Objective: To investigate effect of tumor necrosis factor-alpha (TNF-alpha) on the Src-suppressed C kinase substrate (SSeCKS) in C6 glioma cells.
Methods: Cultured C6 glioma cells were randomly divided into two groups. In time-dependent group, cells were cultured with TNF-alpha (2 ng/mL) for 0 h, 1 h, 3 h, 6 h, 12 or 24 h, respectively; in dose-dependent group, cells were cultured with TNF-alpha (0 ng/mL, 0.02 ng/mL, 0.2 ng/mL, or 2 ng/mL) for 6 h. The expression of SSeCKS was detected by Realtime PCR and Western blot analysis, and immunocytochemistry was used to investigate SSeCKS's subcellular localization.
Results: TNF-alpha induced rapid phosphorylations of protein kinase C (PKC) substrates in C6 glioma cells, and upregulated SSeCKS expression in a time and concentration dependent manner. Immunocytochemistry suggested that SSeCKS was localized in the cyroplasm and the leading end of podosomal extensions in control groups, while TNF-alpha induced translocation of SSeCKS perinuclear. This effect could be partly reversed by PKC inhibitor Ro-31-8220.
Conclusion: TNF-alpha activates PKC and upregulates SSeCKS expression in C6 glioma cells. These effects are associated with PKC activity, suggesting that SSeCKS plays a role in response to glia activation in PKC mediated pathway.
目的: 研究肿瘤坏死因子(TNF-α)对培养的C6胶质瘤细胞中Src抑制的蛋白激酶C底物(Src-suppressed C Kinase Substrate, SSeCKS)表达的影响。
方法: 根据 TNF-α 刺激时间与浓度的差异, 将培养的C6 胶质瘤细胞随机分为 TNF-α 时间刺激组与浓度刺激组, 运用实时荧光定量PCR (Realtime PCR)、 免疫印迹和免疫细胞化学法分析SSeCKS的表达变化和亚细胞定位。
结果: 细胞因子TNF-α可引起C6胶质瘤细胞中蛋白激酶C (Protein kinase C, PKC)底物的广泛磷酸化, 并以时间及浓度依赖的方式上调PKC底物SSeCKS的表达。 免疫细胞化学分析显示, 正常情况下, SSeCKS 散在分布于细胞质, 浓集于细胞伸长的足突中。 TNF-α刺激后, SSeCKS 向核周迁移。 这些改变可被PKC的抑制剂Ro-31-8220部分抑制。
结论: TNF-α可诱导C6胶质瘤细胞中PKC的活性, 上调SSeCKS表达, 这些改变与PKC的活性相关, 提示SSeCKS可能参与胶质细胞中炎症信号的转导。