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Endocrinology, doi:10.1210/en.2007-0767
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Endocrinology Vol. 148, No. 12 5913-5924
Copyright © 2007 by The Endocrine Society

Tumor Necrosis Factor-{alpha}-Induced Anterior Pituitary Folliculostellate TtT/GF Cell Uncoupling Is Mediated by Connexin 43 Dephosphorylation

Mélissa-Anne Meilleur, Casimir D. Akpovi, R.-Marc Pelletier and María Leiza Vitale

Département de pathologie et biologie cellulaire, Faculté de médecine, Université de Montréal, Montréal, Québec, Canada H3T 1J4

Address all correspondence and requests for reprints to: María L. Vitale, Department of Pathology and Cell Biology, Faculty of Medicine, Université de Montréal, 2900 Edouard-Montpetit, Montréal, Québec, Canada H3T 1J4. E-mail: maria.leiza.vitale{at}umontreal.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The anterior pituitary folliculostellate (FS) cells are key elements of the paracrine control of the pituitary function. These cells are the source and the target of growth factors and cytokines, and are connected to other pituitary cells via Cx43-mediated gap junctions. Here, we show that acute treatment of the FS TtT/GF cell line with TNF-{alpha} caused a transient cell uncoupling that was accompanied by the dephosphorylation of Cx43 in Ser368. These TNF-{alpha}-evoked effects were dependent on protein phosphatase 2A (PP2A) and protein kinase C (PKC) activities. TNF-{alpha} did not affect total cell Cx43-PP2A catalytic subunit interaction, but it did induce PP2A catalytic subunit recruitment to the Triton X-100 insoluble subcellular fraction, in which Cx43-gap junction plaques are recovered. This recruitment temporally coincided with Cx43 phosphorylated in Ser368-Cx43 dephosphorylation. Cx43 did not interact with the conventional PKC-{alpha}, but it did interact with the atypical PKC-{zeta}. Moreover, this interaction was weakened by TNF-{alpha}. Cx43 dephosphorylation in Ser368 was followed by the tyrosine phosphorylation of the protein. The temporary closure of gap junctions during acute TNF-{alpha} challenge may constitute a protective mechanism to limit or confine the spread of inflammatory signals among the FS cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GAP JUNCTION-MEDIATED intercellular communication is implicated in tissue homeostasis by coordinating and synchronizing the metabolic responses of the many cells composing the tissue. Gap junctions are a collection of channels established across the plasma membrane of apposing cells that permit the communication of the intracellular milieu of the participating cells by allowing the passage of molecules with molecular masses smaller than 1000 (1). Each gap junction channel is formed by a family of proteins called connexins (Cxs). Six Cx molecules associate to form a connexon or hemichannel, and the docking of two hemichannels from neighboring cells forms a gap junction channel (2). In addition, it has been shown that hemichannels on the plasma membrane may have a role in intercellular communication under certain physiological and pathological circumstances (3, 4, 5).

In the anterior pituitary gland, intercellular communication chiefly involves the folliculostellate (FS) cells (6, 7). These are agranular, stellate-shaped cells organized in follicles and surrounding the endocrine cells (8, 9, 10). FS cells express the gap junction protein Cx43 (11, 12, 13). We have demonstrated that the number and localization of Cx43-based gap junctions in FS cells are affected by the activity of the pituitary gland (12). The FS cells play an important role in the paracrine control of anterior pituitary endocrine and immunological responses (10). These anterior pituitary cells are the source as well as the target of proinflammatory cytokines, such as IL-1 and TNF-{alpha} (14, 15, 16, 17). Experimental evidence has revealed that proinflammatory cytokines influence intercellular communication in several cell types (18, 19, 20, 21) and that intracellular communication affects the sensitivity of cells to proinflammatory cytokines (22). We have recently shown that proinflammatory cytokines influence intercellular communication in a FS cell line, the TtT/GF cells, by increasing the synthesis and degradation of Cx43 (23). However, cytokine treatment may lead to different physiological responses depending on the duration of the treatment. This is particularly true when the target is Cx43, a protein characterized by a very rapid turnover.

One way to interfere with Cx43-mediated gap junctions within minutes is by changing the phosphorylation status of the protein (24, 25). Cx43 is the substrate of several kinases (26, 27, 28) and phosphatases (29). Serine residues are the main Cx43 phosphorylation sites (26, 27, 30). Specifically, phosphorylation of serine 368 (Ser368) catalyzed by protein kinase C (PKC) (31) alters Cx43 function (30, 32, 33).

Here, we show that TNF-{alpha} caused a rapid (15 min) and transient decrease in TtT/GF cell-to-cell communication that was accompanied by the dephosphorylation of Cx43 in Ser368. Both effects were dependent on protein phosphatase 2A (PP2A) and PKC activities Our results suggest that TNF-{alpha}-evoked Cx43 dephosphorylation is mediated by alterations in Cx43-PP2A and Cx43-PKC-{zeta} interactions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
TtT/GF cells were kindly provided by Dr. U. Renner (Max-Planck-Institute of Psychiatry, Department of Endocrinology, Munich, Germany). The cells were grown in DMEM supplemented with 5% fetal calf serum, 3.7 g/ml NaHCO3, 10 mM HEPES (pH 7.2), and antibiotics at 37 C under a 95–5% air-CO2 atmosphere. For immunofluorescence and intercellular communication studies, the cells were seeded on glass coverslips. Cells were treated with the cytokines for the time periods and concentrations indicated under each protocol.

Source of antibodies, cytokines, and other compounds
Four different antibodies were used for studies on Cx43. A rabbit polyclonal antibody that recognizes all forms (phosphorylated and nonphosphorylated) of Cx43 (Pan-Cx43) was purchased from Sigma Chemical Co. (Windsor, Ontario, Canada). For loading control in immunoprecipitation (IP) studies, we used a monoclonal antibody to all forms of Cx43 (mPan-Cx43) from Chemicon (Temecula, CA). The rabbit polyclonal antibody that only recognizes Cx43 phosphorylated in Ser368 [P(Ser368)-Cx43] was obtained from Chemicon. This antibody was developed against a synthetic phospho-peptide corresponding to residues surrounding Ser368 of human Cx43. The monoclonal antibody to Cx43 (clone CX-1B1) from Zymed (San Francisco, CA) only recognizes Cx43 when it is not phosphorylated in Ser368 [NP(Ser368)-Cx43], but it recognizes Cx43 if phosphorylated at sites besides Ser368. Monoclonal antibodies to PP2A catalytic subunit (PP2Ac) and PKC-{alpha} were purchased from BD Biosciences (Mississauga, Ontario, Canada). Monoclonal anti-PKC recognizing conventional PKC isoforms {alpha}, ßI-II, and {gamma} was obtained from GeneTex (San Antonio, TX). Monoclonal (H-1) and polyclonal (C-20) anti-PKC-{zeta} were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal anti-phosphotyrosine (clone 4G10) was purchased from Upstate Biotechnology (Lake Placid, NY). Polyclonal anti-nonmuscle actin was from Sigma Chemical Co. Peroxidase-conjugated, tetramethylrhodamine-isothiocyanate conjugated, and fluorescein-isothiocyanate conjugated secondary antibodies were from Jackson Immunochemicals (West Grove, PA).

Recombinant human TNF, aprotinin, phenylmethylsulfonyl fluoride (PMSF), and chemiluminescence detection kit Lumilight were obtained from Roche (Laval, Québec, Canada). Materials for cell culture and Lucifer yellow were purchased from GIBCO-Invitrogen (Burlington, Ontario, Canada). Protein A-Sepharose 4B beads and rhodamine-dextran were from Sigma Chemical Co. Phorbol myristate acetate (PMA), the PKA inhibitor KT5720, the MAPK kinase (MEK) inhibitor U0126, the PKC inhibitor (isoforms {alpha}, ßI-II, {gamma}, {delta}, {zeta}) Gö6983, and the protein phosphatase inhibitor okadaic acid (OA) were purchased from Calbiochem (San Diego, CA). The protein phosphatase inhibitor calyculin A (Cal A) was from Biomol (Plymouth Meeting, PA).

Preparation of Triton X-100 soluble and insoluble subcellular fractions
TtT/GF cells were rinsed with cold PBS [137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4 (pH 7.4)], and next incubated with PBS containing 1% Triton X-100, 10 mM MgCl2, and protein phosphatase and protease inhibitors [2 mM PMSF, 1 mM EGTA, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 4 mM Na3VO4, 80 mM NaF, 20 mM Na4P2O7, and 10 µM bpV (phen)]. After incubation, the cells were scraped off and homogenized. Cell lysates were centrifuged at 15,000 x g for 25 min (Beckman Microfuge E; Beckman Canada, Mississauga, Ontario, Canada). The pellet was considered the Triton X-100 insoluble fraction and the supernatant the Triton X-100 soluble fraction. The Triton X-100 insoluble fractions were dissolved in 1.5 volumes of radioimmunoprecipitation assay lysis buffer [150 mM NaCl, 1.0% NP-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate, and 50 mM Tris (pH 8.0)]. Proteins in the samples were measured by the Bradford dye binding assay with materials obtained from Bio-Rad (Mississauga, Ontario, Canada). The purity of the subcellular fraction has been shown elsewhere (34).

Electrophoresis and Western blot analyses
Cells were scraped and homogenized by sonication in PBS containing phosphatase and protease inhibitors. Cell lysates were centrifuged at low speed to remove unbroken cells and cell debris. The pellets were discarded, and the protein content in the supernatant was measured by the method of Bradford. Ten to 20 µg proteins were loaded onto 12% polyacrylamide gels. After electrophoresis, the proteins were transferred onto nitrocellulose membranes. The membranes were quickly stained with ponceau red to verify equal loading, rinsed with PBS and blocked with skim milk in PBS, and incubated with the first antibody at 37 C for 2 h. The membranes were incubated with the corresponding second antibody coupled to horseradish peroxidase at room temperature for 1 h. After incubation with the antibodies, membranes were stripped and reprobed with anti-nonmuscle actin. Antibody dilutions were: anti-Pan-Cx43, 1/20,000; anti-mPan-Cx43, 1:1000; anti-P(Ser368)-Cx43, 1/300; anti-NP(Ser368)-Cx43, 1/200; antiactin, 1/500; anti-PP2Ac, 1/1000; anti-PKC-{alpha}, 1/500; and anti-PKC-{zeta}, 1/ 2500.

Bands on the films were scanned, and the intensity of the bands was quantified using the Scion Image Program (Scion Corp., Frederick, MD). To compare results from different experiments, the intensity values were normalized by dividing them by the intensity of the actin band of the corresponding sample.

Co-IP and IP studies
The protocol used was an adaptation of the method of Carlile et al. (35). Forty to 50 µl Protein A-Sepharose 4B bead suspension was washed twice with 0.2 M Na-borate (pH 9.0) and mixed with 50 µg/ml of either Pan-Cx43, PKC-{alpha}, or phosphotyrosine antibodies. The mixture was incubated on a rotation wheel at 4 C overnight. The beads-antibody suspension was washed twice with 0.2 M Na-borate (pH 9.0). Control and TNF-{alpha}-treated TtT/GF cells were harvested and incubated while on ice with 1 ml cold IP buffer [150 mM NaCl, 20 mM Tris-HCl (pH 7.4), 1% NP-40, 100 µM PMSF, 25 µg/ml aprotinin, 25 µg/liter leupeptin, and 10 µM bpV (phen)] for 1 h with occasional vortexing. After centrifugation at 15,000 rpm for 30 min (Beckman Microfuge E) to remove nonsoluble material, the supernatants obtained were recovered, and the protein content was determined according to Bradford. After protein measurement, 10–20 µg cell lysate protein was removed from the supernatants to determine the level of expression of the protein under study (see Figs. 6–9GoGoGoGo, lane TtT/GF). Next, 1600 µg cell lysate proteins was incubated with the immunoprecipitating antibody-Protein A-Sepharose bead mixture overnight at 4 C with continual rotation. After this step, the beads were recovered by centrifugation and washed with the IP buffer 10 times. The pellet was extracted with 20 µl electrophoresis buffer and boiled for 3 min. Proteins were subjected to Western blot analysis with different antibodies. Controls for the co-IP studies included incubation of the cell lysates with Sepharose 4B beads preincubated with either medium alone (see Figs. 6–9GoGoGoGo, lane b), or rabbit or mouse IgG. Neither PP2Ac, PKC-{zeta}, Cx43, nor N(Ser368)-Cx43 bound to the beads or IgGs.


Figure 6
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  		FIG. 6. Studies on the interaction between Cx43 and PP2A. A and B, Co-IP studies. TtT/GF cell cultures were incubated with TNF-{alpha} for increasing periods of time. Next, the cells were harvested, lysed, and incubated with the Protein A-Sepharose 4B-Pan Cx43 mixture. The pellets (IP) were subjected to SDS-PAGE, followed by immunoblotting with PP2Ac antibodies. A, Representative Western blots showing the presence of PP2Ac in total TtT/GF cell lysates (TtT/GF lane). Protein A-Sepharose 4B beads not preincubated with the Pan-Cx43 antibody did not pull down PP2Ac (b lane). However, PP2Ac was recovered in the pellet from cell lysates incubated with Protein A-Sepharose 4B beads preincubated with the Pan-Cx43 polyclonal antibody (IP lanes). As loading control for the co-IP experiments, the membranes were stripped and reprobed with a monoclonal Pan-Cx43 antibody (mPan-Cx43, lanes b and IP). B, The bands were scanned, and the PP2Ac band intensity values were normalized to the corresponding mPan-Cx43 band intensity value. Values shown are the mean ± SEM of three independent experiments. C, Confocal microscopy studies. TtT/GF cell cultures were incubated with TNF-{alpha} for increasing periods of time. Next, the cells were processed for fluorescence microscopy with Pan-Cx43 and PP2Ac antibodies. The confocal microscopy images show the distribution of total Cx43 (green) and PP2Ac (red) in TtT/GF cells. Colocalization (yellow) was observed in all incubation times. D and E, Association of PP2Ac with the Triton X-100 insoluble and soluble subcellular fractions. TtT/GF cell cultures were incubated with TNF-{alpha} for increasing periods of time. Next, the cells were harvested, and the Triton X-100 insoluble and soluble fractions were prepared. Equal amounts of proteins from the subcellular fractions were subjected to SDS-PAGE, followed by immunoblotting with PP2Ac antibodies. Membranes were stripped and reprobed with antiactin. D, Representative Western blots. E, The bands were scanned, and PP2Ac immunoreactive band intensity values were normalized to the corresponding actin band intensity value. Values shown are the mean ± SEM of three independent experiments. *, P < 0.05 15 min vs. 30 min (Triton X-100 soluble fraction). **, P < 0.01 0 min vs. 15 min (Triton X-100 insoluble fraction).

 

Figure 7
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  		FIG. 7. Studies on the interaction between Cx43 and PKC-{alpha}. A and B, Co-IP studies. TtT/GF cell cultures were incubated with TNF-{alpha} for increasing periods of time. Next, the cells were harvested, lysed, and incubated with Protein A-Sepharose 4B beads preincubated or not with the Pan-Cx43antibody. The pellets were subjected to SDS-PAGE, followed by immunoblotting with PKC-{alpha} antibodies (A), and membranes were stripped and reprobed with anti-PKC-({alpha}ß{gamma}) (B). TtT/GF lane, Total cell lysates. B lane, Pellets from cell lysates incubated with Protein A-Sepharose 4B beads not preincubated with Pan-Cx43 antibody. IP lane, Pellets from cell lysates incubated with Protein A-Sepharose 4B beads preincubated with Pan-Cx43 antibody. C, Confocal microscopy studies of TtT/GF cells incubated for increasing periods of time with TNF-{alpha}, and next subjected to fluorescence microscopy with Pan-Cx43 antibody (green) and PKC-{alpha} antibody (red). Colocalization (yellow) was apparent in cells treated for 30 min with TNF-{alpha}. D and E, Association of PKC-{alpha} with the Triton X-100 insoluble and soluble subcellular fractions. TtT/GF cell cultures were incubated with TNF-{alpha} for increasing periods of time. Next, the cells were harvested, and the Triton X-100 insoluble and soluble fractions were prepared. Equal amounts of proteins in the samples were subjected to SDS-PAGE, followed by immunoblotting with PKC-{alpha} antibodies. Representative Western blots are shown in D. Membranes were stripped and reprobed with antiactin. The bands were scanned, and PKC-{alpha} immunoreactive band intensity values were normalized to the corresponding actin band intensity value. Values shown in E are the mean ± SEM of three independent experiments. *, P < 0.05 15 min vs. 30 min (Triton X-100 soluble fraction). **, P < 0.01 15 min vs. 30 min (Triton X-100 insoluble fraction).

 

Figure 8
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  		FIG. 8. Studies on the interaction between Cx43 and PKC-{zeta}. TtT/GF cell cultures were incubated with TNF-{alpha} for increasing periods of time (0–60 min). Next, the cells were harvested, lysed, and incubated with the Protein A-Sepharose 4B-Pan-Cx43 antibody mixture. The pellets were subjected to SDS-PAGE, followed by immunoblotting with PKC-{zeta} antibodies. The figure shows a representative Western blot from three independent experiments. TtT/GF lane, Total cell lysates. b lane, Cell lysates incubated with Protein A-Sepharose 4B beads not preincubated with the Pan-Cx43 antibody. IP lane, Immunoprecipitates from cell lysates incubated with Protein A-Sepharose 4B beads preincubated with the Pan-Cx43 antibody.

 

Figure 9
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  		FIG. 9. Tyrosine phosphorylation of Ser368-dephosphorylated Cx43. TtT/GF cell cultures were incubated with TNF-{alpha} for increasing periods of time. Next, the cells were harvested, lysed, and incubated with the Protein A-Sepharose 4B-anti-phosphotyrosine mixture. The pellets were subjected to SDS-PAGE, followed by immunoblotting with Pan-Cx43 and NP(Ser368)-Cx43 antibodies. The figure shows representative Western blots from three independent experiments. TtT/GF lane, Total cell lysates. b lane, Cell lysates incubated with Protein A-Sepharose 4B beads not preincubated with the phosphotyrosine antibody. IP lane, Immunoprecipitates from ell lysates incubated with Protein A-Sepharose 4B beads preincubated with the phosphotyrosine antibody.

 
Immunofluorescence microscopy
After treatments the TtT/GF cells were fixed with 3.7% formaldehyde for 20 min and permeabilized in acetone, as described previously (12). The cell preparations were blocked with milk and incubated with the first antibodies. Next, cells were incubated with the mixture of secondary antibodies coupled to different fluorescent dyes. The cells were viewed with a Carl Zeiss Axioskop microscope (Carl Zeiss, Inc., Thornwood, NY) and visualized using the Northern Eclipse program (Microsoft Corp., Redmond, WA). The quantitative analysis of P(Ser368)-Cx43 immunolabeling was performed on six microphotographs per experimental condition and obtained from three independent experiments, as previously described (12, 23). The immunopositive dots were counted without knowing from which experimental condition the photographs were taken. Colocalization studies were performed using a Leica TCS SP confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany).

Cell-to-cell communication
The scrape-loading method was used with some modifications to measure intercellular communication (23, 36). Briefly, TtT/GF cells were grown on glass coverslips till confluence and incubated with culture medium alone (control) or containing TNF-{alpha} for increasing periods of time. When inhibitors of protein kinases and phosphatases were used, the cells were preincubated for 30 min with the inhibitors and then challenged with TNF-{alpha} in the presence of the inhibitors. After treatments the cells were rinsed with PBS. Cells were bathed in a mixture of 1 ml Lucifer yellow solution (1 mg/ml in PBS) plus 0.5 ml rhodamine-dextran solution (50 mg/ml in PBS). Three cuts were applied on the cell monolayer with a needle, and the cell cultures were left in the dark for 3 min. The solution was removed, and the cells were incubated for an additional 10 min to permit diffusion of the dye. Next, the cells were rinsed three times with PBS and fixed with 3.7% formaldehyde. Images were acquired with a Carl Zeiss Axioskop microscope and visualized using the Northern Eclipse program.

Statistical analyses
Data were evaluated by the Student’s t test using the Sigma plot statistical analysis software (Systat Software, Inc., San Jose, CA). Quantitative data are expressed as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TNF-{alpha} transiently disrupts cell-to-cell coupling and decreases Cx43 phosphorylation in TtT/GF cells
Short-term treatment of TtT/GF cells with TNF-{alpha} decreased the diffusion of Lucifer yellow within the cell layer (Fig. 1AGo). Quantification of the effect showed a significant decrease in gap junction-mediated intercellular communication after 15-min incubation with TNF-{alpha}. However, this effect was transient, and cell coupling control levels were restored after 60 min in the presence of the cytokine (Fig. 1BGo).


Figure 1
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  		FIG. 1. Effect of TNF-{alpha} on cell-to-cell communication in TtT/GF cells. TtT/GF cells were incubated with culture medium alone (control) or containing 20 ng/ml TNF-{alpha} for 15, 30, and 60 min, and cell-to-cell communication was measured with the scrape and loading technique. A, Representative images of cell cultures incubated with the cell membrane impermeable dyes Lucifer yellow (green) and rhodamine-dextran (red). The diffusion of Lucifer yellow is an indication of the level of cell coupling. Rhodamine-dextran labeled cells are damaged cells that allowed the uptake of the dyes. Bar, 250 µm. B, The graph depicts cell-to-cell coupling within the TtT/GF cell cultures treated with medium alone (control) or TNF-{alpha} for increasing periods of time. The number of cell rows containing rhodamine phalloidin-labeled cells (damaged cells) was subtracted from the number of cell rows containing Lucifer yellow-labeled cells (damaged and communicating cells) to establish the cells coupled by gap junctions. Each value represents the mean ± SEM of at least 10 independent experiments. #, P < 0.001 TNF-{alpha} vs. control (15 min).

 
Because alterations in gap junction behavior are frequently associated with changes in Cx43 phosphorylation status, we tested whether changes in Cx43 phosphorylation levels contributed to TNF-{alpha}-induced cell coupling inhibition. Assessment of Cx43 phosphorylation was done using an antibody that detects Cx43 only when the protein is phosphorylated in Ser368 (P(Ser368)-Cx43). Incubation of TtT/GF cells with TNF-{alpha} for 15 min caused a significant decrease in P(Ser368)-Cx43 levels (Fig. 2Go, A and B). The effect of TNF-{alpha} on Cx43 phosphorylation at Ser368 was transient, and control P(Ser368)-Cx43 levels were recovered at 30 min and increased after 60 min in the presence of the cytokine [Fig. 2Go, A and B, P(Ser368)-Cx43]. Total Cx43 levels were not affected by the treatment (Fig. 2Go, A and B, Pan-Cx43), indicating that the reduction of P(Ser368)-Cx43 levels was due to the dephosphorylation of Cx43 in Ser368 and not to a decrease in total Cx43 protein concentration.


Figure 2
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  		FIG. 2. Effect of TNF-{alpha} on Cx43 levels and phosphorylation state. TtT/GF cells were incubated with 20 ng/ml TNF-{alpha} for increasing periods of time. A and B, Cells were scraped off and homogenized. Total cell proteins were subjected to SDS-PAGE, followed by immunoblotting with either the Pan-Cx43 antibody or P(Ser368)-Cx43 antibody. Next, the membranes were stripped and reprobed with antiactin as loading control. A, Representative Western blots. B, The bands were scanned, and immunoreactive band intensity values were normalized to the corresponding actin band intensity value. Values shown are the mean ± SEM of at least six independent experiments [P < 0.05 P(Ser368)-Cx43; 15 min TNF-{alpha} vs. 0 min]. C and D, After incubations with TNF-{alpha}, Triton X-100 insoluble and soluble fractions were prepared and subjected to electrophoresis and Western blots with P(Ser368)-Cx43 antibodies. Next, the membranes were stripped and reprobed with antiactin as loading control. C, Representative Western blots. D, The bands were scanned, and the P(Ser368)-Cx43 band intensity values were normalized to the corresponding actin band intensity value. Values shown are the mean ± SEM of at least three independent experiments. *, P < 0.05 15 min TNF-{alpha}; Triton X-100 insoluble and soluble fractions vs. the corresponding control. ##, P < 0.005 Triton X-100 insoluble fractions 30 vs. 15 min. E, Fluorescence microscopy studies. After treatment of TtT/GF cells with TNF-{alpha}, the cells were processed for fluorescence microscopy with P(Ser368)-Cx43 antibodies. In control cells (0 min), P(Ser368)-Cx43 labeling is associated with the cell membrane and the cytoplasm. Treatment of the cells with TNF-{alpha} for 15 min decreased P(Ser368)-Cx43 labeling, particularly at the cell membrane and, to a lesser extent, in the cytoplasm (15 min). Longer incubation periods with TNF-{alpha} showed increased P(Ser368)-Cx43 labeling at the cell membrane (30 min). Bar, 50 µm.

 
Because incorporation of Cx43 gap junctions into membrane plaques renders the protein insoluble in 1% Triton X-100 (37), we investigated whether the TNF-{alpha}-induced changes in Cx43 phosphorylation in Ser368 preferentially occurred in Cx43 molecules that had or had not been incorporated into plaques. Triton X-100 insoluble and soluble fractions were prepared from cells treated for increasing periods of time with TNF-{alpha}. The decrease in P(Ser368)-Cx43 levels after a 15-min incubation period with the cytokine occurred in both Triton X-100 insoluble and soluble fractions (Fig. 2Go, C and D). However, the increase of P(Ser368)-Cx43 levels after 30- and 60-min incubation periods with TNF-{alpha} was only recorded in the Triton X-100 insoluble fraction.

Fluorescence microscopy studies showed that P(Ser368)-Cx43 labeling was associated with the plasma membrane and the cytoplasm in nontreated cells (Fig. 2EGo, 0 min). After a 15-min treatment with TNF-{alpha}, P(Ser368)-Cx43 labeling decreased principally from the cell periphery and to a lesser extent from the cytoplasm (Fig. 2EGo, 15 min). Quantification of the immunopositive spots showed a significant reduction in total P(Ser368)-Cx43 labeling in 15-min treated cells when compared with control cells: fluorescent spots/10,000 µm2, control cells 39.1 ± 4.2 (n = 6); 15-min TNF-{alpha}-treated cells 28.4 ± 2.1 (n = 6); P < 0.05 vs. control cells; 30-min TNF-{alpha}-treated cells 30.5 ± 5.6 (n = 6). Reoccurrence of P(Ser368)-Cx43 labeling was predominantly observed at the cell membrane (Fig. 2EGo, 30 min).

Cx43 dephosphorylation affected cell-to-cell communication in TtT/GF cells
TNF-{alpha} treatment caused a rapid and concomitant reduction of cell-to-cell communication as well as of P(Ser368)-Cx43 levels, suggesting a link between the TNF-{alpha} effect on cell coupling and phosphorylation status of Cx43. This was evaluated by assessing the effect of protein phosphatase and protein kinase inhibitors on TNF-{alpha}-induced decrease in cell-to-cell communication. Pretreatment of the cells with two different concentrations of the phosphatase inhibitor OA, 10 nM preferentially inhibiting PP2A, and 200 nM inhibiting both PP1 and PP2A, significantly blocked the TNF-{alpha}-induced decrease in cell-to-cell communication (Fig. 3Go, upper panel). Pretreatment with the PKC inhibitor Gö6983 decreased TtT/GF cell coupling and blocked a further decrease by TNF-{alpha} (Fig. 3Go, lower panel).


Figure 3
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  		FIG. 3. Effect of protein phosphatase and PKC inhibition on TNF-{alpha}-induced inhibition of cell-to-cell communication in TtT/GF cells. TtT/GF cells were preincubated for 30 min with medium alone or containing either the protein phosphatase inhibitor OA (10 or 200 nM; upper panel) or PKC inhibitor Gö6983 (Gö) (25 nM; lower panel). Next, TNF-{alpha} (20 ng/ml final concentration) was added to the medium, and the cells were further incubated for 15 min. Control cells (C) were incubated neither with phosphatase nor PKC inhibitors, nor with TNF-{alpha}. Cell-to-cell communication was measured with the scrape and loading technique. The number of communicating cells was calculated by subtracting the number of cell rows containing rhodamine phalloidin-labeled cells (damaged cells) to the cell rows containing Lucifer yellow-labeled cells (damaged plus communicating cells) in each experimental condition. Each value represents the mean ± SEM of at least seven independent experiments. Upper panel, #, P < 0.001 phosphatase inhibitor-treated cells vs. TNF-{alpha}; lower panel, #, P < 0.001 treated cells vs. control cells.

 
Participation of Ser/Thr protein phosphatases and protein kinases in TNF-{alpha}-induced changes in Cx43 phosphorylation
To determine the intercellular pathways involved in the TNF-{alpha}-induced decrease in P(Ser368)-Cx43, the involvement of protein phosphatases was assessed. The treatment of TtT/GF cells with Cal A, an inhibitor of protein phosphatases PP1and PP2A, and with different concentrations of OA, increased the basal P(Ser368)-Cx43 levels (data not shown) and completely prevented TNF-{alpha}-induced dephosphorylation of Cx43 at Ser368 (Fig. 4Go). Low concentrations of OA still blocked the effect of TNF-{alpha}, indicating the involvement of at least PP2A in the TNF-{alpha}-induced decrease of P(Ser368)-Cx43 levels.


Figure 4
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  		FIG. 4. Participation of protein phosphatases in the TNF-{alpha}-induced dephosphorylation of Cx43 in Se368. TtT/GF cells were preincubated for 30 min with medium alone or containing the protein phosphatase inhibitors: Cal A 10 nM; or OA 10, 100, and 200 nM. Next, TNF-{alpha} was added to the medium, and the cells were further incubated for 15 min. Control cells (C) were incubated neither with phosphatase inhibitors nor with TNF-{alpha}. After treatments the cells were scraped off and homogenized. Total cell proteins were subjected to SDS-PAGE, followed by immunoblotting with the P(Ser368)-Cx43 antibody. The figure shows a representative Western blot. The membranes were stripped and reprobed with antiactin as loading control. The bands were scanned, and P(Ser368)-Cx43 band intensity values were normalized to the corresponding actin band intensity value. Values shown are the mean ± SEM of more than six independent experiments. TNF-{alpha}-treated cells + phosphatase inhibitors vs. TNF-{alpha}-treated cells: *, P < 0.05, **, P < 0.01; {ddagger}, P < 0.001.

 
Next, we investigated whether the inhibition of protein kinases was involved in TNF-{alpha}-induced reduction of P(Ser368)-Cx43 levels. The inhibition of PKC with the specific inhibitor Gö6983 lowered P(Ser368)-Cx43 basal levels (Fig. 5Go and Table 1Go, PKC inhibitor). This observation is consistent with the fact that Cx43 phosphorylation in Ser368 is dependent on the activity of PKC (30, 31). Moreover, in the presence of the PKC inhibitor, TNF-{alpha} failed to reduce further P(Ser368)-Cx43 levels (Fig. 5Go and Table 1Go, PKC inhibitor). Inhibition of PKC also blocked the recovery of control P(Ser368)-Cx43 levels after 30- to 60-min incubation in the presence of TNF-{alpha}. Inhibition of PKA or MEK had no impact on basal P(Ser368)-Cx43 levels (Table 1Go), however, inhibition of PKA, but not of MEK, partially blocked TNF-{alpha}-induced decrease in P(Ser368)-Cx43 (Table 1Go, PKA inhibitor). Inhibition of PKA and MEK slowed down the rephosphorylation of Cx43 in Ser368 observed after a 30- to 60-min incubation period with TNF-{alpha} in the control (Fig. 5Go and Table 1Go).


Figure 5
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  		FIG. 5. Involvement of protein kinases in TNF-{alpha}-induced changes in P(Ser368)-Cx43 levels. TtT/GF cells were preincubated for 30 min with medium alone or containing the protein kinase inhibitors: PKC inhibitor Gö6983, 25 nM; PKA inhibitor KT5720, 100 nM; and MEK inhibitor U0126, 20 µM. Next, TNF-{alpha} (20 ng/ml final concentration) was added to the medium, and the cell cultures were further incubated for 0–60 min. After treatments the cells were scraped off and homogenized. Total cell proteins were subjected to SDS-PAGE, followed by immunoblotting with the P(Ser368)-Cx43 antibody. The figure shows representative Western blots. P(Ser368)-Cx43 levels were transiently diminished by TNF-{alpha}. Preincubation with the PKC inhibitor reduced basal (0 min) and TNF-{alpha} induced changes. Blockade of PKA partially blocked TNF-{alpha}-induced decrease of P(Ser368)-Cx43 at 15-min incubation. Preincubation with the MEK inhibitor did not block TNF-{alpha} effects but slowed down the rephosphorylation of Cx43.

 

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  		TABLE 1. Involvement of protein kinases in TNF-{alpha}-induced changes in P(Ser368)-Cx43 levels

 
Studies on the Cx43-PP2A interaction
The present results provide evidence that PP2A and PKC contribute to TNF-{alpha}-induced changes in P(Ser368)-Cx43 levels. Enzyme-substrate interaction is an important modulatory step for the activity of these enzymes, therefore, we tested whether TNF-{alpha} affected the interaction between Cx43 and PP2A and/or PKC. Because Cx43 directly interacts with PP2A (29) and because PP2A is part of the TNF-{alpha}-activated signaling pathway (38), we assessed whether TNF-{alpha} modified Cx43- PP2A interactions.

Untreated TtT/GF cells express PP2Ac (Fig. 6AGo, TtT/GF lane). Co-IP studies performed with the Pan-Cx43 antibody revealed that PP2Ac was pulled down by this antibody (Fig. 6AGo, IP lanes). Yet, TNF-{alpha} treatment did not influence the extent of the association of the two proteins (Fig. 6BGo). Confocal microscopy performed with the same antibodies show that PP2Ac was mainly cytoplasmic and that some staining was associated with the periphery of TtT/GF cells (Fig. 6CGo, 0 min). There was a partial colocalization of Cx43 and PP2A in the cytoplasm and cell membrane. Because the co-IP studies were performed on whole cell lysates, they do not provide information on whether TNF-{alpha} affected the interaction of PP2Ac with a particular type of Cx43 molecule, i.e. Cx43 molecules assembled in gap junctions. Triton X-100 insoluble and soluble fractions were then generated for sorting Cx43 molecules. TNF-{alpha} significantly increased the association of PP2Ac with the Triton X-100 insoluble fraction by 15-min incubation and with the Triton X-100 soluble fraction by 30-min incubation (Fig. 6Go, D and E).

Studies on the interaction between Cx43 and the PKC isoforms {alpha} and {zeta}
Data obtained in the present study suggest the involvement of PKC in the modulation of P(Ser368)-Cx43 levels by TNF-{alpha}. The PKC inhibitor we used here blocks the activity of the conventional PKC isoforms {alpha}, ßI-II, {gamma}, of the novel PKC isoform {delta}, and of the atypical isoform {zeta}. Because, within the PKC family, the {alpha} and {zeta}-isoforms have been implicated in mediating TNF-{alpha} effects (39, 40, 41), the interaction between Cx43 and these PKC isoforms and the modifications by TNF-{alpha} of these interactions were evaluated.

PKC-{alpha} was expressed in TtT/GF cells (Fig. 7AGo, TtT/GF lane). The Pan-Cx43 antibody did not coimmunoprecipitate PKC-{alpha} in either the control or the TNF-{alpha}-treated cells at all incubation times examined. This was confirmed by using another PKC antibody that detects the conventional PKC isoforms ({alpha}, ßI-II, and {gamma}) (Fig. 7BGo). However, confocal microscopy performed with Pan-Cx43 and PKC-{alpha} antibodies showed some degree of proximity between the two proteins in cells incubated with TNF-{alpha} for periods longer than 30 min that temporally coincided with the recovery of P(Ser368)-Cx43 levels (Fig. 7CGo). This result was expanded by measuring the association of PKC-{alpha} with the Triton X-100 insoluble and soluble fractions. The PKC-{alpha} association with the Triton X-100 insoluble fraction significantly increased by 30-min incubation with TNF-{alpha} (Fig. 7Go, D and E).

Because PKC-{alpha} has been associated with PP2Ac (40), whether this interaction took place in TtT/GF cells or was affected by TNF-{alpha} was investigated. Co-IP studies with PKC-{alpha} antibody showed no interaction between PKC-{alpha} and PP2Ac, either in control or TNF-{alpha}-treated cells (data not shown). Collectively, the results indicate that neither PP2Ac nor Cx43 interacts with PKC-{alpha} in TtT/GF cells or that such interactions would be weak.

The atypical PKC-{zeta} isoform is not only part of the signaling pathway activated by TNF-{alpha}, but, in addition, it has interacted with cell junction components (42). Thus, the possibility of a Cx43-PKC-{zeta} interaction and a modulation of this interaction by TNF-{alpha} was assessed by co-IP analyses. TtT/GF cells express this atypical PKC isoform (Fig. 8Go, TtT/GF lane). Moreover, PKC-{zeta} was coimmunoprecipitated by the Pan Cx43 antibody under basal conditions (Fig. 8Go, IP lane, 0 min). TNF-{alpha} treatment decreased Cx43-PKC-{zeta} interaction. A partial recovery started after 60-min incubation with TNF-{alpha} (Fig. 8Go, IP lanes).

Tyrosine phosphorylation of NP(Ser368)-Cx43 during TNF-{alpha} treatment of TtT/GF cells
Ser/Thr dephosphorylation of Cx43 was accompanied by its tyrosine phosphorylation (43). Because our results show that TNF-{alpha} induced the dephosphorylation of Cx43 in Ser368, whether this particular dephosphorylation was followed by the tyrosine phosphorylation of Cx43 was investigated by immunoprecipitating tyrosine phosphorylated proteins with an antiphosphotyrosine antibody. Next, Western blots of the immunoprecipitated proteins were performed with the Pan-Cx43 antibody and with an antibody that recognizes NP(Ser368)-Cx43. In control cells the Pan-Cx43 antibody recognized Cx43 among the immunoprecipitated proteins, which indicates a low, yet measurable level of tyrosine phosphorylation of the protein [Fig. 9Go, Western blot (WB), Pan-Cx43; IP lane, 0 min). After TNF-{alpha} treatment there was a discrete and transient increase in the intensity of the Cx43 immunoreactive band from 15- to 30-min incubation intervals (Fig. 9Go, WB, Pan-Cx43). However, only the faster migrating Cx43 immunoreactive band immunoprecipitated with the phosphotyrosine antibody [Fig. 9Go, WB, Pan-Cx43, compare TtT/GF lane (total cell lysate) with IP lanes], suggesting that some "non-phosphorylated" Cx43 molecules in that band were in fact "phosphorylated" in tyrosine. This finding was supported by Western blots of the phosphotyrosine immunoprecipitated proteins performed with the NP(Ser368)-Cx43 antibody. Analysis of the blots revealed that very little amounts of NP(Ser368)-Cx43 were coimmunoprecipitated with antiphosphotyrosine when compared with total cell NP(Ser368)-Cx43 [Fig. 9Go, WB, NP(Ser368)-Cx43, TtT/GF lane vs. IP lane, 0 min]. This observation indicates that most NP-Cx43 was not phosphorylated in tyrosine under basal conditions. However, after incubation with TNF-{alpha}, a sharp and sustained increase in the NP(Ser368)-Cx43 immunoreactive band intensity was apparent [Fig. 9Go, WB, NP(Ser368)-Cx43, IP lanes]. This strongly suggests that the TNF-{alpha}-induced dephosphorylation of Cx43 in Ser368 was followed by the tyrosine phosphorylation of the protein.

Effect of Cx43 phosphorylation status on cell-to-cell communication: a time course study
The effect of Cx43 phosphorylation status on gap junction physiology is controversial. Likely, due to the fact that many residues in the Cx43 molecule are phosphorylated by different kinases and that Cx43 phosphorylation is associated with different phases of the life of the Cx43 molecule, both parameters must be considered when studying the effect of any compound on Cx43 phosphorylation status and gap junction function. To address the relationship between Cx43 phosphorylation in Ser368 (a PKC target) (30, 31) and cell-to cell communication, a time course study on the effect of PMA, a stimulator of the conventional and novel PKC isoforms, and of the PKC inhibitor, Gö6983, on the phosphorylation status of Cx43 in Ser368, as well as on cell-to-cell communication was performed. The stimulation of PKC with PMA was followed by a rapid (within 5 min) and sustained (up to 60 min) increase in phosphorylation of Cx43 in Ser368 (Fig. 10AGo, PMA), whereas inhibition of PKC with Gö6983 caused a rapid and sustained decrease in P(Ser368)-Cx43 levels (Fig. 10AGo, Gö6983). PKC stimulation had a biphasic effect on cell-to-cell communication under the same experimental conditions. First, there was a fast (within 5 min) and very short-lived (less than 5 min) increase in intercellular communication, followed by a rapid decrease in cell coupling (Fig. 10BGo, PMA). This observation shows that the same stimulus had a completely different effect on cell-to-cell coupling, depending on the time frame when the measurements were performed. On the other hand, inhibition of PKC caused a sharp and sustained decrease in cell-to-cell communication at all time points studied (Fig. 10BGo, Gö6983).


Figure 10
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  		FIG. 10. Impact of the Ser368 phosphorylation of Cx43 in cell-to-cell communication in TtT/GF cells. A time course study. TtT/GF cells were incubated with medium alone (control) or containing either the PKC stimulator PMA (10 ng/ml) or the PKC inhibitor Gö6983 (25 nM) for increasing periods of time (0–60 min). After treatment the cells were processed as follows. A, Cells were scraped off, homogenized, and total proteins were subjected to SPS-PAGE, followed by immunoblotting with P(Ser368)-Cx43 antibody. The figure shows representative Western blots. Next, the membranes were stripped and reprobed with actin antibodies as loading control. The bands were scanned, and the intensities of the P(Ser368)-Cx43 immunoreactive bands were normalized to the corresponding actin band intensity value. Data shown are the mean ± SEM of five independent experiments. *, P < 0.05 5 min PMA- and Gö6983-treated cells vs. control cells. B, After treatment, cell-to-cell communication was measured with the scrape and loading technique. The figure shows representative micrographs of Lucifer yellow diffusion in control (0 min) and treated cells. Quantification of the effects of the PKC stimulator and inhibitor on cell-cell coupling was performed by counting the cell rows containing Lucifer yellow-labeled cells and subtracting from these values the number of cell rows containing rhodamine phalloidin-labeled cells in each experimental condition. Each value represents the mean ± SEM of at least five independent experiments. {dagger}, P < 0.0001 5-min Gö6983 vs. control; {ddagger}, P < 0.0005 5-min PMA vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that short-term treatment of the FS cell line TtT/GF with the proinflammatory cytokine TNF-{alpha} induced a rapid and transient disruption of cell coupling. TNF-{alpha}-induced cell uncoupling was accompanied by the dephosphorylation of Cx43 in Ser368. Our results suggest that the dephosphorylation of Cx43 in Ser368 is one mechanism for TtT/GF cell uncoupling. Changes in PP2Ac-Cx43 and PKC-{zeta}-Cx43 interactions may contribute to TNF-{alpha}-induced Cx43 dephosphorylation in Ser368. Moreover, the serine dephosphorylated Cx43 was rapidly tyrosine phosphorylated. In addition, we showed that TtT/GF cell coupling recovery was mediated by PKC-catalyzed rephosphorylation of the Ser368 residue of Cx43.

Here, the phosphorylation status of Cx43 was assessed using an antibody that recognizes the protein only when it is phosphorylated in Ser368. Phosphorylation of Ser368 is catalyzed by PKC, which only phosphorylates Ser368, Ser372, and Ser262 in Cx43 (44). Phosphorylation of the Ser368 residue has a significant impact on the turnover and functioning of Cx43-mediated gap junctions (30, 45, 46, 47). The use of this antibody allows to evaluate the contribution of the phosphorylation status of this particular residue on the TNF-{alpha}-mediated effects. In addition, it represents an advantage over the survey of the appearance/disappearance of slower migrating Cx43 immunoreactive bands recorded in Western blots because phosphorylated Cx43 molecules do not necessarily exhibit an electrophoretic mobility shift (32). Nonetheless, this experimental approach does inform on the participation of other phosphorylation sites on the Cx43 molecule that have been also shown to influence Cx43-mediated gap junctions (26, 27, 48).

TNF-{alpha} treatment of TtT/GF cells transiently decreased P(Ser368)-Cx43 levels. Because the total Cx43 was not affected by the treatment, lower P(Ser368)-Cx43 levels mean the dephosphorylation of the protein. This dephosphorylation is required for TNF-{alpha}-induced cell uncoupling. First, P(Ser368)-Cx43 dephosphorylation temporally coincided with a decrease in cell-to-cell communication. Second, reduced P(Ser368)-Cx43 occurred mainly, although not exclusively, at the plasma membrane, indicating that Cx43 dephosphorylation primarily affected active Cx43-mediated gap junctions. Third, inhibition of protein phosphatases and PKC blocked TNF-{alpha}-induced cell uncoupling.

TNF-{alpha}-induced P(Ser368)-Cx43 dephosphorylation was dependent on the activity of both protein phosphatases and protein kinases. Two inhibitors of protein phosphatases PP1 and PP2A, Cal A and OA (49, 50), prevented TNF-{alpha}-induced Cx43 dephosphorylation. The observation that low OA concentrations were as effective as higher concentrations of the inhibitor reveals that the TNF-{alpha}-induced dephosphorylation involved PP2A activity. Phosphatase catalyzed-Cx43 dephosphorylation in response to stress stimuli such as ischemia or hypoxia has been reported (51, 52, 53). Moreover, PP2A activation has been related to a signaling pathway activated by TNF-{alpha} (49, 54, 55, 56). On the other hand, inhibition of PKC decreased basal P(Ser368)-Cx43 levels and prevented further dephosphorylation of Cx43 by TNF-{alpha}, showing that a lower PKC activity toward Cx43 also contributed to TNF-{alpha}-evoked Ser368 dephosphorylation. Our results also revealed a certain degree of cross talk with other kinases, which indicates that the phosphorylation status of the Ser368 residue may be affected by the phosphorylation of other residues catalyzed by kinases other than PKC.

Protein-protein interaction is central to the activity of protein kinases and phosphatases because these enzymes localize close to their substrates by anchoring or scaffolding proteins to ensure a strict control of the phosphorylation events in either restricted subcellular compartments or a subset of phosphoprotein molecules. It is also crucial in the modulation of Cx43-mediated gap junction function. Cx43 physically interacts with several kinases (43, 44, 57, 58, 59) and phosphatases (29, 52). Here, co-IP and confocal microscopy studies show that total cell Cx43 and PP2Ac physically interact but that TNF-{alpha} did not modify the extent of the interaction in TtT/GF cells. Nevertheless, the increased PP2Ac association with the Triton X-100 insoluble fraction during Cx43 dephosphorylation suggests PP2Ac recruitment to dephosphorylate Cx43 molecules already assembled into gap junction plaques. The observation that the disappearance of P(Ser368)-Cx43 labeling chiefly occurred at the plasma membrane provides support to this notion.

TNF-{alpha}-induced Cx43 dephosphorylation in TtT/GF cells also involved PKC, the enzyme responsible for the phosphorylation of the Ser368 residue (30, 31). Based on the reports that among the different PKC isoforms, the {alpha} (39) and the {zeta} (41) isoforms are affected by TNF-{alpha}, the potential link between these two PKC isoforms and the Cx43 phosphorylation status in TNF-{alpha}-treated TtT/GF cells was explored. Cx43 and PKC-{alpha} did not coimmunoprecipitate either under basal or stimulated conditions, indicating that in TtT/GF cells, Cx43-PKC-{alpha} interaction does not exist, or is either weak or indirect. For instance, PKC-{alpha} may interact with a protein that itself interacts with Cx43. This possibility is interesting because PKC-{alpha} may interact with PP2A (40) and been dephosphorylated, and thereby inactivated by this phosphatase (60, 61, 62). Furthermore, TNF-{alpha} induced PKC-{alpha} inactivation implicates the activation of a protein phosphatase (39). However, no interaction between PKC-{alpha} and PP2Ac, either in control or TNF-{alpha}-treated cells, was detected in our cell system. Moreover, PP2Ac and PKC-{alpha} translocation from the Triton X-100 soluble to the Triton X-100 insoluble subcellular fractions did not temporally coincide. Collectively, the results suggest that PP2Ac did not interact with PKC-{alpha} during TNF-{alpha} treatment and that PKC-{alpha} was not involved in TNF-{alpha}-induced reduction of P(Ser368)-Cx43. Contrarily to PKC-{alpha}, the atypical isoform {zeta} did interact with Cx43 under basal conditions, which explains why inhibition of PKC activity reduced the basal P(Ser368)-Cx43 levels. Moreover, after treatment with TNF-{alpha}, PKC-{zeta} and Cx43 interaction decreased, showing that TNF-{alpha} maintained the enzyme apart from Cx43 to prevent the phosphorylation of Ser368.

Ischemia/hypoxia-induced serine/threonine dephosphorylation of Cx43 is followed by the tyrosine phosphorylation of the protein (43). Our present results agree with that report and extend those findings by showing that the NP(Ser368)-Cx43 molecule was phosphorylated at tyrosine residues after TNF-{alpha} treatment. Tyrosine phosphorylation of NP(Ser368)-Cx43 may contribute to TtT/GF cell uncoupling, as it has been reported in other cell systems (28, 63). However, contrarily to TNF-{alpha}-induced P(Ser368)-Cx43 dephosphorylation, tyrosine phosphorylation of Cx43 was sustained, not transient. Moreover, the TNF-{alpha}-induced changes in the phosphorylation state of Ser368 closely paralleled the ones recorded in cell coupling, whereas the changes in tyrosine phosphorylation of Cx43 did not. This experimental evidence strongly suggests that the main signal to reduce cell coupling in TNF-{alpha}-treated TtT/GF cells was the dephosphorylation of Ser368. In addition, these results indicate that P(Ser368)-Cx43 is not a good substrate for tyrosine kinases and that it must, therefore, be dephosphorylated for a subsequent tyrosine phosphorylation. On the contrary, tyrosine phosphorylation of NP(Ser368)-Cx43 may not impair the rephosphorylation of the Ser368 residue. The rapid increase in P(Ser368)-Cx43 after 30- to 60-min incubation with TNF-{alpha} supports this idea.

Rephosphorylation of Cx43 in Ser368 involves PKC activity because it is blocked by a PKC inhibitor. However, PKC-{zeta} does not seem to be the isoform implicated here because its interaction with Cx43 decreased with time in TNF-{alpha}-treated cells. On the other hand, the increased PKC-{alpha} recruitment to the Triton X-100 insoluble fraction during Ser368 rephosphorylation suggests first that PKC-{alpha} is the isoform involved in the phosphorylation of the Ser368 residue in Cx43 and, second, that Cx43 molecules assembled in gap junctional plaques were the preferred target of PKC-{alpha}. In agreement with this suggestion, the confocal microscopy studies also showed an increased proximity of PKC-{alpha} and Cx43 after a 30-min exposure to TNF-{alpha}.

The influence of the Cx43 phosphorylation status on Cx43-mediated gap junction intercellular communication has been extensively studied. Cx43 phosphorylation occurs at different moments of the life of a gap junction, and influences channel assembly, turnover, and function (25, 29). More importantly, depending on the kinase, thus on the phosphorylated residue(s) involved, the impact on channel behavior greatly differs (28, 44). Therefore, establishing a correlation between "Cx43 phosphorylation" and cell coupling requires great caution. The phosphorylation of the specific residue Ser368, catalyzed by PKC, results in the inhibition of intercellular communication (for a review, see Refs. 25 and 44). Dephosphorylation of Cx43 has generally been associated with decreased cell-to-cell coupling (43, 52, 53), although the process may be a consequence of cell uncoupling (51). There is no indication on whether Ser368 dephosphorylation has any particular effect on cell coupling. We show that a decrease in P(Ser368)-Cx43 levels correlated with a decreased TtT/GF cell coupling. Contrarily, an increase in P(Ser368)-Cx43 levels was accompanied by a short-lived increase in cell coupling itself, followed by a sharp decrease in cell-to-cell communication. These results stress the fact that the length of the treatment must be considered in the interpretation of the response of intercellular communication to changes in the phosphorylation levels of Cx43.

The effect of cytokines on intercellular communication is unpredictable. Depending on the cell type, experimental cell environment, and exposure time, these physiological compounds either stimulate, inhibit, or cause no effect on cell-to-cell coupling (18, 19, 64, 65). FS cell Cx43-mediated gap junctions are sensitive to growth factors, cytokines, and other extracellular signaling molecules (12, 23, 66, 67). We show that a short-term incubation with the proinflammatory cytokine TNF-{alpha} transiently decreased cell-cell communication in the FS cell line TtT/GF. FS cells participate in the paracrine regulation of the anterior pituitary hormone secretion (10), and they influence and are influenced by endocrine cells of the anterior pituitary gland (66, 67). In the anterior pituitary, FS cells constitute an organized cellular network (6, 7) in which individual cells communicate via gap junctions with other FS cells (12, 68) and with anterior pituitary endocrine cells (69, 70). Our studies clearly show that TNF-{alpha} has a biphasic effect on Cx43-mediated TtT/GF cell coupling: an acute inhibition (this work) followed by an increase (23). These opposite effects on TNF-{alpha} are likely the consequence of the role of the intrapituitary FS cell network in the cellular response to an inflammatory signal. First, there is a temporary closure of gap junctions as an initial protective measure of FS cells reacting to the inflammatory signal, to stop or confine the propagation of the signal. Then, with the persistence of the signal, the increased cell coupling that follows may reflect the coordinated synchronization of the FS cells’ responses within the network in reaction to the deleterious actions of TNF-{alpha}.


   Footnotes
 
This work was funded by the Natural Sciences and Engineering Research Council of Canada (to M.L.V.). M.L.V. is supported by a scholarship from Fonds de la recherche en santé du Québec.

Disclosure Summary: The authors have nothing to disclose.

First Published Online September 13, 2007

Abbreviations: Cal A, Calyculin A; Cx, connexin; FS, folliculostellate; IP, immunoprecipitation; MEK, MAPK kinase; NP(Ser368)-Cx43, Cx43 not phosphorylated in serine 368; OA, okadaic acid; P(Ser368)-Cx43, Cx43 phosphorylated in serine 368; PKC, protein kinase C; PMA, phorbol myristate acetate; PMSF, phenylmethylsulfonyl fluoride; PP2A, protein phosphatase 2A; PP2Ac, protein phosphatase catalytic subunit; Ser368, serine 368; WB, Western blot.

Received June 8, 2007.

Accepted for publication September 6, 2007.


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 Introduction
 Materials and Methods
 Results
 Discussion
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