T Tanaka-Kagawa1, N Hanioka, H Yoshida, H Jinno, M Ando. 1. Division of Environmental Chemistry, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
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.
BACKGROUND:Inorganic arsenic is an environmental contaminant and is associated with the increased risk of humanskin 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 tyrphostinAG1478 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.
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