R-Ras Alters Ca2+ Homeostasis by Increasing the Ca2+ Leak across the Endoplasmic Reticular Membrane*

Werner J. H. KoopmanDagger §, Remko R. BoschDagger §, Sjenet E. van Emst-de VriesDagger , Marcel Spaargaren||, Jan Joep H. H. M. De PontDagger , and Peter H. G. M. WillemsDagger **

From the Dagger  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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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- 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.

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- 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).

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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- 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.


                              
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Table I
Cell morphology in control and R-Ras-expressing cells

To demonstrate that GFP expression reports expression of R-Ras, GFP+ and GFP- 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.

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- 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·pi ·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.

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- 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.


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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.

The duration of the Tg-induced Ca2+ transient was shortened significantly from 190 ± 12 s (n = 38 cells) in GFP- 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).

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- 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.

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 - A2)/(1 + exp((x - x0)/tau ))) (Fig. 2B). The time constant tau , 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.

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-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.

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- cells and N43R-ras-expressing cells, respectively, to 93 ± 8 arbitrary units (n = 24) in V38R-Ras-expressing cells (Table II).


                              
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Table II
Calcium dynamics in control and R-Ras-expressing cells

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- 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.

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- 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 tau  value that was increased for V38R-Ras-expressing cells (tau  = 0.12 ± 0.01 s; r2 = 0.99; n = 4) compared with the corresponding GFP- cells (tau  = 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).

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- 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).

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- cells and N43R-ras-expressing cells, respectively, to 133 ± 7 arbitrary units (n = 44) in V38R-Ras-expressing cells (Table II).

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.


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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.

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.


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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."

Analysis of the first oscillatory Ca2+ rise revealed no significant differences in amplitude and width between V38R-Ras-expressing cells and GFP- 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).

Analysis of the rate of Ca2+ rise revealed tau  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).

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- 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.

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-, V38R-Ras, and N43R-Ras, respectively).


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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

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- cells by FACS followed by Western blot analysis revealed that GFP+ cells indeed expressed the HA-tagged R-Ras protein.

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-) 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).

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- 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.

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 (tau  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.

The integrated area underneath the cytosolic Ca2+ peak was decreased significantly in V38R-Ras-expressing cells compared with N43R-Ras-expressing cells and GFP- 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.

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 (tau  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).

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- 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.

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.

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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