Cystic fibrosis (CF) is a genetic disease characterized by CF transmembrane regulator (CFTR) dysfunction. With over 2000 CFTR variants identified, in addition to known patient to patient variability, there is a need for personalized treatment. The discovery of CFTR modulators has shown efficacy in certain CF populations, however there are still CF populations without valid therapeutic options. With evidence suggesting that single drug therapeutics are insufficient for optimal management of CF disease, there has been an increased pursuit of combinatorial therapies. Our aim was to test cyclic AMP (cAMP) modulation, through ATP Binding Cassette Transporter C4 (ABCC4) and phosphodiesterase-4 (PDE-4) inhibition, as a potential add-on therapeutic to a clinically approved CFTR modulator, VX-770, as a method for increasing CFTR activity. Human airway epithelial cells (Calu-3) were used to test the efficacy of cAMP modulation by ABCC4 and PDE-4 inhibition through a series of concentration–response studies. Our results showed that cAMP modulation, in combination with VX-770, led to an increase in CFTR activity via an increase in sensitivity when compared to treatment of VX-770 alone. Our study suggests that cAMP modulation has potential to be pursued as an add-on therapy for the optimal management of CF disease.
Cystic fibrosis (CF) is a recessive genetic disease characterized by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), a phosphorylation-regulated ion channel responsible for conducting chloride and bicarbonate ions across epithelial cell membranes1,2,3,4. CFTR is localized at the apical membrane of epithelial cells that line primary conducting airways and submucosal glands and contributes to the regulation of airway surface lining (ASL) fluid volume and composition5. In CF, disease-causing mutations in CFTR lead to dysregulated ASL fluid volume and composition in the lung which is associated with increased mucus viscosity, increased susceptibility to pathogens, and modulated lung immunity6,7,8,9.
There are over 2000 known CFTR variants identified that are associated with a wide range of biological and functional consequences (https://www.genet.sickkids.on.ca). These variants have traditionally been classified into six classes based on their phenotype, including impairments to protein maturation, protein gating, and ion conductance10,11,12. However, evidence has suggested that this traditional classification is outdated since there exists CFTR variants with complex phenotypes that cannot be adequately classified into a single class13. Moreover, there has been evidence demonstrating that there are phenotypic differences between CF patients carrying the same genotypic CF variant14. This emphasizes the need for combinational therapies that will target several biological and functional defects.
Recent advancements in the molecular understanding of the functional impacts of specific CFTR variants has revolutionized drug development. As a result of discovery research focused on the function of specific CFTR variants, the Federal Drug Administration has approved four CFTR modulators15,16,17,18. The first CFTR modulator approved was CFTR potentiator ivacaftor (VX-770) which directly binds to CFTR to increase opening of the channel thus increasing chloride conductance19. In G551D-CFTR variants, VX-770 has demonstrated significant increases in predicted forced expiratory volume in 1 s (FEV1) from baseline at various doses and treatment durations20,21. VX-770 was subsequently assessed in combination with the CFTR corrector lumacaftor (VX-809), which increases the amount of CFTR reaching the cell surface, and showed mild improvements in FEV1 (2.6–4 percent points) and decreases in pulmonary exacerbations (30–39%) in the F508del homozygous population, which represents the global majority of CF patients22,23,24. Additional CFTR modulators approved include CFTR correctors tezacaftor (VX-661) and elexacaftor (VX-445)25,26,27,28. On-going combinatorial studies with VX-770, novel potentiators, VX-561—which is an altered form of VX-770, ABBV-3067, and PTI-808; novel correctors, VX-661, VX-445, VX-659, VX-121, ABBV-2222, and PTI-801; and amplifier, PTI-428, which increases the amount of CFTR produced, suggest that a single drug therapy will not be sufficient for optimal management of CF disease (NCT03912233, NCT03969888, NCT03500263)25,26,27,28,29,30. Furthermore, although combinatorial CFTR modulator therapies have shown efficacy in previously poorly managed CF populations, many CFTR variants have not been studied at an individual level. In addition, these CFTR modulator therapies directly target CFTR defects and do not take into account mechanisms implicated in CFTR activity, such as phosphorylation of the regulatory domain of CFTR31,32,33. As such, new drugs and drug combinations that align with precision medicine approaches should be developed to ensure that there are valid therapeutic options for each individual with CF.
While understanding the genetic variation in CF patients is important for developing combinatorial therapies, there are many factors that can influence CFTR potentiation, including cyclic AMP (cAMP) metabolism. CFTR is phosphorylated by the cAMP-dependent enzyme protein kinase A (PKA)31,32. Following a rise in intracellular cAMP, PKA is activated and can phosphorylate CFTR, increasing open channel probability31,32,33. There are multiple mechanisms contributing to cAMP regulation, including cAMP-efflux transporters, phosphodiesterases (PDE), and β2 adrenergic receptors14,34,35,36,37,38,39. A key cAMP transporter is ATP Binding Cassette Transporter C4 (ABCC4), formerly known as multi-drug resistance protein-434,40. ABCC4 is functionally and physically coupled with CFTR through the scaffolding protein PDZK134. Inhibition of ABCC4 with MK-571 was first shown to be able to attenuate cAMP transport and potentiate CFTR activity in gut epithelial cells, however this has been later demonstrated in airway epithelial cells14,34,41,42. In the context of CF, we have demonstrated that pharmacological inhibition of ABCC4 in human airway epithelial cells is able to potentiate CFTR in G551D-variants above and beyond VX-77014. Aside from ABCC4 inhibition, there has also been evidence demonstrating that pharmacological inhibition of PDE-4 was able to potentiate CFTR activity through the elevation of cAMP levels37,38,43. The interaction of ABCC4 and PDE-4 inhibition to increase cAMP levels to augment CFTR potentiation remains to be explored.
Currently there are several investigational or clinically available therapeutics that have potential applications in influencing CFTR activity through cAMP elevation. MK-571 has been previously used as an ABCC4 inhibitor to prevent cAMP-efflux, resulting in an increase in CFTR activity in human airway epithelial cells14,41,42. MK-571 was designed as a cysteinyl leukotriene receptor antagonist and is exploited as a research compound due to off-target effects that include ABCC4 and PDE-4 inhibition and has also been used in humans safely44. Conversely, Ceefourin-1 and 2 have been reported as ABCC4 inhibitors with demonstrated in vitro efficacy and selectivity45. Roflumilast is a clinically available selective PDE-4 inhibitor with anti-inflammatory effects that is commonly administered to chronic obstructive pulmonary disease (COPD) patients suffering from chronic bronchitis46,47,48,49. Similar to Roflumilast, Rolipram is also known to be a selective PDE-4 inhibitor. Originally it was investigated as a potential antidepressant drug, however, it is not used clinically due to adverse side-effects and its small therapeutic window50,51,52.
Due to our previous demonstration that pharmacological inhibition of ABCC4, in the presence of CFTR modulator VX-770, leads to CFTR potentiation beyond VX-770 alone and evidence demonstrating the use of PDE-4 inhibitors is also able to potentiate CFTR activity, we hypothesize that cAMP modulation with ABCC4 and PDE-4 inhibitors, in the presence of VX-770, will potentiate CFTR activity. In order to begin defining the efficacy of cAMP modulation by ABCC4 and PDE-4 inhibitors on CFTR activity, we pursued a series of concentration–response studies on Calu-3 human airway epithelial cells with wild-type CFTR53,54,55. We demonstrate that combinatorial additions of CFTR modulator VX-770 with either an ABCC4 or PDE-4 inhibitor led to CFTR potentiation via an increase in sensitivity. Our results suggest that combinatorial additions of VX-770 with ABCC4 or PDE-4 inhibitors may increase sensitivity and efficacy of VX-770 alone.
In vitro extracellular cAMP assay analysis of ABCC4 inhibitor compounds in human airway epithelial cells
An in vitro extracellular cAMP assay of two commercially available ABCC4 inhibitors, MK-571 and Ceefourin-1 (Fig. 1a,b), was performed in human airway epithelial Calu-3 cells, and human bronchial epithelial cells (HBEC6-KT, Supplementary Fig. 1), to define their efficacy in decreasing extracellular cAMP levels. Concentration-dependent ABCC4 inhibition using MK-571 and Ceefourin-1 (Fig. 1) caused a decrease in extracellular cAMP levels in both cell lines. The half maximal inhibitory concentration (IC50) values of MK-571 and Ceefourin-1 were found to be 2.6 μM and 0.7 μM respectively and defined a role for ABCC4-mediated cAMP transport in human airway epithelial cells that may be leveraged for CFTR potentiation.
Detection of ABCC4 and CFTR in human airway epithelial cell lines
The expression and function of ABCC4 has been confirmed in HBEC6-KT cells (Supplementary Fig. 1) although CFTR expression and function remain to be defined41,42. In contrast, human airway epithelial Calu-3 cells are well documented for CFTR expression and function but not for ABCC453,54,55.
ABCC4 and CFTR protein expression levels were therefore probed via immunoblot in HBEC6-KT and Calu-3 cell lines with total protein stain performed as a loading control (Fig. 2). Due to reported time-dependent differentiation and polarization of Calu-3 cells, protein was extracted at 4 different time points (0, 7, 14, and 21 days post-confluency)53,56,57,58. ABCC4 was present in both HBEC6-KT and Calu-3 cells at all-time points, indicated by the bands present at 150 kDa. CFTR was present at all-time points in Calu-3 cells. No CFTR was detected in HBEC6-KT cells. Due to co-expression of ABCC4 and CFTR, Calu-3 cells were used for subsequent studies exploring the interrelationships between ABCC4, cAMP modulation, and CFTR.
Receptor-dependent and -independent activation of CFTR activity—impact of VX-770
A concentration–response analysis was performed with forskolin (FSK) and isoproterenol (ISO), G protein-coupled receptor-independent and -dependent cAMP inducers, respectively (Fig. 3). The half maximal effective concentration (EC50) value for FSK and ISO were determined to be 0.19 μM and 0.07 μM respectively, with both compounds demonstrating ability to induce CFTR activity as measured by a validated membrane potential sensitive assay14.
Concentration–response curves with CFTR modulator VX-770, using both receptor-independent FSK and receptor-dependent ISO cAMP inducers (Fig. 4a,e), demonstrated an upward and leftward shift in the curve, indicating an increase in CFTR activity. Area under the curve (AUC) analysis showed a significant increase with the addition of VX-770 with either cAMP inducer (Fig. 4b,f; **P ≤ 0.01 and ****P ≤ 0.0001). VX-770 increased the sensitivity of FSK-induced CFTR activity as measured by EC50 analysis (Fig. 4c; **P ≤ 0.01). However, the maximal effect concentration (Emax) remained unchanged (Fig. 4d). Despite changes in AUC, no changes in EC50 or Emax were observed with the combination of VX-770 and ISO (Fig. 4g,h).
Collectively, these results demonstrate that both receptor-dependent and -independent mechanisms of cAMP elevation are able to induce CFTR activity in Calu-3 cells, which in turn is potentiated with VX-770.
Effect of ABCC4 and PDE-4 inhibition on CFTR activity
Following our quantification of CFTR activity with the clinically approved CFTR potentiator, VX-770, we next investigated the effect of ABCC4 inhibition and PDE-4 inhibition in Calu-3 cells.
For pharmacological inhibition of ABCC4, we used MK-571 and Ceefourin-1 (see Fig. 1) with FSK and ISO (Fig. 5a–d). No combination of ABCC4 inhibitor and cAMP-elevating agent changed CFTR activity in Calu-3 cells when examining AUC, EC50, Emax, and baseline values (Fig. 5e–h).
For pharmacological inhibition of PDE-4 we used Roflumilast and Rolipram with FSK and ISO (Fig. 6a–d). In contrast to ABCC4 inhibition, the concentration–response curves of PDE-4 inhibitors showed leftward shifts with ISO (Fig. 6c,d). Similarly, AUC analysis revealed significant increases with the addition of either Roflumilast or Rolipram with ISO but not FSK (Fig. 6e; *P ≤ 0.05). EC50, Emax, and baseline values for PDE-4 inhibitors with either cAMP inducer were not different (Fig. 6f–h). In addition to Roflumilast and Rolipram, pharmacological inhibition using non-specific PDE inhibitor IBMX was also tested (Supplementary Fig. 2). Significant increases in AUC (Supplementary Fig. 2c,i; *P ≤ 0.05; **P ≤ 0.01), with either cAMP inducer, were seen and significant increases in baseline values for IBMX with ISO only was revealed (Supplementary Fig. 2l; *P ≤ 0.05).
Effect of cAMP modulation with VX-770 on CFTR activity
Due to our observations that ABCC4 and PDE-4 inhibition alone showed minor potentiation of CFTR activity relative to VX-770, we next investigated whether a combinatorial approach could be more efficacious13.
Concentration–response curves of VX-770 + MK-571 or Roflumilast combinations with FSK stimulation led to an upward shift of the curve at all concentrations, indicating an increase in CFTR activity (Fig. 7a,b). VX-770 + MK-571 or Roflumilast combinations with ISO also led to shifts in the curves, however only at lower concentrations (Fig. 7c,d). For the AUC analysis of each combination, increases were observed with FSK but not ISO (Fig. 7e; **P ≤ 0.01, and ***P ≤ 0.001). EC50 and Emax analysis for each combination with both cAMP inducers showed no difference (Fig. 7f,g).
The consequences of these pharmacological intervention combinations with FSK, in addition to Roflumilast + MK-571 + VX-770 combinations, were also tested in primary human bronchial epithelial cells (HBECs), from two independent donors, grown under air–liquid interface culture conditions (Supplementary Fig. 3). In one donor sample, significant increases in AUC and max peak analysis, beyond VX-770 alone were observed. For AUC, Roflumilast ± VX-770, Roflumilast + MK-571, and Roflumilast + MK-571 + VX-770 combinations were significantly greater than VX-770 alone (Supplementary Fig. 3c; **P ≤ 0.001; ****P ≤ 0.0001). Similarly, the max peak achieved with Roflumilast ± VX-770, MK-571 + VX-770, Roflumilast + MK-571, and Roflumilast + MK-571 + VX-770 were significantly greater than VX-770 alone as well (Supplementary Fig. 3d; *P ≤ 0.05; ****P ≤ 0.0001). These effects were not observed in the other donor sample (Supplementary Fig. 3g,h).
To compare our series of cAMP modulation interventions with and without VX-770 (Figs. 4, 5, 6, 7), fold-change comparisons of AUC, EC50, and Emax, relative to control conditions were performed for all experiments (Fig. 8). Fold-change analysis of AUC with FSK stimulation showed that VX-770 was superior to either ABCC4 (MK-571) or PDE-4 (Roflumilast) inhibition alone, and that combinatorial approaches did not potentiate CFTR AUC values (Fig. 8a, **P ≤ 0.01 and ***P ≤ 0.001). ISO stimulation resulted in similar modest trends that were not significant (Fig. 8d). Fold-change EC50 analysis between VX-770 alone to VX-770 with ABCC4 or PDE-4 inhibition showed a significant decrease with ISO for both combinatorial approaches, observations that were absent with FSK stimulation. (Fig. 8b,e, *P ≤ 0.05). Lastly, fold-change Emax analysis performed with either FSK or ISO showed no changes for any combinatorial approach (Fig. 8c,f).
There is an increased need for combinatorial therapies for CF subjects to optimally treat all subjects at a personalized level13,25,27,29. CFTR potentiation with VX-770 represents a high-water mark for increased chloride conductance in CF patients. In the present study, we quantified the capacity of combinatorial approaches that modulate intracellular cAMP levels to activate CFTR. Specifically, we inhibited ABCC4, a cAMP-efflux pathway, and PDE-4, a cAMP metabolism pathway. Using Calu-3 cells, we demonstrate that interventions targeting intracellular cAMP are unable to potentiate CFTR to levels observed with VX-770. Combinatorial approaches of cAMP modulation with VX-770 suggest that increases in CFTR activity as measured by AUC and EC50 values, are possible and warrant further exploration in primary human airway epithelial cells expressing loss of function CFTR variants14.
The addition of VX-770 to Calu-3 cells induced shifts in the curve with both FSK and ISO, providing context for the amount of CFTR potentiation possible in Calu-3 cells with a clinically approved CFTR potentiator. While there was a significant change to EC50 with FSK, no changes were seen with ISO, suggesting that mechanisms governing cAMP elevations may influence downstream analyses of CFTR activities59,60,61,62,63. For both cAMP inducers, the Emax values were not significant and while there were no significant changes to baseline with FSK, there was a significant increase in baseline with ISO (data not shown). Collectively, this suggests not only is the mechanism of action in elevating cAMP levels affecting sensitivity, but also efficacy59,60,61,62,63. Collectively, these results suggest that it is possible to potentiate CFTR activity in Calu-3 cells by modulating cAMP levels.
Combinatorial approaches have been suggested as the future for modulation of CFTR activity and may include direct or indirect approaches13. Modulation of cAMP levels functions as an indirect mechanism to modulate CFTR and could potentially be combined with direct CFTR modulator approaches. We therefore decided to investigate ABCC4 and PDE-4 inhibitors which are able to differentially modulate cAMP levels by blocking extracellular transport and intracellular breakdown, respectively.
It has been previously demonstrated that pharmacological inhibition of ABCC4 by MK-571, in combination with VX-770, was able to improve CFTR activity in primary nasal epithelial cells from CF patients heterozygous for the G551D-variant14. In addition, in primary non-CF HBECs, it has been demonstrated that the administration of Roflumilast led to CFTR activation38. However, in this same study, the addition of Roflumilast to Calu-3 cells did not potentiate CFTR activity beyond the max stimulation that resulted from the addition of FSK38.
In our concentration–response analysis of ABCC4 and PDE-4 inhibitors in Calu-3 cells, only mild CFTR potentiation was observed alone or in combination with VX-770. Our results for ABCC4 inhibition contrast our previous demonstration of ABCC4 inhibitor MK-571 in potentiating CFTR activity in G551D-variant expressing primary human airway epithelial cells14. Conversely, our results of PDE-4 inhibitors align with previous findings in primary HBECs that were exposed to whole cigarette smoke in which Roflumilast, in combination with VX-770, demonstrated an increase in short-circuit current, suggesting this combination leads to an increase in CFTR activity38. We highlight that it may be possible that wild-type CFTR expressed in Calu-3 cells is maximally active and difficult to further potentiate with cAMP modulating agents. Our data showing shifts in the left of the curve (increased sensitivity of CFTR activity) without changes in maximal response support this concept. Specifically, the addition of VX-770 with either cAMP inducer to Calu-3 cells led to an upward shift in the curve, a significant increase in AUC but no significant changes to Emax. However, VX-770 with FSK showed a significant decrease in EC50 and VX-770 with ISO showed a significant increase in baseline values. When VX-770 was combined with an ABCC4 or PDE-4 inhibitor and cAMP inducer FSK, there were significant increases in AUC, indicating an increase in CFTR activity, but again no change in Emax. In order to determine whether ABCC4 and PDE-4 inhibitor with VX-770 had an additive effect beyond VX-770 alone, a fold-change analysis for AUC, EC50, and Emax values of the independent experiments was performed for indirect comparisons. Combinatorial additions of VX-770 with ABCC4 or PDE-4 inhibitor, in the presence of cAMP inducer ISO, led to a decrease in fold EC50 values compared to VX-770 alone, suggesting that ABCC4 and PDE-4 inhibitors may increase the sensitivity to VX-770. In addition, with cAMP inducer FSK, VX-770 with Roflumilast had a significant increase in fold baseline values, suggesting that there may also be an increase in efficacy. With these fold-change comparison analyses in mind, further investigation of these combinatorial treatments should be performed.
Our interrogation of the role of ABCC4 in airway epithelial cell modulation of cAMP levels is grounded in reports from primary human airway epithelial cells and cell lines, while examining CFTR activity in Calu-3 cells has a strong foundation35,41,42,53,54. To determine the optimal cell system to perform ABCC4 interventions with outcomes of CFTR activity, we interrogated both HBEC-6KT and Calu-3 cells for ABCC4 and CFTR protein expression levels. To our surprise, while ABCC4 is present in both cell lines, CFTR is only present in the Calu-3 cells, leading to the selection of Calu-3 cells as the model of study. Our observation highlights the importance of basal expression analysis for CFTR protein in any human airway epithelial cells or lines prior to performing functional experiments.
Elevations in intracellular cAMP can be induced via multiple G protein-coupled receptor-dependent (β-adrenergic receptor and adenosine receptor agonists—ISO and adenosine respectively) and independent mechanisms (direct activation of the enzyme adenylyl cyclase—FSK)64,65,66,67,68. The compartmentalization of cAMP signalling may be important for downstream signalling pathways within the cell—with specific implications for CFTR activity34,69. To determine whether the mechanism of cAMP production impacts CFTR activity, Calu-3 cells were treated with FSK and ISO at varying concentrations. Sigmoidal shape concentration–response curves were observed for both cAMP-elevating agents with the maximal degree of change for CFTR activity greater with ISO. The interpretation of this observation is that the compartmentalization of cAMP to the plasma membrane may more effectively couple protein kinase A activation to CFTR phosphorylation, which could be masked by global cytosolic increases in cAMP induced by FSK34,69.
In our previous studies, we have not performed complete concentration–response analysis experiments for the leukotriene receptor antagonist MK-571, which has off-target ABCC4 inhibition effects, in parallel to the more selective ABCC4 inhibitor Ceefourin-1, thus we wanted to explore both in greater detail14,42,45. In our concentration–response analysis experiments utilizing an in vitro extracellular cAMP assay platform, both commercially available Ceefourin-1 and MK-571 were able to decrease extracellular cAMP levels, thus both were used in downstream in vitro CFTR membrane potential assays to investigate the consequences of ABCC4 inhibition on CFTR function. Due to similar findings using either compound in the in vitro CFTR membrane potential assay and previous demonstrations of MK-571, in combination with VX-770, potentiating CFTR activity beyond VX-770 alone, MK-571 was selected for combinational studies14.
While our findings suggest that cAMP modulation has the potential to be used as an add-on therapy with existing therapeutics, limitations of the study should be noted. Although Calu-3 cells expressed both ABCC4 and CFTR, it might not have been an appropriate model of study due to its WT-CFTR expression. Potentiation of CFTR activity by ABCC4 or PDE-4 inhibitors in the literature were performed in primary cells, suggesting that a potential add-on combinatorial therapy may only show an effect in primary cell cultures, especially ones with CFTR defects. This emphasizes that further investigation of ABCC4 and PDE-4 inhibitors as a potential add-on combinatorial therapy should be performed in primary human airway epithelial cells from a large subset of CF subjects, covering a wide-range of CFTR variants with compromised CFTR expression or function, in order to determine which patient populations could benefit from cAMP modulation therapy. In addition, further investigations of cAMP modulation with other drugs that modulate CFTR, such as VX-770, VX-809, VX-661, VX-445, and genistein, should be tested as it may be more efficacious.
The human bronchial epithelial cell (HBEC6-KT) line was cultured in Keratinocyte Serum-Free Medium 1X (Gibco) according to the manufacturers directions and supplemented with P/S (VWR) at 1X70. Human airway epithelial cell line Calu-3 (ATCC HTB-55) were cultured in Minimum Essential Medium Alpha Medium (Corning) and supplemented with Premium Grade FBS (VWR) at 10%, HEPES (Corning) at 1X, and P/S (VWR) at 1X. The following chemical reagents were dissolved in DMSO, cAMP elevating agents Forskolin (Cayman Chemical) and Isoproterenol (Cayman Chemical), ABCC4 inhibitors MK-571 (Cayman Chemical) and Ceefourin 1 (Abcam), PDE inhibitors Roflumilast (Cayman Chemical) and Rolipram (Cayman Chemical), and CFTR Inhibitor (Selleck Chemicals).
Human airway epithelial cell lines
Two human airway epithelial cell lines were used for in vitro experiments. HBEC6-KT cells, provided by Dr. John Minna and Dr. Darryl Knight, are derived from healthy non-smoking individuals and immortalized through human telomerase reverse transcriptase and cyclin-dependent kinase 4 expression was used for in vitro concentration–response analysis of ABCC4 inhibitors through an extracellular cAMP assay41,42,70,71,72. Calu-3 cells derived from the metastatic site of lung adenocarcinoma tissue was used for both in vitro concentration–response analysis of ABCC4 inhibitors and in vitro analysis of CFTR function.
ABCC4 and CFTR protein expression analysis
HBEC6-KT and Calu-3 cells were lysed using RIPA Lysis Buffer containing Protease Inhibitor Cocktail powder (Sigma-Aldrich) for 60–90 min at 4 °C on a rocker. Lysates were then centrifuged at 16,000xg for 15 min with the supernatants collected for downstream immunoblots for ABCC4 and CFTR. Protein quantification was performed using a BCA protein assay. Twenty micrograms of protein were loaded on 4–15% gradient TGX Stain-Free Protein Gels and transferred to Immun-Blot LF PVDF membrane (Bio-Rad). The membranes were blocked with 1X TBS with 0.05% Tween 20 and 5% skim milk powder for 2 h at 25 °C. The membranes were then incubated with primary antibody ABCC4/MRP4 (1:40, Abcam, AB15602) or CFTR (1:5000, UNC-Chapel Hill, AB596) overnight. The membranes were then washed in 1X TBS with 0.05% Tween 20 and incubated with HRP-linked anti-rat secondary antibody (1:3000, Cell Signaling Technology, 7077S) or anti-mouse secondary antibody (1:3000, Cell Signaling Technology, 7076S) for 2 h at 25 °C. A chemiluminescence image of the blot was taken using the Bio-Rad Image Lab software. Full western blot images have been included and can be found in the supplementary information (Supplementary Fig. 4).
In vitro extracellular cAMP assay
Calu-3 and HBEC6-KT cells were cultured as described previously41,42,71,72. Cells were pretreated with IBMX (20 μM) for 2 h prior to experimental conditions. After the pre-treatment of IBMX, cells were treated with ABCC4 inhibitors (0.01–100 μM) or DMSO for 30 min. Following these exposures, the cells were treated with forskolin (10 μM) for 6 h and cell-culture supernatants were collected for analysis of extracellular cAMP. The negative control was IBMX alone and the positive control was IBMX and forskolin.
In vitro CFTR membrane potential assay
An in vitro CFTR membrane potential assay was performed on Calu-3 cells and primary human bronchial epithelial cells, from two independent donors, cultured at 37 °C as previously described14. All human samples were collected from consented individuals under ethics approval granted by Hamilton Integrated Research Ethics Board (Project Number 5099-T).
Calu-3 cells were grown for 21 days on 96-well plates and primary HBECs were grown for 14 days under air–liquid interface conditions on collagen-coated (Sigma-Aldrich) 6.5 mm Transwell Inserts (Corning). All cells were washed with HBSS prior to experimental conditions. After washing with HBSS, cells were loaded with BLUE Membrane Potential Dye (Molecular Devices, #R8042) dissolved in 37 °C chloride-free buffer (NMDG Gluconate Buffer – 150 mM NMDG-gluconate, 3 mM KCl, 10 mM HEPES, pH 7.35, 300 mOsm). Immediately after dye loading, CFTR activity measurements were taken. CFTR activity measurements are fluorescent readouts for detecting ion channel activity. CFTR inhibition was performed with CFTRinh-172 (10 μM) for all experiments to attribute changes in fluorescence as CFTR channel activity. This assay has been validated using proof of concept studies where results obtained from this assay were able to recapitulate findings obtained from direct measurements of ion channel activity using Ussing chambers14. At the end of this assay, raw data was exported for statistical analysis. Tracings demonstrating the variation in CFTR Activity as a function of time, for key experiments, have been included in the supplementary information (Supplementary Fig. 5).
For Calu-3 cells, measurements were taken during baseline (40 min), cAMP elevation (30 min), and CFTR inhibition (20 min). cAMP elevation was performed using forskolin or isoproterenol (0.0005–50 μM). For assays with ABCC4 or PDE-4 inhibitors, a pre-incubation with the inhibitors (30 min) in the plate reader was performed prior to cAMP elevation. CFTR modulator VX-770 was used at 1 μM. CFTR activity was determined by dividing a single membrane potential peak measurement after the ABCC4 or PDE-4 inhibition and cAMP elevation additions to the stabilized baseline over DMSO control.
For primary HBECs, measurements were taken during baseline (8 min), drug additions (40 min), and CFTR inhibitions (18 min). cAMP elevation was performed using forskolin (10 μM). The same concentrations used in Calu-3 cells for ABCC4 and PDE4 inhibitors, and VX-770 were used for primary HBECs, however the pre-incubation was skipped and instead, all drug combinations were added once at the beginning of the drug addition step. CFTR activity was determined by averaging six independent measurements within a single Transwell insert. Measurements were normalized to the averaged baseline over DMSO control.
For the in vitro extracellular cAMP assay, values were normalized to the positive control (IBMX and forskolin) and the half maximal inhibitory concentration (IC50) was determined. SD was calculated using data from biological replicates (n = 4–5).
For in vitro CFTR membrane potential assays, in Calu-3 cells, values were normalized to the averaged baseline over averaged vehicle (DMSO) control of all biological replicates. SD was calculated using data from biological replicates (n = 4–8). An area under the curve (AUC), half-maximal concentration (EC50), maximum concentration (Emax), and baseline analysis was performed for each individual replicate then averaged together. The AUC analysis is an aggregate measure of sensitivity and maximal response, allowing for the observation of the total net responsiveness of the cells. The AUC analysis was done by normalizing the individual replicates to the bottom most value of the averaged vehicle control. In primary HBECs, six independent measurements were taken within a single Transwell insert. The value is the average of at least six measurements. These values were normalized to the averaged baseline over the averaged vehicle (DMSO) control. SD was calculated by treating the six measurements as independent, with additional technical replicates (n = 6–18). An AUC and max peak analysis were performed for each measurement, then averaged together. The max peak analysis was the max CFTR activity achieved during the drug addition step.
In order to compare the independent experiments performed, a fold-change analysis using individual AUC, EC50, and Emax, determined from their respective concentration–response curves, was performed and was normalized to the averaged DMSO vehicle control. A one-way ANOVA with subsequent post-hoc test or a paired t-test was performed. Statistical analysis was performed using GraphPad Prism 6.
The datasets generated and analysed during the course of the study are available from the corresponding author on reasonable request.
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Nguyen, J.P., Bianca, M., Huff, R.D. et al. Modulation of cAMP metabolism for CFTR potentiation in human airway epithelial cells. Sci Rep 11, 904 (2021). https://doi.org/10.1038/s41598-020-79555-w
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