Differential regulation of epidermal growth factor receptor by hydrogen peroxide and flagellin in cultured lung alveolar epithelial cells
Differential regulation of epidermal growth factor receptor by hydrogen peroxide and flagellin in cultured lung alveolar epithelial cells
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培养肺泡上皮细胞中过氧化氢和鞭毛蛋白对表皮生长因子受体的差异调节
DOI：
10.1016/j.ejphar.2014.12.016
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发表时间：
2015
影响因子：
5
通讯作者：
Kazuhiro Sugahara and Hideyuki Yamamoto
中科院分区：
文献类型：
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作者：
Hiroyuki Nishi;Noriko Maeda,Shunsuke Izumi;Sayomi Higa-Nakamine;Seikichi Toku;Manabu Kakinohana; Kazuhiro Sugahara and Hideyuki Yamamoto
在以前的研究中，我们发现，Toll样受体5（TLR 5）的鞭毛蛋白刺激诱导的有丝分裂原活化蛋白激酶（MAPK）激活的蛋白激酶2（MAPKAPK-2）通过激活p38 MAPK通路在培养的肺泡上皮细胞A549细胞的激活。我们的研究强烈表明MAPKAPK-2磷酸化表皮生长因子受体（EGFR）的Ser 1047。已有研究表明，肿瘤坏死因子α（TNFα）处理后，Ser 1047的磷酸化可诱导EGFR的内化。在本研究中，我们首次发现过氧化氢处理A549细胞30分钟内可诱导MAPKAPK-2活化和EGFR Ser 1047磷酸化。这与鞭毛蛋白处理不同，因为过氧化氢处理诱导EGFR在Tyr 1173以及Ser 1047处的磷酸化，表明EGFR的活化。我们还发现，KN 93，CaM激酶II的抑制剂，抑制过氧化氢诱导的EGFR的Ser 1047磷酸化，通过抑制p38 MAPK通路的激活。此外，我们还通过三种不同的方法研究了EGFR的内化。流式细胞术与抗体对EGFR和细胞表面蛋白的生物素化的细胞外结构域显示，鞭毛蛋白，但不是过氧化氢，细胞表面EGFR的量减少。此外，鞭毛蛋白预处理降低了EGF处理对细胞外信号调节激酶的激活。这些结果有力地表明，过氧化氢通过激活CaM激酶II激活p38 MAPK通路，并且鞭毛蛋白和过氧化氢通过不同的机制调节EGFR的功能。
In previous studies, we found that stimulation of Toll-like receptor 5 (TLR5) by flagellin induced the activation of mitogen-activated protein kinase (MAPK)-activated protein kinase-2 (MAPKAPK-2) through activation of the p38 MAPK pathway in cultured alveolar epithelial A549 cells. Our studies strongly suggested that MAPKAPK-2 phosphorylated epidermal growth factor receptor (EGFR) at Ser1047. It has been reported that phosphorylation of Ser1047 after treatment with tumor necrosis factor α (TNFα) induced the internalization of EGFR. In the present study, we first found that treatment of A549 cells with hydrogen peroxide induced the activation of MAPKAPK-2 and phosphorylation of EGFR at Ser1047 within 30 min. This was different from flagellin treatment because hydrogen peroxide treatment induced the phosphorylation of EGFR at Tyr1173 as well as Ser1047, indicating the activation of EGFR. We also found that KN93, an inhibitor of CaM kinase II, inhibited the hydrogen peroxide-induced phosphorylation of EGFR at Ser1047 through inhibition of the activation of the p38 MAPK pathway. Furthermore, we examined the internalization of EGFR by three different methods. Flow cytometry with an antibody against the extracellular domain of EGFR and biotinylation of cell surface proteins revealed that flagellin, but not hydrogen peroxide, decreased the amount of cell-surface EGFR. In addition, activation of extracellular signal-regulated kinase by EGF treatment was reduced by flagellin pre-treatment. These results strongly suggested that hydrogen peroxide activated the p38 MAPK pathway via activation of CaM kinase II and that flagellin and hydrogen peroxide regulate the functions of EGFR by different mechanisms.