platelet-derived growth factor-mediated Ca'' mobilization (ras/g proteins/phospholipase)

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1 Proc. Nail. Acad. Sci. USA Vol. 85, pp , June 1988 Cell Biology NIH-3T3 cells transformed by the EJ-ras oncogene exhibit reduced platelet-derived growth factor-mediated Ca'' mobilization (ras/g proteins/phospholipase) C. W. BENJAMIN*, J. A. CONNORt, W. G. TARPLEYt, AND R. R. GORMAN* Departments of *Cell Biology and tcancer Research, The Upjohn Company, Kalamazoo, MI 49001; and tat&t Bell Laboratories, Murray Hill, NJ Communicated by Philip Needleman, February 22, 1988 ABSTRACT NIH-3T3 cells transformed by the EJ-ras oncogene synthesize only 10-15% as much inositol 1,4,5- trisphosphate (InsP3) as control cells after stimulation with platelet-derived growth factor (PDGF). This is despite the fact that the basal (unstimulated) levels of InsP3 synthesized in control and EJ-ras-transformed cells are not significantly different. Using the fluorescent indicator fura-2 and digitalimaging techniques, we have visualized and quantified changes in intracellular Ca2+ concentrations in control and EJ-rastransformed NIH-3T3 cells in response to PDGF. Within 3 min after exposure of control cells to PDGF, intracellular Ca2 + levels are increased 3- to 9-fold, paralleling the increase in InsP3. In contrast, the majority (>90%) of the EJ-rastransformed cells show no increase in Ca2+ levels after PDGF exposure and the few that did respond exhibited only a small transient increase. Pronounced differences in the intracellular localization of Ca2+ increases in control and the responding EJ-ras-transformed cells were also observed. Despite the inhibition of InsP3 synthesis and subsequent Ca2+ mobilization, the EJ-ras-transformed cells respond mitogenically to PDGF. These data do not support the hypothesis that the EJ-ras gene product (p21) stimulates a phosphatidylinositol 4,5-bisphosphate-specific phospholipase C in NIH-3T3 cells; instead they suggest that the EJ-ras p21 may uncouple the PDGF receptor from phospholipase C resulting in inhibition of PDGFstimulated activity of phospholipase C, InsP3 synthesis, and Ca2+ mobilization. Platelet-derived growth factor (PDGF) stimulates phospholipase C (PLC), resulting in hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2), formation of inositol 1,4,5- trisphosphate (InsP3) and diacylglycerol (acyl2gro), and an increase in Ca2 +fluxes in mouse fibroblasts (1). This cascade of events has been linked to c-fos and c-myc induction and subsequent PDGF-stimulated mitogenesis (2-5). The regulation of PLC is thought to be controlled by guanine nucleotide regulatory proteins (G proteins) (6). The recent finding that the ras gene product, p21, shares sequence similarity with the G proteins (7), and the enhanced growth characteristics of ras-transformed cells, has suggested that p21 might modulate PLC activity in eukaryotic cells. A sustained activation of PLC is associated with stimulation of growth (8, 9), and the p21 encoded by transforming ras genes may accelerate PLC activity or enhance the coupling between PLC and growth factor receptors (10-12). However, reports from several laboratories have yielded conflicting results concerning PLC activity in ras-transformed cells. Wakelam et al. (12) found no significant change in basal InsP3 levels in control cells when compared to cells expressing high levels of N-ras p21, while others reported a small increase in InsP3 or acyl2gro levels after ras transformation (10, 13), suggesting an in- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. creased PLC activity (14, 15). Recently, we found that NIH-3T3 cells expressing high levels of the EJ-ras oncogene display markedly reduced PDGF-stimulated PLC and phospholipase A2 activities as determined by InsP3 and prostaglandin E2 synthesis as well as phospholipid mass and specific activity measurements (16). Subsequent work from our laboratory has shown that EJ-ras-transformed cells have phospholipase A2 activity equivalent to control cells if the PDGF receptor-plc complex is bypassed (17). These data suggested that the EJ-ras p21 uncouples the PDGF receptor from a PtdInsP2-specific PLC. This work has been confirmed by Parries et al. (18) who also found markedly reduced PDGF-stimulated inositol phospholipid turnover in rastransformed fibroblasts. Because of the relationship between PDGF-stimulated InsP3 synthesis, Ca2+ mobilization, and growth, we have examined Ca2+ mobilization in EJ-rastransformed cells under conditions where PDGF-stimulated InsP3 synthesis is attenuated, but PDGF can still stimulate mitogenesis (16). In this report we show by digital-imaging techniques and fura-2 fluorescence measurements that PDGF-stimulated Ca2 + mobilization is diminished in EJ-rastransformed cells, indicating that the entire sequence of PDGF-stimulated PtdInsP2 hydrolysis, InsP3 synthesis, and Ca2+ mobilization is inhibited. These data appear to dissociate inositol phospholipid turnover and Ca2+ mobilization from PDGF-stimulated mitogenesis in these transformed cells. MATERIALS AND METHODS Materials were obtained as follows: Dulbecco's modified Eagle's medium (DMEM), fetal calf serum, and trypsin/ EDTA in Hanks' balanced salt solution (HBSS) were from Irvine Scientific; highly purified PDGF was from Oxford Biomedical Research (Oxford, MI) or PDGF was purified according to Raines and Ross (19); [3H]inositol phosphate (InsP), inositol 1,4-bisphosphate (InsP2), InsP3, and myo- [3H]inositol were from Amersham; and fura-2 acetoxymethyl ester (fura-2/am) was from Molecular Probes (Junction City, OR), Plasma-derived serum (PDS) was prepared as described by Habenicht et al. (20). Cell Culture and DNA Transfections. NIH-3T3 cells were maintained in DMEM/10% calf serum (EC-10 medium). Plasmid pej, a pbr322 derivative that carries the EJ human bladder carcinoma oncogene was transfected into NIH-3T3 cells as described (17). Expression of the EJ-ras gene in transfected cells was confirmed by immunoblotting (21). Twelve to 24 hr prior to an experiment, EC-10 medium was replaced with DMEM/1.25% PDS. This protocol was used Abbreviations: InsP3, inositol 1,4,5-trisphosphate; InsP2, inositol 1,4-bisphosphate; InsP, inositol phosphate; PtdInsP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; PDGF, plateletderived growth factor; acyl2gro, diacylglycerol; PDS, plasmaderived serum; fura-2/am, fura-2 acetoxymethyl ester.

2 4346 Cell Biology: Benjamin et al. for all experiments involving InsP3 determination, Ca2 + flux and mitogenesis. HPLC Analysis of Inositol Phospholipids. Levels of watersoluble inositol phosphates were determined by HPLC (22). Cells were grown in EC-10 medium in 35-mm culture wells to about 75% confluency, and then 1.0 ml of DMEM/1.25% PDS containing 10 GCi (1 Ci = 37 GBq) of myo-[3h]inositol was added to each well, and the cells were incubated at 370C for 24 hr. The cells were washed three times with HBSS and incubated for 30 min at 370C in 1.0 ml of HBSS/10 mm LiCl. The reactions were terminated by addition of S ml of 15% trichloroacetic acid. After ether extraction, the aqueous samples were subjected to HPLC and the levels of [3H]inositol phosphates were determined (22). Digital Imaging and Quantitation of Intracellular Ca2+. NIH-3T3 cells were plated onto no. 1 glass coverslips coated with poly(d-lysine) and incubated for hr with 1.25% PDS-containing medium. The cells were loaded with the Ca2+ indicator fura-2/am (6 um) (23) by incubation for min at 370C in serum-free DMEM. The cells were then washed with DMEM and incubated for at least 20 min to allow deesterification of the indicator. Intracellular Ca2+ levels were determined by taking the ratio of fura-2 fluorescence at 340 and 380 nm. Details of the experimental method have been published (23-27). RESULTS Exposure of control myo[3h]inositol-labeled NIH-3T3 cells to PDGF at 10 ng/ml results in marked stimulation of [3H]InsP3 synthesis (Fig. 1). A significant increase in InsP3 levels is observed by 3 min (237 cpm), and the response is maximal at 10 min. InsP and InsP2 synthesis lag behind InsP3 synthesis and are still elevated 15 min after PDGF exposure (data not shown). An essentially identical InsP3 time course has been reported in PDGF-stimulated Swiss mouse 3T3 cells (28). When EJ-ras-transformed cells are exposed to PDGF under identical conditions, InsP3 synthesis is only 2-14% of that observed in control cells. Moreover, the increase in InsP3 levels is transient in the ras-transformed cells, lasting only a few minutes (Fig. 1). Similarly, only slight increases in InsP and InsP2 levels were measured in the EJ-rastransformed cells throughout the time course (data not shown). These data indicate that PDGF-stimulated PLC activity is inhibited in EJ-ras-transformed cells. CLV X TIME IN MINUTES FIG. 1. Time course of PDGF-stimulated InsP3 synthesis. NIH-3T3 cells were grown in EC-10 medium. Then, the medium was removed and replaced with 1.0 ml of DMEM 1.25% PDS containing 10 ACi of myo-[3h]inositol. After 24 hr the cells were washed and exposed to PDGF at 10 ng/ml, and ['H]InsP3 synthesis was quantitated. Data are reported as mean + SEM of triplicate samples and are representative of three confirmatory experiments. 0, Control cell InsP3 levels; o, EJ-ras-transformed cell InsP3 levels. A direct consequence of PDGF-stimulated PLC activation is Ca2" mobilization (1). Measurements on single cells and populations of cells using fura-2 have shown an increase in intracellular Ca2+ and a delayed acidification in response to PDGF (29-32). In principle then, reduced PDGF-stimulated PLC activity might result in diminished changes in intracellular Ca2+ since InsP3 is not synthesized. Consequently, we quantitated the time course of intracellular Ca2+ levels in control and EJ-ras-transformed cells exposed to PDGF. The typical response in control NIH-3T3 cells exposed to PDGF is shown in Fig. 2, in which Ca2+ levels are mapped in false color with blue signifying low and red, high. The distribution of Ca2 + in unstimulated serum-starved cells indicates pronounced nonuniformities in Ca2+ concentration across individual cells, with the nuclear region being significantly lower than cytoplasmic regions (Fig. 2A). The Ca2+ levels in these same cells 15 s after the addition of PDGF at 10 ng/ml are shown in Fig. 2B; there was an immediate nm decrease in Ca2+ levels. Only in rare cases did Ca2+ levels show significant increases during the first min after addition of PDGF. Fig. 2 C and D shows the development of the Ca2' response at 2.5 and 3 min, respectively; during this time, clear "hot spots," developed where an up to 6-fold increase in Ca2+ concentration was measured around the perimeter ofthe nucleus, corresponding to the regions where prestimulus levels were highest (Fig. 2A). The response then swept into the nuclear region as shown in Fig. 2 E and F, taken 4 and 4.5 min after exposure to PDGF; during this time the nuclear region showed the highest Ca2+ levels (approaching 600 nm), a characteristic feature of the responses in control cells. By 7 min the response had waned in both cells to about 200 nm Ca2 + (Fig. 2G) and by 12 min Ca2+ levels were nm below the prestimulus values; such undershoots were commonly observed. It was not unusual for Ca2+ levels to oscillate in cells following exposure to PDGF. These oscillations were observed in the 5- to 15-min period following PDGF exposure and had periods of min. The two cells illustrated here had no oscillatory activity. There is a large difference in the magnitude of the response (about 2-fold) in the two cells of Fig. 2, reflecting the heterogeneity of responses observed. Ca2" levels at three locations in each cell are plotted in Fig. 2H to include additional time values not shown pictorially. Exact locations where the levels were measured are indicated in Fig. 2A. The response to PDGF at 10 ng/ml in the EJ-rastransformed cells was significantly different. Time sequences in two fields of these cells are shown in Fig. 3A and B. Fig. 3A shows five cells (reading left to right) before stimulation, 10 s after stimulation, at 1 min (corresponding to the peak of the response), and at 11 min, when the cells had recovered from exposure to PDGF. It should be emphasized that no large increase in fluorescence could be detected at any point during the 11 min. Fig. Proc. Natl. Acad. Sci. USA 85 (1988) 3C shows that Ca2+ levels rise to nm in the cells in response to PDGF, a response that is only about 10% of that of control cells, more transient, and relatively less in the immediate nuclear area. Although EJ-ras-transformed cells showed a greatly attenuated PDGFstimulated Ca2' mobilization, they did display a PDGF mitogenic response (Table 1). After culture in PDS, ['H]thymidine incorporation was markedly stimulated by PDGF in both control and EJ-ras-transformed cells with similar degrees of stimulation, 6.32-fold for control cells and 5.08-fold for EJ-ras-transformed cells. Similar data were obtained when actual cell counts were made (16). Fig. 3B is taken from a different coverslip of cells than Fig. 3A and illustrates the limiting ends of the responses in EJ-ras-transformed cells-i.e., no response and a large Ca2" increase in one cell, similar to the response of control cells.

3 Cell Biology: Benjamin et al. Proc. Natl. Acad. Sci. USA 85 (1988) nm 475 nm 20 pm 250 nm J 80 nm For this series, the color map is expanded to, accommodate the larger dynamic range of the response, since the resting levels in these cells are higher than those in Fig. 3A: Reading from left to right, a Nomarski image of the cells is shown to indicate the positions of the nuclei in the cells; Ca2+ levels in unstimulated cells; the response to PDGF at 4 min, when Ca2+ levels have increased almost everywhere except the nuclear area; and the maximum response in a single cell, with the nuclear area nearly as high as the rest of the cell. The single cell that responded to PDGF had completely recovered by 7 min after stimulation (data not shown). It should be emphasized that the remaining cells in the field responded only slightly to PDGF. The actual intracellular Ca2" levels in the responder and two nonresponders are shown in Fig. 3D. These data highlight the fact that, although a few EJ-rastransformed cells respond to PDGF, they do so more transiently than control cells, and any response in such cells was unusual. A large Ca2" increase in an EJ-ras-transformed cell such as that shown in Fig. 3B was observed in only 8 of 109 cells examined (7%). The majority of EJ-ras-transformed cells (about 75%) failed to respond at all to PDGF and only about 16% produced the limited response illustrated in Fig. 3A. All of the EJ-ras-transformed cells that gave large responses showed prestimulus Ca2+ levels in the extranuclear areas that were above the population average. This behavior was in marked contrast to the control cells where 100% of the cells responded to PDGF. It is appropriate to note that there was no significant difference in basal resting Ca2+ levels in control and EJ-ras cells. Fig. 3B illustrates an important point regarding fura-2 behavior in the nuclear environment; that is, it could be argued that fura-2 in the nucleus or some perinuclear envelope was merely unresponsive to Ca2" changes. The fact that we have observed occasional large nuclear signals in EJ-rastransformed cells implies that modified fura-2 characteristics H O 200 oo A FIG. 2. Digital imaging of PDGF-stimulated intracellular Ca2" levels in control NIH-3T3 cells. (A) Resting cells in DMEM containing 1.8 mm Ca2". (B-G) Cells 15 s and 2.5, 3.0, 4.0, 4.5, and 12 min, respectively, after PDGF exposure. (H) Actual intracellular Ca2+ levels. Ca2+ levels were monitored at two locations in the cytoplasm and one location in the nucleus in each cell. e and o, The righthand cell; * and A, the lefthand cell. * and *, nuclear Ca2" levels; o and, cytoplasmic Ca2" levels. do not explain the differences observed in EJ-ras-transformed cells. We have also used the Ca2" ionophore ionomycin on three sets of EJ-ras-transformed cells (34 cells total) that gave responses similar to those shown in Fig. 3B. Addition of 2 AM ionomycin to the cells sent the indicator ratio to its limiting high value in all regions of all cells within s, showing that the indicator was responsive in all parts of the cells (data not shown). Fig. 3B also shows the inadequacy of making averaged measurements on populations of cells, as is the case in conventional spectroscopy or 45Ca flux studies. A population average using either technique would have reported a very minor Ca2 + change due to the large percentage of unresponsive cells, whereas in actuality some of the cells gave a large response. DISCUSSION These experiments document an interruption of PDGF signal transduction in mouse fibroblasts transformed by the EJ-ras oncogene. Our data suggest that one function of the EJ-ras p21 may be to block PDGF-stimulated PLC activation, InsP3 synthesis, and consequent elevation of intracellular Ca2+ levels. The results of the use of the fluorescent probe fura-2 to quantify and visually observe the distribution of intracellular Ca2 + levels in intact NIH-3T3 cells correlates well with our measurements of the levels of PDGF-stimulated watersoluble inositol phosphate(s) synthesized in these cells. Control cells, which are stimulated by PDGF to synthesize InsP3, showed prolonged and marked elevation in intracellular Ca2 +levels. Importantly, the rise in InsP3 levels always preceded the elevation of intracellular Ca2+ levels and the first increases in Ca2 + were observed in the cytoplasm, with later, more profound localization in the nucleus. In contrast, in EJ-ras-transformed cells, the lack of measurable PDGF-

4 4348 Cell Biology: Benjamin et al. Proc. Natl. Acad. Sci. USA 85 (1988) C E.a 300r 15 plm nm 20 Fm _...I_ ilr-s4:g =t-h L = = =: ---=-=Z t- - to o i nm D E 2 e3 500 r stimulated InsP3 synthesis was accompanied by a severely reduced or absent increase in intracellular Ca2 + levels. In the few transformed cells that did respond to PDGF, the increases in intracellular Ca2 + were of reduced magnitude and shorter duration. It should be emphasized that, although PDGF-stimulated PLC activation, InsP3 synthesis, and Ca2" accumulation is attenuated in EJ-ras-transformed cells, these cells still grow in response to PDGF (16). In fact, PDGFstimulated [3H]thymidine incorporation is nearly identical in Table 1. PDGF-stimulated [3H]thymidine incorporation cpm/3 x 104 cells EJ-ras Control transformed Basal 31,903 ± 4,229 46,826 ± 2,770 PDGF stimulated 201,746 ± 19, ,047 ± 7,483 Cells were incubated in 35-mm wells for hr with medium containing 1.25% PDS and then for an additional 18 hr with fresh medium/1.25% PDS with or without PDGF at 10 ng/ml. Then, they were exposed to [3H]thymidine at 2.0,uCi/ml at 37TC, after which the medium was aspirated, and the cells were washed with two 1-ml portions of ice-cold 5% trichloroacetic acid. The acid-insoluble material was solubilized in 0.25 M NaOH (0.8 ml), and radioactivity was determined by liquid scintillation spectroscopy [ ==+a I '% FIG. 3. Digital imaging of PDGFstimulated intracellular Ca2l levels in EJ-ras-transformed NIH-3T3 cells. (A) EJ-ras-transformed cells were loaded with fura-2/am and, then, at time 0, exposed to PDGF at 10 ng/ml and followed for 11 min. Reading left to right: resting EJ-ras-transformed cells, cells 10 s after PDGF exposure, cells 1 min after PDGF exposure, cells 11 min after PDGF exposure. (B) EJ-rastransformed cells were loaded with fura-2/am as described. Reading left to right; a Nomarski image of the resting cells, Ca2" levels in resting cells, cells 4 min after addition of PDGF at 10 ng/ml, cells 5 min after PDGF exposure. (C) Actual intracellular Ca2" levels following PDGF stimulation were measured in nuclear and cytoplasmic regions of four of the cells shown in A., Nuclear Ca2+ levels,- - -, cytoplasmic Ca2" levels. *, The bottom left cell; c, the adjacent cell; o, the cell second from the top; A, the uppermost cell. (D) Actual intracellular Ca2" levels were measured in the one responding cell and two nonresponding cells from B. A, o, o, cytoplasmic Ca2+ levels; A, *, *, nuclear Ca2+ levels. A and A, the responding cell; o, *, o, and *, the nonresponding cells. control and EJ-ras-transformed cells. These data, as well as direct binding experiments, indicate the presence of functional growth-promoting PDGF receptors in the EJ-rastransformed cells (16). Thus, our data suggest that PDGFstimulated inositol phospholipid turnover and elevation of intracellular Ca2+ levels can be dissociated from the mitogenic activity of PDGF in EJ-ras DNA-transformed cells. The source of the intracellular Ca2+ following PDGF stimulation is not established. Although most reports suggest that InsP3 releases Ca2" from intracellular stores (33-35), we have been unable to elevate intracellular Ca2+ levels with PDGF in the presence of EGTA and Ca2" -free medium. This is not likely due to an interference with PDGF binding, since EGTA-treated cells exposed to PDGF that are subsequently washed free of PDGF showed rapid elevation of intracellular Ca2+ levels following the addition of Ca2+ to the medium (data not shown). It is tempting to speculate that the PDGF response in NIH-3T3 cells is analogous to that in the sea urchin egg, where InsP3 is converted to inositol 1,3,4,5- tetrakisphosphate, which subsequently controls entry of Ca2+ from the outside of the cell (36). The possible modulation of PLC in ras-transformed cells has been the focus of much recent experimentation, but the data from these studies have been confusing and contradic-

5 Cell Biology: Benjamin et al. tory. Initially, Fleischman et al. (10) using NRK cells reported a 23-29% increase in acyl2gro levels but no significant change in InsP3 levels; however, total water-soluble inositol phospholipids were slightly elevated (10). That paper was followed closely by Wolfman and Macara (14), who also found elevated acyl2gro levels in ras-transformed fibroblasts without any change in either InsP3 or total water-soluble inositol phospholipids. The specificity of these acyl2gro increases in transformed cells is, however, now in question since Preiss et al. (15) have found elevated acyl2gro levels in both ras and sis-transformed NRK cells. Furthermore, Lacal et al. (13) have reported elevated acyl2gro and InsP3 levels in Xenopus oocytes microinjected with EJ-ras p21 but not with cellular p21 and concluded that an increased level of inositol phospholipid is an early event after transforming p21 microinjection. However, a subsequent study from this group using fibroblasts transformed by Ha-ras found an elevation in acyl2gro level and no change in inositol phosphate level (37), and they then postulated that the acyl2gro came from phosphatidylcholine and phosphatidylethanolamine. However, the method used (radiolabeling) precluded interpretation, since no measurements of phospholipid mass or specific activity were made. Previous results from our laboratory (16, 17), as well as this report, argue against elevated inositol phospholipid metabolism in EJ-ras-transformed cells. Instead we find that PDGFstimulated PLC activity is inhibited in EJ-ras-transformed cells. This conclusion is based on three results: no stimulation of InsP3 levels, no change in the specific activity of phosphatidylinositol (16), and no mobilization of Ca2+ in EJ-ras-transformed cells stimulated with PDGF. Our data suggest that, if there is an elevation in acyl2gro in EJ-rastransformed fibroblast it must come from an inositol phospholipid pool other than that activated by PDGF or from another phospholipid pool. Persistent elevations of InsP3 and Ca2+ levels that would result from increased PLC activity might be expected to activate compensatory growth signals that could actually arrest rather than stimulate growth. It follows that if a cell became refractory to PDGF signalling and regulation, as we believe EJ-ras-transformed cells are, they may actually become more likely to grow in a nonregulated manner. The actual relationship between reduced PDGF-stimulated inositol phospholipid metabolism, Ca2 + mobilization, growth control, and transformation is not known. Whatever the exact mechanism, our data suggest that the EJ-rastransformed cells have reduced PDGF-stimulated PLC activation and intracellular Ca2 + levels but can still grow in response to PDGF. 1. Berridge, M. J., Heslop, J. 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