JNK and ERK MAP kinases mediate induction of IL-1β, TNF-α and IL-8 following hyperosmolar stress in human limbal epithelial cells
JNK and ERK MAP kinases mediate induction of IL-1β, TNF-α and IL-8 following hyperosmolar stress in human limbal epithelial cells
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DOI：
10.1016/j.exer.2005.08.019
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发表时间：
2006-04-01
影响因子：
3.4
通讯作者：
Pflugfelder, SC
中科院分区：
文献类型：
--
作者：
Li, DQ;Luo, LH;Pflugfelder, SC
高渗透压已被认为是对角膜上皮的促炎性应激。高渗性应激与炎症之间的细胞信号通路尚未得到很好的阐明。本研究调查了人角膜缘上皮细胞暴露于高渗应激是否激活丝裂原活化蛋白激酶（MAPK）通路并诱导促炎细胞因子白细胞介素（IL）-1 β、肿瘤坏死因子（TNF）α和C-X-C趋化因子IL-8的产生。正常渗透介质中的原代人角膜缘上皮培养物（312 mOsM）暴露于具有较高渗透压的培养基（400-500 mOsM），加入50-90 mM NaCl，加入或不加入SB 202190（c-Jun N-末端激酶（JNK）途径抑制剂），PD 98059（细胞外调节激酶（ERK）途径抑制剂），地塞米松或多西环素持续不同的时间长度。处理24小时后收集条件培养基用于ELISA。从处理6小时的培养物中提取总RNA用于半定量RT-PCR。将处理15-60分钟的细胞在RIPA缓冲液中裂解，并用针对p-JNK和p-ERK的磷酸（p）特异性抗体进行蛋白质印迹。角膜缘上皮细胞24小时条件培养基中IL-1 β、TNF-α和IL-8蛋白的浓度随着培养基渗透压从312增加到500 mOsM而逐渐增加。Western blot检测到p-JNK-1/p-JNK-2和p-ERK-1/p-ERK-2的活性，并在暴露于高渗培养基的细胞中在60 min时达到峰值。p-JNK-1/p-JNK-2和pERK-1/p-ERK-2的表达水平与中渗浓度呈正相关。SB 202190、PD 98059和多西环素显著抑制由高渗介质刺激的p-JNK-1/p-JNK-2和/或p-ERK-1/p-ERK-2以及IL-1 β、TNF-α和IL-8 mRNA和蛋白的水平。这些发现提供了直接的证据，即高渗通过增加促炎细胞因子和趋化因子的表达和产生来诱导人角膜缘上皮细胞的炎症，这一过程似乎是通过激活JNK和ERK MAPK信号通路介导的。多西环素治疗眼表疾病的功效可能是由于其抑制JNK和ERK信号传导激活和角膜缘上皮中炎症介质产生的能力。(c)2005爱思唯尔有限公司保留所有权利。
Hyperosmolarity has been recognized to be a pro-inflammatory stress to the corneal epithelium. The cell signalling pathways linking hyperosmolar stress and inflammation have not been well elucidated. This study investigated whether exposure of human limbal epithelial cells to hyperosmotic stress activates the mitogen-activated protein kinase (MAPK) pathways and induces production of pro-inflammatory cytokines, interleukin (IL)-1 beta, tumor necrosis factor (TNF) alpha, and the C-X-C chemokine IL-8. Primary human limbal epithelial cultures in normal osmolar media (312 mOsM) were exposed to media with higher osmolarity (400-500 mOsM) by adding 50-90 mM NaCl, with or without SB202190, an inhibitor of c-Jun N-terminal kinases (JNK) pathway, PD 98059, an inhibitor of extracellular-regulated kinase (ERK) pathway, dexamethasone or doxycycline for different lengths of time. The conditioned media were collected after 24 hr of treatment for ELISA. Total RNA was extracted from cultures treated for 6 hr for semi-quantitative RT-PCR. Cells treated for 15-60 min were lysed in RIPA buffer and subjected to Western blot with phospho (p)-specific antibodies against p-JNK and p-ERK. The concentrations of IL-1 beta, TNF-alpha and IL-8 proteins in 24 hr conditioned media of limbal epithelial cells progressively increased as the media osmolarity increased from 312 to 500 mOsM. Active p-JNK-1/p-JNK-2 and p-ERK-1/p-ERK-2 were detected by Western blot and peaked at 60 min in cells exposed to hyperosmolar media. The levels of p-JNK-1/p-JNK-2 and pERK-1/p-ERK2 were positively correlated with the medium osmolarity. SB202190, PD98059 and doxycycline markedly suppressed the levels of p-JNK-1/p-JNK-2 and/or p-ERK-1/p-ERK-2, as well as IL-1 beta, TNF-alpha and IL-8 mRNAs and proteins stimulated by hyperosmolar media. These findings provide direct evidence that hyperosmolarity induces inflammation in human limbal epithelial cells by increasing expression and production of pro-inflammatory cytokines and chemokines, a process that appears to be mediated through activation of the JNK and ERK MAPK signalling pathways. The efficacy of doxycycline in treating ocular surface diseases may be due to its ability to suppress JNK and ERK signalling activation and inflammatory mediator production in the limbal epithelium. (c) 2005 Elsevier Ltd. All rights reserved.