Tài liệu Báo cáo khoa học: A novel splice variant of occludin deleted in exon 9 and its role in cell apoptosis and invasion docx

A novel splice variant of occludin deleted in exon 9 and its
role in cell apoptosis and invasion
Jin-Mo Gu1, Seung Oe Lim1, Young Min Park2 and Guhung Jung1
1 Department of Biological Sciences and Seoul National University, Korea
2 Hepatology Center and Laboratory of Hepatocarcinogenesis, Bundang Jesaeng General Hospital, Kyungkido, Korea

Keywords
apoptosis; calcium; invasion; occludin; splice
variant
Correspondence
G. Jung, Department of Biological Sciences
and Seoul National University, 56-1 Shillimdong, Kwanak-gu, Seoul 151-747, Korea
Fax: +82 2 872 1993
Tel: +82 2 880 7773
E-mail:
(Received 18 February 2008, revised 11
April 2008, accepted 15 April 2008)
doi:10.1111/j.1742-4658.2008.06467.x

The tight junction protein occludin participates in cell adhesion and migration and has been shown to possess antitumorigenic properties; however,
the exact mechanism underlying these effects is poorly understood. In liver
cell lines, we identified an occludin splice variant deleted in exon 9
(OccDE9). Furthermore, comparison analysis of wild-type occludin (OccWT)
and OccDE9 revealed that exon 9 played important roles in the induction of
mitochondria-mediated apoptosis and the inhibition of invasion, along with
the downregulation of matrix metalloproteinase expression. In addition, by
using the calcium indicator X-rhod-1, and the inositol trisphosphate receptor inhibitor 2-aminoethoxydiphenyl borate, we found that OccWT but not
OccDE9 increased calcium release from the endoplasmic reticulum. In conclusion, our results showed that occludin mediates apoptosis and invasion
by elevating the cytoplasmic calcium concentration and that exon 9 of
occludin is an important region that mediates these effects.

Occludin is a tight junction (TJ) protein and the first
identified TJ-associated molecule [1]. Occludin is the
product of a single gene located on human chromosome band 5q13.1 and produces several different
mRNAs as a result of alternative splicing [2].
Recently, several variants of occludin, such as
occludin 1B and a fourth transmembrane domain
(TM4)-deleted variant, have been discovered [3,4].
Occludin 1B contains a 193 bp insertion corresponding to an alternatively spliced exon in the gene
encoding a unique N-terminus. Conversely, the TM4deleted variant has a missing fourth TM corresponding to exon 4. In addition to these variants, there is
evidence to show that two distinct promoters, P1 and
P2, confer separate transcriptional start sites [5]. Promoter P2 is located downstream of promoter P1, and

both promoter regions are regulated by tumor necrosis factor-a [5].
Occludin ( 65 kDa) is composed of two extracellular loops that form four membrane-spanning domains;
occludin specifically forms TJ complexes with phosphoproteins such as zonula occludens protein 1, zonula
occludens protein 2, and P-130. Other possible signaling molecules include Ga subunits, tyrosine kinase,
small GTPases (Rab ⁄ Rho), and junctional adhesion
molecules [6]. Interactions of these proteins with the
actin cytoskeleton are major determinants of the TJ
complex and play significant roles not only in cell–cell
contact but also in signal transmission [7].
Accumulating evidence points to TJ disruption in
malignant phenotypes, including local tumor growth,
invasion, and metastasis [8]. In humans, TJ disruption

Abbreviations
2-APB, 2-aminoethoxydiphenyl borate; 5¢-aza-dC, 5¢-aza-2¢-deoxycytidine; BrdUTP, 5-bromo-2¢-deoxyuridine-5¢-triphosphate; ER, endoplasmic
reticulum; ERK1 ⁄ 2, extracellular signal-regulated kinase 1 ⁄ 2; HA, hemagglutinin; IP3, inositol trisphosphate; IP3R, inositol trisphosphate
receptor; JNK, Jun N-terminal kinase; MMP, matrix metalloproteinase; MSP, methylation-specific PCR; shRNA, short hairpin RNA; TJ, tight
junction; TM, transmembrane domain; TSA, trichostatin A; TUNEL, terminal deoxyribonucleotide transferase-mediated nick-end labeling.

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is a distinguishing characteristic of epithelial and many
other types of cancers, where decreases in TJ expression are associated with cancer stage and metastatic
potential [9]. Furthermore, the overexpression of TJ
proteins such as claudin-1 and claudin-4 induces apoptosis and suppresses metastatic potencies [10,11].
Occludin has also been reported to induce apoptosis
and apoptotic sensitization, which are regarded as
antitumorigenic activities [12]. Moreover, various studies have investigated how specific occludin domain
deletions affect the localization and function of occludin [13,14]. Overexpression of deletion mutants
resulted in a lack of capability for membrane localization and induction of apoptosis [12,15]. However, the
mechanism underlying the specific role of occludin in
cancer cell apoptosis remains poorly understood. Furthermore, most studies on multiple signal pathways
involving the TJ complex have focused on the effects
of TJ biogenesis, including occludin [16,17]. For example, membrane-localized kinases, calcium, Ga proteins,
calmodulin and phospholipase were analyzed in several
types of cancer cells from the perspective of TJ formation [18,19].
Gaq protein, as a subunit of Ga, induces the formation of inositol trisphosphate (IP3), which binds to the
IP3 receptor (IP3R) located on the endoplasmic reticulum (ER), thereby releasing calcium into the cytoplasm
[20,21]. When calcium concentrations in the cytoplasm
increase, calcium accumulation occurs along the inner
membrane of mitochondria. This destabilizes membrane potentials and facilitates the initiation of apoptosis [22]. In addition, elevated calcium levels

contribute to cell invasion [23,24]. In contrast, in the
case of occludin, an ER-mediated increase in intracellular calcium levels is not considered to be the
mechanism underlying its effects in cancer cell apoptosis and invasion [21].
In this article, we provide evidence of an additional
occludin variant deleted in exon 9 (OccDE9). On the
basis of a comparative analysis of the involvement of
wild-type occludin (OccWT) and variant occludin in
apoptosis and invasion, as determined by assay, we
revealed that exon 9 played a major role in the induction of mitochondria-mediated apoptosis and the
reduction of cell invasiveness, along with the downregulation of matrix metalloproteinase (MMP) expression. In addition, on the basis of an analysis using
X-rhod-1, a calcium indicator, and 2-aminoethoxydiphenyl borate (2-APB), which inhibits IP3R and thus
calcium release from the ER, we suggest that an
increase in the intracellular calcium level is a possible
explanation for occludin-induced apoptosis and
reduced invasiveness.
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Results
Identification of the human occludin splice
variant
We detected a splice variant of occludin in liver cell
lines by using an antibody to the occludin N-terminus,
which detected two specific bands, one of which had
the expected molecular mass for OccWT ( 65 kDa),
and the other of which had a smaller molecular mass
(Fig. 1A). OccWT was not expressed only in Chang
cells. On the basis of these observations, we selected
Chang cells for the identification of the splice variant.
The PCR product of Chang cell-derived cDNA was
amplified by primers bound to each exon, which

showed that exon 9 was deleted in this occludin variant (OccDE9) (Fig. 1B). To verify whether this deletion
occurred due to the loss of genomic DNA, where
exon 9 is located, we amplified genomic DNA with the
primers bound to the exon 9 region; the size of the
resulting PCR product was as expected (data not
shown). To determine the detailed occludin variant
sequence, we performed 3¢-RACE and obtained an
exon 9-deleted splice variant, which was compatible
with the results of PCR (Fig. 1C).
In a previous report, occludin was shown to involve
two different promoters [5]. Depending on the promoter, either exon 1 or exon 1a was selected for the
alternative splicing process [5]. Using primers designed
for exon 1 and exon 1a, we compared the promoters
involved in Chang and Huh7 cells. As shown in
Fig. 1D, PCR products containing exon 1 were produced in Huh7 cells but not in Chang cells. To determine whether the different splice variants were caused
by a difference in the boundary sequence deciding the
splicing points between exon 8 and exon 9, we compared the sequences of these regions in Huh7 and
Chang cells; no difference was found (Fig. 1E).
Figure 1F depicts the schematic of OccDE9 obtained
from Chang cells as compared to OccWT, based on
PCR analysis.
OccWT is epigenetically silenced by promoter
hypermethylation in Chang cells
On the basis of the different usages of exon 1 and
exon 1a in Huh7 and Chang cells, we decided to analyze the relationship between usage of exon 1a and P1
promoter methylation. In liver cell lines, we initially
performed methylation specific-PCR (MSP) with primers amplifying the CpG island of the occludin P1 promoter. We observed methylated DNA only in Chang
cells. Unmethylated DNA was not detected (Fig. 2A).

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Exon 9 of occludin in apoptosis and invasion

A

C

B

D

E

F

Fig. 1. Identification of a human occludin splice variant in liver cell lines. (A) Occludin protein expression was examined with an antibody to
the N-terminus of occludin in HepG2, Hep3B, Huh7, Chang and HLE cells. (B) cDNA synthesized from Chang cells was amplified with primers matching each exon of occludin. (C) 3¢-RACE was performed on the cDNA of Chang cells, and the PCR product was sequenced.
(D) PCRs for exon 1 and exon 1a were performed in Huh7 and Chang cells. (E) The boundary regions of exon 8 and exon 9 in the genomic
DNA of Chang cells were sequenced and aligned with OccWT. (F) Schematic of mRNA coding sequence for human occludin, showing the
comparison of OccWT and OccDE9. E, exon; P, promoter; CpG, CpG island.

A

B

C

trichostatin A (TSA), an inhibitor of histone deacetylase (Fig. 2B). In the same treated samples, OccWT
expression was detected by measuring protein and
mRNA levels, using primers for exon 9 (Fig. 2C).
Furthermore, we confirmed the use of exon 1 by using
RT-PCR in Chang cells treated with TSA and 5¢-azadC (Fig. 2B).
Different localizations of OccWT and OccDE9

Fig. 2. OccWT expression is epigenetically silenced by methylation
in Chang cells. (A) The methylation status of the occludin promoter
was analyzed in HepG2, Hep3B, Huh7, Chang and HLE cells by
MSP. (B) The demethylating agents 5¢-aza-dC (5 lM) and TSA
(300 nM) were used to treat Chang cells. MSP analysis for the
occludin promoter and PCR with primers for exon 1 and exon 1a
were performed using demethylating agent-treated cells. (C) OccWT
expression in Chang cells treated with 5 lM 5¢-aza-dC and 300 nM
TSA was analyzed using immunoblotting (upper) and RT-PCR
(lower). U, unmethylated DNA; M, methylated DNA; T, 5¢-aza-dC
and TSA treatment.

As expected,
was induced
with 5 lm
inhibitor of

demethylation of the occludin promoter
in Chang cells by combined treatment
5¢-aza-2¢-deoxycytidine (5¢-aza-dC), an
DNA methyltransferase 1, and 300 nm

To investigate whether or not the occludin variant was

localized in the membrane, we cloned the OccWT and
OccDE9 genes into pCMV ⁄ HA and performed immunocytochemistry. The expression of each construct in
Chang, Hep3B and Huh7 cells was analyzed using
immunoblotting (Fig. 3A). As shown in Hep3B cells
transfected with a control plasmid, endogenous occludin was intermittently localized in the membrane and
diffused through the cytoplasm. Occludin in the membrane of Huh7 cells was localized in a more disconnected fashion than that in Hep3B cells. In Chang
cells, stained occludin was observed in the cytoplasmic
region. Additionally, in OccWT-overexpressing Hep3B
and Huh7 cells, regions stained with antibody to hemagglutinin (HA), indicating the expression of exogenous occludin, were located in the cell membranes. In
OccDE9-overexpressing cells, however, no regions
stained with antibody to HA were observed in the
membrane (Fig. 3B). In contrast, neither OccWT nor

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(Fig. 4C). Caspase 3 activity was likewise induced by
OccWT but not by OccDE9 (Fig. 4D). These data
implied that OccWT induced mitochondrial apoptosis
[25], and that exon 9 played a major role in occludininduced apoptosis. In a previous study, the phosphorylation of mitogen-activated protein kinase was shown
to be altered in occludin-overexpressing cells [12]. To
test whether similar modulation occurred in liver cells,
we examined the expression and phosphorylation of
extracellular signal-regulated kinase 1 ⁄ 2 (ERK1 ⁄ 2),

p38, and Jun N-terminal kinase (JNK). The phosphorylation levels of ERK1 ⁄ 2 were high in OccWT-overexpressing cells but not in OccDE9-overexpressing cells
(Fig. 5). No differences were observed in the phosphorylation levels of p38 and JNK (data not shown).

A

B

Invasiveness is reduced in OccWT-overexpressing
cells but not in OccDE9-overexpressing cells

Fig. 3. Localization of OccWT and OccDE9. (A) OccWT and OccDE9
were cloned into pCMV ⁄ HA, a mammalian overexpression vector
(pCMV ⁄ HA-OccWT and pCMV ⁄ HA-OccDE9), and their expression
was confirmed by immunoblot analysis. (B) Green fluorescence
indicates endogenous occludin stained with antibodies to occludin
in Chang, Hep3B and Huh7 cells transfected with pCMV ⁄ HA. Red
fluorescence indicates exogenous occludin stained with antibodies
to HA in Chang, Hep3B and Huh7 cells transfected with pCMV ⁄ HAOccWT or pCMV ⁄ HA-OccDE9. Cont, pCMV ⁄ HA-transfected
cells; WT, OccWT-overexpressing cells; DE9, OccDE9-overexpressing
cells.

OccDE9 were localized in the membranes of Chang cells
(Fig. 3B).
Mitochondrial apoptosis is induced by OccWT
overexpression but not OccDE9 overexpression
Consistent with previous observations [12], in OccWToverexpressing cells, cell proliferation decreased, and
the number of apoptotic cells increased as compared
to findings in control cells. These changes, however,
were not observed in OccDE9-overexpressing cells
(Fig. 4A,B).

To identify the pathways linked to apoptosis
induced by OccWT, we analyzed caspase 3 activity and
the expression of several apoptosis-related genes.
Owing to the overexpression of OccWT, the expression
of the apoptotic genes BAX and Apaf-1 increased, and
that of the antiapoptotic gene Bcl-2 decreased. OccDE9overexpressing cells, on the other hand, did not show
any alteration in the expression of these genes
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Using a Matrigel invasion assay and analysis of MMP
expression, we next determined whether occludin
expression in cancer cells was responsible for invasion.
The Matrigel invasion assay revealed that significantly
fewer OccWT-overexpressing Chang and Huh7 cells
were invasive as compared to the number of invasive
control and OccDE9-overexpressing cells (Fig. 6A). Specifically, in Chang and Huh7 cells, OccWT significantly
decreased the expression of MMP2, MMP7, MMP9
and MMP14, and of MMP1, MMP2, MMP3, MMP7,
MMP9 and MMP14, respectively (Fig. 6B).
To determine the function of occludin, we reduced
occludin expression in Huh7 cells by using occludin
short hairpin RNA (shRNA). After determining the
reduced expression level of occludin with immunoblotting (Fig. 7A), we analyzed the effects of downregulated occludin on apoptotic sensitization for H2O2
treatment and on invasiveness. Occludin shRNA-transfected Huh7 cells were more resistant to apoptosisinducing signal (H2O2), and were more invasive than
control shRNA-transfected cells (Fig. 7B,C).
Occludin-mediated increase in calcium
concentration increases apoptosis and decreases
invasiveness
On the basis of the differences between OccWT and
OccDE9 with regard to mitochondria-dependent apoptosis and reduction of invasion, along with downregulation of MMP expression, we postulated that

increased intracellular calcium might be involved in
the effects of occludin. In previous reports, the modulation of calcium concentration has been shown to be
related to cell apoptosis and invasion [21,22].

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Exon 9 of occludin in apoptosis and invasion

A

B

C

D

Fig. 4. OccWT but not OccDE9 induces mitochondria-mediated apoptosis by the modulation of expression of apoptosis-related genes and
caspase 3 activity. Chang, Hep3B and Huh7 cells transfected with pCMV ⁄ HA, pCMV ⁄ HA-OccWT or pCMV ⁄ HA-OccDE9. (A) The viability of
cells was determined by CCK-8 assay. (B) Apoptotic cell numbers were calculated by counting BrdUTP-incorporating cells. (C) The levels of
BAX, Bcl-2 and Apaf-1 were determined using real-time RT-PCR. (D) Caspase 3 activity was determined using the CaspACE colorimetric
assay. All results in (A), (B), (C) and (D) are expressed as the fold ratio relative to control (Cont). All numerical data represent mean and standard deviation of three independent experiments. Cont, pCMV ⁄ HA-transfected cells; WT, OccWT-overexpressing cells; DE9, OccDE9-overexpressing cells; *P < 0.05.

Fig. 5. Phosphorylation of ERK1 ⁄ 2 in OccWT-overexpressing cells.
Immunoblot analyses using specific antibodies against occludin,
ERK1 ⁄ 2, pERK1 ⁄ 2 and b-actin were performed in Chang, Hep3B
and Huh7 cells transfected with pCMV ⁄ HA, pCMV ⁄ HA-OccWT or
pCMV ⁄ HA-OccDE9. Cont, pCMV ⁄ HA-transfected cells; WT, OccWToverexpressing cells; DE9, OccDE9-overexpressing cells.

To confirm the effects of occludin on the cytoplasmic calcium concentration, we analyzed calcium
concentrations by using X-rhod-1, a calcium indicator.

X-rhod-1 has been developed from rhodamine, and
emits red fluorescence [26]. Therefore, we cotransfected
pCMV ⁄ HA, pCMV ⁄ HA-OccWT or pCMV ⁄ HA-OccDE9
with pEGFP-N1 vector. As shown in Fig. 8A, OccWTtransfected cells showed increased cytoplasmic calcium
concentrations, whereas OccDE9-transfected and control-transfected cell did not show any changes in
calcium concentration (Fig. 8A,B).
We tested whether the OccWT-mediated increase in
calcium levels was caused by calcium release from the
ER – one of the calcium metabolism pathways related
to the TJ complex [20]. We examined the effects of
blocking calcium release from the ER by using 2-APB,
an inhibitor of the IP3R, which is located in the ER
membrane. Chang cells were treated with 20 lm 2-APB
12 h after transfection with pCMV ⁄ HA, pCMV ⁄ HAOccWT or pCMV ⁄ HA-OccDE9. The concentration of
2-APB was determined by a proliferation assay, and
20 lm 2-APB did not induce apoptosis in Chang cells
(data not shown). In 2-APB-treated Chang cells, no
difference in apoptosis was seen among the control and

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A

B

Fig. 6. Decreased invasiveness of OccWT-overexpressing cells but not of OccDE9-overexpressing cells. (A) A Matrigel assay was performed
in Chang and Huh7 cells transfected with pCMV ⁄ HA, pCMV ⁄ HA-OccWT or pCMV ⁄ HA-OccDE9. Cells penetrating the Matrigel-coated inserts
were stained with crystal violet. The dots are stained cells on the bottom of the Matrigel-coated inserts. The number of invasive cells was
determined by counting the number of stained cells and then normalizing it to the number of control (Cont) cells. (B) Real-time RT-PCR analysis was performed to determine the levels of MMP1, MMP2, MMP3, MMP7, MMP9 and MMP14 in Chang and Huh7 cells transfected
with pCMV ⁄ HA, pCMV ⁄ HA-OccWT or pCMV ⁄ HA-OccDE9. All numerical data represent the mean and standard deviation of three independent
experiments. Cont, pCMV ⁄ HA-transfected cells; WT, OccWT-overexpressing cells; DE9, OccDE9-overexpressing cells; *P < 0.05.

the OccWT-overexpressing and OccDE9-overexpressing
cells (Fig. 7C). In addition, 2-APB abrogated the effects
of OccWT overexpression, namely, decreased invasiveness, ERK1 ⁄ 2 activation, and modulation of apoptoticrelated gene expression levels (Fig. 8D–F).

Discussion
TJs are involved in cell adhesion and migration; their
downregulation can cause epithelial transformation,
whereas their upregulation induces apoptosis and
inhibits invasion [8]. Occludin, one of the TJ proteins,
has also been shown to have several types of splice
variant and antitumorigenic activity [3,4,27]; however,
its effects on and implications for tumorigenesis are
poorly understood. Here, we present a novel occludin
splice variant deleted in exon 9; in Chang cells, this
variant acts via the P2 promoter. As the P2 promoter
was involved in occludin action, and as no wild-type
form was observed in Chang cells, the methylation

status of the occludin promoter P1 could be
examined. Therefore, we tested the occludin promoter
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P1-containing CpG island region in liver cell lines, and
observed that the occludin promoter was strongly
methylated only in Chang cells.
Osanai et al. reported that an occludin mutant
deleted in region 478–522, which contains exon 9,
could not localize in the membrane or induce apoptosis in mammary cell lines [12]. In addition, Wang et al.
described another occludin mutant deleted in the
whole cytoplasmic tail. This mutant did not exert
a potent inhibitory effect on Raf1-mediated tumorigenesis [15]. In liver cells, as in mammary cell lines, we
observed that OccWT was localized in the membrane,
whereas OccDE9 was localized in the cytoplasm. In previous reports, several factors, such as the proteins
involved in TJ complexes, affected occludin localization [16,17]. On the basis of this, the inability of
OccWT to associate with the membrane in Chang cells
could be possibly explained by cell-type-dependent
events.
In liver cell lines, OccWT but not OccDE9 induced
apoptosis and reduced invasiveness (Figs 4 and 6). The
modulation of the expression levels of BAX, Bcl-2 and

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Exon 9 of occludin in apoptosis and invasion

A

B

Fig. 7. Effects of occludin shRNA on apoptosis and invasiveness. Huh7 cells were transfected with five constructs producing predesigned
occludin shRNA. (A) Immunoblot analysis confirmed the silencing effects of occludin shRNA. The degree of occludin downregulation is represented as a bar graph in the right panel. (B) Apoptosis of Huh7 cells transfected with occludin shRNA or control shRNA was analyzed using
a TUNEL assay in the presence of 400 and 800 lM H2O2. The number of apoptotic cells is expressed as the ratio of these cells to the number of apoptotic control shRNA-transfected cells (Cont shRNA). (C) The invasiveness of Huh7 cells transfected with occludin shRNA or control shRNA was analyzed using a Matrigel assay. The number of invasive cells was determined by counting stained cells and then
normalizing to the number in the case of control shRNA-transfected cells. All numerical data represent the means and standard deviations of
three independent experiments. –, control shRNA-transfected cells; +, occludin shRNA-transfected cells; *P < 0.05.

Apaf-1 and the induction of caspase 3 activity in
OccWT-overexpressing cells indicated that occludin
affected mitochondria-mediated apoptosis [25]. The
mitogen-activated protein kinase pathway, which is
known to exhibit some correlation with occludin
expression, is inactivated by increased expression of
this protein in HeLa cells [12]. Our data showed that
OccWT activated only ERK1 ⁄ 2 and not p38 and JNK.
On the basis of our data, exon 9 of occludin may play
a role in altering cell behavior via the internal cellular
signaling pathway. Furthermore, we newly confirmed
that OccWT located in the cytoplasmic region induced
the same effects (Fig. 3).
In view of mitochondria-mediated apoptosis,
decreased invasiveness, and upregulated phosphorylation of ERK1 ⁄ 2, we speculated that the effects of
occludin were controlled in one respect by the intracellular concentration of calcium [21,22]. Of various
signal pathways that involve calcium, those that are
mediated by Gaq protein were examined first [20].
We observed that the intracellular calcium level
increased in OccWT-overexpressing cells, and that

2-APB inhibited the effects of OccWT. Therefore,

the increase in intracellular calcium may explain the
induction of apoptosis and reduction of invasiveness mediated by occludin. In previous reports,
intracellular calcium concentrations were reported to
influence the onset of apoptosis and invasion
[22]; however, the relationship between increased calcium levels and the mechanisms underlying the
above-mentioned effects of occludin has not been
investigated.
In conclusion, we have discovered a new occludin
splice variant deleted in exon 9 in liver cell lines.
Moreover, the occludin promoter P1 was found to be
methylated, and the effects of demethylating agents on
the expression of wild-type occludin were examined in
Chang cells. Furthermore, in a comparative analysis
between OccWT and OccDE9, the exon 9 region played
a significant role in promoting apoptosis and inhibiting
invasion by regulating signaling pathways. Calcium
release from the ER has been described as one of the
mechanisms of this regulation. On the basis of the
above findings, our research provides new insights into
the role of exon 9 in the regulation of apoptosis and
invasion in liver cell lines.

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A

B

C

D

E

F

Fig. 8. Occludin increases the intracellular calcium concentration in Chang cells. (A) Representative cell images obtained using a confocal
microscope. Chang cells were cotransfected with pCMV ⁄ HA, pCMV ⁄ HA-OccWT or pCMV ⁄ HA-OccDE9 and with pEGFP-N1 vector. The cells
were stained with X-rhod-1, a calcium indicator, and then analyzed. Each image shows one cell in the center. (B) The relative fluorescence
intensity is represented as a bar graph. Chang cells were transfected with pCMV ⁄ HA, pCMV ⁄ HA-OccWT or pCMV ⁄ HA-OccDE9, and then treated with 20 lM 2-APB. (C) Apoptotic cell numbers were calculated by counting BrdUTP-incorporating cells. (D) The levels of BAX, Bcl-2 and
Apaf-1 were determined using real-time RT-PCR analysis. The relative expression levels are represented as a bar graph. (E) Immunoblot analyses using specific antibodies against ERK1 ⁄ 2, pERK1 ⁄ 2 and b-actin were performed. (F) Invasiveness of cells was analyzed using a Matrigel
assay. Cells penetrating the Matrigel-coated inserts were stained with crystal violet. The number of invasive cells was determined by counting the number of stained cells. All results in (B), (C), (D), (E) and (F) are expressed as the fold ratio relative to control (Cont). All numerical
data represent the means and standard deviations of three independent experiments. Cont, pCMV ⁄ HA-transfected cells; WT, OccWT-overexpressing cells; DE9, OccDE9-overexpressing cells; +, 2-APB-treated cells; *P < 0.05.

were incubated for another 24 h. H2O2 was administered in
DMEM with 10% fetal bovine serum for 24 h.

Experimental procedures
Cell cultures and treatment
Human liver cell lines Huh7, HepG2, Hep3B, and HLE,
and Chang cells, were cultured in DMEM with 10% fetal

bovine serum (Invitrogen, Carlsbad, CA, USA). To examine the effect of a demethylating agent, the cells were treated with 5 lm 5¢-aza-dC (Sigma Aldrich, St Louis, MO,
USA) for 48 h, and then with a histone deacetylase inhibitor, TSA (300 nm) (Sigma Aldrich) for 24 h. 2-APB
(20 lm) (Sigma Aldrich) was administered in DMEM with
10% fetal bovine serum at 12 h after transfection. Cells

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Construction of expression vectors and
transfection
cDNA fragments representing the complete ORFs of occludin (GenBank accession no. NM002538) and occludin
exon 9 deletion variant were cloned into the eukaryotic
expression vector pCMV ⁄ HA (Clontech, Carlsbad, CA,
USA). One microgram of each plasmid was transfected
using FuGENE 6 (Roche, Indianapolis, IN, USA) transfection reagent in six-well plates. MISSION pLKO.1 vector

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J.-M. Gu et al.

constructs expressing occludin shRNA were purchased from
Sigma and transfected with FuGENE 6 (Roche). Transfected cells were selected with puromycin (0.5 lgỈmL)1)
(Sigma). The pLKO.1 vector that does not contain an
shRNA insert was used as a control.

3¢-RACE
For 3¢-RACE, modified protocols from Scotto-Lavio et al.
were used [28]. First-strand cDNA synthesis was performed
using the adaptor primer 5¢-CCAGTGAGCAGAGTGAC
GAGGACTCGAGCTCAAGCTTTTTTTTTTTTTTTTT-3¢,

and then two rounds of 3¢-RACE PCR were performed,
using forward gene-specific primer 1 (GSP1), 5¢-TGGGAG
TGAACCCAACTGCT-3¢ (sense) and 5¢-CCAGTGAGCA
GAGTGACG-3¢ (antisense), for the first round, and genespecific primer 2 (GSP2), 5¢-CTCGTTACAGCAGCGGTG
GTAACTT-3¢ (sense) and 5¢-GAGGACTCGAGCTCAA
GC-3¢ (antisense), for the second round. The PCRs were
performed using the following conditions: 35 cycles of
95 °C for 30 s, 53 °C for 30 s and 72 °C for 1 min for the
first round PCR; and 35 cycles of 95 °C for 30 s, 53 °C for
30 s and 72 °C for 1 min for the second round.

Immunocytochemistry
For immunocytochemistry, cells were fixed in acetone ⁄ methanol (1 : 1) at )20 °C for 10 min and stained
using mouse anti-HA IgG (1 : 100) (Sigma Aldrich) and
rabbit anti-occludin IgG (1 : 100) (Zymed, San Francisco,
CA, USA). The secondary antibodies were anti-(mouse
Alexa 594) IgG (1 : 200) and anti-(rabbit Alexa 488) IgG
(1 : 200) (Molecular Probes, Carlsbad, CA, USA). Nuclei
were stained with 4¢,6-diamidino-2-phenylindole (Molecular
Probes). After mounting, cells were visualized using a multiphoton confocal laser scanning microscope equipped with
a 40· water immersion lens (Carl Zeiss, Thornwood, NY,
USA).

RT-PCR
Total RNA was isolated with Trizol (Invitrogen) according
to the manufacturer’s instructions. RNA (1 lg) was reverse
transcribed with oligo-dT by using avian myeloblastosis
virus (AMV) reverse transcriptase (Promega, Madison, WI,
USA). For PCR of occludin variants, the primers used were:
5¢-ACTCGACAATGAACAATCCGTCAGAA-3¢

(sense)
and 5¢-AGAGTATGCCATGGGACTGTCA-3¢ (antisense)
for exon 5; 5¢-TGCAGG-TGCTCTTTTTGAAGGT-3¢
(antisense) for exon 6; 5¢-GC-TCTTGTATTCCTGTAGGC
CAG-3¢ (antisense) for exon 7; 5¢-GTATTCATCAGCAG
CAGCC-3¢ (antisense) for exon 8; and 5¢-CTGTCTATCA
TAGTCTCCAACCAT-CTTC-3¢ (antisense) for exon 9.
PCR was carried out at 53 °C for 32 cycles.

Exon 9 of occludin in apoptosis and invasion

Genomic DNA was extracted from the cells using a
standard phenol protocol. For PCR, the primers
used were: 5¢-CAGCAATTGTCACACATCAAGAA-3¢
(sense) and 5¢-T-ACATGTAGGTATGAAGACATCGTC
T-3¢ (antisense) for exon 9; 5¢-TCCCTGCTTCCTCTGGC
GGA-3¢ (sense) and 5¢-AGCCATAGCCATAGCCACTTC
C-3¢ (antisense) for exon 1; 5¢-CCGGAGGGTCGGGCC
CAGTT-3¢ (sense) and 5¢-AGCCATAGCCATAGCCACT
TCC-3¢ (antisense) for exon 1a; 5¢-TAATAGGCTGCTGC
TGATGAATA-3¢ (sense) and 5¢-GGTATGTGGTCACAT
TGTGAAAATT-3¢ (antisense) for the exon 8–intron
boundary; and 5¢-ACTGCCAGGCACCTTGCGTATTT-3¢
(sense) and 5¢-TATCATAGTCTCCAACCATCTTCTTGA
-3¢ (antisense) for the intron–exon 9 boundary. PCR was
carried out at 58 °C for 30 cycles. All PCR products
were analyzed on agarose gels and stained with ethidium
bromide.

Real-time RT-PCR analysis

Real-time RT-PCR analysis was performed with specifically
designed primers using primer express software (Applied
Biosystems, Foster City, CA, USA). Primers for b-actin
were: 5¢-GCAAAGACCTGTACGCCAACA-3¢ (sense)
and 5¢-TGCATCCTGTCGGCAATG-3¢ (antisense). Primers for MMP1 were: 5¢-TGTGGCTCAGTTTGTCCTC
ACT-3¢ (sense) and 5¢-TTGGCAAATCTGGCGTGTA
A-3¢ (antisense). Primers for MMP2 were: 5¢-TGTGACGC
CACGTGACAAG-3¢ (sense) and 5¢-GCCTCGTATACCG
CATC-AATC-3¢ (antisense). Primers for MMP3 were:
5¢-TCG-TTGCTGCTCATGAAATTG-3¢ (sense) and 5¢-AC
AGGCGGAACCGAGTCA-3¢ (antisense). Primers for
MMP7 were: 5¢-TGCTGACATCATGATTGGCTTT-3¢
(sense)
and
5¢-TCCTCATCGAAGTGAGCATCTC-3¢
(antisense). Primers for MMP9 were: 5¢-ATGCGTGGAG
AGTCGAAATCTC-3¢ (sense) and 5¢-GGTTCGCATG
GCCTTCAG-3¢ (antisense). Primers for MMP14 were:
5¢-GACTACCTCCCGGCCTTCTG-3¢ (sense) and 5¢-ATGG
CCACGGTGTCAAAGTT-3¢ (antisense). To test expression levels of BAX, Bcl-2 and Apaf-1 with real-time PCR,
the primers used were: 5¢-TGGAGCTGCAGAGGATG
ATTG-3¢ (sense) and 5¢-CCAGTTGAAGTTGCCGTCA
GA-3¢ (antisense) for BAX; 5¢-GGATTGTGGCCTTCTTT
GAGTT-3¢ (sense) and 5¢-CGGTTCAGGTACTCAGT
CATCCA-3¢ (antisense) for Bcl-2; and 5¢-ACGGGAGAT
GACAATGGAGAAAT-3¢ (sense) and 5¢-CATGGGTAG
CAGCTCCTTCTTC-3¢ (antisense) for Apaf-1. Total RNA
was extracted from cultured cells using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. cDNA
was synthesized using 1 lg of RNA with AMV reverse transcriptase (Promega) and oligo-dT primers. Transcript levels
were assessed by quantitative real-time PCR (ABI 7300;

Applied Biosystems); all experiments were normalized to
b-actin.

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J.-M. Gu et al.

Immunoblot analysis
Cells were washed three times in NaCl ⁄ Pi and scraped with
lysis buffer (ReadyPrep Sequential extraction kit, Reagent 3;
Bio-Rad, Hercules, CA, USA). Next, the protein concentration was determined by measuring with Bradford reagent
(Bio-Rad). Cell lysates (20 lg) were resolved by SDS ⁄ PAGE
and transferred onto poly(vinylidene difluoride) membranes.
The blots were blocked with 5% nonfat milk in NaCl ⁄ Pi
containing 0.1% Tween-20, and probed with antibodies to
occludin (Zymed), ERK1 ⁄ 2 (pERK1 ⁄ 2; Cell Signaling Technology, Danvers, MA), pERK1 ⁄ 2 (Cell Signaling Technology), and b-actin (Sigma Aldrich). After being washed with
NaCl ⁄ Tris containing 0.1% Tween-20, the membranes were
incubated for 1 h with horseradish peroxidase-conjugated
secondary antibodies. Detection of peroxidase-coupled antibodies was performed using the Western Lightning chemiluminescence kit (Perkin-Elmer, Boston, MA, USA).

ium [29]. Boyden chambers were incubated for 48 h. Following removal of noninvading cells from the upper surface
with a cotton swab, invading cells were fixed, counted, and
normalized to control sample.

MSP

One microgram of genomic DNA was treated with sodium
bisulfate using the One Day MSP Kit (IN2GEN, Seoul,
Korea). To analyze the occludin promoter, we designed
MSP primers using the Methyl Primer Express system (Applied Biosystems). Primers for methylated DNA
were: 5¢-AAGTAGGCGGAGTATCGAAC-3¢ (sense) and
5¢-GAAAAAACGCGATCCTACTT-3¢ (antisense). Primers
for unmethylated DNA were: 5¢-GAAGTAGGTGGAGT
ATTGAAT-3¢ (sense) and 5¢-CAAAAAAACACAATCCT
ACTT-3¢ (antisense).

Caspase 3 activity
Cell proliferation assay
For the cell proliferation assay, cells were seeded on the
96-well plates, transfected using FuGENE 6 according to
the manufacturer’s manual, and incubated in 5% CO2 at
37 °C for 48 h. Cell proliferation was analyzed using Cell
Counting Kit-8 as described by the manufacturer (Dojindo
Laboratories, Kumamoto, Japan).

Terminal deoxyribonucleotide transferasemediated nick-end labeling (TUNEL) assay
Cells subjected to the TUNEL assay were seeded on coverslips and transfected using FuGENE 6. The DeadEND
Fluorometric TUNEL assay kit was used as described by
the manufacturer (Promega) after 48 h of transfection. Cells
were fixed in 4% formaldehyde for 25 min at 4 °C and permeabilized with 0.2% Triton X-100 for 5 min at room temperature. After being washed with NaCl ⁄ Pi, coverslips were
incubated with fluorescently labeled 5-bromo-2¢-deoxyuridine-5¢-triphosphate (BrdUTP) (Molecular Probes). Nuclei
were stained with 4¢,6-diamidino-2-phenylindole. After
mounting, cells were visualized with a fluorescence microscope (Olympus, Thornwood, NY, USA).

Invasion assays
Invasion assays were performed as described, with modifications [29–31]. An 8 lm cell culture insert (BD Biosciences) was briefly coated with reconstituted Growth Factor

Reduced BD Matrigel (10 lgỈcm)2) (BD Biosciences)
according to the manufacturer’s instructions. Transfected
cells (1.0 · 105) were resuspended in serum-free medium
and then plated in the upper part of the chamber. The
lower chamber was filled with NIH ⁄ 3T3 conditioned med-

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Cells subjected to the caspase assay were seeded on a
24-well plate and transfected with FuGENE 6. The caspase
assay was performed using the CaspACE colorimetric assay
kit as described by the manufacturer (Promega). Twentyfour hours after transfection, cells were harvested and lysed
with supplied lysis buffer by freeze–thawing. Ac-DEVDpNA, caspase 3 substrate, was added to the cell extract and
incubated for 4 h. Measurement of the caspase activity was
done at 405 nm.

Green fluorescent protein expression construct
and fluorescence imaging of calcium
Cells were seeded on coverslips coated with poly(l-lysine)
(Sigma Aldrich) and cotransfected with pCMV ⁄ HA containing occludin genes and pEGFP-N1 (Clontech) at a ratio of
10 : 1 using FuGENE 6. Coverslips with attached cells were
incubated in growth medium supplemented with 10% fetal
bovine serum for 24 h. After washing with Hepes buffer
(120 mm NaCl, 5.5 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2,
25 mm glucose, 20 mm Hepes, pH 7.2), 1 lm X-rhod-1
(Molecular Probes) was added, and cells were incubated for
45 min at room temperature. After mounting, cells were visualized with a multiphoton confocal laser scanning microscope (Carl Zeiss, Thornwood, NY, USA). Relative
fluorescence intensity was determined using imagemaster
2d elite software 4.01 (Amersham Bioscience, Uppsala,
Sweden).

Statistical analysis
Data in bar graphs are expressed as the mean and standard
deviation of three independent experiments. Student’s
t-tests were used in order to compare the differences

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J.-M. Gu et al.

Exon 9 of occludin in apoptosis and invasion

between different groups. A P-value < 0.05 was considered
to be statistically significant.
11

Acknowledgements
This study was supported by a grant from the
National R&D Program for Cancer Control, the Ministry of Health & Welfare, Korea (0520020). Seung Oe
Lim and Jin-Mo Gu were supported by a BK21
Research Fellowship from the Korea Ministry of
Education and Human Resources Development.

12

13

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