Developmental expression of a mucinlike glycoprotein (MUCLIN)
in pancreas and small intestine of CF mice
Robert C.
De Lisle,
Matthew
Petitt,
Kathryn S.
Isom, and
Donna
Ziemer
Department of Anatomy and Cell Biology, University of Kansas Medical
Center, Kansas City, Kansas 66160
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ABSTRACT |
The mucinlike glycoprotein MUCLIN, one
of two protein products of the
CRP-ductin gene, was used to study
changes in the expression of sulfated glycoconjugates during the
pathogenesis of cystic fibrosis, using the cystic fibrosis
transmembrane conductance regulator (CFTR) knockout mouse (CF mouse).
We assessed the appearance of dilated lumina containing protein or
mucus plugs in pancreatic acini and crypts of the small intestine and
quantified MUCLIN protein and
CRP-ductin mRNA during postnatal
development. In CF mice, the pancreatic acinar lumen was dilated by
postnatal day 16 (P16), but MUCLIN protein was first
significantly increased by P23 and
remained elevated through adulthood compared with normal mice.
Similarly, intestinal crypts had CF-like mucus plugs by P16, but MUCLIN protein was first
elevated by P23 and remained elevated
through adulthood compared with normal mice. In both organs, MUCLIN
labeling of the luminal surface was increased concomitantly with
dilation and protein or mucus plugging but before upregulation of
expression. The morphological changes were then followed by upregulation of MUCLIN protein and
CRP-ductin mRNA expression. This is
the first direct study of CF pathogenesis and the resultant increase in
glycoconjugate gene expression. The data are consistent with CF
pathogenesis progressing from an initial alteration in protein
secretory dynamics (increased luminal MUCLIN and protein/mucus plugs)
to an upregulation of glycoprotein/mucin gene expression, which is
expected to exacerbate obstruction of the luminal spaces.
CRP-ductin; cystic fibrosis
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INTRODUCTION |
CYSTIC FIBROSIS (CF) is caused by mutations in the gene
for the cystic fibrosis transmembrane conductance regulator (CFTR), a
cAMP-regulated Cl
channel
(19). Although it is clear that mutations in the CFTR gene that affect
Cl
channel function of the
CFTR protein cause CF, the pathogenesis of this disease is unclear. A
hallmark of CF is the alteration in the amount and composition of mucus
in exocrine secretions. The increase and altered composition of these
glycoconjugates are believed to contribute to obstruction of the
lumina, leading to tissue damage (22). How the loss of functional CFTR
results in the increase in glycoconjugate secretion and altered
carbohydrate composition in affected epithelia is not known.
An animal model for CF is the CFTR knockout mouse
(cftrm1Unc; CF
mouse) (24), which exhibits aggregated secretory material in some
lumina of pancreatic acini (8, 9) and virtually all small intestinal
crypts (12, 23, 24), similar to the pathologies observed in CF patients
(17, 22). In this study we used the recently cloned
CRP-ductin gene and its sulfated
mucinlike glycoprotein product, MUCLIN, to investigate these CF-related
changes. MUCLIN is expressed in several mouse gastrointestinal organs
and exhibits increased expression in the adult CF mouse pancreas and
intestine (8, 9). In both organs of the adult CF mouse, the luminal spaces are dilated and filled with aggregated protein. The luminal membranes are more highly immunoreactive to MUCLIN, and
CRP-ductin mRNA is increased compared
with normal. Thus increased MUCLIN expression is related to the CF-like
morphological changes.
To investigate the relationship between CF pathogenesis and altered
MUCLIN expression, we studied MUCLIN protein and
CRP-ductin mRNA expression and the
appearance of CF-like morphologies during postnatal development in
normal and CF mice. The relative timing of the appearance of CF
pathologies and increased MUCLIN expression should be informative as to
the role of glycoconjugate gene overexpression in the pathogenesis of
CF. Our observations indicate that MUCLIN and its mRNA are upregulated
after the appearance of the dilated lumina in pancreatic acini and
small intestinal crypts. Thus upregulation of this glycoconjugate is
secondary to the loss of functional CFTR, likely in response to
alterations in the affected lumina where these CF-like pathologies
develop. That MUCLIN is upregulated subsequent to dilation of the
acinar lumina and mucus accumulation in the crypt lumina strengthens
the idea that MUCLIN has a mucinlike protective function, as predicted
by its biochemical composition (7-9). Overexpression of
MUCLIN in these circumstances may be inappropriate and may
contribute to the progression of CF by exacerbating obstruction of
these luminal spaces.
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MATERIALS AND METHODS |
Animals.
CFTR(+/
) mice, developed at the University of North Carolina
(cftrm1Unc)
(24), were obtained from Jackson Laboratories (Bar Harbor, ME) and
maintained in our animal care facility. All experiments were performed
in accordance with National Institutes of Health guidelines and were
approved by our Institutional Animal Care and Use Committee.
Heterozygotes were bred to obtain CFTR(+/+) and CFTR(
/
)
mice, which are referred to as normal and CF, respectively. The morning
of birth was considered postnatal day
1 (P1). Both CF and
normal mice were maintained from P10
on Peptamen (Clintek, Deerfield, IL) to prevent intestinal obstruction,
which otherwise results in the death of the majority of the CFTR
knockout animals (8, 10). Genotypes of the mice were determined by PCR
analysis of DNA prepared from tail snips of postweaning animals or from a piece of liver from younger animals (9).
Tissue homogenization and immuno-dot-blot analysis of MUCLIN.
The pancreas and the proximal half of the small intestine (referred to
as small intestine) were homogenized in 10 mM Tris, pH 7.4, with added
protease inhibitors (phenylmethylsulfonyl fluoride, leupeptin,
pepstatin A, and benzamidine). Because the small intestine of young
animals is fragile, we homogenized the whole organ from all time points
rather than obtaining mucosal scrapings as was done previously (9).
This fact accounts for the differences in intestinal MUCLIN levels for
adult tissue reported here compared with our previous work. Protein in
the homogenates was determined by the Bradford method (5), using
reagents from Bio-Rad (Hercules, CA) and BSA as standards. MUCLIN
levels were quantified using an immuno-dot-blot assay with a
monospecific anti-MUCLIN antiserum as previously described (6), with
modifications detailed in Ref. 9.
Glycan detection and Western immunoblot.
The total glycoconjugate composition of pancreas and intestinal tissues
was determined with the use of a glycan detection kit (Boehringer
Mannheim, Indianapolis, IN). Tissue homogenates were separated by
SDS-PAGE, transferred to polyvinylidine difluoride membranes, and
probed for the presence of glycans. Briefly, the blots were treated
with sodium metaperiodate to oxidize carbohydrates, derivatized with
digoxigenin hydrazide, and probed with an antidigoxigenin alkaline
phosphatase-conjugated antibody. Color was developed with
5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. For
Western immunoblots, proteins were separated by SDS-PAGE, transferred
to nitrocellulose, and detected with the anti-MUCLIN antibody and a
goat anti-rabbit alkaline phosphatase secondary antibody.
RNA extraction and Northern blot analysis of
CRP-ductin and CFTR mRNA.
Total RNA was prepared, and Northern blots were performed as previously
described (9). Briefly, blots were probed sequentially with
32P-labeled single-stranded DNA
probes to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and
CRP-ductin (the mRNA that encodes
MUCLIN and a second smaller protein). Radioactivity on the blots was determined with an Ambis radioanalytic detector (Ambis, San Diego, CA)
to quantify 32P. Representative
blots were exposed to X-ray film at
70°C with an
intensifying screen. Northern blots were also performed with a CFTR
single-stranded DNA probe generated from a mouse CFTR cDNA (obtained
from the American Type Culture Collection, Rockville, MD; ATCC no.
63165; see Ref. 26). Probes were generated by PCR (2), using purified
plasmid DNA as a template as previously described (9).
Histology and immunocytochemistry.
Tissue was fixed by immersion in 10% neutral buffered Formalin. For
standard histology, tissue was processed for paraffin sections, which
were stained with hematoxylin and eosin or with periodic acid Schiff
(PAS) for neutral mucus. Immunocytochemistry was performed on 2.5-µm
frozen sections prepared at
40°C on an RMC CR-21 cryostat
(RMC, Tucson, AZ). Frozen sections were sequentially incubated with
anti-MUCLIN antiserum (1:500 in 2% normal goat serum) and goat
anti-rabbit-FITC (Jackson ImmunoResearch, West Grove, PA). The labeled
slides were mounted with SlowFade (Molecular Probes, Eugene, OR) and
observed on a Nikon Diaphot with an FITC filter cube.
Statistical analysis.
Data were analyzed by one-way ANOVA with a post hoc pairwise Tukey
comparison or by paired t-test, using
SYSTAT 7.0 software (Evanston, IL).
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RESULTS |
We have previously reported that in adult CF mice there is a dilation
of some pancreatic acinar lumina and the lumen exhibits protein
aggregates (8, 9). Even more striking, most or all intestinal crypts of
CF mice are dilated and filled with mucus plugs (9, 12, 23). To
determine the time course of CF pathogenesis in the knockout mice, we
examined the morphology of these organs in normal and CF mice from late
fetal development (embryonic day 18.5) through postnatal development and into
adulthood (8-12 wk). Dilations of acinar lumina in CF mice first
became apparent at P8 but were more
common by P16 (Fig.
1,
A' and
B', respectively). These
dilations persisted in the CF pancreas through
P40 (Fig. 1D') and were also observed in
the adult, as previously reported (8, 9). It should be noted that
dilations of acinar lumina were variable and seemed to occur locally in
individual lobules, with some lobules having no evidence of luminal
dilations, as previously reported (9).

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Fig. 1.
Pancreatic morphology and MUCLIN immunocytochemistry in cystic fibrosis
transmembrane conductance regulator (CFTR) knockout mice (CF mice)
during postnatal development. Cryosections were immunostained for
MUCLIN at postnatal day 8 (P8)
(A),
P16
(B),
P23
(C),
P32
(D), and
P40
(E). Tissue was prepared without
permeabilization such that cell surface labeling is predominant and
labeling of zymogen granules is minimal.
A'-E',
corresponding phase-contrast micrographs. MUCLIN is expressed along
apical plasma membrane and on occasional accessible zymogen granules.
Note dilation of the acinar lumen from
P23 to
P40
(C'-E'),
which is strongly labeled for MUCLIN
(C-E). A decrease in the number of
zymogen granules compared with normal (cf. Fig. 2) is apparent at
P32 and
P40
(D' and
E'). Bar = 20 µm.
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Our previous work showed that MUCLIN labeling was strongly enhanced on
the acinar luminal surface of CF mice (8, 9). To assess when this
redistribution of MUCLIN from its predominantly zymogen granule
localization in the normal adult mouse (7) to the luminal membrane in
the CF mouse occurs, we performed immunostaining for MUCLIN. The tissue
was prepared in a manner that does not permeabilize the zymogen
granules and reveals primarily luminal membrane MUCLIN. At all
postnatal times, the luminal membrane of CF pancreatic acini was more
highly immunoreactive for MUCLIN than that of normal pancreatic acini,
and the labeling highlighted the distended lumina and protein
aggregates in the lumen (Fig. 1,
A-E), as previously shown for adult
CF mouse pancreas (8, 9).
We also examined the appearance of normal pancreatic tissue at the same
developmental times. Unexpectedly, the luminal membrane of normal mice
showed an equal degree of dilation at
P16 and
P23 (Fig.
2,
B' and
C', respectively) compared with
the CF mice (Fig. 1, B' and
C', respectively). By
P32 in normal mice, the lumina looked
like those seen in normal adult mice, that is, the lumina were of small
diameter (Fig. 2D'); this was
also true at P40 (Fig. 2E'). As shown in Fig. 2,
A-E, throughout normal postnatal
pancreatic development MUCLIN is localized to the luminal membrane and
the occasionally accessible granule membrane. During the times when the
luminal membranes are dilated in the normal mouse pancreas, MUCLIN is
more patchy along the dilated luminal surfaces, and there is not a
dramatic increase in the level of luminal labeling (Fig. 2,
B and
C). This is in contrast to the
strong labeling observed in the CF mouse pancreas at the same ages
along the dilated luminal surfaces (Fig. 1,
B and
C).

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Fig. 2.
Pancreatic morphology and MUCLIN immunocytochemistry in normal mice
during postnatal development. Cryosections were immunostained for
MUCLIN at P8
(A),
P16
(B),
P23
(C),
P32
(D), and
P40
(E).
A'-E',
corresponding phase-contrast micrographs. MUCLIN is expressed at
moderate levels along apical plasma membrane and on occasional zymogen
granules. Note dilation of the acinar lumen at
P16 and
P23
(B' and
C') but no increase in MUCLIN
immunoreactivity (B and
C). Bar = 20 µm.
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Since the normal and CF mice used were ~95% C57BL/6J and were
maintained on an unusual diet (Peptamen), we determined whether the
observation of dilation of the acinar lumen at
P16-P23 was related to strain or
diet. We evaluated tissue from normal Swiss Webster and 129/SVJ mice
maintained on either standard mouse chow or on a fiber-free pellet diet
(custom formulation in which the fiber was replaced by starch; ICN,
Costa Mesa, CA). In all cases, observations of dilated acinar lumina at
P16-P23 were as common in these
other strains and on different diets as in the normal C57BL/6J mice on
Peptamen (data not shown). Thus transient dilation of the acinar lumen
appears to be a normal postnatal developmental event in mice.
We next measured MUCLIN and CRP-ductin
mRNA levels in pancreata from these same developmental time points.
Pancreatic MUCLIN levels were identical when normal and CF mice were
compared at P8-P16 (Fig.
3). By
P23, there was a small but significant
increase in MUCLIN in the CF pancreas compared with normal. MUCLIN in
the CF mice continued to increase to
P40, and the level was still significantly elevated at adulthood in the CF mouse compared with normal (Fig. 3). In the normal mice at
P32-P40, MUCLIN levels were
slightly and significantly elevated compared with normal P23 and adult mice, but these values
were significantly less than MUCLIN levels in CF mice at the same ages
(Fig. 3). Likewise, CRP-ductin mRNA in
the CF pancreas was slightly elevated at
P23, but the increase was only
significant at P32, as shown in the representative Northern blot in Fig. 4, and
remained significantly elevated through adulthood (Fig.
5). Thus upregulation of MUCLIN and its
mRNA (CRP-ductin) occurs after
dilation of the acinar lumen.

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Fig. 3.
MUCLIN protein expression in normal and CF pancreas during postnatal
development. MUCLIN protein was quantified by immuno-dot-blot analysis
as described in MATERIALS AND METHODS;
n = 5 P8, 8 P16, 5 P23, 7 P32, 5 P40, and 8 adult normal mice, and
n = 3 P8, 7 P16, 6 P23, 7 P32, 7 P40, and 8 adult CF mice. Data are
means ± SE. * P < 0.01 comparing CF with normal mice of same age.
# P < 0.05 comparing normal
mice with normal P16 and adult mice.
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Fig. 4.
Representative Northern blot of
CRP-ductin and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in normal (N) and
CF pancreas at P32. Also shown is blot
probed for GAPDH to demonstrate equal RNA loading.
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Fig. 5.
Quantitation of CRP-ductin mRNA
expression in normal and CF pancreas during postnatal development.
Ambis scanning of Northern blots of total RNA probed with a
single-stranded 32P-labeled
antisense DNA to CRP-ductin. Counts
were normalized to GAPDH mRNA levels obtained on the same blots after
stripping and reprobing with an antisense GAPDH probe;
n = 4 P16, 4 P23, 3 P32, 5 P40, and 5 adult normal mice, and
n = 4 P16, 4 P23, 3 P32, 4 P40, and 5 adult CF mice. Data are
means ± SE. * P < 0.01 comparing CF with normal mice of same age.
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We next examined crypts of the small intestine during development to
determine if the temporal relationship between CF pathology and MUCLIN
expression was the same as in the pancreas. In mice, the intestinal
crypt develops as a morphologically identifiable entity after birth
(13). In our studies, although villi were well developed early on, very
few fully formed crypts were observed at
P8 in either normal or CF mice (Fig.
6, A and
F). By
P16 shallow crypts were evident, and
they increased in depth with age (Fig. 6,
B-E). In normal mice, the crypt
lumina were clear and, although the luminal membrane was PAS reactive,
the lumen itself was clear of PAS-reactive material (Fig. 6,
B-E). In contrast, by
P16 the crypt lumina in CF mice were
PAS reactive (Fig. 6G). The CF
crypts also increased in size, and the degree of mucus accumulation
increased with age (Fig. 6, H-J).

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Fig. 6.
Intestinal morphology during postnatal development and in adult normal
and CF mice. Paraffin sections were processed for routine periodic acid
Schiff (PAS) staining. A-E: normal
mice at P8
(A),
P16
(B),
P23
(C),
P32
(D), and adult
(E).
F-J: CF mice at
P8
(F),
P16
(G),
P23
(H),
P32
(I), and adult
(J). Note that although villi are
present at P8
(A and
F), typical crypts are not apparent
until P16. Normal mice have crypts
that are small and clear throughout development (arrows in
B-E). CF mice from
P16 to adult exhibit PAS-reactive
mucus plugs (arrows in G-J). Bar = 50 µm.
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As determined by immunocytochemistry, there was little or no MUCLIN
labeling of intestine in either normal or CF mice before P16 (data not shown). From
P16 through adulthood in the CF
intestine, there was strong labeling for MUCLIN along the distended
luminal membrane (Fig. 7,
A-C), similar to that shown
previously for adult CF mouse intestine (9). MUCLIN labeling of normal
small intestine was consistently low and associated with the luminal
surface of crypts and the Golgi area of the crypt enterocytes (data not
shown), as in the normal adult intestine (9).

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Fig. 7.
Intestinal morphology and MUCLIN immunocytochemistry in CF mice during
postnatal development. Cryosections were immunostained for MUCLIN at
P16
(A),
P23
(B), and
P32
(C).
A'-C',
corresponding phase-contrast micrographs.
A and
C are longitudinal sections and
B is a cross-section through the
crypts. At all ages past P8 (not
shown), MUCLIN is strongly expressed along the crypt apical plasma
membrane, and there are mucus plugs in the crypt lumina. Bar = 50 µm.
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Expression of MUCLIN and CRP-ductin
mRNA were quantified at the same developmental times in the intestines
of normal and CF mice. MUCLIN levels were identical in normal and CF
intestines between P8 and
P16 (Fig.
8). Thereafter, MUCLIN remained at a low
constant level in normal mice. In the CF intestine, MUCLIN was
significantly increased at P23
compared with normal and increased further through
P40 (Fig. 8). The adult CF intestine
level of MUCLIN was the same as at
P40.
CRP-ductin mRNA levels were slightly and significantly increased at P16
comparing CF with normal (Fig. 9).
Thereafter, CRP-ductin mRNA levels in
the CF intestine remained significantly elevated through adulthood
compared with normal.

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Fig. 8.
MUCLIN expression in normal and CF small intestine during postnatal
development. MUCLIN was quantified by immuno-dot-blot analysis as
described in MATERIALS AND METHODS;
n = 3 P8, 3 P16, 3 P23, 4 P32, 5 P40, and 6 adult normal mice, and
n = 3 P8, 3 P16, 3 P23, 4 P32, 7 P40, and 6 adult CF mice. Data are
means ± SE. * P < 0.01 comparing CF with normal mice of same age.
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Fig. 9.
Quantitation of CRP-ductin mRNA
expression in normal and CF small intestine during postnatal
development. Ambis scanning of Northern blots of total RNA probed with
a single-stranded 32P-labeled DNA
antisense to CRP-ductin and normalized
to GAPDH; n = 5 P16, 4 P23, 8 P32, 6 P40, and 5 adult normal mice, and
n = 4 P16, 5 P23, 4 P32, 3 P40, and 4 adult CF mice. Data are
means ± SE. * P < 0.05;
** P < 0.01 comparing CF with
normal mice of same age.
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Thus, as in the pancreas, the appearance of CF pathology in the small
intestine preceded increases in MUCLIN and
CRP-ductin mRNA. Unlike the pancreas,
there was no transient dilation of crypt lumina or increased MUCLIN
expression in the normal mouse.
To determine whether glycoconjugates other than MUCLIN were affected in
the CF tissues, we used a total glycan detection method on Western
blotted protein. In the pancreas, the most obviously changed
glycoconjugate was MUCLIN (Fig.
10A).
There were some more subtle changes in a broad band of ~155-165
kDa, which was more abundant in P23 CF
tissue, but these changes were not observed at all ages. In addition, a
band of ~95 kDa appeared to be stronger in CF tissue at
P16 through adulthood, although the
differences between normal and CF mice were less than for MUCLIN.

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Fig. 10.
Total glycan detection in normal and CF pancreas and small intestine
during postnatal development. Samples were separated on 5% acrylamide
reducing gels, blotted, and probed for total glycans or MUCLIN.
A: pancreas samples probed for total
glycans (lanes 1-12: 10 µg of
indicated age and genotype per lane) and for MUCLIN (MUC;
lane 13: 1 µg of normal adult pancreas).
B: small intestinal samples probed for
total glycans (10 µg of indicated age and genotype per lane).
C: small intestinal samples probed for
MUCLIN (10 µg indicated age and genotype per lane). Molecular weight
standards are as indicated. Ad, adult.
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In the intestine, again the most apparent change was in the levels of
MUCLIN (Fig. 10B). That the bands
near the top of the blot exhibiting the largest changes were MUCLIN was
verified by immunoblot analysis (Fig.
10C). As previously reported, the
migration of MUCLIN in the CF intestine was slower than in normal
intestine, and this apparent increase in the mass of MUCLIN was most
apparent at P32-P40. More clearly
than in the pancreas, there was an increase in a broad glycoconjugate
band of ~155-165 kDa in the CF intestine (Fig.
10B), which, similar to MUCLIN, was
strongest at P40. This band was not
MUCLIN immunoreactive, so it is likely a distinct glycoprotein. It has
been shown that there is an increase in the number of goblet cells in
the intestine of CF mice (12, 24), and one of the mucins expressed by
these cells is MUC2 (15). The gels we ran were 5% acrylamide, and they
resolved proteins up to ~400 kDa. MUC2 is 550-600 kDa and
therefore would not be expected to enter the gels we used. We attempted
to visualize glycoconjugates with a higher mass using 3% acrylamide
gels, but no distinct bands were observed after the blots were probed
for glycans (data not shown).
Because the CF pathologies examined in this study are due to loss of
the CFTR gene by gene targeting, we wanted to determine the normal
developmental time course of CFTR expression in the mouse. In the mouse
pancreas, CFTR mRNA levels are very low and require use of RT-PCR to
obtain measurable signals (9, 18). In contrast, CFTR mRNA is relatively
abundant in the intestine (9, 12, 25) and is therefore readily
measured. We quantified CFTR mRNA in normal mouse intestinal
development by Northern blot as described in MATERIALS
AND METHODS. As shown in the representative samples in
Fig. 11 and quantitatively in Fig.
12, CFTR mRNA was not detected by
Northern blot in the mouse small intestine at P8. By
P16, CFTR mRNA is measurable, and the
levels increase strongly by P23 and
remain fairly constant thereafter through adulthood (Figs. 11 and 12).
Thus a CF-like pathology in the mouse small intestine quickly appears
at the time when CFTR would normally be expressed in postnatal
development. This indicates that the events we studied are temporally
closely related to the time at which CFTR should first be expressed in
the mouse intestine.

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Fig. 11.
Representative Northern blot of CFTR and GAPDH mRNA in normal small
intestine during postnatal development. CFTR Northern blot of 5 µg
total RNA from mice of indicated ages exposed for 24 h; even after a
2-wk exposure no signal was detected at
P8. Also shown are blot probed for
GAPDH and blot stained with methylene blue before probing to
demonstrate equal total RNA loading.
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Fig. 12.
Quantitation of CFTR mRNA expression in normal small intestine during
postnatal development. Ambis scanning of Northern blots of total RNA
probed with single-stranded
32P-labeled DNA antisense probe to
CFTR and normalized to GAPDH; n = 3 P8, 5 P16, 4 P23, 10 P32, 7 P40, and 5 adult samples. CFTR mRNA was not detectable at
P8.
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DISCUSSION |
CF is well described as a clinically manifested disease, but the early
events in the pathogenesis of CF are largely unknown, principally
because the disease in humans begins in utero (17, 22). To begin
exploring the earliest events in the pathogenesis of CF, we used the
CFTR knockout mouse and examined expression of the mucinlike
glycoprotein MUCLIN in the pancreas and intestine during postnatal
development. MUCLIN is a sulfated mucinlike glycoprotein, which is
abundant in the normal pancreas and intestine (7-9). We have
postulated that MUCLIN is involved in protein packaging in the zymogen
granule by interactions of zymogen aggregates with the negatively
charged sulfates on MUCLIN in the acidic milieu of the trans-Golgi
network (7). In addition, MUCLIN is localized to the luminal plasma
membranes of epithelial cells in several gastrointestinal organs,
including the gallbladder, the pancreas, and crypts throughout the
entire intestinal tract (9). With the exception of the pancreatic
acinar cell, these other cells are not known to have major storage
pools of regulated secretory granules. Therefore, it is likely that
MUCLIN has an additional function when localized to these luminal
membranes. Because of its mucinlike biochemical characteristics
(7, 9), it is probable that MUCLIN serves as a protective
molecule on the mucosal surfaces of digestive organs. The majority of
the peptide motifs in the protein is either predicted to be heavily
O-glycosylated or has numerous
intrachain disulfide bonds, both of which confer protease resistance
(3, 16).
We have documented for the first time that CF-like pathologies first
appear in the pancreas and small intestine during postnatal development
in the mouse (P16-P23), whereas
in humans CF develops in utero (17, 22). The earliest observed changes
were morphological: dilation of the luminal plasma membranes and
increased labeling for MUCLIN and neutral mucus (PAS reactivity) in the
luminal spaces. Subsequent to the morphological changes there were
increases in MUCLIN protein and its mRNA
(CRP-ductin). The morphological
criteria we used cannot reveal any changes in gene expression during
the pathogenesis of CF in this mouse model. Although we were not able to measure an increase in MUCLIN expression at early ages, it is also
not known whether mucin gene expression was increased at the earliest
appearance of CF pathologies. Using glycan detection on Western blots,
we attempted to see if mucins were increased, but we were unable to
resolve very high molecular weight glycoconjugates (>400 kDa) on
polyacrylamide gels (data not shown). This emphasizes the utility of
MUCLIN in these studies, as it is readily resolved on gels
and its mRNA, unlike those of known mucin genes, is also reasonably
easily quantified.
It may be that at these early times both mucins and MUCLIN simply
accumulate on the luminal plasma membrane. It has been proposed that an
alteration in the balance of exocytosis and endocytosis occurs in CF
(4, 21). In CF, endocytosis may not remove luminal plasma membrane to
adequately match the insertion of membrane by exocytosis. This
imbalance would result in dilation of the lumina. It was proposed that
endocytosis is inhibited by abnormal acidity in the luminal spaces due
to loss of CFTR function (21). Alternatively, the luminal membrane may
become dilated due to the accumulation of aggregated protein/mucus in
the lumen which would physically expand this space.
In the CF mouse we observed higher luminal labeling for MUCLIN in both
pancreas and intestine than in normal mice even before there was a
measurable increase in total MUCLIN protein. At the same time, there
was noticeable mucus plug accumulation in the intestine. In the CF
pancreas, MUCLIN labeling appeared more intense than in normal pancreas
as early as P8 (Figs.
1A and
2A, respectively). In the intestine
MUCLIN labeling was weak or absent at
P8 (data not shown) but was strongly
elevated along the luminal surface by
P16 (Fig.
7A) at the same time that mucus
plugs became apparent (Fig. 6G).
Thus our data using MUCLIN labeling as a marker of the luminal membrane
are consistent with the idea that there is an imbalance in exocytosis
and endocytosis in CF. An alternative explanation is that exocytosed
MUCLIN is cleared more slowly in the CF organs at these early
developmental times. This is less likely because there was no increase
in total MUCLIN protein, which would occur if MUCLIN were accumulating
rather than being redistributed from an intracellular site to the
luminal plasma membrane. In support of this hypothesis, we previously
noted that there are fewer zymogen granules in the CF pancreas (8), and this is also apparent by comparison of Figs. 1 and 2. These
observations are consistent with a decrease in recycling of zymogen
granule membrane in the CF pancreas.
Our analysis of CF-like morphological changes in the pancreas was
somewhat less than straightforward in that we discovered that acinar
lumina dilations are a normal but transient event in postnatal
pancreatic development in the mouse. The cause of this dilation is
unknown, but one possibility is that pancreatic ductal function may lag
behind that of the acinar tissue and the luminal space may be
overwhelmed by protein secretion as the mice go through weaning, a time
known to involve significant developmental maturation in mouse
gastrointestinal organs (11). If ductal function lags behind acinar
function, this might be a time when events occur during normal
development that are similar to those that happen in CF, namely,
insufficient ductal bicarbonate and fluid secretion and perturbation of
the environment at the acinar luminal space. However, the normal
pancreas was distinct from the CF in that the dilated lumina of the
normal mice did not exhibit increased labeling for MUCLIN. Despite this
complication, it is clear that in the CF pancreas the dilation persists
and is followed by upregulation of
CRP-ductin mRNA and MUCLIN protein
expression.
On the basis of these new data on MUCLIN expression and CF, our working
hypothesis is that the initial event in CF is altered fluid and
electrolyte secretion by the affected epithelia, which probably results
in a more acidic luminal environment. Initially, this acidic
environment perturbs the dynamics of protein secretion, resulting in
protein redistribution from an intracellular site into the luminal
space and dilation of the luminal plasma membrane. Then, in some way,
this perturbation of the luminal space signals to the epithelial cells
to increase their expression of protective molecules such as MUCLIN.
The increased expression of such glycoconjugates is probably
inappropriate under these circumstances and is believed to contribute
to the progression of the CF pathogenesis (22). Eventually, the luminal
space becomes occluded, resulting in acinar damage in the pancreas (17,
22) and meconium ileus in the intestine (22).
Future goals are to understand how loss of CFTR alters the cellular
distribution of glycoprotein and mucin gene products from intracellular
sites to the luminal plasma membrane and how expression of these genes
is subsequently upregulated in the affected epithelial cells. In
humans, CF pathogenesis in the gastrointestinal system begins in utero,
and CF patients often have severe pancreatic destruction (17, 22) and
meconium ileus at birth (22), whereas in the knockout mouse intestinal
problems become severe only at weaning. This may be explained by the
fact that maturation of the gastrointestinal system is more complete in
utero in humans than in rodents (13). The mildness of pancreatic CF in
the mouse is likely related to the expression of additional genes in
the mouse pancreas, which allows it to be relatively unaffected when CFTR is lost. It has been demonstrated that mouse pancreatic ducts expresses a Ca2+-regulated
Cl
conductance whose
activity can compensate for loss of CFTR (1). Also, there is evidence
that there are other, as yet unidentified, interacting genes in the
mouse that can profoundly modulate the severity of CF in the knockout
mice (12, 14, 20). The fact that CF pathogenesis in the mouse occurs
postnatally is an experimental advantage, as it allows investigation of
the earliest events in CF pathogenesis. Studies of MUCLIN in the CF
mouse should continue to be informative about the pathogenesis of
CF.
 |
ACKNOWLEDGEMENTS |
We thank Amanda Bhattachan for caring for the mouse colony and
genotyping the offspring, and we thank Rosetta Barkley for expert
technical assistance with paraffin sections and histochemical staining.
 |
FOOTNOTES |
This study was supported by the Cystic Fibrosis Foundation and by
National Institute of Diabetes and Digestive and Kidney Diseases Grant
DK-46594. R. C. De Lisle is a Pew Scholar in the Biomedical Sciences.
Address reprint requests to R. C. De Lisle.
Received 1 December 1997; accepted in final form 9 April 1998.
 |
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