GSK-3β对小鼠骨髓树突状细胞成熟和功能的调控作用

摘要

目的：探讨GSK-3β对小鼠骨髓树突状细胞（BMDC）成熟和功能的调控作用。方法脂多糖催熟BMDC，Western blotting检测刺激前后糖原合成酶激酶-3β（GSK-3β）的磷酸化水平的改变；利用GSK-3β的选择性抑制剂SB216763处理BMDC，检测表型、细胞因子表达和混合淋巴反应（MLR）的变化情况；构建过表达小鼠GSK-3β的慢病毒载体并转染DC2.4细胞，Western blotting检测过表达GSK-3β对调控树突状细胞（DC）成熟的关键调控因子鸟的网状内皮组织增生病毒癌基因相关B（RelB）蛋白水平的影响。结果经脂多糖处理后，GSK-3βY216磷酸化水平下调，Ser9磷酸化水平显著上调，表明GSK-3β的活性显著下降。抑制GSK-3β的活性能上调DC表面共刺激分子CD40和CD86的表达，削弱脂多糖诱导的促炎细胞因子IL-6和IL-12表达上调，而对抗炎细胞因子IL-10的表达有促进作用，同时降低脂多糖诱导成熟的DC刺激同种异基因T细胞增殖的能力。在未成熟的DC2.4细胞中，过表达GSK-3β能够下调RelB的水平。结论 GSK-3β参与调控DC的成熟和功能，高活性的GSK-3β抑制未成熟树突状细胞（iDC）的自发性成熟，GSK-3β失活后促进DC表型的成熟，而在脂多糖诱导DC分化的过程中GSK-3β发挥促炎作用。GSK-3β能够下调RelB的蛋白水平，有望成为构建新型耐受性DC的新靶点。%Objective To investigate the role of glycogen synthase kinase 3β(GSK-3β) in the maturation and function of murine bone marrow-derived dendritic cells (BMDCs). Methods Mature DCs (mDCs) induced by LPS were examined for GSK-3βphosphorylation level with Western blotting before and after LPS exposure. To explore the role of GSK-3βin maturation and function of DCs, we added SB216763, a selective inhibitor of GSK-3β, in the cell culture of immature DCs (iDCs), and examined CD40 and CD86 expressions in the cells by flow cytometry and the expression of IL-6, IL-12 and IL-10 mRNA by real-time PCR;the changes of the immunogenicity of the cells was evaluated by mixed lymphocyte reaction. The expression of GSK-3βand RelB was examined by Western blotting in DC2.4 cells transfected with a lentiviral vector over-expressing murine GSK-3βgene. Results LPS exposure significantly lowered GSK-3βactivity in iDCs as demonstrated by increased Ser9 phosphorylation and reduced Tyr216 phosphorylation. GSK-3β inhibition induced DC maturation by increasing the expression of surface costimulatory molecules CD40 and CD86, lowered the expressions of IL-6 and IL-12 while enhanced the expression of IL-10 in iDCs, and impaired mixed lymphocyte reaction of the cells. In DC2.4 cells, lentivirus-mediated over-expression of GSK-3βobviously down-regulated the expression of RelB. Conclusion GSK-3βis a crucial enzyme involved in the differentiation and maintenance of an immature phenotype of DCs. GSK-3β is constitutively active in iDCs to inhibit their spontaneous maturation. DCs become phenotypically mature after inhibition of GSK-3β, which also executes a proinflammatory task in DC activation. The reduction of RelB protein levels as a result of GSK-3β overexpression supports GSK-3β as a new target for inducing tolerogenic DCs.