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

Janus kinase-2Signal transducer and activator of transcription 3 Glioma Glial fibrillary acidic protein Cholera toxin A B S T R A C TDifferentiation-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 (À1518 w À1510) and found that it was unmethylated in C6 glioma cells. In addition, neither DNA methyltransferase1 (DNMT1) inhibitor 5-Aza-2 0 -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.ª 2011 Federation of European Biochemical Societies.Published by Elsevier B.V. All rights reserved.Abbreviations: IL-6, Interleukin-6; JAK2, Janus kinase-2; STAT3, Signal transducer and activator of transcription 3; GFAP, Glial fibrillary acidic protein; ELISA, enzyme-linked immunosorbent assay; 5-AZA, 5-Aza-2 0 -deoxycytidine; DNMT1, DNA methyltransferase1; siDNMT1, siRNA-mediated knockdown of DNMT1.

MicroRNA 335 Is Required for Differentiation of Malignant Glioma Cells Induced by Activation of cAMP/Protein Kinase A Pathway

“…We have reported that activation of interleukin-6 (IL-6)/Janus tyrosine kinase2 (JAK2)/ signal transducer and activator of transcription3 (STAT3) pathway partially mediates cholera toxin-activated GFAP expression (Shu et al, 2011b), suggesting that endogenous cytokines might be potential candidates for differentiation agents against malignant gliomas. In this study, we found that exogenous miR-335 clearly induced GFAP expression, whereas knockdown of endogenous miR-335 markedly abrogated GFAP accumulation by cholera toxin, indicating that miR-335 is potently required for cAMP/ PKA activation-induced astrocytic differentiation of malignant gliomas and miRNA analogs might work as differentiation agents or synergistic ones against malignant gliomas.…”

Pathophysiological mechanisms regulated by cytokines in gliomas

“…Partial activation of Ras/ p38 MAPK and PI3K/Akt/mTOR pathways has been reported [67]. Oncogenes c-myc and pim [68], as well as src [66] are identified as possible targets and their activation may extend cell survival, triggering the S phase of the cell cycle. The study by Saidi et al [69] in IL-6 knockdown glioma cells revealed decreased transcription of key oncogenic proteins such as mitotic kinases Aurora-A, -B and survivin.…”

“…First of all, IL-6/STAT3 signaling cascade increases the expression of VEGF [64] that strongly stimulates neoangiogenesis by increasing the activity of Sp1 transcription factor that binds to GC rich -88/-50 nt sequence of its promoter [67]. In addition, STAT3 transcription factor binds to the promoter of Glial Fibrillary Acidic Protein (GFAP) [68], an intermediate filament which offers cytoskeleton stability in astrocytes and whose expression could prevent the migration of malignant cells. However, the multiple interactions that occur between the cancer cell and its environment silence this gene and do not allow activation of GFAP.…”

Transgenic mice with increased astrocyte expression of IL-6 show altered effects of acute ethanol on synaptic function

“…Immunohistochemical studies showed increased levels of activated STAT3 (i.e., pSTAT3), primarily in astrocytes and microglia of the IL-6 tg CNS, indicative of activation of IL-6R and consistent with elevated levels of IL-6 in the IL-6 tg CNS (Sanz et al, 2008). The IL-6 tg CNS shows increase expression of IL-6 regulated genes (e.g., GFAP, eb22, Socs3), increased levels of GFAP, and increased levels of activated STAT3 (pSTAT3), the signal transduction molecule through which IL-6 acts to increase GFAP (Gruol et al, 2014; Herrmann et al, 2008; Nelson et al, 2012; Sanz et al, 2008; Shu et al, 2011). The increased sensitivity to seizure activity observed in the IL-6 tg mice was specific for IL-6 in that a similar sensitivity was not observed in transgenic mice that express elevated levels of TNF-α in the CNS through astrocyte expression (Samland et al, 2003).…”