Department of Anesthesiology and Pain Medicine, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan.
J Neuroinflammation. 2010 Mar 6;7:16. doi: 10.1186/1742-2094-7-16.
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.
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.
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.
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.
白细胞介素 (IL)-6 在多种中枢神经系统功能中发挥关键作用，如诱导和调节反应性星形胶质细胞增生、病理性炎症反应和神经保护。肿瘤坏死因子 (TNF)-α 通过抑制κB (IkappaB)-核因子 κB (NFkappaB) 途径、p38 丝裂原激活蛋白 (MAP) 激酶和应激激活蛋白激酶 (SAPK)/c-Jun N-末端激酶 (JNK) 诱导大鼠 C6 神经胶质瘤细胞释放 IL-6。本研究比以往的研究更详细地研究了 TNF-α诱导 IL-6 释放的机制。
用 TNF-α刺激培养的 C6 细胞。通过酶联免疫吸附试验测量细胞中 IL-6 的释放，并通过 Western 印迹分析 IkappaB、NFkappaB、MAP 激酶超家族和信号转导和转录激活因子 (STAT)3 的磷酸化。通过实时逆转录聚合酶链反应评估细胞中 IL-6 mRNA 的水平。
TNF-α显著诱导 NFkappaB 在 Ser 536 和 Ser 468 处磷酸化，但不在 Ser 529 或 Ser 276 处磷酸化。 Wedelolactone，一种 IkappaB 激酶抑制剂，抑制 TNF-α诱导的 IkappaB 磷酸化和 NFkappaB 在 Ser 536 和 Ser 468 处的磷酸化。Wedelolactone 抑制 TNF-α刺激的 IL-6 水平增加。TNF-α诱导 STAT3 磷酸化。Janus 家族酪氨酸激酶 (JAK) 抑制剂 I，一种 JAK1、2 和 3 的抑制剂，减弱 TNF-α诱导的 STAT3 磷酸化，并显著降低 TNF-α刺激的 IL-6 释放。NADPH 氧化酶抑制剂 Apocynin 抑制细胞内活性氧，显著抑制 TNF-α诱导的 IL-6 释放和 mRNA 表达。然而，Apocynin 不影响 IkappaB、NFkappaB、p38 MAP 激酶、SAPK/JNK 或 STAT3 的磷酸化。
这些结果强烈表明，TNF-α在 C6 神经胶质瘤细胞中通过 p38 MAP 激酶和 SAPK/JNK 以外的 JAK/STAT3 途径诱导 IL-6 合成，并且 NFkappaB 在 Ser 536 和 Ser 468 的磷酸化和 NADPH 氧化酶参与 TNF-α 刺激的 IL-6 合成。
