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The Effect of SSeCKS in Schwann Cells under Inflammation

Author: TaoTao
Tutor: JiYuHong
School: Nantong University
Course: Immunology
Keywords: Shwann cell Inflammation Src-suppressed C kinasesubstrate Secretion Demyelination Rattus
CLC: R392
Type: Master's thesis
Year: 2010
Downloads: 1
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Abstract

ObjectiveTo investigate the role of Src-suppressed protein kinase Csubstrate (SSeCKS) in the Shwann cells’ tumor necrosis factor-alpha(TNF-α) autocine, growth arrest and myelination disorderunderlying inflammation condition.Methods1. To analyses the effect of SSeCKS in TNF-α autocrine,primary Schwann cells were treated with TNF-α, the secretion ofTNF-α were detected by enzyme linked immunosorbent assay(ELISA), the expression of SSeCKS were analysis by Western Blotand reverse transcription polymerase chain reaction (RT-PCR), theactivation of p38and Jun N-terminal kinase (JNK) were analysisby Western Blot. After that, Schwann cells were transfected withthe SSeCKS α isoform siRNA and overexpression vector, and thenthe secretion of TNF-α and the activation of p38and JNK wereanalyses.2. To analyses the effect of SSeCKS in Schwann cells growtharrest caused by TNF-α, concentrations recombined rat TNF-α were used, and5’-bromodeoxyuridine (BrdU) labeling shown theproliferation and RT-PCR and Western Blot shown the expressionand phosphorylation of SSeCK. Then siRNA vector were usedknockdown SSeCKS α isoform expression and the expression ofcyclin D1, the activation of extracellular signal-regulated kinase(ERK1/2), the localization of cyclin D1were detected.3. To analyze the function of SSeCKS in Shwann cellsdifferentiation and myelination. Primary Schwann cells and dorsalroot ganglion (DRG) were cultured in vitro. Shwann cells weretreated with cAMP and the expression of SSeCKS was detected byWestern Blot. And then the EGFP-SSeCKS siRNA lentvirus wereused to knockdown SSeCKS expression. After that themorphological changes were detected by immunofluorescencestaining, the expression of differentiated and myelinated marked,the activation of protein kinase B (PKB/Akt) was analyzed byWestern Blot.Results1. Tumor necrosis factor-alpha (TNF-α) derived fromactivated Shawn cells could induced the increasing of TNF-αmRNA and secretion, accompany with the increase of the mRNAand protein levels of SSeCKS α isoform and the activation of p38and JNK pathways. Knockdown SSeCKS expression suppressedTNF-α production induced by TNF-α, and overexpression SSeCKScould promoted TNF-α autocrine in Shawn cells. Western Blotshown knockdown SSeCKS expression could suppressed thepromote p38and JNK activation in Schwann cells treated byTNF-α. And the inhibitors of p38and JNK could prevent theincreased autocine caused by SSeCKS overexpression.2. TNF-α induced both SSeCKS α isoform expression,phosphorlation and Shwann cells growth arrest in adose-dependent manner. By knocking down SSeCKS α isoform expression, TNF-α-induced growth arrest in Shwann cells waspartially rescued. Concurrently, the expression of cyclin D1wasreduced and the activity of ERK1/2was decreased. A luciferaseactivity assay showed that cyclin D1expression was regulated bySSeCKS at the transcription level. In addition, the cell fragmentsassay and immunofluorescence revealed that TNF-α prevented thetranslocation of cyclin D1into the nucleus, while knocking downSSeCKS α isoform expression prompted cyclin D1redistributionto the nucleus.3. SSeCKS were decreased in differentiated Schwann cells. Inlong-term SSeCKS-reduced Schwann cells, cell morphologychanged and myelin gene expression induced by cAMP wasaccelerated. Myelination was also enhanced inSSeCKS-suppressed Schwann cells co-culture with DRG. Inaddition, we found suppression of SSeCKS expression promotedAkt serine473phosphorylation in cAMP treated Schwann cells.Conclusions1. TNF-α autocine were existed in Shwann cells, and theexpression of SSeCKS could promoted TNF-α secretion activatingof p38and JNK pathways.2. SSeCKS may play a critical role in TNF-α-induced Shwanncells growth arrest. In the process, SSeCKS could inhibit cyclin D1expression via preventing ERK1/2activation and thus preventingits nuclear translocation via increasing the binding ability withcyclin D1.3. SSeCKS is a negative regulator in Shwann cellsdifferentiation and myelination, which were caused by inhabitingthe activation of Akt.

The Effect of SSeCKS in Schwann Cells under Inflammation

Abstract

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