CD40 ligation stimulates MCP-1 and IL-8 production, TRAF6 recruitment, and MAPK activation in proximal tubule cells
CD40 ligation stimulates MCP-1 and IL-8 production, TRAF6 recruitment, and MAPK activation in proximal tubule cells
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DOI：
10.1152/ajprenal.00291.2001
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发表时间：
2002-06-01
影响因子：
4.2
通讯作者：
Nord, EP
中科院分区：
文献类型：
--
作者：
Li, H;Nord, EP
探讨了CD40/ cd154诱导趋化因子产生的机制及其在肾脏炎性疾病中的潜在作用。以原代培养的人近端小管细胞为实验模型。利用免疫细胞化学、共聚焦显微镜和细胞分离实验，我们发现CD40受体在上皮细胞的细胞膜中表达，当CD154与其同源配体接触时，CD40受体会转移到细胞质区室。CD154参与CD40刺激了白细胞介素-8 （IL-8）和单核细胞趋化蛋白-1 （MCP-1）的产生，这一过程通过受体激活细胞外信号调节激酶（ERK）1/2、应激激活蛋白激酶(SAPK)/c-Jun nh2末端激酶（JNK）和p38丝裂原激活蛋白激酶（MAPK）途径进行。CD40连接还涉及肿瘤坏死因子受体激活因子6 (TRAF6)，这可以通过激活受体与TRAF6在细胞质室中的共定位，两种蛋白从不溶性细胞部分易位到可溶性细胞部分，以及仅在配体刺激条件下两种蛋白的共免疫沉淀来证明。此外，针对TRAF6 mRNA的反义寡脱氧核糖核苷酸会减弱p38和SAPK/JNK的活性，但不会减弱ERK1/2 MAPK的活性，以及IL-8和MCP-1的产生，这表明TRAF6是上游激活剂。锌螯合剂TPEN，而不是钙螯合剂BAPTA，可以消除cd154诱导的MAPK活性和趋化因子的产生，这为蛋白质-蛋白质相互作用在这些细胞中CD40信号传导中起关键作用提供了间接证据。我们得出结论，在人类近端小管细胞中，CD40和TRAF6存在于不同的低密度、洗涤剂不溶性膜微结构域或筏中，并且在激活时，可能通过可溶性或细胞质室中的锌指结构域相互转运和结合。TRAF6反过来激活SAPK/JNK和p38 MAPK磷酸化，进而刺激这些细胞中IL-8和MCP-1的产生。
The mechanism of CD40/CD154-induced chemokine production and its potential role in renal inflammatory disease were explored. Human proximal tubule cells maintained in primary culture were used as the experimental model. With the use of immunocytochemistry, confocal microscopy, and a cell fractionation assay, the CD40 receptor was found to be expressed in the cell membrane of the epithelial cell, and, on engagement by CD154, its cognate ligand, translocated to the cytoplasmic compartment. Engagement of CD40 by CD154 stimulated interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) production, which proceeded via receptor activation of the extracellular signal-regulated kinase (ERK)1/2, stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) pathways. CD40 ligation also engaged tumor necrosis factor receptor-activating factor 6 (TRAF6), as evidenced by colocalization of the activated receptor with TRAF6 in the cytoplasmic compartment, translocation of both proteins from the insoluble to the soluble cell fraction, and coimmunoprecipitation of the two proteins only under ligand-stimulated conditions. Furthermore, an antisense oligodeoxyribonucleotide targeted against TRAF6 mRNA blunted p38 and SAPK/JNK but not ERK1/2 MAPK activities, as well as IL-8 and MCP-1 production, arguing that TRAF6 is an upstream activator. The zinc chelator TPEN, but not the calcium chelator BAPTA, obliterated CD154-evoked MAPK activity and chemokine production, providing indirect evidence for protein-protein interactions playing a critical role in CD40 signaling in these cells. We conclude that in human proximal tubule cells, CD40 and TRAF6 reside in separate low-density, detergent-insoluble membrane microdomains, or rafts, and on activation translocate and associate with one another probably via zinc-finger domains in the soluble or cytoplasmic compartment. TRAF6, in turn, activates SAPK/JNK and p38 MAPK phosphorylation, which in turn stimulates IL-8 and MCP-1 production in these cells.