Trophic activity of a naturally occurring truncated ... - The FASEB Journal

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Elena Adinolfi,* Maria Cirillo,* Ronja Woltersdorf,† Simonetta Falzoni,* Paola Chiozzi,*. Patrizia Pellegatti,* Maria Giulia Callegari,* Doriana Sandona`,‡ Fritz ...
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Trophic activity of a naturally occurring truncated isoform of the P2X7 receptor Elena Adinolfi,* Maria Cirillo,* Ronja Woltersdorf,† Simonetta Falzoni,* Paola Chiozzi,* Patrizia Pellegatti,* Maria Giulia Callegari,* Doriana Sandona`,‡ Fritz Markwardt,§ Gu¨nther Schmalzing,† and Francesco Di Virgilio*,1 *Department of Experimental and Diagnostic Medicine, Section of General Pathology, Interdisciplinary Center for the Study of Inflammation, University of Ferrara, Ferrara, Italy; † Department of Molecular Pharmacology, RWTH Aachen University, Aachen, Germany; ‡Department of Biomedical Sciences, University of Padova, Padova, Italy; and §Julius Bernstein Institute for Physiology, Martin Luther University Halle, Halle/Saale, Germany P2X7 is the largest member of the P2X subfamily of purinergic receptors. A typical feature is the carboxyl tail, which allows formation of a large pore. Recently a naturally occurring truncated P2X7 splice variant, isoform B (P2X7B), has been identified. Here we show that P2X7B expression in HEK293 cells, a cell type lacking endogenous P2X receptors, mediated ATP-stimulated channel activity but not plasma membrane permeabilization, raised endoplasmic reticulum Ca2ⴙ content, activated the transcription factor NFATc1, increased the cellular ATP content, and stimulated growth. In addition, P2X7B-transfected HEK293 cells (HEK293-P2X7B), like most tumor cells, showed strong soft agarinfiltrating ability. When coexpressed with full-length P2X7 (P2X7A), P2X7B coassembled with P2X7A into a heterotrimer and potentiated all known responses mediated by this latter receptor. P2X7B mRNA was found to be widely distributed in human tissues, especially in the immune and nervous systems, and to a much higher level than P2X7A. Finally, P2X7B expression was increased on mitogenic stimulation of peripheral blood lymphocyte. Altogether, these data show that P2X7B is widely expressed in several human tissues, modulates P2X7A functions, participates in the control of cell growth, and may help understand the role of the P2X7 receptor in the control of normal and cancer cell proliferation.— Adinolfi, E., Cirillo, M., Woltersdorf, R., Falzoni, S., Chiozzi, P., Pellegatti, P., Callegari, M. G., Sandona`, D., Markwardt, F., Schmalzing, G., Di Virgilio, F. Trophic activity of a naturally occurring truncated isoform of the P2X7 receptor. FASEB J. 24, 3393–3404 (2010). www.fasebj.org ABSTRACT

Key Words: extracellular ATP 䡠 purinergic receptors 䡠 cell growth 䡠 ion channels

hexamers (5, 6). P2X subunits range in length from 362 (mouse P2X6 short isoform) to 595 aa (P2X7) and have 2 transmembrane domains, a large extracellular loop, and intracellular N and C termini (7). The carboxyl-terminal domain is the most crucial for the functional properties of P2X channels, as mutations or deletions in this region strongly affect channels kinetics, permeability, and desensitization (8). This is particularly true of the P2X7 subunit, which, depending on the presence and activity of its unusually long (⬃200 aa) carboxyl tail, may generate an ion channel or a nonselective plasma membrane pore (9, 10). The P2X7 receptor has become a focus of increasing attention because several key cell responses depend on its activity. Such responses range from cytokine secretion to mycobacterial killing, from modulation of bone formation to macrophage and osteoclasts fusion, and from cell death to cell proliferation (see refs. 11–13 for recent reviews). Most of these functions have been assigned to the large, nonselective P2X7 pore, but with the recent discovery of carboxyl-terminal truncated P2X7 isoforms (14) the question arises whether such isoforms might be responsible for some of the functions assigned to the full-length receptor. The carboxyl tail, besides being responsible for pore formation, is also endowed with an intriguing regulatory activity (4), which suggests that the it might serve as link between the P2X7 receptor itself and cytoplasmic or plasma membrane proteins (15, 16). This possible function of the P2X7 carboxyl tail is highlighted by the presence of numerous potential protein-protein and protein-lipid interaction motifs (17), and by the recent demonstration that a tail fragment (residues 434 –595) modulates P2X7 receptor activity in a reconstituted system (4). Among membrane proteins that the carboxyl tail might interact with, of particular interest is pannexin-1, as this plasma membrane channel might be the molecular 1

P2X receptors are ATP-gated ion channels made by the assembly of multiple subunits (P2X1–7). There is substantial evidence to support a trimeric structure for all P2X channels (1– 4), although scattered evidence suggests that P2X7 subunits might aggregate to form 0892-6638/10/0024-3393 © FASEB

Correspondence: Department of Experimental and Diagnostic Medicine, Section of General Pathology, Interdisciplinary Center for the Study of Inflammation, University of Ferrara, via L. Borsari 46, 44100, Ferrara, Italy. E-mail: [email protected] doi: 10.1096/fj.09-153601 3393

structure responsible of formation of the large P2X7 pore (18). Nine different naturally occurring human P2X7 splice variants, named P2X7A–J, have been identified, P2X7A being the well-characterized full-length P2X7 receptor (14, 19). Of the 9 splice variants, 4, P2X7B, P2X7E, P2X7G, and P2X7J, lack the C terminus. Among these, P2X7J carries deletions in the TM2 domain and in the extracellular loop (19) and acts as dominant negative, whereas P2X7B forms a functional plasma membrane channel (14, 19). P2X7B retains an intron between exons 10 and 11, which causes the insertion of a stop codon that completely abolishes translation of the last 249 aa of the carboxyl terminus and the addition of an extra 18 aa after residue 346. These modifications do not prevent receptor stimulation by ATP or benzoyl-ATP (BzATP). P2X7B is of particular interest for its wide tissue expression, sequence similarity to other P2X receptors, and loss of the proapoptotic activity typical of P2X7 (14, 19). This last feature is of particular interest to us because in previous studies we have investigated in depth the growth-promoting activity of P2X7 as opposed to its well-known cytotoxic effect (10, 20, 21). Presence of a shorter P2X7 isoform lacking a frank cytotoxic activity suggested to us that P2X7 responses might be finely tuned via expression of different P2X7 isoforms, with obvious implications for the control of cell growth. Here we show that transfection of P2X7B conferred most of the positive but not the negative responses associated to P2X7A, and in particular it triggered enhanced NFATc1 activation, increased total cellular ATP content, and supported growth, but did not render cells susceptible to ATP-triggered apoptosis. Moreover, P2X7B coassembled with P2X7A to a P2X7A⫹B heterotrimer and directly modulated its functions, pore formation included.

MATERIALS AND METHODS Reagents Apyrase grade IV, oxidized ATP (oATP), BzATP, and ionomycin were purchased from Sigma-Aldrich (Milan, Italy). Fura-2 AM and bisoxonol were obtained from Invitrogen (Tema Ricerca, Bologna, Italy). Primers and oligonucleotides were obtained from MWG (MWG Biotech/M-Medical, Milan, Italy) or Applied Biosystems (Monza, Milan, Italy). Restriction enzymes and ligase were purchased from New England Biolabs (Celbio S.P.A., Milan, Italy). Qiaprep Spin Miniprep Kit and Mini Elute Gel Extraction Kit were obtained from Qiagen (Milan, Italy). All experiments shown were performed with BzATP as a stimulant. However, similar results were also obtained with ATP, albeit at higher agonist concentrations.

(nt 1004 –1095 of hP2X7B, GenBank AY847298) were synthesized at MWG Biotech. Annealed oligonucleotides were then ligated into the BsrGI-XhoI-digested P2X7A plasmid. To select the double transfectants, the P2X7B sequence was subcloned in pcDNA3.1 Hygro, carrying an hygromicin B resistance. The construct sequence was checked by sequence analysis carried out at the CRIBI Biomolecular Research Sequencing Core, University of Padova, Italy.

Cell culture, transfection, and stable clone selection HEK293 cells and peripheral blood lymphocytes (PBLs) were cultured in DMEM-F12 (Sigma-Aldrich) and RPMI (GE Health Care, Little Chalfont, UK), respectively, complemented with 10% fetal calf serum (FCS; Life Technologies, Milan, Italy), 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen). Experiments, unless otherwise indicated, were performed in the following saline solution, also referred to as “standard saline” in the text: 125 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM NaH2PO4, 20 mM HEPES, 5.5 mM glucose, 5 mM NaHCO3, and 1 mM CaCl2, pH 7.4. When indicated, CaCl2 was omitted and 0.5 mM EGTA was added. To exclude the effects of clonal selection, all the experiments were performed with different stable clones, obtained as described below. HEK293 cells were transfected with 1 ␮g of DNA/␮l of lipofectamine 2000 (Invitrogen) as described by the manufacturers. After transfection cells were kept in the presence of increasing concentations (0.2– 0.8 mg/ml) of the selection marker G418 sulfate (Calbiochem, La Jolla, CA, USA), alone or with hygromicin B from 0.1 to 0.2 mg/ml (Sigma-Aldrich) for selection of the double transfectants. Single cell-derived clones were isolated into 90-well plates after serial dilution.

RT-PCR P2X7B expression was checked by RT-PCR. RNA was extracted from 5 ⫻ 106 cells/sample with TRIZOL according to the manufacturer’s instructions (Invitrogen). RNA integrity and concentration were checked by electrophoresis on 1.5% agarose gel and spectrophotometric analysis, respectively. RT-PCR was performed with the Promega RT-PCR access kit (Promega, Milan, Italy) using 200 ng of RNA/sample as template. Briefly, the mRNA was retrotranscribed for 45 min at 45°C with AMV reverse transcriptase. The following 40 cycles included 1 min at 94°C for denaturation, 1 min at 50°C for primer annealing, and 2 min at 68°C for elongation with TFL polymerase. Primers used were forward 5⬘ CCCATCGAGGCAGTGGA 3⬘, reverse 5⬘ TAAAGCATGGAAAAGAGAATCTC 3⬘, for P2X7B; forward 5⬘ AGATCGTGGAGAATGGAGTG 3⬘, reverse 5⬘ TTCTCGTGGTGTAGTTGTGG 3⬘ for pan P2X7 (isoform A and B); forward 5⬘ CCTCTGACTTCAACAGCGAC 3⬘ reverse 5⬘ CATGACAAGGTGCGGCTCCC 3⬘ for G3PDH.

Immunofluorescence Cloning To obtain the P2X7B construct we engineered a plasmid containing the P2X7A receptor sequence cloned in pcDNA 3.1 (GenBank NM_002562) (22, 23) by removing the sequence corresponding to the C-terminus of P2X7A thanks to the single cutters BsrGI-XhoI. Sense and antisense oligonucleotides corresponding to the missing extra region 3394

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Cells were fixed with 2% paraformaldehyde in PBS, pH 7.2, for 1 h at room temperature, rinsed twice with PBS, incubated for 30 min in 100 mM NH4Cl to quench paraformaldehyde autofluorescence, further rinsed with PBS, and finally incubated for 30 min in a blocking buffer containing 1% BSA and 50 mM l-lysine in PBS. The same blocking buffer was used for an overnight incubation at 4°C with the primary anti-P2X7

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mAb (20 ␮g/ml), kindly provided by Professor James Wiley (Penrith Hospital, University of Sydney, Penrith, NSW, Australia). Cells were then rinsed 3 times with PBS and incubated with the secondary Ab at a 1:200 dilution (TRITC-conjugated goat anti-mouse; Sigma-Aldrich) for 1 h. Cells were mounted with prolong gold antifade (Invitrogen) and analyzed with a Zeiss LS-510 confocal microscope (Carl Zeiss, Arese, Italy).

Measurement of plasma membrane potential Changes in plasma membrane potential were measured with the fluorescent dye bisoxonol (Invitrogen) at the wavelength pair 450/580 nm, as described previously (24).

[Ca2ⴙ]i measurement [Ca2⫹]i measurements were performed in a thermostat-controlled and magnetically stirred Perkin Elmer (Monza, Italy) fluorimeter with the fluorescent indicator Fura-2/acetoxymethylester (Fura-2/AM) as described previously (10). Briefly, cells were loaded with 4 ␮M Fura-2/AM for 20 min in the presence of 250 ␮M sulfinpyrazone in standard saline, rinsed, and resuspended at a final concentration of 106/ml either in standard saline solution or, when required, in Ca2⫹-free saline. Excitation ratio and emission wavelength were 340/380 and 505 nm, respectively. For ER Ca2⫹ release experiments (see Fig. 8B), the total amount of calcium release was calculated from the time vs. [Ca2⫹] graph as secondary derivative with Origin software (OriginLab, Northampton, MA, USA).

Changes in plasma membrane permeability ATP-dependent increases in plasma membrane permeability were measured by monitoring the uptake of ethidium bromide. Briefly, 5 ⫻ 105 cells/ml were incubated in a thermostat-controlled and magnetically stirred fluorimetric cuvette at 37°C in the presence of 20 ␮M ethidium bromide and challenged with the agonist. Full permeabilization was achieved by adding digitonin (100 ␮M) at the end of the experiment. Fluorescence emission was measured at an excitation/emission wavelength couple of 360/580 nm.

Microscopy Microscopic analysis was performed with a Nikon Eclipse TE300 inverted microscope (Nikon, Tokyo, Japan) equipped with a thermostat-controlled chamber (Bioptechs, Butler, PA, USA) and an ⫻63 immersion objective. The microscope was equipped with the following devices: a computer-controlled light shutter, a 6-position filter wheel, a piezoelectric z-axis focus device, a back-illuminated 1000 ⫻ 800 charge-coupleddevice camera (Princeton Instruments, Trenton, NJ, US), and a computer equipped with MetaMorph software (Universal Imaging, Downingtown, PA, USA) for image acquisition, 2and 3-dimensional (3-D) visualization, and analysis. All experiments were performed at 37°C in standard saline solution. Cells were plated onto 40-mm coverslips, precoated with polylysine, and challenged with Bz-ATP. Images were acquired every 5 min for a total time length of 30 min. P2X7B-STIMULATED CELL GROWTH

ATP measurement Intracellular ATP concentration was measured with the Enliten ATP assay system (Promega). Briefly 105 cells/sample were lysed with 10 ␮l lysis buffer (FireZyme, San Diego, CA, USA), and supplemented with 90 ␮l of diluent buffer (FireZyme) to stabilize ATP. Samples were then placed in a Victor 3 multilabel counter (Perkin Elmer) equipped with a Wallac liquid injector (Perkin Elmer) that allowed rapid injection of the luciferin-luciferase solution (100 ␮l). Total protein content of samples was determined with Bradford assay. Extracellular ATP release was measured with the pmeLUC construct previously engineered in our laboratory (25). Briefly, cells plated onto 13-mm coverslips were transfected with pmeLUC DNA (0.8 ␮g/␮l of lipofectamine 2000; Invitrogen) as described by the manufacturer. Forty-eight hours after transfection coverslips were placed in the thermostatcontrolled (37°C) chamber hosted in a custom-made highsensitivity luminometer equipped with a low-noise photomultiplier with built-in amplifier discriminator (25), and perfused with standard saline solution supplemented with luciferin at a concentration of 5 ␮M. NFATc1 activation assay The NFATc1 activation assay was performed with the TransAM NFATc1 ELISA kit (Active Motif, Vinci Biochem, Vinci, Italy) as described previously (21). Proliferation and soft agar infiltration assays For cell growth and soft agar infiltration assays cells, ⬃105/ml, were seeded in DMEM-F12 medium (BD Biosciences, Lincoln Park, NJ, USA) in 6-well Falcon plates and placed in a CO2 incubator at 37°C. Cell numbers were assessed at various time intervals in a Burker chamber with a phase-contrast Olympus microscope (Olympus Life Science Europe, Hamburg, Germany). To perform the soft agar assay, a layer of 0.6% agarose (electrophoresis grade; Invitrogen) dissolved in DMEM-F12 medium was placed in 36 mm Petri dishes and allowed to solidify. At this stage, a second layer of DMEM-F12/ 0.4% agarose in which ⬃5000 cells had been previously dispersed was stratified on top of the first. Dishes were then transferred to a CO2 incubator at 37°C. Number and dimension of the cell colonies were evaluated after 10 and 15 d by microscopy. Real-time PCR and lymphocyte stimulation P2X7A and B expression in human tissues and PBL was evaluated by real-time PCR in a Step One Real-Time PCR system (Applied Biosystems). Human normal tissue scan cDNA panel was obtained by Origene (Rockville, MD, USA). PBLs were obtained from healthy donors as described previously (26). Proliferation was evaluated after a 72 h incubation in the presence of either phytohemoagglutinin (PHA, 1 ␮g/ml) or medium alone (controls). Total RNA was extracted with TRIZOL reagent (Invitrogen). Reverse transcription was performed starting from 1 ␮g of total RNA/sample, with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems) as described by the manufacturer. Two microliters of cDNA were used as template for real-time PCR. Amplification was performed with custom-made primers and Taqman probes (Applied Biosystems), as detailed below, and with an internal GAPDH reference (Pre developed Taq Man assay reagents, human GAPDH; Applied Biosystems). For P2X7A, forward primer: 5⬘CGGCTCAACCCTCTCCTACT-3⬘; reverse primer: 5⬘GGAGTAAGTGTCGATGAGGAAGTC 3⬘; 3⬘FAM probe: 5⬘ CACAGCGGCCAGACCG 3⬘. For P2X7B, forward primer: 5⬘ GGAAAATGGTTTGGAGAAGGAAGTG 3395

3⬘; reverse primer: 5⬘ CGATGAGGAAGTCGATGAACACA 3⬘; 3⬘FAM probe: 5⬘ ACAAGCGCTGCGTTAGT 3⬘. A comparative CT experiment (⌬⌬CT) was run to allow determination of the change of expression (fold increase) of the target cDNA in the test sample relative to the reference sample. Reference sample was HEK293 cells for the tissue scan and lymphocytes at time 0 for the lymphocyte proliferation experiment.

Data analysis All data are shown as mean ⫾ se, except for the real-time PCR data, where sd is reported. Tests of significance were performed by Student’s t test using GraphPad InStat software (GraphPad, San Diego, CA, USA).

RESULTS

Biochemical characterization of P2X7A and B P2X7A and P2X7B plasmids were subcloned into the oocyte expression vector pNKS2 (27) and C-terminally fused to the coding sequence for a hexahistidine (His) tag or a StrepII tag (NWSHPQFEK) alone or for a His tag followed by a StrepII tag (His-StrepII). The constructs were expressed singly or in pairwise combination by injection of capped cRNAs into defolliculated Xenopus laevis oocytes as described previously (4). After overnight labeling with l-[35S]methionine and an additional 24-h chase period, membrane proteins were extracted with digitonin (1%), followed by receptor purification by Ni-NTAagarose (Qiagen, Hilden, Germany) or by Strep-Tactin Sepharose (IBA, Go¨ttingen, Germany) affinity chromatography as described previously (4). Proteins were resolved in the native and the SDS-denatured state by blue native (BN-PAGE) and SDS-PAGE, respectively, as described previously (1, 3, 4), and visualized by scanning with a Storm 820 PhosphorImager (GE Healthcare). Individual bands were quantified with ImageQuant software.

Generation of HEK293 cell clones stably transfected with P2X7B and double P2X7A/P2X7B transfectants Based on the published sequence (GenBank AY847298), human P2X7B is 231 aa shorter than P2X7A and bears an insertion of an extra 18 aa that modifies the sequence of TM2 and of the short carboxyl terminus (Fig. 1). P2X7B was cloned into 2 pcDNA3.1 vectors carrying different antibiotics resistances, as described in Materials and Methods. These plasmids were transfected into HEK293 cells alone or together with a P2X7A-expressing vector. Several different stable clones were obtained by gentamicin or gentamicin plus hygromicin selection and serial dilution. Figure 2A shows RT-PCR amplification of mRNA from different transfectants with primers specific for P2X7B, both P2X7B and P2X7A (pan P2X7 primers) or the housekeeping gene G3PDH. Expression of P2X7A or B protein at the plasma membrane was confirmed by

Figure 1. Membrane topology and alignment of full-length P2X7A and truncated P2X7B receptors. Sequences were compared with the DIALIGN program (Morgenstern 2004). Lowercase indicates differing residues between P2X7A and P2X7B. Yellow highlighted residues, TM1; green highlighted residues, TM2; red highlighted residues, extra 18 aa present in the P2X7B C terminus.

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Figure 2. P2X7B expression in HEK293 cells. A) Amplification by RT-PCR of P2X7B (top panel), P2X7A and B (middle panel), and the housekeeping gene G3PDH (bottom panel). B) Membrane expression of P2X7A and P2X7B. Staining and analysis were performed as detailed in Materials and Methods.

immunofluorescence with a mAb raised against the extracellular domain of P2X7, and thus recognizing both P2X7A and B (Fig. 2B). Activation of P2X7A with the ATP analog BzATP caused Ca2⫹ and Na⫹ uptake, as well as the associated collapse of plasma membrane potential (Fig. 3A, red trace). HEK293P2X7B cells were also sensitive to BzATP-triggered depolarization (blue trace), although to a lesser extent than HEK293-P2X7A. HEK293-mock were fully refractory to BzATP-induced depolarization (green trace). As expected, BzATP stimulated a large increase in intracellular Ca2⫹ in HEK293-P2X7A, but a much smaller increase in the HEK293-P2X7B clones, and no response in HEK293-mock (Fig. 3B). Accordingly, dye uptake was completely lost in the

P2X7B transfectants, as indicated by lack of BzATPstimulated ethidium bromide uptake (Fig. 3C). These data show that P2XB generates an ion channel but is unable to form the typical P2X7 pore. Expression of P2X7B potentiates P2X7A responses BzATP dose dependency for plasma membrane depolarization in HEK293-P2X7B cells (Fig. 4A, blue trace) was shifted to the right by almost 2 log units compared to HEK293-P2X7A (Fig. 4A, red trace). Coexpression of P2X7A and B generated a depolarization response shifted to the left of the P2X7A curve, especially at low agonist concentrations (Fig. 4A, gray trace). Potentia-

Figure 3. Permeability properties of HEK293-P2X7B clones. A) Membrane potential changes. Cells were incubated at 37°C in the fluorimeter cuvette at a concentration of 2 ⫻ 105/ml in standard saline supplemented with 100 nM bisoxonol. Here 30 ␮M KCl was added twice at the end of each experiment to fully collapse membrane potential. B) [Ca2⫹]i changes. See Materials and Methods for experimental details. C) Large pore activation. Ethidium bromide uptake was monitored as described in Materials and Methods. BzATP concentration was 500 ␮M. Color coding: green, HEK293-mock; red, HEK293-P2X7A; blue, HEK293-P2X7B. P2X7B-STIMULATED CELL GROWTH

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Figure 4. Potentiation of P2X7A-dependent responses by P2X7B. A) Bz-ATP dose dependency of plasma membrane depolarization. EC50 for P2X7B, P2X7A, and P2X7A⫹B was 500, 25, and 10 ␮M, respectively. B) Plasma membrane depolarization in response to low (10 ␮M) BzATP concentration. C) [Ca2⫹]i changes. BzATP was added at the concentration of 100 ␮M. D) Average ⌬[Ca2⫹]i evoked by 100 ␮M Bz-ATP. Data are from 9 separate experiments replicated on 3 different occasions. ***P ⬍ 0.001 vs. other treatments; ###P ⬍ 0.01 vs. HEK293-P2X7B. E) Large pore activation monitored by measuring ethidium bromide uptake. BzATP concentration was 500 ␮M. F) Average ethidium bromide uptake evoked by 500 ␮M Bz-ATP. ***P ⬍ 0.001 vs. other treatments; ###P ⬍ 0.001 vs. HEK293-P2X7B and HEK293-mock. Data are from 3 separate experiments replicated on 3 different occasions. Color coding: green, HEK293-mock; red, HEK293-P2X7A; blue, HEK293-P2X7B; gray, HEK293-P2X7A⫹B (double transfectants).

tion of the response especially occurred at low agonist concentrations (Fig. 4A, B), as in double P2X7A and B transfectants 10 ␮M BzATP triggered a plasma membrane potential collapse almost as large as that caused by a 3-fold higher agonist concentration (30 ␮M) applied to single P2X7A transfectants. A similar potentiation was also observed in the cytoplasmic Ca2⫹ ([Ca2⫹]i) response (Fig. 4C). Figure 4D reports average [Ca2⫹]i peak levels measured in HEK293-P2X7A, HEK293-P2X7B, and HEK293-P2X7A/P2X7B, respectively, stimulated with a submaximal concentration of BzATP. Potentiation of BzATP-stimulated ion fluxes by coexpression of P2X7A and B might not be surprising simply because double transfectants might express a higher density of P2X7 channels on the plasma membrane. However, it is more difficult to reconcile this interpretation with the potentiation of ethidium bromide uptake reported in Fig. 4E, F. As shown in these experiments, P2X7B alone was unable to generate the large conductance pore, thus we anticipated that when coexpressed together with P2X7A, P2X7B should not interfere with P2X7A-mediated pore formation or, alternatively, might antagonize pore formation if it assembled together with P2X7A in an heteromeric receptor and acted as a dominant negative. Contrary to this 3398

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predictions, transfection of P2X7B strongly increased P2X7A pore formation. The enhancing effect by P2X7B on P2X7A-mediated responses is also shown by plasma membrane blebbing. Plasma membrane blebbing is one of the earliest morphological changes following P2X7stimulation and often precedes cell death. HEK293-P2X7B did not bleb in response to stimulation with BzATP (Fig. 5B), while HEK293-P2X7A showed the typical membrane blebbing. In HEK293-P2X7A/P2X7B cells time of onset was not changed, but number of blebs formed per cell was much larger compared to HEK293-P2X7A. P2X7A and B isoforms homotrimerize and heterotrimerize efficiently Experiments shown in Figs. 4 and 5 suggested that P2X7A and B might interact. To assess coassembly of P2X7A and B isoforms at the protein level, we coexpressed in oocytes double-tagged P2X7A-His -StrepII (bearing a 9-residue StrepII sequence Cterminal to the C-terminal hexahistidine tag) together with the His-P2X7B as bait and prey, respectively. Proteins were purified from aliquots of the same digitonin extracts of cells using metal affinity

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Figure 5. Plasma membrane blebbing in P2X7 transfectants. HEK293-P2X7B (A, B), HEK293P2X7A (C, D), and HEK293-P2X7A⫹B (E, F) cells were plated on glass coverslips at a concentration of 105/ml in standard saline solution. Coverslips were then transferred to the thermostat-controlled stage of a Nikon Eclipse 300 microscope, and images were acquired at time 0 (A, C, E) and at 30 min (B, D, F). BzATP concentration was 500 ␮M.

chromatography or Strep-Tactin chromatography to verify the expression of the 2 P2X7 isoforms (Fig. 6A, lanes 1–5) and to screen for the presence of the copurified His-P2X7B isoform (Fig. 6A, lanes 7–9), respectively. P2X7A-His-StrepII (calculated protein core 71 kDa, i.e., without N-glycans) and His-P2X7B (calculated protein core 43 kDa) could be isolated as N-glycosylated proteins of 85 kDa (P2X7A) and 57 kDa (P2X7B) from the cells in which they were expressed (Fig. 6A, lanes 1–5). Purification by StrepTactin chromatography led to the coisolation of the non-StrepII-tagged His-P2X7B (Fig. 6A, lanes 7–9). Reciprocal copurification using P2X7A-His and P2X7B-His-StrepII as prey and bait proteins, respectively, resulted in the coisolation of P2X7A-His (Fig. 6B, lanes 2– 4). We further analyzed the homo- and heterooligomerization of P2X7A and B subunits by Blue Native (BN)-PAGE, which has been shown to reliably display the trimeric structure of P2X receptors (1, 3, 4). Both singly expressed P2X7A-StrepII and P2X7BStrepII subunits purified by nondenaturing StrepTactin chromatography migrated as defined homotrimers in the BN-PAGE gel (Fig. 6C, lanes 1 and 7). Partial denaturation by low SDS treatment caused the homotrimers to disassemble into dimers and monomers (Fig. 6C, lanes 2 and 8). Coexpression of P2X7A-StrepII with P2X7B as bait and prey, respectively, resulted in the appearance of an additional protein complex (Fig. 6C, lane 3) that migrated at a slightly lower molecular mass than the P2X7A homotrimer and significantly above that of the P2X7B P2X7B-STIMULATED CELL GROWTH

homotrimer. We assessed a molecular mass of 215 kDa for this additionally formed complex, using as BN-PAGE mass markers the 246-kDa P2X7A homotrimer and 164-kDa P2X7A homodimer (Fig. 6C, lane 2), calculated as multiples of 82 kDa/P2X7A monomer (the latter estimated from the Fig. 6B). Taking further an SDS-PAGE mass of 54 kDa of the P2X7B monomer into account (Fig. 6B), the 215 kDa complex fits best to a (P2X7A)2/P2X7B heterotrimer, which has an expected mass of 218 kDa (2⫻82⫹54 kDa). Also the reciprocal coexpression of P2X7BStrepII with P2X7A as bait and prey, respectively, led to the appearance of new distinct band in the BN-PAGE gel at ⬃187 kDa (Fig. 6C, lane 5), close to the 190 kDa expected for a P2X7A/(P2X7B)2 heterotrimer. To further assess the stoichiometry of P2X7A⫹B heterotrimer, we quantified the ratio of coisolated P2X7A polypeptide to the P2X7B polypeptide and vice versa at the various mixing ratios of P2X7A to P2X7B from Fig. 6A, B (taking into consideration the different numbers of methionine residues of the 2 polypeptides). The molar P2X7A/P2X7B ratio varied between 0.50 and 1.10. The ratios of 1.08 and 0.71 determined at low bait expression levels (cf. Fig. 6A, B, respectively) should be more reliable than the others because of a lower probability of the formation of bait homotrimers. A fair conclusion from the mean value of these 2 ratios (0.90) is that P2X7A and P2X7B coassemble with variable 1:2 and 2:1 stoichiometry to form both P2X7A/ (P2X7B)2 and (P2X7A)2/P2X7B heterotrimers. These data are fully consistent with the detection of hetero3399

Figure 6. Biochemical evidence for coassembly of P2X7A and P2X7B. X. laevis oocytes expressing the indicated P2X7 constructs singly or in combination as indicated were [35S]methionine-labeled, chased, and then extracted with digitonin. Proteins were purified by Ni-NTA chromatography or Strep-Tactin chromatography, as indicated, resolved by reducing SDS-PAGE (A, B) or BN-PAGE (C) and visualized by phosphorimaging. A) Strep-Tactin chromatography resulted in the coisolation of the non-StrepII-tagged P2X7B subunit (prey) with the StrepII-tagged P2X7A subunit used as bait. B) Conversely, using the StrepII-tagged P2X7B as bait, the non-StrepII-tagged P2X7A subunit was isolated. In contrast, the singly expressed non-StrepII-tagged P2X7A subunit was not isolated by Strep-Tactin affinity chromatography. Numbers in the margins in panels A and B indicate molecular mass markers (kDa); solid and open arrows indicate coisolated P2X7A and P2X7B subunits, respectively. C) Protein migration both under native conditions and after partial denaturation produced by a 1 h incubation with 0.1% SDS, as indicated. For better visibility of weak and strong protein bands, individual lanes from the same, but differently enhanced, ImageQuant image were cropped and positioned using Photoshop (Adobe, San Jose, CA, USA). Solid and open circles indicate numbers of P2X7A and/or P2X7B subunits, respectively, incorporated in the respective protein band. Dashed arrows assign protein bands to defined heteromeric assembly states. Numbers in margins indicate molecular masses deduced by referring to the partially denatured P2X7A receptor states in lane 2 as a mass marker; corresponding numbers given in parentheses were calculated on the basis of the SDS-PAGE-derived masses of 82 and 54 kDa for the P2X7A and P2X7B isoform, respectively. Number symbol indicates position where the coisolated disassembled P2X7B isoform is visible on contrast-enhanced images.

trimers of both possible stoichiometries in the BNPAGE gel (Fig. 6C). Taken together, these findings indicate that P2X7A and P2X7B coassemble efficiently as detergent-resistant heterotrimeric complexes. This can be easily reconciled with the known location of the P2X subunit assembly domains in the ectodomain and part of the second transmembrane domain (28), which are preserved between P2X7A and B isoforms. Expression of P2X7B increases intracellular ATP content and potentiates P2X7A-mediated ATP secretion We have recently shown that the growth advantage conferred by P2X7A expression is largely due to a trophic effect on mitochondrial oxidative phosphorylation and the consequent increase in intracellular ATP levels (10). Figure 7A shows that HEK293-P2X7B cells (blue bar) had an almost 3-fold higher intracellular ATP level compared to HEK293-mock cells, and not dissimilar from the levels measured in HEK293-P2X7A cells (red bar). Interestingly, intracellular ATP levels were almost doubled in HEK293-P2X7A/P2X7B compared to either single transfectants (gray bar). Higher intracellular ATP content was associated with an in3400

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creased ability to release ATP into the extracellular milieu (Fig. 7B and inset). Whether such an enhanced ATP-secreting activity was due to an increased ATP transport through the P2X7A pore (see ref. 25) or to an enhanced ATP leakage as a consequence of the higher intracellular ATP content is an open question. On the other hand, expression of P2X7B alone did not cause ATP secretion in response to BzATP (compare blue and green traces in Fig. 7B), thus suggesting that an increased intracellular ATP level in the absence of a functional P2X7A pore is not sufficient to support ATP secretion. P2X7B expression increases endoplasmic reticulum Ca2ⴙ levels and NFATc1 activation and stimulates growth rate One of key intracellular pathways responsible for the growth advantage conferred by P2X7A is NFATc1. We have recently shown that enhanced NFATc1 activity in HEK293-P2X7A cells is associated to, and likely dependent on, a higher ER Ca2⫹ content (21). Figure 8A, B shows that expression of P2X7B increased thapsigarginreleasable ER Ca2⫹ calcium content to about the same level as that found in HEK293 cells expressing the full-length isoform, and expression of both subunits caused a further increase in the amount of released

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P2X7B is widely expressed in human tissues and is up-regulated by mitogenic stimulation Previous data suggested that P2X7B is expressed in human tissues to about the same level as P2X7A (14). We carried out a screening of a wide range of human tissues, which showed that P2X7B is in fact expressed to a several-fold higher level than P2X7A in most tissue examined. Particularly striking was the several-fold higher expression of P2X7B in lymphoid tissue and in the lymphocytes themselves (Fig. 9A). This intriguing finding suggested to us that expression of P2X7B might correlate with lymphocyte proliferation. Figure 9B shows that this is indeed the case, as expression of P2X7B, and to a lesser extent of P2X7A, was increased ⬃12-fold by mitogenic stimulation of human lymphocytes.

DISCUSSION

Figure 7. Intracellular ATP levels and ATP secretion in P2X7 transfectants. A) Intracellular ATP was measured with the luciferin/luciferase assay as described in Materials and Methods. Data are averages from 15 separate determinations performed on different occasions. B) Extracellular ATP was measured with the pmeLUC probe as described in Materials and Methods. BzATP was added at a concentration of 300 ␮M. Inset: average BzATP-evoked ATP release from HEK293P2X7A and HEK293-P2X7A⫹B as measured with pmeLUC (n⫽10). **P ⬍ 0.01, ***P ⬍ 0.001 vs. other treatments; ### P ⬍ 0.001 vs. HEK293-mock. Color coding: green, HEK293-mock; red, HEK293-P2X7A; blue, HEK293-P2X7B; gray, HEK293-P2X7A⫹B (double transfectants).

Ca2⫹. The increase in releasable ER Ca2⫹ was paralleled by an increase in NFATc1 nuclear translocation (Fig. 8C). Increased ER Ca2⫹ content and NFATc1 activation are related to increased survival in HEK293P2X7A cells (21). The truncated P2X7B isoform conferred a similar growth advantage (Fig. 8D), which was even larger when cotransfected with the full-length isoform (Fig. 8D, gray trace). Growth of P2X7 transfectants was fully dependent on the presence of extracellular ATP as was inhibited by addition of apyrase (Fig. 8E). P2X7 transfectants, showed not only an increased growth rate but also higher efficiency to infiltrate soft agar, an index of tumor invasiveness (Fig. 8F). Double transfectants showed a slightly higher infiltrative activity, which, however, was not significantly different from that of single transfectants. P2X7B-STIMULATED CELL GROWTH

P2X7 is an intriguing receptor. It was originally described as a nonselective, ATP-gated, plasma membrane pore (29), and later shown to be characterized by 2 distinct conductance states, as a cation-selective channel and a large conductance nonselective pore (9). It was immediately apparent that as a pore its activation would have dire effects on cell physiology, and therefore general consensus viewed P2X7 as a cytotoxic receptor (whether pronecrotic or proapoptotic really depended on the experimental conditions and the given cell type) (9, 30, 31). However, over the years it has become apparent that pharmacological activation of P2X7 by ATP does not faithfully mimic physiological stimulation. It is clear that P2X7 in the absence of added ATP is characterized by a tonic state of activation (likely due to autocrine/paracrine stimulation by tonically released ATP), which far from being cytotoxic is in fact growth promoting (10, 13, 31). We have investigated in detail the growth-promoting activity of P2X7, showing that only the full-length receptor is endowed with the ability to promote growth, thus implying that the “pore function” is needed (10). In these early experiments we compared the activity of the full-length receptor (P2X7A) to that of a defective rat receptor truncated to aa 415 (P2X7⌬C). P2X7⌬C, like P2X7B mediates ATP-stimulated ion fluxes but not plasma membrane permeabilization to normally impermeant aqueous solutes. However, P2X7⌬C does not faithfully mimic the naturally existing P2X7 truncated isoform (P2X7B) as, besides being of different species origin, it is also 51 aa longer. These differences turned out to be of crucial relevance, as when, as in the present study, we used the natural truncated receptor, we were able to unveil a growthpromoting activity in the absence of pore formation. P2X7B is ubiquitously expressed, often to a much higher level than P2X7A (see, e.g., Fig. 9). We were intrigued by the high expression level of P2X7B in lymphocytes, a cell type that is known to express P2X7, but to be refractory to ATP-mediated plasma membrane permeabilization (13, 32), the functional signature of the P2X7 receptor. This observation lead us to hypothesize some time ago that lymphocytes might 3401

Figure 8. P2X7B expression increases Ca2⫹ release from the ER, potentiates NFATc1 activation, and promotes growth. A) Fura-2-loaded cells were incubated in calcium-free standard solution at a concentration of 106/ml. The SERCA inhibitor thapsigargin was added at a concentration of 2 ␮M. B) Summary data of repeated (n⫽6) [Ca2⫹]i determinations. Absolute amount of Ca2⫹ released by thapsigargin was determined by measuring the area under the peak, as described in Materials and Methods. ***P ⬍ 0.001 vs. other treatments; ###P ⬍ 0.001 vs. HEK293-mock. C) Nuclear NFATc1 was determined by ELISA (n⫽9). *P ⬍ 0.05, **P ⬍ 0.01 vs. HEK293-mock; ***P ⬍ 0.001 vs. other treatments. D, E) Cells were plated at a concentration of 105/ml and incubated for in serum-free DMEM F12 in the absence (D) or presence (E) of apyrase (0.1 U/ml). F) Cells, 5 ⫻ 103/ml, were plated in agarose/DMEM-F12 medium in the absence of serum, as described in Materials and Methods. Infiltration was evaluated by counting by microscopy the number of colonies in the lower layer after 10 and 15 d (n⫽9). **P ⬍ 0.01, ***P ⬍ 0.001 vs. HEK293-mock; ##P ⬍ 0.01 vs. HEK293-P2X7B. Color coding: green, HEK293-mock; red, HEK293-P2X7A; blue, HEK293-P2X7B; gray, HEK293-P2X7A⫹B (double transfectants).

express an “atypical” P2X7 receptor (32, 33). The present data showing that the truncated isoform B is highly expressed in lymphocytes suggest that our hypothesis was not too far from the truth. Other tissues also express high P2X7B levels, for example, nerve tissue. Nerve tissue includes neurons and glial cells, therefore while we cannot assign P2X7B to any specific cell type, it is possible that P2X7B might be responsible for some ill-defined “P2X7-like” responses observed in the CNS in the absence of overt plasma membrane permeabilization. In the P2X receptor subfamily, P2X7A (the fulllength P2X7 receptor subunit) stands out for its size. Six of the 7 members of this family are comprised within 362 (mouse P2X6 short isoform) and 472 aa (P2X2), while P2X7A is 595 aa long. Without the long COOH tail (over 200 aa), P2X7 subunits would be within the size of the other P2X subunits, and the P2X7 receptor would also share the same ion permeability properties. Therefore it is tempting to hypothesize that P2X7B appeared earlier in evolution, may be by duplication of the p2x4 gene, and that P2X7A arose later as a result of acquisition by the p2x4 gene of the exons encoding the “pore-forming” tail. This hypothesis is supported by the close chromosomal location of the human p2x7 and p2x4 genes (12q24 and 12q24.32, 3402

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respectively), and the high identity and similarity between P2X7B and P2X4 (45 and 71%, respectively). Our data suggest that P2X7A and B can heterotrimerize in the plasma membrane generating a P2X7A⫹B receptor with a variable stoichiometry of 1:2 or 2:1, and with distinct permeability properties with respect to the P2X7A or B homotrimers. Furthermore, expression of P2X7B together with P2X7A strongly potentiated all P2X7A functions, large pore formation included, thus implicating that, rather intriguingly, P2X7A⫹B heterotrimers generate better dye-uptake pathways than P2X7A homotrimers. This may depend on an increased affinity for ATP (or BzATP) of the heteromeric receptor, as shown in Fig. 4, or on an increased level of expression on the plasma membrane due to a reduced recycling of the subunits. Preliminary experiments in a heterologous transfection system suggest that coexpression of P2X7A and B generates a channel with distinct electrophysiological properties. Despite uncertainty about the heteromer stoichiometry, the high expression level of P2X7B relative to P2X7A, especially in lymphoid tissue, and its trophic activity raise the issue of the physiological role of this truncated isoform. In fact, except for large pore forma-

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Figure 9. Tissue distribution and PHA-stimulated expression of P2X7A and B. A) P2X7A and B mRNA levels were measured by real-time PCR in a human tissue cDNA panel, as described in Materials and Methods. B) PBLs were seeded in 24-well plates at 106/ml density in FCS (10%)-supplemented RPMI medium in the absence or presence of 1 ␮g/ml PHA. After 72 h of incubation, total RNA was isolated, and real-time PCR was performed. P2X7A and P2X7B mRNA expression was evaluated with specific primers and probes (see Materials and Methods) and is indicated as fold increase over expression levels of HEK293-mock (A) or PBL at time zero (B). **P ⬍ 0.01 vs. controls; ##P ⬍ 0.01 vs. P2X7A. Color coding: red, P2X7A; blue, P2X7B.

tion, P2X7B reproduces all other P2X7A-dependent responses, such as increase in intracellular Ca2⫹ and ATP, activation of NFATc1 and growth stimulation. Of particular relevance is the ability of P2X7B to increase ER Ca2⫹ content, a crucial factor in the activation of the NFATc1 pathway and in the stimulation of growth, as shown in our recent study (21). We hypothesize that P2X7B, like its longer isoform, might have a primary role in cell proliferation. In this respect, P2X7B is clearly a better, less “dangerous,” growth-promoting receptor than P2X7A, as it is devoid of the known cytotoxic activity linked to the extended carboxyl-terminal tail present in P2X7A. Although under basal conditions of stimulation P2X7A stimulates growth (10, 34, 35), the associated plasma membrane pore that may undergo prolonged opening in the presence of high extracellular ATP levels makes P2X7A a risky receptor, as we now know that under several physiological circumstances extracellular ATP levels can be high enough to trigger sustained P2X7 pore activity (36). We hypothesize that at basal interstitial ATP levels, both P2X7A and P2X7B can be activated by transient, restricted, local increases in the extracellular ATP concentration, and thus both receptors can sustain cell growth. However, when the extracellular ATP concentration increases, cells can respond in 2 different ways, depending on the level of expression of either isoforms. If the predominant isoform is P2X7B, as in lymphocytes, then even high ATP concentrations will fail to cause pore formation and cytotoxicity, but will on the contrary provide a strong, additional stimulus for growth. If P2X7A predominates, or both subunits are present in comparable amount, than pore opening will occur, thus precipitating cell death. Modulation of P2X7A and P2X7B expression would therefore confer a P2X7B-STIMULATED CELL GROWTH

high degree of plasticity in cell responses to extracellular ATP, allowing immune cells to exploit the high extracellular ATP concentration found at inflammatory sites as a growth stimulus, or alternatively as a death signal. Furthermore, stimulation of cell functions by P2X7A and B extends to other responses besides proliferation, as shown by the higher infiltrating capacity of cells expressing either or both receptors (see Fig. 8). Although this might depend on many different factors, it is clear that the higher cellular energy content might well be a crucial determinant of the higher motility of the P2X7 transfectants. One may ask whether this life/death discrimination power afforded by the balance of expression of different isoforms is also shared by other P2X receptors besides P2X7. No other P2X have been shown so far to be endowed with the ability to support growth or cytotoxicity, and thus we believe that this “Jekyll/Hyde” behavior is specific to the P2X7 receptor and due to its peculiar COOH extensions. In fact, although there is an indication, albeit circumstantial, that some P2X receptors other than P2X7 might be involved in cell proliferation (37), for no P2X receptor besides P2X7 has a cytotoxic effect been consistently described. Thus P2X7A might be thought of as the “deadly” molecular evolution of an otherwise harmless P2X receptor (P2X7B). This research was supported by grants from the Italian Association for Cancer Research (IG 5354), Telethon of Italy (GGP06070), the Italian Space Agency (ASI-OSMA), the Commission of European Communities (7th Framework Program HEALTH-F2-2007-202231), the Regione Emilia Romagna (Research Programs “Innovative Approaches to the Diagnosis of Inflammatory Diseases” and “Monitor”), the Young Researchers Project of the Univer3403

sity of Ferrara (to E.A.), the Deutsche Forschungsgemeinschaft (Schm536/9-1 to G.S. and Ma1581/15-1 to F.M.), and institutional funds from the University of Ferrara. F.D.V. acts as consultant to Cordex Pharma Inc. and Affectis Pharma AG, biotech companies involved in the development of ATP-based drugs.

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Received for publication January 7, 2010. Accepted for publication April 22, 2010.

ADINOLFI ET AL.