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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