Polychlorinated biphenyls impair dibutyryl cAMP-induced astrocytic differentiation in rat C6 glial cell line

Abstract

In the central nervous system, alteration of glial cell differentiation can affect brain functions. Polychlorinated biphenyls (PCBs) are persistent environmental chemical contaminants that exert neurotoxic effects in glial and neuronal cells. We examined the effects of a commercial mixture of PCBs, Aroclor1254 (A1254) on astrocytic differentiation of glial cells, using the rat C6 cell line as in vitro model. The exposure for 24 h to sub-toxic concentrations of A1254 (3 or 9 μM) impaired dibutyryl cAMP-induced astrocytic differentiation as showed by the decrease of glial fibrillary acidic protein (GFAP) protein levels and inhibition in change of cell morphology toward an astrocytic phenotype. The A1254 inhibition was restored by the addition of a protein kinase C (PKC) inhibitor, bisindolylmaleimide (bis), therefore indicating that PCBs disturbed the cAMP-induced astrocytic differentiation of C6 cells via the PKC pathway. The phosphorylation of signal transducer and activator of transcription 3 (STAT3) is essential for cAMP-induced transcription of GFAP promoter in C6 cells. Our results indicated that the exposure to A1254 (3 or 9 μM) for 24 h suppressed cAMP-induced STAT3 phosphorylation. Moreover, A1254 reduced cAMP-dependent phosphorylation of STAT3 requires inhibition of PKC activity. Together, our results suggest that PCBs induce perturbation in cAMP/PKA and PKC signaling pathway during astrocytic differentiation of glial cells.

Keywords: A1254, Aroclor 1254; Aroclor1254; Astrocytic differentiation; C6 glial cell line; CNS, central nervous system; CRE, cAMP responsive element; CREB, cAMP-response element binding protein; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco’s Modified Eagle’s Medium; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; Glial fibrillary acidic protein (GFAP); MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; NMDA, N-methyl-d-aspartate; PCBs, polychlorinated biphenyls; PKA, protein kinase A; PKC, protein kinase C; Protein kinase C (PKC); ROS, reactive oxygen species; STAT3, signal transducer and activator of transcription 3; Signal transducer and activator of transcription 3 (STAT3); TRE, CRE transcriptional response element; bis, 2-[1-(3-dimethylamino-propyl)indol-3-yl]-3-(indol-3-yl) maleimide; dbcAMP, N6,2′-O-dibutyryl cAMP; nNOS, neuronal nitric oxide.

Fig. 1

Exposure to A1254 affected cell viability in a concentration and time-dependent manner in C6 cells. (A) Cells were exposed to increasing concentrations of A1254 (0.05–90 μM) for 24 h in serum-free medium with 0.1% DMSO (vehicle). (B) C6 cells were treated with A1254 (3 or 9 μM) for 48 and 72 h. After incubation, cell viability was evaluated using the MTT assay, as described in Section 2.3. The cell viability was calculated vs untreated control cells, cultured in serum-free-DMEM with 0.1% DMSO (vehicle) at the respective time of incubation (24, 48 or 72 h) set as 100%. Results are presented as percentage (mean ± SEM) (n = 3) of the control cells. Significant difference from the untreated control; ^*p < 0.05; ^§p < 0.01; ^#p < 0.001.

Fig. 2

Exposure to A1254 decreased dbcAMP-induced GFAP protein expression levels in C6 cells. Control and C6 cells treated with A1254 (3 or 9 μM) in presence or absence of dbcAMP (1 mM) were harvested after 24 h and assayed for GFAP protein expression levels. Equal amounts of protein cell lysates (20 μg) were subjected to protein analysis by 12% SDS–PAGE. (A) Western blotting showing GFAP protein expression levels. GAPDH was used as loading control for cell lysates. Signals were revealed by immunostaining and ECL, as described in Section 2.5. (B) Densitometric analysis of GFAP expression levels. Fold change in GFAP levels was calculated by first normalizing to GAPDH levels in individual samples and then relative to untreated control (cells cultured in serum-free DMEM with 0.1% (v/v) DMSO, vehicle) set as 1. Each bar represents the mean ± SEM (n = 3). Columns with (#) were statistically different from control and dbcAMP-differentiated cells (^#p < 0.001).

Fig. 3

Effects of A1254 exposure on GFAP immunoreactivity in dbcAMP differentiating C6 cells. C6 cells were subjected to different treatments, fixed and then immunocytochemistry was performed as detailed in Section 2.4. C6 cells were cultured in DMEM with 10% FBS (A–C) or kept in serum-free DMEM in presence (G–I) or absence (D–F) of 0.1% (v/v) DMSO (vehicle). Cells exposed to: 3 μM A1254 (J–L); 9 μM A1254 (M–O); 1 mM dbcAMP (P–R). dbcAMP-differentiating cells exposed to A1254 3 μM (S–U) or 9 μM (V–X). After the treatments, cells were subjected to GFAP immunostaining (green) (A, D, G, J, M, P, S and V). DAPI-nuclear stain (blue) of the same field (B, E, H, K, N, Q, T and W). Merge for composite images (C, F, I, L, O, R, U and X). All the treatments were performed for 24 h under serum-free conditions in presence of 0.1% (v/v) DMSO used as vehicle for A1254. Scale bar = 50 μm. (For interpretation of color in Fig. 3, the reader is referred to the web version of this article.)

Fig. 4

Effects of A1254 exposure on morphological change in dbcAMP-differentiating C6 cells. C6 cells were subjected to different treatments, fixed and then subjected to phase- contrast analysis, as detailed in Section 2.4. Phase-contrast micrographs of C6 cells cultured in DMEM with 10% serum (A) or kept in serum-free DMEM with (C) or without (B) of 0.1% (v/v) DMSO (vehicle). Cells exposed to: 3 μM A1254 (D); 9 μM A1254 (E); 1 mM dbcAMP (F). dbcAMP-differentiating cells exposed to A1254 3 μM (G) or 9 μM (H). All the treatments were performed for 24 h under serum-free conditions in presence of 0.1% (v/v) DMSO, used as vehicle for A1254. Scale bar = 25 μm.

Fig. 5

Effect of exposure to the protein kinase C inhibitor, bisindolylmaleimide, (bis) on C6 cell survival. (A) Concentration-dependent cytotoxic effects were evaluated by exposure for 24 h to increasing concentrations of A1254 (0.05–90 μM) in serum-free medium with 0.1% (v/v) DMSO (vehicle). (B) Effects of co-exposure to bis (0.125 μM) and A1254 (3 or 9 μM) of dbcAMP stimulated cells. Control treatments were performed as reported in the figure. After the treatments, cell survival was determined by MTT assay, as reported in Section 2.3. Results are presented as percentage (mean ± SEM) (n = 3) of the control cells cultured in serum-free DMEM with 0.1% (v/v) DMSO (vehicle) set as 100%. Significant difference from the control; ^*p < 0.05; ^§p < 0.01.

Fig. 6

Effect of the protein kinase C inhibitor, bisindolylmaleimide (bis) on GFAP protein expression levels in dbcAMP-stimulated C6 cells exposed to A1254. Cells were treated with bis (0.125 μM) or co-exposed to bis (0.125 μM) and A1254 (3 or 9 μM) in presence of dbcAMP (1 mM) in serum-deprived medium containing 0.1% (v/v) DMSO, used as vehicle for A1254 and bis. After 24 h incubation, treated and untreated control cells were harvested and assayed for GFAP protein expression levels. Equal amounts of protein cell lysates (20 μg) were subjected to protein analysis by 12% SDS–PAGE. (A) Western blotting showing GFAP protein expression levels. GAPDH was used as loading control for cell lysates. Signals were revealed by immunostaining and ECL, as described in Section 2.5. (B) Densitometric analysis of GFAP protein expression levels. Fold change in GFAP protein levels was calculated by first normalizing to GAPDH levels in individual samples and then relative to untreated control cells cultured in serum-free DMEM with 0.1% (v/v) DMSO, (vehicle) set as 1. Each bar represents the mean ± SEM (n = 3). Columns with (#) were statistically different from untreated control cells or dbcAMP-differentiated cells (# p<0.001).

Fig. 7

Effect of co-exposure to A1254 and PKC inhibitor, bis, on dbcAMP-induced GFAP immunoreactivity and morphological change in C6 cells. C6 cells were subjected to different treatments, fixed and then immunocytochemistry (A) and phase-contrast (B) analysis were performed as detailed in Section 2.4. Cells were treated with bis (0.125 μM) or co-exposed to bis (0.125 μM) and A1254 (3 or 9 μM) in presence of dbcAMP (1 mM) in serum-deprived medium containing 0.1% (v/v) DMSO. After 24 h incubation, cells were subjected to immunofluorescence analysis to reveal GFAP immunoreactivity (green). Immunocytochemistry of cells treated with 0.1% (v/v) DMSO (vehicle) (A–C); cells exposed to bis (0.125 μM) (D–F). Astrocytic differentiation induced by treatment with dbcAMP (1 mM) in presence of bis (0.125 μM) (G–I). dbcAMP-differentiated cells co-exposed to: bis (0.125 μM) and 3 μM A1254 (J–L): bis (0.125 μM) and 9 μM A1254 (M–O). GFAP immunostaining (A, D, G, J and M). DAPI-nuclear stain (blue) of the same field (B, E, H, K, and N). Merge for composite images (C, F, I, L and O). Scale bar = 50 μm. (B) Phase-contrast micrographs of C6 cells cultured in serum-free DMEM in presence (B) or absence (A) of bis (0.125 μM). dbcAMP-differentiating cells were treated with bis (0.125 μM) alone (C) or in combination with A1254 3 μM (D) or 9 μM (E). All the treatments were performed for 24 h under serum-free conditions in presence of 0.1% (v/v) DMSO used as vehicle for A1254 and bis. Scale bar = 25 μm. (For interpretation of color in Fig. 7, the reader is referred to the web version of this article.)

Fig. 8

Effects of A1254 treatment on PKC activity (A) and activation status of STAT3 (B) in bis co-treated dbcAMP C6 cells. C6 cells were treated or not with A1254 (3 or 9 μM) in presence or absence of the PKC inhibitor, bis (0.125 μM) during dbcAMP (1 mM) stimulation in serum-deprived medium containing 0.1% (v/v) DMSO, used as vehicle for A1254 and bis. After 24 h incubation, control and treated cells were harvested and equal amounts of protein cell lysates (20 μg) were separated by 12% SDS–PAGE and subjected to immunostaining, as described in Section 2.5. (A) Western blotting using an anti-phospho-(Ser) PKC substrates showing PKC substrates phosphorylation. Arrows indicated the more abundant phosphorylated protein bands (Mr of 200, 78, 65 and 28 kDa). (B) Western blotting showing phospho-Serine727 form of STAT3 and STAT3 total protein expression levels. GAPDH was used as loading control for cell lysates. Signals were revealed by immunostaining and ECL, as described in Section 2.5. (C) Densitometric analysis of phospho-STAT3 and total protein expression levels; fold changes were calculated by first normalizing to GAPDH levels in individual samples and then relative to untreated control (cells cultured in serum-free DMEM with 0.1% (v/v) DMSO, vehicle) set as 1. Each bar represents the mean ± SEM (n = 3). Columns with (#) were statistically different from untreated control or dbcAMP-differentiated cells (^#p < 0.001).