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Logo of ajrcmbIssue Featuring ArticlePublisher's Version of ArticleSubmissionsAmerican Thoracic SocietyAmerican Thoracic SocietyAmerican Journal of Respiratory Cell and Molecular Biology
 
Am J Respir Cell Mol Biol. Nov 2010; 43(5): 607–616.
Published online Dec 30, 2009. doi:  10.1165/rcmb.2009-0281OC
PMCID: PMC2970857
Activation of the Cystic Fibrosis Transmembrane Conductance Regulator by the Flavonoid Quercetin
Potential Use as a Biomarker of ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator Rescue
Louise C. Pyle,46* Jennifer C. Fulton,1* Peter A. Sloane,16 Kyle Backer,16 Marina Mazur,16 Jeevan Prasain,36 Stephen Barnes,36 J. P. Clancy,26 and Steven M. Rowe1256
Departments of 1Medicine, 2Pediatrics, 3Pharmacology, 4Genetics, and 5Physiology, and 6Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama
Correspondence and requests for reprints should be addressed to Steven M. Rowe, M.D., M.S.P.H., MCLM 768, 1918 University Blvd., Birmingham, AL 35294-0006. E-mail: smrowe/at/uab.edu
*These authors contributed equally to this manuscript.
Received August 5, 2009; Accepted October 24, 2009.
Therapies to correct the ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) folding defect require sensitive methods to detect channel activity in vivo. The β2 adrenergic receptor agonists, which provide the CFTR stimuli commonly used in nasal potential difference assays, may not overcome the channel gating defects seen in ΔF508 CFTR after plasma membrane localization. In this study, we identify an agent, quercetin, that enhances the detection of surface ΔF508 CFTR, and is suitable for nasal perfusion. A screen of flavonoids in CFBE41o cells stably transduced with ΔF508 CFTR, corrected to the cell surface with low temperature growth, revealed that quercetin stimulated an increase in the short-circuit current. This increase was dose-dependent in both Fisher rat thyroid and CFBE41o cells. High concentrations inhibited Cl conductance. In CFBE41o airway cells, quercetin (20 μg/ml) activated ΔF508 CFTR, whereas the β2 adrenergic receptor agonist isoproterenol did not. Quercetin had limited effects on cAMP levels, but did not produce detectable phosphorylation of the isolated CFTR R-domain, suggesting an activation independent of channel phosphorylation. When perfused in the nares of Cftr+ mice, quercetin (20 μg/ml) produced a hyperpolarization of the potential difference that was absent in Cftr−/− mice. Finally, quercetin-induced, dose-dependent hyperpolarization of the nasal potential difference was also seen in normal human subjects. Quercetin activates CFTR-mediated anion transport in respiratory epithelia in vitro and in vivo, and may be useful in studies intended to detect the rescue of ΔF508 CFTR by nasal potential difference.
Keywords: cystic fibrosis, short-circuit current, airway epithelia, nasal potential difference
CLINICAL RELEVANCE
This article describes the use of the flavonoid quercetin as an important adjunct for detecting surface-localized ΔF508 CFTR by nasal potential difference, a topic of considerable interest in the field of cystic fibrosis (CF) therapeutics, and relevant to emerging therapies in clinical testing. Our findings elucidate important mechanistic features of quercetin-mediated CFTR activation in vitro, and provide data in mice and humans that support the use of the agent in subjects with CF homozygous for ΔF508 CFTR, to identify individuals with residual surface protein, and in the evaluation of correctors of ΔF508 CFTR processing.
The most common cause of cystic fibrosis (CF) is attributable to the deletion of phenylalanine at position 508 of the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR), which causes misfolding of the protein product in the endoplasmic reticulum (ER) and accelerated degradation in the proteosome. At least one copy of the ΔF508 mutation is found in approximately 90% of patients with CF, and is therefore a logical target for treating the disease. The absence of CFTR at the cell plasma membrane results in defective ion transport and subsequent clinical manifestations of CF (1). Several maneuvers were identified that correct ΔF508 CFTR misprocessing, including cell growth at low temperature (2), treatment with chemical chaperones that promote ER escape (35), and exposure to small molecules (identified by high-throughput screening programs and other means) that promote protein localization to the plasma cell membrane (69).
Numerous laboratories showed that the ΔF508 CFTR demonstrates defective channel gating (1012). These findings are evident in airway cell monolayers, because ΔF508 CFTR rescue using low temperatures or small molecule correctors of misprocessing do not consistently restore the cAMP-dependent activation of Cl transport, a finding that is also germane to measures of ion transport across the human nasal membrane (13, 14). Thus, proof-of-concept studies testing agents that correct ΔF508 CFTR processing may be hampered if assays of CFTR activity, such as nasal potential difference (NPD), fail to stimulate membrane-localized ΔF508 CFTR adequately. Recently, an evaluation of the ΔF508 CFTR processing corrector (VX-809) has entered Phase 2 testing in human subjects, and includes assessment via NPD, further emphasizing the importance of an adequate stimulus within the measurement of NPD (NCT00865904).
The standard stimulus of CFTR-dependent Cl transport is perfusion with β2 adrenergic receptor (β2 AR) agonists (e.g., isoproterenol) in the presence of a Cl secretory gradient. If cAMP-dependent activation is defective in human airway cells expressing surface-localized ΔF508 CFTR, the identification of a molecule capable of activating the mutant channel that is also suitable for use in NPD protocols may improve the sensitivity of the assay to detect ΔF508 CFTR at the cell surface. Because flavonoids are among the best-described activators of CFTR activity (1517), we sought to determine the most suitable agent that could be applied to the measurement of NPD. Some results of these studies were previously reported in abstracts (18, 19).
Voltage Clamp Studies in Ussing Chambers on Cultured Monolayers
The CFBE41o and Fisher rat thyroid (FRT) cells stably transduced with ΔF508 or WT CFTR cDNA were grown on Costar permeable inserts and mounted in Ussing chambers, and the short-circuit current (Isc) was measured under voltage clamp conditions, as previously described (13, 20). Briefly, cells were seeded on 0.4-μm permeable supports (5 × 105 cells/filter, 6.5-mm diameter; Costar, Bethesda, MD) after coating with fibronectin. Cells were grown to confluence, and then switched to at an air–liquid interface 48 hours before mounting in modified Ussing chambers (Jim's Instruments, Iowa City, IA), and initially bathed on both sides with identical Ringer solution. Measurements of Isc were obtained by using an epithelial voltage clamp (University of Iowa Bioengineering, Iowa City, IA). A 3-mV pulse of 1-second duration was imposed every 100 seconds to monitor resistance, which was calculated using Ohm's law. To measure stimulated Isc, the mucosal bathing solution was changed to a low Cl sodium gluconate solution plus 100 μM amiloride. Agonists (flavonoids, isoproterenol, and forskolin) were added to the bathing solutions as indicated (for a minimum 5 min of observation under each condition). Glibenclamide (200 μM) or CFTRInh-172 (10 μM) was added to the mucosal bathing solution at the end of the experiments to block CFTR-dependent Isc. All chambers were maintained at 37°C, vigorously stirred, and gassed with 95%O2/5% CO2.
Detection of Regulatory-Domain Phosphorylation and cAMP Concentrations
Polyclonal NIH-3T3 cells expressing the hemagglutinin (HA)-tagged R-domain were treated with quercetin for 20 minutes, and compared with forskolin (20 μM) × 5 minutes as a positive control. After lysis, equal amounts (50 μg) of total cell lysate were electrophoresed through a 12% SDS-PAGE gel, and immunoblotted with antibody to the HA tag (Covance, Cumberland, VA). Phosphorylation of the R-domain was visualized as a 2- to 4-kD shift in migration, as previously described (21). Cellular cAMP was measured using an ELISA-based detection kit (Cayman Chemicals, Ann Arbor, MI) according to published methods (13).
Mouse NPD Measurements
Mice were anesthetized with ketamine, and each mouse's tail was gently abraded, placed in lactated Ringer solution, and connected through a calomel cell to a high-impedance voltmeter (VF-1; World Precision Instruments, Sarasota, FL), as previously described (22, 23). Perfusion solutions included Ringer lactate plus amiloride (100 μM), a low Cl concentration solution with amiloride (100 μM), and an agonist in low Cl solution. Each condition was studied for 5 to 10 minutes until a stable signal was achieved before changing to the next solution (23). Additional details are described in the online supplement.
Human NPD Measurements
All subjects provided written, informed consent, and the study was approved by the Institutional Review Board of the University of Alabama at Birmingham. The NPD studies were performed in accordance with the report by Knowles and colleagues (24) and the standard operating procedure of the CF-Therapeutics Development Network (perfusion method) using electronic data capture (Biopac, San Francisco, CA), KCl calomel electrodes (Fischer Scientific, Pittsburgh, PA), and 3% agar bridges. Additional details are described in the online supplement.
Reagents
Forskolin was obtained from Calbiochem (San Diego, CA), and genistein from Sigma-Aldrich (St. Louis, MO). Quercetin (Jarrow Formulas, Los Angeles, CA) was freshly reconstituted for each experiment in DMSO at a 20 mg/ml stock concentration, and passed through a 0.2-μM filter to remove particulates. Quercetin from an alternative source (Sigma-Aldrich) was used for confirmation.
Statistics
For Isc, cAMP, and Western blot measurements, descriptive statistics (mean, SD, and SEM) were compared using the Student t test or ANOVA, as appropriate. All statistical tests were two-sided, and were performed at a 5% significance level (i.e., α = 0.05), using SPSS software (Chicago, IL) and Microsoft Excel (Seattle, WA).
Screen of Flavonoids Identifies Activators of Surface-Localized ΔF508 CFTR
To identify compounds that potentiate ΔF508 CFTR activity, 20 flavonoid agents were screened in ΔF508 CFTR-transduced CFBE41o cells after temperature correction of aberrant protein processing (growth at 27°C × 48 h). As shown in Figure 1, cell monolayers were evaluated in modified Ussing chambers after prestimulation with forskolin (20 μM) and exposure to incrementally increasing doses (1, 10, and 50–100 μM) of the respective flavonoid agents. The concentrations of flavonoids tested were based on previous experience with genistein and other flavonoids on CF processing and maturation (13, 17, 25, 26). Several agents stimulated Cl conductance above that produced by forskolin and other cAMP agonists, which are poor stimuli of ΔF508 CFTR activity in this airway cell type (13). Genistein, a well-characterized flavonoid activator of ΔF508 CFTR, was found to be the most potent stimulus in our screen, but was not considered further because of potential human safety concerns with repeated nasal administration and off-target effects (27). Equol and apigenin were avoided because of their observed effects on ΔF508 CFTR processing. Of the agents that activated ΔF508 CFTR residing at the cell surface, quercetin was selected for additional evaluation because of its well-established safety profile for human use, including clinical trials in a variety of disorders encompassing cancer and heart disease, and because of its usefulness as an anti-inflammatory agent (2832).
Figure 1.
Figure 1.
Flavonoid activation of ΔF508 CFTR-dependent Cl transport. Screen of flavonoid compounds for activation of temperature-corrected ΔF508 CFTR in CFBE41o monolayers. Dose–response experiments in modified Ussing chambers (more ...)
Quercetin Activates ΔF508 CFTR despite Minimal Effects on cAMP and R-Domain Phosphorylation
The protein kinase A (PKA)/cAMP-dependent phosphorylation of the CFTR R-domain is a critical step in channel activation, but this stimulus was shown to be insufficient to activate ΔF508 CFTR fully at the plasma membrane of airway epithelial cells (13, 33). To examine the mechanistic basis of CFTR activation by quercetin, we measured the effects of quercetin on cAMP levels and on R-domain phosphorylation. Quercetin had small but detectable effects on cell cAMP concentrations (126 pmol over vehicle control) that were less than 10% of that observed with forskolin treatment but greater than those seen with genistein (Figure 2A). As opposed to the potent cAMP agonist forskolin, quercetin had no detectable effect on the phosphorylation-dependent mobility shift of the recombinant CFTR R-domain (Figure 2B), suggesting that its effects were independent of CFTR phosphorylation, despite reports of quercetin as a phosphatase inhibitor and/or PKA activator in other cell types (34, 35). Together, the results of Figure 2 provide evidence that the quercetin-induced activation of ΔF508 CFTR differs from classic cAMP-signaling agonists, and is consistent with a mechanism of action that imparts direct effects on CFTR that are independent of R-domain phosphorylation, as reported with other flavonoid agents including genistein (3638).
Figure 2.
Figure 2.
Quercetin activates CFTR through a mechanism independent of PKA and R-domain phosphorylation. (A) Concentrations of cAMP after forskolin, genistein, or quercetin exposure. CFBE41o cells were exposed to quercetin (50 μg/ml), genistein (more ...)
Dose–Response Relationships between Quercetin and ΔF508 CFTR Activity
To define the dose–response relationship between quercetin and ΔF508 CFTR activation, and to confirm its effects in a distinct ΔF508 CFTR-expressing cell model, additional studies were performed in ΔF508 CFTR-transduced FRT monolayers after the temperature correction of ΔF508 misprocessing (27°C × 48 h). This cell type is frequently used to examine ΔF508 CFTR function and processing, including high-throughput screens of CFTR potentiators and ΔF508 CFTR correctors (7, 39). Quercetin activated CFTR-dependent Cl conductance at 1 and 10 μg/ml, but inhibited Isc after the addition of higher concentrations (>20 μg/ml; Figures 3A and 3B). The Cl transport stimulated by quercetin (10 μg/ml) was not affected by forskolin (20 μM) pretreatment, insofar as quercetin stimulated Isc by 57.6 ± 2.7 μA/cm2 in the presence of forskolin, compared with 53.4 ± 3.8 μA/cm2 produced by quercetin alone (P = NS, n = 4, data not shown). In monolayers without temperature correction of ΔF508 CFTR processing (i.e., grown at 37°C), the effect of quercetin on Cl conductance was minimal, establishing the specificity of its effect on ΔF508 CFTR residing at the cell surface. Experiments in wild-type CFTR-expressing FRT monolayers suggested a peak effect at 10 μg/ml (Figure 3C). Confirmatory experiments performed in ΔF508 CFTR-transduced CFBE41o cells after low temperature treatment showed a similar dose–response relationship (Figure 3D) and specificity for surface-residing ΔF508 CFTR (no activation seen with growth at 37°C). As seen in FRT cells, the quercetin-dependent activation of Cl transport did not require forskolin preactivation in temperature-corrected ΔF508 CFBE41o cells. The dose–response curve in CFBE41o cells transduced with wild-type CFTR was similar to that of ΔF508 CFTR-transduced cells (Figure 3E). Experiments using parental CFBE41o cells (without ΔF508 or wild-type CFTR transduction and undetectable expression of endogenous ΔF508 CFTR) (13) showed no activation by quercetin (0 ± 0 μA/cm2, data not shown), confirming the specificity of the agonist for CFTR.
Figure 3.
Figure 3.
Dose–response effects of quercetin on Isc in ΔF508 CFTR-transduced FRT and CFBE41o monolayers. Cells were studied in modified Ussing chambers, and stimulated Isc was determined after treatment with a low chloride gradient + (more ...)
Quercetin Enhances ΔF508 CFTR Activity Above That Produced by B2 Adrenergic Receptor Stimulation
We next examined whether quercetin increased ΔF508 CFTR Cl conductance above that produced by the β2 AR agonist isoproterenol, a well-established regulatory pathway to activate CFTR in vivo and a standard agonist for NPD studies. These experiments were designed to mirror CFTR detection in humans using NPD protocols (40), and used isoproterenol (10 μM) and a Cl secretory gradient in the presence of amiloride. These studies were limited to ΔF508 CFTR-transduced CFBE41o cells after low temperature growth (Figure 4), because FRT cells do not express β2 ARs. Quercetin (20 μg/ml) significantly increased the Cl conductance above isoproterenol stimulation, increasing Isc 12.3 μA/cm2 above that produced by isoproterenol alone. To confirm that pre-exposure to quercetin did not induce tachyphylaxis (an effect that would impede its use in human NPD protocols requiring repeated measurements), monolayers were treated with 20 μg/ml quercetin for two 30-minute intervals, 24 and 48 hours before evaluation in Ussing chambers. Pre-exposure to quercetin had no measurable effect on the subsequent activation of corrected ΔF508 CFTR by the compound (Figure 4D). The activation of CFTR-dependent ion transport was confirmed in primary human airway cells from a normal (non-CF) donor (Figures 4E and 4F).
Figure 4.
Figure 4.
Quercetin stimulates CFTR-dependent Isc in conditions simulating human NPD protocols. A representative short-circuit current tracing in temperature-corrected ΔF508 CFTR CFBE41o monolayers shows a poor response to isoproterenol perfusion, (more ...)
Quercetin Activates CFTR-Dependent Cl Conductance across the Nasal Epithelium
We next examined the effects of quercetin on anion transport in vivo via standardized murine NPD measurements. As shown in Figure 5, mice expressing wild-type CFTR (Cftr+/ and Cftr+/+ mice of a Cftrtm1Kth C57BL6/J genetic background) demonstrated significant hyperpolarization after perfusion with quercetin (20 μg/ml) in the presence of amiloride (100 μM) and a Cl secretory gradient (−2.9 ± 0.7 mV), compared with the continued depolarization seen in mice perfused with vehicle and otherwise identical conditions (+0.9 ± 0.5 mV). The Cftr−/− mice did not demonstrate repolarization after quercetin perfusion, confirming the specificity of quercetin's effects in CFTR-dependent anion transport in vivo (Figure 5C). Consistent with the effects of quercetin on ΔF508 CFTR cells without temperature correction, quercetin perfusion did not hyperpolarize the nasal epithelial membrane of mice homozygous for murine ΔF508 CFTR, compared with isoproterenol or vehicle control (Figure 5D). In normal (non-CF) human subjects, sequential perfusion with quercetin at 5, 10, and 20 μg/ml was well tolerated, and demonstrated a sequential dose-dependent polarization of the nasal membrane, indicative of CFTR-dependent anion transport (Figure 6). No adverse events were reported, and no changes were seen in the nasal examination rating or total nasal symptom score before and after the NPD procedure. The results confirm findings observed in polarized epithelial model systems, and demonstrate that quercetin retains bioactivity to stimulate CFTR Cl transport when topically applied to the nasal mucosa in an NPD protocol.
Figure 5.
Figure 5.
Quercetin activates CFTR-dependent ion transport across the murine nasal mucosa in vivo. Mice underwent a standardized NPD protocol with the addition of quercetin (or vehicle control) in the final perfusion solution, that is, 20 μg/ml in Cl (more ...)
Figure 6.
Figure 6.
Quercetin activates CFTR in humans by nasal potential difference. (A) Representative NPD tracing after sequential perfusion of Ringer solution, Ringer solution with amiloride (Amil), Cl-free solution (Zero Cl), Cl-free solution (more ...)
The detection of plasma membrane-localized ΔF508 CFTR is a vital step in the development of therapies designed to treat the underlying cause of CF in the majority of patients with CF. In this study, we evaluated several flavonoid compounds for the activation of surface ΔF508 CFTR, and identified a number of agents that robustly activate ΔF508 CFTR-dependent Cl transport in airway monolayers after temperature correction of aberrant processing. One agent in particular, quercetin, was selected for further investigation because it demonstrated efficient activation of ΔF508 CFTR-dependent Isc, and has a well-established safety profile in human subjects. Two other flavonoids, equol and apigenin, also demonstrated potent ΔF508 CFTR activation, but were not studied further because of their inferior safety profile in the literature. Furthermore, additional studies indicated that these agents affect ΔF508 CFTR misprocessing, a characteristic that could confound studies intended to detected ΔF508 CFTR correction. Using two polarized epithelial models, we showed that quercetin produces a dose-dependent activation of ΔF508 CFTR after cell surface localization (low temperature rescue) (Figure 3). In both ΔF508 CFTR expressing FRT and CFBE41o cells, the effect was specific for CFTR at concentrations below 100 μg/mL, as indicated by the lack of effect on anion transport in ΔF508 CFTR-transduced cells without temperature correction (or in cells lacking the ΔF508 CFTR transgene). When repeated in a sequence analogous to a standard NPD protocol, quercetin stimulated CFTR-dependent Isc without evidence of tachyphylaxis, indicating that the compound may be suitable for clinical protocols requiring repeated exposure (Figure 4D). Together with results demonstrating the stimulation of CFTR-dependent anion transport across the nasal epithelium in vivo (Figures 5 and and6),6), the findings provide support for further studies of this agent in human NPD protocols as a means to enhance the detection of ΔF508 CFTR at the plasma membrane. Based on these findings, a multicenter study is being conducted to examine the response of ΔF508 homozygotes to quercetin perfusion compared with isoproterenol, and is intended to determine whether the response to quercetin is associated with a more mild pulmonary phenotype.
In the Calu-3 serous glandular cell line, Illek and Fischer showed that quercetin (at concentrations >40 μM, equivalent to 12.1 μg/ml) stimulated Cl secretion (half-maximal doses were 22.1 ± 4.5 μM) in vitro, whereas higher doses were inhibitory (26). Our findings indicate that quercetin produced maximal Isc at approximately 10 μg/ml (33 μM) in vitro, and hyperpolarized the NPD response between 5 to 20 μg/ml. These results concerning the effects of quercetin in the murine airway extend earlier studies, and firmly establish the CFTR dependence of observations at the concentrations tested. Interestingly, Illek and Fischer (26) suggested that pretreatment of Calu-3 cells with forskolin may sensitize to subsequent quercetin stimulation. Our results in ΔF508 CFTR-expressing FRT cells did not show this effect, insofar as quercetin stimulated Isc to a similar degree, regardless of forskolin pretreatment. This could indicate differences in the response of wild-type CFTR versus the ΔF508 protein, or other variables attributable to the cell types being tested (26). The inhibition of anion conductance at higher concentrations observed in our studies was reported with other flavonoids (26) and in studies of colonic epithelia (41). These results emphasize the importance of using the appropriate concentration for studies intended to activate CFTR. Moreover, tissue-specific differences may mediate anion conductance in response to quercetin. These concerns may be abrogated through the use of the NPD assay, which requires topical administration directly to the nasal epithelia, as opposed to systemic treatment in which pharmacokinetics are more difficult to control, and unintended consequences of CFTR inhibition may be more likely in off-target tissues.
In other studies, Lim and colleagues reported no activation of chloride efflux with the addition of quercetin (1 or 5 μM) to forskolin (13 μM) in IB3–1 bronchial epithelial cells after the correction of endogenous ΔF508 CFTR with 4-phenylbuturate (42). This disparity may be attributable to the low concentrations of quercetin tested, because our experiments also showed that no ΔF508 CFTR activation occurred with 3.3 μM quercetin. Plasma membrane ΔF508 CFTR levels can also be markedly influenced by cell polarity and by growth at an air–liquid interface (13, 43, 44). Because the studies with IB3–1 cells were conducted under nonpolarizing conditions, this fundamental difference may have contributed to the previously reported negative results. These effects may also represent additional cell model-specific responses to flavonoid exposure, as reported by Hansen and colleagues regarding the quercetin modulation of heat shock protein-70 synthesis (45).
Although previous work with quercetin in Calu-3 cells demonstrated a cAMP/PKA-dependent activation of CFTR (26), our results provide evidence that the quercetin signaling that promotes corrected ΔF508 CFTR activation may differ from that produced by classic PKA-dependent stimuli, and this effect may be unique to flavonoids. Quercetin produced a small increase in cAMP relative to forskolin, despite potent stimulation of ion transport compared with cAMP agonist (Figure 2). The cAMP levels resulting from quercetin treatment, however, were greater than those produced by genistein. Previous work from our laboratory has shown that ΔF508 CFTR channel activation (by genistein) can be partially blocked by inhibiting PKA in CFBE41o cells (13). This implies that ΔF508 CFTR requires some level of PKA activity (and thus PKA dependent RD phosphorylation) for genistein to confer Cl transport. While no RD phosphorylation was detected following quercetin treatment (further distinguishing the mechanistic basis of flavonoid versus PKA activation pathways; see Figure 2B), we speculate that quercetin may provide two stimuli that together optimize ΔF508-CFTR activation (including both cAMP-dependent and -independent effects), particularly in cells where endogenous cAMP levels could be limiting.
Several laboratories showed that ΔF508 CFTR, in addition to a well-described cellular processing defect, also exhibits significant abnormalities in channel gating at the cell surface. For example, ΔF508 CFTR exhibits reduced sensitivity to phosphorylation dependent activation in membrane patch and whole-cell studies (46). More recent findings by Wang and colleagues using a series of molecules (including genistein, NPPB-AM, and curcumin), indicate that ΔF508 CFTR activation required stimuli independent of cAMP and PKA to rescue mutant channel function fully (47). Studies from our laboratory and from others using intact airway monolayers established that low temperature or pharmacologically corrected ΔF508 CFTR is not fully activated by cAMP stimuli, including the β2 agonists albuterol and isoproterenol or the A2B adenosine receptor agonists adenosine or 5′-N-ethylcarboxamide adenosine. This defect appears to be more pronounced in pulmonary epithelia, including human alveolar and airway cells (13, 33). These findings provide a compelling rationale to investigate more sensitive biomarkers of ΔF508 CFTR activity after processing correction, including agents such as quercetin. For example, current methods may not detect effective biochemical corrections of ΔF508 CFTR, insofar as chemically corrected ΔF508 CFTR may not be activated by the cAMP pathway because of persistent gating defects. As such, improved surface activation of CFTR by an agent such as quercetin would be expected to decrease the number of subjects required to observe a relevant improvement in CFTR-dependent NPD response, simplifying the designs of clinical trials.
In addition, agents such as quercetin could help detect and discriminate the ΔF508 CFTR that resides in the plasma membrane, but that is unresponsive to endogenous activation because of a persistent gating defect, thus helping define the mechanistic basis underlying a ΔF508 CFTR processing corrector under study. Although the mechanism by which quercetin and other flavonoids activate CFTR is unknown, our findings support a direct effect on the CFTR Cl channel that can overcome the defective gating of cell surface-localized ΔF508 CFTR. These results are further supported by observations from whole-cell studies with quercetin (26) and single-channel studies with other flavonoids (47).
Because the endoplasmic reticulum-associated degradation of ΔF508 CFTR was postulated to be less than 100% efficient in vitro (45), and because small amounts of ΔF508 CFTR at the cell membrane were speculated to account for phenotypic differences among ΔF508 CFTR homozygous individuals (48), the evaluation of CFTR-dependent Cl conductance by NPD using quercetin (or other potentiating stimuli) could be used as an approach to detect individuals with small but relevant amounts of ΔF508 CFTR at the plasma membrane. Such an approach could identify ΔF508 CFTR homozygotes who are potential candidates for treatment with other CFTR potentiators, including the agent VX-770, currently under investigation in patients with CF harboring the G551D CFTR mutation (49). The relative safety of the approach, as confirmed during the human NPD studies described here, makes this assessment feasible.
Flavonoids are ubiquitous in the human diet, are highly bioavailable, and have been extensively studied with regard to their pharmacology and toxicology (50). Multiple laboratory clinical trials point to an excellent safety profile of quercetin, including studies using systemic, oral, and intravenous administration at levels far exceeding those found to activate ΔF508 CFTR (5153). For example, Shoskes and colleagues reported that oral quercetin (500 mg, twice daily) was well tolerated and significantly improved symptoms in men with chronic prostatitis (54). The intravenous administration of high doses of quercetin was well tolerated over a 6- to 7-month period in a study examining quercetin as a tyrosine kinase inhibitor (51). More recently, oral quercetin (730 mg daily) for 28 days produced no significant adverse events in a hypertension trial (55). Carcinogenic testing in hamsters by Morino and colleagues (56), and additional studies involving systemic exposure in rats (57, 58), further indicate the safety of the agent in animal models. This extensive preclinical and clinical experience, coupled with our results in vitro and in vivo, provide strong support for further studies to examine quercetin in subjects with CF as a means to activate ΔF508 CFTR.
Supplementary Material
[Online Supplement]
Acknowledgments
The authors thank Dr. Eric J. Sorscher for reviewing the manuscript and for infrastructural support, and Dr. Melissa Ashlock for critical appraisal and support of the work. The authors are also grateful to Ms. Heather Hathorne and Ms. Ginger Reeves for assistance in performing NPD procedures, Ms. Meenakshi Sthanam for technical expertise, and Ms. Cheryl Owens for assistance preparing the manuscript.
Notes
This research was funded by National Institutes of Health grants 1K23DK075788–01 (S.M.R.), 1R03DK084110-01 (S.M.R.), and 1P30DK072482–01A1 (to Eric J Sorscher; see Acknowledgments), and Cystic Fibrosis Foundation grants ROWE08XX (S.M.R.), R464-CR07 (to Eric J Sorscher), and CLANCY02YO (J.P.C.). This study also benefited from National Institutes of Health funding to the Purdue University-University of Alabama at Birmingham Botanicals Center for Age-Related Disease from the National Center for Complementary and Alternative Medicine, and b funding from the National Institutes of Health Office of Dietary Supplements (P50 AT00477, C. Weaver, principal investigator).
The contents of this article are solely the responsibility of the authors, and do not necessarily represent the official views of the National Institute of Diabetes and Digestive and Kidney Diseases, the National Heart, Lung, and Blood Institute, or the National Institutes of Health.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10. 1165/rcmb.2009-0281OC on December 30, 2009
Author Disclosure: L.C.P. has received sponsored grants from the National Heart, Lung, and Blood Institute of the National Institutes of Health (in excess of $100,000). S.M.R. has served on the advisory boards for Vertex Pharmaceuticals (up to $1,000) and PTC Therapeutics ($1,001–$5,000), has received industry-sponsored grants from Novartis Pharmaceuticals (in excess of $100,000), Vertex Pharmaceuticals (in excess of $100,000), and PTC Therapeutics (in excess of $100,000), and has received sponsored grants from the Cystic Fibrosis Foundation ($50,000–$100,000) and the National Institutes of Health (in excess of $100,000). None of the other authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
1. Rowe SM, Miller S, Sorscher EJ. Cystic fibrosis. N Engl J Med 2005;352:1992–2001. [PubMed]
2. Denning GM, Anderson MP, Amara JF, Marshall J, Smith AE, Welsh MJ. Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive. Nature 1992;358:761–764. [PubMed]
3. Egan ME, Glockner-Pagel J, Ambrose C, Cahill PA, Pappoe L, Balamuth N, Cho E, Canny S, Wagner CA, Geibel J, et al. Calcium-pump inhibitors induce functional surface expression of delta F508-CFTR protein in cystic fibrosis epithelial cells. Nat Med 2002;8:485–492. [PubMed]
4. Egan ME, Pearson M, Weiner SA, Rajendran V, Rubin D, Glockner-Pagel J, Canny S, Du K, Lukacs GL, Caplan MJ. Curcumin, a major constituent of turmeric, corrects cystic fibrosis defects. Science 2004;304:600–602. [PubMed]
5. Brown CR, Hong-Brown LQ, Biwersi J, Verkman AS, Welch WJ. Chemical chaperones correct the mutant phenotype of the delta F508 cystic fibrosis transmembrane conductance regulator protein. Cell Stress Chaperones 1996;1:117–125. [PMC free article] [PubMed]
6. Van Goor F, Straley KS, Cao D, Gonzalez J, Hadida S, Hazlewood A, Joubran J, Knapp T, Makings LR, Miller M, et al. Rescue of deltaF508-CFTR trafficking and gating in human cystic fibrosis airway primary cultures by small molecules. Am J Physiol Lung Cell Mol Physiol 2006;290:L1117–L1130. [PubMed]
7. Pedemonte N, Lukacs GL, Du K, Caci E, Zegarra-Moran O, Galietta LJ, Verkman AS. Small-molecule correctors of defective deltaF508-CFTR cellular processing identified by high-throughput screening. J Clin Invest 2005;115:2564–2571. [PMC free article] [PubMed]
8. Sato S, Ward CL, Krouse ME, Wine JJ, Kopito RR. Glycerol reverses the misfolding phenotype of the most common cystic fibrosis mutation. J Biol Chem 1996;271:635–638. [PubMed]
9. Bebok Z, Venglarik CJ, Panczel Z, Jilling T, Kirk KL, Sorscher EJ. Activation of deltaF508 CFTR in an epithelial monolayer. Am J Physiol 1998;275:C599–C607. [PubMed]
10. Hwang TC, Wang F, Yang IC, Reenstra WW. Genistein potentiates wild-type and delta F508-CFTR channel activity. Am J Physiol 1997;273:C988–C998. [PubMed]
11. Wang F, Zeltwanger S, Yang IC, Nairn AC, Hwang TC. Actions of genistein on cystic fibrosis transmembrane conductance regulator channel gating. Evidence for two binding sites with opposite effects. J Gen Physiol 1998;111:477–490. [PMC free article] [PubMed]
12. Al-Nakkash L, Hwang TC. Activation of wild-type and deltaF508-CFTR by phosphodiesterase inhibitors through cAMP-dependent and -independent mechanisms. Pflugers Arch 1999;437:553–561. [PubMed]
13. Bebok Z, Collawn JF, Wakefield J, Parker W, Li Y, Varga K, Sorscher EJ, Clancy JP. Failure of cAMP agonists to activate rescued deltaF508 CFTR in CFBE41o airway epithelial monolayers. J Physiol 2005;569:601–615. [PubMed]
14. Hentchel-Franks K, Lozano D, Eubanks-Tarn V, Cobb B, Fan L, Oster R, Sorscher E, Clancy JP. Activation of airway Cl- secretion in human subjects by adenosine. Am J Respir Cell Mol Biol 2004;31:140–146. [PubMed]
15. Galietta LJ, Springsteel MF, Eda M, Niedzinski EJ, By K, Haddadin MJ, Kurth MJ, Nantz MH, Verkman AS. Novel CFTR chloride channel activators identified by screening of combinatorial libraries based on flavone and benzoquinolizinium lead compounds. J Biol Chem 2001;276:19723–19728. [PubMed]
16. Illek B, Lizarzaburu ME, Lee V, Nantz MH, Kurth MJ, Fischer H. Structural determinants for activation and block of CFTR-mediated chloride currents by apigenin. Am J Physiol Cell Physiol 2000;279:C1838–C1846. [PubMed]
17. Springsteel MF, Galietta LJ, Ma T, By K, Berger GO, Yang H, Dicus CW, Choung W, Quan C, Shelat AA, et al. Benzoflavone activators of the cystic fibrosis transmembrane conductance regulator: towards a pharmacophore model for the nucleotide-binding domain. Bioorg Med Chem 2003;11:4113–4120. [PubMed]
18. Fulton JC, Pyle LC, Fan L, Fortenberry J, Sthanam M, Clancy JP, Rowe SM. The flavonoid quercetin activates wild-type and dF508 CFTR at concentrations suitable for in vivo detection of rescued protein. Pediatr Pulmonol Suppl 2008;43:249. (abstract).
19. Fulton JC, Fortenberry JA, Fan L, Sthanam M, Clancy JP, Rowe SM. The flavonoid quercetin activates wild type and mutant CFTR at concentrations suitable for in vivo detection of rescued protein. Am J Respir Crit Care Med 2008;177:A457. (abstract).
20. Rowe SM, Varga K, Rab A, Bebok Z, Byram K, Li Y, Sorscher EJ, Clancy JP. Restoration of w1282x CFTR activity by enhanced expression. Am J Respir Cell Mol Biol 2007;37:347–356. [PMC free article] [PubMed]
21. Pyle L, Ehrhardt A, Nowotarski K, Rowe SM, Sorscher EJ. The role of R-domain phosphorylation in CFTR activation by potentiators. North American Cystic Fibrosis Foundation Annual Meeting. Denver, CO; 2006.
22. Kelley TJ, Thomas K, Milgram LJ, Drumm ML. In vivo activation of the cystic fibrosis transmembrane conductance regulator mutant deltaF508 in murine nasal epithelium. Proc Natl Acad Sci USA 1997;94:2604–2608. [PubMed]
23. Cobb BR, Ruiz F, King CM, Fortenberry J, Greer H, Kovacs T, Sorscher EJ, Clancy JPA. (2) Adenosine receptors regulate CFTR through PKA and PLA(2). Am J Physiol Lung Cell Mol Physiol 2002;282:L12–L25. [PubMed]
24. Knowles MR, Paradiso AM, Boucher RC. In vivo nasal potential difference: techniques and protocols for assessing efficacy of gene transfer in cystic fibrosis. Hum Gene Ther 1995;6:445–455. [PubMed]
25. Schmidt A, Hughes LK, Cai Z, Mendes F, Li H, Sheppard DN, Amaral MD. Prolonged treatment of cells with genistein modulates the expression and function of the cystic fibrosis transmembrane conductance regulator. Br J Pharmacol 2008;153:1311–1323. [PubMed]
26. Illek B, Fischer H. Flavonoids stimulate Cl conductance of human airway epithelium in vitro and in vivo. Am J Physiol 1998;275:L902–L910. [PubMed]
27. Mall M, Wissner A, Seydewitz HH, Hubner M, Kuehr J, Brandis M, Greger R, Kunzelmann K. Effect of genistein on native epithelial tissue from normal individuals and CF patients and on ion channels expressed in Xenopus oocytes. Br J Pharmacol 2000;130:1884–1892. [PubMed]
28. Wang L, Tu YC, Lian TW, Hung JT, Yen JH, Wu MJ. Distinctive antioxidant and antiinflammatory effects of flavonols. J Agric Food Chem 2006;54:9798–9804. [PubMed]
29. Mamani-Matsuda M, Kauss T, Al-Kharrat A, Rambert J, Fawaz F, Thiolat D, Moynet D, Coves S, Malvy D, Mossalayi MD. Therapeutic and preventive properties of quercetin in experimental arthritis correlate with decreased macrophage inflammatory mediators. Biochem Pharmacol 2006;72:1304–1310. [PubMed]
30. Jackson JK, Higo T, Hunter WL, Burt HM. The antioxidants curcumin and quercetin inhibit inflammatory processes associated with arthritis. Inflamm Res 2006;55:168–175. [PubMed]
31. Hung H. Dietary quercetin inhibits proliferation of lung carcinoma cells. Forum Nutr 2007;60:146–157. [PubMed]
32. Jalili T, Carlstrom J, Kim S, Freeman D, Jin H, Wu TC, Litwin SE, David Symons J. Quercetin-supplemented diets lower blood pressure and attenuate cardiac hypertrophy in rats with aortic constriction. J Cardiovasc Pharmacol 2006;47:531–541. [PubMed]
33. Pedemonte N, Sondo E, Galietta LJ. Evaluation of potentiators and correctors for the functional rescue of dF508 CFTR protein. Pediatr Pulmonol Suppl 2007;42:A268.
34. Chan AL, Huang HL, Chien HC, Chen CM, Lin CN, Ko WC. Inhibitory effects of quercetin derivatives on phosphodiesterase isozymes and high-affinity [(3) H]-rolipram binding in guinea pig tissues. Invest New Drugs 2008;26:417–424. [PubMed]
35. Ko WC, Shih CM, Lai YH, Chen JH, Huang HL. Inhibitory effects of flavonoids on phosphodiesterase isozymes from guinea pig and their structure-activity relationships. Biochem Pharmacol 2004;68:2087–2094. [PubMed]
36. Moran O, Galietta LJ, Zegarra-Moran O. Binding site of activators of the cystic fibrosis transmembrane conductance regulator in the nucleotide binding domains. Cell Mol Life Sci 2005;62:446–460. [PubMed]
37. Al-Nakkash L, Hu S, Li M, Hwang TC. A common mechanism for cystic fibrosis transmembrane conductance regulator protein activation by genistein and benzimidazolone analogs. J Pharmacol Exp Ther 2001;296:464–472. [PubMed]
38. Weinreich F, Wood PG, Riordan JR, Nagel G. Direct action of genistein on CFTR. Pflugers Arch 1997;434:484–491. [PubMed]
39. Yang H, Shelat AA, Guy RK, Gopinath VS, Ma T, Du K, Lukacs GL, Taddei A, Folli C, Pedemonte N, et al. Nanomolar affinity small molecule correctors of defective delta F508-CFTR chloride channel gating. J Biol Chem 2003;278:35079–35085. [PubMed]
40. Standaert TA, Boitano L, Emerson J, Milgram LJ, Konstan MW, Hunter J, Berclaz PY, Brass L, Zeitlin PL, Hammond K, et al. Standardized procedure for measurement of nasal potential difference: an outcome measure in multicenter cystic fibrosis clinical trials. Pediatr Pulmonol 2004;37:385–392. [PubMed]
41. Schuier M, Sies H, Illek B, Fischer H. Cocoa-related flavonoids inhibit CFTR-mediated chloride transport across t84 human colon epithelia. J Nutr 2005;135:2320–2325. [PubMed]
42. Lim M, McKenzie K, Floyd AD, Kwon E, Zeitlin PL. Modulation of deltaF508 cystic fibrosis transmembrane regulator trafficking and function with 4-phenylbutyrate and flavonoids. Am J Respir Cell Mol Biol 2004;31:351–357. [PubMed]
43. Varga K, Jurkuvenaite A, Wakefield J, Hong JS, Guimbellot JS, Venglarik CJ, Niraj A, Mazur M, Sorscher EJ, Collawn JF, et al. Efficient intracellular processing of the endogenous cystic fibrosis transmembrane conductance regulator in epithelial cell lines. J Biol Chem 2004;279:22578–22584. [PubMed]
44. Swiatecka-Urban A, Brown A, Moreau-Marquis S, Renuka J, Coutermarsh B, Barnaby R, Karlson KH, Flotte TR, Fukuda M, Langford GM, et al. The short apical membrane half-life of rescued {delta}F508-cystic fibrosis transmembrane conductance regulator (CFTR) results from accelerated endocytosis of {delta}F508-CFTR in polarized human airway epithelial cells. J Biol Chem 2005;280:36762–36772. [PubMed]
45. Hansen RK, Oesterreich S, Lemieux P, Sarge KD, Fuqua SA. Quercetin inhibits heat shock protein induction but not heat shock factor DNA-binding in human breast carcinoma cells. Biochem Biophys Res Commun 1997;239:851–856. [PubMed]
46. Wang F, Zeltwanger S, Hu S, Hwang TC. Deletion of phenylalanine 508 causes attenuated phosphorylation-dependent activation of CFTR chloride channels. J Physiol 2000;524:637–648. [PubMed]
47. Wang W, Bernard K, Li G, Kirk KL. Curcumin opens cystic fibrosis transmembrane conductance regulator channels by a novel mechanism that requires neither ATP binding nor dimerization of the nucleotide-binding domains. J Biol Chem 2007;282:4533–4544. [PubMed]
48. Derichs N, Mekus F, Bronsveld I, Bijman J, Veeze HJ, von der Hardt H, Tummler B, Ballmann M. Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated residual chloride secretion does not protect against early chronic Pseudomonas aeruginosa infection in F508del homozygous cystic fibrosis patients. Pediatr Res 2004;55:69–75. [PubMed]
49. Accurso F, Rowe SM, Durie PR, Konstan MW, Dunitz J, Hornick DB, Sagel SD, Boyle MP, Uluer AZ, D. U, et al. Interim results of Phase 2a study of VX-770 to evaluate safety, pharmacokinetics, and biomarkers of CFTR activity in cystic fibrosis subjects with G551D. European CF Meeting 2008.
50. Ross JA, Kasum CM. Dietary flavonoids: bioavailability, metabolic effects, and safety. Annu Rev Nutr 2002;22:19–34. [PubMed]
51. Ferry DR, Smith A, Malkhandi J, Fyfe DW, deTakats PG, Anderson D, Baker J, Kerr DJ. Phase I clinical trial of the flavonoid quercetin: pharmacokinetics and evidence for in vivo tyrosine kinase inhibition. Clin Cancer Res 1996;2:659–668. [PubMed]
52. Harwood M, Danielewska-Nikiel B, Borzelleca JF, Flamm GW, Williams GM, Lines TC. A critical review of the data related to the safety of quercetin and lack of evidence of in vivo toxicity, including lack of genotoxic/carcinogenic properties. Food Chem Toxicol 2007;45:2179–2205. [PubMed]
53. Matsuo M, Sasaki N, Saga K, Kaneko T. Cytotoxicity of flavonoids toward cultured normal human cells. Biol Pharm Bull 2005;28:253–259. [PubMed]
54. Shoskes DA, Zeitlin SI, Shahed A, Rajfer J. Quercetin in men with Category III chronic prostatitis: a preliminary prospective, double-blind, placebo-controlled trial. Urology 1999;54:960–963. [PubMed]
55. Edwards RL, Lyon T, Litwin SE, Rabovsky A, Symons JD, Jalili T. Quercetin reduces blood pressure in hypertensive subjects. J Nutr 2007;137:2405–2411. [PubMed]
56. Morino K, Matsukara N, Kawachi T, Ohgaki H, Sugimura T, Hirono I. Carcinogenicity test of quercetin and rutin in golden hamsters by oral administration. Carcinogenesis 1982;3:93–97. [PubMed]
57. Ito N, Hagiwara A, Tamano S, Kagawa M, Shibata M, Kurata Y, Fukushima S. Lack of carcinogenicity of quercetin in f344/DUCRJ rats. Jpn J Cancer Res 1989;80:317–325. [PubMed]
58. Hard GC, Seely JC, Betz LJ, Hayashi SM. Re-evaluation of the kidney tumors and renal histopathology occurring in a 2-year rat carcinogenicity bioassay of quercetin. Food Chem Toxicol 2007;45:600–608. [PubMed]
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