The airway epithelial cells (AECs) lining the conducting passageways of the lung secrete a variety of immunomodulatory factors. Among these, PGE2 limits lung inflammation and promotes bronchodilation. By contrast, IL-6 drives intense airway inflammation, remodeling, and fibrosis. The signaling that differentiates the production of these opposing mediators is not understood. In this study, we find that the production of PGE2 and IL-6 following stimulation of human AECs by the damage-associated molecular pattern extracellular ATP shares a common requirement for Ca2+ release-activated Ca2+ (CRAC) channels. ATP-mediated synthesis of PGE2 required activation of metabotropic P2Y2 receptors and CRAC channel-mediated cytosolic phospholipase A2 signaling. By contrast, ATP-evoked synthesis of IL-6 occurred via activation of ionotropic P2X receptors and CRAC channel–mediated calcineurin/NFAT signaling. In contrast to ATP, which elicited the production of both PGE2 and IL-6, the uridine nucleotide, UTP, stimulated PGE2 but not IL-6 production. These results reveal that human AECs employ unique receptor-specific signaling mechanisms with CRAC channels as a signaling nexus to regulate release of opposing immunomodulatory mediators. Collectively, our results identify P2Y2 receptors, CRAC channels, and P2X receptors as potential intervention targets for airway diseases.
The airway epithelial cells (AECs) lining the conducting airways of the lung form the first line of defense against a variety of inhaled pathogens, allergens, and environmental irritants. AECs not only provide a physical barrier, but also actively orchestrate immune responses to inhaled substances through the production of a wide array of secreted factors that include alarmins, chemokines, cytokines, and eicosanoids (1). Among these alarmins, extracellular ATP, a damage-associated molecular pattern, is of growing interest due to its ability to drive airway inflammation (2–4). Elevated ATP in the bronchoalveolar lavage (BAL) fluid stimulates cytokine release, activates the inflammasome, and recruits immune cells (2–5). Moreover, elevated ATP in the BAL fluid is a key characteristic of many lung disorders, including asthma, acute respiratory distress syndrome (ARDS), and chronic obstructive pulmonary disease (COPD) (2, 6–9). In contrast, ATP signaling also plays important roles in physiological processes in the healthy lung related to wound healing, rejection of tumors, killing of bacterial pathogens, and mucociliary clearance (MCC) (3, 10–14). This latter response is also evoked by the uridine nucleotide, UTP, which is released in the lung airways and can stimulate numerous physiologically important airway functions, including MCC, ion transport, and mucin secretion (15). How these nucleotides elicit such broad effects on airway function is not well understood.
Two of the most prominent mediators produced by AECs are PGE2 and IL-6. PGE2 is synthesized through an enzymatic cascade tied to the activities of phospholipase A2, cyclooxygenase 1 (COX-1) and COX-2 enzymes, and a terminal PGE synthase (16). PGE2 stimulates dilation of the lung airways, thereby protecting the lung against severe bronchoconstriction that is a hallmark of diseases such as asthma (17, 18). PGE2 inhibits several inflammatory processes, including the release of histamine and cysteinyl leukotrienes from mast cells (17), T cell migration (19), ILC2 function (20), and the production of proinflammatory TNF-α and IL-12β by dendritic cells (21), while also enhancing the synthesis of the anti-inflammatory cytokine IL-10 (21) and promoting wound healing (22). Further, growing evidence suggests that PGE2 can drive reverse migration and removal of neutrophils from sites of inflammation to dissipate inflammation in vivo (23). Failure of resolution of inflammation can lead to inflammatory diseases such as COPD and ARDS. Thus, within the lung, PGE2 exerts predominantly bronchoprotective and anti-inflammatory effects (24).
By contrast, IL-6 is a potent inflammatory cytokine that elicits a wide range of inflammatory effects, including T cell expansion, pulmonary neutrophil infiltration, airway mucus secretion, lung fibrosis, and the hyperplasia and hypertrophy of airway smooth muscle cells (25–28). Further, IL-6 is linked to numerous airway diseases from asthma to COPD (25–28), and recent studies in patients with severe coronavirus disease 2019 (COVID-19) have revealed massive increases in IL-6 in the lung airways, a feature thought to contribute to microvascular thrombosis in the lung and multiple organ dysfunction in these patients (29). Conversely, blockade of IL-6 signaling in mouse models of asthma protects against airway inflammation (26, 30), and early reports indicate that targeting IL-6 may be a viable therapy to decrease mortality in patients with severe COVID-19 (31).
The mechanisms by which ATP and UTP signaling regulates the synthesis of these distinct mediators (PGE2 versus IL-6) in AECs are largely unclear. There is compelling evidence, however, that cytosolic Ca2+ elevations mediated by the opening of store-operated Ca2+ release-activated Ca2+ (CRAC) channels formed by the ORAI1 protein regulates both factors (32, 33). As a multifunctional second messenger, Ca2+ activates distinct genetic programs to regulate many cellular functions, including gene transcription, cytokine production, and activation of numerous enzymes (34). Although the necessity for Ca2+ elevations is suggestive of a unified mechanism driving the synthesis of both mediators, there are many unanswered questions. Are the upstream receptors driving ATP-mediated synthesis of PGE2 and IL-6 identical? What are the signaling mechanisms downstream of receptor activation in their synthesis? And what is the precise role of CRAC channels in the synthesis of these mediators? In this study, we addressed these and other questions in primary human AECs. We find that ATP induction of PGE2 and IL-6 requires different ATP concentrations and involves distinct upstream receptors (P2Y versus P2X receptors). In contrast to ATP, extracellular UTP acting via P2Y2 receptors exclusively stimulated PGE2 synthesis without concurrent induction of IL-6. Synthesis of PGE2 involved activation of cytosolic phospholipase A2 (cPLA2) by local Ca2+ signals near CRAC channels and activity of COX-2, whereas IL-6 induction required activation of calcineurin/NFAT signaling by CRAC channels. These findings reveal the key signaling checkpoints that mediate the synthesis of distinct immunomodulatory products from the same ligand but working through distinct upstream receptors and involving Ca2+ signals through CRAC channels as the final common pathway.
Materials and Methods
Cells, media, and solutions
Normal human bronchial epithelial (NHBE) cells were purchased from Lonza (catalog number CC-2540) and grown in bronchial epithelial growth medium (BEGM; CC-3170). All experiments that used media for the stimulation phase used Lonza bronchial epithelial basal medium (BEBM) supplemented with Ca2+ to bring the total concentration up to 2 mM. Cells were grown in 37°C and 5% CO2. Ringer solutions were as follows: 2 mM Ca2+ Ringer solution: 155 mM NaCl, 4.5 mM KCl, 10 mM d-glucose, 5 mM HEPES, 2 mM CaCl2, and 1 mM MgCl2; and 0 mM Ca2+ Ringer solution: 155 mM NaCl, 4.5 mM KCl, 10 mM d-glucose, 5 mM HEPES, 1 mM EGTA, and 3 mM MgCl2.
Abs and pharmacological tools
Primary Abs and their sources were as follows: COX-2 (12282; Cell Signaling Technology), β-actin (3700; Cell Signaling Technology), phospho-ERK (4370; Cell Signaling Technology), total ERK (9102; Cell Signaling Technology), cPLA2 (2832; Cell Signaling Technology), STIM1 (3917; Feske Lab), and α-tubulin (ab52866; Abcam). Pharmacological tools used in the study were: UTP (U6875; Sigma-Aldrich), SLIGKV-NH2 (4153; Tocris Bioscience), FK-506 (3631; Tocris Bioscience), BTP2 (203890; Sigma-Aldrich), ATP (A6419; Sigma-Aldrich), diclofenac (4454; Tocris Bioscience), apocynin (4663; Tocris Bioscience), NAC (106425; Sigma-Aldrich), U0126 (1144; Tocris Bioscience), ATPγS (4080; Tocris Bioscience), AACOCF3 (1462; Tocris Bioscience), AR-C 118925XX (4890; Tocris), NF546 (3892; Tocris Bioscience), S3QEL 2 (5735; Tocris Bioscience), ADPβS (A8016; Sigma-Aldrich), UDP (U4125; Sigma-Aldrich), NF157 (2450; Tocris Bioscience), Apyrase (A6410 Grade VI, High ATPase/ADPase activity; Sigma-Aldrich), TNP-ATP (2464; Tocris), 5-BDBD (3579; Tocris), A740003 (3701; Tocris Bioscience), Suramin (AC328540500; Acros Organics-Fisher), and PPADS (0625; Tocris Bioscience). CM4620 was a kind gift from CalciMedica.
On day 0, NHBE cells were plated onto 24-well plates in the morning in BEGM lacking antibiotics. Six to 8 h later, the cells were transfected using Lipofectamine RNAiMAX reagent and small interfering RNA (siRNA) of interest at final concentrations of 10 nM. On day 1, cells were given fresh BEGM lacking antibiotics and retransfected using identical conditions as day 0. On day 2, cells were given fresh BEGM lacking antibiotics and lacking hydrocortisone and retransfected using identical conditions as day 0. On day 3 (72 h after initial transfection), cells were either collected for analysis of protein levels or mRNA levels or stimulated with ATP for 2 h for PGE2 analysis according to the standard PGE2 stimulation protocol (see PGE2 measurements section). siRNAs used included “siRNA Universal Negative Control #1” SICOO1 (Sigma-Aldrich), hereafter termed “siCon,” siSTIM1: SASI_Hs01_00107803 (Sigma-Aldrich), and siORAI1: 4392420, assay ID s228396 (Thermo Fisher Scientific). For short hairpin RNA (shRNA) experiments, cells were infected with multiplicity of infection of 10. Two days later, puromycin was added to the culture media at a final dose of 4 μg/ml, and selection was allowed to occur for 3 d. Nontransduced cells were always handled in parallel to confirm puromycin’s ability to induce selection. Cells were then plated for experiments and maintained in 1 μg/ml puromycin until the time of stimulation when puromycin was removed from the culture media, and cells were stimulated in BEBM without growth factors. Lentiviral particles expressing shRNA against P2RX4 were purchased from Sigma-Aldrich with the clone identification number TRCN0000044962.
Intracellular Ca2+ measurements
NHBE cells were grown on poly-l-lysine–coated glass-bottom dishes purchased from MatTek. Cells were loaded with 2 μM Fura-2-AM (F1221; Thermo Fisher Scientific) in BEGM with 5% FBS added to increase loading. Cells were loaded for 40 min at room temperature in the dark. Cells were washed three times with 2 mM Ca2+ Ringer solution and then incubated for an additional 15 min in the dark before initiating Ca2+ imaging. Experiments were performed at room temperature. Dishes were mounted on the stage of an Olympus IX71 inverted microscope. Images were acquired every 6 s at excitation wavelengths of 340 nm and 380 nm and an emission wavelength of 510 nm. Image acquisition and analysis were performed using SlideBook software. For data analysis, regions of interest were drawn around individual cells, background fluorescence was subtracted, and the F340/F380 ratios were calculated for each time point. An increase in the ratio of F340/F380 indicates a rise in intracellular Ca2+ concentration ([Ca2+]i). The F340/F380 ratios were then converted to an estimate of [Ca2+]i through the equation: [Ca2+]i = β × Kd × (R − Rmin)/(Rmax − R), where R is the F340/F380 ratio and the values of β, Rmin, and Rmax were determined from an in vitro calibration with Fura-2 pentapotassium salt. β is determined from the Fmin/Fmax ratio at 380 nm, and Kd is the dissociation constant of Fura-2 binding to Ca2+ (135 nM). The determined values were β = 23.152, Rmin = 0.2092, and Rmax = 6.954.
COX-2 and ERK1/2 activation assays
On day 0, NHBE cells were plated onto 24-well plates. On day 1, BEGM was replaced with BEGM lacking hydrocortisone. On day 2, cells were stimulated. If cells were pretreated with drugs, half of the BEGM lacking hydrocortisone was taken off the cells, the drug was added to that media at 2× final concentration, vortexed, and added back to the relevant wells. Cells were activated for either 15 min (for ERK1/2 measurements) or 2 h to overnight (for COX-2 upregulation measurements) as indicated in the figure legends. Following stimulation, cells were lysed using 1× Cell Signaling Lysis Buffer (9803S) containing protease and phosphatase inhibitors (PPIs) (78440; Thermo Fisher Scientific). Cell lysates were incubated on ice for 30 min and vortexed every 10 min. Samples were spun at 12,000 rpm for 15 min at 4°C, and supernatants were collected and analyzed via Western blotting.
Lysates were boiled for 5 min in 1× Laemmli Sample Buffer (1610747; Bio-Rad) containing 2-ME. Samples were then subjected to SDS-PAGE. Transfer occurred at 4°C for 1.5 h and at 100 V. Polyvinylidene difluoride membranes were used for the transfer. Following transfer, membranes were washed in TBST (0.1% Tween 20), blocked for 1 h at room temperature using 5% BSA dissolved in TBST, and then incubated overnight at 4°C with primary Abs. Dilutions for primary Abs were as follows: anti-STIM1: 1:1000; anti–COX-2: 1:1000; anti–phospho-ERK1/2: 1:2000; anti-total ERK1/2: 1:1000; anti–β-actin: 1:2000; anti-cPLA2: 1:1000; anti-histone H3: 1:1000; and anti–α-tubulin: 1:5000. The following day, membranes were washed three times for 5 min each using TBST and incubated with secondary Abs for 1 h at room temperature in 5% BSA dissolved in TBST. Li-Cor secondary Abs were used (IRDye 800CW or IRDye 680RD) at dilutions of 1:10,000. Membranes were washed three times for 5 min each using TBST and immediately imaged using an Odyssey CLx imaging system.
Nuclear and cytosolic fractionation assays
On day 0, NHBE cells were plated onto six-well plates. On day 1, BEGM was replaced with BEGM lacking hydrocortisone. On day 2, cells were stimulated. If cells were pretreated with drugs, half of the BEGM lacking hydrocortisone was taken off the cells, and the drug was added to that media at 2× final concentration, vortexed, and added back to the relevant wells. This protocol was adapted from Chang et al. (35). Cells were stimulated for 5 min in BEBM lacking all growth factors. Media was immediately aspirated, cells were placed on ice, and cells were scrapped in hypotonic lysis buffer containing PPIs (78440; Thermo Fisher Scientific) and collected into prechilled Eppendorf tubes. Lysates from two wells were combined into one tube to ensure sufficient protein content. Samples were incubated on ice for 10 min. Samples were then spun at 1000 rpm for 4 min at 4°C. The supernatant was collected as the crude cytosolic fraction, and the pellet was resuspended in hypertonic lysis buffer containing PPIs. Both the crude cytosolic fractions and the resuspended pellet were incubated on ice for another 30 min and periodically vortexed every 10 min to ensure full lysis. Next, both crude cytosolic fraction samples and the resuspended pellet samples were centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatants were collected and termed “cytosolic fraction” and “nuclear fraction.” The hypotonic buffer consisted of: 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.5 mM EDTA, and PPIs added fresh. The hypertonic buffer consisted of: 20 mM HEPES (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, and PPIs added fresh.
On day 0, NHBE cells were plated onto 24-well plates. On day 1, BEGM was replaced with BEGM lacking hydrocortisone. On day 2, cells were stimulated. If cells were pretreated with drugs, half of the BEGM lacking hydrocortisone was taken off the cells, and the drug was added to that media at 2× final concentration, vortexed, and added back to the relevant wells. At the time of stimulation, media was removed, and cells were stimulated for 2 h in BEBM lacking all growth factors. Following this stimulation phase, the supernatants were collected, spun at 300 × g for 4 min to remove cellular debris, and the supernatant from this sample was then collected and stored at −80°C until the time of analysis. To perform analysis of PGE2 levels, the Cayman Chemical kit (514010) was used, and the manufacturer’s protocols were followed. All samples were diluted at least 10-fold in ELISA buffer prior to analysis. All samples that were compared statistically had the same concentration of organic solvents (such as DMSO and ethanol) as organic solvents can interfere with the assay.
On day 0, NHBE cells were plated onto 24-well plates. On day 1, BEGM was replaced with BEGM lacking hydrocortisone. On day 2, cells were stimulated. If cells were pretreated with drugs, half of the BEGM lacking hydrocortisone was taken off the cells, and the drug was added to that media at 2× final concentration, vortexed, and added back to the relevant wells. At the time of stimulation, media was removed, and cells were stimulated overnight (16 h) in BEBM lacking all growth factors. Following this stimulation phase, the supernatants were collected, spun at 300 × g for 4 min to remove cellular debris, and the supernatant from this sample was then collected and stored at −80°C until the time of analysis. To perform analysis of IL-6 levels, the RayBiotech Human IL-6 ELISA kit (ELH-IL6-1) was used, and the manufacturer’s protocols were followed. All samples were diluted typically 5-fold in ELISA buffer prior to analysis. Samples that were compared statistically had the same concentration of organic solvents (DMSO).
Quantitative RT-PCR analysis
Total RNA was extraction was performed using RNeasy Plus Mini Kit (74134; Qiagen). cDNA generation was performed using iScript Reverse Transcription Supermix for RT-qPCR (1708841; BioRad). Quantitative PCR (qPCR) was performed using PowerUp SYBR Green Master Mix (A25741). For qPCR, the final concentration of primers was 500 nM, and cDNA was used at 6 ng/well. Primer sequences were as follows: ORAI1 forward, 5′-GATGAGCCTCAACGAGCACT-3′, ORAI1 reverse, 5′-ATTGCCACCAT-GGCGAAGC-3′; P2RY2 forward, 5′-CCGCTTCAACGAGGACTTCAA-3′, P2RY2 reverse, 5′-GCGGGCGTAGTAATAGACCA-3′; P2RX4 forward, 5′-CTACCAGGAAACTGACTCCGT-3′, P2RX4 reverse, 5′-GGTATCACATAATCCGCCACAT-3′; HPRT1 forward, 5′-ACCCTTTCCAAATCCTCAGC-3′, HPRT1 reverse, 5′-GTTATGGCGACCCGCAG-3′; RPLP0 forward, 5′-AGCCCAGAACACTGGTCTC-3′, RPLP0 reverse, 5′-ACTCAGGATTTCAATGGTGCC-3′; PPIA forward, 5′-CCCACCGTGTTC-TTCGACATT-3′, PPIA reverse, 5′-GGACCCGTATGCTTTAGGATGA-3′; P2RY11 forward, 5′-GTAGCAGACACAGGCTGA-3′, P2RY11 reverse, 5′-CCTGGAACCCACTGAGTTTG-3′; P2RX1 forward, 5′-CGTTATCTTCCGACTGATCCAG-3′, P2RX1 reverse, 5′-CACAGAGACACTGCTGATGAG-3′; P2RX2 forward, 5′-GAGGTGTTCGGCTGGTG-3′, P2RX2 reverse, 5′-GGTAGTGGATGCTGTTCTTGA-3′; P2RX3 forward, 5′-CAACATCATC-CCCACCATCA-3′, P2RX3 reverse, 5′-CTCATTCACCTCCTCAAACTTCT-3′; P2RX5 forward, 5′-GGAAGCAGCAGTCAGAAGG-3′, P2RX5 reverse, 5′-AAAGGCATGGGATCACTGG-3′; P2RX6 forward, 5′-TGGCCTCACTACTCCTTCC-3′, P2RX6 reverse, 5′-ATGTCGAAGCGGATTCCATAG-3′; and P2RX7 forward, 5′-CCCTGTGTGTGGTCAACGAAT-3′, P2RX7 reverse, 5′-TGCAGACTTCTCCCTAGTAGC-3′.
All bar graphs summarizing data are represented as mean ± SEM. Individual points are always indicative of biological replicates. For data sets involving more than two groups, an initial one-way ANOVA was performed followed by the Tukey multiple-comparison test. For data sets with only two groups, a two-tailed unpaired Student t test was performed. Dose-response curves were created using a four parameter (variable slope) nonlinear regression. Extra sum-of-squares F test was used to statistically compare the PGE2 and IL-6 dose-response curves. Statistical analysis and data analysis were performed using Prism 8 (GraphPad Software). When statistical comparisons are made: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Data and materials availability
All data needed to evaluate the conclusions in the study are present in the article or Supplemental Figs. 1, 2, 3 and 4. CM4620 was obtained via a material transfer agreement from CalciMedica.
P2Y2 receptors mediate ATP- and UTP-induced Ca2+ elevations in human AECs
ATP acts on two broad classes of membrane receptors: G-protein–coupled P2Y receptors and ionotropic P2X receptors (36, 37). Previous studies have shown that AECs express the Gq/phospholipase C–coupled P2Y2 and P2Y6 receptors as well as several classes of P2X receptors (38, 39). Because both P2X and P2Y receptors can initiate Ca2+ signaling, we first investigated the identity of the purinergic receptors contributing to Ca2+ signals evoked by extracellular ATP in primary NHBE cells using the ratiometric dye, Fura-2, as previously described (32, 33). These experiments revealed that administration of a saturating dose of ATP (100 µM) evoked a biphasic elevation in [Ca2+]i consisting of an initial rapid phase of [Ca2+]i elevation followed by a sustained phase of [Ca2+]i elevation (Fig. 1A). Removing extracellular Ca2+ resulted in the selective loss of the sustained [Ca2+]i rise with virtually no effect on the initial [Ca2+]i signal (Fig. 1B), indicating that the early, transient increase of [Ca2+]i results from release of Ca2+ from intracellular stores, whereas the sustained late phase requires Ca2+ entry across the plasma membrane. This kinetic signature of the ATP-induced Ca2+ response is consistent with nucleotide-evoked Gq/phospholipase C–inositol 1,4,5-triphosphate signaling and store-operated calcium entry (SOCE) that we and others have previously described in AECs and other cell types (32, 40–42). In agreement with this interpretation, UTP, a uridine nucleotide ligand that is selective for the metabotropic P2Y purinergic receptors (P2Y2 and P2Y4), also induced Ca2+ elevations with amplitude and kinetics similar to Ca2+ elevations evoked by ATP (Fig. 1C). These results indicate that the P2Y receptors are important mediators of the ATP-mediated [Ca2+]i response.
Among the various P2Y receptors, P2Y2 is highly expressed by AECs and shares a similar potency and efficacy for ATP and UTP (39, 43). To examine the role of P2Y2 in mediating the [Ca2+]i rises, we applied the selective P2Y2 receptor antagonist AR-C 118925XX (44) (henceforth referred to as “AR-C”). Pretreatment with AR-C abolished the ATP and UTP-mediated Ca2+ signals (Fig. 1D–F). By contrast, the Ca2+ elevation elicited by activation of PAR2, an unrelated Gq receptor, which also evokes SOCE (32), was unaffected (Fig. 1D, 1E). These results indicate that the nucleotide triphosphate agonists ATP and UTP stimulate Ca2+ signaling in human bronchial epithelial cells via activation of Gq-coupled P2Y2 receptors, reminiscent of SOCE activated by ATP and UTP in astrocytes (40).
CRAC channels mediate the sustained P2Y2 receptor–evoked Ca2+ response
The finding that P2Y2 receptor activation by ATP and UTP elicits a sustained phase of Ca2+ entry (Fig. 1A) led to us to next consider whether this secondary phase requires the activation of CRAC channels. Previous work has demonstrated ORAI1 and STIM1, components of CRAC channels, are required for PAR2-mediated SOCE in AECs (32, 33, 41). To address the molecular identity of the channels responsible for SOCE following P2Y2 receptor activation, we examined the consequences of siRNA-mediated knockdown of ORAI1 and STIM1 on ATP-mediated Ca2+ rises. The effectiveness of knockdown was assessed to be >90% based on Western blots for STIM1 and by qPCR for ORAI1 (Supplemental Fig. 1A, 1B). Knockdown of either ORAI1 or STIM1 significantly decreased the sustained Ca2+ entry phase but did not substantively affect the initial Ca2+ release from intracellular stores (Fig. 2A, 2B). Moreover, the sustained phase of the Ca2+ rise was abolished by the CRAC channel inhibitors, BTP2 and CM4620 (Fig. 2C, 2D) (45–49). Ca2+ add-back experiments following depletion of endoplasmic reticulum Ca2+ stores in Ca2+-free Ringer solution revealed robust SOCE in NHBE cells, which was completely abolished by the ORAI1 inhibitor CM4620 (Supplemental Fig. 1C, 1D). Together, these results, summarized in (Fig. 2E, indicate that CRAC channels composed of ORAI1 and STIM1 are essential for SOCE that is activated by stimulation of P2Y2 receptors in bronchial epithelial cells.
P2Y2 receptor activation evokes PGE2 synthesis
As described above, a major mediator produced by AECs that limits the immune–inflammatory response and promotes resolution of inflammation is PGE2 (24). PGE2 induction is stimulated by ATP (50), but the purinergic receptors mediating this effect are unclear. We therefore sought to identify the ATP receptors driving synthesis of PGE2. Administration of ATP resulted in rapid synthesis of PGE2 (Fig. 3A), and analysis of the dose-response relationship indicated an EC50 of ∼7 μM (Fig. 3B). Likewise, UTP, which stimulates P2Y2 and P2Y4 receptors, also induced robust and rapid PGE2 synthesis within 2 h (Fig. 3C). The agonist efficacies of ATP and UTP were similar (Fig. 3C), suggesting that both ligands act on a common membrane receptor. Consistent with this interpretation, pretreatment with the selective P2Y2 receptor antagonist AR-C completely blocked the PGE2 induction by both ATP and UTP (Fig. 3C). Further, siRNA-mediated knockdown of P2RY2 mRNA (Supplemental Fig. 2A) resulted in complete abrogation of the ATP-induced PGE2 response, confirming the involvement of P2Y2 receptors (Fig. 3D). AR-C and knockdown of P2RY2 also inhibited basal PGE2 synthesis (Fig. 3E, 3F), indicating that activation of P2Y2 receptors by AEC-derived nucleotides drives the constitutive PGE2 synthesis in resting cells. Collectively, these results demonstrate that nucleotide-driven PGE2 synthesis in AECs occurs via activation of P2Y2 receptors, implicating epithelial P2Y2 receptors in the beneficial effects of PGE2 in the lung airways.
P2X receptors drive IL-6 release
As noted above, in addition to its ability to stimulate PGE2 synthesis, extracellular ATP is also linked to the production of the proinflammatory cytokine IL-6 (51). To begin to understand how signaling underlying induction of IL-6 contrasts with that of PGE2, we examined the pathways regulating ATP-mediated induction of IL-6. Administration of ATP to AECs induced IL-6 secretion, albeit with slower kinetics than PGE2 (compare (Figs. 4A and (3A). Moreover, ATP-mediated synthesis of IL-6 occurred with an EC50 of ∼16 μM (Fig. 4B), ∼2-fold higher than the EC50 required for PGE2 induction (∼7 μM), raising the possibility that divergent signaling mechanisms are involved in ATP-mediated PGE2 and IL-6 induction. ATPγS, an ATP variant that is resistant to degradation by ecto-ATPases, also significantly induced IL-6 ruling out ATP metabolites such as ADP or adenosine in inducing the IL-6 response (Fig. 4C). Additionally, preincubating ATP with apyrase, an enzyme that degrades ATP, abolished IL-6 induction, indicating ATP is necessary for the IL-6 release (Fig. 4D). Together, these results indicate that ATP-induced IL-6 production occurs at higher ligand doses and requires longer periods of cell stimulation than the synthesis of PGE2.
Several lines of evidence indicated that ATP-mediated induction of IL-6 is not mediated by activation of P2Y receptors. First, unlike the phenotype for PGE2 synthesis, ATP-evoked IL-6 secretion was not affected by the P2Y2 antagonist AR-C (Fig. 4E). Second, UTP, which acts on P2Y2 and P2Y4 receptors, failed to induce IL-6 (Fig. 4E). This is in striking contrast with UTP’s ability to strongly induce PGE2 (Fig. 3C). Third, ADPβS, a ligand for P2Y1, P2Y12, and P2Y13 receptors, also had no effect (Supplemental Fig. 2B). Fourth, UDP and NF546 (agonists for P2Y6, P2Y11, and P2Y14 receptors) were also unable to drive IL-6 secretion (Supplemental Fig. 2C, 2D). Puzzlingly, NF157, a P2Y11 receptor antagonist, significantly enhanced the potency of ATPγS-induced IL-6 synthesis (Supplemental Fig. 2E). We did not further investigate the mechanism of this synergism but continued our focus on the receptor subtype that mediates ATP-driven IL-6 induction. Collectively, these results indicate that in contrast to PGE2 synthesis, ATP-driven IL-6 synthesis is not mediated by P2Y2 receptors.
By contrast, the broad-spectrum P2X antagonists suramin and PPADS (36) both blocked ATPγS-mediated IL-6 production (Fig. 4F). mRNA analysis by real-time PCR revealed significant expression of the P2X receptor subtype transcripts P2X4, P2X5, and P2X7, which agrees well with previous reports (52, 53) (Supplemental Fig. 2F). In an attempt to further narrow down the receptor subtypes involved, we next examined the effects of low doses of TNP-ATP, which inhibits P2X1, P2X3, and P2X2/3 heteromeric receptors, on IL-6 production (36). However, TNP-ATP had no effect on IL-6 production (Fig. 4G). Likewise, the selective P2X4 and P2X7 receptor antagonists 5-BDBD and A740003 also did not inhibit ATPγS-induced IL-6 induction (Fig. 4H, 4I). shRNA-mediated knockdown of P2RX4 also failed to inhibit of IL-6 production (Supplemental Fig. 2G, 2H) consistent with the above pharmacological results. Endogenous P2X receptors are known for their ability to oligomerize to form channels with biophysical and pharmacological properties that are distinct from homomeric P2X receptor subunits (36). For example, a previous characterization of the biophysical properties of P2X receptors in AECs has concluded that P2X channels have unique features potentially indicative of heteromers of P2X4/P2X7 subunits (54). Thus, although our tests did not implicate specific P2X receptor subtypes, the failure of P2Y receptor agonists and antagonists to affect IL-6 production, the strong expression of P2X receptor subtypes in AECs, and the ability of the broad-spectrum P2X receptor inhibitors suramin and PPADS to block IL-6 production implicates P2X receptors in driving ATP-mediated IL-6 production.
CRAC channels are essential for both PGE2 synthesis and IL-6 secretion
The results presented thus far indicate that ATP stimulates the production of bronchoprotective PGE2 and proinflammatory IL-6 through distinct membrane receptors. Consistent with previous evidence indicating that PGE2 synthesis is a Ca2+-regulated process (50), siRNA-mediated knockdown of ORAI1 or STIM1 abrogated the ATP-evoked PGE2 synthesis (Fig. 5A). Likewise, inhibiting SOCE with the CRAC channel inhibitors CM4620 and BTP2 effectively blocked both ATP- and UTP-induced PGE2 synthesis (Fig. 5B, 5C). These results indicate that CRAC channels are essential for ATP-evoked PGE2 synthesis. Similarly, pharmacological inhibition of CRAC channels with BTP2 or CM4620 inhibited ATPγS-evoked IL-6 induction (Fig. 5D). CM4620 has recently been shown to improve outcomes in hospitalized patients with severe COVID-19 infections (55). Thus, the ability of CM4620 to block IL-6 induction suggests that CRAC channel blockade may alleviate IL-6–mediated inflammation in the lung. Taken together, these results indicate that activation of CRAC channels is essential for both ATP-mediated PGE2 and IL-6 production in AECs.
To further understand the mechanism by which CRAC channels regulate PGE2 production, we examined the role of local Ca2+ signaling by using Ca2+ buffers EGTA and BAPTA. Local Ca2+ signals in the vicinity of CRAC channels are implicated in many downstream signaling events, including the synthesis of the arachidonic acid metabolite leukotriene C4 in mast cells (56), calcineurin/NFAT activation in neural stem cells (57), and gliotransmitter release in astrocytes (40). We found that BAPTA, which binds Ca2+ with rapid kinetics, abolished the P2Y2 receptor–mediated increases in PGE2 synthesis, but EGTA, a Ca2+ chelator with comparable Ca2+ affinity to BAPTA but with 100-fold slower binding kinetics (58), was far less effective ((Fig. 5E). BAPTA also inhibited basal PGE2 synthesis, whereas EGTA had no effect (Fig. 5F). Attempts to examine EGTA/BAPTA regulation of IL-6 production were unsuccessful due to high cell death in buffer-loaded cells over the 16-h measurement period for IL-6. Collectively, these results indicate that CRAC channels are spatially coupled to the PGE2 synthesis machinery (likely cPLA2, see below), analogous to the functional coupling described in other cells between CRAC channels and calcineurin/NFAT signaling and gliotransmitter vesicles (40, 57).
P2Y2 receptor–mediated PGE2 synthesis requires COX-2 activity but not COX-2 induction
To further determine how P2Y2–CRAC channel signaling stimulates PGE2 synthesis, we next examined signaling downstream of P2Y2 receptor activation. Previous work in the alveolar type II cancer cell line A549 has indicated that extracellular ATP and UTP activate and increase the expression of the inducible COX isoform COX-2 (59). However, whether primary human bronchial epithelial cells share the same mechanism is unknown. We found that low doses of FR122047 and celecoxib, which are selective inhibitors of the COX-1 and COX-2 enzymes, respectively, abolished ATP-mediated PGE2 synthesis (Supplemental Fig. 3A), indicating that COX enzyme activity is essential for ATP-mediated PGE2 synthesis. However, examination of COX-2 protein levels revealed only a very modest increase in COX-2 protein levels following ATP stimulation at 2 h, which returned to baseline levels by 6 h (Supplemental Fig. 3B). This slight increase in COX-2 expression was inhibited with the P2Y2 receptor antagonist AR-C (Supplemental Fig. 3C, 3D), but was not affected by the CRAC channel inhibitors CM4620 and BTP2 (Supplemental Fig. 3E). Thus, P2Y2 receptor–CRAC channel–mediated synthesis of PGE2 requires COX-2 activity but does not substantively involve increased expression of COX-2 protein.
ERK1/2 activation is essential for both PGE2 synthesis and IL-6 secretion
A key molecule implicated in the induction of PGE2 by growth factors and cytokines is ERK1/2 (60, 61). In this signaling, ERK1/2 phosphorylates the enzyme cPLA2, and this step has been shown to be essential for the synthesis of PGE2 by epidermal growth factor, IL-1β, and PAR2 ligands (60, 62). ERK1/2 activation is regulated by its own phosphorylation by the kinase MEK1/2 (63). Thus, to examine the necessity of ERK1/2 involvement for ATP-induced PGE2 synthesis, we probed the phosphorylation status of ERK1/2 following ATP administration. These experiments showed that extracellular ATP rapidly induced ERK1/2 activation within 15 min (Fig. 6A, 6B). The P2Y2 antagonist AR-C completely blocked this step (Fig. 6A, 6B). Consistent with a requirement for MEK1/2-mediated phosphorylation, the selective MEK1/2 inhibitor U0126 (20 μM) abrogated ERK1/2 activation (Supplemental Fig. 3F). Importantly, ATP- and UTP-induced PGE2 synthesis was strongly inhibited by MEK1/2 inhibition (Fig. 6C, 6D), reaffirming the importance of ERK1/2 activation for P2Y2-induced rapid PGE2 synthesis. Further, MEK1/2 inhibition did not affect the modest upregulation of COX-2 expression (Fig. 6E, 6F), suggesting that COX-2 upregulation is not a major contributor to ATP-induced PGE2 synthesis. Interestingly, the CRAC channel inhibitors BTP2 or CM4620 failed to attenuate ERK1/2 activation (Supplemental Fig. 3G). Thus, although both CRAC channels and ERK1/2 activation are required for PGE2 synthesis by ATP and UTP, they appear to be independent pathways converging on enzymes in the PGE2 synthesis pathway, likely cPLA2 (60). Finally, inhibiting the MEK1/2-ERK1/2 kinase cascade with U0126 also impaired ATPγS-induced IL-6 induction (Fig. 6G). Thus, MEK1/2-ERK1/2 kinase signaling appears to be necessary for the synthesis of both PGE2 and IL-6 by extracellular ATP.
Distinct reactive oxygen species are necessary for PGE2 synthesis and IL-6 secretion
Previous work has shown that, in addition to Ca2+ signaling, the induction of PGE2 and IL-6 is regulated by numerous other signaling pathways, including reactive oxygen species (ROS) (59, 64, 65). The two primary sources of ROS in most cells are the plasma membrane–associated NADPH enzymes and mitochondria (66). To examine the potential involvement of these ROS sources for ATP-induced synthesis of PGE2 and IL-6, we tested the mitochondrial complex III ROS inhibitor S3QEL-2 and the NADPH oxidase inhibitor apocynin. These experiments showed that apocynin abolished nucleotide-driven PGE2 synthesis, whereas S3QEL-2 had no effect (Supplemental Fig. 4A, 4B). NADPH oxidase inhibition did not attenuate ERK1/2 activation downstream of P2Y2 (Supplemental Fig. 4C, 4D). Thus, production of ROS by NADPH oxidase, but not mitochondria, is necessary for PGE2 synthesis. By contrast, both S3QEL-2 and apocynin decreased ATPγS-induced production of IL-6, indicating that both sources of ROS regulate IL-6 induction (Supplemental Fig. 4E). These results reveal an essential role of ROS for both PGE2 synthesis and IL-6 secretion by ATP, but suggest that the sources of ROS differ for the induction of the two mediators.
Distinct Ca2+-sensing enzymes are necessary for PGE2 and IL-6 induction
We next interrogated the role of Ca2+-dependent enzymes in purinergic-driven PGE2 synthesis and IL-6 secretion. A key Ca2+-dependent enzyme involved in agonist-driven transcriptional responses is calcineurin. To examine a role for calcineurin/NFAT signaling in the ATP-mediated IL-6 response, we used FK-506, a widely used small-molecule inhibitor of calcineurin/NFAT signaling. FK-506 abrogated ATPγS-induced IL-6 secretion (Fig. 7A), indicating that the ATP-induced IL-6 response is mediated by NFAT-dependent gene transcription, as previously shown for PAR2-mediated induction of IL-6 (32).
By contrast, FK-506 did not inhibit ATP-induced PGE2 synthesis (Fig. 7B), consistent with the evidence presented above indicating that acute PGE2 production is not substantively influenced by calcineurin (Supplemental Fig. 3A–E). Another key Ca2+-dependent enzyme linked to PGE2 synthesis is cPLA2, which liberates arachidonic acid from lipid membranes and is regulated independently both by ERK1/2 phosphorylation and Ca2+ binding to its C2 domain (67, 68). ATP-induced PGE2 production was blocked by the cPLA2 inhibitor AACOCF3, whereas ATPγS-induced IL-6 secretion was not inhibited (Fig. 7C, 7D). Upon Ca2+ binding, cPLA2 translocates to intracellular organelle membranes of the endoplasmic reticulum, Golgi, and nucleus, where it liberates arachidonic acid from the resident phospholipids (67, 68). Consistent with this signaling, cell fractionation analysis (35) revealed that ATP stimulation of AECs induced rapid enrichment of cPLA2 in the nuclear fraction (Fig. 7E, 7F), a response that was blocked by the P2Y2 receptor antagonist AR-C (Fig. 7E 7F). The CRAC channel inhibitors CM4620 and BTP2 abrogated the P2Y2 receptor–mediated recruitment of cPLA2 to the nuclear fraction (Fig. 7G–J). Collectively, these data indicate that P2Y2 receptors stimulate PGE2 synthesis via CRAC channel–mediated activation of cPLA2 (Fig. 8).
In the airways, ATP and UTP signaling is linked to a wide range of immune effects. High concentrations of extracellular ATP are linked to the production of inflammatory cytokines, leading to immune cell infiltration, airway remodeling, and hyperresponsiveness (2, 4, 5, 51). Patients with asthma, COPD, and ARDS show increased airway ATP levels in the BAL fluid, and elevated extracellular ATP is thought to contribute to disease pathology (2, 8, 9). However, although high concentrations of extracellular ATP are considered proinflammatory, ATP (and UTP) can also stimulate many anti-inflammatory (69) and physiologically beneficial effects in the lung, including MCC, PGE2 synthesis, and wound healing (10–12, 14, 50). The differential cellular pathways mediating these potentially beneficial and harmful effects are not well understood.
In this study, we describe the cell signaling mechanisms underlying the divergent effects of ATP and UTP on PGE2 and IL-6 production from AECs involving distinct purinergic receptor subtypes and downstream effectors (Fig. 8). Our results indicate that ATP/UTP signaling through P2Y2 receptors activates CRAC channels and ERK1/2 and subsequently cPLA2, resulting in rapid synthesis of PGE2, which is known to evoke bronchoprotective and immune-suppressive effects (17–21, 24, 70). To our knowledge, this is the first identification of AEC P2Y2 receptors in stimulating protective PGE2 synthesis. By contrast, ATP-mediated induction of IL-6, which is implicated in proinflammatory responses in the airways (25–28, 30, 31), appears to occur through stimulation of P2X receptors through a process requiring CRAC channels and ERK1/2 activation, but also distinctly involving calcineurin/NFAT activation (Fig. 8).
Several key features both upstream and downstream of CRAC channels underscore the differences in the two pathways (Fig. 8). First, although UTP strongly induces PGE2, it is completely ineffective in evoking IL-6 synthesis (Fig. 3C versus (Fig. 4E). ATP by contrast evokes both PGE2 and IL-6 synthesis, albeit at different potencies (7 μM versus ∼16 μM; (Fig. 4B) and over significantly different time courses (Fig. 3A versus (Fig. 4A). As noted above, this difference is related to the different receptors (P2Y2 versus P2X receptors) involved in stimulating PGE2 and IL-6. Second, the Ca2+-dependent signaling pathways downstream of receptor activation significantly differ: P2Y2 receptor–driven PGE2 synthesis involves Ca2+ activation of cPLA2, whereas P2X-driven IL-6 secretion requires Ca2+-dependent transcription via the calcineurin/NFAT pathway. As well, distinct sources of ROS appear to be involved in regulating the production of these mediators (Supplemental Fig. 4).
What are the functional implications of these findings, and under what conditions could these differing outcomes become apparent? Although speculative, we can envision two scenarios. First, the observation that PGE2 synthesis by ATP occurs at lower ATP doses than that required for IL-6 suggests that protective versus proinflammatory outcomes for ATP may be dictated in part by the concentration and duration of ATP signaling in the airways. In the healthy lung, low micromolar levels of ATP that are naturally released from cells may activate tonic baseline P2Y2 signaling conducive to selective PGE2 induction and its bronchoprotective effects. By contrast, higher levels of ATP occurring over longer durations, such as those found under conditions of severe damage and/or cellular necrosis, would be predicted to elicit strong IL-6 synthesis and IL-6–mediated inflammation. High-dose ATP is also known to engage the NLRP3 inflammasome, leading to additional pathways for inflammation (71). Second, extracellular UTP secretion would be predicted to exclusively induce PGE2. Growing evidence points to UTP as a physiologically relevant signaling molecule linked to numerous cellular processes, including ion transport, ciliary beat frequency, and mucin release (15). Although regulated secretion of UTP is very poorly understood likely due to lack of tools to easily detect extracellular UTP (72), release of UTP in vitro following mechanical stress, apoptosis, and solution exchange has been demonstrated using HPLC from a number of tissues, including AECs (73–76). UTP release under these conditions would be expected to preferentially evoke bronchoprotective responses in the lung. Future in vivo studies using mouse models in which P2Y2 receptors are selectively deleted in the lung epithelium could shed light on this important question.
An interesting feature of PGE2 synthesis is its exquisite sensitivity to blockade by the rapid Ca2+ buffer BAPTA but not the slower buffer EGTA (Fig. 5E, 5F). This finding implies that PGE2 synthesis relies on local Ca2+ signaling likely through Ca2+ microdomains near CRAC channels that are functionally linked to cPLA2 activation. Previous studies have shown that Ca2+ microdomains arising from CRAC channels can stimulate arachidonic acid release and leukotriene production in mast cells (35). Ca2+ microdomains around CRAC channels are also linked to NFAT-dependent gene transcription and exocytosis in neuronal stem cells and astrocytes (40, 57). Thus, the finding that local Ca2+ signals around CRAC channels are essential for cPLA2-mediated synthesis of PGE2 broadens the role of Ca2+ microdomains linked to enzyme activation in different cell types.
Two important mechanistic questions raised by our study that remain to be addressed relate to the nature of the Ca2+ signal activated by CRAC channels that drives IL-6 induction and the mechanism of how P2X receptor stimulation is coupled to activation of CRAC channels and IL-6 synthesis. The data indicate that antagonism of P2Y2 receptors blocks both ATP- and UTP-evoked Ca2+ elevations to similar extents (Fig. 1F). Yet, unlike ATP, UTP is completely ineffective in inducing IL-6 (Fig. 4E). Because CRAC channel blockade strongly inhibits induction of IL-6 by ATP (Fig. 5D), this result suggests that ATP-mediated induction of IL-6 requires Ca2+ influx reliant on CRAC channels that is not readily detected by Fura-2. We speculate that P2X receptors may cause downstream activation of CRAC channels over longer time periods than was evaluated in our Ca2+ imaging experiments, and this step is sufficiently local so as to evade detection by the bulk Ca2+ indicator, Fura-2. In this scenario, the functional coupling between P2X receptors and CRAC channels could involve localized ryanodine receptor–mediated calcium-induced calcium release events of the type that have been described in skeletal muscle and T cells (77, 78). Additional mechanistic studies using low-affinity and membrane-tethered Ca2+ indicators and genetic tools to manipulate specific P2X receptors are needed to help address the unknown links among P2X receptors, CRAC channels, and IL-6 induction.
Although P2X receptors have been widely investigated in many physiological contexts, their physiological roles and effector signaling mechanisms in AECs are not well understood. One study described a key role for P2X receptor activation in inducing IL-8 production via Ca2+ signaling and NF-κB (52), suggesting that P2X receptors may have an important role in AECs to drive proinflammatory responses. Our finding that the P2X receptor antagonists suramin and PPADS strongly suppress ATP-evoked synthesis of IL-6 in AECs is in agreement with this suggestion and expands the potential roles of P2X receptors in the airways to include the inflammatory cytokine IL-6.
Finally, airway P2Y2 receptors and CRAC channels have attracted significant therapeutic interest in recent years. A stable P2Y2 receptor agonist (denufosol) was investigated in patients with cystic fibrosis based on its ability to stimulate MCC (79). Unfortunately, long-term treatment with denufosol showed no improvement of lung function in patients with cystic fibrosis (80). CRAC channel inhibitors have also shown efficacy in preclinical models of asthma (81–83), and a new-generation CRAC channel inhibitor, CM4620, is currently being tested in human patients for relieving the cytokine storm in seriously ill patients with COVID-19 (55). Our results showing that CM4620 is very effective in occluding IL-6 production may provide the mechanistic explanation for the benefits of this small molecule in improving patient survival. Although CRAC channels also play a key role in agonist-evoked PGE2 synthesis, this may be counterbalanced under chronic inflammatory conditions in vivo due to its role in driving proinflammatory cytokine production, thus providing a therapeutic window to dampen chronic inflammation in the lung airways. More studies are needed to address these scenarios, but the results of this study provide a framework for testing these and other models.
This work used resources of the Northwestern University Structural Biology Facility at the Robert H. Lurie Comprehensive Cancer Center. We thank members of the Prakriya laboratory for helpful discussions and Michaela Novakovic and Mohammad Mehdi Maneshi for technical advice.
T.S.K., A.J., and M.P. were responsible for conceptualization; T.S.K. was responsible for methodology; T.S.K., A.J., and C.D.K. performed the investigation; T.S.K., A.J., K.A.S., R.P.S., and M.P. were responsible for writing; T.S.K. was responsible for visualization; M.P. was responsible for supervision; T.S.K. and M.P. were responsible for funding acquisition.
This work was supported by National Institutes of Health Fellowships (National Institute of Allergy and Infectious Diseases 5T32AI007476 and National Heart, Lung, and Blood Institute F31HL151170) to T.S.K. and National Heart, Lung, and Blood Institute Grant R01 HL149385 to M.P. The Northwestern University Structural Biology Facility was supported by National Cancer Institute Cancer Center Support Grant P30 CA060553.
The online version of this article contains supplemental material.
Abbreviations used in this article
airway epithelial cell
acute respiratory distress syndrome
bronchial epithelial basal medium
bronchial epithelial growth medium
intracellular Ca2+ concentration
chronic obstructive pulmonary disease
coronavirus disease 2019
cytosolic phospholipase A2
Ca2+ release-activated Ca2+
normal human bronchial epithelial
protease and phosphatase inhibitor
reactive oxygen species
short hairpin RNA
small interfering RNA
store-operated calcium entry
K.A.S. is an employee of, and holds stock in, CalciMedica, Inc. The other authors neither received payment from CalciMedica for participation as investigators in the study nor for the development of the present manuscript.