Dehydrocostuslactone, a sesquiterpene lactone activates wild-type and ΔF508 mutant CFTR chloride channel
Cystic fibrosis transmembrane conductance regulator (CFTR) represents the main cAMP-activated Cl2 channel expressed in the apical membrane of serous epithelial cells. Both deficiency and overactivation of CFTR may cause fluid and salt secretion related diseases. The aim of this study was to identify natural compounds that are able to stimulate wild-type (wt) and DF508 mutant CFTR channel activities in CFTR- expressing Fischer rat thyroid (FRT) cells. We found that dehydrocostuslactone [DHC, (3aS, 6a R, 9a R, 9bS)-decahydro-3,6,9-tris (methylene) azuleno [4,5-b ] furan-2(3H)- one)] dose dependently potentiates both wt and DF508 mutant CFTR-mediated iodide influx in cell-based fluorescent assays and CFTR-mediated Cl2 currents in short-circuit current studies, and the activations could be reversed by the CFTR inhibitor CFTRinh-172. Maximal CFTR-mediated apical Cl2 current secretion in CFTR-expressing FRT cells was stimulated by 100 mM DHC. Determination of intracellular cAMP content showed that DHC modestly but significantly increased cAMP level in FRT cells, but cAMP elevation effects contributed little to DHC-stimulated iodide influx. DHC also stimulated CFTR-mediated apical Cl2 current secretion in FRT cells expressing DF508-CFTR. Subsequent studies demonstrated that activation of CFTR by DHC is forskolin dependent. DHC represents a new class of CFTR potentiators that may have therapeutic potential in CFTR-related diseases.
Keywords: cystic fibrosis transmembrane conductance regulator (CFTR); cystic fibrosis (CF); DF508-CFTR; dehydrocostuslactone; potentiator
1. Introduction
Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated anion channel permeable to Cl2 and HCO2 [1], ubiquitously expressed in apical membrane of serous epithelial cells. CFTR mediates fluid and electrolyte transport in secretory epithelia in airways, pancreas, sweat glands, bile ducts, and intestines [2]. Reduced function of CFTR is involved in the most common lethal disease such as cystic fibrosis (CF) [3] and other diseases such as bronchiectasis [4], CF-associated liver disease [5], keratoconjunctivitis sicca
[6], idiopathic chronic pancreatiti [7], and habitual constipation [8], whereas poly- cystic kidney disease [9] and secretory diarrhea [10] are regarded to be associated with overactivation of CFTR. Therefore, CFTR is a potential molecular therapeutic target in treating the diseases mentioned earlier. In recent years, several selective CFTR regulators (including both activators and inhibitors) from the combinatorial library [11 – 13] have been identified. But none seems to be ideal for therapeutic use in the treatment of CFTR-related diseases.
It has been generally accepted that natural compounds are more beneficial than the combinatorial compounds in drug discoveries. Previously, in screening of CFTR activators from naturally occurring compounds, we found a large number of CFTR potentiators [14] including dehy- drocostuslactone [DHC, (3aS, 6a R, 9a R, 9bS)-decahydro-3,6,9-tris (methylene) azuleno [4,5-b ] furan-2(3H)-one)]. The aim of this study was to investigate in more detail the effect of DHC on wild-type (wt) and mutant CFTR in cell-based experiments in Floustar and Ussing chambers. Our study provides a new leading compound for developing potential drugs for the treatment of CFTR-related diseases such as CF and bronchiectasis.
2. Results
2.1 DHC activates wt-CFTR-mediated Cl2 transport
DHC (shown in Figure 1(a)) was identified as an effective wt-CFTR activator when we applied the cell-based iodide influx fluor- escence assay by recording CFTR-mediated iodide influx stimulated by 386 natural compounds. Studies were performed in the presence of 50 nM forskolin (FSK), and DHC was tested at 10, 50, 100, and 200 mM. Original time-course fluorescence curves for DHC, along with genistein (positive control), phosphate-buffered saline (PBS) (negative control) were shown in Figure 1(b). DHC was then evaluated at a series of concentrations to generate a dose– response relationship. As shown in Figure 1(c), DHC showed lower efficiency and affinity than those of genistein: Kd and Vmax values are ,30 mM and ,0.6 mM/S, respectively.
CFTR activation by DHC was further confirmed using short-circuit current recording tests. Measurements were car- ried out after the basolateral membrane of FRT cells was permeabilized with 250 mg/ml amphatericin B in the presence of transepithelial Cl2 gradient, so the recorded currents represent apical Cl2 currents (Isc). As typical dose– response data shown in Figure 2(a) and summarized in Figure 2(b), DHC induced significant Isc increases in the FRT cells expressing CFTR with EC50 of about 20 mM. The known CFTR blocker CFTRinh-172 added at the end of tests completely abolished the activities.
2.2 DHC stimulates submucosal fluid secretion in mouse trachea mucosa
Effect of DHC was also tested on live tissue. Single submucosal gland fluid secretion was measured in cartilaginous airways from Kunming mice. Figure 3(a) shows significant gland fluid secretion is stimulated by the addition of 10 mM pilocarpin or 100 mM DHC to the serosal bathing solution, and the DHC-stimulated fluid secretion was inhibited by CFTRinh- 172 and GlyH 101. Summarized data are shown in Figure 3(b).
2.3 DHC activates DF508 mutant CFTR Cl2 transport
DHC was also tested on FRT cells expressing DF508-CFTR and G551D- CFTR. DF508-CFTR belongs to the second class CFTR mutant that has both protein processing and channel gating defects [15], whereas G551D-CFTR is a third class CFTR mutant that has only decreased channel gating activity [16]. In the DF508-CFTR potentiating activity studies, the FRT cells were cultured under low temperature (278C) condition to allow sufficient mutant CFTR located in the apical membrane before assays [17]. Tests were performed in the presence of 20 mM FSK to stimulate maximal cellular cAMP concentration. As original time- course fluorescence curves shown in Figure 4(a), significant stimulation activi- ties were detected in the FRT cells treated with DHC. Dose – response analysis showed DHC activation of DF508-CFTR with Kd and Vmax (expressed as d[I2]/dt) values of 50 mM and 0.05 mmM/s, respect- ively (Figure 4(b)). Short-circuit current analysis confirmed the results. Typical short- circuit current recordings are shown in Figure 4(c) and summarized in Figure 4(d). DHC was also tested on G551D-CFTR. Both fluorescence assay (Figure 4(e)) and Ussing chamber study (Figure 4(f)) gave negative results.
CFTRinh-172 and Gly H101 are known CFTR blockers [12,18]. To further confirm that activation of iodide influx in the fluorescence assay was CFTR-mediated, CFTRinh-172 and Gly H101 were tested at various concentrations. As shown in Figure 5(a), (c), both blockers significantly inhibited iodide influx in FRT cells expressing wt-CFTR. Maximal inhibitions were achieved by 10 mM CFTRinh-172 and 10 mM GlyH 101. Similar results were seen in FRT cells expressing DF508- CFTR (Figure 5(b), (d)).
2.4 Mechanism of CFTR activation by DHC
To evaluate the mechanism of CFTR activation by DHC, DHC was tested at different concentrations on FRT cells expressing wt-CFTR to assess the depen- dence on CFTR phosphorylation level. We found that DHC was also effective with non- maximal FSK stimulation, although its potency was relatively lower. In addition, when concentration .200 mM, even in the absence of FSK, DHC could still activate CFTR-mediated iodide influx though its efficiency was relatively lower (Figure 6(a)). We further measured the cellular cAMP concentration stimulated by DHC. In the presence of 100 nM FSK, genistein (50 mM) significantly increased cAMP concentration in the FRT cells, which correlates well with the results of others. Similarly, in the presence of 100 nM FSK, 200 mM DHC caused modest but significant cAMP increase ( p , 0.05) (Figure 6(b)). To further evaluate the contribution of DHC-stimulated iodide influx by cAMP elevation pathway, we measured cellular cAMP level in FRT cells after incubated with different concentrations (10, 50, and 200 mM) of DHC in the presence or absence of FSK for 10 min. Cellular cAMP concen- trations were normalized to that of 20 mM FSK-stimulated cAMP. We found that in the presence of 100 nM FSK, the concen- tration of cAMP produced by 200 mM DHC was similar to that of produced by 150 nM FSK (Figure 6(c)). So, we compared the efficiency of iodide influx stimulated by 200 mM DHC and 150 nM FSK to that stimulated by 150 nM FSK and 200 mM DHC. As shown in Figure 6(d), neither 150 nM FSK nor 200 mM DHC alone stimulated significant iodide influx in the fluorescence assay, whereas 150 nM FSK plus 200 mM DHC vigorously increased CFTR-mediated iodide influx with d[I2]/ dt < 0.9 mM/s ( p , 0.01). In addition, when CFTR protein maximal phosphory- lated with 20 mM FSK and 100 mM 3- isobutyl-1-methylxanthine (IBMX) prior, 200 mM DHC could further increase iodide influx rate by ,30%. 2.5 Effect of DHC on DF508-CFTR misprocessing defect rescuing The observations mentioned earlier pro- vided a rationale to investigate the effect of DHC on DF508-CFTR misprocessing defect rescuing. Iodide influx tests were performed after the incubation of 24 h (at 378C) of the FRT cells expressing DF508-CFTR with different concen- trations of DHC. Cells were washed with PBS after incubation, and I2 influx was measured 15 min after the addition of FSK (20 mM) and genistein (50 mM). No significant increase in the rate of I2 influx (d[I2]/dt) was observed as representative time-course fluorescence curves shown in Figure 7(a) and quantified in Figure 7(b). In the low temperature (278C incubated for 18 – 24 h) rescued group, significant increase of I2 influx rates were recorded. DF508-CFTR mislocalization defects res- cuing effect of DHC was further analyzed by Ussing chamber assay. The low temperature rescue group gave positive results, whereas in the FRT cells incubated with different concentrations of DHC at 378C for .24 h, FSK and genistein still did not stimulate the change of significant short-circuit currents when compared with dimethyl sulfoxide (DMSO) control group as shown in Figure 7(c) and summarized in Figure 7(d). 2.6 Kinetics and reversible characteristics of CFTR activation by DHC The cell-based fluorescence assay was performed to evaluate kinetics and rever- sibility of DHC-induced CFTR activation. CFTR activation manifested rapid and reversible characteristics, in which maximal activation was acquired in 20 min and abolished in 30 min after DHC was washed out (Figure 8(a)). Similar results were found in DF508-CFTR activation (Figure 8(b)). 2.7 Statistics Data were presented as mean ^ SE or as representative traces. Student’s t test was used to compare test and control values, p values of , 0.05 were considered statistically significant. 3. Discussion DHC, a naturally occurring sesquiterpene lactone derivative, is rich in the traditional Chinese medicine Saussurea costus (Falc.) Lipschitz. S. costus is one of the most popular traditional Chinese herbs that show potential anti-angiogenesis [19] and anti-tumor [20] effects, which is widely used in smooth muscle relaxation therapy, antispasmodic therapy, cholago- gic therapy, and anti-ulcerogenic therapy in both prescription and non-prescription forms. In this study, we investigated the effects of DHC on CFTR chloride channel activities using cell-based fluorescence assays and short-circuit current measure- ments. DHC dose dependently potentiates wt-CFTR Cl2 channel activities, which were reversed by CFTR blockers, CFTRinh-172, and GlyH101. CFTR-mediated apical Cl2 current in FRT cells expressing CFTR was stimulated by DHC with half maximal concentration of about 20 mM. Determination of intracellular cAMP content showed that DHC modestly but significantly increased cAMP concen- tration in FRT cells, and cAMP elevation effects contributed little to DHC-stimu- lated iodide influx. DHC also potentiates DF508-CFTR Cl2 channel in fluorescence assays and CFTR-mediated apical short- circuit current measurements, although potency of the activation was relatively lower. CFTR is a cAMP-activated Cl2 channel, it has been well-defined that the mechanisms of CFTR activation may include elevating phosphorylation at R- domain and interacting directly with CFTR proteins [21]. Although DHC slightly increased cellular cAMP level that may lead to CFTR activation, our data supported a direct interaction mechanism for the following reasons: First, DHC- stimulated iodide influx did not correlate closely with changes in total cAMP concentration. The results from Figure 6 demonstrated that 100 nM FSK plus 200 mM DHC mixture produced compara- tively similar increases in cAMP to that of 150 nM FSK, and the cAMP concentration stimulated only marginal iodide influx of <0.2 mM/s, yet the mixture stimulated an increased iodide influx rate of greater than fourfold (d[I2]/dt < 0.9 mM/s), which suggested that FSK and DHC work in a different way. Second, although FSK (100 nM) plus DHC (200 mM) could increase cAMP, this elevation in cAMP had no significant effect on CFTR Cl2 channel activities. Further studies mani- fested that even if phosphorylation of CFTR was saturated by FSK and IBMX, DHC further increased CFTR iodide influx that also suggested a direct binding activation mechanism. Third, DF508- CFTR lost cAMP-dependent activation properties, and phosphorylation alone is not able to potentiate channel opening. Conformational change induced by small molecules like DHC in CFTR protein is essential for DF508-CFTR activation. Airway submucosal glands lie beneath the epithelium and play key roles in the pathophysiology of CF. CFTR is expressed in serous cells in submucosal glands. It has been well documented that CFTR function contributes to several airway diseases like CF and bronchiectasis [3,4]. Our results clearly indicated that DHC can effectively stimulate fluid secretion in live epithelial tissues, which suggests that potential therapeutic use of DHC in the treatment of CFTR-related disease such as bronchiectasis. 4. Materials and methods 4.1 Chemicals DHC was purchased from NICPBP (National Institute for the Control of Pharmaceutical and Biological Products in China, Beijing, China). Purity was confirmed (. 99%) by high-performance liquid chromatography/mass spec- troscopy. FSK, genistein, F12 Coon’s medium, and L-glutamine were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum (characterized) was purchased from HyClone (Thermo Scientific HyClone, Logan, UT, USA). CFTRinh-172 was synthesized in our laboratory as reported previously [22]. All other analytical inorganic salts were purchased from BBI (Shanghai Sangon Biological Engineering Technology & Services Co. Ltd, Shanghai, China). Compounds were dissolved as 50 mM parent solution in DMSO and stored at 2808C. All compounds were diluted in PBS before experiment, and the final concentration of DMSO was ,0.1% to ensure no significant effect on tests.
4.2 Cell lines
Human wt, G551D, and DF508 mutant CFTR-expressing Fischer rat thyroid epithelial cells (FRT/wt-CFTR/EYFP- H148Q, FRT/G551D-CFTR/EYFP- H148Q/I152L, and FRT/DF508-CFTR/ EYFP-H148Q/I152L) were prepared as described in Yang et al. [13] and Galietta et al. [23]. For iodide influx fluorescence assays, the cells were plated into black- walled clear-bottomed 96-well tissue culture plates (Corning-Costar 3904, Corning Life Sciences, Oneonta, NY, USA), and served with F-12 Coon’s medium (supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin). For short-circuit current measurements, the cells were seeded into Snapwell porous support inserts (0.4 mm pore diameter and 12 mm insert diameter; Corning-Costar) at ,1 £ 106 cells per well. Once the cells were confluent, the apical surface medium was removed. The cells were tested when the tight junction was formed.
4.3 Iodide influx fluorescence assays The FRT cells grown in 96-well micro- plate were washed three times with PBS (containing in: 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, 1 mM CaCl2, and 0.5 mM MgCl2) and incubated with FSK and test compounds in a final volume of 40 ml. PBS with same concentration of FSK was used as negative control. Iodide influx rates (d[I2]/dt) were measured using a microplate reader (Fluostar Optima, BMG Lab Technologies, Offenburg, Germany), and the plate reader was equipped with two pumps and customized excitation (HQ500/20X: 500 ^ 10 nm) and emission (HQ535/30M: 535 ^ 15 nm) filters (Chroma Technology Corp., Bellows Falls, VT, USA) for enhanced yellow fluorescent protein (EYFP) fluorescence. Each well was recorded for 14 s (five pionts per second) continuously with 2 s before and 12 s after injection of 120 ml I2-containing solution (PBS with 137 mM Cl2 replaced by I2). Iodide influx rates were computed from initial time-course fluorescence as described in Ma, Vetrivel et al. [11].
4.4 Short-circuit current measurements
FRT cells grown as monolayer in the Snapwell inserts were mounted in a Ussing chamber system (Vertical diffusion chamber, Costar, Corning Life Sciences). Measurements were carried out in the presence of transepithelial Cl2 gradient (basolateral side solution contained in: 130 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 1 mM CaCl2, 0.5 mM MgCl2, 10 mM Na-Hepes, pH 7.3, and 10 mM glucose; apical side solution contained the same solution except that 65 mM NaCl was replaced by sodium gluconate, and CaCl2 was increased to 2 mM. Bath solutions were vigorously bubbled with 95% O2 and 5% CO2. The basolateral membrane was permeabilized with 250 mg/ml amphotericin B. All measure- ments were carried out at 378C. Short- circuit current (Isc) was measured using voltage clamp (DVC-1000 voltage clamp, World Precision Instruments, Sarasota, FL, USA) as reported in He et al. [24].
4.5 Submucosal gland fluid secretion
Mouse trachea submucosal fluid secretion tests were performed as in Song et al. [25]. In general, freshly excised Kunming mice (Central Research Laboratory, Jilin University Bethune Second Hospital, Changchun, Jilin, China) mucosa was mounted on a perfusion chamber with the mucosal side up. After cleared with PBS, the mucosa was covered with PBS saturated mineral oil. Gland fluid droplets were imaged using a light microscopy (Olympus Micro DP Controller, Olympus Co., Shinjuku-ku, Tokyo, Japan) after exposed to test compounds. Rates of fluid secretion from submucosal glands were computed from fluid droplet diameter assuming semi-spherical droplet geometry as reference.
4.6 DF508-CFTR misprocessing rescuing analysis FRT/DF508-CFTR/EYFP-H148Q/I152L cells were incubated with different con- centrations of test compounds for , 24 h at 378C before applied to the iodide influx fluorescence assays and transepithelial short-circuit current measurements as described in Pedemonte et al. [17]. Reduced-temperature (incubation at 278C for 18 – 24 h) rescue was used as positive control.
4.7 cAMP assay
Cellular cAMP concentration was deter- mined using a cAMP radio active immu- noassay kit (Shanghai Traditional Chinese Medicine University, Shanghai, China). Measurements were carried out in hex- iplicates according to manufacture’s instruction. Cell lysates prepared from CFTR-expressing FRT cells were assayed for cAMP level after incubation with test compounds for 10 min in the presence or absence of FSK.
In conclusion, our data demonstrated that DHC can stimulate CFTR-dependent Cl2 transport in a rapid, reversible, and FSK-dependent manner. It is suggested that DHC activates CFTR Cl2 channel by directly binding to the protein itself instead of by indirect pathways. DHC is useful for probing CFTR channel gating mechanisms and as a lead compound for developing pharmacological therapies for CFTR-related diseases such as CF and bronchiectasis.