# Bronchial Epithelial Calcium Metabolism Impairment in Smokers and Chronic Obstructive Pulmonary Disease Decreased ORAI3 Signaling

Aurelie Petit, Lucie Knabe, Kamel Khelloufi, Myriam Jory, Delphine Gras, Yann Cabon, Malcolm Begg, Sylvain Richard, Gladys Massiera, Pascal Chanez, et al.

► To cite this version:

Aurelie Petit, Lucie Knabe, Kamel Khelloufi, Myriam Jory, Delphine Gras, et al. Bronchial Epithelial Calcium Metabolism Impairment in Smokers and Chronic Obstructive Pulmonary Disease Decreased ORAI3 Signaling. American Journal of Respiratory Cell and Molecular Biology, 2019, 61 (4), pp.501-511. 10.1165/rcmb.2018-0228OC. hal-02091961

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# Bronchial epithelial calcium metabolism impairment in smokers and COPD: decreased ORAI3 signaling

Aurelie Petit<fnref n="1" />, Lucie Knabe<fnref n="1" />,<fnref n="2" />, Kamel Khelloufi<fnref n="3" />, Myriam Jory<fnref n="4" />, Delphine Gras<fnref n="5" />, Yann Cabon<fnref n="6" />, Malcolm Begg<fnref n="7" />, Sylvain Richard<fnref n="2" />, Gladys Massiera<fnref n="4" />, Pascal Chanez<fnref n="5" />, Isabelle Vachier<fnref n="1" />, Arnaud Bourdin<fnref n="1" />,<fnref n="2" />,*

**\*Correspondence**

Aurelie Petit, PhD

Department of Respiratory Diseases,

CHU Arnaud de Villeneuve

371 avenue du Doyen Gaston Giraud

34295 Montpellier cedex 05

phone: +33467042020

fax: +33467042000

e-mail: aurelie.fort@inserm.fr

**Running title:** ORAI3 impairment in bronchial epithelium from smokers.

**Author contributions:** A.P., I.V. and A.B. conceived and designed the experiments; A.P. performed the experiments; L.K. performed cell culture; K.K and M.J. performed ciliary beating experiments and analyzes; A.P. analyzed the data; Y.C. performed statistical analyses; A.B. and I.V. supervised research; G.M. supervised ciliary beating experiments; D.G., P.C. and S.R. participated in the data discussion and interpretation of results. M.B. provided GSK-7975A. A.P. wrote the paper and all authors contributed to the final drafting of the manuscript.

This work was supported by the PHRC (IR 2013-14), ANR-13-BSV5-0015-02, APARD 2012 and CHU de Montpellier n°ID-RCB: 2013-A01405-40.

<footnote n="1">¹Department of Respiratory Diseases and Addictology, Hôpital Arnaud de Villeneuve, CHU Montpellier, France.</footnote>

<footnote n="2">²INSERM U1046, CNRS UMR 9214, University of Montpellier, Montpellier, France.</footnote>

<footnote n="3">³Aix Marseille University, CNRS, CINaM UMR7325, Marseille, France</footnote>

<footnote n="4">⁴Laboratoire Charles Coulomb (L2C), UMR5221 CNRS-UM F34095, Montpellier, France</footnote>

<footnote n="5">⁵Aix Marseille University, APHM, CV2N, Inserm U1263 INRA 1260, Clinique des bronches allergies et sommeil, Marseille, France.</footnote>

<footnote n="6">⁶Department of Medical Information, Montpellier University Hospital, Montpellier, France.</footnote>

<footnote n="7">⁷Refractory Respiratory Inflammation DPU, Respiratory TAU, GlaxoSmithKline, Stevenage, UK.</footnote>
**Abstract:** The airway epithelium represents a fragile environmental interface potentially disturbed by cigarette smoke, the major risk factor for developing Chronic Obstructive Pulmonary Disease (COPD). Cigarette smoke leads to bronchial epithelial damage on ciliated, goblet and club cells, which could involve calcium signaling. Calcium is a key messenger involved in virtually all fundamental physiological functions, including mucus and cytokine secretion, cilia beating and epithelial repair. In this study, we analyzed calcium signaling in Air-Liquid Interface reconstituted bronchial epithelium from control subjects and smokers (with and without COPD). We further aimed to determine how smoking impaired calcium signaling. Firstly, we showed that the endoplasmic reticulum (ER) depletion of calcium stores was decreased in COPD patients and that the calcium influx was decreased in epithelial cells from smokers (regardless of COPD status). In addition, acute cigarette smoke exposition lead to a decrease in ER calcium release, significant in smoker subjects, and to a decrease in calcium influx only in control subjects. Furthermore, the differential expression of 55 genes involved in calcium signaling highlighted that only ORAI3 expression was significantly altered in smokers (regardless of COPD status). Finally, we incubated epithelial cells with an ORAI antagonist (GSK-7975A). GSK-7975A altered calcium influx and ciliary beating but not mucus and cytokine secretion or epithelial repair in control subjects. Our data suggest that calcium signaling is impaired in smoker epithelia (regardless of COPD status) and involves ORAI3. Moreover, ORAI3 is additionally involved in ciliary beating.

**Key words:** bronchial epithelial cells, calcium signaling, cigarette smoke, ORAI3
Introduction

The airway epithelium represents the first line of defense for the bronchi and its function as a
barrier is paramount for preventing airway damage (1, 2). Much evidence supports that
dysregulation of bronchial epithelial homeostasis contributes to airway disease. COPD is the
third cause of death in the world and treatments relieve symptoms but do not cure the disease.
Cigarette smoking, the main environmental risk factor associated with COPD, negatively
affects the innate defense system of the lung (3-5). Cigarette smoke exposure leads to airway
epithelium damage, including cilia loss and impaired beating (6), metaplasia and hyperplasia
of goblet cells (7), as well as decreased numbers of club cells (8, 9). Tobacco is also involved
in calcium (Ca²⁺) signaling dysregulation in many cell types and processes (10-13). Cytosolic
Ca²⁺ signals control a wide range of cellular functions ranging from short-term (contraction
and secretion) to longer-term responses (cell growth and proliferation) (14, 15). In response to
the activation of different receptors, Ca²⁺ generates signals involving two closely coupled
processes: a rapid transient release of Ca²⁺ stores (mainly from endoplasmic reticulum (ER)),
followed by a slow progressive extracellular Ca²⁺ entry (14, 16-18). The resulting depletion of
Ca²⁺ stored within the ER lumen is the primary trigger for a message that is returned to the
plasma membrane, resulting in the activation of store-operated channels (SOCs), which
mediate Ca²⁺ entry (16-18). The activation of SOCs is relatively slow (10–100 sec), and the
Ca²⁺ entry phase mediates longer-term cytosolic Ca²⁺ elevations and helps to replenish
intracellular stores (16-18). In bronchial epithelial cells, Ca²⁺ is involved in mucus and
cytokine secretion (19), ciliary beating (20) and epithelial repair. Most studies concerning
Ca²⁺ signaling in bronchial epithelium were carried out either in animal models (21-23) or in
human bronchial cell lines (24-27). Moreover, Ca²⁺ signaling in primary bronchial epithelial
cells has never been studied as a whole. Instead, studies have usually focused on a specific
protein involved in this signaling (28-30). In addition, the majority of studies within the lung
are related to compartments other than the epithelium, such as nerves (31), macrophages (32) or smooth muscle cells (33). Obtaining a global understanding of Ca²⁺ signaling, particularly ER- Ca²⁺ release and Ca²⁺ entry, and the nature of the channels mediating Ca²⁺signaling, remains crucial but unresolved in human primary bronchial epithelial cells.

In this work, we aimed to elucidate Ca²⁺ signaling and to investigate whether Ca²⁺ signaling dysfunction is a critical, initiating step in cigarette smoke related disorders. We used human primary bronchial epithelial ALI cultures obtained from bronchial biopsies from controls, smoking subjects and COPD patients.

## Materials and Methods

*Materials*: see online data supplement.

*Bronchial biopsies and human bronchial epithelium:* Bronchial biopsies from control subjects (n=6), smokers (n=8) and smokers with COPD (n=14) were collected during fiberoptic bronchoscopy on a subsegmental bronchus of the *left lower lobe*, as previously described (34). Subjects were recruited at the Arnaud de Villeneuve Hospital, Montpellier, France under a protocol approved by an independent ethics committee and all patients gave their informed consent in writing. Biopsies were processed and primary human bronchial epithelial cells isolated and cultured under air-liquid interface (ALI) conditions, as previously described (35). Briefly, bronchial epithelial biopsies were mechanically dissociated and suspended in bronchial epithelial growth medium (BEGM, Lonza, reference CC-3170, unavailable for France since November 2017). After the expansion phase in monolayer culture, cells were plated on uncoated nucleopore membranes (24-mm dia., 0.4 µm pore size, Transwell Clear, Costar) in a 1:1mixture of BEGM and Dulbecco's modified Eagle's medium (DMEM, Lonza reference BE12-707F)) applied only at the basal side to establish the ALI. In this study, we worked with 28 day-old differentiated ALI cultures

*Measurement of calcium signals using fura-2:* see online data supplement.
*Acute smoke exposition of ALI cultured airway epithelia:* To evaluate acute smoke exposition effects on Ca²⁺ signaling, 28 day-old ALI cultures were exposed to the smoke of one cigarette (Red Marlboro (Philip Morris)) in gaseous phase during 5 minutes just after Fura2 AM incubation and before Ca²⁺ signal recording.

ALI cultures were placed in a homemade system consisting of a 250 ml exposition chamber connected with a pipe to a pump where the cigarette can be attached. The pump enabled continuous cigarette aspiration during 5 min, allowing smoke to fill the chamber and expose ALI cultures.

*Gene expression analysis:* Total RNA from 28 day-old ALI cultures and quantitative RT-PCR were performed. See online data supplement.

*Analysis of ORAI3 protein expression:* Protein extracts from 28 day-old ALI cultures were analyzed by western-blot. See online data supplement.

*Measurement of ciliary activity:* The percentage of epithelial surface covered by beating cilia and the mean ciliary beat frequency were determined by transmission light microscopy (Nikon Eclipse Ti microscope at 37°C and 5% CO₂) on 28 day-old ALI cultures from 8 control subjects, 7 smokers and 5 smokers with COPD, before and after either DMSO (diluted 1/1000, 30 min) or GSK-7975A (10µM, 30 min) incubation. See online data supplement.

*Measurement of IL8 concentration:* IL8 concentration was assessed in the apical lavage of 28 day-old ALI cultures before and 24 hours after 10µM GSK-7975A incubation using ELISA Kits (Dial clone).

*Measurement of Muc5AC secretion:* Muc5AC secretion was measured before and 24 hours after 10µM GSK-7975A in a dot blot assay using a primary mouse antibody (anti Muc5Ac (45M1) ThermoFisher Scientific). See online data supplement.
*Analysis of epithelial repair:* 28-day differentiated ALI culture bronchial epithelium from 6 control subjects, 7 smokers and 10 COPD subjects were considered for epithelial repair analysis. See online data supplement.

*Statistical analysis:* Most of the data are represented by box plots (with whiskers spanning min to max). Data within Figure 5 are represented either in scatter plot (5A) or in individual data (before/after 5B). Data from Figure 6C represent means+/-SEM for the GSK-7975A effect on Muc5AC secretion and data from Figure 6E means+/-SEM for the GSK-7975A effect on IL8 secretion. Statistical comparisons were performed (Mann and Whitney test or Wilcoxon signed rank test) and graphics were generated using GraphPad Prism version 5.1 (GraphPad Software Inc., San Diego, CA). Each statistical test is specified on each figure legend.

Figures 3A and B: Due to the large number of gene expressions analyzed, a Principal Component Analysis (PCA) was performed. The PCA was performed on the patient-space in order to identify outlying genes. Linear combinations of the variables formed new independent variables or components (36). Results were visualized via scatterplots, in which the original variables or individuals are displayed, allowing visual interpretation of their relationships (37).

No transformation was performed on the data used for the PCA, implemented using R 3.4.3 software and the package FactoMineR.

p values <0.05 were considered as statistically significant.

# Results

## Subject characteristics

Bronchial biopsies from 6 controls, 8 smokers without COPD and 14 smokers with COPD were successfully grown in ALI cultures and considered for analysis. The clinical characteristics of these subjects are reported in Table 1. There were no differences between
the three groups except that FEV1/FVC and FEV1 are decreased in COPD according to the
disease definition (GOLD).

*Calcium signaling is impaired in the airway epithelium of smokers with or without COPD*

Cigarette smoke is the first risk factor for developing COPD. What is the effect of cigarette smoke on airway epithelia Ca²⁺ signaling?

We first compared Ca²⁺ metabolism between controls and smokers with or without COPD.
This experiment consisted in comparing the effect of the susceptibility to CHRONIC smoke
exposure (COPD vs smokers) on Ca²⁺ signaling.

To discriminate between the two main sources of Ca²⁺ (namely the ER and the extracellular
space), we worked without Ca²⁺ in the extracellular space (Locke buffer without Ca²⁺ and
with 2mM EGTA). The SERCA-blocker thapsigargin (sarco/endoplasmic reticulum calcium
ATPases) (1µM) was applied to assess the ER-Ca²⁺ release into the intracellular space. We
are conscious that because external Ca²⁺ is null, the thapsigargin effect reflects not only ER-
Ca²⁺ release but also Ca²⁺ leak across the plasma membrane. For simplicity, we summarize
the thapsigargin response to ER-Ca²⁺ release. ER depletion activates plasma membrane
channels. Then, Ca²⁺ (1.8mM) was applied to assess Ca²⁺ re-entry (Ca²⁺ influx). The protocol
and Ca²⁺ movements are presented in Figure1A. Baseline cytosolic Ca²⁺ concentration was
similar between the 3 groups (① and Figure 1B). The ER-Ca²⁺ release (② and Figure 1C)
and Ca²⁺-influx components (③ and Figure 1D) were investigated. Ca²⁺ release from the ER
was significantly decreased only in smokers with COPD (p=0.043). Ca²⁺ influx was
significantly decreased in reconstituted airway epithelium from smokers with or without
COPD (p=0.043 and p=0.007, respectively).

*Calcium signaling is impaired in the cigarette-smoke-exposed airway epithelium*
In addition to "natural" chronic exposition, we exposed ALI cultures (from control subjects and smokers with and without COPD) to an acute cigarette smoke protocol and then investigated Ca²⁺ release and Ca²⁺ influx. Baseline cytosolic Ca²⁺ concentration is unaffected in control subjects exposed to cigarette smoke (p=1) but is significantly decreased in smoker subjects (p=0.0131) and tended to decrease in COPD patients (p=0.0685). Ca²⁺ release from the ER tended to decrease in all groups of subjects exposed to cigarette smoke but was significantly decreased only in smoker subjects (controls vs CS-controls: p=0.15; smokers vs CS-smokers: p=0.039; COPD vs CS-COPD: p=0.058) (Figure 2A) whereas Ca²⁺ influx was decreased only in control subjects (p=0.031) (Figure 2B).

*Genes involved in calcium signaling*

In parallel, we examined the gene expression of 55 proteins potentially involved in Ca²⁺ signaling (44 expressed at the plasma membrane and 11 at the ER membrane) in the ALI cultures from our 3 groups of subjects (see online data supplement Tables E3 and E4). ALI bronchial epithelium was characterized by the absence of expression of several genes including all RyR (Ryanodine Receptor) isoforms, all voltage-gated Ca²⁺ channels and some SOCs (TRPM1, TRPM2, TRPM3, TRPV5, TRPC3, TRPC7, TRPA1, TRPP3 and TRPP5). A principal component analysis (PCA) was carried out to visualize how the expression of the 55 normalized genes differed across our sample. The PCA showed a clear dichotomy among the three subject groups: control subjects on the one hand and smokers with or without COPD on the other hand (Figure 3A). The individual factor map highlighted three genes expressed differently compared to the others: SERCA2, TRPV4 and ORAI3 (Figure 3B). Only ORAI3 gene expression appeared significantly lower in smokers with or without COPD (p=0.004 and p=0.020) compared to controls (Figure 3E). The expression of the 52 other genes studied was summarized in Tables E3 and E4. We then focused on ORAI3 since it was the only differentially expressed gene involved in Ca²⁺ signaling between the three groups of subjects.
We found that ORAI3 protein expression was also reduced in ALI culture cells from smokers with or without COPD (p=0.035 and p=0.029 respectively, compared to controls, Figure 3F).

*ORAI is involved in epithelial cell calcium signaling*

We assessed the role of ORAI by investigating Ca²⁺ metabolism and several biological roles of the bronchial epithelium. For that purpose, we used the ORAI channel inhibitor (GSK-7975A (38)). ALI culture pre-incubation with GSK-7975A (10µM) had no effect on baseline Ca²⁺ concentration in controls and smokers with or without COPD (Figure 4A). Moreover, GSK-7975A pre-incubation on control-subject ALI cultures led to a decreased Ca²⁺ influx (Figure 4B, p=0.039), whereas the epithelium from smokers with or without COPD remained unaffected. The GSK-7975A effect on Ca²⁺ influx of control ALI epithelium mimicked the Ca²⁺ influx profiles of smokers with or without COPD, which were already low.

*ORAI is involved in ciliated cell function (ciliary beats) but not in goblet cell function (mucus secretion), basal cell function (epithelial repair) or inflammation (IL8 secretion)*

Ca²⁺ is likely to be involved in ciliary beating, mucus secretion, bronchial epithelium repair and IL8 secretion, all of which are affected by cigarette smoke. To determine whether ORAI participates in these cellular functions, we analyzed cilia beats, Muc5AC secretion, epithelium repair and IL8 secretion with and without GSK-7975A, in reconstituted epithelia from controls and from smokers with or without COPD.

At steady state, the epithelia of smokers with or without COPD tended to present a lower percentage of epithelial surface covered by beating cilia tufts compared to controls (Figure 5A). Cilia beat frequencies were similar among the three groups (Figure 5B). When ALI epithelia were treated with 2mM of EGTA, the percentage of epithelial surface covered by beating cilia seemed to decrease in all the 3 groups of subjects and ciliary beat frequency tended to decrease in controls and smokers subjects but not significantly (Figure 5 C-D).
When cultured ALI epithelia were pre-incubated with GSK-7975A (10µM), the percentage of epithelial surface covered by beating cilia (p=0.0234) and ciliary beat frequency (p=0.0078) were decreased in control subjects, whereas there was no significant effect in smokers with or without COPD (Figure 5E-F).

Epithelia Muc5AC secretion from smokers without COPD was increased when compared to those from control subjects (p=0.015) and tended to increase in epithelia from smokers with COPD (Figure 6B) in agreement with the literature (39). Pre-incubation with the ORAI inhibitor (GSK-7975A) did not affect this secretion (Figure 6C).

IL8 release tended to increase in the supernatant of culture epithelium from smokers with or without COPD (Figure 6D), but remained unaffected by pre-incubation with GSK-7975A (Figure6E).

Epithelia from smokers with or without COPD were not totally repaired 24 hours after injury in contrast to controls, suggesting impaired repair in smokers with or without COPD (p=0.004 and p=0.010 respectively) (Figure 7). The ORAI inhibitor had no related effects.

Discussion

Cigarette smoking is the main COPD risk factor leading to airway epithelium damage and involved in the deregulation of Ca²⁺ signaling in many cell types and processes. We report the Ca²⁺ signaling of control subjects, smokers and COPD bronchial epithelial cell ALI cultures derived from donor biopsies. The majority of studies on airway epithelia are conducted on animals or cell lines (21-27) that don't correspond to an ideal model. Indeed, working with an animal model is easier because of availability, but interspecies differences are found with respect to the cellular composition of the airways, which varies along the proximal-distal axis.

Also, cell lines do not systematically result in a differentiated epithelium, but rather a
monolayer culture that does not reflect in vivo structure. Finally, ALI cultures are possible
with cell lines, but previous experiments were carried out after only 2 weeks of culture (40-
43), probably before the complete differentiation of the epithelium. Working with ALI
cultures from human donor biopsies is currently the closest match for an experimental model
representing the specific bronchial-epithelia-cell phenotype for each category of subjects.

Moreover, previous studies (28-30, 44) have focused on one protein or group of proteins
involved in Ca²⁺ signaling, but Ca²⁺ signaling in bronchial epithelial cells has never been
studied as a whole. In this work, we analyzed Ca²⁺ signaling divided into two closely coupled
processes: endoplasmic reticulum depletion and Ca²⁺ influx through the plasma membrane.

We also depicted the mapping of 55 genes involved. We assessed two situations in relation to
smoking: the first consisted of chronic cigarette smoke exposure corresponding to the study
on controls and smokers with or without COPD and the second of acute cigarette smoke
exposure corresponding to the exposition of ALI cultures (again from controls and smokers
with or without COPD) to the smoke of one cigarette. The Ca²⁺ signaling profiles of the two
exposition modes were different: acute exposition caused a decrease in ER- Ca²⁺ release in
smokers and a decrease in Ca²⁺ influx in controls, whereas chronic exposition causes a
decrease of Ca²⁺ influx in smokers and a decrease of Ca²⁺ influx and ER depletion in COPD
subjects. Because external Ca²⁺ is null before thapsigargin stimulation, the data named ER-
Ca²⁺ release reflect the release of Ca²⁺ from the ER but also the leak of Ca²⁺ out of the cell
across the plasma membrane. Thereby, the decrease of "ER-Ca²⁺ release" could be partially
attributed to an enhanced extrusion of Ca²⁺ from cytosol via NCX transporters. The
expression of NCX transporters did not differ among the 3 groups of subjects suggesting that
NCXs influence could be modest but their activity could be altered especially in smoker and
COPD subjects. Given that NCX transporter activity is enhanced because of the extracellular
space is Ca²⁺-free, we must therefore remain cautious about the conclusions to be drawn.

Future works focused on "depletion/leak" injury could answer this question.

Ca²⁺ entry can be achieved by different mechanisms including voltage-operated Ca²⁺ entry through voltage-sensitive Ca²⁺ channels and receptor-operated Ca²⁺ entry following receptor occupation using mechanisms other than a change in membrane potential (45). The latter may take several forms and is conducted by different types of channels: receptor-operated channels (ROC), second messenger-operated channels (SMOC) and, finally, store-operated (or capacitive) Ca²⁺ channels (SOC/CRAC). SOCs are activated when the luminal Ca²⁺ concentration in the intracellular Ca²⁺ stores is reduced as a result of receptor occupation and of the subsequent generation of a Ca²⁺-mobilizing second messenger (46, 47). In non-excitable cells, as well as in certain excitable cells, store-operated Ca²⁺ entry (SOCE) is the major mechanism for Ca²⁺ influx (18). In this study, the difference in Ca²⁺ entry is not due to voltage-gated Ca²⁺ channels since non-excitable cells, as our model of human primary epithelial cells in ALI culture, probably do not express them. This is confirmed by the absence of mRNA of all voltage-gated Ca²⁺ channels in our model of human bronchial epithelium.

Analysis of mRNA for the genes involved in Ca²⁺ signaling showed no significant differences between the three subject groups, except for ORAI3 which decreased in smokers regardless of COPD status. ORAI proteins are considered as the essential pore-forming components of the low conductance, highly Ca²⁺-selective CRAC channels whose activation is dependent on depletion of intracellular stores. In smokers with or without COPD, the decrease in ORAI3 expression on the one hand and the decrease in Ca²⁺ influx caused by chronic exposure to cigarette smoke on the other hand, suggests that ORAI3 is involved in this alteration of Ca²⁺ influx. Moreover, acute cigarette smoke exposure has no effect on the Ca²⁺ influx of smokers with or without COPD and leads to a decrease in Ca²⁺ influx among control subjects, thus mimicking smoker and COPD phenotype Ca²⁺ influx. In the same way, GSK 7975A
incubation of control subject experiments decreased Ca²⁺ influx but not with smoker or COPD subjects. In smokers and COPD, there is a lack of effect of GSK 7975A on Ca²⁺ influx but they still do have a Ca²⁺ influx. This point could be explained by the involvement of another pathway. Indeed, some studies showed that TRP channels (Transient Receptor Potential) especially TRPCs (TRPC1, TRPC4, TRPC5) would be SOC and could play a role similar to ORAI (48, 49). Because of the decreased ORAI3 protein in smokers and COPD, a switch could occur in chronic exposition to cigarette smoke to compensate with another pathway like TRP channels. Even if there were no differences in TRP gene expression between the 3 groups of subjects in this work (table E3), the channel activity could be enhanced.

All these data suggest that ORAI3 is involved in bronchial epithelial cell Ca²⁺ signaling and that it is a tobacco target.

To summarize, one of the physiological injuries provoked by cigarette smoke is the impairment of Ca²⁺ influx, whereas COPD phenotypes are additionally associated with ER depletion injury.

In the human bronchial epithelium, Ca²⁺ is involved in ciliary beating (20). Moreover, cigarette smoke exposure leads to airway epithelium damage such as cilia loss and impaired beating (6). In our model, smokers (regardless of COPD status) tended to have fewer ciliated cells with beating cilia, but the average ciliary beat frequency is the same as in controls. Furthermore, the pre-incubation of ALI cultures from control subjects with an ORAI antagonist and the pre-incubation of epithelia from control and smoker subjects (regardless of COPD status) with EGTA decreased the percentage of epithelial surface covered by beating cilia tufts and ciliary beat frequency, confirming the implication of Ca²⁺ and ORAI in ciliary beating. GSK 7975A had no effect on smokers (regardless of COPD status), where ORAI3 expression is decreased, confirming the implication of this isoform in ciliary beating. We can speculate that the ciliated cells from smokers (regardless of COPD status) have compensated
the lack of Ca²⁺ influx by Ca²⁺ channels other than ORAI3 in order to maintain normal beat frequency. TRPV4 has been shown to participate to epithelial ciliary activity (50). TRP channels could again be involved in the substitution of ORAI channels by TRP channels in the ciliary beating process.

Although the results of our study provide new insights into smoking pathologies, especially COPD, there are limitations and unknowns as well. So far, we have not identified the specific Ca²⁺ pathway affected by COPD, although Ca²⁺ store release is clearly impaired only in smokers with COPD. Smokers without COPD seem to be protected from the action of cigarette smoke on ER- Ca²⁺ release, whereas smokers with COPD are not. This component of the ER- Ca²⁺ signaling impairment in smokers with COPD seems to be directly linked to the pathology. Future work will concentrate on determining the specificities of exactly how Ca²⁺ signaling is altered in the bronchial epithelia of COPD patients. We focused on impaired Ca²⁺ influx because we found an alteration of expression of only ORAI3 in smokers with and without COPD. Moreover, the chosen ORAI antagonist (GSK-7975A) is not specific to the ORAI3 isoform. In fact, there is no specific antagonist of ORAI3. A siRNA approach is possible, but this technique is complex, especially as concerns air-liquid interface models. Certain previous studies (51-54) have used the siRNA approach on monolayer cultures and ALI models, but mostly using cell lines or with experiments carried out at early time frames (two weeks), at which time the complete differentiation of the epithelium is uncertain.

Finally we have chosen to apply CaCl₂ at the peak of thapsigargin response without waiting Ca²⁺ level drop back to basal level. In the literature, methods used to apply CaCl₂ described two main ways to study SOCE after thapsigargin store depletion. In most studies the authors waited Ca²⁺ level drop back to basal level after thapsigargin response (55, 56) while in others they did not wait and CaCl₂ was applied at the peak response of thapsigargin (57, 58). Finally, some authors like Peel et al used both methods (59, 60) which consisted to wait Ca²⁺ level
drop back to basal level after thapsigargin response but did not wait and applied CaCl₂ at the peak when CPA (Cyclo Piazonic Acid) was used instead of thapsigargin. At the peak of thapsigargin response, Ca²⁺ transporters should work at their maximum activity. In order to overcome the hypothetical influence of transporters (such as NCXs) on Ca²⁺ elevation, we performed preliminary experiments to compare the influx of Ca²⁺ either when applying CaCl₂ at the peak of the thapsigargin response, or after waiting for Ca²⁺ level drop back to basal level. No significant difference were observed between the two conditions (Figure E1). Working at the peak resulted to reduce the duration of the experiment and allowed us to complete the experiment on the same patient within the same day for all conditions.

In conclusion, this study aimed to improve the understanding of Ca²⁺ signaling in bronchial epithelium and, specifically, how smoking is involved. It represents the first time that Ca²⁺ signaling is described in *ex vivo* reconstituted bronchial epithelium. Ca²⁺ signaling was demonstrated to be impaired in smokers (regardless of COPD status) compared to healthy subjects, and ORAI3 appears implicated in Ca²⁺ influx alteration. Impaired ER Ca²⁺ depletion was specific to COPD, and understanding Ca²⁺ signaling differences between healthy smokers and COPD patients may provide new therapeutic targets.

**Acknowledgments:**

The authors are grateful to Dr Carey Suehs for her English corrections.
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**Competing interests:** The authors declare that they have no competing interests.
<table><caption>Table 1</caption><thead><tr><th>Characteristics</th><th>Controls<br>(n=6)</th><th>Smokers<br>(n=8)</th><th>COPD<br>(n=14)</th><th>p-value</th></tr></thead><tbody><tr><td>age, years<br>(mean ± SD)</td><td>50.83±19.8</td><td>51±18.06</td><td>64.93±9.64</td><td>0.1047</td></tr><tr><td>Gender<br>(% male)</td><td>50</td><td>50</td><td>85.71</td><td>0.1291</td></tr><tr><td>Cumulative<br>smoking, Pack<br>years (mean ±<br>SD)</td><td>NA</td><td>40.83±34.7</td><td>31.08±18.87</td><td>0.7781</td></tr><tr><td>FEV1/FVC<br>(mean ± SD)</td><td>0.900±0.117</td><td>0.900±0.136</td><td>0.600±0.097</td><td>0.00034</td></tr><tr><td>FEV1, % th<br>(mean ± SD)</td><td>99.73±4.25</td><td>95.61±9.93</td><td>66.51±22.07</td><td>0.0069</td></tr><tr><td>GOLD “2011”<br>status<br>(% I-II)</td><td>NA</td><td>NA</td><td>81.82</td><td>NA</td></tr><tr><td>Smoking status<br>(% weaned > 1<br>year)</td><td>NA</td><td>28.57</td><td>50</td><td>0.7018</td></tr></tbody></table>
FIGURE LEGENDS

**FIGURE 1.** Cytosolic Ca²⁺ was measured in human air-liquid interface bronchial epithelium cultures from 6 control subjects, 8 smokers and 14 smokers with COPD, using Fura-2 AM. (A) Representative traces of cytosolic Ca²⁺, in the absence of extracellular Ca²⁺, in response to thapsigargin (1µM) followed by adding of CaCl₂ (1.8mM). Measurements taken from these Ca²⁺ responses were : baseline Ca²⁺ concentration = ①, change in F340/F380 ratio after application of 1µM of thapsigargin (Tg), corresponding to ER-Ca²⁺ release and Ca²⁺ leak across plasma membrane = ② and change in F340/F380 ratio after supplementation with 1.8mM of CaCl₂ corresponding to Ca²⁺ influx = ③. The baseline Ca²⁺ concentration(①) (B), maximum response following thapsigargin stimulation② (C) and maximum response following Ca²⁺ supplementation③ (D) were measured for each subject and these values were plotted for control, smoker and COPD subjects. Data displayed as median and whiskers min to max and analyzed using Mann-Whitney test. * p≤0.05; ** p≤ 0.005

**FIGURE 2.** Cytosolic Ca²⁺ was measured in human air-liquid interface bronchial epithelium cultures, exposed or not to smoke of one cigarette (CS for Cigarette Smoke), from 6 control subjects, 8 smokers and 14 smokers with COPD, using Fura-2 AM. (A) Baseline cytosolic Ca²⁺ concentration. (B) Maximum peak responses after activation of store release by 1µM thapsigargin in the absence of extracellular calcium corresponding to ER-Ca²⁺ release and Ca²⁺ leak across plasma membrane (C) Maximum peak responses after reintroduction of 1.8mM CaCl₂ in extracellular space, corresponding to Ca²⁺ influx. Data displayed as median and whiskers min to max and analyzed using Mann and Whitney test. * p≤0.05.
**FIGURE 3.** Analysis of 55 gene expressions in the ALI epithelia of 6 control subjects, 8 smokers and 14 smokers with COPD. Principal Component Analysis (PCA) was performed to identify genes which have "outlier" behavior. PCA was done regarding patient space definition in order to identify outlier in gene description. No transformation have been done on the data used for the PCA. (A) Variable factor map of PCA analysis, (B) individual factor map of PCA analysis. PCA analysis highlighted 3 genes which have "outlier" comportment: SERCA2, TRPV4 and ORAI3. (C) Real time (q)PCR of SERCA2, (D) TRPV4 and (E) ORAI3 expression relative to GAPDH in 6 control subjects, 8 smokers and 14 smokers with COPD. (F) a) Expression of ORAI3 and β actin protein in total cell lysates using western immunoblot in 1 representative experiment; b) ORAI3 protein expression normalized to β actin expression.

Data were plotted as median and whiskers min to max and analyzed using Mann and Whitney test. \*p\le0.05, \*\*p\le0.005.

**FIGURE 4.** ORAI involvement in Ca²⁺ signaling. Cytosolic Ca²⁺ was measured using fura-2 AM in 4 control subjects, 4 smokers and 3 smokers with COPD with or without 30 min pre-incubation of 10µM GSK 7975A. (A) Baseline Ca²⁺ concentration. GSK 7975A did not affect steady state cytosolic Ca²⁺ concentration. (B) Ca²⁺ influx recorded after ER-Ca²⁺ release by 1µM thapsigargin stimulation. GSK 7975A decreased Ca²⁺ entry in control subjects and had no effect on smokers with and without COPD. Data were plotted as median and whiskers min to max. The comparisons of the responses with and without GSK 7975A were analyzed using Wilcoxon signed rank test. \*p\le0.05.

**FIGURE 5.** ORAI is involved in ciliary beats. (A and B) steady state percentage of epithelial surface covered by beating cilia tufts and ciliary beat frequencies respectively in 8 control
subjects, 7 smokers and 5 smokers with COPD. Data were represented with scatter plot and analyzed with Mann and Whitney test. (C and D) epithelial surface covered by beating cilia tufts and ciliary beat frequencies respectively in 3 control subjects, 3 smokers and 3 smokers with COPD before (●) and after (■) 30min preincubation of 2mM EGTA. (E and F) epithelial surface covered by beating cilia tufts and ciliary beat frequencies respectively in 8 control subjects, 7 smokers and 5 smokers with COPD before (●) and after (■) 30min preincubation of 10µM GSK 7975A. Data were plotted as individual data before and after GSK7975A and comparisons were analyzed with Wilcoxon signed rank test.*p≤0.05, **p≤0.01. Three replicates of each condition for each subject were recorded.

**FIGURE 6.** GSK7975A did not affect Muc5ac and IL8 secretion. (A,B,C) Dot blot assay to measure Muc5AC secretion using mouse primary antibodies that react specifically with Muc5AC protein. (A) Representative picture of Muc5AC dot blot of 6 control subjects, 6 smokers and 4 smokers with COPD with or without 10µM GSK 7975A preincubation (B) Steady state Muc5AC secretion in ALI culture of controls and smokers with and without COPD. Data were plotted as median and whiskers min to max and analyzed with Mann and Whitney test. (C) Effect of 24H preincubation of 10µM GSK 7975A on Muc5AC secretion. Bar graph represent ratio of Muc5AC secretion after GSK 7975A preincubation/Muc5AC secretion before GSK 7975A incubation. Data were analyzed with Wilcoxon signed rank test in comparison to 1. (D,E) IL8 secretion measured by ELISA assay. (D) Steady state IL8 secretion in 6 control subjects, 7 smokers and 4 smokers with COPD. Data were plotted as median and whiskers min to max and analyzed with Mann and Whitney test. (E) Effect of 24H preincubation of 10µM GSK 7975A on IL8 secretion. Bar graph represent ratio of IL8 secretion after GSK 7975A preincubation/IL8 secretion before GSK 7975A incubation. Data were analyzed with Wilcoxon signed rank test in comparison to 1.**P≤0.005.
**FIGURE 7.** ORAI is not involved in epithelium reparation. ALI bronchial epithelium was damaged with a pipette tip and area of wound was measured just after and 24 hours after the scratch. (A) representative pictures of damaged epithelium just after the scratch (0H), 24 hours after the scratch (24H) and 24 hours after the scratch with 24 hours preincubation with 10µM GSK7975A. (B) % of reparation of ALI bronchial epithelium from 6 control subjects, 7 smokers and 10 smokers with COPD at 24 hours post scratch, with or without GSK7975A preincubation, compared to initial wound. Data were plotted as median and whiskers min to max and analyzed with Mann and Whitney test for the % of reparation between controls, smokers and COPD, and with Wilcoxon signed rank test between with and without GSK 7975A comparison. \*p≤0.05, \*\*p≤0.005.
<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>

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<figure><img src="image_10.png" /><figcaption>Figure 7
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## ONLINE DATA SUPPLEMENT

# Bronchial epithelial calcium metabolism impairment in smokers and COPD: decreased ORAI3 signaling

Aurelie Petit<fnref n="1" />, Lucie Knabe<fnref n="1" />,<fnref n="2" />, Kamel Khelloufi<fnref n="3" />, Myriam Jory<fnref n="4" />, Delphine Gras<fnref n="5" />, Yann Cabon<fnref n="6" />, Malcolm Begg<fnref n="7" />, Sylvain Richard<fnref n="2" />, Gladys Massiera<fnref n="4" />, Pascal Chanez<fnref n="5" />, Isabelle Vachier<fnref n="1" />, Arnaud Bourdin<fnref n="1" />,<fnref n="2" />,*

## Material and methods

*Materials:* Cell culture media and components were purchased from Lonza (Basel, Switzerland), Fura-2 AM was purchased from Euromedex (Souffelweyersheim, France) and thapsigargin was purchased from Tocris-Biotechne (Lille, France), All prep DNA/RNA/protein Mini Kits were purchased from Qiagen (Courtaboeuf, France), SYBR Green was purchased from Roche Diagnostics GmbH (Mannheim, Germany), primary antibodies against ORAI3 and beta actin were purchased from Santa Cruz biotechnology (Heidelberg, Germany), primary antibodies

<footnote n="1">¹Department of Respiratory Diseases and Addictology, Hôpital Arnaud de Villeneuve, CHU Montpellier, France.</footnote>

<footnote n="2">²INSERM U1046, CNRS UMR 9214, University of Montpellier, Montpellier, France.</footnote>

<footnote n="3">³Aix Marseille University, CNRS, CINaM UMR7325, Marseille, France</footnote>

<footnote n="4">⁴Laboratoire Charles Coulomb (L2C), UMR5221 CNRS-UM F34095, Montpellier, France</footnote>

<footnote n="5">⁵Aix Marseille University, APHM, CV2N, Inserm U1263 INRA 1260, Clinique des bronches allergies et sommeil, Marseille, France.</footnote>

<footnote n="6">⁶Department of Medical Information, Montpellier University Hospital, Montpellier, France.</footnote>

<footnote n="7">⁷Refractory Respiratory Inflammation DPU, Respiratory TAU, GlaxoSmithKline, Stevenage, UK.</footnote>
against Muc5AC and secondary antibodies were purchased from Fisher Scientific (Illkirch, France). GSK-7975A was a generous gift from Malcom Begg at GlaxoSmithKline (Stevenage, United Kingdom). Human IL8 elisa kits were purchased from Eurobio (Courtaboeuf, France). All other drugs and chemicals were purchased from Sigma Aldrich (Schnelldorf, Germany).

*Measurement of calcium signals using fura-2:* Fura-2 is a ratiometric and sensitive indicator dye for measuring cytosolic Ca²⁺. Ratiometric measurements of cytosolic Ca²⁺ signals significantly reduce the effects of differences in dye loading, leakage, and photobleaching and minimize problems with Ca²⁺ measurements in cells of uneven thickness, such as primary cultures of airway epithelia. Polarized 28 day-old cultures of human bronchial epithelia are loaded with 2.5µM fura-2/AM plus 0.02% Pluronic 127 by exposing their mucosal and serosal compartments to 0.5 and 1 ml, respectively, of Locke buffer (140mM NaCl, 5mM KCl, 1.2mM KH2PO4, 1.2mM MgSO4, 10mM HEPES, 10mM glucose and 1.8mM CaCl₂) containing Fura-2 and Pluronic 127 and incubating them at 37°C for 30 min in 5% CO₂/95% O₂. Cultures are subsequently washed with Locke buffer and kept in Locke buffer (only at the serosal compartment) for ~20–30 min to allow for de-esterification of fura-2 by endogenous esterases.

Buffers and drugs were either pre-incubated (10µM GSK-7975A and Locke buffer 0 Ca²⁺+2mM EGTA) or applied directly (1µM thapsigargin, 1.8 mM CaCl₂) on top of the imaged cells. Images were captured digitally every 2 s with a cooled CCD camera (Photometrics ; Roper Scientific, Evry, France). Cells loaded with Fura-2 were illuminated by excitation with a dual UV light source at 340 nm (Ca²⁺-bound) and 380 nm (Ca²⁺-free) using a lambda DG-4 excitation system (Sutter Instrument Company, Novato, CA, USA). Fluorescence emission was measured at 510 nm and analyzed (Metafluor software, Universal Imaging Corporation, Downingtown, PA,
USA). Changes in [Ca²⁺]ᵢ were deduced from variations in the F340/F380 ratio after correction for background and dark currents. Data were averaged with n representing the number subjects.

Ca²⁺ release from intracellular stores was evaluated by switching the normal Locke buffer to a Ca²⁺-free EGTA buffer and measuring the cytosolic Ca²⁺ increase induced by 1µM thapsigargin.

Ca²⁺ influx was evaluated by measuring the increase in cytosolic Ca²⁺ induced by addition of 1.8mM of CaCl₂ after thapsigargin effect. First experiments were conducted to compare Ca²⁺ influx when Ca²⁺ was added at two different time-points. First, Ca²⁺ was added at the maximum increase of cytosolic Ca²⁺ induced by thapsigargin (Figure E1 red data). Secondly, Ca²⁺ was added after thapsigargin Ca²⁺ increase dropped back to basal level (Figure E1 blue data).

Roughly, thapsigargin induced similar Ca²⁺ influx whenever it was added (Figure E1B). Therefore, in this study, to evaluate Ca²⁺ influx, we always added Ca²⁺ at the maximum increase of thapsigargin-induced cytosolic Ca²⁺.

*Gene expression analysis:* Total RNA from 28 day-old ALI cultures was extracted using All prep DNA/RNA/protein Mini kits (Qiagen), and 0.4µg of RNA was reverse transcripted with verso one step RT-PCR kits (Thermofischer scientific). Real-time PCR was performed using a Lightcycler DNA master SYBR green (Roche applied science). The comparative Ct method for relative quantification of gene expression was used (2ΔCt, where ΔCt represents the difference in the threshold cycle between the target and control genes). Data were normalized to GAPDH mRNA levels. Primer sequences are detailed thereafter:
<table><caption>Table E1. Primer sequences for plasma membrane calcium signaling genes.</caption><thead><tr><th>Plasma membrane GENES</th><th>Forward (5'-3')</th><th>Reverse (5'-3')</th><th>Size (bp)</th></tr></thead><tbody><tr><td>hGAPDH</td><td>CCATCTTCCAGGAGCGAG</td><td>CTTGAGGCTGTTGTCATACT</td><td>212</td></tr><tr><td>hTRPM1</td><td>GCACAGGTGTTATCAGCCA</td><td>GAGCACAGAGAGCTTACTTAGA</td><td>185</td></tr><tr><td>hTRPM2</td><td>TACATCGACGGTGTGAACT</td><td>TGTTGGTGAAGAGCAGGTAGA</td><td>160</td></tr><tr><td>hTRPM3</td><td>CAGGCAAGCCATCCTTT</td><td>GAGGGTCTATCTGGTCCG</td><td>116</td></tr><tr><td>hTRPM4</td><td>CGCCCTTTATCGTCATCT</td><td>CTCTCCCGCTTGTCCCTA</td><td>199</td></tr><tr><td>hTRPM5</td><td>CCGAGAAGCAGTTTAAGGAGA</td><td>AAGTCATACACGGTGAGC</td><td>121</td></tr><tr><td>hTRPM6</td><td>GGCTCTTCAGAAATAGGGC</td><td>CTGTTATGATTGGCACCTGG</td><td>115</td></tr><tr><td>hTRPM7</td><td>CAGCACAGCCCTACCGA</td><td>CTTGGTTTCTGGATCATCAATGTCTA</td><td>101</td></tr><tr><td>hTRPM8</td><td>GTATTCGGCTCCACTCTTC</td><td>TCTTGGGTCCTAAGTTTCTGC</td><td>130</td></tr><tr><td>hTRPV1</td><td>AGTGGGACAGATTCGTCA</td><td>CCAGTAACTCGGAAATAGTCTC</td><td>154</td></tr><tr><td>hTRPV2</td><td>GACACCGAATGGTCGTT</td><td>GTAGGCTGATGGTAGGCA</td><td>142</td></tr><tr><td>hTRPV3</td><td>TCCCAAAGACAACAAGGACT</td><td>GGTGAGGATGACATAGGTGAT</td><td>160</td></tr><tr><td>hTRPV4</td><td>GTGGAACTGAACAAGAACTCGAA</td><td>CTGAAATGCGGCTGGACTA</td><td>188</td></tr><tr><td>hTRPV5</td><td>GACCTGTCTGTTCTTAGGCAA</td><td>TTGTCATAGAGGGCTGCT</td><td>104</td></tr><tr><td>hTRPV6</td><td>CAGGACCAATAACCGCAC</td><td>CTGAAGATGTCTGGAACCTCTA</td><td>165</td></tr><tr><td>hTRPC1</td><td>CGTAGATGTGCTTGGGAG</td><td>TCCATTAGTTTCTGACAACCG</td><td>102</td></tr><tr><td>hTRPC3</td><td>TTACCTCTCATTGTCCAGCG</td><td>CTTCCGATAGTCATTCTTGAACTC</td><td>109</td></tr><tr><td>hTRPC4</td><td>CTTACCAACTGATTGCTGAC</td><td>GACATTGAAGGGAGTAGGC</td><td>110</td></tr><tr><td>hTRPC5</td><td>CAAGACATCTCCAGCTTTTCG</td><td>CCACCACTGCCATCATTATTATC</td><td>125</td></tr><tr><td>hTRPC6</td><td>CAGGTTGGGCACAATAAACA</td><td>CTTCGTTCACTTCATCACTCT</td><td>163</td></tr><tr><td>hTRPC7</td><td>AGGCTTACGTCAACAGTCTA</td><td>ACCTTCAAATCGGTCAGATG</td><td>214</td></tr><tr><td>hTRPA1</td><td>GAAGACAAGTCCTGCCGA</td><td>GGATGATTGAGAAGCTCTATGC</td><td>164</td></tr><tr><td>hTRPML1</td><td>CCCTATCATGTGAAGTTCCG</td><td>TAAAGGTAGAGCTGGGAGAAGA</td><td>152</td></tr><tr><td>hTRPML2</td><td>GTGGGAGTTCATCAACGG</td><td>ACCCAAACCAAGAGCGTA</td><td>156</td></tr><tr><td>hTRPML3</td><td>AATCTGCTATGGCAATCTGT</td><td>AGTCCAGTGTTAAGTTCAGTTTAT</td><td>171</td></tr><tr><td>hTRPP2</td><td>GTTCTTTACCACGTCCCAT</td><td>GGTCCACTCGTCTCACC</td><td>162</td></tr><tr><td>hTRPP3</td><td>AGTCTACAATGCCAATATCAATCT</td><td>TGACCTCACAGCCAACG</td><td>155</td></tr><tr><td>hTRPP5</td><td>AGTTCCCAGAGTTCGTCA</td><td>TTCGGTAAACACCAAGAAATCC</td><td>209</td></tr><tr><td>hTMEM16A</td><td>TGATTAAGCAGGAAACAGCAC</td><td>CCCATAAGGAAGATGAACAAC</td><td>113</td></tr><tr><td>hNCX1</td><td>CATTTGCGTGTCTTCTTTGTGA</td><td>GTCTCCTATCCGCTACCC</td><td>169</td></tr><tr><td>hNCX2</td><td>AGACCCGTTACCCACTAC</td><td>CAGTCAGAGACCTTATTAGCC</td><td>164</td></tr><tr><td>hNCX3</td><td>TTCTGCGGACATCAGGT</td><td>CCTTTCGTATTCCCTCCTCATCT</td><td>176</td></tr><tr><td>hCACNA1A</td><td>CCCGAGAATAGCCTTATCGTCA</td><td>GTTGGCGTTTTTGTTCACTT</td><td>153</td></tr></tbody></table>
<table><tr><td>hCACNA1B</td><td>ACATCAATACCATCAAGTCTCTG</td><td>CCGCAATGACGGCAAAT</td><td>174</td></tr><tr><td>hCACNA1C</td><td>CCAACCATTGTGTATGCCC</td><td>GCCAGTTACCTTTCCATTAGA</td><td>100</td></tr><tr><td>hCACNA1D</td><td>CCATTTCCCTCTGAGCACTAC</td><td>CATTTTTACCAGCATCTCGCA</td><td>107</td></tr><tr><td>hCACNA1E</td><td>CACCCACCTCCTCTACTA</td><td>TTGCCCAGACCACTTCAAAGATA</td><td>164</td></tr><tr><td>hCACNA1F</td><td>GAAACTAACAAAGCCACGATG</td><td>GCCCAAAGGAGAGTGAAT</td><td>109</td></tr><tr><td>hCACNA1G</td><td>ACTGAAACCCATCGGCAA</td><td>GATGTTCCTGGTATCCTCG</td><td>124</td></tr><tr><td>hCACNA1H</td><td>GAGTTTTGCTACCAGCCGA</td><td>CCCAGCGAGTGTCCCTGTAA</td><td>158</td></tr><tr><td>hCACNA1I</td><td>CTTCTTCATCTTTGGCATCATAG</td><td>CCCATTATCCCATTGTCGC</td><td>180</td></tr><tr><td>hCACNA1S</td><td>CAGTTCATCCGACATTGGAG</td><td>CTGAGGCGATAGACAGG</td><td>130</td></tr><tr><td>hORAI 1</td><td>TATCGTCTTCGCCGTCCA</td><td>GTCCTGTAAGCGGGCAAA</td><td>103</td></tr><tr><td>hORAI 2</td><td>GGGTAGTTCAAGACCAAAGT</td><td>CAACGCTCTTAGCAACACA</td><td>112</td></tr><tr><td>hORAI3</td><td>CATCAGTCTGTCCCTTGG</td><td>GGAAATGGTGGCTAAAGTCA</td><td>133</td></tr></table>
<table><caption>Table E2. Primer sequences for endoplasmic reticulum calcium signaling genes.</caption><thead><tr><td>ER GENES</td><td>Forward (5'-3')</td><td>Reverse (5'-3')</td><td>Size (bp)</td></tr></thead><tbody><tr><td>hITPR 1</td><td>GGCGTGTTCAGGGTTTAC</td><td>GACTTGAGAAAGAGGTTGTTGAC</td><td>155</td></tr><tr><td>hITPR 2</td><td>CAAGCCTGTGGGTATTCG</td><td>CAGGATGACTGCTCGGT</td><td>181</td></tr><tr><td>hITPR 3</td><td>TGCTCAAGTCCTCAGCG</td><td>CAGTAGTGGTGAAGAGCC</td><td>183</td></tr><tr><td>hSERCA 1</td><td>CGCCAGCTAATGAAGAAGGA</td><td>CACATAGTTACAGCGGTCG</td><td>159</td></tr><tr><td>hSERCA 2</td><td>GTGACAAAGTTCCTGCTGATA</td><td>GTGTGCTTGATGACAGAGAC</td><td>106</td></tr><tr><td>hSERCA 3</td><td>CGACTCGGCTCTGGACTA</td><td>GCTGCTTGATGACCGTGTTA</td><td>170</td></tr><tr><td>hRYR 1</td><td>GGCCCTACAAGACCTTTTC</td><td>GCCTTCTCTATCGTCCATTC</td><td>103</td></tr><tr><td>hRYR 2</td><td>CCTTTACAACCGGACTCG</td><td>CAGCCATCATTTCTGCCATA</td><td>140</td></tr><tr><td>hRYR 3</td><td>CAAAGGATACCCTGATGTCG</td><td>TTTCTTCCACACTCTCACTG</td><td>101</td></tr><tr><td>hSTIM1</td><td>TCCTCTCTTGACTCGCCATA</td><td>TCCTTGGAGTAACGGTTCTG</td><td>111</td></tr><tr><td>hSTIM2</td><td>TCCCGACTGTGTAGGTCT</td><td>ATGATGTGTGGCGAGGTTTA</td><td>143</td></tr></tbody></table>

*Analysis of ORAI3 protein expression:* Protein extracts from 28 day-old ALI cultures were resolved by SDS-PAGE on a 10% polyacrylamide gel, immunoblotted onto nitrocellulose membranes (Bio-Rad, Hercules, CA) and visualized by fluorescence using odyssey protocols. The following antibodies were used: anti-rabbit ORAI3 (Santa Cruz) and anti-mouse beta-actin (Santa Cruz). Membranes were scanned and densitometric measurements were performed using ImageJ software (NIH). The optical density of ORAI3 bands was normalized to that of beta actin. ORAI3 protein levels were expressed as the ratio of their optical density relative to that of beta actin.

*Measurement of ciliary activity:* The percentage of epithelial surface covered by beating cilia and the mean ciliary beat frequency were determined by transmission light microscopy (Nikon Eclipse Ti microscope at 37°C and 5% CO₂) on 28 day-old ALI cultures from 8 control subjects, 7 smokers and 5 smokers with COPD, before and after either DMSO (diluted 1/1000, 30 min)
GSK-7975A (10µM, 30 min) or EGTA (2mM, 30 min, for 3 control, 3 smoker and 3 COPD subjects) incubation.

In each culture well, the central zone and two opposite side zones, each one 255µm² in area, were observed (x60 objective). In each zone, ciliary motion was recorded at 250 fps for 3 seconds with a Photron SA4 camera. Using Fiji software, the average intensity of each pixel obtained from all images in the stack of 250x3 images was determined and subtracted, pixel per pixel, to each image of the stack, in order to enhance the visualization of mobile cilia. In each zone, the fraction of surface area covered by motile cilia tufts was determined. Ciliary beat frequencies were determined over 20 cycles on 5 randomly chosen ciliated cells. Cilia on a same ciliated cell were found to beat at the same frequency. Mean ciliary beat frequencies were determined by averaging the three zones.

*Measurement of Muc5AC secretion:* Muc5AC secretion was measured before and 24 hours after 10µM GSK-7975A in a dot blot assay using a primary mouse antibody (anti Muc5Ac (45M1) thermoFisher Scientific). Apical lavages from 28 day-old ALI cultures were spotted onto a nitrocellulose membrane. The membrane was then incubated in Odyssey blocking solution. After washing, the membrane was incubated with the primary antibody at room temperature for 2 hours. A secondary fluorescent anti-mouse antibody was then added to the membrane. Positive signals were detected using an Odyssey imager. Optical densities were measured with Alphaview software.

*Analysis of epithelial repair:* 28-day differentiated ALI culture bronchial epithelium from 6 control subjects, 7 smokers and 10 COPD subjects were considered for analysis. After the cells were incubated with or without 10µM GSK-7975A for 24 hours, the cells were scratched with a 100µl pipette tip and cultured in the presence or not of 10µM GSK-7975A for an additional 24 hours.
hours. Images of the wound were recorded using a microscope, immediately after wounding (0H) and after culturing (24H). The wound area of each epithelium was noted and subsequently measured using image J analysis software.
## Results

<table><caption>Table E3. The gene expression of plasma membrane proteins involved in calcium signaling.</caption><thead><tr><th>Plasma membrane GENES</th><th>CONTROLS<br>N=6<br>mean±SD</th><th>SMOKERS<br>N=8<br>mean±SD</th><th>COPD<br>N=14<br>mean±SD</th></tr></thead><tbody><tr><td>TRPM1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>TRPM2</td><td>0</td><td>0</td><td>0</td></tr><tr><td>TRPM3</td><td>0</td><td>0</td><td>0</td></tr><tr><td>TRPM4</td><td>0.0016 ± 0,0007</td><td>0.0018 ± 0,0004</td><td>0.0013 ± 0,0006</td></tr><tr><td>TRPM5</td><td>3.503E-6 ± 5,008E-6</td><td>4.398E-6 ± 7,409E-6</td><td>5.009E-6 ± 1,385E-5</td></tr><tr><td>TRPM6</td><td>3.687E-5 ± 1,875E-5</td><td>5.543E-5 ± 3,795E-5</td><td>5.840E-5 ± 3,939E-5</td></tr><tr><td>TRPM7</td><td>0.0058 ± 0,0012</td><td>0.0045 ± 0,0018</td><td>0.0033 ± 0,0017</td></tr><tr><td>TRPM8</td><td>0.00015 ± 0,00020</td><td>2.807E-5 ± 3,214E-5</td><td>2.258E-5 ± 2,836E-5</td></tr><tr><td>TRPV1</td><td>0.0004 ± 0,00039</td><td>0.0003 ± 0,00023</td><td>0.0002 ± 0,00016</td></tr><tr><td>TRPV2</td><td>3.566E-6 ± 7,399E-6</td><td>2.910E-6 ± 2,580E-6</td><td>2.348E-6 ± 3,157E-6</td></tr><tr><td>TRPV3</td><td>6.383E-5 ± 5,440E-5</td><td>2.922E-5 ± 1,962E-5</td><td>2.785E-5 ± 3,426E-5</td></tr><tr><td>TRPV4</td><td>0.037 ± 0,0338</td><td>0.013 ± 0,0136</td><td>0.023 ± 0,0308</td></tr><tr><td>TRPV5</td><td>0</td><td>0</td><td>0</td></tr><tr><td>TRPV6</td><td>0.00076 ± 0,00055</td><td>0.00031 ± 0,00026</td><td>0.00054 ± 0,00067</td></tr><tr><td>TRPC1</td><td>0.00037 ± 0,00025</td><td>0.0006 ± 0,00046</td><td>0.00036 ± 0,00030</td></tr><tr><td>TRPC2</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>TRPC3</td><td>0</td><td>0</td><td>0</td></tr><tr><td>TRPC4</td><td>6.411E-7 ± 9,599E-7</td><td>5.1009E-7 ± 8,340E-7</td><td>1.575E-6 ± 2,544E-6</td></tr><tr><td>TRPC5</td><td>0.00020 ± 0,00042</td><td>0.00024 ± 0,00066</td><td>3.33E-5 ± 4,730E-5</td></tr><tr><td>TRPC6</td><td>7.181E-5 ± 12,69E-5</td><td>6.74E-5 ± 7,141E-5</td><td>8.286E-5 ± 8,069E-5</td></tr><tr><td>TRPC7</td><td>0</td><td>0</td><td>0</td></tr><tr><td>hTRPA1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>hTRPML1</td><td>0.0025 ± 0,0010</td><td>0.0027 ± 0,0013</td><td>0.0036 ± 0,0021</td></tr><tr><td>hTRPML2</td><td>7.2176E-5 ± 10,59E-5</td><td>3.4775E-5 ± 3,262E-5</td><td>4.8786E-5 ± 4,483E-5</td></tr><tr><td>hTRPML3</td><td>0.00066 ± 0,00078</td><td>0.00045 ± 0,00024</td><td>0.00063 ± 0,00063</td></tr><tr><td>hTRPP2</td><td>0.00508 ± 0,00441</td><td>0.00297 ± 0,00096</td><td>0.00350 ± 0,00241</td></tr><tr><td>hTRPP3</td><td>0</td><td>0</td><td>0</td></tr><tr><td>hTRPP5</td><td>0</td><td>0</td><td>0</td></tr><tr><td>hTMEM16A</td><td>0.021 ± 0,0517</td><td>0.036 ± 0,0999</td><td>0.033 ± 0,1303</td></tr><tr><td>hNCX1</td><td>1.592E-5 ± 1,683E-5</td><td>1.739E-5 ± 1,973E-5</td><td>2.407E-5 ± 2,055E-5</td></tr></tbody></table>
<table><tr><td>hNCX2</td><td>3.176E-6 ± 2,286E-6</td><td>4.889E-6 ± 4,260E-6</td><td>5.173E-6 ± 5,130E-6</td></tr><tr><td>hNCX3</td><td>1.921E-6 ± 2,280E-6</td><td>3.642E-5 ± 5,504E-6</td><td>4.182E-5 ± 1,094E-5</td></tr><tr><td>hCACNA1A</td><td>0</td><td>0</td><td>0</td></tr><tr><td>hCACNA1B</td><td>0</td><td>0</td><td>0</td></tr><tr><td>hCACNA1C</td><td>0</td><td>0</td><td>0</td></tr><tr><td>hCACNA1D</td><td>0</td><td>0</td><td>0</td></tr><tr><td>hCACNA1E</td><td>0</td><td>0</td><td>0</td></tr><tr><td>hCACNA1F</td><td>0</td><td>0</td><td>0</td></tr><tr><td>hCACNA1G</td><td>0</td><td>0</td><td>0</td></tr><tr><td>hCACNA1H</td><td>0</td><td>0</td><td>0</td></tr><tr><td>hCACNA1I</td><td>0</td><td>0</td><td>0</td></tr><tr><td>hCACNA1S</td><td>0</td><td>0</td><td>0</td></tr><tr><td>hORAI 1</td><td>0.0036 ± 0,0016</td><td>0.0023 ± 0,0011</td><td>0.0028 ± 0,0012</td></tr><tr><td>hORAI 2</td><td>0.0024 ± 0,0030</td><td>0.0013 ± 0,0005</td><td>0.0013 ± 0,0010</td></tr><tr><td>hORAI3</td><td>0.207 ± 0,162</td><td>0.038 ± 0,049</td><td>0.033 ± 0,042</td></tr></table>
<table><caption>Table E4. The gene expression of endoplasmic reticulum proteins involved in calcium signaling.</caption><thead><tr><th>ER GENES</th><th>CONTROLS<br>N=6<br>mean±SD</th><th>SMOKERS<br>N=8<br>mean±SD</th><th>COPD<br>N=14<br>mean±SD</th></tr></thead><tbody><tr><td>ITPR 1</td><td>0.0022 ± 0,0017</td><td>0.0023 ± 0,0022</td><td>0.0019 ± 0,0015</td></tr><tr><td>ITPR 2</td><td>0.00042 ± 0,00045</td><td>0.00065 ± 0,00084</td><td>0.00047 ± 0,00044</td></tr><tr><td>ITPR 3</td><td>0.0159 ± 0,0104</td><td>0.0153 ± 0,0133</td><td>0.0186 ± 0,0092</td></tr><tr><td>SERCA 1</td><td>0.00010 ± 0,00012</td><td>0.00013 ± 0,00025</td><td>0.00012 ± 0,00019</td></tr><tr><td>SERCA 2</td><td>0.0443 ± 0,0216</td><td>0.0649 ± 0,0376</td><td>0.0720 ± 0,0508</td></tr><tr><td>SERCA 3</td><td>0.00043 ± 0,00040</td><td>0.00017 ± 0,00016</td><td>0.00077 ± 0,00121</td></tr><tr><td>RYR 1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>RYR 2</td><td>0</td><td>0</td><td>0</td></tr><tr><td>RYR 3</td><td>0</td><td>0</td><td>0</td></tr><tr><td>STIM1</td><td>0.0160 ± 0,0200</td><td>0.0158 ± 0,0089</td><td>0.0229 ± 0,0111</td></tr><tr><td>STIM2</td><td>0.0146 ± 0,0106</td><td>0.0111 ± 0,0053</td><td>0.0126 ± 0,0084</td></tr></tbody></table>
<figure><img src="image_11.png" /><figcaption>Figure E1</figcaption></figure>

**FIGURE E1:** Cytosolic Ca²⁺ was measured in human air-liquid interface bronchial epithelium cultures from 6 COPD subjects using Fura-2 AM. (A) Representative traces of cytosolic Ca²⁺, in the absence of extracellular Ca²⁺, in response to thapsigargin (1µM) followed by adding of CaCl₂ (1.8mM). CaCl₂ was applied either at maximum cytosolic Ca²⁺ increase induced by thapsigargin (red curve) or after thapsigargin Ca²⁺ increase dropped back to basal level (blue curve) (B) Maximum response following Ca²⁺ supplementation were measured for each subject in the two conditions and were plotted. Data displayed as median and whiskers min to max and analyzed using Mann-Whitney test.