Research Summary: Mitochondrial Dysfunction Links Ceramide Activated HRK Expression and Cell Death



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


Publisher: Public Library of Science

Date Published: 31-March-2011

Author(s): Rizvi F., Heimann T., Herrnreiter A., O’Brien W.


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