Immunology Division, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, J1H 4N5, Canada.
Agriculture and Agri-Food Canada, Dairy and Swine Research and Development Center, 2000 College Street, Sherbrooke, QC, Canada.
Cell Commun Signal. 2019 Mar 4;17(1):21. doi: 10.1186/s12964-019-0334-6.
Platelet-activating factor (PAF) is a potent lipid mediator whose involvement in the onset and progression of atherosclerosis is mediated by, among others, the modulation of cytokine expression patterns. The presence of multiple potential protein-tyrosine phosphatase (PTP) 1B substrates in PAF receptor signaling pathways brought us to investigate its involvement in PAF-induced cytokine expression in monocyte-derived dendritic cells (Mo-DCs) and to study the pathways involved in this modulation.
We used in-vitro-matured human dendritic cells and the HEK-293 cell line in our studies. PTP1B inhibition was though siRNAs and a selective inhibitor. Cytokine expression was studied with RT-PCR, luciferase assays and ELISA. Phosphorylation status of kinases and transcription factors was studied with western blotting.
Here, we report that PTP1B was involved in the modulation of cytokine expression in PAF-stimulated Mo-DCs. A study of the down-regulation of PAF-induced IL-8 expression, by PTP1B, showed modulation of PAF-induced transactivation of the IL-8 promoter which was dependent on the presence of the C/EBPß -binding site. Results also suggested that PTP1B decreased PAF-induced IL-8 production by a glycogen synthase kinase (GSK)-3-dependent pathway via activation of the Src family kinases (SFK). These kinases activated an unidentified pathway at early stimulation times and the PI3K/Akt signaling pathway in a later phase. This change in GSK-3 activity decreased the C/EBPß phosphorylation levels of the threonine 235, a residue whose phosphorylation is known to increase C/EBPß transactivation potential, and consequently modified IL-8 expression.
The negative regulation of GSK-3 activity by PTP1B and the consequent decrease in phosphorylation of the C/EBPß transactivation domain could be an important negative feedback loop by which cells control their cytokine production after PAF stimulation.
血小板激活因子（PAF）是一种强效的脂类介质，其在动脉粥样硬化的发生和发展中的作用是通过调节细胞因子表达模式等多种机制介导的。在 PAF 受体信号通路中存在多种潜在的蛋白酪氨酸磷酸酶（PTP）1B 底物，这促使我们研究其在单核细胞衍生的树突状细胞（Mo-DC）中 PAF 诱导的细胞因子表达中的作用，并研究参与这种调节的途径。
我们在研究中使用了体外成熟的人树突状细胞和 HEK-293 细胞系。通过 siRNA 和选择性抑制剂抑制 PTP1B。用 RT-PCR、荧光素酶测定和 ELISA 研究细胞因子表达。用 Western blot 研究激酶和转录因子的磷酸化状态。
在这里，我们报告 PTP1B 参与了 PAF 刺激的 Mo-DC 中细胞因子表达的调节。通过 PTP1B 下调 PAF 诱导的 IL-8 表达的研究表明，PAF 诱导的 IL-8 启动子的反式激活受到 C/EBPß 结合位点存在的调节。结果还表明，PTP1B 通过激活Src 家族激酶（SFK），通过糖原合酶激酶（GSK）-3 依赖性途径减少 PAF 诱导的 IL-8 产生。这些激酶在早期刺激时间激活了一条未知的途径，而在后期阶段则激活了 PI3K/Akt 信号通路。这种 GSK-3 活性的变化降低了 C/EBPß 丝氨酸 235 的磷酸化水平，该残基的磷酸化已知会增加 C/EBPß 的反式激活潜能，从而改变 IL-8 的表达。
PTP1B 对 GSK-3 活性的负调节以及 C/EBPß 反式激活结构域磷酸化水平的降低可能是细胞在 PAF 刺激后控制其细胞因子产生的重要负反馈回路。
