Introduction

Pendrin (SLC26A4) is a member of the SLC26 (solute carrier 26) family and functions as an anion exchanger for monovalent anions including iodide, chloride, bicarbonate, hydroxide, and thiocyanate [41, 53]. This protein is mainly expressed in the inner ear, thyroid, and kidney [32, 43, 51]. A relatively lower expression of pendrin has been described in other organs such as the mammary gland, testis, endometrium, liver, and lung [15, 17, 53]. Lung expression of pendrin was first described in transgenic mouse models of asthma induced by the overexpression of the Th2 cytokine interleukin-13 (IL13) [31]. A correlation between pendrin expression and several respiratory disorders such as asthma, chronic obstructive pulmonary disease (COPD), rhinovirus, and Bordetella pertussis infection has been described [27, 39, 49]. In the respiratory system, pendrin reabsorbs Cl− against HCO3 − or thiocyanate (SCN−), which is oxidized by H2O2 in hypothiocyanite, having an antimicrobial activity in airway surface liquid (ASL) [19, 41]. Balanced chloride and bicarbonate transport is important to modulate fluid secretion and to pH regulation. In fact, loss of activity of cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel that facilitates HCO3 − transport, decreases airway surface pH, thus preventing bacterial killing [42]. Within the respiratory epithelium, airway surface hydration depends closely on the expression of selective water and ion channels [21, 30]. Deregulated expression and/or channel activity may contribute to the lung diseases [3]. COPD and asthma both damage lung functions. COPD is the third leading cause of death worldwide after ischaemic heart disease and stroke [35] and is associated with obstruction of the lung due to a chronic and irreversible inflammatory condition [52]. Asthma is a heterogeneous syndrome displaying different clinical phenotypes characterized by reversible lung obstruction, bronchial hyperresponsiveness, and airway inflammation [36].

Besides IL4 and IL5, IL13 plays a crucial role in determining airway inflammation and hyperresponsiveness [29], and increased airway narrowing is the main symptom characterizing these disorders. However, the molecular mechanisms promoting airway inflammation and hyperresponsiveness remain unclarified. Abnormal contractile properties of airway smooth muscle cells play a pivotal role in the development of these diseases. IL13 is a key mediator of Rho-dependent calcium sensitization in smooth muscle contraction [9]. Importantly, IL13 can act also on airway epithelial cells, where it stimulates the signal transducer and activator of transcription factor 6 (STAT6) [31]. STAT6 activation, via IL13 pathway, results in a significant increase of the expression level of pendrin [39, 40].

Management of the abovementioned respiratory diseases is very complex due to the heterogeneity of responses to treatment. Antibodies targeting the IL13 receptor have been tested in patients with severe asthma, though clinical follow-up demonstrated their inefficacy [5]. On the other hand, lebrikizumab, a monoclonal antibody to IL13, ameliorated lung functions within 12 weeks [11]. Alternatively, a selective inhibitor of Rho signals have been also preliminary tested because Rho kinase inhibition decreases inflammation and mucus hypersecretion [50, 63], even though the molecular signals controlled by the Rho-dependent pathway are not clarified yet. In this scenario, an improved understanding of the downstream effectors in the IL13 pathway is an unmet need, and is essential to the identification of novel therapeutic targets. Here, we found that IL13 treatment increases the abundance of pendrin at the plasma membrane via Rho/actin signaling, an effect counteracted by theophylline, a known bronchodilator compound used to treat asthma. These observations may contribute to elucidate the pathogenesis of lung diseases in which pendrin may represent a novel therapeutic target.

Methods

Chemicals and reagents

All chemicals were purchased from Sigma (Sigma-Aldrich, Milan, Italy). 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), media, sera for cell culture, Alexa 555 and Alexa 488-conjugated antibodies, were purchased from Life Technologies (Life Technologies, Monza, Italy). Mirus TransIT®-2020 transfection reagent was purchased from Tema Ricerca (Tema Ricerca s.r.l. Bologna, Italy). Biocytin hydrazide and streptavidin beads were purchased from EZ-Link® Pierce. Anti-Pendrin (H-195), anti-G-alpha-13, and HRP-linked goat anti-rabbit antibodies were from Santa Cruz (D.B.A. Italia s.r.l. Milano).

Plasmids

Plasmids were already described [59]. Briefly, the open reading frame (ORF) of human pendrin (PDS) isolated from the thyroid was subcloned into the XhoI and BamHI restriction sites of the pECFPN1 (Clontech) vector. When transfected into mammalian cells, this construct leads to the production of pendrin with a C-terminal ECFP tag (PDS-ECFP). Alternatively, pECFPC1-pendrin, coding for pendrin with an N-terminal ECFP tag (ECFP-PDS), was used. The pEYFP-Mem construct was from Clontech. Transfection of cells with this construct results in a strong labeling of the plasma membrane. The construct pEYFPN1-Actin was kindly gifted by Dr. Bazzini (University of Milan, Italy). Rho activity was evaluated using the probe Raichiu-RBD (RhoA-binding domain with ECFP and EYFP fused to the N and C terminus, respectively) [65].

Cell culture and transfection

NCI-H292 cells (from ATCC) were grown in Advanced RPMI 1640 Media supplemented with 10% (v/v) fetal bovine serum, 100 i.u./ml penicillin and 100 μg/ml streptomycin at 37 °C with 5% CO2. Cells were seeded and grown on filters for cell surface biotinylation and for spectrophotometric measurements of bicarbonate. Otherwise, cells were seeded on plastic dishes or coverslips. After seeding for 24 h (~80% confluence), cells were transiently transfected with plasmids (0.4 μg of DNA/cm2) using TransIT®-2020 transfection reagent (0.75 μl/cm2) according to the protocol provided by the manufacturer (Mirus, Mirus Bio LLC, USA). Experiments were performed 48 h post-transfection.

Treatments

NCI-H292 cells were incubated with IL13 (50 ng/ml) for 24 h, theophylline (10 μg/ml) for 8 h, or the vehicle (control). Further, cells were treated with IL13 (50 ng/ml) for 24 h in the presence of theophylline (10 μg/ml) for the last 8 h.

Video imaging measurements

For intracellular pH measurements, the coverslips with cells were mounted in a perfusion chamber (FCS2 Closed Chamber System, BIOPTECHS, Butler, U.S.A.) and measurements were performed using an inverted microscope (Nikon Eclipse TE2000-S), equipped for single-cell fluorescence measurements and imaging analysis. The sample was illuminated through a ×40 oil immersion objective (NA = 1.30).

FRET measurements

NCI-H292 cells were co-transfected (transiently) with PDS-ECFP and EYFP-Mem or with ECFP-PDS and Actin-EYFP. Alternatively, cells were transfected with ECFP-Raichu-RBD-EYFP. Forty-eight hours after transfection and treatments, cells were fixed with 4% paraformaldehyde (PFA) in PBS (pH 7.4) for 20 min at room temperature and washed three times in PBS. Afterwards, coverslips with cells were mounted in Mowiol (polyvinyl alcohol 4-88; Sigma-Aldrich, Milan, Italy). Visualization of ECFP- and/or EYFP-expressing cells and detection of FRET were performed on an inverted microscope (Nikon Eclipse TE2000-S), equipped with a monochromator controlled by MetaMorph/MetaFluor software. ECFP was excited at 433 nm and EYFP at 512 nm. All images were aligned and corrected for background in the emission windows for FRET (535/30 nm), ECFP (475/30 nm), and EYFP (535/26 nm). Each image was further corrected for ECFP crosstalk and EYFP cross-excitation as shown by Rodighiero et al. [47]. Thus, netFRET = [IFRETbg − ICFPbg × k1 − IYFPbg (K2 − αK1)]/(1 − K1δ), where IFRETbg, ICFPbg, and IYFPbg are the background-corrected pixel gray values measured in the FRET, ECFP, and EYFP windows, respectively; K1, K2, α, and δ are calculated to evaluate the crosstalk between donor and acceptor. The obtained netFRET values were normalized for the expression levels of ECFP and EYFP (NFRET = netFRET × 100/(ICFPbg × IYFPbg)1/2). The integrated fluorescence density values of the images from ten regions of interest in each cell [46], close to the cells’ outer margins, were analyzed using MetaMorph and Microsoft Excel softwares.

Cell surface biotinylation

Biotinylation was carried out as already described [57]. Shortly, NCI-H292 cells were cultured on six-well filters in complete growing medium and transfected with PDS-ECFP. After treatments, cells were washed thoroughly with an ice-cold coupling buffer (0.1 M NaPO3, 0.15 M NaCl, pH 7.2) before being subjected to oxidation with 20 mM Na-meta-periodate in an oxidation buffer for 30 min on ice in the dark. Following three rounds of washing with the coupling buffer, cell surface glycoproteins were labeled with 5 mM Biocytin hydrazide (EZ-Link® Pierce) for 30 min. The biotinylation buffer was removed and cells were incubated with a quenching solution (50 mM NH4Cl in PBS, pH 7.2) for 5 min and washed three times with the coupling buffer. Cells were solubilized with lysis buffer (1% Triton X-100, 0.01% SDS in PBS supplemented with 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 2 mM PMSF) for 30 min. The lysates were homogenized using an ultrasonic homogenizer at 40 Hz for 20 s and centrifuged at 12,000×g for 20 min. An aliquot of the supernatants (30 μl) was diluted in Laemmli buffer containing 8 M Urea. The remaining biotinylated proteins were pulled down with immobilized streptavidin beads. The complexes were washed 3 times with wash buffer (0.5% Triton X-100, 0.01% SDS in PBS), denatured in a Laemmli buffer containing 8 M Urea for 5 min at 90 °C, separated by SDS-PAGE and immunoblotted with anti-pendrin antibodies (1:500 dilution).

Intracellular pH measurements

NCI-H292 cells were loaded with 2 μM BCECF-AM for 30 min at 37 °C in Dulbecco’s modified Eagle’s medium and then washed in Ringer’s solution containing (in mmol/L) 130 NaCl, 3 KCl, 0.5 MgCl2, 1.2 NaHCO3, 10 glucose, 1.2 CaCl2, 10 HEPES, and pH 7.4. BCECF was excited at 490 and 440 nm. Emitted fluorescence was passed through a single-band dichroic mirror and filtered at 535 nm (Omega Optical, Brattleboro, VT, USA). The BCECF fluorescence intensity ratio was calibrated with solutions containing 10 μM nigericin and high potassium (in mmol/L 5 NaCl, 130 KCl, 0.1 MgCl2, 10 glucose, 1.2 CaCl2, 10 HEPES, and pH 5.30, 6.2, 7.4, and 8.45 adjusted with KOH or HCl).

Immunofluorescence and F-actin visualization

Immunocytochemistry was performed as previously described [58]. Briefly, NCI-H292 cells were grown, transfected with PDS-ECFP, treated as described, and fixed for 30 min with 4% paraformaldehyde in PBS. Cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min, blocked with 1% BSA in PBS for 30 min, and incubated for 2 h with anti-pendrin antibodies (raised in rabbit, 1:500 dilution). After washing, cells were incubated with goat anti-rabbit antibodies coupled to Alexa-555 (1:3000 dilution).

To detect pendrin in co-localization experiments, the secondary antibody was coupled to Alexa-488 (1:3000 dilution). To detect F-actin, specimens were further incubated with TRITC-conjugated phalloidin (400 μg/ml) for 45 min and subsequently washed.

The coverslips were mounted on glass slides with Mowiol mounting medium. The fluorescence signals were detected by a confocal microscope (Leica TCS SP2, Leica Microsystems, Heerbrugg, Switzerland).

F-actin co-sedimentation assay

F-actin co-sedimentation was performed as described previously [60]. Briefly, total membrane and cytosol fractions were prepared from NCI-H292 cells. Cells were scraped and resuspended in homogenization buffer that contained 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1 mM dithiothreitol, and protease inhibitors. Cells were homogenized using a 27-gauge needle, and nuclei were removed by centrifugation at 800×g for 10 min. Membrane and cytosol fractions were obtained by centrifugation for 1 h at 4 °C at 150,000×g. Cytosolic proteins (800 μg each conditions) were used for F-actin polymerization. Specifically, formation of F-actin was initiated using a 50-fold polymerization buffer that contained 200 mM MgCl2, 4 M KCl, and 100 mM ATP. The samples were incubated for 1 h at 37 °C, and F-actin was pelleted by ultracentrifugation for 1 h at 4 °C at 150,000×g. The F-actin-containing pellets were rinsed with homogenization buffer.

Cytosol and F-actin fractions were separated by 13% SDS-PAGE and immunoblotted with G-alpha 13-specific antibodies.

Statistical analysis

One-way ANOVA followed by multiple comparison tests was used for the statistical analysis. When applicable, t test was also used. All values are expressed as means ± SEM. A value of P < 0.05 was considered as statistically significant.

Results

IL13 stimulates pendrin trafficking to the plasma membrane in bronchiolar NCI-H292 cells

The effect of IL13 exposure on pendrin localization was evaluated in epithelial NCI-H292 cells by confocal microscopy (Fig. 1). Treatment with IL13 (50 ng/ml) increased the cell surface abundance of pendrin (white arrows indicate the plasma membrane in IL13-treated cells) compared with untreated cells (control). Co-treatment with theophylline (10 μg/ml), a classical bronchodilator drug, abolished IL13 action, and pendrin staining appeared similar to that observed in control cells. Incubation with theophylline alone did not alter the cellular distribution of pendrin compared to control.

Fig. 1
figure 1

Confocal analysis of pendrin localization in NCI-H292 cells. Cells were transfected with pendrin, treated with the vehicle (CTR), IL13 (50 ng/ml for 24 h), theophylline (Theo, 10 μg/ml for 8 h), or IL13 in the presence of theophylline (IL13-Theo; 50 ng/ml IL13 for 24 h in the presence of 10 μg/ml theophylline for the last 8 h) and processed for immunocytochemistry. Scale bar 5 μM

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To quantify pendrin abundance at the plasma membrane, FRET studies were performed as previously described [59]. NCI-H292 cells were co-transfected with constructs leading to the expression of pendrin C-terminally tagged with ECFP (PDS-ECFP), and EYFP-Mem (a marker of the plasma membrane, see the “Methods” section) and treated with IL13 and/or theophylline. Experiments were executed 48 h post-transfection on specimens showing a transfection efficacy of 40–45%. NFRET was calculated in fixed cells in ten regions near the outer perimeter of each cell; the values were corrected as described by Rodighiero [47]. When the two fusion proteins PDS-ECFP and EYFP-Mem are in close proximity to each another, FRET signal increases due to the transfer of resonance energy from the donor (PDS-ECFP) to the acceptor (EYFP-Mem). With the ECFP-EYFP FRET pair, statistically significant NFRET variations ranging from 10 to 20% are generally considered biologically relevant [7]. Compared to control, exposure of cells to IL13 significantly increased FRET signal, consistent with an augmented abundance of pendrin at the plasma membrane (IL13, 109.9 ± 2.319, n = 211 cells vs. CTR, 100 ± 1.347, n = 250 cells, *p < 0.001 vs. CTR Fig. 2a). Theophylline reversed IL13-dependent accumulation of pendrin at the plasma membrane (IL13-Theo, 97.03 ± 3.480, n = 67 cells, Fig. 2a). Incubation with theophylline alone did not exert any relevant change on the cellular distribution of pendrin (Theo, 94.87 ± 3.480, n = 84 cells, Fig. 2a). Data are also shown as frequency distribution histogram of the relative changes of NFRET under the described conditions (Fig. 2b).

Fig. 2
figure 2

Evaluation of pendrin expression at the plasma membrane by FRET studies. a NCI-H292 cells were transiently transfected with PDS-ECFP and EYFP-Mem. FRET experiments were performed 48 h after transfection. Histograms show NFRET measured in cells treated with the vehicle (CTR) or stimulated with IL13 (50 ng/ml for 24 h), theophylline (Theo, 10 μg/ml for 8 h), or IL13 and theophylline (IL13-Theo; 50 ng/ml IL13 for 24 h in the presence of 10 μg/ml theophylline for the last 8 h). (*p < 0.001 vs. CTR). b Frequency distribution histograms of the relative changes of NFRET in percent, under basal condition (CTR), IL13 stimulation, theophylline (Theo), and co-treatment with IL13 and theophylline (IL13-Theo). The obtained data were collected from ten regions of interest (ROI) located near the plasma membrane

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To verify whether the IL13-dependent increase in the localization of pendrin at the cell surface observed by confocal imaging and FRET analysis involved the insertion into the plasma membrane, cell surface biotinylation experiments were performed in cells transiently expressing pendrin (Fig. 3a). To increase the yield of the detectable membrane-inserted pendrin [4], the cell surface was labeled with biocytin hydrazide which binds glycoproteins specifically. In contrast, conventional biotinylation reagents bind lysine residues, which may be poorly accessible from the extracellular side [57–59, 62]. The amount of membrane-inserted pendrin, labeled with biocytin hydrazide, was normalized to the total cellular amount of pendrin. In line with immunofluorescence and FRET data, IL13 significantly increased the cell surface expression of pendrin (IL13, 1.879 ± 0.2550, p < 0.05 vs. CTR, n = 5 *p = 0.0045). Importantly, the IL13-dependent increase of pendrin abundance at the plasma membrane was abolished when cells were co-treated with theophylline (IL13-Theo, 0.9079 ± 0.1226, n = 5). Compared to untreated cells, incubation with theophylline alone (Theo, 1.083 ± 0.2084, n = 5) did not affect the membrane insertion of pendrin (Fig. 3a–b). The specificity of anti-pendrin antibodies was evaluated by Western blotting on protein extracts from untransfected (mock) and pendrin expressing cells (Fig. 3c).

Fig. 3
figure 3

a Left, representative pendrin immunoblot on biotinylated (top panel) and whole cell (bottom panel) proteins in NCI-H292 cells transiently transfected with PDS-ECFP. Cells were treated with the vehicle (CTR), IL13 (50 ng/ml for 24 h), theophylline (Theo, 10 μg/ml for 8 h), or IL13 and theophylline (IL13-Theo; 50 ng/ml IL13 for 24 h in the presence of 10 μg/ml theophylline for the last 8 h). b The histogram on the right shows the densitometry of specific bands in each lane (*p = 0.0045 vs. CTR). The relative amounts of pendrin upon each treatment were quantified from five independent experiments. c Western blotting to assess the specificity of the anti-pendrin antibody. Untransfected (mock) and pendrin expressing cells were assayed with or without (negative control) the anti-pendrin antibody. The black arrows indicate the specific bands used to semi-quantify the abundance of pendrin in the plasma membrane

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IL13 stimulates pendrin activity in bronchiolar NCI-H292 cells

Previous studies documented a functional involvement of pendrin in controlling intracellular pH, owing on its activity as a Cl−/HCO3 − exchanger [21, 48, 59]. To evaluate whether the increased abundance of pendrin at the plasma membrane coincides with an increase in its activity, intracellular pH was measured (Fig. 4). To this end, NCI-H292 cells were transiently transfected with PDS-ECFP and loaded with the pH-sensitive dye BCECF-AM (2 μM for 30 min). Surprisingly, treatment with IL13 resulted in a significant intracellular alkalinization (IL13, 7.97 ± 0.0822, n = 56, vs. CTR, 7.37 ± 0.053, n = 33 *p < 0.0001) in pendrin-expressing cells. Co-treatment with theophylline counteracted IL13 by decreasing intracellular pH to a mean value not significantly different from that observed in control cells (IL13-Theo, 7.66 ± 0.073, n = 28). Theophylline alone did not alter intracellular pH. Moreover, mentioned treatments did not alter the intracellular pH in cells expressing the transfection marker ECFP (data not shown).

Fig. 4
figure 4

Intracellular pH measured in NCI-H292 cells transiently transfected with PDS-ECFP. Cells were loaded with BCECF-AM (2 μM for 30 min). Histograms represent the intracellular pH after exposure of cells to the vehicle (CTR), IL13 (50 ng/ml for 24 h), theophylline (Theo, 10 μg/ml for 8 h), or IL13 and theophylline (IL13-Theo; (50 ng/ml IL13 for 24 h in the presence of 10 μg/ml theophylline for the last 8 h) (*p < 0.0001 vs. CTR)

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In bronchial epithelial cells, IL13 promotes a secretory phenotype by reducing Na+ reabsorption via ENaC and stimulating Cl− secretion via CFTR [2], but the molecular mechanism modulating intracellular pH is not clarified yet. In hematopoietic FL5.12 and COS cells, IL7 and IL3 caused a significant cellular alkalinization by stimulating the activity of Na+/H+ exchanger NHE1 via p38 MAPK [28]. To evaluate the possible involvement of Na+/H+ exchange (NHE) on intracellular alkalinization following exposure to IL13, we measured changes of intracellular pH in the presence of 5-(N,N-dimethyl)-amiloride (DMA), a selective NHE inhibitor. NCI-H292 cells were treated as described above in the presence of DMA for 30 min and assayed for intracellular pH (Fig. 5). The difference between the intracellular pH measured in cells treated with DMA and IL13-DMA was significantly higher in PDS-expressing cells compared to untransfected cells (ΔpH = 0.442 ± 0.0473, n = 29 vs. untransfected cells ΔupH = 0.22 ± 0.007, n = 19 *p = 0.0001), therefore indicating that, following IL13 exposure, a greater intracellular acidification occurs in PDS-transfected cells. These findings led us to hypothesize that cells expressing pendrin display a higher IL13-stimulated Cl−/HCO3 − exchange activity that only becomes apparent following inhibition of a compensatory Na+/H+ exchange activity.

Fig. 5
figure 5

Effect of IL13 (50 ng/ml for 24 h) on intracellular pH (pHi) under exposure to 5-(N,N-dimethyl)-amiloride (DMA). The pHi was measured in NCI-H292 cells untransfected or transiently transfected with PDS-ECFP loaded with BCECF (2 μM for 30 min). DMA (20 μM) was added 30 min before the experiment. Histograms represent the differences of intracellular pH variation calculated from cells treated with DMA alone and DMA in the presence of IL13 (ΔupH) (*p = 0.0001)

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IL13 stimulates Rho/actin signaling in bronchiolar NCI-H292 cells

The heterotrimeric G 13 proteins (G-alpha-13) stimulate NHE activity in several cell types [26, 61]. Therefore, to investigate the possible pathway by which IL13 causes intracellular alkalinization via NHE stimulation, G-alpha-13 involvement was evaluated by analyzing its affinity with F-actin, an index of G-alpha-13 activity [55, 60]. Cytosolic fractions of NCI-H292 cells were prepared, F-actin polymerization was induced, and F-actin-interacting proteins were analyzed by Western blotting (Fig. 6). The amount of G-alpha-13 in the F-actin-enriched fraction (active G-alpha-13) was normalized to the total amount of G-alpha-13 in the soluble fraction from which the F-actin-enriched fraction was obtained (see the “Methods” section). Relative to the control (Fig. 6), IL13 caused a significant increase in immune-detectable G-alpha-13 in the F-actin-enriched fraction (IL13, 1.617 ± 0.1930, *p = 0.027 vs. CTR, n = 3). Theophylline abolished IL13 action on G-alpha-13 affinity to F-actin (IL13, 1.617 ± 0.1930 vs. IL13-Theo, 0.7823 ± 0.1987, n = 3 #p = 0.0027).

Fig. 6
figure 6

G-alpha-13 and F-actin co-sedimentation. a Cytosolic fractions of NCI-H292 cells exposed to the vehicle (CTR), IL13 (50 ng/ml for 24 h), theophylline (Theo, 10 μg/ml for 8 h), or IL13 and theophylline (IL13-Theo; 50 ng/ml IL13 for 24 h in the presence of 10 μg/ml theophylline for the last 8 h) were prepared and F-actin polymerization was induced. F-actin-interacting proteins were analyzed by Western blotting. Equal amounts of F-actin fractions (30 μg/lane) were separated by SDS-PAGE and immunoblotted with an anti-G-alpha-13 antibody. b On the right, densitometric analysis of four independent experiments is shown (*p = 0.027 vs. CTR; # p = 0.0027 vs. IL13)

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It is known that G-alpha 12/13 activates RhoA, a small GTPase of the Rho family, which plays a role in controlling actin dynamics [54–56]. Rho activity was evaluated with FRET studies using a fluorescent probe (Raichu-RBD, see the “Methods” section) consisting of the Rho-binding domain (RBD) of Rhotekin sandwiched between EYFP and ECFP [65]. Binding of endogenous GTP-RhoA to RBD distances the FRET donor ECFP from the FRET acceptor EYFP, thereby decreasing FRET efficiency. IL13 decreased FRET signal, consistent with a significant upregulation of Rho activity (Fig. 7a). Theophylline prevented IL13 response on Rho functionality, as revealed by FRET values which were comparable to those measured in untreated cells. Data are also represented as frequency distribution histogram of the relative changes of netFRET under the described conditions (Fig 7b).

Fig. 7
figure 7

Evaluation of Rho activity by FRET experiments. a NCI-H292 cells were transiently transfected with Raichiu-RBD as described in the “Methods” section. FRET experiments were performed 48 h after transfection. Histograms represent netFRET measured in cells treated with the vehicle (CTR) or stimulated with IL13 (50 ng/ml for 24 h), theophylline (Theo, 10 μg/ml for 8 h), or IL13 and theophylline (IL13-Theo; 50 ng/ml IL13 for 24 h in the presence of 10 μg/ml theophylline for the last 8 h). (*p = 0.008 vs. CTR). b Frequency distribution histograms of the relative changes of netFRET in percent, under basal condition (CTR), IL13 stimulation, theophylline (Theo), and co-treatment with IL13 and theophylline (IL13-Theo). The obtained data were collected from ten regions of interest (ROI) located near the plasma membrane

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Theophylline treatment alone resulted in a significant increase in FRET signal, indicating downregulation of Rho activity. These data suggest that Rho proteins are involved in the signal transduction cascade activated by IL13.

Next, we tested the hypothesis that Rho proteins might play a role in regulating the cellular distribution of pendrin. To this end, the membrane expression of pendrin was evaluated by FRET with a membrane probe (EYFP-Mem, see the “Methods” section). NCI-H292 cells were left untreated of stimulated with IL13 (50 ng/ml). Alternatively, cells were treated with IL13 in the presence of Y27632 (100 μM for 30 min), a selective inhibitor of Rho kinase, a downstream effector of Rho proteins. A significant decrease of NFRET signal was detected in cells co-treated with Y27632 and IL13 compared with cells incubated with IL13 alone (IL13-Y27632, 91.81 ± 2.392, n = 65 cells, vs. IL13, 109.9 ± 2.319, n = 211 cells, #p < 0.0001 Fig. 8a), therefore indicating that Y27632 abolished the effect of IL13 on pendrin abundance at the plasma membrane. These findings are also indicated as frequency distribution histogram of the relative changes of NFRET under the described conditions (Fig. 8b).

Fig. 8
figure 8

Evaluation of pendrin expression at the plasma membrane by FRET studies in the presence or absence of IL3 and Y27632. a NCI-H292 cells were transiently transfected with PDS-ECFP and EYFP-Mem. FRET experiments were performed 48 h after transfection. Histograms represent NFRET determined in cells exposed to the vehicle (CTR), IL13 (50 ng/ml for 24 h), Y27632 (100 μM for 30 min), or IL13 and Y27632 (*p < 0.001 vs. CTR; # p < 0.0001 vs. IL13; & p < 0.05 vs. Y27632). b Frequency distribution histograms of the relative changes of NFRET in percent, under basal condition (CTR), IL13 stimulation, Y27632, and co-treatment with IL13 and Y27632. The obtained data were collected from ten regions of interest (ROI) located near the plasma membrane

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To better understand the molecular mechanism bridging pendrin, Rho signaling, and actin dynamics, we next investigated the co-immunolocalization of pendrin and actin by confocal microscopy (Fig. 9). These studies indicated that, compared with untreated cells, IL13 exposure stabilized actin cytoskeleton (red), which colocalized with pendrin (green). No relevant co-localization between pendrin and actin was observed following treatment with IL13 in the presence of theophylline, therefore indicating that theophylline prevented IL13 effect (Fig. 9).

Fig. 9
figure 9

Confocal analysis of F-actin and pendrin localization in NCI-H292 cells. Cells were transfected with PDS-ECFP and treated with the vehicle (CTR), IL13 (50 ng/ml for 24 h), theophylline (Theo, 10 μg/ml for 8 h), or IL13 and theophylline (IL13-Theo; 50 ng/ml IL13 for 24 h in the presence of 10 μg/ml theophylline for the last 8 h). Specimens were subjected to immunocytochemistry to detect pendrin and then incubated with Phalloidin-TRITC (400 μg/ml) for 45 min at room temperature to detect F-actin. Pendrin is visualized in green and actin in red. Scale bar 5 μM

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To quantify the interaction between actin and pendrin, we performed FRET studies using pendrin N-terminally tagged with ECFP and actin C-terminally tagged with EYFP. In line with co-immunolocalization findings, IL13 increased the NFRET signal, which is consistent with an increased interaction between pendrin and actin. Exposure to theophylline decreased the binding of pendrin to actin induced by IL13 (Fig. 10).

Fig. 10
figure 10

Analysis of pendrin interaction with actin by FRET experiments. a NCI-H292 cells were co-transfected with ECFP-PDS and Actin-EYFP for 48 h and treated with the vehicle (CTR), IL13 (50 ng/ml for 24 h), theophylline (Theo, 10 μg/ml for 8 h), or IL13 and theophylline (IL13-Theo; 50 ng/ml IL13 for 24 h in the presence of 10 μg/ml theophylline for the last 8 h). (*p < 0.05 vs. CTR; # p < 0.0001 vs. IL13). b Frequency distribution histograms of the relative changes of NFRET in percent, under basal condition (CTR), IL13 stimulation, theophylline (Theo), and co-treatment with IL13 and theophylline (IL13-Theo). The obtained data were collected from ten regions of interest (ROI) located near the plasma membrane

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Discussion

In this study, we provide evidence that IL13 stimulates the trafficking of the epithelial anion exchanger pendrin to the plasma membrane in NCI-H292 cells and that this effect is reversed by theophylline treatment. Slc26a4/pendrin expression was shown to be enhanced by IL4/IL13 and IL17A dependently or independently of STAT6 signaling in HEK-Blue cells and in the lungs of B. pertussis-infected mice, respectively [40, 49]. Here, in NCI-H292 cells, treatment with IL13 for 24 h did not promote the expression of pendrin (no specific band corresponding to pendrin has been detected by immunoblotting analysis either in the presence or absence of IL13 stimulation, data not shown).

In the past few years, increasing evidence for the involvement of pendrin in respiratory disorders have been reported [27, 39, 49]. Pendrin knockout mice displayed a lower allergen-induced airway hyperreactivity and inflammation compared to control mice [38], despite the higher expression of inflammatory cytokines. During allergic response, IL13 alters chloride conductance [13], promotes mucus production, and electrolyte and water secretion into the airway surface liquid (ASL). Reduction of the ASL thickness, due to dehydration and/or abnormal ion transport, is associated with airway inflammation and obstruction. A recent report demonstrated that pendrin is involved in controlling ASL thickness, since pendrin deficiency significantly increased ASL thickness in IL13-stimulated mouse tracheal epithelial cells [38]. In line, selective pendrin inhibitors increased ASL in cystic fibrosis [23].

As previously mentioned, pendrin can exchange chloride for other anions such as iodide, hydroxide, thiocyanate, and bicarbonate [41, 53]. Bicarbonate has many important functions in the human body. In the respiratory system, bicarbonate secretion is needed for mucus secretion, mucin expansion, and mucosal protection [18, 22]. Altered pH/bicarbonate secretion may play a role in determining cystic fibrosis lung phenotype since low pH, due to aberrant bicarbonate secretion, decreases ciliary beat frequency [10], thus preventing bacterial removal by phagocytic cells [1]. Surprisingly, here we found that exposure to IL13 increased intracellular pH (Fig. 4), despite the increase of the cell surface expression of pendrin (Figs. 1, 2, and 3). We could think of two explanations for this finding. The first hypothesis is that exposure to IL13, similarly to IL4, changed the transport features of pendrin, thus promoting thiocyanate (SCN−) instead of bicarbonate transport [41]. However, as assessed by anionic chromatography, SCN− ions were not detected in the apical fluid of untreated and IL13-treated cells (data not shown).

Alternatively, IL13, similarly to other cytokines such as IL7 and IL3, may promote cellular alkalinization by stimulating NHE1 activity [28], consequently increasing reactive oxygen species (ROS) production via NADPH oxidase [33, 34]. In line, an increase of oxidant activity under IL13 exposure has been described in airway epithelial cells [14, 25] and in NCI-H292 cells (data not shown). Here, treatment with DMA, which inhibits NHE activity, revealed that cells expressing pendrin and treated with IL13 displayed a high extent of intracellular acidification, due to sustained bicarbonate extrusion. Importantly, IL13 increased intracellular pH only in cells expressing pendrin. These results let to conclude that IL13-dependent over-activation of pendrin may be associated with activation of NHE.

Upregulation of pendrin accompanied by elevated expression levels of inflammatory cytokines can be actually considered a signature of lung inflammation [16]. In lung diseases, the increased oxidative stress caused lack of response to glucocorticoids and decrease of the histone deacetylase (HDAC) activity [12]. Independently of phosphodiesterases inhibition, theophylline, at lower concentration, restored corticosteroids sensitivity and displayed an anti-inflammatory effect in asthma and COPD, by modulating the activation of the inflammatory mediator NF-kB and increasing the activity of HDAC [6]. In the present study, theophylline did not alter the basal intracellular cAMP level (data not shown), likely suggesting that the counteracting effect of theophylline on IL13 action may be due to its anti-inflammatory and antioxidant features.

Notably, in bronchial smooth muscle cells, IL13/STAT6 signaling increases cell contractility by augmenting the Rho pathway [9], which is actually considered an important therapeutic target for asthma [50]. Selective inhibitors of Rho kinase decreases IL13-induced airway inflammation and mucus secretion by modulating STAT6 [20, 50, 63]. In NCI-H292 cells, IL13 stimulates Rho proteins (Fig. 7), possibly via G-alpha-13 that activates NHE1 through a Rho-dependent pathway [26]. Interestingly, treatment with Y27632, an inhibitor of Rho kinase, reduces IL13-induced pendrin abundance at the plasma membrane (Fig. 8), indicating the involvement of Rho signaling in modulating the cellular distribution of pendrin. At high concentration (100 μM), Y27632 not only inhibits Rho kinases but promotes the loss of actin stress fibers in human airway epithelial cells [43].

Rho-GTPases are known regulators of actin cytoskeleton [24, 44]. When active, Rho proteins can bind cell membranes and promote actin polymerization at discrete sites on the plasma membrane, thereby controlling membrane remodeling and trafficking of cellular proteins [45, 56]. To date, no actin-binding sites have been described in pendrin, even though its C-terminal domain holds a putative actin-binding sequence (DxI/LKKxT) similar to that observed in other actin-binding proteins such as cofilin [8, 64]. Our data demonstrate that IL13 stabilizes pendrin at the plasma membrane through the interaction with actin filaments, which are partially depolymerized with theophylline. A possible explanation is that the formation of cortical actin filaments, under L13 treatment, may slow the constitutive endocytosis of pendrin whereby stabilizing its plasma membrane localization (Figs. 1, 2, and 3). Indeed, actin depolymerization, obtained with theophylline or with Y27632 under IL13 treatment, is associated with a significant reduction of the abundance of pendrin at the plasma membrane.

In this respect, we have previously showed that inhibition of Rho, obtained after forskolin stimulation, resulted in intracellular acidification due to a significant increase of pendrin trafficking at the plasma membrane. These apparent discrepancies can be explained considering the dual and bidirectional role of actin cytoskeleton in controlling the constitutive exocytosis and endocytosis. Specifically, a partial depolymerization of the “cortical actin cage” may facilitate the constitutive exocytosis [37, 46, 56]. Conversely, IL13, by stimulating the formation of actin filaments (Fig. 9), might reduce the rate and the extent of the constitutive endocytosis. Therefore, the plasma membrane abundance of pendrin may be considered the result of a precise balance between the constitutive exocytosis and endocytosis. Nevertheless, an increase in the pendrin abundance at the cell membrane due to increased exocytosis or to decreased endocytosis did not modify the pendrin transport property as bicarbonate exchanger.

To conclude, the present studies highlight the pivotal role of IL13 in controlling airway remodeling, which is regulated by the integrated cooperation of different cells types that are target of IL13 action and includes the Rho signal pathway activation. Moreover, our observations propose a role of pendrin in modulating the intracellular pH and promoting respiratory inflammation.