Department of Ophthalmology, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.
PLoS One. 2011 Mar 31;6(3):e18137. doi: 10.1371/journal.pone.0018137.
Cell death is an essential process in normal development and homeostasis. In eyes, corneal epithelial injury leads to the death of cells in underlying stroma, an event believed to initiate corneal wound healing. The molecular basis of wound induced corneal stromal cell death is not understood in detail. Studies of others have indicated that ceramide may play significant role in stromal cell death following LASIK surgery. We have undertaken the present study to investigate the mechanism of death induced by C6 ceramide in cultures of human corneal stromal (HCSF) fibroblasts.
Cultures of HCSF were established from freshly excised corneas. Cell death was induced in low passage (p<4) cultures of HCSF by treating the cells with C6 ceramide or C6 dihydroceramide as a control. Cell death was assessed by Live/Dead cell staining with calcein AM and ethidium homodimer-1 as well as Annexin V staining, caspase activation and TUNEL staining Mitochondrial dysfunction was assessed by Mito Sox Red, JC-1 and cytochrome C release Gene expression was examined by qPCR and western blotting.
Our data demonstrate ceramide caused mitochondrial dysfunction as evident from reduced MTT staining, cyto c release from mitochondria, enhanced generation of ROS, and loss in mitochondrial membrane potential (ΔΨm). Cell death was evident from Live -Dead Cell staining and the inability to reestablish cultures from detached cells. Ceramide induced the expression of the harikari gene(HRK) and up-regulated JNK phosphorylation. In ceramide treated cells HRK was translocated to mitochondria, where it was found to interact with mitochondrial protein p32. The data also demonstrated HRK, p32 and BAD interaction. Ceramide-induced expression of HRK, mitochondrial dysfunction and cell death were reduced by HRK knockdown with HRK siRNA.
Our data document that ceramide is capable of inducing death of corneal stromal fibroblasts through the induction of HRK mediated mitochondria dysfunction.
细胞死亡是正常发育和内稳态的一个必要过程。在眼睛中，角膜上皮损伤导致其下基质细胞的死亡，这一事件被认为启动了角膜伤口愈合。诱导性角膜基质细胞死亡的分子基础尚未得到详细阐明。其他研究表明，神经酰胺可能在 LASIK 手术后的基质细胞死亡中发挥重要作用。我们进行了本研究，以探讨 C6 神经酰胺在人角膜基质（HCSF）成纤维细胞培养物中诱导死亡的机制。
从新鲜切取的角膜中建立 HCSF 培养物。用 C6 神经酰胺或 C6 二氢神经酰胺（作为对照）处理低传代（p<4）的 HCSF 培养物，诱导细胞死亡。通过 Calcein AM 和 ethidium homodimer-1 的 Live/Dead 细胞染色以及 Annexin V 染色、半胱天冬酶激活和 TUNEL 染色评估细胞死亡。通过 Mito Sox Red、JC-1 和细胞色素 C 释放评估线粒体功能障碍。通过 qPCR 和 Western blot 检查基因表达。
我们的数据表明，神经酰胺引起线粒体功能障碍，表现为 MTT 染色减少、线粒体细胞色素 c 释放增加、ROS 生成增强以及线粒体膜电位（ΔΨm）丧失。通过 Live-Dead 细胞染色和无法从分离的细胞重新建立培养物来证明细胞死亡。神经酰胺诱导 harikari 基因（HRK）的表达并上调 JNK 磷酸化。在神经酰胺处理的细胞中，HRK 易位到线粒体，在那里它被发现与线粒体蛋白 p32 相互作用。数据还表明 HRK、p32 和 BAD 相互作用。用 HRK siRNA 进行 HRK 敲低可降低 HRK 诱导的表达、线粒体功能障碍和细胞死亡。
我们的数据表明，神经酰胺能够通过诱导 HRK 介导的线粒体功能障碍诱导角膜基质成纤维细胞死亡。
