American Journal of Respiratory Cell and Molecular Biology

Acute respiratory distress syndrome is often associated with elevated levels of CO2 (hypercapnia) and impaired alveolar fluid clearance. Misfolding of the Na,K-ATPase (NKA), a key molecule involved in both alveolar epithelial barrier tightness and resolution of alveolar edema, in the endoplasmic reticulum (ER) may decrease plasma membrane abundance of the transporter. Here, we investigated how hypercapnia affects the NKA β-subunit (NKA-β) in the ER. Exposing murine precision-cut lung slices and human alveolar epithelial A549 cells to elevated CO2 levels led to a rapid decrease of NKA-β abundance in the ER and at the cell surface. Knockdown of ER mannosidase α class 1B member 1 and ER degradation-enhancing α-mannosidase like protein 1 by siRNA or treatment with the mannosidase α class 1B member 1 inhibitor kifunensine rescued loss of NKA-β in the ER, suggesting ER-associated degradation (ERAD) of the enzyme. Furthermore, hypercapnia activated the unfolded protein response by promoting phosphorylation of inositol-requiring enzyme 1α (IRE1α), and treatment with an siRNA against IRE1α prevented the decrease of NKA-β in the ER. Of note, the hypercapnia-induced phosphorylation of IRE1α was triggered by a Ca2+-dependent mechanism. In addition, inhibition of the inositol trisphosphate receptor decreased phosphorylation levels of IRE1α in precision-cut lung slices and A549 cells, suggesting that Ca2+ efflux from the ER might be responsible for IRE1α activation and ERAD of NKA-β. In conclusion, here we provide evidence that hypercapnia attenuates maturation of the regulatory subunit of NKA by activating IRE1α and promoting ERAD, which may contribute to impaired alveolar epithelial integrity in patients with acute respiratory distress syndrome and hypercapnia.

Carbon dioxide (CO2) is a product of oxidative metabolism during cellular respiration. It is now increasingly evident that especially in the lung, CO2 acts as a signaling molecule by activating specific pathways, altering mitochondrial and immune function (16). Different mechanisms have been proposed; however, the exact mechanisms of CO2 sensing have not yet been determined (7, 8). Although physiologically CO2 is eliminated from the body by breathing, during alveolar epithelial barrier failure, a significant elevation of CO2 levels is often observed (9). Clinically, elevation of arterial CO2 tension levels above 45 mm Hg is considered hypercapnia, which occurs in patients with various acute and chronic lung diseases such as acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease, or cystic fibrosis (1013). One of the hallmarks of ARDS is the formation and persistence of alveolar edema, which is a consequence of alveolar epithelial barrier dysfunction and impaired alveolar fluid clearance (14). It is widely accepted that in patients with ARDS, removal of the excess alveolar fluid is of critical importance and is a key determinant of survival (9). Importantly, elevated levels of CO2 (independently of the associated acidosis) have been shown to increase mortality in patients with ARDS and sepsis (15, 16).

Under normal conditions, excess alveolar fluid is cleared by a Na+ gradient, which is generated by the coordinated activity of apically localized epithelial sodium channels and the basolateral Na,K-ATPase (NKA) (17). NKA is a ubiquitously expressed transporter that consists of a catalytic α-, a regulatory β-, and axillar γ-subunits. Of note, the β-subunit of the enzyme drives proper trafficking of the Na+ pump to the cell surface (18). NKA is widely expressed in different cell types, tissues, and organs, where its function is not limited to establishing ion gradients and membrane potential; it also modulates glucose and amino acid transport and regulates cell adhesion properties, cell motility, and polarity (1820). The main function of the NKA β-subunit (NKA-β) is to act as a regulatory chaperon for the NKA α-subunit by driving assembly and delivery of the enzyme to the plasma membrane (PM). Moreover, it plays an important role as an adherens junction molecule in the epithelium, which is critical for maintaining tightness of the epithelial barrier (20). In a recent study, it was shown that knocking out NKA-β in alveolar epithelial cells (AEC) results in a significant decrease of alveolar fluid clearance, confirming its important role in regulating the function of the catalytic subunit of the transporter and thus maintaining alveolar epithelial barrier function (21). We have previously demonstrated that hypercapnia impairs alveolar fluid clearance by promoting endocytosis of the NKA α-subunit by activation of a specific signaling cascade, involving AMPK (AMP-activated protein kinase), PKC-ζ (protein kinase C-ζ), and JNK (c-Jun-N-terminal kinase), leading to phosphorylation and subsequent endocytosis of the NKA α-subunit from the cell surface (1, 5, 6, 22, 23).

The endoplasmic reticulum (ER) is centrally involved in maturation of PM proteins. A fully functional NKA molecule, to be delivered to the PM, must undergo various maturation and folding steps after protein translation, including glycosylation in the ER and the Golgi apparatus before delivery to the PM (24, 25). Importantly, only NKA α:β complexes assembled in ER at 1:1 stoichiometry reach the PM, whereas unassembled α-subunits are retained in the ER and are rapidly degraded (24). It has been previously shown that sustained hypercapnia causes metabolic stress and decreases cellular ATP (26). As the ER folding machinery highly depends on cellular homeostasis (27, 28), we hypothesized that elevated CO2 levels may interfere with NKA-β maturation in the ER and thus delivery of the NKA α:β complex to the PM. Because NKA function is critical for maintaining alveolar epithelial tightness and optimal lung fluid balance, alterations in the maturation of NKA-β may be deleterious in the context of ARDS.

Please see the data supplement for full details regarding material, Western blot analysis, cell surface biotinylation, quantitative real-time PCR, transfection of cells with siRNA, immunofluorescent staining, and Fluo-4 fluorescent calcium measurements.

Precision-Cut Lung Slices and Cell Culture

Murine precision-cut lung slices (PCLS) were prepared as described previously (29). Briefly, lungs were filled intratracheally with 0.3% agarose and lung lobes were cut into 200-μm sections using a Leica VT 1200S vibratome (Leica Microsystems), placed in PBS, and kept in Dulbecco’s modified Eagle medium (DMEM)/nutrient mixture F12 (Thermo Fisher) supplemented with 10% FBS (PAA Laboratories), 100 U/ml penicillin, 100 μg/ml streptomycin, and 1% amphotericin B. Human epithelial A549 cells (ATCC, CCL 185) were grown in DMEM media supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin as previously described (3). Experiments with A549 cells were performed on subconfluent cellular monolayers. PCLS were used for experiments on Day 3. PCLS and A549 cells were incubated in a humidified incubator with an atmosphere of 5% CO2/95% air at 37°C.

CO2 Exposure

Murine PCLS and human A549 cells were exposed to 40 mm Hg (normocapnia, Ctrl) or 120 mm Hg (hypercapnia, CO2) CO2 unless otherwise indicated. Before each experiment, media solutions were prepared fresh with DMEM or DMEM/F12 (Thermo Fisher) and MOPS base (Sigma Aldrich) to obtain a final pH of 7.4 at 40 or 120 mm Hg of CO2, as described previously (26). Normocapnic and hypercapnic solutions were kept overnight in a humidified chamber and CO2 levels were controlled by an RO-CO2 carbon dioxide controller (BioSpherix Ltd). Before each experiment, media pH, pCO2 and pO2 levels were measured using a Rapidlab gas analyzer (Siemens).

Statistics

Data are presented as mean ± SD unless otherwise indicated. Statistical analysis and plotting of the data were performed by using GraphPad Prism version 6.0 (GraphPad Software). Comparisons between two groups were done by Student’s t test. When comparisons were performed between more than two groups, the one- or two-way ANOVA was used. Unless otherwise specified, the significance is shown while comparing the asterisk-labeled group with the control. A P value of <0.05 was considered to be statistically significant.

Hypercapnic Exposure Decreases PM Abundance of NKA-β in Murine PCLS and AEC

To determine whether elevated CO2 decreases cell surface abundance of NKA-β, murine PCLS and human A549 AEC were exposed to physiological (pCO2 = 40 mm Hg; normocapnia) or elevated (pCO2 = 120 mm Hg; hypercapnia) CO2 levels at a constant extracellular pH of 7.4 for 60 minutes. As assessed by confocal microscopy, elevation in CO2 levels markedly decreased NKA-β abundance at the PM in three-dimensional (3D) cultures (Figure 1A). Similarly, in AEC, cell surface biotinylation and streptavidin pull-down showed a marked reduction in PM abundance of both α- and β-subunits of NKA (Figures 1B and 1C). These findings are in agreement with previous reports showing that elevated CO2 significantly decreases PM abundance of the NKA α-subunit in AEC (5, 22, 30).

Elevated CO2 Levels Induce Reduction of the ER Fraction of NKA-β

Next, we addressed the question whether elevated levels of CO2 affect cellular protein levels and distribution of NKA-β. During maturation, NKA-β undergoes high-mannose glycosylation in the ER, followed by more complex glycosylation forms in the ER and Golgi that reach the PM (20). Thus, we assessed complex-type and exclusively ER-resident high-mannose glycosylated fractions of NKA-β in PCLS and AEC upon exposure to normocapnia or hypercapnia for 60 minutes, and subsequently, total protein abundance of NKA-β was determined by immunoblotting. In both cell culture models, elevated CO2 levels markedly decreased the ER-resident high-mannose NKA-β. In contrast, complex-type total NKA-β remained unchanged within the first 60 minutes of CO2 exposure (Figures 2A and 2B). In addition, exposure of AEC for different durations or levels of CO2 with a constant extracellular pH 7.4 revealed that the reduction of NKA-β abundance in the ER was time and dose dependent (Figures 2C and 2D). To test whether the hypercapnia-induced decrease of NKA-β was transcriptionally regulated, we next measured mRNA levels of NKA subunits by quantitative PCR after 60 minutes of hypercapnia and found no significant changes within normo- and hypercapnia, suggesting that the ER reduction of NKA-β was mediated in a post-translational manner (Figure 2E). Together, these data indicate that elevated levels of CO2 rapidly induce reduction of NKA-β abundance in the ER, which may contribute to the overall decrease in cell surface expression of NKA.

Hypercapnia Induces ER-associated Degradation of NKA-β

A decrease in the ER high-mannose fraction of the NKA-β may be a consequence of ER-associated degradation (ERAD) of the enzyme (19). An initial step of ERAD is trimming of mannose residues on the nascent peptide by a mannosidase enzyme (31). To test whether ERAD played a role in the reduction of NKA-β in the ER, we next treated AEC with the mannosidase inhibitor kifunensine and exposed the cells to 40 or 120 mm Hg of CO2 for 60 minutes. Of note, we found that kifunensine treatment fully prevented the decrease of NKA-β abundance in the ER (Figure 3A). To further confirm that hypercapnia induces ERAD, AEC were transfected with an siRNA against mannosidase α class 1B member 1 (MAN1B1) or against ER degradation-enhancing α-mannosidase-like protein 1 (EDEM1) and were exposed to normal or elevated CO2 levels. In line with our findings using a pharmacological inhibitor of mannosidase, knockdown of MAN1B1 or EDEM1 was also sufficient to completely prevent the hypercapnia-induced loss of NKA-β in the ER (Figures 3B and 3C). After being transferred out of the ER during ERAD, proteins usually undergo proteasomal or lysosomal degradation (32). To assess the potential involvement of these processes, we next treated AEC with the proteasomal inhibitor MG-132 and observed prevention of NKA-β degradation in the ER (Figure 3D). In contrast, treatment of cells with the lysosomal inhibitor chloroquine showed no effect (Figure 3E). Collectively, these data suggest that ERAD of NKA-β was mediated by a proteasomal process.

IRE1α Activation Is Required for Hypercapnia-induced ERAD of NKA-β

Protein folding in the ER is tightly controlled, and upon disturbances, the unfolded protein response (UPR) may be activated (27). UPR consists of several receptors and their downstream pathways—IRE1α, PERK, and ATF6—by which protein homeostasis can be maintained or restored (33). Because IRE1α is one of the most conserved ER quality control pathways and its activation has been implicated in the degradation of misfolded proteins in the ER (3436), we next investigated the effects of IRE1α phosphorylation in hypercapnia-exposed AEC and PCLS (Figures 4A and 4B). Of note, we observed a rapid and time-dependent IRE1α phosphorylation upon exposure to elevated CO2 levels in AEC (Figure 4A). Moreover, treatment of these cells with 1 μM thapsigargin, an irreversible inhibitor of sarco/endoplasmic reticulum Ca-ATPase (SERCA) and ER stress inducer, similarly to hypercapnia, increased IRE1α phosphorylation and promoted a decrease of NKA-β abundance in the ER (see Figure E1 in the data supplement). To further investigate whether IRE1α phosphorylation is responsible for the hypercapnia-induced ERAD of NKA-β, we next transfected AEC with an siRNA against IRE1α and exposed cells to normal or elevated CO2 levels for 60 minutes. In line with our abovementioned findings, knockdown of IRE1α prevented the hypercapnia-induced reduction of NKA-β in the ER (Figure 4C). It has previously been reported that IRE1α affects protein degradation in the ER either by its kinase activity or through splicing of XBP1, a downstream target of IRE1α, resulting in the activated XBP1 s form of the protein (36, 37). To differentiate between these mechanisms, we next treated AEC with the IRE1α kinase inhibitor KIRA6 before exposing the cells to elevated levels of CO2 and observed stabilization of ER-resident NKA-β (Figure 4D). In contrast, transfecting AEC with an siRNA against XBP1 s did not affect ERAD of NKA-β upon hypercapnia (Figure 4E). In line with these findings, in cells treated with thapsigargin, levels of XBP1 s were increased after the temporal phosphorylation of IRE1α (Figure E1). Finally, and in line with the above-described findings, pretreatment of AEC with STF-083010, an inhibitor of IRE1α splicing activity, did not affect hypercapnia-induced ERAD of NKA-β either (Figure 4F).

Accumulating evidence suggests that JNK1/2, another downstream target of IRE1α, is centrally involved in the hypercapnia-induced pathways that lead to downregulation of NKA PM abundance by promoting endocytosis of the transporter from the cell surface (3, 22, 23). To investigate the potential role of JNK1/2 in the ERAD of NKA-β, AEC were treated with the specific JNK inhibitor SP600125 and were then exposed to elevated CO2 concentrations for 60 minutes (Figure E2). Consistent with previous reports, elevated CO2 levels induced phosphorylation of JNK1/2. However, inhibition of the activity of the kinase by SP600125 was not sufficient to prevent the decrease in the levels of ER-resident NKA-β. Finally, it has been previously reported that flavonoid compounds, such as quercetin, may activate IRE1α (38). Importantly, treatment of AEC with quercetin increased phosphorylation of IRE1α and subsequently induced degradation of ER-resident NKA-β, without affecting XBP1 s expression or JNK1/2 activation (Figure E3), further underlining the direct role of IRE1α activity in the hypercapnia-induced loss of ER-resident NKA-β.

Intracellular Calcium Efflux from the ER through IP3R Receptors Mediates Hypercapnia-induced ERAD

Hypercapnia has been described to rapidly increase cytosolic Ca2+ concentrations, thus activating intracellular signaling pathways (4, 5). In addition, IRE1α has been shown to be involved in maintaining calcium homeostasis of the ER (39). Thus, we next investigated whether Ca2+ signaling was related to IRE1α phosphorylation and degradation of NKA-β. Thus, PCLS and AEC were loaded with the calcium fluorescent dye Fluo-4 indicator and exposed to 40 or 120 mm Hg at an extracellular pH 7.4 for 60 minutes. In line with previously published observations, hypercapnia significantly increased intracellular levels of Ca2+ in PCLS and AEC (Figures 5A and 5B). To further investigate whether depletion of cytoplasmatic Ca2+ affects ERAD, AEC were treated with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and exposed to 40 or 120 mm Hg of CO2 for 60 minutes (Figures 5C and 5D). Intriguingly, chelation of Ca2+ by BAPTA-AM markedly increased IRE1α phosphorylation and ERAD of NKA-β. Therefore, we next hypothesized that elevated CO2 levels induce intracellular Ca2+ levels by increasing Ca2+ efflux from the ER. To test whether the depletion of extra- and/or intracellular Ca2+ and ER Ca2+ stores affected phosphorylation of IRE1α, we exposed cells to 40 or 120 mm Hg of CO2 in normal DMEM media, DMEM without Ca2+ (Figure E4A), and in presence or absence of BAPTA-AM and thapsigargin (Figure E4B). Our results revealed that hypercapnic exposure in the absence of extracellular calcium or its depletion by BAPTA-AM markedly increased the phosphorylation levels of IRE1α. Moreover, additional application of thapsigargin aggravated the activation of IRE1α. These results suggest that hypercapnia induces a depletion of intra-ER Ca2+ stores. Ca2+ uptake by the ER is mainly mediated by SERCA and Ca2+ release is regulated by receptors, such as inositol trisphosphate receptors (IP3R) that are highly abundant in epithelial cells (40). To further investigate whether the increase in the levels of intracellular Ca2+ is due to its release through IP3R, we next treated PCLS and AEC with the IP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) and exposed the cultures to normocapnia and hypercapnia for 60 minutes (Figures 6A and 6B). Of note, pretreatment with 2-APB was sufficient to decrease intracellular Ca2+ levels in both culture models. Moreover, and further highlighting the central role of IP3R in hypercapnia-induced calcium release, preincubation with 2-APB of either PCLS or AEC significantly decreased phosphorylation levels of IRE1α (Figures 6C and 6D).

Treatment with 2-APB Decreases ERAD and Restores NKA-β PM Abundance

After establishing that IRE1α plays a central role in ERAD and that blocking calcium release through IP3R inhibits IRE1α phosphorylation, we next investigated the effects of 2-APB treatment on NKA-β. PCLS and AEC were pretreated with a 2-APB inhibitor and were then exposed to elevated CO2 concentrations for 60 minutes. Our results showed that inhibition of IP3R activity prevented ERAD of NKA-β in both PCLS and AEC (Figures 7A and 7B). To further prove that the hypercapnia-induced decrease of NKA-β PM abundance is also dependent on Ca2+ release through IP3R, AEC were treated with 2-APB and exposed to normo- or hypercapnia as described above. Importantly, blocking the activity of IP3R restored NKA-β expression at the PM upon hypercapnia (Figure 7C). Finally, and in line with the above-described findings, pretreatment with 2-APB prevented the hypercapnia-induced reduction of NKA-β cell surface abundance in PCLS, as assessed by confocal microscopy (Figure 7D).

In the present study, we provide evidence that in 3D murine PCLS and two-dimensional human AEC cultures, exposure to elevated CO2 levels results in a rapid degradation of the ER-resident, high-mannose glycosylated form of NKA-β. This hypercapnia-induced signaling pathway is dependent on intracellular influx of Ca2+ from the ER through IP3R channels and involves activation of IRE1α that triggers mannose trimming of NKA-β by MAN1B1, causing proteasomal degradation and thus reducing PM abundance of NKA. This novel mechanism may have a deleterious impact in patients with ARDS because impairing PM abundance of NKA leads to dysfunction of the Na pump, impairing vectorial sodium transport and thus aggravating alveolar edema, which is clearly associated with worse outcomes in this patient group (4143). Our results may also partially explain data generated in recent clinical studies showing that in patients with ARDS, hypercapnia is an independent risk factor that worsens clinical outcome (15, 44).

Effective gas exchange requires relatively dry alveoli, a condition that is achieved by clearance of excess alveolar fluid. Alveolar fluid clearance is mediated by coordinated action of ion transporters. Among them, apically localized epithelial sodium channels (ENaC) and the basolateral NKA have been characterized as primary drivers of fluid reabsorption (17). Previously, we and others have shown that hypercapnia decreases PM abundance of both ENaC and NKA by inducing endocytosis of the transporters secondary to phosphorylation and ubiquitination by activation of AMPK, PKC-ζ, ERK, and JNK (3, 5, 6, 22, 30). The cellular effects of hypercapnia are not limited to activation of signaling pathways but also involve a decrease in proliferation, mitochondrial dysfunction, decrease in ATP production, inhibition of inflammatory responses, and changes in adipogenesis (26, 4548). NKA is a heterodimeric protein consisting of three subunits: a catalytic α-subunit, a regulatory β-subunit, and an axillar γ-subunit. Before delivery to the PM, the subunits must be transcribed, translated, and properly folded in the ER and the Golgi (18, 25). Especially, the ER is involved in secretory and transmembrane protein synthesis, folding, maturation, quality control, and degradation and ensures that only properly folded proteins are delivered to their sites of action. As a glycoprotein, NKA-β undergoes extensive glycosylation processes in the ER (24), and proper assembly of this subunit with the α-subunit of NKA is critical for further maturation, trafficking, membrane insertion, and, thus, transport activity of the NKA complex (25). Previous studies associated elevated levels of CO2 with decreased PM abundance of the NKA α-subunit (5, 22). As NKA-β is critically required for delivery of NKA-α to the PM, changes in ER abundance of NKA-β may alter cell surface expression of the NKA-α:β complex (24, 25). Importantly, the ER is a highly vulnerable environment, and alterations in ER homeostasis may have a negative impact on protein folding and thus on function of PM proteins (49).

Therefore, we first investigated whether elevated levels of CO2 affect the ER-resident fraction of NKA-β and the PM abundance of the protein. Of note, we found that hypercapnia induced a rapid, marked, and dose-dependent decrease in the high-mannose glycosylated ER-resident forms of NKA-β, suggesting that the ER is indeed sensitive to changes in CO2 levels. In addition, and in agreement with this notion, our data showed that an increase in CO2 concentrations caused a significant reduction of NKA-β cell-surface abundance in the 3D lung culture model, PCLS, and AEC. In contrast, mRNA and total protein levels of NKA-β remained unchanged during short-term CO2 exposure, suggesting that post-translational modifications were primarily responsible for the changes observed.

It has been shown that in mammalian cells, after being transcribed and translated, glycoproteins are cotranslationally translocated into the ER, where mannose and glucose tags are added to the nascent protein, thus assisting in proper protein folding by the calnexin–calreticulin cycle (32). Misfolded or unassembled proteins in the ER may undergo a specific degradation process, also known as ERAD, which consists of three steps: recognition and targeting, retrotranslocation of substrates from the ER to the cytosol, and degradation of proteins (50). During the initial step of recognition and targeting, in case a glycosylated protein is permanently misfolded, the mannose residue in the middle branch of the oligosaccharide is removed by MAN1B1 in the ER (32). To uncover the potential role of ERAD and particularly of MAN1B1 in the decrease of ER-resident NKA-β, both a pharmacological and a genetic approach to inhibit the activity or expression of MAN1B1 were sufficient to prevent the hypercapnia-induced reduction of NKA-β abundance in the ER. Moreover, silencing EDEM1, which assists in retrotranslocation of substrates from the ER to the cytosol before degradation (51), also prevented the CO2-induced reduction in the high-mannose glycosylated forms of NKA-β. Recently, it has been shown that the mannosidase activity of EDEM1 depends on the folding state of the target protein (31); thus, further studies focusing on the folding state of NKA molecules that undergo ERAD may be of interest. Finally, inhibiting proteasomal but not lysosomal activity was effective in preventing degradation of the ER-resident NKA-β, highlighting the role of proteasomes in ERAD. Collectively, here we demonstrate for the first time that NKA-β is an endogenous substrate for hypercapnia-induced ERAD.

Protein folding in the ER is tightly regulated by UPR pathway proteins and receptors of those, namely, IRE1α, PERK, and ATF6 (52). It has been previously shown that IRE1α actively degrades proteins by directing proteasomal or lysosomal degradation of the targets (35, 52, 53). Our data demonstrate that hypercapnia causes a rapid and transient phosphorylation, and thus activation, of IRE1α at Ser724. Moreover, genetic ablation or direct inhibition of the kinase activity of IRE1α prevents ERAD of NKA-β. Furthermore, treatment of cells with the ER stress inducer thapsigargin or the IRE1α activator quercetin resulted in ERAD of NKA-β, further confirming the central role of IRE1α kinase activity in these processes. These findings are consistent with recent reports, which demonstrated that IRE1α controls degradation of misfolded receptors during eicosanoid protein synthesis (53, 54).

Interestingly, the cytosolic domain of activated IRE1α possesses RNase activity and may activate XBP1 signaling and a mechanism termed regulated IRE1-dependent decay (RIDD), which results in cleavage and degradation of the target mRNA, thus reducing protein abundance (55, 56). Of note, recently it has been reported that NKA-α mRNA might be a substrate for RIDD (37). In contrast, in the current study, we did not detect any changes in the mRNA expression of either NKA-α or -β. In line with this notion, neither inhibiting the endonuclease and mRNA splicing activity of IRE1α nor genetically ablating XPB1 s siRNA affected the hypercapnia-induced ERAD of NKA-β, suggesting that RIDD was not involved in the process of ER-resident NKA-β degradation. Interestingly, treatment with thapsigargin, in contrast to hypercapnia alone, resulted in a shift of the molecular weight of the phospho- and total forms of IRE1α, thus resulting in the “double-band” when assessed by Western blot analysis. This might be a consequence of oligomerization and dimerization of IRE1α domains, leading to subsequent autophosphorylation of IRE1α and activation of the classical UPR response, which involves splicing of XBP1 (57, 58). These findings suggest that hypercapnia, in contrast to classical chemical activators of ER stress, may induce a different IRE1α response, which is independent of XBP1 activity.

Of note, another downstream target of activated IRE1α is JNK (27), which is of particular interest as we and other have previously shown the key role of this kinase in the CO2-induced downregulation of ENaC and NKA-α (3, 22, 23). The mechanism by which hypercapnia reduces PM abundance of NKA-α involves JNK-driven reorganization of the actin cytoskeleton and enhanced interaction of NKA with LMO7b, thereby promoting retrieval of the transporter from the cell surface by endocytosis (22). In addition, IRE1α has been shown to regulate cytoskeleton remodeling and cell migration by affecting filamin A signaling (59). Although in the current setting, we confirmed activation of JNK upon hypercapnic exposure, inhibition of the kinase did not prevent ERAD of NKA-β, suggesting that these signaling events do not contribute to CO2-induced UPR.

Calcium signaling plays a central role in maintaining cellular and, in particular, ER homeostasis (40, 60). Our data establish that hypercapnia-induced activation of IRE1α and ERAD of NKA-β are triggered by Ca2+ efflux from the ER via IP3R channels. Indeed, recent studies demonstrated that hypercapnia may alter Ca2+ homeostasis in either a rapid (up to 1 min) or prolonged (up to 6 h) manner (4, 5). Under physiological conditions, the release of Ca2+ from the ER does not alter luminal ER Ca2+ concentrations. This is controlled by “store-operated” Ca2+ entry mechanisms, in which upon reduced ER Ca2+ concentrations STIM1/STIM2 proteins oligomerize and cooperate with the PM-localized ORAI1 channel, thus activating Ca2+ efflux to the cell and filling of ER stores by SERCA (60, 61). Interestingly, hypoxia-sensing mechanisms involving an interaction of STIM1 and ORAI1 and subsequent Ca2+ influx have been recently reported (62, 63). Regarding the hypercapnia-induced activation of IRE1α and degradation of NKA-β, we observed similarities between exposure to elevated CO2 and thapsigargin treatment, the latter of which is known to deplete ER Ca2+ levels by inhibiting SERCA (39, 64). It is well documented that IRE1α is involved in the regulation of ER calcium homeostasis by controlling IP3R Ca2+ release and mitochondrial calcium uptake (39, 65). In addition, it has been recently shown that elevated CO2 levels may increase Ca2+ release through IP3R (66, 67). Furthermore, it has been demonstrated that NKA, by binding to the scaffolding protein ankyrin, creates a contact between the NKA α-subunit and IP3R in the ER, suggesting Ca2+ sensing by the transporter in epithelial cells (6870). By using Ca2+-free DMEM and thus excluding the extracellular Ca2+ source, we observed a marked upregulation of IRE1α phosphorylation, which was augmented in the presence of the SERCA inhibitor thapsigargin. Therefore, elevated CO2 levels might deplete ER Ca2+ concentrations by two distinct mechanisms: 1) by increasing Ca2+ release from the ER through IP3R and/or 2) by decreasing Ca2+ influx by impairing the function of SERCA or of store-operated channels. Indeed, inhibition of IP3R and thus Ca2+ release from the ER stabilized NKA at the PM upon hypercapnic exposure. However, whether the hypercapnia-induced cellular responses and depletion of Ca2+ stores involve decreased mitochondrial function, dysfunction of ATP-dependent ER Ca2+ and store-operated channels will need to be addressed in future studies. In addition, elevated CO2 levels have been shown to decrease NKA abundance at the PM by calcium-dependent stimulation of AMPK (5). Whether AMPK activation is downstream of Ca2+ release by IP3R receptors and thus affects NKA PM abundance as well needs to be further investigated.

Our study has some limitations. It remains unknown to what extent the above-described elevated CO2-induced signals are adaptive or maladaptive, which is of high relevance when one aims to interfere with these pathways in order the rescue the hypercapnia-induced effects. Moreover, we have recently demonstrated that prolonged exposure to elevated CO2 levels (as opposed to short-term, which is the focus of the current manuscript) induces ER-retention of NKA-β by a distinct mechanism, which may also contribute to decreased NKA α:β complex formation (71). Thus, it will be necessary to tease out the exact time-course and relative contributions of these signaling cascades to altered NKA maturation upon hypercapnia. Moreover, the cell culture systems used in this study have some limitations. Although PCLS is an excellent 3D culture system, in which the architecture and function of cells and cellular interactions remain intact over weeks, the data generated in this model does not exclusively represent AEC, as various other cell types are also present. Similarly, A549 cells, which are often used to study membrane transporters, such as the NKA, are a transformed cell type; thus, calcium dynamics in these cells may not necessarily replicate the situation in the primary alveolar epithelium. However, as the hypercapnia-induced signals appear to be identical regarding the regulation of NKA-β in the two model systems, we believe that our data are of translational relevance. Furthermore, a direct assessment of ER Ca2+ levels and SERCA activity should be performed. Finally, further research in an animal model of acute lung injury is warranted to study the role of IRE1α in the hypercapnia-induced signaling pathways identified in the current manuscript and, particularly, in alveolar fluid clearance.

Conclusions

Collectively, our study demonstrates for the first time that elevated levels of CO2 promote rapid ERAD of the regulatory NKA-β and decrease PM abundance of the enzyme in human AEC and murine PCLS. This mechanism is triggered by Ca2+ release through IP3R receptors und subsequent activation of IRE1α and involves activity of MAN1B1 and EDEM1 (Figure 8). This novel signaling pathway may contribute to impairment of alveolar fluid balance, and thus, its specific inhibition may ameliorate alveolar edema and improve clinical outcomes in patients with ARDS and hypercapnia.

The authors thank Athanasios Fysikopoulos and Andrés Alberro Brage for providing precision-cut lung slices.

1. Briva A, Vadász I, Lecuona E, Welch LC, Chen J, Dada LA, et al. High CO2 levels impair alveolar epithelial function independently of pH. PLoS One 2007;2:e1238.
2. Gates KL, Howell HA, Nair A, Vohwinkel CU, Welch LC, Beitel GJ, et al. Hypercapnia impairs lung neutrophil function and increases mortality in murine pseudomonas pneumonia. Am J Respir Cell Mol Biol 2013;49:821828.
3. Gwoździńska P, Buchbinder BA, Mayer K, Herold S, Morty RE, Seeger W, et al. Hypercapnia impairs ENaC cell surface stability by promoting phosphorylation, polyubiquitination and endocytosis of beta-ENaC in a human alveolar epithelial cell line. Front Immunol 2017;8:591.
4. Shigemura M, Lecuona E, Angulo M, Homma T, Rodríguez DA, Gonzalez-Gonzalez FJ, et al. Hypercapnia increases airway smooth muscle contractility via caspase-7-mediated miR-133a-RhoA signaling. Sci Transl Med 2018;10:eaat1662.
5. Vadász I, Dada LA, Briva A, Trejo HE, Welch LC, Chen J, et al. AMP-activated protein kinase regulates CO2-induced alveolar epithelial dysfunction in rats and human cells by promoting Na,K-ATPase endocytosis. J Clin Invest 2008;118:752762.
6. Welch LC, Lecuona E, Briva A, Trejo HE, Dada LA, Sznajder JI. Extracellular signal-regulated kinase (ERK) participates in the hypercapnia-induced Na,K-ATPase downregulation. FEBS Lett 2010;584:39853989.
7. Cummins EP, Selfridge AC, Sporn PH, Sznajder JI, Taylor CT. Carbon dioxide-sensing in organisms and its implications for human disease. Cell Mol Life Sci 2014;71:831845.
8. Endeward V, Al-Samir S, Itel F, Gros G. How does carbon dioxide permeate cell membranes? A discussion of concepts, results and methods. Front Physiol 2014;4:382.
9. Vadász I, Sznajder JI. Gas exchange disturbances regulate alveolar fluid clearance during acute lung injury. Front Immunol 2017;8:757.
10. Barnes T, Zochios V, Parhar K. Re-examining permissive hypercapnia in ARDS: a narrative review. Chest 2018;154:185195.
11. Shigemura M, Lecuona E, Sznajder JI. Effects of hypercapnia on the lung. J Physiol 2017;595:24312437.
12. Vadász I, Hubmayr RD, Nin N, Sporn PH, Sznajder JI. Hypercapnia: a nonpermissive environment for the lung. Am J Respir Cell Mol Biol 2012;46:417421.
13. Yang H, Xiang P, Zhang E, Guo W, Shi Y, Zhang S, et al. Is hypercapnia associated with poor prognosis in chronic obstructive pulmonary disease? A long-term follow-up cohort study. BMJ Open 2015;5:e008909.
14. Matthay MA. Resolution of pulmonary edema. Thirty years of progress. Am J Respir Crit Care Med 2014;189:13011308.
15. Nin N, Angulo M, Briva A. Effects of hypercapnia in acute respiratory distress syndrome. Ann Transl Med 2018;6:37.
16. Tiruvoipati R, Pilcher D, Buscher H, Botha J, Bailey M. Effects of hypercapnia and hypercapnic acidosis on hospital mortality in mechanically ventilated patients. Crit Care Med 2017;45:e649e656.
17. Matalon S, Bartoszewski R, Collawn JF. Role of epithelial sodium channels in the regulation of lung fluid homeostasis. Am J Physiol Lung Cell Mol Physiol 2015;309:L1229L1238.
18. Clausen MV, Hilbers F, Poulsen H. The structure and function of the Na,K-ATPase isoforms in health and disease. Front Physiol 2017;8:371.
19. Tokhtaeva E, Sachs G, Souda P, Bassilian S, Whitelegge JP, Shoshani L, et al. Epithelial junctions depend on intercellular trans-interactions between the Na,K-ATPase β1 subunits. J Biol Chem 2011;286:2580125812.
20. Vagin O, Dada LA, Tokhtaeva E, Sachs G. The Na-K-ATPase α1β1 heterodimer as a cell adhesion molecule in epithelia. Am J Physiol Cell Physiol 2012;302:C1271C1281.
21. Flodby P, Kim YH, Beard LL, Gao D, Ji Y, Kage H, et al. Knockout mice reveal a major role for alveolar epithelial type I cells in alveolar fluid clearance. Am J Respir Cell Mol Biol 2016;55:395406.
22. Dada LA, Trejo Bittar HE, Welch LC, Vagin O, Deiss-Yehiely N, Kelly AM, et al. High CO2 leads to Na,K-ATPase endocytosis via c-Jun amino-terminal kinase-induced LMO7b phosphorylation. Mol Cell Biol 2015;35:39623973.
23. Vadász I, Dada LA, Briva A, Helenius IT, Sharabi K, Welch LC, et al. Evolutionary conserved role of c-Jun-N-terminal kinase in CO2-induced epithelial dysfunction. PLoS One 2012;7:e46696.
24. Tokhtaeva E, Sachs G, Vagin O. Assembly with the Na,K-ATPase alpha(1) subunit is required for export of beta(1) and beta(2) subunits from the endoplasmic reticulum. Biochemistry 2009;48:1142111431.
25. Tokhtaeva E, Sachs G, Vagin O. Diverse pathways for maturation of the Na,K-ATPase β1 and β2 subunits in the endoplasmic reticulum of Madin-Darby canine kidney cells. J Biol Chem 2010;285: 3928939302.
26. Vohwinkel CU, Lecuona E, Sun H, Sommer N, Vadász I, Chandel NS, et al. Elevated CO(2) levels cause mitochondrial dysfunction and impair cell proliferation. J Biol Chem 2011;286:3706737076.
27. Almanza A, Carlesso A, Chintha C, Creedican S, Doultsinos D, Leuzzi B, et al. Endoplasmic reticulum stress signalling - from basic mechanisms to clinical applications. FEBS J 2019;286:241278.
28. Depaoli MR, Hay JC, Graier WF, Malli R. The enigmatic ATP supply of the endoplasmic reticulum. Biol Rev Camb Philos Soc 2019;94: 610628.
29. Niehof M, Hildebrandt T, Danov O, Arndt K, Koschmann J, Dahlmann F, et al. RNA isolation from precision-cut lung slices (PCLS) from different species. BMC Res Notes 2017;10:121.
30. Lecuona E, Sun H, Chen J, Trejo HE, Baker MA, Sznajder JI. Protein kinase A-Iα regulates Na,K-ATPase endocytosis in alveolar epithelial cells exposed to high CO(2) concentrations. Am J Respir Cell Mol Biol 2013;48:626634.
31. Shenkman M, Ron E, Yehuda R, Benyair R, Khalaila I, Lederkremer GZ. Mannosidase activity of EDEM1 and EDEM2 depends on an unfolded state of their glycoprotein substrates. Commun Biol 2018;1:172.
32. Ellgaard L, Helenius A. Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol 2003;4:181191.
33. Hetz C, Chevet E, Oakes SA. Proteostasis control by the unfolded protein response. Nat Cell Biol 2015;17:829838.
34. Chen Y, Brandizzi F. IRE1: ER stress sensor and cell fate executor. Trends Cell Biol 2013;23:547555.
35. Hwang J, Qi L. Quality control in the endoplasmic reticulum: Crosstalk between ERaD and UPR pathways. Trends Biochem Sci 2018;43:593605.
36. Adams CJ, Kopp MC, Larburu N, Nowak PR, Ali MMU. Structure and molecular mechanism of ER stress signaling by the unfolded protein response signal activator IRE1. Front Mol Biosci 2019;6:11.
37. Acosta-Alvear D, Karagöz GE, Fröhlich F, Li H, Walther TC, Walter P. The unfolded protein response and endoplasmic reticulum protein targeting machineries converge on the stress sensor IRE1. eLife 2018;7:e43036.
38. Wiseman RL, Zhang Y, Lee KP, Harding HP, Haynes CM, Price J, et al. Flavonol activation defines an unanticipated ligand-binding site in the kinase-RNase domain of IRE1. Mol Cell 2010;38:291304.
39. Son SM, Byun J, Roh SE, Kim SJ, Mook-Jung I. Reduced IRE1α mediates apoptotic cell death by disrupting calcium homeostasis via the InsP3 receptor. Cell Death Dis 2014;5:e1188.
40. Bagur R, Hajnóczky G. Intracellular Ca(2+) sensing: Its role in calcium homeostasis and signaling. Mol Cell 2017;66:780788.
41. Matthay MA, Zemans RL, Zimmerman GA, Arabi YM, Beitler JR, Mercat A, et al. Acute respiratory distress syndrome. Nat Rev Dis Primers 2019;5:18.
42. Sznajder JI. Alveolar edema must be cleared for the acute respiratory distress syndrome patient to survive. Am J Respir Crit Care Med 2001;163:12931294.
43. Sznajder JI, Factor P, Ingbar DH. Invited review: lung edema clearance: Role of Na(+)-K(+)-ATPase. J Appl Physiol (1985) 2002;93:18601866.
44. Nin N, Muriel A, Peñuelas O, Brochard L, Lorente JA, Ferguson ND, et al.; VENTILA Group. Severe hypercapnia and outcome of mechanically ventilated patients with moderate or severe acute respiratory distress syndrome. Intensive Care Med 2017;43:200208.
45. Casalino-Matsuda SM, Nair A, Beitel GJ, Gates KL, Sporn PH. Hypercapnia inhibits autophagy and bacterial killing in human macrophages by increasing expression of bcl-2 and bcl-xl. J Immunol 2015;194:53885396.
46. Casalino-Matsuda SM, Wang N, Ruhoff PT, Matsuda H, Nlend MC, Nair A, et al. Hypercapnia alters expression of immune response, nucleosome assembly and lipid metabolism genes in differentiated human bronchial epithelial cells. Sci Rep 2018;8:13508.
47. Keogh CE, Scholz CC, Rodriguez J, Selfridge AC, von Kriegsheim A, Cummins EP. Carbon dioxide-dependent regulation of NF-κB family members RelB and p100 gives molecular insight into CO2-dependent immune regulation. J Biol Chem 2017;292:1156111571.
48. Kikuchi R, Tsuji T, Watanabe O, Yamaguchi K, Furukawa K, Nakamura H, et al. Hypercapnia accelerates adipogenesis: a novel role of high CO2 in exacerbating obesity. Am J Respir Cell Mol Biol 2017;57:570580.
49. Wang M, Kaufman RJ. Protein misfolding in the endoplasmic reticulum as a conduit to human disease. Nature 2016;529:326335.
50. Araki K, Nagata K. Protein folding and quality control in the ER. Cold Spring Harb Perspect Biol 2012;4:a015438.
51. Olivari S, Molinari M. Glycoprotein folding and the role of EDEM1, EDEM2 and EDEM3 in degradation of folding-defective glycoproteins. FEBS Lett 2007;581:36583664.
52. Frakes AE, Dillin A. The UPR(ER): sensor and coordinator of organismal homeostasis. Mol Cell 2017;66:761771.
53. Chiang WC, Messah C, Lin JH. IRE1 directs proteasomal and lysosomal degradation of misfolded rhodopsin. Mol Biol Cell 2012;23:758770.
54. Chopra S, Giovanelli P, Alvarado-Vazquez PA, Alonso S, Song M, Sandoval TA, et al. IRE1α-XBP1 signaling in leukocytes controls prostaglandin biosynthesis and pain. Science 2019;365:eaau6499.
55. Maurel M, Chevet E, Tavernier J, Gerlo S. Getting RIDD of RNA: IRE1 in cell fate regulation. Trends Biochem Sci 2014;39:245254.
56. Sun S, Shi G, Sha H, Ji Y, Han X, Shu X, et al. IRE1α is an endogenous substrate of endoplasmic-reticulum-associated degradation. Nat Cell Biol 2015;17:15461555.
57. Ghosh R, Wang L, Wang ES, Perera BG, Igbaria A, Morita S, et al. Allosteric inhibition of the IRE1α RNase preserves cell viability and function during endoplasmic reticulum stress. Cell 2014;158:534548.
58. Prischi F, Nowak PR, Carrara M, Ali MM. Phosphoregulation of Ire1 RNase splicing activity. Nat Commun 2014;5:3554.
59. Urra H, Henriquez DR, Cánovas J, Villarroel-Campos D, Carreras-Sureda A, Pulgar E, et al. IRE1α governs cytoskeleton remodelling and cell migration through a direct interaction with filamin A. Nat Cell Biol 2018;20:942953.
60. Mekahli D, Bultynck G, Parys JB, De Smedt H, Missiaen L. Endoplasmic-reticulum calcium depletion and disease. Cold Spring Harb Perspect Biol 2011;3:a004317.
61. van Vliet AR, Giordano F, Gerlo S, Segura I, Van Eygen S, Molenberghs G, et al. The ER stress sensor PERK coordinates ER-plasma membrane contact site formation through interaction with filamin-A and F-actin remodeling. Mol Cell 2017;65:885899.e6.
62. Gusarova GA, Trejo HE, Dada LA, Briva A, Welch LC, Hamanaka RB, et al. Hypoxia leads to Na,K-ATPase downregulation via Ca(2+) release-activated Ca(2+) channels and AMPK activation. Mol Cell Biol 2011;31:35463556.
63. Lahiri S, Roy A, Li J, Mokashi A, Baby SM. Ca2+ responses to hypoxia are mediated by IP3-R on Ca2+ store depletion. Adv Exp Med Biol 2003;536:2532.
64. Jones KT, Sharpe GR. Thapsigargin raises intracellular free calcium levels in human keratinocytes and inhibits the coordinated expression of differentiation markers. Exp Cell Res 1994;210:7176.
65. Carreras-Sureda A, Jaña F, Urra H, Durand S, Mortenson DE, Sagredo A, et al. Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics. Nat Cell Biol 2019;21:755767.
66. Cook ZC, Gray MA, Cann MJ. Elevated carbon dioxide blunts mammalian cAMP signaling dependent on inositol 1,4,5-triphosphate receptor-mediated Ca2+ release. J Biol Chem 2012;287:2629126301.
67. Turner MJ, Saint-Criq V, Patel W, Ibrahim SH, Verdon B, Ward C, et al. Hypercapnia modulates cAMP signalling and cystic fibrosis transmembrane conductance regulator-dependent anion and fluid secretion in airway epithelia. J Physiol 2016;594:16431661.
68. Tian J, Xie ZJ. The Na-K-ATPase and calcium-signaling microdomains. Physiology (Bethesda) 2008;23:205211.
69. Liu X, Spicarová Z, Rydholm S, Li J, Brismar H, Aperia A. Ankyrin B modulates the function of Na,K-ATPase/inositol 1,4,5-trisphosphate receptor signaling microdomain. J Biol Chem 2008;283:1146111468.
70. Aperia A, Brismar H, Uhlén P. Mending fences: Na,K-ATPase signaling via Ca2+ in the maintenance of epithelium integrity. Cell Calcium 2020;88:102210.
71. Kryvenko V, Wessendorf M, Morty RE, Herold S, Seeger W, Vagin O, et al. Hypercapnia impairs Na,K-ATPase function by inducing endoplasmic reticulum retention of the beta-subunit of the enzyme in alveolar epithelial cells. Int J Mol Sci 2020;21:1467.
Correspondence and requests for reprints should be addressed to István Vadász, M.D., Ph.D., Department of Internal Medicine, Justus Liebig University, Universities of Giessen and Marburg Lung Center, Klinikstrasse 33, 35392 Giessen, Germany. E-mail: .

*S.H. is Deputy Editor of AJRCMB. Her participation complies with American Thoracic Society requirements for recusal from review and decisions for authored works.

Supported by Bundesministerium für Bildung und Forschung grant DZL/ALI (S.H., R.E.M., W.S., and I.V.); the Hessen State Ministry of Higher Education, Research and the Arts (Landes-Offensive zur Entwicklung Wissenschaftlichökonomischer Exzellenz) (S.H., R.E.M., W.S., and I.V.); Von-Behring-Röntgen-Stiftung grant Project 66-LV07 (I.V.); Deutsche Forschungsgemeinschaft grant CRU309/P5 (S.H., R.E.M., W.S., and I.V.); The Cardio-Pulmonary Institute (EXC 2026; Project ID: 390649896) (K.T., S.H., R.E.M., W.S., and I.V.); and an M.D./Ph.D. start-up grant (DFG/KFO309, M.D./Ph.D.) (V.K.).

Author Contributions: V.K. and I.V. designed the study and drafted the manuscript. V.K. and M.W. executed the study. V.K., K.T., S.H., R.E.M., W.S., and I.V. analyzed and interpreted results. All authors contributed significant edits, gave final approval for publication, and agree to be accountable for the integrity of the information contained in this manuscript.

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Originally Published in Press as DOI: 10.1165/rcmb.2021-0114OC on June 30, 2021

Author disclosures are available with the text of this article at www.atsjournals.org.

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