CD40 ligand induces expression of vascular cell adhesion molecule 1 and E-selectin in orbital fibroblasts from patients with Graves’ orbitopathy
CD40 ligand induces expression of vascular cell adhesion molecule 1 and E-selectin in orbital fibroblasts from patients with Graves’ orbitopathy
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CD40配体诱导格雷夫斯眼眶病患者眼眶成纤维细胞表达血管细胞粘附分子1和E-选择素
DOI：
10.1007/s00417-014-2902-1
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
R. Wei
中科院分区：
文献类型：
--
作者：
Hao Wang;Li;Jin;Ji;You Li;Xiao;R. Wei
目的探讨CD40配体(CD40L)对Graves眼眶病(GO)患者眼眶成纤维细胞血管细胞黏附分子-1(VCAM-1)和E-选择素(E-选择素)表达的影响及其信号转导途径。检测可溶性CD40L(SCD40L)刺激的OFs中VCAM-1和E-选择素的RNA和蛋白表达水平。采用实时定量聚合酶链式反应(PCR)和蛋白质印迹分析进行RNA和蛋白质定量。分离胞浆和胞核，以检测核因子-κB(NF-κB)的核转位。结果与未刺激的OFs相比，sCD40L作用后OFs中VCAM-1和E-选择素的表达水平显著升高。这是在剂量和时间-过程实验中观察到的，而sCD40L对来自健康捐赠者的OFs的诱导作用要弱得多。同时，我们观察到CD40L诱导的NF-κB核转位也呈剂量和时间依赖关系。针对丝裂原活化蛋白激酶(MAPK)、磷脂酰肌醇3-激酶(PI3K)和核因子-κB的抑制剂可显著抑制CD40L诱导的VCAM1和E-选择素的上调以及对NF-κB的核转位。结论CD40L能够在翻译前水平上调GO患者OFs中VCAM1和E-选择素的表达。MAPK和PI3K信号通路及NF-κB可能在CD40L诱导的血管细胞黏附分子-1和E-选择素的表达中起重要作用。
PurposeThe aim of this study was to detect the effect of the CD40 ligand (CD40L) on the expression of vascular cell adhesion molecule 1 (VCAM-1) and E-Selectin in orbital fibroblasts (OFs) from patients with Graves’ orbitopathy (GO), as well as the signaling pathways involved in this effect.MethodsOFs were isolated from orbital tissues obtained from patients with severe GO who were undergoing orbital decompression surgery. VCAM-1 and E-selectin RNA and protein expression levels were quantified in OFs stimulated with soluble CD40L (sCD40L). RNA and protein quantification was performed with real-time polymerase chain reaction (PCR) and western blot analysis. Cytoplasmic and nuclear fractions were isolated in order to detect the nuclear translocation of nuclear factor-κB (NF-κB). Signaling pathway inhibitors were applied to determine the pathways involved.ResultsCompared to unstimulated OFs, the mRNA and protein levels of VCAM-1 and E-selectin in OFs incubated with sCD40L were significantly increased. This was observed in dose- and time-course experiments, and the inductive effects of sCD40L were much weaker in OFs from healthy donors. At the same time, we observed that CD40L induced nuclear translocation of NF-κB, also in a dose- and time-dependent manner. The up-regulation of VCAM-1 and E-selectin, as well as the NF-κB nuclear translocation induced by CD40L, was significantly attenuated by inhibitors targeting mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K), and NF-κB.ConclusionsCD40L demonstrated the ability to up-regulate the expression of VCAM-1 and E-selectin at the pre-translational level in OFs from patients with GO. The MAPK and PI3K pathways and NF-κB may play important roles in CD40L-induced VCAM-1 and E-selectin expression.
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