Post on 11-May-2023
TOXICOLOGICAL SCIENCES 107(1), 9–18 (2009)
doi:10.1093/toxsci/kfn202
Advance Access publication October 1, 2008
The 2,2#,4,4#,5,5#-Hexachlorobiphenyl–Enhanced Degradation ofConnexin 43 Involves Both Proteasomal and Lysosomal Activities
Pavlına Simeckova,* Jan Vondracek,*,† Zdenek Andrysık,*,† Jirina Zatloukalova,*,† Pavel Krcmar,* Alois Kozubık,† and
Miroslav Machala*,1
*Department of Chemistry and Toxicology, Veterinary Research Institute, 62100 Brno, Czech Republic; and †Department of Cytokinetics, Institute of Biophysics,
62165 Brno, Czech Republic
Received May 27, 2008; accepted September 18, 2008
One of the toxic effects of non-dioxin–like polychlorinated
biphenyls (NDL-PCBs) is the acute inhibition of gap junctional
intercellular communication (GJIC), an event possibly associated
with tumor promotion. The model NDL-PCB-2,2#,4,4#,5,5#-hexachlorobiphenyl (PCB 153)—induces a sustained GJIC
inhibition in rat liver epithelial WB-F344 cells. As this effect
might be related to deregulation of connexin 43 (Cx43) synthesis,
trafficking, or degradation, we investigated the impact of PCB 153
on these events. Although PCB 153 had no effect on Cx43 mRNA
levels, it induced a gradual loss of Cx43 protein and significantly
decreased the amount of gap junction plaques in plasma
membrane. PCB 153 contributed to extracellular signal–regulated
kinases 1 and 2 (ERK1/2)–dependent accumulation of hyper-
phosphorylated Cx43-P3 form, thus indicating that ERK1/2
activation by PCB 153 might contribute to its effects on Cx43
internalization or degradation. Inhibition of either proteasomes or
lysosomes with their specific inhibitors largely restored total Cx43
protein levels, thus suggesting that both proteasomes and
lysosomes may participate in the PCB 153–enhanced Cx43
internalization and degradation. However, neither the proteaso-
mal nor the lysosomal inhibitors restored normal GJIC or
number/size of gap junction plaques. Finally, PCB 153 also
interfered with restoration of gap junction plaques following the
inhibition of Cx43 transport to plasma membrane. Taken together,
multiple modes of action seem to contribute to downregulation of
Cx43 in PCB 153–treated rat liver epithelial cells. The enhanced
degradation of Cx43, together with persistent inhibition of GJIC,
might contribute to tumor-promoting effects of NDL-PCBs.
Key Words: PCBs; connexin; tumor promotion; gap junctions;
lysosomes; proteasome.
Polychlorinated biphenyls (PCBs) are environmental pollu-
tants that have become ubiquitously distributed throughout
environment and food web due to their widespread industrial
use. PCBs can be classified based on their structure and the
structure-related toxic modes of action. Non-ortho-substituted
coplanar PCBs, sometimes termed as dioxin-like PCBs, have
been shown to elicit a set of adverse effects associated with the
activation of the aryl hydrocarbon receptor (AhR), resulting in
liver damage, thymus atrophy, skin lesions, a wasting
syndrome, and tumor promotion (Robertson and Hansen,
2001; Safe, 1994). The ortho-substituted PCBs, which tend to
form noncoplanar structures of the biphenyl molecule that does
not significantly activate AhR, exhibit a different spectrum of
toxic modes of action, which have been also linked to
neurotoxicity, immunotoxicity, endocrine disruption, or tumor
promotion (Glauert et al., 2001; Hansen, 1998; Pessah et al.,2006; van der Plas et al., 2000). A widespread distribution and
persistence of non-dioxin–like polychlorinated biphenyls
(NDL-PCBs) has led to numerous studies on their potential
role in carcinogenesis (Silberhorn et al., 1990). Various
mixtures of PCBs have been demonstrated to cause liver
cancer in rodents, and purified NDL-PCBs have been shown to
act as tumor promoters (Glauert et al., 2001). However, the
precise mechanisms underlying various modes of toxic action
of the NDL-PCBs are still not fully described.
The inhibition of gap junctional intercellular communication
(GJIC) has been suggested to provide a useful tool for detection
of tumor-promoting agents (Rosenkranz et al., 2000; Yama-
saki, 1996). The removal of an initiated cell from the growth
suppression by neighboring cells inflicted by the intercellular
transfer of signal messengers via gap junctions might play an
important role in cancer development (Trosko and Upham,
2005). The available data seem to suggest that NDL-PCBs can
inhibit GJIC in various types of cells (Hemming et al., 1991;
Kang et al., 1996; Machala et al., 2003; Ruch and Klaunig,
1986; Swierenga et al., 1990). The inhibitory activity of NDL-
PCBs seems to involve alterations of lipid signaling (Machala
et al., 2003; Umannova et al., 2008); however, this toxic mode
of action of NDL-PCBs is still not understood in detail. In
contrast to other tumor promoters or environmental toxicants,
which induce a rapid downregulation of GJIC followed by
its gradual recovery, the model NDL-PCB—2,2#,4,4#,5,5#-hexachlorobiphenyl (PCB 153)—has been found to induce
a sustained GJIC inhibition in rat liver epithelial WB-F344 cells
1 To whom correspondence should be addressed at Department of Chemistry
and Toxicology, Veterinary Research Institute, Hudcova 70, 62100 Brno,
Czech Republic. Fax: (420) 541211229. E-mail: machala@vri.cz.
� The Author 2008. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.For permissions, please email: journals.permissions@oxfordjournals.org
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(Machala et al., 2003). These results seem to suggest that PCB
153 may interfere also with degradation or synthesis of connexin
43 (Cx43), a major connexin species forming connexons and gap
junctions in rat liver epithelial WB-F344 cells.
The life cycle of Cx43 is a complex, highly regulated, and
a very dynamic process (Berthoud et al., 2004). Following
translation, Cx43 proteins are translocated into endoplasmic
reticulum (ER) membrane, where they oligomerize to form
hexameric connexons that are delivered through Golgi
apparatus to plasma membrane (Segretain and Falk, 2004).
They subsequently join with connexons in adjacent cells and
form tightly packed groups of channels, known as the gap
junction plaques (Segretain and Falk, 2004). Cx43 has a rapid
turnover rate with half-life of 1–4 h in most tissues (Berthoud
et al., 2004). The older channels are removed from the center,
by invagination of both connexon into one of the adjacent cells,
followed by lysosomal degradation (Berthoud et al., 2004;
Laird, 2005). However, both lysosomal and proteasomal
pathways can play an important role in the degradation of
Cx43, with internalized gap junctions being degraded via
lysosomes, whereas active proteasomal degradation may
destabilize phosphorylated gap junctions at the plasma
membrane or participate in ER-assisted degradation of Cx43
(Berthoud et al., 2004; Qin et al., 2003; Segretain and Falk,
2004). Both phosphorylation and ubiquitination of Cx43 are
important steps in regulation of Cx43 endocytosis and
degradation (Laird, 2005; Leithe and Rivedal, 2004a,b,
2007). Tumor promoters, such as 12-O-tetradecanoylphorbol
13-acetate (TPA), or growth factors, such as epidermal growth
factor, have been shown to promote internalization and
degradation of Cx43 as a part of their GJIC inhibitory effects
(Leithe and Rivedal, 2004a,b; Rivedal and Leithe, 2005).
However, nothing is currently known about the effects on
NDL-PCBs on processes leading to Cx43 degradation.
The principal aim of the present study was thus to analyze
the impact of PCB 153 (the most abundant NDL-PCB) on the
levels and cellular distribution of Cx43. Using selective
inhibitors of lysosomal and proteasomal pathways, protein
trafficking, and the inhibitor of extracellular signal–regulated
kinases 1 and 2 (ERK1/2) activation, we then investigated the
role of the respective mechanisms in the PCB 153–induced
downregulation of Cx43. Our results seem to suggest that PCB
153 induces a gradual loss of Cx43 in WB-F344 cells,
proceeding via lysosomal pathway, with active participation of
both ERK1/2-induced Cx43 phosphorylation and proteasome
in regulation of Cx43 degradation. Moreover, PCB 153 seems
also to interfere with restoration of gap junction plaques
following inhibition of Cx43 trafficking to plasma membrane.
MATERIALS AND METHODS
Chemicals. PCB 153 was purchased from Ehrenstorfer (Augsburg,
Germany). Stock solutions were prepared in dimethyl sulfoxide (DMSO)
(Merck, Darmstadt, Germany) and stored in the dark. Polyvinylidene difluoride
(PVDF) membrane Hybond-P and chemiluminescence ECL Plus detection
reagents were purchased from GE Healthcare (Buckinghamshire, UK).
Leupeptin was from AppliChem (Darmstadt, Germany). MG132, lactacystin,
chloroquine, TPA, brefeldin A, and all other chemicals were provided by
Sigma-Aldrich (Prague, Czech Republic).
Cells and treatment. Nontumorigenic, diploid rat liver WB-F344
epithelial cells (Tsao et al., 1984) were kindly provided by Dr. James
E. Trosko (Michigan State University, East Lansing, MI). Cells were maintained
in Dulbecco’s modified Eagle’s medium, supplemented with 10mM N-2-
hydroxyethylpiperazine-N#-2-ethanesulfonic acid, 24mM NaHCO3, and 5% fetal
bovine serum. All tissue culture reagents were from Sigma-Aldrich. Cells were
incubated in a humidified atmosphere of 5% CO2 at 37�C, and they were routinely
maintained in 75 cm2 flasks and subcultured twice a week. For detection of effects
of PCB 153 on GJIC inhibition, Cx43 degradation, and mitogen-activated protein
kinase (MAPK) activation, cells were treated with 40lM PCB 153 for time
intervals indicated in legends to figures. This concentration, leading to 80%
inhibition of GJIC in WB-F344 cells, was based on our previous study (Machala
et al., 2003). The specific treatments with inhibitors are indicated below.
Cells were treated with inhibitors of proteasomal (10lM MG132 or 10lM
lactacystin) and lysosomal degradation (100lM chloroquine and 100lM
leupeptin) for 30 min before 40lM PCB 153 was added for 6 h. Inhibition of
MAPK/ERK kinases 1 and 2 (MEK1/2) was performed by a 30-min
preincubation with 10lM U0126. For the experiments addressing restoration
of gap junction plaques, we exposed confluent cells grown on coverslips to 100
ng/ml brefeldin A, an inhibitor of protein transport to plasma membrane, for 3
h. After washing the cells three times with warm PBS, serum-free medium with
or without PCB 153 was added for another 3 h.
GJIC inhibition assay. Confluent cells, grown in 24-well plates, were
exposed to 40lM PCB 153 or 20nM TPA for 1, 24, and 48 h. After the
exposure, a modified protocol of scrape-loading/dye transfer technique (Blaha
et al., 2002) was used to assess in vitro modulations of GJIC. The cells were
washed twice by PBS solution, fluorescent dye was added (lucifer yellow,
0.05% w/v in PBS), and the cells were scraped using a surgical steel blade.
After 2 min of the dye diffusion between the adjacent cells via gap junctions,
the cells were washed by PBS and fixed with 4% (v/v) formaldehyde. The dye
transfer from the scrape line was measured with an epifluorescent microscope.
At least three independent experiments were carried out in duplicates; at least
three scrapes per well were evaluated using Lucia image analysis software
(Laboratory Imaging, Prague, Czech Republic). The GJIC inhibition was
expressed as a percentage of GJIC in cells treated with vehicle (DMSO).
Western blotting. Cells were grown to reach confluence, medium was then
changed, and cells were exposed to 40lM PCB 153 for indicated time. The 1-h
treatment with 20nM TPA or 0.2% DMSO was used as a positive control and
vehicle control, respectively. The cells were harvested in 90 ll of the lysis
buffer (1% SDS, 10% glycerol, 100mM Tris pH 7.4, 1mM NaF, 1mM Na3VO4,
1mM PMSF). The samples were then sonicated, and the protein concentration
was determined using the bicinchoninic acid method. The samples were then
diluted to equal protein levels and boiled for 4 min. Following SDS-
polyacrylamide gel electrophoresis (PAGE) in 10% gels, proteins were
transferred onto a PVDF membrane using a semidry blotter. The proteins
were detected using the following primary antibodies: polyclonal rabbit anti-
Cx43 antibody (Sigma-Aldrich; cat. no. C6219), anti-phospho-Cx43 antibody
(Ser 279/282) (Santa Cruz Biotechnology, Santa Cruz, CA; cat. no. 12900),
anti-phospho-p44/42 MAPK (ERK1/2) antibody (Cell Signaling Technology,
Beverly, MA; cat. no. 9101), or anti b-actin (Sigma-Aldrich; cat. no. A1978).
The immunoreactive bands were visualized using chemiluminescence detection
with ECL Plus reagent. The densitometric analysis of selected Western blots
was performed using Scanner 3 equipped with winCATS software (CAMAG,
Muttenz, Switzerland).
Real-time reverse transcription-PCR. The level of Cx43 mRNA was
determined by quantitative real-time reverse transcription (RT)-PCR. Cells
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were harvested with cell lysis buffer (provided with RNA isolation kit), and
total RNA was isolated using NucleoSpin RNA II Purification Kit (Macherey-
Nagel, Duren, Germany), according to the manufacturer’s instructions. The
primers were designed to flank the exon junctions of the transcripts for
amplification of cDNA only. The sequences of primers and TaqMan probe for
rat Cx43 were forward 5#-ACGATGGCTAATGGCTGGAG-3#, reverse 5#-GCAAAACTGGGCGAACTACA-3#, probe 5#-CTGGTTGTCGTCGGGGA-
AATCGA AC-3#; GenBank Accession No. NM_012567. The sequences of
primers and probe for the reference gene porphobilinogen deaminase have been
published previously (Vondracek et al., 2005). The RT-PCR amplifications of
the samples were carried out using QuantiTect Probe RT-PCR Kit (Qiagen
GmbH, Hilden, Germany) according to the manufacturer’s specifications, using
the LightCycler thermocycler (Roche Diagnostics GmbH, Mannheim,
Germany). All PCR reactions were performed in triplicates, and the changes
in gene expression were calculated using the comparative threshold cycle
method (Livak and Schmittgen, 2001).
Immunostaining. Cells were grown until reaching confluence on glass
coverslips in four-well plates and treated with 40lM PCB 153 for 1, 6, and 24 h.
The cells were then rinsed in cold PBS, fixed in methanol/acetone 1:1 for 20
min in �20�C, washed three times for 10 min in TBS-T (20mM Tris, 100mM
NaCl, 0.1% Tween 20, pH 7.4), and blocked in TBS-T containing 3% bovine
serum albumin and 5% dried nonfat milk for 1 h. The cells were incubated with
anti-Cx43 antibody overnight and, after another washing, with fluorescein-
labeled anti-rabbit IgG (GE Healthcare) for 1 h. The coverslips were mounted
on glass slides with the Vectashield Hard Set mounting medium (Vector
Laboratories, Inc., Burlingame, CA). Immunofluorescence images were captured
using the inverted epifluorescent microscope T200 equipped with a digital camera
(CCD-1300, Nikon, Japan) and the Lucia software.
Statistical analyses. The results of GJIC analyses and Cx43 densitometry
were expressed as means ± SD. Multiple comparisons were made with one-way
ANOVA and Tukey’s post hoc test or with the two-way ANOVA, followed by
Dunnett’s post hoc test versus a respective control group. For two group
comparisons (Figs. 3 and 4D), unpaired Student’s t-test was used. Values of p <0.05 and/or 0.01 (as indicated in legends to figures) were considered significant.
RESULTS
PCB 153 Reduced the Level of Cx43 Protein and Amount ofGap Junction Plaques in WB-F344 Cells
As shown in Figure 1A, unlike TPA, PCB 153 induced
a sustained downregulation of GJIC in WB-F344 cells. As the
restoration of GJIC in TPA-treated cells has been related to
renewal of membrane pool of phosphorylated Cx43 (Leithe
and Rivedal, 2004b; Rivedal and Leithe, 2005), we in-
vestigated the impact of PCB 153 on Cx43 amount and
phosphorylation pattern. When WB-F344 cell lysates were
separated on SDS-PAGE, Cx43 protein migrated as three
separate bands according to phosphorylation state. According
to previous studies, the faster migrating band (P0) represents
intracellular nonphosphorylated Cx43. Two slower migrating
phosphorylated forms are termed P1 and P2, with the second
one being reportedly located in gap junctions (Musil and
Goodenough, 1991). We found that PCB 153 reduced levels of
both P1- and P2-form of Cx43 in WB-F344 (Fig. 1B). In
contrast to TPA, PCB 153 did not induce the rapid hyper-
phosphorylation of Cx43, observed as the P3-Cx43 band.
Since these observations seemed to imply that PCB 153
induced a preferential loss of gap junction–localized Cx43, we
next used immunofluorescence staining of Cx43 in fixed WB-
F344 cells, in order to examine whether the alterations in Cx43
phosphorylation correspond with changes in Cx43 localization.
A significant part of Cx43 protein in untreated cells was found
in plasma membrane forming gap junction plaques, with some
perinuclear staining, corresponding to ER and Golgi apparatus
Cx43 pool (Fig. 2). The exposure to PCB 153 caused
a progressive reduction of both size and number of gap
junction plaques (Fig. 2). The maximum effect was reached
already after 6-h treatment, when we also observed an increase
of the intracellular Cx43 amount. This corresponded with the
effects of PCB 153 on phosphorylated forms of Cx43 detected
by Western blotting. Nevertheless, there was still a certain
amount of gap junction plaques present in plasma membrane
even after 24 h, which corresponded with the approximately
20% level of GJIC relative to control (Fig. 1A).
In order to find out whether the decrease of Cx43 protein
might be also due to reduced transcription, we used the
FIG. 1. PCB 153 induced a sustained inhibition of GJIC and Cx43
degradation in WB-F344 cells. (A) The effect of TPA (20nM) and PCB 153
(40lM) on GJIC in WB-F344 cells after 1-, 24-, and 48-h exposure periods, as
determined by the dye transfer/scrape-loading assay. The results represent
means ± SD of three independent experiments. **A significant inhibition of
GJIC, as compared to respective control cells at time 0 h (p < 0.01), as
determined by one-way ANOVA followed by Tukey’s post hoc test. (B)
Western blotting detection of Cx43 protein in WB-F344 cells. Cells were
treated with 0.2% DMSO for 24 h (vehicle control), 40lM PCB 153 for
indicated time, or with 20nM TPA for 1 h. Equal amounts of cell lysates (10 lg
per lane) were subjected to SDS-PAGE and immunoblotted with anti-Cx43
antibody or with anti-b-actin antibody (loading control). The positions of
nonphosphorylated (P0-Cx43) and phosphorylated (P1-Cx43, P2-Cx43,
P3-Cx43) forms of Cx43 are indicated. The results are representative of three
independent experiments.
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real-time RT-PCR to determine the levels of Cx43 mRNA. As
shown in Figure 3, treatment with PCB 153 did not reduce the
Cx43 mRNA amount in WB-F344 cells as compared to cells
treated with DMSO (vehicle control). This confirmed that the
observed decrease of Cx43 occurs at the protein level.
Effects of Proteasomal and MEK1/2 Inhibitors onDegradation of Cx43
Cx43 phosphorylation and ubiquitination have been linked
to its degradation (Laird, 2005; Leithe and Rivedal, 2004a,b,
2007) following the treatment of cells with either growth
factors or TPA. Our previous studies have suggested that PCB
153 is a potent inducer of activation of ERK1/2 (Machala et al.,2003; Umannova et al., 2008). However, no hyperphosphor-
ylation of Cx43 was observed in cells treated with PCB 153
(Fig. 1B). Therefore, we next investigated ERK1/2-specific
phosphorylation using antibody specifically recognizing Cx43
phosphorylated at Ser 279/282. However, as shown in Figure 4A,
although PCB 153 efficiently activated ERK1/2 at both 1 and
6 h time intervals, in contrast to TPA, it induced only a faint
increase of Ser 279/282 phosphorylation (Fig. 4A).
In order to examine more closely the role of ERK1/2 and
proteasome in the degradation of Cx43, we used specific
chemical inhibitors of MEK1/2 (upstream ERK1/2–activating
kinases) and proteasome, U0126 and MG132, respectively
(Fig. 4B). As summarized in Figure 4C, the results of
densitometric analysis of Western blots showed that total
levels of Cx43 were similar in control cells and cells pretreated
with either MG132 or MG132 þ U0126, prior to PCB 153
treatment, suggesting that both types of treatment preserved
total Cx43 levels. U0126 alone was not sufficient to prevent
Cx43 degradation, and it had no effect on Cx43 levels itself.
Nevertheless, there were significant differences among
effects of inhibitors regarding the levels of individual Cx43
forms. As shown in Figure 4B, MG132 alone or
in combination with PCB 153 induced accumulation of
hyperphosphorylated P3 band, whereas hypophosphorylated
P0-form almost disappeared. When U0126 was applied
simultaneously with MG132, the accumulation of hyper-
phosphorylated band was prevented, suggesting that it depends
on ERK1/2 activity. As the results of Western blotting also
suggested that PCB 153 further potentiated accumulation of
Cx43-P3 form in cells pretreated with MG132, we performed
densitometric analysis of P3 bands. As shown in Figure 4D,
significantly more Cx43 was present as hyperphosphorylated
P3-form in PCB 153 þ MG132–treated cells as compared to
cells treated with MG132 alone (p < 0.05). These results seem
to suggest that there is a pool of hyperphosphorylated Cx43,
which is only observable after inhibition of proteasome and which
might be enhanced through the PCB 153–induced ERK1/2
activation. Therefore, PCB 153 might contribute to increased
internalization of Cx43, which would be also dependent on
proteasomal activity. Similar data were obtained also with another
proteasome inhibitor, lactacystin (data not shown).
Lysosomal Pathway Is Involved in PCB 153-InducedDegradation of Cx43
Nevertheless, it has been suggested that proteasome plays
only a partial role in Cx43 degradation, regulating, e.g., Cx43
internalization (Berthoud et al., 2004; Leithe and Rivedal,
2004a,b). The internalized protein would then be degraded via
lysosomal pathway. Therefore, in the next step, we studied the
role of lysosomal degradation in the PCB 153–induced
decrease of Cx43 protein. Inhibition of lysosomal pathway
FIG. 2. PCB 153 decreased amount of gap junction plaques in WB-F344 cells.
Confluent WB-F344 cells were grown on glass coverslips and incubated with 0.2%
DMSO for 24 h (vehicle control) or with 40lM PCB 153 for indicated time. Cells
were then fixed and stained for Cx43 using anti-Cx43 antibody and a fluorescein-
labeled anti-rabbit IgG, as described in the ‘‘Materials and Methods’’ section. The
results are representative of three independent experiments.
FIG. 3. PCB 153 did not reduce Cx43 mRNA levels. Confluent WB-F344
cells were incubated with 0.2% DMSO for 24 h (vehicle control) or with 40lM
PCB 153 for 24 h. The levels of Cx43 mRNA were determined by quantitative
real-time RT-PCR, as described in the ‘‘Materials and Methods’’ section. The
results represent means ± SD of three independent experiments. There was no
significant difference between the groups, as assessed by the Student’s t-test.
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by leupeptin or chloroquine completely suppressed Cx43
degradation, which is also supported by the results of
densitometric analysis of Western blots (Fig. 5). These results
seem to indicate that both proteasomes and lysosomes are
involved in enhanced degradation of Cx43 in cells treated with
PCB 153.
Inhibitors of Proteasomal and Lysosomal ActivitiesDifferentially Modulated Gap Junction Plaques and GJICin WB-F344 Cells
As the above data suggested that proteasomal and lysosomal
inhibitors may differentially affect Cx43 forms in WB-F344
cells, we next examined their impact on Cx43 levels and
cellular distribution in both control- and PCB 153–treated cells,
using immunofluorescent staining of Cx43 in fixed WB-F344
cells. MG132 induced accumulation of Cx43 within cell
membrane in both control- and PCB 153–treated cells (Fig. 6).
These results, similar to pretreatment with another inhibitor of
proteasomal degradation lactacystin (data not shown), sug-
gested that proteasomal inhibitors might counteract the PCB
153–enhanced internalization of Cx43. The accumulation of
hyperphosphorylated Cx43-P3 form in WB-F344 cells treated
with both MG132 and PCB 153 (see Fig. 4B) seems also to
provide further support for this hypothesis, suggesting that the
membrane-accumulated Cx43 indeed corresponded to
the Cx43-P3 form. Moreover, in cells cotreated with both the
proteasomal inhibitors MG132 and U0126, the MEK1/2
inhibitor which prevented the increased formation of P3-form
(Fig. 4B), we observed a control-like pattern of gap junction
plaques (Fig. 6), thus suggesting that ERK1/2-dependent
phosphorylation may contribute to the PCB 153–enhanced
internalization and degradation of Cx43. However, U0126
alone was not sufficient to prevent the PCB 153–induced
degradation of gap junction plaques, thus indicating that
additional mechanisms might be involved in deleterious effects
of PCB 153 on Cx43 protein and gap junction plaques in
membranes of rat liver epithelial cells.
FIG. 4. Effects of inhibitors of proteasomal degradation (MG132) and ERK1/2 activation (U0126) on Cx43 degradation in WB-F344 cells. (A) PCB 153 did
not induce a major phosphorylation of Cx43 Ser 279/282 residues, despite activating ERK1/2. Cells were treated with 0.2% DMSO for 24 h (vehicle control),
40lM PCB 153 for indicated time, or with 20nM TPA for 1 h. Equal amounts of cell lysates (30 lg per lane) were subjected to SDS-PAGE and immunoblotted
with anti-phospho-ERK1/2 or anti-phospho-Cx43 antibodies. The results are representative of three independent experiments. (B) Cells were preincubated with
MG132, U0126, or their combination for 30 min, followed by 6-h incubation with either 0.2% DMSO or 40lM PCB 153. Equal amounts of cell lysates (10 lg per
lane) were subjected to SDS-PAGE and immunoblotted with anti-Cx43 antibody or with anti-b-actin antibody (loading control). The results are representative of
five independent experiments. (C) The results of densitometric analysis of total Cx43 protein detection were expressed as percentage of control samples (treated
with DMSO) and are presented as means ± SD of five independent experiments. *A significant decrease of Cx43 protein in comparison with control cells (DMSO)
(p < 0.05), as determined by two-way ANOVA followed by Dunnett’s post hoc test. **A significant decrease of Cx43 protein in comparison with control cells
(DMSO) (p < 0.01), as determined by two-way ANOVA followed by Dunnett’s post hoc test. (D) Comparison of relative abundance of Cx43-P3 band between
samples treated with MG132 and MG132 þ PCB 153. The results of densitometric analysis of Cx43-P3 bands were expressed as percentage of total Cx43 protein
in a given sample and are presented as means ± SD of five independent experiments. *A significant difference between MG132 and MG132 þ PCB 153 samples
(p < 0.05), as determined by Student’s t-test.
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In contrast to proteasomal inhibitors, pretreatment with
lysosomal inhibitors leupeptin (Fig. 6) or chloroquine (data not
shown) in control cells led to accumulation of intracellular
Cx43. Moreover, leupeptin prevented the PCB 153–assisted
Cx43 degradation; however, despite a strong intracellular
staining of Cx43, the pretreatment with leupeptin failed to
restore the gap junction plaques in cells treated with PCB 153
(Fig. 6).
As the pretreatment with the above inhibitors prevented the
PCB 153–induced degradation of Cx43 and in some cases also
contributed to restoration of gap junction plaques, we next
examined their impact on GJIC in both control- and PCB 153–
treated cells. However, none of these inhibitors restored GJIC
in PCB 153–treated cells (Table 1). Moreover, inhibitors of
proteasomal degradation alone significantly reduced the GJIC
FIG. 5. Lysosomal inhibitors prevented degradation of Cx43 protein in
WB-F344 cells. Cells were preincubated with leupeptin (A) or chloroquine (B)
for 30 min, followed by 6-h incubation with either 0.2% DMSO or 40lM PCB
153. Equal amounts of cell lysates (10 lg per lane) were subjected to SDS-
PAGE and immunoblotted with anti-Cx43 antibody or anti-b-actin antibody
(loading control). The results are representative of three independent experi-
ments. (C) The results of densitometric analysis of total Cx43 protein detection
were expressed as percentage of control samples (treated with DMSO) and are
presented as means ± SD of three independent experiments. *A significant
decrease of Cx43 protein in comparison with control cells (DMSO) (p < 0.05),
as determined by two-way ANOVA followed by Dunnett’s post hoc test. FIG. 6. Pretreatment with inhibitors of proteasomal and lysosomal
degradation differentially modulated the amount and localization of Cx43
protein in PCB 153–treated WB-F344 cells. Confluent WB-F344 cells were
grown on glass coverslips and preincubated with 10lM MG132, 10lM MG132
and 10lM U0126, and 10lM U0126 or 100lM leupeptin. After 30 min,
0.2% DMSO (left column) or 40lM PCB 153 (right column) was added for
6 h. Cells were then fixed and stained with anti-Cx43 antibody, followed
with a fluorescein-labeled anti-rabbit IgG, as described in the ‘‘Materials
and Methods’’ section. The results are representative of three independent
experiments.
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levels in WB-F344 cells either in control cells or in cells treated
with PCB 153. These results seem to indicate that simple
accumulation of either intracellular Cx43 (lysosomal inhib-
itors) or membrane-associated Cx43 forms (proteasomal
inhibitors) is not sufficient to restore GJIC function in rat liver
epithelial cells.
PCB153 May Interfere with Restoration of Gap JunctionPlaques in WB-F344 Cells
Finally, as a recent study suggested that Cx43 could be
degraded not only from its membrane pool but also directly after
transportation from early secretory compartments to lysosomes
(Qin et al., 2003), we next investigated whether PCB 153 may
interfere with restoration of gap junction plaques in cells
pretreated with brefeldin A, an inhibitor of transport of membrane
proteins to plasma membrane. When the protein transport to cell
surface was inhibited, most of the gap junction plaques dis-
appeared from the membrane (Fig. 7B). The plaques were restored
when cells were allowed to recover for 3 h in medium without
brefeldin A (Fig. 7C). Using this approach, we found that PCB 153
disrupted the restoration of gap junction plaques and Cx43
remained localized intracellularly (Fig. 7D). Thus, PCB 153 might
interfere also with processes involved in intracellular transport of
Cx43 or contribute to its direct lysosomal degradation.
DISCUSSION
Gap junctions play important roles in growth regulation and
neoplastic transformation (Mesnil et al., 2005). Many toxic
agents have been reported to inhibit GJIC both in vitro and
in vivo, thus suggesting that inhibition of GJIC might
contribute to their carcinogenicity (Rosenkranz et al., 2000;
Yamasaki, 1996). However, the mechanisms of actions of
many of these agents on GJIC and connexin functions are still
only partly understood. While some of these toxic compounds,
such as polyaromatic compounds, induce only a transient
inhibition of GJIC (Blaha et al., 2002; Upham et al., 2008),
others, such as pesticides or PCBs, may induce a sustained
downregulation of intercellular communication (Bager et al.,1997; Guan and Ruch, 1996; Machala et al., 2003). Our
previous studies in WB-F344 rat liver epithelial cells have
shown that both NDL-PCBs and their hydroxylated metabolites
inhibit GJIC and that this is a phenomenon persisting for
several days (Machala et al., 2003, 2004), suggesting that it
may involve changes in Cx43 life cycle. Therefore, in the
present study, we investigated effects of PCB 153, the most
abundant environmental NDL-PCB, on phosphorylation and
degradation of Cx43, using established inhibitors of ERK1/2
activation, proteasomal and lysosomal degradation, or Cx43
transportation to plasma membrane.
The mechanisms underlying Cx43 degradation have
received a considerable attention because its half-life is
relatively short (1–4 h) and modulation of its turnover rate
may significantly modify GJIC in response to model inhibitory
TABLE 1
Effects of Selected Proteasomal and Lysosomal Inhibitors on
GJIC in WB-F344 Cells (Expressed as Percentage of Control)
Treatment Inhibitors
GJIC (expressed as % of
control ± SD)a
DMSO — 100 ± 9
PCB 153 — 26 ± 6**
DMSO MG132 40 ± 11**
PCB 153 MG132 6 ± 3**
DMSO U0126 85 ± 13
PCB 153 U0126 30 ± 10**
DMSO MG132 þ U0126 67 ± 11**
PCB 153 MG132 þ U0126 5 ± 3**
DMSO Leupeptin 92 ± 10
PCB 153 Leupeptin 22 ± 14**
aAll data represent means of at least three independent experiments.
**A significant decrease of GJIC in comparison with control cells (DMSO)
(p < 0.01), as determined by two-way ANOVA followed by Dunnett’s post hoc
test versus a control group. The effects of MG132 (10lM), U0126 (10lM),
leupeptin (100lM), and PCB 153 (40lM) on GJIC in WB-F344 cells were
determined by the dye transfer/scrape-loading assay. Cells were treated with
PCB 153 (40lM) or DMSO (0.2%; control) for 6 h; the respective inhibitors
were added to cell culture 30 min prior to treatment. See ‘‘Materials and
Methods’’ section for details of treatment and other abbreviations.
FIG. 7. PCB 153 delayed the restoration of gap junction plaques.
Confluent WB-F344 cells were grown on glass coverslips and incubated for
3 h with 100 ng/ml brefeldin A (BFA), an inhibitor of protein transport to Golgi
apparatus. After washing the cells three times with warm PBS, serum-free
medium with or without 40lM PCB 153 was added for another 3 h. Cells were
then fixed and stained for Cx43 using anti-Cx43 antibody and a fluorescein-
labeled anti-rabbit IgG, as described in the ‘‘Materials and Methods’’ section.
(A) Control; (B) 3-h treatment with BFA; (C) 3-h treatment with BFA,
followed by 3-h recovery in medium with DMSO 0.2% (vehicle control); (D)
3-h treatment with BFA, followed by 3-h recovery in medium with 40lM PCB
153. The results are representative of three independent experiments.
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stimuli, such as growth factors or phorbol esters, as reviewed in
Berthoud et al. (2004), Laird (2005), Leithe and Rivedal
(2007), and Segretain and Falk (2004). The regulation of
connexin degradation is also an important mechanism
regulating gap junction assembly and function (Musil et al.,2000). In the present study, we found that PCB 153 did not
modify Cx43 transcription but instead induced a preferential
degradation of phosphorylated P1- and P2-forms of Cx43, the
latter one representing the mature form of Cx43-forming gap
junctions (Musil and Goodenough, 1991). The results of
Western blotting analysis matched those of immunofluores-
cence detection of gap junction plaques as PCB 153 apparently
reduced both size and number of gap junction plaques. These
effects were similar to the impact of other toxic compounds on
gap junctions in WB-F344 cells, thus suggesting that both
PCBs and, e.g., chlorinated pesticides might share at least some
mechanisms leading to GJIC downregulation (Guan and Ruch,
1996; Hakulinen et al., 2006).
Both proteasomal and lysosomal pathways have been
suggested to take part in degradation of connexins (Berthoud
et al., 2004; Laing et al., 1997; Leithe and Rivedal, 2007). In
the present study, the proteasomal inhibitors both restored the
total Cx43 levels in cells treated with PCB 153 and induced
accumulation of hyperphosphorylated Cx43-P3 form in
control- and PCB 153–treated cells. Previously, a similar
accumulation of phosphorylated Cx43 at the expense of
hypophosphorylated P0-form, which is thought to represent
the intracellular pool of Cx43 (Berthoud et al., 2004; Segretain
and Falk, 2004), has been observed, e.g., in human breast
carcinoma cells overexpressing Cx43 (Qin et al., 2003).
As shown in Figure 4, PCB 153 enhanced the ERK1/2-
dependent formation of the hyperphosphorylated form of
Cx43. This seemed to indicate that PCB 153 might enhance
Cx43 internalization and degradation via ERK1/2 activation.
However, unlike TPA, PCB 153 did not induce a major
phosphorylation of Cx43 at Ser 279/282 residues or accumu-
lation of Cx43-P3 form in PCB 153–treated cells. The principal
reason for the absence of hyperphosphorylated Cx43-P3 forms
in cells treated with PCB 153 might be its rapid internalization
and degradation. Therefore, this form of Cx43 might only be
observed in cells treated with proteasomal inhibitors (e.g.,
MG132) which may prevent Cx43 internalization (Laing et al.,1997; Leithe and Rivedal, 2004b; Qin et al., 2003). Indeed, the
results shown in both Figures 4 and 6 seem to support this
hypothesis—application of MG132 significantly increased both
the P3-form levels and the membrane Cx43 staining, which
was prevented by U0126 inhibitor. Therefore, despite having
no role in acute inhibition of GJIC by NDL-PCBs (Machala
et al., 2003), ERK1/2 activation might still contribute to NDL-
PCB-induced Cx43 degradation. Our data also suggested that
although proteasomal inhibitor restored total Cx43 levels,
a preferential accumulation of Cx43-P3 form did not lead to
restoration of GJIC, which is in line with the evidence that
phosphorylation of Cx43 by ERK1/2 blocks gap junction
channels (reviewed in Lampe and Lau, 2004). Both phosphor-
ylation and ubiquitination of Cx43 have been suggested to play
a role of a signal in internalization and degradation of Cx43 in
rat liver epithelial cells (Leithe and Rivedal, 2004a,b; Leykauf
et al., 2006). Active proteasomal degradation may also
destabilize phosphorylated gap junctions at the plasma
membrane (Segretain and Falk, 2004). Nevertheless, the
precise role of proteasome in these events still remains unclear
as it has been also suggested that the proteasomal inhibitors
may alter also ubiquitination pattern of Cx43 (Leithe and
Rivedal, 2004a), thus preventing the ubiquitination signaling
for Cx43 internalization and subsequent degradation (Leykauf
et al., 2006).
Following their internalization, gap junctions (connexo-
somes) may be fused with and degraded in lysosomes. It has
been suggested that Nedd4, an ubiquitin-protein ligase,
controls the ubiquitination of Cx43 protein, prior to its
endocytosis (Leykauf et al., 2006). Like other membrane
proteins, Cx43 contains sorting signal, which might mediate
both Cx43 internalization and its targeting to lysosomes
(Bonifacino and Traub, 2003; Thomas et al., 2003). Proteaso-
mal and ERK1/2 inhibitors may thus prevent signaling leading
to lysosomal degradation of Cx43 in WB-F344 cells. When
using lysosomal inhibitors, we observed that both leupeptin
and chloroquine prevented degradation of Cx43 in response to
PCB 153 treatment, similar to, e.g., chlorinated pesticides
(Guan and Ruch, 1996). However, this Cx43 was predomi-
nantly intracellular (Fig. 6) and the application of lysosomal
inhibitors thus failed to restore GJIC function as shown in
Table 1. This, together with the results summarized above,
seems to suggest that both proteasomes and lysosomes are
involved in the effects of PCB 153 on Cx43 degradation in
WB-F344 cells, which is, in the former case, probably related
to internalization and sorting signaling.
Finally, it has been recently suggested that Cx43 may bypass
transport to membrane and become directly degraded in
lysosomes (Qin et al., 2003). In order to analyze possible
impact of PCB 153 on Cx43 trafficking to the surface of the
cells, we employed WB-F344 cells, where Cx43 transport was
blocked by the fungal antibiotic brefeldin A (Laird et al., 1995;
VanSlyke and Musil, 2000). We found that PCB 153 inhibited
restoration of gap junction plaques following the inhibition of
transport of synthesized Cx43 to plasma membrane. This
seems to indicate that PCB 153 might also interfere with the
proper transport of Cx43 to cell membrane, which might be
related to direct lysosomal degradation of ER/Golgi compart-
ment–trapped Cx43. These results might perhaps help to
explain why the MEK1/2 inhibitor U0126 failed to prevent the
PCB 153–induced Cx43 degradation.
Taken together, the present data seem to suggest that PCB
153 induces degradation of phosphorylated forms of Cx43
in WB-F344 cells. Its degradation probably proceeds via
lysosomes; however, both ERK1/2-regulated pathways and
the proteasome seem to contribute to regulation of Cx43
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internalization and degradation. PCB 153 seems also to
interfere with restoration of gap junction plaques following
inhibition of Cx43 trafficking to plasma membrane. Degrada-
tion of Cx43 might contribute to prolonged inhibitory effects of
NDL-PCBs on GJIC in epithelial cells. Moreover, it may also
interfere with Cx43-mediated regulation of genes linked to
regulation of cell proliferation (Kardami et al., 2007). As
a recent study has suggested that some of nodular hepatic
lesions may originate from the liver epithelial cells following
a chronic treatment with organochlorine contaminants (Hailey
et al., 2005), the impact of NDL-PCBs on GJIC in this cell
population deserves further attention. Nevertheless, as principal
liver cells—hepatocytes—express predominantly other con-
nexin species, Cx26 and Cx32, their deregulation via similar
mechanisms would be also of interest. However, only little is
currently known about the mechanisms of degradation re-
garding these connexin species. Cx26 is not phosphorylated
due to its lack of cytoplasmic tail, although it has a short half-
life similar to Cx43, suggesting that the mechanisms regulating
its degradation might differ from Cx43 (Laird, 2005; Lampe
and Lau, 2004). Cx32 has been shown to be highly related to
liver cancer development as Cx32 knockout mice show high
levels of both spontaneous and chemically induced liver tumors
(Temme et al., 1997). However, like Cx26, also Cx32 could be
degraded through this connexin species–specific mechanism
(Laird, 2005). Therefore, possible similar effects of NDL-PCBs
on other liver connexins remain unclear. Future studies should
also aim to establish a link between the PCB-induced Cx43
degradation observed in vitro and its in vivo relevance.
FUNDING
ATHON (EU Framework Programme 6, Priority 5: ‘‘Food
Quality and Safety’’; EU Contract No. FOOD-CT-2005-
022923); Czech Science Foundation (grant No. 524/06/0517);
The institutional support was provided by the Academy of
Sciences of the Czech Republic (Research Plans AV0Z50040507
and AV0Z50040702) and the Czech Ministry of Agriculture
(MZE0002716201).
ACKNOWLEDGMENTS
The authors thank Josef Slavik for his expert assistance with
densitometry.
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