SSeCKS promotes tumor necrosis factor-alpha autocrine via activating p38 and JNK pathways in Schwann cells

Abstract

Tumor necrosis factor-alpha (TNF-alpha) derived from activated Schwann cells (SCs) plays a critical role as an inflammatory mediator in the peripheral nervous system disease. TNF-alpha could act as an autocrine mediator in SC activation. In this study, we found knockdown Src-suppressed protein kinase C substrate (SSeCKS) expression suppressed TNF-alpha production induced by TNF-alpha, overexpression of SSeCKS could promoted TNF-alpha autocrine in SCs. Such effects might be resulted in SSeCKS promoted p38 and JNK activation in SCs treated by TNF-alpha. Thus present data show that while SCs activation, SSeCKS may plays an important role in the release of inflammatory mediators.

Fig. 1

TNF-α induces the expression of TNF-α and SSeCKS in cultured SCs. Cultures were untreated or treated with TNF-α (50 ng/ml) for 24 h. The expression of TNF-α mRNA (a) and SSeCKS mRNA (c) were detected by RT-PCR. At the same time, the secretion of TNF-α was detected by ELISA (b), and the change of SSeCKS protein level was analyzed by immunoblot (d). GAPDH was used as an internal control. Statistical differences compared with the controls (untreated) were given as * P < 0.05

Fig. 2

SSeCKS expression impaired TNF-α secretion induced by TNF-α. SCs were transfected with a non-specific siRNA or SSeCKS siRNA and then treated with TNF-α (50 ng/ml) for 24 h. a RT-PCR was used to assess the efficiency of the siRNA vector transfection. b ELISA was used to detect the secretion of TNF-α. c Light microscope shown EGFP–SSeCKS were expressed in SCs. d ELISA shown the secretion of TNF-α in SSeCKS-overexpressed SCs. Data were expressed as mean ± SEM of the maximum response observed. Statistical differences compared with the normal SCs were given as * P < 0.05 and with the TNF-α treated normal SCs group as ^# P < 0.05

Fig. 3

Effect of p38 and JNK signal pathways in TNF-α-induced TNF-α production. a Primary SCs were treated with TNF-α (50 ng/ml) for 24 h, and then cells were lysed and proceeded for analysis of the expression of phosphorylated p38 and JNK (p-p38 and p-JNK) and total p38 and JNK (t-p38 and t-JNK). b Cells were pretreated with SB202190 (SB) and SP600125 (SP) for 40 min, and then stimulated with TNF-α for 24 h. Culture mediums were harvested and ELISA was used to assay the level of TNF-α. These data were means ± SEM. * P < 0.05 comparing with the untreated groups. ^# P < 0.05 comparing with the TNF-α-treated group

Fig. 4

Knockdown SSeCKS expression reduces phosphorylation of p38 and JNK induced by TNF-α. a SCs were transfected with a non-specific siRNA or SSeCKS siRNA and then exposed to 50 ng/ml TNF-α for 24 h. Whole-cell lysates were prepared and immunoblotted with the antibodies against the p-p38, p-JNK, t-p38, t-JNK. Densitometric values were plotted and values represented the means ± SEM. * P < 0.05 vs. TNF-α untreated non-specific siRNA-transfected SCs. ^# P < 0.05 comparing with the TNF-α-treated non-specific siRNA-transfected SCs. b SCs were transfected with EGFP or EGFP–SSeCKS overexpression vectors and pretreated with SB202190 (SB) and SP600125 (SP) for 40 min following by TNF-α treatment for 24 h. Densitometric values were plotted and values represented the means ± SEM. * P < 0.05 vs. TNF-α untreated SCs. ^# P < 0.05 comparing with the TNF-α treated SSeCKS over-expressed SCs