The mechanism of cleavage of EGFR ligands induced by inflammatory cytokines in gastric cancer cells
The mechanism of cleavage of EGFR ligands induced by inflammatory cytokines in gastric cancer cells
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DOI：
10.1053/j.gastro.2004.05.017
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发表时间：
2004-08-01
期刊：
影响因子：
29.4
通讯作者：
Itoh, M
中科院分区：
文献类型：
--
作者：
Tanida, S;Joh, T;Itoh, M
背景和目的：表皮生长因子 (EGF) 受体 (EGFR) 可以被许多因素反式激活，包括 G 蛋白偶联受体激动剂和细胞因子。尽管据报道这种 EGFR 反式激活需要解整合素和金属蛋白酶 (ADAM) 来释放 EGFR 配体的胞外域，但详细机制仍不清楚。本研究评估了 KATO III 细胞中白细胞介素 (IL)-8 和 IL-1β 依赖性 EGFR 配体脱落的机制。方法：我们使用针对 ADAM10、12 或 17 的短干扰 RNA，建立了稳定表达碱性磷酸酶标记的肝素结合 EGF 样生长因子 (HB-EGF)、转化生长因子 α 或双调蛋白前体以及耗尽 ADAM 蛋白的转染子。通过测量 IL-1beta 或 IL-8 刺激后条件培养基中的 AP 活性来评估 EGFR 配体的脱落。使用抗磷酸酪氨酸抗体通过免疫沉淀和蛋白质印迹检查 EGFR 激活。 KB-R7785和抗IL-8中和抗体分别用于抑制ADAMs的活性和IL-8的作用。结果：IL-8剂量依赖性地释放EGFR配体并瞬时磷酸化EGFR，在15分钟处达到峰值。 KB-R7785 完全阻断 IL-8 诱导的脱落和 EGFR 反式激活。 ADAM10 的消耗也显着减少了 IL-8 诱导的脱落和 EGFR 反式激活，但 ADAM12 和 17 的消耗却没有。 IL-1β 剂量依赖性地增强 HB-EGF 的脱落，而 KB-R7785 在早期并未阻断这种脱落。然而，在晚期，EGFR 反式激活被 Kbeta-R7785 阻断，并被抗 IL-8 中和抗体消除。 结论：在胃癌细胞中，IL-8 通过 ADAM10 依赖性途径诱导 EGFR 配体脱落，而 IL-1beta 主要通过 ADAM 独立途径发挥作用。 IL-1β 依赖性延长 EGFR 反式激活涉及多种途径，包括 IL-8 依赖性途径。
Background & Aims: The epidermal growth factor (EGF) receptor (EGFR) can be transactivated by many factors including G-protein-coupled receptor agonists and cytokines. Although this EGFR transactivation reportedly requires a disintegrin and metalloproteinase (ADAM) that sheds the ectodomain of EGFR ligands, the detailed mechanisms are still unknown. This study evaluated the mechanism of interleukin (IL)-8- and IL-1beta-dependent shedding of the EGFR ligand in KATO III cells.Methods: We established transfectants stably expressing alkaline phosphatase-tagged heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor alpha, or amphiregulin precursors, and depleted ADAM proteins, using short interfering RNA against ADAM10, 12, or 17. We assessed shedding of EGFR ligands by measuring AP activities in the conditioned media after IL-1beta or IL-8 stimulation. EGFR activation was examined by immuno-precipitation and Western blotting using antiphosphotyrosine antibody. KB-R7785 and anti-IL-8 neutralizing antibody were used to inhibit activities of ADAMs and IL-8 action, respectively.Results: IL-8 dose dependently released the EGFR ligands and transiently phosphorylated EGFR, with a peak at 15 minutes. KB-R7785 completely blocked IL-8-induced shedding and EGFR transactivation. Depletion of ADAM10 also dramatically reduced IL-8-induced shedding and EGFR transactivation, but depletion of ADAM12 and 17 did not. IL-1beta dose dependently enhanced shedding of HB-EGF, which was not blocked by KB-R7785 in the early phase. In the late phase, however, the EGFR transactivation was blocked by Kbeta-R7785 and abrogated by anti-IL-8 neutralizing antibody.Conclusion : IL-8 induces shedding of EGFR ligands because of an ADAM10-dependent pathway in gastric cancer cells, whereas IL-1beta acts principally by an ADAM-independent pathway. IL-1beta-dependent prolonged EGFR transactivation involves multiple pathways, including an IL-8-dependent pathway.