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
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
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).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
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).
|
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.
|
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.
|
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).
|
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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Allen, A,
and
Hoskins LC.
Colonic mucus in health and disease.
In: Diseases of the Colon, Rectum and Anal Canal, , edited by Kusner R,
and Shorter RG.. Baltimore, MD: Williams and Wilkins, 1988, p. 65-94.
2.
Audie, JP,
Janin A,
Porchet N,
Copin MC,
Gosselin B,
and
Aubert JP.
Expression of human mucin genes in respiratory, digestive, and reproductive tracts ascertained by in situ hybridization.
J Histochem Cytochem
41:
1479-1485,
1993
3.
Chang, SK,
Dohram AF,
Basbaum CB,
Ho SB,
Tsuda T,
Toribara NW,
Gum JR,
and
Kim SY.
Localization of Mucin (MUC2 and MUC3) messenger RNA and peptide expression in human normal intestine and colon cancer.
Gastroenterology
107:
28-36,
1994[ISI][Medline].
4.
Cohen, JC,
Morrow SL,
Cork RJ,
Delcarpio JB,
and
Larson JE.
Molecular pathophysiology of cystic fibrosis based on the rescued knockout mouse model.
Mol Genet Metab
64:
108-118,
1998[ISI][Medline].
5.
Crawley, SC,
Gum Jr JR,
Hicks JW,
Pratt WS,
Aubert JP,
Swallow DM,
and
Kim YS.
Genomic organization and structure of the 3' region of human MUC3: alternative splicing predicts membrane bound and soluble forms of the mucin.
Biochem Biophys Res Commun
263:
728-736,
1999[ISI][Medline].
6.
Ferretti, E,
Li S,
Wang J,
Post M,
and
Moore A.
Mesenchymal regulation of differentiation of intestinal epithelial cells.
J Pediatr Gastroenterol Nutr
23:
65-73,
1996[ISI][Medline].
7.
Forstner, JF,
and
Forstner GG.
Gastrointestinal mucus.
In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by Johnson LR.. New York: Raven, 1994, p. 1255-1283.
8.
Gum, JR, Jr,
Ho JJL,
Pratt WS,
Hicks JW,
Hill AS,
Vinall LE,
Roberton AM,
Swallow DM,
and
Kim YS.
MUC3 human intestinal mucin analysis of gene structure, the carboxyl terminus, and a novel upstream repetitive region.
J Biol Chem
272:
26678-26686,
1997
9.
Halm, DR,
and
Halm ST.
Secretagogue response of goblet cells and columnar cells in human colonic crypts.
Am J Physiol Cell Physiol
278:
C212-C233,
2000
10.
Henning, SJ.
Functional development of the gastrointestinal tract.
In: Physiology of the Gastrointestinal Tract, edited by Johnson LR.. New York: Raven, 1987, p. 285-300.
11.
Ho, SB,
Niehans GA,
Lyftogt C,
Yan PS,
Cherwitz DL,
and
Gum ET.
Heterogeneity of mucin gene expression in normal and neoplastic tissues.
Cancer Res
53:
641-651,
1993[Abstract].
12.
Kent, G,
Oliver M,
Foskett JK,
Frndova H,
Durie P,
Forstner JF,
Forstner GG,
Riordan JR,
Percy D,
and
Buchwald M.
Phenotypic abnormalities in long-term surviving cystic fibrosis mice.
Pediatr Res
40:
233-241,
1996[Abstract].
13.
Kessler, M,
Acuto O,
Storelli C,
Murer H,
Muller M,
and
Semenza G.
A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes. Their use in investigating some properties of D-glucose and choline transport systems.
Biochim Biophys Acta
506:
136-154,
1978[ISI][Medline].
14.
Khatri, I,
Forstner G,
and
Forstner J.
Preparation of polyclonal antibodies to native and modified mucin antigens.
In: Methods in Molecular Biology. Vol 14. Glycoprotein Analysis in Biomedicine, edited by Hounsell E.. Totowa, NJ: Humana, 1993, p. 225-235.
15.
Khatri, IA,
Forstner GG,
and
Forstner JF.
The carboxyl-terminal sequence of rat intestinal mucin RMuc3 contains a putative transmembrane region and two EGF-like motifs.
Biochim Biophys Acta
1326:
7-11,
1997[ISI][Medline].
16.
Khatri, IA,
Forstner GG,
and
Forstner JF.
Susceptibility of the cysteine-rich N-terminal and C-terminal ends of rat intestinal mucin Muc2 to proteolytic cleavage.
Biochem J
331:
323-330,
1998[ISI][Medline].
17.
Komatsu, M,
Carraway CA,
Fregien NL,
and
Carraway KL.
Reversible disruption of cell-matrix and cell-cell interactions by overexpression of sialomucin complex.
J Biol Chem
272:
33245-33254,
1997
18.
Larson, JE,
Delcarpio JB,
Farberman MM,
Morrow SL,
and
Cohen JC.
CFTR modulates lung secretory cell proliferation and differentiation.
Am J Physiol Lung Cell Mol Physiol
279:
L333-L341,
2000
19.
Larson, JE,
Morrow SL,
Happel L,
Sharp JF,
and
Cohen JC.
Reversal of cystic fibrosis phenotype in mice gene therapy in utero.
Lancet
349:
619-620,
1997[ISI][Medline].
20.
Mantle, M,
and
Allen A.
A colorimetric assay for glycoproteins based on the periodic acid/Schiff stain.
Biochem Soc Trans
6:
607-609,
1978[Medline].
21.
McNeer, RR,
Huang D,
Fregien NL,
and
Carraway KL.
Sialomucin complex in the rat respiratory tract: a model for its role in epithelial protection.
Biochem J
330:
737-744,
1998[ISI][Medline].
22.
Moniaux, N,
Nollet S,
Porchett N,
Degand P,
Laine A,
and
Aubert J.
Complete sequence of the human mucin MUC4: a putative cell membrane-associated mucin.
Biochem J
338:
325-333,
1999[ISI][Medline].
23.
Parmley, RR,
and
Gendler SJ.
Cystic fibrosis mice lacking Muc1 have reduced amounts of intestinal mucus.
J Clin Invest
102:
1798-1806,
1998
24.
Perez-Vilar, J,
and
Hill RL.
The structure and assembly of secreted mucins.
J Biol Chem
274:
31751-31754,
1999
25.
Posnett, DN,
McGrath H,
and
Tam JP.
A novel method for producing anti-peptide antibodies. Production of site-specific antibodies to the T cell antigen receptor beta-chain.
J Biol Chem
263:
1719-1725,
1988
26.
Redinbaugh, MG,
and
Turley BB.
Adaptation of the bicinchoninic acid protein assay for use with microtiter plates and sucrose gradient fractions.
Anal Biochem
153:
267-271,
1986[ISI][Medline].
27.
Rossi, EA,
McNeer RR,
Price-Schiavi SA,
Van den Brande JMH,
Komatsu M,
Thompson JF,
Carraway CAC,
Fregien NL,
and
Carraway KL.
Sialomucin complex, a heterodimeric glycoprotein complex. Expression as a soluble, secretable form in lactating mammary gland and colon.
J Biol Chem
271:
33476-33485,
1996
28.
Schmitz, J,
Preiser H,
Maestracci D,
Ghosh BK,
Cerda JJ,
and
Crane RK.
Purification of the human intestinal brush border membrane.
Biochim Biophys Acta
323:
98-112,
1973[ISI][Medline].
29.
Shekels, LL,
Hunninghake DA,
Tisdales AS,
Gipson IK,
and
Kieliszewski M.
Cloning and characterization of mouse intestinal MUC3 mucin: 3' sequence contains epidermal-growth factor-like domains.
Biochem J
330:
1301-1308,
1998[ISI][Medline].
30.
Sheng, Z,
Wu K,
Carraway KL,
and
Fregien N.
Molecular cloning of the transmembrane component of the 13762 mammary adenocarcinoma sialomucin complex. A new member of the epidermal growth factor superfamily.
J Biol Chem
267:
16341-16346,
1992
31.
Sherblom, AP,
and
Moody CE.
Cell surface sialomucin and resistance to natural cell-mediated cytotoxicity of rat mammary tumor ascites cells.
Cancer Res
46:
4543-4546,
1986[Abstract].
32.
Shi, S,
Key ME,
and
Kalra KL.
Antigen retrieval in Formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.
J Histochem Cytochem
39:
741-748,
1991[Abstract].
33.
Snouwaert, JN,
Brigman KK,
Latour AM,
Malouf NN,
Boucher RC,
Smithies O,
and
Koller BH.
An animal model for cystic fibrosis made by gene targeting.
Science
257:
1083-1088,
1992[ISI][Medline].
34.
Starkey, BJ,
Snary D,
and
Allen A.
Characterization of gastric mucoproteins isolated by equilibrium density-gradient centrifugation in caesium chloride.
Biochem J
141:
633-639,
1974[ISI][Medline].
35.
Steck, PA,
and
Nicolson GL.
Cell surface glycoproteins of 13762NF mammary adenocarcinoma clones of differing metastatic potentials.
Exp Cell Res
147:
255-267,
1983[ISI][Medline].
36.
Stefanini, M,
De Martino C,
and
Zamboni L.
Fixation of ejaculated spermatozoa for electron microscopy.
Nature
216:
173-174,
1967[ISI][Medline].
37.
Weiss, AA,
Babyatsky MW,
Ogata S,
Chen A,
and
Itzkowitz SH.
Expression of MUC2 and MUC3 mRNA in human normal, malignant, and inflammatory intestinal tissues.
J Histochem Cytochem
44:
1161-1166,
1996
38.
Williams, SJ,
McGuckin MA,
Gotley DC,
Eyre HJ,
Sutherland GR,
and
Antalis TM.
Two novel mucin genes down-regulated in colorectal cancer identified by differential display.
Cancer Res
59:
4083-4089,
1999
39.
Williams, SJ,
Munster DJ,
Quin RJ,
Gotley DC,
and
McGuckin MA.
The MUC3 gene encodes a transmembrane mucin and is alternatively spliced.
Biochem Biophys Res Commun
261:
83-89,
1999[ISI][Medline].
40.
Wu, K,
Salas PJ,
Yee L,
Fregien N,
and
Carraway KL.
Tissue and tumor expression of a cell surface glycoprotein complex containing an integral membrane glycoprotein activator of p185neu.
Oncogene
9:
3139-3147,
1994[ISI][Medline].
41.
Xu, G,
Huan L,
Khatri IA,
Wang D,
Bennick A,
Fahim REF,
Forstner GG,
and
Forstner JF.
cDNA for the carboxyl-terminal region of a rat intestinal mucin-like peptide.
J Biol Chem
267:
5401-5407,
1992