Activation of a pro-survival pathway IL-6/JAK2/STAT3 contributes to glial fibrillary acidic protein induction during the cholera toxin-induced differentiation of C6 malignant glioma cells

Abstract

Differentiation-inducing therapy has been proposed to be a novel potential approach to treat malignant gliomas. Glial fibrillary acidic protein (GFAP) is a well-known specific astrocyte biomarker and acts as a tumor suppressor gene (TSG) in glioma pathogenesis. Previously we reported that a traditional biotoxin cholera toxin could induce malignant glioma cell differentiation characterized by morphologic changes and dramatic GFAP expression. However, the molecular mechanisms underlying GFAP induction are still largely unknown. Here we demonstrate that an oncogenic pathway interleukin-6/janus kinase-2/signal transducer and activator of transcription 3 (IL-6/JAK2/STAT3) cascade mediates the cholera toxin-induced GFAP expression. Cholera toxin dramatically stimulated GFAP expression at the transcriptional level in C6 glioma cells. Meanwhile, phosphorylation of STAT3 and JAK2 was highly induced in a time-dependent manner after cholera toxin incubation, whereas no changes of STAT3 and JAK2 were observed. Furthermore, the IL-6 gene was quickly induced by cholera toxin and subsequent IL-6 protein secretion was stimulated. Importantly, exogenous recombinant rat IL-6 can also induce phosphorylation of STAT3 concomitant with GFAP expression while JAK2 specific inhibitor AG490 could effectively block both cholera toxin- and IL-6-induced GFAP expression. Given that the methylation of the STAT3 binding element can suppress GFAP expression, we detected the methylation status of the critical recognition sequence of STAT3 in the promoter of GFAP gene (-1518 ∼ -1510) and found that it was unmethylated in C6 glioma cells. In addition, neither DNA methyltransferase1 (DNMT1) inhibitor 5-Aza-2'-deoxycytidine (5-AZa-CdR) nor silencing DNMT1 can stimulate GFAP expression, indicating that the loss of GFAP expression in C6 cells is not caused by its promoter hypermethylation. Taken together, our findings suggest that activation of a pro-survival IL-6/JAK2/STAT3 cascade contributes to cholera toxin-induced GFAP expression, which implies that a survival-promoting signal may also play a differentiation-supporting role in malignant gliomas.

Figure 1

Cholera toxin induces differentiation of C6 malignant glioma cells. (A), morphologic transformation (Original magnification: ×200). C6 cells were incubated with 10 ng/ml cholera toxin for 48 h. (B), Time‐dependent effect of GFAP protein expression. (C), Time‐dependent effect of GFAP mRNA level. C6 cells were incubated with 10 ng/ml cholera toxin for the indicated times.

Figure 2

Cholera toxin activates IL‐6/JAK2/STAT3 pathway in C6 glioma cells. Time‐dependent effects of phosphorylation of STAT3(tyr705). (A), phosphorylation of JAK2(tyr1007/1008). (B), IL‐6 protein expression. (C) and mRNA level. (D). C6 cells were incubated with 10 ng/ml cholera toxin for the indicated times, ∗∗, P < 0.01.

Figure 3

IL‐6/JAK2/STAT3 pathway is involved in cholera toxin‐induced up‐regulation of GFAP. (A), C6 cells were incubated with recombinant rat IL‐6 (100 ng/ml) or 10 ng/ml cholera toxin for 48h. (B), C6 cells were pretreated with 12.5 μM AG490 for 2 h and then treated with 10 ng/ml cholera toxin for an additional 48 h. (C), C6 cells were pretreated with 12.5 μM AG490 for 2 h and then treated with 100 ng/ml recombinant rat IL‐6 for further 48 h.

Figure 4

Cholera toxin‐induced GFAP expression is independent on its promoter demethylation in C6 glioma cells. (A), A schematic of the particular binding side of STAT3 in the promoter of GFAP gene. (B), C6 glioma cells were treated with or without cholera toxin for 48 h, then the methylation status was measured by bisulfite sequencing analysis. (C), C6 glioma cells were treated with 10 μM 5‐AZA‐CdR or transfected with 50 nM siDNMT1 oligonucleotides for 48 h for western blotting to evaluate DNMT1 and GFAP expression.