Aberrant Connexin 43 endocytosis by the carcinogen lindane involves activation of the ERK/mitogen-activated protein kinase pathway
Baharia Mograbi1,4,
Elisabeth Corcelle1,
Norah Defamie1,
Michel Samson2,
Marielle Nebout1,
Dominique Segretain1,3,
Patrick Fénichel1 and
Georges Pointis1
1 INSERM EMI 00-09, IFR 50, Faculté de Médecine, Avenue de Valombrose, F-06107 Nice Cedex 02, France
2 INSERM U 364, IFR 50, F-06107 Nice Cedex 02, France
3 Université Paris 5, 45 rue des St Pères, F-75006 Paris, France
4 To whom correspondence should be addressed Email: mograbi{at}unice.fr
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Abstract
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Although worldwide concerns have emerged about environmental factors that display carcinogenic and reprotoxic effects, little is known about the mechanism(s) by which these chemicals alter testicular function. Using the 42GPA9 Sertoli cell line, we recently reported that one widely used lipid-soluble pesticide, Lindane impairs gap junctional intercellular communication by promoting the intracellular localization of Connexin 43 (Cx43), a tumor suppressor. We showed here that this chemical triggered the accumulation of Cx43 within Rab5 positive endosomes. Interestingly, evidence is provided that Lindane-induced Cx43 endocytosis did not stem on alteration of Cx43 partition in lipid rafts. Lindane induced concomitantly Cx43 phosphorylation and activation of extracellular signal-regulated kinases (ERK) but not of JNK and p38 mitogen- activated protein kinases. Inhibition of ERK pathway by PD98059, a MEK1-specific inhibitor, prevented Lindane-induced Cx43 phosphorylation, restored Cx43 membranous localization and gap junction coupling. Altogether, these findings provide the first evidence that Lindane-altered Cx43 endocytosis requires ERK activation. Such inappropriate activation of the mitogenic MAPK pathway and inactivation of the tumor suppressor Cx43 by Lindane may participate in the promotion of neoplastic cell growth.
Abbreviations: Cx43, Connexin 43; ERK, extracellular signal-regulated kinases; GJIC, gap junction intercellular communication; PBS, phosphate-buffered saline
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Introduction
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Worldwide concerns have recently emerged about the increased incidence of several cancers in industrialized countries (1). In particular, the incidence of testicular cancer, which is the most common malignancy of young men, has risen dramatically in Europe and North America (2). Because of this consistent trend, a chronic exposure to environmental chemicals has been suspected (3). Indeed, widely used pesticides are able to promote carcinogenic effects in rodents and are known to concentrate in testis (4,5). However, the mechanism(s) by which these chemicals might lead to testicular neoplasia remain(s) unknown. Much effort has been, therefore, devoted to unravelling the targets of these chemicals. It turned out that some of these carcinogens possess in vitro weak estrogenic activities, as they are able to bind to estrogen nuclear receptors and to induce the proliferation of MCF-7 breast cancer cells (6). Based on these features, the prevailing model is that these chemicals mimic or interfere with the action of sexual steroid hormones and by inference they are referred to as endocrine disrupters (3,7). Beyond the steroid hormones, understanding of the complexity of signaling pathways activated by hormones has provided many other attractive targets for pesticides such as downstream signaling molecules that remain to be determined (8).
Other major targets of pesticides are gap junction channels, known to play a critical role in the regulation of cell growth (911). Indeed, these channels are intercellular channels, formed of connexins (Cx), which allow the direct exchange of small signaling molecules (<1 kDa) between the cytoplasm of two adjacent cells (for review see refs 12,13). It is well documented that this cellcell communication is tightly regulated by growth factors and consistently aberrant control of gap junction intercellular communication (GJIC) is a hallmark of malignant cell growth in tumors, in transformed cell lines or in response to carcinogens (9,14). Although an increasing number of chemicals are able to decrease this cellcell communication, the underlying mechanism(s) is not identified. It has been proposed that some compounds particularly the lipid-soluble pesticides may disturb the fluidity of the plasma membrane and thereby gap junction channel functions (15).
Within the testis, Sertoli cells control germ cell growth and differentiation by establishing paracrine and gap junction communications (16). The notion that Connexin 43 (Cx43)-mediated GJIC is absolutely required for spermatogenesis came from the recent demonstration in mice that Cx43 gene targeted deletion results in male infertility (17). Moreover, altered Cx43 expression has been correlated with impaired human infertility (18) and testicular cancer (19). We recently reported that one widely used lipid-soluble pesticide, Lindane (
-hexachlorocyclohexane) impairs GJIC between Sertoli cells (20).
The present study demonstrates that this pesticide induces the aberrant endocytosis of Cx43 within Rab5 positive endosomes. We show that Cx43 is present in lipid rafts and Lindane-induced Cx43 endocytosis does not stem from lipid raft alteration. Rather, we demonstrate that Lindane selectively activates the extracellular signal-regulated kinases (ERK) and that this pathway is required for Lindane-induced-Cx43 phosphorylation, -endocytosis and -impairment of gap junction coupling. Implications of these findings in the understanding of the carcinogenic power of this chemical are discussed.
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Materials and methods
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Cell culture and treatments
The mouse 42GPA9 Sertoli cell line (21) was maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum at 32°C. For all the experiments, cells were serum starved for 16 h in fresh DMEM supplemented 0.1% bovine serum albumin (BSA A7030, Sigma, St Louis, MO). The serum-starved cells were then activated by addition of Lindane (50 µM, Sigma), U18666A [(22); 3 µg/ml; Biomol Research Laboratories, Plymouth, PE] or 17ß-estradiol (10-6 M, Sigma). The Park Davis Inhibitor PD98059 (MEK1-specific inhibitor, 10 µM, Calbiochem, La Jolla, CA) or PP2 (inhibitor of the src tyrosine kinase family, 30 µM, Calbiochem) were added to the starvation medium for 90 min, prior to the addition of Lindane. In control conditions, cells were incubated with Me2SO (excipient; 1:5000) or left untreated.
Western blotting
Cells were washed with phosphate-buffered saline (PBS) and lysed in NP-40/Brij buffer [50 mM TrisHCl pH 7.5, 1% NP-40, 1% Brij 96 (Fluka, Saint Quentin Fallavier, France), 1 mM Na3VO4, 10 mM ß-glycerophosphate, 10 mM NaF, 2 mM EDTA, protease inhibitors (CompleteTM, Roche, Meylan, France)] to solubilize raft- and non-raft-associated proteins. Cell lysates were analyzed by western blotting as described previously (20) with antibodies directed against the phosphorylated active forms of ERK (anti-phospho-Thr202/Tyr204 ERK; 1:2000), JNK (c-jun N-terminal protein kinase, anti-phospho-Thr183/Tyr185 JNK; 1:1000) and p38 (anti-phospho-Thr180/Tyr182 p38; 1:1000; New England Biolabs, Boston, MA). After stripping, equal loadings of proteins were verified by reprobing the same blots with anti-ERK1 (Santa Cruz Biotechnology, Santa Cruz, CA; data not shown). When indicated, densitometric scannings of p42ERK2 phosphorylation were realized with UltroScan Laser densitometer. Results were expressed relative to the level of p42ERK2 phosphorylation in Lindane-stimulated cells for 15 min, which was given an arbitrary value of 100. The phosphorylation state of Cx43 was analyzed by pre-incubating protein extracts with alkaline phosphatase (Roche) before anti-Cx43 western blotting (1:2000; Transduction Laboratories, Lexington, KY).
Purification of lipid rafts
Lipid rafts were isolated by a detergent-free method to avoid the unspecific association of proteins with light membrane fractions induced by detergent (23). This method of raft isolation disrupts membrane by sonication followed by sucrose density gradient centrifugation. Sertoli cells (from two 150 mm confluent culture dishes) were stimulated with Lindane (50 µg/ml for 2 or 24 h), washed twice and collected by scraping in ice-cold PBS. The cells were pelleted by centrifugation at 800 g for 5 min, resuspended in 5 ml of homogenization buffer (250 mM sucrose, 10 mM HEPESKOH pH 7.5, 1 mM EDTA, supplemented with protease and phosphatase inhibitors) and lysed with 80 strokes of a tight fitting pestle of a Dounce homogenizer. The post-nuclear supernatant fraction was prepared by spinning the lysates at 1000 g for 10 min. After centrifugation at 8000 g for 30 min, the post-mitochondrial supernatant fraction (cytosol and microsomal membranes) was collected and centrifuged at 100 000 g for 60 min. The pellet containing the microsomal fraction was suspended in 1 ml of HEPES buffer (10 mM HEPESKOH pH 7.5, 1 mM EDTA, supplemented with protease and phosphatase inhibitors), briefly sonicated (3 x 10 s bursts at 32 W each) and then adjusted to 40% w/w sucrose by adding 2 ml of a 60% w/w sucrose in HEPES buffer, poured in the bottom of a centrifuge tube and overlaid with 4.5 ml of 30% w/w sucrose and 4.5 ml of 5% w/w sucrose solution in HEPES buffer. The sucrose gradient was then centrifuged at 200 000 g for 16 h. The pellet of the gradient was collected and designated fraction 3. The membrane fractions (10.5 ml) were collected at the 530% (corresponding to lipid rafts, fraction 1) and the 3040% sucrose interfaces (non-lipid rafts, fraction 2) and diluted in HEPES buffer. After centrifugation at 100 000 g for 1 h, the precipitated proteins were dissolved in 0.1 ml of Laemmli buffer for western blot experiments with antibodies directed against Cx43 and Caveolin-1 [raft marker (24); 1:5000; Transduction Laboratories].
Preparation of Triton X-100-soluble and -insoluble membranes
Confluent Sertoli cells were treated with methyl-ß cyclodextrin (Me-ß CD, 2.4% w/v), Lindane (50 µM) or U18666A (3 µg/ml) for 2 h. Cells were harvested by scraping in PBS, pelleted by centrifugation at 800 g for 5 min and lysed in 500 µl of Triton X-100 buffer (1% Triton X-100; 50 mM TrisHCl pH 7.4; 150 mM NaCl; 2 mM EDTA; protease and phosphatase inhibitors). Supernatant and pellet were recovered following centrifugation (100 000 g; 30 min), and insoluble membranes in the pellet were then dissolved in Laemmli buffer. Soluble and insoluble membranes were subjected to western blotting.
Scrape-loading/dye transfer assay
GJIC was assessed by the scrape-loading/dye transfer assay (25). Briefly, Sertoli cells were grown to confluency on glass coverslips after which a blade was used to create two longitudinal scratches through the cell monolayer in the presence of DMEM containing the fluorescent dye Lucifer yellow (0.05% w/v, MW: 457.3; Molecular Probes, Eugene, OR). After 5 min at 32°C, the cells were quickly rinsed three times with PBS, fixed in 3.7% paraformaldehyde and dye diffusion was visualized by fluorescent microscopy.
Immunofluorescence staining
Indirect immunofluorescence was performed as described previously (26) using antibodies against Cx43 (Transduction Laboratories), specific markers of lipid rafts (Caveolin-1, Transduction Laboratories), endoplasmic reticulum (27), Golgi apparatus (CTR433, a gift from M.Bornens, Curie Institute, Paris, France) or early endosomes (Rab5, 1/100, gift from M.Zerial, Max Planck Institute, Dresden, Germany). For cholesterol labeling, cells were fixed with paraformaldehyde and labeled with filipin (50 µg/ml, Sigma), as described (22). Pictures were taken with a 63x magnification lens using a confocal laser-scanning microscope (Leica) fitted with a 488 or 543 nm krypton/argon laser allowing simultaneous analysis of the fluorescein and rhodamine chromophores.
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Results
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Lindane promotes Cx43 endocytosis
To identify the subcellular localization of Cx43 in Lindane-treated Sertoli cells, cells were treated for 2 or 24 h with 50 µM of Lindane, a dose established previously to inhibit GJIC (20), and stained with antibodies raised against Cx43 and various exocytic and endocytic markers. As shown in Figure 1A, Cx43 was present at the appositional plasma membranes between untreated cells. In accordance with the ability of Lindane to inhibit GJIC, this carcinogen induced the progressive cytoplasmic localization of Cx43 that was detected by 2 h. After 24 h, we failed to detect any trace of membrane staining in treated cells. In this latter condition, Cx43 exhibited an intracellular punctuate localization that co-localized with Rab5 (Figure 1B), a specific marker of early endosomes. No co-localization was observed with Golgi apparatus, endoplasmic reticulum or late endosomal/lysosomal compartments (data not shown).

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Fig. 1. Lindane induces Cx43-internalization and -accumulation in early endosomes of Sertoli cells. (A) Indirect immunofluorescence analysis shows a linear Cx43 staining at appositional plasma membranes in untreated cells and a cytoplasmic Cx43 signal in Lindane-treated cells (50 µM) for 2 and 24 h (x400). (B) Deconvolution microscopy analysis of dual immunolabeling of Lindane-treated cells for 24 h indicates that all Cx43 signal co-localized with Rab5 in the merged image (x900). Representative of three separate experiments.
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Lindane does not induce Cx43 endocytosis via alteration of lipid rafts
Given the critical role played by lipid rafts in channel trafficking and assembly (28,29), we addressed the possibility that Lindane could alter Cx43 localization by targeting lipid rafts. We first investigated whether Cx43 is present in these membrane microdomains. As shown in Figure 2A, a punctuate linear staining for the raft marker Caveolin-1 could be readily detected at plasma membrane between adjacent cells that co-localized with Cx43 at this location. Consistently, western blot analysis of sucrose fractions demonstrated that a significant proportion of Cx43 was present in the low-density fraction together with Caveolin-1 (Figure 2B). Interestingly, three immunoreactive bands of Cx43 were detected
43 kDa and almost all the high molecular mass of Cx43 was recovered from this raft-enriched fraction. Alkaline phosphatase treatment of this fraction indicated that this band represented the phosphorylated P2 isoform of Cx43 (Figure 2B). Likewise, the non-raft-associated fractions contained most of the low molecular mass of Cx43, which corresponded to the unphosphorylated (P0) Cx43 isoform. Consistently, we found that the phosphorylated Cx43 isoforms (P1 and P2) were insoluble in the non-ionic detergent Triton X-100 (Figure 2C), another feature of raft-associated proteins (30). Altogether, our findings indicate that the phosphorylated Cx43 P2 isoform was exclusively associated with lipid rafts, whereas almost all the unphosphorylated P0 isoform was localized in non-raft fractions.

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Fig. 2. Cx43 is targeted to lipid rafts. (A) Co-localization of Cx43 and Caveolin-1. Double immunolabeling with anti-Cx43 and anti-Caveolin-1 antibodies reveals that Cx43 co-localized with this raft marker at appositional membranes between adjacent Sertoli cells (see high magnification in the insert). (B) Partitioning of Cx43 in lipid rafts. Rafts were isolated from confluent Sertoli cells by their light buoyant density after centrifugation on a sucrose gradient and the distributions of Cx43 and Caveolin-1 (raft marker) were analyzed by western blotting. Note that the majority of Cx43 phosphorylated isoforms were found in the raft-enriched fraction (fraction 1 from sucrose gradient). Unphosphorylated (P0) and phosphorylated (P1 and P2) isoforms of Cx43 were identified by alkaline phosphatase treatment (Pase). The positions of the phosphorylated and unphosphorylated Cx43, as well as of Caveolin-1 are indicated by arrowheads. (C) Triton X-100 insolubility of Cx43. Cells were treated with Me-ß CD (2.4%) for 2 h before being subjected to Triton X-100 lysis. Cx43 and Caveolin-1 distributions were analyzed by western blotting of Triton X-100-soluble (supernatant, S) and -insoluble cell lysates (pellet, P; raft-enriched fraction). Representative of three separate experiments.
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Agents that bind and/or extract raft lipids (cholesterol and sphingolipids) are known to alter the localization, the trafficking or the function of the raft-associated proteins (31). Consistently, the phosphorylated Cx43 were completely solubilized in Triton X-100 when cellular cholesterol was depleted by Me-ß CD (Figure 2C). Moreover, we found that incubation of Sertoli cells for 24 h with the hydrophobic amine U18666A (3 µg/ml) promoted intracellular cholesterol accumulation and concomitantly Cx43 delocalization (Figure 3A) as observed with Lindane. Interestingly, both U18666A and Lindane treatments for 2 or 24 h did not alter the partition of Cx43 isoforms and Caveolin-1 in lipid raft-containing fractions (Figure 3A and B). At that stage, it was of interest to investigate the distribution of cholesterol in Lindane-treated cells. Figure 3B shows that Lindane did not decrease the level of cholesterol at the cell surface nor did it lead to intracellular accumulation. These data therefore highly suggest that Lindane-induced Cx43 endocytosis did not result from an alteration of lipid rafts.

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Fig. 3. Lindane does not alter the association of Cx43 and Caveolin-1 with lipid rafts as well as the distribution of cholesterol. Cells were treated with 3 µg/ml U18666A (A) or 50 µM Lindane (B) for the indicated times before being subjected to either Triton X-100 lysis, sucrose gradient centrifugation, cholesterol or Cx43 staining (x400). Representative of three separate experiments.
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Lindane induces activation of the MAPK pathway ERK in Sertoli cells
We therefore considered the possibility that the Lindane-induced effects were instead due to the activation of signaling pathways. MAP kinases are key signaling molecules involved in the control of cell proliferation (32). To gain insight into their possible involvement in the mediation of Lindane-induced effects, activation of MAPK was assessed by western blotting with antibodies directed against the phosphorylated and active forms of ERK, p38 and JNK. As indicated in Figure 4A, Lindane induced the rapid and sustained activation of both p42ERK2 and p44ERK1 in Sertoli cells. ERK activation peaked at 715 min and lasted for 24 h. In contrast, neither JNK nor p38 phosphorylation was affected by Lindane (Figure 4B). The upstream chain of events that activates ERK following Lindane treatment remains to be identified. One possibility would be that Lindane might activate signaling molecules such as the GTPase Ras, the tyrosine kinase c-src or other kinases that lie upstream of ERK. In support of this hypothesis, we have shown that pre-treatment with PP2 (30 µM), a selective src inhibitor completely abrogated Lindane-induced ERK phosphorylation (Figure 4B).

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Fig. 4. Lindane induces the selective activation of the MAPK pathway ERK. Sertoli cells were pre-treated for 90 min with PP2 (30 µM, src tyrosine kinase inhibitor) or vehicle, before the addition of Lindane (50 µM). (A) Kinetics of Lindane-induced ERK activation. At the indicated time, cells were lysed and ERK activation was assessed by western blotting with anti-phospho-ERK antibodies. Densitometric scanning of p42ERK2 phosphorylation is shown in the lower panel. Similar quantification was obtained for p44ERK1 phosphorylation (data not shown). (B) In the same conditions, no p38 and JNK phosphorylations were detected in Lindane-treated cells. As a positive control, cells that were stimulated for 10 min by anisomycin (A, 10 µg/ml) showed high p38 and JNK phosphorylations. The positions of the phosphorylated ERK (p42ERK2 and p44ERK1), p38 and JNK are indicated by arrowheads. After stripping, loading of equal amounts of p42ERK2 and p44ERK1 was verified by reprobing the same blot with anti-ERK (data not shown). Representative of three separate experiments.
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Activation of ERK pathway is required for mediating Lindane-induced Cx43 phosphorylation and endocytosis
We found that Lindane-induced Cx43 endocytosis was associated with an increased level of Cx43 phosphorylated P2 isoform (Figure 5A). We therefore investigated whether Lindane-induced Cx43 hyperphosphorylation and endocytosis were the results of ERK activation. For this purpose, we used PD98059, a potent cell permeable and specific inhibitor of MEK1/2, the kinases immediately upstream of ERK in the MAPK cascade (33). Pre-treatment of Sertoli cells with PD98059, which completely blocked the ability of Lindane to activate ERK (Figure 5A), prevented Cx43 hyperphosphorylation (Figure 5A) and restored its localization at cellcell interfaces (Figure 5B). We therefore assessed Cx43 functionality in gap junction coupling using the scrape-loading/dye transfer assay. As shown in Figure 5C, Lindane-treated cells were not coupled by gap junction channels as indicated by the absence of Lucifer yellow transfer from wounded cells at scrape border to neighbouring cells. In contrast, intercellular dye transfer was restored when cells were pre-treated with PD98059 before Lindane addition. Taken together, the data underscore that activation of MAPK/ERK pathway was mandatory for Lindane-induced Cx43- phosphorylation, -endocytosis and thereby -GJIC impairment.

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Fig. 5. Activation of ERK pathway is essential for Lindane-induced Cx43 phosphorylation, -delocalization and GJIC impairment. Sertoli cells were pre-treated with PD98059 (MEK1 inhibitor, 10 µM, 90 min) before the addition of Lindane. (A) Western blot analyses of Cx43 and ERK phosphorylations of cells treated for 15 min. (B) Cx43 localization and (C) GJIC analyses of Lindane and PD98059 treated cells for 24 h. Representative of three separate experiments.
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Discussion
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The reported rising incidences of testicular cancer and of several other human malignancies together with their potential link with environmental factors raise important health concerns. There is strong evidence that most environmental chemicals, such as pesticides, target gap junction communication (9,10); however, the underlying mechanism(s) remain unknown.
The present study provides the first evidence that Lindane, a widely used pesticide, impairs GJIC in Sertoli cells by promoting the sustained sequestration of Cx43 within early endosomes via the activation of the ERK pathway. At least two mechanisms should be considered to explain the Lindane-induced intracellular Cx43 accumulation. One possibility would be that Cx43 transport to the cell surface could be disturbed by Lindane, addressing newly synthesized Cx43 to the endosomes instead of the gap junction plaques. However, we found in the same condition, that Lindane did not alter the biosynthetic trafficking of a secreted GFP or a GPI-anchored GFP (data not shown). This suggested strongly that Cx43 accumulation in the endosomes might be rather consecutive to Lindane-induced Cx43 internalization and a blockage in early endosomes.
Although critical, very little information is currently available about the mechanism(s) that target(s) Cx43 to endocytosis and thereby to degradation. While considerable progress has been made in defining Cx trafficking to plasma membrane (34,35), there is only indirect evidence showing that lysosomes and/or proteasomes are the main degradative pathways involved in Cx turnover; based on the use of chemical inhibitors (3638). Moreover, in most of the studies carried out so far, the precise subcellular localization of Cx remains unclear, probably because low Cx levels are present in the endosomes, below the limit of detection (39). Consistently, electron microscopy studies have allowed localization of internalized gap junction plaques in lysosomes (40). This unusual failure to detect Cx in the endosomes might be explained by their short half-life (37). Therefore, delineation of the mechanism involved in Lindane-induced Cx43 accumulation in endosomes may provide important insight into the deleterious effect of this carcinogen but also into the regulation of Cx43 endocytosis.
Lipid rafts have emerged as unique platforms for the assembly and the trafficking of many ion channels along the exocytic and endocytic pathways (28,29). Recently, one partner of Cx43, Zonula Occludens-1 (41,42) was demonstrated to be distributed within these membrane microdomains (43). We have shown previously that Lindane induces the intracellular sequestration of Zonula Occludens-1, which co-localizes with Cx43 in Sertoli cells (20). This prompted us to consider the possibility that Cx43 may be present in lipid rafts. In agreement with this hypothesis, we showed that Cx43 displayed four features of raft-associated proteins: it (i) co-localized with Caveolin-1; (ii) co-fractionated with this raft marker in low-density raft fractions; (iii) was resistant to Triton X100 solubilization; and (iv) the association of Cx43/rafts was dependent on cholesterol content. These findings are in agreement with original biochemical studies identifying some isoforms of Cx43 as Triton X-100-insoluble proteins (44) and the particular cholesterol-rich content of gap junctions (45,46). Of particular interest, we found that the phosphorylated Cx43 P2 isoform was exclusively targeted to the raft fractions while the P1 isoform was equally distributed in the raft and non-raft domains and almost all the unphosphorylated P0 isoform was excluded from these lipid microdomains. The molecular mechanisms responsible for the specific Cx43 P2/raft association remain unknown. One possibility would be that the unphosphorylated Cx43 is targeted outside the lipid rafts and moves into lipid rafts upon phosphorylation, as has been observed for T-cell receptor (47). Alternatively, the P0 isoform might be efficiently addressed to these microdomains where it could be phosphorylated. In this regard, evidence that several kinases, such as PKC and MAPK, are localized within lipid rafts is of great interest (48). While this work was in progress, it was shown that the phosphorylated and unphosphorylated Cx43 isoforms are distributed to lipid rafts with similar efficiencies when transfected in HEK-293 cells (49). This discrepancy with the current result in Sertoli cells may be the consequence of different Cx43 expression levels and/or lipid raft compositions between the two cell types examined. Taken together, the association of Cx43 with Caveolin-1 and rafts points to the important notion that gap junctions are distributed at least in part in these microdomains. The ordered structure of lipid rafts, enriched in cholesterol, sphingolipids and signaling proteins might favor trafficking and assembly of Cx43 channels into gap junction as well as regulate Cx43 channel gating. In support of this hypothesis, increased gap junction assembly and permeability have been reported upon cholesterol supplementation (50,51). This is further strengthened by our observation that incubation of Sertoli cells with U18666A, a hydrophobic amine that induced intracellular cholesterol accumulation, promoted concomitantly intracellular Cx43 delocalization. Owing to its highly lipophilic propriety, it has been proposed that Lindane could insert into the plasma membrane, alter membrane fluidity, and thereby directly modulate the activity of the integrated membrane proteins (15). Consistently, it was demonstrated that a widely used GJIC inhibitor heptanol decreases the fluidity of cholesterol rich domains (52). But at odds with this proposal, we demonstrated that Lindane did not affect the level of cholesterol at the cell surface nor did it alter partition of Cx43 isoforms and Caveolin in lipid rafts. We therefore assume that Lindane may indirectly promote Cx43 endocytosis.
Other mechanism(s) that can be considered in Lindane-induced Cx43 endocytosis are the activation of signaling pathways. Indeed, there is growing evidence that some environmental chemicals exert their deleterious effects by mimicking the action of estrogens. Of particular interest, these hormones have been shown to induce beyond their genomic effects the mitogen-activated protein kinases ERK in normal and transformed cells (53). We provide here the first evidence that Lindane induced the activation of ERK whereas no induction of stress kinases JNK and p38 was observed. This specific activation of ERK is consistent with the carcinogenic potency of Lindane as ERKs promote cell growth, survival and transformation (32,54). In response to growth factors and tumor promoters, ERK and several other kinases (src, PKC, PKA and p38) are known to phosphorylate Cx and thereby to control channel trafficking, assembly, gating, and turnover (5558). We provide here new evidence that endocytosis of Cx43 is induced by the ERK pathway. This is supported by our current observations that: (i) Lindane promoted ERK activation with a time course that preceded Cx43 phosphorylation and endocytosis; and (ii) inhibition of the MAPK pathway by PD98059 prevented Lindane-induced Cx43-phosphorylation, -endocytosis and -GJIC impairment.
Decreased expression and/or aberrant cytoplasmic localization of Cx have been shown to correlate with neoplastic transformation of several human tissues including breast (59), lung (60), brain (61), endometrium (62), skin (63), prostate (64) and ovary (65). Recently, an aberrant cytoplasmic Cx localization in Sertoli cells of human testes with seminoma has been reported (19). The rising incidence of this testicular cancer in industrialized countries raises important issues about the molecular targets of environmental chemicals. In agreement with the concentration range of Lindane reported in blood and tissue samples of exposed humans and rodents (66,67), the present study demonstrated that this carcinogen induces in Sertoli cells the activation of mitogen-activated protein kinase ERK pathway which down-regulates GJIC through sequestration of Cx43 in early endosomes. Consistently, we found that Lindane could induce this pathway in another sensitive cell-type (as kidney, data not shown), raising the relevance of this carcinogenic pathway to other tissues. Based on these findings, it would be expected that exposure to chemicals as Lindane that both activate mitogenic signaling pathways and inhibit the function of tumor-suppressor genes as Cx, would result in uncontrolled cell growth and tumor promotion.
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Acknowledgments
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We thank A.Doye and A.Spadafora for microscopy analysis, and N.Gauthier for reagents and helpful advices. This work was supported by funds from the Institut National de la Santé et de la Recherche Médicale, European Chemical Industry Council (CEFIC), Ligue Nationale contre le Cancer and the Association pour la Recherche sur le Cancer (no. 98-80). B.M. is a recipient of a post-doctoral fellowship from Fondation Fertilité Stérilité and Fondation Aide à la Recherche Organon. E.C. and N.D. contributed equally to this work and are recipient of a fellowship from the Agence de lEnvironnement et de la Maîtrise de l'Energie'.
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Received February 26, 2003;
revised April 18, 2003;
accepted May 16, 2003.