Abstract
purpose. To investigate the expression and localization of the human gene
encoding uroplakin Ib in ocular surface epithelium.
methods. The full-length cDNA of human uroplakin Ib was isolated from a cDNA
library of human corneal epithelium, and the expression of uroplakin Ib
in various tissues was examined by reverse transcription–polymerase
chain reaction (RT-PCR). In cornea and conjunctiva, the expressions of
uroplakin Ia, II, and III were also examined by RT-PCR. Finally, the
localization of uroplakin Ib in the ocular surface was analyzed by
immunofluorescence confocal microscopy, by using an antiserum against a
synthetic peptide.
results. Two mRNA isoforms, arising through two polyadenylation sites, were
isolated. RT-PCR detected uroplakin Ib in cornea, conjunctiva, bladder,
placenta, and kidney. Among other uroplakins, uroplakin II was also
faintly detected in cornea and conjunctiva. Immunofluorescence confocal
microscopy documented uroplakin Ib protein in the cell membranes of
superficial and wing cells in the corneal epithelium. It was not found,
however, in the most apical corneal epithelial cells. In limbus and
conjunctiva, uroplakin Ib was also localized in the cell membranes of
all epithelial layers, apart from the most apical cells.
conclusions. Uroplakin Ib is highly expressed in ocular surface epithelia. As in
bladder epithelium, uroplakin Ib may protect the ocular surface from
bacterial infection.
In previous experiments we identified several genes uniquely and
abundantly active in human corneal epithelium. This was achieved by a
random 3′-directed expressed sequence tag (EST) collection that we
called a gene expression profile.
1 2 3 4 Among the expressed
genes,
GS8103 appeared twice and
GS8043 once in
1062 isolates; both encoded human uroplakin Ib.
Uroplakins Ia, Ib, II, and III are transmembrane proteins constituting
the asymmetrical unit membrane of urothelial umbrella cells. They are
believed to play a major role in stabilizing the apical surface of the
mammalian urothelium, thereby preventing it from rupturing during
bladder distension.
5 6 7 Uroplakin Ia and Ib are members of
the transmembrane 4 (TM4) superfamily that contains many leukocyte
differentiation-related surface proteins including CD9, CD37, CD53,
CD63, and CD81.
8 9 10 11 12 Recent work has shown that uroplakin
Ia and Ib can bind specifically to
Escherichia coli,
expressing type 1 pili that can result in the exfoliation of bladder
epithelial cells as part of an innate host defense system through an
apoptosis-like mechanism.
13 14
In this study, we determined the complete nucleotide sequence of
uroplakin Ib cDNA tagged by GS8103. Moreover, the tissue
distribution of uroplakin Ib mRNA was determined by reverse
transcription–polymerase chain reaction (RT-PCR), and the ocular
localization of the protein was examined by immunofluorescence confocal
microscopy.
By use of the cDNA library of the human corneal epithelium,
approximately 80,000 plaques were screened with
32P-labeled random primed fragments of
GS8103 (331 bp) after cutting off Alu-repetitive
sequences. Among the positive plaques, the longest cDNA insert was
sequenced by the shotgun method.
15 16
All investigations followed the tenets of the Declaration of
Helsinki, informed consent was gained from the patients, and approval
was obtained from the Research Committee of the Kyoto Prefectural
University of Medicine. Normal cornea and conjunctiva were obtained at
autopsy; skin was excised during orthopedic surgery. Total RNA from
other tissues (bladder, brain, heart, kidney, liver, lung, placenta,
skeletal muscles, small intestine, and uterus) was purchased from
Clontech (Palo Alto, CA). cDNA of each tissue was generated from 1 μg
of total RNA according to standard procedures.
17 The mRNA
tissue distribution of uroplakin Ib was examined by RT-PCR using a
protocol described previously.
1 The expressions of
uroplakin Ia, II, and III mRNA in corneal epithelium and conjunctival
epithelium were also examined by RT-PCR in comparison with the
expressions in bladder.
Specific primers for uroplakin Ia, Ib, II, and III were produced by
selecting specific nucleotide sequences from among the published
sequences
(Table 1) .
18 19 20 The expression of the extended 3′-untranslated
region (UTR) of uroplakin Ib was examined using additional primers
produced from the sequence of
GS8103 (Table 1) .
Glyceraldehyde 3-phosphate dehydrogenase(G3PDH) primers were used as
controls.
Western Blot Analysis and Immunofluorescence Confocal Microscopy of
Uroplakin Ib Protein in the Ocular Surface
Normal human cornea, limbus, and conjunctiva were obtained from
eyes at autopsy. For Western blot analysis, scraped corneal epithelial
cells were solubilized, after which approximately 50 μg of protein
was fractionated by 12% sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS-PAGE), blotted to a polyvinylidene difluoride
membrane, and analyzed as described previously.
1
To prepare antiserum for uroplakin Ib, a synthetic peptide that
corresponds to amino acid residues 2 to 12 (AKDNSTVRCFQ;
Fig. 1 ) in uroplakin Ib was conjugated to keyhole limpet hemocyanin and used
to immunize the rabbit.
1
The localization of uroplakin Ib in normal human cornea, limbus, and
conjunctiva was investigated by immunofluorescence confocal microscopy.
Tissue specimens were snap frozen, sectioned into 7-μm-thick slices
and fixed in cold acetone at 4°C for 10 minutes. To block nonspecific
binding, the tissues were incubated with 10% goat serum at room
temperature for 20 minutes. Subsequently, the sections were incubated
at room temperature for 30 minutes with diluted 1:4000 anti-uroplakin
Ib antiserum. Preabsorbed anti-uroplakin Ib antiserum with 5 μg/ml
synthetic peptides was used for controls after a 2-hour incubation at
room temperature. Sections were incubated further with fluorescein
isothiocyanate–conjugated swine anti-rabbit immunoglobulins (working
dilution 1:500; Dako, Carpinteria, CA) for 30 minutes at room
temperature and mounted with antifading medium containing propidium
iodide (Vectashield; Vector, Burlingame, CA). Between each of these
steps, the tissue sections were rinsed thoroughly with 0.1 M
phosphate-buffered saline. Slides were examined using a laser scanning
confocal microscope (Fluoview; Olympus, Tokyo, Japan).
The longest cDNA clone corresponding to
GS8103 comprised 2018 bp of nucleotides and contained an open reading frame
encoding 260 amino acids of human uroplakin Ib (GenBank accession
number, AB002155). The nucleotide sequence 977 to 1135 corresponded to
another gene signature (GS),
GS8043, that matched the 3′-UTR
of human uroplakin Ib in GenBank (accession numbers AB015234 and
AF0422331). These results imply that the uroplakin Ib gene has two
polyadenylation sites. A canonical polyadenylation signal (AATAAA) was
found in the region of
GS8043, but not in the region of
GS8103. In the coding region, it was 92% identical with
bovine uroplakin Ib and 93% identical with mink TI1 (mink uroplakin
Ib), as described by Finch et al.
19 In the 3′-UTR, the
nucleotide sequence 1462 to 1741 of human uroplakin Ib corresponded to
the Alu-repetitive sequence. Bovine uroplakin Ib also contained a
bovine Alu-like repetitive sequence that was not present in mink
TI1.
8 21 Compared with bovine uroplakin Ib in the 3′-UTR,
the nucleotide sequence 1984 to 1989 (AGTAAA) of human uroplakin Ib may
be a noncanonical polyadenylation site of
GS8103.
The mRNA expression of uroplakin Ib was examined in various
tissues by RT-PCR. Uroplakin Ib transcript was clearly detected in
bladder, corneal epithelium, conjunctival epithelium and placenta. It
was detected faintly in kidney and not at all in skin, brain, heart,
liver, lung, skeletal muscle, small intestine, and uterus (
Fig. 2A ). Among other uroplakins, uroplakin II was also detected weakly in
cornea and conjunctiva, although at a much lower level than in bladder
(Fig. 2B) . Uroplakin Ia and III were not detected in corneal epithelium
or conjunctival epithelium
(Fig. 2B) . The results of RT-PCR using
uroplakin Ib primers and RT-PCR using uroplakin Ib extended 3′-UTR
primers showed that two mRNA isoforms of uroplakin Ib were present in
cornea, conjunctiva, and bladder
(Fig. 2B) .
Western blot analysis of human corneal epithelium revealed an
immunoreactive band with a molecular weight of approximately 28 kDa
that corresponds to uroplakin Ib protein
8 (
Fig. 3 , lane 1). The specificity of this reaction was established by the fact
that the staining was completely blocked by preincubating the
antiserum with the peptide antigens (5 μg/ml;
Fig. 3 , lane 2).
The localization of uroplakin Ib protein was investigated by
immunofluorescence confocal microscopy using the same antiserum with
specificity confirmed by Western blot analysis. In corneal epithelium,
the cell membranes of the superficial and wing cells immunoreacted
intensely with the antibody. The most apical cells, however, did not
stain (
Fig. 4A ). In conjunctival epithelium, the pattern of positive immunostaining
was little different from that in corneal epithelium. Although the
staining was less than that in corneal epithelium, it was seen in all
the epithelial layers except for the most apical cells
(Fig. 4E) . In
limbal epithelium, the staining pattern was similar to that in
conjunctival epithelium
(Fig. 4C) . All the control slides showed
negative staining
(Figs. 4B 4D 4F) .
Herein, we report how we cloned human uroplakin Ib cDNA and found
that it has two polyadenylation sites, tagged by
GS8043 and
GS8103. These two transcripts are present in approximately
equal intensities in normal human urothelium examined by Northern blot
analysis.
19 By RT-PCR, we found that these two transcripts
are also present in the normal ocular surface epithelia of humans. When
we searched against database (db) EST, however, we found that
the expressions of the two GS sequences were quite different.
GS8043 matched five of EST’s material sources: invasive
kidney tumor (GenBank accession numbers AA513869 and AA484313),
well-differentiated endometrial adenocarcinoma (AI632869), poorly
differentiated endometrial adenocarcinoma (AI811548), and gallbladder
(AA345,951);
GS8103 was not found at all. Judging by this,
the transcript corresponding to
GS8043 may be more highly
expressed in carcinoma tissues than in normal tissues.
Using RT-PCR, we clearly detected uroplakin Ib and faintly detected
uroplakin II in corneal and conjunctival epithelia. Uroplakins Ia and
III, were not detected. Our results therefore seem to indicate a
difference between ocular surface epithelia and bladder epithelium,
because it is known that uroplakin Ib makes a heterodimer with
uroplakin III in bladder epithelium.
22 Uroplakins Ia
and Ib are members of the TM4 family of proteins and have been
implicated in diverse cellular processes, including cell activation,
proliferation, differentiation, adhesion, and motility by facilitating
specific interactions between cell surface proteins.
23 24 Recent studies have shown that CD9, CD63, and CD81 form complexes with
integrins on the cell surface.
25 26 To clarify the
possible function of uroplakin Ib in the ocular surface, it would be
useful to investigate its possible association with other proteins.
In the corneal epithelium, uroplakin Ib was identified by
immunofluorescence confocal microscopy in the membranes of cells in the
superficial half of this multicellular layer. This localization is
generally similar to that in bladder epithelium, except that in bladder
it is the superficial epithelial cells that label the
strongest.
27 As in corneal epithelium, in conjunctival
epithelium and limbal epithelium, uroplakin Ib was found in the cell
membranes in all epithelial layers, albeit with lower levels of
immunostaining. It is interesting that uroplakin Ib protein was not
detected in the most superficial cells in the ocular surface
epithelium, even when immunofluorescence confocal microscopy was
performed after treatment with neuraminidase or acetylcysteine to avoid
an effect of heavily glycosylated proteins (data not shown).
When we consider the tissue distribution of uroplakin Ib as described
in dbEST, it is apparent that it exists in several nonkeratinized
epithelia at the tissue–fluid interface. In the ocular surface,
apolipoprotein J and mucin proteins are known to localize at the
tissue–fluid interface.
28 29 Similar to these proteins,
uroplakin Ib may function to maintain ocular surface homeostasis at the
tear–ocular surface interface. Uroplakin Ib is one of the major
components of bladder, where it is thought to prevent rupturing during
distension.
5 6 7 We reason that if uroplakin Ib has a
similar mechanical function in ocular surface, it may help resist
mechanical forces on the cornea by intraocular pressure or external
pressures such as those due to blinking.
As an ocular surface defense system and to prevent bacterial infection,
tear fluid contains secretory IgA and IgG, complement components,
lactoferrins, and lysozyme.
30 Recently, it has been
indicated that antimicrobial peptides called defensins are produced by
the cornea, conjunctiva, and lacrimal gland.
31 In the
corneal epithelium, there are numerous solid junctions, and the
permeability of this cellular layer is much less than that of
conjunctival epithelium.
32 However, if the defense system
is compromised and bacteria invade the corneal epithelium, the
immunologic defense of the cornea is lower than that of the
conjunctiva.
30 With this in mind, it is interesting to
note that uroplakin Ia and Ib are able to bind to
E. coli type I pili and that this results in exfoliation of host bladder
epithelial cells as part of an innate host defense
system.
13 14 We postulate that uroplakin Ib expressed in
ocular surface epithelium, especially the apical side of corneal
epithelium, may prevent bacterial invasion into deeper tissues. The
most superficial cells may not need uroplakin Ib, because they fall off
in a short time, whether bacteria attach them or not. The reason the
uroplakin Ib, not uroplakin Ia, is necessary in ocular surface
epithelium is not clear at present and is the subject of proposed
studies.
The authors thank Satoshi Kawasaki for technical help and Tung-Tien
Sun, New York University, and Andrew Quantock, Cardiff University,
United Kingdom, for critical reading and comments on the manuscript.