EGFR ligands trafficking into the nucleus in gastric cancer cells

我们最近的研究表明，作为胃癌的诱因，幽门螺杆菌诱导胃上皮细胞产生IL-8， IL-8随后通过金属蛋白酶裂解proHB-EGF促进细胞迁移和增殖。HB-EGF C末端通过转运进入细胞核，对成纤维细胞瘤细胞的生长产生一定的调控作用。然而，这一机制是否存在于胃上皮细胞中尚不清楚。因此，我们在胃上皮和胃癌组织中检测了HB-EGF C末端转运进入细胞核的存在。（方法）用含或不含ADAM抑制剂（KB-R7785）的6OnM磷酸酯处理胃细胞系（KATO III, gcity）。用抗egfr抗体免疫沉淀后，用抗磷酸化抗体检测细胞裂解物。将表达YFP的载体与proHB-EGF的C端融合，转染细胞。用含或不含KB-R7785的60nM荧光酯处理细胞，然后在荧光显微镜下观察。切除的胃标本用HB-EGF c端抗体免疫组织化学染色。（结果）在KATO III细胞中，磷酸酯对EGFR的磷酸化在15 min内达到峰值。KB-R7785消除了磷酸化。HB-EGF C末端随时间从膜转运到细胞核。在KATO III和gcity细胞中，刺激60min后细胞核中染色HB-EGF C末端。在切除的晚期胃癌的癌成分中，HB-EGF C末端在细胞核中部分染色。（结论）ADAM在EGF信号通路中发挥重要作用，在胃癌细胞中，ADAM切割proHB-EGF的胞外结构域，并将proHB-EGF的C端转运到细胞核中。因此，调节ADAM可能有助于控制细胞的生长、迁移和细胞周期，并可能导致对抗胃癌的策略。

We have recently shown that Helicobacter pylori as a causal of gastric cancer induces IL-8 production from gastric epithelia, IL-8 subsequently promotes cell migration and proliferation through metalloproteinase-cleavage proHB-EGF. HB-EGF C terminus, trafficking into nucleus, subsequently exerts some effects on regulation of cell growth in fibroblastoma cells. However, little is known about whether this mechanism exists in gastric epithelia. Thus, the presence of HB-EGF C terminus trafficking into nucleus was examined with gastric epithelia and gastric cancer tissues.(method)Gastric cell lines(KATO III, GCIY)were treated with a 6OnM of phorbor ester with or without ADAM inhibitor(KB-R7785). Cell lysates were probed with antibodies against phosphorylation after immunoprecipitation with anti-EGFR antibodies. Cells were transfected with vectors expressing YFP fused to C terminus of proHB-EGF. Cells were treated with a 60nM of phorbor ester with or without KB-R7785, and then were observed under a fluorescent microscopy. Excided stomach samples were immunohistochemically stained with anti-bodies against HB-EGF C-terminus.(result)In KATO III cells, EGFR phosphorylation by phorbor ester peaked within 15 min. That phosphorylation was abolished by KB-R7785. HB-EGF C terminus trafficked into nucleus from membrane with time. In KATO III and GCIY cells, HB-EGF C terminus was stained in the nuclei 60min after stimulation. In carcinomatous components of excided advanced gastric cancer, HB-EGF C terminus was partially stained in the nuclei.(conclusion)ADAM plays an important roles in the EGF signaling pathways, where ADAM cleaves extracellular domain of proHB-EGF and C terminus of proHB-EGF trafficks into the nucleus in gastric cancer cells. Thus, regulation of ADAM may be helpful for control of cell growth, migration and cell cycle, and leads to possible strategy against gastric cancer.