Trimethylangelicin promotes the functional rescue of mutant F508del CFTR protein in cystic fibrosis...

Trimethylangelicin promotes the functional rescue of mutant F508del CFTRprotein in cystic fibrosis airway cells

Maria Favia,1 Maria T. Mancini,1 Valentino Bezzerri,2 Lorenzo Guerra,1 Onofrio Laselva,1

Anna C. Abbattiscianni,1 Lucantonio Debellis,1 Stephan J. Reshkin,1 Roberto Gambari,3* Giulio Cabrini,2*and Valeria Casavola1,4*1Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy; 2Laboratory of MolecularPathology, Department of Pathology and Diagnostics, University Hospital Verona, Verona, Italy; 3Department of LifeSciences and Biotechnology, University of Ferrara, Ferrara, Italy; and 4Centre of Excellence in Comparative Genomics,University of Bari, Bari, Italy

Submitted 28 October 2013; accepted in final form 5 May 2014

Favia M, Mancini MT, Bezzerri V, Guerra L, Laselva O,Abbattiscianni AC, Debellis L, Reshkin SJ, Gambari R, CabriniG, Casavola V. Trimethylangelicin promotes the functional rescue ofmutant F508del CFTR protein in cystic fibrosis airway cells. Am JPhysiol Lung Cell Mol Physiol 307: L48–L61, 2014. First publishedMay 9, 2014; doi:10.1152/ajplung.00305.2013.—Cystic fibrosistransmembrane conductance regulator (CFTR) carrying the F508delmutation is retained in endoplasmic reticulum and fails to traffic to thecell surface where it functions as a protein kinase A (PKA)-activatedchloride channel. Pharmacological correctors that rescue the traffick-ing of F508del CFTR may overcome this defect; however, the rescuedF508del CFTR still displays reduced chloride permeability. There-fore, a combined administration of correctors and potentiators of thegating defect is ideal. We recently found that 4,6,4=-trimethylangelicin(TMA), besides inhibiting the expression of the IL-8 gene in airwaycells in which the inflammatory response was challenged with Pseu-domonas aeruginosa, also potentiates the cAMP/PKA-dependent ac-tivation of wild-type CFTR or F508del CFTR that has been restoredto the plasma membrane. Here, we demonstrate that long preincuba-tion with nanomolar concentrations of TMA is able to effectivelyrescue both F508del CFTR-dependent chloride secretion and F508delCFTR cell surface expression in both primary or secondary airwaycell monolayers homozygous for F508del mutation. The correctioneffect of TMA seems to be selective for CFTR and persisted for 24 hafter washout. Altogether, the results suggest that TMA, besides itsanti-inflammatory and potentiator activities, also displays correctorproperties.

cystic fibrosis; F508del cystic fibrosis transmembrane conductanceregulator corrector; F508del cystic fibrosis transmembrane conduc-tance regulator rescue; chloride transport

CYSTIC FIBROSIS TRANSMEMBRANE conductance regulator (CFTR)is a glycosylated protein that functions as a plasma membranechloride and bicarbonate channel (2) and modulates salt andwater transport across epithelial cell membranes of varioustissues (24, 34). CFTR is comprised of two membrane-span-ning domains (MSD1 and MSD2), two nucleotide-bindingdomains (NBD1 and NBD2), and a regulatory domain (R) (49).CFTR activation requires both ATP binding to the interfacebetween NBD1 and NBD2 and protein kinase A (PKA)-mediated phosphorylation of the R domain (6, 11, 13, 64). Alarge number of cellular chaperones and cochaperones partic-

ipate in regulating the correct folding of CFTR (31). Thedeletion of the phenylalanine residue at position 508 (F508del)in the NBD1 domain is the most common class II mutation incystic fibrosis (CF) and results in a folding defect of F508delCFTR, its retention in the endoplasmic reticulum (ER), and thepremature degradation by the ubiquitin-proteasome system(UPS). The small amount of F508del CFTR that escapes fromUPS and reaches the plasma membrane functions poorly and isunstable on the plasma membrane with a half-life on the cellsurface significantly shorter than that of the wild-type (wt)protein (22, 37, 54). The consequent F508del CFTR-dependentionic imbalance and the aberrant fluid homeostasis at epithelialsurfaces in the lung result in the secretion of thick mucus,which impairs the mucociliary clearance favoring the bacterialinfections, inflammation, and consequent impairment of lungfunction (7).

It has been shown that culturing cells at low temperature(27°C) can rescue the trafficking defect of F508del CFTR andits expression at the cell surface, although still with alteredgating of the channel and reduced cell surface density andrecycling efficiency (12, 15, 27). In addition to low tempera-ture culture, chemical compounds such as DMSO (5), glycerol(53), or organic solutes (73) increase the processing of thecore-glycosylated, endoplasmic reticulum-arrested F508delCFTR into the fully glycosylated mature form of CFTR. Thesefindings, together with the evidence that restoration of smallamounts of functional CFTR protein (20–30% of normallevels) (72) can greatly ameliorate the disease severity, havestimulated a great effort to identify membrane-permeablesmall-molecule compounds that could either rescue the biosyn-thetic defect of F508del CFTR thus restoring its folding,trafficking, and insertion into the plasma membrane (correc-tors) and/or enhance its regulated function once rescued to thesurface (potentiators). The potentiators, interacting directlywith the NBD1 and NBD2 of CFTR (9), rapidly increaseCFTR channel activation by either an ATP-dependent (25) or-independent (17) mechanism, whereas the correctors requireseveral hours to rescue the functional expression of CFTR.

Most of the presently identified potential correctors havebeen selected by high-throughput screening, which allows arapid screening of thousands of small molecules before beingvalidated in “in vitro” culture systems (65, 66). Some of themhave displayed some efficacy in restoring F508del CFTRplasma membrane localization and improving chloride trans-port (10, 29, 43, 52, 67). In particular, VX-809 (60), developedby Vertex Pharmaceuticals, has resulted to be efficacious in

* R. Gambari, G. Cabrini, and V. Casavola contributed equally to this work.Address for reprint requests and other correspondence: V. Casavola, Dept.

of Biosciences, Biotechnologies and Biopharmaceutics, Univ. of Bari, ViaAmendola 165/A, Bari 70126, Italy (e-mail: [email protected]).

Am J Physiol Lung Cell Mol Physiol 307: L48–L61, 2014.First published May 9, 2014; doi:10.1152/ajplung.00305.2013.

1040-0605/14 Copyright © 2014 the American Physiological Society http://www.ajplung.orgL48

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improving F508del CFTR surface expression and chloridetransport in cultured human bronchial epithelial cells isolatedfrom CF patients homozygous for the F508del mutation and ispresently considered suitable for advancement into Phase IIIclinical studies in combination with the potentiator VX-770“Kalydeco” (59). This combined administration of a potentia-tor and a corrector together is required to rescue a sufficientfunctional expression of F508del CFTR at the cell surface,since the F508del mutation displays both trafficking and gatingdefects.

The mechanism of action of many correctors remains rela-tively poorly known, and studies to better understand thestructure of the CFTR protein and the conformational destabi-lization of F508del NBD1 are useful to develop new com-pounds that can bind directly to the mutant protein. In thisrespect, the identification of a number of folding correctors ofF508del CFTR has been discovered in in silico structure-basedscreening utilizing homology models of CFTR (29).

Correctors may rescue the functional expression of F508delCFTR either acting as pharmacological chaperones that inter-act directly with the misfolded F508del CFTR protein favoringits folding and trafficking to the cell surface (36, 52, 68, 70) oras proteostasis regulators modulating the activity of a plethoraof pathways involved in F508del CFTR folding and degrada-tion (3, 21, 39). However, because F508del CFTR trafficking isinhibited by various quality control systems, it is becomingevident that a combination of two different correctors or acorrector and a potentiator together might be necessary toachieve a significant increase in F508del CFTR functionalexpression and restore the CF symptoms. In this respect,compounds having both corrector and potentiator functionswould alleviate this need for multiple combined administra-tions, but, until now, very few compounds have been identifiedwith both corrector and potentiator activities (32, 44, 46).

We recently demonstrated that the psoralen-related compound,4,6,4=-trimethylangelicin (TMA), potentiates the cAMP/PKA-de-pendent activation of wt CFTR in airway cells (58). Impor-tantly, here we report that long-term incubation with TMAsignificantly rescues F508del CFTR functional expression tothe cell surface of primary and secondary airway CF cellmonolayers, suggesting that, as observed for a few othermolecules, TMA may act both as potentiator and corrector.Moreover, previous findings (58) demonstrating that TMAsignificantly inhibits the expression of the IL-8 gene in airwaycells in which the inflammatory response has been challengedwith Pseudomonas aeruginosa suggest that TMA may repre-sent a promising lead compound for a potential development ofa single drug therapy useful for overcoming the CF defect.

MATERIALS AND METHODS

Cell culture. Experiments were performed in two human immor-talized CF bronchial epithelial cell lines: the CFBE41o� cells, stablyoverexpressing F508del CFTR, (CFBE�F) homozygous for theF508del allele (F508del/F508del) (a generous gift from Dr. J. P.Clancy, University of Cincinnati, Children’s Hospital Medical Center,Cincinnati, OH) (4), and the CuFi-1 cells (F508del/F508del CFTRmutant genotype) (a generous gift of Dr. J. Zabner, University ofIowa); the 16HBE14o� (HBE) expressing endogenous wt CFTR andthe CFBE wtCFTR cells (CFBE41o�, with stable expression ofwild-type CFTR) were a generous gift from Dr. D. Gruenert (Univer-sity of California, San Francisco, CA). HBE, CFBE�F, and CFBE

wtCFTR cells were grown in complete media (E-MEM, 10% FBS,L-glutamine, and penicillin/streptomycin) at 37°C under 5% CO2. Thestably transfected cells were maintained in the presence of 2 �g/mlPuromicin (for CFBE�F) or 200 �g/ml Hygromycin B (for CFBEwtCFTR) positive selection. Cells were routinely grown on flaskscoated with an extracellular matrix containing fibronectin/vitrogen/BSA. CuFi-1 cells were grown on human placental collagen type VI(Sigma)-coated flasks in BEGM medium (Cambrex Bio Science), aspreviously described (71).

Fischer rat thyroid (FRT) epithelial cells, stably coexpressinghuman F508del CFTR and the high-sensitivity halide-sensing greenfluorescent analog yellow fluorescent protein (HS-YFP) YFP-H148Q/I152L (FRT-F508del) or stably transfected with a plasmid coding forP574H-CFTR and carrying the resistance gene for zeocin (FRT-P574H) were a generous gift from Dr. L. J. Galietta (Gaslini Institute,Genoa, Italy). FRT-F508del and FRT-P574H cells were grown inCoon’s modified Ham’s F-12 medium plus 10% FBS, L-glutamine,and penicillin/streptomycin at 37°C under 5% CO2.

Human Embryonic Kidney 293 (HEK293) cells were grown inDMEM (with 5% FBS, penicillin/streptomycin). HEK293 cells,grown on 35-mm-diameter culture dishes, were transfected with 1 �gof plasmid DNA coding for 3HA-wt “human ether-a-go-go-relatedgene” (hERG) or for 3HA-hERG R752W (a generous gift from Dr. E.Ficker, MetroHealth Medical Center, Case Western Reserve Univer-sity, Cleveland, OH) using Lipofectamine 2000 (Invitrogen).

Human airway epithelial cells, differentiated in a polarized epithe-lium primary culture (MucilAir), are derived from CF (homozygousfor the F508del mutation) and non-CF bronchial biopsies and wereprovided by Epithelix Sarl (Geneva, Switzerland). “Italian CysticFibrosis Foundation Primary Culture Core Facility” provided otherprimary cultures of human bronchial epithelial cells derived eitherfrom F508del CFTR homozygous donors, CF-BE, and from non-CFdonors. All primary cells were maintained at an air-liquid interface(ALI) according to the manufacturer’s instructions.

For iodide influx experiments, 20,000 FRT-F508del cells wereseeded on each round glass cover slip. For chloride efflux experi-ments, confocal immunofluorescence analysis, and biotinylation ex-periments, cells were seeded on 0.4-�m-pore size polyethylene te-rephthalate filter inserts (Falcon BD). For transepithelial currentmeasurements, cells were seeded on 0.4-�m-pore size Snapwellpolyester membrane inserts (Costar, Corning, NY). The functionalexperiments were performed when the cell monolayers reached atransepithelial electrical resistance �1,000 ohm � cm2 (primary cells)and �500 ohm � cm2 (immortalized cells) measured with theMillicell-Electrical Resistance System (Millipore).

The drug TMA was dissolved in 3% DMSO in methanol (vehicle)at a concentration of 2.5 mM (stock solution), whereas VX-809 andVX-770 were dissolved in DMSO at concentrations of 20 and 50 mM,respectively.

The vitality of cells was evaluated after 24 h treatment with vehiclealone or TMA at all tested concentrations by trypan blue exclusionassay and resulted to be �98%.

Fluorescence measurements of iodide influx. FRT-F508del cellswere preincubated for 48 h with vehicle, TMA, or VX-809 (Selleck).At the time of assay, cells were washed in Dulbecco’s PBS (in mM:137 NaCl, 2.7 KCl, 8.1 Na2HPO4, 1.5 KH2PO4, 1 CaCl2, and 0.5MgCl2, pH 7.4) and subsequently incubated with stimulation cocktail[20 �M forskolin, 100 �M isobutyl methylxanthine (IBMX), and 50�M genistein] in the presence and in the absence of 10 �M CFTRinhibitor, CFTRInh-172 (Sigma) (38, 57), for 30 min. The cover slipswere then transferred on a Nikon TMD inverted microscope througha Nikon Fluor �40 objective. The signal was acquired with aHamamatsu C2400–97 charge-coupled intensified video camera at arate of 1 frame/3 s. Fluorescence coming from each single cell wasanalyzed by customized software (Spin, Vicenza, Italy). Results arepresented as transformed data to obtain the percentage signal variation(Fx) relative to the time of addition of the stimulus, according to the

L49TMA CORRECTS F508del CFTR FUNCTIONAL EXPRESSION

AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00305.2013 • www.ajplung.org

equation: Fx � ([Ft � Fo]/Fo) � 100, where Ft and Fo are thefluorescence values at the time t and at the time of addition of thestimulus, respectively. The assay of each sample consists of a con-tinuous 120-s fluorescence reading with 40 s before and 80 s afterinjection of the iodide-rich Dulbecco’s PBS (in mM: 137 NaI, 2.7KCl, 8.1 Na2HPO4, 1.5 KH2PO4, 1 CaCl2, and 0.5 MgCl2, pH 7.4) toreach a final iodide concentration of 50 mM.

Transepithelial current measurements. Airway cells (1.5 � 105)seeded on Snapwell inserts after reaching the confluence wereswitched to air-liquid interface for 48 h and then incubated with TMA(100 nM) for 24 h. Primary or secondary cell monolayers weremounted in modified Ussing chambers (Mussler Scientific Instru-ments, Aachen, Germany). Each half-chamber consisted of a circularfluid canal of 3.5 ml total volume filled with Ringer solution that wasconstantly recirculated by means of a bubble lift. The cells wereinitially bathed on both sides with identical Ringer solutions contain-ing (in mM): 115 NaCl, 25 NaHCO3, 2.4 KH2PO4, 1.24 K2HPO4, 1.2CaCl2, 1.2 MgCl2, and 10 D-glucose (pH 7.4). Bath solutions werecontinuously gassed with a gas mixture containing 95% O2-5% CO2.Solutions and chambers were maintained at 37°C. The transepithelialvoltage was monitored with a voltmeter using two calomel half-cellsconnected to each bath solution via agar bridges (Ringer solution in1% agar). Short-circuit current (Isc) was applied by a voltage-clampdevice for 4 s every 3 min via a pair of platinum electrodes.Transepithelial resistance was calculated using Ohm’s law from Isc

and transepithelial voltage. To measure stimulated Isc, only in sec-ondary cell monolayers the mucosal bathing solution was changed toa low-Cl� solution containing (in mM) 1.2 NaCl, 115 sodium glu-conate, 25 NaHCO3, 2.4 KH2PO4, 1.24 K2HPO4, 2.4 CaCl2, 1.2MgCl2, and 10 D-glucose (pH 7.4), and subsequently 100 �Mamiloride was added. The CaCl2 concentration was increased to 2.4mM to compensate calcium chelation by the gluconate. The CFTR-dependent chloride secretion was stimulated by the concomitantaddition of 10 �M forskolin (FSK) plus 500 �M IBMX to bothbathing solutions and, subsequently, by the addition of the potentiatorgenistein (50 �M). At the end of the experiment, CFTRInh-172 (10�M) was added to the apical bath solution to inhibit F508del CFTR-mediated Isc.

Fluorescence measurements of apical chloride efflux. Chlorideefflux was measured using the Cl�-sensitive dye N-(ethoxy-carbon-ylmethyl)-6-methoxyquinolinium bromide, MQAE (Sigma). Conflu-ent cell monolayers were loaded overnight in culture medium con-taining 5 mM MQAE at 37°C in a CO2 incubator and then inserted ina perfusion chamber that allowed independent perfusion of apical andbasolateral cell surfaces. Fluorescence was recorded with a CaryEclipse spectrofluorometer (Varian). To measure chloride efflux rateacross the apical membrane, the apical perfusion medium waschanged with a medium in which chloride was substituted withiso-osmotic nitrate. All experiments were performed in HEPES-buffered bicarbonate-free media, Cl� medium (in mM: 135 NaCl, 3KCl, 1.8 CaCl2, 0.8 MgSO4, 20 HEPES, 1 KH2PO4, and 11 glucose),and Cl�-free medium [in mM: 135 NaNO3, 3 KNO3, 0.8 MgSO4, 1KH2PO4, 20 HEPES, 5 Ca(NO3)2, and 11 glucose]. At the end of eachexperiment, a two-point calibration procedure was performed: themaximal intensity of fluorescence (F0) was determined by perfusingthe cells with the Cl�-free medium on both sides of the monolayer;the minimal fluorescence was obtained by then exposing the cells toa solution containing KSCN (in mM: 110 KSCN, 1 MgSO4, 10HEPES, 1 CaSO4, 5 glucose, and 0.005 valinomycin). For dataanalysis, the value for minimal fluorescence was subtracted from theexperimentally measured fluorescence, and the resulting fluorescencewas divided by the value of F0. The rate of Cl� efflux was determinedby linear regression analysis of 30 points taken at 4-s intervals andexpressed in arbitrary slope changes in (F/F0)/min. We measured theapical CFTR-dependent chloride secretion as the difference in the rateof change of FSK plus IBMX-stimulated fluorescence in the absence

or presence of apical treatment with CFTRInh-172 [�(F/F0)/min] asdescribed previously (20).

RNA extraction and quantitative RT-PCR. Total RNA extractedfrom CFBE�F cells treated for 24 h with 100 nM TMA or its vehiclewas purified using a High Pure RNA Isolation Kit (Roche, Mannheim,Germany), and 2.0 mg RNA were reverse transcribed to cDNA usingthe High Capacity cDNA Archive Kit and random primers (AppliedBiosystems, Foster City, CA) in a final reaction volume of 20 �l.cDNA (5 �l) was mixed with TaqMan Fast Advanced Master Mix(Applied Biosystems) and TaqMan gene expression assay (AppliedBiosystems) for CFTR (Hs00357011_m1, NM_000492.3) and GAPDH(Hs02758991_g1, NM_002046.4) as normalizer gene. Real-time PCRwas performed in duplicate for both target and normalizer genes usingthe ABI Prism 7900 HT Fast real-time PCR system (Applied Biosys-tems) as follows: 50°C for 2 min, 95°C for 20 s, and 40 two-stepcycles of 95°C for 1 s and 60°C for 20 s. Results were collected withSDS 2.3 software (Applied Biosystems), and relative quantificationwas performed using the Ct method. Data were analyzed with RQManager software 1.2 (Applied Biosystems). Changes in mRNAexpression levels were calculated after normalization to calibratorgene.

CFTR immunoblot analysis. Immunoblot analysis was performedas described by Van Goor et al. (60). Briefly, cells were incubated for24 h at 37°C with vehicle or TMA or VX-809 or for 24 h at 27°C inculture media. After incubation, cells were harvested in ice-coldD-PBS solution (without calcium and magnesium) and pelleted at1,000 g at 4°C. Cell pellets were lysed in 1% Nonidet P-40, 0.5%sodium deoxycholate, 200 mM NaCl, 10 mM Tris, pH 7.8, and 1 mMEDTA plus protease inhibitor cocktail (Sigma) for 30 min on ice.Lysates were spun for 10 min at 10,000 g at 4°C to pellet insolublematerial. Approximately 30 �g total proteins were heated in Laemmlibuffer with 5% �-mercaptoethanol at 37°C for 5 min and loaded on3–8% Tris-acetate gel (Bio-Rad). The gel was transferred to polyvi-nylidene difluoride (PVDF) membranes (GE Healthcare) and pro-cessed for Western blotting by using monoclonal CFTR antibody(dilution 1:500; clone: 24-1, MAB25031 R&D Systems) or monoclo-nal �-tubulin antibody (dilution 1:1,000; Sigma). Blots were devel-oped by enhanced chemiluminescence (GE Healthcare). F508delCFTR expression was not significantly altered by the 24-h treatmentwith the vehicles of TMA or VX-809. Densitometric quantificationand image processing were carried out using Photoshop (AdobeSystems) and the NIH Image software package version 1.61 (NationalInstitutes of Health).

Internalization assay and cell surface stability. CFTR internaliza-tion was performed as described (28) using a two-step biotinylationprotocol. Polarized cell monolayers were cultured at 27°C for 48 h tofacilitate F508del CFTR rescue and delivery to the cell surface (62).In the last 3 h of this incubation, one-third of the monolayers wastreated with 100 nM TMA, one-third was treated with the vehicle, andthe last third was left untreated. Next, surface carbohydrate groups onthe cell surface were oxidized with sodium periodate (NaIO4, 10mM), washed with ice-cold PBS containing 0.1 mM CaCl2 and 1 mMMgCl2 (PBS-CM), and then warmed to 37°C for 2.5 min. Oxidizedsurface carbohydrate groups remaining on the cell surface after awarm-up period were labeled with biotin-LC-hydrazide (1 mg/ml;Pierce) in PBS-CM for 30 min with gentle agitation. Excess biotinwas removed by washing cells two times at 4°C with 0.1 M glycinein PBS-CM. Cells were lysed in biotinylation lysis buffer (BLB: 0.4%sodium deoxycholate, 1% NP-40, 50 mM EGTA, 10 mM Tris-Cl, pH7.4, and protease inhibitor cocktail). Lysates were centrifuged (10,000g) to remove cell debris, and total protein levels were determined.Equivalent amounts of cell lysate proteins (400 �g) were incubatedovernight with 150 �l of streptavidin (Pierce). Precipitated proteinswere washed three times with BLB, solubilized with Laemmli samplebuffer, separated on a 4–12% SDS-PAGE (Bio-Rad), and blotted forCFTR.

L50 TMA CORRECTS F508del CFTR FUNCTIONAL EXPRESSION


To analyze the TMA effect on rF508del CFTR cell surface stabil-ity, cell monolayers were cultured at 27°C for 48 h and incubated inthe last 3 h with 100 nM TMA, or its vehicle was then transferred at37°C and incubated for 0 up to 8 h in the presence of the corrector.Next, cell surface glycoproteins were biotinylated and lysed in BLBbuffer and processed as described above.

hERG immunoblot analysis. HEK293 cells, grown on 35-mm-diameter culture dishes, were transiently transfected with 1 �g ofplasmid DNA coding for 3HA-wt hERG or for 3HA-hERG R752Wusing Lipofectamine 2000 and were subjected to treatment withcorrectors 24 h after transfection. After incubation, cells were homog-enized in lysis buffer (110 mM NaCl, 50 mM Tris, 0.5% TritonX-100, and 0.5% Igepal CA-630, pH 8, with an added proteaseinhibitor mixture), sonicated for 10 s, and centrifuged for 10 min(16,000 g), and then the pellet was discarded. Supernatant proteinconcentration was measured, and an aliquot of 30 �g of proteins wasdiluted in Laemmli buffer, heated at 100°C for 5 min, and separatedby Tris/HCl SDS/PAGE (7% gel). The gel was transferred to PVDFmembranes (GE Healthcare) and processed for Western blotting byusing monoclonal HA antibody (dilution 1:1,000; HA.11 clone:16B12; Covance) or monoclonal �-tubulin antibody (dilution 1:1,000;Sigma).

Immunofluorescence and confocal analysis. Cell monolayers weretreated with TMA or VX-809 24 h before analysis. Cells were fixedin 3.7% paraformaldehyde and stained with an anti-CFTR (CF3)mouse monoclonal antibody (dilution 1:500; Abcam) as previouslydescribed by us (18). Goat anti-mouse IgG conjugated to Alexa Fluor568 (dilution 1:1,000; Invitrogen) was used as secondary antibody.Monolayers were then mounted on slides with Prolong Gold antifadereagent with DAPI (Invitrogen) and examined with a Leica TCS SP5II microscope equipped with a laser-scanning confocal unit containingan He-Ne argon laser (Leica). Specimens were viewed through aPlanapo �63/1.25 oil immersion objective, and images were acquiredby the LAS-AF version 2.2.1 software.

Data analysis. Data are presented as means SE for the numberof samples indicated. Statistical comparisons were made using pairedor unpaired data Student’s t-test. Differences were considered signif-icant when P 0.05.

RESULTS

We have previously demonstrated that the psoralen-relatedcompound, TMA, at nanomolar concentrations inhibits theexpression of the IL-8 gene in bronchial epithelial cells inwhich the inflammatory response has been challenged with P.aeruginosa (58). In the same study, we observed that acuteaddition (15 min treatment) of 250 nM TMA potentiatedFSK-stimulated chloride secretion in airway cell monolayersexpressing wt CFTR or in CF cells in which F508del CFTR hasalready been rescued to the apical membrane by overexpress-ing the interacting protein NHERF1 (58).

The main goal of the present study was to test if TMA, inaddition to being a CFTR “potentiator,” may also function as a“corrector” of F508del CFTR mistrafficking.

Characterization of TMA as a F508del CFTR corrector inFRT-F508del cells. FRT epithelial cells, coexpressing F508delCFTR and a fluorescent YFP halide sensor (FRT-F508del),were used as an initial screen to test and evaluate long-termTMA effects by measuring iodide influx (43). TMA (200 nM)or its vehicle was added to each well for 48 h at 37°C, andF508del CFTR-facilitated iodide influx was then determinedfrom the kinetics of decreasing YFP fluorescence followingaddition of extracellular iodide in the presence of the adenylatecyclase activator FSK (20 �M), the phosphodiesterase inhibi-tor IBMX (100 �M), and the well-known CFTR potentiator

genistein (50 �M) (26). Examples of representative tracesshowing TMA-induced iodide influx are shown in Fig. 1A,where it can be seen that TMA produced a large YFP fluores-cence decrease corresponding to a significant increase of iodideinflux with respect to cells treated only with the vehicle. Thespecificity of TMA effect on F508del CFTR-dependent iodideinflux was verified by inhibition of the TMA-stimulated in-crease of iodide influx by the specific CFTR inhibitor CFTRInh-172 (10 �M) (Fig. 1, A and C). Similar results were obtainedin FRT-F508del cells treated for 48 h with the establishedcorrector VX-809 (10 �M) (60) (Fig. 1, B and C). TMA (200nM) started increasing iodide influx in FRT-F508del after 12 hof preincubation, with a maximal effect at 48 h of treatment(data not shown).

We then tested the dose-response effect (50–500 nM) of48 h preincubation with TMA and, as shown in Fig. 1D, 50 nMTMA treatment already significantly produced a maximumlevel of increase of F508del CFTR-facilitated iodide influx. Atconcentrations �200 nM, the TMA-induced response de-creased even if still greater than control values.

Functional correction of F508del CFTR by TMA in immor-talized airway CF cell monolayers. To verify the possibilitythat TMA may act as a corrector in CF cell monolayers, wethen examined the effect of a 24-h preincubation with TMA onF508del CFTR-mediated chloride transport in polarized humanairway cells homozygous for the F508del mutation, CFBE�F,grown on permeable supports. These cells have been exten-sively used in the study of other correctors (50, 51, 56, 62).

After 24 h preincubation with TMA or the vehicle, the rateof chloride efflux was measured by following the change influorescence [(F/F0)/min] of the chloride-sensitive dye MQAEafter replacement of chloride by nitrate in the apical perfusionmedium as previously described (20) (Fig. 2, A and B). Theapical F508del CFTR-dependent chloride secretion was calcu-lated as the difference in alterations of 10 �M FSK plus 100�M IBMX-stimulated fluorescence in the absence or presenceof apical treatment with 5 �M CFTRInh-172 (Fig. 2, C and D).Dose-response (50–500 nM TMA) functional experimentswere performed to determine the minimum TMA concentrationthat rescues F508del CFTR-dependent chloride efflux. Asshown in Fig. 2E, 50 nM TMA preincubation already signifi-cantly restored the F508del CFTR-dependent chloride efflux,and 100 nM TMA produced a maximum level of the rescue ofF508del CFTR activity that was 29.3 4.36% of CFTR-dependent chloride transport measured in polarized monolay-ers of 16HBE14o� (HBE) cells expressing wt CFTR. More-over, experiments performed after a 48-h preincubation of 100nM TMA gave similar results to those obtained after 24 h ofTMA treatment [0.0169 0.002 �(F/F0)/min, n � 11 for 24 hand 0.0160 0.004 �(F/F0)/min, n � 4 for 48 h; not signif-icant (NS)].

As found in FRT-F508del cells, at higher TMA concentra-tions, the response was reduced, resulting in a bell-shapeddose-response relationship as also previously reported for VX-809 (60) and other correctors (50). The efficacy of TMA as acorrector was analyzed by comparing the effects of TMApreincubation with those obtained by preincubation for 24 hwith VX-809 under the identical experimental conditions. Wefound that 100 nM TMA induced an increase of F508delCFTR-dependent chloride efflux similar to that produced by 5�M VX-809 [0.0179 0.003, �(F/F0)/min, n � 6; NS],



suggesting a potential advantage of TMA in terms of reducedtherapeutic dosage.

To explore the persistence of functional correction promotedby TMA, F508del CFTR-dependent chloride efflux measure-ments were performed in CFBE�F cell monolayers at differenttimes after wash-out of the compound. CFBE�F cells weretreated with 100 nM TMA for 24 h and then washed with freshmedium and assayed for 10 �M FSK plus 100 �M IBMX-stimulated chloride efflux at different time points up to 24 h.As shown in Fig. 2F, 3 h after wash-out, F508del CFTR-dependent chloride efflux was reduced by about 20% of themaximum and remained relatively constant at this level for theremainder of the 24-h period.

Moreover, to verify the effect of TMA preincubation inanother immortalized airway CF cell line, we analyzed theCFTR-dependent chloride secretion in CuFi-1 cells, exten-sively used by various laboratories to study F508del CFTRactivity, therapeutic interventions for cystic fibrosis, and innate

immunity (14, 58). We found that 24 h preincubation with 100nM TMA significantly rescued F508del CFTR-dependent chlo-ride efflux also in polarized CuFi-1 cell monolayers [0.009 0.001, n � 4 vs. 0.002 0.001, n � 3 �(F/F0)/min after 24 hpreincubation with TMA or vehicle, respectively, P 0.01] to asimilar level of that induced by 5 �M VX-809 (0.010 0.0002,n � 3).

In a parallel series of functional experiments, polarizedCFBE�F cell monolayers incubated for 24 h in the presence of100 nM TMA or its vehicle alone were mounted in Ussingchambers for measurements of chloride efflux by Isc analysis.Amiloride (100 �M) was added to the apical bath solution toinhibit electrogenic Na� absorption. As shown in Fig. 3A, inCFBE�F monolayers treated with TMA, the addition of 10�M FSK plus 500 �M IBMX elicited an increase in the Isc thatwas further increased by the potentiator genistein (50 �M).The subsequent addition of the selective CFTR inhibitorCFTRInh-172 (10 �M) caused a drastic decrease of the Isc,

Fig. 1. Characterization of 4,6,4=-trimethylangelicin (TMA) as F508del cystic fibrosis transmembrane conductance regulator (CFTR) corrector in FRT-F508delcells. Representative traces showing iodide influx in control conditions (treated with vehicle) or after 48 h incubation with the correctors 200 nM TMA (A) or10 �M VX-809 (B) in the absence or presence of 10 �M CFTRInh-172. FRT-F508del cells, coexpressing F508del CFTR and a halide-sensitive YFP, wereincubated with TMA or VX-809 at 37°C. F508del CFTR function was assayed at 48 h by single-cell fluorescence imaging analysis of YFP fluorescencequenching by iodide in the presence of forskolin (FSK, 20 �M) and isobutyl methylxanthine (IBMX, 100 �M) plus genistein (50 �M). C: summary of thesedata from 5 independent experiments where the fluorescence decay rate corresponding to iodide influx stimulated by correctors in the absence of (gray bar) andpresence of (black bar) CFTRInh-172 is represented. D: dose-response data of the fluorescence decay rate corresponding to F508del CFTR facilitated iodide influxto increasing concentrations from 50 to 500 nM of TMA. Each bar represents the mean SE. Statistical comparisons were made using an unpaired Student’st-test (*P 0.05, **P 0.01, and ****P 0.0001).



Fig. 2. Functional analysis of F508del CFTR-dependent chloride efflux in polarized CFBE�F cell monolayers. A and B: typical recordings showing changes inintracellular Cl�-dependent N-(ethoxy-carbonylmethyl)-6-methoxyquinolinium bromide (MQAE) fluorescence (expressed as the F/F0 ratio where F0 is maximalintensity of fluorescence) in cell monolayers, preincubated either with the vehicle (A) or with 100 nM TMA (B). The monolayers were stimulated for 3 min with10 �M FSK plus 100 �M IBMX before substitution of apical chloride by nitrate in the absence (gray dotted trace) or presence (black dotted trace) of the specificCFTR inhibitor CFTRInh-172 (5 �M) added apically (ap) 5 min before nitrate substitution and remained for the entire chloride efflux. C and D: summary of thedata from 11 independent experiments in cells treated with vehicle (C) or with TMA (D) in which the F508del CFTR-dependent chloride efflux rates[�(F/F0)/min] across the apical membrane (open bar) were calculated as the difference in the F/F0 ratio per minute [(F/F0)/min] obtained in the absence (graybar) and presence of (black bar) CFTRInh-172. E: F508del CFTR-dependent chloride transport measured in HBE (dark bar) and CFBE�F (open bars) cellmonolayers. CFBE�F cell monolayers were treated for 24 h with increasing concentrations of TMA. Statistical comparisons were made with respect to the valuesobtained in monolayers incubated for 24 h with the vehicle alone indicated as 0 in the histogram. Each bar represents the mean SE. F: persistence ofTMA-rescued F508del CFTR activity after TMA removal in CFBE�F cell monolayers. After 24 h of 100 nM TMA incubation, CFBE�F cells were fed withfresh medium without TMA, and CFTR activity was followed for a further 24 h, and F508del CFTR activity was expressed as a percentage of rescued F508delCFTR activity. Statistical comparisons were made with respect to the values obtained in monolayers incubated for 24 h with TMA designed as 100%. *P 0.05,**P 0.01, ***P 0.001, and ****P 0.0001.



demonstrating a specific F508del-CFTR-mediated chloride ef-flux. Figure 3B summarizes the CFTRinh-172-inhibited short-circuit current (�Isc) measurements obtained in CFBE�F cellmonolayers preincubated with TMA or its vehicle, demonstrat-ing that 100 nM TMA produces a significant rescue of F508delCFTR-dependent chloride secretion that was equivalent to26.54 2.30% of that recorded in HBE cell monolayers.

Effect of treatment combinations in rescuing F508del CFTR-dependent chloride secretion in CFBE�F polarized monolayers.We then analyzed if the addition of the two correctors TMAand VX-809 together had greater effect compared with thesingle treatments. F508del CFTR-dependent chloride effluxmeasurements were performed in CFBE�F cell monolayers,by spectrofluorometry, after treatment with 100 nM TMA plus5 �M VX-809 for 24 h. As shown in Fig. 4A, the combinedeffect of two correctors was not significantly different from theeffect induced by each single treatment, suggesting that TMAand VX-809 share a similar mechanism of action to performtheir corrector function. On the contrary, TMA correctionactivity has an additive effect with low temperature (27°C)preincubation (Fig. 4B).

Evaluation of acute potentiator activity of TMA after itsprolonged corrector effect. Next, to determine if the long-termTMA corrector effect on F508del CFTR-dependent chlorideefflux can be further increased with the acute addition of aCFTR potentiator, CFBE�F monolayers were preincubated for24 h with 100 nM TMA and then treated for 15 min with eitherthe well-known CFTR potentiator VX-770 (10 �M) (59) orwith TMA (250 nM) that we previously described to be apotentiator of the wt CFTR or F508del CFTR that has beenrescued to the cell membrane by the interacting protein NHERF1(58). Indeed, as shown in Fig. 4C, the acute addition of eitherVX-770 or TMA significantly potentiated the FSK-inducedF508del CFTR-dependent chloride secretion, whereas the ad-dition of only the vehicle for either VX-770 or TMA did notsignificantly alter the effect of TMA preincubation for 24 h[0.01 0.0007, n � 8 vs. 0.008 0.001, n � 3 and 0.008 0.001, n � 3 �(F/F0)/min, for TMA-preincubated control andfor vehicles for VX-770 and TMA, respectively]. Interestingly,we found that, as reported for other potentiators (46), thepotentiator effect of TMA or VX-770 was unmasked onlywhen the activation of F508del CFTR-dependent chlorideefflux in CFBE�F cell monolayers was performed with a lowerconcentration of FSK (1 �M) and in the absence of IBMX.

Indeed, the potentiator effect of either VX-770 or TMA wasnot significantly evident in response to 10 �M forskolin stim-ulation (data not shown).

Effect of TMA treatment on F508del CFTR transcription,expression, and localization in CFBE�F cells. To determine ifTMA affects CFTR transcription, we used quantitative real-time RT-PCR to assess the effect of TMA treatment onF508del CFTR transcript levels in polarized CFBE�F cells,and we observed (Fig. 5A) that the relative expression of CFTRmRNA was not altered by 100 nM TMA treatment for 24 h,suggesting that this compound may act at the posttranscrip-tional level.

The biosynthetic processing of F508del CFTR induced byTMA was, therefore, assayed by immunoblot analysis of theaccumulation of mature, complex-glycosylated F508del CFTRin CFBE�F cells. Figure 5B, left, shows a typical Western blotperformed in both CFBE cells stably overexpressing wt CFTR(CFBE wtCFTR) and in CFBE�F cells that were preincubatedfor 24 h with vehicle alone or either 100 nM TMA or 5 �MVX-809. In CFBE wtCFTR cells, the mature band C of CFTRwas strongly expressed. On the contrary, in the CFBE�F cellstreated with only vehicle, F508del CFTR was almost com-pletely expressed as the immature band B, and preincubationwith either TMA or VX-809 resulted in both the appearance ofthe mature band C and in an increase of immature band Bexpression of F508del CFTR protein. Densitometry analysis(Fig. 5B, right) revealed that 24 h pretreatment with TMA orVX-809 increased the mature band C of the F508del CFTR byabout 70% with respect to their vehicles, which were normal-ized to one. It is noteworthy that also the band B was increasedto the same extent when cells were exposed to TMA orVX-809 for 24 h. The increase in the core-glycosylated band Bcould be the result of inhibition of the ubiquitin-proteasomepathway, which has been demonstrated to regulate CFTR degra-dation (69), or to its stabilization and/or retention in the ER (61).Further studies are required to clarify these hypotheses.

Because F508del CFTR cannot mature conformationally at37°C but can avoid ER quality control and proceed to theplasma membrane at 27°C (15), we next compared the TMAeffect on F508del CFTR rescue with that induced by 24 hlow-temperature treatment (Fig. 5C). In control conditions at37°C, F508del CFTR is mostly revealed as band B (�160kDa), representing the form of the protein residing in the ER.Low-temperature treatment significantly increased the relative

Fig. 3. A: representative short-circuit current record-ings in airway HBE (broken line) and CFBE�F cellmonolayers preincubated for 24 h at 37°C with 100nM TMA (solid line) or with its vehicle (dotted line)showing the response to the sequential application of10 �M FSK plus 500 �M IBMX, 50 �M genistein,and 10 �M CFTRInh-172. Before adding FSK,amiloride was added to block the epithelial Na� chan-nel. B: summary of CFTRInh-172-inhibited short-cir-cuit current (�Isc) for a series of experiments con-ducted as described in A. CFTR-dependent componentof the transepithelial Isc was calculated as the differ-ence of FSK plus genistein-stimulated increase in Isc

before and after CFTRInh-172 treatment. Each barrepresents the mean SE. Statistical comparison wasmade with respect to the values obtained in monolay-ers incubated for 24 h with the vehicle alone (****P 0.0001).



abundance of band C and B to the same extent as that producedby TMA incubation.

The finding that TMA incubation increased F508del CFTRexpression and, in particular, the mature band C, does not

necessarily mean that more F508del CFTR is present in theapical region of the plasma membrane, since band C onlyindicates the passage of F508del CFTR through the Golgi.Therefore, to determine whether TMA treatment causesF508del CFTR to be redistributed to the apical membrane, weperformed immunofluorescence studies by confocal micros-copy analysis in nonpermeabilized polarized CFBE�F mono-layers immunolabeled with a monoclonal primary antibody,CF3, that recognizes a sequence in the first extracellular loopof the human CFTR protein as previously performed by us(18). As shown in Fig. 5D, TMA preincubation rescuedF508del CFTR in the apical membrane of CFBE�F cells asspecifically detected by CF3 antibody. A similar effect wasobtained in the same cell monolayers incubated with 5 �MVX-809 for 24 h. Altogether, these data suggest that TMA isable to induce F508del CFTR trafficking from the ER com-partment to the apical membrane where it functions as acAMP-responsive channel with a significant increase in chlo-ride secretion.

We then investigated if TMA preincubation may affect theF508del CFTR endocytosis rate from the cell surface of po-larized CFBE�F cell monolayers. As it is known, low temper-ature-rescued F508del CFTR (rF508del CFTR) is unstable onthe cell surface and, at 37°C, is rapidly internalized anddegraded (12, 54, 55). We measured the TMA effect onrF508del CFTR endocytosis rates following the two-step bi-otinylation protocol previously described (28). CFBE�F cellmonolayers, cultured for 48 h at 27°C and incubated in the last3 h with 100 nM TMA or its vehicle, were warmed up at 37°Cfor 2.5 min to allow the proteins to be internalized. Theinternalization time of 2.5 min was chosen since in various celllines it has been found that periodate-oxidized CFTR did notrecycle back to the cell surface (62). Figure 5E shows thatTMA (100 nM) significantly slowed down the internalizationof rF508del CFTR. Next, we monitored the effect of TMA onthe cell surface stability of rF508del CFTR by following thesurface pool of rF508del CFTR using the surface biotinylationassay as previously described (27) (Fig. 5F). We observed thatTMA stabilized the glycosylated band C of rF508del CFTR upto 8 h at 37°C with respect to cell CFBE�F monolayers nottreated (data not shown) or treated with the only vehicle of

Fig. 4. A and B: effect of treatment combinations in rescuing F508delCFTR-dependent chloride secretion in CFBE�F polarized monolayers. A:CFBE�F were treated for 24 h with either 100 nM TMA or 5 �M VX-809 orthe combination of the two correctors. B: CFBE�F were incubated for 24 h at27°C in the presence or not of TMA. Statistical comparisons were made usingan unpaired Student’s t-test with respect to the values obtained in monolayersincubated for 24 h with TMA alone at 37°C. Each bar represents the mean SE of the indicated no. of experiments (*P 0.05). C: effect of acute additionof VX-770 or TMA on F508del CFTR-chloride secretion already rescued by24 h incubation with 100 nM TMA. CFBE�F cell monolayers, grown onpermeable filters, were incubated with 100 nM TMA for 24 h. After this time,10 �M VX-770 or 250 nM TMA was acutely added for 15 min before nitratesubstitution and remained for the entire chloride efflux measurement. F508delCFTR-dependent chloride transport was calculated from the difference inalterations of 1 �M FSK-stimulated fluorescence measurements in the absenceand presence of CFTRInh-172 (5 �M). Each bar represents the mean SE ofthe indicated no. of experiments. Statistical comparisons were made using anunpaired Student’s t-test with respect to the values obtained in monolayersincubated with 100 nM TMA for 24 h after 1 �M FSK stimulation (ctrl)(**P 0.01).



Fig. 5. Effect of TMA treatment on F508del CFTR transcription, expression, and localization in CFBE�F cells. A: CFTR mRNA expression was measured byquantitative RT-PCR in CFBE�F cells preincubated with TMA (100 nM) or with vehicle alone for 24 h. Each bar represents the mean SE of 3 experiments.B and C: expression levels of F508del CFTR protein were analyzed in CFBE�F cells by Western blotting using anti-hCFTR antibody against theCOOH-terminus, and anti-�-tubulin was used to normalize protein loading. B: a representative Western blot after 24 h incubation with either 100 nM TMA or5 �M VX-809 is shown (left). The lysate of CFBE wild-type (wt) CFTR is shown in the first lane as a reference to the location of band C and band B of wtCFTR. The histogram (right) summarizes the relative change in the expression of band B or band C in corrector-treated cells with respect to F508del CFTRprotein band B or band C expression in cells treated with vehicle normalized as 1. Mean SE of 5 independent experiments (*P 0.05). C: left, representativeWestern blot of the effect of TMA preincubation or low-temperature treatment on F508del CFTR expression levels in CFBE�F cells. Right, the histogramsummarizes the relative change in the expression of band B or band C in cells incubated at low temperature or with TMA corrector with respect to F508del CFTRprotein band B or band C expression in untreated cells incubated at 37°C set at 1. Mean SE of 6 independent experiments (*P 0.05 and **P 0.01).D: representative confocal immunofluorescence analysis of polarized CFBE�F monolayers treated or not with TMA (100 nM) or VX-809 (5 �M) for 24 h.Unpermeabilized cells were immunolabeled with a primary mouse monoclonal antibody (CF3) raised against the first extracellular loop of CFTR. Nuclei werestained with DAPI. Confocal scans are shown in the vertical cross section (xz) plane (ap, apical membrane; bl, basolateral region. Scale bar: 10 �m). E: effectof TMA treatment on the internalization rate of rF508del CFTR. A representative blot of cell-surface biotinylated F508del CFTR at 0 and 2.5 min ofinternalization (warm-up time) is shown (top). rF508del CFTR remaining on the cell surface is reported in the histogram as the percentage change in band Cdensity after a 2.5-min internalization step compared with 0 min (n � 4) (bottom) (****P 0.0001). F: effect of TMA on rF508del CFTR cell surface stability.Polarized CFBE�F cells, cultured at 27°C for 48 h and treated with TMA in the last 3 h, were then transferred to 37°C up to 8 h in the presence of the corrector(red) or its vehicle (black). Cell surface CFTR levels were measured after cell surface biotinylation, and a representative blot is shown (top). Quantification ofcell surface CFTR is plotted as the percentage change in density of band C CFTR at different time points (bottom; n � 4).



TMA, suggesting that TMA may partially protect the thermallydestabilized F508del CFTR by the peripheral degradation path-ways.

Correction of functional expression of F508del CFTR inprimary cultures of CF human airway epithelial cells. It hasbeen observed that the efficacy of correctors may be dependenton the cell type used (45). The reason for the different efficacyof the correctors may be due to the various mechanismscontrolling CFTR maturation, trafficking, or degradation thatcould vary from one cell type to another. In this respect,increasing evidence indicates that primary airway epithelialcells provide the closest “in vitro” representation of the airwayepithelium by providing a microenvironment or architecturecloser to in vivo situations (35). Because primary cells derivedfrom different patients can behave differently depending on thegenetics and age of individuals from whom the tissue wasderived, multiple donors need to be investigated to take intoaccount donor to donor variability. We therefore performedfluorimetric measurements of TMA action on polarized mono-layers of primary human bronchial epithelial cells (CF-BE)derived from four different F508del CFTR homozygous indi-viduals (3 CF donors supplied by Italian Cystic Fibrosis Foun-dation Primary Culture Core Facility and 1 CF donor fromEpithelix Sarl). To simulate the “in vivo” lung conditions, theCF-BE polarized cells were grown on permeable supports atthe ALI. As shown in Fig. 6A, 24 h incubation with 100 nMTMA resulted in a significant increase in F508del CFTR-dependent chloride secretion in all examined CF-BE monolay-ers. The increase in F508del CFTR-dependent chloride secre-tion was 39.60 1.69% of that measured in non-CF cellmonolayers derived from four non-CF donor lungs (3 non-CFdonors supplied by Italian Cystic Fibrosis Foundation PrimaryCulture Core Facility and 1 non-CF donor from Epithelix Sarl).

In a parallel series of functional experiments in Ussingchambers, CFTR-dependent chloride efflux was measured byIsc analysis in primary CF-BE and non-CF cell monolayersderived from two independent donors in each case. In CF-BEcell monolayers, the F508del CFTR-dependent chloride efflux(estimated from the amplitude of CFTRinh-172-inhibited Isc) inresponse to 10 �M FSK plus 500 �M IBMX and 50 �Mgenistein was significantly rescued by TMA treatment com-pared with vehicle-treated cells [1.75 0.07 �A/cm2 (n � 8)vs. 0.16 0.04 �A/cm2 (n � 3), respectively; P 0.0001].This level of rescued Isc was 35.14 2.95% of that measuredin the non-CF cell monolayers [4.98 0.32 �A/cm2 (n � 12)].

Moreover, to analyze whether the rescue of chloride effluxinduced by TMA treatment was accompanied by translocationof F508del CFTR to the apical membrane, as seen above forimmortalized CFBE�F cells, we performed immunofluores-cence studies followed by confocal analysis in the same pri-mary CF-BE monolayers in which we had measured thechloride effluxes. Cell monolayers were immunolabeled with amonoclonal primary antibody for the first extracellular loop ofCFTR protein (18). As shown in Fig. 6B, preincubation with100 nM TMA for 24 h significantly rescued the localization ofF508del CFTR to the apical membrane.

Specific effect of TMA on F508del CFTR correction. Last, toanalyze if the correction effect of TMA was specific for theF508del mutation of CFTR, TMA corrector activity was testedon FRT cells stably expressing P574H-CFTR (FRT-P574H), amutant of CFTR that, like F508del CFTR, is retained at the ERand can be rescued by incubation for 24 h at reduced temper-ature (42). FRT-P574H cell monolayers were incubated for24 h with 100 nM TMA or at 27°C, and the chloride efflux inresponse to FSK plus IBMX was measured as described inMATERIALS AND METHODS. We found that, indeed, TMA did not

Fig. 6. Effect of TMA treatment on F508del CFTR activ-ity and localization in primary cultures of cystic fibrosis(CF) human airway epithelial polarized cell monolayersgrown in air-liquid interface culture. A: dark bar repre-sents the pooled CFTR-dependent chloride transportmeasured in non-CF cell monolayers derived from 4healthy donors, whereas the open bars and gray barsrepresent F508del bronchial CF-BE cell monolayersderived from 4 CF donors treated for 24 h with eithervehicle or TMA, respectively, and stimulated with 10�M FSK plus 100 �M IBMX. Each bar represents themean SE of the indicated no. of experiments. The setof red-lined bars is the transport data pooled from all ofthe CF-BE cell monolayer experiments (*P 0.05,**P 0.01, and ****P 0.0001). B: confocal immu-nofluorescence microscopy images of F508del bronchialCF-BE cells show that, after TMA treatment, F508delCFTR expression at the apical membrane was greatlyincreased (scale bar: 10 �m).



produce a significant correction in FRT-P574H cells [0.0005 0.0003 �(F/F0)/min, n � 4], with respect to the positivelow-temperature rescue response [0.011 0.001 �(F/F0)/min,n � 5], suggesting that the TMA correction effect is specificfor F508del mutation of CFTR.

We next analyzed the specificity of TMA correction effectby testing the effect of 24 h preincubation with 100 nM TMAon the trafficking to the cell surface of the hERG K� proteinchannel. Under physiological conditions, wt hERG exhibitstwo bands of Western blot analysis: the core glycosylatedimmature form present in ER, represented by a 135-kDa band,and the fully glycosylated mature protein observable as a155-kDa band, representing the protein trafficked in the Golgiapparatus or at the cell surface. The trafficking mutant R752WhERG is retained in the ER and is rescued to the cell surface bytemperature reduction (19). We found that TMA did notimprove the trafficking process of this mutant form of hERG(Fig. 7), suggesting that, indeed, TMA correction effect wasselective for F508del CFTR.

DISCUSSION

Recently, an intensive effort has been made toward theidentification of correctors that may act to rescue the process-ing defect in F508del CFTR (1). Up to now, the best level ofcorrection has been obtained with VX-809, which is selectivefor F508del CFTR and enhances chloride secretion to �15% ofthat of non-CF human bronchial cells (60). The difficulty inachieving higher levels of correction is due to the fact that theF508del mutation induces multiple conformational defects inthe mutant protein. Biophysical and biochemical studies have,in fact, shown that the F508del mutation, besides causing adefect in the conformation stability of NBD1 (33), also influ-ences the interdomain assembly of NBD1 with NBD2 (16) aswell as the interaction between NBD1 with MSD2 (36). There-fore, to reach the efficacious level of F508del CFTR correctionrequired to achieve substantial clinical benefit in CF, it isbecoming evident that it is necessary to make a combinedadministration of two correctors that display synergistic action(23) or a corrector with a potentiator that counteracts distinctconformational defects in trafficking and activation of F508delCFTR (41, 65). The inherent difficulties in this combined

compound strategy for CF have led to studies to identifydual-acting molecules that may both correct F508del CFTRand potentiate the F508del CFTR-dependent chloride permea-bility to avoid the need for multiple combined administration.However, as yet, very few dual-acting compounds have beenidentified (44, 46).

We have previously found that the acute addition of TMA,at the nanomolar concentrations, potentiates the cAMP/PKA-dependent activation of CFTR in airway cells expressing wtCFTR as well as in CF airway cells in which the F508delCFTR has been rescued to the plasma membrane by overex-pressing the interacting protein NHERF1 (58). In the presentstudy, we demonstrated that TMA significantly rescued chlo-ride secretion in FRT cells expressing F508del CFTR as wellas in polarized cell monolayers of secondary airway cells suchas the CuFi-1 or the CFBE�F overexpressing F508del CFTR.Furthermore, in CFBE�F cells preincubated with TMA, wefound that the F508del CFTR-dependent chloride efflux inresponse to a submaximal FSK concentration was furtherincreased by acute addition of the CFTR potentiator, VX-770(59), or by the acute addition of TMA itself (Fig. 4C). Thisfinding is in agreement with a previously reported study dem-onstrating that TMA is a potentiator of CFTR function (58).

The TMA-dependent functional rescue of F508del CFTR inCFBE�F was accompanied by a significant increase of theamount of fully glycosylated mature band C that represents thealiquot of F508del CFTR that acquires a more stable confor-mation that is not recognized as defective by the ER qualitycontrol (16) and may traffic to the Golgi and be delivered to theplasma membrane. Confocal immunolocalization analysis per-formed in polarized CFBE�F cell monolayers confirmed thatF508del CFTR rescued by TMA is significantly expressed onthe apical membrane region. Moreover, when we compared,under identical experimental conditions, the TMA preincuba-tion effects on the functional expression of F508del CFTR withthose observed in the presence of the established correctorVX-809 (60), we found that TMA displays a similar correctoreffectiveness at lower concentrations.

The findings that corrector activity is strongly influenced bythe cell background indicate that the corrector effect needs tobe tested in different cell systems, including primary cultures

Fig. 7. TMA preincubation did not improve the processing of the mutant misfolded form human ether-a-go-go-related gene (R752W hERG). Expression levelsof R752W hERG transiently transfected in the HEK293 were analyzed by Western blotting using anti-HA antibody after 24 h incubation with 100 nM TMAor 5 �M VX-809 or after 24 h incubation at 27°C. A: representative Western blot of a typical experiment. The lysate of HEK293 transfected with wt hERG isshown in the first lane as a reference to the location of mature band and immature band. B: the histogram summarizes the relative change in the expression oftwo bands of R752W hERG treated with TMA, VX-809, or low temperature. Results represent means SE of 3 independent experiments (*P 0.05).



of bronchial epithelial cells (45). We found that the TMAeffect is not influenced by the cell background since its cor-rector activity was also detected in primary human bronchialepithelial cells isolated from lungs of patients homozygous forF508del mutation. In primary CF-BE cell monolayers, derivedfrom different CF donors homozygous for the F508del muta-tion, we reported that 100 nM TMA produced a significantincrease in 1) chloride secretion in response to cAMP/PKAstimulation and 2) F508del CFTR expression on the apicalmembrane of polarized monolayers. Importantly, we found thatthe extent of F508del CFTR-dependent chloride efflux rescuedby TMA preincubation in primary CF-BE cell monolayersreached �35–39% of the CFTR-dependent chloride secretionmeasured in primary non-CF cell monolayers under the samecAMP/PKA-dependent CFTR activation conditions.

The correctors discovered to date may be functionally char-acterized either as pharmacological chaperones that interactdirectly with the misfolded F508del CFTR protein favoring itsfolding or as proteostasis regulators that act by modulating thepathways involved in F508del CFTR biogenesis and/or degra-dation. That the TMA corrector effect was not influenced bythe cell background, together with the results demonstratingboth the persistence of the TMA corrector effect on theF508del CFTR-dependent chloride secretion (Fig. 2F) and theTMA effect in enhancing the cell surface stability of F508delCFTR (Fig. 5, E and F), suggest that TMA, by interacting withand improving F508del CFTR folding, protects it from theperipheral degradation pathways (40). Moreover, we foundthat, when the CFBE�F cells were treated with TMA incombination with VX-809, which displays some characteristicsof a pharmacological chaperone (60), no additional effect wasobserved, whereas, on the contrary, TMA displayed an additiveeffect with low temperature preincubation, which has beenconsidered to be a proteostasis regulator since it causes aprofound alteration in the cell transcriptome (56).

Whereas the dual corrector-potentiator activity of TMAcould be consistent with a direct binding of TMA with F508delCFTR, on the basis of our results, we cannot establish themolecular mechanism by which TMA exerts the corrector andpotentiator activities since these TMA effects could involvedistinct TMA-binding sites on F508del CFTR protein or dif-ferent targets involved in cellular protein homeostasis, andfurther studies are needed to clarify this point.

Importantly, we found that TMA corrector activity is spe-cific for the F508del mutation since TMA incubation fails toactivate the temperature-sensitive mutant P574H-CFTR (42).Moreover, the findings demonstrating that TMA does notimprove the processing of mutant form R752W of hERG, aprotein that uses a similar trafficking pathway as CFTR (19),are consistent with the hypothesis that TMA selectively im-proves the F508del CFTR processing even if further studies arerequired to evaluate other possible off-target specificities.

Our previous findings demonstrated that preincubation ofTMA is able to decrease the expression of the IL-8 gene inairway cells in which the inflammatory response has beenchallenged with P. aeruginosa (58), at the same concentrationrange and time length (24 h) that rescues F508del CFTR.Defective CFTR protein has been found associated with asterile lung inflammation in early cystic fibrosis infants (30).This has been related to the intracellular signals induced bymutated CFTR and/or because of altered electrolyte transport

(8, 47), although the relationship between CFTR protein defectand the pathogenesis of inflammation in early CF lung diseaseis still under scrutiny (48). Interestingly, it has been reportedthat correction of chloride transport itself, mediated by CFTRor TMEM16A, suppresses the secretion of IL-8 and proinflam-matory cytokines in bronchial airway cells in vitro (63). Al-though it is conceivable that TMA can reduce the release of IL-8also because of its correction of the ion transport defect, which canresult in downmodulation of the different steps of the proinflam-matory signaling, including the activity of NF-kB, this issue stillrequires further investigation.

In conclusion, our results suggest that TMA, besides havingan anti-inflammatory action, acts as a F508del CFTR correctorand potentiator at nanomolar concentrations. A deeper under-standing of its mechanism of action will be important in thedevelopment of more active analogs.

ACKNOWLEDGMENTS

We thank Dr. D. Gruenert for providing 16HBE14o� and CFBE41o� cellsstably expressing wild-type CFTR, Dr. J. P. Clancy for providing theCFBE41o� overexpressing F508del CFTR, Dr. J. Zabner for providing CuFi-1cells, Dr. L. J. Galietta for providing FRT cells stably coexpressing humanF508del CFTR and high-sensitivity halide-sensing GFP analog YFP H148Q/I152L and the P574H-CFTR FRT cells, and Dr. E. Ficker for providing themutated constructs of hERG. We thank Dr. Domenico Marzulli of the Instituteof Biomembranes and Bioenergetics C.N.R. Bari (Italy) for helpful assistancein the morphological analysis and Dr. Rosa Angela Cardone for culturingairway primary cells.

GRANTS

This work was supported by the Italian Cystic Fibrosis Foundation (GrantsFFC nos. 1/2011 and 1/2013) with the contribution of “Delegazione FFC diVicenza” and by the Fondazione Cassa di Risparmio di Puglia (grant for theproject “Studio sulle proprieta del TMA come correttore della CFTR-CysticFibrosis Transmembrane conductance Regulator”).

DISCLOSURES

M. Favia and M. T. Mancini have been PostDoc fellows of the ItalianCystic Fibrosis Foundation. No conflicts of interest, financial or otherwise aredeclared by the authors.

AUTHOR CONTRIBUTIONS

M.F., R.G., G.C., and V.C. conception and design of research; M.F.,M.T.M., V.B., L.G., and O.L. analyzed data; M.F., L.G., L.D., S.J.R., R.G.,G.C., and V.C. interpreted results of experiments; M.T.M., V.B., O.L., A.C.A.,and L.D. performed experiments; V.B. and L.G. prepared figures; S.J.R. andG.C. drafted manuscript; S.J.R. and V.C. edited and revised manuscript; R.G.,G.C., and V.C. approved final version of manuscript.

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Trimethylangelicin promotes the functional rescue of mutant F508del CFTR protein in cystic fibrosis...

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