Mechanisms of tumor necrosis factor-α-induced interleukin-6 synthesis in glioma cells

Background: interleukin (il)-6 plays a pivotal role in a variety of cns functions such as the induction and modulation of reactive astrogliosis,pathological inflammatory responses and neuroprotection. tumor necrosis factor (tnf)-α induces il-6 release from rat c6 glioma cells through the inhibitory kappa b (iκb)-nuclear factor kappa b (nfκb) pathway,p38 mitogen-activated protein (map) kinase and stress-activated protein kinase (sapk)/c-jun n-terminal kinase (jnk). the present study investigated the mechanism of tnf-α-induced il-6 release in more detail than has previously been reported.methods: cultured c6 cells were stimulated by tnf-α. il-6 release from the cells was measured by an enzyme-linked immunosorbent assay,and the phosphorylation of iκb,nfκb,the map kinase superfamily,and signal transducer and activator of transcription (stat)3 was analyzed by western blotting. levels of il-6 mrna in cells were evaluated by real-time reverse transcription-polymerase chain reaction.results: tnf-α significantly induced phosphorylation of nfκb at ser 536 and ser 468,but not at ser 529 or ser 276. wedelolactone,an inhibitor of iκb kinase,suppressed both tnf-α-induced iκb phosphorylation and nfκb phosphorylation at ser 536 and ser 468. tnf-α-stimulated increases in il-6 levels were suppressed by wedelolactone. tnf-α induced phosphorylation of stat3. the janus family of tyrosine kinase (jak) inhibitor i,an inhibitor of jak 1,2 and 3,attenuated tnf-α-induced phosphorylation of stat3 and significantly reduced tnf-α-stimulated il-6 release. apocynin,an inhibitor of nadph oxidase that suppresses intracellular reactive oxygen species,significantly suppressed tnf-α-induced il-6 release and mrna expression. however,apocynin failed to affect the phosphorylation of iκb,nfκb,p38 map kinase,sapk/jnk or stat3.conclusion: these results strongly suggest that tnf-α induces il-6 synthesis through the jak/stat3 pathway in addition to p38 map kinase and sapk/jnk in c6 glioma cells,and that phosphorylation of nfκb at ser 536 and ser 468,and nadph oxidase are involved in tnf-α-stimulated il-6 synthesis. © 2010 tanabe et al; licensee biomed central ltd.