OBJECTIVETo investigate effect of tumor necrosis factor-alpha (TNF-alpha) on the Src-suppressed C kinase substrate (SSeCKS) in C6 glioma cells.

METHODSCultured 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.

RESULTSTNF-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.

CONCLUSIONTNF-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.