R - RAS ALTERS C A 2+ HOMEOSTASIS BY INCREASING THE C A 2+ LEAK ACROSS THE ENDOPLASMIC RETICULAR MEMBRANE

The involvement of R-Ras and intracellular Ca 2+ 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 Ca 2+ handling. Here we show that constitutively active V38R-Ras decreases the ER Ca 2+ content in a manner similar to the pro-apoptotic protein Bcl-2 and slows down the frequency of stimulus-induced periodic Ca 2+ rises. We propose that the reduction in ER Ca 2+ content may underly the anti-apoptotic effect of R-Ras Furthermore, we propose that the decrease in frequency of the stimulus-induced cytosolic Ca 2+ rises may inhibit stimulus-induced activation of calpain thus causing inhibition of cell detachment and migration and favoring integrin-mediated cell attachment and spreading.

These functions appear to be mediated by few, if any, of the signaling pathways taken by Ras (4,6,9,10,11). 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 anti-apoptotic Bcl-2 (14). Thusfar, 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). (19). These findings explain previously reported effects of alterations in intracellular Ca 2+ concentration on integrin-mediated adhesion (20). A role for Ca 2+ in apoptosis became apparent when it was shown that a modest reduction in endoplasmic reticulum (ER) Ca 2+ content prevented cell death (21,22).
The involvement of R-Ras and intracellular Ca 2+ in both programmed cell death and integrinmediated adhesion prompted us to investigate the possibility that R-Ras might exert its actions through an effect on cellular Ca 2+ handling. Here we show that constitutively active V38R-Ras decreases the ER Ca 2+ content in a manner similar to the pro-apoptotic protein Bcl-2 and slows down the frequency of stimulus-induced periodic Ca 2+ rises. We propose that the reduction in ER Ca 2+ content may underly the anti-apoptotic effect of R-Ras described by Suzuki and coworkers (16,17). Furthermore, we propose that the decrease in frequency of the stimulusinduced cytosolic Ca 2+ 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 CCK A receptor (CHO-CCK A ) has been described in detail elsewhere (23). CHO-CCK A cells were grown to confluence in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) in a humidified atmosphere of 5% CO 2 at 37°C. For transfection, cells were trypsinized (5x10 6 cells/300 µl) and electroporated (280 V, 975 µF) in the presence of 2 µg of plasmid pGFP-N1 (Clontech, Palo Alto, CA, USA) and 18 µg of either pMT2-HA-V38R-Ras or pMT2-HA-N43R-Ras (24). Subsequently, cells were seeded on a glass cover slip (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 and GFP-negative cells were separated by means of fluorescence activated cell sorting 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 polyvinylidine difluoride membranes (Immobilon P, Millipore, Bedford, MA, USA). 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. 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. Cover slips 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)   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 watersaturated diethyl ether. Subsequently, 75 µl was taken to which 2 µl KHCO 3 (50%) was added to increase the pH above 7.5. The inositol 1,4,5-trisphosphate content of the extract was determined by isotope dilution assay as previously described (28).

Data analysis
Data was analyzed using Origin Pro 6.1 (Microcal, Northampton, MA, USA) and Image Pro

Co-expression of R-Ras and GFP in CHO cells
CHO cells expressing the rat CCK A receptor (CHO-CCK A cells) were co-transfected with GFP and either constitutively active HA-V38R-Ras or dominant negative HA-N43R-Ras. At 48 hrs post-transfection, cells were loaded with the fluorescent Ca 2+ indicator indo-1 and visualized by confocal microscopy. Figure 1A shows a representative example of a cluster of GFP-positive (GFP + ) cells co-transfected with V38R-Ras. When identical hardware settings were used during acquisition, no significant differences in GFP intensity between V38R-Ras-and N43R-Rastransfected cells were observed ( Table 1). The corresponding indo-1 image (Fig. 1B) shows a second cluster of GFP-negative (GFP -) cells. Importantly, these GFPcells, present on the same cover slip as the GFP + cells, were used as a control for the effect of R-Ras expression on cellular Ca 2+ handling. Under the experimental conditions used, no cross-talk between GFP and indo-1 fluorescence signals was observed.
To demonstrate that GFP expression reports expression of R-Ras, GFP + and GFPcells were separated by fluorescence-activated cell sorting (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. Figure 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 GFPand GFP + cells. Table 1 shows that the cross-sectional area was significantly increased in N43R-Ras-expressing cells (p<0.01). To detect more subtle morphological alterations we calculated the 'formfactor' F (perimeter 2 /4·π·area). For a round cell, the numerical value of F is one. The data presented show that F was significantly decreased in the R-Ras-expressing cells ( Table 1, p<0.01).
Importantly, however, F was significantly smaller in N43R-Ras-expressing cells as compared to V38R-Ras-expressing cells (p<0.05). Taken together, these findings demonstrate that CHO cells which express N43R-Ras and, to a lesser extent, V38R-Ras are larger and rounder than GFPcells.

V38R-Ras alters the kinetics of the thapsigargin-induced Ca 2+ rise
To assess possible effects of R-Ras on cellular Ca 2+ handling, CHO-CCK A cells transiently expressing either N43R-Ras or V38R-Ras were treated with thapsigargin (Tg), a specific inhibitor of the sarco-and endoplasmic reticulum Ca 2+ -ATPase (SERCA). The cells were loaded with indo-1 and the Tg-induced changes in cytosolic Ca 2+ concentration were monitored by means of high-speed confocal microscopy. Tg increases the cytosolic Ca 2+ concentration by preventing the active re-uptake of the Ca 2+ ions that continuously leak out of the endoplasmic reticulum (ER) Ca 2+ store. In the absence of extracellular Ca 2+ , these Ca 2+ ions are removed from the cytosol by the action of the plasma membrane Ca 2+ -ATPase (PMCA). and 169 ± 15 AU.s (n = 10) in GFPcells and N43R-ras-expressing cells, respectively, to 93 ± 8 AU.s (n = 24) in V38R-Ras-expressing cells (Table 1).

V38R-Ras alters the kinetics of the CCK 8 (100 nM)-induced single Ca 2+ rise
In CHO-CCK A cells, CCK-induced cytosolic Ca 2+ signals arise from Ins(1,4,5)P 3 -mediated Ca 2+ release from the endoplasmic reticulum (23), paralleled by capacitative Ca 2+ entry across the plasma membrane (29). To investigate whether R-Ras affects stimulus-induced Ca 2+ release from the endoplasmic reticulum, cells were stimulated with 100 nM CCK 8 in the absence of extracellular Ca 2+ . At this concentration, CCK 8 evokes a single Ca 2+ transient that is not followed by repetitive Ca 2+ transients (Ca 2+ oscillations). Figure 2 (D and F) shows that both GFP + and GFPcells displayed a single Ca 2+ 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.
However, this decrease was not statistically significant. Detailed analysis of the rate of Ca 2+ rise revealed a τ value that was increased for V38R-Ras-expressing cells (τ = 0.12 ± 0.01 s; R 2 = 0.99; n = 4) as compared to the corresponding GFPcells (τ = 0.07 ± 0.01 s; R 2 = 0.99; n = 4) present on the same cover slip (Fig. 2E). This demonstrates that the rate of Ca 2+ 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 Ca 2+ transient were identical between the GFP + and GFPcells. This shows that GFP expression in itself had no effect on the shape of the CCK 8 -induced Ca 2+ transient. Importantly, 100 nM CCK 8 completely released the thapsigargin-sensitive intracellular Ca 2+ store (Fig. 2H).  (Table 1). Analysis of the first oscillatory Ca 2+ rise revealed no significant differences in amplitude and width between V38R-Ras-expressing cells and GFPcells ( Table 1)

Reduced frequency of stimulus-induced repetitive Ca 2+ rises in V38R-Ras-expressing cells
To demonstrate a possible effect of V38R-Ras-expression on the temporal characteristics of the cytosolic Ca 2+ oscillations, we stimulated the cells with 0.1 nM CCK 8 in the presence of 1 mM extracellular Ca 2+ . The latter prevented depletion of the endoplasmic reticulum Ca 2+ store and allowed recording of Ca 2+ oscillations during prolonged periods of time. Cells were loaded with fura-2 and video-imaging microscopy was used to monitor the CCK 8 -induced Ca 2+ changes. Figure 5 shows that the oscillation frequency was significantly reduced 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).

Evidence in the literature has implicated both R-Ras and intracellular Ca 2+ in programmed
cell death (1,12,13,16,17,21,31) 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 Ca 2+ handling. To study the effects of R-Ras, CHO cells stably expressing the cholecystokinin (CCK)-A receptor were cotransfected with GFP and either constitutively active V38R-Ras or dominant negative N43R-

Ras. Separation of GFP + and GFPcells by fluorescence-activated cell sorting 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 cover slip immediately after transfection and grown for 48 h. This procedure provided us with the unique opportunity to simultaneously monitor the cytosolic Ca 2+ changes in R-Ras-expressing (GFP + ) cells and the corresponding sham-transfected (GFP -) cells present on the same cover slip. 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 Ca 2+ content and the frequency of the CCK 8induced cytosolic Ca 2+ rises
Cells transiently expressing either V38R-Ras or N43R-Ras were loaded with indo-1 or fura-2 and the changes in cytosolic free Ca 2+ concentration were monitored by means of high-speed confocal or conventional video-imaging microscopy, respectively. To start with, the cells were treated with Tg, a potent and selective inhibitor of the Ca 2+ pump of the ER Ca 2+ store (SERCA). Inhibition of this pump prevents re-uptake of Ca 2+ ions that continuously leak out of the ER into the cytosol. We have previously shown that in the absence of active Ca 2+ pumping this Ca 2+ leak process is adequately described by a monoexponential equation This indicates that at this concentration it causes the sustained opening of the inositol 1,4,5trisphosphate-operated Ca 2+ release channels. The rate of Ca 2+ rise obtained with CCK 8 was considerably higher than that obtained with Tg (τ values of 0.07 s and 7.5 s for CCK 8 and Tg, respectively). This difference in rate of Ca 2+ rise is in agreement with the idea that CCK 8 induces a significantly larger leak than Tg. The down-stroke of the CCK 8 -induced Ca 2+ transient consisted of a first phase of slow decay and a second phase of fast decay to prestimulatory levels. The rate of Ca 2+ decay during the second (fast) phase was markedly higher in CCK 8 -stimulated cells (µ values of 30-60 s and 200 s for CCK 8 and Tg, respectively). This result is compatible with the idea that in these cells, due to a faster depletion of the ER Ca 2+ store, no Ca 2+ is released during the second (fast) phase of Ca 2+ decay.
The integrated area underneath the cytosolic Ca 2+ peak was significantly decreased in V38R-

Ras-expressing cells as compared to N43R-Ras-expressing cells and GFPcells. This
substantiates our conclusion that V38R-Ras causes a reduction of the ER Ca 2+ content.
Expression of V38R-Ras decreased rather than increased the rate of Ca 2+ rise during the under oscillatory conditions Ca 2+ is largely pumped back into the ER. V38R-Ras expression did not alter the kinetics of the CCK 8 -induced Ca 2+ oscillations but significantly reduced their frequency. The lack of effect of V38R-Ras on the rate of Ca 2+ rise and amplitude of the oscillatory Ca 2+ rises is most likely explained by the cytosolic Ca 2+ dependence of the SERCA pumping Ca 2+ back into the ER at a rate depending on the ambient Ca 2+ concentration. But, whereas the ER Ca 2+ content has no effect on the kinetics of the oscillatory Ca 2+ rises it decreases their frequency (35,36).

Possible implications of the V38R-Ras-induced reduction in ER Ca 2+ content
The present study provides evidence that V38R-Ras expression reduces the ER Ca 2+ content by increasing the passive Ca 2+ leak across the ER membrane. A similar observation was reached following overexpression of the anti-apoptotic 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 Ca 2+ leak rather than decreasing the activity of the ER Ca 2+ 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 Ca 2+ content, realized by SERCA overexpression, increased spontaneous apoptosis (39), strengthens the idea that the anti-apoptotic action of Bcl-2 is mediated through its effect on the ER Ca 2+ content. In this context, the present finding that V38R-Ras decreases the ER Ca 2+ content provides a good explanation for the anti-apoptotic 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 upregulation of Bcl-2 (40.41) and the caspase inhibitor cIAP-2 (41). Based on these findings, it is temping to speculate that activation of R-Ras promotes the phosphatidylinositol 3-kinasemediated upregulation of Bcl-2, which, in turn, causes a decrease in ER Ca 2+ content by increasing the ER Ca 2+ 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 pro-apoptotic effect of R-Ras.
Lowering of the ER Ca 2+ content has been demonstrated to trigger the process of capacitative Ca 2+ entry across the plasma membrane (42). In case the reduced ER Ca 2+ content is due to an increased ER Ca 2+ leak, this would lead to an elevation of the cytoplasmic Ca 2+ concentration.
However, in the case of Bcl-2 overexpression it has been demonstrated that the capacitative Ca 2+ entry was also down-regulated thus preventing a sustained increase of the resting cytosolic Ca 2+ concentration (37).

Possible implications of the V38R-Ras-induced decrease in frequency of stimulus-induced cytosolic Ca 2+ oscillations
V38R-Ras expression did not significantly alter the amplitude and duration of the CCK 8 -induced cytosolic Ca 2+ oscillations. This means that R-Ras does not signal to its downstream effectors through modulation of the amplitude and/or duration of the cytosolic Ca 2+ rises. However, the frequency of the cytosolic Ca 2+ oscillations appeared to be reduced by 30% in V38R-Rasexpressing cells. This is in agreement with theoretical studies predicting a decrease in oscillation frequency when the ER Ca 2+ content is reduced at a constant inositol 1,4,5-trisphosphate concentration (35,36). In accordance with this idea, measurement of the CCK 8 -stimulated production of inositol 1,4,5-trisphophate revealed no differences between V38R-Ras-expressing cells and GFPor N43R-Ras-expressing cells. Interference with frequency-encoded Ca 2+ 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 by guest on March 24, 2020 http://www.jbc.org/ Downloaded from 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 Ca 2+ -dependent enzyme calpain cleaves RhoA and that cleaved RhoA inhibits integrininduced stress fiber assembly and cell spreading (19). Because of the Ca 2+ -dependence of calpain and previously reported effects of alterations in intracellular Ca 2+ 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 Ca 2+ 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 Ca 2+ leak across the endoplasmic reticulum membrane thus decreasing both the Ca 2+ content of this intracellular Ca 2+ store and, as a consequence, the frequency of the stimulus-induced oscillatory Ca 2+ rises.
We propose that the reduction in endoplasmic reticulum Ca 2+ content may underly the antiapoptotic effect of R-Ras described by Suzuki and co-workers (16,17). Furthermore, we propose that the decrease in frequency of the stimulus-induced cytosolic Ca 2+ rises may inhibit calpain activation, which, in turn, leads to inhibition of cell detachment and migration thus favouring integrin-mediated cell attachment and spreading.       2 /4·π·area] and has a minimal value of one for a perfect circular shape. The amplitude was defined as Rmax/Ro with Ro and Rmax being the pre-stimulatory and maximal indo-1 ratio respectively. Legend: $ only first transient, a compared to control, b compared to V38R-Ras cells, c compared to N43R-Ras cells, d compared to control cells treated with 100 nM CCK 8 , *: p< 0.05, **: p<0.01, ***: p<0.001.