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亚砷酸盐和砷酸盐通过表皮生长因子受体介导的途径激活正常人角质形成细胞中的细胞外信号调节激酶1/2。

Arsenite and arsenate activate extracellular signal-regulated kinases 1/2 by an epidermal growth factor receptor-mediated pathway in normal human keratinocytes.

作者信息

Tanaka-Kagawa T, Hanioka N, Yoshida H, Jinno H, Ando M

机构信息

Division of Environmental Chemistry, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.

出版信息

Br J Dermatol. 2003 Dec;149(6):1116-27. doi: 10.1111/j.1365-2133.2003.05704.x.

DOI:10.1111/j.1365-2133.2003.05704.x

PMID:14674888

Abstract

BACKGROUND

Inorganic arsenic is an environmental contaminant and is associated with the increased risk of human skin cancer. Arsenic has been reported to activate or inhibit a variety of cellular signalling pathways which has effects on cell growth, differentiation and apoptosis. However, the molecular mechanisms of these arsenic-induced biological effects are not completely understood.

OBJECTIVES

To understand the molecular basis for the mode of action of arsenicals, we examined the effect of arsenite and arsenate on the activation of mitogen-activated protein kinases (MAPK) and the upstream signalling cascade in normal human epidermal keratinocytes (NHEK).

METHODS

NHEK were exposed to arsenite or arsenate. Western blot analysis was performed to determine the activation of extracellular signal-regulated kinases (ERK) 1/2, c-jun N-terminal kinases (JNK), p38, and MAPK or ERK kinases (MEK) 1/2. Epidermal growth factor receptor (EGFR) tyrosine phosphorylation and recruitment of its adaptor proteins, Shc and Grb2, to EGFR were detected by immunoprecipitation and Western blot analysis.

RESULTS

Both arsenicals activated ERK1/2, which are most highly activated in response to mitogenic stimulation, in addition to JNK and p38, which show greater activation in response to cellular stresses. The kinetics of ERK1/2 activation differed from those of JNK and p38 activation. Both arsenicals transiently activated ERK1/2 prior to JNK and p38 activation. MEK1/2, upstream kinases of ERK1/2, were also activated by arsenicals with similar time kinetics to that of ERK1/2 activation. To investigate a signalling pathway leading to activation of MEK1/2-ERK1/2, we examined the tyrosine phosphorylation of EGFR and Shc adapter protein. Both arsenicals stimulated tyrosine phosphorylation of EGFR and Shc. After arsenical treatment, Shc immunoprecipitates contained coprecipitated EGFR and Grb2, suggesting that both arsenicals induce the assembly of EGFR-Shc-Grb2 complexes. Both the EGFR inhibitor tyrphostin AG1478 and anti-EGFR blocking antibody markedly attenuated ERK1/2 activation induced by arsenicals, but did not affect JNK and p38 activation.

CONCLUSIONS

Our data indicate that both arsenite and arsenate activate the EGFR-Shc-Grb2-MEK1/2-ERK1/2 signalling cascade in NHEK.

摘要

背景

无机砷是一种环境污染物，与人类皮肤癌风险增加相关。据报道，砷可激活或抑制多种细胞信号通路，对细胞生长、分化和凋亡产生影响。然而，这些砷诱导的生物学效应的分子机制尚未完全明确。

目的

为了解砷化合物作用模式的分子基础，我们研究了亚砷酸盐和砷酸盐对正常人表皮角质形成细胞（NHEK）中丝裂原活化蛋白激酶（MAPK）及其上游信号级联激活的影响。

方法

将NHEK暴露于亚砷酸盐或砷酸盐。采用蛋白质免疫印迹分析来确定细胞外信号调节激酶（ERK）1/2、c-jun氨基末端激酶（JNK）、p38和MAPK或ERK激酶（MEK）1/2的激活情况。通过免疫沉淀和蛋白质免疫印迹分析检测表皮生长因子受体（EGFR）的酪氨酸磷酸化及其衔接蛋白Shc和Grb2向EGFR的募集情况。

结果

两种砷化合物均可激活ERK1/2（在有丝分裂原刺激下激活程度最高），此外还可激活JNK和p38（在细胞应激时激活程度更高）。ERK1/2激活的动力学与JNK和p38激活的动力学不同。两种砷化合物在激活JNK和p38之前均短暂激活ERK1/2。ERK1/2的上游激酶MEK1/2也被砷化合物激活，其时间动力学与ERK1/2激活相似。为研究导致MEK1/2-ERK1/2激活的信号通路，我们检测了EGFR和Shc衔接蛋白的酪氨酸磷酸化。两种砷化合物均刺激EGFR和Shc的酪氨酸磷酸化。砷化合物处理后，Shc免疫沉淀物中含有共沉淀的EGFR和Grb2，提示两种砷化合物均可诱导EGFR-Shc-Grb2复合物的组装。EGFR抑制剂 tyrphostin AG1478和抗EGFR阻断抗体均显著减弱砷化合物诱导的ERK1/2激活，但不影响JNK和p38激活。