Rescue of F508-CFTR (Cystic Fibrosis Transmembrane ...

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Rescue of F508-CFTR (Cystic Fibrosis Transmembrane. Conductance Regulator) by Curcumin: Involvement of the. Keratin 18 Network. Joanna Lipecka ...
0022-3565/06/3172-500–505$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics JPET 317:500–505, 2006

Vol. 317, No. 2 97667/3100537 Printed in U.S.A.

Rescue of ⌬F508-CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) by Curcumin: Involvement of the Keratin 18 Network Joanna Lipecka, Caroline Norez, Noura Bensalem, Maryvonne Baudouin-Legros, Gabrielle Planelles, Fre´de´ric Becq, Aleksander Edelman, and Noe´lie Davezac

Received October 27, 2005; accepted January 18, 2006

ABSTRACT The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, ⌬F508, causes retention of ⌬F508-CFTR in the endoplasmic reticulum and leads to the absence of CFTR Cl⫺ channels in the plasma membrane. ⌬F508-CFTR retains some Cl⫺ channel activity so increased expression of ⌬F508-CFTR in the plasma membrane can restore Cl⫺ secretion deficiency. Recently, curcumin was shown to rescue ⌬F508-CFTR localization and function. In our previous work, the keratin 18 (K18) network was implicated in ⌬F508-CFTR trafficking. Here, we hypothesized that curcumin could restore a functional ⌬F508-CFTR to the plasma membrane acting via the K18 network. First, we analyzed the effects of curcumin on the localization of ⌬F508-CFTR in different cell lines (HeLa cells stably transfected with wild-type CFTR or ⌬F508-CFTR, CALU-3 cells, or cystic fibrosis pancreatic epi-

Cystic fibrosis (CF) is a lethal disease caused by defective function of the cftr gene product, the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) (Riordan et al., 1989). CF is characterized by abnormal chloride transport in many tissues, including lung, pancreas, gastrointestinal tract, liver, sweat glands, and male reproductive ducts. The most common mutation, ⌬F508, results in the production of an immature protein, which is retained in the endoplasmic

This work was supported by grants from INSERM and the French cystic fibrosis associations Vaincre la Mucoviscidose (VLM), Mucoviscidose: ABCF2, and Europrocf (EC Grant QLRT 2001-1335). C.N. is supported by studentships from VLM. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.097667.

thelial cells CFPAC-1) and found that it was significantly delocalized toward the plasma membrane in ⌬F508-CFTR-expressing cells. We also performed a functional assay for the CFTR chloride channel in CFPAC-1 cells treated or not with curcumin and detected an increase in a cAMP-dependent chloride efflux in treated ⌬F508-CFTR-expressing cells. The K18 network then was analyzed by immunocytochemistry and immunoblot exclusively in curcumin-treated or untreated CFPAC-1 cells because of their endogenic ⌬F508-CFTR expression. After curcumin treatment, we observed a remodeling of the K18 network and a significant increase in K18 Ser52 phosphorylation, a site directly implicated in the reorganization of intermediate filaments. With these results, we propose that K18 as a new therapeutic target and curcumin, and/or its analogs, might be considered as potential therapeutic agents for cystic fibrosis.

reticulum (ER) for subsequent proteolytic degradation (Ward et al., 1995), preventing the correct localization of functional protein (Cheng et al., 1990; Ward and Kopito, 1994; Ward et al., 1995). However, it has been shown that, in certain tissues, in vivo, a small amount of ⌬F508-CFTR may reach the plasma membrane (Kalin et al., 1999) and function as a cAMP-activated Cl⫺ channel but with a decreased chloride channel open probability compared with WT-CFTR (Dalemans et al., 1991; Benharouga et al., 2001). Among the different strategies that increase the delivery of ⌬F508-CFTR to the plasma membrane are low temperature, chemical chaperones, such as glycerol, and molecular chaperones, such as 4-phenylbutyrate (Buphenyl) or benzo[c]quinolizinium components (MPB-07 and MPB-91) (for review see Kerem, 2005). Although these molecules are

ABBREVIATIONS: CF, cystic fibrosis; K18, keratin 18; CFTR, cystic fibrosis transmembrane conductance regulator; CFPAC, cystic fibrosis pancreatic epithelial cell; ER, endoplasmic reticulum; MPB-07, 6-hydroxy-10-chlorobenzo[c]quinolizinium chloride; MPB-91, 5-butyl-10-chloro6-hydroxybenzo[c]quinolizinium chloride; WT, wild type; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; PBS-T, 0.1% Triton X-100 in PBS; ANOVA, analysis of variance; IF, intermediate filament; A23187, calcimycin; Hsp, heat shock protein. 500

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Institut National de la Sante et de la Recherche Medicale U467, Universite´ Rene´ Descartes Paris 5, Faculte´ de Me´decine Paris 5, Paris, France (J.L., N.B., M.B.-L., G.P., A.E., N.D.); Institut de Physiologie et Biologie Cellulaires, Unite´ Mixte de Recherche 6187 Centre National de la Recherche Scientifique, Universite´ de Poitiers, Poitiers Cedex, France (C.N., F.B.); and Universite´ Paris 11, Faculte´ des Sciences d’Orsay, Orsay Cedex, France (N.D.)

Curcumin Rescues ⌬F508-CFTR: Involvement of the Keratin 18

Materials and Methods Cell Culture and Treatment. CFPAC-1 (a pancreatic duct cell line derived from a cystic fibrosis patient bearing a ⌬F508 mutation) and CALU-3 (human airway cells derived from serous cells of submucosal glands) were obtained from the American Type Culture

Collection (ATCC, Manassas, VA). CALU-3 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM Glutamax; Invitrogen, Carlsbad, CA) containing 1% nonessential amino acids and 1 mM sodium pyruvate. CFPAC-1 cells were grown in Iscove’s modified Dulbecco’s medium (IMDM Glutamax; Invitrogen). Stably transfected HeLa cells coding for WT-CFTR and ⌬F508-CFTR (kindly provided by Dr. Pascale Fanen, INSERM U.654, Cre´teil, France) were cultured as described previously (Jungas et al., 2002). All cell lines were cultured at 37°C with 5% CO2/95% humidified air, and all of the media were supplemented with 10% fetal calf serum and 100 IU/ml penicillin and streptomycin. Curcumin (Sigma-Aldrich, St. Louis, MO) was dissolved in DMSO at a concentration of 50 mM and was stored aliquoted in a darkcolored bottle at ⫺20°C as a stock solution. The stock was diluted to the required concentration with growth media immediately before use. The stability of stock solution was verified during the study by high-performance liquid chromatography analysis. Controls consisted of cells grown in culture medium containing equivalent amounts of DMSO. Prior to drug treatment, the cells were seeded at a density of 1 ⫻ 106 per 60 cm2 for 24 h. Curcumin was added to the cell medium at concentrations of 25 and 50 ␮M for 2, 4, and 16 h. Immunocytochemistry. Cells were fixed in ice-cold acetone (⫺20°C) for 10 min and stored at ⫺20°C until use. After fixation, cells on glass slides were rehydrated in PBS, pH 7.4 and then incubated with 0.25% Triton X-100 in PBS for 10 min for permeabilization. Nonspecific binding was prevented by blocking with 10% fetal calf serum in PBS-T (0.1% Triton X-100 in PBS) for 1 h at room temperature. Primary antibodies against CFTR (monoclonal antibody 24-1; R&D Systems, Minneapolis, MN; dilution 1:100) and K18 (DC10; Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:100) were applied for 1 h at 37°C. After three 10-min washes with PBS-T, cells were incubated for 1 h at room temperature with Alexa 594conjugated goat anti-mouse IgG (Invitrogen) secondary antibody diluted 1:1000 in PBS-T. Slides were mounted with Vectashield medium (Vector Laboratories, Burlingame, CA) and examined under a Zeiss LSM 510 confocal laser-scanning microscope (Zeiss, Jena, Germany) using helium-neon laser appropriate for Alexa 594 fluorochrome. Images were collected with Zeiss 63⫻ or 100⫻ objectives. Immunoblot. Total proteins from CFPAC-1 cells were solubilized in radioimmunoprecipitation buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, and 0.1% SDS) containing protease inhibitors (protease inhibitor cocktail; Roche Diagnostics, Basel, Switzerland) and phosphatase inhibitors (phosphatase inhibitors cocktail II; Sigma-Aldrich). Identical amounts (20 ␮g) were separated by SDS-polyacrylamide gel electrophoresis on 10% polyacrylamide gels and transferred onto nitrocellulose filters (TransBlot; Bio-Rad, Hercules, CA). Membranes were probed with anti-K18 monoclonal antibody (DC10; Santa Cruz Biotechnology; dilution 1:500) and anti-K18 (Ser52) rabbit polyclonal antibody (Santa Cruz Biotechnology; dilution 1:300) followed by incubation with goat anti-mouse IgG or goat anti-rabbit IgG HRP-conjugated secondary antibodies (ABCYS, Paris, France). Proteins were detected using ECL Plus Western Blotting detection system (GE Healthcare, Little Chalfont, Buckinghamshire, UK) according to the manufacturer’s instructions. Quantification of the signal was performed using an ImageMaster VSD (GE Healthcare). Iodide Efflux. CFTR Cl⫺ channel activity was determined on a population of cultured CFPAC-1 cells by measuring the rate of iodide (125I) efflux as described previously (Marivingt-Mounir et al., 2004). All experiments were performed with a MultiPROBEIIex robotic liquid handling system (Perkin Elmer Life Sciences, Courtaboeuf, France). To compare sets of data from separate experiments, the relative rates r ⫽ kpeak ⫺ kbasal (minute⫺1) were calculated. In all of the experiments, the peak rate (kpeak) corresponds to the maximal value of the rate of efflux. Other details are given by MarivingtMounir et al (2004). The CFTR chloride channel activator, benzo[c]quinolizinium compound MPB-91 [5-butyl-10-chloro-6-hydroxybenzo[c]quinolizinium

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effective on ⌬F508-CFTR function, their precise sites of action are not well known. The search for new drugs and/or mechanisms to direct ⌬F508-CFTR protein to the plasma membrane is still an important strategy in establishing a new pharmacotherapy for CF patients with the ⌬F508 mutation. Recently, several studies reported the use of curcumin to correct defective processing of ⌬F508-CFTR in cell and mouse models (Berger et al., 2004; Dragomir et al., 2004; Egan et al., 2004; Loo et al., 2004; Song et al., 2004). Curcumin (diferuloylmethane) is a major phenolic compound of the plant Curcuma longa L., commonly known as turmeric. Apart from its daily use as a condiment and spice, turmeric has been widely used in traditional Oriental medicine to treat a number of ailments, e.g., gastrointestinal and respiratory problems, such as chronic diarrhea, cough, and asthma (Chainani-Wu, 2003; Gilani et al., 2005). In the last few decades, there has been considerable interest in the biological activities of turmeric and its active components, curcuminoids. Many studies report significant anti-inflammatory, antioxidant, anticancer, and immunomodulatory properties of curcumin in vitro and in animal models and humans (Chainani-Wu, 2003). The origin of curcumin use as a remedy in CF comes from the observation that the sarcoplasmic/endoplasmic reticulum calcium pump inhibitor thapsigargin restores functional surface expression of ⌬F508-CFTR (Egan et al., 2002). Curcumin is also an inhibitor of the sarcoplasmic/endoplasmic reticulum calcium pump (Bilmen et al., 2001) and thus was tested in ⌬F508-CFTR-transfected baby hamster kidney cells and in homozygous ⌬F508-CFTR mice, and it was shown to correct the trafficking defect in baby hamster kidney cells and to improve the survival rate in transgenic mice (Egan et al., 2004). However, the precise site of curcumin action is not presently known. In our previous work, we have shown that K18 is implicated in the trafficking of ⌬F508-CFTR. We analyzed the expression of ⌬F508-CFTR in the plasma membrane after silencing K18 expression and found that changes in K18 were accompanied by delivery of functional CFTR to the plasma membrane (Davezac et al., 2004). No specific inhibitors or remodeling agents are known for intermediate filaments and, in particular, for the K8/K18 network. We hypothesized that, while acting through the K18 network, curcumin could restore functional ⌬F508-CFTR in the plasma membrane and postulated that K18 might be a target for new cystic fibrosis therapy. In the present study, we analyzed the effect of curcumin treatment in a time- and dose-dependent manner on the K18 network, on the one hand, and on ⌬F508-CFTR trafficking and function, on the other, using two cell lines expressing ⌬F508-CFTR. The results show important changes in the K18 network in cells treated with curcumin. These changes are correlated with the functional expression of ⌬F508-CFTR in the plasma membrane.

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chloride], was prepared as described previously (Marivingt-Mounir et al., 2004). All other chemicals were purchased from SigmaAldrich. All compounds were dissolved in dimethyl sulfoxide DMSO (final DMSO concentration ⬍0.1%). Statistics. Results are expressed as means ⫾ S.E.M. of n observations. Sets of data were compared with analysis of variance (ANOVA) or a Student’s t test. Differences were considered statistically significant when P ⬍ 0.05. For curcumin-induced phosphorylation of keratin 18, the statistical analysis was performed using Mann-Whitney nonparametric test, and a P value ⬍0.05 was considered statistically significant. All statistical tests were performed using Prism version 4.0 for Windows (GraphPad Software, Inc., San Diego, CA) and StatView 512⫹.

Results The effect of curcumin was tested on cultured CFPAC-1 and on stably transfected HeLa cells, both expressing ⌬F508CFTR. WT-CFTR-expressing cells were lung adenocarcinoma cells (CALU-3), and HeLa cells stably transfected with WT-CFTR. The use of the CFPAC-1 line expressing endogenous ⌬F508-CFTR was of particular interest, because these

Fig. 2. Keratin 18 network organization in curcumin-treated CFPAC-1 cells. a and b, nontreated cells; c and d, cells treated with 25 ␮M curcumin; e and f, cells treated with 50 ␮M curcumin. b, d, and f, high magnification views. Bar: 20 (a, c, and e) and 10 ␮m (b, d, and f). Images are representative of 16-h treatment with curcumin. The same effect was observed after 2- and 4-h treatment.

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Fig. 1. CFTR distribution in different cell lines after curcumin treatment. A, CFPAC-1 cells. a and b, not treated; c and d, curcumin-treated. B, ⌬F508-HeLa cells. a and b, nontreated; c and d, curcumin-treated. Bar: 20 (a and c) and 10 ␮m (b and d). C, WT-HeLa cells. a, nontreated; b, curcumin-treated. Bar: 20 ␮m. D, CALU-3 cells. a, nontreated; b, curcumin-treated. Bar: 20 ␮m. Arrowheads in Ac, Ad, Bc, and Bd indicate examples of curcumin-treated cells, where CFTR localization was cytoplasmic and/or membranous, compared with Aa, Ab, Ba, and Bb, where only perinuclear staining could be observed. Shown images are representative of 4-h treatment with 50 ␮M curcumin. The same results were obtained at all incubation times (2 and 16 h) and at both curcumin concentrations (25 and 50 ␮M).

cells are from the pancreas of a CF patient, an organ directly implicated in cystic fibrosis. The choice of curcumin concentrations and incubation times was based on previous observations obtained by other groups, who used the concentrations between 1 and 100 ␮M curcumin during 3 to 24 h. We also wanted to investigate intermediate/high and short/longterm effects of curcumin on ⌬F508-CFTR functional expression. The preliminary series of cell-survival experiments revealed a high cytotoxicity of curcumin after 16 h of treatment of the sole HeLa cells. The results of cytotoxicity test are as follows: 292%ⴱ ⫾ 15 and 309%ⴱ ⫾ 22 for WT-CFTR cells treated with 25 and 50 ␮M, respectively; and 143% ⫾ 28 and 194%ⴱ ⫾ 12 for ⌬F508-CFTR cells treated with 25 and 50 ␮M, respectively (ⴱ, significant at 95% with ANOVA analysis; n ⫽ 4). In view of these results, the experiments on HeLa cells were performed using 2- and 4-h treatment only. Curcumin was used at the concentrations of 25 and 50 ␮M, and the incubation times were as follows: CFPAC-1 cells, 2, 4, and 16 h; ⌬F508-CFTR and WT-CFTR HeLa cells, 2 and 4 h; and CALU-3 cells, 16 h. CFTR Distribution after Curcumin Treatment. To investigate whether curcumin affects ⌬F508-CFTR localization, CFTR immunolabeling was first performed. In nontreated CFPAC-1 and ⌬F508-CFTR HeLa cells, CFTR staining was concentrated in the perinuclear region (Fig. 1, A, a and b, and B, a and b). After curcumin treatment, we observed a considerable shift of ⌬F508-CFTR toward the plasma membrane in both cell lines (Fig. 1, A, c and d, and B, c and d). In most cells, the CFTR signal was spread all over the cytoplasm, and in some cells, the staining was detected in or close to the plasma membrane [Fig. 1, A, c and d (magnified view), and B, c and d (magnified view)]. The microscopical observation revealed that this effect appeared at all incubation times (2, 4, and 16 h) and at 25 and 50 ␮M curcumin. Curcumin had no effect on the localization of WTCFTR as revealed by immunocytochemistry of CALU-3 cells (Fig. 1D, a and b), and WT-CFTR stably transfected HeLa cells (Fig. 1C, a and b) treated with 25 and 50 ␮M for 16 and 4 h, respectively. Effect of Curcumin on the K18 Network. Previously, we have shown that the keratin 18 network is involved in ⌬F508-CFTR trafficking (Davezac et al., 2004). Thus, we investigated whether ⌬F508-CFTR delocalization by curcumin was correlated with changes in K18 network organization in CFPAC-1 cells. In nontreated CFPAC-1 cells, we observed dense and reg-

Curcumin Rescues ⌬F508-CFTR: Involvement of the Keratin 18

Fig. 3. Effect of curcumin on K18 Ser52 phosphorylation in CFPAC-1 cells. A representative immunoblot of keratin 18 Ser52 phosphorylation in the presence of the indicated concentrations of curcumin and for different incubation times is shown. Each experiment was repeated at least three times. The percentages of Ser52 phosphorylation increase were as follows: 25 ␮M for 2 h, 127 to 174% (Z ⫽ ⫺2.341, P ⫽ 0.0107); 25 ␮M for 4 h, 160 to 172% (Z ⫽ ⫺2.087, P ⫽ 0.0179); 50 ␮M for 2 h, 115 to 215% (Z ⫽ ⫺2.46, P ⫽ 0.0062); 50 ␮M for 4 h, 148 to 306% (Z ⫽ ⫺2.46, P ⫽ 0.0062); 25 ␮M for 16 h, 101 to 152% (Z ⫽ ⫺2.341, P ⫽ 0.0107); and 50 ␮M for 16 h, 145 to 291% (Z ⫽ ⫺2.46, P ⫽ 0.0062) of control (DMSOtreated cells). Statistical analysis was assessed using Mann-Whitney U test, and differences were considered statistically significant when P ⬍ 0.05.

Fig. 4. Iodide efflux in CFPAC-1 cells. A, endogenous chloride transport stimulated by hypotonic medium and by the calcium ionophore A23187 compared with resting cells (noted basal). Note that no efflux was measured with forskolin ⫹ genistein (noted Fsk and Gst, respectively). B, CFPAC-1 cells were incubated for 2 h at 37°C with MPB-91 and curcumin at the concentrations indicated. The efflux was then stimulated by Fsk ⫹ Gst. Vehicle was DMSO. C, summary of the relative rates in the different experimental conditions as indicated. Results are expressed as means ⫾ S.E.M. of four experiments. Sets of data were compared by ANOVA or Student’s t test. Differences were considered statistically significant when P ⬍ 0.05. Ns, nonsignificant difference, ⴱⴱⴱ, P ⬍ 0.001.

curred in CFPAC-1 stimulated with the CFTR agonists forskolin and genistein (Fig. 4A), confirming the trafficking defect of ⌬F508 in this cell line. CFPAC-1 cells then were incubated at 37°C with the CFTR corrector MPB-91 (Dormer et al., 2001) or with curcumin at two different concentrations and for two different times of incubation. In treated CFPAC-1 cells, ⌬F508-CFTR-dependent efflux was stimulated by forskolin/genistein. Figure 4B shows the results obtained after a 2-h incubation with the correctors, and the corresponding calculated relative rates are presented in Fig. 4C. Results indicated that ⌬F508-CFTR was rescued by MPB-91 (Becq and Mettey, 2004) to the same level at 2 or 4 h of treatment. Curcumin rescued ⌬F508-CFTR to 50% of the level reached with MPB-91. No significant differences were obtained with

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ular cytoplasmic staining of K18 structures. The filaments were rather thick and well organized along the long cell axis as shown in Fig. 2 (a and b). Curcumin treatment (25 and 50 ␮M) induced a considerable alteration in the K18 network organization, as shown by indirect immunofluorescence experiments (Fig. 2, c and d), reminiscent of the filament depolymerization described by Omary et al. (1998). We observed that the filament bundles became shorter and thinner and seemed to be entangled (Fig. 2, d and f, high magnification). These changes were observed after all incubation times. The phenomenon of intermediate filament network reorganization in vivo is associated with its enhanced phosphorylation at serine and threonine residues and can be associated with increased solubility of these proteins (Ku et al., 1996). The major physiologic phosphorylation site involved in the keratin 18 filament reorganization and solubility in vivo is Ser52 (Ku and Omary, 1994; Omary et al., 1998). Thus, we wanted to confirm the observed reorganization of the K18 network by analyzing the effect of curcumin on the Ser52 phosphorylation state in CFPAC-1 cells. The immunoblot experiments were performed using 25 and 50 ␮M curcumin after 2, 4, and 16 h of treatment. Analysis of protein extracts from CFPAC-1 cells revealed that the K18 network alterations described above were indeed associated with changes in K18 phosphorylation. A marked increase in Ser52 phosphorylation was observed in treated cells compared with control cells (without changes in K18 expression, data not shown) (Fig. 3). Functional Test for CFTR Chloride Channel. The redistribution of ⌬F508-CFTR protein observed after curcumin treatment prompted us to explore the possibility that some CFTR molecules could reach the plasma membrane. The functional assay thus was performed on curcumin-treated human pancreatic duct CF cells to test for the presence of cAMP-dependent chloride conductance. Chloride transport in CFPAC-1 was first analyzed in response to various treatments at 37°C aimed at stimulating the calcium- and cAMPdependent pathways. Figure 4 shows iodide efflux responses in CFPAC-1 cells stimulated by a hypotonic medium and by the calcium ionophore A23187 (Fig. 4A). The stimulation of iodide efflux resulted in a rapid increase in the rate of efflux (k) within the first minute after hypotonic challenge or the addition of A23187. The peak efflux occurred at the same time with either condition. In contrast, no stimulation oc-

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cells incubated for 2 or 4 h at 25 or 50 ␮M (Fig. 4C). However, after 16 h of curcumin treatment, no CFTR function was detected in CFPAC-1 cells (data not shown).

Discussion

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In this study, we analyzed the effects of curcumin on ⌬F508-CFTR localization and on the keratin 18 network. We show that 1) ⌬F508-CFTR is functionally present in the plasma membrane in curcumin-treated cells and that 2) ⌬F508-CFTR delocalization is accompanied with reorganization of the K18 network. Our results add to the evidence that curcumin and/or its derivatives (analogs or metabolites) might be potentially used to deliver ⌬F508-CFTR to the plasma membrane. The effect of curcumin on ⌬F508-CFTR processing was shown for the first time by Egan et al. (2004) and raised a lot of interest in the potential of curcumin in the treatment of cystic fibrosis. However, subsequent studies failed to reproduce these results; neither maturation of ⌬F508-CFTR nor cAMP-activated Cl⫺ fluxes was observed in different cell models (Dragomir et al., 2004; Loo et al., 2004; Song et al., 2004). On the other hand, two recent studies reported that curcumin influences CFTR channel function. Curcumin was shown to stimulate currents mediated by temperature-rescued ⌬F508CFTR in excised membrane patches from transiently transfected human embryonic kidney cells and stably transfected CF bronchial epithelial cells (CFBE41o⫺) and to increase Cl⫺ transport in differentiated non-CF airway epithelia but not in CF epithelia (Berger et al., 2004; Wang et al., 2005). The reason for these contradictions remains obscure but may be linked to the different genetic background of the animals or to a cell type-dependent efficiency of curcumin. The present study does not support this latter hypothesis given that ⌬F508-CFTR transfer to the plasma membrane after curcumin treatment was seen in two different cell lines, especially in CFPAC-1 cells derived from a cystic fibrosis patient carrying a ⌬F508 mutation. Differences in the observed curcumin effects might also be due to the pharmacological action of its metabolites, such as tetrahydrocurcumin or glucoronide conjugates that were suggested to serve as the available forms of curcumin in vivo (Lin et al., 2000). It is an important goal in CF therapy to identify compounds that restore ⌬F508-CFTR function in secretory epithelia, by acting either on its chloride channel function and/or on correct processing of misfolded protein. Within this field, it is known that ⌬F508-CFTR interacts with molecular chaperones that are responsible for its biogenesis and folding in the ER. The best studied are the 70-kDa heat shock proteins, including inducible Hsp70 and constitutively expressed Hsc70 (heat-shock cognate). Depending on the amount, Hsp70/Hsc70 can promote either folding or degradation of CFTR protein. Biological actions of curcumin include the ability to induce the expression of Hsp70 at concentrations and incubation times similar to those used in this study (Chen et al., 1996; Dunsmore et al., 2001). It is possible that, by raising the Hsp70 levels, curcumin leads to increased Hsp70/⌬F508-CFTR complex formation and ⌬F508-CFTR maturation, which could explain the presence of ⌬F508CFTR near/in the plasma membrane. The positive effect of Hsp70 up-regulation on ⌬F508-CFTR maturation has been shown by Choo-Kang and Zeitlin (2001) for phenyl butyrate.

Furthermore, curcumin was also shown to inhibit proteasome expression (Wyke et al., 2004). These two effects can be cumulative, thus increasing the amount of stabilized ⌬F508CFTR. Our functional assay data for ⌬F508-CFTR recorded in curcumin-treated cells allow us to speculate that curcumin can indeed rescue ⌬F508-CFTR but can also stimulate its function once it reaches the plasma membrane, as suggested previously (Berger et al., 2004; Wang et al., 2005). An interesting point is the time-response effect of curcumin treatment on ⌬F508-CFTR function. CFPAC-1 cells display a functional CFTR after short treatment, i.e., 2 and 4 h but not after prolonged exposure of 16 h. The lack of response to curcumin treatment observed in this study and by others, where curcumin was added for 18 and 24 h prior to functional assay (Loo et al., 2004; Song et al., 2004), could be related to a possible curcumin cytotoxicity or might also be due to curcumin-mediated induction of the heat shock response. Alternatively, a prolonged curcumin treatment can alter the protein kinase A activity. Berger and colleagues showed that increased concentrations of curcumin significantly decrease protein kinase A phosphorylation of CFTR after 18 h of exposure. On the other hand, it cannot be excluded that curcumin might inhibit channel activity at high concentrations and/or longer incubation times. Such an effect was reported for genistein, which stimulated CFTR current at low concentrations but inhibited it at concentrations above 20 ␮M (Lansdell et al., 2000). Structural similarities between curcumin and genistein suggest that the two compounds may share common mechanisms (Berger et al., 2004). It has been shown, for example, that both curcumin and genistein have important anti-inflammatory effects that can be beneficial for the treatment of cystic fibrosis. Like genistein, curcumin was found to inhibit the nuclear factor ␬B, leading to decreased interleukin-8 production (Tabary et al., 1999; Li et al., 2004). Our study further suggests that reorganization of the K18 network may be an important step in the delocalization of ⌬F508-CFTR toward the plasma membrane. Several studies have pointed out novel functions of intermediate filaments (IFs). The role of microtubules and actin networks in protein sorting and vesicle trafficking is well established, but no data have indicated the role of intermediate filaments in this process, especially because of lack of specific motor proteins. However, IFs are integrated with actin and microtubule cytoskeletons through interactions with the microtubule motors dynein and kinesin and with actin motor myosin Va (Styers et al., 2005). Recent studies implicate IFs in the control of organelle position and in membrane protein composition via sorting mechanisms. The first indications came from the study reporting alterations in protein delivery to the apical membrane after depletion of epithelial cells in keratin 19 (Salas et al., 1997). Consistent with this study are two other reports showing the mistargeting of membrane proteins, i.e., CFTR, and altered ion transport in small intestine and colonic epithelia of keratin 8-deficient mice (Ameen et al., 2000; Toivola et al., 2004). Another example of the interaction between IFs and vesicular trafficking and protein sorting is an association between intermediate filament vimentin and adaptor complex AP3, which regulates sorting of membrane proteins from endosomes to lysosomes (Styers et al., 2004). This study shows that perturbation of the vimentin network induces changes in the

Curcumin Rescues ⌬F508-CFTR: Involvement of the Keratin 18

Acknowledgments

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Address correspondence to: Dr. Noe´lie Davezac, U467 INSERM, 156 Rue de Vaugirard, 75015 Paris, France. E-mail: [email protected]

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subcellular distribution of AP3 complex and late endosome/ lysosome compartments. In addition, two AP3-sorted membrane proteins were mistargeted in cells lacking vimentin filaments. It is of note that the apical turnover of CFTR protein is dependent on the AP2 adaptor complex, which facilitates endocytosis of CFTR. The reorganization of the K18 cytoskeleton observed here may lead to either inhibition of endocytosis and/or to enhanced exocytosis, which in consequence would increase the amount of ⌬F508-CFTR in the membrane. Our present results support the hypothesis formulated in a recent study (Davezac et al., 2004) and specify the possible targets of curcumin action. In addition, the involvement of keratin 18 in ⌬F508-CFTR trafficking to the plasma membrane increased the growing body of data concerning novel functions of intermediate filaments. In conclusion, our study shows that curcumin treatment induced an important change in the K18 network and, in parallel, allowed ⌬F508-CFTR to escape from ER and to anchor in the plasma membrane. Although we do not know the precise targets of curcumin action, we postulate that keratin 18 may be a possible target for patients with the ⌬F508 mutation.

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