Mechanisms of tumor necrosis factor-alpha-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)-alpha induces IL-6 release from rat C6 glioma cells through the inhibitory kappa B (IkappaB)-nuclear factor kappa B (NFkappaB) 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-alpha-induced IL-6 release in more detail than has previously been reported.


METHODS
Cultured C6 cells were stimulated by TNF-alpha. IL-6 release from the cells was measured by an enzyme-linked immunosorbent assay, and the phosphorylation of IkappaB, NFkappaB, 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-alpha significantly induced phosphorylation of NFkappaB at Ser 536 and Ser 468, but not at Ser 529 or Ser 276. Wedelolactone, an inhibitor of IkappaB kinase, suppressed both TNF-alpha-induced IkappaB phosphorylation and NFkappaB phosphorylation at Ser 536 and Ser 468. TNF-alpha-stimulated increases in IL-6 levels were suppressed by wedelolactone. TNF-alpha induced phosphorylation of STAT3. The Janus family of tyrosine kinase (JAK) inhibitor I, an inhibitor of JAK 1, 2 and 3, attenuated TNF-alpha-induced phosphorylation of STAT3 and significantly reduced TNF-alpha-stimulated IL-6 release. Apocynin, an inhibitor of NADPH oxidase that suppresses intracellular reactive oxygen species, significantly suppressed TNF-alpha-induced IL-6 release and mRNA expression. However, apocynin failed to affect the phosphorylation of IkappaB, NFkappaB, p38 MAP kinase, SAPK/JNK or STAT3.


CONCLUSIONS
These results strongly suggest that TNF-alpha 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 NFkappaB at Ser 536 and Ser 468, and NADPH oxidase are involved in TNF-alpha-stimulated IL-6 synthesis.
Tanabe
Kumiko
K
Department of Anesthesiology and Pain Medicine, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan. kumiko-t@m2.gyao.ne.jp
Matsushima-Nishiwaki
Rie
R
Yamaguchi
Shinobu
S
Iida
Hiroki
H
Dohi
Shuji
S
Kozawa
Osamu
O
D000098:Acetophenones / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D045744:Cell Line, Tumor; D003374:Coumarins / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D005910:Glioma / Q000378:metabolism* / Q000473:pathology; D020672:I-kappa B Proteins / Q000378:metabolism; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D050796:STAT3 Transcription Factor / Q000378:metabolism; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D000097782:NF-kappaB-Inducing Kinase
D000098:Acetophenones; D003374:Coumarins; D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D015850:Interleukin-6; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger; D050796:STAT3 Transcription Factor; D014409:Tumor Necrosis Factor-alpha; D000097782:NF-kappaB-Inducing Kinase; C083487:I kappa B beta protein; C051122:wedelolactone; C056165:acetovanillone