Department of Ophthalmology, Kansai Medical University, Takii Hospital, Osaka, Japan.
Curr Eye Res. 2011 Dec;36(12):1153-63. doi: 10.3109/02713683.2011.606592. Epub 2011 Oct 6.
To investigate the effects of endoplasmic reticulum (ER) stress on the tight junctions of the retinal pigment epithelial (RPE) cells in vitro.
ER stress was induced in cultured ARPE-19 cells, a human RPE cell line, by exposure to tunicamycin (TM) or to thapsigargin (TG). After 6, 12, 24 and 48 hours of exposure, the expressions of GRP78/Bip (Bip), C/EBP-homologous protein (CHOP), vascular endothelial growth factor (VEGF), zonula occludens (ZO)-1, occludin and claudin-1 were determined by real-time RT-PCR. Immunoblot analysis and/or immunohistochemistry for proteins of tight junctions and ER stress markers, viz., Bip, activating transcription factor (ATF) 6, CHOP, and caspase-4, were performed at 48 hours after the exposure. Enzyme-linked immunosorbent assay was used to determine the concentration of VEGF165. Transepithelial electrical resistance (TER) of the ARPE-19 cells was determined to measure the permeability.
The expressions of the mRNAs and/or proteins of Bip, CHOP, ATF6 and caspase-4 were significantly increased in ARPE-19 cells under ER stress induced by TM and TG. The mRNAs of VEGF were also increased by both TM and TG. However, the concentration of VEGF165 was not significantly increased after 48 hours exposure to TM and TG compared to that of the control in the apical chamber medium. The proteins and mRNAs of occludin and claudin-1 were significantly increased by TM and TG, and that of ZO-1 was significantly increased by TG. Immunohistochemistry showed that the staining of ZO-1, occludin and claudin-1 under ER stress was stronger than that of the control. A significant increase of TER was observed after exposure to TM and TG.
The increased expressions of tight junction molecules by TM- or TG-exposed ARPE-19 cells indicate that ER stress can alter the function of RPE cells and may be involved in the pathogenesis of age-related macular degeneration.
研究内质网（ER）应激对体外视网膜色素上皮（RPE）细胞紧密连接的影响。
用衣霉素（TM）或他普西龙（TG）孵育培养的 ARPE-19 细胞（人 RPE 细胞系），诱导 ER 应激。孵育 6、12、24 和 48 小时后，通过实时 RT-PCR 测定葡萄糖调节蛋白 78/结合免疫球蛋白（Bip）、C/EBP 同源蛋白（CHOP）、血管内皮生长因子（VEGF）、封闭蛋白（ZO）-1、occludin 和 claudin-1 的表达。孵育 48 小时后，通过免疫印迹分析和/或免疫组织化学检测紧密连接和 ER 应激标志物，即 Bip、激活转录因子（ATF）6、CHOP 和半胱天冬酶-4 的蛋白表达。酶联免疫吸附试验用于测定 VEGF165 的浓度。通过测定 ARPE-19 细胞的跨上皮电阻（TER）来测量通透性。
在 TM 和 TG 诱导的 ER 应激下，ARPE-19 细胞中 Bip、CHOP、ATF6 和 caspase-4 的 mRNA 和/或蛋白表达显著增加。TM 和 TG 也增加了 VEGF 的 mRNA。然而，与对照相比，TM 和 TG 孵育 48 小时后，顶端腔室培养基中 VEGF165 的浓度没有显著增加。TM 和 TG 显著增加了 occludin 和 claudin-1 的蛋白和 mRNA，TG 显著增加了 ZO-1 的蛋白。免疫组化显示，在 ER 应激下，ZO-1、occludin 和 claudin-1 的染色比对照更强。孵育 TM 和 TG 后，TER 显著增加。
TM 或 TG 暴露的 ARPE-19 细胞中紧密连接分子的表达增加表明 ER 应激可改变 RPE 细胞的功能，并可能参与年龄相关性黄斑变性的发病机制。
