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Am J Physiol Gastrointest Liver Physiol 291: G1148-G1154, 2006. First published July 6, 2006; doi:10.1152/ajpgi.00547.2005
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NEUROREGULATION AND MOTILITY

Absence of CFTR is associated with pleiotropic effects on mucins in mouse gallbladder epithelial cells

Rahul Kuver, Thomas Wong, Johanne Henriette Klinkspoor, and Sum P. Lee

Division of Gastroenterology, Department of Medicine, University of Washington, and the Puget Sound Veterans Affairs Health Care System, Seattle Division, Seattle, Washington

Submitted 1 December 2005 ; accepted in final form 29 June 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mucus of cystic fibrosis patients exhibits altered biochemical composition and biophysical behavior, but the causal relationships between altered cystic fibrosis transmembrane conductance regulator (CFTR) function and the abnormal mucus seen in various organ systems remain unclear. We used cultured gallbladder epithelial cells (GBEC) from wild-type and Cftr(–/–) mice to investigate mucin gene and protein expression, kinetics of postexocytotic mucous granule content expansion, and biochemical and ionic compositions of secreted mucins. Muc1, Muc3, Muc4, Muc5ac, and Muc5b mRNA levels were significantly lower in Cftr(–/–) GBEC compared with wild-type cells, whereas Muc2 mRNA levels were higher in Cftr(–/–) cells. Quantitative immunoblotting demonstrated a trend toward lower MUC1, MUC2, MUC3, MUC5AC, and MUC5B mucin levels in Cftr(–/–) cells compared with cells from wild-type mice. In contrast, the levels of secreted MUC1, MUC3, MUC5B, and MUC6 mucins were significantly higher from Cftr(–/–) cells; a trend toward higher levels of secreted MUC2 and MUC5AC was also noted from Cftr(–/–) cells. Cftr(–/–) cells demonstrated slower postexocytotic mucous granule content expansion. Calcium concentration was significantly elevated in the mucous gel secreted by Cftr(–/–) cells compared with wild-type cells. Secreted mucins from Cftr(–/–) cells contained higher sulfate concentrations. Thus absence of CFTR is associated with pleiotropic effects on mucins in murine GBEC.

biliary tract; cystic fibrosis transmembrane conductance regulator; exocytosis

THICK, INSPISSATED MUCUS IN the airways, intestinal lumen, pancreatic ducts, and biliary ducts is a phenotypic hallmark of cystic fibrosis (CF). This mucoviscidosis points to a web of causal and incidental relationships between the defective protein in CF patients, the cystic fibrosis transmembrane conductance regulator (CFTR), and the various cellular processes that lead to the formation of a mucous gel on epithelial surfaces. CFTR functions have been implicated in mucous granule exocytosis (1, 14, 22) and in modifications of the ionic compositions of mucous secretions via alterations in chloride secretion, plasma membrane recycling, intracellular acidification, and sodium reabsorption (3, 6, 39). Indirect effects, such as alterations in mucin gene expression due to chronic infection (27) and alterations in airway surface liquid pH, have also been identified (10). Furthermore, changes in glycosylation profiles, particularly with respect to higher levels of sulfation, have been observed in airway and intestinal mucins in CF (9, 42, 45). Despite these studies, no consensus has emerged regarding a unifying mechanism whereby viscous mucus accumulates in diverse organs in this disease (30, 33).

Our previous work has focused on CFTR effects on mucins in the extrahepatic biliary system. While another group had reported that loss of CFTR expression in mouse gallbladder epithelial cells (GBEC) did not result in changes in the total amount of secreted mucus (32), several observations led us to ask whether specific steps along the pathway from mucin gene expression to the formation of the extracellular mucous gel were altered. First, intrahepatic bile duct epithelial cells from CF patients secrete proteoglycans with an altered glycosylation profile (5). Second, overexpression of CFTR is associated with increased rates of mucin secretion in cultured canine GBEC (20). Third, CFTR localizes to intracellular mucous granules in cultured canine GBEC (22). Finally, calcium binding to biliary mucins is dependent on the sulfate content of mucins and is decreased with increasing sodium content (23), suggesting that highly sulfated mucins would be more viscous under conditions in which sodium content was low, as predicted in an epithelium with abnormal CFTR function. These findings suggested that the effects of CFTR on the mucous gel were likely to be multifaceted. We therefore undertook a more comprehensive analysis of CFTR effects on mucins by assessing mucin gene and protein expression profiles, kinetics of postexocytotic mucous granule content expansion, and biochemical and ionic compositions of the mucous gel in wild-type and Cftr(–/–) murine GBEC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. Mouse GBEC were isolated and cultured as previously described (21). Mice with targeted disruption of the Cftr gene created at the University of North Carolina [Cftrm1UNC(–/–)] (36) were maintained at the University of Washington Animal Facility. Isolation of mouse GBEC was carried out under a protocol approved by the institutional Animal Care Committee.

Mouse GBEC were cultured in Eagle's modified essential medium with the following supplements: 10% fetal bovine serum, 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, 20 mM HEPES pH 7.0, 1.0 g/l glucose, 5 µg/ml insulin from bovine pancreas, 5 µg/ml human transferrin, 5 ng/ml sodium selenite (insulin-transferrin-sodium selenite supplement), 1 x vitamins solution, and 1 x nonessential amino acids solution (Sigma, St. Louis, MO). Cell culture reagents were from Life Technologies (Gaithersburg, MD) unless stated otherwise. Human gallbladder myofibroblasts were used as a feeder layer on six-well plates, and mouse GBEC were grown on overlying Transwell inserts (24 mm diameter, 3 µm pore size, Costar, Cambridge, MA) coated with Vitrogen (Celtrix Labs, Palo Alto, CA). Cells were grown at 37°C in a 5% CO2 incubator. When cells were grown to confluency, we observed a clear layer of mucous gel adherent to the apical surface of polarized sheets of columnar epithelial cells.

Quantitative real-time PCR. Muc1, Muc2, Muc3, Muc4, Muc5ac, Muc5b, and Muc6 mRNA levels were evaluated by real-time PCR. Relative quantitative real-time PCR was performed using the ABI 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). The primers and fluorogenic probes of mouse origin were designed and synthesized by Applied Biosystems: Muc1 (Mm00449604_m1); Muc2 (Mm00458299_m1); Muc3 (Mm01207058_m1); Muc4 (Mm00466886_m1); Muc5ac (Mm01276725_g1); Muc5b (Mm00466376_m1); Muc6 (Mm00725165_m1) based on GeneBank accession numbers NM_013605, BC030862, AF027131, NM_080457.1, L42292, NM_028801.1, and NM_181729.1, respectively. Reactions were set up in triplicate. Briefly, 1 µl of cDNA in 8 µl of water was mixed with 1 µl of primer and probe mix and 10 µl of 2x TAQMAN universal master mix (Applied Biosystems) in a 96-well plate. Wild-type mouse GBEC cDNA served as a positive control, and three cDNA negative controls (no reverse transcriptase, no RNA, and water control) were run in parallel with the sample cDNA. The threshold cycle and the standard curve method were used to calculate the relative amount of the target RNA. The PCR sequence was hold at 50°C for 2 min, 95°C for 10 min, followed by 50 cycle repeats at 95°C for 15 s and 60°C for 1 min. SDS version 1.0 software (Applied Biosystems) was used to analyze real-time and end-point fluorescence. The expression levels of the target genes were normalized to internal beta-actin control samples.

Sample collection for immunoblotting. Cells harvested from one Transwell were washed three times with PBS to remove secreted mucins, pelleted, and solubilized in 200 µl of RIPA lysis buffer (Santa Cruz). After 13,000-rpm centrifugation for 15 min at 4°C, the supernatant was collected for protein quantification (Bio-Rad). For secreted mucins, monolayers were washed x 3 with PBS then incubated for 1 h with 2 ml of serum-free media under normal culture conditions. Monolayers were then flushed gently x 10 with the same 2 ml of serum-free media. Cells and debris were removed by centrifugation of the collected fractions at 1,600 rpm x 15 min. Samples were concentrated by Centricon Plus-20 (Amicon), washed with 2 ml of PBS twice, lyophilized overnight, and resuspended in 200 µl of RIPA buffer.

Immunoblotting. Cytosolic fractions were mixed with 2x electrophoresis sample buffer (Santa Cruz), and 20-µg samples were separated on a 7.5% SDS-PAGE gel (Bio-Rad). RIPA-resuspended secreted fractions were mixed with 2x electrophoresis sample buffer, and 2-µl samples were separated on a 7.5% SDS-PAGE gel. After a 40-V overnight electrophoretic transfer at 4°C onto polyvinylidene difluoride membranes (Bio-Rad), the immunoblots were incubated with Super Block T20 (TBS) blocking buffer (Pierce) with 0.05% Tween-20 for 1 h at room temperature. Primary antibody conjugated with horseradish peroxidase (HRP) at 200 ng/ml was added, and the immunoblots were further incubated overnight at 4°C, followed by 6 x 10 min room temperature wash with TBS/0.05% Tween-20. The immunoblots were then incubated for 60 min at room temperature with a corresponding secondary antibody conjugated with HRP at 10 ng/ml, followed by 6 x 10 min room temperature wash with TBS/0.05% Tween-20. Immunoblots were processed using SuperSignal West Femto chemiluminescent substrate (Pierce) and BioMax XAR film (Kodak). All primary antibodies used were from Santa Cruz Biotechnology: polyclonal goat anti-mouse MUC1 (sc-6826), polyclonal goat anti-rat MUC2 (sc-13312), polyclonal goat anti-human MUC3 (sc-13313), polyclonal rabbit anti-human MUC4 (sc-20117), polyclonal goat anti-mouse MUC5AC (sc-16903), polyclonal goat anti-human MUC5B (sc-16911), and polyclonal goat anti-human MUC6 (sc-16914). Corresponding secondary antibodies were donkey anti-goat IgG-HRP (sc-2020, Santa Cruz) and goat anti-rabbit IgG-HRP (1858415, Pierce). Blots were stripped and reprobed with polyclonal goat anti-human actin antibody (sc-1615 Santa Cruz). Gels were scanned and then analyzed via NIH Image J Density Analysis software.

Video-enhanced phase microscopy. In the study of postexocytosis expansion of mucous granule contents, we used video-enhanced phase microscopy as described by Verdugo et al. (43). Newly released mucous granule contents from mouse GBEC apical membranes could be easily observed and followed. Mucous granule contents were imaged by phase contrast light microscopy at a magnification of x500 and video recorded at a rate of 30 pictures/s. Images of expanding mucous granule contents were digitized at a rate of 5 samples/s. Previous observations have indicated that the swelling of mucus is governed by the same physical principles that govern the swelling of synthetic polymer gels (41, 43). A linear deformation of the expanding microspheres of mucins is observed during degranulation in murine GBEC analogous to what has been described for goblet cells isolated from rabbit trachea (43). This expansion follows typical first order kinetics, where the time course of the radial expansion r(t) is related to the initial radius (ri) and final radius (rf) by:

Formula 1(1)
Also in agreement with the linear theory of swelling of gels (41) is the proportional relationship between the characteristic time of the swelling kinetics ({tau}, the reciprocal value of the kinetic constant) and the square of the final radius rf2, where the diffusivity (D; i.e., the ability to expand) of the mucin network is:

Formula 2(2)
Measurements of the radius of the newly exocytosed granule contents as a function of time were fitted to Eq. 1. Diffusivity was calculated from Eq. 2.

To investigate the expansion of the condensed polyionic network inside the mucous granule in relationship to the ionic concentration of the medium, we chose a medium with ionic concentrations within the physiological range (Na+ 140 mM, K+ 5 mM, and Ca2+ 4 mM).

X-ray elemental microanalysis. We obtained cryosections at –110°C by cutting mouse gallbladders, glued with toluene to metal chucks, using a Sorvall MT2B ultramicrotome with FS 1000 cryokit. Thin cryosections were then transferred dry to carbon-coated Formvar support films on folding grids and freeze-dried in an oil-free vacuum system. Freeze-dried cryosections were viewed. The mucous gel was identified and analyzed in STEM mode in a JEOL 1200 EX electron microscope. X-ray spectra were collected and processed by using a Link AN 10,000 and 30-mm2 detector. Quantitation procedures, which have been described elsewhere (17), make use of protein and salt standards (34). We made at least five measurements of the mucous gel for Na+, K+, and Ca2+ with each of the five samples from the wild-type and Cftr(–/–) cells. Square rasters were placed over regions of granules (200 x 200 nm), cytosol (600 x 600 nm), and nuclei (1 x 1 µm). Electron-dense structures consistent with mucous granules were identified on the basis of their size and location (apical to nucleus) in the cells. Larger perinuclear electron dense mitochondria were also studied. Spectra were collected from samples at room temperature and corrections were made for beam-induced mass loss (8).

Biochemical analysis of biliary mucins. Soluble mucin from cell culture media was separated from small molecular weight proteins using Sepharose CL-2B (Pharmacia, Uppsala, Sweden). A column, 200 x 3.5 cm, was preequilibrated with 0.01 M K2PO4 buffer at pH 7.0, containing 3.5 mM sodium taurocholate (Calbiochem, La Jolla, CA), and fractions were eluted with the same buffer. The void volume peaks containing the macromolecular mucin were pooled (25 cell culture plates were pooled, 5 each, into 5 samples) to yield wild-type and Cftr(–/–) mucin fractions. Samples of pooled void volume fractions containing mucin were pooled, dialyzed against distilled water at 4°C for 48 h, freeze-dried, and further purified by CsCl density gradient ultracentrifugation as described by Starkey et al. (38). A 20 mg/ml preparation was treated with 60% (wt/vol) CsCl and centrifuged at 100,000 g at 4°C for 48 h. After centrifugation, the contents of these tubes were fractioned, dialyzed against distilled water, and assayed for protein (absorbance 280 nm) and mucin by the periodic acid-Schiff (PAS) method (28, 37). The fractions containing the PAS-reactive peak corresponding to a density of 1.4–1.5 g/ml were collected and subjected to another CsCl ultracentrifugation step. The resultant purified mucin was analyzed for carbohydrates and sialic acid by gas-liquid chromatography as previously described (25). Amino acid analysis was performed using a Beckman Multichrom analyzer after hydrolysis of glycoproteins in 6 M HCl at 110°C for 24 h. Sulfate was assayed by the method of Silvestri et al. (35).

Cells were fixed for histological examination. Confluent sheets of cells with the attached apical mucous gel layer and the basal collagen matrix were separated from the culture dish and plunged into Freon slush at –160 to –180°C for cryosection and elemental microanalysis. In other cell culture preparations, the surface mucous gel was carefully removed by pipetting a strong stream of culture medium over it. Our previous work using scanning electron microscopy has shown that if this layer of mucous gel were removed, new mucin spherules representing exocytosis could be observed from the apical membrane. The cell preparations were then rapidly transferred to a video enhanced phase microscope stage to examine exocytosis.

Statistical analysis. Data are expressed as means (SD). Student's unpaired t-test was used to assess the significance of difference between two groups. Significance levels were established at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Differential mucin mRNA expression in wild-type and Cftr(–/–) GBEC. We performed quantitative real-time PCR using primers specific for Muc1, Muc2, Muc3, Muc4, Muc5ac, and Muc5b. As shown in Fig. 1, the mRNA levels of these mucin genes were significantly decreased in the Cftr(–/–) cells compared with the wild type, with Muc2 being the exception. Muc6 expression was also analyzed in this manner, but expression levels were extremely low in both cell types, precluding quantitative analysis (data not shown).


Figure 1
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  		Fig. 1. Quantitative real-time PCR analysis of mucin gene expression. Wild-type and Cftr(–/–) gallbladder epithelial cell (GBEC) mRNA were analyzed for Muc1, Muc2, Muc3, Muc4, Muc5ac, and Muc5b expression levels. Shown are the results of n = 36 samples. Relative mRNA levels of each mucin gene are normalized to the wild-type GBEC Muc1 value. *P = 0.03; **P = 0.009; ***P = 0.005; #P = 0.003; +P = 0.00009.

 
MUC1, MUC2, MUC3, MUC5AC, and MUC5B levels trend lower in Cftr(–/–) compared with wild-type GBEC. We performed immunoblotting using antibodies against various mucins on cell lysates following removal of the extracellular mucous gel as a measure of cellular mucin content. These studies were performed in a quantitative fashion, with the signal as measured densitometrically normalized to the signal for the housekeeping protein actin. Representative samples of these results are shown in the left two lanes in Fig. 2. Figure 3 shows the results of the densitometric analyses from three experiments using three successive passages of matched wild-type and Cftr(–/–) cells. MUC1, MUC2, MUC3, MUC5AC, and MUC5B mucins showed a trend toward lower levels of expression in the Cftr(–/–) cells (P > 0.05). MUC6 expression was not appreciably different between the two cell types. We were not able to detect a signal for MUC4.


Figure 2
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  		Fig. 2. Representative immunoblots of wild-type (WT) and Cftr(–/–) cell lysates and of their respective media using mucin antibodies. The immunoblots are representative of 3 experiments performed with cells matched for passage number done on 3 successive passages for each mucin antibody.

 

Figure 3
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  		Fig. 3. Mucin glycoprotein expression in Cftr(–/–) compared with wild-type GBEC. Densitometric values are for 3 immunoblots for each mucin antibody, each taken from cells matched for passage number from 3 successive passages. Data were normalized to the density of actin bands. The results are depicted as the Cftr(–/–) GBEC band density expressed as a % of the wild-type GBEC band density from the same experiment. P = not significant for all mucins.

 
Secreted mucin levels are higher Cftr(–/–) compared with wild-type GBEC. We also isolated secreted mucins for immunoblot analysis from the two cell types. MUC2, MUC5AC, MUC5B, and MUC6 mucins are classified as secreted, gel-forming mucins, and, with the exception of MUC2 and MUC5AC, each type was present at significantly higher levels in the media from Cftr(–/–) GBEC compared with wild-type cells. Figure 2 shows representative immunoblots, with the right two lanes, labeled media, representing secreted mucins. Figure 4 shows the pooled densitometric analyses from three experiments using three successive passages of matched wild-type and Cftr(–/–) cells. The epithelial mucins MUC1 and MUC3 were also found at significantly higher levels in the media from Cftr(–/–) GBEC compared with wild-type cells. As for the immunoblots on cell lysates, we were not able to detect a signal for MUC4. Cftr(–/–) cells exocytosed a significantly greater proportion of the mucins measured in our experiments when the data were pooled (Fig. 4, inset; P = 0.0002). No actin signal was detectable in the media (Fig. 2), indicating that cellular contents did not leak at the time of sample collection.


Figure 4
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  		Fig. 4. Mucin glycoprotein expression in the media from Cftr(–/–) compared with wild-type GBEC. The densitometric analysis and the depiction of results are as noted for Fig. 3. Inset: percentage of total measured mucins secreted [i.e., extracellular measured mucins/total (cellular + extracellular) measured mucins x 100] for the 2 cell types. *P = 0.0002. **P = 0.025. ***P = 0.04. #P = 0.03. +P = 0.006.

 
Differential postexocytotic mucous granule content hydration kinetics in wild-type and Cftr(–/–) GBEC. Postexocytotic mucous granule contents could be easily identified and their subsequent expansion followed and analyzed using video enhanced microscopy. Mucous granule contents from both wild-type and Cftr(–/–) cells followed first order swelling kinetics, driven by a Donnan equilibrium process (40). The final volume expansion of both wild-type and Cftr(–/–) mucous granule contents did not differ significantly and was ~1:650. The mucous granule contents from Cftr(–/–) cells were slower to expand (Fig. 5A). The characteristic time of swelling is proportional to the square of the linear size of the secreted boluses (Fig. 5B) as predicted by the linear theory of swelling of gels (41) and as shown previously for rabbit tracheal goblet cells (43). The diffusivity of the mucus gel, as calculated by Eq. 2, decreased from 3.12 x 10–5 cm2/s in the wild-type cells to 1.92 x 10–5 cm2/s in the Cftr(–/–) cells, as measured at the 2-s time point following exocytosis. The difference between the two curves was significant (P < 0.025).


Figure 5
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  		Fig. 5. A: swelling kinetics of mucin granules from mouse GBEC. Representative plot of the time in seconds of the course of swelling of exocytosed mucin granules from wild-type ({blacksquare}) and Cftr(–/–) ({blacktriangleup}) GBEC. The change in radius as a function of time follows first order kinetics. The continuous line is a nonlinear least-square fit of experimental measurements of radius to Eq. 1. B: the characteristic time of swelling of spherical boluses of exocytosed mucins ({tau}), as a function of the square of their final radius r2 from wild-type ({blacksquare}) and Cftr(–/–) ({blacktriangledown}) GBEC showing a linear relationship obtained by least-square fitting of experimental data. The regression equations are:

Formula 2

Formula 2

 
Biochemical characterization of mucins secreted by wild-type and Cftr(–/–) GBEC. Despite the difference in the quantity of individual mucins in the secreted mucous gel as measured by immunoblotting, we found no difference in the total amount of mucus secreted by wild-type and Cftr(–/–) mouse GBEC (Table 1), consistent with a previous report (32). The biochemical profile of mouse GBEC mucins was characteristic of other gastrointestinal mucins, with a high carbohydrate component (~70%). There was no discernible difference between the amino acid and oligosaccharides profiles (data not shown). There was a significant increase in sulfation of mucins secreted by Cftr(–/–) cells compared with wild-type cells [wild-type 1.1 (0.3); Cftr(–/–) 2.9 (0.8); P < 0.05].


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  		Table 1. Biochemical analysis of secreted mucins from mouse GBEC

 
Elemental microprobe analysis of ionic concentration of mouse GBEC and of the mucus gel adherent to the apical membrane of these cells. Our laboratory (22) has previously reported that mouse gallbladder epithelium showed a striking accumulation of calcium in intracellular mucous granules compared with the calcium contents in the cytoplasm. We extended these studies by comparing the ionic concentrations in the mucous gel isolated from cultured wild-type and Cftr(–/–) cells. As shown in Table 2, the mucous gel from wild-type cells contained lower levels of calcium than did the gel from Cftr(–/–) cells [4.5 (0.5) vs. 7.7 (0.6), respectively; P = 0.015]. No significant differences in sodium and potassium concentrations were measured in the mucous gels isolated from wild-type and Cftr(–/–) cells. Calcium concentrations in the nuclei and in the mucous granules did not significantly differ between the wild-type and Cftr(–/–) cells.


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  		Table 2. Elemental microprobe analysis of ionic concentration of mouse GBEC and of the mucus gel adherent to the apical membrane of these cells

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The relationship between CFTR and the altered mucus phenotype in CF may be explained by intracellular effects and/or extracellular processes (26). Support for the former is provided by reports that mucin gene expression (15, 29) and glycosylation profiles (9, 42, 45) of mucins from CF patients or rodent models of CF are altered. Evidence in support of the latter is provided by reports demonstrating that features of the extracellular milieu such as ionic concentration and pH affect the rheology of the secreted mucous gel (10, 30, 33). Secondary effects due to infection, inflammation, or drugs further cloud the relationship (27), as does the use of in vitro systems of neoplastic origin or expressing nonnative mucins (26). Conversely, several studies have shown no direct relationship between CFTR expression and mucin characteristics such as sulfation (7, 18, 26). We sought to address these issues by investigating mucin characteristics in nonneoplastic murine GBEC that natively express mucins, wherein the only difference between cell types was the presence or absence of CFTR (21).

Previous studies with cultured GBEC provided evidence for a relationship between CFTR expression and mucin characteristics. High calcium concentrations were measured within intracellular mucin granules, and CFTR colocalized with mucin in these granules (22). These findings supported a model in which influx of chloride into mucin granules disrupted calcium-mediated shielding of anionic charges on mucin carbohydrate groups, leading to rapid swelling, exocytosis, and hydration of mucins (22). We subsequently showed that calcium binding to biliary mucins is dependent on sodium concentration (23) and is effected mainly through sulfate, a finding that has relevance to CF because sodium hyperabsorption and mucus hypersulfation are reported to be consequences of aberrant CFTR function (9, 39, 42, 45). We hypothesized that, in CF, the relative decrease in the levels of luminal sodium would lead to a more viscous mucous gel due to stronger binding of calcium with sulfate groups. Indeed, the mechanism through which inhaled hypertonic saline improves lung function in patients with CF might be due, in part, to such an effect (12, 13, 24).

We found that mucin mRNA levels were significantly lower in Cftr(–/–) compared with wild-type cells, with the exception of the gel-forming mucin Muc2. The immunoblot results showed that both epithelial (MUC1, MUC3) and gel-forming (MUC2, MUC5AC, and MUC5B) mucins trended toward lower levels of expression in Cftr(–/–) cells. More intriguingly, the media from Cftr(–/–) cells contained significantly higher levels of most mucins, suggesting that mucin granule exocytosis and/or the physical and chemical transformations of mucins during granule expansion and release were altered. Immunoblot analysis of media is a measure of constitutive mucin secretion, because no secretogogues were applied to the cells, although it is conceivable that mechanical forces applied during sample collection could have accelerated mucous granule exocytosis. The lack of cellular actin in the media argues against cell lysis. Regardless of whether mucin secretion was constitutive or triggered, the higher amounts of mucins detected in the Cftr(–/–) samples suggest that the threshold for mucin release was lower in these cells.

Studies in airway cells support the concept of alterations in mucin granule composition in CF. Baconnais et al. (1) demonstrated in human airway submucosal cell lines that the CF phenotype was associated with higher secretory granule ionic content and lower secretory granule water content. This, in association with the hyperabsorption of sodium that occurs in cells with altered CFTR function (39), supports the concept that both the mucus and surface hydration characteristics of CF epithelia are altered, leading to a more highly viscous mucus. Our findings are in agreement with such a model. We found a decreased capacity of Cftr(–/–) mucous granules to swell from the condensed to an expanded gel phase. This may account for decreased CF mucus transportability and its subsequent accumulation following exocytosis in the extracellular mucous gel, as suggested by Puchelle et al. (33). Mucin granule architecture is also dependent on the characteristics of the carbohydrate species attached to mucin polypeptides. Studies with a GFP-mucin fusion protein have shown that inhibition of mucin type O-glycosylation, sialylation, or sulfation altered the characteristic diffusion time (31). Therefore, altered posttranslational processing of mucins, due to carbohydrate content or orientation, could affect the granular organization of mucins, which in turn would alter swelling characteristics and exocytosis.

The presence of the transmembrane epithelial mucins MUC1 and MUC3 in the media would seem unexpected; in fact, there is precedence for this observation. In the Cftr(–/–) mouse, MUC1 accumulates in the intestinal lumen (16, 29). Similarly, the extracellular domain of MUC3 (44) is found in the small intestinal lumens of Cftr(–/–) mice (19). These studies on intestinal mucins also provide a useful comparison with respect to the mucin genes that were differentially expressed in wild-type and Cftr(–/–) cells. A sixfold increase in Muc1 RNA expression was reported in the colons of Cftr(–/–) mice with a moderate increase in MUC1 protein (29). In contrast, MUC2, MUC3, and MUC5AC exhibited similar glycoprotein expression with lower levels of mRNA. MUC2 and MUC3 also contribute to the excess intestinal luminal mucus of Cftr(–/–) mice (19). MUC2 and MUC3 signals were higher in goblet cells and columnar cells, respectively, of wild-type mice, whereas higher signals of each were noted in the intestinal lumens of Cftr(–/–) mice (19). This is consistent with our findings that cellular mucin levels tended to be higher in wild-type GBEC, yet the amount of most secreted mucins was higher from Cftr(–/–) cells.

Although our study is the most comprehensive analysis of mucin mRNA and protein expression in murine GBEC to date, certain anomalies in our results deserve comment. MUC6 mucin was detected by immunoblotting despite low mRNA levels by real-time PCR. We suspect that technical issues related to the primers used account for this finding. Conversely, although Muc4 mRNA levels were detected and followed the general pattern showing significantly lower levels of expression in Cftr(–/–) cells, we could not detect MUC4 by immunoblotting in cells or in the media. Poor cross-reactivity of murine MUC4 with the anti-human MUC4 antibody is a likely explanation.

We propose the following model to explain enhanced mucous gel viscosity in CF. In the absence of functional CFTR, sodium hyperabsorption (39), higher mucin sulfate content and a higher concentration of calcium after mucous granule expansion, exocytosis, and annealing conspire to result in a stiffer mucous gel because it is less hydrated and possesses stronger sulfate-calcium bridges. This occurs because even a small increase in the gel fluid calcium concentration dramatically increases the viscoelasticity of the gel (2, 4). Gastrointestinal mucins bind to calcium through sialic acid and sulfate groups (11), with binding of sulfate groups providing the most rigid bridging effect. This binding is easily displaced by NaCl because the binding affinity of sodium to mucin is much higher than that of calcium (23). If there is an excessive removal of NaCl and water from the gel fluid, there is an increase in binding of mucins with calcium, especially if the mucin is high in sulfate content as in CF. The resultant mucus gel is highly viscous. A more concentrated stiffer gel in turn slows the gel {rightleftarrows} sol transition into the aqueous phase, resulting in a gel with thicker dimensions. The final result is more mucus retention with mucus that is highly viscoelastic. We speculate that the epithelial cell senses these changes and compensates by downregulating expression of certain mucin genes. Alterations in the mucin gene expression profiles, in concert with hypersulfated carbohydrate groups, in turn lead to mucous granule contents with altered hydration kinetics and a lower threshold for exocytosis.


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This work was supported by a Merit Review Award from the Department of Veterans Affairs.


   ACKNOWLEDGMENTS
 
We thank Jurgen Seppen and William R. A. Osborne for helpful discussions.

Present address for J. H. Klinkspoor: Klinisch Chemicus, Hoofd Laboratorium Speciële Hematologie, Staflid Laboratorium Algemene, F1-217.3, Academisch Medisch Centrum, Meibergdreef 9, Postbus 22660, 1100 DD Amsterdam, The Netherlands.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Kuver, Division of Gastroenterology, Box 356424, Univ. of Washington School of Medicine, 1959 NE Pacific St., Seattle, WA 98195 (e-mail: kuver{at}u.washington.edu)

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.


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Mucin Granule Intraluminal Organization
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