The Role Of RAGE In The Secretion Of TNF-α And IFN-γ Induced By MGO In Jurkat Cells

Objective: Methylglyoxal (MGO) is a reactive metablite of glucose and is efficientlycatabolised to D-lactate by glyoxalase-1. The receptor for advanced glycation endproducts(RAGE) regulates the level of glyoxalase-1(GLO-1). The plasma concentration of MGOand the expression of RAGE are increased in diabetic patients. Both MGO and thereceptor for advanced glycation endproducts (RAGE) have been shown to play a causativerole in atherosclerosis. However, whether they function interactively in the processremains uncertain. Recently, we have demonstrated enhanced expression of tumornecrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) in Jurkat cells induced byMGO. We therefore studied the effects of RAGE on the methylglyoxal induced expressionof TNF-α and IFN-γ in Jurkat cells.Methods:①Jurkat cells which prestimulated by PHA (0.25μg/ml) for24h were incubatedin different concentrations of MGO (0,15,30,60μM) for another24h, then the expressionof RAGE was determined by Western blot.②Jurkat cells were incubated in differentconcentrations of MGO for24h, then the releases of TNF-а and IFN-γ were determined byELISA. To further explore the role of RAGE in the expression of cytokines, neutralizingantibody for RAGE was added to the cells60min before the addition of30μM MGO.③Jurkat cells were treated with different concentrations of MGO for24h, the expression ofCaMKIV was determined by Western blot. In order to study the nuclear translocation ofCaMKIV, Jurkat cells were treated with30μM MGO for0,15,30and60min, and thenuclear translocation of CaMKIV was observed by immunofluorescence assay.④Jurkatcells were treated with30uM MGO for0-60min, the expression of pp38MAPK/p38MAPK was determined by Western blot. Neutralizing antibody for RAGE was usedbefore MGO treated in addition.⑤For the purpose of exploring the role of CaMKIV andp38MAPK in expression of cytokines, the inhibitor KN62and SB203580were added tothe cells30min before the addition of30uM MGO, the productions of TNF-α and IFN-γ were investigated by ELSIA.Results:①E xposure of PHA-actived Jurkat cells to MGO enhanced the expression ofRAGE.②From ELISA, we found a significant increase in TNF-α and IFN-γ expressionsin Jurkat cells exposed to MGO; Furthermore, the MGO-stimulated TNF-α and IFN-γexpressions were significantly suppressed by a neutralizing Abs for RAGE.③Exposure ofPHA-actived Jurkat cells to MGO enhanced the expression of CaMKIV. Furthermore, theMGO-stimulated CaMKIV was significantly suppressed by Abs for RAGE. Afterexposure to the30μM MGO for0-60min, the translocation of CaMKIV protein wasaccelerated by fluorescence microscopy; In addition, the nuclear translocation of CaMKIVinduced by MGO were blocked by a neutralizing RAGE antibody.④After exposure to the30μM MGO for0-60min, the expression of pp38/p38MAPK increases, but were groweddownwards by RAGE antibody.⑤TNF-α and IFN-γ expression were significantlysuppressed in the presence of the specific inhibitors SB203580and KN62respectively.Conclusion: The findings from this study suggested that MGO can up-regulate theexpression of RAGE in Jurkat cells, and promote the secretion of inflammatory cytokinesTNF-α and IFN-γ via RAGE, p38MAPK and CaMKIV signal pathways.