Trafficking of GFP-tagged Delta F508-CFTR to the plasma membrane in a polarized epithelial cell line

Dominique Loffing-Cueni, Jan Loffing, Collin Shaw, Amilyn M. Taplin, Malu Govindan, Caitlin R. Stanton, and Bruce A. Stanton

Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The Delta F508 mutation reduces the amount of cystic fibrosis transmembrane conductance regulator (CFTR) expressed in the plasma membrane of epithelial cells. However, a reduced temperature, butyrate compounds, and "chemical chaperones" allow Delta F508-CFTR to traffic to the plasma membrane and increase Cl- permeability in heterologous and nonpolarized cells. Because trafficking is affected by the polarized state of epithelial cells and is cell-type dependent, our goal was to determine whether these maneuvers induce Delta F508-CFTR trafficking to the apical plasma membrane in polarized epithelial cells. To this end, we generated and characterized a line of polarized Madin-Darby canine kidney (MDCK) cells stably expressing Delta F508-CFTR tagged with green fluorescent protein (GFP). A reduced temperature, glycerol, butyrate, or DMSO had no effect on 8-(4-chlorophenylthio)-cAMP (CPT-cAMP)-stimulated transepithelial Cl- secretion across polarized monolayers. However, when the basolateral membrane was permeabilized, butyrate, but not the other experimental maneuvers, increased the CPT-cAMP-stimulated Cl- current across the apical plasma membrane. Thus butyrate increased the amount of functional Delta F508-CFTR in the apical plasma membrane. Butyrate failed to stimulate transepithelial Cl- secretion because of inhibitory effects on Cl- uptake across the basolateral membrane. These observations suggest that studies on heterologous and nonpolarized cells should be interpreted cautiously. The GFP tag on Delta F508-CFTR will allow investigation of Delta F508-CFTR trafficking in living, polarized MDCK epithelial cells in real time.

cystic fibrosis; Madin-Darby canine kidney; butyrate; glycerol; dimethyl sulfoxide; green fluorescent protein; cystic fibrosis transmembrane conductance regulator


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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REFERENCES

THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR), a cAMP-activated Cl- channel, is expressed in a wide variety of epithelial cells, including airway and kidney (13, 46). Mutations in the CFTR gene lead to the genetic disease cystic fibrosis (CF), a lethal autosomal recessive disorder (30, 40). Approximately 70% of individuals with CF are homozygous for the Delta F508-CFTR mutation, which accounts for approximately 90% of all mutant CFTR alleles (30). Trafficking of Delta F508-CFTR from the endoplasmic reticulum (ER) to the apical plasma membrane of epithelial cells is extremely inefficient. Moreover, Delta F508-CFTR has a relatively short residence time in the plasma membrane compared with wild-type CFTR (wt-CFTR) (20, 29). Thus little Delta F508-CFTR is expressed in the plasma membrane (8). Because Delta F508-CFTR retains partial function as a cAMP-activated Cl- channel (11, 28, 39), identification of drugs that increase the amount and activity of Delta F508-CFTR in the apical plasma membrane of airway epithelial cells would have important implications for the treatment of CF.

Several experimental maneuvers increase Delta F508-CFTR expression in the plasma membrane. For example, growing cells in culture treated with reduced temperature (9, 12, 14, 16, 17, 24, 41), butyrate compounds (19, 24, 41), and so-called "chemical chaperones" including DMSO and glycerol (41, 43) facilitate the expression of Delta F508-CFTR in the plasma membrane and allow cAMP to enhance Delta F508-CFTR-mediated Cl- permeability (reviewed in Refs. 3 and 47). However, because many of these studies were conducted in either nonpolarized or heterologous cells, the results should be interpreted cautiously, since it is well known that protein trafficking is cell-type specific and depends on the polarized state of the epithelium (reviewed in Refs. 5 and 6). For example, the human low-density lipoprotein (LDL) receptor, when expressed in transgenic mice, is located in the apical membrane of renal tubules. By contrast, the LDL receptor is expressed in the basolateral membrane of colonocytes and enterocytes. In addition, in nonpolarized HT-29 intestinal cells, wt-CFTR is localized in an intracellular compartment, whereas in polarized HT-29 cells, wt-CFTR is expressed in the apical plasma membrane (32, 33). Moreover, cAMP induces the trafficking of wt-CFTR from an intracellular compartment to the plasma membrane in polarized, but not nonpolarized, HT-29 cells (33). Several laboratories have studied Delta F508-CFTR trafficking in polarized epithelial cells. Many of these studies have failed to confirm observations made in nonpolarized and/or heterologous cells. For example, in polarized LLC-PK1 cells expressing Delta F508-CFTR, glycerol, sodium butyrate, or a reduced temperature (27°C) did not stimulate cAMP-stimulated Cl- secretion (2). In addition, sodium butyrate had no effect on Cl- currents across polarized human nasal airway epithelial cells expressing Delta F508-CFTR (7). Thus studies on polarized epithelial cells have not consistently confirmed results obtained in nonpolarized and/or heterologous cells.

Although LLC-PK1 cells expressing Delta F508-CFTR form polarized monolayers (2, 10, 20), additional polarized cell lines expressing Delta F508-CFTR would be valuable for secondary drug screening studies and to study Delta F508-CFTR trafficking. Moreover, addition of a green fluorescent protein (GFP) tag on Delta F508-CFTR would allow investigation of Delta F508-CFTR trafficking in living, polarized epithelial cells in real time. In preliminary studies, we were unable to identify a robust human airway epithelial cell line that consistently forms polarized monolayers. Thus we have stably expressed Delta F508-CFTR with a GFP tag in Madin-Darby canine kidney (MDCK) cells, a well-established cell model in which to study protein trafficking in polarized epithelial cells (35), and have conducted studies to determine whether a reduced temperature, glycerol, butyrate, or DMSO stimulate Cl- secretion in polarized cells. We attached GFP to the NH2 terminus of Delta F508-CFTR to monitor the subcellular localization of Delta F508-CFTR. GFP, a 27-kDa protein from the jellyfish Aequorea victoria, generates a striking green fluorescence, is resistant to photobleaching, and does not require any exogenous cofactors or substrates to fluoresce (37). We report that a reduced temperature, glycerol, sodium butyrate, or DMSO has no effect on 8-(4-chlorophenylthio)adenosine 3'5'-cyclic monophosphate (CPT-cAMP)- stimulated transepithelial Cl- secretion. However, when the basolateral membrane was permeabilized with nystatin, sodium butyrate increased the CPT-cAMP-stimulated Cl- current across the apical plasma membrane. Sodium butyrate failed to stimulate transepithelial Cl- secretion because of inhibitory effects on Cl- uptake across the basolateral membrane. Reduced temperature, DMSO, and glycerol had no effect on the CPT-cAMP-stimulated Cl- current across the apical plasma membrane. These observations demonstrate that data obtained in heterologous, nonpolarized cell models should be interpreted cautiously. The GFP tag on Delta F508-CFTR will allow investigation of Delta F508-CFTR trafficking in living, polarized MDCK epithelial cells in real time.


    METHODS
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METHODS
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Expression vector. The pGFP-Delta F508-CFTR mammalian expression vector, driven by the cytomegalovirus promoter, was constructed by methods described previously (34, 35). Proceeding from the NH2 to the COOH terminus, the resultant chimeric protein consists of enhanced GFP, a flexible linker sequence of 23 amino acids, and Delta F508-CFTR. The cDNA coding for human Delta F508-CFTR was a generous gift of Dr. William Guggino (Johns Hopkins University School of Medicine, Baltimore, MD).

Cell culture and stable cell lines. MDCK type I cells (C7 clone, a generous gift of Dr. Hans Oberleithner) stably expressing GFP-Delta F508-CFTR were established by methods described previously (35). The C7 clonal line expresses very low levels of cAMP-stimulated Cl- currents attributable to wt-CFTR (18, 31, 35). Three independent stable cell lines expressing GFP-Delta F508-CFTR were studied to exclude the possibility that results were attributable to clonal variation. MDCK I cells stably expressing GFP-wt-CFTR and parental MDCK I cells (C7) have been characterized and described in detail (34, 35). Previously, we demonstrated that addition of GFP to the NH2 terminus of wt-CFTR had no effect on CFTR localization, trafficking, or biophysical properties (35). Moreover, others have shown that GFP has no effect on the synthesis and degradation of wt-CFTR (25). In preliminary pulse-chase studies, we observed that GFP does not affect the rate of biosynthesis or degradation of Delta F508-CFTR. For simplicity, we refer to the chimeric GFP proteins as Delta F508-CFTR and wt-CFTR.

Immunocytochemistry. Cells grown on glass slides or Transwell filters were fixed in paraformaldehyde and prepared for laser scanning confocal microscopy as described previously (35). The ER was identified by indirect immunofluoresence with the use of an anti-BiP polyclonal antibody (StressGen Biotechnology, Victoria, BC, Canada) to detect the ER-resident protein BiP, followed by an anti-rabbit Texas Red-labeled secondary antibody (Molecular Probes, Eugene, OR), as described previously (35).

SDS-PAGE and Western blotting. Delta F508-CFTR and wt-CFTR were detected on membranes blocked with 5% nonfat dry milk in Tris-buffered saline (TBS)-0.02% Tween 20 using a monoclonal CFTR antibody (M3A7; Chemicon International, Temecula, CA) (26, 27), followed by anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (Amersham, Arlington Heights, IL) as described previously (35). Blots were developed by enhanced chemiluminescence (ECL) using Hyperfilm ECL (Amersham).

Cell surface biotinylation. Selective apical membrane biotinylation and immunoprecipitation of GFP-CFTR and Delta F508-CFTR were performed as described in detail previously (34, 35). All steps were performed at 4°C. Proteins were separated by SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes as described previously (35). Biotinylated GFP-CFTR fusion proteins were immunoprecipitated with a GFP antibody (1:1,000; Clontech, Palo Alto, CA) and were detected on membranes blocked with 5% nonfat dry milk in TBS-0.02% Tween 20 using a monoclonal CFTR antibody (M3A7) followed by anti-mouse HRP-conjugated secondary antibody (1:5,000; Amersham).

Short-circuit current. Short-circuit current (Isc) was measured across MDCK cell monolayers stably expressing Delta F508-CFTR or wt-CFTR or across MDCK cells not expressing the CFTR transgene, as described previously (35). In all experiments, amiloride (10-5 M) was present in the apical bath solution to inhibit electrogenic Na+ absorption. Under these conditions, cAMP-stimulated Isc is referable to CFTR-mediated Cl- secretion from the basolateral to the apical solution (35, 36). To measure the Cl- currents across the apical membrane, we permeabilized the basolateral membrane with nystatin (200 µg/ml). When the basolateral membrane was permeabilized, Isc measured in the presence of a transepithelial Cl- ion gradient directed from the basolateral to the apical solution (140 vs. 14 mM: Cl- was replaced on an equimolar basis with gluconate; Ca2+ was increased from 0.5 to 2 mM in the gluconate-containing solution to maintain Ca2+ activity similar in both solutions) represented the Cl- current across the apical membrane, as described previously (35). Cl- currents are reported as the difference between the peak value of Isc following CPT-cAMP (100 µM) minus the steady-state Isc after inhibition of the CPT-cAMP-stimulated CFTR Cl- current by diphenylamine-2-carboxylic acid (DPC; 3 mM).

Statistical analyses. Differences between means were compared by using the unpaired, two-tailed Student's t-test or by ANOVA followed by a post hoc test (Dunnett's or Tukey's test) as appropriate using Instat v2.01 statistical software (GraphPad, San Diego, CA). Data are expressed as means ± SE. P < 0.05 is considered significant.


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Characterization of Delta F508-CFTR stable cell lines. We characterized three independent clonal lines stably expressing Delta F508-CFTR. First, we conducted Western blots on cell lysates to confirm CFTR protein expression. As demonstrated previously (35), in cells expressing wt-CFTR, but not in parental nontransfected cells (i.e., C7), we detected two protein bands on Western blots using a monoclonal CFTR antibody (Fig. 1A). Core glycosylated wt-CFTR had an apparent molecular mass of 210 kDa (the so-called "band B"), and maturely glycosylated wt-CFTR had an apparent molecular mass of 240 kDa (the so-called "band C"). These results confirm our previous study demonstrating that GFP-wt-CFTR runs ~30 kDa larger than anticipated by SDS-PAGE analysis (35). By contrast, in cells expressing Delta F508-CFTR, we only detected core glycosylated band B Delta F508-CFTR in cells treated with sodium butyrate (Fig. 1B). We could not detect Delta F508-CFTR expression in cells not treated with butyrate (Fig. 1B). Moreover, Delta F508-CFTR expression was low, even in butyrate-treated cells, relative to the robust expression of wt-CFTR. Lower expression of Delta F508-CFTR relative to that of wt-CFTR is due in part to the shorter half-life of Delta F508-CFTR (<4 h) relative to wt-CFTR (>24 h) (20, 29). As expected, Delta F508-CFTR was expressed in an intracellular compartment, primarily in the ER (Fig. 2). Delta F508-CFTR could not be detected in the apical plasma membrane of cells treated with sodium butyrate by selective cell-surface biotinylation. By contrast, the maturely glycosylated band C of wt-CFTR was detected in the apical plasma membrane as described previously (35, 36).


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Fig. 1.   A: Western blot analysis of whole cell lysates from Madin-Darby canine kidney (MDCK) parental cells (C7) and MDCK cells expressing wild-type cystic fibrosis transmembrane conductance regulator (wt-CFTR) grown on Transwell filters. Cells were not treated with sodium butyrate. CFTR was not detected in parental C7 cells not expressing the CFTR transgene. Bands B and C of wt-CFTR were detected in MDCK cells stably expressing the wt-CFTR transgene using monoclonal antibody M3A7. When the membranes were stripped and reprobed with an anti-green fluorescent protein (GFP) monoclonal antibody, as described previously (35), similar results were obtained (data not shown). B: band B of Delta F508-CFTR, but not band C, is evident only in cells treated with sodium butyrate (Delta F508 + NaB; 5 mM) overnight, not in untreated cells (Delta F508). The predicted molecular masses of GFP (27 kDa plus a 3-kDa flexible linker sequence) fused to core glycosylated (150 kDa) and mature glycosylated (180 kDa) forms of CFTR are predicted to run at 180 and 210 kDa, respectively (35). The molecular mass of band B of CFTR was ~210 kDa, and the mature glycosylated band C of CFTR was ~240 kDa. Thus, as shown previously (35), GFP-CFTR fusion proteins run ~30 kDa larger than anticipated by SDS-PAGE. Glycoproteins, including CFTR, frequently migrate slower than predicted because SDS does not bind sugar moieties, and, as a result, migration toward the positive electrode is hindered (31, 35). In A and B, 50 µg/protein were loaded per lane.



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Fig. 2.   Laser scanning confocal microscopy of MDCK cells stably expressing Delta F508-CFTR linked to GFP. A: overview of several cells expressing GFP-Delta F508-CFTR (green) in a diffuse, intracellular endoplasmic reticulum (ER)-like location. B: higher magnification of a cell expressing GFP-Delta F508-CFTR. C: same image as B except the ER is shown as detected by indirect immunofluoresence using an anti-BiP monoclonal antibody and an anti-mouse-Texas red secondary antibody (red). D: superimposition of B and C. Yellow areas in C indicate that GFP-Delta F508-CFTR is expressed in the ER. Cells were treated with sodium butyrate (5 mM) overnight. Images are from cells grown on glass slides. A similar ER localization of Delta F508-CFTR was observed in polarized MDCK cells grown on Transwell filters.

We also examined the electrophysiological properties of parental MDCK I cells as well as cells stably expressing wt-CFTR and Delta F508-CFTR. Compared with the parental nontransfected cells, stable expression of wt-CFTR increased basal Isc (i.e., unstimulated Isc) and CPT-cAMP stimulated Isc (Table 1). These data confirm previous observations that wt-CFTR functions as a cAMP-stimulated Cl- channel and mediates Cl- secretion across polarized MDCK I cells (35). Stable transfection of Delta F508-CFTR had no effect on basal or CPT-cAMP-stimulated Isc compared with parental nontransfected cells (Table 1). These functional data are consistent with the view that Delta F508-CFTR does not traffic to the apical plasma membrane. Together, Western blot analysis, laser scanning microscopy, and electrophysiological studies demonstrate that in polarized MDCK cells, Delta F508-CFTR is expressed primarily in the ER.

                              
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Table 1.   Electrophysiological properties of MDCK cells

Low temperature, butyrate, glycerol, and DMSO do not increase CPT-cAMP-stimulated transepithelial Cl- secretion. We examined the effect of reduced temperature, sodium butyrate, glycerol, and DMSO on the ability of CPT-cAMP to stimulate Cl- secretion in polarized MDCK cells stably expressing Delta F508-CFTR. The effect of sodium butyrate on the CPT-cAMP-stimulated Isc is summarized in Fig. 3. Cells were exposed to sodium butyrate (1 or 5 mM) for 1, 3, or 5 days. Similar experimental maneuvers stimulate Cl- permeability in several other cell types expressing Delta F508-CFTR (1, 7, 19, 41). Stock solutions of sodium butyrate were made immediately before addition to the cell culture medium, and the butyrate containing cell culture medium was changed daily. Although sodium butyrate dramatically increased Delta F508-CFTR protein expression (see Fig. 1B), butyrate had no effect on CPT-cAMP-stimulated Isc (Fig. 3).


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Fig. 3.   Transepithelial Cl- secretion, reported as the 8-(4-chlorophenylthio)-cAMP (CPT-cAMP; 100 µM) stimulated and diphenylamine-2-carboxylic acid (DPC; 3 mM)-inhibited short-circuit currents (Isc) across MDCK I cells stably expressing Delta F508-CFTR. Control, n = 29 monolayers; NaB (sodium butyrate), n = 10 monolayers on day 1, 13 monolayers on day 3, and 13 monolayers on day 5; 27°C, n = 14 monolayers on day 1, 14 monolayers on day 2, and 10 monolayers on day 3; 27°C + NaB, n = 8 monolayers on day 1 and 7 monolayers on day 2; glycerol, n = 3 monolayers with 7.5, 5.0, and 2.5%; and DMSO, n = 15 monolayers. Details on experimental treatments are described in RESULTS. Isc are reported as the difference between the peak value of Isc following CPT-cAMP minus the steady-state Isc after DPC (3 mM) inhibition of CPT-cAMP-stimulated Isc.

The effect of reducing the temperature to 27°C for 1, 2, or 3 days on the CPT-cAMP-stimulated Isc also is summarized in Fig. 3. Similar experimental maneuvers stimulate membrane Cl- permeability in numerous cell types expressing Delta F508-CFTR (12, 16, 17, 19, 41). However, reducing the temperature to 27°C had no effect on the CPT-cAMP-stimulated Isc across polarized MDCK cells expressing Delta F508-CFTR (Fig. 3). In a recent study, plasma membrane expression of Delta F508-CFTR was markedly enhanced by combination treatment with sodium butyrate and low temperature (19). Sodium butyrate (5 mM) and 25°C for 48-60 h had a maximum stimulatory effect. However, as demonstrated in Fig. 3, a combination of sodium butyrate (5 mM) and 27°C for 1 or 2 days had no effect on the CPT-cAMP-stimulated Isc across polarized MDCK cells expressing Delta F508-CFTR.

We also examined the effect of glycerol in the cell culture medium on the ability of CPT-cAMP to stimulate Isc. Glycerol (10, 7.5, 5, or 2.5%) was added to the cell culture medium for 2 days, a protocol that stimulates Cl- permeability in a variety of cells expressing Delta F508-CFTR (4, 43). High concentrations of glycerol (10%) disrupted the integrity of MDCK monolayers such that we could not measure Isc or transepithelial resistance (Rt) (n = 9 monolayers). Lower concentrations of glycerol (7.5, 5, or 2.5%) had no effect on the CPT-cAMP-stimulated Isc (Fig. 3). DMSO (2% for 3 days) also enhances cAMP-stimulated Isc across polarized LLC-PK1 cells expressing Delta F508-CFTR (2). However, DMSO treatment for 3 days had no effect on the CPT-cAMP-stimulated Isc (Fig. 3). Together, these data suggest that a reduced temperature, butyrate, or "chemical chaperones" including glycerol and DMSO do not facilitate the trafficking of functional Delta F508-CFTR channels to the apical membrane.

Delta F508-CFTR channel activity and Cl- electrochemical driving force. The inability to detect an increase in CPT-cAMP-stimulated Cl- secretion in cells treated with sodium butyrate in the present study may be due to a low open probability of Delta F508-CFTR channels in the plasma membrane (21, 22). To test this possibility, we added genistein, a flavinoid that increases the open probability of Delta F508-CFTR channels (1, 14), to the apical bathing solution after addition of CPT-cAMP. Genistein (50 µM) had no effect on Isc across control or sodium butyrate-treated monolayers (5 mM for 2 days). The change in Isc with genistein was 0.1 ± 1.1 µA/cm2 in control monolayers and 2.1 ± 0.8 µA/cm2 in monolayers treated with sodium butyrate [n = 6 monolayers/group, P = NS (not significant)]. Thus the lack of effect of sodium butyrate on CPT-cAMP-stimulated Cl- secretion did not appear to be due to a reduced activity of Delta F508-CFTR channels in the apical plasma membrane.

Additional studies were conducted to determine whether the inability of butyrate compounds to stimulate CPT-cAMP-stimulated Isc was due to a lack of a sufficient electrochemical gradient to drive Cl- across the apical plasma membrane. To increase the driving force for Cl- movement across the apical membrane, we treated monolayers with 1-EBIO (300 µM), a drug that activates K+ channels in the basolateral membrane and hyperpolarizes the basolateral and apical membrane potentials (14, 15). This, in turn, enhances the electrochemical driving force promoting Cl- secretion across the apical plasma membrane (14, 15). 1-EBIO also stimulates CFTR (14, 15). 1-EBIO (300 µM) added to the basolateral bath solution after CPT-cAMP had no significant effect on Isc, which changed by 0.4 ± 0.2 µA/cm2 in untreated cells and 2.8 ± 2.3 µA/cm2 in sodium butyrate-treated cells (5 mM for 2 days: n = 6 monolayers/group, P = NS). Thus it is unlikely that the lack of effect of sodium butyrate on Cl- secretion was due to a lack of a sufficient electrochemical gradient to drive Cl- secretion across the apical membrane. However, these data should be interpreted cautiously because we did not test whether 1-EBIO activates K+ channels in the basolateral membrane and increases the electrochemical gradient for Cl- across the apical plasma membrane. Thus additional studies were conducted to determine whether sodium butyrate and the other experimental maneuvers described above increased the amount of functional Delta F508-CFTR in the apical plasma membrane.

Apical membrane Delta F508-CFTR currents. To examine the effect of sodium butyrate, reduced temperature, glycerol, and DMSO on Cl- currents across the apical membrane of cells expressing Delta F508-CFTR, we permeabilized the basolateral membrane with nystatin and measured CPT-cAMP-stimulated Isc across the apical membrane as described in METHODS. The data are summarized in Fig. 4, and representative experiments are illustrated in Fig. 5. Only sodium butyrate significantly increased the Cl- currents across the apical membrane. In untreated monolayers expressing Delta F508-CFTR, the CPT-cAMP-stimulated Isc was 11.8 ± 2.7 µA/cm2. Sodium butyrate increased the CPT-cAMP-stimulated Isc in monolayers expressing Delta F508-CFTR to 33.6 ± 5.4 µA/cm2 (P < 0.05 compared with control). However, a reduced temperature, DMSO, or glycerol did not increase the Cl- current across the apical membrane (Fig. 4). Moreover, a combination of sodium butyrate and reduced temperature did not increase the Cl- current compared with sodium butyrate treatment alone (Fig. 4). In addition, a combination of sodium butyrate and glycerol or sodium butyrate and DMSO failed to increased the Cl- current compared with sodium butyrate treatment alone (Fig. 4). These data, in combination with studies described below, demonstrate that sodium butyrate increased the amount of functional Delta F508-CFTR in the apical plasma membrane of polarized MDCK cells.


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Fig. 4.   Cl- currents across the apical plasma membrane of cells stably expressing Delta F508-CFTR. Control (no treatment), n = 14 monolayers; NaB, n = 16 monolayers; 27°C, n = 11 monolayers; glycerol, n = 9 monolayers; DMSO, n = 7 monolayers; NaB + 27°C, n = 7 monolayers; NaB + glycerol, n = 9 monolayers; and NaB + DMSO, n = 7 monolayers. Details on experimental treatments are presented in RESULTS. Isc are reported as the difference between the peak value of Isc following CPT-cAMP minus the steady-state Isc after DPC (3 mM) inhibition of CPT-cAMP-stimulated Isc. A transepithelial Cl- ion gradient was directed from the basolateral to the apical solution (140 vs. 14 mM). *P < 0.05 vs. control.



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Fig. 5.   Representative experiments illustrating the effect of sodium butyrate on the Cl- current across the apical membrane in cells expressing Delta F508-CFTR. Records are from monolayers at passage 12 treated with vehicle (distilled water) or sodium butyrate (5 mM for 24 h). Nystatin (200 µg/ml) was added to the basolateral bath solution of both monolayers as indicated by arrow, and ~20 min later CPT-cAMP (100 µM) was added to the apical and basolateral solutions of control (solid line) and sodium butyrate-treated monolayers (shaded line). In these representative experiments, CPT-cAMP increased Isc by 39.1 µA/cm2 in the monolayer treated with sodium butyrate and by 10.2 µA/cm2 in the control monolayer. Genistein (50 µM), which activates Delta F508-CFTR, added to the apical bath solution increased Isc by 13.4 µA/cm2 in the sodium butyrate-treated monolayer and had no effect in the control monolayer. Finally, DPC (3 mM) added to the apical and basolateral solutions completely inhibited the CPT-cAMP-stimulated Isc in both monolayers. These results are consistent with the view that sodium butyrate increases the amount of functional Delta F508-CFTR in the apical plasma membrane. By comparison, in a previous study we reported that in MDCK cells stably expressing wt-CFTR, CPT-cAMP increased Isc by 9.2 µA/cm2 in untreated monolayers and by 274.6 µA/cm2 in monolayers treated with sodium butyrate (36). Moreover, as stated in RESULTS, sodium butyrate had no effect on the CPT-cAMP-stimulated Cl- current across the apical membrane in parental C7 cells. Thus the effect of butyrate to increase Cl- current across the apical plasma membrane can be attributed to GFP-Delta F508-CFTR.

Sodium butyrate had no effect on endogenous wt-CFTR Cl- currents in parental MDCK I cells. Because the parental MDCK I (C7) cell line expresses low levels of endogenous wt-CFTR (31), it is possible that sodium butyrate stimulated Cl- currents across the apical membrane of cells stably expressing Delta F508-CFTR by increasing endogenous wt-CFTR expression. To examine this possibility, we treated MDCK I parental cells with sodium butyrate (5 mM for 2 days). CPT-cAMP-stimulated transepithelial Cl- secretion was 2.5 ± 0.5 µA/cm2 in control untreated monolayers and 0.5 ± 0.3 µA/cm2 in sodium butyrate-treated monolayers (n = 6, P < 0.05). Next, the Cl- current across the apical plasma membrane was measured in the presence of nystatin to permeabilize the basolateral membrane. Sodium butyrate had no effect on the CPT-cAMP-stimulated Cl- current across the apical membrane, which was 9.0 ± 1.5 µA/cm2 in untreated monolayers and 11.6 ± 0.9 µA/cm2 in sodium butyrate-treated monolayers (5 mM for 2 days: n = 7 monolayers/group, P = NS). These data demonstrate that sodium butyrate does not increase the functional expression of endogenous wt-CFTR in the apical membrane of parental MDCK cells. Thus the effects of sodium butyrate in MDCK cells expressing Delta F508-CFTR, described above, are due to increased functional expression of Delta F508-CFTR in the apical plasma membrane and not to the enhanced expression of endogenous wt-CFTR.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A new polarized epithelial cell line in which to study Delta F508-CFTR trafficking. We have established and characterized a new polarized cell line stably expressing GFP-tagged Delta F508-CFTR. MDCK I cells form polarized monolayers, are robust and easy to maintain in culture, express low levels of endogenous CFTR, have Rt values ~400 Omega  · cm2, and are a well-established model in which to study trafficking of ion channels. In addition, Ussing chamber studies can be performed to monitor transepithelial Cl- secretion as well as Cl- secretion across the apical plasma membrane. As expected, Delta F508-CFTR was expressed primarily in the ER. However, sodium butyrate increased the functional expression of Delta F508-CFTR in the apical plasma membrane. The GFP tag on Delta F508-CFTR will allow, in future studies, investigation of Delta F508-CFTR trafficking in living, polarized MDCK epithelial cells in real time.

In extensive preliminary studies, we examined the ability of several human airway epithelial cell lines expressing endogenous Delta F508-CFTR to polarize and establish a Rt in culture, including, for example, CFT1, CFBE14o- and Sigma CFTE-29o- (23, 24, 38). Although others have reported that these cells lines develop a Rt, we found these cell lines, relative to MDCK cells, difficult to maintain in culture and were unable to obtain Rt values above 100 Omega  · cm2 on a consistent basis (i.e., <10% of monolayers tested had Rt values >100 Omega  · cm2). Thus, although polarized monolayers of human airway epithelial cells would be the most preferable model in which to study Delta F508-CFTR trafficking, we could not identify a robust human cell line that polarized in a reproducible manner.

Butyrate increases functional Delta F508-CFTR expression in the apical plasma membrane. Sodium butyrate increased the amount of functional Delta F508-CFTR in the apical plasma membrane as determined by measuring the CPT-cAMP-stimulated, DPC-sensitive Cl- current across the membrane. Thus, as in many cell types, sodium butyrate increases the plasma membrane expression of Delta F508-CFTR. However, we did not detect Delta F508-CFTR in the apical membrane by cell surface biotinylation, and we did not detect maturely glycosylated (band C) Delta F508-CFTR by Western blot analysis. Similar results were observed in LLC-PK1 cells stably expressing Delta F508-CFTR (20). Thus it is not surprising that Delta F508-CFTR in the apical membrane was below the detection limit of Western blot analysis and selective cell surface biotinylation. Plasma membrane expression of Delta F508-CFTR in vivo is also very low compared with the expression of wt-CFTR.

The results in this study support a growing body of evidence to support the view that Delta F508-CFTR trafficking is cell-type dependent. In addition, we speculate that Delta F508-CFTR trafficking, like wt-CFTR trafficking (33), may depend on the state of cell polarization. Of the experimental maneuvers tested, only sodium butyrate increased the functional expression of Delta F508-CFTR in the apical plasma membrane of polarized MDCK cells. A reduced temperature, glycerol, or DMSO did not increase the amount of Delta F508-CFTR in the apical plasma membrane, even when Delta F508-CFTR expression was increased by pretreatment with sodium butyrate. By contrast, in several heterologous cells and/or nonpolarized epithelial cells, these experimental maneuvers increased the amount of Delta F508-CFTR in the plasma membrane (see Introduction). It is unlikely that the lack of effect of these experimental maneuvers on polarized MDCK cells is due to differences in protocol between this and previous studies, since our protocols, including concentrations and times of exposure, were similar to those reported previously. Moreover, other investigators have noted a similar lack of effect of these experimental maneuvers on Cl- transport in polarized epithelial cells. For example, glycerol, sodium butyrate, or a reduced temperature had no effect on cAMP-stimulated Cl- secretion in polarized LLC-PK1 cells expressing Delta F508-CFTR (2). Finally, sodium butyrate had no effect on Cl- currents across polarized human nasal airway epithelial cells expressing Delta F508-CFTR (7). However, several experimental maneuvers including DMSO, 4-phenylbutyrate, and reduced temperature have been shown to increase Delta F508-CFTR in the plasma membrane of polarized epithelial cells (2, 14, 42). Thus, together, the data support the view that the effect of a reduced temperature, glycerol, or DMSO on Delta F508-CFTR trafficking may be cell-type dependent (i.e., kidney, airway, fibroblast) and may also depend on the state of polarization.1 Additional studies are required to test these hypotheses directly.

Untoward effects of sodium butyrate on transepithelial Cl- secretion. Why did sodium butyrate have no effect on CPT-cAMP-stimulated transepithelial Cl- secretion but increase CPT-cAMP-stimulated Cl- secretion across the apical plasma membrane in MDCK cells expressing Delta F508-CFTR? Previously, we reported that sodium butyrate increased the expression of wt-CFTR in the apical membrane of MDCK I cells 25-fold and stimulated Cl- currents across the apical membrane 30-fold (36). However, transepithelial CPT-cAMP-stimulated Cl- secretion was reduced because butyrate reduced the activity of the Na+-K+-ATPase. In epithelial cells, the Na+-K+-ATPase maintains a low intracellular concentration of Na+, which is important for providing the driving force for Cl- entry into the cell across the basolateral membrane via the Na+-K+-2Cl- cotransporter. Cl- then exits the cell across the apical membrane through CFTR Cl- channels. By reducing the activity of the Na+-K+-ATPase, we speculated that sodium butyrate increases intracellular Na+ concentration and thereby reduces Cl- entry into the cell across the basolateral membrane via the Na+-K+-2Cl- cotransporter. The decrease in Cl- entry would reduce intracellular Cl- concentration and thereby inhibit Cl- secretion across the apical membrane. The present study also supports the view that despite an increase in the amount of Delta F508-CFTR in the apical membrane, sodium butyrate does not increase transepithelial CPT-cAMP-stimulated Cl- secretion, most likely because sodium butyrate inhibits the Na+-K+-ATPase and indirectly reduces Cl- uptake across the basolateral membrane. It is possible, but not proven, that the inability of sodium butyrate to stimulate Cl- secretion across polarized epithelial MDCK cells expressing Delta F508-CFTR could be due to a similar effect of butyrate on the Na+-K+-ATPase. Additional experiments, beyond the scope of this report, are required to elucidate the cellular mechanism(s) whereby butyrate inhibits Na+-K+-ATPase activity in MDCK cells expressing Delta F508-CFTR.

In conclusion, our data, as well as other data in the literature, suggest that CF drug studies conducted on heterologous and/or nonpolarized cells should be interpreted cautiously. This is not to say that nonpolarized and/or heterologous cell models are not valuable tools for drug discovery. Many nonpolarized and/or heterologous models are ideal for high-throughput screening and rapid identification of candidate drugs to either move Delta F508-CFTR to the plasma membrane or increase Delta F508-CFTR channel activity. However, our results, together with other data in the literature indicating that CFTR trafficking is cell-type dependent and may depend on the state of polarization, suggest that once a drug is identified, secondary assays should be performed, ideally in polarized human airway epithelial cells lines expressing Delta F508-CFTR. At the present time, however, we are not aware of any robust models currently available. Until such cells lines are available, MDCK (present study) and LLC-PK1 cells (2, 10, 20), and perhaps FRT cells (44, 45), expressing Delta F508-CFTR may prove useful in secondary drug screens.


    ACKNOWLEDGEMENTS

We gratefully acknowledge Katherine Karlson and Bonita Coutermarsh for assistance with cell culture and Western blots, Dr. Alice Givan and Ken Orndorff for assistance with confocal microscopy, and Dr. Sandra Guggino for the suggestion to conduct the nystatin experiments.


    FOOTNOTES

These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-45881 and Cystic Fibrosis Foundation Grant STANTO97RO. J. Loffing was supported by a fellowship from the Swiss National Science Foundation. Confocal microscopy was performed at Dartmouth Medical School, in the Herbert C. Englert Cell Analysis Laboratory, which was established by a grant from the Fannie E. Rippel Foundation and is supported in part by the Core Grant of the Norris Cotton Cancer Center (CA 23108). The Dartmouth Cystic Fibrosis Cell Biology/Cell Culture Core (STANTO97RO) provided scientific and technical support.

Present address of D. Loffing-Cueni and J. Loffing: Institute of Anatomy, University of Zurich, 8006 Zurich, Switzerland.

Address for reprint requests and other correspondence: B. A. Stanton, Dept. of Physiology, 615 Remsen Bldg., Dartmouth Medical School, Hanover, NH 03755 (E-mail: bruce.a.stanton{at}dartmouth.edu).

1 We cannot formally exclude the possibility that the GFP tag on triangle F508-CFTR prevented glycerol, DMSO, or low temperature from increasing triangle F508-CFTR expression in the plasma membrane of MDCK cells. However, as noted above, our data are consistent with reports by others that these maneuvers fail to increase plasma membrane expression of untagged triangle F508-CFTR in polarized epithelial cells.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 15 March 2001; accepted in final form 1 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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