Thrombospondin-1 induces differential response in human corneal and conjunctival epithelial cells lines under in vitro inflammatory and apoptotic conditions
Thrombospondin-1 induces differential response in human corneal and conjunctival epithelial cells lines under in vitro inflammatory and apoptotic conditions
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DOI：
10.1016/j.exer.2015.03.004
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发表时间：
2015-05-01
影响因子：
3.4
通讯作者：
Diebold, Yolanda
中科院分区：
文献类型：
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作者：
Soriano-Romani, Laura;Garcia-Posadas, Laura;Diebold, Yolanda
最近，据报道，血小板反应蛋白-1 (TSP-1) 对于维持健康的眼表至关重要。本研究的目的是表征角膜和结膜上皮细胞中 TSP-1 及其受体 CD36 和 CD47 的表达，并确定外源性 TSP-1 治疗在诱导炎症和凋亡相关变化后对这些细胞的影响。首先通过ELISA、免疫荧光分析、Western blotting和逆转录聚合酶链反应（RT-PCR）对角膜和结膜细胞系中TSP-1、CD36和CD47的表达进行表征。苯扎氯铵 (BAC) 暴露 5 或 15 分钟分别用作角膜或结膜上皮细胞的促炎和促凋亡刺激。为了分析炎症和细胞凋亡相关的变化，通过ELISA测定的IL-6和TGF-β2分泌物被用作炎症标志物，而分别通过CellEvent（TM）Caspase-3/7绿色检测试剂和XTT细胞毒性测定测定的活化的caspase-3/7水平和细胞活力被用作细胞凋亡标志物。通过实时 RT-PCR 定量 CD36 和 CD47 mRNA 表达的变化。角膜上皮细胞分泌和表达的 TSP-1 蛋白水平高于结膜上皮细胞，尽管 TSP-1 mRNA 表达水平相似，并且在蛋白和 mRNA 水平上具有较低的 CD36 和 CD47。两种细胞系对外源 TSP-1 处理均有反应，增加 CD36 的蛋白质和 mRNA 水平。阻断实验揭示了 TSP-1/CD47 而非 TSP-1/CD36 相互作用主导结膜上皮细胞中 CD36 水平的上调，但在角膜上皮细胞中则不然。 BAC 暴露增加了 IL-6 分泌和 caspase-3/7 水平，并降低了角膜和结膜上皮细胞的细胞活力。此外，BAC 暴露会增加结膜上皮细胞中潜在的 TGF-β2 水平。有趣的是，两种细胞系暴露于 BAC 后 CD36 mRNA 表达均下调。外源性 TSP-1 治疗可降低结膜上皮细胞中因 BAC 暴露而上调的 TGF-β 2 水平，并不太明显地降低 BAC 暴露的角膜上皮细胞中的 IL-6。对 CD36 和 CD47 调节的影响不太明显，甚至相反，具体取决于测试的炎症和细胞凋亡相关标记。我们的结果显示了角膜和结膜上皮细胞通过 CD36 或 CD47 对 TSP-1 做出反应的能力。炎症和细胞凋亡相关条件的实验模拟改变了 TSP-1 对角膜和结膜上皮细胞引起的不同影响，表明 TSP-1 对眼表稳态调节具有意想不到的相关贡献。 (C) 2015 Elsevier Ltd. 保留所有权利。
Recently, thrombospondin-1 (TSP-1) has been reported to be critical for maintaining a healthy ocular surface. The purpose of the study was to characterize the expression of TSP-1 and of its receptors CD36 and CD47 in corneal and conjunctival epithelial cells and determine the effect of exogenous TSP-1 treatment on these cells, following the induction of inflammation- and apoptosis-related changes. The expression of TSP-1, CD36 and CD47 by corneal and conjunctival cell lines was firstly characterized by ELISA, immunofluorescence analysis, Western blotting and reverse transcription polymerase chain reaction (RT-PCR). Benzalkonium chloride (BAC) exposure for 5 or 15 min was used as pro-inflammatory and pro-apoptotic stimulus for corneal or conjunctival epithelial cells, respectively. To analyze inflammation and apoptosis-related changes, IL-6 and TGF-beta 2 secretion determined by ELISA was used as inflammatory markers, while activated caspase-3/7 levels and cell viability, determined by CellEvent (TM) Caspase-3/7 Green Detection Reagent and XTT cytotoxicity assay, respectively, were used as apoptotic markers. Changes in CD36 and CD47 mRNA expression were quantified by real time RT-PCR. Corneal epithelial cells secreted and expressed higher protein levels of TSP-1 than conjunctival epithelial cells, although TSP-1 mRNA expression levels were similar and had lower CD36 and CD47, both at protein and mRNA levels. Both cell lines responded to exogenous TSP-1 treatment increasing CD36 at protein and mRNA levels. Blocking experiments revealed a predominance of TSP-1/CD47 rather than TSP-1/CD36 interactions to up-regulate CD36 levels in conjunctival epithelial cells, but not in corneal epithelial cells. BAC exposure increased IL-6 secretion and caspase-3/7 levels and decreased cell viability in both, corneal and conjunctival epithelial cells. Moreover, BAC exposure increased latent TGF-beta 2 levels in conjunctival epithelial cells. Interestingly, CD36 mRNA expression was down-regulated after BAC exposure in both cell lines. Exogenous TSP-1 treatment reduced TGF-beta 2 up-regulated levels by BAC exposure in conjunctival epithelial cells and less pronounced reduced IL-6 in BAC-exposed corneal epithelial cells. The effect on CD36 and CD47 regulation was less pronounced or even opposite depending on the inflammation- and apoptosis-related markers tested. Our results show evidence of the capacity of corneal and conjunctival epithelial cells to respond to TSP-1 via CD36 or CD47. Experimental simulation of inflammation- and apoptosis-related conditions changed the effects differentially elicited by TSP-1 on corneal and conjunctival epithelial cells, suggesting an unexpected and relevant contribution of TSP-1 on ocular surface homeostasis regulation. (C) 2015 Elsevier Ltd. All rights reserved.