Corticosterone Inhibits the Proliferation of C6 Glioma Cells via the Translocation of Unphosphorylated Glucocorticoid Receptor

Abstract

Astroglial cells have been considered to have passive brain function by helping to maintain neurons. However, recent studies have revealed that the dysfunction of such passive functions may be associated with various neuropathological diseases, such as schizophrenia, Alzheimer’s disease, amyotrophic lateral sclerosis and major depression. Corticosterone (CORT), which is often referred to as the stress hormone, is a well-known regulator of peripheral immune responses and also shows anti-inflammatory properties in the brain. However, it is still obscure how CORT affects astroglial cell function. In this study, we investigated the effects of CORT on the proliferation and survival of astroglial cells using C6 glioma cells. Under treatment with CORT for 24h, the proliferation of C6 glioma cells decreased in a dose-dependent manner. Moreover, this inhibition was diminised by treatment with mifepristone, a glucocorticoid receptor (GR) antagonist, but not by spironolactone, a mineralocorticoid receptor (MR) antagonist, and was independent of GR phosphorylation and other GR-related intracellular signaling cascades. Furthermore, it was observed that the translocation of GR from the cytosol to the nucleus was promoted by the treatment with CORT. These results indicate that CORT decreases the proliferation of C6 glioma cells by modifying the transcription of a particular gene related to cell proliferation independent of GR phosphorylation.

Glucocorticoid, which is sorted as a steroid hormone, is an adrenal corticosteroid that works in wide phenomena, such as the stress response. Various glucocorticoids are known to exist, such as hydrocortisone, cortisone and corticosterone (CORT). Generally, both glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) are localized in the cytosol of a cell body. After a homodimer is formed, it translocates from the cytosol to the nucleus and binds to a specific DNA motif to regulate DNA transcription.¹⁾ Usually, intracellular GR is inactivated by heat-shock proteins.^2,3) After the ligand-receptor complex forms a homodimer and translocates to the nucleus, it mediates the transcription of various genes, either by binding to DNA or by regulating intracellular signal cascades, such as those involving nuclear factor-kappaB (NF-κB),⁴⁾ Jun N-terminal kinase,⁵⁾ p38 mitogen-activated protein kinase⁶⁾ or cAMP response element-binding protein (CREB).⁷⁾ As a result, CORT can exhibit various effects by modulation of a cytokine production, such as tumor necrosis factor-α (TNF-α) or interleukin (IL-1β).⁸⁾

Usually, CORT concentration increases in various place of body including both blood and tissue under stressful conditions. Although CORT shows a potent anti-inflammatory effect, many studies have demonstrated that CORT also has inhibitory effect of cell proliferation and a cytotoxic effect toward various cell types. A large number of studies have indicated that prolonged and massive secretion of CORT modifies the brain neural network. It has been shown that the stress accompanied with chronic secretion of CORT promotes hippocampal neuronal damages in rats.⁹⁾

Moreover, it has been reported that hippocampal cell proliferation, which might related to neurogenesis or gliogenesis, decreased under chronic treatment with CORT and chronic stress stimulation.^10–12) These findings indicate that CORT may regulate to the proliferation and survival of various types of cells in the brain.

While neurons have been researched in various pathological conditions, astroglial cells have just been considered to aid in maintaining neurons. Recent studies have reported that the dysfunction of these may lead to the development of various neuropathological diseases, such as major depression.¹³⁾ Actually, it has been demonstrated that both proliferations of neurons and astrocytes are inhibited in the cortex.¹⁴⁾ Moreover, histopathological studies have demonstrated a decrease in the density of astrocytes in postmortem brain tissue in major depression.¹⁵⁾ We also previously reported that CORT impaired the proliferation of a cell line of microglia, which are resident macrophage cells that have proliferative potency like astroglial cells in the brain, accompanied by a cytotoxic effect.¹⁶⁾

These findings suggest that CORT induces the disturbance of cell cycle regulation and cell survivability in astroglial cells and leads to the development of neuropathological disorders such as major depression. In this study, we investigated the effects of CORT on the proliferation and survival of astroglia cells using C6 glioma cells, as well as those cell signalings.

MATERIALS AND METHODS

Cell Culture

C6 glioma cells were cultured in Ham’s F12 medium (Wako Pure Chemical Industries, Ltd., Osaka, Japan) with 10% heat-inactivated fetal bovine serum (FBS; Biowest, Nuaillé, France) containing 100 U/mL penicillin and 100 µg/mL streptomycin (Wako Pure Chemical Industries, Ltd.). Cells were generally passaged every 6 days.

WST-8 Assay

C6 glioma cell proliferation was measured by a WST-8 assay using a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). Briefly, cells were plated into 96-well microplates at a density of 5×10⁴/mL (5×10³/well) with CORT and its antagonists. CORT, mifepristone, spironolactone (Sigma-Aldrich, St. Louis, MO, U.S.A.) and 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) (Dojindo) were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich). After culture in various time courses, WST-8 solution were added to cells, and then cells were incubated at 37°C for 2 h. The spectrophotometric absorbance was measured at a wavelength of 450 nm using a VersaMax (Molecular Devices, Tokyo, Japan). Absorbance at 650 nm was also measured as a reference.

Lactate Dehydrogenase (LDH) Assay

The cytotoxicity of CORT against C6 glioma cells was analyzed by a LDH assay using an LDH Cytotoxicity Detection Kit (TaKaRa, Otsu, Japan). Cells were plated into 96-well microplates at a density of 5×10⁴/mL (1×10⁴/well) with CORT and antagonists. To avoid a high background signal induced by FBS, cells were cultured with 1% FBS culture medium. Cells were also cultured in culture medium with 1% NP-40 (MP Biomedicals, Solon, OH, U.S.A.) to determine the maximum cytotoxicity. After 24 h, the supernatant of the culture medium was collected by centrifugation and incubated with reagents at room temperature for 30 min. The spectrophotometric absorbance was measured at a wavelength of 492 nm. Absorbance at a wavelength of 600 nm was also measured as a reference.

Immunocytochemistry

Immunocytochemistry was performed using the method in previous.¹⁶⁾ Cells were plated onto poly-L-lysine (Sigma-Aldrich) coated-slide glass (Matsunami Glass Ind., Osaka, Japan). After 5 d from plating cells, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS). After the addition of 3% FBS containing phosphate-buffered saline for blocking to avoid unspecific binding of antibodies, cells were permeabilized with 0.1% Triton X-100 in PBS for 30 min. As a primary antibody, anti-GR polyclonal antibody (Santa Cruz, Dallas, TX, U.S.A, 1 : 250), anti-phospho-GR (Ser²³²) antibody (Abcam, Cambridge, U.K., 1 : 250), anti-MR polyclonal antibody (Santa Cruz, 1 : 250) and anti-glial fibrillary acidic protein (GFAP) antibody (Chemicon, Billerica, MA, U.S.A., 1 : 250) were used and incubated overnight at 4°C. To detect a primary antibody, Alexa Fluor 546-conjugated anti-rabbit immunoglobulin G (IgG) antibody (Invitrogen, Carlsbad, CA, U.S.A., 1 : 500) was used and incubated for 2 h in dark place. Stained cells were observed by use of an Olympus DP70 fluorescent microscope.

JC-1 Staining

To investigate the effect of CORT on the mitochondrial membrane potential, JC-1 mitochondrial probe was used (Invitrogen). After cells were plated onto with poly-L-lysine-coated slide glass and cultured for 24 and 72 h with CORT and mifepristone, they were then incubated with 10 µM JC-1 at 37°C for 30 min. After washing to remove extra dye, stained cells were observed by use of an Olympus DP70 fluorescent microscope.

Western Blotting

To prepare whole, cytosol and nuclear fraction protein extracts from C6 glioma cells, the methods which we reported in previous were preformed. Protein concentrations of those were determined by using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, U.S.A.). For Western blotting, protein extracts were placed on sodium dodecyl sulfate (SDS)-polyacrylamide gel, transferred to a polyvinylidene difluoride membrane (BioRad, Hercules, CA, U.S.A) and immunoblotted with anti-GR antibody (Santa Cruz, M-20, 1 : 2000), anti-MR antibody (Santa Cruz, H-300, 1 : 2000), anti-phospho-GR (Ser²³²) antibody (Abcam, 1 : 2000), anti-phospho-GR (Ser²⁴⁶) antibody (Abcam, 1 : 2000), anti-c-fos antibody (Santa Cruz, 1 : 2000), anti-p38 mitogen-activated protein kinase (MAPK) antibody (Cell Signaling Technology, Danvers, MA, U.S.A., 1 : 2000), anti-phospho-p38 antibody (Cell Signaling Technology, 1 : 2000), anti-CREB antibody (Cell Signaling Technology, 1 : 2000), anti-phospho CREB antibody (Cell Signaling Technology, 1 : 2000), anti-p44/42 MAPK antibody (Cell Signaling Technology, 1 : 2000), anti-phospho-p44/42 MAPK antibody (Cell Signaling Technology, 1 : 2000), anti-NF-κB p65 subunit antibody (Chemicon, 1 : 2000), anti-inhibitor of κB (IκB) antibody (Santa Cruz, 1 : 1000), anti-cleaved caspase-3 antibody (Cell Signaling Technology, 1 : 2000), anti-caspase-3 antibody (Cell Signaling Technology, 1 : 2000), anti-B-cell lymphoma 2 antibody (Cell Signaling Technology, 1 : 2000), anti-Bcl-2-associated X protein antibody (Cell Signaling Technology, 1 : 2000), anti-GFAP antibody (Sigma-Aldrich, 1 : 1000) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Millipore, 1 : 10000). The blots were developed with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, U.S.A., 1 : 20000) and visualized by chemiluminescence using an ECL Prime Western blotting Detection System (Amersham, Piscataway, NJ, U.S.A.).

Statistics

The results are presented as the mean±standard deviation (S.D.). A statistical analysis was performed using the Dunnett multiple comparisons test or Student’s paired t-test. A p value <0.05 was considered to be statistically significant.

RESULTS

Inhibitory Effect of CORT on the Proliferation of C6 Glioma Cells

First, we investigated that CORT affected the proliferation of C6 glioma cells. As shown in Fig. 1A, the proliferation of C6 glioma cells was inhibited by treatment with 10 µM CORT for 24 h compared with the control. Figure 1B shows the results to evaluate the effect of CORT on the proliferation of C6 glioma cells. The proliferation of C6 glioma cells under treatment of CORT was significantly decreased to less than 80% of that in the control within the initial 24 h of incubation. This decrease persisted for at least 72 h. In addition, this inhibition of the proliferation of C6 glioma cells resulted in dose-dependent manner.

Fig. 1. Effects of CORT on the Proliferation of C6 Glioma Cells

(A) Images of C6 glioma cells cultured with and without 10 µM CORT. Pictures were taken after treatment with CORT for 24 h. Scale bars represent 200 µm (upper) and 50 µm (lower), respectively. (B) WST-8 cell proliferation assay of C6 glioma cells cultured with and without CORT. The vertical axis shows the proliferation rate relative to that in the control. DMSO was used as a vehicle to prepare CORT solution. The histogram shows the mean±S.D. (n=12, * p<0.05 vs. vehicle).

Effects of Mifepristone and Spironolactone on the Proliferation of C6 Glioma Cells Induced by Corticosterone

Figure 2A shows the results of immunostaining by use of anti-GR and MR antibodies. Whole somata of C6 glioma cells were broadly stained with both anti-GR and anti-MR antibody, which means that C6 glioma cells express both GR and MR. To show which receptor could inhibit the effect of CORT on the proliferation of C6 glioma cells, antagonist competition experiments were carried out using mifepristone and spironolactone. First, we examined the appropriate concentrations of both antagonists for this experiment to exclude those cytotoxic concentrations. As shown in Fig. 2B, treatment with 0.5, 2.5, and 5 µM spironolactone, and 0.1, 0.5, 1, 5 and 10 µM mifepristone did not show any effects on the proliferation of C6 glioma cells. However, treatment with 25 and 50 µM spironolactone decreased the proliferation of C6 glioma cells to 88 and 74% of that in the control, respectively. In the antagonist competition experiments, treatment with all concentrations of mifepristone abolished the inhibitory effect induced by 10 µM CORT on C6 glioma cell proliferation in dose-dependent manner. On the other hand, spironolactone did not influence the inhibitory effect of 10 µM CORT on C6 glioma cell proliferation at all.

Fig. 2. Competitive Effect of Mifepristone on the Inhibitory Effect of CORT on the Proliferation of C6 Glioma Cells

(A) Immunocytochemical analysis of C6 glioma cells as determined with the use of anti-GR antibody and anti-MR antibody under normal culture conditions. Scale bars represent 50 µm. (B) The effects of mifepristone and spironolactone on the proliferation of C6 glioma cells that were co-cultured with 10 µM CORT for 24 h. The vertical axis shows the proliferation rate relative to that in the control. The histogram shows the mean±S.D. (n=3, * p<0.05 vs. without 10 µM CORT).

Cytotoxic Effect of Corticosterone on C6 Glioma Cells

Figure 3 shows the results of an LDH assay for C6 glioma cells treated with CORT for 24 h. Considering that cells were cultured with 1% FBS culture medium, it might be reasonable that percentage of dead cells was approximately 10% even under the control culture condition. Compared with the control culture condition, 0.1, 1, and 10 µM CORT including vehicle did not show any cytotoxic effect on C6 glioma cells.

Fig. 3. Cytotoxic Effects of CORT on C6 Glioma Cells

LDH cytotoxicity assay for C6 glioma cells cultured with and without CORT for 24 h. The vertical axis shows the percentage of dead cells as calculated using treatment with 1% NP-40 as complete cell death. The histogram shows the mean±S.D. (n=4, * p<0.05 vs. control).

Effect of Corticosterone on the Mitochondrial Membrane Potential in C6 Glioma Cells

Several studies have reported that CORT affects mitochondrial function which has been known to be related to energy metabolism.^17,18) In this experiment, we examined whether or not CORT affect the mitochondrial membrane potential in living C6 glioma cells. Figure 4 shows the results of JC-1 staining under various culture conditions for 24 and 72 h. Unexpectedly, under treatment with 10 µM CORT did not show a large difference from those in the control and vehicle-treatment conditions in the mitochondrial membrane potential. In addition, treatment with both 10 µM CORT and 1 µM mifepristone also did not show any difference.

Fig. 4. JC-1 Staining of C6 Glioma Cells Treated with CORT

The cells were stained and observed after being cultured with vehicle, 10 µM CORT or 10 µM CORT plus 1 µM mifepristone for 24 and 72 h. An orange signal indicates a high mitochondrial membrane potential in cells. In contrast, a green signal indicates a low mitochondrial membrane potential. Scale bars represent 100 µm, respectively.

Relationship between the Inhibitory Effect of Corticosterone on Proliferation and the Expression of Various Proteins Related to the Corticosteroid Signaling Cascade in C6 Glioma Cells

First, we performed a Western blotting analysis to investigate both GR and MR expressions in the whole-cell fraction of C6 glioma cells treated with 10 µM CORT under various time courses. The expression level of GR in the whole-cell fraction significantly decreased to 80 and 63% upon treatment with CORT for 24 and 72 h, respectively (Fig. 5A). While treatment with 1 µM mifepristone partially recovered the down-regulation of GR induced by CORT, treatment with 1 µM mifepristone alone showed a decrease in GR expression compared with vehicle-treatment conditions. In contrast, treatment of CORT did not affect the expression of MR significantly (Fig. 5A). It has been known that several signaling cascades related to cell proliferation are regulated by corticosteroids.^5–7) Therefore, we also investigated whether CORT affects the expression of proteins that are involved in cell proliferation, such as MAPK cascade-related protein. The expression levels of p44/42, p38 MAPK and those phosphorylated forms did not change under treatment with 10 µM CORT. In addition, there were no changes in the expression of c-fos. The expression levels of CREB and its phosphorylated form were also not affected by the treatment of CORT though they interact with corticoids and regulate cell proliferation.¹⁹⁾ NF-κB is considered as a protein which regulates anti-apoptotic genes transcription.²⁰⁾ Unexpectedly, the expression levels of NF-κB and IκB, which is a protein that inactivates the function of NF-κB in the steady state, also did not change. Furthermore, caspase-3 and Bcl family-related proteins also did not change at all under treatment with CORT (Fig. 5B). It has been reported that GFAP is associated with the proliferation of C6 glioma cells.²¹⁾ However, no changes were observed in the expression of GFAP when C6 glioma cells were treated with CORT (Figs. 6A, B).

Fig. 5. The Expression of GR, MR and Various Proteins That Are Related to the GR Signal Cascade in Whole-Cell Fractions of C6 Glioma Cells

(A) Western blot analysis was performed to investigate the expression levels of GR and MR. Protein extracts of whole-cell fractions were collected from cells cultured with 10 µM CORT for 1, 3, 6, 24, and 72 h. Otherwise, the protein extracts were obtained after 72 h of culture under each culture condition. The vertical axis shows the protein expression rate relative to that in the control. The histogram shows the mean±S.D. (n=21–22, * p<0.05 vs. vehicle). (B) Western blot analysis was performed to investigate the expression levels of proteins that are related to GR signaling, such as NF-κB and IκB, and proteins that are involved in cell proliferation or apoptosis signaling cascades, such as p44/42 MAPK, p38 MAPK, c-fos, JNK and CREB. Western blot analysis was also performed to investigate the expression levels of proteins that are related to the apoptosis cascade, such as cleaved-caspase-3, caspase-3, Bcl-2 and Bax. GAPDH was used as an internal control. Protein extracts of whole-cell fractions were collected from cells cultured with 10 µM CORT for 1, 3, 6, 24, and 72 h. Otherwise, the protein extracts were obtained after 72 h of culture under each culture condition.

Fig. 6. The Expression of GFAP on C6 Glioma Cells

(A) Immunocytochemical analysis of C6 glioma cells with the use of anti-GFAP antibody under culture with vehicle, 10 µM CORT or 10 µM CORT plus 1 µM mifepristone for 72 h. Scale bars represent 200 µm (left column) and 50 µm (right column), respectively. (B) Western blotting of the whole-cell fraction of C6 glioma cells with the use of anti-GFAP antibody. Protein extracts of whole-cell fractions were collected from cells cultured with 10 µM CORT for 1, 3, 6, 24, and 72 h. Otherwise, the protein extracts were obtained after 72 h of culture under each culture condition.

Role of GR Phosphorylation in the Inhibitory Effect of Corticosterone on the Proliferation of C6 Glioma Cells

It has been known that GR is phosphorylated after it forms a ligand-receptor complex. To confirm whether GR phosphorylation by treatment with CORT is involved in its ability to inhibit cell proliferation, Western blotting analysis was performed. When we investigated which site was phosphorylated by treatment with CORT, the expression of phospho-GR (Ser²³²) increased remarkably for 1 h after treatment with CORT and persisted until 72 h (Fig. 7A). In contrast, the expression of phospho-GR (Ser²⁴⁶) was not observed at all, though one study reported that various ligands could accelerate GR phosphorylation.²²⁾ To determine whether the phosphorylation of GR (Ser²³²) was directly related to the inhibitory effect of CORT on cell proliferation, a competition experiment was performed using BAPTA-AM, an intracellular calcium-chelating agent, because it has been reported that BAPTA-AM inhibited GR phosphorylation.²²⁾ When C6 glioma cells were exposed to BAPTA-AM at various concentrations for 1 h before passage and cultured with CORT, the phosphorylation of GR (Ser²³²) induced by treatment with CORT was dramatically decreased (Fig. 7B). In addition, the abolishment of the phosphorylation of GR (Ser²³²) induced by 20 µM BAPTA-AM persisted for at least 72 h compared to that without 20 µM BAPTA-AM. When a WST-8 assay was performed in C6 glioma cells, cell proliferation significantly decreased under treatment with CORT in a dose-dependent manner though treatment with BAPTA-AM abolished the phosphorylation of GR (Ser²³²) (Fig. 7D). In addition, while treatment with CORT had an inhibitory effect on cell proliferation despite pretreatment with BAPTA-AM, this effect was reduced by treatment with mifepristone, but not spironolactone (Fig. 7E).

Fig. 7. Relationship between GR Phosphorylation and Cell Proliferation in C6 Glioma Cells

(A) The difference in GR phosphorylation between Ser²³² and Ser²⁴⁶ in whole-cell fraction of C6 glioma cells. The histogram shows the mean±S.D. (n=3–4, * p<0.05 vs. control). The relative expressions of both phospho-GR (Ser²³²) and phospho-GR (Ser²⁴⁶) were calculated with respect to the value of the control as 100%. The picture under each graph shows a typical Western blotting image. Protein extracts of both cytosol and nuclear fractions were collected from cells cultured with 10 µM CORT for 1, 3, 6, 24, and 72 h. Otherwise, the protein extracts were obtained after 72 h of culture under each culture condition. (B) The inhibitory effect of BAPTA-AM on GR phosphorylation in C6 glioma cells. After the cells were incubated with various concentrations of BAPTA-AM for 1 h, the cells were cultured with 10 µM CORT for 1 h. The extracted whole-cell fractions were then immunoblotted with anti-phospho-GR (Ser²³²) and anti-phospho-GR (Ser²⁴⁶) antibodies. Pre-treatment with 20 µM BAPTA-AM completely inhibited the GR phosphorylation of Ser²³² which was induced by treatment with 10 µM CORT. (C) Time–course inhibition of GR phosphorylation of Ser²³² inC6 glioma cells by 20 µM BAPTA-AM. Cells were incubated with or without 20 µM BAPTA-AM for 1 h before passage. Protein extracts of whole-cell fractions were then collected from cells cultured with 10 µM CORT for 1, 3, 6, 24, and 72 h. Otherwise, the protein extracts were obtained after 72 h of culture under each culture condition. (D) WST-8 cell proliferation assay of C6 glioma cells cultured with and without CORT for 72 h. Cells were incubated with or without 20 µM BAPTA-AM for 1 h before passage. The vertical axis shows the proliferation rate relative to that in the control. The histogram shows the mean±S.D. (n=3, * p<0.05 vs. vehicle). (E) The effects of 1 µM mifepristone and 5 µM spironolactone on the proliferation of C6 glioma cells that were co-cultured with 0.1, 1, and 10 µM CORT for 72 h. Cells were incubated with 20 µM BAPTA-AM for 1 h before passage. The vertical axis shows the proliferation rate relative to that in the control. The histogram shows the mean±S.D. (n=3, * p<0.05 vs. vehicle).

Subcellular Translocation of GR and Phosphorylated GR from Cytosol to Nucleus by Treatment with Corticosterone in C6 Glioma Cells

To investigate the localization and expression levels of GR and phospho-GR in C6 glioma cells after treatment with CORT, Western blotting and immunocytochemical analysis were performed. Under treatment with CORT, the GR expression level in the cytosol fraction decreased gradually, but significantly, in a time-dependent manner. In contrast, the GR expression level in the nuclear fraction was transiently increased to 175% of that under vehicle treatment at 1 h after treatment with CORT, and then gradually decreased in a time-dependent manner (Fig. 8A). On the other hand, the expression level of phospho-GR (Ser²³²) drastically increased to 180 and 350% of that under vehicle treatment in both the cytosol and nuclear fractions, respectively. Similar to GR expression in the nuclear fraction, the peak expression level of phospho-GR (Ser²³²) in both fractions was observed 1 h after treatment with CORT. This expression level then gradually decreased in a time-dependent manner, although the phospho-GR (Ser²³²) expression level in both fractions was significantly higher than that under vehicle treatment for up to 6 h after treatment with CORT (Fig. 8A). In the results of the immunocytochemical analysis, treatment of C6 glioma cells with CORT induced the translocation of GR from the periphery to the center of the cell body and these cells were stained with dot patterns (Fig. 8B). In contrast, phosphorylated GR was present throughout the whole cell body in the immunocytochemical analysis, and there was almost no change in the staining pattern of phosphorylated GR despite treatment with CORT (Fig. 8B).

Fig. 8. Subcellular Localization of Phosphorylated (Ser²³²) and Unphosphorylated GR in C6 Glioma Cells

(A) The protein expression of GR and phospho-GR (Ser²³²) in cytosol and nuclear fractions of C6 glioma cells. The histogram shows the mean±S.D. (n=3–10, * p<0.05 vs. vehicle). The relative expressions of both GR and phospho-GR (Ser²³²) were calculated with respect to the value of the control as 100%. Protein extracts of both cytosol and nuclear fractions were collected from cells cultured with 10 µM CORT for 1, 3, 6, 24, and 72 h. Otherwise, the protein extracts were obtained after 72 h of culture under each culture condition. (B) Immunocytochemical analysis of C6 glioma cells as determined with the use of anti-GR (left column) and phospho-GR (Ser²³²) (right column) antibody under culture with vehicle, 10 µM CORT or 10 µM CORT plus 1 µM mifepristone for 72 h. Scale bars represent 20 µm.

DISCUSSION

In this study, we investigated the effects of CORT on cell proliferation, survival and signaling cascade in astroglial cells using C6 glioma cells.

We demonstrated that C6 glioma cells express both GR and MR and that the proliferation of C6 glioma cells was inhibited by treatment with CORT via GR, but not MR, by using the antagonists mifepristone and spironolactone. Several studies have suggested that stress can attenuate the function of cells in the brain, including both neurons and astroglial cells, in most animals, including humans. It has been reported that glucocorticoids have a cytotoxic effect on hippocampal neurons both in vivo and in vitro.^23,24) We previously reported that CORT exhibited a cytotoxic effect on hippocampal neurons from mice embryo and in BV2 microglia cell lines.^16,25) These reports indicated that the function of astroglial cells may also be affected by treatment with CORT. It has been demonstrated that glucocorticoid inhibited the proliferation of primary cultured astrocytes in vitro.^14,26) In addition, it has been shown that chronic treatment with CORT decreased the number of astrocytes in the hippocampus in rats.²⁷⁾ Furthermore, chronic social stress has been shown to inhibit cell proliferation in the adult medial prefrontal cortex and this may be related to the abnormality in the number of glial cells induced by gliogenesis.¹²⁾

As these studies suggest, the secretion of CORT that accompanies stress stimuli may affect the function of astroglial cells. Actually, it is well known that astroglia cells have various transporters and ion channels which can regulate the potassium concentration in the intercellular gap, which is called potassium buffering.²⁸⁾ Astroglial cells can also attenuate the cytotoxic effect of glutamic acid, which is particularly potent in neurons.^29,30) As mentioned above, we observed a decrease in the proliferation of C6 glioma cells under treatment with CORT. These studies suggest that the decrease in the number of astroglial cells induced by glucocorticoid might increase the vulnerability of neurons to glutamic acid via a reduction in the buffering of glutamic acid toxicity. With regard to the effect of CORT on the number of C6 glioma cells, the results of our LDH assay revealed that CORT did not have a cytotoxic effect on C6 glioma cells. In addition, JC-1 staining showed that treatment with CORT did not affect the mitochondria membrane potential, which reflects energy metabolism and mitochondrial apoptosis. Moreover, treatment with CORT did not change the expression of Bcl-2 or Bax. Several reports have indicated that the CORT-induced change in mitochondrial function caused a decrease in cell-protective ability and energy metabolism or the activation of cell-apoptotic signaling such as that involving Bcl-2 or Bax.^17,18,31,32) A possible explanation for this discrepancy is that CORT, which influences not only cell survival but also intracellular signaling cascades, provides different effects in astrocytes compared to neurons.

To confirm which signaling cascades are involved in the inhibitory effect of CORT on cell proliferation, we investigated proteins that are related to cell proliferation. In the whole-cell fraction, treatment with CORT drastically decreased the expression of GR in a time-dependent manner. On the other hand, there was no change in the expression of MR. These data corresponded to the results of a cell proliferation assay with antagonists, which showed that GR played a role in the inhibitory effect of CORT on the proliferation of C6 glioma cells. In contrast, the expression levels of MAPK cascade-related proteins, which related to cell proliferation or stress responses, did not change. The expression of CREB and its phosphorylated form also did not change at all. In addition, CORT did not influence the expression of mitochondria-controlled apoptosis-related proteins. Unexpectedly, CORT did not show any effects in the expression levels of NF-κB and IκB, although the NF-κB signaling pathway is considered as one of the major pathways for the glucocorticoid receptor signaling cascade. Furthermore, the expression of GFAP did not change at all, even though GFAP plays a role in the proliferation of C6 glioma cells.²¹⁾ These results indicate that major signaling cascades, which are known to be regulated by glucocoritcoid, and other protein expression related to cell proliferation were also not involved in the cell proliferative inhibition in C6 glioma cells.

As next, we investigated whether or not GR phosphorylation was involved in the inhibitory effect of CORT on the proliferation of C6 glioma cells because GR phosphorylation differentially affects target gene expression.²²⁾ Under treatment with CORT, the expression of phospho-GR (Ser²³²) was drastically increased in C6 glioma cells, while the expression of phospho-GR (Ser²⁴⁶) did not change at all. To observe whether or not phospho-GR (Ser²³²) is related to the inhibitory effect of CORT on the proliferation of C6 glioma cells, the inhibitory effect of CORT on cell proliferation was checked under treatment with BAPTA-AM, which can inhibit the phosphorylation of GR. Unexpectedly, treatment with BAPTA-AM did not affect the inhibitory effect of CORT on cell proliferation at all. In addition, mifepristone, but not spironolactone, completely negated the inhibitory effect of CORT on the proliferation of C6 glioma cells in which the phosphorylation of GR was inhibited by BAPTA-AM. These results indicate that the GR phosphorylation of Ser²³² is not involved in the inhibitory effect of CORT on the proliferation of C6 glioma cells. Finally, to reveal whether phosphorylated (Ser²³²) or unphosphorylated-GR is involved in the inhibitory effect of CORT on cell proliferation, a Western blotting analysis using cytosol and nuclear fractions and an immunocytochemical analysis were performed to clarify the subcellular localization. Western blotting analysis showed that the expression of phospho-GR (Ser²³²) was significantly increased in both cytosol and nuclear fractions by treatment with CORT. In contrast, the expression of unphosphorylated-GR increased acutely, while the expression levels in both the cytosol and whole-cell fractions decreased significantly and sustainably. Moreover, the results of the immunocytochemical analysis demonstrated that unphosphorylated-GR, but not phosphorylated-GR (Ser²³²), was translocated and aggregated as dots under treatment with CORT. These results demonstrate that a CORT unphosphorylated-GR complex plays in the inhibitory effect of CORT on cell proliferation at least. As a candidate which is regulated its expression by CORT, cyclin-related proteins, such as cyclin D1, D3, cyclin-dependent kinase 4 or 6, might play a role here, since they are related to cell proliferation and their function is modulated by glucocorticoid.^33–35)

In conclusion, we demonstrated that CORT inhibited the proliferation of C6 glioma cells via GR, but not MR, by using their respective antagonists, mifepristone and spironolactone. In addition, this inhibitory effect of cell proliferation was unrelated to apoptosis or mitochondrial dysfunction. Moreover, our results also indicated a possibility that this inhibitory effect of cell proliferation was correlated to the ligand–receptor complex translocation from the cytosol to the nucleus without the participation of a signaling cascade related to glucocorticoids or GR phosphorylation. Further studies will be needed to elucidate the mechanisms of GR-regulated cell proliferation in astroglial cells and to identify a novel molecular mechanism of the development of depression via astroglial dysfunction related to cell cycle progression.

Acknowledgments

We thank Rina Takiguchi, Masumi Kato and Ikumi Karasawa for their help with the experiments, and the members of the Department of Pharmacotherapeutics, School of Pharmacy, International University of Health and Welfare, for their helpful discussions throughout this study. This work was supported in part by Grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant-in-Aid for Scientific Research (C), 26460703, 2014) (Grant-in-Aid for Young Scientists (B), 15K18878, 2015).

Conflict of Interest

The authors declare no conflict of interest.

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