Inflammation-induced upregulation of P2X₄ expression augments mucin secretion in airway epithelia

Abstract

Mucus clearance provides an essential innate defense mechanism to keep the airways and lungs free of particles and pathogens. Baseline and stimulated mucin secretion from secretory airway epithelial cells need to be tightly regulated to prevent mucus hypersecretion and mucus plugging of the airways. It is well established that extracellular ATP is a potent stimulus for regulated mucus secretion. Previous studies revealed that ATP acts via metabotropic P2Y₂ purinoreceptors on goblet cells. Extracellular ATP, however, is also a potent agonist for ionotropic P2X purinoreceptors. Expression of several P2X isoforms has been reported in airways, but cell type-specific expression and the function thereof remained elusive. With this study, we now provide evidence that P2X₄ is the predominant P2X isoform expressed in secretory airway epithelial cells. After IL-13 treatment of either human primary tracheal epithelial cells or mice, P2X₄ expression is upregulated in vitro and in vivo under conditions of chronic inflammation, mucous metaplasia, and hyperplasia. Upregulation of P2X₄ is strongest in MUC5AC-positive goblet cells. Moreover, activation of P2X₄ by extracellular ATP augments intracellular Ca²⁺ signals and mucin secretion, whereas Ca²⁺ signals and mucin secretion are dampened by inhibition of P2X₄ receptors. These data provide new insights into the purinergic regulation of mucin secretion and add to the emerging picture that P2X receptors modulate exocytosis of large secretory organelles and secretion of macromolecular vesicle cargo.

INTRODUCTION

Mucus secretion in the airways is an essential mechanism for maintaining airway function and integrity. The mucus layer in the airways and mucociliary clearance provide a first line of defense and keep the airways and lungs free of particles and pathogens (33). To maintain and replenish the mucus layer in the airways, mucins are continuously synthesized and secreted from secretory cells along the airways (3, 15, 73, 74). In the upper airways goblet cells are the main secretory cells secreting mucins (MUC5AC and MUC5B), whereas club cells are the principal secretory cells in the distal airways, where goblet cells are absent under healthy, noninflammatory conditions (30). Mucin secretion (and clearance) needs to be tightly regulated under baseline as well as stimulated conditions (3, 73). Excessive secretion (or impaired clearance) leads to mucin aggregation and mucus plugging of the airways. This is particularly prevalent in inflammatory lung diseases including asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and chronic bronchitis when club-to-goblet cell transformation, mucous metaplasia, and hyperplasia result in mucin hypersecretion (16). The mechanisms regulating mucin secretion have therefore been major research areas for almost half a century.

Stimulated mucin secretion from goblet cells occurs via Ca²⁺-dependent exocytosis of large secretory granules. Within recent years substantial progress has been achieved in identifying key molecular mechanisms of the trafficking and core and regulatory exocytic machinery (see reviews in Refs. 3, 10). It is now also well established that extracellular ATP is a potent agonist for stimulated mucin secretion (1, 32). ATP activates P2Y₂ purinoreceptors on the apical membranes of goblet cells via phospholipase C pathways, resulting in generation of the second messengers inositol 1,4,5-trisphosphate and diacylglycerol and release of Ca²⁺ from intracellular stores (6).

Extracellular ATP, however, is also a potent agonist for P2X purinoreceptors, ligand-gated ion channels. Expression of several P2X receptors has been reported in lung tissues, with P2X₄ and P2X₇ being the most widely expressed isoforms (6, 21). To date, information on cell type-specific expression is mostly lacking. Also, apart from a well-established proinflammatory/proapoptotic role for P2X₇ (41, 46, 56), little is known of the function of P2X receptors in airway epithelia. In recent years P2X receptors have attracted increasing attention as regulators of exocytosis and cellular secretion (24, 25, 28, 29, 45, 69). It has been shown that P2X receptor activation stimulates exocytosis directly via influx of Ca²⁺ from the extracellular space and elevation of cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c) (27, 29, 35, 60). More recently, we have reported that activation of P2X₄ receptors after vesicle-plasma membrane (PM) fusion modulates the secretion and activation of pulmonary surfactant from alveolar type II (ATII) epithelial cells (12, 44, 65). Secretion of pulmonary surfactant in many aspects resembles mucin secretion. First, surfactant, similar to mucins, is a large macromolecular complex stored in large, lysosome-related secretory organelles termed lamellar bodies, and second, the regulatory machinery governing lamellar body exocytosis is similar to the regulatory machinery in mucin granule secretion (13, 19, 26, 36, 55). Therefore, it was the aim of this study to investigate whether P2X receptors are expressed in goblet cells and if activation of P2X receptors impacts mucin secretion.

With this study, we now provide evidence that P2X₄ is the predominant P2X isoform expressed in secretory airway epithelial cells. Moreover, we show that P2X₄ expression is upregulated in vitro and in vivo under conditions of chronic inflammation with mucous metaplasia and hyperplasia. Upregulation is strongest in MUC5AC⁺ cells. Activation and/or potentiation of P2X₄ augments mucin secretion, whereas inhibition of P2X₄ receptors dampens mucin secretion. These data provide new insights into the purinergic regulation of mucin secretion and add to the emerging picture that P2X receptors modulate exocytosis of large secretory organelles and secretion of macromolecular vesicle cargo.

METHODS

Materials.

The α-P2X₄ antibody was designed by us against the human protein sequence CKKYKYVEDYEQGLASELDQ, raised in rabbits and affinity purified by Pineda (Berlin, Germany); α-tubulin β-IV mouse monoclonal antibody (catalog no. T7941) was purchased from Sigma-Aldrich (Steinheim, Germany), α-Club Cell Secretory Protein (CCSP) was either rabbit polyclonal antibody (catalog no. sc-9772) from Santa Cruz (Dallas, TX) or rat monoclonal antibody (catalog no. MAB4218) from R&D Systems (Minneapolis, MN), and the α-MUC5AC mouse monoclonal antibody (catalog no. MA1-21907) was from ThermoScientific/Pierce (Rockford, IL). Fluorescently labeled secondary antibodies were obtained from Molecular Probes (Life Technologies, Karlsruhe, Germany). All other chemicals were purchased from Sigma-Aldrich unless otherwise indicated.

Cell culture.

Human primary tracheal epithelial cells (HTECs) and growth medium were purchased from Promocell (Heidelberg, Germany). For differentiation, a 50:50 mixture of DMEM-H and LHC Basal (both from Thermo Fisher, Waltham, MA) was supplemented with Airway Epithelial Cell Growth Medium SupplementPack (Promocell) and different trace elements as described previously (20). Briefly, frozen primary HTECs derived from several donors were obtained at passage 2, thawed, and expanded in a T75 flask. Growth medium was replaced every 2 days. Once cells were 70–90% confluent, they were passaged with DetachKiT from Promocell and plated at a density of 3.5 × 10⁴ cells per 6.5-mm Transwell filter (Corning Costar 3470; Corning, Corning, NY) precoated with collagen solution (StemCell Technologies, Cologne, Germany). Cells were plated with 200 µl of growth medium apically, and an additional 600 µl of growth medium was added basolaterally. The apical medium was replaced at 48 h and then removed completely at 72 h. The basolateral medium was switched to differentiation medium at the same time and replaced every 3 days. Cells were grown up to 32 days, and after day 14 apical Dulbecco’s phosphate-buffered saline (DPBS) washes were performed in the incubator for 30 min every 3 days. For cells treated with IL-13 (Millipore, Darmstadt, Germany), the IL-13 was added to the basolateral medium every 3 days to a final concentration of 10 ng/ml when air-liquid interphase (ALI) was imposed until day 21, when, after collection of samples, the basolateral medium was switched to normal differentiation medium without IL-13. Calu-3 cells were grown at ALI with supplemented minimal essential medium according to the manufacturer’s recommended conditions (ATCC, Manassas, VA).

HTEC cultures from two to four different donors were used for each experiment.

Mouse IL-13 instillation and FACS sorting.

Mucous metaplasia of tracheal epithelial cells was induced by intrapharyngeal instillation of 2 μg of IL-13 (Biolegend) in 50 μl of water on three consecutive days under isoflurane anesthesia. The lungs were harvested on the fifth day after the final IL-13 instillation. Airway epithelium cell sorting was performed essentially as described previously (8) with BD FACSAria Fusion/BD FACSDiva Software and adult mouse lung tissue digested in 5 mg/ml collagenase type I (Worthington, Lakewood, NJ). A global double-fluorescent Cre reporter mouse (48) crossed to a CCSP-iCre mouse (40) yielded green-only airway epithelial cells, which were selected with the four-way purity precision mode. Mice were bred within our colony, housed in specific pathogen-free conditions, and handled in accordance with protocols reviewed and approved by the Institutional Animal Care and Use Committee of the University of Texas MD Anderson Cancer Center.

Semiquantitative RT-PCR.

Total RNA was isolated with the RNeasy MiniKit (Qiagen, Hilden, Germany). Reverse transcription was performed on 0.8–1.3 µg of total RNA with the SuperScript VILO cDNA Synthesis Kit according to the manufacturer’s protocol. The following validated QuantiTect primer assays (Qiagen) were used: HMBS [QT00014462 (hs), QT00494130 (ms)], MmP2Xr4_1_SG (QT00163149), MmP2X7_1_SG (QT00130900), MmMUC5AC_1_SG (QT001196006), and MmMUC5B_1_SG (QT01067899) for mouse samples and HsP2X₁_1_SG (QT00194320), HsP2X₂_1_SG (QT00427455), HsP2X₃_1_SG (QT01083593), Hs P2X₄_1_SG (QT00049693), HsP2X₅_1_SG (QT00178297), HsP2X₆_1_SG (QT00178297), HsP2X₇_1_SG (QT00083643), HsMUC5AC_1_SG (QT00088991), HsMUC5B_1_SG (QT01529367), and Hs_FoxJ1_1_SG (QT01000797) for human samples. Amplification was performed on a Mastercycler RealPlex² (Eppendorf, Hamburg, Germany) with the EXPRESS SYBR GreenER qPCR supermix. Each reaction was carried out on cDNA from three or more independent biological replicates (cDNAs were used at 1-, 10-, and 100-fold dilutions). Specificity of PCR reactions was confirmed by melting point analysis of PCR products. RealPlex software (Eppendorf) was used for data acquisition and analysis. Correction for PCR performance as well as quantification relative to housekeeping gene hydroxymethylbilane synthase (HMBS) was carried out as described previously (54). Stable expression of HMBS has been confirmed for all time points in control and IL-13 treated samples.

Western blot analysis.

HTECs were washed twice in DPBS, solubilized in lysis buffer, separated by SDS-PAGE, and transferred to nitrocellulose. Ponceau staining of blots was performed before immunolabeling to normalize for sample loading. Immunodetection of P2X₄ was performed with the α-P2X₄ antiserum (1:500) overnight and chromogenic detection of alkaline phosphatase-labeled secondary antibody (WesternBreeze anti-rabbit; Invitrogen, Karlsruhe, Germany).

Cell population analysis by FACS.

To analyze P2X₄ expression in the respective cell populations (ciliated, secretory, and goblet cells), HTECS grown on Transwell filters were trypsinized and detached cells were fixed for 20 min in 4% paraformaldehyde in DPBS. Cells were stained for P2X₄ and tubulin β-IV, MUC5AC, or CCSP as described in Immunofluorescence and analyzed on a BD Canto II flow cytometer with the FACSDiva software for a mean of 1 × 10⁵ events. Cells were gated for different forward (FSC) and sideward light scatter characteristics, and doublet exclusion was achieved by gating in the FSC-A and FSC-W channels. Relative percentages of cell populations were calculated with FlowJo 10.4.1. Median P2X₄ fluorescence intensity was derived from the respective 50% highest-expressing positive and 50% lowest-expressing negative populations.

Immunofluorescence.

For immunofluorescence staining of intact mucin vesicles, HTECs grown on Transwell filters were fixed for 20 min in 4% paraformaldehyde in DPBS containing 30 nM BAPTA to block mucin secretion due to handling (73). Cells were then permeabilized for 10 min with 0.2% saponin and 10% FBS (Thermo Scientific, Bonn, Germany) in DPBS. Cells were stained with primary (1:100) and secondary (1:400) antibodies in PBS, 0.2% saponin, and 10% FBS. Images were taken on an inverted confocal microscope (Leica TCS SP5; Leica) using a ×63 lens (Leica HCX PL APO lambda blue 63.0x1.40 OIL UV). Images for the blue (DAPI), green (Alexa Fluor 488), red (Alexa Fluor 568), and far red (Alexa Fluor 647) channels were taken in sequential mode with appropriate excitation and emission settings.

For formalin-fixed, paraffin-embedded (FFPE) sections of mouse lung samples, 2-μm sections were cut from the blocks for immunostaining. Antigen retrieval was performed with citrate buffer and blocked in Tris-buffered saline solution with 10% bovine serum albumin (BSA). Primary antibodies were added in the diluent of Tris-buffered saline-Tween 20 and 0.5% BSA for 1 h or overnight. Secondary antibodies were added in the diluent for 1 h at a concentration of 1:400.

Mucin ELISA.

The mucin ELISA assay was performed according to the protocol by Abdullah et al. (2), with the application of recently published improvements (73). Briefly, HTECs on filters were washed with DMEM every hour for four times to remove excess mucus. After initial washings, cells were incubated with DMEM two times for 1 h to collect samples for baseline secretion before cells were stimulated (15 min) and samples were collected for analysis. All samples were diluted 1:10 in PBS and treated with neuraminidase (0.625 mU/μg total protein) for 2 h at 37°C (9). One hundred microliters of the diluted sample was plated on an enzyme immunoassay high-binding 96-well plate (Corning) overnight at 4°C. The samples were blocked with 1% (wt/vol) BSA in PBS-Tween 20 (PBST) for 1 h at room temperature and then washed three times with PBST. The anti-MUC5AC antibody (catalog no. MA1-21907; ThermoScientific/Pierce) was added at 1:250 for 2 h at 37°C in 1% (wt/vol) BSA in PBST. Cells were then washed four times with PBST and incubated with horseradish peroxidase-conjugated secondary antibody goat anti-mouse (Jackson ImmunoResearch, West Grove, PA) at a final concentration of 1:1,000 in PBST for 2 h at 37°C. After washing, the samples were developed with 3,3′,5,5′-tetramethylbenzidine substrate (ThermoScientific/Pierce) and stopped with 1 M H₂SO₄ (Sigma-Aldrich). The samples were then immediately read in the ELISA plate reader (Tecan, Salzburg, Austria) for absorbance at 450 nm.

Fluo-4 measurements.

For measurement of changes in intracellular calcium levels, HTECs on filters were loaded with 3 µM fluo-4 AM (ThermoScientific, Karlsruhe, Germany) for 45 min in DMEM, washed twice in bath solution (in mM: 140 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂, 5 glucose, and 10 HEPES; pH 7.4) and maintained in bath solution until the start of the experiments. In addition, HTECs were also stained with LysoTracker Deep Red (50 nM; ThermoScientific) to identify secretory cells. With a two-dimensional imaging system cells were illuminated for 50 ms at a rate of 1 Hz at 480 nm (26) and images were acquired with MetaFluor (Molecular Devices, Ismaning, Germany). Images were analyzed with MetaFluor Analyst (Molecular Devices) as described previously (44) To compare changes in the Ca²⁺ response, the integrated Ca²⁺ signal (area under the curve) was analyzed (19).

Adenoviral transfection.

Adenoviruses expressing either P2X₄-EGFP [wild type (wt)] or P2X₄(C353W)-EGFP [dominant negative (dn)] were produced as recently described (42) with the ViraPower Adenoviral Expression System (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions. HTECs were infected on days 14 and 18 after the start of ALI culture.

Statistics.

All statistics were analyzed and graphs plotted with GraphPad Prism 5 (San Diego, CA). Data are described as means ± SE, unless noted differently. Unless indicated otherwise, an unpaired, nonparametric Mann-Whitney test was applied to compare control vs. IL-13-treated samples. Data were considered significant if the P value was <0.05.

RESULTS

P2X₄ is the dominant P2X isoform expressed in HTECs grown at ALI.

In initial experiments we examined the expression of P2X receptors in HTECs. RT-PCR performed on HTECs revealed that P2X₄ is the most highly expressed isoform in fully differentiated (day 21) HTECs grown at ALI. P2X₁ and P2X₆ receptors were also expressed in differentiated HTEC cultures, whereas expression of P2X₂, P2X₃, P2X₅, and P2X₇ receptors was weak or negligible (<0.1-fold) compared with expression of the housekeeping gene HMBS (Fig. 1). Fully differentiated HTEC cultures contain ciliated as well as secretory cells, and we cannot conclude from these data whether P2X₄ expression exhibits a cell type-specific pattern. Results from Calu-3 cells, however, suggested that P2X₄ is expressed in secretory cells. A large proportion of Calu-3 cells contain MUC5AC granules with a goblet cell-like phenotype, but no ciliated cells are present (23, 37). P2X₄ receptors were the most highly expressed isoform in Calu-3 cells, and relative expression was higher in Calu-3 cells than in fully differentiated HTECs [2.83 ± 0.21 (n = 24)- vs. 0.75 ± 0.08 (n = 12)-fold HMBS expression, respectively; P < 0.001]. Expression of all other P2X isoforms was <0.2-fold expression of housekeeping gene HMBS (Fig. 1), suggesting that P2X₄ receptors are the predominantly expressed P2X isoform in gobletlike cells.

Fig. 1.

P2X₄ is the dominant P2X isoform expressed in human primary tracheal epithelial cells (HTECs) grown at air-liquid interphase (ALI): real-time RT-PCR analysis of P2X purinergic receptor transcripts in HTECs grown at ALI for 21 days and Calu-3 cells, respectively. Data are expressed as fold expression of housekeeping gene hydroxymethylbilane synthase (HMBS). Values are means from 5–24 individual cultures. Significant differences between P2X₄ expression levels and all other P2X isoforms: *HTEC (P < 0.019) and ^#Calu-3 cells (P < 0.015) (statistical significance was assessed by Kruskal-Wallis test for multiple comparisons). In this and subsequent figures, box plots show median, minimum, and maximum.

IL-13 induced goblet cell hyperplasia results in upregulation of P2X₄ expression in HTECs.

In light of these findings we next investigated whether increasing secretory cell numbers and mucous differentiation results in increased P2X₄ expression in HTEC cultures. It is well established that IL-13 treatment induces goblet cell hyperplasia and metaplasia in vitro (4, 37, 68) and in vivo (70, 73). Therefore, confluent HTEC cultures were grown for 21 days in the absence (= control) or presence of 10 ng/ml IL-13 (basolaterally), and then on day 21 IL-13 was removed. Samples were collected at various time points after ALI conditions were established (day 0). In line with previous results, ALI culture induced a significant increase in FOXJ1 expression, a marker for ciliated cells, in control but not IL-13-treated cells (5, 72). IL-13 also significantly delayed the increase of MUC5B expression that occurs during differentiation of control cells (Fig. 2B; Ref. 14) and induced a massive increase in MUC5AC expression from day 8 onward, when goblet cell differentiation begins (66), resulting in >200 times higher MUC5AC expression (compared with baseline or control without IL-13) from day 4 onward. Removal of IL-13 resulted in rapid downregulation of MUC5AC expression (Fig. 2C). Together, these data demonstrate that IL-13 treatment of HTECs results in goblet cell hyperplasia and metaplasia with increased MUC5AC expression.

Fig. 2.

IL-13 induces upregulation of MUC5AC and inhibits expression of FoxJ1 and MUC5B in human primary tracheal epithelial cells (HTECs). Confluent HTEC air-liquid interphase cultures (reaching confluence = day 0) were grown for 21 days (d) in the absence [control (ctrl)] or presence of 10 ng/ml IL-13 basolaterally and then for 4 more days after IL-13 was removed. Cells were collected at various time points for analysis of gene expression by RT-PCR. IL-13 treatment resulted in a sustained decrease in FoxJ1 (A), a transient decrease in MUC5B (B), and a >200-fold increase in MUC5AC expression (C). Removal of IL-13 on day 21 resulted in immediate downregulation of MUC5AC expression. Data are expressed as fold expression of housekeeping gene hydroxymethylbilane synthase (HMBS). Values are means from 6 or 7 individual donors. *Significant differences between control and IL-13-treated cells at individual time points; ^#significant difference between end of IL-13 treatment (day 21) and time point after IL-13 removal (day 25). *P < 0.05; **P < 0.01.

IL-13 treatment of HTECs resulted in significantly increased P2X₄ transcript expression, with a time course similar to the upregulation of mucin gene expression. Akin to MUC5AC expression, removal of IL-13 resulted in downregulation of P2X₄ expression back to control levels (Fig. 3A). P2X₇ receptor expression, which has previously been reported as a marker for airway inflammation (56), however, was unaffected by IL-13 treatment (Fig. 3A). In accordance with the time course of IL-13-induced upregulation of P2X₄ transcription, a significant increase in P2X₄ protein levels was observed by Western blotting from day 14 onward. Again, removal of IL-13 on day 21 resulted in a decrease in P2X₄ protein levels to near-control levels at day 32, whereas cultures maintained for 32 days in the presence of IL-13 maintained high levels of P2X₄ (Fig. 3, B and C).

Fig. 3.

IL-13 induces upregulation of P2X₄ expression in human primary tracheal epithelial cells (HTECs). A: confluent HTEC air-liquid interphase (ALI) cultures were grown for 21 days (d) in the absence [control (ctrl)] or presence of 10 ng/ml IL-13 basolaterally. On day 21 IL-13 was removed. Left: IL-13 treatment significantly increased P2X₄ transcript expression, which returned to control levels upon removal of IL-13. Right: P2X₇ receptor expression was unaffected by IL-13 treatment. Values are the means from 7 or 8 individual donors. *Significant differences between control and IL-13-treated cells at individual time points; ^#significant difference between end of IL-13 treatment (day 21) and time point after IL-13 removal (day 25). B, top: Western blot of confluent HTEC ALI cultures grown for 32 days in the absence (−) or presence (+) of IL-13, except for a sample grown for 21 days in the presence of IL-13 and then another 11 days without IL-13 (day 32, 0–21). P2X₄ receptor expression was detectably increased in IL-13-treated HTECs from day 14 onward, and removal of IL-13 resulted in a return to near control levels [sample day 32 (0–21)]. Bottom: quantitative analysis of P2X₄ protein expression from 5 independent experiments (donors) as illustrated at top. P2X₄ expression was normalized to total protein. a.u., Arbitrary units. C: representative immunoblot to validate specificity of α-P2X4 antibody. Left: Ponceau staining of blot to control for equal loading of lanes. Right: Western blot for P2X₄ from untreated HeLa cells (control) and HeLa cells infected with P2X₄ short hairpin (sh)RNA for 48 h. P2X₄ shRNA adenovirus was constructed based on the shRNA cassette design described by Gou et al. (22) and recently utilized by Fois et al. (18). *Significant differences between control and IL-13-treated cells at individual time points. *P < 0.05; **P < 0.01.

To further delineate the correlation between increases in MUC5AC expression, goblet cell metaplasia, and increased P2X₄ expression, we used FACS analysis to investigate relative changes in P2X₄ expression in goblet (MUC5AC⁺), club (CCSP⁺) and ciliated (tub IV⁺) cells upon IL-13 treatment. Initial FACS analysis of the percentage of the respective cell populations further confirmed that IL-13 treatment results in a significant increase in MUC5AC⁺ cells [25.64 ± 4.53% (n = 8) (IL-13) vs. 8.28% ± 2.10 (control), P = 0.007] (Fig. 4A). The percentage of tub IV⁺ and CCSP⁺ cells, on the other hand, either was significantly downregulated or showed a trend toward reduction upon IL-13 treatment, respectively. Our data further revealed that P2X₄ expression significantly increased in MUC5AC⁺ cells (n = 8) after IL-13 treatment, whereas P2X₄ expression was not significantly altered in MUC5AC⁻ cells (Fig. 4B). In summary, these data provide strong evidence that the IL-13-induced increase of P2X₄ expression in HTECs results from increased goblet cell numbers and increased P2X₄ expression within these goblet cells.

Fig. 4.

IL-13-induced upregulation of P2X₄ expression is primarily induced in MUC5AC⁺ cells. A, left: FACS analysis histograms for MUC5AC fluorescence intensity of human primary tracheal epithelial cells (HTECs) cultured in the absence (control) or presence of IL-13. Overlay illustrates that the proportion of MUC5AC⁺ cells is increased in IL-13-treated samples. Right: quantitative analyses of goblet (MUC5AC⁺), club cell [α-Club Cell Secretory Protein (CCSP)⁺], and ciliated cell [tubulin β-IV (tub IV)⁺] populations in control and IL-13-treated HTEC cultures reveal a significant upregulation of MUC5AC⁺ cells and downregulation of tub IV⁺ cells after IL-13 treatment. The percentage of CCSP⁺ was not significantly changed. pos, Positive. B: P2X₄ median intensity is increased in MUC5AC⁺ cells. Left: FACS blot depicting P2X₄ vs. MUC5AC intensity in IL-13-treated HTECs. High MUC5AC expression in cells is associated with high P2X₄ expression. Center: P2X₄ expression levels in MUC5AC- and MUC5AC⁺ cells from blot on left. Right: quantitative analysis of P2X₄ expression in MUC5AC⁻ and MUC5AC⁺ cell populations in control and IL-13-treated HTEC cultures. Median P2X₄ expression is significantly increased in MUC5AC⁺ cells after IL-13 treatment. n, No. of individual experiments. *P < 0.05; **P < 0.01.

P2X₄ is expressed on PM and in vesicular structures in goblet cells.

We next investigated the subcellular localization of P2X₄ receptors in goblet cells. P2X₄ receptors have been shown to be expressed at the PM but predominantly reside on lysosomes and lysosome-related organelles (47), including lamellar bodies in ATII epithelial cells (42), affecting lysosomal trafficking and secretion. First of all, immunofluorescence staining of HTECs confirmed that the strongest P2X₄ signal is found in MUC5AC⁺ cells (Fig. 5A). Again, IL-13 treatment significantly increased the percentage of MUC5AC⁺ cells and accordingly P2X₄⁺ cells (Fig. 5A). High-resolution confocal microscopy further revealed that the MUC5AC⁺ structures in IL-13-treated HTECs indeed resemble granular structures identical in size and shape to mucin granules in goblet cells (∼1–3 μm in diameter). However, at close examination, P2X₄ receptors were closely associated with the mucin vesicles but did not strictly colocalize with the granular structures (Fig. 5B, top). Analysis of individual confocal sections confirmed that P2X₄ receptors are localized on circular structures devoid of MUC5AC (Fig. 5B, middle), and xy-sectioning further supported that P2X₄ receptors are, in addition to some PM staining, localized on intracellular organelles located basolaterally of the apical MUC5AC⁺ mucin granule pool (Fig. 5B, bottom). In line with this, when P2X₄-EGFP is expressed in IL-13-treated cultures of HTECs, P2X₄-EGFP is localized in vesicular, intracellular structures distinct from Muc5AC-containing granules (Fig. 5C).

Fig. 5.

P2X₄ is expressed on the plasma membrane (PM) and in vesicular structures in goblet cells. A: immunofluorescence staining of human primary tracheal epithelial cells (HTECs) cultured in the absence (control) or presence of IL-13 for 21 days. IL-13 treatment significantly increased % of MUC5AC-positive cells. Overlay reveals that P2X₄ (red) expression is almost exclusively restricted to MUC5AC (green)-positive cells. Scale bars, 25 μm. B: confocal images reveal that P2X₄ (red) is, in addition to PM localization (arrows), expressed on vesicular structures (arrowheads) devoid of MUC5AC (green) residing basolateral to mucin granules. Top: maximum projection of confocal z-sections through apical region of a single goblet cell. Middle: individual confocal section beneath the apical pool of mucin granules (as indicated in scheme on left). Bottom: xz-section of the same cell. Scale bars, 5 μm. C: confocal cross section of HTECs, expressing wild-type (wt) P2X₄-EGFP (green) and treated for 21 days with IL-13. Cells were stained for MUC5AC (red). Scale bar, 10 μm.

IL-13 treatment induces P2X₄ expression in goblet cells in vivo.

We next confirmed whether this goblet cell-specific expression of P2X₄ could also be observed in vivo. Goblet cell metaplasia was induced in a murine model using IL-13 instillation. Subsequent RT-PCR analysis of mucin expression on FACS-sorted secretory cells confirmed that many of the club cells had transitioned to mucin-producing goblet cells. Compared with naive animals, IL-13 induced a significant increase in Muc5ac expression [1.11 ± 0.20 (n = 7)-fold vs. 3.30 ± 0.95 (n = 5)-fold Hmbs expression, respectively; P = 0.01] and an increase in Muc5b expression [1.37 ± 0.42 (n = 7)-fold vs. 5.09 ± 2.97 (n = 5)-fold Hmbs expression, respectively; P = 0.11] (Fig. 6A). In line with findings in HTECs, P2X₄ expression increased after IL-13 treatment [1.02 ± 0.07 (n = 7)-fold vs. 1.47 ± 0.18 (n = 5)-fold Hmbs expression, respectively; P = 0.04], whereas no change was detected for P2X₇ expression [1.16 ± 0.27 (n = 7)-fold vs. 1.30 ± 0.33 (n = 5)-fold Hmbs expression, respectively; P = 0.76] (Fig. 6B).

Fig. 6.

IL-13 treatment in vivo induces P2X₄ expression in mouse secretory cells. A: RT-PCR analysis of mucin gene expression in airway epithelial cells isolated by FACS from control mice and mice treated with intratracheal instillation of IL-13. B: RT-PCR analysis of P2X₄ and P2X₇ expression in the same cells as those in A reveal that P2X₄ but not P2X₇ expression is upregulated in animals treated with IL-13. C: immunofluorescence staining of airway epithelia from control (top) and IL-13 treated (middle and bottom) animals. Sections were stained for P2X₄ and markers of club cells [open arrows, α-Club Cell Secretory Protein (CCSP)], goblet cells (filled arrows, Muc5ac), and ciliated cells [arrowheads, tubulin β-IV (tubIV)]. P2X₄ receptors are localized to secretory cells with both club (CCSP) and goblet (Muc5ac) phenotypes but not to ciliated cells. Asterisk denotes alveolar type II cell. Scale bars, 15 μm. *P < 0.05, significant difference between groups.

To verify these findings, we also performed additional immunofluorescence staining on tissue sections from control and IL-13-treated animals. Immunofluorescence confirmed the absence of Muc5ac⁺ goblet cells in control animals and revealed expression of P2X₄ in CCSP⁺ club cells (Fig. 6C, top). After IL-13 treatment several of the CCSP⁺ cells were also positive for Muc5ac (Fig. 6C, middle), and CCSP staining was less pronounced (being restricted to the apical region), indicating club-to-goblet cell transition. These goblet cells were positive for P2X₄ receptors. with P2X₄ receptor distribution resembling a vesicular distribution, basolateral from the Muc5ac⁺ granules. In comparison, P2X₄ receptor staining was exclusive of tubulin β-IV staining (Fig. 6C, bottom), a marker for ciliated cells. In keeping with previous findings (44, 65), P2X₄ expression can also be detected in ATII cells. In summary, these data further substantiate that P2X₄ receptors are expressed in secretory cells of the airways and that inflammation-induced mucous metaplasia entails upregulation of P2X₄ expression.

Upregulation and activation of P2X₄ augment intracellular Ca²⁺ signals and mucin secretion.

We previously reported that P2X₄ activation facilitates secretion of pulmonary surfactant in primary ATII epithelial cells (42, 45). Therefore, we reasoned that P2X₄ expression in goblet cells might also be involved in regulating mucin secretion. Mucins are released upon Ca²⁺-dependent exocytosis of large secretory granules, and it has been shown that PM P2X receptor activation stimulates exocytosis directly via Ca²⁺ entry and elevation of [Ca²⁺]_c.

We therefore analyzed the contribution of P2X₄ activation to the generation of intracellular Ca²⁺ signals in individual secretory cells (Fig. 7A) as well as its impact on overall mucin secretion.

Fig. 7.

Upregulation and activation of P2X₄ augments ATP-induced Ca²⁺ signals. A: staining of human primary tracheal epithelial cells (HTECs) with fluo-4 and LysoTracker Deep Red (LTR) to analyze Ca²⁺ signals in secretory cells (dashed line) upon stimulation with ATP. Scale bar, 15 μm. B: fluo-4 traces from individual untreated (top) or IL-13-treated (bottom) secretory HTECs. Cells were grown at air-liquid interphase conditions, treated with respective inhibitors of P2Y₂ (suramin) or P2X₄ [expression of P2X₄(C353W)-mcherry], and stimulated with 100 μM ATP at time = 5 s. a.u., Arbitrary units; dn, dominant negative. C: quantitative analysis of changes in the intracellular Ca²⁺ signal under different conditions. Changes in intracellular Ca²⁺ were analyzed by calculating the integrated increase in fluo-4 fluorescence (area under the curve in B) upon stimulation with 100 μM ATP. Data are from 12–726 individual cells per condition. Statistical significance was assessed by nonparametric tests (Mann-Whitney for comparison of 2 groups or Kruskal-Wallis test for multiple comparisons as indicated by ### and ***, respectively).

Under control conditions, stimulation with ATP resulted in a transient rise in intracellular Ca²⁺ that was almost completely inhibited (>94.7%) by the P2Y₂ receptor inhibitor suramin (Fig. 7, B and C). In IL-13 treated cells, the ATP-induced integrated Ca²⁺ signal was significantly higher than in control cells [21.3 ± 0.52 arbitrary units (n = 726 cells) and 16.7 ± 0.55 arbitrary units (n = 323 cells), respectively]. Moreover, in IL-13-treated cells, suramin treatment resulted in partial inhibition of the intracellular Ca²⁺ signal (54.0%) only. The remaining Ca²⁺ increase likely resulted from activation of P2X₄ receptors. Overexpression of the dn mutant P2X₄(C353W)-EGFP, which dramatically decreases currents of P2X₄ receptor units when coexpressed with wt P2X₄ (61), resulted in significant inhibition (55.4%) of the Ca²⁺ signal. Combined inhibition of P2Y₂ (suramin) and P2X₄ [P2X₄(C353W)-EGFP] receptors almost completely abolished (>93%) the ATP-induced rise in intracellular Ca²⁺ (Fig. 7, B and C).

To assess mucin secretion from HTEC cultures we used a previously developed MUC5AC ELISA assay (2, 73). Initially, baseline secretion was determined for untreated HTECs and HTECs treated with IL-13 for 21 days, and then stimulated secretion was compared to baseline secretion (Fig. 8A). Not surprisingly, in control cultures with almost no expression of mucins (see Fig. 2, B and C), stimulation with 100 μM ATP or 100 μM UTP, known agonists for mucin secretion, resulted in no to weak increase in MUC5AC secretion [1.14 ± 0.17-fold (P = 0.50) and 1.55 ± 0.11-fold (P = 0.03) increase over baseline, respectively; n = 6 for all]. In IL-13-treated cultures, both agonists resulted in significantly increased MUC5AC secretion [1.80 ± 0.18-fold (P < 0.01) and 2.59 ± 0.21-fold (P < 0.001), respectively; n = 8 for all], again confirming that IL-13 induces goblet cell metaplasia and that increased mucin expression leads to increased mucus secretion after stimulation.

Fig. 8.

Upregulation and activation of P2X₄ augment mucin secretion. MUC5AC secretion was assessed by ELISA. A: stimulation of mucin secretion with 100 μM ATP or 100 μM UTP in control (untreated) and IL-13-treated cultures. Agonist treatment resulted in significantly increased MUC5AC secretion in IL-13-treated cells. B: stimulation of mucin secretion with 100 μM ATP in control and IL-13-treated cultures where P2X₄ activity was modulated by overexpression of wild-type (wt) P2X₄-EGFP, the P2X₄ potentiator ivermectin (iverm, 3 μM), the P2X₄ inhibitor 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD, 30 μM), or overexpression of the dominant-negative (dn) mutant P2X₄(C353W)-EGFP. No effects were observed in control cultures, whereas P2X₄ activation resulted in a significant increase and P2X₄ inhibition in a significant decrease of mucin secretion in IL-13-treated cultures. C: stimulation of mucin secretion with 100 μM UTP in control and IL-13-treated cultures where P2X₄ activity was modulated. The effects of modulating P2X₄ activity on mucin secretion in IL-13 treated cultures were absent in cells stimulated with 100 μM UTP, which stimulates mucin secretion but does not activate P2X₄ receptors. Means are derived from 5–12 individual experiments. Statistical significance was assessed by Kruskal-Wallis test for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001.

We next aimed at elucidating the impact of P2X₄ receptor activation on stimulated mucin secretion. Not surprisingly, modulation of P2X₄ activity had no impact on mucin secretion in control cultures after stimulation with either 100 μM ATP (Fig. 8B) or 100 μM UTP (Fig. 8C). However, increasing P2X₄ activity by either overexpression of wt P2X₄-EGFP or treatment with the P2X₄ potentiator ivermectin (3 μM) (34, 42, 65) resulted in significantly increased mucin secretion in IL-13-treated HTECs after stimulation with 100 μM ATP [4.40 ± 1.30-fold (n = 6, P < 0.05) and 4.43 ± 0.79-fold (n = 8, P < 0.01) increase over baseline, respectively] compared with stimulation with 100 μM ATP alone (Fig. 8B). In contrast, inhibition of P2X₄ activity with either 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (30 μM) or overexpression of the dn mutant P2X₄(C353W)-EGFP, which significantly reduced Ca²⁺ signals in these cultures (Fig. 7B), resulted in dampened mucin secretion after stimulation with 100 μM ATP (0.57 ± 0.21-fold and 0.99 ± 0.34-fold increase over baseline, respectively; n = 6 for all) compared with stimulation with 100 μM ATP alone (Fig. 8B). The effects of modulating P2X₄ activity on mucin secretion in IL-13-treated cultures were absent in cells stimulated with 100 μM UTP (Fig. 8C), which stimulates mucin secretion via P2Y₂ receptors but does not activate P2X₄ receptors.

Together, these data support that upregulation and activation of P2X₄ receptors in IL-13-treated cells augment ATP-induced Ca²⁺ signals, modulate the rate of mucin secretion, and result in a rapid and increased mucin secretion response.

DISCUSSION

It is well established that extracellular ATP stimulates mucin release from airway goblet cells via P2Y₂ purinergic receptors (6, 32). Ion-gated P2X purinergic receptors, on the other hand, have so far been neglected in regulation of mucin secretion. With this study we now provide evidence that P2X₄ receptors are expressed in secretory airway epithelial cells, in particular in goblet cells, and activation of P2X₄ receptors augments mucin secretion. In recent years P2X receptors have attracted increasing attention as regulators of exocytosis and cellular secretion (24, 25, 28, 29, 45, 69). Activation of P2X can stimulate granule exocytosis either directly via influx of Ca²⁺ from the extracellular space and elevation of [Ca²⁺]_c (27, 29, 35, 60) or by modulating vesicle content release from fused granules during the so-called exocytic “postfusion” phase (12, 44, 65). From our data we cannot discriminate the exact role of P2X₄ activation in mucin secretion.

Our data suggest that P2X₄ receptors are localized at the PM and in vesicular structures inside the goblet cells that are devoid of MUC5AC and located basolaterally of the apical MUC5AC⁺ mucin granule pool. Our data further indicate that activation of P2X₄ augments the ATP-induced rise in intracellular Ca²⁺ in cells where P2X₄ receptor expression is upregulated upon IL-13 treatment. It is easily conceivable that this results from Ca²⁺ entry via P2X₄ receptors at the PM, which amplifies the Ca²⁺ signal generated by P2Y activation. However, it is also tempting to speculate whether the vesicular P2X₄ receptors contribute to generating a Ca²⁺ signal. This has been reported from various secretory systems where vesicular P2X₄ receptors modulate secretion (18, 42, 44, 57). However, the identity of the P2X₄-expressing organelles in MUC5AC⁺ cells is still elusive and the subject of ongoing studies. P2X₄ receptors are predominantly expressed on intracellular, mainly lysosomal or lysosome-related, organelles (47, 57). The size of the P2X₄⁺ structures (∼1 μm) in goblet cells is much larger than the average size of “classical” lysosomes (100–500 nm) (71), indicating that these vesicles represent a distinct class of organelles. Recently, a VNUT-dependent, ATP-loaded vesicular pool, different from the pool associated with mucin granules, has been reported in Calu-3 cells (59). It is hence possible that the P2X₄⁺ vesicles observed in this study contain the P2X₄ agonist ATP. Alkalinization of these vesicles could therefore lead to P2X₄-dependent Ca²⁺ release facilitating mucus secretion similar to mechanisms recently described in ATII cells (18). Alternatively, it is possible that the P2X₄⁺ organelles observed in our images, although completely devoid of MUC5AC, belong to the mucin granule maturation pathway containing MUC5AC precursors that are not recognized by the 45M1 antibody used or simply constitute a compartment of the recycling pathway to resensitize P2X4 receptors again (17).

The intracellular localization potentially also indicates a different role for P2X₄ receptor activation in addition to amplifying the Ca²⁺ signal for the initial exocytotic response to extracellular ATP. After stimulation of the exocytic response, P2X₄⁺ granules could fuse with the PM, resulting in surface expression of P2X₄ receptors and, in the continued presence of extracellular ATP, also activation of P2X₄ receptors. The relatively high Ca²⁺ permeability of P2X₄ receptors (50) will then result in a substantial rise of subplasmalemmal [Ca²⁺]_c. Such a Ca²⁺ signal could then either induce a sustained exocytotic response in an amplifying feedback mechanism or, alternatively, facilitate release of the macromolecular mucin glycoproteins from fused mucin granules. Similar to ATII cells, the P2X₄ receptor on vesicles may control the kinetics of mucin release during the exocytic postfusion phase. In ATII cells P2X₄-mediated Ca²⁺ signals around fused lamellar bodies induce dilation of the exocytic fusion pore and thereby facilitate secretion of pulmonary surfactant (43–45, 49, 65). Such a model requires sufficiently high levels of extracellular ATP at the time of P2X₄ surface expression. The ATP concentration outside resting cells has been shown to be in the 5–20 nM range (38, 39, 51), well below the EC₅₀ values for P2X₄ activation (50). However, it has been shown that ATP is released from mucin granules (53) as well as via vesicular mechanisms not associated with mucin granule secretion (52). The tight spatio-temporal coupling of mucin granule exocytosis, ATP release, and P2X₄ surface expression could therefore result in sufficiently high ATP concentrations within microdomains around newly inserted P2X₄ receptors on the apical side of goblet cells to activate the P2X₄ receptors. Alternatively, ATP could be released directly from the P2X₄⁺, MUC5AC⁻, granules (59). In both scenarios, P2X₄ activation would be tightly linked and locally restricted to mucin granule exocytosis and provide an ideal feedback loop to induce a “fusion-activated,” local Ca²⁺ signal to impact on the postfusion phase of mucin secretion (42, 65).

Irrespective of the molecular mechanisms linking P2X₄ activation and increased mucin secretion, upregulation of P2X₄ during inflammatory and hypersecretion pathologies may facilitate rapid secretion of MUC5AC into the airway surface liquid (ASL), thereby contributing to mucus plaque formation. Additionally, an inward flux of Na⁺ into the cell may promote water absorption from the ASL as well, furthering this probability (31, 50, 65). Effective mucociliary clearance depends on maintaining a thin (7 μm) periciliary layer lining airway surfaces, which provides a viscous solution for ciliary beating and acts as a lubricant layer for mucus transport (7, 11, 63). ASL volume production reflects a balance between Cl⁻ secretion and Na⁺ absorption. Cl⁻ secretion via cystic fibrosis transmembrane regulator (CFTR), a cAMP-regulated Cl⁻ channel (62), is a major element in regulating ASL levels. CFTR activity is also controlled by the A_2b adenosine receptor, and, under resting conditions, rapid degradation of extracellular ATP results in adenosine levels capable of promoting A_2b receptor activation (64). Therefore, fine control mechanisms are required in the lung to establish local nucleotide/nucleoside concentrations that maintain mucociliary clearance. Under inflammatory conditions, however, ATP release is enhanced (52, 53), which, in combination with increased P2X₄ expression, could result in increased Na⁺ currents via P2X₄ receptors, counteracting fluid secretion and leading to decreases in ASL, mucus hypohydration, and mucus plaque formation—prominent features of chronic lung diseases including asthma, COPD, and CF (58, 67).

In summary, our data provide strong evidence that P2X₄ receptors are predominantly expressed in secretory cells of the airways. Furthermore, P2X₄ expression is upregulated in those cells under inflammatory conditions, and activation of P2X₄ receptors augments mucin secretion and potentially contributes to mucus hypersecretion and mucus plaque formation. Although the molecular mechanisms by which P2X₄ activation leads to increased mucin secretion are still elusive and currently under investigation, P2X₄ could constitute a potential pharmaceutical target to attenuate mucus hypersecretion in patients with mucous metaplasia.

GRANTS

This work was supported by a grant from the Ministry of Science, Research and the Arts of Baden-Württemberg, Grant Az: 32-7533.-6-10/15/5 (to M. Frick) and by the Deutsche Forschungsgemeinschaft Grants D-1402/3-1 (to P. Dietl) and SFB1149/1 (A05) (to M. Frick).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

V.E.W., K.E.T., B.F.D., and M.F. conceived and designed study; V.E.W., K.E.T., K.N., A.-M.J., G.F., H.S., W.H., M.J.T., and M.F. performed experiments; V.E.W., K.E.T., K.N., G.F., and M.F. analyzed data; V.E.W., K.E.T., G.F., O.H.W., B.F.D., and M.F. interpreted results of experiments; V.E.W., K.E.T., and M.F. prepared figures; V.E.W., K.E.T., P.D., and M.F. drafted manuscript; V.E.W., K.E.T., O.H.W., M.J.T., B.F.D., P.D., and M.F. edited and revised manuscript; V.E.W., G.F., B.F.D., and M.F. approved final version of manuscript.