Glotin Anne-Lise, Calipel Armelle, Brossas Jean-Yves, Faussat Anne-Marie, Tréton Jacques, Mascarelli Frédéric
Institut Biomédical des Cordeliers, INSERM (Institut National de la Santé et de la Recherche Médicale) Unité 598, Paris, France.
Invest Ophthalmol Vis Sci. 2006 Oct;47(10):4614-23. doi: 10.1167/iovs.06-0297.
Oxidative stress is thought to contribute to the pathogenesis of age-related macular degeneration (AMD), which involves retinal pigmented epithelial (RPE) cell death. However, signaling pathways involved in the oxidative-stress-induced RPE cell death are poorly understood. This study was conducted to investigate the involvement of the MAP kinase pathways during the induction of RPE cell death by oxidative stress.
ARPE-19 cells were exposed to the oxidant tert-butyl hydroperoxide (t-BHP). Cell viability was assessed by cell counting and MTT-staining, and apoptosis was quantified by TUNEL and flow cytometry. Activation of JNK1/3, p38 alphabeta MAPKs and ERK1/2 and their potential targets was detected by Western blot analysis and immunochemistry with specific anti-phospho protein antibodies. Specific pharmacologic inhibitors directed against the MAPKs were used to analyze the signaling involved in cell death of RPE cells exposed to t-BHP.
Exposure of RPE cells to t-BHP, associated with increase in reactive oxygen species and intracellular glutathione depletion, induced time- and concentration-dependent apoptosis, which was associated with the accumulation of inactive ERK1/2 in cell nuclei and a transient and weak ERK1/2 activation. This activation was accompanied by a deactivation of P90(RSK), the major target of ERK1/2 and consequently by the delayed activation of its transcription factor CREB. MEK1/2 inhibition completely suppressed the transient activation of ERK1/2 and completely blocked apoptosis, demonstrating the role of the MEK-ERK module in mediating oxidative-stress-induced RPE cell death. In contrast, neither JNKs nor p38 alphabeta MAPKs were involved in mediating t-BHP-induced apoptotic signaling in RPE cells.
The results suggest that inhibiting the MEK-ERK module may allow the development of selective methods for treating oxidative-stress-induced RPE degeneration, such as AMD.
氧化应激被认为与年龄相关性黄斑变性（AMD）的发病机制有关，AMD涉及视网膜色素上皮（RPE）细胞死亡。然而，氧化应激诱导RPE细胞死亡所涉及的信号通路尚不清楚。本研究旨在探讨丝裂原活化蛋白激酶（MAP）通路在氧化应激诱导RPE细胞死亡过程中的作用。
将ARPE-19细胞暴露于氧化剂叔丁基过氧化氢（t-BHP）。通过细胞计数和MTT染色评估细胞活力，通过TUNEL和流式细胞术定量细胞凋亡。用特异性抗磷酸化蛋白抗体通过蛋白质印迹分析和免疫化学检测JNK1/3、p38αβ MAPK和ERK1/2及其潜在靶点的激活情况。使用针对MAPK的特异性药理抑制剂分析暴露于t-BHP的RPE细胞死亡所涉及的信号传导。
RPE细胞暴露于t-BHP后，伴随着活性氧增加和细胞内谷胱甘肽消耗，诱导了时间和浓度依赖性细胞凋亡，这与细胞核中无活性ERK1/2的积累以及ERK1/2的短暂弱激活有关。这种激活伴随着ERK1/2的主要靶点P90（RSK）的失活，进而伴随着其转录因子CREB的延迟激活。MEK1/2抑制完全抑制了ERK1/2的短暂激活并完全阻断了细胞凋亡，证明了MEK-ERK模块在介导氧化应激诱导的RPE细胞死亡中的作用。相反，JNK和p38αβ MAPK均未参与介导RPE细胞中t-BHP诱导的凋亡信号传导。
结果表明，抑制MEK-ERK模块可能有助于开发治疗氧化应激诱导的RPE变性（如AMD）的选择性方法。
