Ruffels James, Griffin Martin, Dickenson John M
School of Science, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK.
Eur J Pharmacol. 2004 Jan 12;483(2-3):163-73. doi: 10.1016/j.ejphar.2003.10.032.
Reactive oxygen species including H(2)O(2) activate an array of intracellular signalling cascades that are closely associated with cell death and cell survival pathways. The human neuroblastoma SH-SY5Y cell line is widely used as model cell system for studying neuronal cell death induced by oxidative stress. However, at present very little is known about the signalling pathways activated by H(2)O(2) in SH-SY5Y cells. Therefore, in this study we have investigated the effect of H(2)O(2) on extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase B (PKB) activation in undifferentiated and differentiated SH-SY5Y cells. H(2)O(2) stimulated time and concentration increases in ERK1/2, JNK and PKB phosphorylation in undifferentiated and differentiated SH-SY5Y cells. No increases in p38 MAPK phosphorylation were observed following H(2)O(2) treatment. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin and LY 294002 ((2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) inhibited H(2)O(2)-induced increases in ERK1/2 and PKB phosphorylation. Furthermore, H(2)O(2)-mediated increases in ERK1/2 activation were sensitive to the MAPK kinase 1 (MEK1) inhibitor PD 98059 (2'-amino-3'-methoxyflavone), whereas JNK responses were blocked by the JNK inhibitor SP 600125 (anthra[1-9-cd]pyrazol-6(2H)-one). Treatment of SH-SY5Y cells with H(2)O(2) (1 mM; 16 h) significantly increased the release of lactate dehydrogenase (LDH) into the culture medium indicative of a decrease in cell viability. Pre-treatment with wortmannin, SP 600125 or SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; p38 MAPK inhibitor) had no effect on H(2)O(2)-induced LDH release from undifferentiated or differentiated SH-SY5Y cells. In contrast, PD 98059 and LY 294002 significantly decreased H(2)O(2)-induced cell death in both undifferentiated and differentiated SH-SY5Y cells. In conclusion, we have shown that H(2)O(2) stimulates robust increases in ERK1/2, JNK and PKB in undifferentiated and differentiated SH-SY5Y cells. Furthermore, the data presented clearly suggest that inhibition of the ERK1/2 pathway protects SH-SY5Y cells from H(2)O(2)-induced cell death.
包括过氧化氢（H₂O₂）在内的活性氧会激活一系列与细胞死亡和细胞存活途径密切相关的细胞内信号级联反应。人神经母细胞瘤SH-SY5Y细胞系被广泛用作研究氧化应激诱导的神经元细胞死亡的模型细胞系统。然而，目前对于H₂O₂在SH-SY5Y细胞中激活的信号通路知之甚少。因此，在本研究中，我们研究了H₂O₂对未分化和分化的SH-SY5Y细胞中细胞外信号调节激酶1/2（ERK1/2）、c-Jun氨基末端激酶（JNK）、p38丝裂原活化蛋白激酶（p38 MAPK）和蛋白激酶B（PKB）激活的影响。H₂O₂刺激未分化和分化的SH-SY5Y细胞中ERK1/2、JNK和PKB磷酸化的时间和浓度增加。H₂O₂处理后未观察到p38 MAPK磷酸化增加。磷脂酰肌醇3激酶（PI-3K）抑制剂渥曼青霉素和LY 294002（2-（4-吗啉基）-8-苯基-4H-1-苯并吡喃-4-酮）抑制H₂O₂诱导的ERK1/2和PKB磷酸化增加。此外，H₂O₂介导的ERK1/2激活增加对丝裂原活化蛋白激酶激酶1（MEK1）抑制剂PD 98059（2'-氨基-3'-甲氧基黄酮）敏感，而JNK反应被JNK抑制剂SP 600125（蒽[1-9-cd]吡唑-6（2H）-酮）阻断。用H₂O₂（1 mM；16小时）处理SH-SY5Y细胞显著增加了乳酸脱氢酶（LDH）释放到培养基中，这表明细胞活力下降。用渥曼青霉素、SP 600125或SB 203580（4-（4-氟苯基）-2-（4-甲基亚磺酰基苯基）-5-（4-吡啶基）-1H-咪唑；p38 MAPK抑制剂）预处理对未分化或分化的SH-SY5Y细胞中H₂O₂诱导的LDH释放没有影响。相反，PD 98059和LY 294002显著降低了未分化和分化的SH-SY5Y细胞中H₂O₂诱导的细胞死亡。总之，我们已经表明H₂O₂刺激未分化和分化的SH-SY5Y细胞中ERK1/2、JNK和PKB的显著增加。此外，所呈现的数据清楚地表明，抑制ERK1/2途径可保护SH-SY5Y细胞免受H₂O₂诱导的细胞死亡。
