1 Division of Cell Sciences, School of Biological Sciences, University of
Southampton, Bassett Crescent East, Southampton SO16 7PX, UK
2 Department of Biology, University of York, PO Box 373, York YO10 5YW, UK
* Present address: Division for Infection, Inflammation and Repair, School of
Medicine, University of Southampton, Southampton General Hospital, Southampton
SO16 6YD, UK
These authors contributed equally to this work
Author for correspondence (e-mail:
tpf{at}soton.ac.uk
)
Accepted 6 May 2002
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Summary |
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Key words: Occludin, Tight junction, Epithelium, Isoform, Alternative splicing, Embryo
![]() |
Introduction |
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TJs contain several interacting constituents comprising at least three
types of transmembrane proteins, which contribute to the intercellular sealing
process, and a series of cytoplasmic `plaque' proteins with signalling and
regulative properties, which also mediate linkage to the cytoskeleton
(Stevenson and Keon, 1998;
Matter and Balda, 1999
). The
first transmembrane protein of the TJ to be discovered was occludin
(Furuse et al., 1993
),
followed by claudins (Furuse et al.,
1998a
; Morita et al.,
1999
) and JAM [junction adhesion molecule
(Martin-Padura et al., 1998
)].
Occludin (
65 kDa) has four membrane-spanning domains creating two
extracellular loops, characteristically rich in glycine and tyrosine residues,
and cytoplasmically located N- and C-termini
(Furuse et al., 1993
;
Ando-Akatsuka et al., 1996
).
The longer C-terminal domain contains binding sites for plaque proteins
including ZO-1, ZO-2, ZO-3 and cingulin
(Furuse et al., 1994
;
Haskins et al., 1998
;
Fanning et al., 1998
;
Cordenonsi et al., 1999
;
Wittchen et al., 1999
;
Itoh et al., 1999
) and is
essential for occludin assembly at the TJ, mediated by ZO-1 association
(Furuse et al., 1994
;
Chen et al., 1997
;
Matter and Balda, 1998
;
Mitic et al., 1999
;
Sheth et al., 1997
;
Sheth et al., 2000
;
Medina et al., 2000
). Several
studies have provided evidence that occludin contributes to both the structure
and the sealing function of the TJ and to its role in maintaining epithelial
membrane polarity (Furuse et al.,
1996
; Balda et al.,
1996
; McCarthy et al.,
1996
; Chen et al.,
1997
; Van Itallie and
Anderson, 1997
; Wong and
Gumbiner, 1997
; Bamforth et
al., 1999
; Lacaz-Vieira et
al., 1999
; Balda et al.,
2000
; Medina et al.,
2000
). However, since occludin null embryonic stem cells and mice
are capable of differentiating TJs (Saitou
et al., 1998
; Saitou et al.,
2000
), other TJ transmembrane proteins, notably claudins
(Furuse et al., 1998b
;
Tsukita and Furuse, 1999
;
Sonoda et al., 1999
;
Furuse et al., 2002
), may have
a more direct role in regulating TJ activity.
Occludin is the product of a single gene located on human chromosome band
5q13.1 (Saitou et al., 1997).
However, several protein forms of occludin may be evident in epithelial cells,
representing different states of phosphorylation or other post-translational
modifications. These occludin forms have been implicated in the regulation of
occludin assembly at the TJ (Cordenonsi et
al., 1997
; Sakakibara et al.,
1997
; Wong, 1997
;
Antonetti et al., 1999
;
Farshori and Kachar, 1999
;
Sheth et al., 2000
). In
addition to post-translational modifications, there is evidence that distinct
occludin isoforms may be expressed as a result of alternative splicing. Thus,
several occludin mRNA bands have been identified in northern blots of mouse
cultured epithelial cells and tissues
(Saitou et al., 1997
).
Occludin 1B variant has been discovered recently in canine cells, containing a
193 bp insertion corresponding to an alternatively spliced exon in the gene
encoding a unique N-terminus sequence of 56 amino acids
(Muresan et al., 2000
).
In this paper, we provide evidence for an additional site for alternative splicing of occludin, located at the fourth transmembrane domain (TM4). From reverse transcription-PCR analysis of human, monkey, dog and mouse occludin mRNA and genomic analysis of the human occludin gene, together with western blotting and immunofluorescence data, we report an isoform of occludin detected only in primates in which the TM4 and immediate 3'-flanking sequence is deleted (TM4 minus isoform; TM4-). In the human, both isoforms are expressed ubiquitously and from the preimplantation stage of development when the trophectoderm epithelium differentiates. Our data also indicate that occludin TM4- is expressed as a protein, albeit at low levels and in particular culture conditions associated with subconfluent cells. The potential for this isoform, in which the C-terminus becomes extracellular, as a regulator of occludin functional activity at the TJ is briefly discussed.
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Materials and Methods |
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RT-PCR analysis
Total RNA was extracted from tissues using the RNeasy Midi kit (Qiagen) and
from cells using Tri-Reagent (Sigma), according to manufacturer's
instructions. In addition, commercially available total RNA from human kidney,
cervix and uterus (Ambion) was used. Poly A+ RNA was extracted from
single human and mouse embryos using the Dynabeads mRNA DIRECT kit (Dynal
A.S., Norway) as described previously
(Eckert and Niemann, 1998;
Holding et al., 2000
), with
modifications. Individual embryos were lysed in 150 µl lysis buffer for 10
minutes and transferred to tubes containing 20 µl Dynabeads previously
washed in lysis buffer, and roller incubated for 15 minutes. Bead-mRNA complex
was removed by magnet and washed twice in kit buffers A and B before cDNA
synthesis either after mRNA elution (mouse embryos) in 20 µl RNase-free
water at 65°C for 2 minutes or by solid-phase method (human embryos, see
below). Total RNA (200-500 ng) and 80% of the PolyA+ mRNA from
single mouse embryos was reverse transcribed in 20-50 µl RT reactions into
cDNA using 0.5 mM of each dNTP, 5 µM random hexamers (Promega), 40 U
RNAGuard (Pharmacia) and 200 U Superscript II reverse transcriptase
(Gibco-BRL). To control for genomic DNA contamination of PolyA+
mRNA, 20% of the fraction eluted from the Dynabeads was treated similarly but
in the absence of reverse transcriptase. Conditions for reverse transcription
were 10 minutes at 25°C, 1 hour at 42°C and 5 minutes at 95°C. For
solid-phase cDNA synthesis of single human embryos, a 50 µl RT reaction was
used as above except containing 50 U RNAGuard and 250 U Superscript II.
Supernatant was removed by magnet and bead-cDNA complexes washed in
Tris-HCl.
cDNA derived from 50 ng total RNA or 95% mouse embryo cDNA was then amplified in 50 µl PCR reactions using 0.2 µM of each sequence-specific primer for tissue/cell-line RNA and 1 µM for mouse embryos (Table 1), 2 mM MgCl2, 0.2 mM dNTPs and 2.5 U native Taq Polymerase (Gibco-BRL). PCR conditions were as follows: 95°C for 5 minutes followed by 72°C for 1 minute. After addition of the enzyme at 72°C (hot start), cDNAs were amplified for 40-45 cycles at 95°C for 30 seconds; 58°C for 60 seconds and 72°C for 90 seconds. A two-stage PCR reaction was also employed, using nested primers and generally 2 µl (4%) of the first-stage product. For solid-phase PCR (human embryos), 50 µl PCR reactions comprised 0.2 µM of each primer in Dynabead PCR buffer (x1), 1 mM dNTPs and 2.5 U Taq Polymerase and were heated at 94°C for 3 minutes before two cycles at 94°C for 30 seconds, 58°C for 1 minute, 72°C for 1 minute, and finally 94°C for 2 minutes before placing on ice to prevent reannealing of strands. Beads were removed using the magnet and sufficient supernatant placed in a new tube for a further 40 cycles of amplification.
|
For detection of human occludin mRNA, four pairs of primers were designed,
two pairs flanking the fourth transmembrane domain (TM4) including the
beginning of the C-terminal domain, and two pairs exclusively within the
C-terminus (Table 1). Human
primers were also used successfully to amplify monkey occludin. Mouse and dog
primers included two pairs flanking the TM4 domain and adjacent region of
C-terminus, in each case (Table
1). Primers for RNA polymerase A were used as a positive control
in embryo samples (Eckert and Niemann,
1998). PCR products were directly sequenced using a BigDye
Terminator kit (Applied Biosystems) and automated sequencing.
Antibodies and western blotting
Rabbit polyclonal antibody to the C-terminus of human occludin
(Van Itallie and Anderson,
1997) was kindly provided by J. M. Anderson (Yale University, CT)
and was used in immunoblotting of cells and tissues. Cells were washed in PBS
and solubilised in boiling SDS-sample buffer for 5 minutes. Tissue extract for
electrophoresis was generated using frozen tissue powder boiled for 5 minutes
in PBS:1% SDS and centrifuged at 10,000 g for 3 minutes.
Samples were run on 4-12% polyacrylamide gradient gels (Invitrogen) and
blotted onto Hybond-C nitrocellulose (Amersham) at 300 mA overnight before
immunoblotting and ECL chemiluminescence (Amersham) as described previously
(Sheth et al., 2000
).
Densitometric analysis of immunoblots was performed using AlphaImager (Alpha
Innotech Corporation).
Immunocytochemistry
Caco-2 cells were cultured on coverslips at
3x106/cm2 and
6x106/cm2 for up to 4 days to induce confluent and
sub-confluent monolayers. Confluent layers were also wounded with a fine
sterile forceps tip to create cell islands, washed in EMEM and cultured
further for 30 minutes, 1 hour and 2 hours. Confluent, sub-confluent and
wounded layers were processed for localisation of occludin using a rabbit
polyclonal antibody recognising the C-terminal domain of human occludin.
Rabbit polyclonal antibody to mouse ZO-1
(Sheth et al., 1997) and ZO-2
(Zymed) were also used for immunostaining. Cells were processed either living
(using EMEM for antibody and washing steps at 4°C, fixing finally in
paraformaldehyde) or after 10 minutes fixation in 1% paraformaldehyde in PBS,
permeabilised in 0.25% Triton X-100 in PBS, washed three times in PBS:1.8 mM
CaCl2 and treated with occludin antibody (1:1000 in PBS:1.8 mM
CaCl2:0.1% NaN3, 1 hour) followed by washing three times
in PBS:1.8 mM CaCl2 and labelling with Alexa 488-conjugated
secondary antibody (1:500 in PBS:1.8 mM CaCl2; Cambridge
Bioscience). Specimens were viewed on an MRC-600 series confocal imaging
system (BioRad, UK) or a Leitz Fluovert epifluorescence microscope using
x63 objectives and appropriate filters.
![]() |
Results |
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|
The sequence of the larger RT-PCR product from human tissues, embryos and cells using the primer sets spanning the TM4 region corresponded exactly to the published coding sequence. However, in the sequence for the smaller product, a 162 bp region from 1166 to 1327 bp of the published occuldin cDNA sequence was deleted. The deleted region encodes the entire TM4 segment and adjacent C-terminal sequence (Fig. 2A,B). We designate these two forms of occludin as TM4 plus (+) and TM4 minus (-) variants.
|
Human occludin genomic sequence analysis
In order to determine whether the TM4- variant occludin cDNA was
due to differential splicing, we initially compared the published human
occludin cDNA sequence to the working draft of the human genome using the
Blast program at the NCBI website. This revealed the presence of nine exons in
the human occludin mRNA (not shown). The positioning of these exons is
confirmed by the current annotation of the pertinent genomic contig
(NT_006497.6) as viewed on the NCBI website. Remarkably, the sequence
alignment showed that human occludin exon 4 of 162 nucleotides precisely
coincided with the 162 bp region deleted in the smaller TM4- RT-PCR
product observed with primers H1/H2 and H3/H4
(Fig. 3A). This observation
strongly supported the view that the TM4- variant is a genuine mRNA
isoform due to alternative splicing. In addition, the data indicated skipping
of exon 4 as the mechanism to generate the TM4- isoform
(Fig. 3B). While the
TM4- mRNA isoform is predicted to encode a shorter protein product,
the deletion of the 162 bp of coding sequence contained in exon 4 does
preserve the reading frame in the downstream exon 5
(Fig. 3A). The predicted
peptide sequence of the TM4--encoded protein would therefore be
identical to canonical occludin, except for the deletion of the 54 amino acids
encoded by exon 4.
|
Species variation in occludin TM4 isoforms
We next investigated whether the presence of the TM4- isoform
could be demonstrated in species other than human. RNA extracted from the
African Green Monkey (Cercopithecus aethiops) kidney epithelial cell
line BSC1 was employed in single and nested RT-PCR using human occludin primer
pairs H1/H2 and H3/H4. Two products were amplified, the larger being
predominant, and which corresponded in size to the human TM4+ and
TM4- cDNAs, respectively (Fig.
4). Both of the Cercopithecus putative TM4+
and TM4- products were sequenced. Alignment to each other and to
the corresponding human sequences confirmed that size and position of the 162
bp deletion are precisely conserved between Cercopithecus and
Homo (Fig. 2B). We
concluded that the TM4- isoform in monkey cells was homologous to
the human and likely generated by an exon skipping mechanism similar to the
one in human tissues. There was divergence between the monkey and human
nucleotide sequences, ruling out human contamination as a source of the monkey
cDNAs and predicting three amino acid substitutions in the 54 amino acid
sequence encoded by occludin exon 4 (Fig.
2B).
|
We investigated whether the TM4- occludin isoform was present in two other mammalian orders, rodents and carnivores. Based on the published murine occludin cDNA sequence (accession no. NM_008756.1), a primer pair spanning the 162 bp TM4 was synthesized (M1/M2, Table 1). When these primers were used in RT-PCR experiments on mouse tissue (kidney, lung and liver), embryos and cell line (CMT64/61 cells) RNA, a band corresponding in size to the canonical TM4+ occludin mRNA was observed (and confirmed by sequencing), but no smaller product corresponding to the hypothetical mouse occludin TM4- isoform was detectable (Fig. 4). The same result was found using M3/M4 primers either alone or in nested PCR after M1/M2. Likewise, we designed suitable primers (C1/C2 and C3/C4, Table 1) based on the published canine occludin cDNA sequence (accession no. U49221.1) and used them on MDCK cell line RNA. Again, while the product expected from the canonical TM4+ mRNA was readily amplified as a strong band (and confirmed by sequencing), the smaller product predicted for the hypothetical TM4- isoform could not be detected (Fig. 4).
Immunocytochemistry
Preliminary experiments were conducted on human cells to investigate
whether the occludin TM4- isoform was expressed as protein.
Conceptual translation of the TM4- isoform indicates extracellular
localisation of the C-terminus. Confluent and subconfluent monolayers of fixed
and permeabilised Caco-2 cells showed strong staining for occludin by confocal
microscopy at apicolateral contact sites using the C-terminal antibody
(Fig. 5A,B). A similar
junctional staining pattern was evident for cytoplasmic plaque TJ proteins,
ZO-1 (Fig. 5C,D) and ZO-2.
Junctional staining using these antibodies was not evident in living confluent
monolayers but in subconfluent islands, the occludin antibody resulted in
intermittent, weak staining around the periphery of the island in 13/17 (76%)
islands examined (Fig. 5E,F).
This pattern of staining was not observed using either the ZO-1
(Fig. 5G,H) or ZO-2 antibodies
(0/15), or in the negative control of the secondary antibody alone
(Fig. 5I,J), employed in all
staining reactions. In addition, when confluent monolayers were wounded to
generate islands of cells and subsequently washed and cultured for up to 2
hours, peripheral staining of occludin was observed in living cultures,
appearing to increase in intensity between 30 minutes and 1 hour of culture
(Fig. 5K,L). At high
magnification, discontinuous spots of occludin staining were present in
outermost and adjacent enclosed cells of the islands, at both contact-free and
cell-cell contact surfaces (Fig.
5K,L). In the absence of a specific antibody for occludin
TM4-, these data, showing weak extracellular exposure of occludin
C-terminus in living cells, indicate the potential for protein expression of
this isoform in specific conditions.
|
Western blotting
Human, monkey, mouse and canine epithelial cells and mouse lung were probed
with occludin antibody in immunoblots. Several bands of occludin were
identified around 58-72 kDa, as shown previously (e.g.
Sakakibara et al., 1997;
Wong, 1997
;
Sheth et al., 2000
), and
represent potential post-translational states
(Fig. 6). The lowest band of
the complex migrated at
58 kDa, some 7 kDa below the predominant band at
65 kDa, corresponding to the reduction in protein mass anticipated for the
TM4- isoform. A similar pattern of occludin bands was evident
across species although the position of individual bands varied slightly. We
investigated the level of expression of the lowest band in Caco-2 cells from
confluent and subconfluent culture using equal protein loading and
densitometry. While the expression of the predominant 65 kDa band was
equivalent in both confluent and subconfluent cells, the 58 kDa band was
significantly upregulated in subconfluent culture
(Fig. 6). Collectively, these
data indicate that occludin TM4- in human and monkey cells may
migrate as a component of the 58 kDa band and be expressed in subconfluent
cells.
|
![]() |
Discussion |
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Recent analysis of the human genome indicates that a large proportion
(around 42%) of genes are alternatively spliced, in particular for cell
surface receptors (Modrek et al.,
2001). The vast majority of detected splice variants appeared to
produce highly specific, biologically meaningful control of function forms
(Modrek et al., 2001
).
Modulation of intercellular adhesion by expression of alternate splice
variants of adhesion molecules is well documented, including, in particular,
the deletion of TM domains. Mouse neural cell adhesion molecule (N-CAM) of the
immunoglobulin superfamily, is expressed as at least three splice variants,
one of which lacks the TM domain, altering localisation and adhesive function
(Santoni et al., 1987
;
Owens et al., 1987
;
Powell et al., 1991
). Other
examples of adhesion molecules in which TM domain deletion by alternative
splicing has been identified include integrin
IIb subunit
(Trikha et al., 1998
), the
leukocyte selectin adhesion molecules GMP-140 and P-selectin
(Johnston et al., 1990
;
Ushiyama et al., 1993
), kit
ligand (Flanagan et al.,
1991
), vascular cell adhesion molecule
(Terry et al., 1993
;
Cybulsky et al., 1993
;
Pirozzi et al., 1994
),
receptor tyrosine phosphatase ß
(Barnea et al., 1994
;
Maurel et al., 1995
) placental
sialo-adhesion molecule CD33L (Takei et
al., 1997
), PECAM-1
(Goldberger et al., 1994
) and
ICAM-1 (Wakatsuki et al.,
1995
). However, our data is the first report of a TM deletion
within a TJ membrane protein. The importance of adhesion systems in
intercellular signalling further raises the possibility of alternative splice
forms acting as functional regulators.
Comparative analysis of human, monkey, mouse and canine occludin cDNA
illustrated that the TM4- isoform was detectable in the primate
species examined but not elsewhere. A plausible explanation is that such a
splicing mechanism was present in the common ancestor of Homo and
Cercopithecus, which is thought to have lived 23.3 million years ago
(Kumar and Hedges, 1998).
Since Homo and Cercopithecus belong to different
superfamilies within the infraorder Catarrhini
(Fleagle, 1999
), it appears
that the occurrence of occludin TM4 differential splicing may be conserved
among the Catarrhini, which comprise humans, great apes, gibbons, and Old
World monkeys.
Although the occurrence of species-specific differences in alternative
splicing of genes amongst mammals is uncommon (reviewed by
Lu et al., 1999), a growing
number of human- or primate-specific splicing events mediated by exon skipping
and with functional implications have been reported. Thus, a splice variant of
hormone-sensitive lipase generated by skipping of exon 4 has been detected in
human but not rat, mouse, dog or rabbit tissues
(Laurell et al., 1997
).
Similarly, deletion of exon 4 resulting in a truncated splice variant of
5-aminolevulinate synthase mRNA is evident in human differentiating erythroid
cells but not in dog or mouse (Conboy et
al., 1992
). The estrogen receptor-
also exhibits
exon-deleted variants in the human but not mouse
(Lu et al., 1999
). Other
examples of human-specific alternative splicing by exon skipping include the
EGF1 domain of cartilage aggrecan (Fulop
et al., 1996
) and glucocorticoid receptor-ß
(Otto et al., 1997
). It would
appear, therefore, that exon skipping has remained a viable mechanism for
modifying protein function during mammalian evolution.
Alternative splicing by exon skipping has also been identified in
TJ-associated proteins, notably in the MAGUK proteins ZO-1
(Balda and Anderson, 1993) and
ZO-2 (Chlenski et al., 2000
).
Moreover, the species-specific nature of exon skipping to generate the
TM4- isoform of occludin is not unique for this gene. Thus, the 1B
variant of canine occludin that results in a unique N-terminus is generated by
insertion of exon 2B, not expressed in canonical occludin
(Muresan et al., 2000
). This
exon is not present in the human genome database and is undetectable in mouse
following extensive sequence analysis of cDNAs generated by RT-PCR (B.S.,
unpublished), indicating a canine-specific modification. Given the breadth of
modulation that occurs at the TJ to regulate transcellular electrical
resistance, paracellular transport and signalling activity
(Stevenson and Keon, 1998
;
Matter and Balda, 1999
), it is
not surprising that alternative splicing may be used to expand the repertoire
of protein function. This may be true in particular for occludin as a single
copy gene while variability in claudin TJ transmembrane protein activity is
known to be derived from gene multiplicity.
We have made a preliminary investigation of the potential for human
occludin TM4- isoform to be expressed as a protein, which would be
indicative of a possible functional role. In the absence of a specific
antibody recognising occludin TM4-, we used a C-terminal antibody
on living cells since, if TM4- is expressed as a protein, the
C-terminus switches from cytoplasmic to extracellular domains. This might be
an interesting biological phenomenon since the occludin C-terminus is
recognised as a site of interaction with several TJ cytoplasmic plaque
proteins including ZO-1, ZO-2, ZO-3 and cingulin
Furuse et al., 1994;
Haskins et al., 1998
;
Fanning et al., 1998
;
Cordenonsi et al., 1999
;
Wittchen et al., 1999
;
Itoh et al., 1999
). Thus,
adhesive activity of occludin TM4- in the absence of plaque protein
interaction might act to negatively regulate TJ integrity.
We found no convincing evidence of occludin immunostaining in living confluent Caco-2 cell cultures using the C-terminal antibody. However, living islands of cells in subconfluent or wounded cultures showed weak, intermittent membrane staining in the periphery corresponding to the outermost cells, a staining pattern not reproduced in controls using other TJ antibodies with the same secondary antibody. The extent of peripheral staining in wounded islands also appeared to increase with time. These data indicate that TM4- occludin may be expressed at low levels in certain cellular conditions such as subconfluency, although it is possible that reduced antibody accessibility may also contribute to the failure to detect expression in living confluent cells. Further support for expression of TM4- at the protein level was provided by immunoblotting analysis. Here, a weak positive band for occludin at the size appropriate for the TM4- splice variant (58 kDa) was present in both primate and nonprimate cells. Significantly, the level of expression of the 58 kDa band was upregulated in subconfluent versus confluent culture of human epithelial cells.
Epithelial cells are known to become more motile and proliferative in
response to wounding, which may involve changing their adhesive state
(Martin, 1997). For example,
in a culture model similar to the one employed here, peripheral cells in
subconfluent islands after wounding have been shown to alter their state of
desmosome adhesion (Wallis et al.,
2000
). We speculate that induction of increased motility and
proliferative potential in epithelial cells will also include mechanisms to
downregulate TJ integrity. Our data indicate that such a response may in part
be mediated by expression of occludin TM4- to modulate
intercellular adhesion. In conclusion, we have identified a new mRNA splice
variant of the TJ membrane protein, occludin, within human and monkey
epithelial cells in which the fourth TM domain is deleted, resulting in
misalignment of the C-terminus with respect to the membrane. Preliminary
evidence suggests weak expression of this isoform also occurs at the protein
level in conditions of cellular subconfluency, which may regulate TJ
intercellular adhesion.
![]() |
Acknowledgments |
---|
![]() |
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