Characterization of mucins in human middle ear and Eustachian
tube
Jizhen
Lin1,
Vladimir
Tsuprun1,
Hirokazu
Kawano1,
Michael M.
Paparella2,
Zhiqiang
Zhang3,
Ruth
Anway4, and
Samuel B.
Ho4,5
University of Minnesota Otitis Media Research Center, Departments
of 1 Otolaryngology, 3 Microbiology, and
5 Internal Medicine, University of Minnesota School of
Medicine, and 2 Minnesota Ear, Head and Neck Clinic, Minneapolis
55455; and 4 Veterans Administration Medical Center,
Minneapolis, Minnesota 55417
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ABSTRACT |
Mucins are important glycoproteins in the mucociliary transport
system of the middle ear and Eustachian tube. Little is known about
mucin expression within this system under physiological and
pathological conditions. This study demonstrated the expression of
MUC5B, MUC5AC, MUC4, and MUC1 in the human Eustachian tube, whereas
only MUC5B mucin expression was demonstrated in noninflamed middle
ears. MUC5B and MUC4 mucin genes were upregulated
4.2- and 6-fold, respectively, in middle ears with chronic otitis media (COM) or mucoid otitis media (MOM). This upregulation of mucin genes
was accompanied by an increase of MUC5B- and MUC4-producing cells in
the middle ear mucosa. Electron microscopy of the secretions from COM
and MOM showed the presence of chainlike polymeric mucin. These data
indicate that the epithelium of the middle ear and Eustachian tube
expresses distinct mucin profiles and that MUC5B and MUC4 mucins are
highly produced and secreted in the diseased middle ear. These mucins
may form thick mucous effusion in the middle ear cavity and compromise
the function of the middle ear.
mucin biology; otitis media; epithelial pathology; hearing
impairment
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INTRODUCTION |
MUCINS ARE A
FAMILY of glycoproteins with distinct biological structure and
physiological functions. Mucins are involved in host cellular
protection (29), proliferation (29, 40),
host-pathogen adhesion (32), and tumor biology
(1). There are two classes of mucins, membrane-bound and
secretory, which differ in biological structure and cellular location.
Mucins with a transmembrane domain are designated as membrane-bound
mucins. Membrane-bound mucins anchor directly to the bilayer of a
membrane and play a role in cellular shielding, adherence, and cellular
proliferation of the mucosal epithelium. Secretory mucins lacking a
transmembrane domain are synthesized in a dimeric or trimeric manner,
are packed into mucous granules, and are secreted on an apical surface
to form a mucus layer that protects the epithelium and defends against invading microorganisms. Membrane-bound mucins may also be produced as
a secreted or soluble form in certain tissues. Detailed information on
mucins and mucin genes has appeared in several review articles (16, 26, 34, 35).
To date, at least nine distinct mucins from the human body have been
identified and designated as MUC1-4, MUC5AC, MUC5B, or MUC6-8
(16, 26, 35, 39), depending on the date at which they were
first described. MUC1, MUC3, and MUC4 are considered to be
membrane-bound mucins; MUC2, MUC5AC, MUC5B, MUC6, and MUC7 are thought
to be secretory mucins; MUC8 is still unclassified.
Genes for secretory mucins MUC2, MUC5AC, MUC5B, and MUC6 are known to
be clustered at the locus on chromosome 11p15.5 (3, 14, 18,
42) and are believed to be derived from the same ancestral gene,
given their genetic organization and structural similarity.
MUC1 is located on 1q21-24 (15, 41),
MUC3 on 7q22 (19), MUC4 on 3q29
(31), MUC7 on 4q13-21 (7),
and MUC8 on 12q24.3 (39).
In the middle ear, mucins are important structural components of the
mucociliary transport system that covers cellular surfaces, provides
transport toward the nasopharyngeal orifice, and cleans the middle ear
cavity. Because middle ear mucins are able to bind to proteins in the
outer membrane of bacteria (32), they are thought to play
an essential role in evacuating middle ear pathogens that ascend along
the Eustachian tube. Under disease conditions in the middle ear,
however, alterations in mucin metabolism are thought to contribute to
dysfunction of the mucociliary transport system (36, 37).
The profile of mucin genes that are expressed in the normal human
Eustachian tube and middle ear is unknown at the present time, as are
possible alterations that may occur in mucin gene(s) under disease
conditions. Studies of mucin gene expression in these two structures
are hindered by difficulties in obtaining normal human tissues and are
limited by the extremely small amounts of middle ear effusion available
for study. Understanding mucin metabolism under normal physiological
conditions and the alterations that occur during disease states is key
to understanding the molecular mechanisms of middle ear diseases. This
study was designed to characterize mucin gene expression in the normal
middle ear and Eustachian tube and in the middle ear of humans with
otitis media, which is the most common cause of acquired hearing loss
in adults and impaired language development and communication disorders in children.
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MATERIALS AND METHODS |
Procurement of human middle ear and Eustachian tube tissues and
middle ear effusions.
Eight middle ear and Eustachian tube tissue specimens were obtained at
immediate autopsy (3-6 h after death) of the temporal bones of
four male patients, ages 50-70 yr, who died from heptocellular carcinoma, pneumonia, coronary artery disease, and infarcted bowel, respectively. None of the patients had a history of middle ear disease.
Middle ear specimens were dissected from the inferior area of the
promontory, and Eustachian tube specimens were dissected from the
orifice of the nasopharynx to the orifice of the tympanium. Twenty
middle ear tissue specimens from the same areas described above were
biopsied from the middle ears of 20 patients diagnosed with otitis
media [chronic otitis media (COM) or mucoid otitis media (MOM)]. A
total of 3 ml of middle ear effusion collected at surgery from six of
the patients with COM or MOM was pooled for mucin analyses. Very little
or no middle ear effusion was evident in 14 patients as a result of
perforation of the tympanic membrane.
Middle ear effusion (3 ml) was used for purification of mucins.
Identity of the purified middle ear mucins was determined by ELISA.
Morphometric analysis of the purified middle ear mucin was completed by
electron microscopy.
Ten middle ear biopsies (8 COM and 2 MOM), three middle ear autopsy
specimens, and three autopsy Eustachian tube autopsy specimens were
fixed in 10% formalin overnight, embedded in paraffin, and sectioned
at a thickness of 5 µm using the method described previously (27). Surgical tissues of human gallbladder, small
intestine, stomach, and submaxillary glands served as positive or
negative controls for mucins. Ten middle ear biopsies (8 COM and 2 MOM), five middle ear autopsy specimens, and five Eustachian tube
autopsy specimens were used for total RNA isolation using the method
described by Chomczynski and Sacchi (10). Surgical
specimens of human stomach, colon, submaxillary glands, and fibroblast
tissues served as positive or negative controls for mucins in these specimens.
To study the expression of mucin mRNA and proteins, in situ
hybridization, Northern blot, or immunohistochemistry was performed as
described below. Mucin cDNA probes and antibodies used in this study
are listed in Tables 1 and
2. The antibodies MRP and M3P and the
cDNA probes SIB139 and SMUC-41 were gifts from Drs. Young S. Kim, James
R. Gum, Jr., and Carol Basbaum. The 139H2 antibody and cDNA probe pum
24p were donated by Drs. John Hilkens and Dallas M. Swallow,
respectively.
Analyses of mucin mRNA by in situ hybridization and Northern
blot.
For in situ hybridization, linearized vectors with cDNA probes for
MUC1, MUC2, MUC3, MUC5AC, MUC5B, and MUC6 tandem
repeat units were transcribed into sense or antisense riboprobes with isotope labeling with 35S in vitro by T7/T3/Sp6 phage RNA
polymerase, using the MAXIscript kit (Ambion, Austin, TX). MUC4, MUC7,
and MUC8 oligo cDNA probes were linked with T7/T3 promoter sequences
and then transcribed into sense or anti-sense riboprobes using the same
kit. In situ hybridization of middle ear, Eustachian tube, and control
tissues was performed as described previously (27).
Briefly, middle ear and Eustachian tube sections were deparaffinized,
digested with protease K, hybridized with mucin MUC1-8 riboprobes,
washed under stringent conditions [5× sodium chloride and sodium
citrate (SSC, pH 7.0) and 10 mM dithiothreitol (DTT) at 42°C for 30 min; 2× SSC, 50% formamide, and 10 mM DTT at 60°C for 20 min; and
RNA washing solution (0.1 M Tris · HCl at pH 7.5, 0.4 M NaCl,
and 50 mM EDTA) for 10 min, two times], exposed to emulsion solution, and counterstained with hematoxylin.
For Northern analysis, the same cDNA probes as for tandem repeat units
were used. Vectors with cDNA probes were digested with EcoRI, BamHI, or PstI endonuclease and
labeled with 32P using the Prime-a-Gene labeling system
(Promega, Madison, WI). Northern analysis of middle ear mRNA
was performed as described previously (27). Total RNA
(5-20 µg) was denatured and size-separated by electrophoresis on
a 1.0% agarose gel containing 2.2 M formaldehyde, blotted on a nylon
membrane, cross-linked by ultraviolet light, hybridized by
32P-labeled mucin MUC1 cDNA probe at a
radioactivity of 2 × 106
counts · min
1 · ml
1, and
washed under stringent conditions. The blot was stripped with 0.1% SDS
and reprobed with MUC2, MUC3, MUC4,
MUC5AC, MUC5B, MUC6, MUC7,
MUC8, and
-actin cDNA probes. To make certain that Northern analysis was quantitative, MUC5B and
MUC4 nontandem repeat cDNA probes were also used.
Analyses of mucin glycoprotein by immunohistochemistry, ELISA,
and electron microscopy.
To study the expression of mucins in middle ear and Eustachian tube
tissues, specific mucin antibodies (Table 2) were used for
identification of their mucin glycoproteins. Immunohistochemistry was
performed as described previously (22).
To study the expression of mucins in the middle ear effusion, mucins
were purified using the method described previously (28). Briefly, middle ear effusion was suspended in buffer (containing 4 M
guanidine hydrochloride, 50 mM Tris · HCl, 5 mM EDTA, and 2 mM
phenylmethylsulfonyl fluoride), sonicated for 1 min, homogenized for 1 min, and incubated with 100 mM DTT for 24 h and 250 mM of iodoamylamide for 24 h. Debris and insoluble substances were
removed by centrifugation at 8,000 g for 20 min. Effusions
were supplemented with cesium chloride at 1.4 g/ml, centrifuged at
1.6 × 105 g for 72 h, and then
fractionated at 1 ml/tube. Density, protein, and hexose were determined
by weight, bicinchoninic acid assay, and hexose assay for each tube,
according to the method described previously (28).
Fractions with a density of ~1.4 g/ml and a hexose-to-protein ratio
of at least 2:1 were defined as mucins.
Mucins were refined by repeating the cesium chloride gradient
centrifugation three times under the same conditions as above, filtered
through an AP-100 device (Amecon) to cut off molecules (<100 kDa), and
dialyzed against distilled water at 4°C with stirring. To confirm the
identity of purified middle ear mucins, ELISA was performed using the
method described previously (38). Briefly, 1.5 µg of
middle ear mucin and 1 µg of cervix mucin (control) were loaded on
96-well plates for 2 h at room temperature. Plates were blocked
with 5% BSA in Tris-buffered saline (TBS: 25 mM Tris, pH 7.4, 140 mM
NaCl, and 3 mM KCl) overnight at 4°C. The plates were washed with
0.02% Tween 20 in TBS and incubated with the primary antibodies
(chicken anti-human MUC5B and MUC4 in 1:1,000 dilution) for 3 h at
room temperature. After washing as before, peroxidase-conjugated rabbit
anti-chicken IgY (1:2,000) was added for 1.5 h. Color development
was performed with 3,3',5,5'-tetramethylbenzidine and quenched with 8 M
H2SO4. Bound antibody was quantified by measuring the absorbance at 450 nm with a TiterTek spectrophotometer. Preimmune antibodies were used as negative controls.
To determine the molecular image of the purified middle ear mucins,
middle ear mucin was applied to thin carbon-coated grids and stained
with 1% (wt/vol) uranyl acetate. Micrographs were recorded with
a Jeol 1010 electron microscope at 60 kV at a magnification of
×50,000. The printed images were digitalized on a Linotype-Hell flatbed scanner interfaced to a Macintosh computer at a pixel size of
~0.4 nm at specimen level. Adobe Photoshop software was used to apply
a high-pass filter to suppress the very low spatial frequencies and
thus increase the signal-to-noise ratio of the images.
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RESULTS |
Mucin gene expression in human Eustachian tubes.
The profile and cellular location of mucin gene expression in the
Eustachian tube are presented in Fig.
1. Mucin gene
antisense riboprobes (MUC1, MUC4, MUC5AC, and
MUC5B) hybridized positively with the apical surface of the
Eustachian tube epithelium (Fig. 1, M1, b), the
whole epithelial layer (Fig. 1, M4, b),
epithelium (Fig. 1, M5AC, b and M5B,
b), and submucosal glands (Fig. 1, M5B, b), respectively. Expression of MUC4 and
MUC1 was not as abundant as that of MUC5B and
MUC5AC. No other mucin genes (MUC2, MUC3, MUC6,
MUC7, and MUC8) were expressed in the epithelium of the Eustachian tubes. No mucin riboprobes other than MUC5B
hybridized with the submucosal glands of the Eustachian tube.
MUC-positive control tissues demonstrated hybridizing signals for
MUC1 (gallbladder), MUC2 (small intestine),
MUC3 (small intestine), MUC5AC and
MUC6 (stomach), and MUC5B and MUC7
(submaxillary glands) antisense riboprobes, respectively. Northern
analysis of Eustachian tube specimens is presented in Fig.
2. Mucin mRNA transcripts in the Eustachian tube were hybridized with MUC5B, MUC5AC, MUC4,
and MUC1 but not with MUC2, MUC3, MUC6, MUC7, or
MUC8 cDNA probes.
-Actin housekeeping gene expression for
loading controls is shown in Fig. 2, bottom.

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Fig. 1.
Expression of mucin genes in the human Eustachian tube
(ET). ET sections were hybridized with mucin antisense riboprobes
(second and fourth columns, positive signals) and with mucin sense
riboprobes (first and third columns, negative signals). MUC1
(M1), MUC4 (M4), MUC5AC (M5AC), and
MUC5B (M5B) mucin genes were expressed in the epithelium of
the ET (arrows, M1, b; M4,
b; M5AC, b; and M5B,
b), whereas MUC2 (M2), MUC3
(M3), MUC6 (M6), MUC7 (M7), or MUC8
(M8) were not (M2, b; M3,
b; M6, b; M7, b;
and M8, d). Note the distribution of mucin gene
expression: MUC5AC mucin mRNA was distributed exclusively in
the epithelium (M5AC, b, arrows), and the
MUC5B mucin mRNA was found in both epithelium (arrows) and
submucosal glands (M5B, b, arrowheads). Positive
controls of MUC1 [M1, d, gallbladder
epithelium (GB)], MUC2 [M2, d,
goblet cells of the small intestine (SI)], MUC3
(M3, d, SI), MUC5AC [M5AC,
d, stomach epithelium (SM)], MUC5B
[M5B, d, submaxillary mucus glands (SG)],
MUC6 (M6, d, SM), and MUC7
(M7, d, SG) all demonstrated hybridizing signals.
M1, a and b, represents transverse
sections of the ET; all others were parallel sections of the ET.
Original magnification was ×400 for M1, M4,
M7, and M8 and ×80 for all others.
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Fig. 2.
Northern analysis of middle ear and ET mucin gene expression.
Lane 1, middle ear (M); lane 2, ET (E);
lane 3, stomach (S); lane 4, colon (C);
lane 5, submaxilary glands (G); lane 6,
fibroblast (F, negative control for mucin genes). MUC1 (M1)
was weakly expressed in the ET but not in the middle ear.
MUC4 (M4) was expressed in the ET but not in the middle ear,
as was MUC5AC (M5AC). MUC5B (M5B) was expressed
in both the middle ear and ET. None of the MUC2 (M2),
MUC3 (M3), MUC6 (M6), MUC7 (M7), or
MUC8 (M8) mucin genes was expressed in the middle ear or ET. Note that
controls for MUC1 (S or G), MUC2 (C), MUC3
(C), MUC4 (C), MUC5AC (S), MUC5B
(G), MUC6 (S), and MUC7 (G) were
positive, whereas controls for MUC1 (C), MUC2 (S
or G), MUC3 (S or G), MUC4 (S or G), MUC5AC
(C and G), MUC5B (S and C), MUC6 (C or
G), MUC7 (S or C), and MUC8 (S, C, or G) were
negative. The size of MUC1, MUC4, MUC5AC, and
MUC5B mucin mRNA transcripts in the ET was ~6 kb,
14-15 kb, 17-18 kb, and 17.5 kb, respectively. Note that
MUC4 mucin mRNA transcript in autopsy specimens appeared to
be partially degraded. -Actin gene expression (bottom)
was used as a loading control for RNA.
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Immunohistochemistry demonstrated positive staining of Eustachian tube
tissue sections with MUC5B, MUC5AC, MUC4, and MUC1 mucin antibodies
(Fig. 3) but not with their preimmune
sera. The cellular distribution of the mucins was consistent with that
of mucin mRNA. MUC5B mucin glycoprotein was found in the epithelium and
submucosal glands of the Eustachian tube (Fig. 3i). MUC5AC mucin glycoprotein was found in the epithelium but not submucosal glands of the Eustachian tube (Fig. 3f). MUC4 mucin
glycoprotein was found throughout the epithelium (Fig. 3d).
Low levels of MUC1 mucin glycoprotein were found in the surface
epithelium. Neither MUC4 nor MUC1 mucin glycoproteins were found in
submucosal glands of the Eustachian tube.

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Fig. 3.
Immunohistochemistry of the ET
and middle ear tissues. ET tissues stained positively to MUC1
(b, arrows), MUC4 (d, arrows), MUC5AC
(f, arrows), and MUC5B (i, arrows for
goblet cells and arrowheads for submucosal glands) polyclonal
antibodies, but no staining was evident with preimmune sera for MUC1
(a), MUC4 (c), or MUC5AC (e). Middle
ear tissues from chronic otitis media (COM) patients stained positively
to MUC4 (h, arrows) and MUC5B (k, arrows).
Tissues from noninflamed middle ears did not react with MUC4
(g) or MUC5B (j) polyclonal
antibodies. Note that MUC5B/MUC4 mucin-producing cells were highly
populated in middle ears with COM (h and k,
arrows) compared with noninflamed middle ears (j
and g). Immunohistochemistry of MUC2, MUC3, and MUC6 was
negative, as expected (data not shown). The ET and middle ear tissues
stained negatively to MUC5B preimmune serum (data not shown). ME,
middle ear; Ctrl, control noninflamed middle ear. Original
magnification was ×800 for left and ×400 for
right.
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Expression of mucin genes in noninflamed middle ears and
upregulation of MUC5B and MUC4 mucin genes in inflamed middle ears.
Expression of MUC5B mucin gene was detected in the
noninflamed middle ears. In situ hybridization showed spotty positive
signals with MUC5B mucin antisense riboprobe (Fig.
4A, M5B,
b). Consistent with this, Northern analysis demonstrated
only MUC5B mucin gene expression in middle ear epithelium
(Fig. 2f). No other mucin gene expression was
detected by in situ hybridization or Northern blot (data not shown).
Immunohistochemistry detected only very weak expression of MUC5B (Fig.
3j) mucin, supporting the findings of in situ hybridization
and Northern analysis.

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Fig. 4.
A: upregulation of MUC5B and MUC4
mucin genes in otitis media middle ears. In situ hybridization
indicated intensive and extensive expression of the MUC5B
mucin gene in the middle ears with COM (M5B, d)
compared with noninflamed middle ears (M5B, b).
Note that hybridizing signals of the MUC5B mucin gene in the
middle ears with COM were not limited to goblet cells but appeared
within the entire layer of the middle ear epithelium (arrows), whereas
only spotty hybridizing signals (M5B, b, arrows)
were demonstrated in the noninflamed middle ears. MUC4 mucin
gene expression was not detected in noninflamed middle ears
(M4, b), but its expression was detected in the
middle ear with COM (M4, d, arrows).
Left: hybridization with sense probe; right:
hybridization with antisense probe. Original magnification was ×800
for bottom and ×400 for the others. B:
upregulation of the MUC5B and MUC4 mucin genes in
middle ears with COM. Northern analysis indicated an increase in
MUC5B mucin gene expression in middle ears with COM
(lane 2) compared with noninflamed middle ears (lane
1) after normalization with -actin gene expression. Hybridizing
signals for the MUC4 mucin gene were detected in middle ears
with COM (lane 4), whereas none was detected in noninflamed
middle ears (lane 3). Note partial degradation of
MUC5B mucin mRNA in the autopsy specimens. Northern analysis
using both tandem repeat (this figure) and nontandem repeat (data not
shown) cDNA probes showed the same results; however, weak hybridization
of the entire blot with nontandem repeat cDNA probes was noted,
although the ratio of inflamed vs. noninflamed specimens remained the
same. 5B, MUC5B mRNA transcript (17.5 kb); M4, MUC4
mRNA transcript (17-19 kb in middle ear biopsies).
C: densitometry for MUC5B and MUC4
mucin gene expression. The magnitude of increase in MUC5B
and MUC4 mucin mRNA expression was 4.2 and 6.0, respectively, in middle ears with COM compared with noninflamed middle
ears after normalizing to expression of the -actin
gene.
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In the inflamed middle ears, expression of not only MUC5B
but also MUC4 mucin genes was detected. In situ
hybridization demonstrated extensive and strong positive signals with
MUC5B mucin antisense riboprobe (Fig. 4A,
M5B, d) and extensive positive signals with MUC4 mucin antisense riboprobe (Fig. 4A,
M4, d). Semiquantitative Northern analysis (Fig.
4B) with both tandem repeat and nontandem repeat cDNA probes
showed MUC5B and MUC4 mucin gene expression to be ~4.2-
and 6-fold, respectively, higher in the inflamed middle ear specimens
than in the noninflamed specimens after normalization of expression by
the housekeeping gene
-actin (Fig. 4C). No mucin gene
expression other than MUC5B and MUC4 was detected
by Northern analysis and in situ hybridization (data not shown).
Immunohistochemistry demonstrated extensive expression of MUC5B and
MUC4 mucins in the entire middle ear epithelium with COM (Fig.
3k, COM), supporting the findings of Northern and in situ hybridization.
An increase of the MUC5B- and MUC4-producing cells was obvious in the
inflamed middle ears (Fig. 3, h and k, COM) but
not in the noninflamed middle ears (Fig. 3, g and
j, control).
Identity and morphology of the middle ear mucins.
ELISA and electron microscopy were used to identify the mucins in
middle ear secretions and study their morphology. After ultracentrifugation of middle ear effusion in cesium chloride solution,
the hexose assay demonstrated two peaks among the 12 fractions of
middle ear effusion: one at fraction 7 and another at
fraction 10 (Fig.
5A). Both fractions were
considered to be mucins, since they displayed the following
characteristic features of a mucin: a high density between 1.35 and
1.45 g/ml and a high hexose-to-protein ratio of ~2:1 or above. Mucins
in both fractions were recognized by MUC5B and MUC4 mucin antibodies
(Fig. 5B). Fraction 10 was rich in MUC5B, in
contrast to fraction 7; therefore, fraction 10 was submitted to further examination by electron microscopy. The middle
ear mucins in fraction 10 demonstrated a branched strand containing linear portions and thickened bulb-like portions in the
central region. The linear portions of the strands between bulbs varied
between 60 and 180 nm in length, with a thickness of 4-5 nm.
Globular domains 4-5 nm in diameter were interspersed along the
linear portions of the mucin strands (Fig.
6, inset).

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Fig. 5.
A: identification of mucins in middle ear effusions. One
hexose peak (fraction 10) was identified
after three spins of the effusion in a cesium chloride solution.
Fraction 10 had a high density of 1.41 g/ml and a
hexose-to-protein ratio of ~3:1, which is characteristic of mucins.
Fraction 7 had a density of 1.37 g/ml and a
hexose-to-protein ratio of ~1.9:1, which may also be indicative of
mucins. B: identities of the mucins in both fractions
7 (F7) and 10 (F10) by ELISA. Morphological analysis of
mucins in fraction 10 is presented in Fig. 6. Mucins in
fraction 10 were clearly recognized by MUC5B mucin antibody
and MUC4 antibody (MUC4-1), whereas mucins in fraction
7 were less clearly recognized by MUC5B antibody. This indicates
that 1) mucins in middle ear (ME) effusions are a mixture of
MUC5B and MUC4, although MUC5B is predominant, and 2) MUC4
mucin, a membrane-bound mucin, has to be released in the middle ear
cavity and is present in middle ear effusions with COM. OD, optical
density.
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Fig. 6.
Morphology of middle ear mucins by electron microscopy.
The middle ear mucins in fraction 10 demonstrated branched
strands with linear portions and thickened bulblike portions
(arrowheads). The linear portions of the strands between bulbs varied
between 60 and 180 nm in length. Thickness of the linear portions is
~4-5 nm. There were globular domains ("beads"), 4-5 nm
in diameter, interspersed along the linear portions of the mucin
strands (arrows in inset). Bars, 50 nm and 10 nm
(inset).
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A summary of findings on mucin mRNA and glycoprotein of noninflamed
Eustachian tube and middle ear specimens from immediate autopsies and
inflamed middle ear specimens from biopsies is presented in Table
3.
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DISCUSSION |
The human Eustachian tube, also known as an auditory tube because
of its role in hearing, is deeply embedded in the temporal bone and
inaccessible in living humans for the study of its gene profiles in
physiological and pathological states. Recently, we designed a method
for acquiring immediate-autopsy specimens of the Eustachian tube in
which RNA was well preserved for in situ hybridization and Northern
analysis (27). This newly developed method was used for
characterization of mucin gene expression in the Eustachian tube of humans.
The middle ear and Eustachian tube are integral parts of the auditory
system and are involved in sound transduction from the tympanic
membrane to the inner ear. Proper functioning of the auditory system is
reliant on the mucociliary transport system, which originates in the
inferior tympanum and covers the entire Eustachian tube
(5), evacuating invading microorganisms and clearing the
middle ear cavity. This maintains an air- and pressure-balanced environment, free of infection, for the sound-conducting structures (the ossicular chain). The function of the mucociliary transport system
is largely dependent upon interactions between mucins, cilia, and
periciliary fluid.
It is clear from this study that the Eustachian tube possesses a
profile of MUC5B, MUC5AC, MUC4, and MUC1 mucins. Secretory MUC5B and
MUC5AC mucins are major structural components of the mucociliary
transport system in the Eustachian tube under noninflamed conditions.
Membrane-bound MUC1 and MUC4 mucins may play a role in cellular
protection in the Eustachian tube. Because MUC4 is expressed in
ciliated, intermediate, and basal cells within the entire epithelium
and contains epidermal growth factor-like domains, it may be involved
in protein-protein interaction and play a role in cellular renewal of
the epithelium. MUC4 has been shown to act as a ligand and modulator
for the receptor tyrosine kinase ErbB2 (8, 9),
which is involved in cellular growth and proliferation. It is still
unknown, however, how mucins in the Eustachian tube are altered by
inflammation, as none of the eight Eustachian tubes harvested for this
study had evidence of otitis media.
The lining of the middle ear cavity is thought to be a modified
respiratory epithelium, a transitional epithelium ranging from squamous
cells at the superior tympanum to columnar cells at the inferior
tympanum. A ciliated tract rings the tympanic orifice and extends up to
the inferior promontory area. Goblet cells are distributed in the
ciliated tract under normal conditions. This study demonstrated that
some cells in the promontory area of the middle ear were labeled with
the MUC5B mucin gene probe, suggestive of MUC5B mucin
productivity. The primary mucin involved in the mucociliary transport
system of the middle ear appears to be MUC5B. MUC5AC was not detectable
in the middle ear, nor were MUC4, MUC1, or other MUCs detectable in the
noninflamed ears.
The middle ear has a unique or modified mucin gene expression pattern
when compared with that of the Eustachian tube or airway. Tracheobronchial epithelium mainly expresses MUC5AC (4,
33) and weakly expresses MUC5B (4);
Eustachian tube epithelium expresses MUC5AC and MUC5B equally, whereas
middle ear epithelium expresses mainly MUC5B. There appears to be an
alteration in the mucin pattern of the epithelium as it ascends from
the airway to the middle ear; expression of MUC5AC is weakened or
profoundly downregulated in epithelium as it ascends from the airway to
the middle ear.
MUC5B and MUC5AC mucins share regions of oligonucleotide similarity at
their 3'- and 5'-ends, and their DNA loci are close to each other (at
11p15.5) and believed to be derived from the same ancestral gene. The
functional significance of this double distribution of secretory mucins
in the Eustachian tube is not clear. MUC5B has the largest central
tandem repeat region (13) among known mucins. Because the
adhesive portion of mucin is within the central area (tandem repeat
units), one would expect that the length of the tandem repeat region
would be a contributing factor toward efficient entrapment and adhesion
to pathogens. It was suggested in a von Willebrand factor molecule
study that tandem repeat units originated to catch moving molecules or
objects (11).
Secreted middle ear mucin is a branched, chainlike strand interspersed
with bulblike portions (Fig. 6). The linear portion of the strand may
represent the tandem repeat region of mucin, which is heavily
glycosylated and therefore rigid. The "bulbs" may represent
nontandem repeat regions that are less glycosylated and rich in
cysteine residues, likely areas in which disulfide bonds could link
inter- and intramolecularly between mucin monomers to form mucous strands.
The amino acid sequence of MUC5B deduced from cDNA indicates abundant
cysteine residues at both ends of the molecule (25). Some
disulfide bonds between MUC5B mucin monomers were noted to survive
reduction with DTT in this study, indicative of protected disulfide
bonds between MUC5B monomers. Dimerization of mucin monomers occurs at
the endoplasmic reticulum (2, 12); however, it is not
clear where dimerized mucins polymerize. On the basis of the
biochemical properties, structural characteristics, and gene
organization, it is highly plausible that MUC5B in the human body is an
extremely viscous mucin, characteristic of a high density [1.4 g/ml, a
very large peptide backbone (8,000 amino acids) with abundant
carbohydrates], gel-forming capability (a chainlike strand capable of
further linkage), and resistance to reduction (remaining polymerized
after DTT treatment). It would be of interest to know whether MUC5B
mucin accumulation in the middle ear cavity is a major cause of "glue
ear," a type of otitis media in which mucus gel plugs the middle ear.
This study indicated that MUC5B mucin is a major structural component
of the mucociliary transport system that maintains homeostasis of the
middle ear. Under inflamed conditions, MUC5B and MUC4 mucins were
highly upregulated in the middle ear in association with an increase in
MUC5B/MUC4 mucin-producing cells (Fig. 3, h and k), suggestive of a transition from normal middle ear
epithelium to a hypersecretory epithelium. Because MUC5B and MUC4 are
evident in the middle ear effusion, it is clear that they contribute to formation of mucoid effusion, which can cause dysfunction of the mucociliary transport system and conductive hearing loss. MUC4 may
participate in proliferation and differentiation of middle ear
epithelial cells, as suggested in other studies (6). In this study, MUC5B mucin gene expression in the middle ear was not shown
to be limited to goblet cells under inflamed conditions; it appeared
that the entire middle ear epithelium is involved in MUC5B mucin
production (Fig. 4A, M5B, d). Two
recent studies that reported MUC5B to be present in middle ear
epithelium and effusion in patients with COM (23, 24)
support these findings. Therefore, highly active production of MUC5B
and MUC4 mucins in the middle ear mucosa and accumulation in the middle
ear cavity of mucins capable of forming a mucus gel may be key to
compromised middle ear function and resultant clinical disease.
The mechanism for upregulation of MUC5B and
MUC4 in middle ear epithelium is not currently understood,
since the promoter area of these mucin genes has not been studied.
Also, the factors that upregulate these mucin genes in the middle ear
with otitis media have not been identified. It is essential to
investigate the molecular mechanisms responsible for upregulation of
MUC5B and MUC4 mucins and mucus hyperproduction. Insight into these mechanisms will facilitate the development of pharmaceutical
intervention strategies against mucus hyperproduction in COM or MOM.
 |
ACKNOWLEDGEMENTS |
We thank JoAnn Knox for editing the manuscript.
 |
FOOTNOTES |
This work was supported in part by National Institute on Deafness and
Other Communication Disorders Grant R01-DC-03433, the Lions 5M Multiple
District Hearing Foundation, and the Veterans Affairs Research Service.
Address for reprint requests and other correspondence: J. Lin,
Lions Research Bldg., Rm. 216, 2001 Sixth St. S.E., Minneapolis, MN
55455 (E-mail: linxx004{at}tc.umn.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 25 April 2000; accepted in final form 3 January 2001.
 |
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