Hyperosmolarity-induced apoptosis in human corneal epithelial cells is mediated by cytochrome c and MAPK pathways
Hyperosmolarity-induced apoptosis in human corneal epithelial cells is mediated by cytochrome c and MAPK pathways
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DOI：
10.1097/ico.0b013e318030d259
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发表时间：
2007-05-01
期刊：
影响因子：
2.8
通讯作者：
Pflugfelder, Stephen C.
中科院分区：
文献类型：
--
作者：
Luo, Lihui;Li, De-Quan;Pflugfelder, Stephen C.
目的：研究高渗透压是否通过细胞色素 c 介导的死亡途径和丝裂原激活蛋白激酶 (MAPK) 的激活诱导人角膜上皮细胞凋亡。方法：将在正常渗透压培养基 (312 mOsM) 中培养的原代人角膜上皮细胞通过添加 70、90 和 120 mOsM 转换为高渗透压培养基（450、500 和 550 mOsM）。 mM NaCl，分别含有或不含 c-jun N 末端激酶 (JNK) 抑制剂 SB202190 或细胞外调节激酶 (ERK) 抑制剂 PD98059。通过 ApopTag 原位寡核苷酸连接 (ISOL) 测定评估细胞凋亡。使用共聚焦显微镜检测细胞色素c和活性caspase-3。提取总RNA并进行逆转录酶-聚合酶链式反应以获得凋亡相关基因。对细胞提取物进行蛋白质印迹，以检测凋亡分子细胞色素 c 和 Smac/DIABLO，以及磷酸化 JNK 和 ERK。结果：ISOL 阳性凋亡细胞从对照培养基中的 3.3 +/- 1.6% 显着增加到 70、90 和 70、90 和 90 培养基中的 11.4 +/- 5.8%、18.9 +/- 4.8% 和 43.9 +/- 8.8%。分别添加 120 mM NaCl 介质。 90 mM NaCl 高盐培养基显着增加了线粒体中细胞色素 c 和 Smac/DIABLO 的释放；激活 caspase-3、JNK 和 ERK；刺激白细胞介素 1 转换酶和 Bax 的 mRNA 表达；并减少 Bcl2 表达。 SB202190 和 PD98059 显着抑制高渗透压诱导的 JNK/ERK 激活和 ISOL 阳性细胞。此外，PD98059还抑制线粒体释放细胞色素c和Smac/DIABLO。结论：这些研究结果表明，高渗透压通过细胞色素c介导的死亡途径诱导人角膜上皮细胞凋亡，该途径可能是由JNK和ERK MAPK信号通路介导的。
Purpose: To study whether hyperosmolarity induces apoptosis in human corneal epithelial cells through cytochrome c-mediated death pathways and by activation of mitogen-activated protein kinases (MAPKs).Methods: Primary human corneal epithelial cells cultured in normal osmolar media (312 mOsM) were switched to hyperosmolar media (450, 500, and 550 mOsM) by adding 70, 90, and 120 mM NaCl, respectively, with or without the c-jun N-terminal kinase (JNK) inhibitor SB202190 or the extracellular-regulated kinase (ERK) inhibitor PD98059. Apoptosis was assessed by the ApopTag In Situ Oligo Ligation (ISOL) assay. Confocal microscopy was used to detect cytochrome c and active caspase-3. Total RNA was extracted and subjected to reverse transcriptase-polymerase chain reaction for apoptosis-associated genes. Western blots were performed on cell extracts for the apoptogenic molecules cytochrome c and Smac/DIABLO, and phospho-JNK and ERK.Results: ISOL-positive apoptotic cells significantly increased from 3.3 +/- 1.6% in control medium to 11.4 +/- 5.8%, 18.9 +/- 4.8%, and 43.9 +/- 8.8% in 70, 90, and 120 mM NaCl added media, respectively. The 90 mM NaCl high saline medium notably increased release of cytochrome c and Smac/DIABLO from mitochondria; activated caspase-3, JNK and ERK; stimulated mRNA expression of interleukin-1-converting enzyme and Bax; and reduced Bcl2 expression. SB202190 and PD98059 significantly suppressed hyperosmolarity-induced JNK/ERK activation and ISOL-positive cells. In addition, PD98059 inhibited the release of cytochrome c and Smac/DIABLO from mitochondria.Conclusions: These findings show that hyperosmolarity induces apoptosis of human corneal epithelial cells through a cytochrome c-mediated death pathway, which may be mediated by JNK and ERK MAPK signaling pathways.