Loss of plasma membrane phospholipid asymmetry requires raft integrity. Role of transient receptor potential channels and ERK pathway.

Cholesterol-rich membrane microdomains, also termed lipid rafts, are implicated in the recruitment of essential proteins for intracellular signal transduction. In nonstimulated cells, phosphatidylserine, an anionic aminophospholipid essential for the hemostatic response, is mostly sequestered in the inner leaflet of the plasma membrane. Cell stimulation by Ca(2+)-mobilizing or apoptogenic agents induces the migration of phosphatidylserine to the exoplasmic leaflet, allowing the assembly and activation of several key enzyme complexes of the coagulation cascade and phagocyte recognition of stimulated or senescent cells. We have recently proposed that store-operated Ca(2+) entry regulates externalization of phosphatidylserine at the cell surface (Kunzelmann-Marche, C., Freyssinet, J.-M., and Martinez, M. C. (2001) J. Biol. Chem. 276, 5134-5139). Here, we show that store-operated Ca(2+) entry and phosphatidylserine exposure are dramatically reduced after raft disruption by methyl-beta-cyclodextrin. In addition, transient receptor potential channel 1-specific antibody was able to significantly decrease Ca(2+)-induced redistribution of phosphatidylserine. Furthermore, store-operated Ca(2+) entry and phosphatidylserine exposure were dependent in part on the extracellular signal-regulated kinase pathway associated with rafts. Hence, raft integrity and store-operated Ca(2+) entry involving transient receptor potential channel 1 channels are essential for completion of the phosphatidylserine transmembrane redistribution process.

Store-operated Ca 2ϩ entry (SOCE) 1 represents an essential source of Ca 2ϩ in nonexcitable cells. When internal Ca 2ϩ stores are depleted, an as yet unknown signal triggers the opening of Ca 2ϩ channels localized in the plasma membrane, allowing subsequent Ca 2ϩ influx (1,2). Although the mechanisms governing SOCE are not completely elucidated, recent studies in human platelets suggest the involvement of actin polymeriza-tion, phosphorylation cascades, and coupling between the endoplasmic reticulum and the plasma membrane through TRPC1 (3,4). In nonstimulated cells, phosphatidylserine (PS) is mostly sequestered in the inner leaflet of the plasma membrane (5). After cell stimulation, including apoptosis, and subsequent Ca 2ϩ increase, cells lose their plasma membrane phospholipid asymmetry and expose PS in the exoplasmic leaflet (6, 7). Once exposed, PS promotes the assembly and activation of several key enzyme complexes of the coagulation cascade (8,9) and becomes a determinant for recognition by phagocytes (10 -12). In the HEL cell line, a human megakaryoblastic cell line taken as a model because it expresses platelet-specific membrane glycoproteins (13,14), we have recently proposed that SOCE regulates PS transmembrane movements and that cytoskeleton architectural organization participates in both processes (15).
Lipid rafts are membrane microdomains implicated in the recruitment of specialized proteins for intracellular signal transduction (16 -18). Among the various pathways known to be associated with raft domains, Ca 2ϩ regulation seems to be dependent on rafts to facilitate the association of appropriate signaling proteins (19,20). For example, recruitment of several members of the mitogen-activated protein kinase (MAPK) family into these microdomains leads to the activation of other downstream kinases (21)(22)(23). Recently, the MAPK pathway has been found to be able to regulate SOCE in platelets (4). TRPC1 has been localized in the membrane rafts, where it interacts, either directly or indirectly, with a complex of Ca 2ϩ signaling proteins (24,25). Here, we have investigated the contribution of lipid rafts and TRPC1 in both SOCE and PS outward translocation and their regulation by the mitogenactivated protein kinase/ERK kinase (MEK) cascade. Perturbation of raft integrity dramatically reduced both SOCE and PS transmembrane redistribution, with participation of the MEK pathway, which is also modulated by cholesterol depletion.
Cell Culture-Human erythroleukemia (HEL, ATCC TIB-180) cells were cultured in RPMI 1640 medium supplemented as described previously (15) plus 10% (v/v) heat-inactivated fetal calf serum at 37°C in a humidified 5% CO 2 atmosphere. All experiments were performed with the maximal concentrations of inhibitors at which no cytotoxicity was observed, as deduced from trypan blue exclusion.
Measurements of [Ca 2ϩ ] i by Flow Cytometry-Experimental procedures were the same as those detailed in a previous study (15). Briefly, HEL cells were loaded with 3 M Fluo-3 in RPMI 1640 medium supplemented with CaCl 2 (final Ca 2ϩ concentration, ϳ1 mM). To study Ca 2ϩ release and Ca 2ϩ entry separately, experiments were performed in the presence of EGTA (1 mM) in the extracellular medium. Cells were then stimulated by the different agents, and, when indicated, CaCl 2 was added to restore an extracellular Ca 2ϩ concentration of ϳ1 mM, allowing SOCE. Fluo-3 fluorescence was monitored using a FACScan BD PharMingen flow cytometer.
Subcellular Fractionation-After stimulation, HEL cells were pelleted in a microcentrifuge, and the pellets were resuspended in 0.5 ml of ice-cold 10 mM Tris/HCl buffer, pH 7.2, containing 158 mM NaCl, 1 mM EGTA, 5 g/ml leupeptin, 5 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, and 1 mM Na 3 VO 4 . The suspensions were lysed by three freezing-thawing steps, and intact cells were removed by centrifugation at 1500 ϫ g. The cell lysate was centrifuged at 100,000 ϫ g at 4°C for 60 min to obtain the membrane and cytosolic fractions. Membranes were washed with cold Tris buffer solution containing 1 mM Na 3 VO 4 and resuspended in 10 mM Tris/HCl buffer, pH 7.2, containing 158 mM NaCl, 1 mM EGTA, 0.1% SDS, 1% (w/v) sodium deoxycholate, 1% (v/v) Triton X-100, 5 g/ml leupeptin, 5 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, and 1 mM Na 3 VO 4 . Lysates were centrifuged at 16,000 ϫ g for 5 min to remove insoluble substances. Lysates (cytoplasm fraction) were used to evaluate ERK phosphorylation, and Ras protein was detected in the membrane counterpart. Proteins were separated by SDS-PAGE. Blots were probed with monoclonal antibodies to phospho-ERK1/2 or Ras and developed with horseradish peroxidase-conjugated anti-mouse antibody. Proteins were visualized by enhanced chemiluminescence reagents and then exposed to CL-XPosure films.
Functional Detection of Procoagulant PS-Procoagulant PS exposure in stimulated cells was detected using a human prothrombinase assay in which this phospholipid is the rate-limiting parameter promoting the activation of prothrombin by factor Xa in the presence of factor Va (26).
Thrombin generated by the assembled prothrombinase complex was measured using a chromogenic assay, as described previously (15).
Determination of PS Exposure by Fluorescence Microscopy-Cells were activated under the same conditions described above and then centrifuged at 12,000 ϫ g for 2 min, resuspended in Hanks' balanced salt solution, and visualized by fluorescence laser scanning microscopy. The fluorescence was acquired by using Spot Software from Diagnostic Instruments, Inc. (Sterling Heights, MI). To enable comparison, all images were recorded using the same parameters of laser power and photomultiplier sensitivity. Images shown are representative of at least three separate experiments for each condition and were processed by using identical values for contrast and brightness.
Statistical Analysis-Results were expressed as the mean Ϯ S.E. of at least four separate experiments performed at different culture stages and/or passages, with no obvious difference attributable to the culture conditions. Paired Student's t test was used for statistical analysis. p Ͻ 0.05 was considered significant.

Changes of [Ca 2ϩ
] i -To determine whether Ca 2ϩ signaling is modulated by lipid rafts, cells were treated with MCD, an agent able to extract cholesterol from the plasma membrane, leading to raft disruption (27). As illustrated in Fig. 1, A and B, MCD treatment resulted in a reduction of 59 Ϯ 4% of the amplitude of the Ca 2ϩ response in A23187-stimulated cells. To establish whether disruption of lipid raft domains affects Ca 2ϩ release from intracellular stores or SOCE, control and MCD-treated cells were initially stimulated with TG, an inhibitor of reticulum endoplasmic Ca 2ϩ -ATPases, in the presence of EGTA in the extracellular medium ( Fig. 1, C and D). Under these conditions, MCD reduced the TG-induced Ca 2ϩ response (47 Ϯ 9% inhibition compared with the value in its absence), providing evidence that Ca 2ϩ release requires interaction between Ca 2ϩ stores and the plasma membrane. The inhibition of Ca 2ϩ release by MCD resulted in the reduction of the magnitude of SOCE upon CaCl 2 reintroduction, as reported recently in other cell types (24,28,29). With respect to controls, the inhibition due to MCD was 69 Ϯ 7%.
In contrast, stimulation by the combination of NE (30) and THR (31), two agonists of G-protein-coupled receptors, evoked Ca 2ϩ signal with two distinct phases: a rapid rise in [Ca 2ϩ ] i within a few seconds, followed by an exponential decrease lasting several minutes ( Fig. 2A). The latter was more rapid in the presence of EGTA, indicating that this phase of Ca 2ϩ re- These results demonstrate that disruption of lipid raft domains accounts for the regulation of Ca 2ϩ signaling in general and SOCE in particular.
Effect of Cholesterol Depletion on PS Externalization-The transbilayer movement of endogenous PS was assessed by the procoagulant prothrombinase functional assay and by annexin V labeling (32); the former gave a quantitative estimation, and the latter gave an indication of the proportion of PS-positive cells. In the absence of stimulation (Fig. 3A), the cells presented a basal PS-dependent prothrombinase activity of 0.24 Ϯ 0.07 NIH units of thrombin generated/min/ml/2 ϫ 10 5 cells. After treatment with 10 mM MCD, this activity was not significantly modified, testifying to nonperturbed PS asymmetric distribution. The association of NE and thrombin did not elicit a noticeable increase in prothrombinase activity. After Ca 2ϩ ionophore treatment, prothrombinase activity was enhanced ϳ4.2fold. MCD significantly reduced the development of prothrombinase activity induced by Ca 2ϩ ionophore with an inhibition of ϳ68% (Fig. 3A). These results were confirmed by fluorescence microscopy with annexin V-FITC as a specific probe for PS (Fig. 3B). Unstimulated (control) cells were poorly labeled, indicating a low basal exposure of PS currently observed (15). Ionophore A23187 induced extensive PS exposure, as evidenced by the high fluorescence intensity. Disruption of raft integrity by MCD in A23187-stimulated cells resulted in a more scattered annexin V labeling, corresponding to an important reduction of the degree of PS transbilayer redistribution at the cell surface. Collectively, these results show that PS externalization requires the integrity of membrane rafts.
Implication of TRPC1 in SOCE and PS Exposure in HEL Cells-Prompted by recent work demonstrating the expression of TRPC1 in platelets and megakaryocytic cells (3,33), we confirmed this observation in HEL cells using a polyclonal antibody targeted to a mammalian TRPC1-specific peptide (data not shown). Because TRPC1 is a candidate for the mediation of SOCE in normal human cells (3), the effect of TRPC1specific antibody was assessed with respect to alteration of Ca 2ϩ signal. In the presence of anti-TRPC1, Ca 2ϩ response induced by A23187 was reduced (Fig. 4A). This antibody also interfered with Ca 2ϩ store release and entry in response to TG (Fig. 4B). Although the blocking effect of antibody to TRPC1 was only partial at the low concentration used (1:100 dilution, corresponding to a final concentration of 3 g/ml in the incubation mixture), it was statistically significant in seven independent experiments and was peptide-specific because it was abolished after preadsorption to antigenic peptide (data not shown). This indicates that TRPC1 mediates SOCE, at least in part. In addition, the fact that this antibody reduced TG-in- duced response suggests that the coupling between Ca 2ϩ stores and the plasma membrane occurs, at least in part, through TRPC1. Fig. 4C shows the effect of TRPC1-specific antibody on PS externalization at the HEL cell membrane surface. This antibody by itself did not significantly modify prothrombinase activity. However, in A23187-stimulated cells, the development of prothrombinase activity was significantly attenuated (ϳ25% of inhibition) after incubation with the TRPC1-specific antibody (Fig. 4C). This supports the view that Ca 2ϩ entry through TRPC1 is involved in PS transmembrane redistribution.
It must be emphasized that this polyclonal antibody was deliberately used at a low concentration to avoid artifactual membrane perturbation, which may explain the observation that inhibition of SOCE and prothrombinase activity was only partial.
Involvement of the ERK Cascade in Ca 2ϩ -induced Redistribution of PS-To investigate the role of the ERK cascade in Ca 2ϩ response, the effect of two structurally unrelated inhibitors of MEK, PD98059 (34) and U0126 (35), was examined. These agents were deliberately used at a low concentration to prevent artificial perturbation of lipid organization in the plasma membrane. Both inhibitors reduced the Ca 2ϩ ionophore-induced response, with an inhibition of 47 Ϯ 4% and 54 Ϯ 4% for PD98059 and U0126, respectively (Fig. 5A). In the presence of EGTA, both inhibitors also diminished the TGevoked Ca 2ϩ signal (52 Ϯ 7% and 65 Ϯ 2% inhibition for PD98059 and U0126, respectively), indicating that Ca 2ϩ release from stores involves coupling with the MEK pathway (Fig. 5B). In addition, both inhibitors reduced the CaCl 2 -induced response in TG-stimulated cells (49 Ϯ 9% and 59 Ϯ 8% inhibition for PD98059 and U0126, respectively), in agreement with observations in platelets (4). These results suggest that SOCE is partially dependent on ERK activation in HEL cells.
MEK inhibition resulted in a reduction of A23187-induced PS exposure by ϳ25% as measured by the prothrombinase assay (Fig. 5C). Hence, it is likely that the diminution of Ca 2ϩ influx across the plasma membrane in relation to MEK inhibition results in the reduction of PS exposure. Indeed, these results confirm that SOCE regulates PS externalization at the cell surface and suggest that activation of the ERK cascade modulates, at least in part, PS transmembrane redistribution. The activation of ERK was analyzed by Western blotting. As shown in Fig. 5D, in A23187-stimulated HEL cells, phosphorylation/activation of both isoforms of ERK (p44-ERK1 and p42-ERK2) was detected in the cytosolic fraction. Phosphorylation of ERK1/2 was partly inhibited by the MEK inhibitor U0126, which was again used at a low concentration to avoid artifactual membrane perturbation. Cholesterol depletion by MCD resulted in almost complete inhibition of A23187-induced activation of ERK1 with a concomitant reduction of ERK2 phosphorylation, testifying to the participation of the ERK pathway in the organization of the plasma membrane and, more particularly, of lipid microdomains. To determine whether Ras protein is recruited at the plasma membrane, we therefore investigated whether raft disruption by MCD was responsible for alterations of its subcellular localization. Ca 2ϩ ionophore A23187 induced an enhancement of Ras translocation to the membrane, but cholesterol depletion severely impaired this association (Fig. 5E), confirming that the integrity of lipid rafts is important for the translocation of Ras and activation of the ERK pathway. Here, we provide direct evidence that the activation of the ERK cascade is coupled with membrane rafts. Altogether, PS externalization requires the integrity of rafts, SOCE, and participation of the ERK pathway. DISCUSSION Recent data suggest that SOCE is essential for the regulation of a variety of cell functions and the maintenance of Ca 2ϩ homeostasis. We have shown previously that PS exposure in stimulated cell is related to the level of SOCE (15,36). However, the mechanisms controlling SOCE and PS externalization remain obscure. The data presented here show that SOCE and PS externalization are dramatically reduced after disruption of raft integrity in the plasma membrane and in the presence of TRPC1-specific antibody. Furthermore, SOCE and PS exposure are regulated, at least in part, by activation of the ERK pathway associated with rafts. Hence, raft integrity and SOCE involving TRPC1 are essential for completion of the PS transmembrane redistribution process.
The potential function of lipid rafts in Ca 2ϩ signaling was investigated after partial cholesterol depletion by noninvasive extraction by MCD (27). MCD is a widely used tool to study the function of membrane cholesterol. Without incorporating itself into the membrane, MCD selectively extracts cholesterol from the surface of the cell (37)(38)(39). This probably explains why it has no deleterious effect on cell viability and on membrane asymmetry as verified here. MCD treatment reduced Ca 2ϩ ionophore-and TG-induced Ca 2ϩ release and entry, suggesting that raft integrity is essential for normal Ca 2ϩ handling in HEL cells. Consistent with these observations, recent studies have shown that disruption of raft microdomains reduced Ca 2ϩ influx triggered by receptor activation in T and B cells or by TG treatment in salivary epithelial cells (24,28,29). These data suggest that rafts play a crucial role in the regulation of Ca 2ϩ signaling in general and SOCE in particular. In this respect, it has been shown that TRPC1, a candidate protein for storeoperated Ca 2ϩ channels, is localized in the lipid raft domains in

FIG. 4. TRPC1 is involved in SOCE and PS externalization.
Fluo-3 fluorescence variations were measured by flow cytometry analysis as described previously (15). HEL cells were preincubated in the absence or presence of anti-TRPC1 antibody (@-TRPC1; 3 g/ml, 90 min at 37°C) and then stimulated with A23187 (2 M; A) or TG (1 M; B) in the presence of EGTA, and 1 mM CaCl 2 (final concentration) was reintroduced in the external medium. C, PS exposure was revealed by functional prothrombinase assay. Cells were preincubated with anti-TRPC1 (@-TRPC1; 1:100) and nonstimulated or stimulated by A23187 (2 M, 10 min at 37°C). Histogram data are from 10 independent determinations (each with triplicate samples). ‫,ء‬ p Ͻ 0.05; ‫,ءء‬ p Ͻ 0.01; ‫,ءءء‬ p Ͻ 0.001. the plasma membrane, where it interacts, either directly or indirectly, with a complex of Ca 2ϩ signaling proteins such as the inositol trisphosphate receptor (24,25). Because HEL cells express TRPC1, a similar interaction between this channel and lipid raft domains can be reasonably hypothesized in these megakaryocytic cells.
Because MEK inhibitors significantly reduced Ca 2ϩ ionophore-mediated Ca 2ϩ influx, it is highly probable that the significant reduction due to both agents PD98059 and U0126 occurs via SOCE. In this respect, it has been shown that the Ca 2ϩ signal induced by Ca 2ϩ ionophores, thrombin, or epinephrine elicits ERK1/2 phosphorylation in several cell types, including HEL cells (31,40). In addition, both MEK inhibitors reduced the Ca 2ϩ response elicited to A23187 and Ca 2ϩ entry to the same extent in TG-treated cells. The data are in agreement with those obtained in platelets, where the MAPK pathway has been described to regulate SOCE (4). Furthermore, we show that the ERK pathway is closely associated with raft integrity/ formation, and mobilization of Ras to the membrane compartment is required for activation of the MEK kinase pathway in HEL cells. In this respect, a recent study has reported that depletion of plasma membrane cholesterol also prevented insulin-dependent phosphorylation of MEK and Ras translocation to the plasma membrane in HIRcB fibroblasts (41). In contrast, extensive cholesterol depletion in adipocytes had no effect on ERK1/2 phosphorylation (42). This discrepancy is probably related to a wide variability in the activation of the ERK pathway as a function of the cell type.
In the present study, A23187 Ca 2ϩ ionophore was used at 2 M for 10 min at 37°C to preserve cell viability while inducing PS exposure at the cell surface and expression of membraneassociated procoagulant activity. It has been shown that Ca 2ϩ ionophores promote intracellular Ca 2ϩ store depletion and subsequent SOCE (43)(44)(45). In this context, we can reasonably assume that the effects of the different compounds used here on A23187-induced Ca 2ϩ response are relevant to Ca 2ϩ entry mediated by store depletion or to Ca 2ϩ release from intracellular stores. This view is further supported by the results obtained with TG treatment, which triggers Ca 2ϩ entry but does not form channels in the plasma membrane. In addition, raft disruption by MCD also resulted in reduced Ca 2ϩ response to thrombin and NE, two agonists of G-protein-coupled receptors, although these agonists were not able to promote PS externalization. This confirms that an elevated and sustained [Ca 2ϩ ] i is absolutely necessary for PS exposure, in agreement with other reports, and explains why ionophores are the commonly used activators (9). In Scott syndrome, a genetic defect of PS transmembrane redistribution, blood cells are nonresponsive to Ca 2ϩ ionophores (9) and precisely show defective SOCE (46). MEK inhibitors reduced prothrombinase activity induced by Ca 2ϩ ionophore. Hence, it is likely that the diminution of Ca 2ϩ influx across the plasma membrane due to MEK inhibition results in reduction of PS exposure. Indeed, these results confirm that SOCE regulates PS externalization and suggest that activation of the MAPK pathway plays a role in the transmembrane redistribution of PS at the cell surface. As discussed above, there is direct evidence that the activation of the MAPK cascade is coupled with membrane rafts. PS externalization requires both the integrity of membrane rafts and activation of MAPK pathway.
The dramatic raft-disrupting effect of MCD on the ability of cells to externalize PS suggests that the transporter(s) or the regulatory elements involved in PS externalization could be localized into or near to lipid rafts. In this context, it has been shown that PS can be found in membrane lipid microdomains in association with B-cell receptor, suggesting a role for this aminophospholipid in the interaction of signaling molecules with their intracellular targets (47). Besides this, ABCA1, a lipid transporter recently described to favor the transmembrane redistribution of PS and cholesterol efflux (48), is thought to be preferentially distributed in membrane domains distinct from cholesterol-rich rafts (49). In addition, Fielding et al. (50) have observed several differences in the phospholipid composition of the membrane domains where ABCA1 is found, depending on the cellular model. Thus, ABCA1 could be localized not in but near to the lipid rafts.
Our previous results (15,46,48) and the present study point to the complexity of the mechanisms governing the plasma membrane PS redistribution process and show the implication of various membrane (rafts, transporter(s), and store-operated Ca 2ϩ channels) and cytoplasmic (cytoskeleton and the MEK pathway) actors. In this context, lipid rafts appear to provide an appropriate environment for the assembly of some of these effector complexes, and the search for further links between elements involved in the maintenance of membrane homeostasis and plasticity remains an obvious challenge.