1 INSERM EMI 00-09, IFR 50, Faculté de Médecine, Avenue de Valombrose, 06107 Nice CEDEX 02, France
2 LBSC, CNRS UMR 6558, Université de Poitiers, 86022 Poitiers CEDEX, France
3 INSERM U 364, IFR 50 Faculté de Médecine, 06107 Nice CEDEX 02, France
4 Université Paris V, 45 rue des Saint-Pères, 75006 Paris, France
5 GRIPL, EA 2675, Faculté de Médecine, 06107 Nice CEDEX 02, France
6 INSERM U 427, Faculté de Pharmacie, 75270 Paris, France
* Author for correspondence (e-mail: pointis{at}unice.fr)
Accepted 27 May 2004
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Summary |
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Key words: Cx33, Cx43, Dominant negative, Cx33-Cx43 interaction, Testis
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Introduction |
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A key observation indicates that coexpression of Cx33 with other Cxs inhibits the junctional conductance (Chang et al., 1996). However, the molecular mechanism(s) by which Cx33 impairs GJIC is unknown. We hypothesized that Cx33 might interfere with Cx function by associating with them. As the expression of Cx33 is mainly restricted to the testis (Haefliger et al., 1992
; Tan et al., 1996
; Chung et al., 1999
) and to differentiating neuroblasts (Rozental et al., 1998
), this prompted us to analyze the direct association of Cx33 with other Cxs expressed in the testis, in particular Cx43. This issue is of primordial importance because gap junctional coupling via Cx43 is essential for spermatogenesis (Juneja et al., 1999
; Plum et al., 2000
; Roscoe et al., 2001
). In the present study, we show that Cx33 is able to associate selectively with Cx43 but not with Cx26 and Cx32. We further demonstrate that this selective complex occurred between Cx33 and two phosphorylated isoforms of Cx43 and is intracellularly sequestered within early endosomes. Most importantly, the association formed by Cx33 and Cx43 causes a complete inhibition of the GJIC. Our data emphasize a new physiological control of gap junctional communication by intracellular sequestration of a functional Cx.
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Materials and Methods |
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Cell culture and transfection
The 42GPA9 Sertoli cell line was maintained in Dulbecco's Modified Eagle's Medium (GIBCO BRL) containing 10% fetal calf serum at 32°C (Bourdon et al., 1998). Sertoli and COS-7 cells, seeded on glass coverslips were transfected with expression plasmids for Cx33, Cx33-myc, Cx43, EGFP-Rab5, EGFP-Rab7 and EGFP-Lamp1 alone or in combination (3 µg, at a ratio of 1:1) using Fugene reagent (Roche Molecular Biochemicals) or DEAE/Dextran (Amersham Biosciences) (Seed and Aruffo, 1987
) and grown to confluency. To generate a Sertoli cell line expressing Cx33, cells were transfected with 1 µg Cx33 or Cx33-myc plasmid cDNA using Fugene reagent. Control cells were transfected with empty vector. After 48 hours, transfected cells were selected in complete medium containing 0.8 mg/ml of G418 (Geneticin, Gibco BRL). After 3 weeks, more than 100 G418-resistant clones were individually picked, expanded and assayed for Cx33 or myc expression by western blotting.
Preparation of tissue and cell extracts
Mature rat testes, brain, heart, ovary and uterus were solubilized in NP40/Brij lysis buffer (50 mM Tris-HCl pH 7.5, 1% NP40, 1% Brij 96, 1 mM Na3VO4, 1 mM ß-glycerophosphate, 50 mM sodium fluoride, 1 mM EDTA, 1 mM aprotinin, 25 mM leupeptin, 1 mM pepstatin, 2 mM phenylmethylsulfonyl fluoride). Triton X-100 solubilization of connexons was prepared as previously described (Koval et al., 1997). Briefly, confluent cells were collected by scraping in ice-cold PBS and centrifuged at 500 g for 5 minutes. The cell pellet was resuspended in detergent-free lysis buffer and lysed with a Dounce homogenizer. The post nuclear supernatant fraction was collected by centrifugation at 500 g for 5 minutes and solubilized with Triton X-100 (final concentration 0.1%) for 30 minutes at 4°C. After centrifugation at 100,000 g for 1 hour, Triton X-100 soluble fractions were subjected to immunoprecipitation.
Immunoprecipitation and western blotting
Lysates and Triton X-100 soluble fractions were immunoprecipitated with 2 µg of either anti-Cx43 (Transduction Laboratories), anti-Cx33 or anti-myc (gift from JF Tanti, Inserm U568, Nice, France) antibodies for 16 hours at 4°C. The immunocomplexes were precipitated by incubation with protein A-Sepharose (Pharmacia) for 1 hour at 4°C. Immunopellets were washed four times in PBS 0.5% NP 40, once in PBS, and then eluted with 40 µl reduced Laemmli buffer at 100°C for 5 minutes. Proteins were separated on 5-15% SDS-PAGE, electroblotted onto a polyvinylidene fluoride membrane (PVDF Immobilon-P, Millipore) and analyzed by western blotting with antibodies against Cx43 (1:2000), myc (1:2000), Cx33 (1:1000), Cx26 or Cx32 (1:2000, Santa Cruz Biotechnology) as previously described (Defamie et al., 2001).
Analysis of Cx33 phosphorylation status
The phosphorylation status of Cx33 was analyzed on testicular immunoprecipitates either by pre-incubating for 4 hours with alkaline phosphatase (Roche) or by western blotting with anti-phosphotyrosine (1:6000, Upstate Biotechnology Incorporated), anti-phosphoserine and anti-phosphothreonine antibodies (1:1000, Zymed). In other experiments, COS-7 cells were transiently transfected with Cx33-myc and Cx43. Two days after transfection, the confluent cells were labeled with [32P]orthophosphate (500 µCi) in phosphate-free DMEM for 5 hours, before being subjected to cell lysis with NP40/Bridj buffer as described above and supplemented with phosphatase and protease inhibitor cocktail. Lysates were then immunoprecipitated with myc or Cx43 antibodies. The [32P]-phosphorylated proteins were resolved by SDS-PAGE, electroblotted onto PVDF membrane and visualized by autoradiography. The presence of Cx43 and Cx33-myc in immunoprecipitates was verified by western blotting.
Immunofluorescence staining
The localization of Cx33 and Cx43 in cells or in testis sections was assessed as previously described (Batias et al., 2000; Defamie et al., 2001
). Briefly, 5 µm testis sections or cells were fixed with cold methanol, permeabilized for 5 minutes with saponin buffer (0.5% saponin, 2.5% goat serum, 1% bovine serum albumin and 0.2% gelatin in PBS) and incubated overnight with Cx33, Cx43 and myc (1/100) antibodies at 4°C. The slides were rinsed with 0.5% saponin-PBS, incubated with FITC-conjugated anti-rabbit (DAKO) and Texas red-conjugated anti-mouse (Pharmacia) antibodies and mounted in Mowiol medium (Calbiochem). Pictures were taken with a x63 magnification lens using a confocal microscope (Leica) fitted with a 488 or 543 nm krypton/argon laser allowing simultaneous analysis of the fluorescein and rhodamine chromophores. One representative section was chosen from the total series of 20 optical sections. Testes sections or cells incubated with the control rabbit and mouse IgG showed no staining.
For determining the subcellular localization of Cx43 and Cx33, Sertoli cells and COS-7 cells were cotransfected with EGFP-Rab5, EGFP-Rab7 or EGFP-Lamp1 and with Cx33-myc. Cells at confluency were fixed with cold methanol and then incubated with a mixture containing anti-Cx43 (1:100) and anti-myc (1:1000) antibodies. A polyclonal antibody (1:300) (Louvard et al., 1982) that was a gift from H. Zahraoui (Institut Curie, Paris France) was used to detect the endoplasmic reticulum (ER). To detect the Golgi apparatus, an anti-Golgi CTR433 monoclonal antibody (1:10, a gift from M. Bornens, Institut Curie, Paris France) was used. Antibodies were revealed with the anti-mouse IgG coupled to rhodamine, anti-rabbit IgG coupled to Cyan-5, and anti-rabbit IgG coupled to FITC. Deconvolution microscopy analysis of the immunoreactive signals was performed as described (Savino et al., 2001
; Segretain et al., 2003
).
Dye transfer analysis
GJIC was assessed by the microinjection dye transfer method. Briefly, stably Cx33-myc transfected cells were microinjected with 10% Lucifer Yellow CH (Sigma) and 1% rhodamine dextran (Sigma) dissolved in 1 mM LiCl using a Narishige microinjector (IM300, Narishige Co., Japan) and an Eppendorf micromanipulator (5171, Eppendorf, Germany). Fluorescence emission and dye transfer were examined after 5 minutes by confocal laser scanning microscopy using a Biorad MRC 1024 equipped with a 15 mW argon-krypton gas laser. The confocal unit was attached to an inverted microscope (Olympus IX70, Japan). For each condition, at least 20 microinjections were performed and the average number of communicating cells was estimated. Analysis of variance (ANOVA) with additional post hoc analysis (Newman-Keuls test) was applied to compare coupling between control cells (transfected with the empty vector) and cells stably transfected with the vector containing Cx33-myc cDNA. Differences between means were considered significant when P<0.05.
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Results |
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Specific association of Cx33 with Cx43
To determine if Cx33 and Cx43 were able to interact, Cx33 was immunoprecipitated from testis lysates and Cx33-containing complexes were analyzed by western blotting with antibodies against Cx43, Cx26 and Cx32. As shown in Fig. 2A, Cx33 co-immunoprecipitated with Cx43. The association of Cx33 and Cx43 was further supported by the presence of Cx33 in Cx43 immunoprecipitates (left panels). The specificity of this physical interaction was evidenced by the observations that Cx26 and Cx32 did not co-immunoprecipitate with Cx33 (Fig. 2A, right panels). This was also supported by the observations that Cx26 and Cx32 were unable to co-immunoprecipitate Cx33 (data not shown). Interestingly, two bands instead of four were detected by the anti-Cx43 antibody in Cx33 immunoprecipitates (Fig. 2A, left panel). Alkaline phosphatase treatment indicates that the upper band corresponded to the P1 Cx43 isoform (data not shown). Cx33 also coprecipitated, at a lesser level with a lower molecular weight band corresponding to the unphosphorylated (P0) Cx43 isoform.
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To verify the existence of direct physical interaction between Cx33 and Cx43, immunoprecipitation experiments were performed on Triton X-100 soluble Cx oligomers from Sertoli cells. As low levels of Cx33 were found in whole testis homogenates and the Sertoli cell line expressed Cx33 at a barely level detectable only by RT-PCR (data not shown), Sertoli cells were transfected with the vector containing Cx33. As indicated in Fig. 2B (left panels) western blot analysis of Cx33 transfected Sertoli cells reveals the presence of a band at about 33 kDa. Sertoli cells transfected with Cx33-myc shows the presence of a band with the same molecular weight revealed with both anti-Cx33 and anti-myc antibodies consistent with the finding that the myc epitope does not alter Cx33 molecular conformation. The 33 kDa protein was not detected with anti-Cx33 and anti-myc antibodies when cells were transfected with the empty vector (data not shown). The detergent-solubilized fractions from Cx33-myc transfected Sertoli cells were then immunoprecipitated with either anti-Cx43 or anti-myc antibodies. As indicated in Fig. 2B (middle panels), the anti-myc antibody co-immunoprecipitated Cx43. The interaction between Cx33 and Cx43 was further supported by the presence of a band of about 33 kDa revealed with the anti-myc antibody in Cx43 immunoprecipitates. Only two isoforms of Cx43 corresponding to P0 and P1 were detected in Cx33-myc transfected cells whereas three isoforms of Cx43 were observed in both the wild type and Sertoli cells transfected with the vector without Cx33-myc (Fig. 2B, right panel).
Sequestration of Cx43 by Cx33 in early endosomes
To determine the subcellular localization of the Cx33/Cx43 complex, Sertoli cells were transfected with Cx33-myc cDNA. Punctuate linear staining for Cx43 was observed at appositional plasma membranes between adjacent Sertoli cells transfected with empty vector (Fig. 3A) or in wild-type Sertoli cells (data not shown). No immunoreactive signal was found with the antibody directed against myc (Fig. 3B). In contrast, in Cx33-myc transfected Sertoli cells, Cx43 was no longer detected at the cell surface but mainly in the perinuclear region (Fig. 3D). A similar localization was found for Cx33 detected with the anti-myc antibody (Fig. 3E) or with anti-Cx33 antibody (data not shown). Merging Cx43 and Cx33-myc micrographs reveals a complete colocalization of the two immunoreactive signals (Fig. 3F). Similarly, Cx33 or Cx33-myc intracellularly colocalized with Cx43 in COS-7 cells cotransfected with Cx43 and Cx33 (Fig. 4G-I) or Cx33-myc (Fig. 4J-L). Control experiments were performed in which COS-7 cells were transfected with vectors containing Cx43, Cx33-myc or Cx33 cDNAs. In COS-7 cells transfected only with Cx43, the Cx43 signal was present as expected at the plasma membrane (Fig. 4A-C). In cells transfected with Cx33-myc cDNA, the immunoreactive signal detected with Cx33 antibody was restricted to a region around the nucleus (Fig. 4E) and colocalized with the myc signal (Fig. 4D,F). A similar localization for Cx33 detected with the anti-Cx33 antibody was observed in Cx33 transfected cells (data not shown). Together, the co-immunoprecipitation and microscopy results strongly suggested that Cx33 sequestered Cx43 intracellularly via a protein-protein interaction.
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In order to localize finely the Cx33/Cx43 complex, immunofluorescence microscopy with various markers of the intracellular compartments was performed in Cx33-myc transfected Sertoli cells as previously described (Segretain et al., 2003). High-resolution deconvolution fluorescent microscopy of the signals in transfected Sertoli cells indicates that myc and Cx43 did not colocalize with markers of the Golgi apparatus, lysosomes, late endosomes (Fig. 5E-H,M-P,Q-T) or with the ubiquitin/proteasomal markers (data not shown). A faint colocalization of Cx33-myc/Cx43 was however detected with a marker of the ER (Fig. 5D). In contrast, Cx43 mainly colocalized with Cx33-myc on vesicle positively stained for the early endosomal marker Rab5 (Fig. 5L). In wild-type untransfected Sertoli cells or cells transfected with the empty vector, Cx43 staining was always present at the plasma membrane level (Fig. 3A) and was undetectable in early endosomes.
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Inhibition of gap junctional coupling by Cx33
We therefore investigated the effect of Cx33 on gap junctional coupling. Sertoli cells transfected with the vector without Cx33-myc cDNA were able to communicate through gap junctions as demonstrated by the diffusion of Lucifer yellow from one microinjected cell to neighboring cells (Fig. 6C). Rhodamine dextran, a high molecular weight dye, was localized only inside the microinjected cells (Fig. 6B). Similar results were obtained in wild-type untransfected Sertoli cells. In this condition, the number of coupled cells was 18.15±4.9 (mean±s.d.; number of microinjections=20). In contrast, in Sertoli cells stably overexpressing Cx33-myc no coupling occurred as indicated by the absence of Lucifer yellow transfer from the microinjected cell to adjacent cells (Fig. 6F). Quantitative analysis demonstrates that the number of communicating cells was markedly reduced (1.05±0.22, n=20, P<0.001).
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Discussion |
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Spermatogenesis is a complex process of germ cell multiplication and differentiation that takes place under the control of Sertoli cells. Within the seminiferous tubules, germ cells are closely associated with supporting Sertoli cells and cross talk between these cells is mediated through paracrine regulations, direct cell-cell interactions and communication via gap junctions. In the testis, ten Cx mRNAs are presumably translated within the seminiferous epithelium (Risley, 2000). We and others reported the presence of Cx43 in the basal compartment of seminiferous tubules in both Sertoli and germ cells (Risley et al., 1992
; Pelletier, 1995
; Batias et al., 2000
) and its physiological implication in male fertility (Juneja et al., 1999
; Plum et al., 2000
; Defamie et al., 2001
). More recently, we have characterized the presence of Cx43-based gap junctions between neighboring Sertoli cells, between Sertoli cells and spermatogonia and between Sertoli cells and spermatocytes (Decrouy et al., 2004
). The current study demonstrates that Cx33 traps Cx43 intracellularly and inhibits Cx43 gap junctional coupling. Thus, it is tempting to suggest that Cx33, through this process, could alter the formation of Cx43 gap junctions within the seminiferous epithelium. Our results also show that Cx33 was unable to immunoprecipitate and colocalize with Cx26 and Cx32, two other Cxs expressed in the testis but differently localized within the seminiferous epithelium (Risley et al., 1992
). Together these data suggest that the selective association of Cx33 with Cx43 plays an essential role in the control of spermatogenesis.
There is growing evidence that phosphorylation may play a role in Cx43 trafficking and/or assembly (Lampe and Lau, 2000). Interestingly, our data indicate that Cx33 was mainly associated with the P1 phosphorylated isoform of Cx43. The current results also indicate that Cx43, which was normally localized at the plasma membrane, was intracellularly colocalized with Cx33 in Sertoli cells expressing Cx33. This raises the possibility that the interaction between Cx33 and Cx43 contributed to the regulation of Cx43 phosphorylation and subsequent Cx43 trafficking.
Another interesting feature is that Cx33, in contrast to other identified Cxs except Cx26, appeared as an unphosphorylated Cx (Traub et al., 1989; Saez et al., 1990
). The reason(s) for such a characteristic are presently unknown. It is well recognized that the length of the cytoplasmic C-terminal tail is essential in the regulation of the protein function since several recognition sites for different protein kinases are present in this domain of the protein (Lampe and Lau, 2000
). Among the Cxs identified, Cx26 is the smallest. It has almost no C-terminal tail and is unphosphorylated (Traub et al., 1989
; Saez et al., 1990
). After Cx26, Cx33 has the shortest C-terminal tail (Haefliger et al., 1992
). Thus, it is possible that lack of the phosphorylated isoform of Cx33 may be due to the structural characteristics of this Cx.
Previous studies have shown that in normal or pathological conditions Cx may accumulate in intracellular stores that correspond to the ER-Golgi intermediate compartment (ERGIC) or Golgi apparatus (George et al., 1999). The reasons for the current observation that the Cx33/Cx43 complex colocalized in the early endosomes are presently unknown. Recently, it has been suggested that early endosomes are the first sorting platform from which endocytosed materials are targeted to various intracellular destinations (Deneka and van der Sluijs, 2002
). Thus, the possibility that Cx33 acts as a chaperone that alters the stability of the gap junction plaques by maintaining Cx43 in the early endosome before recycling to the plasma membrane or to other cytoplasmic components can be hypothesized. Altered trafficking of the Cx33/Cx43 complex to the plasma membrane could be also considered since it has been demonstrated that early/recycling endosomes can also be part of the exocytic pathway (Laird and Spiess, 2000
). In addition, a role of Cx in processes independent of the formation of gap junctional channels has been suggested (Huang et al., 1998
; Krutovskikh et al., 2000
; Plotkin et al., 2002
; Olbina and Eckhart, 2003
). Whether the Cx43/Cx33 trapped in the early endosomes could play such roles remains to be elucidated.
Our data also demonstrate that the presence of Cx33 caused a dramatic inhibition of GJIC in Sertoli cells. These results are consistent with previous observations that coexpression of Cx33 with Cx43 dramatically reduces or inhibits the junctional conductance in the paired Xenopus oocyte system (Chang et al., 1996). From our data, one can infer that the inhibitory effect of Cx33 on GJIC stems from intracellular sequestration of the Cx33/Cx43 complex. Whether this model is applicable to other Cxs remains to be determined. In this regard, it is noteworthy that the transfection of dominant negative Cx43 constructs leads to aberrant cytoplasmic localization of Cx46 or P0/P1 Cx43 isoforms (Krutovskikh et al., 2000
; Lampe and Lau, 2000
). So far, such behavior has only been described for mutated Cx constructs. The current findings provide a new mechanism for the control of gap junctions in which a native Cx (like Cx33) can have dominant negative effects by sequestering Cxs that associate with it. Thus, further studies on the function(s) of Cx33 within the seminiferous epithelium will provide new insights into the role of this Cx in the control of the spermatogenic process.
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Acknowledgments |
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