Abnormal spatial diffusion of Ca2+ in F508del-CFTR airway epithelial cells

Background In airway epithelial cells, calcium mobilization can be elicited by selective autocrine and/or paracrine activation of apical or basolateral membrane heterotrimeric G protein-coupled receptors linked to phospholipase C (PLC) stimulation, which generates inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG) and induces Ca2+ release from endoplasmic reticulum (ER) stores. Methods In the present study, we monitored the cytosolic Ca2+ transients using the UV light photolysis technique to uncage caged Ca2+ or caged IP3 into the cytosol of loaded airway epithelial cells of cystic fibrosis (CF) and non-CF origin. We compared in these cells the types of Ca2+ receptors present in the ER, and measured their Ca2+ dependent activity before and after correction of F508del-CFTR abnormal trafficking either by low temperature or by the pharmacological corrector miglustat (N-butyldeoxynojirimycin). Results We showed reduction of the inositol 1,4,5-trisphosphate receptors (IP3R) dependent-Ca2+ response following both correcting treatments compared to uncorrected cells in such a way that Ca2+ responses (CF+treatment vs wild-type cells) were normalized. This normalization of the Ca2+ rate does not affect the activity of Ca2+-dependent chloride channel in miglustat-treated CF cells. Using two inhibitors of IP3R1, we observed a decrease of the implication of IP3R1 in the Ca2+ response in CF corrected cells. We observed a similar Ca2+ mobilization between CF-KM4 cells and CFTR-cDNA transfected CF cells (CF-KM4-reverted). When we restored the F508del-CFTR trafficking in CFTR-reverted cells, the specific IP3R activity was also reduced to a similar level as in non CF cells. At the structural level, the ER morphology of CF cells was highly condensed around the nucleus while in non CF cells or corrected CF cells the ER was extended at the totality of cell. Conclusion These results suggest reversal of the IP3R dysfunction in F508del-CFTR epithelial cells by correction of the abnormal trafficking of F508del-CFTR in cystic fibrosis cells. Moreover, using CFTR cDNA-transfected CF cells, we demonstrated that abnormal increase of IP3R Ca2+ release in CF human epithelial cells could be the consequence of F508del-CFTR retention in ER compartment.


Introduction
The existence of distinct membrane localizations and multiple isoforms of inositol 1,4,5-trisphosphate (IP 3 ) receptors (IP 3 R) within the same cell type may explain the complex spatiotemporal patterns of Ca 2+ release from IP 3sensitive calcium pools in epithelial cells. In addition to requiring IP 3 , IP 3 R are regulated in a biphasic manner by direct interaction with Ca 2+ , i.e. activation at low concentrations (up to 0.3 μM) and inhibition at higher concentrations (0.5-1 μM) [1]. The different modes of interaction of IP 3 R with Ca 2+ are involved in the complex feedback regulation of the Ca 2+ release [2]. IP 3 R activity is also regulated by Ca 2+ -independent accessory proteins, Mg 2+ , redox potential and ATP [3]. Furthermore, a local Ca 2+ discharge by photolysis of NP-EGTA technique can activate the IP 3 Rs Ca 2+ release. For example, the type 3 IP 3 R remaining open in the presence of high Ca 2+ concentration, initiates a rapid, large and almost total release of Ca 2+ from intracellular stores [4]. These properties place IP 3 Rs at the heart of calcium signalling pathways.
Recent studies have demonstrated higher intracellular Ca 2+ mobilization in Cystic Fibrosis (CF) compared to normal human nasal [5] or bronchial [6] epithelia. Cystic Fibrosis is the most frequent lethal autosomal recessive genetic disease in Caucasian population. The most common mutation in CF is a deletion of phenylalanine at position 508 in the Cystic Fibrosis Transmembrane conductance Regulator protein (F508del-CFTR). F508del-CFTR protein is misfolded, trapped in the endoplasmic reticulum (ER) by the ER quality control (ERQC) [7] and subsequently submitted to proteasomal degradation [8].
In this report we monitored the cytosolic Ca 2+ transients using the flash photolysis technique to uncage caged Ca 2+ into the cytosol of nitrophenyl-EGTA (NP-EGTA) loaded human CF nasal epithelial CF15 cells [9], human CF tracheal gland CF-KM4 cells [10] and human non-CF tracheal gland epithelial MM39 cells [11]. We also used the membrane-permeable UV light photolysis caged IP 3 analogue (iso-Ins(1,4,5)P3/PM) to examine the consequence on the local IP 3 R Ca 2+ release of rescuing F508del-CFTR by the pharmacological corrector miglustat [12] and after culturing cells at low temperature [13].

Extraction of IP 3 R mRNA and reverse transcription
Total RNA was extracted using RNABle ® (Eurobio), according to the protocol provided by the manufacturer and mRNA was reverse transcribed to cDNA as described elsewhere [15]. The specific oligonucleotide primers used for each subtype of the IP 3 Rs are presented Table 1. The temperature cycling conditions were initial melting at 94°C for 5 min, annealing at 56°C for 2 min followed by 30 cycles of 72°C for 30 s, 94°C for 30 s, annealing of 56°C for 30 s and a final extension at 72°C for 5 min.

Quantification of IP 3 R mRNA by RT-PCR
Quantitative PCR was used to determine the copy numbers of IP 3 R1, IP 3 R2, and IP 3 R3 in mRNA extracted from CF15 cells in different conditions. The IP 3 R mRNA quantities were normalized against β-actin. Quantitative PCR were performed on the ABI Prism 7700. The specific oligonucleotide primer used for each subtype of the IP 3 Rs is presented Table 1. For β-actin-cDNA, the primers were 5'-TGTGGATCGGCGGCTC-3' and 5'-ACTCCTGCTTGCT-GCTGATCCAT-3' (900 nM for each primer). The probe taqman FAM used was 5'FAM-TGGCCTCGCTGTCCAC-CTTCCA-TAMRA3' (200 nM). The temperature cycling conditions were: initial melting at 94°C for 5 min, annealing at 56°C for 2 min followed by 30 cycles of 72°C for 30 s, 94°C for 30 s, annealing of 56°C for 30 s and a final extension at 72°C for 30 s. Each sample was analysed in triplicate. After PCR was completed, the FAM fluorescent signal (490 nm) was analysed and converted into a relative number of copies of target molecules. These results were expressed by threshold cycle value (Ct = number of necessary amplification cycle that emitted the fluorescent signal superior at non specific fluorescence). To allow comparison between different experimental conditions, uncaging pulses of the same intensity were delivered with 5% of 405 nm diode for 500 ms with tornado scanning mode in a region of interest of 10 pixels diameter (= 2 μm). Simultaneous scanner system of Olympus FV1000 station allows laser stimulation in a restricted region while recording Fluo-4 fluorescence images with no delay and high resolution. As shown on XY images, laser stimulation with 405 nm diode applied on a restricted region of interest (yellow circle in Fig. 1A) induced a localized Ca 2+ increase that propagated throughout the cell. For high time resolution, intracellular Ca 2+ images were acquired in a line scan mode during 3 s (XT image, Fig. 1B) with line scan defined in the center of stimulation region (XY reference image, Fig. 1A). 500 ms duration of laser stimulation was chosen for its efficacy to induce large response with no sign of bleach or saturation of cellular response. Typical intensity profile of Ca 2+ variation was then extracted from XT images with FV10-ASW v1.3 software within a 10 pixels width region to reduce noise (Fig. 1C). Intensity profiles were normalized by dividing the fluorescence intensity of each pixel (F) by the average resting value before stimulation (F0) to generate an (F-F0/F0) image. With this intensity profile, we compared the different Ca 2+ responses by measuring the area under the curve (AUC) and the peak value (Fig. 1C).

Caged IP 3 experiments
To activate directly the IP 3

Statistics
Results are expressed as mean ± SEM of n observations. Sets of data were compared with a Student's t test. Differences were considered statistically significant when P < 0.05. ns: non significant difference, * P < 0.05, ** P < 0.01, *** P < 0.001. All statistical tests were performed using GraphPad Prism version 4.0 for Windows (Graphpad Software) and Origin version 5.0.

Role of IP 3 receptors in local ER Ca 2+ mobilization in human epithelial cells
We first characterized IP3R isoforms in human nasal epithelial CF15 cells. Using reverse transcription-PCR technique, we found mRNA for the three isoforms of IP 3 R ( Fig. 2A). Moreover, confocal immunofluorescence microscopy studies of IP 3 Rs indicated for each isoform a punctiform and diffuse immunostaining in the cytoplasm of CF15 cells (Fig. 2B top images). No immunostaining of IP 3 Rs was detected when the primary antibodies were omitted ( Fig. 2B bottom images). Then, to directly investigate IP 3 R activity, we used the flash photolysis technique to uncage caged Ca 2+ into the cytosol of NP-EGTA loaded CF15 cells [17]. Because the capacity of IP 3 receptors to release Ca 2+ into the cytosol is influenced, in part, by the cytosolic local Ca 2+ concentration, a confined discharge of Ca 2+ by NP-EGTA photolysis induced an activation of Ca 2+ release by IP 3 receptors. To eliminate Ca 2+ influx, we performed all experiments in absence of extracellular Ca 2+ (Ca 2+ -free). As described in the method section, images were acquired in a line scan mode during 3 s (XT image) with CF15 cells cultured at 37°C (Fig. 3A). The corresponding normalized fluorescence and AUC are shown Fig. 3B (black line) and C (black bar). To study the contri-

Figure 1
Determination of localized Ca 2+ mobilization by Ca 2+ caged technique Figure 1 Determination of localized Ca 2+ mobilization by Ca 2+ caged technique. A Confocal XY images illustrating Ca 2+ release by photolysis of NP-EGTA molecule. The uncaging pulses were delivered with 5% of 405 nm diode for 500 ms with tornado scanning mode in a region of interest of 10 pixels diameter (yellow circle). Scale bars 25 μm. B XT images were obtained by acquisition in line scan mode (green line in A) during 3 s. C Typical intensity profile of Ca 2+ variation was extracted from XT images presented in B, the grey area represents the measure of area under the curve (AUC). The number 1 to 4 represented the Ca 2+ response induce by the photolysis at different time (in figure 1A and 1C). All the parameters automatically measured with a computer program developed in our laboratory under IDL 5.3 structured language were represented on the typical intensity profile (peak and kinetics parameters). Results are presented as mean ± SEM and the number of experiments is noted on each bar graph. * P < 0.05; ** P < 0.01*** P < 0.001; ns, non significant difference. bution of IP 3 receptors into the local Ca 2+ release in CF15 cells, we used 2-APB and decavanadate [18], two non specific inhibitors of IP 3 R isoforms. In these experimental conditions, we observed a decrease by more than 70% of the Ca 2+ response when we used either 100 μM 2-APB or 100 μM decavanadate ( Fig. 3A-C). To prevent the release of Ca 2+ by IP 3 Rs from the ER, we measured the response that had been only induced by the flash. We treated cells 2 h with 10 μM thapsigargin (TG) to release the whole Ca 2+ store. The light stimulation in presence of TG produced a very small response corresponding only to ~20% of the response obtained at 37°C (Fig. 3A-B). In presence of the receptor blockers, these Ca 2+ responses were similar to the response induced only by the photolysis flash (represented in Fig. 3C by a dashed black line). These experiments demonstrate that the total Ca 2+ response in human nasal epithelial CF15 cells is due to the activity of IP 3 receptors.

Characterization of IP 3 Rs isoforms in human nasal epithelial cells
To discriminate between the different isoforms of IP 3 Rs implicated in Ca 2+ release, we used two inhibitors of IP 3 R1 (caffeine, cyclosporine A) in absence of extracellular Ca 2+ (Fig. 3). Caffeine is known to inhibit the IP 3 R type 1 and to inhibit this isoform at millimolar concentrations [19]. In our hand, 20 mM caffeine induced an inhibition of Ca 2+ response limited to the peak intensity (Fig. 3B).
The Ca 2+ quantity mobilized in presence of caffeine decreased by 30% (Fig. 3C). We also compared the uncaged Ca 2+ response induced by UV flash photolysis in presence of cyclosporine A (CsA), an agent known to abolish type 1 IP 3 R [20]. Cyclosporine A induced a decrease of peak fluorescence intensity and a decrease of Ca 2+ quantity mobilization by 45% ( Fig. 3B-C). Since, we have shown previously the absence of ryanodine receptors in human nasal epithelial cells line [5], the fraction of Ca 2+ response not inhibited by cyclosporine A or caffeine probably arose from the two other isoforms of IP 3 R (type 2 and 3) activity.

Consequence on local IP3Rs Ca 2+ activity of rescuing F508del-CFTR in CF cells
To study the consequence of F508del-CFTR rescue on the IP 3 R activity, before loading with NP-EGTA, CF15 cells were either cultured at 27°C during 24 h or incubated 2 h with a culture medium containing 100 μM miglustat. We compared the mRNA quantity of each IP 3 R isoform by quantitative RT-PCR (Fig. 4A), and found no variation of mRNA for each IP 3 R isoforms whatever the experimental conditions (Fig. 4A). The activity of IP 3 receptors was then evaluated. Example of intracellular Ca 2+ XT images are provided for each experimental condition (Fig. 4B). By analysis of the XT images, we observed a decrease by ≈ 40% and 50% in temperature-(24 h at 27°C, grey trace and bar) and miglustat-corrected CF15 cells (2 h at 100 μM, green trace and bar), respectively, compared to uncor-rected CF15 cells (37°C, black trace and bar) (Fig. 4C-D). We used NB-DGJ, because this compound is not able to rescue the abnormal trafficking of F508del-CFTR [16]. It is remarkable that treating CF15 cells with NB-DGJ (2 h at 100 μM) did not modify the Ca 2+ response compared to untreated CF15 cells as shown by the XT images (Fig. 4B) and the histograms (blue trace and bar Fig. 4C-D). Fig.  4D also provides the corresponding statistical analysis for all these experiments. Therefore, these results show that the rescue of F508del-CFTR either by miglustat or by low temperature deeply affects the capacity of the ER to release Ca 2+ into the cytosol of CF15 cells. In each treatment condition, IP 3 R1 inhibition by 10 μM CsA induced a significant decrease of Ca 2+ response by 40% in control or NB-DGJ treated cells (Fig. 4E). In contrast, 10 μM CsA did not modify the Ca 2+ response in CF15 corrected cells (low temperature or miglustat treatments) (Fig. 4E).
To complement this study and to confirm our results, we used two other epithelial cell lines which have another tissue origin: the human tracheal gland serous CF cells (CF-KM4) and non CF cells (MM39). As in CF15 cells, the RT PCR technique shows the presence of IP 3 R1, IP 3 R2 and IP 3 R3 in both human tracheal CF-KM4 and MM39 cells (Fig. 5A). To confirm the exacerbated ER Ca 2+ release in CF cells, we also applied the NP-EGTA technique to examined IP 3 R Ca 2+ dependent activity in MM39 and CF-KM4 cells. The Ca 2+ responses (Fig. 5B-D) showed 40% increase of the Ca 2+ response in CF-KM4 cells versus non-CF MM39 cells (black and red traces and histograms, respectively). Figure 5B shows line-scan XT images recorded in the absence of extracellular Ca 2+ in MM39 cells and in CF-KM4 cells maintained either at 37°C, or at 37°C for 2 h in presence of miglustat or NB-DGJ. The Ca 2+ response in CF cells was decreased by ≈ 40% after miglustat treatment (Fig. 5B-E, green traces and histograms). The local Ca 2+ response obtained following miglustat treatment was similar to that obtained with the non-CF MM39 cells (Fig. 5C, red traces and histograms). As for CF15 cells, NB-DGJ did not induce any variation of Ca 2+ response in CF-KM4 cells compared to uncorrected CF-KM4 cells (Fig. 5D). In fact, the peak of the Ca 2+ responses was decreased in non CF MM39 cells and miglustat-corrected CF-KM4 cells compared to uncorrected CF-KM4 cells (Fig. 5C, E). Thus, correction of the abnormal F508del-CFTR trafficking by miglustat induces a profound modification of IP 3 R Ca 2+ dependent activity in CF cells.
Then, we measured the global ER Ca 2+ release (in absence of extracellular Ca 2+ ) by 100 μM histamine in control or NB-DGJ treated CF-KM4 or corrected CF-KM4 (by low temperature or miglustat), and on untreated or miglustattreated MM39 cells (Fig. 6A-B). These experiments show that the Ca 2+ response induced by histamine was decreased in CF-KM4 cells corrected either by temperature Modification of local stimulation of caged Ca 2+ in corrected F508del-CFTR CF15 cells (by 25%) or miglustat (by 30%), compared to uncorrected CF-KM4 cells (Fig. 6A-B). Example tracings are performed Figure 6A. Again, these ER Ca 2+ mobilizations are similar to that observed with MM39 cells. To emphasize the specificity of the effect of miglustat, we also noted that NB-DGJ treatment has no effect on histamine-ER Ca 2+ release (Fig. 6B). Furthermore, the histamine-ER Ca 2+ release in miglustat-treated MM39 cells was similar to the response observed in untreated MM39 cells (Fig. 6B). Therefore, the decrease of ER Ca 2+ release observed in miglustat corrected CF-KM4 cells is not a side effect of miglustat on Ca 2+ homeostasis but rather the consequence of F508del-CFTR ER escape.

Direct activation of the IP 3 Rs using cell-permeable IP3 in human tracheal gland cells
Calcium is known to directly activate IP 3 R and ryanodine receptors (RYRs), but the sensitivity of the IP 3 receptors to Ca 2+ depending on a process CICR (Ca 2+ increase Ca 2+ release) requires IP 3 [1]. However, in absence of agonist stimulation, the level of intracellular IP 3 remains very low. When we used 10 mM caffeine to activate specifically RYRs, we did not observe Ca 2+ mobilization, on the contrary of 100 μM histamine stimulation (Fig. 7A). These results indicate that RYRs are absent or not functional in these human epithelial cell models (CF15, CF-KM4 and MM39 cells). Then, the Ca 2+ response produced by the NP-EGTA photolysis was the consequence of the presence and activity of IP 3 Rs. This explains that the Ca 2+ increase observed is only measured during the UV photolysis (500 ms). To eliminate these limitation of the NP-EGTA technique, and to study, more directly, the IP 3 R activity, we examined the ER Ca 2+ release by UV light photolysis of a cell-permeable caged iso-Ins(1,4,5)P3/PM in absence of extracellular Ca 2+ . In CF-KM4 cells, preloaded with iso-Ins(1,4,5)P3/PM, short exposure (100 ms) to flash photolysis induced a biphasic increase of [Ca 2+ ] i . We observed an initial peak of Ca 2+ release which stabilized during 1 or 2 s, and an increase of Ca 2+ release by a propagation of this Ca 2+ response at the whole cell level (Fig. 7C). In MM39 and miglustat-treated CF-KM4 cells, the UV photolysis stimulated a biphasic increase of [Ca 2+ ] i , but the amplitude of the first peak and Ca 2+ mobilization was reduced compared to untreated CF-KM4 cells (Fig. 7C and 7D). Moreover, the second part of Ca 2+ response was stabilized, and the rise of Ca 2+ release was lower than the response measured for untreated CF-KM4 cells (Fig. 7C). We observed the [Ca 2+ ] i return to a basal concentration approximately after 15 to 20 s after the UV flash (not shown). To ensure that the response evoked by exposing the cells to UV light was not due to phototoxicity or to a non-specific effect, the experiments were repeated with CF-KM4 cells loaded with fluo-4 without iso-Ins(1,4,5)P3/PM. In this experimental condition, exposure to UV flash did not induce an increase in [Ca 2+ ] i (Fig.   7B). This experimental procedure confirms that the correction of the abnormal F508del-CFTR trafficking by miglustat induces a profound modification of IP 3 R Ca 2+ dependent activity in CF cells.

Consequence on IP 3 Rs Ca 2+ activity of F508del-CFTR ER retention in CF cells
Finally, we used cells derived from CF-KM4 that were stably transfected to achieve low-level expression of fulllength wild-type CFTR (wt-CFTR) (CF-KM4-reverted). These CF-KM4-reverted cells have been shown to have phenotypic correction of a wide range of CF phenotypes [14]. In fact, this cell line possesses both CFTR proteins: endogenous F508del-CFTR and transfected wild-type CFTR (wt-CFTR). When we measured the Ca 2+ mobilization induced by a solution of 100 μM histamine in absence of extracellular Ca 2+ , this Ca 2+ response was also similar to CF-KM4 cells (Fig. 6B). The Ca 2+ mobilization induced by UV photolysis of iso-Ins(1,4,5)P3/PM in CF-KM4-reverted was similar to CF-KM4 cells (Fig. 7C). The plasma membrane localization of wt-CFTR did not disrupt the sensitivity of IP 3 Rs to the photolysis of iso-Ins(1,4,5)P3/PM and to agonist response. To restore the endogenous F508del-CFTR trafficking, we treated these cells 2 h at 37°C with 100 μM miglustat. In this condition, the specific IP 3 R activity, measured by Ca 2+ response to agonist stimulation and by iso-Ins(1,4,5)P3/PM photolysis was reduced to the level measured in non CF cells ( Fig.  6B and 7C). This abnormal increase of IP 3 R Ca 2+ release in CF human epithelial cells compared to non CF cells appear thus to be the consequence of F508del-CFTR retention in ER compartment.

Morphology of the ER in non CF and CF cells
To begin to understand the cause of the ER Ca 2+ release abnormal in CF cells, we examined the ER morphology in our experimental conditions. In a first set of experiments, the ER structure was investigated by calreticulin immunofluorescence (Fig. 8A). Calreticulin is an intraluminal ER protein involved in Ca 2+ sequestration [21]. Figure 8A shows that the ER remains highly concentrated around the nucleus in untreated or NB-DGJ-treated CF-KM4 cells.
On the contrary, in MM39 and in miglustat-corrected-CF-KM4 cells, the ER is spreaded throughout the cells. No immunostaining of calreticulin was detected when the primary antibodies were omitted (data not shown). To verify whether this difference in ER morphology observed between CF and non CF cells is due to the ER structure or to a change of calreticulin localization, we also stained the ER with a specific fluorescent probes (ER tracker) (Fig.  8B). Again, the ER was also found highly concentrated around the nucleus in untreated and NB-DGJ-treated CF-KM4 cells, whereas in periphery of the cells the ER network was very thin. On the contrary, in non CF cells or corrected CF cells, the ER was clearly extended throughout the cell (Fig. 8B). Thus treatment of CF cells with the corrector miglustat induces an ER spreading throughout the cells.

What is the consequence of the ER Ca 2+ decreased on the CaCC activity?
Since intracellular Ca 2+ regulates the functionality of numerous proteins and because the ER Ca 2+ mobilization was decreased in miglustat-CF cells, we determined whether these changes in Ca 2+ signalling lead to changes in the Ca 2+ mediated Cltransport. The Ca 2+ -activated Clchannels (CaCC) are functionally expressed in many non excitable cells [22,23]. We performed iodide efflux experiments in untreated and miglustat-treated MM39 (Fig. 9A) and CF-KM4 (Fig. 9B) cells and stimulated the activity of CaCC by the Ca 2+ ionophore A23187. No variation was detected following the treatment of cells by miglustat (Fig.  9). Then we examined the activity of CaCC stimulated by ER Ca 2+ release using two different agonists (ATP and histamine). Again no difference was observed between untreated vs miglustat treated cells. Taken altogether, and in spite of the decreased ER Ca 2+ mobilization in miglustat-corrected-CF cells, the activity of CaCC remained unaffected by miglustat.

Discussion
Our study on the regulation of Ca 2+ signalling in human F508del-CFTR and in corrected CF cells reveals that (i) the ER morphology in uncorrected or corrected CF human tracheal gland cells compared to non CF human tracheal gland cells release of ER Ca 2+ store is dependent on the presence of the three isoforms of IP 3  We propose that Ca 2+ homeostasis in cystic fibrosis airway epithelial cells is disturbed and related to the retention in the ER of F508del-CFTR proteins.
Epithelium from trachea to distal intrapulmonary airways (bronchioles) presented positive immunoreactivity for all types of IP 3 Rs [24]. All three isoforms of IP 3 Rs are also expressed in Madin-Darby canine kidney cells, a well studied tight polarized epithelial cell type [25]. Thus, in epithelial cell models, multiple isoforms of IP 3 R appeared to be present in a single cell. In our epithelial models, we showed the presence of the three isoforms. In CF15 cells their localisation is comparable, i.e. diffuse in the cytoplasm of the cells. Moreover, no variation of IP 3 Rs mRNA was observed. The three subtypes of IP 3 R Ca 2+ release channels share basic properties but differ in term of regulation. Type 1 IP 3 R, with both Ca 2+ -dependent activation and inhibition, is well suited for establishing Ca 2+ oscillations [1, 26,27], where the frequency of Ca 2+ transients can be modulated when IP 3 concentrations are increased [27,28]. The effects of CsA are lower in CF15 corrected cells than in uncorrected CF15 cells; it suggests that the CsA-sensitive IP 3 R participation in Ca 2+ response was decreased in CF15 corrected cells.
Human CF primary bronchial epithelial cells and respiratory cell lines were reported to produce an exaggerated proinflammatory cytokine response associated with an activation of NF-κB [29][30][31]. Intracellular Ca 2+ is known to play a central role in production and secretion of Il-8 [32,33]. The IL-1β stimulation induces a prolonged [Ca 2+ ] i in IB3-1 cells which was correlated to NF-κB activation [34]. The deregulation of IP 3 R Ca 2+ release observed in human nasal and tracheal epithelial cells could be implicated in increasing inflammatory response observed in numerous CF cell lines in particular in CF epithelial cells [6,34].
The apical ER network is expended in human CF bronchial epithelial cells compared to ER volume in human non CF bronchial epithelial cells [6]. In this present study, the ER staining (by calreticulin immunostainning or ER tracker probe) shows that the ER structure is highly different in CF compared to non CF or CF-corrected cells. The ER volume seems to be concentrated around the nucleus in CF cells and expanded throughout the cytoplasm of non CF and CF-corrected cells. This expansion could be responsible for the variation in IP 3 R Ca 2+ dependent activity observed in this present study. Indeed, the display of ER web could induce probably an augmentation of distance between IP 3 receptors which would induce a decrease in the propagation of the Ca 2+ response. Furthermore, the F508del-CFTR correction is causing a potential redistribution of IP 3 Rs at the ER membrane. We believed that in corrected CF and non CF cells, IP 3 Rs are more distant from each others, leading to reduce propagation of the Ca 2+ wave. Then IP 3 Rs clustering of the ER could facilitate the formation of highly sensitive Ca 2+ release sites, thereby stimulating the Ca 2+ wave initiation and propagation [35]. For example, in maturation of oocytes, the modifications in ER clusters are accompanied by an increase in the sensitivity of Ca 2+ release by IP 3 [36,37]. Moreover, in CF cells IP 3 Rs are closer to each other and the photolysis induces a long Ca 2+ wave due to a better propagation. Our interpretation of the data obtained with CF-KM4-reverted cells is that the IP 3 Rs deregulation is not due to the CFTR absence at the plasma membrane but is more likely due to the abnormal ER F508del-CFTR retention. The F508del-CFTR escape of ER by pharmacological corrector treatment induces the normalization of IP 3 R Ca 2+ release.
In the airways, pleiotropic consequences accompanied the production of F508del-CFTR protein generating vicious cycle of airway obstruction, infection, and inflammation at the origin of most of the morbidity and mortality in cystic fibrosis. The pro-inflammatory mediator bradykinin triggers Ca 2+ mobilization [38,39] and induces interleukin-8 secretion in non-CF and CF human airway epithelia [40,41]. A mechanism has been proposed to explain the increase of Ca 2+ signals at the apical membrane in human CF airway epithelia; it results from the expansion of the ER Ca 2+ store compartment rather than from a greater number of purinergic receptors [6,40]. We can confirm these results in our cellular models because we observed that the kinetics of activation of the Ca 2+ response obtained in NB-DNJ-treated CF cells do not mimic the effect of the inhibitors of the IP 3 R and we observed a variation of the ER morphology between non CF or corrected and uncorrected epithelial cells.
We recently provided evidence that the pharmacological correction of abnormal trafficking of F508del-CFTR [12] induces a restoration of Ca 2+ mobilization in CF cells [5].
Here, we showed reduction of the inositol 1,4,5-trisphosphate receptors (IP 3 R) dependent-Ca 2+ response following two different correcting treatments compared to uncorrected cells in such a way that Ca 2+ responses (CF+treatment vs wild-type cells) were normalized. Altogether, these results suggest reversal of the IP 3 R dysfunction in F508del-CFTR epithelial cells by a pharmacological correction of the abnormal trafficking of F508del-CFTR in cystic fibrosis cells.
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