Am J Physiol Gastrointest Liver Physiol 284: G853–G862, 2003.
First published January 15, 2003; 10.1152/ajpgi.00326.2002.
Novel MUC1 splice variants contribute to mucin
overexpression in CFTR-deficient mice
A. Marina Hinojosa-Kurtzberg,1 Malin E. V. Johansson,2 Cathy S. Madsen,1
Gunnar C. Hansson,2 and Sandra J. Gendler1
1
Mayo Clinic Scottsdale, Department of Biochemistry and Molecular Biology, Scottsdale, Arizona 85259;
and 2Göteborg University, Department of Medical Biochemistry, 413 90 Gothenburg, Sweden
Submitted 6 August 2002; accepted in final form 8 January 2003
Hinojosa-Kurtzberg, A. Marina, Malin E. V. Johans-
son, Cathy S. Madsen, Gunnar C. Hansson, and Sandra
J. Gendler. Novel MUC1 splice variants contribute to mucin
overexpressioninCFTR-deficientmice. AmJPhysiolGastroin-
test Liver Physiol 284: G853–G862, 2003. First published
January 15, 2003; 10.1152/ajpgi.00326.2002.—A cystic fibro-
sis (CF) mouse expressing the human mucin MUC1 trans-
gene (CFM) reverted the CF/Muc1⫺/⫺ phenotype (little mu-
cus accumulated in the intestine) to that of CF mice express-
ing mouse Muc1, which exhibited increased mucus
accumulation. Western blots and immunohistochemical anal-
ysis showed that the MUC1 protein was markedly increased
in CFM mice in which it was both membrane bound and
secreted into the intestinal lumen. Studies to determine the
reason for increased levels of the extracellular domain of
MUC1 mucin identified mRNA and protein of two novel
splice variants and the previously described secreted MUC1
lacking the cytoplasmic tail (MUC1/SEC). Novel MUC1
splice variants, CT80 and CT58, were both transmembrane
proteins with cytoplasmic tails different from the normal
MUC1. The MUC1-CT80 and MUC1/SEC forms are found
expressed mainly in the CFM mice intestines. Thus MUC1
expression is increased, and it appears that alternate cyto-
plasmic tails may change its role in signaling. MUC1 could be
an important contributor to the CF intestinal phenotype.
cystic fibrosis; intestinal obstruction; small intestine; gene
splicing
CYSTIC FIBROSIS (CF) is caused by defects in the CF
transmembrane conductance regulator (CFTR) ion
channel. Some of the clinical manifestations of the
disease are mucus accumulation in the respiratory and
gastrointestinal tracts. Normally, a layer of mucus
covers the surface of the gastrointestinal tract, which
is indispensable to maintain the integrity of the intes-
tinal mucosa. In CF, the mucus deposits (seemingly a
mixture of several mucins) are one of the pleiotropic
manifestations of the disease. The excessive mucus
results in severe intestinal obstruction at birth, known
as meconium ileus. Intestinal obstruction can also oc-
cur later in life. Several mucins have been found to be
important in the normal or diseased intestine. These
include the gel-forming secreted mucins MUC2,
MUC5B, and MUC5AC (4, 10, 11, 49) and the nongel-
Address for reprint requests and other correspondence: S. J. Gen-
dler, Mayo Clinic Scottsdale, Dept. of Biochemistry and Molecular
Biology, S. C. Johnson Medical Research Center, 13400 E. Shea
Blvd., Scottsdale, AZ 85259 (E-mail: gendler.sandra@mayo.edu).
http://www.ajpgi.org 0193-1857/03 $5.00 Copyright © 2003 the American Physiological Society
forming membrane bound or secreted mucins MUC1,
-3, -4, -12, -13, and -17 (6, 13, 19, 39, 44, 50, 52). Some
biochemical and immunological analyses of mucus ob-
tained from CF patients suggest abnormalities in mu-
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cus glycosylation, production, and secretion, but there
is a lack of knowledge of specific alterations in mucin
expression. It is unclear how the absence of a func-
tional CFTR protein could modify the pattern of ex-
pression of some epithelial mucins. The defective ion
transport in CF may contribute to a reduction in the
volume of airway surface liquid (29) or alter the salt
content of the airway surface liquid (43), and either of
these situations would change the milieu into which
the mucus is secreted. Other studies on CF mice have
suggested that the absence of CFTR in the lungs and
intestines perturbs normal differentiation of a secre-
tory cell population (7, 12, 22). These situations would
potentially facilitate abnormal mucin/mucus produc-
tion.
CFTR mutant mice (Cftrtm1UNC/Cftrtm1UNC), called
CF mice, exhibit intestinal goblet cell hyperplasia and
dilated crypts with excessive mucus accumulation and
obstruction mimicking human meconium ileus, but
they do not show mucus accumulation in the lungs (45,
55). Interestingly, when CF mice lacking the mucin
Muc1 were analyzed, the intestinal mucus accumula-
tion was decreased compared with Muc1-expressing
CF mice (34). The human mucin is designated as
MUC1, whereas the mouse homologue is designated
Muc1. Despite the fact that Muc1 is expressed at low
levels in the normal intestine and that it was originally
described as a transmembrane mucin, it appears to
play a role in the accumulation of intestinal mucus in
CF mice (34). MUC1 has a large extracellular domain
(1,000–2,000 amino acids), a hydrophobic membrane-
spanning domain, and a tyrosine-phosphorylated cyto-
plasmic domain of 72 amino acids (17, 18, 27). MUC1 is
overexpressed in mammary tumors (18, 27), in colon
cancer (3, 10), and in other malignancies (56), and
soluble forms exist in tissue culture supernatants and
body fluids (1, 6). The MUC1 cytoplasmic tail (CT)
interacts with many proteins involved in signal trans-
duction and cell adhesion, including ␤-catenin, p120ctr,
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.
G853
G854 MUC1 SPLICE VARIANTS IN CF MUCUS
glycogen synthase kinase-3␤ (GSK-3␤), c-src, Grb2/
SOS, PKC␦, epidermal growth factor receptor (EGFR),
erbB2, erbB3, and erbB4 (23–26, 33, 37, 38, 41, 53).
Our hypothesis is that Muc1 is participating in the
intestinal mucus obstruction in the CF mice, it is
consequently increased in the CF intestines, and an
alternative shed or secreted MUC1 form is aberrantly
produced under the altered environment generated by
the absence of a functional CFTR. Here, we demon-
strate the contribution of MUC1 to the CF intestinal
phenotype. We show 1) the differential expression of
MUC1 at the mRNA and protein level in CF MUC1
transgenic (CFM) mice vs. the control MUC1 trans-
genic (MUC1.Tg) mice with an intact CFTR, 2) histo-
logical localization of MUC1 in the intestinal lumen of
CFM mice, 3) the presence of increased MUC1/SEC
(message and protein) in the CFM mice, and 4) the
existence and characterization of a new MUC1 splice
variant (MUC1-CT80) that shows elevated mRNA and
protein levels in the CFM mice. Additionally, the dif-
ferential expression of the MUC1 forms along the in-
testinal tract consistently demonstrate that MUC1 is
aberrantly expressed in the small intestine of CFM
mice in which expression resembles large intestine
autochthonous MUC1 expression. These findings are
consistent not only with a MUC1 role in the CF intes-
tinal phenotype but also suggest that CFTR has the
ability to affect the nature and amount of mucus secre-
tion of certain epithelial cells. Whether this is directly
related to CFTR function or indirectly due to secondary
phenomena caused by the lack of CFTR is still to be
defined.
MATERIALS AND METHODS
Animals. All studies were performed on inbred C57BL/6
mice containing either wild-type CFTR or mutant CFTR
(Cftrtm1UNC/Cftrtm1UNC) (45), the latter referring to the CF
mouse. Mice lacking Muc1 (46) were crossed with CF mice.
The resulting CF/Muc1⫺/⫺ mice were mated with MUC1.Tg
mice (40) in a Muc1⫺/⫺ background, to produce the CFM
mouse (Cftrtm1UNC/Cftrtm1UNC, Muc1⫺/⫺, MUC1⫹). The con-
trol mice were MUC1.Tg in a Muc1⫺/⫺ background with a
functional CFTR (40). All mice were maintained in specific
pathogen-free conditions in a barrier facility at Mayo Clinic
Scottsdale (Natalie Schafer Transgenic Animal Facility).
Mice 3- to 4-wk-old were weaned onto a mouse liquid diet
(product no. F3017; BioServ, Frenchtown, NJ), and sterile
water was provided ad libitum. All experimental procedures
were conducted according to Institutional Animal Care and
Use Committee guidelines. Sixty-five animals were studied,
ages 2 wk to 11 mo. CFM, MUC1.Tg, and CF/Muc1⫺/⫺ mice
were age matched within 1–2 days when younger than 8 wk,
and older animals were matched within 1–2 wk.
Intestinal lysates. Intestinal lysates were prepared from
CFM mice (controls MUC1.Tg and CF/Muc1⫺/⫺). The small
intestine (defined as the portion directly below the duodenum
and above the cecum) was divided into two equal parts; the
proximal part was taken as jejunum and the distal part as
the ileum. The colon was the portion between the cecum and
the anus. Intestinal segments were opened longitudinally
and cleaned with cold PBS. The lumenal mucus was scraped
away from the intestinal lumen and homogenized in Triton
X-100 lysis buffer of (in mM): 20 HEPES (pH 8.0), 150 NaCl,
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2 EDTA, 2 sodium orthovanadate, and 10 sodium fluoride,
with 50 ␮M ammonium molybdate and 1% Triton X-100, plus
complete inhibitor mixture (Sigma, St. Louis, MO). Protein
lysates were centrifuged to separate cell debris. Protein con-
centration was determined using the bicinchoninic acid assay
(Pierce) and samples were stored at ⫺80°C.
Antibodies. We used three MAbs to recognize MUC1.
HMFG-2 (kindly provided by Dr. Joyce Taylor-Papadimi-
triou, Imperial Cancer Research Fund, London, UK) recog-
nizes the epitope DTR (9) in the extracellular domain
(MUC1-EX). B27.29 (kindly provided by Biomira) recognizes
the epitope PDTRPAP (36) also in the MUC1-EX. CT2 rec-
ognizes an epitope within the last 17 amino acids of the
MUC1 cytoplasmic domain (MUC1-CT) (41). A chicken poly-
clonal antibody (PAb) directed to a synthetic peptide of a
unique (underlined) MUC1/SEC sequence (5⬘-CGGVSIGLS-
FMPLP-3⬘) (44) and a rabbit PAb (CT80) directed to unique
(underlined) COOH-terminal sequence of the splice variant
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MUC1-CT80 (5⬘-CGKDSEGGTWKTQRAWKR-3⬘) were pro-
duced. The amino acids C or CGG were added to the amino
terminal peptide sequences to facilitate their conjugation to
keyhole limpet hemocyanin.
Western blots were performed using 25 ␮g of protein lysate
per sample detected with the antibodies HMFG-2, B27.29,
and MUC1/SEC, and 200 ␮g of protein lysate per sample for
CT2 and CT80. Samples were separated by SDS-PAGE and
transferred to polyvinylidene diflouride membrane (Immo-
bilon-P; Millipore) for Western blot analysis. Negative con-
trols included antibody-specific peptide blocking and Muc1
knockout tissues. Dilutions of the antibodies were as follows:
CT2 (supernatant) 1:100, HMFG-2 (supernatant) 1:20,
MUC1-SEC 1:1,000 (serum), and CT80 1:1,000 (serum).
Mucin deglycosylation. Partial deglycosylation of MUC1
was carried out by immersing the immobilized protein on
Immobilon-P membrane in liquid trifluoromethanesulfonic
acid (TFMSA; Aldrich) for 6 h at 4°C (2). The membrane was
neutralized by washing in 50 mM Tris 䡠 HCl pH 8 for 30 s and
blocked before Western analysis as described above.
Immunohistochemistry. Four longitudinal sections of 2 cm
each and four transverse sections each of jejunum, ileum, and
colon of CFM mice ranging in age from 2 wk to 11 mo were
analyzed. Control MUC1.Tg and CF/Muc1⫺/⫺ mice were an-
alyzed the same way. These tissues were carefully handled to
prevent mechanical shearing of the intestinal mucus. Tissues
were fixed in methacarn, embedded in paraffin, and 5-␮m
sections were cut from different areas of the small intestine.
Before immunostaining with MAb CT2, tissues were treated
with antigen retrieval reagent BD Retrievagen A, pH 6.5,
according to the manufacturer’s instructions (Pharmingen,
San Diego, CA), blocked for endogenous peroxidase activity,
blocked in normal goat serum, and incubated with primary
antibodies overnight at 4°C. Slides were washed in enhanc-
ing wash buffer (Immunex), incubated with horseradish
peroxidase-conjugated secondary antibodies, washed in en-
hancing wash buffer again, and developed with 3⬘,3⬘-diami-
nobenzidine (Santa Cruz Biotechnology, Santa Cruz, CA) and
counterstained with Meyers hematoxylin (Sigma). Negative
controls included antibody-specific peptide blocking and
Muc1⫺/⫺ intestinal tissues. Antibodies were diluted as fol-
lows: CT2, 1:100 and MUC1/SEC, 1:300.
RNA extraction, RT-PCR, cloning, and DNA sequence
analysis. Total RNA was extracted from the jejunum, ileum,
and colon of CFM and MUC1.Tg mice, using the TRIzol
reagent (Invitrogen, Carlsbad, CA). Samples of 20 ␮g of total
RNA were treated with DNase (DNA-free kit; Ambion, Aus-
tin, TX) before cDNA synthesis. RT was done as previously
described (34). The cDNA was aliquoted and stored at ⫺70°C.
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 MUC1 SPLICE VARIANTS IN CF MUCUS
A single batch of cDNA was used for all the PCR results
shown here. Each experiment was repeated three times with
different batches of cDNA to confirm the consistency of the
results. The Expand High Fidelity PCR System (Roche Diag-
nostics, Indianapolis, IN) was used for all PCR reactions, as
recommended by the manufacturer for a 100-␮l volume. To
compare the presence or absence of transcripts compared
with a known RNA, we used a semiquantitative RT-PCR
approach featuring low-cycle number amplification (25 cy-
cles) and a mixture of actin primers/actin competimers (Am-
bion) as amplification reference. For this calibration control,
a 2:7.6 ratio of primers/competimers (0.5 ␮l of premixed actin
primers and 1.9 ␮l of actin competimers) was used for all the
RT-PCRs. Amplification conditions included an initial dena-
turing at 94°C for 5 min followed by 25 cycles of denaturing
94°C (30 s), annealing 57°C (1 min), and terminating 72°C (2
min) for MUC1, and MUC1 splice variants. For MUC1/SEC
the annealing temperature was 61°C (1 min). Routine con-
trols for all RT-PCRs included omission of the RT. Amplified
products were cloned and sequenced to confirm their identi-
ties.
The following primers were used for RT-PCR analysis: For
MUC1/SEC, primers were designed from the MUC1 (Gen-
Bank accession no. X52228) sequence: P2 (sense), 5⬘-GGTAC-
CTCCTCTCACCTCCTCCAA-3⬘ (900–923 bp) (32), and
P7 (antisense), 5⬘-GGGGAAGGAAAGGCCGATACTCAC-3⬘
(1089–1066 bp); a 190-bp product was amplified. For classic
MUC1 and MUC1 splice variants, primers were designed
from the MUC1 genomic sequence (GenBank accession no.
M61170 version gi:2055365). For classic MUC1, the primers
were Ex5 (sense), 5⬘-TGCTGGTCTGTGTTCTGGTTGC-3⬘
(4986–5007 bps) and Ex7 (antisense), 5⬘-TTGGCAGAAGTG-
GCTGCCACT-3⬘ (complementary to 6361–6341 bp), gener-
ating a 265-bp fragment. To detect MUC1 splice variants, the
primers were Ex5 (described above) and InVI (antisense), 5⬘-
CTTCTTCTTGCTTCTACCTGGG-3⬘ (5755–5734 bp).
RESULTS
Phenotype of the CFM mouse corresponds to the orig-
inal phenotype of the CF mouse. We (34) previously
described that Cftrtm1UNC/Cftrtm1UNC (CF) mice lack-
ing Muc1 exhibited a phenotype with decreased in-
testinal mucus accumulation compared with the CF
mice. To further pursue the role of MUC1 in the mu-
cus accumulation, we introduced the human MUC1
gene into the Muc1⫺/⫺ mice by mating the CF/Muc1⫺/⫺
with the MUC1.Tg/Muc1⫺/⫺ mice to generate the
Cftrtm1UNC/CftrtmUNC, Muc1⫺/⫺, MUC1⫹ (CFM) mice.
This facilitated the detection of MUC1, because there
is a lack of antibodies for the extracellular domain of
Muc1, and several well-characterized antibodies to hu-
man MUC1 are available. The MUC1.Tg mouse con-
tains a 10-kb piece of genomic DNA comprising the
entire MUC1 gene and regulatory sequences, and it
expresses MUC1 at normal levels, conserving its tissue
specificity (40). Intestinal sections of CFM and the
control MUC1.Tg mice were stained with Alcian blue
and compared with the sections previously obtained
from CF and wild-type mice (34). Intestinal mucus
accumulation was similar between CFM and CF mice
(Fig. 1). The CFM mouse also presents goblet cell
hyperplasia and dilation of the crypts filled with mucus
as well as the physiological phenotype of early death of
the pups as documented with the CF mouse (data not
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Fig. 1. Replacement of mouse Muc1 with human MUC1 in cystic
fibrosis (CF) mice. Representative Alcian blue-stained intestinal
sections of the CF mice expressing the MUC1 gene (CFM) mice
(Cftrtm1UNC/Cftrtm1UNC, MUC1⫹, Muc1⫺/⫺) (C and D) display the
same intestinal phenotype of lumenal mucus accumulation as the CF
mouse with its native Muc1 (Cftrtm1UNC/Cftrtm1UNC) (A and B).
shown) (45, 55). Likewise, the life span, percentage of
survival, and general health of the CFM mice housed
under the same conditions as the CF mice have proven
to be similar for both colonies over a period of 2.5 yr
(data not shown).
MUC1-EX is substantially increased in the intestines
of the CFM mice. We examined the CFM mice vs.
MUC1.Tg mice for MUC1 protein expression along the
intestinal lumen. We performed Western blots from
intestinal mucus lysates with MUC1 MAbs directed
either to the MUC1-EX (B27.29 and HMFG-2) or to the
MUC1-CT (CT2). Immunoblotting with MAbs to the
MUC1-EX revealed a noticeable increase in the levels
of MUC1 protein mainly in the jejunum and ileum of
the CFM mice (Fig. 2, A and B). The detected MUC1
protein appears heterogeneous in size, probably due to
variable O-linked glycosylation. The MAb B27.29,
which reacts with the heavily glycosylated MUC1-EX
(42), shows the most dramatic MUC1 increase in the
small intestine of the CFM mice (Fig. 2A). Further-
more, an identical Western blot probed with MAb
HMFG-2, which reacts with an epitope influenced by
the length of the oligosaccharide side chains (8),
showed no reaction (data not shown). However, when a
similarly prepared membrane blot was first partially
deglycosylated with TFMSA and then immunoreacted
with MAb HMFG-2, MUC1 reactivity was found (Fig.
2B). This reaction with MAb HMFG-2 also showed that
MUC1 is augmented in the jejunum of the CFM mice
and that the ileum and colon of both animals present
very similar amounts of MUC1 (Fig. 2B). The immu-
noblots with MAb CT2 (Fig. 2C) revealed that the
MUC1-CT is also increased in the CFM intestines. In
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G856 MUC1 SPLICE VARIANTS IN CF MUCUS
Fig. 2. Immunoblotting evidence of in-
creased MUC1 in the CFM intestines.
Intestinal mucosal lysates (25 ␮g) from
CFM and MUC1 transgenic (MUC1.Tg)
mice were immunoblotted with either
MUC1 extracellular domain (MUC1-
EX) MAb B27.29 (A) or (200 ␮g) with
MUC1 cytoplasmic tail (CT) MAb CT2
(C). A polyvinylidene diflouride mem-
brane containing the same lysates
shown on (A) was deglycosylated and
immunoblotted with the MUC1-EX
MAb HMFG2 (B), the binding of which
is influenced by the length of oligosac-
charide chains on MUC1.
this case, MUC1-CT was increased in jejunum, ileum,
and colon of the CFM mice. Furthermore, in CFM mice
MUC1-CT was more abundant in the colon, whereas
MUC1-EX was more copious in the jejunum. In the
jejunum of CFM mice, when MUC1-EX showed the
highest accumulation (Fig. 2A), MUC1-CT was barely
detectable (Fig. 2C).
MUC1-EX is localized abundantly in the crypts and
in the mucus secreted into the intestinal lumen. To
study the accumulation of MUC1 in the intestine, im-
munostaining with several MUC1 MAbs was per-
formed. Figure 3, A and E, shows a representative
Alcian blue stain of the mucus accumulated in the
lumen of the small intestine in the CFM and CF/
Muc1⫺/⫺ mice. The immunohistochemical analysis of
intestinal sections of CFM mice with MAb B27.29 dis-
tinctly showed MUC1 protein, not only on the cell
membrane of the villi and crypts, but also in the se-
creted mucus accumulated in the lumen of the small
intestine (Fig. 3B). That the reactivity was not due to
nonspecific antibody-mucus interaction was shown by
the lack of staining in CF/Muc1⫺/⫺ mice containing
some accumulated mucus but not MUC1 (Fig. 3F).
Immunostaining with CT2 also revealed MUC1 in the
cell membrane of villi and crypt cells with very faint
staining of the lumenal mucus (Fig. 3C). Also in this
case, the mucus lacking MUC1 did not react with MAb
CT2 (Fig. 3G). These results are in agreement with the
results obtained in the Western blots of intestinal
mucus lysates with MAb CT2. In both, a strong reac-
tion in the small intestine with MUC1-EX was associ-
ated with a weak reaction with MUC1-CT (Fig. 2, A
and C). Comparable stains performed in colon sections
of CFM and MUC1.Tg animals showed no clear differ-
ence (data not shown).
Two novel MUC1 splice variants, MUC1-CT80 and
MUC1-CT58, were identified. Because the small intes-
tine of CFM mice showed a strong reaction with
MUC1-EX accompanied with a weak reaction of its
cytoplasmic domain (by immunoblotting and immuno-
histochemistry), we postulated the presence of a se-
creted MUC1 form as one of the constituents of the
lumenal mucus. We used a semiquantitative RT-PCR
approach, featuring low cycle number amplification (25
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cycles) and actin for amplification reference, to search
for transcripts of MUC1 alternate forms and to com-
pare their expression profiles in CFM and MUC1.Tg
mice. First, the normal transmembrane form of MUC1
was amplified by using primers unique to this sequence
(Ex5-FOR, Ex7-REV). MUC1 levels appear to be in-
creased in both jejunum and ileum of the CFM mice,
but not in the colon.
One of the known MUC1 splice variants is MUC1/
SEC, which lacks the cytoplasmic tail and has been
described in breast cancer cells (44) and in human
endometrial glands (1). The MUC1/SEC mucin is
partly encoded by intron 2, which results in 11 unique
amino acids (VSIGLSFPMLP) at its COOH terminus.
We used the P2 and P7 primers to amplify MUC1/SEC,
which would not amplify the classic transmembrane
MUC1 (Fig. 5A). RT-PCR using 25 cycles revealed
MUC1/SEC mRNA in the jejunum, ileum, and colon of
CFM mice, but only in the ileum and colon of the
MUC1.Tg mice (Fig. 4B). When the RT-PCR number of
cycles was taken up to 40, the MUC1/SEC message was
also found in jejunum of MUC1.Tg mice (not shown).
The expression pattern of MUC1/SEC and MUC1 ap-
peared fairly similar along the small and large intes-
tines of the CFM mice (Fig. 4, A and B). This pattern
differed from the one obtained in the MUC1.Tg mice, in
which MUC1 and MUC1/SEC messages were ex-
pressed in the colon, very little were expressed in the
ileum, and no expression could be detected in the
jejunum (Fig. 4, A and B).
Using the same RT-PCR approach, we further
screened the region between the MUC1 transmem-
brane domain in exon 5 and sequences in intron 6 (Fig.
5A). The classic MUC1-CT is comprised of 48 amino
acids in exon 6 and 24 amino acids in exon 7. We
detected two novel splice variants of the MUC1-CT, a
689-bp transcript called MUC1-CT80 and a 280-bp
transcript called MUC1-CT58 (Fig. 4C). The MUC1-
CT80 transcript was found in the jejunum, ileum, and
colon of the CFM mice but only in the colon of
MUC1.Tg mice (Fig. 4C). RT-PCR reactions taken to 40
cycles failed to produce the MUC1-CT80 transcript in
small intestines of MUC1.Tg mice (data not shown).
The shorter transcript, MUC1-CT58, was observed in
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 MUC1 SPLICE VARIANTS IN CF MUCUS
Fig. 3. Immunohistochemical localization of MUC1-EX in the
CFM small intestine. Representative small intestine sections of
CFM (A–D), and CF/Muc1⫺/⫺(E–H) mice were stained with Alcian
blue (A and E) to detect the localization of lumenal mucus (L) or
were immunostained with the MUC1-EX MAb B27.29 (B and F),
MUC1-CT MAb CT2 (C and G), and MUC1/SEC PAb (D and H).
The CF/Muc1⫺/⫺ mice displayed no staining with either antibody
(F, G, and H). Higher magnification of B shows abundant
MUC1-EX secreted into the intestinal lumen (red arrow) as well
as its localization on the cell membrane (blue arrow). The higher
magnification of C shows the intense staining of cell membrane-
associated MUC1-CT (blue arrow), which appears scarce in the
lumen. Higher magnification of D indicates MUC1/SEC present in
the intestinal lumen (red arrows).
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Fig. 4. Semiquantitative RT-PCR of transcripts for MUC1 and 3 of
its isoforms differentially expressed in the CFM and MUC1.Tg in-
testines. Representative reactions of a semiquantitative RT-PCR
approach featuring low-cycle amplification (25 cycles) and actin as an
amplification reference are presented in A–C to show the expression
of different MUC1 transcripts in MUC1.Tg and CFM intestines. The
arrows point to the RT-PCR amplicons for 265-bp MUC1 (A), 190-bp
MUC1/SEC (B), and 689-bp MUC1-CT80 and 280-bp MUC1-CT58
(C). Increased expression of MUC1 (A) and MUC1/SEC (B) is ob-
served in the jejunum, ileum, and colon in the CFM mice, but only in
the ileum and colon of MUC1.Tg mice. MUC1-CT80 (C) is observed in
the CFM intestines, but only in the colon in the MUC1.Tg mice.
MUC1-CT58 is present along the intestines of CFM and MUC1.Tg
mice.
the jejunum, ileum, and colon of both types of mice
with higher expression in MUC1.Tg mice (Fig. 4C).
The predicted proteins for the splice variants would
have the following general characteristics (shown
in Fig. 5). MUC1-CT58 (GenBank accession no.
AF423031) and MUC1-CT80 (GenBank accession no.
AF423030) share identical coding sequences with
MUC1, from 1 to 5265 bp (GenBank accession no.
M61170.2 GI: 15321729). Thereafter, the clone MUC1-
CT58 has a new exon (exon 6b) located between MUC1
exon 6 and MUC1 exon 7 (Fig. 5A). In MUC1-CT58, the
first 420 bp of MUC1 intron 6 are spliced out and the
new exon 6b starts at 5686 bps and continues at least
60 more residues within the same MUC1 intron 6
sequence. The clone MUC1-CT80 has a new exon in
place of MUC1 exon 6, named 6a (Fig. 5A). MUC1 exon
6 ends at 5265 bp but the new exon 6a continues to
5746 bp. The splice donor and acceptor sites conform to
known consensus sequences (see GenBank accession
nos. AF423031 and AF423030 for specific sites). The
first 48 amino acids at the 5⬘ end of the cytoplasmic tail
are the same in MUC1, MUC1-CT58, and MUC1-
CT80, because they are coded within exon 6 that is
unaltered in the splice variants (Fig. 5B). Exon 6b in
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G858 MUC1 SPLICE VARIANTS IN CF MUCUS

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Fig. 5. Gene structure and nucleotide sequence for MUC1, MUC1/SEC, and the novel MUC1-CT splice variants
MUC1-CT58 and MUC1-CT80. A: Arabic numbers designate exons and the Roman numerals denote introns.
Primers used for RT-PCR are marked. UTR, 3⬘ untranslated region; VNTR, variable number of tandem repeats;
TM, transmembrane domain. B: comparison of MUC1-CT and the predicted sequences for the MUC1-CT splice
variants. Sites for characterized binding proteins are shown on the MUC1 sequences.

MUC1-CT58 generates a 3⬘ end of the cytoplasmic tail

with 10 unique amino acids substituting for the 3⬘ end
24 amino acids of the cytoplasmic tail (Fig. 5B). Exon
6a in MUC1-CT80 generates a cytoplasmic tail with 32
unique amino acids substituting for the 3⬘ end 24
amino acids of the cytoplasmic tail (Fig. 5B). The sites
for ␤-catenin and Grb2 interactions are therefore lack-
ing in both of the splice variants.
Sequencing the newly found transcripts revealed
several differences with the published sequence of
MUC1 intron 6 (GenBank accession no. M61170 ver-
sion GI: 2055365). We did extensive resequencing of
that region of the genomic DNA with several sense and
antisense primers and confirmed that the differences
were errors in the published intron 6 sequence. The
correct sequence has been submitted to GenBank and
the update can be viewed as accession no. M61170.2
GI: 15321729.
MUC1-CT80 is translated into a protein expressed
mainly in CFM intestines. To confirm that the MUC1-
CT80 transcript was translated into the corresponding
protein, a rabbit PAb (CT80) directed to a unique
sequence of MUC1-CT80 was used. This antiserum
was tested by immunoblotting and immunostainning
of CFM, MUC1.Tg, and CF/Muc1⫺/⫺ intestinal lysates
and fixed tissues, respectively. Intestinal lysates were
immunoblotted with the CT80 antiserum, revealing Fig. 6. Immunoblotting evidence of MUC1-CT80 and MUC1/SEC
several forms of the protein ranging between ⬃27 and differential expression in CFM and MUC1.Tg intestines. A: intesti-
45 kDa (Fig. 6A). Antiserum was proven to be specific nal lysates were immunoblotted (IB) either with polyclonal antibody
when its reactivity was partially or completely inhib- (PAb) CT80 or with nonimmune serum. Jejunum, ileum, and colon of
ited by preincubating the CT80 antiserum with the CFM mice are compared with MUC1.Tg mice. MUC1-CT80 bands
are designated with arrows and a bracket and bands that appear to
immunizing peptide and by the absence of reactivity in be nonspecific are designated NS. B: immunoblot with MUC1/SEC
CF/Muc1⫺/⫺ mice (not shown). Nonimmune rabbit se- PAb comparing CFM and MUC1.Tg intestines. The arrow indicates
rum lacked the MUC1-CT80 bands but showed nonspe- the MUC1/SEC protein.

AJP-Gastrointest Liver Physiol • VOL 284 • MAY 2003 • www.ajpgi.org

 MUC1 SPLICE VARIANTS IN CF MUCUS
cific bands below 30 kDa (Fig. 6A). To further deter-
mine the specificity of the CT80 antiserum, MUC1 was
immunoprecipitated with an antibody to MUC1-EX
and blotted with CT80 antiserum and a similar band-
ing pattern was observed (data not shown). The differ-
ent MUC1-CT80 forms observed in the immunoblot
likely represent different levels of MUC1-CT80 glyco-
sylation, phosphorylation, or proteolytic processing.
The phenomenon with multiple bands containing the
cytoplasmic tail is also observed for the normal MUC1
by using the CT1 and CT2 antibodies (Fig. 2) (41).
Multiple bands for MUC1-CT80 were expressed along
the whole CFM intestinal tract but only in the colon of
MUC1.Tg mice in which a single, diffuse band of ⬃30
kDa was observed (Fig. 6A). Even with increased
amounts of protein, still no CT80 reactivity was found
in jejunum and ileum of MUC1.Tg mice. This observa-
tion was consistent with the pattern of MUC1-CT80
mRNA seen in both types of mice.
MUC1/SEC protein expression and histological lo-
calization patterns coincide with MUC1-EX. A chicken
PAb (MUC1/SEC) directed to a unique peptide se-
quence in MUC1/SEC detected protein on immuno-
blots of intestinal lysates of CFM and MUC1.Tg mice
(Fig. 6B). The amount of MUC1/SEC protein decreased
from jejunum to colon in CFM intestines. In MUC1.Tg
mice, no protein was seen in the small intestine but
there is some reaction in the colon. Testing up to 100
␮g of protein did not give a positive reaction in the
MUC1.Tg small intestine (not shown). CF/Muc1⫺/⫺
lysates tested with MUC1/SEC were always negative
as expected (not shown). We investigated the histolog-
ical localization of MUC1/SEC in the CFM and CF/
Muc1⫺/⫺ intestines. Figure 3D shows that MUC1/SEC
was localized in the intestinal lumen similarly to
MUC1-EX (Fig. 3B). The specificity of the MUC1/SEC
antibody was corroborated by the absence of staining
in the lumenal mucus present in the CF/Muc1⫺/⫺ in-
testines (Fig. 3H).
DISCUSSION
In CF, the major pathological problems are the result
of an accumulation of mucus within the respiratory
and digestive systems. Despite progress in the identi-
fication and study of mucins and their obvious rele-
vance as components of mucus, there is a lack of knowl-
edge of specific alterations in mucin expression in CF.
This is due, in part, to the fact that new mucin genes
are still being identified (MUC12, -13, -17) (19, 50, 51).
In addition, the rodent homologues of all the human
mucins have not yet been obtained, which would facil-
itate the generation of animal models. We have used
analyses of both RNA and protein to investigate the
participation of MUC1 in the intestinal mucus accu-
mulation in the CFM mouse compared with the
MUC1.Tg mouse. Results showed that the extracellu-
lar domain of MUC1 is an intrinsic part of the mucus
accumulation observed in the intestines of the CFM
mice. Western blots revealed an increase in the levels
of the MUC1-EX, particularly evident in the jejunum of
AJP-Gastrointest Liver Physiol • VOL
G859
the CFM mice. This increase was less prominent as one
moved distally to the ileum and colon. Because there
was no concordant increase in the cytoplasmic tail of
MUC1, we investigated the expression of alternate
forms of MUC1 mRNA that could account for the ap-
parent inverse proportion of MUC1-EX and MUC1-CT
in the small intestine. Both mRNAs and protein ex-
pression of novel and previously described MUC1 iso-
forms matched those of increased MUC1-EX protein in
the small intestine of CFM mice.
Alternate splicing is a common event among mucins
and several splice variants have been described for
MUC1 (5, 30, 32, 44). Therefore, one way to explain the
large amount of MUC1-EX in the small intestine of the
CFM mice is by the presence of a MUC1 splice variant
lacking the cytoplasmic tail, such as MUC1/SEC (44) or
any other cytoplasmic tail variant either lacking some
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or all of the CT2 epitopes. We found roughly similar
levels of MUC1/SEC and MUC1 mRNA and protein in
the CFM mice, whereas MUC1/SEC was very scarce in
MUC1.Tg mice. Messages for the two novel splice vari-
ants (MUC1-CT58 and MUC1-CT80) lacking the CT2
epitope were found in CFM and MUC1.Tg mice; the
message for MUC1-CT80 showed increased expression
in CFM small intestine vs. MUC1.Tg small intestine.
Thus it appears that the large increase of MUC1-EX in
the small intestine of the CFM mouse may be due to
increased transcription of classic MUC1 as well as to
MUC1 isoforms expressing variant cytoplasmic tails
and at least one secreted MUC1 form lacking the cyto-
plasmic tail altogether. Immunoblots corroborated that
MUC1-CT80 and MUC1/SEC proteins were expressed.
The MUC1-CT80 and MUC1/SEC expression patterns
matched that of the MUC1-EX pattern of expression in
the small intestine of CFM mice. The fact that MUC1/
SEC is overexpressed in CFM intestines, and it is
present in the intestinal lumen, indicates that this
secreted form is a contributor to the mucus accumu-
lated in such mice. Thus it appears that the large
increase of MUC1-EX in the small intestine of the CFM
mouse is mediated by increased transcription and
translation of classic transmembrane MUC1 and
MUC1/SEC as well as the novel cytoplasmic domain
variant MUC1-CT80.
The functional significance of alternate splice vari-
ants in mucins has not been studied, but other trans-
membrane proteins use this strategy with alternate
cytoplasmic domains to switch signaling pathways (14,
31). MUC1-CT80 is lacking the region known to di-
rectly interact with ␤-catenin (53), which potentially
could have a large impact on signaling events involving
the MUC1-CT. Because MUC1-CT80 is overexpressed
in the CFM small intestine in which the main
MUC1-EX and MUC1/SEC increase is noticed, it seems
possible that MUC1-CT80 is participating in some sig-
naling leading to an atypical mucus secretion into the
intestinal lumen. However, more studies are necessary
to assess the function of the MUC1-CT splice variants.
It is known that the MUC1-CT can participate in a
wide variety of signaling events, but until now, these
studies have mainly dealt with adhesion and transfor-
284 • MAY 2003 • www.ajpgi.org
G860 MUC1 SPLICE VARIANTS IN CF MUCUS
mation of cancer cells. One can suggest that the altered
CFM intestine epithelium may trigger distinct signal-
ing events via alternative splicing of the MUC1 gene.
The discordant MUC1-EX and MUC1-CT in the
small intestine of CFM mice could also be due to
shedding or proteolytic processing of the classic MUC1.
Mature MUC1 results from the posttranscriptional
processing of a large precursor that is cleaved in the
endoplasmic reticulum, producing a noncovalently
bound heterodimer (28, 35). Although this cleavage
does not directly lead to the release of the MUC1-EX, it
may provide a mechanism for MUC1 shedding and a
concomitant degradation of the cytoplasmic tail. A sec-
ond proteolytic cleavage could also liberate MUC1-EX
from the cell membrane. The feature of a proteolytic
cleavage associated with posttranslational processing
of membrane-associated mucins is shared by at least
two other mucins of the same class, MUC4 and epigly-
canin (47). Accordingly, the heterodimeric presentation
of MUC1 and the other transmembrane mucins may
provide a mechanism for rapid shedding of the mucin
domain into the lumen where it appears to participate
in the accumulation of mucus detected in CF.
The major phenotype in the CF mouse occurs in the
small intestine, an organ also affected in human CF
patients (15). How the lack of a functional CFTR favors
the increase of MUC1, in particular, and the increase
of mucus secretion in general, remains unresolved.
Considering our results of MUC1 message and protein
expression in the CF intestines, it appears that the
CFM small intestine takes on a resemblance to the
normal colon, but it does not present histological signs
of colonic differentiation. We have found that mucin
fucosylation was also altered in the CF mouse small
intestine and resembled fucosylation in the normal
colon (48). This resemblance of the CFM small intes-
tine to the colon could be reflecting a subtle alteration
in the differentiation of some epithelial cells, leading to
atypical mucus secretion. Crypt enterocytes of the
small intestine are the cells most abundantly express-
ing CFTR, although it is expressed in the villus cells as
well. One would expect that the lack of a functional
CFTR could affect water secretion. It has been sug-
gested that reduced airway-surface liquid in CF pa-
tients may create underhydrated mucus and impaired
mucociliary clearance. By inference, any reduced secre-
tion of water into the intestinal lumen in the CFM
mouse could result in impaired mucus clearance. How-
ever, a dehydrated environment could, in principle,
also generate a stress signal with the potential power
to activate a cascade of protective events to maintain
the integrity of the lumenal epithelium. Mucus produc-
tion may be increased as a primary line of defense
within such a protective response. Our observations
and those of other researchers favor the idea that there
is increased expression of mucins and increased secre-
tion of mucus contributing to the intestinal mucus
accumulation as well as an altered environment in
which such mucus is less readily cleared. Our results
clearly point out that MUC1 is part of the mucus
accumulated. Recent findings of Khatri et al. (20),
AJP-Gastrointest Liver Physiol • VOL
using quantitative immunoassays and immunohisto-
chemistry with a variety of anti-Muc3 antibodies, re-
vealed that the ectodomain, but not the cytoplasmic
domain, of Muc3 is increased largely in the small
intestine of CF mice. This pattern of expression for
Muc3 is similar to what we observed for MUC1. Given
the identification of message and protein for Muc4, -12,
and -13 in the intestines of control mice (39, 50, 52), it
is likely that these mucins also participate in the mu-
cus accumulation in CF intestines.
The exact composition of the intestinal mucus accu-
mulated as a result of a nonfunctional CFTR is cur-
rently not known, but it is clear that such mucus
contains a mixture of mucins, some of them tradition-
ally considered not important intestinal mucins, such
as MUC1. Considering our present data of increased
MUC1 mRNA and protein and that MUC1 expression
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is regulated at the level of transcription (16, 21, 54), we
establish that MUC1 is not only accumulated in the
CFM intestinal lumen due to an impaired mucus clear-
ance but to its increased expression as well. We hy-
pothesize that MUC1 in the CF intestines is not only
contributing to the mucus accumulation through the
shedding and/or secretion of its extracellular domain
but also via its proven role in signal transduction
(23–26, 33, 37, 41, 53). MUC1 may signal to the cell
interior an altered extracellular milieu, which would
provoke a response intended to maintain the integrity
of the intestinal mucosa under the stressful environ-
ment generated by the absence of CFTR.
We thank Marvin Ruona for help with computer graphics and
Carol Williams for administrative assistance and manuscript sub-
mission.
This work was supported by the Cystic Fibrosis Foundation, the
Swedish Foundation for International Cooperation in Research and
Higher Education, The Swedish Research Council, and National
Institutes of Health Grant T32-H107897.
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