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BOR - Papers in Press, published online ahead of print August 31, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.044313
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BIOLOGY OF REPRODUCTION 74, 2–12 (2006)
DOI: 10.1095/biolreprod.105.044313
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Modulation of GJA1 Turnover and Intercellular Communication by Proinflammatory Cytokines in the Anterior Pituitary Folliculostellate Cell Line TtT/GF1

Marie-Ève Fortin, R.-Marc Pelletier, Mélissa-Anne Meilleur, and Maria Leiza Vitale 2

Department de Pathology and Cell Biology, Faculty of Medicine, Université de Montréal, Montréal, Québec, Canada H3T 1J4

ABSTRACT

Our previous studies have advanced the idea that the folliculostellate cell GJA1 (gap junction membrane channel protein alpha1; previously known as connexin 43)-mediated gap junctions contribute to the establishment of an intercellular network that regulates the paracrine messages and the endocrine response within the anterior pituitary. The folliculostellate cells are targets for growth factors and cytokines that modulate hormone secretion. Proinflammatory cytokines modulate the cell-to-cell communication in many tissues of the body. The present study measured the effect of the proinflammatory cytokines tumor necrosis factor and interleukin-1 on the GJA1-mediated intercellular communication, specifically the expression, localization, degradation, and phosphorylation status of GJA1 in the folliculostellate cell line TtT/GF. The GJA1 localized to the plasma membrane and to minute cytoplasmic vesicles in the perinuclear area. Using different antibodies that recognize distinctly the nonphosphorylated from the phosphorylated forms of GJA1, we showed that nonphosphorylated GJA1 in Ser-368 (NP-GJA1) localized chiefly in the cytoplasm, whereas GJA1 phosphorylated in Ser-368 (P-GJA1) localized to the plasma membrane in controls. The cytokine treatment transiently increased 1) GJA1, NP-GJA1, and P-GJA1 levels; 2) NP-GJA1 and P-GJA1 degradation by both the lysosomal and proteasomal pathways; and 3) cell-to-cell communication in TtT/GF cells. The results suggest that the cytokine-evoked, transient enhancement of folliculostellate cell-mediated intercellular communication contributes to the coordination of the response among folliculostellate cells.

anterior pituitary, cytokines, pituitary

INTRODUCTION

The anterior pituitary folliculostellate (FS) cells are agranular, nonendocrine, fibroblast-like cells with many lysosomes. First believed to be POMC1 (previously known as ACTH)-secreting cells [1], the FS cells are now considered to be a distinctive cellular group with a typical physiological profile [2]. The cells are targets of cytokines and hormones, such as interleukin (IL)-1, tumour necrosis factor (TNF) [3–6], glucocorticoids [7, 8], angiotensin II [9], and estrogen [10, 11]. In response to these systemic factors, FS cells synthesize and secrete cytokines and growth factors, such as follistatin, IL-6, annexin A1, transforming growth factor b3, and fibroblast growth factor 2 [12–15], that affect their own growth, that of the anterior pituitary endocrine cells, and the secretion of the anterior pituitary hormones, follicle-stimulating hormone, prolactin, and PMOC1 [4, 6, 16–19]. Because FS cells are both the target and the source of cytokines, they are ideal candidates for the regulation of the function of the anterior pituitary.

The FS cells are arranged in clusters with long cytoplasmic processes in contact with FS cells and endocrine cells [20–22]. By establishing gap junction-mediated cell-to-cell communication with neighboring FS cells [21, 23, 24] and endocrine cells [25], the FS cells form a cellular network within the anterior pituitary [20, 26, 27]. The gap junctions are intercellular channels formed by tansmembrane proteins. We and others have shown that FS cells express the gap junction membrane channel protein alpha1 (GJA1), formerly known as connexin 43, both in vivo and in vitro [23, 28, 29] and that the expression and localization of the protein in the anterior pituitary change with the endocrine status [24, 28].

Because FS cells establish GJA1-mediated gap junctions and possess the capacity to respond to modulators of anterior pituitary hormone secretion, we assessed the influence of cytokines that modify the anterior pituitary endocrine response on the expression and metabolism of the GJA1 protein in the FS cells. The GJA1 levels and GJA1-mediated gap junctions have been shown to be affected by proinflammatory cytokines in different tissues [30–35]. We used the TtT/GF cells as the experimental cell model. The TtT/GF cells belong to an FS cell line derived from a mouse pituitary tumor [36]; they show the morphological and biochemical features of normal FS cells [36, 37].

In the present study, we assessed the effect of the proinflammatory TNF and IL-1 on the expression, localization, and phosphorylation status of GJA1 by immunofluorescence microscopy and immunoblotting in the TtT/GF cells. In addition, the impact of the cytokines on cell-to-cell communication was evaluated. We show that TNF and IL-1 modulate the GJA1 turnover as well as the cell-to-cell communication in TtT/GF cells.

MATERIALS AND METHODS

Cell Culture

The TtT/GF cells were kindly provided by Dr. U. Renner (Department of Endocrinology, Max Planck Institute of Psychiatry, Munich, Germany). The cells were grown in Dulbecco modified Eagle medium (DMEM) supplemented with 5% fetal calf serum, 3.7 g/ml of NaHCO3, 10 mM Hepes (pH 7.2), and antibiotics (penicillin 0.2 mg/ml and streptomycin 50 µg/ml) at 37°C under a 95%:5% air:CO2 atmosphere. For immunofluorescence studies, the cells were seeded on glass coverslips. Cell were treated with the cytokines for the time periods and concentrations indicated in each protocol. Pilot studies carried out with different cytokine concentrations (5–100 ng/ml) showed that for a 2-h incubation period, maximal responses were obtained with 10–25 ng/ml for TNF and 10 ng/ml for IL-1.

Source of Antibodies and Organelle Markers

Three different antibodies were used for GJA1 identification and labeling. The first antibody recognizes all forms (phosphorylated and nonphosphorylated) of GJA1 (Pan-GJA1) was purchased from Sigma Chemical Co. The second antibody only recognizes the GJA1 nonphosphorylated in serine 368 (NP-GJA1) and was obtained from Zymed. The third antibody only recognizes the GJA1 phosphorylated in serine 368 (P-GJA1) and was obtained from Chemicon. Polyclonal anti-nonmuscle actin and antimyosin heavy chain 2B were purchased from Sigma Chemical Co. The Golgi markers, wheat germ agglutinin-rhodamine (WGA), and the antibody to TGN38 were purchased from Molecular Probes and Serotec, respectively. The antibody against LAMP1 (1D4B), which was used as a lysosome marker, was developed by Dr. Thomas August and obtained from the Developmental Studies Hybridoma Bank (Department of Biological Science, University of Iowa, Iowa City, IA) developed under the auspices of the National Institute of Child Health and Human Development. The endoplasmic reticulum marker rhodamine-labeled concanavalin A was obtained from Molecular Probes. The secondary antibodies from fluorescence microscopy and for Western blots were from Bio/Can.

Source of Cytokines and Other Compounds

Recombinant mouse TNF, aprotinin, and PMSF were obtained from Roche. Recombinant mouse IL-1, cycloheximide, leupeptin, lactacystin, biotin-dextran acid, Na3VO4, NaF, and Na4P2O7 were from Sigma Chemical Co. Calcein and rhodamine-streptavidin were purchased from Molecular Probes. Potassium Bisperoxo (1,10-phenanthroline) oxovanadate (V) [bpV (phen)] was purchased from Calbiochem.

Membrane- and Cytosol-Enriched Fraction Preparations

Following treatments, the cells were collected by centrifugation at 1500 x g (Beckman GS-6R; Beckman Canada) for 10 min at 4°C. The pellet was rinsed and resuspended in PBS (137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4; pH 7.4) containing phosphatase and protease inhibitors (2 mM PMSF, 1 mM EGTA, 2 µg/ml of leupeptin, 2 µg/ml of aprotinin, 4 mM Na3VO4, 80 mM NaF, 20 mM Na4P2O7, and 10 µM bpV (phen). The cell suspension was sonicated, and the homogenate was centrifuged at 1500 x g (Beckman GS-6R) for 10 min at 4°C to remove the nuclei. The supernatant was then centrifuged at 15000 x g (Beckman Microfuge E; Beckman Canada) for 25 min at 4°C. The pellet was considered to be the membrane-enriched fraction and the supernatant to be the cytosol-enriched fraction. The membrane-enriched fraction contained plasma membrane proteins (scavenger-receptor class B member 1 [SCARB1] and epidermal growth factor receptor [EGFR]), endoplasmic reticulum (calnexin), Golgi (TGN38), and lysosome (LAMP1) membrane proteins, yet the cytosolic enzyme hormone-sensitive lipase (LIPE) was absent. On the other hand, the cytosol-enriched fraction contained LIPE and trace amounts of calnexin, TGN38, LAMP1, and EGFR, but it did not contain SCARB1. The protein content of the fractions was measured by the Bradford method with materials provided by Bio-Rad.

Electrophoresis and Western Blot Analyses

The total cell homogenates and subcellular fractions were homogenized in PBS containing phosphatase and protease inhibitors, and aliquots containing 10 µg of 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, 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 GJA1 antibodies, membranes were stripped using the reagent Re-blot plus (Chemicon, Temecula, CA) and reprobed with anti-nonmuscle actin; in some cases, the membranes were incubated with antimyosin heavy chain 2B without stripping. The antigen-antibody reactions were revealed by chemiluminescence (Lumilight; Roche).

Bands on the films were scanned, and the intensity of the bands was quantified using the Scion Image Program (Scion Corporation, Frederick, MD). To compare results from different experiments, the intensity values were normalized by dividing them by the intensity of the control value (time, 0 h) within the same experiment and by the actin or myosin content of the sample.

Immunofluorescence Microscopy

Following treatments, the TtT/GF cells were fixed with 3.7% formaldehyde for 20 min and permeabilized in acetone as described previously [28]. 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 or with fluorescent probes. The cells were viewed with an Axiophot II fluorescence microscope (Carl Zeiss). Micrographs were taken with T-MAX films (Eastman Kodak, Rochester, NY). Confocal microscopy images were obtained with an LSM510 Axiovert (Carl Zeiss) from the Institut de Recherches Cliniques de Montréal (PQ, Canada). The quantitative analyses of GJA1 immunolabeling were carried out on 10 microphotographs per experimental condition and obtained from three independent experiments. The micrograph negatives were magnified 10-fold. Both NP-GJA1- and P-GJA1-immunopositive dots were counted in a 20- x 25-cm area of the enlarged micrographs without knowledge of the experimental condition from which the photographs were taken [28].

Cell-to-Cell Communication

The scrape-loading method was used with some modifications to measure intercellular communication [38–40]. Briefly, TtT/GF cells were grown on glass coverslips until confluence and incubated with culture medium alone (control) or culture medium containing the cytokines for 1, 2, and 24 h. Following treatments, the cells were rinsed and incubated in DMEM (pH 8.0). Three scratches were made with a needle through the cell culture, and calcein (final dilution, 10 mM) was added to the medium bathing the cells for 1 min. The solution was then removed, and the cells were incubated for an additional 10 min in DMEM to permit diffusion of the dye. Next, the cells were rinsed three times with PBS and fixed with 3.7% formaldehyde. Controls were carried out using biotin-dextran and rhodamine-streptavidin. Images were acquired with an Axiophot microscope (Carl Zeiss) and visualized by using the Northern Eclipse program.

Statistical Analyses

Data were evaluated by either one-way ANOVA, with the differences between means analysed by the Keuls multiple-range test (see Fig. 15), or by two-way ANOVA followed by t-test for each condition studied (see Figs. 1–14). All analyses were carried out using the Stata software (Stata Corporation, College Station, TX).


Figure 15
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  		FIG. 15. Intercellular communication in proinflammatory cytokine-treated TtT/GF cells. A) Fluorescence photomicrographs of TtT/GF after scrape-loading. The cells were incubated with culture medium alone (control) or culture medium containing 25 ng/ml of TNF for 1, 2, or 24 h. The data are representative of six independent experiments. Original magnification x80. B) Quantification of the number of fluorescent cell rows after scrape-loading in control cells and in cells treated with 25 ng/ml of TNF or 10 ng/ml of IL-1 for 1, 2, or 24 h. Data shown are the mean ± SEM of six independent experiments. *P < 0.05 for IL-1 vs. C (1 h) and IL-1 vs. C (2 h); {ddagger}P < 0.005 for TNF vs. C (1 h) and TNF vs. C (2 h)


Figure 1
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  		FIG. 1. Expression and distribution of Pan-GJA1, NP-GJA1, and P-GJA1 in TtT/GF cells. A) Representative immunoblots showing expression levels of (a) Pan-GJA1, (b) NP-GJA1, and (c) P-GJA1 in TtT/GF cells. Ten micrograms of proteins of total cell homogenates (t), membrane (m)-enriched, and cytosol (c)-enriched fractions were subjected to immunoblotting with antibodies that recognize either all forms of GJA1 (Pan-GJA1), the form not phosphorylated in serine 368 (NP-GJA1), or the form phosphorylated in serine 368 (P-GJA1). B) The same antibodies were used to carry out immunofluorescence microscopy studies. a) Cells stained with anti-Pan-GJA1 show intense labeling at the plasma membrane regardless of whether the membrane was (arrowhead) or was not (arrow) involved in cell-to-cell contacts. In addition, a spotty labeling was observed in the cytoplasm, particularly in the perinuclear region (open arrow). b) Labeling of the cells with anti-NP-GJA1 showed that NP-GJA1 immunolabeling was mostly intracellular, with some accumulation in the perinuclear area (open arrow). Little labeling was associated with the plasma membrane (arrowhead). c) Staining with anti-P-GJA1 revealed that in TtT/GF cells, P-GJA1 was associated with the plasma membrane regardless of whether the membranes were (arrowhead) or were not (arrow) involved in intercellular contacts. At the plasma membrane, large P-GJA1-positive plaques were apparent. Little labeling was observed in the cytoplasm (open arrow). Original magnification x425


Figure 14
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  		FIG. 14. Effect of the inhibition of proteasomal activity on P-GJA1 distribution in control and cytokine-treated TtT/GF cells. The TtT/GF cells were pretreated with either culture medium alone (Lac (–)) or culture medium containing 15 µM of the proteasomal activity-inhibitor lactacystin (Lac (+)) for 30 min. Next, the cells were challenged for 2 h with culture medium alone (control) or culture medium containing 25 ng/ml of TNF or 10 ng/ml of IL-1. The cells were double-labeled with anti-NPGJA1 and anti-P-GJA1. Lactacystin treatment increased the P-GJA1 labeling in the cytoplasm and at the plasma membrane in control cells and, particularly, in the cytokine-treated cells. Original magnification x425

RESULTS

Expression and Distribution of GJA1 in TtT/GF Cells

The TtT/GF cells express the gap junction protein GJA1 (Fig. 1). Using the Pan-GJA1 antibody, several bands were detected in TtT/GF cell homogenates (Fig. 1A, a). The slower-migrating bands have been shown to correspond to the phosphorylated forms of GJA1 [41], whereas the faster-migrating band corresponds to the nonphosphorylated form of GJA1 [41] but also may contain phosphorylated forms of GJA1 [42]. The phosphorylated forms were preferentially recovered in the membrane-enriched fraction that contains plasma membranes and membranes from the endoplasmic reticulum, the Golgi, and the lysosomes (Fig. 1A, a, m). Labeling of the cells with the Pan-GJA1 antibody showed immunoreactivity at the plasma membrane regardless of whether cell-to-cell contacts were present or absent (Fig. 1B, a). In addition, spotty labeling was found in the cytoplasm, particularly in the perinuclear region (Fig. 1B, a, open arrow). Because phosphorylation of GJA1, particularly in Ser-368, affects the behavior of GJA1-mediated gap junctions [43, 44], we assessed the influence of the phosphorylation of this site in GJA1 distribution. Two distinct antibodies were used to distinguish the nonphosphorylated from the phosphorylated in Ser-368 forms of GJA1. One antibody recognizes GJA1 molecules not phosphorylated in Ser-368 (henceforth termed NP-GJA1), and the other recognizes the Ser-368-phosphorylated GJA1 (henceforth called P-GJA1). Western blot analyses using the NP-GJA1 antibody showed only one band that was more intense in the membrane-enriched fraction than in the cytosol-enriched fraction (Fig. 1A, b). The labeling with this antibody showed that NP-GJA1 was mainly intracellular (Fig. 1B, b, open arrow), with faint spots at the plasma membrane (Fig. 1B, b, arrowhead). Western blot analyses with the P-GJA1 antibody identified only one band that was more intense in the membrane-enriched fraction than in the cytosol-enriched fraction (Fig. 1A, c). Large immunoreactive plaques were labeled with P-GAJ1 antibody in regions of the plasma membrane that were (Fig. 1B, c, arrowhead) or were not (Fig. 1B, c, arrow) involved in intercellular contacts. Little labeling was found in the cytoplasm (Fig. 1B, c, open arrow). The patterns of distribution of NP-GJA1 and P-GJA1 were different when taken separately but appeared to be similar to that detected with the Pan-GJA1 antibody when taken together.

Effect of Proinflammatory Cytokines TNF and IL-1 on GJA1 Expression and Localization

Incubation of TtT/GF cells with proinflammatory cytokines for 1 or 2 h induced an increase in GJA1 levels (Fig. 2A). Double-labeling fluorescence microscopy with Pan-GJA1 (Fig. 2B, a, b) and the Golgi marker WGA (Fig. 2B) revealed that proinflammatory cytokine treatment increased Pan-GAJ1 labelling, particularly in the perinuclear region (Fig. 2B, b).


Figure 2
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  		FIG. 2. Effect of the proinflammatory cytokines TNF and IL-1 on the distribution and expression of GJA1. The TtT/GF cells were incubated with culture medium alone or with culture medium containing either 25 ng/ml of TNF or 10 ng/ml of IL-1 for 1 and 2 h. A) Representative Western blot analyses of GJA1 content (Pan-GJA1 antibody) in total TtT/GF cell homogenates (t) and in subcellular fractions (membrane [m]-enriched fraction and cytosol [c]-enriched fraction) obtained from control cells (C) and cells treated with cytokines for 2 h. B) The cells were double-labeled with Pan-GJA1 antibody and the Golgi marker rhodamine-labeled WGA. The representative micrographs show the distribution of GJA1 and the Golgi apparatus in control cells and cytokine-treated cells. Proinflammatory cytokines enhanced GJA1 labeling in the Golgi region. Original magnification x300

The same series of experiments was conducted with the NP-GJA1 and P-GJA1 antibodies. Following TNF and IL-1 treatment, a strong NP-GJA1 immunolabeling appeared, primarily in the perinuclear area as large immunoreactive vesicles and to a lesser extent at the plasma membrane, when compared to controls (Fig. 3, NP-GJA1). The P-GJA1 labeling also was heavier at the plasma membrane and in the cytoplasm after cytokine treatment compared to controls (Fig. 3, P-GJA1). Confocal microscopy showed that NP-GJA1 and P-GJA1 colocalized predominantly at the plasma membrane in nontreated cells, but a partial colocalization of NP-GJA1 and P-GJA1 also was found at the plasma membrane and in the cytoplasm during the first 2 h of incubation with the cytokines (data not shown).


Figure 3
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  		FIG. 3. Fluorescence microscopy studies on the effect of TNF and IL-1 on NP-GJA1 and P-GJA1 distribution in TtT/GF cells. The TtT/GF cells were incubated with culture medium alone or with culture medium containing either 25 ng/ml of TNF or 10 ng/ml of IL-1 for 1 and 2 h. The cells were double-labeled with anti-NP-GJA1 and anti-P-GJA1. The NP-GJA1 labeling was mainly cytoplasmic in control cells and increased chiefly in the perinuclear area following treatment with the cytokines. The P-GJA1 labeling localized preferentially to the plasma membrane in control cells. Proinflammatory cytokines enhanced P-GJA1 labeling in the cytoplasm and at the plasma membrane. Original magnification x425

Incubation of the cells with the cytokines for at least 1 h increased NP-GJA1 expression in the membrane-enriched fraction (Fig. 4, NP-GJA1), but the increase was transient. After a 24-h incubation with either TNF or IL-1, the NP-GJA1 levels were lower than the control values (Fig. 4, NP-GJA1). The P-GJA1 levels also transiently increased in both the membrane- and cytosol-enriched fractions after cytokine treatment (Fig. 4, P-GJA1). The P-GJA1 levels in membrane- and cytosol-enriched fractions were lower in cells treated with the cytokines for 24 h than in control cells (Fig. 4).


Figure 4
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  		FIG. 4. Time-course studies on the effect of the proinflammatory cytokines TNF and IL-1 on the levels of NP-GJA1 and P-GJA1 TtT/GF subcellular fractions. The TtT/GF cells were incubated for increasing periods of time with 25 ng/ml of TNF or with 10 ng/ml of IL-1. Following treatments, the cells were scraped off, and membrane (m)- and cytosol (c)-enriched fractions were prepared. Five-microgram (m) and 10-µg (c) proteins were subjected to electrophoresis followed by immunoblotting either with anti-NP-GJA1 or anti-P-GJA1. The figure shows representative membranes of six independent experiments. Treatment of the cells with cytokines induced a transient increase in the intensity of NP-GJA1 and P-GJA1 immunoreactive bands in both m- and c-enriched fractions

Effect of TNF and IL-1 on GJA1 Protein Synthesis in TtT/GF Cells

The increased levels of GJA1 and the accumulation of GJA1 staining in the Golgi region following proinflammatory cytokine treatment suggested increased protein synthesis. The following series of experiments was carried out to test this possibility.

First, confocal microscopy showed a partial colocalization of NP-GJA1 with the trans-Golgi marker TGN-38 at 1 h after exposure to IL-1 (Fig. 5) or to TNF (data not shown), suggesting increased GJA1 synthesis. The colocalization decreased by 2 h after treatment (Fig. 5).


Figure 5
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  		FIG. 5. Colocalization of NP-GJA1 and the Golgi marker TGN38. The TtT/GF cells were incubated with culture medium alone or with culture medium containing 10 ng/ml for IL-1 for 1 and 2 h. The cells were double-labeled with anti-NP-GJA1 and anti-TGN-38. The figure shows the merged confocal images of NP-GJA1 labeling (green) and TGN-38 labeling (red). Colocalization of both markers (yellow) is observed after 1 h of treatment with the cytokine and decreased thereafter. Original magnification x637

Second, TtT/GF cells were incubated with or without the protein synthesis-inhibitor cycloheximide for 30 min before being exposed to medium alone (Fig. 6, control) or containing the cytokines for 1 and 2 h (Fig. 6, TNF and IL-1). In control cells, cycloheximide first increased (1 h) but then decreased (2 h) NP-GJA1 and P-GAJ1 levels in the membrane fraction, whereas only decreases were seen in the cytosolic fraction (Fig. 6, control). Cycloheximide blocked the rise in NP-GJA1 in the membrane and cytosolic fractions in cells treated with TNF for 2 h (Fig. 6, TNF). Cycloheximide also decreased P-GJA1 levels in the membrane fraction of cells treated with TNF for 2 h and in the cytosolic fraction in cells treated for 1 and 2 h with TNF (Fig. 6, TNF). The increased NP-GJA1 levels in the membrane fraction of cells treated for 1 h with IL-1 were reduced by cycloheximide pretreatment (Fig. 6, IL-1). In the cytosolic fraction, the NP-GJA1 level increase that was measured after 2-h incubation with IL-1 was reduced by cycloheximide (Fig. 6, IL-1). Cycloheximide also diminished P-GJA1 levels in the cytosolic fraction of IL-1-treated cells (Fig. 6, IL-1).


Figure 6
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  		FIG. 6. Effect of protein synthesis inhibition on the levels of NP-GJA1 and of P-GJA1 in membrane- and cytosol-enriched fractions of control and cytokine-treated cells. The TtT/GF cells were pretreated with either culture medium alone (black bars and dark grey bars) or containing 20 µg/ml of cycloheximide (medium and light grey bars) for 30 min, then challenged for 1 or 2 h with culture medium alone (C) or with culture medium containing 25 ng/ml of TNF or 10 ng/ml of IL-1. Membrane- and cytosol-enriched fractions were obtained, homogenized, and subjected to electrophoresis followed by immunoblotting with anti-NP-GJA1 or with anti-P-GJA1. The immunoreactive bands on the membranes were scanned and their intensities calculated using a computer program. Values shown in the figure are the mean ± SEM of six independent experiments. NP-GJA1, membrane-enriched fraction: *P < 0.05 for C + CHX vs. C (2 h), TNF vs. C (2 h), IL-1 vs. C (1 h), IL-1 vs. C (2 h), and IL-1 + CHX vs. IL-1 (1 h); **P < 0.01 for TNF vs. C (1 h) and TNF + CHX vs. TNF (2 h), {ddagger}{ddagger}P < 0.001 for C + CHX vs. C (1 h); cytosol-enriched fraction: *P < 0.05 for TNF vs. C (2 h); {ddagger}P < 0.005 C + CHX vs. C (1 h) and IL-1 vs. C (2 h); {ddagger}{ddagger}P < 0.001 for C + CHX vs. C (2 h), TNF + CHX vs. TNF (2 h), and IL-1 + CHX vs. IL-1 (2 h). P-GJA1, membrane-enriched fraction: *P < 0.05 for C + CHX vs. C (1 h), C + CHX vs. C (2 h), and TNF vs. C (1 h); {ddagger}P < 0.005 for TNF vs. C (2 h), TNF + CHX (2 h s), and IL-1 vs. C (2 h); cytosol-enriched fraction: *P < 0.05 for TNF + CHX vs. TNF (1 h) and IL-1 + CHX vs. IL-1 (1 h); **P < 0.01 for C + CHX vs. C (2 h), TNF + CHX vs. TNF (2 h), IL-1 + CHX vs. IL-1 (2 h)

Third, immunocytochemistry studies showed that cycloheximide blocked the enhanced NP-GJA1 labeling usually observed in the perinuclear area of TNF- and IL-1-treated cells (Fig. 7, TNF ± CHX, IL-1 ± CHX). In addition, the NP-GJA1 cytoplasmic labeling was more diffuse in cytokine-treated cells pre-exposed to cycloheximide than in cytokine-treated cells not exposed to the protein synthesis inhibitor (Fig. 7, TNF ± CHX, IL-1 ± CHX). Cycloheximide slightly reduced P-GJA1 membrane labeling in control cells (Fig. 7, P-GJA1 control). In TNF-treated cells, cycloheximide also decreased P-GJA1 labeling (Fig. 7, TNF ± CHX). In these cells, P-GJA1-positive spots appeared smaller and more dispersed than those in cells treated with TNF alone (Fig. 7, P-GJA1, TNF ± CHX). Inhibition of protein synthesis in IL-1-treated cells caused the appearance of large P-GJA1-positive spots (Fig. 7, IL-1 ± CHX).


Figure 7
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  		FIG. 7. Effect of protein synthesis inhibition on the cellular distribution of NP-GJA1 and of P-GJA1 in control and cytokine-treated cells. The TtT/GF cells were pretreated with either culture medium alone (CHX (–)) or culture medium containing 20 µg/ml (CHX (+)) of cycloheximide for 30 min, then challenged for 2 h with culture medium alone (control) or with culture medium containing 25 ng/ml of TNF or 10 ng/ml of IL-1. The cells were double-labeled with anti-GJA1 and anti-P-GJA1. Cycloheximide reduced NP-GJA1 labeling particularly in the perinuclear area in control and cytokine-treated cells. The P-GJA1 labeling also decreased in control and TNF-treated cells. In IL-1-treated cells, large P-GJA1-positive spots were apparent in the cytoplasm of cycloheximide-pretreated cells. Original magnification x425

Effect of TNF and IL-1 on GJA1 Degradation in TtT/GF Cells

The increased NP-GJA1 and P-GJA1 levels measured following cytokine treatment could be the result, in part, of a decrease in protein degradation. It has been shown that GJA1-mediated cell-to-cell communication is enhanced by stabilization of the GJA1 protein with proteasome and lysosome inhibitors [38]. Our results show that cytokine treatment increased Pan-GJA1 and NP-GJA1 labeling in the perinuclear area. In this localization, NP-GJA1 only transiently colocalized with the trans-Golgi network marker. Therefore, we investigated whether the buildup of NP-GJA1 in the perinuclear area during incubation periods longer than 2 h was the result of the accumulation of the protein in lysosomes. Confocal microscopy studies showed a strong colocalization of NP-GJA1 with the lysosome marker LAMP1 in the perinuclear area following treatment of the cells with TNF or IL-1 (Fig. 8). The peak of colocalization appeared within 2–6 h after addition of cytokines to the medium (Fig. 8).


Figure 8
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  		FIG. 8. Colocalization of NP-GJA1 and the lysosome marker LAMP1 in control and cytokine-treated TtT/GF cells. The TtT/GF cells were incubated with culture medium alone (control) or with culture medium and either 25 ng/ml of TNF or 10 ng/ml of IL-1 for 1, 2, 6, and 24 h. The cells were next double-labeled with anti-NP-GJA1 and anti-LAMP1. Micrographs are the merged confocal images of NP-GJA1 (red) and LAMP1 (green) labeling. Colocalization (yellow) of NP-GJA1 with LAMP1 was low in control cells but increased after cytokine treatment, reaching a maximum after 6 h of incubation. After 24 h in the presence of TNF or IL-1, the colocalization levels went back to control values. Original magnification x425

To evaluate the significance of this increased association of NP-GJA1 with lysosomes following treatment with the proinflammatory cytokines, we challenged the cells with drugs known to affect GJA1 degradation. Inhibition of lysosomal activity with the protease-inhibitor leupeptin caused little effect on the distribution and the intensity of NP-GJA1 labeling in either control or cytokine-treated cells (Figs. 9 and 10). Nevertheless, in cytokine-treated cells, NP-GJA1 labeling was more diffuse in cells pretreated with leupeptin (Fig. 10, TNF and IL-1). In addition, an increased association of NP-GJA1 with the plasma membrane in cells treated with leupeptin and IL-1 was found (Fig. 10, arrow). The same observation was apparent in TNF-treated cells only after 4 h of incubation with leupeptin and TNF (data not shown). Leupeptin treatment induced an increase in P-GJA1 labeling (Fig. 9) both in the cytoplasm and at the plasma membrane in control and cytokine-treated cells (Fig. 11).


Figure 9
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  		FIG. 9. Quantification of the effect of the lysosomal protease-inhibitor leupeptin in NP-GJA1 and P-GJA1 immunolabeling in control and in cytokine-treated cells. The TtT/GF cells were pretreated with either culture medium alone (Leu (–)) or culture medium containing 100 µM of the lysosomal protease-inhibitor leupeptin (Leu (+)) for 30 min. Next, the cells were challenged for 2 h with culture medium alone (C) or culture medium containing 25 ng/ml of TNF or 10 ng/ml of IL-1. The cells were labeled with either anti-NP-GJA1 or anti-P-GJA1. Ten micrographs per experimental condition were obtained. The negatives were magnified 10-fold, and immunofluorescent spots in the enlarged micrographs were counted in a 20- x 25-cm area. The images were from three independent experiments. Values shown are the mean ± SEM. NP-GJA1: {ddagger}P < 0.005 for IL-1 vs. C; {ddagger}{ddagger}P < 0.001 for TNF vs. C and IL-1 vs. C; P-GJA1: *P < 0.05 for C + Leu vs. C, TNF +Leu vs. TNF, and IL-1 + Leu vs. IL-1; {ddagger}{ddagger}P < 0.001 for TNF vs. C IL-1 vs. C


Figure 10
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  		FIG. 10. Effect of the inhibition of lysosomal activity on NP-GJA1 distribution in control and cytokine-treated TtT/GF cells. The TtT/GF cells were pretreated with either culture medium alone (Leu (–)) or culture medium containing 100 µM of the lysosomal activity-inhibitor leupeptin (Leu (+)) for 30 min. Next, the cells were challenged for 2 h with culture medium alone (control) or culture medium containing 25 ng/ml of TNF or 10 ng/ml of IL-1. The cells were labeled with anti-NP-GJA1. Leupeptin treatments did not affect NP-GJA1 labeling in the perinuclear area of both control and cytokine-treated cells, although the labeling was more dispersed in cells treated with the combination of drugs. Leupeptin slightly increased NP-GJA1 associated with the plasma membrane in cytokine-treated cells (arrow). Original magnification x425


Figure 11
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  		FIG. 11. Effect of the inhibition of lysosomal activity on P-GJA1 distribution in control and cytokine-treated TtT/GF cells. The TtT/GF cells were pretreated with either culture medium alone (Leu (–)) or culture medium containing 100 µM of the lysosomal activity-inhibitor leupeptin (Leu (+)) for 30 min. Next, the cells were challenged for 2 h with culture medium alone (control) or culture medium containing 25 ng/ml of TNF or 10 ng/ml of IL-1. The cells were labeled with anti-P-GJA1. In leupeptin-treated cells, a stronger labeling of P-GJA1 was observed in the cytoplasm and plasma membrane in both control and cytokine-treated cells when compared to cells not incubated with leupeptin. In addition, in cells treated with leupeptin and cytokines treated cells, large P-GJA1 immunopositive plaques were apparent in the cytoplasm and at the plasma membrane. Original magnification x425

In addition, GJA1 is degraded by the proteasome. Inhibition of proteasomal activity in TtT/GF cells with lactacystin increased NP-GJA1 labeling in control and cytokine-treated cells (Fig. 12). The most intense labeling was recorded in the cytoplasm, particularly in the perinuclear region of control and cytokine-treated cells (Fig. 13). The NP-GJA1 accumulated in the perinuclear region colocalized with lysosomes (data not shown). In addition, in lactacystin cytokine-treated cells, less NP-GJA1 labeling was observed in the plasma membrane compared to cells incubated with cytokines alone (Fig. 13). Lactacystin increased P-GJA1 labeling in control and cytokine-treated cells (Fig. 12). The increased labeling was detected at the plasma membrane of control cells and at the plasma membrane and the cytoplasm in cytokine-treated cells (Fig. 14).


Figure 12
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  		FIG. 12. Quantification of the effect of the proteasome-inhibitor lactacystin in NP-GJA1 and P-GJA1 immunolabeling in control and in cytokine-treated cells. The TtT/GF cells were pretreated with either culture medium alone (Lac (–)) or culture medium containing 15 µM of the proteasome activity-inhibitor lactacystin (Lac (+)) for 30 min. Next, the cells were challenged for 2 h with culture medium alone (C) or culture medium containing 25 ng/ml of TNF or 10 ng/ml of IL-1. The cells were labeled with either anti-NPGJA1 or anti-P-GJA1. Ten micrographs per experimental condition were obtained, and the negatives were magnified 10-fold. Immunofluorescent spots in the enlarged micrographs were counted in a 20- x 25-cm area. The images were obtained from three independent experiments. Values shown are the mean ± SEM. NP-GJA1: *P < 0.05 for IL-1 + Lac vs. IL-1; {ddagger}P < 0.005 for TNF + Lac vs. TNF; {ddagger}{ddagger}P < 0.001 for C + Lac vs. C, TNF vs. C, and IL-1 vs. C; P-GJA1: *P < 0.05 for TNF + Lac vs. TNF and IL-1 + Lac vs. IL-1; {ddagger}{ddagger}P < 0.001 for C + Lac vs. C, TNF vs. C, and IL-1 vs. C


Figure 13
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  		FIG. 13. Effect of the inhibition of proteasomal activity on NP-GJA1 distribution. The TtT/GF cells were pretreated with either culture medium alone (Lac (–)) or culture medium containing 15 µM of the proteasomal activity-inhibitor lactacystin (Lac (+)) for 30 min. Next, the cells were challenged for 2 h with culture medium alone (control) or culture medium containing 25 ng/ml of TNF or 10 ng/ml of IL-1. The cells were labeled with anti-NPGJA1. Lactacystin treatment increased the NP-GJA1 labeling associated with the perinuclear area in control cells. The already strong perinuclear labeling in cytokine-treated cells was not affected by the treatment, but a decrease was observed in the NP-GJA1 associated with the plasma membrane. Original magnification x425

Effect of TNF and IL-1 on Cell-to-Cell Communication in TtT/GF Cells

The transient cytokine-induced increase in GJA1 levels in TtT/GF cells may affect intercellular communication. When nontreated cells were incubated with the nonpermeable fluorescent dye calcein after cutting the cell culture with a needle, few cell rows adjacent to the cuts were labeled, indicating a remaining degree of intercellular communication in control cells (Fig. 15, A and B, control). The same procedure performed in cells treated with TNF or IL-1 (not shown) for 1–2 h showed an increased number of labelled cells adjacent to the cut, indicating that the cytokines caused a transient increase in cell-to-cell communication (Fig. 15, A, TNF 1 and 2 h, and B). The level of intercellular communication of cells exposed for 24 h to the cytokines was similar to that of control cells (Fig. 15, A, TNF 24 h, and B).

DISCUSSION

The FS cells are both the target and the effector of cytokines involved in the regulation of anterior pituitary endocrine cell function [2]. Because the FS cells express the gap junction protein GJA1 [23, 28, 29] and form functional gap junctions [25, 27], they contribute to the establishment of a functional network of interconnected cells that can modulate the anterior pituitary endocrine response [26, 28]. In the present study, we measured the effect of the cytokines on FS cell-mediated intercellular communication, specifically the effects of the proinflammatory cytokines TNF and IL-1 on GJA1-mediated gap junctions in the FS cell line TtT/GF. Our results showed that the cytokines transiently increased the GJA1 turnover and cell-to-cell communication in TtT/GF cells.

We began by characterizing the expression and localization of the gap junction protein GJA1 in TtT/GF cells using an antibody that recognizes different GJA1 isoforms simultaneously. The GJA1 labeling in nontreated TtT/GF cells was associated with small vesicles in the cytoplasm and in the perinuclear region. Moreover, our observation that large GJA1-positive plaques were present in plasma membrane regions involved in cell-to-cell contacts as well as in membrane regions that were not involved suggests the presence of GJA1 hemichannels in TtT/GF cells. The Pan-GJA1 antibody detected three immunoreactive bands, indicating the presence of phosphorylated forms of GJA1 in TtT/GF cells, as has been shown in other cell systems [41]. Our finding of phosphorylated bands being stronger in the membrane-enriched than in the cytosol-enriched fraction of TtT/GF cells agrees with the report that GJA1 phosphorylation occurs preferentially in the vicinity of the plasma membrane [38, 45]. Yet, the phosphorylation status of the protein, which appears to be essential for neither the assembly nor the functioning of GJA1-mediated gap junctions [42, 46], seems instead to reflect different moments in the life of the gap junction. The GJA1 phosphorylation has been reported during connexon assembly [47], GJA1 degradation [39], and removal from the plasma membrane [47]. In addition, rapid (15–30 min) GJA1 phosphorylation inhibits gap junction function [48–50].

The GJA1 is phosphorylated at multiple residues, primarily at serine residues [42, 46, 51] and to a lesser extent at tyrosine residues [52]. Phosphorylation of GJA1 in Ser-368 has been reported to modify the turnover and function of GJA1-mediated gap junctions [43, 44]. Significantly, phosphorylation in Ser-368 is not always accompanied by an electrophoretic shift [42]. Therefore, use of one antibody that recognizes the nonphosphorylated Ser-368 GJA1 (NP GJA1) and of another antibody that recognizes the phosphorylated Ser-368 GJA1 (P-GJA1) allowed us to study the effect of proinflammatory cytokines on GJA1 expression and localization in TtT/GF cells. Treatment of the cells with TNF and IL-1 transiently increased NP-GJA1 expression. The finding of an increased GJA1 and NP-GJA1 immunoreactivity in the perinuclear region and of a partial colocalization of NP-GJA1 with the Golgi apparatus marker TGN-38 during the first hour of cytokine treatment strongly suggest an enhanced synthesis of GJA1 in the presence of the cytokines. The decreased NP-GJA1 levels and the reduced NP-GJA1 labeling in the perinuclear area induced by cycloheximide in TNF- and IL-1-treated cells also indicates that the cytokines enhanced GJA1 synthesis. The P-GJA1 levels were found here to increase during cytokine treatment. Phosphorylation of GJA1 occurs while the molecules are assembled into connexons [47]; on the other hand, GJA1 phosphorylation is a tag for connexon degradation [53, 54]. Cycloheximide reduced P-GJA1 in the membrane fraction in TNF-treated, but not in IL-1-treated, cells. This observation may be the result of a faster connexon assembly and the subsequent tagging for degradation in the presence of IL-1 than in the presence of TNF. The large P-GJA1-positive spots in cycloheximide- and IL-1-treated cells support this view. On the other hand, the fact that cytokine-induced increase in P-GJA1 levels in the cytosol were significantly reduced by cycloheximide pretreatment indicates that the high levels of P-GJA1 in cytokine-treated cells also were a consequence of enhanced protein synthesis.

Our protein synthesis-inhibition studies revealed yet another aspect of the effect of cytokines on GJA1. The transient increase in NP-GJA1 and P-GJA1 levels in the membrane fraction and the concomitant decrease in the cytosolic fraction measured in control cells exposed to cycloheximide may be the consequence of a rapid turnover of the protein in TtT/GF cells and could represent the assembly into gap junctions of GAJ1 molecules already present in the cells before the inhibition of protein synthesis. Because the GJA1 connexons are less soluble than isolated GJA1 molecules [55], this could explain why we recovered most NP-GJA1 in the membrane-enriched fraction. This view is supported by our observation of decreased GJA1 in both membrane and cytosolic fractions in the sustained absence of protein synthesis.

Our observation that cytokine-induced enhancement of GJA1 protein synthesis was transient suggests that the protein was rapidly degraded. Two results support this contention. First, cytokines increased both NP-GJA1 and P-GJA1, and GJA1 phosphorylation is a known tag for degradation [53, 54]. Second, colocalization of NP-GJA1 with the lysosomes increased after 2–6 h of incubation with cytokines. It has been established that GJA1 is degraded by both the proteosomal and lysosomal pathways [56]. Inhibition of lysosomal activity with leupeptin affected neither the levels nor the distribution of NP-GJA1, but it did cause an accumulation of P-GJA1 at the plasma membrane and in the cytoplasm of control TtT/GF cells, suggesting that P-GJA1 normally is broken down by lysosomes. In other cell systems, inhibition of lysosomal activity is accompanied by higher total GJA1 levels [55, 56] and by increased GJA1 labeling at the plasma membrane and in intracellular vesicles [55, 57, 58]. Other reports, however, showed that inhibition of the lysosomal activity affects neither the gap junction assembly [38] nor the phosphorylated GJA1 levels [39]. Clearly, the impact of the lysosome-mediated degradation of GJA1 varies with the cell type. Inhibition of the proteasomal pathway led to the accumulation of NP-GJA1 in lysosomes and to an increase of P-GJA1 at the plasma membrane in control TtT/GF cells, suggesting that blocking of proteasome activity completely stopped GJA1 turnover. Work done in other cell systems showed that inhibition of the proteasomal pathway stabilized the phosphorylated forms of GJA1 [39, 56, 58], augmenting GJA1 labeling, likely of the phosphorylated form, at the plasma membrane [38, 55]. Taken together, our results show that GJA1 is degraded in lysosomes and proteasomes in TtT/GF cells. Nevertheless, the present study assessed the absence or presence of phosphorylation in Ser-368 of GJA1, without providing information on other phosphorylated forms that would not be recognized by the antibodies and that may exhibit different characteristics and behavior.

In cytokine-treated cells, inhibition of the lysosomal activity increased P-GJA1 accumulation at the plasma membrane and in the cytoplasm. In these cells, we showed that the association of NP-GJA1 with the plasma membrane increased and that the NP-GJA1 cytoplasmic labeling was more diffuse than in control cells. Therefore, the blockage of lysosomal activity not only inhibited P-GJA1 degradation but also increased the incorporation of NP-GJA1 into connexons in cytokine-treated cells. On the other hand, inhibition of the proteasomal pathway caused NP-GJA1 to accumulate in lysosomes and to almost disappear from the plasma membrane in cytokine-treated cells. In the absence of proteasomal activity, P-GJA1 accumulated not only at the plasma membrane but also in cytoplasmic vesicles in cytokine-treated cells. These results demonstrate that cytokines enhanced GJA1 degradation in TtT/GF cells via the proteasome and lysosomal pathways.

We showed in the present study that TNF and IL-1 transiently increased GJA1 turnover in TtT/GF cells. These results are in partial agreement with previous reports that long-term treatment with proinflammatory cytokines decreases GJA1 levels in astrocytes [45, 59] and cardiac cells [30] and also blocks de novo gap junction formation by inhibiting GJA1 synthesis and increasing internalization of the protein in Hela cells [60]. It also has been reported that TNF promotes insertion of GJA1 into the neutrophil cell surface within 1 h [61]. The present findings do not, however, support the claim that IL-1 does not affect GJA1 levels [53]. From the above studies and the present results, the effect of the proinflammatory cytokines on the GJA1 synthesis and degradation clearly depends on the moment at which the observation of the phenomenon is carried out. Here, we showed that TNF and IL-1 induced a transient increase in GJA1 turnover, even in cells that remained exposed to cytokines. This short-lived, cytokine-induced increase in GJA1 synthesis, assembly into connexons, and degradation may be a triggering factor for the modulation of other physiological cellular activities. Our finding that the cytokine-induced increase in GJA1 turnover was accompanied by an increase in cell-to-cell communication leads us to propose that the proinflammatory cytokines transiently increase cell-to-cell communication within the anterior pituitary FS cell network by increasing GJA1 synthesis and assembly into connexons and enhancing GJA1 degradation. The network of interconnected FS cells could provide coordination of the response to stimuli within the anterior pituitary gland.

ACKNOWLEDGMENTS

We gratefully acknowledge the generous gift of the TtT/GF cell line from Dr. U. Renner (Department of Endocrinology, Max Planck Institute of Psychiatry, Munich, Germany). We thank the Developmental Studies Hybridoma Bank (Department of Biological Science, University of Iowa, Iowa City, IA) for kindly providing us with the antibody against LAMP1. We also appreciate very much the technical assistance of C. Charbonneau for the confocal microscopy.

FOOTNOTES

1 Supported by the Natural Sciences and Engineering Research Council of Canada grant OGP0194652 (to M.L.V.) and by a scholarship from Fonds de la recherche en santé du Québec to M.L.V. Back

2 Correspondence: Maria L. Vitale, Department of Pathology and Cell Biology, Faculty of Medicine, Université de Montréal, 2900 boulevard Edouard-Montpetit, Montréal, Québec H3T 1J4, Canada. FAX: 514 343 2459; maria.leiza.vitale{at}umontreal.ca Back

Received: 1 June 2005.

First decision: 7 July 2005.

Accepted: 31 August 2005.

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