ORIGINAL RESEARCH Activation of P2RY11 and ATP Release by Lipoxin A4 Restores the Airway Surface Liquid Layer and Epithelial Repair in Cystic Fibrosis Gerard Higgins1, Paul Buchanan1, Marianne Perriere1, Mazen Al-Alawi2, Richard W. Costello2, Valia Verriere2, Paul McNally1, Brian J. Harvey2, and Valerie ´ Urbach1,2,3 1

National Children Research Centre, Dublin 12, Ireland; 2Royal College of Surgeons in Ireland, Education and Research Centre, Beaumont Hospital, Dublin 9, Ireland; and 3INSERM U845, Paris, France

Abstract In cystic fibrosis (CF), the airway surface liquid (ASL) height is reduced as a result of impaired ion transport, which favors bacterial colonization and inflammation of the airway and leads to progressive lung destruction. Lipoxin (LX)A4, which promotes resolution of inflammation, is inadequately produced in the airways of patients with CF. We previously demonstrated that LXA4 stimulates an ASL height increase and epithelial repair. Here we report the molecular mechanisms involved in these processes. We found that LXA4 (1 nM) induced an apical ATP release from non-CF (NuLi-1) and CF (CuFi-1) airway epithelial cell lines and CF primary cultures. The ATP release induced by LXA4 was completely inhibited by antagonists of the ALX/FPR2 receptor and Pannexin-1 channels. LXA4 induced an increase in intracellular cAMP and calcium, which were abolished by the selective inhibition of the P2RY11 purinoreceptor. Pannexin-1 and ATP hydrolysis inhibition and P2RY11 purinoreceptor knockdown all abolished the increase of ASL height induced by LXA4. Inhibition of the A2b adenosine receptor did not affect the ASL height increase induced by LXA4, whereas the PKA

Cystic fibrosis (CF) is a lethal genetic disorder arising from the mutation of the CF transmembrane conductance regulator (CFTR) gene coding for a Cl2 channel (1) and results in a reduced airway surface liquid (ASL) layer (2). The decreased ASL height leads to impaired mucociliary clearance, resulting in chronic bacterial infection, persistent inflammation, and progressive lung destruction.

inhibitor partially inhibited this response. The stimulation of NuLi-1 and CuFi-1 cell proliferation, migration, and wound repair by LXA4 was inhibited by the antagonists of Pannexin-1 channel and P2RY11 purinoreceptor. Taken together, our results provide evidence for a novel role of LXA4 in stimulating apical ATP secretion via Pannexin-1 channels and P2RY11 purinoreceptors activation leading to an ASL height increase and epithelial repair. Keywords: lipoxin A4; cystic fibrosis; ATP release; Pannexin-1; P2RY11 receptor

Clinical Relevance We reported here multiple lipoxin A4 functions in enhancing airway surface liquid layer height and restoring epithelial barrier function in cystic fibrosis. This might provide significant advances and therapeutic benefit in improving quality of life and longevity for patients with cystic fibrosis.

Lipoxin (LX)A4, first identified by Serhan and colleagues (3), promotes resolution of inflammation and has been proposed as a novel regulator of adaptive immunity that may have therapeutic potential in chronic immune disorders (4–6). LXA4 was shown to inhibit proinflammatory cytokine IL-8 release, to arrest neutrophilic inflammation, and to decrease infection in a mouse model of chronic airway inflammation and infection,

which suggested that its inadequate production in CF could contribute to the persistence of airway inflammation (7). Our group reported that LXA4 targets airway epithelium, inducing intracellular Ca21 mobilization, enhanced Cl2 secretion and ASL height (8, 9), and stimulating airway epithelial repair and tight-junction formation (10, 11). The activation of airway epithelial Cl2 secretion and inhibition of Na1 absorption

( Received in original form October 22, 2012; accepted in final form February 9, 2014 ) This work was supported by the Institut National de la Sante´ et de la Recherche Medicale ´ (INSERM, France), by Vaincre La Mucoviscidose (VLM, France), by Career Enhancement and Mobility Programme Marie Curie Fellowship, by the Higher Education Authority of Ireland under the Programme for Research in Third Level Institutions (PRTLI) Cycle 4, and by the National Children’s Research Centre (NCRC, Ireland). Correspondence and requests for reprints should be addressed to Valerie Urbach, Ph.D., National Children Research Centre, Dublin 12, Ireland. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Cell Mol Biol Vol 51, Iss 2, pp 178–190, Aug 2014 Copyright © 2014 by the American Thoracic Society Originally Published in Press as DOI: 10.1165/rcmb.2012-0424OC on March 3, 2014 Internet address: www.atsjournals.org

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ORIGINAL RESEARCH by extracellular nucleotides has triggered interest for CF therapy in targeting purinoreceptors, which are abundantly expressed in respiratory epithelium (12–16). The P2RY11 receptor, which has been identified in tracheal epithelium, has the unique property of acting through Gq and Gs proteins to stimulate the calcium and cAMP signaling pathways (17, 18). Receptors for adenosine, the final product of ATP hydrolysis, also stimulate Cl2 secretion in airway epithelial cells (15, 19). The role of LXA4 in epithelial repair of corneal and airway epithelia has been previously reported, but the cellular mechanisms involved are not well understood (10, 20–22). Epithelial repair requires cell proliferation and migration, which are triggered by a variety of stimuli, including nucleotides and purinoreceptors (23–27). Stimulation of P2Y purinoreceptors induces calcium release and ERK phosphorylation, both of which play a key role in initiating cell proliferation and migration (28–31). Furthermore, a role of purinoreceptors in cell migration and proliferation, and thus wound repair, has been reported (32–35). In this study, we investigated the cellular mechanisms for the action of extracellular ATP in mediating the stimulatory effect of LXA4 on ASL height and epithelial repair. We found that LXA4 induced an autocrine ATP response in stimulating apical ATP release by bronchial airway epithelial cells in non-CF cells (NuLi-1 cell line) and CF cells (CuFi-1 cell line and CF primary culture). The LXA4–induced ATP secretion resulted in P2RY11 activation, ASL height increase, stimulation of cell proliferation and migration, and epithelial wound repair. This paper reports new roles for the inflammation resolution mediator LXA4 in the regulation of epithelial functions that are altered in CF airway. These findings strongly support a potential therapeutic role for LXA4 as a novel regulator of airway purinoreceptors restoring ASL height and the recovery from airway epithelial damage in CF.

Materials and Methods

For primary culture, epithelial cells were obtained from bronchial brushings (, 6 yr old), grown as previously described, and used at passage , P2 (9) (details are provided in the online supplement). Donors were recruited through the SHIELD CF study (Study of Host Immunity and Early Lung Disease in CF). Local ethics committee approval (Our Lady’s Children’s Hospital, Dublin) was granted. Written informed consent was obtained from parents or from legal guardian for minor donors (n = 5 patients).

Cell Proliferation and Migration

A MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] colorimetric assay and a Boyden chamber (8 mm pore, Becton, Dickinson and Company, Dublin, Ireland, UK) were used to quantify cell proliferation and migration, respectively (details are provided in the online supplement). cAMP Assay

Live cell confocal microscopy (LSM 510; Zeiss, Cambridge, UK) was used to visualized the ASL using Texas red-dextran (10 kD) (Invitrogen, Auckland, New Zealand) as previously described (9). Details are provided in the online supplement.

cAMP was measured directly from cell culture medium using a cAMP Enzyme Immunoassay Kit (CA200; Sigma-Aldrich, St. Louis, MO) to inhibit phosphodiesterase as per kit instructions. Absorbance was recorded using a Synergy Mx plate reader (Biotek Instruments).

Extracellular ATP

Material

A luciferin-luciferase luminometric assay (Molecular Probes, Eugene, OR) was used to determine ATP levels. Luminescence was read on a Synergy Mx plate reader (Biotek Instruments, Winooski, VT). Samples were analyzed in triplicate in a blackened 96-well plate. The ATP detection limit was 0.1 pM ATP.

LXA4 was obtained from Calbiochem (Merck KGaA, Darmstad, Germany), and aliquots (1024 M in ethanol) were stored at 2808 C. The peptide Boc-Phe-Leu-PheLeu-Phe (Boc-2) was obtained from Phoenix Pharmaceutical (Belmont, CA). 10 Panx (Panx-1 mimetic inhibitory peptide) was supplied by Tocris Bioscience (Abingdon, UK). BAPTA-AM was obtained from Molecular Probes (Leiden, The Netherlands). All other reagents were obtained from Sigma-Aldrich.

ASL Height

P2RY11 and P2RY2 Receptor Detection

For immunofluorescence experiments, a rabbit polyclonal anti-P2RY11 primary antibody (Alamone Labs, Jerusalem, Israel), the Alexafluor 488-conjugated anti-rabbit secondary antibody (Invitrogen, Christchurch, New Zealand), and the Vectashield mount (Vector Laboratories, Peterborough, UK) containing DAPI were used. For Western blotting, anti-P2RY11 primary antibody (Abcam, Cambridge, UK) (1:5,000) in 5% milk and anti-rabbit HRP secondary antiobody (Cell Signaling, Danvers, MA) (1:5,000) were used (see Figure E1 in the online supplement). Betaactin was used as loading control. For this purpose, the same membranes were stripped and reprobed with antiactin antibodies. The P2RY11 receptor silencing method is described in the online supplement (Figure E3).

Cell Culture

Human non-CF (NuLi-1) and CF (CuFi-1; homozygous Phe508 del) bronchial epithelial cell lines were used at passage , P15 and grown as previously described (9).

Wound closure was determined using ImageJ software at different time points to give a percentage wound closure value.

Epithelial Repair Assay

A wound was induced mechanically using a P200 pipette tip. Photomicrographs were captured from a Nikon Diaphot microscope.

Higgins, Buchanan, Perriere, et al.: Lipoxin Enhances CF Epithelium ASL and Repair

Statistical Analysis

All “n” refer to the number of wells or inserts used in experiments, which were repeated three times on different days and represented as mean 6 SEM. For comparisons between two groups, a Student’s t test was conducted. For three or more groups, a one-way ANOVA was used with a post hoc Newman-Keuls test to allow for multiple comparisons and performed using Graphpad Prism (v5.0). P , 0.05 was treated as significant.

Results LXA4 Stimulation of ATP Release

The release of ATP from non-CF (NuLi-1) and CF (CuFi-1) airway epithelial cell lines and primary cultures grown under an air–liquid interface (ALI) was conducted at room temperature on an antivibration table (Figure 1). Under unstimulated control conditions, the amounts of ATP measured 179

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Figure 1. ATP release induced by lipoxin (LX)A4 through pannexin-1 channels. (A and B) Basal levels of ATP where measured in apical and basolateral culture compartments NuLi-1 (n = 7) and CuFi-1 (n = 5) grown in air–liquid interface cultures. Effect of carbenoxolone (CBX) (10 mM) (NuLi-1, n = 4; CuFi-1, n = 4) and Probenecid (PROB) (10 mM) (NuLi-1, n = 5; CuFi-1, n = 5) on spontaneous ATP release. (C–E) LXA4 (1 nM) stimulation of a significant apical ATP release in NuLi-1 (n = 8), CuFi-1 (n = 10), and cystic fibrosis bronchial epithelial (CFBE) (n = 8) cells. Inhibition of the LXA4–induced ATP release by Boc-2 (10 mM) in non-CF (n = 6) and cystic fibrosis (CF) cell lines (n = 5) and primary cultures (n = 6), by CBX (n = 3), by PROB (n = 3), by 10Panx (100 mM; n = 6), and by Nocodozole (NOCO) (20 mM; n = 3). Data are representative of at least three independent experiments run in duplicate. One-way ANOVA with Newman-Keuls post hoc test was used for analysis of all data. *P , 0.05; **P , 0.01; ***P , 0.001. NS, not stimulated.

in the culture medium of basolateral and apical compartments of NuLi-1 and CuFi-1 cells were not significantly different (Figures 1A and 1B) (NuLi-1, P . 0.1 [n = 7]; CuFi-1, P . 0.1 [n = 5]). Under control conditions, carbenoxolone (10 mM), a general connexin inhibitor, and 180

probenecid (10 mM), a pannexin-1 inhibitor, significantly reduced the amount of ATP appearing apically in NuLi-1 epithelia (P , 0.05 [n = 4] and P , 0.05 [n = 3], respectively) but not in CuFi-1 epithelia. In the non-CF and CF cell lines and in primary cultures of CF bronchial

epithelia (CFBE), LXA4 induced a significant release of ATP in the apical compartment without affecting the amount of ATP released into the basolateral compartment (Figures 1C–1E). In NuLi-1 cells, the amount of ATP in the apical compartment increased from 11.01 6 0.48

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ORIGINAL RESEARCH nM (n = 9) to 186.4 6 51.04 nM (P , 0.001 [n = 8]) on exposure to LXA4 (1 nM, 10 min) (Figure 1C). In CuFi-1 cells, LXA4 also stimulated a highly significant apical ATP release. The amount of ATP measured in the apical compartment of CuFi-1 cells increased from 11.98 6 1.62 nM (n = 9) to 279.3 6 61.02 nM (n = 10) on exposure to LXA4 (Figure 1D). The ATP measured in the apical compartment of CFBE primary cultures increased from 16.81 6 2.00 nM (n = 8) to 291.1 6 73.91 (n = 8) on exposure to LXA4 (Figure 1E). Used as an inhibitor of the ALX/FPR2 receptor, Boc-2 (10 mM) has been shown to mediate the stimulatory effect of LXA4 on Cl2 secretion and ASL height. In this study, Boc-2 completely abolished the ATP increase induced by LXA4 in non-CF (n = 6) and CF cell lines (n = 5) and in primary cultures (n = 6). The antagonists of pannexin-1 channels, carbenoxolone (n = 3) and probenecid (n = 3), and the pannexin-1 mimetic inhibitory peptide, 10Panx (100 mM [n = 6]), completely abolished the stimulatory effect of LXA4 on apical ATP release in NuLi-1 and CuFi-1 cell line and in in CFBE primary cultures (Figures 1C–1E). Nocodozole (20 mM [n = 3]) used as a microtubule disrupting agent also inhibited LXA4–induced ATP release (Figures 1C and 1D). The Role of ATP in Regulating ASL Height

The effect of LXA4–induced ATP secretion on ASL height was investigated using live cell confocal microscopy. In non-CF and CF cell lines, the hydrolysis of external ATP using hexokinase and inhibition of ATP release using carbenoxolone and probenecid did not significantly affect ASL height under basal conditions but did inhibit the effect of LXA4 to stimulate ASL height (Figures 2A and 2B). LXA4 (1 nM) significantly increased ASL height from 7.2 6 0.11 mm (n = 4) to 8.8 6 0.24 mm (n = 4; P , 0.001) in NuLi-1 cells (Figure 2A), from 5.7 6 0.11 mm (n = 5) to 7.5 6 0.06 mm (n = 5; P , 0.001) in CuFi-1 cells (Figure 2B), and from 5.67 6 0.12 mm (n = 9) to 7.89 6 0.45 mm (n = 10) (P , 0.001) in CFBE primary cultures (Figure 2E). Apical ATP (1025 M) mimicked the effect of LXA4 by increasing the ASL height to 9.1 6 0.19 mm in NuLi-1 cells (n = 4) (P , 0.001) (Figure 2A) and 8.2 6 0.05 mm in CuFi-1 cells (n = 5) (P = 0.001) (Figure 2B). In NuLi-1 cells, ASL height measured when cells were pretreated by

hexokinase, carbenoxolone, and probenecid before LXA4 stimulation was 6.9 6 0.15 mm (n = 4), 7.1 6 0.16 mm (n = 4), and 6.8 6 0.19 mm (n = 4), respectively. In CuFi-1 cells, ASL height measured on exposure to hexokinase, carbenoxolone, and probenecid before LXA4 stimulation was 5.9 6 0.15 mm (n = 4), 5.9 6 0.18 mm (n = 4), and 5.4 6 0.12 mm (n = 4), respectively (Figures 2A and 2B). Taken together, these results indicate that the apical release of ATP induced by LXA4 mediates the stimulatory effects of LXA4 on ASL height in the non-CF and CF differentiated bronchial cell lines. Purinoreceptors as Transducers for LXA4 Effects on ASL Height

The role of P2Y receptors in transducing the ASL response to LXA4 was tested using the nonspecific antagonist of the P2Y receptor family, reactive blue 2 (RB2). RB2 did not significantly affect basal ASL height in NuLi-1 (n = 12) or CuFi-1 (n = 9) cells but completely abolished the effect of LXA4 on ASL height in NuLi-1 and CuFi-1 epithelia (Figures 2C and 2D). Specifically, the role of the P2RY11 receptor in the LXA4 effect on ASL was tested using a selective inhibitor (NF340) and small interfering RNA (siRNA) to knockdown the expression of P2YR11. NF340 (100 nM and 1 mM) did not significantly affect basal ASL height in NuLi-1 (n = 6 for 100 nM; n = 7 for 1 mM) or CuFi-1 cells (n = 4 for 100 nM; n = 4 for 1 mM) (P . 0.05). However, NF340 completely abolished the effect of LXA4 on ASL height in NuLi-1 and CuFi-1 epithelia (Figures 2C and 2D). NF340 also abolished the effect of LXA4 on ASL height in CFBE primary culture (Figure 2E). Knockdown of the P2RY11 receptor significantly reduced the ASL height increase induced by LXA4 in NuLi-1 cells from 8.9 6 0.08 mm (n = 4; P , 0.001) in scrambled RNA control conditions to 6.06 6 0.22 mm (n = 4) in siRNA-inhibited cultures (Figure 2F) and in CuFi-1 cells from 8.9 6 0.09 mm (n = 3) in scrambled control conditions to 5.04 6 0.09 mm (n = 3; P , 0.001) in siRNA-treated cells (Figure 2G). The P2RY11 knockdown was confirmed by RT-PCR and immunostaining (details are provided in the online supplement). These results strongly suggest a role for a P2RY11 receptor in the ASL height response to LXA4. Because cAMP-dependent protein kinase activity is a potent regulator of epithelial ion transport and P2RY11

Higgins, Buchanan, Perriere, et al.: Lipoxin Enhances CF Epithelium ASL and Repair

potentially stimulates the cAMP signaling pathway, we tested the effect of the PKA inhibitor H89 on the ASL response to LXA4. The PKA antagonist partially inhibited the up-regulation of ASL height by LXA4, suggesting a role for PKA in this effect. Adenosine receptor stimulation might also regulate airway epithelial ASL height via a cAMP-dependent signaling pathway. However, alloxazine (10 mM) used as an inhibitor of A2b receptor did not affect the response to LXA4 (Figures 2C and 2D). The Role of ATP Release and P2RY11 Receptor in LXA4–Induced Cell Proliferation

We tested the consequence of the LXA4–induced ATP release on cell proliferation of CF and non-CF bronchial epithelial cells. Under basal conditions, cell proliferation was significantly slower in CuFi-1 cells (0.551 6 0.03 AU [n = 18]) compared with NuLi-1 (0.665 6 0.02 AU [n = 18]) (P , 0.001) (Figures 3A and 3B). LXA4 significantly stimulated cell proliferation in non-CF (0.773 6 0.02 AU; P , 0.05 [n = 18]) and CF epithelial cells (0.672 6 0.03 AU; P , 0.05 [n = 18]) (Figures 3A and 3B). The pannexin-1 inhibitor CBX (1 mM) decreased basal cell proliferation and completely blocked the induction of cell proliferation by LXA4 in non-CF (0.255 6 0.05 AU; P , 0.001 [n = 12]) and CF cells (0.304 6 0.04 AU; P , 0.001 [n = 12]) (Figures 3A and 3B). The P2RY11 antagonist NF340 (1 mM) had no effect on basal cell proliferation but significantly blocked the LXA4–induced effect in non-CF (0.669 6 0.01 AU; P , 0.05 [n = 18]) and CF cell lines (0.545 6 0.02 AU; P , 0.05 [n = 18]) (Figures 3A and 3B). The calcium chelator BAPTA-M (10 mM) significantly reduced basal and LXA4–induced cell proliferation in the nonCF (P , 0.001 [n = 18]) and CF epithelial cells (P , 0.001 [n = 18]) (Figures 3A and 3B). The PKA inhibitor H89 910 mM) had no effect on basal cell proliferation; however, H89 entirely blocked the LXA4–induced increase in cell proliferation of NuLi-1 (0.605 6 0.01 AU; P , 0.05 [n = 12]) and CuFi-1 cells (0.443 6 0.01 AU; P , 0.05 [n = 12]) (Figures 3A and 3B). P2RY11 Activation and Calcium Mobilization Are Required for LXA4–Induced Cell Migration

We investigated the effect of LXA4 on cell migration and the role of purinoreceptor 181

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Figure 2. Consequences of LXA4–induced ATP release on airway surface liquid (ASL) height in CF airway epithelial cell lines. Mean height of ASL stained with dextran Texas Red in NuLi-1 (A and C), in CuFi-1 (B and D) cells, and in CFBE in primary culture (E). Effect of LXA4 (1 nM) on ASL height in NuLi-1 (n = 4), in CuFi-1 (n = 5), and in CFBE primary cultures (n = 10). Apical ATP (1025 M) effect on ASL height in NuLi-1 (n = 4) and CuFi-1 (n = 5) cells. Effect

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ORIGINAL RESEARCH signaling. We found that the basal level of cell migration was significantly lower in CF epithelial cells (118.3 6 3.7 [n = 18]) compared with non-CF cells (149.3 6 16.3 [n = 18]) (P , 0.05) (Figure 3C). Furthermore, LXA4 significantly stimulated cell migration in non-CF (191.3 6 8.2; P , 0.05 [n = 18]) and CF (157.6 6 4.2; P , 0.05 [n = 18]) epithelial cells (Figure 3C). The P2RY11 antagonist NF340 (1 mM) prevented the increase in cell migration induced by LXA4 to 90.8 6 12.5 NuLi-1 cells (P , 0.001 [n = 18]) and 91.7 6 10.3 CuFi-1 (P , 0.001 [n = 18]) (Figures 3C and 3D). BAPTA-M (10 mM) completely blocked the effect of LXA4 on cell migration of NuLi-1 (101.4 6 12.6; P , 0.001 [n = 18]) and CuFi-1 cells (87.6 6 6.5; P , 0.001 [n = 18]) (Figure 3C).

decreased the LXA4–induced wound repair to 25.8 6 5.3% in NuLi-1 (P , 0.01 [n = 8]) and 35.8 6 3.0% in CuFi-1 cells (P , 0.01 [n = 8]) grown on plastic (Figure 3E). Furthermore, BAPTA-AM significantly reduced the wound repair induced by LXA4 to 17.2 6 3.4% in NuLi-1 (P , 0.01 [n = 8]) and in to 22.2 6 1.2% CuFi-1 cells grown on plastic (P , 0.01 [n = 8]) (Figure 3E). BAPTA-AM significantly reduced the wound repair induced by LXA4 to 13.9 6 3.0% for NuLi-1 (P , 0.05 [n = 10]) and to 19.4 6 7.5% for CuFi-1 cells grown under ALI (P , 0.05 [n = 10]) (Figure 3F). Taken together, these results support a role for P2RY11 receptor signaling and calcium mobilization induced by LXA4 in the enhancement of cell proliferation, cell migration, and epithelial repair.

and the possible involvement of P2RY11 in generating this response. In NuLi-1 and CuFi-1 cells, LXA4 induced a significant increase in intracellular cAMP. The most significant cAMP rise was obtained at 100 nM LXA4 in NuLi-1 cells, whereas the highest rise in cAMP was obtained at 1 nM LXA4 in CuFi-1 cells. Furthermore, the increase in intracellular cAMP levels induced by LXA4 at 1 and 100 nM were completely inhibited when cells were pretreated with the P2RY11 inhibitor NF340 in NuLi-1 and CuFi-1 cell cultures (Figures 5A and 5B). These results indicate that LXA4 generates a cAMP increase mediated by the stimulation of P2RY11 receptors.

LXA4–Induced Wound Repair Is Mediated through Purinoreceptor Signaling

Expression of P2RY11 in Bronchial Epithelial Cells

In previous studies, we described the effect of LXA4 in triggering a transient intracellular calcium increase in non-CF and CF airway epithelial cells (9, 36). Because P2Y receptors, including P2RY11, can generate a rise in intracellular calcium, we tested the role for P2Y and more specifically P2RY11 receptors in the calcium signal induced by LXA4. As shown in Figure 5, LXA4 (1 nM) produced a transient intracellular calcium increase. Pretreatment with RB2 or NF340 to inhibit P2Y receptor family or P2RY11, selectively, did not affect the basal intracellular calcium levels. However, both inhibitors completely abolished the calcium increase induced by LXA4 (1 nM) (Figure 5). These results suggest that the calcium signal induced by LXA4 is mediated by P2RY11 receptor activation.

We investigated if cell proliferation and migration induced by the ATP release on exposure to LXA4 resulted in epithelial wound repair (Figures 3D–3F). We observed a reduced repair in CuFi-1 cells (CF) compared with, NuLi-1 (non-CF) cells cultured as monolayers on plastic or as a fully differentiated epithelium under ALI (Figures 3E and 3F). When grown on plastic, the CF cells achieved 21.32 6 1.3% of repair within 8 hours (P , 0.05 [n = 14]), whereas non-CF, NuLi-1 cells reached 38.5 6 2.2% repair within the same period (P , 0.05 [n = 14]) (Figures 3D and 3E). Similarly, when grown under ALI, NuLi-1 achieved 46.2 6 3.8% of repair (P , 0.05 [n = 12]) and CuFi-1 significantly less (25.0 6 4.4%; P , 0.05 [n = 8]) (Figure 3F). LXA4 induced a significant increase in wound repair in NuLi-1 and CuFi-1 cells grown on plastic or as fully differentiated epithelial cells (Figures 3D–3F). On plastic, wound repair was enhanced by LXA4 to 50.1 6 1.1% in NuLi-1 (P , 0.01 [n = 14]) and 48.7 6 6.2% in CuFi-1 (P , 0.001 [n = 14]) (Figure 3E). Under ALI, LXA4 increased wound repair of NuLi-1 to 52.7 6 1.1% (n = 10) and to 43.0 6 8.7% in CuFi-1 cells (n = 14) (Figure 3F). Treatment with NF340 (P2RY11 inhibitor) significantly

Our results strongly suggest a role for an apical P2RY11 receptor in transducing LXA4 effects on ASL height, cell proliferation, migration, and epithelial repair. Therefore, we examined the expression and localization of P2RY11 receptors in NuLi-1 and CuFi-1 cells on LXA4 stimulation. As shown by confocal microscopy, in control conditions the P2RY11 receptor appeared diffusely distributed in the cytoplasm of non-CF and CF cell lines and CF bronchial epithelium primary cultures. In contrast, after 15 and 60 minutes of LXA4 (1 nM) exposure, the P2YR11 immunostaining was increased and localized at the apical membrane in all cell preparations (Figures 4B and 4C). However, LXA4 did not significantly affect the overall expression of P2RY11 as assayed by Western blotting, suggesting that LXA4 stimulated the trafficking and/or stabilization of the P2RY11 receptor at the membrane without affecting its total expression (Figure 4A). The Effect of LXA4 on Intracellular cAMP

The H89 (PKA inhibition) sensitivity of the ASL response to LXA4 implicates a signaling role for cAMP. Therefore, we investigated the effect of LXA4 on cAMP

The Role of the P2RY11 Receptor in Calcium Signaling Generated by LXA4

Discussion The numerous reports that airway epithelial cells release ATP and the functional expression of purinergic receptors in airway epithelia suggest that the release of nucleotides may control major epithelial functions, including ion transport, ASL volume homeostasis, and epithelial structural integrity (37–40). We have

Figure 2. (Continued). of hexokinase (10 mM), CBX (10 mM) and PROB (10 mM) in NuLi-1 (n = 4) and CuFi-1 (n = 4) cells. Effect of reactive blue 2 (10 mM) in NuLi-1 (n = 6) and CuFi-1 cells (n = 4). Effect of NF340 in NuLi-1 (n = 6 for 100 nM; n = 7 for 1 mM), in CuFi-1 cells (n = 4 for 100 nM; n = 4 for 1 mM), and in CFBE primary culture (n = 4). Effect of H89 (10 mM) and alloxazine (10 mM) (n = 4). (F and G) Effect of knockdown of the P2RY11 receptor in NuLi-1 (n = 4) and CuFi-1 (n = 3) cells by small interefering RNA. The data are representative of at least three independent experiments run in duplicate. One-way ANOVA with Newman-Keuls post hoc test was used for analysis of all data. ***P , 0.001.

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Figure 3. LXA4 effects on proliferation, migration and repair of NuLi-1 and CuFi-1 cells. Proliferation of NuLi-1 (A) and CuFi-1 (B) cells was determined in nonstimulated conditions (n = 18) or in the presence of vehicle, DMSO (n = 18), or EtOH (n = 18). Effect of CBX (1 mM; n = 18), NF340 (1 mM; n = 12), H89 (10 mM; n = 12), and BAPTA-AM (10 mM; n = 18) on cell proliferation in the presence or absence of LXA4 (n = 18). (C) Mean values of the effects of NF340 (1 mM) or BAPTA-AM (10 mM) in NuLi-1 and CuFi-1 on the LXA4–induced cell migration in NuLi-1 and CuFi-1 cells (n = 18). (D) Typical images obtained during the wound repair of NuLi-1 and CuFi-1 cell monolayers cultured on plastic and either nontreated (n = 14), treated with LXA4 alone (n = 14), or after BAPTA-AM (10 mM; n = 8) and NF340 (1 mM; n = 8) pretreatment. (E) Mean changes in wound repair obtained in Nuli-1 and CuFi-1 grown on plastic in the conditions described in (D). The % of repair was obtained by comparing the wound size measured after 8 hours of treatment with the wound size after 8 hours without treatment. (F) Mean value of wound repair performed on Nuli-1 and CuFi-1 cells culture under air–liquid interface in

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ORIGINAL RESEARCH previously shown that LXA4 has a stimulatory effect on airway epithelial Cl2 secretion and on ASL height in CF and non-CF epithelia (9, 36). LXA4 also plays a role in airway epithelial repair and stimulates tight-junction formation (10, 11). In the present study, we provided evidence for the cellular mechanism by which LXA4 induces an increase in the ASL height and airway epithelial repair. We showed that LXA4 causes an apical ATP secretion via pannexin-1 channels, which results in the stimulation of P2RY11 purinoreceptors and subsequent Ca21 and cAMP signaling. The cellular release of ATP and purinoreceptor activation stimulate ASL height increase, cell proliferation, and migration, thereby promoting epithelial repair. When used as an inhibitor of the ALX/ FPR2 receptor, Boc-2 completely abolished the ATP increase induced by LXA4. This finding supports the role of ALX/FPR2 in this response. This result was consistent with other reports showing that LXA4 binds to the ALX/FPR2 receptor to elicit Boc2–sensitive effects in a variety of cells, including airway epithelial cells (9, 11). Whereas LXA4 produced a significant calcium increase in 16HBE14o, NuLi-1, and CuFi-1 airway epithelial cell lines and in primary culture, which expresses the ALX/FPR2 receptor, LXA4 did not affect intracellular calcium in the A549 alveolar cell line that does not express the ALX/ FPR2 receptor (9, 36). Knocking down ALX/FPR2 expression by 81% using siRNA completely abolished the effect of LXA4 on wound repair of NuLi-1 and CuFi-1 cells (41). Although Boc-2 can interact with a number of related receptors, our previous report and this report are consistent with a role for ALX/FPR2 in generating a biological responses to LXA4 in bronchial epithelial cells. In vitro studies have demonstrated that airway epithelia can maintain a constitutive release of ATP (39). In our study, we also reported that under basal conditions, nonCF (NuLi-1) and CF (CuFi-1) bronchial epithelial cells secrete ATP at similar concentrations into the apical and the basolateral compartments. In addition to

a constitutive release, an enhanced ATP secretion from airway epithelial cells can occur under different stimuli, and various cellular mechanisms have been reported. Connexins and pannexins provide a conduction pathway between the cytosol and extracellular space in addition to their constitutive role in the formation of intercellular gap junctions. The Pannexin-1 channel, the most studied of the pannexin family, is mechanosensitive and permeable to second messengers such as ATP, IP3, amino acids, and arachidonic acid and its metabolites (42–44). In our study, inhibition of Pannexin-1 channels reduced the amount of ATP spontaneously released on the apical surface of NuLi-1 epithelium but not in CuFi-1 epithelium. Although the amount of spontaneous ATP released by both cell types was similar, the dissimilar sensitivity to connexin inhibitors suggests that different mechanisms are involved in the constitutive ATP release in resting nonCF and CF cells and could be related to the different secretory phenotypes in the CF and non-CF differentiated cells. For example, CuFi-1 cells secrete more mucus than the non-CF NuLi-1 cells. In contrast, the LXA4–induced apical ATP release was completely inhibited by the selective Pannexin-1 channels inhibitor 10Panx in non-CF (Nuli-1) and CF (CuFi-1 and CFBE primary culture) differentiated epithelia, suggesting that Pannexin-1 channels play a major role in the ATP response to LXA4 compared with other release mechanisms. We also found that microtubule disruption inhibited the ATP release induced by LXA4, suggesting that the LXA4–induced ATP release involved the trafficking of secretory granules at the plasma membrane. However, cytoskeleton disruption also inhibited the trafficking of the ALX/FPR2 receptor and inhibited the ASL height increase induced by LXA4 (data not shown). Therefore, taking into account the low selectivity of cytoskeleton disrupting agent compared with the high specificity of 10 Panx that completely abolished the ATP response to LXA4, we conclude that the main mechanism involved in the ATP release induced by LXA4 was ATP permeability through Pannexin-1 channels.

Extracellular ATP modulates ion transport and intracellular signaling pathways of human airway epithelium (45, 46). Airway epithelial ion transport determines fluid secretion across the epithelium and thereby the hydration of the ASL layer, which is a major component of the mucociliary clearance. Nucleotides were among the first potentially therapeutic agonists to restore Cl2 and fluid secretion independent of CFTR in CF airway (16). Our findings that LXA4 exerts similar effects as ATP on ASL height, which are inhibited by hexokinase and ATP release blockers, indicate that apical ATP release may be a major cellular mediator of the LXA4 effects on ASL dynamics. In contrast, the absence of a significant inhibitory effect on the basal ASL height by hexokinase suggests that constitutive ATP secretion is not a major factor in regulating ASL homeostasis in resting cells, at least in our cellular models. Extracellular ATP actions are mediated by cell surface P2-purinergic receptors: either ligand-gated ion channel P2X receptors that comprise seven species activated by ATP or P2Y family of G protein–coupled receptors composed of eight species that are also activated by adenine and uridine nucleotides (47). In addition, adenosine, the product of ATP hydrolysis, activates a separate family of G protein–coupled receptors, the A1, A2a, A2b, and A3 adenosine receptors (27, 48). In previous studies we have shown that LXA4 stimulates a transient intracellular calcium signal that is consistent with an ATP-mediated effect of LXA4 on ASL involving one or more P2 receptor subtypes, nucleotide hydrolysis, adenosine receptor stimulation, and P2 receptor desensitization. However, the calcium signal induced by LXA4 was due to calcium release from intracellular stores rather than calcium entry (9, 36). Therefore, because ATP binding to P2X receptors results in the opening of a cation-permeable pore, allowing Ca 21 to enter into the cell, the ATPmediated effect of LXA 4 on ASL height most likely did not involve P2X purinoreceptors (49).

Figure 3. (Continued). control conditions (NuLi-1, n = 10; CuFi-1, n = 14) or stimulated with LXA4 (NuLi-1, n = 10; CuFi-1, n = 14) and in the presence or absence of BAPTA-AM (10 mM; NuLi-1, n = 10; CuFi-1, n = 14). Data are representative of at least three independent experiments run in duplicate. One-way ANOVA with Newman-Keuls post hoc test was used for analysis of all data. *P , 0.05; **P , 0.01; ***P , 0.001.

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Figure 4. Expression of P2RY11 in NuLi-1 and CuFi-1 cell lines and CFBE primary cultures. (A) Protein expression levels of P2RY11 in Nuli-1 (n = 3) and CuFi-1 (n = 3) cell lines and CF primary cultures from two patients (n = 3) with or without LXA4 treatment using anti-P2RY11. (B) Typical immunofluorescence images of the P2RY11 receptor (Green Alexaflour 488) in NuLi-1 and CuFi-1 cell lines and CF bronchial epithelium in primary cultures in control conditions (NS) or after 15 and 60 minutes of exposure to LXA4. Quantification of P2RY11 staining (fluorescence intensity) measured at the epithelial apex in control conditions or after 15 and 60 minutes of exposure to LXA4 (n = 6 for each conditions and cell type). One-way ANOVA with Newman-Keuls post hoc test was used for analysis of all data. *P , 0.05; ***P , 0.001.

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Figure 5. LXA4 effect on cAMP and intracellular Ca21 in NuLi-1 and CuFi-1 cells. Mean cAMP concentrations (pM) measured enzyme immunoassay in NuLi-1 (A) and CuFi-1 (B) cells after treatment with LXA4 at different concentrations: 1029 M (Nuli-1, n = 12; CuFi-1, n = 12), 1028 M (n = 8 in Nuli-1 and n = 9 in CuFi-1), 1027 M (Nuli-1, n = 10; CuFi-1, n = 10). Effect of NF340 in cAMP induced by LXA4 in NuLi-1 (n = 13 for LXA4 1029 M; n = 11 for LXA4 1027 M) and CuFi-1 cells (n = 13 for LXA4 1029 M; n = 11 for LXA4 1027 M) (*P , 0.05). (C) Typical calcium responses obtained on exposure of NuLi-1 to

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ORIGINAL RESEARCH Tarran and colleagues found that luminal shear stress induced an increase in ASL height due to increased ATP levels within the ASL, which stimulated P2RY2 receptors and activated an outward rectifying Cl2 current (50). The results presented here and in our previous reports strongly indicate that the ASL height increase induced by LXA4 involves P2Y receptors because RB2 used as a nonspecific P2Y inhibitor completely inhibited the response (9, 36). Therefore, a role for P2RY2 could not be excluded in the ATP-mediated effect of LXA4 on the ASL height and epithelial repair. Indeed, the human P2RY2 was initially cloned in the CF/T43 tracheal cell line (51), and numerous reports provided evidence for the functional role for a P2RY2 receptors in the airway, showing in particular equipotency of ATP and UTP in stimulating physiological responses (intracellular calcium increase and Cl2 secretion) (15). However, the complete inhibition of the LXA4 effects on ASL height and epithelial repair using NF340, a selective P2RY11 antagonist (52), suggests a major role for P2RY11 in transducing LX responses in bronchial epithelium. Furthermore, P2RY11 silencing using siRNA in fully differentiated NuLi-1 and CuFi-1 cell lines grown under ALI completely inhibited the ASL height induced by LXA4. The knockdown of the P2RY11 receptor provides strong evidence for the involvement of P2RY11 in the ASL response to LXA4. After P2RY11 knockdown, it appears that other P2Y receptors cannot compensate to sustain a LXA4 effect on ASL height, which suggests a primary role for P2RY11 receptors compared with other expressed receptors, such as P2RY2. The dominant role for P2RY11 in the response to LXA4 might results from the effect of LXA4 on P2RY11 trafficking at the plasma membrane that we reported in nonCF and CF bronchial epithelial cell lines and for primary HBE cultures. This rapid effect of LXA4 on receptor trafficking is consistent with the nongenomic effect of LXA4 (8, 9, 11, 41) and with the

LXA4 effects on cytoskeleton (53–55). In contrast, LXA4 does not affect P2RY2 immunostaining or protein expression in NuLi-1, CuFi-1, and CF primary cultures (Figure E2). The P2RY11 receptor has the unique property of signal transduction through the Gq and Gs proteins (56, 57). Pharmacological data have shown that the P2RY11 receptor is preferentially activated by ATP and is coupled to phosphoinostide and cAMP pathways (57). Evidence is available that ATP and ADP, two endogenous nucleotides, can raise cAMP levels in native cells via activation of P2RY11 receptors. Thus, P2RY11 receptors provide a mechanism, in addition to activation of P2RY2 or adenosine receptors, by which exogenous or endogenously secreted nucleotides can increase cellular levels of calcium and cAMP. Nucleotidemediated activation of P2RY11 receptors provides a means for autocrine regulation of epithelial cell function types (58). In airway epithelial cells, LXA4 stimulated an intracellular calcium signal and a cAMP increase, which are consistent with activation of Gq and Gs signaling pathways via ATP binding to an apical P2RY11 receptor. These results, taken together with the inhibition of the LXA4 effects on ASL by silencing P2YR11 and inhibiting PKA, strongly implicate the P2RY11 receptor subtype in transducing the epithelial responses to LXA4. Additional support for this conclusion was the finding of LXA4 stimulation of the apical expression of the P2RY11 receptor in NuLi-1 and CuFi-1 cells. Nucleotide-mediated epithelial responses are modified by surface ectonucleotidases that convert ATP into adenosine, thus providing a possible stimulation of adenosine receptors (59, 60) and cAMP/PKA signal transduction pathways and subsequent regulation of the epithelial sodium channel and CFTR (19, 61). In CF, the A2b receptor and PKA are functional, but the absence of functional CFTR renders the Cl2 prosecretory and Na2 inhibitory effects of adenosine signaling ineffective (61). In this study, we found that LXA4 induced an increase in

ASL height, which was dependent on the cAMP and PKA signaling pathways. Therefore, we cannot exclude the possibility that LXA4 mediates its ASL response via activation of an adenosine receptor. However, the ineffectiveness of the A2b receptor antagonist alloxazine to inhibit LXA4 effects on ASL does not support a substantial role for adenosine receptors in transducing ASL responses to LXA4. The P2Y receptor family and intracellular calcium signal have been shown to play a role in proliferation, migration, and wound repair in several epithelia, including airway (28, 29, 33–35, 62, 63). ATP released in response to cellular damage activates P2Y signaling pathways to trigger epithelial repair (29, 31, 64). Consistent with previous reports, our studies in reconstituted airway epithelia grown under an ALI show that epithelial repair is reduced and delayed in CF (CuFi1) cells compared with non-CF (Nuli-1) cells (65–69). Given the critical role ATP plays in epithelial repair, our results suggest that the reduced constitutive ATP release from CuFi-1 cells in vitro could account for the slower wound repair in CF airway observed in vivo. In the present study, the ATP release induced by LXA4 led to stimulation of wound repair. Furthermore, we showed that this pathway triggers two key steps of epithelial repair—cell migration and proliferation—in non-CF and CF airway epithelial cell lines. The role of LXA4 in epithelial repair of corneal and in non-CF and CF airway epithelial cell lines and primary cultures has been reported (37–39); however, the role of ATP release as a key mechanism in the repair induced by LXA4 was unknown. We provide evidence for a novel role for LXA4 in airway epithelial proliferation, migration, and wound repair, which is dependent on ATP release by Pannexin-1 channel and specific P2RY11 purinoreceptor–mediated calcium signaling. In monocytes, LXA4 also induces an intracellular calcium mobilization and plays a role of chemoattractant, which is consistent with LXA4 effects on airway epithelial

Figure 5. (Continued). LXA4 (1 nM) with or without pretreatment with NF340. Maximum calcium change (F340/F380) obtained on LXA4 exposure alone and when reactive blue 2 (10 mM) or NF340 (1 mM) was applied for 2 minutes before LXA4 in NuLi-1 (D) and CuFi-1 (E) cells. Data are representative of at least three independent experiments run in duplicate. One-way ANOVA with Newman-Keuls post hoc test was used for analysis of all data. *P , 0.05; ***P , 0.001.

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ORIGINAL RESEARCH cell migration (70). In contrast, in human polymorphonuclear leukocytes, LXA4 prevents intracellular calcium mobilization and suppresses polymorphonuclear leukocyte migration (71). Therefore, LXA4 seems to induce different signal transduction pathways according to the targeted cells to stimulate or prevent cell migration.

Extracellular nucleotides and nucleosides have been reported to regulate other major components of the mucocilary clearance machinery, including secretion of mucins by goblet cells or submucosal glands and cilia beating (72, 73). These other possible physiological outcomes of LXA4–induced ATP release are under investigation.

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In conclusion, this study provides molecular insights into a novel role for LXA4 in restoring normal ASL dynamics, correcting ion transport dysfunction, and enhancing epithelial repair in welldifferentiated CF airway cell cultures. n Author disclosures are available with the text of this article at www.atsjournals.org.

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