The Effect Of Ssecks In Schwann Cells Under Inflammation

ObjectiveTo investigate the role of Src-suppressed protein kinase C substrate (SSeCKS) in the Shwann cells'tumor necrosis factor-alpha (TNF-α) autocine, growth arrest and myelination disorder underlying inflammation condition.Methods1. To analyses the effect of SSeCKS in TNF-αautocrine, primary Schwann cells were treated with TNF-α, the secretion of TNF-αwere detected by enzyme linked immunosorbent assay (ELISA), the expression of SSeCKS were analysis by Western Blot and reverse transcription polymerase chain reaction (RT-PCR), the activation of p38 and Jun N-terminal kinase (JNK) were analysis by Western Blot. After that, Schwann cells were transfected with the SSeCKSαisoform siRNA and overexpression vector, and then the secretion of TNF-αand the activation of p38 and JNK were analyses.2. To analyses the effect of SSeCKS in Schwann cells growth arrest caused by TNF-α, concentrations recombined rat TNF-α were used, and 5'-bromodeoxyuridine (BrdU) labeling shown the proliferation and RT-PCR and Western Blot shown the expression and phosphorylation of SSeCK. Then siRNA vector were used knockdown SSeCKSαisoform expression and the expression of cyclin D1, the activation of extracellular signal-regulated kinase (ERK1/2), the localization of cyclin D1 were detected.3. To analyze the function of SSeCKS in Shwann cells differentiation and myelination. Primary Schwann cells and dorsal root ganglion (DRG) were cultured in vitro. Shwann cells were treated with cAMP and the expression of SSeCKS was detected by Western Blot. And then the EGFP-SSeCKS siRNA lentvirus were used to knockdown SSeCKS expression. After that the morphological changes were detected by immunofluorescence staining, the expression of differentiated and myelinated marked, the activation of protein kinase B (PKB/Akt) was analyzed by Western Blot.Results1. Tumor necrosis factor-alpha (TNF-α) derived from activated Shawn cells could induced the increasing of TNF-αmRNA and secretion, accompany with the increase of the mRNA and protein levels of SSeCKSαisoform and the activation of p38 and JNK pathways. Knockdown SSeCKS expression suppressed TNF-αproduction induced by TNF-α, and overexpression SSeCKS could promoted TNF-αautocrine in Shawn cells. Western Blot shown knockdown SSeCKS expression could suppressed the promote p38 and JNK activation in Schwann cells treated by TNF-α. And the inhibitors of p38 and JNK could prevent the increased autocine caused by SSeCKS overexpression.2. TNF-αinduced both SSeCKSαisoform expression, phosphorlation and Shwann cells growth arrest in a dose-dependent manner. By knocking down SSeCKSαisoform expression, TNF-α-induced growth arrest in Shwann cells was partially rescued. Concurrently, the expression of cyclin D1 was reduced and the activity of ERK1/2 was decreased. A luciferase activity assay showed that cyclin D1 expression was regulated by SSeCKS at the transcription level. In addition, the cell fragments assay and immunofluorescence revealed that TNF-αprevented the translocation of cyclin D1 into the nucleus, while knocking down SSeCKSαisoform expression prompted cyclin D1 redistribution to the nucleus.3. SSeCKS were decreased in differentiated Schwann cells. In long-term SSeCKS-reduced Schwann cells, cell morphology changed and myelin gene expression induced by cAMP was accelerated. Myelination was also enhanced in SSeCKS-suppressed Schwann cells co-culture with DRG. In addition, we found suppression of SSeCKS expression promoted Akt serine 473 phosphorylation in cAMP treated Schwann cells.Conclusions1. TNF-αautocine were existed in Shwann cells, and the expression of SSeCKS could promoted TNF-αsecretion activating of p38 and JNK pathways.2. SSeCKS may play a critical role in TNF-α-induced Shwann cells growth arrest. In the process, SSeCKS could inhibit cyclin D1 expression via preventing ERK1/2 activation and thus preventing its nuclear translocation via increasing the binding ability withcyclin D1. 3. SSeCKS is a negative regulator in Shwann cells differentiation and myelination, which were caused by inhabiting the activation of Akt.