Mitochondrial dysfunction links ceramide activated HRK expression and cell death

Purpose

Cell death is an essential process in normal development and homeostasis. Ineyes, corneal epithelial injury leads to the death of cells in underlyingstroma, an event believed to initiate corneal wound healing. The molecularbasis of wound induced corneal stromal cell death is not understood indetail. Studies of others have indicated that ceramide may play significantrole in stromal cell death following LASIK surgery. We have undertaken thepresent study to investigate the mechanism of death induced by C6 ceramidein cultures of human corneal stromal (HCSF) fibroblasts.

Methods

Cultures of HCSF were established from freshly excised corneas. Cell deathwas induced in low passage (p<4) cultures of HCSF by treating the cellswith C6 ceramide or C6 dihydroceramide as a control. Cell death was assessedby Live/Dead cell staining with calcein AM and ethidium homodimer-1 as wellas Annexin V staining, caspase activation and TUNEL staining Mitochondrialdysfunction was assessed by Mito Sox Red, JC-1 and cytochrome C release Geneexpression was examined by qPCR and western blotting.

Results

Our data demonstrate ceramide caused mitochondrial dysfunction as evidentfrom reduced MTT staining, cyto c release frommitochondria, enhanced generation of ROS, and loss in mitochondrial membranepotential (ΔΨ_m). Cell death was evident from Live -DeadCell staining and the inability to reestablish cultures from detached cells.Ceramide induced the expression of the harikari gene(HRK) and up-regulatedJNK phosphorylation. In ceramide treated cells HRK was translocated tomitochondria, where it was found to interact with mitochondrial protein p32.The data also demonstrated HRK, p32 and BAD interaction. Ceramide-inducedexpression of HRK, mitochondrial dysfunction and cell death were reduced byHRK knockdown with HRK siRNA.

Conclusion

Our data document that ceramide is capable of inducing death of cornealstromal fibroblasts through the induction of HRK mediated mitochondriadysfunction.