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Originally published online as doi:10.1189/jlb.1203648 on April 9, 2004

Published online before print April 9, 2004
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(Journal of Leukocyte Biology. 2004;75:1173-1182.)
© 2004 by Society for Leukocyte Biology

The nucleotide receptor P2X7 mediates actin reorganization and membrane blebbing in RAW 264.7 macrophages via p38 MAP kinase and Rho

Zachary A. Pfeiffer, Mini Aga, Usha Prabhu, Jyoti J. Watters1, David J. Hall2 and Paul J. Bertics3

Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison

3 Correspondence: Department of Biomolecular Chemistry, University of Wisconsin, 1300 University Avenue, Madison, WI 53706-0450. E-mail: pbertics{at}wisc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Extracellular nucleotides regulate macrophage function via P2X nucleotide receptors that form ligand-gated ion channels. In particular, P2X7 activation is characterized by pore formation, membrane blebbing, and cytokine release. P2X7 is also linked to mitogen-activated protein kinases (MAPK) and Rho-dependent pathways, which are known to affect cytoskeletal structure in other systems. As cytoskeletal function is critical for macrophage behavior, we have tested the importance of these pathways in actin filament reorganization during P2X7 stimulation in RAW 264.7 macrophages. We observed that the P2X7 agonists adenosine 5'-triphosphate (ATP) and 3'-O-(4-benzoylbenzoyl) ATP (BzATP) stimulated actin reorganization and concomitant membrane blebbing within 5 min. Disruption of actin filaments with cytochalasin D attenuated membrane blebbing but not P2X7-dependent pore formation or extracellular-regulated kinase (ERK)1/ERK2 and p38 activation, suggesting that these latter processes do not require intact actin filaments. However, we provide evidence that p38 MAPK and Rho activation but not ERK1/ERK2 activation is important for P2X7-mediated actin reorganization and membrane blebbing. First, activation of p38 and Rho was detected within 5 min of BzATP treatment, which is coincident with membrane blebbing. Second, the p38 inhibitors SB202190 and SB203580 reduced nucleotide-induced blebbing and actin reorganization, whereas the MAPK kinase-1/2 inhibitor U0126, which blocks ERK1/ERK2 activation, had no discernable effect. Third, the Rho-selective inhibitor C3 exoenzyme and the Rho effector kinase, Rho-associated coiled-coil kinase, inhibitor Y-27632, markedly attenuated BzATP-stimulated actin reorganization and membrane blebbing. These data support a model wherein p38- and Rho-dependent pathways are critical for P2X7-dependent actin reorganization and membrane blebbing, thereby facilitating P2X7 involvement in macrophage inflammatory responses.

Key Words: mitogen-activated protein kinases • cytoskeleton • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
P2X7 is a member of the P2 family of nucleotide receptors and is known to be important for macrophage inflammatory functions including cell fusion, intracellular bacterial killing, and cytokine release [1 , 2 ]. P2X7 forms an ion channel upon activation by extracellular adenosine 5'-triphosphate (ATP) or the synthetic agonist 3'-O-(4-benzoylbenzoyl) ATP (BzATP), resulting in Ca2+ and Na+ influx and K+ efflux [3 ]. P2X7 also forms a nonselective pore that is permeable to fluorescent dyes less than 900 Da in size such as YO-PRO. The P2X7 C terminus, which is over 100 amino acids longer than other P2X receptors, is thought to be necessary for pore formation [4 ]. Prolonged stimulation of this pore activity can be cytotoxic [5 ] but has also been proposed to be involved in physiological processes such as cell fusion and phagocytosis [1 , 4 ]. In addition, P2X7 activation in macrophages has been linked to increased protein tyrosine phosphorylation and the regulation of the mitogen-activated protein kinases (MAPK) extracellular-regulated kinase (ERK)1/ERK2 and p38 [6 , 7 ]. Modulation of signaling pathways by P2X7 has been proposed to play an important role in the control of macrophage inflammatory responses, especially in response to lipopolysaccharide (LPS) [2 , 6 , 8 ].

Protein–protein interactions between ion channels and cytoskeletal components can facilitate receptor clustering and downstream signaling events [9 ]. It is interesting that several lines of evidence suggest that P2X7 can interact with the cytoskeleton, thereby influencing macrophage function. Considering that the macrophage cytoskeleton is important for cell morphology, migration, and exocytosis [10 , 11 ], P2X7-mediated cytoskeletal interactions are likely to have broad implications in macrophage biology. In this regard, the C terminus of P2X7 contains protein–protein interaction motifs that could facilitate interaction with cytoskeletal and signaling components [12 ]. Furthermore, several regions in the C terminus appear to be critical for receptor trafficking and surface expression [13 , 14 ], strongly suggesting the involvement of the cytoskeleton in receptor localization. P2X7 heterologously expressed in human embryonic kidney (HEK) cells coimmunoprecipitates with many cytoskeletal components and interacting proteins, including the ß2 integrin subunit, {alpha}-actinin, and ß-actin [15 ]. Ion fluxes can also influence the organization of the cytoskeleton, in part by Ca2+-dependent actin-binding proteins, which can serve to cross-link or cleave actin filaments [16 ]. Therefore, in the current study, we tested the idea that P2X7 regulates cytoskeletal reorganization and that intact actin filaments are important for certain nucleotide-mediated biological activities.

Several studies have established that extracellular nucleotides acting via P2X7 can influence cell morphology, but very little is known about the underlying signaling pathways that are responsible. For example, HEK cells transfected with P2X7 undergo extensive morphological changes and membrane blebbing upon stimulation with extracellular ATP [17 ]. Cell rounding and membrane blebbing are also induced by extracellular nucleotides in many cell types that endogenously express P2X7, including dendritic cells (DC) [18 ] and macrophages [19 , 20 ]. Although the functional consequences of P2X7-mediated membrane blebbing are unknown, in other systems, blebbing is thought to involve the cytoskeleton and is associated with physiological functions such as apoptosis, mitosis, cell motility, and secretion [21 ]. Actin filaments localized to membrane blebs are proposed to be necessary for bleb formation and retraction [21 , 22 ]. Furthermore, membrane blebbing may be a coordinated response involving multiple pathways including the actin cytoskeleton, the p38 MAPK, and members of the Rho small molecular weight (MW) G-protein family [23 ]. It is interesting that a very recent study has linked P2X7 action to RhoA activation and membrane blebbing via the Rho-dependent kinase, Rho-associated coiled-coil kinase (ROCK), in BAC1 murine macrophages [19 ]. However, it is unknown whether alterations in p38 and the actin cytoskeleton are also essential for P2X7-dependent, morphological changes and membrane blebbing.

Given that P2X7 markedly affects cell morphology, membrane blebbing, and macrophage biology, we hypothesized that P2X7 can induce actin reorganization in RAW 264.7 murine macrophages and sought to determine the critical, intracellular signaling events involved in this process. We present for the first time that P2X7 agonists induce dramatic changes, which are dependent on the activation of p38 and Rho, in macrophage actin reorganization.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
The nucleotides BzATP, ATP, uridine 5'-triphosphate (UTP), and {alpha},ß-methylene ATP were obtained from Sigma Chemical Co. (St. Louis, MO). LPS (Escherichia coli, serotype 0111:B4), cytochalasin D, phorbol 12-myristate 13-acetate (PMA), and anisomycin were also purchased from Sigma Chemical Co. Texas Red-X phalloidin and YO-PRO were obtained from Molecular Probes (Eugene, OR). PreferTM fixative solution was obtained from Anatech, Ltd. (Battle Creek, MI). The p38 inhibitors SB202190 and SB203580, the inactive analog SB202474, the phosphatidylinositol-3 kinase (PI-3K) inhibitor LY294002, and the ROCK inhibitor Y-27632 were obtained from Calbiochem (San Diego, CA), whereas the MAPK kinase (MEK)1/2 inhibitor U0126 was purchased from Promega (Madison, WI). Antibodies for immunoblotting were purchased from the following sources: anti-active ERK1/ERK2 (which recognizes the dually phosphorylated TXY motif, Promega and Biosource, Camarillo, CA), antiactive p38 (which recognizes the dually phosphorylated TXY motif, Promega), pan-reactive ERK1/ERK2 and anti-Rho (-A, -B, -C, Upstate Biotechnology, Waltham, MA), antiphospho Ser473 Akt (Cell Signaling Technology, Beverly, MA), and anti-Grb2 (Santa Cruz Biotechnology, Santa Cruz, CA). Granulocyte macrophage-colony stimulating factor (GM-CSF) was purchased from PeproTech (Rocky Hill, NJ). Purified C3 exoenzyme was a gift from Dr. Patricia Keely (University of Wisconsin, Madison), and the glutathione S-transferase (GST)–Rhotekin Rho-binding domain (RBD) construct was a gift from Dr. Anna Huttenlocher (University of Wisconsin).

Cell culture
RAW 264.7 murine macrophages were maintained in RPMI supplemented with 5% cosmic calf serum (Mediatech, Herndon, VA), 2 mM sodium pyruvate, 2 mM L-glutamine, and 100 U/ml penicillin/streptomycin. The cells were grown in 10 cm tissue-culture dishes at 37°C in a humidified atmosphere with 5% CO2. In general, cells were split 1:10 every 2 days.

Fluorescence microscopy
RAW 264.7 macrophages (5x105 or 1x106 cells/ml) were plated on glass coverslips in six-well plates 1 day before the experiments were performed. Following treatments with inhibitors and nucleotides, cells were washed once with phosphate-buffered saline (PBS). Cells were fixed with Prefer solution for 10 min at room temperature, washed three times with PBS + 0.1% Triton X-100 (PBST), and stained for actin filaments using 66 nM Texas Red-X phalloidin in PBST for 20 min at room temperature. Following three 10-min washes with PBST, the glass coverslips were mounted on slides with 60% glycerol in PBS. Cells were visualized for fluorescence or differential interference contrast (DIC) with a 100x or 63x objective using an Axioplan 2 fluorescence microscope (Zeiss, Chester, VA). Images were captured with QED (Pittsburgh, PA) imaging software and processed in Adobe Photoshop. For quantification of membrane blebbing, nine random DIC images were captured for each treatment and scored for the presence of any membrane blebbing versus the absence of any membrane blebbing.

Immunoblotting
Murine RAW 264.7 macrophages were plated at a density of 3 x 105 cells/well in 24-well tissue-culture plates the day before each experiment. Following each experiment and subsequent cell lysis with sodium dodecyl sulfate (SDS) sample buffer, the proteins were resolved on 10% SDS-polyacrylamide gel electrophoresis (PAGE) gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA). The membranes were blocked in 5% milk in Tris-buffered saline/Tween 20 (TBST; 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) or 5% bovine serum albumin (BSA)/TBST for antiphospho-Akt. Immunoblotting was performed by incubating the membranes with the primary antibody in blocking buffer for 1 h at 37°C (1:2000 dilution for antiactive ERK and antiactive p38, 1:5000 dilution for anti-Grb2, and antipan ERK1/ERK2 for loading controls) or 4°C overnight (1:1000 for antiphospho-Akt), followed by washing twice for 5 min with TBST and incubating with the appropriate horseradish peroxidase-conjugated secondary antibody (1:8000 dilution for goat anti-rabbit and 1:5000 for goat anti-mouse) for 1 h at 37°C. The membranes were then washed with TBST and visualized using SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL). Membranes were exposed to film or visualized using the Epichemi II darkroom (UVP, Upland, CA) equipped with a 12-bit cooled charged-coupled device camera. Image processing and analysis were performed using LabWorks 4.0 software (UVP).

Measurement of YO-PRO uptake by flow cytometry
RAW 264.7 murine macrophages were lifted from 10 cm tissue-culture dishes using cell dissociation medium (Sigma Chemical Co.) and resuspended in potassium-glutamate buffer (25 mM K-HEPES, pH 7.4, 130 mM K-glutamate, 5 mM KCl, 1.5 mM CaCl2, 0.5% BSA, and 10 mM glucose) at a concentration of ~1 x 106 cells/ml. YO-PRO (1 µM) was added, and the cells were treated with or without 2 µM cytochalasin D for 30 min followed by treatment with control buffer or 250 µM BzATP for 5 min. Flow cytometry was performed by measuring 10,000 events on a BD Biosciences FACScan flow cytometer (Mountain View, CA). Propidium iodide-stained cells (dead cells) were not included in the analysis.

Rho activation assay
Cell lysates (500 µl), prepared from E. coli expressing GST–Rhotekin RBD fusion protein, were incubated with 150 µl of a 50% slurry of glutathione-coupled agarose beads (Pierce) for 45 min at 4°C. After complex formation, the beads were washed five times with GST lysis buffer (10% glycerol, 50 mM Tris, pH 7.4, 200 mM NaCl, 1% Nonidet P-40, 2 mM MgCl2, supplemented with 100 µM sodium vanadate, 100 µM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin) and resuspended in 250 µl GST lysis buffer. RAW 264.7 macrophages (1x106 cells/well) plated in 12-well plates were stimulated the next day as indicated in the figure legends. Cells were lysed in 500 µl GST lysis buffer and centrifuged at 14,000 rpm for 10 min at 4°C. Cell lysates were incubated with 50 µl GST–Rhotekin beads for 45 min at 4°C. Proteins bound to the beads were eluted with 40 µl SDS sample buffer, and the total volume was electrophoresed on a 15% SDS-PAGE gel and transferred to PVDF membranes. Immunoblotting was performed as stated above with a 1:1000 dilution of anti-Rho antibodies in 5% milk/TBST at 4°C overnight. The blot was subsequently probed with antirabbit secondary antibody (1:8000) in 5% milk/TBST at room temperature for 1.5 h.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
P2X7-mediated cytoskeletal reorganization and membrane blebbing
P2X7 activation has been linked to the induction of changes in cell morphology, cytoskeletal architecture, and membrane blebbing when heterologously expressed in HEK cells [17 ]. However, as most studies on membrane blebbing have used P2X7-transfected HEK cells [19 , 24 ], we tested the idea that activation of naturally occurring P2X7 leads to morphological changes and membrane blebbing. To this end, we monitored the reorganization of the actin cytoskeleton in RAW 264.7 murine macrophages following a brief stimulus with the P2X7 agonist BzATP using fluorescence and DIC microscopy. As shown in Figure 1A , RAW 264.7 macrophages stimulated with 250 µM BzATP for 5 min exhibit a substantial change in cell morphology and the formation of membrane blebs. It is interesting that cortical actin rings near the plasma membrane surface are clearly discernable and colocalize with membrane blebs protruding from the cell surface. The cells also appear rounded and have generally retracted the filopodia that are observed in cells treated with control buffer. In contrast, cells treated with control buffer have numerous filopodia, they appear more elongated, and they do not have membrane blebs. These data indicate that ligands for endogenously expressed P2X7 can promote changes in macrophage morphology, actin cytoskeletal structure, and membrane blebbing.



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  		Figure 1. Actin reorganization and membrane blebbing by P2X7 agonists. (A) RAW 264.7 macrophages (5x105 cells/well) were plated on glass coverslips and treated with vehicle (Control) or 250 µM BzATP for 5 min, fixed, and then stained with Texas Red-X phalloidin as stated in Materials and Methods. The cells were visualized for fluorescence and DIC with a 100x objective. The arrowheads highlight a membrane bleb, which is clearly seen in the DIC image and contains a ring of cortical actin. The original scale bar = 10 µm. (B) Membrane blebbing was quantified by treating cells in triplicate with vehicle (Control), 250 µM BzATP, UTP, {alpha},ß-methylene ATP, or 3 mM ATP for 5 min. Cells were fixed and stained as stated above, and cells from nine random fields were counted and scored for blebbing. The graph represents the mean percentage of cells that were positive for blebbing ± SD. The data are representative of two independent experiments.

 
To test whether other P2X and P2Y receptors could stimulate actin reorganization and membrane blebbing, we treated RAW 264.7 macrophages with 250 µM UTP, which is an agonist for P2Y2 and P2Y4 receptors, or 250 µM {alpha},ß-methylene-ATP, which is a preferential agonist of P2X1 and P2X3. As shown in Figure 1B , neither of these nucleotides induced membrane blebbing or actin reorganization. Furthermore, administration of 250 µM BzATP or 3 mM ATP stimulated membrane blebbing in RAW 264.7 macrophages, whereas cell treatment with 250 µM ATP did not promote blebbing (data not shown). This pharmacological behavior is consistent with the agonist profile of P2X7, wherein BzATP is more potent than ATP at activating this receptor [25 ]. These data also suggest that the actin reorganization observed here is a P2X7-dependent process, as other P2X and P2Y agonists have no discernable effect on the actin cytoskeleton or membrane blebbing.

Involvement of the actin cytoskeleton in P2X7-dependent membrane blebbing, MAPK activation, and pore formation
The actin cytoskeleton is important for cell morphology and has been proposed to be critical for membrane bleb formation [21 , 22 ]. As the generation of membrane blebs stimulated by P2X7 agonists contained a ring of cortical actin (Fig. 1) , we examined the role of the actin cytoskeleton on bleb formation using cytochalasin D, which is a toxin that caps the barbed end of actin filaments [26 ], thereby inhibiting association and dissociation of subunits and disrupting cytoskeletal organization. Treatment of RAW 264.7 macrophages with 2 µM cytochalasin D for 30 min caused the actin cytoskeleton to become disorganized, and the presence of filopodia was reduced (Fig. 2 ). Furthermore, BzATP-stimulated cell-shape changes, actin reorganization, and membrane blebbing were markedly attenuated by cytochalasin D, as cortical actin rings and membrane protrusions were not observed following this treatment. These results are consistent with a model wherein actin filaments are necessary for P2X7 ligands to initiate cell-shape changes and membrane blebbing.



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  		Figure 2. Cytochalasin D attenuates P2X7-dependent membrane blebbing. RAW 264.7 macrophages (5x105 cells/well) plated on glass coverslips were pretreated with vehicle (Control) or 2 µM cytochalasin D for 30 min followed by treatment with control buffer or 250 µM BzATP for 5 min. The cells were washed, fixed, and stained with Texas Red-X phalloidin as stated in Materials and Methods. Cell fluorescence was visualized with a 100x objective, and the original scale bar = 10 µm. The arrowheads show membrane blebbing and actin rings that are not observed in the cytochalasin D-treated cells. The data are representative of three independent experiments.

 
As a result of the ability of P2X7 agonists to induce dramatic cytoskeletal changes in RAW 264.7 macrophages, we hypothesized that the actin cytoskeleton is important for P2X7-dependent functions and can contribute to the activation of ERK1/ERK2, p38, and pore formation. As shown in Figure 3A , BzATP stimulated the detectable activation of ERK1/ERK2 within 5 min and p38, within 2 min of treatment. However, cytochalasin D pretreatment did not attenuate P2X7-mediated ERK1/ERK2 or p38 activation (Fig. 3B) . Cytochalasin D itself also did not stimulate detectable MAPK activation, and it did not affect the ability of PMA treatment to induce ERK1/ERK2 activation or for anisomycin administration to induce p38 activation. These data suggest that the actin cytoskeleton is not critical for P2X7-dependent MAPK activation and that MAPK activation alone is not sufficient for membrane blebbing.



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  		Figure 3. Cytochalasin D does not affect BzATP-stimulated MAPK activation or pore formation. (A) RAW 264.7 macrophages (3x105 cells/well) were treated with vehicle (Control), 100 nM PMA, or 250 µM BzATP for the indicated times. Cell lysates were prepared and following SDS-PAGE, were immunoblotted with antiactive ERK1/ERK2 or antiactive p38 antibodies. Pan-reactive ERK antibodies were used to show equal protein loading. The immunoblots are representative of three independent experiments. (B) RAW 264.7 macrophages were pretreated with or without 2 µM cytochalasin D (Cyto D) for 30 min followed by treatment with vehicle (Control), 100 nM PMA for 5 min, 10 µg/ml anisomycin (Ani) for 10 min, or 250 µM BzATP for 15 min. Active ERK1/ERK2 and p38 were analyzed by immunoblotting as above. Anti-Grb2 antibodies were used to show equal protein loading. (C) RAW 264.7 macrophages (1x106 cells/ml) were resuspended in potassium-glutamate buffer with or without 1 µM YO-PRO as stated in Materials and Methods. Cells were pretreated with or without 2 µM cytochalasin D for 30 min followed by treatment with vehicle (Control) or 250 µM BzATP for 5 min. Cell fluorescence was analyzed by flow cytometry and displayed as histograms representing 10,000 events.

 
P2X7 agonists stimulate pore formation that allows the passage of molecules <900 Da, and this process is measurable by monitoring the uptake of the dye YO-PRO. Proposed interactions between the P2X7 C terminus and cytoskeletal proteins could provide a mechanism by which P2X7 mediates pore formation, although no detectable changes in P2X7 distribution are detected upon ligand stimulation [27 ]. We hypothesized that actin filaments have a role in the assembly of P2X7 into the nonspecific pore or facilitate protein–protein interactions for pore assembly. Accordingly, to test this idea, RAW 264.7 macrophages were incubated with or without 2 µM cytochalasin D in the presence of 1 µM YO-PRO followed by stimulation with control buffer or BzATP for 5 min. The cell fluorescence, indicating YO-PRO uptake, was measured by flow cytometry. As shown in Figure 3C , pretreatment with cytochalasin D did not attenuate BzATP-induced pore formation. P2X7-stimulated Ca2+ fluxes were also not affected by cytochalasin D (data not shown). From these results, we conclude that extensive actin rearrangement is not necessary for P2X7-mediated MAPK activation, pore formation, and ion fluxes.

Influence of MAPK pathways on BzATP-stimulated membrane blebbing
The mechanism of P2X7-mediated actin reorganization and membrane blebbing is undefined. MAPK activation alone is not sufficient to induce membrane blebbing, as cytochalasin D attenuates blebbing but not MAPK activation (Fig. 3) . However, as MAPK activation can participate in actin-filament reorganization in other systems, we tested the idea that MAPK activation acts upstream of P2X7-dependent actin reorganization and membrane blebbing. RAW 264.7 macrophages were pretreated for 15 min with the MEK antagonist U0126 (10 µM) or the p38 antagonist SB202190 (10 µM). The cells were then treated with P2X7 ligands followed by fluorescence and DIC microscopy to monitor the actin cytoskeleton (Fig. 4 ). The MEK antagonist did not attenuate membrane blebbing, actin reorganization, or cell morphological changes induced by the P2X7 agonist BzATP, suggesting that the MEK/ERK pathway is not involved in these processes. However, the p38 antagonist effectively blocked P2X7-mediated membrane blebbing. The effects of cytochalasin D, U0126, and SB202190 on membrane blebbing are quantified in Figure 5A , and these studies revealed that BzATP stimulated 47 ± 5% of the cells to bleb, which was markedly attenuated by cytochalasin D (8±4%) and the p38 inhibitor SB202190 (11±4%). These results suggest that p38 activation is upstream of cytoskeletal reorganization and membrane blebbing, whereas the ERK1/ERK2 pathway is not critical for these events.



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  		Figure 4. Activation of p38 but not ERK contributes to cytoskeletal reorganization and membrane blebbing. RAW 264.7 macrophages (5x105 cells/well) were plated on glass coverslips and pretreated with vehicle (Control), the MEK1/2 inhibitor U0126 (10 µM), or the p38 inhibitor SB202190 (10 µM) for 15 min. The cells were then treated with vehicle (Control) or 250 µM BzATP for 5 min, fixed, and then stained with Texas Red-X phalloidin as stated in Materials and Methods. Cell fluorescence was visualized with a 100x objective, and the original scale bar = 10 µm. The arrowheads highlight membrane blebs and actin rings that are clearly visible.

 


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  		Figure 5. P2X7-mediated membrane blebbing in the presence of cytochalasin D or MAPK inhibitors. (A) Membrane blebbing following treatment with vehicle (Control), 2 µM cytochalasin D, 10 µM U0126, or 10 µM SB202190 was quantified by counting and scoring cells from nine random fields for blebbing. The graph represents the mean percentage of blebbing cells from at least three independent experiments ± SD. (B) RAW 264.7 macrophages were pretreated with the indicated doses of the selective p38 inhibitor SB202190 for 15 min followed by treatment with 250 µM BzATP for 5 min. Membrane blebbing was quantified by counting and scoring cells from nine random fields for blebbing. The graph represents the mean percentage of blebbing cells from three independent experiments ± SD.

 
To confirm the involvement of p38 in P2X7-mediated membrane blebbing, we analyzed a dose response of SB202190 and the effect of other p38 inhibitors. Figure 5B shows the effect of varying doses of SB202190 on BzATP-stimulated membrane blebbing with maximal inhibition occurring at 10 µM. SB202190 treatment alone did not stimulate the cells to bleb (data not shown). As shown in Figure 6A , the selective p38 antagonists SB202190 and SB203580 attenuated BzATP-stimulated membrane blebbing, whereas an inactive analog, SB202474, did not attenuate membrane blebbing. The inhibitors alone did not affect blebbing (data not shown). It is interesting that pretreatment of RAW cells with the p38 inhibitors SB202190 or SB203580 (10 µM) attenuated the ability of BzATP to stimulate phosphorylation of p38, whereas the inactive analog SB202474 had no discernable effect (Fig. 6B) . Although these pyridinylimidazole compounds bind the ATP-binding site of p38 regardless of phosphorylation status [28 ], the binding of these compounds by p38 has been reported to induce local and global conformational changes in the enzyme. Accordingly, these changes may influence p38 recognition by protein phosphatases or upstream kinases and lead to the reduced p38 phosphorylation status observed in Figure 6B . Consistent with these observations, previous studies have also demonstrated that p38 phosphorylation is reduced in the presence of SB203580 or SB202190 [29 , 30 ]. Furthermore, p38 activation is known to down-regulate certain protein phosphatases [31 ], and the presence of p38 inhibitors leads to increased phosphatase activity, which in turn, may lead to reduced p38 phosphorylation. Taken together, these data obtained with the p38 inhibitors SB202190 and SB203580 support the conclusion that p38-associated pathways are critical for P2X7-mediated membrane blebbing.



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  		Figure 6. Effect of the p38 inhibitor SB203580 and the inactive analog SB202474 on BzATP-stimulated membrane blebbing and p38 activation. (A) RAW 264.7 macrophages (3x105 cells/well) plated on glass coverslips were pretreated with the selective p38 inhibitor SB203580 (10 µM), the inactive analog SB202474 (10 µM), or no inhibitor followed by treatment with vehicle (Control) or 250 µM BzATP. The cells were fixed and analyzed by DIC microscopy with a 100x objective (original scale bar=10 µm). Two fields of BzATP-treated cells are shown, and each treatment is representative of at least three independent experiments. The arrowheads highlight membrane blebs. (B) RAW macrophages were pretreated with vehicle (Control), the selective p38 inhibitors SB202190 or SB203580 (10 µM), or the inactive analog SB202474 (10 µM) for 15 min followed by treatment with vehicle (Control) or 250 µM BzATP for 15 min. Immunoblotting was performed with antiactive p38 antibodies, and the blot is representative of three experiments with identical results.

 
PI-3K-dependent pathways do not appear to be involved in P2X7-mediated responses
PI-3K-dependent pathways have been reported to regulate the actin cytoskeleton in response to various growth factors, cytokines, and chemokines [32 , 33 ]. To ascertain whether PI-3K-dependent pathways were involved in actin reorganization by P2X7 agonists, RAW 264.7 macrophages plated on glass coverslips were pretreated with the PI-3K inhibitor LY294002 (10 µM) followed by a 5-min stimulation with 250 µM BzATP. As shown in Figure 7A , LY294002 did not attenuate BzATP-stimulated actin reorganization and membrane blebbing in RAW 264.7 macrophages.



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  		Figure 7. Effect of a PI-3K inhibitor on membrane blebbing and MAPK activation. (A) RAW 264.7 macrophages (5x105 cells/well) were pretreated with the PI-3K inhibitor LY294002 (10 µM) followed by treatment with vehicle (Control) or 250 µM BzATP for 5 min. Cells were fixed and stained with Texas-Red phalloidin as stated in Materials and Methods. The arrowhead shows a visible membrane bleb, and the original scale bar = 10 µm. (B) RAW 264.7 macrophages were pretreated with vehicle (Control) or 10 µM LY294002 for 15 min before stimulation with vehicle [control (C)] or 250 µM BzATP for the indicated times. Immunoblots are with antiactive ERK1/ERK2 and antiactive p38 antibodies. (C) RAW 264.7 macrophages were pretreated with vehicle (Control) or 10 µM LY294002 for 15 min. Cells were then treated with vehicle (Control), 25 ng/ml GM-CSF for 10 min, 100 ng/ml LPS for 10 min, or 250 µM BzATP for 5 and 15 min. Immunoblotting was performed with antiphospho-Akt antibodies.

 
As the PI-3K inhibitor did not attenuate nucleotide-mediated cytoskeletal reorganization or membrane blebbing, we hypothesized that PI-3K pathways were not essential for BzATP-stimulated MAPK activation. To test this idea, RAW 264.7 macrophages were pretreated with the PI-3K inhibitor LY294002 (10 µM) followed by stimulation with BzATP for 5–15 min. As shown in Figure 7B , the PI-3K inhibitor did not appear to attenuate BzATP-stimulated ERK1/ERK2 or p38 activation. To confirm that LY294002 inhibited PI-3K-dependent pathways, we analyzed activation of the PI-3K downstream effector Akt. Although we observed that GM-CSF and LPS could stimulate Akt activation in RAW 264.7 macrophages, we did not detect Akt phosphorylation in response to BzATP treatment, suggesting that P2X7 does not appreciably stimulate PI-3K at the time-points analyzed (5 and 15 min; Fig. 7C ). Taken together, these results suggest that PI-3K pathways are not critical for P2X7-mediated actin reorganization, membrane blebbing, or MAPK activation in RAW 264.7 cells.

Rho involvement in P2X7-mediated cytoskeletal reorganization
The Rho family of small MW G-proteins is involved in numerous signaling pathways that control the organization of the actin cytoskeleton [34 ]. In particular, Rho has been linked not only to cytoskeletal reorganization but also to membrane blebbing through activation of the effector kinases ROCK-I and ROCK-II [35 , 36 ]. To explore the idea that Rho and the downstream effector kinase ROCK are involved in P2X7-dependent membrane blebbing, we blocked Rho function using the Clostridium botulinum C3 exoenzyme, which can adenosine 5'-diphosphate-ribosylate and inactivate Rho [37 ], and we antagonized ROCK activation by treating cells with the selective ROCK inhibitor Y-27632. RAW 264.7 macrophages were treated with C3 exoenzyme (10 µg/ml) for 18 h followed by treatment with or without BzATP for 5 min. In the absence of nucleotides, C3 exoenzyme treatment caused changes in cell morphology; notably, the cells appeared more spread and generally had more protrusions and filopodia (Fig. 8A ). In C3 exoenzyme-pretreated cells, BzATP treatment did not induce cell rounding, cytoskeletal reorganization, or membrane blebbing, suggesting that Rho activation is a critical step for these processes. Similar results were observed with the ROCK inhibitor Y-27632 and are quantified in Figure 8B . In agreement with Verhoef et al. [19 ] and Morelli et al. [24 ], Y-27632 also inhibited membrane blebbing and cell rounding in BzATP-stimulated HEK/P2X7 cells (data not shown). To confirm Rho activation following cell treatment with P2X7 agonists, Rho activation was monitored via a pull-down assay, which uses a GST–Rhotekin RBD fusion protein to capture active Rho. In these studies, peak Rho activation upon BzATP treatment was observed at 5 min, coinciding with membrane blebbing (Fig. 9A ). Exogenous treatment of RAW cells with purified C3 exoenzyme for 18 h markedly attenuated BzATP-stimulated Rho activation (Fig. 9B) . These results suggest that P2X7 initiates membrane blebbing and actin reorganization via Rho and ROCK-dependent pathways.



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  		Figure 8. Effect of C3 exoenzyme and ROCK inhibitor on membrane blebbing. (A) RAW 264.7 macrophages (1x106 cells/well) were plated on glass coverslips and pretreated with or without C3 exoenzyme (10 µg/ml) for 18 h or the ROCK inhibitor Y-27632 (10 µM) for 30 min followed by treatment with vehicle (Control) or 250 µM BzATP for 5 min. The cells were fixed and stained with Texas Red-X phalloidin as stated in Materials and Methods. The arrowheads highlight visible blebs and actin rings. The original scale bar = 10 µm. (B) Counting and scoring cells from nine random fields for blebbing quantified membrane blebbing following C3 exoenzyme and Y-27632 treatment. The graph represents the mean percentage of blebbing cells from three independent experiments ± SD.

 


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  		Figure 9. Activation of Rho by BzATP treatment and inhibition by C3 exoenzyme. (A) Rho activation was assessed using a pull-down assay as described in Materials and Methods. RAW 264.7 macrophages were treated with control buffer or 250 µM BzATP for the indicated times, lysed with GST buffer, and incubated with glutathione beads coupled with GST–Rhotekin RBD fusion protein. The beads were washed three times with GST buffer, and bound proteins were eluted with SDS-PAGE buffer. Immunoblotting was performed with anti-Rho (-A, -B, -C) antibodies. These data are representative of three independent experiments. (B) RAW macrophages were pretreated with control or 10 µg/ml C3 exoenzyme for 18 h followed by treatment with vehicle (Control) or 250 µM BzATP for 5 min. Rho activation was assessed as described above, and these data are representative of two independent experiments with similar results.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present studies provide data supporting a model, wherein naturally occurring P2X7 in macrophages is linked to actin-filament reorganization and membrane blebbing. It is interesting that P2X7 agonists stimulated a dramatic change in cell morphology; i.e., P2X7 activation promotes the cells to progress from a spread state with numerous filopodia to a round morphology and retracted filopodia. Cortical actin rings colocalized with membrane blebs, suggesting a role for the actin cytoskeleton. Indeed, the cytoskeleton has been shown previously to be involved in bleb formation and retraction [21 , 22 ]. Therefore, to address whether actin filaments were involved in P2X7-dependent membrane blebbing, we used cytochalasin D to inhibit actin polymerization and depolymerization. Cytochalasin D attenuated BzATP-induced actin reorganization and membrane blebbing, leading us to speculate that P2X7-mediated protein–protein interactions are critical for these observations. In fact, truncation of the C terminus of P2X7 disrupts interactions with epithelial membrane proteins, and this process has been shown to attenuate membrane blebbing [38 ]. Furthermore, P2X7 has been reported to exist in a complex with numerous cytoskeletal components and binding proteins including the ß2 integrin subunit, {alpha}-actin, and ß-actin [15 ]. In this regard, we have also identified several protein–protein interaction domains (e.g., an Src homology-3-binding domain and potential cytoskeletal attachment sites) within the P2X7 C terminus that may be important for signaling, actin filament reorganization, and membrane blebbing [12 ].

Numerous studies have linked P2Y and P2X receptor subtypes to actin reorganization in a variety of systems. P2Y receptors have been linked to Ca2+ mobilization and actin reorganization in vascular myocytes via activation of Rho and ROCK [39 ]. P2Y agonists also induce actin polymerization and chemotaxis in immature DC [40 ]. The P2X agonists ATP and {alpha},ß-methylene-ATP stimulate Ca2+-dependent actin disassembly in WRK-1 rat mammary tumor cells [41 ]. However, we did not observe dramatic actin cytoskeleton changes in 5 min with UTP or {alpha},ß-methylene-ATP, as was observed with the P2X7 agonists BzATP and millimolar concentrations of ATP. Our results using pharmacological agonists of P2Y2, P2Y4, P2X1, and P2X3 receptor subtypes suggest that these receptors do not substantially contribute to the dramatic changes in cytoskeletal organization and membrane blebbing observed in RAW 264.7 macrophages.

As membrane receptors and ion channels can interact with components of the cytoskeleton, we hypothesized that P2X7-dependent effects on the actin cytoskeleton may be important for receptor function with regard to pore formation and ion channel activity. Direct effects on ion channel activity by actin disruption have been seen in many different receptor systems [9 ]. For example, P2X1 activation and desensitization are altered following cytoskeletal disruption [42 ]. In the case of P2X7, formation of the nonspecific pore upon receptor activation may involve receptor oligomerization, although changes in cell-surface distribution have not been detected in HEK cells expressing enhanced green fluorescent protein-tagged P2X7 receptors [27 ]. Furthermore, it is unclear whether P2X7 requires accessory proteins for pore formation or is sufficient to initiate pore activity on its own [43 ]. As we did not observe an effect of cytochalasin D on P2X7-dependent pore formation as measured by YO-PRO uptake, this suggests that P2X7 oligomerization does not require interaction with the actin cytoskeleton, or the oligomer is assembled before activation. We also did not detect any changes in P2X7-dependent Ca2+ fluxes following cytochalasin D treatment (data not shown). Furthermore, chelation of extracellular Ca2+ with EGTA did not attenuate BzATP-stimulated actin reorganization or membrane blebbing (data not shown), suggesting that these membrane alterations occur independently of ion fluxes.

The mechanism of P2X7-dependent actin reorganization and membrane blebbing is still unclear. Ligand-gated ion channels have been hypothesized to interact with cytoskeletal components to form signaling complexes [44 ]. P2X7-mediated signaling events such as activation of ERK1/ERK2 and p38 were not affected by cytochalasin D treatment, suggesting that actin integrity is not necessary for certain P2X7-dependent signaling events. Conversely, P2X7-dependent MAPK activation appears to be upstream of membrane blebbing, given that the selective p38 inhibitor SB202190 attenuated BzATP-stimulated membrane blebbing and actin reorganization. This inhibition is dose-dependent and is also observed with another selective p38 antagonist, SB203580. Furthermore, an inactive analog, SB202474, did not attenuate membrane blebbing. Previous evidence has linked p38 activation to membrane blebbing via the phosphorylation of the heat shock protein (HSP)27 and subsequent translocation to membrane blebs in fibroblasts [45 ]. The initiation of p38-dependent HSP27 phosphorylation is mediated by MAPK-activated protein kinase 2 [46 ], but whether these events occur in macrophages is still in question. In contrast, we found that the MEK inhibitor U0126 does not attenuate membrane blebbing and cytoskeletal reorganization. These results are consistent with work in other systems, which support the conclusion that the ERK1/ERK2 pathway is not essential for membrane blebbing [45 ].

PI lipids can contribute to changes in cytoskeletal architecture [16 ]. Activation of PI-3K-dependent pathways has also been linked to nucleotide-stimulated MAPK signaling pathways in astrocytes [47 ], leading us to hypothesize that these pathways are involved in P2X7-mediated actin reorganization and MAPK activation. Indeed, PI-3K pathways have been linked to membrane blebbing in other systems [48 ]. We analyzed activation of the PI-3K effector Akt and did not detect stimulation by BzATP within 15 min of treatment. This suggests that P2X7 is not coupled to the PI-3K pathway in this time-frame and is consistent with our other observations that PI-3K inhibitors do not attenuate P2X7-mediated actin reorganization, membrane blebbing, or MAPK activation in RAW 264.7 macrophages.

The Rho family of small MW G-proteins modulates cell morphology and cytoskeletal organization. For example, active RhoA maintains human monocytes in a rounded state and is therefore thought to be a negative regulator of cell spreading [49 ]. Previous studies have also indicated that exogenous treatment of cells with C3 exoenzyme can enter leukocytes and inactivate Rho [49 , 50 ]. In the current study, we demonstrate that exogenous addition of C3 exoenzyme reduces basal Rho activation and attenuates BzATP-stimulated Rho activation (Fig. 8B) . We also observed that C3 exoenzyme increases cell spreading and filopodia formation in RAW 264.7 macrophages, suggesting that C3 exoenzyme is taken up and functional. The time-course of Rho activation following BzATP treatment coincides with the onset of blebbing and is consistent with our observation that C3 exoenzyme attenuates P2X7-mediated membrane blebbing and actin reorganization. The Rho-effector kinases ROCK-I and ROCK-II have been implicated in membrane blebbing and cell rounding [35 , 36 ]. The compound Y-27632 is cell-permeable, highly selective for ROCK kinases at 10 µM [51 ], and has been shown to inhibit membrane blebbing in BAC1 murine macrophages and HEK/P2X7 cells [19 ]. Our data support a model, where p38 and ROCK-dependent pathways contribute to membrane blebbing. We propose that membrane blebbing requires several distinct steps: Cell rounding and filopodia retraction occur in a Rho/ROCK and cytoskeleton-dependent manner followed by activation of p38-dependent pathways, which lead to the initiation of membrane blebbing. A similar model has been proposed in which p38 and Rho regulate membrane blebbing in endothelial cells [23 ], but the cross-talk between the two pathways remains undefined. A possible mechanism of cross-talk between the p38 and Rho pathways is that HSP27 localization may be dependent on RhoA [52 ], but the contributions of each pathway to different steps in the blebbing process are not currently known.

The actin cytoskeleton is critical for many macrophage functions including cell fusion and phagocytosis. For example, cytochalasin D attenuates interleukin-13-induced macrophage fusion [53 ] and inhibits phagosome-endosome fusion events [54 ]. It is interesting that several studies have linked P2X7 with the formation of multinucleated giant cells and the killing of intracellular mycobacteria [1 , 55 ]. We speculate that the ability of P2X7 to influence cytoskeletal and membrane organization provides a mechanism by which P2X7 influences multiple macrophage inflammatory responses.


   ACKNOWLEDGEMENTS
 
National Institutes of Health Grants HL56396 and AI50500 supported this work. Biotechnology Training Program NIH 5 T32 GM08349 (University of Wisconsin) supported Z. A. P.


   FOOTNOTES
 
1 Current address: Department of Comparative Biosciences, University of Wisconsin School of Veterinary Medicine, Madison, WI 53706. Back

2 Current address: Department of Chemistry, Lawrence University, Appleton, WI 54912. Back

Received December 22, 2003; revised February 9, 2004; accepted February 18, 2004.


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