Characteristics of rodent intestinal mucin Muc3 and alterations in a mouse model of human cystic fibrosis

Ismat A. Khatri1, Catherine Ho1, Robert D. Specian2, and Janet F. Forstner1

1 Division of Biochemistry and Structural Biology, Research Institute, The Hospital for Sick Children, and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5G 1X8; and 2 Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130


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Human mucin MUC3 and rodent Muc3 are widely assumed to represent secretory mucins expressed in columnar and goblet cells of the intestine. Using a 3'-oligonucleotide probe and in situ hybridization, we observed expression of rat Muc3 mostly in columnar cells. Two antibodies specific for COOH-terminal epitopes of Muc3 localized to apical membranes and cytoplasm of columnar cells. An antibody to the tandem repeat (TR) sequence (TTTPDV)3, however, localized to both columnar and goblet cells. On CsCl gradients, Muc3 appeared in both light- and heavy-density fractions. The lighter species was immunoreactive with all three antibodies, whereas the heavier species reacted only with anti-TR antibody. Thus Muc3 is expressed in two forms, a full-length membrane-associated form found in columnar cells (light density) and a carboxyl-truncated soluble form present in goblet cells (heavy density). In a mouse model of human cystic fibrosis, both soluble Muc3 and goblet cell Muc2 were increased in amount and hypersecreted. Thus Muc2 and Muc3 contribute to the excess intestinal luminal mucus of cystic fibrosis mice.

intestine; Muc2; columnar cell; goblet cell


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THIS STUDY WAS DIRECTED AT identifying the cellular origin of rat intestinal mucin Muc3, the subcellular localization of the Muc3 translation product(s), and alterations in the distribution and forms of intestinal Muc3 in a mouse model of human cystic fibrosis (CF).

The major mucins produced in the rodent intestine are Muc2, a goblet cell product, and Muc3. Both are orthologs of human mucins MUC2 and MUC3, respectively. MUC3 has been reported to be expressed in both columnar and goblet cells of the human small intestine and colon (2, 3, 11, 37), and both mucins are usually referred to as "secretory" mucins (24). It seems clear that MUC2 fits this description, because it lacks a transmembrane region, contains typical NH2- and COOH-terminal clusters of cysteines appropriate to oligomerization and gel formation (24), and is released from goblet cells under resting and stimulated conditions (7). The use of the term secretory to describe MUC3 mucin is more problematic. Initial cloning of the COOH-terminal domain of human MUC3 indicated the presence of one epidermal growth factor (EGF)-like motif and no putative transmembrane sequence (8), yet typical cysteine-rich domains characteristic of secretory mucins were not present. More recently, it has been established (5, 39) that MUC3 can exist in more than one form. The dominant allele encodes a COOH-terminal sequence containing two widely spaced EGF-like domains, a putative transmembrane region, and a cytoplasmic tail of 75 amino acids. Soluble forms of MUC3 are predicted to arise from alternate splice sites located between the two EGF motifs (5, 39). In reviewing the original expression studies (3, 11, 37) for human MUC3, it was clear that columnar cells gave a positive mRNA signal, but it was difficult to distinguish specific goblet cell expression in the low-power magnification photomicrographs presented.

Before elucidation of the complete COOH-terminal sequence of human MUC3, our (15) laboratory reported the cloning of a cDNA for the COOH-terminal domain of rat Muc3. We (15) described the presence of two EGF-like domains, a putative transmembrane domain and a cytoplasmic tail of 80 amino acids. We noted a close structural resemblance to the COOH-terminal domain of rat mammary sialomucin complex (30), now recognized as the rodent ortholog of human MUC4 (22). At that time, Muc4 was the only other reported membrane mucin to contain two EGF-like motifs. Rat Muc4 is posttranscriptionally cleaved into two noncovalently bound polypeptide subunits: asialoglycoprotein-1 (ASGP-1), which is a large (>500 kDa) mucin component bearing O-linked oligosaccharides, and ASGP-2, the smaller (120-140 kDa) COOH-terminal domain containing 24 N-glycans, 2 EGF-like motifs, a transmembrane segment, and a short cytoplasmic tail. Rat Muc4 is expressed in a number of normal epithelial tissues and can be found in both nonmembrane (soluble) and membrane-associated forms (21, 27). Unlike typical secretory mucins, Muc4 appears to play important roles in epithelial growth, cell differentiation (27), cell-cell adhesion, metastases, and resistance to natural killer cells (17, 31, 35, 40). Some of these roles are also recognized for MUC1, a membrane mucin found in many epithelial tissues, although in very low abundance in the intestine. Recently, a cDNA for yet another human intestinal membrane mucin, designated MUC12 (38), has been shown to have a double EGF motif, a transmembrane domain, and a cytoplasmic tail pattern similar to those of MUC3 and MUC4, suggesting that there exists a subfamily of similar membrane-associated mucins. Because secretory and membrane mucins may have very different structural and functional properties, it is important to distinguish between the two classes and to identify normal and pathological conditions that alter their expression. These issues motivated us to examine aspects of rodent Muc3 structure and localization more thoroughly.

Small intestinal and colonic pathology is pronounced in patients with CF, particularly with respect to mucins, which cause partial or complete luminal obstruction. This autosomal recessive disease is caused by mutations in the gene encoding the CF transmembrane regulator (CFTR), a protein mediating chloride conductance in epithelial cells. CF has a complex phenotype and is manifested as a multiorgan disease. Some features of the human disease, including the intestinal phenotype, are reproduced by the cftrm1UNC(-/-) mouse model (33). In this model, the cftr gene is rendered nonfunctional by the introduction of a stop codon in exon 10. Prolonged survival of mice homozygous for the cftr gene knockout [cftr(-/-)] is possible with administration of a liquid diet, which reduces but does not remove intestinal obstructive pathology (12). A study carried out by Parmley and Gendler (23) on cftr(-/-) mice developed at the University of North Carolina (UNC) found that intestinal mRNAs for Muc2 and Muc3 were depressed relative to those in normal mice. Muc5ac, another secretory mucin, was unchanged, and Muc1 was elevated. The measured translation products of each mucin did not account for the dramatic increase in periodic acid-Schiff (PAS)-positive material that filled and dilated the crypt lumen. These findings were confusing to interpret, because it is clear that in cftr(-/-) mice goblet cells are in a state of hypersecretion, and one would expect that at least Muc2 would be increased in secretions. Parmley and Gendler (23) recognized that the antibodies used for mucin detection may have contributed to the confusion, because they were prepared against deglycosylated core peptides, and in the case of Muc1, to the nonsecreted cytoplasmic COOH-tail. It was assumed that none of these antibodies would recognize mature (glycosylated) secreted mucins, and thus would not reliably identify the mucins responsible for the increase in PAS-stained (glycosylated) luminal secretions in cftr(-/-) mice.

Using an oligonucleotide probe and two antibodies specific for peptide epitopes located in the unique COOH-terminal region of Muc3, as well as an antibody to the tandem repeats of Muc3, we have investigated the molecular form and localization of Muc3 in normal rat intestine and the intestine of UNC cftr(+/+) and cftr(-/-) mice. Our findings have relevance for the physiology of Muc3 production, Muc3 distribution in intestinal cells, and the pathogenesis of the mucin lesion in CF.


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In situ hybridization. Individual segments (0.5 cm) of jejunum and colon from fasted adult male Wistar rats were embedded in Tissue-Tek O.C.T. (Miles Scientific, Elkhart, IN) and snap frozen in liquid nitrogen. A 1,220-bp EcoR I, Hind III fragment of rat intestinal Muc3 (nt 275-1494), corresponding to amino acids 93 to 380 plus the 354-nt untranslated region (15), was subcloned into the EcoR I, Hind III sites of pBluescript II (SK+) (Stratagene, La Jolla, CA). Both sense and antisense strands were linearized using the appropriate enzyme, purified on Sephacryl S-400 (Pharmacia), and 1 µg was used as a template for the synthesis of digoxigenin (DIG)-labeled sense and antisense probes, respectively. DIG-UTP and T3/T7 polymerase were used as advised in the DIG RNA labeling kit from Roche Diagnostics Canada. After removal of the templates with DNase, the RNA probes were precipitated by 4 M LiCl/ethanol (0.1:3 vol/vol), washed in 70% ethanol, and suspended in diethyl pyrocarbonate-treated water. Tissue sections (5 µm) were fixed with 4% paraformaldehyde in PBS for 2 min and washed twice with PBS, and endogenous alkaline phosphatase was inactivated by heating at 70°C for 30 min in 20% formaldehyde in 2× sodium chloride-sodium citrate (SSC). The DIG-labeled probes (300-500 ng/ml) were heated at 80°C for 2 min, chilled on ice, and added to hybridization buffer containing 50% formamide, 5× SSC, and 2% blocking reagent (Roche Diagnostics Canada). The mixture was incubated with tissue sections for 17 h (100 µl/section) at 37°C in a humidified chamber. Sections were washed twice at 37°C for 20 min with 50% formamide in 2× SSC and incubated for 30 min at 37°C with 20 µg/ml RNase in NTE buffer (500 mM NaCl, 10 mM Tris · HCl, pH 8.0, and 1 mM EDTA). After two more washes (10 min each) with 2× SSC at room temperature, slides were processed according to the Roche Diagnostics Canada, DIG nucleic acid detection kit protocol. Counterstaining was carried out for 1 min with Harris modified hematoxylin stain (Fisher Scientific, Napean, ON, Canada). The slides were washed with water, coverslipped using 50% glycerol, examined by light microscopy, and photographed. Slides were scanned and converted into black and white images by use of Adobe Photoshop software.

Preparation of antisera to rat Muc3. Two peptide sequences chosen within the COOH-terminal region of rat Muc3 were synthesized and used to develop polyclonal antibodies. Residues 135-148 (LKAQYTPGFDNTLD) located between EGF-1 and EGF-2 and residues 317-331 (RWGEEAGRASPGTFH) in the COOH-tail (15) were designated peptides 6279 and 6278, respectively (Fig. 1). The peptides were synthesized by the Hospital for Sick Children Pharmacia Biotechnology Center (Toronto, Ontario, Canada). For enhanced immunogenicity, the peptides were conjugated onto a branched lysine core to give an octameric multiple antigen structure (25). The antigens were injected together with Hunter's TiterMax adjuvant (Sigma Chemical, St. Louis, MO) into two New Zealand rabbits (200 µg/rabbit). Two boosters were given at 2-wk intervals, and antiserum was collected 10 days after the second booster. Peptide 36W, representing three consensus sequences (TTTPDV)3 of tandem repeats of rat Muc3, was synthesized and conjugated to keyhole limpet hemocyanin by BioTools (Edmonton, Alberta, Canada) and used to raise a polyclonal antibody in chickens by Gallina Biotechnology (Edmonton, Alberta, Canada). All three antibodies were specific for their respective peptides and did not show cross reactivity with each other in slot-blot immunoassays. Due to extensive sequence homology of Muc3 in rat and mouse species (15, 29), the antibodies reacted well with both rat and mouse intestinal samples. Immunoreactivity was detected with the appropriate anti-rabbit IgG or anti-chicken IgY conjugated to alkaline phosphatase and nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate substrate from Roche Diagnostics Canada.


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Fig. 1.   COOH terminus of rat Muc3: full-length (a) and putative truncated soluble forms (b and c). Epitopes 36W, 6279, and 6278 represent synthetic peptides to which antibodies were raised. TRs, tandem repeats; TM, transmembrane segment; EGF, epidermal growth factor. Slashes and dotted lines indicate putative deletions.

For detection of goblet cell Muc2, polyclonal anti-D4553 (41) developed against a synthetic peptide in the COOH-terminal region of rat Muc2 was used. A polyclonal antibody specific for rat intestinal brush-border sucrase has been described previously (6).

Immunohistochemistry. Frozen sections of rat and mouse small intestine and rat colon were fixed in methanol, blocked with 5% goat serum, and incubated with preimmune or immune serum (anti-6279 at 1:200 dilution) overnight. After three washes with TBS, the sections were incubated with cyanine-3 (Cy3) fluorophore-tagged goat anti-rabbit IgG (Jackson Laboratories) and counterstained with Mayer's hematoxylin (Sigma Chemical). Slides were coverslipped using VectaShield mounting media (Vector Laboratories, Burlington, CA), examined under dark-field microscopy, and photographed. Images were scanned and converted into black and white images by use of Adobe Photoshop software.

In the case of Formalin-fixed tissues of rats and mice, antigen retrieval was performed by microwaving sections (32) for 20 min in 10 mM citrate buffer, pH 6.0, followed by heat treatment (boiling for 5 min) in TBS containing 10 mM dithiothreitol, followed by two 10-min washes in TBS. All subsequent steps of blocking and incubation with preimmune and immune sera were as described above for methanol-fixed frozen sections. Anti-6279 and anti-6278 antibodies were used at dilutions of 1:200 and 1:100, respectively. Anti-36W was used at a concentration of 100 µg/ml, with Cy3 fluorophore-tagged rabbit anti-chicken IgY (Jackson Laboratories) as the secondary antibody.

Confocal microscopy was performed on intestinal tissues previously fixed in Zamboni's fixative (36) for 24 h, equilibrated in 30% sucrose, and frozen in liquid nitrogen. After blocking, sections (8-10 µm) were incubated with anti-6279 antiserum (1:200 v/v) or its respective preimmune serum (1:100 vol/vol). Samples were imaged with a Bio-Rad MRC 1024 scanning laser confocal system using optical sections at 0.4 µm. Digital images were analyzed using Lasersharp analysis software.

Preparation and assay of brush-border membranes and soluble supernatant. Rat intestinal brush-border membranes (BBM) were prepared by the method of Kessler et al. (13) from jejunum pooled from six fasted adult male Wistar rats. During homogenization of mucosal scrapings in Tris-mannitol, CaCl2 was added (final concn, 10 mM), and the homogenate was centrifuged at 3,000 g. The supernatant was centrifuged at 27,000 g for 30 min, and aliquots of the resulting (second) supernatant were saved at -20°C for later immunoassay. The pellet (containing BBM) was suspended in 2 mM Tris, pH 7.1, containing 50 mM mannitol, and stored at -20°C. Protein in BBM and supernatant was assayed by the bicinchoninic acid assay (26). For immunoassay, samples of BBM and supernatant were boiled for 3 min in a solution of 125 mM Tris, pH 6.8, containing 0.5 M mercaptoethanol and 2% SDS, samples containing 3-12 µg protein were slot blotted onto nitrocellulose membranes, and reactivity measured with anti-sucrase (1:5,000 dilution vol/vol), anti-6279 (anti-Muc3, 1:1,000 dilution vol/vol), and anti-D4553 (anti-Muc2, 1:3,000 dilution vol/vol) antisera. Preimmune sera served as negative controls. Anti-rabbit IgG conjugated alkaline phosphatase and NBT/BCIP substrate from Roche Diagnostics Canada were used for detection. Slots were scanned, and the density of each slot measured using National Institutes of Health gel-plotting macros program.

Preparation of mice tissue. UNC CF knockout mice [cftr(-/-)] (33) and controls [cftr(+/+)] maintained on a liquid diet were supplied by Dr. G. Kent (Animal Facilities, The Hospital for Sick Children, Toronto, ON, Canada). All experiments were carried out under protocols approved by the hospital Animal Care Committee. One group of four test and four control mice ranged from 8 to 10 wk and another group (n = 6) from 6 to 8 wk of age. Jejunal segments from both groups were fixed in 10% Formalin overnight, dehydrated to 95% ethanol, and embedded in paraffin. Serial sections (5 µm) were deparaffinized and processed for immunohistochemistry or stained by PAS and Alcian blue, pH 2.5. Immunolocalization results of rat and mice tissues were the same whether obtained from frozen sections fixed in methanol or Formalin-fixed sections embedded in paraffin.

CsCl density gradient ultracentrifugation. Pooled intestinal scrapings from the jejunum of four fasted adult male rats or pooled jejuneal segments from cftr(-/-) and cftr(+/+) groups of mice were separately homogenized in 50 vol of PBS containing a proteinase inhibitor mixture of Na2EDTA (5 mM), PMSF (1 mM), N-ethyl maleimide (5 mM), leupeptin (1 µM), and pepstatin (1 µM). Insoluble material was removed by centrifugation at 30,000 g for 30 min at 4°C. Supernatants were subjected to CsCl density gradient ultracentrifugation as described earlier (14). Eight fractions were collected ranging in buoyant density from 1.23 to 1.62 g/ml. Each fraction was dialyzed, and equivalent volumes were assayed for Muc3 or Muc2 by slot-blot immunoassays for protein (26) and carbohydrate by the PAS assay (20).


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Intestinal expression of rat Muc3. The DIG-labeled antisense probe for the 3' end of rat Muc3 gave a strong positive signal in the columnar cells of the epithelial cell layer of villi and crypts of rat intestine (Fig. 2b). In the colon, the antisense probe also hybridized to columnar cells, although the signals appeared to be stronger in the crypts than in the surface epithelium (Fig. 2d). Specific signals in goblet cells were rarely detected.


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Fig. 2.   Cellular expression of rat intestinal Muc3 (in situ hybridization). Jejuneal sections (a, b) and colonic sections (c, d) were hybridized to digoxigenin-labeled sense (a, c) and antisense (b, d) probes. Specific signals were seen in enterocytes throughout the small intestine and colon. Magnification: ×400.

Immunofluorescent localization of rat Muc3. Frozen tissue sections (5 µm) of rat and mouse intestine and colon were analyzed by immunofluorescence, initially using only anti-6279 antiserum. Throughout the small intestine, as shown for rat and mouse jejuneum (Fig. 3, a and e), fluorescence was seen in columnar cells of villi and crypts. In the colon (Fig. 3d), surface and crypt cells were also stained. Fluorescence was most pronounced along the apical membrane region of columnar cells (Fig. 3b). The goblet cell granule mass was not stained, but a diffuse signal was observed around the border of the granule mass, suggesting reactivity of either the filamentous theca in the goblet cells or simply reactivity of neighboring columnar cells, which are normally compressed laterally against the bulging granule mass of goblet cells (9). The apical staining of Muc3 was more diffuse than that of sucrase (Fig. 3c), the latter being used as a marker of differentiated BBM (10). This difference, as well as the presence of Muc3 immunostaining in crypts, suggests that Muc3 is not confined to microvillus membranes, and in particular, is not confined to upper villus columnar cells with a well-differentiated microvillus membrane. In intestinal and colonic samples treated with preimmune serum or anti-6279 preincubated with its specific peptide antigen (100 µg/ml), the signals associated with both columnar cells and the halo around the granule mass of goblet cells were eliminated (not presented).


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Fig. 3.   Immunofluorescent localization of rat intestinal Muc3. Antiserum to 6279 (anti-Muc3) was used for immunofluorescent localization in rat jejunum (a, b), rat colon (d, f), and mouse jejunum (e). Confocal microscopy was used for f as described in MATERIALS AND METHODS. Anti-sucrase was used on rat jejuneal sections (c). Magnification: ×400 for a, d, e, and f; ×1,000 for b; and ×250 for c.

More precise localization of anti-6279 was carried out on rat colonic sections using confocal microscopy with 0.4-µm optical sectioning (Fig. 3f). Fluorescence was not detected in goblet cells but was pronounced along the apical membrane and in the apical cytoplasm of columnar cells. Muc3 was also detected over a middle region of each cell presumed to be the endoplasmic reticulum and/or Golgi network. Collectively, these results indicate that the COOH-terminal domain of the Muc3 translation product is confined mainly, if not exclusively, to columnar cells.

Distribution of intestinal Muc3, Muc2, and sucrase in BBM and cytoplasmic fractions. Muc2 (anti-D4553), Muc3 (anti-6279), and sucrase reactivity were measured by slot-blot immunoassays, and the BBM-to-supernatant ratio was calculated for each from densitometry measurements (Table 1). Muc2 was enriched in the supernatant fraction, whereas rat Muc3 was enriched (3- to 4-fold) in the BBM fraction. Sucrase was much more enriched in the membrane fraction, in keeping with its known expression in apical microvillus membranes (13, 28). These results are consistent with the previous immunofluorescence results indicating that Muc3 is distributed in both a soluble (cytoplasmic) compartment as well as in apical membranes.

                              
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Table 1.   Distribution of rat intestinal mucins Muc3 and Muc2 in BBM and soluble supernatant

Distribution of intestinal mucins in CsCl density gradient fractions. CsCl gradients of epithelial scrapings (homogenate supernatants) have been used for many years to separate secretory mucins from lighter serum and membrane glycoproteins and heavier proteoglycans and DNA (1, 16, 34). Typically, secretory mucins appear in a buoyant density range of 1.40 to 1.50 g/ml. In the present study, slot-blot immunoassays of Muc2 and Muc3 were performed on CsCl fractions from homogenized scrapings of rat jejunum. As shown in Fig. 4, immunoreactivity for Muc2 (anti-D4553) was widely distributed, but the major peak was centered together with PAS reactivity at a buoyant density of 1.47 g/ml (Fig. 4a). Anti-6279 (Fig. 4b) and anti-6278 (Fig. 4c) (cytoplasmic tail of Muc3) each gave two peaks, but the majority was present in the lighter density fraction (peak at 1.33 g/ml). Anti-sucrase reactivity was entirely confined to the lighter fraction (not shown). Anti-36W (antibody to tandem repeats of Muc3) reactivity gave a peak in both fractions (Fig. 4d), with ~65% of the total appearing in the heavy-density fraction. Thus Muc3 appears to be present in two forms. The lighter species contains tandem repeats and COOH-terminal epitopes 6279 and 6278 and thus is presumably a full-length form, whereas the heavier species contains tandem repeats and very few COOH-terminal epitopes and thus is presumably a nonmembrane (soluble) form.


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Fig. 4.   Distribution of Muc3 and Muc2 in CsCl density gradient fractions. Intestinal homogenates of rat (), cftr(-/-) (open circle ), and cftr(+/+) mice (triangle ) were subjected to CsCl density gradient ultracentrifugation. Eight fractions were collected in each case. An equal volume of each of the fractions was incubated in slot-blot immunoassays with anti-D4553 (a), anti-6279 (b), anti-6278 (c), and anti-36W (d). Anti-D4553 was used at a dilution (vol/vol) of 1:1,000 and 1:500 for rat and mice fractions, respectively. Anti-6279 was used at a dilution of 1:300 and 1:200 for rat and mice fractions, respectively. Anti-6278 was used at a dilution of 1:200 and anti-36W at a concentration of 10 µg/ml for both rat and mice fractions. Immunoreactivity of each fraction is expressed as % of the total immunoreactivity found in all 8 fractions.

Intestinal mucins in CF mice. The antibodies raised to rat mucin peptides were used to analyze and compare CsCl gradient fractions of similar intestinal homogenates prepared from age-matched cftr(-/-) and cftr(+/+) control mice (Fig. 4). This was possible because the peptide epitopes in the two rodent species differ by only one or two amino acids at most, including the consensus tandem repeat (29). The percent distribution of the various mucin epitopes across the gradient was similar to those of rat homogenates, although in mice, epitopes 6279 and 6278 of Muc3 were confined to the lighter density fraction (Fig. 4, b and c) and the only Muc3 epitope found in the heavier fraction was 36W (Fig. 4d).

Quantitative slot-blot assays were performed on pooled light- and heavy-density fractions of the two different groups of cftr(+/+) and cftr(-/-) mice using equal protein concentrations for each comparison (Table 2). Relative to cftr(+/+) mice (set at 100%), the PAS-to-protein ratio of the heavy-density fraction (1.44-1.54 g/ml) was increased 463% in cftr(-/-) mice, suggesting that mucins in this fraction (Muc2 and/or soluble Muc3) were increased in CF knockout mice. Immunoassays also supported this interpretation, because per unit of protein, both anti-D4553 (Muc2) and particularly anti-36W (Muc3) reactivity, were increased in the heavy-density fraction of cftr(-/-) mice. There was also an increase in the light-density fraction of anti-36W and anti-6279 reactivity, but anti-6278 (COOH-tail) reactivity was not increased. Together, these results suggest that within the light-density fraction, the additional Muc3 of CF mice was due to a form of Muc3 containing tandem repeats and some portion of the COOH terminus (epitope 6279), but lacking the COOH-tail (epitope 6278). Thus the overall increase in Muc3 in cftr(-/-) mice appears to involve two different soluble (nonmembrane) forms: a light-density form lacking the COOH-tail domain and the heavy-density form, which lacks both the COOH-tail and the 6279 epitope (and presumably the entire sequence between these two regions). These two putative soluble forms are shown schematically in Fig. 1, b and c.

                              
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Table 2.   Quantitation of Muc2 and Muc3 in heavy- and light-density fractions of CsCl gradients

Immunofluorescence of Muc3 and Muc2 epitopes in mice intestinal sections. There was no overt clinical evidence of jejuneal obstruction in cftr(-/-) mice, but PAS and Alcian blue staining of multiple upper and mid-jejuneal sections showed many morphological features indicative of partial luminal obstruction, including excessive goblet cell emptying (cavitation), luminal accumulation of PAS-positive material, and dramatic crypt luminal dilatation (not presented). The morphology was very similar to that previously reported for ileal sections by Kent et al. (12) and confirmed by Parmley and Gendler (23). The same mucin hypersecretory pathology was observed in 9 of 10 cftr(-/-) mice examined and was absent in all 10 cftr(+/+) control mice. As shown in Fig. 5, Muc2 (anti-D4553) gave a strong signal in goblet cells of cftr(+/+) mice, but in cftr(-/-) mice fluorescence was mainly shifted into the luminal spaces (Fig. 5, a and b). Anti-6279 fluorescence was apically localized in columnar cells of cftr(+/+) sections, but was much more prominent in the lumen in cftr(-/-) sections (Fig. 5, c and d). Immunofluorescence was completely prevented in these sections by preabsorption of the antisera with appropriate peptide antigens (not presented). Localization of anti-6278 was noted in apical regions of cftr(+/+) columnar cells, but in both membrane and luminal positions in cftr(-/-) sections (Fig. 5, e and f). However, the overall staining intensity of the 6278 epitope was decreased in cftr(-/-) sections. Anti-36W stained apical regions of columnar cells and goblet cell granules in cftr(+/+) sections (Fig. 5g). In cftr(-/-) sections, anti-36W fluorescence in the lumen became much more prominent (Fig. 5h). Preimmune serum controls for all of the antibodies used in the above experiments were negative (not presented). Thus cftr(-/-) mice show excessive secretion of Muc2 and release (secretion) of Muc3 from columnar and goblet cells into the lumen of the intestine. The reduced signal for epitope 6278 may indicate that disproportionately less of the membrane-attached form of Muc3 is made in cftr(-/-) mice.


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Fig. 5.   Jejuneal cross sections of cftr(+/+) (a, c, e, and g) and cftr(-/-) (b, d, f, and h) mice with immunofluorescent localization of anti-D4553 (a and b), anti-6279 (c and d), anti-6278 (e and f), and anti-36W (g and h). Magnification: ×400 for a-f and ×250 for g and h.


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Our results suggest that normal rat intestine expresses two forms of Muc3: an apical membrane-associated form and a soluble nonmembrane form. Goblet cells did not react with antibodies specific for the COOH-terminal 6279 and 6278 antibodies, thus producing only the soluble form containing tandem repeats but lacking the transmembrane and COOH-tail domains. Columnar cells reacted with anti-6279 and anti-6278 and the tandem repeat antibody, thus producing the full-length membrane form. (Our results do not rule out the possibility that columnar cells may also express soluble Muc3). These findings explain why the antisense 1220 nt oligonucleotide probe, which was specific for a 3' sequence of Muc3 downstream of epitope 6279 (15), localized the mucin mRNA to columnar cells. Our results also explain why previous reports (2, 3, 29, 37) based on the use of Muc3 tandem repeat probes showed Muc3 to be expressed in both columnar and goblet cells. CsCl density gradient ultracentrifugation and immunoassays of fractions suggest that the lighter density fraction, which reacted with all of the Muc3 antibodies, contains the full-length Muc3 found in columnar cells, whereas the heavy-density fraction, reactive with the tandem repeat antibody alone, is the COOH-terminally truncated form of Muc3 that was detected in goblet cells. Therefore, similar to mucins Muc1, Muc4, and probably also Muc12 (38), Muc3 can now be classified as a transmembrane mucin with the capability of existing in membrane-associated and nonmembrane soluble forms.

Consideration was given to the possibility that Muc3, like rat mammary ascites Muc4, may be composed of two subunits that arise posttranscriptionally from proteolytic cleavage of a large precursor, but remain associated by noncovalent interactions during transport to the apical membrane. Additional proteolytic cleavage to remove the transmembrane region and cytoplasmic tail of ASGP-2 renders the mucin soluble and able to be released as a secreted product (21, 27). The same type of cleavage mechanism could account theoretically for membrane and soluble Muc3 in columnar cells of rodents but is unlikely to account for the presence of only the soluble form of Muc3 in goblet cells. A more likely possibility is that rodent Muc3 mRNA undergoes splice variation, with goblet cell expression restricted to the soluble form. In human MUC3, variants have been reported to arise from one or more splice sites located at 3' genomic exon-intron boundaries located downstream from the first EGF-like sequence (5, 39). Thus the goblet cell form of rodent Muc3 may represent similar 3'-truncated splice variant products of Muc3 as shown in Fig. 1, b and c. This possibility requires further study.

Although the percent distribution of Muc2 and Muc3 epitopes was similar for both mouse groups in CsCl gradient fractions, quantitative slot-blot immunoassays indicated that per total protein, the concentration of both Muc2 and Muc3 (with the exception of the COOH-tail epitope of Muc3) was increased in the intestine of cftr(-/-) mice. This may be due to retention of hypersecreted mucin products in the lumen rather than an increase in mucin synthesis, because Parmley and Gendler (23) did not find increased mRNA levels of these mucins in cftr(-/-) mice. Indeed, Parmley and Gendler (23) reported reduced levels of Muc2 and Muc3 mRNA compared with controls. These results (34) do not rule out the possibility, however, that alternate splice variants (for the soluble form of Muc3) may be upregulated in cftr(-/-) mice, because the primers used to measure Muc3 expression by RT-PCR may not have been appropriate to detect the truncated form. The present findings make increased expression of a splice variant lacking sequence for much of the COOH terminus an attractive possibility, because the tandem repeats of Muc3 were elevated more than the other (more COOH-terminal) epitopes of Muc3. An additional possibility is that cftr(-/-) mice express more of a specific variable number of tandem repeats allele of (soluble) Muc3 (and/or Muc2), so that the polypeptide products have a greater number of tandem repeats than their normal counterparts. These possibilities will also require further study.

Parmley and Gendler (23) found little or no increase in the amount of mucin products in the lumen of cftr(-/-) mice. It is likely that technical factors are responsible for our measured differences in Muc2 and Muc3 products, because we used antibodies that were more capable of recognizing the unique (nonglycosylated) core peptide epitopes of the mucins and a microwave antigen retrieval technique. Parmley and Gendler (23) also showed that double knockout mice lacking both cftr and Muc1 exhibited greatly diminished luminal mucus obstruction compared with Muc1-expressing CF mice (23). Reversion to an obstructive phenotype was produced by reintroduction of transgenic Muc1 into Muc1-deficient CF mice. Parmley and Gendler (23) have speculated that dysfunctional CFTR may somehow increase the expression of Muc1. The present results suggest that dysfunctional CFTR also leads to an increase in Muc2 and Muc3, but do not discriminate between increased expression vs. reduced luminal clearance.

Increased accumulation of mucus in the intestinal lumen is known to play an important role in the chronic obstructive pathology of CF, both in humans and in CF mouse models. Luminal obstruction is generally thought to arise secondarily from effects of the impaired CFTR chloride channel to produce a combination of altered mucin glycosylation, goblet cell hyperplasia, mucus hypersecretion, defective hydration, and impaired clearance of secreted mucins. Faulty chloride and fluid secretion from intestinal crypt enterocytes could account in theory for defective hydration and stasis of secreted mucins in dilated crypt lumina, but there is no satisfactory theory to link the CFTR-dependent chloride channel with changes in goblet cell number or mucin hypersecretion. Recent studies by Larson and colleagues (4, 18, 19) indicate that there may be another explanation for increased glycoconjugate secretion that is not coupled to the defective, cAMP-dependent CFTR chloride channel. Larson et al. (4, 18, 19) have shown that a single in utero dose of recombinant adenovirus containing the cftr gene, when administered to cftr(-/-) mice, permanently prevented the development of lethal intestinal pathology typical of CF. This was accomplished without restoring the cAMP-dependent CFTR chloride channel to normal. These findings led Larson et al. (4, 18, 19) to suggest that the cftr gene participates at a specific period during gestation in the normal developmental regulation of stem cell differentiation into secretory epithelia, chiefly when the cells are engaged in glycoconjugate secretion in both lungs and intestine. Absence of fetal cftr at this period causes later impairment in intracellular storage of glycoconjugates and response to secretory controls. Larson et al. (4, 18, 19) propose that the result in CF is an epithelial cell with activated mucin genes and continuous synthesis, but faulty storage and excessive, poorly regulated secretion. This is an attractive paradigm to explain hypersecretion and luminal mucus obstruction in CF, without the need to link mucin hypersecretion directly to the CFTR chloride channel dysfunction.


    ACKNOWLEDGEMENTS

We thank Dr. Geraldine Kent (Animal Services Division, The Hospital for Sick Children) for supplying CF and control mice and Dr. Gongqiao Xu for supplying the antibody for rat Muc2.


    FOOTNOTES

Financial support was obtained from the Canadian Cystic Fibrosis Foundation (CCFF) and the Medical Research Council of Canada. Catherine Ho was a 1999 summer student research awardee of the CCFF.

Address for reprint requests and other correspondence: J. Forstner, The Hospital for Sick Children, 555 Univ. Ave., Toronto, Ontario, Canada M5G 1X8 (E-mail: jfforst{at}sickkids.on.ca).

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 25 September 2000; accepted in final form 19 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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