Abstract
Purpose: :
We hypothesize that ceramide is a mediator of cell death during corneal wound healing. The goal of this work was to determine the mechanism of ceramide induced corneal stromal cell death.
Methods: :
Cultures of HCSF were established from corneas excised from human eyes obtained from Wisconsin’s Lion Eye Bank. The corneal epithelium and endothelium were removed by scraping and stroma digested by collagenase. Harvested cells were grown in DMEM with 5%FBS, Mito+, and ciprofloxacin. Cell death was induced in low passage (p < 4) cultures by treating the HCSF with 40µM C6-ceramide. C6-dihydroceramide was used as a negative control. Cell death was assessed by MTT assay, TUNEL assay, and Live/Dead cell staining. Annexin V and Hoechst 33342 staining were also performed. Mitochondrial membrane potential was determined by JC-1 staining using fluorescent microscopy. Fluorescence ratios were established using a plate reader. Production of reactive oxygen species was determined by MitoSOX. Real time RT-qPCR analyses and /or immunoblotting were used to assess cyto c, HRK, JNK-p, RIP1, and RIP3 expression. Statistical analyses were done using SIGMA plot.
Results: :
Our data demonstrate ceramide induced activation of multiple pathways leading to cell death by necroptosis -a combination of apoptosis and programmed necrosis independent of caspases. The pro-apoptotic pathway involved JNK mediated induction of HRK as determined by RT-qPCR and Western. HRK was found to interact with mitochondrial P32, a protein thought to regulate mitochondrial Ca2+ concentration, permeability transition pore complex and OxPhos. The autocrine production of TNF-α and induction of Receptor Interacting Protein kinases 1 and 3 likely contributed to the pro-necrotic pathway. C6-ceramide treatment of HCSF resulted in ER stress and mitochondrial dysfunction as observed by the increased expression of ER stress markers like IRE1,BIP, CHOP, Xbp1 splicing, and the decrease in mitochondrial membrane potential, cyto c release and production of ROS.
Conclusions: :
These observations are the first evidence to suggest that ceramide leads to death of HCSF by activating multiple pathways.
Keywords: apoptosis/cell death • cornea: stroma and keratocytes • cornea: basic science