Objective To investigate effect of tumor necrosis factor-or （TNF-α） 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-α （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-α （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-α 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-α induced translocation of SSeCKS perinuclear. This effect could be partly reversed by PKC inhibitor Ro-31-8220. Conclusion TNF-α 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.
目的研究细菌脂多糖(LPS)对培养的大鼠星形胶质细胞中Src抑制的蛋白激酶C的底物(SSeCKS)表达的影响。方法培养的星形胶质细胞随机分为空白对照组、LPS单一刺激组、LPS联合PKC抑制剂(RO-31-8220)刺激组。运用荧光定量PCR(Real time RT-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Real time RT-PCR显示,LPS可以上调SSeCKS mRNA水平,在作用浓度为100μg/L和1 mg/L时与对照组有显著差异(P<0.01)。Western blotting表明,当LPS作用浓度为100μg/L时,SSeCKS蛋白表达量明显上调;在此浓度作用下,SSeCKS表达于6 h达高峰并广泛磷酸化,至24 h其蛋白表达仍维持于较高水平。免疫细胞化学分析显示,正常情况下,SSeCKS散在分布于胞质,于胞膜略有浓集。在LPS单一刺激组,SSeCKS富集于核周;当LPS联合RO-31-8220共同作用时,SSeCKS的亚细胞定位与正常组无明显差异。结论在体外培养的星形胶质细胞中,LPS可诱导SSeCKS表达,上调其磷酸化水平,影响其细胞内定位。这些改变与PKC的功能相关。提示SSeCKS可能参与星形胶质细胞中炎症信号的转导。
