The mechanism of cleavage of EGFR ligands induced by inflammatory cytokines in gastric epithelial cells ; ADAM10 mediates IL-8-induced EGFR transactivation

表皮生长因子受体（EGFR）可被g蛋白偶联受体（GPCR）激动剂和细胞因子等多种因子反激活。EGFR转激活需要分解素和金属蛋白酶（ADAM），该酶会脱落EGFR配体。为了研究IL-8 （GPCR激动剂）和IL-1β（非GPCR激动剂）在幽门螺杆菌相关胃炎中起关键作用诱导EGFR反激活的机制，我们使用稳定表达碱性磷酸酶（AP）标记的肝素结合egf样生长因子（hbv - egf）、TGF-α或双调节蛋白（AR）前体以及针对ADAM10、12或17的siRNA的KATO III细胞转染物，评估了IL-8和IL-1β依赖的EGFR配体外域脱落。IL-8剂量依赖性地释放EGFR配体（HB-EGF>AR>TGF-α），并在刺激后15分钟瞬时磷酸化EGFR。一种金属蛋白酶抑制剂KB-R7785完全阻断了配体的脱落和EGFR的活化。siRNA缺失ADAM10显著降低EGFR的反式激活，而ADAM12和adam17则无此作用。IL-1β剂量依赖性地增强HB-EGF的脱落，而KB-R7785在早期不抑制这种脱落。然而，晚期EGFR的反活化被KB-R7785阻断，并被抗il -8中和抗体消除。这些结果表明，在胃上皮细胞中，IL-8通过adam10依赖性途径诱导EGFR配体脱落，而IL-1β主要通过adam10非依赖性途径起作用。两种细胞因子均可反激活EGFR，而il -1β依赖性延长EGFR反激活涉及多种途径，包括il -8依赖性途径。

The epidermal growth factor receptor (EGFR) can be transactivated by many factors including G-protein-coupled receptor (GPCR) agonists and cytokines. EGFR transactivation requires a disintegrin and metalloproteinase (ADAM), which sheds EGFR ligands. In order to investigate the mechanism of EGFR transactivation induced by IL-8 (GPCR agonist) and IL-1β(non-GPCR agonist) which play critical roles in Helicobacter pylori-associated gastritis, we assessed IL-8-and IL-1β-dependent ectodomain shedding of EGFR-ligand using KATO III cell transfectants stably expressing alkaline phosphatase (AP)-tagged heparin-binding EGF-like growth factor (HB-EGF), TGF-α, or amphiregulin (AR) precursors as well as siRNA against ADAM10, 12, or 17. IL-8 dose-dependently released the EGFR ligands (HB-EGF>AR>TGF-α), and transiently phosphorylated EGFR with a peak at 15 min after stimulation. A metalloproteinase inhibitor, KB-R7785, completely blocked shedding of the ligands and EGFR transactivation. Depletion of ADAM10 by siRNA significantly reduced the EGFR transactivation, but those of ADAM12 and 17 did not. IL-1β dose-dependently enhanced shedding of HB-EGF, which was not inhibited by KB-R7785 in the early phase. The EGFR transactivation in the late phase was, however, blocked by KB-R7785 and abrogated by anti-IL-8 neutralizing antibody. These results indicate that IL-8 induces shedding of EGFR ligands due to an ADAM 10-dependent pathway in gastric epithelial cells, while IL-1β acts principally by an ADAM-independent pathway. Both cytokines transactivate EGFR, and IL-1β-dependent prolonged EGFR transactivation involves multiple pathways, including an IL-8-dependent pathway.