (SERCA). Inhibition of this pump prevents
reuptake of Ca2+ ions that continuously leak out of the ER
into the cytosol. We have shown previously that in the absence of
active Ca2+ pumping this Ca2+ leak process is
described adequately by a monoexponential equation (33, 34). The
present study shows that Tg evoked a rapid increase in cytosolic
Ca2+ when added in the absence of extracellular
Ca2+ to prevent capacitative Ca2+ uptake. This
indicates that initially the rate of passive Ca2+ leak
exceeds that of active cytosolic Ca2+ removal via the PMCA.
After having reached its maximum, cytosolic Ca2+ slowly
decreases to prestimulatory values. This suggests that during the
entire down-stroke of the Tg-induced Ca2+ transient,
Ca2+ is released from the ER, thus slowing down the rate of
Ca2+ decay.
Expression of V38R-Ras decreased markedly the duration of the
Tg-induced Ca2+ transient. The integrated area underneath
the cytosolic Ca2+ peak, which reflects the amount of
Ca2+ released into the cytosol, was decreased markedly
(40%) in V38R-Ras-expressing cells compared with N43R-Ras-expressing
cells and GFP
CCK8, when added at a relatively high concentration of 100 nM, depleted the ER Ca2+ store. This indicates
that at this concentration it causes the sustained opening of the
Ins(1,4,5)P3-operated Ca2+ release channels.
The rate of Ca2+ rise obtained with CCK8 was
considerably higher than that obtained with Tg (
The integrated area underneath the cytosolic Ca2+ peak was
decreased significantly in V38R-Ras-expressing cells compared with N43R-Ras-expressing cells and GFP
When added at a 1,000-fold lower concentration of 0.1 nM,
CCK8 induced oscillatory changes in Ca2+. This
indicates that at this CCK8 concentration opening of the Ins(1,4,5)P3-operated Ca2+ channels is only
transient. The rate of Ca2+ rise ( Possible Implications of the V38R-Ras-induced Reduction in ER
Ca2+ Content--
The present study provides evidence that
V38R-Ras expression reduces the ER Ca2+ content by
increasing the passive Ca2+ leak across the ER membrane. A
similar observation was reached after overexpression of the
antiapoptotic protein Bcl-2 in HeLa cells (22, 37, 38) and HEK-293
cells (21). It was concluded that Bcl-2 exerted its effect by
increasing the Ca2+ leak rather than decreasing the
activity of the ER Ca2+ pumps. The data presented in this
study provide evidence for a similar mechanism of action of V38R-Ras.
The finding that an increase in ER Ca2+ content, realized
by SERCA overexpression, increased spontaneous apoptosis (39)
strengthens the idea that the antiapoptotic action of Bcl-2 is mediated
through its effect on the ER Ca2+ content. In this context,
the present finding that V38R-Ras decreases the ER Ca2+
content provides a good explanation for the antiapoptotic effect observed with activated mutants of R-Ras under certain experimental conditions (16, 17). The latter study provided evidence for the
involvement of the phosphatidylinositol 3-kinase pathway in the
mechanism of action of R-Ras. Intriguingly, recent studies have
implicated this pathway in agonist-induced up-regulation of Bcl-2 (40. 41) and the caspase inhibitor cIAP-2 (41). Based on these findings, it
is tempting to speculate that activation of R-Ras promotes the
phosphatidylinositol 3-kinase-mediated up-regulation of Bcl-2, which,
in turn, causes a decrease in ER Ca2+ content by increasing
the ER Ca2+ leak via a hitherto unknown mechanism. However,
it should be noted that other studies have shown that R-Ras stimulates
the process of apoptosis under conditions where Ras is protective (1,
12, 13). The present study does not provide an explanation for this
proapoptotic effect of R-Ras.
Lowering of the ER Ca2+ content has been demonstrated to
trigger the process of capacitative Ca2+ entry across the
plasma membrane (31). In case the reduced ER Ca2+
content is caused by an increased ER Ca2+ leak, this would
lead to an elevation of the cytoplasmic Ca2+ concentration.
However, in the case of Bcl-2 overexpression it has been demonstrated
that the capacitative Ca2+ entry was also down-regulated,
thus preventing a sustained increase of the resting cytosolic
Ca2+ concentration (37).
Possible Implications of the V38R-Ras-induced Decrease in Frequency
of Stimulus-induced Cytosolic Ca2+
Oscillations--
V38R-Ras expression did not significantly alter the
amplitude and duration of the CCK8-induced cytosolic
Ca2+ oscillations. This means that R-Ras does not signal to
its downstream effectors through modulation of the amplitude and/or
duration of the cytosolic Ca2+ rises. However, the
frequency of the cytosolic Ca2+ oscillations appeared to be
reduced by 30% in V38R-Ras-expressing cells. This is in agreement with
theoretical studies predicting a decrease in oscillation frequency when
the ER Ca2+ content is reduced at a constant
Ins(1,4,5)P3 concentration (35, 36). In accordance with
this idea, measurement of the CCK8-stimulated production of
Ins(1,4,5)P3 revealed no differences between
V38R-Ras-expressing cells and GFP
In conclusion, the data presented show that activation of R-Ras
increases the Ca2+ leak across the ER membrane thus
decreasing both the Ca2+ content of this intracellular
Ca2+ store and, as a consequence, the frequency of the
stimulus-induced oscillatory Ca2+ rises. We propose that
the reduction in ER Ca2+ content may underlie the
antiapoptotic effect of R-Ras described by Suzuki and co-workers (16)
and Broadway and Engel (17). Furthermore, we propose that the
decrease in frequency of the stimulus-induced cytosolic
Ca2+ rises may inhibit calpain activation, which, in turn,
leads to inhibition of cell detachment and migration thus favoring
integrin-mediated cell attachment and spreading.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells were separated by means
of fluorescence- activated cell sorting (FACS) at 24 h after
electroporation. The cells were cultured for another 24 h,
homogenized, and subjected to SDS-polyacrylamide gel electrophoresis.
Proteins were transferred overnight to polyvinylidene difluoride
membranes (Immobilon P, Millipore, Bedford, MA). For detection of
HA-V38R-Ras and HA-N43R-Ras, blots were incubated overnight with the
anti-HA monoclonal antibody 12ca5. Immunoreactive bands were detected
with alkaline phosphatase-conjugated rabbit anti-mouse IgG.
and GFP+ cells were
separated by means of FACS and plated out in 12-well plates (100,000 cells/well). After another 24 h of culturing, cells were washed in
HEPES/Tris medium containing 1% (w/v) bovine serum albumin and
stimulated by the addition of 125 µl of HEPES/Tris medium containing
the indicated concentration of CCK8. After 20 s, 31 µl of 50% trichloroacetic acid was added to stop the reaction. The
cells were scraped off and transferred to an Eppendorf test tube. The
samples were centrifuged for 4 min at 10,000 × g, and
a 120-µl aliquot of the supernatant was removed. This aliquot was
extracted three times with 2 ml of water-saturated diethyl ether.
Subsequently, 75 µl was taken to which 2 µl of 50%
KHCO3 was added to increase the pH above 7.5. The inositol
1,4,5-trisphosphate (Ins(1,4,5)P3) content of the extract
was determined by isotope dilution assay as described previously
(28).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells. Importantly, these
GFP
cells, present on the same coverslip as the
GFP+ cells, were used as a control for the effect of R-Ras
expression on cellular Ca2+ handling. Under the
experimental conditions used, no cross-talk between GFP and indo-1
fluorescence signals was observed.
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Fig. 1.
Coexpression of R-Ras and GFP in CHO
cells. CHO-CCKA cells, cotransfected with GFP and
V38R-Ras, were loaded with the fluorescent radiometric Ca2+
dye indo-1 and imaged at 48 h post-transfection. A,
fluorescence image recorded at 525 nm (excitation, 488 nm), showing the
GFP+ cells. Dotted lines represent
sham-transfected GFP cells. B, fluorescence
image of the same cells at 405 nm (excitation, 351 nm) depicting
indo-1-loaded cells. C, Western blot (representative of
three independent experiments) of total cell lysates from
GFP
and GFP+ cells showing increased
expression of R-Ras mutants in GFP+ cells.
GFP
and GFP+ cells were separated by means of
FACS. The nt lane represents total cell lysate from
nontransfected cells.
Cell morphology in control and R-Ras-expressing cells
cells were separated by FACS at
24 h post-transfection and cultured for another 24 h. After
this second culturing period, total cell lysates were prepared and
subjected to Western blot analysis using the monoclonal anti-HA
antibody 12ca5. Fig. 1C shows that both R-Ras mutants were
highly expressed in the GFP+ cells.
and
GFP+ cells. Table I shows that the cross-sectional area was
increased significantly in N43R-Ras-expressing cells (p < 0.01). To detect more subtle morphological alterations we calculated
the formfactor F (perimeter2/4·
·area).
For a round cell, the numerical value of F is 1. The data
presented show that F was decreased significantly in the R-Ras-expressing cells (Table I, p < 0.01).
Importantly, however, F was significantly smaller in
N43R-Ras-expressing cells compared with V38R-Ras-expressing cells
(p < 0.05). Taken together, these findings demonstrate
that CHO cells that express N43R-Ras and, to a lesser extent, V38R-Ras,
are larger and rounder than GFP
cells.
cells (open
circles) present on the same coverslip. The experiment was
performed in the absence of extracellular Ca2+. Under these
conditions, Tg transiently increased the cytosolic Ca2+
concentration in both V38R-Ras-expressing cells and the
GFP
cells. This observation shows that the rate of
Ca2+ leak from the ER is considerably faster than the rate
of cytosolic Ca2+ removal via the PMCA.
View larger version (37K):
[in a new window]
Fig. 2.
Tg- and CCK8-induced single
Ca2+ transients in V38R-Ras- and N43R-Ras-expressing
cells. CHO-CCKA cells, cotransfected with GFP and
V38R-Ras, were loaded with the fluorescent radiometric Ca2+
dye indo-1 and monitored at 48 h post-transfection by means of
high speed confocal imaging microscopy. The recordings shown are the
averages (± S.E.) of four GFP cells and either four
N43R-Ras-expressing cells or four V38R-Ras-expressing cells present on
the same coverslip. The experiments were performed in the absence of
extracellular Ca2+. For clarity, only 120 evenly spaced
data points are displayed. For details, see "Results" and
Table II. A, 1 µM Tg evoked a single
Ca2+ rise the duration of which was decreased in
V38R-Ras-expressing cells compared with the corresponding
GFP
cells. B, V38R-Ras expression enhanced the
rate of Ca2+ rise during the rising phase of the
Ca2+ transient. C, the rate of Ca2+
decay was decreased in V38R-Ras-expressing cells. D, the
duration of the CCK8-induced single Ca2+
transient was decreased in V38R-Ras-expressing cells. E,
V38R-Ras expression decreased the rate of Ca2+ rise.
F, 100 nM CCK8 evoked similar
Ca2+ transients in N43R-Ras-expressing cells and
GFP
cells. G, N43R-Ras expression did not
alter the rate of Ca2+ rise. H, 100 nM CCK8 completely emptied the intracellular
Ca2+ store as indicated by the inability of Tg to increase
Ca2+ in CCK8-stimulated cells.
cells to 121 ± 13 s (n = 24 cells; p < 0.001) in V38R-Ras-expressing cells
(Fig. 2A). The effect was specific for V38R-Ras
because the duration of the Tg-induced transient was not altered
significantly in N43R-Ras-expressing cells (173 ± 6 s;
n = 10).
cells
(2.86 ± 0.10; n = 38 cells) and N43R-Ras
expressing cells (3.10 ± 0.19; n = 10 cells), but
this effect was not statistically significant.
A2)/(1 + exp((x
x0)/
))) (Fig.
2B). The time constant
, which is inversely proportional to the rate of Ca2+ rise, was smaller in
V38R-Ras-expressing cells (4.42 ± 0.09 s; r2 = 0.99; n = 4) compared with
the corresponding GFP
cells (7.54 ± 0.012 s;
r2 = 0.99; n = 4) present on the
same coverslip. This shows that the rate of Ca2+ rise is
1.7-fold increased in V38R-Ras-expressing cells.
t/µ) (Fig.
2C). The time constant µ, which is inversely
proportional to the rate of Ca2+ decay, was markedly
smaller in V38R-Ras-expressing cells (49.7 ± 0.4 s;
r2 = 0.93; n = 4) compared with
GFP
cells (200.3 ± 15.0 s;
r2 = 0.98; n = 4). This shows
that the rate of Ca2+ decay is increased 4-fold in
V38R-Ras-expressing cells.
cells and
N43R-ras-expressing cells, respectively, to 93 ± 8 arbitrary
units (n = 24) in V38R-Ras-expressing cells (Table
II).
Calcium dynamics in control and R-Ras-expressing cells
cells displayed a single
Ca2+ transient that consisted of a rapid increase followed
by a first phase of slow decay and a second phase of fast decay to
basal levels.
cells.
The amplitude was only slightly decreased from 3.02 ± 0.10 (n = 58) in GFP
cells to 2.80 ± 0.11 (n = 44) in V38R-Ras-expressing cells or 3.08 ± 0.15 (n = 19) in N43R-Ras-expressing cells
(Table II). However, this decrease was not statistically
significant. Detailed analysis of the rate of Ca2+ rise
revealed a
value that was increased for V38R-Ras-expressing cells
(
= 0.12 ± 0.01 s; r2 = 0.99;
n = 4) compared with the corresponding
GFP
cells (
= 0.07 ± 0.01 s;
r2 = 0.99; n = 4) present on the
same coverslip (Fig. 2E). This demonstrates that the rate of
Ca2+ rise is reduced in V38R-Ras-expressing cells. In
N43R-Ras-transfected cells, the kinetics of the decline (Fig.
2F) and rising phase (Fig. 2G) of
the Ca2+ transient were identical between the
GFP+ and GFP
cells. This shows that GFP
expression in itself had no effect on the shape of the
CCK8-induced Ca2+ transient. Importantly, 100 nM CCK8 completely released the Tg-sensitive intracellular Ca2+ store (Fig.
2H).
cells (63.3 ± 0.8 s;
r2 = 0.96; n = 4) and for
V38R-Ras-expressing cells (25.2 ± 0.4 s;
r2 = 0.98; n = 4) and the
corresponding GFP
cells (31.2 ± 1.2;
r2 = 0.98; n = 4). Of note,
these µ values were similar to that obtained with V38R-Ras-expressing
cells after Tg treatment (49.7 ± 0.4 s;
r2 = 0.93; n = 4) but markedly
lower than those obtained with GFP
cells after Tg
treatment (200.3 ± 15.0 s; r2 = 0.98;
n = 4).
cells
and N43R-ras-expressing cells, respectively, to 133 ± 7 arbitrary
units (n = 44) in V38R-Ras-expressing cells (Table
II).
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Fig. 3.
CCK8-induced
Ins(1,4,5)P3 production in V38R-Ras- and
N43R-Ras-expressing cells. CHO-CCKA cells,
cotransfected with GFP and either N43R-Ras or V38R-Ras, were cultured
for 24 h. GFP and GFP+ cells were
separated by means of FACS and cultured for another 24 h. Cell
were stimulated with the indicated concentration of CCK8
for 20 s, after which the reaction was quenched by the addition of
trichloroacetic acid. The Ins(1,4,5)P3 content of the
extract was determined by isotope dilution assay. V38R-Ras expression
(A)and N43R-Ras expression (B) did not alter
CCK8-induced Ins(1,4,5)P3 production. The data
presented are the mean ± S.E. of three independent
measurements.
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Fig. 4.
CCK8-induced cytosolic
Ca2+ oscillations in CHO-CCKA cells.
CHO-CCKA cells, cotransfected with GFP and V38R-Ras, were
loaded with the fluorescent radiometric Ca2+ dye indo-1 and
monitored at 48 h post-transfection by means of high speed
confocal imaging microscopy. A, superfusion with 0.1 nM CCK8 was started at the indicated time. The
cell was stimulated in the absence of extracellular Ca2+.
Under this condition, the amplitude of the Ca2+
oscillations decreased gradually as a result of store depletion.
Eventually, the Ca2+ oscillations stopped. B,
the rate of Ca2+ increase during the rising phase of
the Ca2+ oscillation, indicated by the dotted
line, was decreased markedly during the later peaks (b)
compared with the first peak (a). This decrease is
compatible with a decreased Ca2+ content of the store.
Details on the kinetics of the first Ca2+ oscillation are
given under "Results."
cells (Table
II). As far as the width is concerned, this is in sharp contrast
with the findings for the single 100 nM-CCK8-induced Ca2+ transient (see
above). However, the kinetics of the 0.1 nM
CCK8-induced oscillatory Ca2+ rise appeared to
be completely different from that of the 100 nM
CCK8-induced single transient. Thus, although the amplitude of the first oscillation was only 0.8-fold lower, its width was 5.8-fold smaller (Table II).
values
0.44 ± 0.09 s (r2 = 0.99;
n = 4) and 0.39 ± 0.05 s
(r2 = 0.96; n = 3) for
V38R-Ras-expressing cells and corresponding GFP
cells,
respectively. These values were markedly higher than those obtained for
the 100 nM CCK8-induced single transient (0.12 s and 0.07 s for V38R-Ras-expressing cells and corresponding
GFP
cells, respectively).
cells which were considerably smaller than those
obtained for the decay phase of the 100 nM
CCK8-induced single transient (25.2 s and 31.2 s for
V38R-Ras-expressing cells and corresponding GFP
cells,
respectively). These differences are compatible with the idea that the
Ins(1,4,5)P3-operated Ca2+ release channels
remain open at 100 nM CCK8, leading to the
removal of all releasable Ca2+ by the action of the
(slower) PMCA, whereas these channels close rapidly at 0.1 nM CCK8, allowing the (faster) SERCA pump to
resequester the larger part of the released Ca2+ in the ER
during each oscillation.
, V38R-Ras, and
N43R-Ras, respectively).
View larger version (10K):
[in a new window]
Fig. 5.
Frequency of CCK8-induced
cytosolic Ca2+ oscillations in V38R-Ras- and
N43R-Ras-expressing cells. CHO-CCKA cells,
cotransfected with GFP and V38R-Ras, were loaded with the fluorescent
radiometric Ca2+ dye fura-2 and monitored at 48 h
post-transfection by means of digital imaging microscopy. The cells
were incubated in the presence of extracellular Ca2+ and
stimulated with 0.1 nM CCK8. The figure shows
that the oscillation frequency was decreased significantly in
V38R-Ras-expressing cells compared with N43R-Ras-expressing cells and
GFP cells. No difference was observed between
N43R-Ras-expressing cells and GFP
cells. The data
presented are the mean ± S.E. of 157 N43R-Ras-expressing cells,
318 V38R-Ras-expressing cells, and 307 GFP
cells. *,
p < 0.01.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells by FACS followed by Western blot analysis
revealed that GFP+ cells indeed expressed the HA-tagged
R-Ras protein.
) cells present on the same coverslip. Detailed
analysis of the size and morphology of the R-Ras-expressing cells
revealed that N43R-Ras and, to a lesser extent, V38R-Ras, caused cell
enlargement and rounding. This is in agreement with the observation
that inactivation of R-Ras by clostridial cytotoxins caused cell
rounding and detachment (32).
cells. This demonstrates that V38R-Ras
expression causes a marked reduction of the ER Ca2+
content. Moreover, V38R-Ras caused a significant increase in the rate
of Ca2+ rise, suggesting an increased Ca2+ leak
across the ER membrane. Finally, V38R-Ras expression increased the rate
of Ca2+ decay. This latter effect is compatible with an
accelerated ER Ca2+ release during the rising phase and
consequently reduced Ca2+ release during the decay phase,
resulting in a reduced slowing down of the rate of Ca2+
decay. Based on the data obtained with Tg we postulate that expression of V38R-Ras increases the ER Ca2+ leak, thereby decreasing
the steady-state ER Ca2+ content.
values of 0.07 s and 7.5 s for CCK8 and Tg, respectively). This
difference in rate of Ca2+ rise is in agreement with the
idea that CCK8 induces a significantly larger leak than Tg.
The down-stroke of the CCK8-induced Ca2+
transient consisted of a first phase of slow decay and a second phase
of fast decay to prestimulatory levels. The rate of Ca2+
decay during the second (fast) phase was markedly higher in
CCK8-stimulated cells (µ values of 30-60 s and 200 s for CCK8 and Tg, respectively). This result is compatible
with the idea that in these cells, because of a faster depletion of the
ER Ca2+ store, no Ca2+ is released during the
second (fast) phase of Ca2+ decay.
cells. This
substantiates our conclusion that V38R-Ras causes a reduction of the ER
Ca2+ content. Expression of V38R-Ras decreased rather than
increased the rate of Ca2+ rise during the 100 nM CCK8-induced single Ca2+
transient. This apparent paradox can be accounted for if it is assumed
that CCK8 induces a significantly larger leak than
V38R-Ras. Because in V38R-Ras-expressing cells the ER Ca2+
content is decreased, less Ca2+ will flow through the
CCK8-induced leak. Evidence that the
CCK8-induced leak is independent of the expression of
V38R-Ras is derived from the observation that the decay rate during the
second (fast) phase is virtually the same for V38R-Ras-expressing
cells, N43R-Ras-expressing cells, and GFP
cells.
value of 0.4 s)
was slower than that obtained with 100 nM CCK8.
When the channels close, Ca2+ is rapidly removed by the
concerted action of the SERCA and the PMCA. The Ca2+
removal rate (µ value of 5.3 s) was markedly faster than that obtained with 100 nM CCK8, demonstrating that
under oscillatory conditions Ca2+ is largely pumped back
into the ER. V38R-Ras expression did not alter the kinetics of the
CCK8-induced Ca2+ oscillations but
significantly reduced their frequency. The lack of effect of V38R-Ras
on the rate of Ca2+ rise and amplitude of the oscillatory
Ca2+ rises is most likely explained by the cytosolic
Ca2+ dependence of the SERCA pumping Ca2+ back
into the ER at a rate depending on the ambient Ca2+
concentration. But, although the ER Ca2+ content has no
effect on the kinetics of the oscillatory Ca2+ rises, it
does decrease their frequency (35, 36).
or N43R-Ras-expressing
cells. Interference with frequency-encoded Ca2+ signals
will lead to altered activation profiles of downstream effectors.
Previous work has shown that constitutively active V38R-Ras keeps
cellular integrins in an active state thus allowing attachment to
surfaces coated with integrin ligands (7). Importantly, the
cytoskeletal reorganizations that occur during integrin-induced cell
adhesion are controlled by cytosolic signals that cause periodic activation and inactivation of Rho GTPases. Recent evidence shows that
the Ca2+-dependent enzyme calpain cleaves RhoA
and that cleaved RhoA inhibits integrin-induced stress fiber assembly
and cell spreading (19). Because of the Ca2+ dependence of
calpain and previously reported effects of alterations in intracellular
Ca2+ concentration on integrin-mediated adhesion (20), it
is tempting to speculate that the periodic activation and inactivation
of RhoA is regulated by a frequency-encoded cytosolic Ca2+
signal. A reduction in frequency of this signal by V38R-Ras might lead
to reduced activation of calpain and, as a consequence, reduced cleavage of RhoA. Cell spreading will no longer be inhibited, and cell
detachment and migration will be inhibited.
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ACKNOWLEDGEMENTS |
---|
We thank Prof. Dr. J. L. Bos (Department of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Centre Utrecht, Utrecht University, The Netherlands) for kindly providing the R-Ras-interfering mutants and A. Pennings and G. Vierwinden (Department of Hematology, University Medical Center Nijmegen, Nijmegen University, The Netherlands) for technical assistance with the FACS analysis.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
¶ Supported by a grant from the Netherlands Organization for Scientific Research.
** To whom correspondence should be addressed: 160 Biochemistry NCMLS, University Medical Centre, Nijmegen University, P.O. Box 9101, Nijmegen NL-6500 HB, The Netherlands. Tel.: 31-24-361-4589; Fax: 31-24-354-0525; E-mail: P.Willems@ncmls.kun.nl.
Published, JBC Papers in Press, February 13, 2003, DOI 10.1074/jbc.M211256200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: R-Ras, Ras-like Ras; [Ca2+]i, intracellular calcium concentration; CCK8, C-terminal octapeptide of cholecystokinin; CHO, Chinese hamster ovary; DAG, 1,2-diacylglycerol; ER, endoplasmic reticulum; FACS, fluorescence-activated cell sorter; GAP, GTPase-activating protein; GFP, green fluorescent protein; HA, hemagglutinin; Ins(1, 4,5)P3, inositol 1,4,5-trisphosphate; PMCA, plasma membrane Ca2+-ATPase; SERCA, sarcoplasmic and ER Ca2+-ATPase; Tg, thapsigargin.
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