Activation of Delta F508 CFTR in an epithelial monolayer

Zsuzsa Bebök1, Charles J. Venglarik1,2, Zita Pánczél3, Tamás Jilling4, Kevin L. Kirk1,2, and Eric J. Sorscher1,2,5

1 Gregory Fleming James Cystic Fibrosis Research Center, Departments of 2 Physiology and Biophysics and 5 Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294; 3 University Medical School of Pécs, Pécs H-7624, Hungary; and 4 Department of Pediatrics, Northwestern University Medical School, Evanston, Illinois 60201

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The Delta F508 mutation leads to retention of cystic fibrosis transmembrane conductance regulator (CFTR) in the endoplasmic reticulum and rapid degradation by the proteasome and other proteolytic systems. In stably transfected LLC-PK1 (porcine kidney) epithelial cells, Delta F508 CFTR conforms to this paradigm and is not present at the plasma membrane. When LLC-PK1 cells or human nasal polyp cells derived from a Delta F508 homozygous patient are grown on plastic dishes and treated with an epithelial differentiating agent (DMSO, 2% for 4 days) or when LLC-PK1 cells are grown as polarized monolayers on permeable supports, plasma membrane Delta F508 CFTR is significantly increased. Moreover, when confluent LLC-PK1 cells expressing Delta F508 CFTR were treated with DMSO and mounted in an Ussing chamber, a further increase in cAMP-activated short-circuit current (i.e., ~7 µA/cm2; P < 0.00025 compared with untreated controls) was observed. No plasma membrane CFTR was detected after DMSO treatment in nonepithelial cells (mouse L cells) expressing Delta F508 CFTR. The experiments describe a way to augment Delta F508 CFTR maturation in epithelial cells that appears to act through a novel mechanism and allows insertion of functional Delta F508 CFTR in the plasma membranes of transporting cell monolayers. The results raise the possibility that increased epithelial differentiation might increase the delivery of Delta F508 CFTR from the endoplasmic reticulum to the Golgi, where the Delta F508 protein is shielded from degradative pathways such as the proteasome and allowed to mature.

short-circuit current; dimethyl sulfoxide; cystic fibrosis transmembrane conductance regulator; cell differentiation

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

APPROXIMATELY 90% of cystic fibrosis (CF) patients possess at least one cystic fibrosis transmembrane conductance regulator (CFTR) allele that lacks a phenylalanine residue at CFTR position 508 (Delta F508). The findings of Cheng et al. (4) indicated that the Delta F508 mutation causes retention of CFTR in the endoplasmic reticulum (ER) and a failure to insert the protein in the plasma membrane. Butyrate, an epithelial differentiating agent, has been shown to augment Delta F508 maturation in nonpolarized cells grown on plastic (3). Another report suggested the possibility that butyrate increases ion transport in CF nasal epithelia, although augmentation of Delta F508 maturation was not directly shown (36). Improvements in Delta F508 CFTR due to butyrate have not been attributed to differentiating effects per se but to increased CFTR mRNA and protein levels. The Delta F508 folding defect is also temperature sensitive and influenced by high concentrations of glycerol (7, 37). CFTR normally functions as a Cl- channel in the context of polarized epithelial monolayers. However, interventions that activate maturation and Cl- secretion through Delta F508 CFTR in physiologically important systems such as polarized epithelial cell monolayers have been difficult to establish.

An effort in many laboratories has been directed toward development of channel openers for the Delta F508 CFTR. This strategy depends on at least some (e.g., small amounts of) Delta F508 protein in the plasma membrane (19). Because studies of Delta F508 processing have focused on undifferentiated cells, less information is available concerning CFTR maturation in polarized epithelia. Previous observations in other laboratories suggest that measurable amounts of Delta F508 protein might be present in highly differentiated Delta F508 human nasal epithelial cells (9), CF sweat ducts in situ (35), or in the nasal airways of Delta F508 CFTR mice (22). In the present experiments, we adapted LLC-PK1 renal epithelial cells transfected with CFTR cDNAs for measurement of transepithelial Cl- transport. This cell line readily forms polarized cell monolayers (30, 34, 39) and may have advantages over primary CF airway epithelial cells (i.e., since lung or nasal tissue from Delta F508 patients is limited or less frequently available) for use in screening drugs for effects on CFTR. Although the kidney-derived LLC-PK1 cell line does not endogenously express wild-type or Delta F508 CFTR, the processing of wild-type and mutant CFTR in this cell type has been carefully studied and found to closely resemble the behavior of other epithelial cells that do endogenously express CFTR (28). Moreover, kidney-derived epithelia have been a useful model system for characterizing effects due to epithelial differentiation. For example, Madin-Darby canine kidney cells grown on filters have been used to study the distribution of membrane proteins in polarized cells (1) and the mechanisms underlying this distribution (12, 29). LLC-PK1 cells have been used to explore specific markers of differentiation (27), ion conductance and membrane voltage (24), chemical induction of differentiation (26), and the differentiation-dependent expression of the Na+-glucose cotransporter (8). We also studied the effect of DMSO on human nasal epithelial cells derived from Delta F508 homozygous patients and on mouse L cells expressing Delta F508 or wild-type CFTR (46). In our studies, we found that growth of epithelial cells under conditions designed to induce a more differentiated phenotype (treatment with an epithelial differentiating agent, DMSO, or growth of the cells as monolayers on permeable supports) led to improvements in Delta F508 CFTR processing. Butyrate (5 mM for 24 h), glycerol (10% for 48 h), or growth at 27°C (for 48 h) had no detectable effect on Delta F508 maturation in these epithelial cell monolayers.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cell culture. Parental LLC-PK1 cells were cultured in M199 media supplemented with 5% fetal bovine serum (FBS). LLC-PK1 cells with stable expression of the wild-type or Delta F508 CFTR (generous gift of S. Cheng, Genzyme; Ref. 28) were cultured in DMEM medium supplemented with 10% FBS and 500 µg/ml G418 (Sigma). DMSO (Sigma) was diluted in medium. Cells were treated with DMSO (2% vol/vol) for 4 days after confluency. Cd2+ was added to induce CFTR expression from the metallothionein promoter at 5 µM in experiments and controls. Mouse fibroblast cells expressing Delta F508 or wild-type CFTR (46) were cultured in DMEM medium supplemented with 10% FBS.

Human nasal polyp cells were isolated and cultured according to an earlier described protocol (47).

Immunoprecipitation. LLC-PK1 cells grown on plastic were washed three times with PBS (pH 7.2, containing 1 mM MgCl2 and 0.1 mM CaCl2) on ice and then lysed in a buffer containing 150 mM NaCl, 20 mM HEPES, 1 mM EDTA, 1% NP-40, 100 µg/ml aprotinin, 100 µg/ml leupeptin, and 2 mM phenylmethylsulfonyl fluoride. Lysate supernatant (300 µg protein) was immunoprecipitated at 4°C for 2 h using an anti-CFTR COOH-terminal monoclonal antibody (Genzyme) and protein G agarose beads (Boehringer Mannheim). Immunoprecipitated proteins were phosphorylated in vitro as described previously (4, 33) and separated on a 7% polyacrylamide gel. Gels were placed on PhosphoScreen and analyzed with PhosphorImager (Molecular Dynamics). Images were further characterized with IPLab spectrum (Signal Analytics).

RT-PCR. RT-PCR was carried out using Promega PolyATtract series 9600 mRNA isolation and cDNA synthesis system, according to the manufacturer's protocol. Briefly, mRNA was extracted from control and DMSO-treated samples (1 × 105 cells) and 50% of each mRNA sample was used to synthesize cDNA. The remainder of the sample was identically treated, but without RT, as a control to exclude DNA contamination. Samples were homogenized and diluted in 40 µl of prewarmed (70°C) manufacturer's hybridization buffer, and mRNA was purified on oligo(dT) matrix. After samples were washed, 10-µl samples were processed to cDNA by adding 5 µl of reverse transcriptase. To ensure that PCR yield in these experiments was roughly dependent on the amount of mRNA starting material, pBluescript containing CFTR (gift of J. Rommens) was transcribed in vitro with T7 polymerase and mRNA added to cell lysates from parental LLC-PK1 cells at known concentrations. First-strand CFTR cDNA was then synthesized exactly as above and studied in parallel with cDNA from cells expressing wild-type or Delta F508 CFTR. In all studies, cDNA synthesis was stopped after 30 min by incubating the samples at 95°C for 5 min. Control experiments in which RT was omitted from the cDNA synthesis step led to no detectable PCR product under any conditions. Quantitation was by densitometry using IPLab spectrum (Signal Analytics).

CFTR detection by confocal immunofluorescence microscopy using anti-first nucleotide binding domain (NBD1) (rabbit) polyclonal antibody. Coverslips were washed three times in PBS containing 1 mM Ca2+ and 0.5 mM Mg2+, fixed in -20°C methanol for 20 min, and air dried at room temperature. The cells were rehydrated in PBS for 5 min. Nonspecific protein binding sites were blocked by incubating with 1% (wt /vol) porcine gamma -globulin in PBS for 30 min at room temperature. Primary antibody [a polyclonal (rabbit) antibody raised against the CFTR NBD1] (17, 33) was applied and incubated at room temperature for 40 min. After subsequent washing (5 × 3-min washes in PBS), the cells were incubated with tetramethylrhodamine isothiocyanate (TRITC)-labeled anti-rabbit IgG antibody for 40 min and then washed, mounted, and visualized as described above. Controls in which the first antibody was replaced by nonimmune rabbit serum were negative in all cases. Cells were viewed with an Olympus IX70 inverted epifluorescence microscope at 623-nm light excitation using UPlanApo ×100 or UApo/340 ×40 objectives. Digital confocal images were captured using a Photometrics SenSys digital camera and IPLab spectrum software with Power Microtome (Signal Analytics).

Detection of tight junction formation by confocal immunofluorescence microscopy using anti-zonula occludens-1 (rabbit) polyclonal antibody. Coverslips were washed three times in PBS supplemented with 1 mM Ca2+ and 0.5 mM Mg2+ and fixed in 4% formaldehyde in PBS for 30 min at room temperature. Nonspecific protein binding sites were blocked by incubating with 1% (wt /vol) porcine gamma -globulin (Sigma) in PBS for 30 min at room temperature. A rabbit polyclonal antibody, anti-zonula occludens-1 (ZO-1) IgG (Zymed Laboratories) (15), was applied and incubated at room temperature for 60 min. After subsequent washing (5 × 3 min washes in PBS), the cells were incubated with TRITC-labeled anti-rabbit IgG antibody (DAKOPATTS) for 40 min at room temperature, washed in PBS, and mounted using Vectashield (Vector Labs) mounting medium. Controls in which the first antibody was replaced by nonimmune rabbit serum were negative in all cases. DMSO treatment (2%) was for 4 days.

Identification of Delta F508 CFTR single-channel activity in the plasma membranes of LLC-PK1 cells following DMSO treatment. DMSO-treated cells grown on plastic were studied in the cell-attached configuration by the patch-clamp technique. The bath and pipette solutions, electronics, pipette fabrication, and further details regarding the patch-clamp technique have been described previously (43). The pipette solution contained an impermeant cation (i.e., N-methyl-D-glutamine) so that inward current could only be due to Cl- flow into the pipette. CFTR channels were activated after excision by 250 U/ml protein kinase A (PKA) plus 200 µM ATP in the bath.

Transepithelial transport through Delta F508 CFTR. LLC-PK1 cell monolayers were grown to confluency on permeable supports (Millipore, Anotec) for at least 5 days and then treated with 2% DMSO for 3-6 additional days. Filters were mounted in an Ussing chamber, and short-circuit current (Isc) measurement was carried out as described in Ref. 42. Bumetanide was added at 100 µM to both the mucosal and serosal surfaces. NaCl Ringer solution contained (in mM) 145 Na+, 5 K+, 124.8 Cl-, 1.2 Ca2+, 1.2 Mg2+, 25 HCO-3, 4.2 PO3-4, and 10 glucose (pH = 7.4). In 6 mM Cl- Ringer, 118.8 mM of Cl- was replaced by the impermeant anion gluconate.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

DMSO treatment of LLC-PK1 cells expressing Delta F508 CFTR. DMSO, like butyrate, displays potent activity as an epithelial differentiating agent (14, 23, 31, 40). Both drugs cause changes in the display of plasma membrane glycoproteins in epithelial cells, although the two compounds are believed to act by independent mechanisms (14, 23, 31, 40). Dose and time dependencies of DMSO treatment (0, 2, 5, and 10% DMSO for 1, 6, or 12 h and 1, 3, 4, and 5 days) were evaluated systematically in LLC-PK1 cells that express recombinant Delta F508 CFTR. We found that treatment for 4 days with 2% DMSO led to maximal effects on Delta F508 maturation and transepithelial Cl- transport in these cells. This 2% DMSO schedule had no visible effect on cell viability, and, in a quantitative assay of cell growth (CellTiter 96, Promega), the well-known differentiating effects of DMSO (which slow cell proliferation) decreased cell proliferation in the LLC-PK1 cells by only ~15%. We therefore settled on this schedule for future studies. We studied LLC-PK1 cells expressing wild-type or Delta F508 CFTR and the parental (non-CFTR expressing) LLC-PK1 cell line (28).

DMSO treatment leads to a mature, band C form of Delta F508 CFTR. Incomplete processing of CFTR can be identified by the absence of a mature, fully glycosylated (band C) form of the protein (4). Lower-molecular-weight forms of CFTR (e.g., band B) represent the high-mannose, endoglycosidase H-sensitive forms of the protein that are established cotranslationally in the ER. The maturation of CFTR from core to complex glycosylation reflects the transition of the protein from the ER to the Golgi, where attachment of carbohydrates and trimming are accomplished as CFTR proceeds to the trans-Golgi network. Immunoprecipitation of CFTR after a 4-day pretreatment with DMSO is shown in Fig. 1. The result shows the appearance of mature, fully glycosylated CFTR after DMSO treatment in Delta F508 cells. This result supports the notion that Delta F508 maturation is augmented by DMSO. Figure 1 also indicates a small increase in total CFTR protein in LLC-PK1 cells after DMSO treatment. To determine whether this increase was due to effects on steady-state levels of mRNA, we performed a semiquantitative RT-PCR from total cellular mRNA. DMSO did not appear to cause increases in CFTR mRNA levels as judged by this RT-PCR assay (Fig. 2). Unlike previous reports concerning butyrate treatment (3), the experiment suggests that increased Delta F508 CFTR mRNA does not account for the modest increase in total Delta F508 protein after DMSO treatment or the increase in band C. These results also suggest that DMSO promotes Delta F508 maturation through a mechanism that is different from that observed with butyrate.


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Fig. 1.   A: immunoprecipitation and in vitro phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR). Location of 170-kDa band C, the fully processed form of CFTR, is shown. A lower-molecular-weight form (band B) is also shown. Band B represents the endoglycosidase H-sensitive, endoplasmic reticulum-localized form of the protein, whereas band C is the complex glycosylated form taken by CFTR at and beyond entry into the Golgi. Total CFTR obtained from ~106 cells (~300 µg total cell lysate protein) is shown in each lane. +, With; -, without. B: densitometric measurement of CFTR after in vitro phosphorylation. Percentage of fully processed band C CFTR compared with total CFTR is shown. Approximately 10% of total Delta F508 CFTR became fully glycosylated after DMSO treatment. Cells in these experiments were grown in plastic dishes. Quantitation was by phosphor imaging.


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Fig. 2.   CFTR mRNA levels after DMSO treatment. A: poly(A) mRNA isolated from ~5 × 105 cells was used to synthesize first-strand cDNA and amplify a segment of the human CFTR first nucleotide binding domain (NBD1; Ref. 16; size ~500 bp, indicated by arrow). PCR products of the predicted size were obtained in all cases. Lane M displays a molecular size standard. WT, wild type. B: no evidence of increased CFTR mRNA was observed under the same DMSO treatment conditions shown in Fig. 1. Known amounts of in vitro-transcribed CFTR mRNA in the reaction mixture (5, 2, 1, or 0 pg/ml) led to a roughly linear PCR yield under conditions shown. Quantitation was by densitometry using IPLab spectrum (Signal Analytics).

Delta F508 CFTR localization in the plasma membrane. We used digital confocal fluorescent microscopy for CFTR localization that was detected by a polyclonal (rabbit) antibody raised against CFTR NBD1. The antibody used in these experiments has been validated previously by its ability to immunoprecipitate either truncated CFTR or full-length CFTR, immunolocalize CFTR to cells overexpressing the protein, and correctly identify the presence or absence of CFTR in vivo from salivary gland sections of CFTR wild-type or knockout mice of the same strain (5, 16, 17, 33). In LLC-PK1 cells expressing the wild-type CFTR, the protein localized mainly to the perinuclear ER compartment and to the region of the plasma membrane (Fig. 3A). DMSO treatment only slightly increased the membrane staining of wild-type CFTR. In contrast, Delta F508 CFTR was detected in the perinuclear (ER) compartment in LLC-PK1 cells with no significant membrane staining. After DMSO treatment, staining in the region of the cell membrane (similar to that observed in wild-type CFTR-expressing cells) could be detected. Delta F508 CFTR localizes to the perinuclear ER region of Delta F508 homozygous human nasal epithelial cells (Fig. 3B). Treatment of these cells with DMSO (2% for 4 days) resulted in a more diffuse cytoplasmic staining that included staining of cells in the plasma membranes. In contrast, DMSO treatment had no significant effect on Delta F508 CFTR staining in mouse fibroblast cells (mouse L cells, Fig. 3C). These studies suggest that the specificity of effects of DMSO on Delta F508 CFTR processing occurs in epithelial cells but not in fibroblast cells.


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Fig. 3.   A: immunofluorescent detection of wild-type or Delta F508 CFTR in recombinant LLC-PK1 cells. B: CFTR staining in Delta F508 homozygous human nasal epithelial cells in primary culture. C: CFTR immunostaining in mouse L cells. A polyclonal (rabbit) antibody directed against NBD1 was used to detect CFTR. In A-C, confocal images are shown of cells under each condition. DMSO treatment (2%) was for 4 days. Controls in which 1) first antibody was replaced by nonimmune rabbit serum and 2) parental (non-CFTR expressing) LLC-PK1 or L cells were studied were negative in all cases.

Identification of Delta F508 channels in the plasma membranes of LLC-PK1 cells. We also examined Delta F508 CFTR activity in the plasma membranes of LLC-PK1 cells following DMSO treatment (Fig. 4) using patch-clamp techniques. Cells grown on plastic were studied in the cell-attached and excised inside-out configurations. The pipette solution contained an impermeant cation (i.e., N-methyl-D-glucamine) so that inward current could only be due to Cl- flow. The holding potential was approximately -60 mV. A current-voltage relationship and typical tracing are shown in Fig. 4, both consistent with the channel activity previously described for Delta F508 CFTR (6, 21, 32). The conductance of the channel is ~8-10 pS, and the channel is activated after excision by 250 U/ml PKA plus 200 µM ATP in the bath, as expected for a functional CFTR protein. We observed CFTR channels in 2 of 27 seals in Delta F508 LLC-PK1 cells and in 12 of 48 seals in the same cells treated with DMSO. The wild-type CFTR was observed in 8 of 25 seals. The open probability of the Delta F508 channels was studied after stimulation by PKA and ATP. In the representative tracing shown in Fig. 4, open probability before activation was 2% (98% closed); open probability after activation was 97% (3% closed). The wild-type CFTR also exhibited strong activation when cells attached (open probability up to 98% following PKA stimulation).


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Fig. 4.   Single Delta F508 CFTR channels in the plasma membrane of LLC-PK1 cells after DMSO treatment. Cells were studied in the cell-attached and excised inside-out (I/O) configuration. Single channels with properties similar to those described previously for Delta F508 CFTR were observed (see text). Holding potential was -60 mV. PKA, protein kinase A. A: typical tracings. B: current-voltage relationship.

Transepithelial Cl- transport mediated by Delta F508 CFTR following DMSO treatment. To extend our findings concerning Delta F508 CFTR in LLC-PK1 cells grown in plastic dishes, we also grew cells on permeable supports to form epithelial monolayers. LLC-PK1 cells exhibit a more differentiated phenotype under these conditions, as indicated by tight junction formation, epithelial resistance measurements, and the capability to perform vectoral ion transport (11, 30, 38, 39). Under these conditions, Delta F508 LLC-PK1 cells had low, but detectable, Cl- transport that was absent in parental (non-CFTR expressing) cells (P < 0.05, Figs. 5 and 6). Resistance in the cell monolayers was ~100 Omega  · cm2. A 3-day DMSO treatment of Delta F508 CFTR-expressing cells led to forskolin-activated currents in the direction of Cl- secretion that were greatly increased compared with untreated Delta F508 LLC-PK1 cells (P < 0.00025). Transport was absent in parental LLC-PK1 cells with or without DMSO treatment, indicating specificity for Delta F508 CFTR expression. When Cl- was omitted from solutions bathing the monolayers, Isc was not detectable either in wild-type or Delta F508 CFTR-expressing cells under any treatment conditions, indicating that the Isc observed in the presence of Cl- was due to vectoral Cl- transport. Studies of wild-type CFTR-expressing cells are shown for comparison. Modest increases in monolayer resistance (e.g., by 15-20%) were noted in most paired experiments as a result of DMSO treatment, a finding that suggests enhancement of tight junction formation in cells grown on permeable supports in the presence of DMSO. A summary of these studies is shown in Fig. 6.


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Fig. 5.   Short-circuit current (Isc) measurements in LLC-PK1 cells. Representative tracings are shown with and without DMSO treatment. Delta F508 LLC-PK1 cell monolayers bathed in NaCl Ringer solution exhibited a rapid, forskolin-activated Isc in the direction of Cl- secretion specifically following DMSO treatment (Delta F508 + DMSO). Isc disappeared when either Cl- (Delta F508 + DMSO, 6 mM Cl- Ringer bath bilaterally) or Delta F508 CFTR (parental + DMSO) was omitted. For comparison, cells expressing the wild-type (wt) CFTR are shown. Effects of bumetanide, a blocker that inhibits transepithelial Cl- transport by inhibiting the basolateral Na+-K+-2Cl- cotransporter, are also shown. Resistance was ~100 Omega  · cm2 in each experiment. Mock-treated cells (Delta F508, top right) represent vehicle treatment without DMSO. Aperture area in these studies was 0.2 cm2.


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Fig. 6.   Summary of Isc activation through Delta F508 CFTR. Maximal Isc stimulation by forskolin in Delta F508 CFTR and parental LLC-PK1 cells under conditions of symmetrical (NaCl) Ringer solution is shown. Statistics were by Student's t-test (compared with parental LLC-PK1 without DMSO, third bar from left); n = 12 paired experiments for each condition. Error bars are SE.

Isc studies of alternative activators of Delta F508 CFTR maturation in LLC-PK1 cells. We performed the same sorts of experiments shown above on Delta F508 LLC-PK1 cells following incubation with 10% glycerol (for 48 h), 5 mM butyrate (for 24 h; longer incubations led to substantial toxicity), or growth at 27°C (for 48 h). Each of these interventions has been shown to augment Delta F508 processing in certain cell types grown on plastic (3, 7, 37). We saw no evidence with any of these approaches of either Isc activation or band C CFTR. These results are significant because they suggest that DMSO acts to potentiate Delta F508 CFTR maturation through a mechanism that 1) is different from that observed with glycerol or butyrate, 2) is more potent than can be obtained with glycerol or butyrate, at least in the LLC-PK1 model, and 3) allows insertion of the Delta F508 CFTR in the plasma membrane of a transporting cellular monolayer in vitro.

DMSO treatment leads to a more differentiated phenotype in recombinant LLC-PK1 cells. To determine whether DMSO altered the phenotype of LLC-PK1 cells, we probed cells grown on plastic with an antibody to ZO-1, a constituent of epithelial tight junctions (15, 41). Organization and assembly of tight junctions is an additional, useful measurement of polarity and differentiation in epithelial cell monolayers. Well-organized tight junctions occurring in an ordered distribution indicate a higher level of polarity and differentiation, whereas tight junction antigens that are absent, poorly developed, or distributed throughout the cytoplasm or plasma membrane may be indicative of a less differentiated phenotype. As shown in Fig. 7, a 4-day treatment with 2% DMSO led to qualitative increases in tight junction organization in LLC-PK1 cells, as detected by ZO-1 staining in the regions of cell-to-cell contact.


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Fig. 7.   Tight junction formation in LLC-PK1 cells after DMSO treatment. Confocal immunofluorescence microscopy of cells without and with DMSO treatment. Increases in the organization of tight junction antigen zonula occludens-1 (ZO-1) were observed as a result of DMSO treatment.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

In LLC-PK1 cells grown on plastic, Delta F508 CFTR is absent from the plasma membrane (Fig. 3) but can be detected at the cell surface when cells are grown as a monolayer on a permeable support (Fig. 5). The maturation of Delta F508 CFTR can be augmented further by treatment with nontoxic concentrations of a differentiation agent known to increase epithelial cell polarity, DMSO (14, 23, 31, 40). The in vitro model described here for cell monolayers (as opposed to previous studies of nonpolarized cells grown on plastic) might be useful in the development of procedures to augment Delta F508 CFTR maturation or to study openers of Delta F508 CFTR Cl- channels present in the plasma membrane.

We tested the possibility that DMSO, like butyrate, increases Delta F508 CFTR mRNA levels (Fig. 2). We saw no evidence of increased CFTR mRNA in either wild-type or Delta F508 LLC-PK1 cells under the same DMSO treatment conditions that partially corrected Delta F508 protein processing. Moreover, butyrate treatment of Delta F508 cells at 5 mM caused no improvement in Cl- transport in an Ussing chamber protocol. These results suggest that DMSO augments the maturation of Delta F508 CFTR by a mechanism that is different from the known effects of butyrate. DMSO has also recently been suggested to act as a "chemical chaperone" and to correct the folding and biogenesis of the pathogenic prion protein in vitro (W. J. Welch, personal communication). Although it is possible that DMSO has effects on CFTR folding, there is no direct evidence in our experiments that DMSO can reach cytoplasmic levels that alter the kinetics, efficiency, or other aspects of CFTR folding in living cells to augment Delta F508 CFTR processing. Therefore, although an effect on CFTR folding remains an important consideration in our experiments, further studies will be necessary to test a direct role for DMSO as a chemical chaperone for CFTR.

In our experiments, interventions that increase epithelial cell polarity and differentiation also conferred increases in Delta F508 CFTR maturation. Growth of epithelial cells on permeable supports is a well-established method of augmenting epithelial cell differentiation and has been described previously as leading to dome formation and a more differentiated phenotype in many cell types (13, 18, 25). LLC-PK1 cells have previously been reported to switch from an undifferentiated to a more differentiated phenotype when grown on filters (48). This method was therefore selected as one way to increase cell polarity in our experiments. We showed that growth on permeable supports also led to functionally detectable Delta F508 CFTR in the plasma membrane by Isc measurements (Fig. 6). DMSO treatment is another well-described method for augmenting cell differentiation in epithelial cells derived from different tissues (14, 23, 31, 40). Accordingly, we tested the influence of DMSO on Delta F508 CFTR maturational processing. DMSO treatment caused increased resistance in LLC-PK1 monolayers (presumably on the basis of greater organization of tight junctions), and the same treatment led to further increases in Delta F508 CFTR activity at the cell surface (Figs. 5 and 6). Moreover, we observed that organization of tight junctions in LLC-PK1 cells increased under the same conditions that led to Delta F508 maturation (Figs. 1, 3, and 7). Finally, vectoral chloride transport (Fig. 5) should only be detectable in a polarized epithelial monolayer and was observed in our studies of anion transport. Together, these experiments indicate that a more differentiated phenotype also confers an increase in Delta F508 CFTR maturation. The effect of DMSO on Delta F508 CFTR processing was also observed in primary human nasal polyp cells but not in nonepithelial cells (mouse fibroblast, L cells; Fig. 3). Experiments in these other cell types suggest the usefulness of the approach for activating Delta F508 processing in human epithelial cells.

Transmembrane proteins are often distributed throughout the plasma membrane when cells are undifferentiated. With the onset of differentiation and the development of adherens junctions, tight junctions, and cell matrix interactions, structural polarity is established. The process by which proteins target to the apical membrane appears to occur by a default mechanism (10). Our results may indicate that cellular polarity helps establish part of this default pathway. The possibility that Delta F508 CFTR enters the apical targeting pathway specifically after an increase in cellular differentiation is suggested by results in Figs. 1, 3, and 5-7. Other contributors to Delta F508 maturation, including stabilization of the folded structure of Delta F508 protein, a change in Delta F508 CFTR interactions with cellular chaperones, or a decrease in the rate of degradation of Delta F508 CFTR in the ER (so as to allow more time to reach the Golgi), are among the alternative explanations for the findings shown here.

In summary, the ER in epithelial cells is a hostile environment, insofar as the CFTR is concerned. Wild-type and Delta F508 molecules, even when folded into functional Cl- channels, are polyubiquitinated and degraded in short order (half time of ~30 min) by the proteasome and possibly other proteolytic pathways (20, 44). CFTR that escapes the ER and enters the Golgi may reach a "safe haven," where half time increases to >12 h for the post-ER form. Our data suggest, after DMSO treatment in LLC-PK1 cells, an increased amount of the post-ER complex-glycosylated and membrane-localized Delta F508 CFTR is observed. Because neither block of ubiquitination nor proteasome inhibition has been reported to enhance Delta F508 maturation, it is less likely that the effects observed here are caused by blockade of the proteasome or of ubiquitination. Our results raise the possibility that induction of polarity in LLC-PK1 cells might increase a transition of Delta F508 CFTR from the ER to the trans-Golgi network (Figs. 1, 3 and 7). If the Delta F508 CFTR quality control mechanism becomes "leaky" in differentiated cells compared with the undifferentiated state, the findings could have important implications concerning the in vivo processing of Delta F508 CFTR, which occurs in well-organized cells within tissues characterized by tight junctions and polarity. Further experiments are needed to test this possibility, as well as to test the effects of DMSO treatment in Delta F508 CF mice.

    ACKNOWLEDGEMENTS

We thank Dr. Jeong Hong, V. K. Gadi, Jan Tidwell, Kynda Roberts and Bonnie Parrott for help with preparation of this manuscript, Eddie Walthall for technical assistance, and Drs. Vytas Bankaitis and Douglas Cyr for useful discussions.

    FOOTNOTES

This work was supported by the National Institutes of Health and the Cystic Fibrosis Foundation.

Address for reprint requests: E. J. Sorscher, Gregory Fleming James Cystic Fibrosis Research Center, Univ. of Alabama at Birmingham, 1918 Univ. Boulevard (BHSB 798), Birmingham, AL 35294-0005.

Received 25 April 1997; accepted in final form 27 April 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Ahn, J., O. Mundigl, T. R. Muth, G. Rudnick, and M. J. Caplan. Polarized expression of GABA transporters in Madin-Darby canine kidney cells and cultured hippocampal neurons. J. Biol. Chem. 271: 6917-6924, 1996[Abstract/Free Full Text].

2.   Brown, C. R., L. Q. Hong-Brown, A. S. Biwersi, W. J. Verkman, and W. J. Welch. Chemical chaperones correct the mutant phenotype of the Delta F508 cystic fibrosis transmembrane conductance regulator protein. Cell Stress Chaperones 1: 117-125, 1996.[Medline]

3.   Cheng, S. H., S. L. Fang, J. Zabner, J. Marshall, S. Piraino, S. C. Schiavi, D. M. Jefferson, M. J. Welsh, and A. E. Smith. Functional activation of the cystic fibrosis trafficking mutant Delta F508-CFTR by overexpression. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L615-L624, 1995[Abstract/Free Full Text].

4.   Cheng, S. H., R. J. Gregory, J. Marshall, S. Paul, D. W. Souza, G. A. White, C. R. O'Riordan, and A. E. Smith. Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell 63: 827-834, 1990[Medline].

5.   Clancy, J. P., L. C. Walker, M. D. DuVall, J. S. Hong, Z. Bebök, S. A. King, D. M. Bedwell, C. J. Venglarik, M. R. Weatherly, N. A. McCarty, B. Lesnick, and E. J. Sorscher. Analysis of the CFTR truncation mutations R553X and G542X in mammalian cell lines, Xenopus oocytes and human subjects (Abstract). Pediatr. Pulmonol. Suppl. 13: 239, 1996.

6.   Dalemans, W., P. Barbry, G. Champigny, S. Jallat, K. Dott, D. Dreyer, R. G. Crystal, A. Pavirani, J. P. Lecocq, and M. Lazdunski. Altered chloride ion channel kinetics associated with the delta F508 cystic fibrosis mutation. Nature 354: 526-528, 1991[Medline].

7.   Denning, G. M., M. P. Anderson, J. F. Amara, J. Marshall, A. E. Smith, and M. J. Welsh. Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive. Nature 358: 761-764, 1992[Medline].

8.   De Toledo, F. G., K. W. Beers, and T. P. Dousa. Pleiotropic upregulation of Na+-dependent cotransporters by retinoic acid in opossum kidney cells. Am. J. Physiol. 273 (Renal Physiol. 42): F438-F444, 1997[Abstract/Free Full Text].

9.   Dupuit, F., N. Kalin, S. Brezillon, J. Hinnrasky, B. Tummler, and E. Puchelle. CFTR and differentiation markers expression in non-CF and delta F508 homozygous CF nasal epithelium. J. Clin. Invest. 96: 1601-1611, 1995[Medline].

10.   Eaton, S., and K. Simons. Apical, basal, and lateral cues for epithelial polarization. Cell 82: 5-8, 1995[Medline].

11.   Evers, R., G. J. Zaman, L. van Deemter, H. Jansen, J. Calafat, L. C. Oomen, R. P. Oude Elferink, P. Borst, and A. H. Schinkel. Basolateral localization and export activity of the human multidrug resistance-associated protein in polarized pig kidney cells. J. Clin. Invest. 97: 1211-1218, 1996[Abstract/Free Full Text].

12.   Fritz, B. A., and A. W. Lowe. Polarized GP2 secretion in MDCK cells via GPI targeting and apical membrane-restricted proteolysis. Am. J. Physiol. 270 (Gastrointest. Liver Physiol. 33): G176-G183, 1996[Abstract/Free Full Text].

13.   Gorodeski, G. I., M. F. Romero, U. Hopfer, E. Rorke, W. H. Utian, and R. L. Eckert. Human uterine cervical epithelial cells grown on permeable support---a new model for the study of differentiation. Differentiation 56: 107-118, 1994[Medline].

14.   Grunt, T. W., H. Oeller, C. Somay, and C. Dittrich. Different propensity for spontaneous differentiation of cell clones isolated from the human ovarian surface epithelial cell line HOC-7. Differentiation 53: 45-50, 1993[Medline].

15.   Guerrier, A., P. Fonlupt, I. Morand, R. Rabilloud, C. Audebet, V. Krutovskikh, D. Gros, B. Rousset, and Y. Munari-Silem. Gap junctions and cell polarity: connexin32 and connexin43 expressed in polarized thyroid epithelial cells assemble into separate gap junctions, which are located in distinct regions of the lateral plasma membrane domain. J. Cell Sci. 108: 2609-2617, 1995[Abstract/Free Full Text].

16.   Hartman, J., Z. Huang, T. A. Rado, S. Peng, T. Jilling, D. D. Muccio, and E. J. Sorscher. Recombinant synthesis, purification, and nucleotide binding characteristics of the first nucleotide binding domain of the cystic fibrosis gene product. J. Biol. Chem. 267: 6455-6458, 1992[Abstract/Free Full Text].

17.   Hasty, P., W. K. O'Neal, K. Q. Liu, A. P. Morris, Z. Bebok, G. B. Shumyatsky, T. Jilling, E. J. Sorscher, A. Bradley, and A. L. Beaudet. Severe phenotype in mice with termination mutation in exon 2 of cystic fibrosis gene. Somat. Cell Mol. Genet. 21: 177-187, 1995[Medline].

18.   Horster, M., J. Fabritius, M. Buttner, R. Maul, and P. Weckwerth. Colonic-crypt-derived epithelia express induced ion transport differentiation in monolayer cultures on permeable matrix substrata. Pflügers Arch. 426: 110-120, 1994[Medline].

19.   Hwang, T.-C., F. Wang, I. C.-H. Yang, and W. W. Reenstra. Genistein potentiates wild-type and Delta F508-CFTR channel activity. Am. J. Physiol. 273 (Cell Physiol. 42): C988-C998, 1997[Abstract/Free Full Text].

20.   Jensen, T. J., M. A. Loo, S. Pind, D. B. Williams, A. L. Goldberg, and J. R. Riordan. Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 83: 129-135, 1995[Medline].

21.   Jilling, T., and K. L. Kirk. The biogenesis, traffic, and function of the cystic fibrosis transmembrane conductance regulator. Int. Rev. Cytol. 172: 193-241, 1997[Medline].

22.   Kelley, T. J., K. Thomas, L. J. Milgram, and M. L. Drumm. In vivo activation of the cystic fibrosis transmembrane conductance regulator mutant deltaF508 in murine nasal epithelium. Proc. Natl. Acad. Sci. USA 94: 2604-2608, 1997[Abstract/Free Full Text].

23.   Kim, Y. S., D. Tsao, B. Siddiqui, J. S. Whitehead, P. Arnstein, J. Bennett, and J. Hicks. Effect of sodium butyrate and dimethylsulfoxide on biochemical properties of human colon cancer cells. Cancer 45: 1185-1192, 1980[Medline].

24.   Kleta, R., M. Mohrmann, and E. Schlatter. Effects of cell differentiation on ion conductances and membrane voltage in LLC-PK1 cells. Pflügers Arch. 429: 370-377, 1995[Medline].

25.   Kunzelmann, K., S. Kathofer, A. Hipper, D. C. Gruenert, and R. Gregner. Culture-dependent expression of Na+ conductances in airway epithelial cells. Pflügers Arch. 431: 578-586, 1996[Medline].

26.   Lever, J. E. Chemical inducers of differentiation in a long-term renal cell line. Environ. Health Perspect. 80: 173-180, 1989[Medline].

27.   Lever, J. E. Expression of differentiated functions in kidney epithelial cell lines. Miner. Electrolyte Metab. 12: 14-19, 1986[Medline].

28.   Marshall, J., S. Fang, L. S. Ostedgaard, C. R. O'Riordan, D. Ferrara, J. F. Amara, H. Hoppe IV, R. K. Scheule, M. J. Welsh, and A. E. Smith. Stoichiometry of recombinant cystic fibrosis transmembrane conductance regulator in epithelial cells and its functional reconstitution into cells in vitro. J. Biol. Chem. 269: 2987-2995, 1994[Abstract/Free Full Text].

29.   Mays, R. W., K. A. Siemers, B. A. Fritz, A. W. Lowe, G. van Meer, and W. J. Nelson. Hierarchy of mechanisms involved in generating Na/K-ATPase polarity in MDCK epithelial cells. J. Cell Biol. 130: 1105-1115, 1995[Abstract].

30.   Mullin, J. M., A. P. Soler, K. V. Laughlin, J. A. Kampherstein, L. M. Russo, D. T. Saladik, K. George, R. D. Shurina, and T. G. O'Brien. Chronic exposure of LLC-PK1 epithelia to the phorbol ester TPA produces polyp-like foci with leaky tight junctions and altered protein kinase C-alpha expression and localization. Exp. Cell Res. 227: 12-22, 1996[Medline].

31.   Omary, M. B., L. de Grandpre, M. McCaffrey, and M. F. Kagnoff. Biochemical and morphological differentiation of the human colonic epithelial cell line SW620 in the presence of dimethylsulfoxide. J. Cell. Biochem. 48: 316-323, 1992[Medline].

32.   Pasyk, E. A., and J. K. Foskett. Mutant (delta F508) cystic fibrosis transmembrane conductance regulator Cl- channel is functional when retained in endoplasmic reticulum of mammalian cells. J. Biol. Chem. 270: 12347-12350, 1995[Abstract/Free Full Text].

33.   Peng, S., M. Sommerfelt, J. Logan, Z. Huang, T. Jilling, K. Kirk, E. Hunter, and E. Sorscher. One-step affinity isolation of recombinant protein using the baculovirus/insect cell expression system. Protein Expr. Purif. 4: 95-100, 1993[Medline].

34.   Pfaller, W., G. Gstraunthaler, and P. Loidl. Morphology of the differentiation and maturation of LLC-PK1 epithelia. J. Cell. Physiol. 142: 247-254, 1990[Medline].

35.   Reddy, M. M., C. L. Bell, and P. M. Quinton. Cystic fibrosis affects specific cell type in sweat gland secretory coil. Am. J. Physiol. 273 (Cell Physiol. 42): C426-C433, 1997[Abstract/Free Full Text].

36.   Rubenstein, R. C., and P. L. Zeitlin. A pilot clinical trial of oral sodium 4-phenyl butyrate (buphenyl) in Delta F508-homozygous cystic fibrosis patients: partial restoration of nasal epithelial CFTR function. Am. J. Respir. Crit. Care Med. 157: 484-490, 1998[Abstract/Free Full Text].

37.   Sato, S., C. L. Ward, M. E. Krouse, J. J. Wine, and R. R. Kopito. Glycerol reverses the misfolding phenotype of the most common cystic fibrosis mutation. J. Biol. Chem. 271: 635-638, 1996[Abstract/Free Full Text].

38.   Shioda, T., T. Ohta, K. J. Isselbacher, and D. B. Rhoads. Differentiation-dependent expression of the Na+/glucose cotransporter (SGLT1) in LLC-PK1 cells: role of protein kinase C activation and ongoing transcription. Proc. Natl. Acad. Sci. USA 91: 11919-11923, 1994[Abstract/Free Full Text].

39.   Takakura, Y., T. Morita, M. Fujikawa, M. Hayashi, H. Sezaki, M. Hashida, and R. T. Borchardt. Characterization of LLC-PK1 kidney epithelial cells as an in vitro model for studying renal tubular reabsorption of protein drugs. Pharm. Res. 12: 1968-1972, 1995[Medline].

40.   Tsao, D., A. Morita, A. Bella, Jr., P. Luu, and Y. S. Kim. Differential effects of sodium butyrate, dimethyl sulfoxide, and retinoic acid on membrane-associated antigen, enzymes, and glycoproteins of human rectal adenocarcinomea cells. Cancer Res. 12: 1968-1972, 1982.

41.   Tsarfaty, I., S. Rong, J. H. Resau, S. Rulong, P. P. da Silva, and G. F. Vande Woude. The Met proto-oncogene mesenchymal to epithelial cell conversion. Science 263: 98-101, 1994[Medline].

42.   Venglarik, C. J., and D. C. Dawson. Cholinergic regulation of Na absorption by turtle colon: role of basolateral K conductance. Am. J. Physiol. 251 (Cell Physiol. 20): C563-C570, 1986[Abstract/Free Full Text].

43.   Venglarik, C. J., B. D. Schultz, R. A. Frizzell, and R. J. Bridges. ATP alters current fluctuations of cystic fibrosis transmembrane conductance regulator: evidence for a three-state activation mechanism. J. Gen. Physiol. 104: 123-146, 1994[Abstract].

44.   Ward, C. L., S. Omura, and R. R. Kopito. Degradation of CFTR by the ubiquitin-proteasome pathway. Cell 83: 121-127, 1995[Medline].

45.   Welch, W. J., and C. R. Brown. Influence of molecular and chemical chaperones on protein folding. Cell Stress Chaperones 1: 109-115, 1996 3: September 1996, p. 207.][Medline]

46.   Yang, Y., D. C. Devor, J. F. Engelhardt, S. A. Ernst, T. V. Strong, F. S. Collins, J. A. Cohn, R. A. Frizzell, and J. M. Wilson. Molecular basis of defective anion transport in L cells expressing recombinant forms of CFTR. Hum. Mol. Genet. 2: 1253-1261, 1993[Abstract].

47.   Yankaskas, J. R., C. U. Cotton, M. R. Knowles, J. T. Gatzy, and R. C. Boucher. Culture of human nasal epithelial cells on collagen matrix supports. A comparison of bioelectric properties of normal and cystic fibrosis epithelia. Am. Rev. Respir. Dis. 132: 1281-1287, 1985[Medline].

48.   Yoneyama, Y., and J. E. Lever. Apical trehalase expression associated with cell patterning after inducer treatment of LLC-PK1 monolayers. J. Cell. Physiol. 131: 330-341, 1987[Medline].


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