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Vol. 14, Issue 7, 2655-2664, July 2003

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Extracellular ATP Causes ROCK I-dependent Bleb Formation in P2X₇-transfected HEK293 Cells

Anna Morelli ^*, Paola Chiozzi ^*, Anna Chiesa ^*, Davide Ferrari ^*, Juana M. Sanz ^*, Simonetta Falzoni ^*, Paolo Pinton ^*, Rosario Rizzuto ^*, Michael F. Olson , and Francesco Di Virgilio ^* 

^*Department of Experimental and Diagnostic Medicine, Section of General Pathology, and Interdisciplinary Center for the Study of Inflammation (ICSI), University of Ferrara, I-44100 Ferrara, Italy; and Abramson Family Cancer Research Institute, BRB II/III, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Submitted April 22, 2002; Revised January 27, 2003; Accepted March 17, 2003
Monitoring Editor: Guido Guidotti

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
The P2X₇ ATP receptor mediates the cytotoxic effect of extracellular ATP. P2X₇-dependent cell death is heralded by dramatic plasma membrane bleb formation. Membrane blebbing is a complex phenomenon involving as yet poorly characterized intracellular pathways. We have investigated the effect of extracellular ATP on HEK293 cells transfected with the cytotoxic/pore-forming P2X₇ receptor. Addition of ATP to P2X₇-transfected, but not to wt P2X₇-less, HEK293 cells caused massive membrane blebbing within 1–2 min. UTP, a nucleotide incapable of activating P2X₇, had no early effects on cell shape and bleb formation. Bleb formation triggered by ATP was reversible and required extracellular Ca²⁺ and an intact cytoskeleton. Furthermore, it was completely prevented by preincubation with the P2X blocker oxidized ATP. It was recently observed that the ROCK protein is a key determinant of bleb formation. Preincubation of HEK293-P2X₇ cells with the ROCK blocker Y-27632 completely prevented P2X₇-dependent blebbing. Although ATP triggered cleavage of the ROCK I isoform in P2X₇-transfected HEK293 cells, the wide range caspase inhibitor z-VAD-fluoromethylketone had no effect. These observations suggest that P2X₇-dependent plasma membrane blebbing depends on the activation of the serine/threonine kinase ROCK I.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
A distinctive feature of apoptosis is the occurrence of coordinated modifications that involve virtually every cell compartment: the plasma membrane, the cytoplasm, the endoplasmic reticulum, the mitochondria, and the nucleus (Ferri and Kroemer, 2001; Yu et al., 2001). One of the earliest changes observed in apoptosis is the formation of plasma membrane blebs, a dramatic phenomenon still poorly understood in its mechanism (Torgerson and McNiven, 1998). Apoptotic blebs consist of membrane expansions that encircle cytoplasm devoid of organelles and filamentous actin. Their formation has been shown to be dependent on myosin light chain (MLC) phosphorylation, a process modulated by the Ca²⁺/calmodulin-dependent MLC kinase and the serine/threonine kinase ROCK (Amano et al., 1996; Kimura et al., 1996; Fukata et al., 2001). It has previously been shown that during apoptosis caspase-3 cleaves the Rho-kinase I (ROCK I), thus leading to a deregulated activity of this kinase and consequently to bleb formation (Coleman et al., 2001).

Accruing evidence suggests that substantial amounts of ATP can accumulate in the pericellular space under several physiological or pathological conditions (Ferrari et al., 1997; Beigi et al., 1999; Schwiebert et al., 2002; Warny et al., 2001). This nucleotide, by acting at plasma membrane P2 receptors, triggers different cell responses, such as secretion, chemotaxis, proliferation, transcription factor activation, or even cytotoxicity (Di Virgilio et al., 2001). In addition, ATP can also be a powerful apoptotic agent via activation of the purinergic P2X₇ receptor, a plasma membrane nucleotidegated ion channel endowed with the peculiar ability to generate a nonselective pore upon sustained stimulation (Zanovello et al., 1990; Zheng et al., 1991; Surprenant et al., 1996). The intracellular pathways responsible for P2X₇-dependent apoptosis are only partially known, although they seem to largely coincide with those activated by other better-known apoptotic receptors such as Fas and the type I TNF receptor (TNFRI). P2X₇ induces a massive depletion of intracellular K⁺, caspase-3 activation, degradation of nuclear lamin, DNA fragmentation, nuclear condensation, and apoptotic body formation (Steinberg and Silverstein, 1987; Ferrari et al., 1999; Morelli et al., 2001). In addition, a typical feature of ATP-stimulated cells is the occurrence of a dramatic membrane blebbing (MacKenzie et al., 2001). The biochemical basis of membrane blebbing has not been studied in detail, nor has it been unequivocally shown to be solely dependent on the activation of the P2X₇ receptor/channel. In the present work, we have investigated the kinetics and biochemical mechanisms of ATP-induced bleb formation in a HEK293 cell clone stably expressing a GFP (green fluorescent protein)-tagged version of the P2X₇ receptor (P2X₇-HEK293). This cell line lacks endogenous P2X receptors and is therefore a good model system to study responses due to stimulation of the transduced P2X₇ receptor.

Our data demonstrate that P2X₇ expression is sufficient to cause membrane blebbing in response to ATP; furthermore, we show that bleb formation 1) is rapid, reversible and Ca²⁺ dependent, 2) is independent of caspase-3, 3) requires an intact cytoskeleton, and 4) is prevented by an inhibitor of ROCK.

Taken together, these data suggest that ROCK activation is a key step in the early membrane modifications triggered by the P2X₇ receptor.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Cells, Solutions, and Reagents
HEK293 cells were cultured in DME/F-12 1:1 medium (Sigma, St. Louis, MO) containing 15% heat-inactivated FCS (Life Technologies, Paisley, Scotland), 100 U/ml penicillin, and 100 µg/ml streptomycin. Stable clones were cultured in the same medium containing G418 sulfate (Geneticin; Calbiochem, La Jolla, CA) at a concentration of 0.2 mg/ml. Visualization of transfected cells was performed in a saline solution (standard saline solution) containing 125 mM NaCl, 5 mM KCl, 1 mM MgSO₄,1 mM Na₂HPO₄, 5.5 mM glucose, 5 mM NaHCO₃, 1 mM CaCl₂, and 20 mM HEPES (pH 7.4).

ATP and UTP were used at a concentration of 3 and 1 mM, respectively, and were both from Roche Diagnostics (Mannheim, Germany). Oxidized ATP (oATP) was synthesized in our laboratory and used at the concentration of 300 µM. Cells were preincubated for 2 h with oATP, rinsed several times with saline solution, and exposed to the different stimuli. Hexokinase (Sigma) was used at the concentration of 100 µg/ml. Cells were preincubated for 45 min and kept in the continuous presence of hexokinase throughout the experiment. z-VAD fluoromethylketone (z-VAD-fmk; 100 µM; Bachem, Bubendorf, Switzerland) and cytochalasin B (10 µM; Sigma) were added to the cell monolayer 10 min before the addition of ATP. The ROCK inhibitor Y-27632 was purchased from Tocris Cookson Ltd. (Bristol, UK). The anti-Fas mAb was a kind gift of Professor Klaus Schulze-Osthoff (University of Munster, Germany) and was used at a concentration of 1 µg/ml.

Transfection of HEK293 Cells and Selection of Stable Clones
HEK293 wt cells were transfected with calcium phosphate. Briefly, the first day, 2.5 x 10⁶ HEK293 cells were plated in Petri dishes. The second day, for each dish, 30 µg of plasmid DNA was resuspended in a total volume of 450 µl TE (10 mM Tris, 1 mM EDTA, pH 8) and then 50 µl of a 2.5 M CaCl₂ solution was added. This solution was added dropwise under vortexing to a tube containing 500 µl of 2x HBS (280 mM NaCl, 50 mM HEPES, 1 mM Na₂HPO₄, pH 7.12, at 25°C). After a 30-min incubation at room temperature, the DNA precipitate was added to the dish dropwise. The third day medium was changed. The fourth day, G418, 0.8 mg/ml, was added to fresh medium to select transfected clones. After clone selection, the selective medium contained G418, 0.2 mg/ml.

Transient transfection was performed using the same procedure directly on coverslips for 0.5 x 10⁶ cells, 4–8 µg of plasmid DNA in a total volume of 90 µl TE, 10 µl of CaCl₂ solution, and 100 µl of 2x HBS. The plasmid containing rat P2X₇-GFP in pcDNA3 used for stable transfection was kindly provided by Dr. Annemarie Surprenant (University of Sheffield, UK).

Collection and Analysis of the GFP Images
Transfected cells seeded on coverslips were observed using a Nikon Eclipse TE-300 fluorescence microscope (Nikon Co., Tokyo, Japan) equipped with a thermostated chamber and the following filter set: excitation HQ480/40, dichroic Q480LP, and emission HQ510LP. The microscope was also equipped with the following devices to form a system for high-speed acquisition and processing of fluorescent images: a computer-controlled light shutter, a six-position filter wheel, a piezoelectric z-axis focus device, a back-illuminated 1000x 800 CCD camera (Princeton Instruments, Princeton, AZ), a computer equipped with Metamorph software (Universal Imaging Corporation, Downingtown, PA) for image acquisition, 2-D and 3-D visualization and analysis. All experiments were performed at 37°C in the standard saline solution described in Cells, Solutions, and Reagents. Microscopic observations were carried out with recently thawed P2X₇-HEK293, never exceeding the 10th in vitro passage. Recordings were performed with at least 10 different recently thawed cell batches, and the effect of inhibitors was consistently reproduced in three or more separate experiments. Criteria for induction of blebbing were 1) that at least one bleb per cell was produced within 1–2 min of ATP addition, 2) that within 3–5 min all cells in the field were actively blebbing, and 3) that, unless ATP was removed, this process continued as long as cells did not detach from the substrate. An inhibitor was considered effective if it was able to fully block blebbing (no blebs at any time points).

Rho Pull-down Assay
The pull-down assay was performed as described in Coleman et al., (2001). Briefly, cells (5 x 10¹⁰) were plated in 10-cm Petri dishes, grown for 48 h, and serum-starved for further 24 h. Lysis was performed in 50 mM Tris, pH 7.2, 500 mM NaCl (TBS), 1% (vol/vol) Triton X-100, 5 mM MgCl₂, 1 mM DTT, and protease inhibitors. An aliquot of cell lysates was used for determination of protein concentration, whereas the remainder was spun at 10,000 x g for 5 min. The supernatant was then mixed with 20 µg of bacterially expressed GST-Rhotekin (murine amino acids 7–89) prebound to 75–100 µl glutathione-Sepharose beads and incubated at 4°C with tumbling for 30 min. Samples were spun, the supernatant was removed, and beads were washed three times in TBS containing 1% (vol/vol) Triton X-100, 5 mM MgCl₂, and 1 mM DTT before mixing with Laemmli buffer and analysis by Western blotting (see below).

Cell-permeable Tat-C3 Botulinum Toxin
The cell-permeable C3 toxin was prepared as described in Coleman et al. (2001). Briefly, the C3 recombinant protein, modified to include the nucleotide sequence 5'-GGA GGA TAC GGC CGA AAG AAG CGA CAG CGA CGC CGT GGA GGA of the thrombin cleavage site, was produced in E. coli BL21 and induced with 0.3 mM isopropyl--thiogalactopyranoside (IPTG) for 3 h at 32°C. Cells were lysed in TBS containing 5 mM MgCl₂, 1 mM DTT, and protease inhibitors by quick freezing followed by sonication. After centrifugation at 10,000 x g for 10 min at 4°C, the supernatant was incubated with glutathione-Sepharose for 2 h at 4°C. The beads were then washed with TBS plus 5 mM MgCl₂ and 1 mM DTT. To cleave Tat-C3, beads were incubated in TBS plus 1 mM MgCl₂, 1 mM CaCl₂, 1 mM DTT, and 30 U of thrombin overnight at 4°C. The supernatant was then removed and incubated with p-aminobenzamidine beads for 1 h. Supernatants containing Tat-C3 were frozen and used at 0.5–1.0 µM in culture medium.

Immunoblotting
Cells were lysated in lysis buffer containing TBS Triton 0.1%, 1 mM benzamidine, 1 mM phenylmethanesulfonyl fluoride (PMSF), 0.2 µg DNase, and 0.2 µg Rnase (only for ROCK I analysis, lysis buffer also contained 1 mM diisopropylfluorophosphate [Fluka Chemie, Buchs, Switzerland], and cells were preincubated in the presence of this inhibitor for 1 h before lysis). Proteins were separated on 6% (ROCK I) or 14% (RhoA) SDS-polyacrylamide gel according to Laemmli, blotted on nitrocellulose paper (Amersham Life Sciences, Cologno Monzese, Italy), and hybridized with anti-ROCK I (clone 46; BD Transduction Laboratories, Los Angeles, CA) or anti-RhoA (Santa Cruz Biotechnology Inc., Santa Cruz, CA) mAbs.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Membrane bleb formation is a widely conserved event occurring during the early phases of apoptosis. It has long been known that extracellular ATP, mainly acting at the P2X₇ receptor (but see Chvatchko et al., 1996), is a powerful proapoptotic agent in several cell types and induces most of the changes that are caused by typical proapoptotic stimuli such as FasL or TNF- (Zanovello et al., 1990; Zheng et al., 1991; Ferrari et al., 1999). Figure 1 shows the effect of ATP on P2X₇-HEK293 cell morphology. The plasma membrane was uniformly stained by the fluorescent P2X₇-GFP chimeric receptor. It is interesting to notice that a substantial amount of receptor also accumulated within cytoplasmic organelles, but not within the nucleus. Furthermore, P2X₇-HEK293 cells appeared rounded and slightly swollen when compared with wt P2X₇-less HEK293 (see Figure 5 below), even in the absence of added stimuli. Within 2 min of the addition of the nucleotide, P2X₇-HEK293 cells underwent a massive process of bleb formation that persisted at a sustained level for >20 min.

View larger version (85K): [in this window] [in a new window]   Figure 1. Extracellular ATP causes fast bleb formation. P2X7-HEK293 cells were incubated in standard saline solution and treated with 3 mM ATP at time zero. Bar, 10 µm.

View larger version (85K): [in this window] [in a new window]   Figure 5. wt HEK293 cells transfected with SNAP25-GFP do not undergo blebbing. Cells were incubated in standard saline and challenged with 3 mM ATP at time zero. Bar, 10 µm.

Blebs originated as projections of the plasma membrane that surrounded an optically empty cytoplasm apparently free of intracellular organelles. Cells appear to undergo a "boiling process" that caused a continuous formation and resorption of blebs of various sizes. The usual pattern included formation of small size blebs during the early phases, followed by protrusion of increasingly larger blebs often generated by fusion of smaller blebs. The observation was continued for >20 min, and at the end of this time cells started to detach from the substrate and disappear from the microscopic field. Removal of ATP stopped bleb formation and allowed full recovery of cell shape and volume (Figure 2, lower panels), thus showing that the process was reversible. We compared blebbing in HEK293 cells transfected with either the chimeric P2X₇-GFP or wtP2X₇ and observed a longer lag in the beginning of blebbing in the former cells, which, after this initial delay, proceeded on a similar time scale in both transfectants. Smart et al. (2002) have recently published a thorough analysis of the effect of GFP fusion at the N or C termini of P2X₇. They reported an approximately threefold decrease in sensitivity to ATP with GFP fused at the P2X₇ C termini, although stimulation with a full dose of ATP (as that used in the present study) caused maximal channel activation and pore formation. Among the few known blockers of the P2X₇ receptor, one of the most useful is oATP, a di-aldehyde derivative that covalently binds and irreversibly blocks this receptor (Murgia et al., 1993). Oxidized ATP could in principle also block other P2X receptors, but this is not a drawback in P2X₇-HEK293 because these cells do not express other P2X subtypes. Figure 3 shows that preincubation with this blocker completely prevented ATP-induced bleb formation throughout the observation time (45 min). After 20–25 min, some cell rounding became detectable but this did not lead to detachment or to major morphological alterations for up to 45 min.

View larger version (103K): [in this window] [in a new window]   Figure 2. ATP-induced bleb formation is reversible. P2X7-HEK293 cells were incubated in standard saline solution and treated with 3 mM ATP at time zero. After 20 min, the ATP-containing solution was replaced with fresh ATP-free solution and the observation was carried for an additional 10 min. Images shown in the bottom left and right panels were taken 2 and 10 min after ATP removal, respectively. Bar, 10 µm

View larger version (128K): [in this window] [in a new window]   Figure 3. Bleb formation is blocked by oxidized ATP. P2X7-HEK293 cells were pretreated for 120 min at 37°C in standard saline solution with 300 µM oATP, rinsed, placed in fresh solution, and stimulated with 3 mM ATP. The image in the top left panel (time zero) was taken soon after ATP addition. Bar, 10 µm.

All cells express powerful ecto-ATPases/nucleotidases that rapidly degrade added ATP to ADP and AMP, which in turn is hydrolized to adenosine by 5'-nucleotidase. Thus, it is possible that some of the effects of extracellular ATP might be mediated by ATP degradation products such as ADP or adenosine. To rule out participation of ATP degradation products, we added ATP in the presence of hexokinase, an enzyme that generates glucose-6 phosphate and ADP and accelerates the final degradation to AMP and adenosine (Figure 4). Hexokinase prevented blebbing throughout the incubation time, but for a brief outburst of bleb formation soon after addition. As in the case of the experiments performed in the presence of oATP, cells underwent some rounding. The HEK293 cell clone used in our laboratory endogenously express two P2Y subtypes: P2Y₁ and P2Y₂ (our unpublished results, see also Schachter et al., 1997). ADP is a much better agonist than ATP for P2Y₁ (Ralevic and Burnstock, 1998; von Kugelgen and Wetter, 2000); thus, we can exclude a main role of this receptor in blebbing because in the presence of hexokinase large amounts of ADP are formed, and yet the cells did not bleb. UTP and ATP are equipotent as agonists of P2Y₂. To test the possible involvement of P2Y₂ receptors in blebbing, we incubated cells in the presence of 3 mM UTP. This treatment caused some blebbing but only after >30 min of incubation (our unpublished results). UTP-induced blebbing was fully abolished by hexokinase. We did not further investigate UTP-induced bleb formation; however, these latter observations show that it is a late event and suggest that it might be secondary to P2Y-triggered release of ATP.

View larger version (107K): [in this window] [in a new window]   Figure 4. Bleb formation is inhibited by hexokinase. P2X7-HEK293 cells were incubated at 37°C in standard saline solution. At time zero, 3 mM ATP and hexokinase (100 µg/ml) were added at the same time, and the observation was carried out for an additional 35 min. Bar, 10 µm.

As a further control for the specificity of the effect of extracellular ATP, we used the parental wt HEK293 cells. To closely compare plasma membrane dynamics of P2X₇less cells to that of P2X₇-GFP transfectants, wt HEK293 cells were transfected with a SNAP-25GFP–containing plasmid. SNAP-25 is a protein that localizes to the cytoplasmic side of the plasma membrane, thus allowing easy detection of cell shape changes and possible occurrence of bleb formation. Figure 5 shows that an incubation of cells lacking P2X₇ with ATP for up to 20 min had no effect on cell morphology, nor did it induce bleb formation.

Formation of membrane blebs is dependent on contraction of the cytoskeleton; therefore it is expected to be inhibited by chelation of Ca²⁺ or administration of the cytoskeletal poison cytochalasin B. Figure 6 shows that in the absence of added Ca²⁺ and in the presence of EGTA (nominal extracellular free Ca²⁺ concentration <10^–7 M) blebbing did not occur, although P2X₇ is known to be fully active under these conditions. Instead, cells swelled and rounded, a process likely due to the massive influx of Na⁺, which is known to occur through P2X₇ in the absence of external Ca²⁺. Thus, ATP-induced bleb formation requires a large and sustained increase in cytoplasmic Ca²⁺ due to influx across the plasma membrane. Bleb formation was also prevented by cell poisoning with cytochalasin, and as in the case of the experiments at low extracellular Ca²⁺, cells underwent a large increase in volume (our unpublished results).

View larger version (127K): [in this window] [in a new window]   Figure 6. ATP-induced bleb formation is prevented by removal of extracellular Ca2+. P2X7-HEK293 cells were incubated in Ca2+-free, 500 µM EGTA-containing saline solution and treated with 3 mM ATP at time zero. Bar, 10 µm.

Bleb formation is a complex event dependent on the stimulation of specific intracellular pathways activated during apoptosis. It has been recently reported that TNFRI and Fas-induced blebbing is mediated by caspase-3 and ROCK I (Coleman et al., 2001; Sebbagh et al., 2001). Thus, we investigated whether these pathways were also stimulated during P2X₇-dependent bleb formation. First, we treated P2X₇-HEK293 cells with the wide range caspase blocker z-VAD-fmk before their exposure to ATP. This treatment did not prevent or delay blebbing (blebs were easily detected 1 min after the addition of ATP; our unpublished results). We then tested the ROCK inhibitor Y-27632, which completely blocked bleb formation (Figure 7). An incidental observation was that in the presence of Y-27632, P2X₇-HEK293 cells lost their rounded morphology and reverted to the usual spindle-like phenotype typical of wt HEK293 cells. An obvious implication of the inhibitory effect of Y-27632 is that extracellular ATP triggers ROCK activation via the P2X₇ receptor, and ROCK in turn mediates the cytoskeletal rearrangement responsible for membrane blebbing. To test this hypothesis, we investigated cleavage of the ROCK I isoform in P2X₇-HEK293 cells. As a control, we tested the effect of an anti-Fas mAb, a treatment shown previously to trigger ROCK I cleavage (Figure 8).

View larger version (81K): [in this window] [in a new window]   Figure 7. ATP-induced bleb formation is prevented by incubation in the presence of the ROCK inhibitor Y-27632. P2X7-HEK293 cells were incubated in serum-free medium, pretreated with 10 µM Y-27632 for 2 h, rinsed, placed in fresh solution supplemented with 10 µM Y-27632, and treated with 3 mM ATP (time zero). Bar, 10 µm.

View larger version (35K): [in this window] [in a new window]   Figure 8. ATP causes ROCK I cleavage. Jurkat or P2X7-HEK293 cells were treated with either anti-Fas mAb, in the presence or absence of z-VAD-fmk (100 µM) or with ATP (3 mM) in the presence or absence of z-VAD-fmk for 20 min (A) or for the times indicated below lanes (B) and then processed for protein extraction and immunoblot analysis (see MATERIALS AND METHODS). Controls received no treatment.

The effect of Fas stimulation was tested in Jurkat as well as in P2X₇-HEK293 cells. Fas activation lead to the formation of the cleaved 130-kDa active form of ROCK I, more extensively in Jurkat compared with P2X₇-HEK293 cells. ATP treatment of P2X₇-HEK293 cells caused accumulation of a 130-kDa band, and rather interestingly but in agreement with the experiments on ATP-induced bleb formation, this process was not inhibited by z-VAD-fmk. We assessed ROCK I proteolysis at various time points, starting 1 min after the addition of ATP. Even at this early time point the 130-kDa fragment was detectable (Figure 8B), suggesting that cleavage of ROCK I might be a very early event after ATP stimulation. ROCK I cleavage stimulated by ATP was z-VAD-fmk insensitive at all time points examined, whereas on the contrary it was z-VAD-sensitive when the stimulus was the anti-Fas antibody both in Jurkat (Figure 8A) and in HEK293 cells (Figure 8B).

Although clearly detectable, evidence of ROCK I cleavage does not rule out the possibility that ROCK I stimulation also occurs by other more conventional pathways, such as activation by Rho GTPases. To test this directly, we performed a pull-down experiment (Figure 9), to assay the RhoA activation status in ATP-stimulated cells. We found that in resting P2X₇-HEK293 cells RhoA was already in a partially activated state that was only very slightly increased by ATP stimulation after 20 min of incubation. We were unable to detect significant differences 1 min after ATP stimulation, when blebbing started. As a control, we show that histamine caused a large RhoA activation. Then, we tested the effect of the C3 botulinum toxin, a selective Rho ADP-ribosylating and -inactivating agent. The toxin was rendered cell permeable by fusion to a portion of the human immunodeficiency virus protein Tat. Figure 10A shows that 500 or 1000 nM Tat-C3 caused a nearly complete ADP-ribosylation of RhoA, as indicated by a MW shift. With 500 nM Tat-C3, ATP-induced blebbing was essentially unimpaired (Figure 10B); however, with 1 µM there was a substantial delay in blebbing that did not start until after 20 min (Figure 10C), as if treatment with a supramaximal dose of toxin was necessary to fully inhibit all residual RhoA-dependent activity. This suggests that the Rho pathway may also play a role in P2X₇-dependent ROCK activation.

View larger version (25K): [in this window] [in a new window]   Figure 9. Effect of ATP stimulation on RhoA activation. Confluent P2X7-HEK293 monolayers incubated in Ca2+-containing saline solution were stimulated with 3 mM ATP or 100 µM histamine (HIST) for the indicated times, rinsed, and lysed. For each time point 1.5 mg of total cell protein was mixed with GST-Rhotekin–bound glutathione-Sepharose beads (100 µl). After this incubation, samples were spun, supernatants were discarded, and beads were washed three times and loaded onto the gel (100 µl/lane) for electrophoresis and immunoblot analysis (see MATERIALS AND METHODS for further details). (A) Immunoblot; (B) densitometric analysis of the bands. DU, arbitrary densitometric units. Density of the bands is expressed as percent variation of control band at the 1-min time point (C, 1').

View larger version (53K): [in this window] [in a new window]   Figure 10. Effect of Tat-C3 treatment on Rho A and ATP-induced bleb formation. Confluent P2X7-HEK293 monolayers were incubated in 15% FCS-containing DME/F-12 medium and treated with 500 nM or 1 µM Tat-C3 for 16 h. At the end of this incubation, cells were rinsed, lysed, and analyzed by SDS-PAGE (A). Treatment with Tat-C3 caused a shift in the mobility of RhoA to a slower mobility ADP-ribosylated form (RhoA'). Alternatively, monolayers were rinsed, incubated in Ca2+-containing saline solution and subjected to microscopic analysis (see MATERIALS AND METHODS). (B and C) Cells treated with 500 nM or 1 µM Tat-C3, respectively. Bar, 10 µm.

Finally, we checked whether P2X₇ might drive ROCK I cleavage by activating other intracellular proteases. Although calpains have been implicated in the control of cytoskeletal dynamics and apoptosis (Chan and Mattson, 1999), we found that specific calpain inhibitors (Calpain Inhibitor I, ALLN, and Calpain Inhibitor II, ALLM), were unable to prevent ATP-induced ROCK I cleavage and activation (our unpublished results).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
The hypothesis that extracellular nucleotides play an important role in cell-to-cell communication has been well substantiated in recent years (Ralevic and Burnstock, 1998; Chizh and Illes, 2001; North, 2002). Several reports have demonstrated that ATP concentrations in the pericellular space attain levels as high as10–20 µM and are modulated by cell activity (Ferrari et al., 1997; Beigi et al., 1999; Schwiebert et al., 2002). Furthermore, a large family of receptors for extracellular nucleotides, the P2 receptors, has been cloned and is currently a focus of intense investigation because of its involvement in many different responses such as pain sensation (Chizh and Illes, 2001), chemotaxis (Oshimi et al., 1999), chloride secretion (Inoue et al., 1997), cytokine and chemokine release (Di Virgilio et al., 2001), mechanosensory transduction in the urinary bladder (Vlaskovska et al., 2001), and cytotoxicity (Di Virgilio et al., 1998). Finally, almost all cell types express very powerful ecto-ATPases that effectively modulate the concentration of extracellular nucleotides (Zimmermann, 2000). Thus, we must add to the known extracellular systems based on more conventional mediators (e.g., growth factors, neurotransmitters, cytokines), a complex and as yet poorly known system based on nucleotides (and nucleosides).

The responses elicited by extracellular ATP depend in the first place on the given P2 receptor subtype(s) expressed by the responding cell and the intensity of stimulation. Among the many different responses produced by ATP stimulation, cytotoxicity is one of the most intriguing and potentially most interesting. This phenomenon was initially described by Mirabelli and coworkers (1986) and later extensively investigated in several laboratories, including our own (Di Virgilio et al., 1989; Filippini et al., 1990; Zanovello et al., 1990; Zheng et al., 1991). Prolonged incubation in the presence of extracellular ATP can cause cell death by either necrosis or apoptosis. Whether one or the other pathway predominates depends on the cell type and the dose and length of exposure to the nucleotide. There are few doubts that the principal (and according to some investigators the only) P2 receptor capable of triggering cell death is P2X₇, a ligand-gated receptor channel, permeable to mono- and di-valent cations, whose only physiological ligand is ATP. This receptor is a homo-oligomer made of subunits (probably three or six) composed of 595 amino acids each (Surprenant et al., 1996; Kim et al., 2001b). In the presence of low ATP concentrations or in response to a single pulse of ATP, P2X₇ behaves as a conventional cation-selective channel. However, when exposed to high ATP concentrations or to repeated pulses of the agonist, it undergoes a channel to pore transition that allows hydrophilic solutes of molecular mass up to 900 Da through the plasma membrane (Di Virgilio, 1995; Surprenant et al., 1996). Although P2X₇ is not the only P2X receptor for which such a peculiar behavior has been described (Khakh et al., 1999; Virginio et al., 1999), it is the receptor for which the channel/pore transition has been most extensively and reproducibly documented. The physiological meaning of this phenomenon is not understood, although some intriguing hypotheses have been forwarded (Di Virgilio, 1995, 2000; Di Virgilio et al., 2001). Whatever the real physiological function of the P2X₇ pore might be, there is no doubt that such a lesion is lethal if it remains patent for any extended time (>10–15 min in most cell types).

P2X₇-mediated cell death occurs either via necrosis or apoptosis. We have observed that cells expressing this receptor to a high level are more prone to undergo fast necrotic death, probably because the rapid upset of intracellular ion homeostasis prevents the initiation of the complex chain of events necessary for apoptosis (Zanovello et al., 1990). On the other hand, cells with a lower expression of P2X₇ or where this receptor forms a smaller sized pore, such as the lymphocytes, are more likely to die by apoptosis. Although seldom acknowledged as a proapoptotic receptor, P2X₇ triggers many of the changes typical of this process: membrane blebbing, phosphatidylserine exposure, cell shrinkage, activation of caspases 1, 3, and 8, cleavage of the caspase substrates PARP and lamin B, chromatin condensation, DNA fragmentation, and apoptotic body formation (Zanovello et al., 1990; Zheng et al., 1991; Ferrari et al., 1999). Although originally bleb formation and phosphatidylserine exposure were regarded as unequivocal indications of apoptosis, it is now increasingly appreciated that these changes are not necessarily followed by cell death (see MacKenzie et al., 2001). Our data also support this view because, provided ATP was removed within 15–20 min, blebbing was reversible. However, a more prolonged exposure to ATP almost invariably committed P2X₇-HEK293 cells to death, in agreement with MacKenzie et al. (2001).

The seemingly simple process of bleb formation is powered by the contractile forces generated by the acto-myosin cytoskeleton through the modulation of a complex array of intracellular signaling molecules that modulate MLC phosphorylation, control myosin ATPase activity, bridge the cytoskeleton with the plasma membrane, and stabilize filamentous actin. Cytoskeletal contractility is critically dependent on the activity of the Rho GTPases, which in their GTP-bound form stimulate the activity of the two ROCK isoforms I and II. Recent data show that ROCK I, but not Rho, activity is necessary for TNF-–stimulated cells to undergo the full sequence of morphological alterations of apoptosis, from membrane blebbing to apoptotic body formation (Coleman et al., 2001; Sebbagh et al., 2001).

Extracellular ATP triggers via P2X₇ dramatic changes in cell shape and volume that are commonly thought to be the mere consequence of the ion unbalance caused by large pore formation. A large Ca²⁺ influx is indeed necessary because our study shows that in the absence of extracellular Ca²⁺ ATP-induced bleb formation is fully inhibited. A functioning cytoskeleton is also obviously required, because the powerful cytoskeletal poison cytochalasin B blocks blebbing. However, our data show that a large Ca²⁺ influx and a functioning cytoskeleton are not by themselves sufficient for bleb formation; rather, a complex chain of events downstream of P2X₇ is very rapidly triggered and is critically required for the full-blown pattern of morphological changes. Like the process triggered by the TNF- receptor, by C2-ceramide, or by cycloheximide, ATP-induced membrane blebbing also is critically dependent on ROCK I activity. However, at variance with these stimuli and despite the fact that P2X₇ has been shown to activate caspases (Ferrari et al., 1999), ATP-induced ROCK I cleavage and membrane blebbing are independent of caspase-3 activation. The identity of the pathway coupling P2X₇ to ROCK I is therefore an open question.

We think it likely that P2X₇ causes ROCK activation via a multiplicity of convergent pathways, some leading to direct ROCK I cleavage and activation, and therefore proapoptotic, and others to Rho-mediated ROCK I stimulation, and therefore nonapoptotic. Generation of a constitutive ROCK I kinase via cleavage of the C-terminal portion with ATP doses and kinetics compatible with blebbing is shown by Western blot analysis, but the identity of the protease responsible for the ATP-induced ROCK I cleavage is as yet unknown. We hypothesized that calpain might be involved, but inhibitors of this protease were unable to prevent the ATP effect. On the other hand, a role for Rho is supported by the delayed blebbing caused by treatment of P2X₇-HEK293 cells with a supramaximal dose of Tat-C3 toxin and by reversibility of bleb formation when ATP is removed within 15–20 min of addition. However, two observations are puzzling: first, blebbing was unaffected by a low Tat-C3 dose (500 nM), which was nevertheless apparently able to modify all available RhoA; second, in the pull-down experiment we observed a minor increase in GTP-RhoA only at a late time point (20 min), when blebbing was well underway. We should also consider other possible pathways. For example, it might be that the P2X₇ is indirectly coupled to ROCK kinases via the Ca²⁺ increase (or the K⁺ decrease; Sanz and Di Virgilio, 2000), or via one of the proteins known to coassemble with this receptor (Kim et al., 2001a). Indirect coupling could also occur through the release of a diffusible soluble messenger generated at the plasma membrane, e.g., arachidonic acid, known to bind the C-terminal regulatory domain and to activate the ROCK I kinase (Feng et al., 1999). Therefore, although our data strongly point to the involvement of ROCK I, the underlying mechanism of activation needs further investigation.

In our hands, sustained activation of P2X₇ beyond 20–30 min leads to P2X₇-HEK293 cell detachment from the substrate and death, not unlike other cell types expressing high P2X₇ levels. MacKenzie et al. (2001) reported that P2X₇-HEK293 stimulated with benzoyl ATP, a more potent and selective P2X₇ agonist, were still alive after 30 min, significant cell loss being detectable only after 6 h. We did observe that after several in vitro passages P2X₇-HEK293 cells became increasingly resistant to ATP (for example, they did not detach but rather became more adherent), but we did not perform a thorough characterization of phenotypic changes shown by P2X₇-HEK293 after prolonged culture.

In conclusion, we have described the dramatic plasma membrane dynamics caused by stimulation of the P2X₇ ATP receptor and have provided clues as to the intracellular mechanisms involved. Future experiments will hopefully disclose the protease(s) responsible for ROCK I cleavage and how it is coupled to P2X₇.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
We thank Dr. Elena Rapizzi for invaluable help. This work was supported by grants by the Italian Ministry of Scientific Research (MIUR), the National Research Council of Italy (Target Project on Biotechnology), the Italian Association for Cancer Research (AIRC), the Italian Space Agency (ASI), Telethon of Italy, and by local funds from the University of Ferrara.

	Footnotes

 
Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.02–04–0061. Article and publication date are available at www.molbiolcell.org/cgi/doi/10.1091/mbc.02-04-0061.

Abbreviations used: GFP, green fluorescent protein; MLC, myosin light chain; oATP, oxidized ATP; TNFRI, type I TNF receptor.

Online version of this article contains video material. Online version is available at www.molbiolcell.org.

Corresponding author. E-mail address: fdv{at}unife.it.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Amano, M., Ito, M., Kimura, K., Fukata, Y., Chihara, K., Nakano, T., Matsuura, Y., and Kaibuchi, K. (1996). Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J. Biol. Chem. 271, 20246–20249.[Abstract/Free Full Text]

Beigi, R., Kobatake, E., Aizawa, M., and Dubyak, G.R. (1999). Detection of local ATP release from activated platelets using cell surface-attached firefly luciferase. Am. J. Physiol. 276, C267–C278.

Chan, S.L., and Mattson, M.P. (1999). Caspase and calpain substrates: roles in synaptic plasticity and cell death. J. Neurosci. Res. 58, 167–190.[CrossRef][Medline]

Chizh, B.A., and Illes, P. (2001). P2X receptors and nociception. Pharmacol. Rev. 53, 553–568.[Abstract/Free Full Text]

Chvatchko, Y., Valera, S., Aubry, J.P., Renno, T., Buell, G., and Bonnefoy, J.Y. (1996). The involvement of an ATP-gated ion channel, P2X₁ in thymocyte apoptosis. Immunity 5, 275–283.[CrossRef][Medline]

Coleman, M.L., Sahai, E.A., Yeo, M., Bosch, M., Dewar, A., and Olson, M.F. (2001). Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I. Nat. Cell Biol. 3, 339–346.[CrossRef][Medline]

Di Virgilio, F. (1995). The P2Z purinoceptor: an intriguing role in immunity, inflammation and cell death. Immunol. Today 16, 524–528.[CrossRef][Medline]

Di Virgilio, F. (2000). Dr. Jekyll/Mr. Hyde: the dual role of extracellular ATP. J. Autonom. Nerv. Syst. 81, 59–63.[CrossRef][Medline]

Di Virgilio, F., Bronte, V., Collavo, D., and Zanovello, P. (1989). Responses of mouse lymphocytes to extracellular adenosine 5'-triphosphate (ATP). Lymphocytes with cytotoxic activity are resistant to the permeabilizing effect of ATP. J. Immunol. 143, 1955–1960.[Abstract]

Di Virgilio, F., Chiozzi, P., Falzoni, S., Ferrari, D., Sanz, J.M., Venketaraman, V., and Baricordi, O.R. (1998). Cytolytic P2X purinoceptors. Cell Death Differ. 5, 191–199.[CrossRef][Medline]

Di Virgilio, F., Chiozzi, P., Ferrari, D., Falzoni, S., Sanz, J.M., Morelli, A., Torboli, M., Bolognesi, G., and Baricordi, O.R. (2001). Nucleotide receptors: an emerging family of regulatory molecules. Blood 97, 587–600.[Abstract/Free Full Text]

Feng, J. et al. (1999). Rho-associated kinase of chicken gizzard smooth muscle. J. Biol. Chem. 274, 3744–3752.[Abstract/Free Full Text]

Ferrari, D., Chiozzi, P., Falzoni, S., Hanau, S., and Di Virgilio, F. (1997). Purinergic modulation of interleukin-1 release from microglial cells stimulated with bacterial endotoxin. J. Exp. Med. 185, 579–582.[Abstract/Free Full Text]

Ferrari, D., Los, M., Bauer, M.K.A., Vandenabeele, P., Wesselborg, S., and Schulze-Osthoff, K. (1999). P2Z purinoceptor ligation induces activation of caspases with distinct roles in apoptotic and necrotic alterations of cell death. FEBS Lett. 447, 71–75.[CrossRef][Medline]

Ferri, K.F., and Kroemer, G. (2001). Organelle-specific initiation of cell death pathways. Nat. Cell Biol. 3, E255–E263.[CrossRef][Medline]

Filippini, A., Taffs, R.E., Agui, T., and Sitkovsky, M.V. (1990). Ecto-ATPase activity in cytolytic T lymphocytes. Protection from the cytotoxic effects of extracellular ATP. J. Biol. Chem. 265, 334–340.[Abstract/Free Full Text]

Fukata, Y., Amano, M., and Kaibuchi, K. (2001). Rho-Rho-kinase pathway in smooth muscle contraction and cytoskeletal reorganization of non muscle cells. Trends Pharmacol. Sci. 22, 32–39.[CrossRef][Medline]

Khakh, B., Bao, X., Labarca, C., and Lester, H. (1999). Neuronal P2X receptor-transmitter-gated cation channels change their ion selectivity in seconds. Nature Neurosci. 2, 322–330.[CrossRef][Medline]

Kim, M., Jiang, L.H., Wilson, H.L., North, R.A., and Surprenant, A. (2001a). Proteomic and functional evidence for a P2X₇ receptor signalling complex. EMBO J. 20, 6347–6358.[CrossRef][Medline]

Kim, M., Spelta, V., Sim, J., North, R.A., and Surprenant, A. (2001b). Differential assembly of rat purinergic P2X₇ receptor in immune cell of the brain and periphery. J. Biol. Chem. 276, 23262–23267.[Abstract/Free Full Text]

Kimura, K. et al. (1996). Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 273, 245–248.[Abstract]

Inoue, C.N., Woo, J.S., Schwiebert, E.M., Morita, T., Hanaoka, K., Guggino, S.E., and Guggino, W.B. (1997). Role of purinergic receptors in chloride secretion in Caco-2 cells. Am. J. Physiol. 272, C1862–C1870.

MacKenzie, A., Wilson, H.L., Kiss-Toth, E., Dower, S.K., North, R.A., and Surprenant, A. (2001). Rapid secretion of interleukin-1 by microvesicle shedding. Immunity, 8, 825–835.

Mirabelli, F., Bellomo, G., Nicotera, P., Moore, M., and Orrenius, S. (1986). Ca²⁺ homeostasis and cytotoxicity in isolated hepatocytes: studies with extracellular adenosine 5'-triphosphate. J. Biochem. Toxicol. 1, 29–39.[CrossRef][Medline]

Morelli, A., Ferrari, D., Bolognesi, G., Rizzuto, R., and Di Virgilio, F. (2001). Proapoptotic plasma membrane pore: P2X₇ receptor. Drug Dev. Res. 52, 571–578.[CrossRef]

Murgia, M., Hanau, S., Pizzo, P., Rippa, M., and Di Virgilio, F. (1993). Oxidized ATP. An irreversible inhibitor of the macrophage purinergic P2Z receptor. J. Biol. Chem. 268, 8199–8203.[Abstract/Free Full Text]

North, R.A. (2002). The molecular physiology of P2X receptors. Physiol. Rev. 82, 1013–1067.[Abstract/Free Full Text]

Oshimi, Y., Miyazaki, S., and Oda, S. (1999). ATP-induced Ca²⁺ response mediated by P2U and P2Y purinoceptors in human macrophages: signalling from dying cells. Immunology 98, 220–227.[CrossRef][Medline]

Ralevic, V., and Burnstock, G. (1998). Receptors for purines and pyrimidines. Pharmacol. Rev. 50, 413–492.[Abstract/Free Full Text]

Sanz, J.M., and Di Virgilio, F. (2000). Kinetics and mechanism of ATP-dependent IL-1 release from microglial cells. J. Immunol. 164, 4893–4898.[Abstract/Free Full Text]

Schachter, J.B., Sromek, S.M., Nicholas, R.A., and Harden, T.K. (1997). HEK293 human embryonic kidney cells endogenously express the P2Y1 and P2Y2 receptors. Neuropharmacology 36, 1181–1187.[CrossRef][Medline]

Schwiebert, L.M., Rice, W.C., Kudlow, B.A., Taylor, A.L., and Schwiebert, E.M. (2002). Extracellular ATP signalling and P2X nucleotide receptors in monolayers of primary human vascular endothelial cells. Am. J. Physiol. 282, C289–C301.

Sebbagh, M., Renvoizè, C., Hamelin, J., Richè, N., Bertoglio, J., and Breard, J. (2001). Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing. Nat. Cell Biol. 3, 346–352.[CrossRef][Medline]

Smart, M.L., Panchal, R.G., Bowser, D.N., Williams, D.A., and Petrou, S. (2002). Pore formation is not associated with macroscopic redistribution of P2X₇ receptors. Am. J. Physiol. 283, C77–C84.

Steinberg, T.H., and Silverstein, S.C. (1987). Extracellular ATP^4– promotes cation fluxes in the J774 mouse macrophage cell line. J. Biol. Chem. 262, 3118–3122.[Abstract/Free Full Text]

Surprenant, A., Rassendren, F., Kawashima, E., North, R.A., and Buell, G. (1996). The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X₇). Science 272, 735–738.[Abstract]

Torgerson, R.R., and McNiven, M.A. (1998). The actin-myosin cytoskeleton mediates reversible agonist-induced membrane blebbing. J. Cell Sci. 111, 2911–2922.[Abstract]

Virginio, C., MacKenzie, A., Rassendren, F.A., North, R.A., and Surprenant, A. (1999). Pore dilation of neuronal P2X receptor channels. Nat. Neurosci. 2, 315–321.[CrossRef][Medline]

Vlaskovska, M., Kasakov, L., Rong, W., Bodin, P., Bardini, M., Cockaine, D.A., Ford, A.P.D.W., and Burnstock, G. (2001). P2X₃ knock-out mice reveal a major sensory role for urothelially-released ATP. J. Neurosci. 21, 5670–5677.[Abstract/Free Full Text]

von Kugelgen, I., and Wetter, A. (2000). Molecular pharmacology of P2Y-receptors. Naunyn-Schmiedeberg's Arch. Pharmacol. 362, 310–323.[CrossRef][Medline]

Warny, M., Aboudola, S., Robson, S.C., Sevigny, J., Communi, D., Soltoff, S.P., and Kelly, C.P. (2001). P2Y₆ nucleotide receptor mediates monocyte IL-8 production in response to UDP or lipopolysaccharide. J. Biol. Chem. 276, 26051–26056.[Abstract/Free Full Text]

Yu, S.P., Canzoniero, L.M., and Choi, D.W. (2001). Ion homeostasis and apoptosis. Curr. Opin. Cell Biol. 13, 405–411.[CrossRef][Medline]

Zanovello, P., Bronte, V., Rosato, A., Pizzo, P., and Di Virgilio, F. (1990). Responses of mouse lymphocytes to extracellular ATP. II. Extracellular ATP causes cell type-dependent lysis and DNA fragmentation. J. Immunol. 145, 1545–1550.[Abstract]

Zheng, L.M., Zychlinsky, A., Liu, C-C., Ojcius, D.M., and Young, J.D-E. (1991). Extracellular ATP as a trigger for apoptosis or programmed cell death. J. Cell Biol. 112, 279–288.[Abstract/Free Full Text]

Zimmermann, H. (2000). Extracellular metabolism of ATP and other nucleotides. Naunyn-Schmiedberg's Arch. Pharmacol. 362, 299–309.[CrossRef][Medline]

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