title: Mitochondrial dysfunction links ceramide activated HRK expression and cell death.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  Our data document that ceramide is capable of inducing death of corneal stromal fibroblasts through the induction of HRK mediated mitochondria dysfunction.
authors.affiliation: Department of Ophthalmology, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America. frizvi@mcw.edu
authors.forename: Farhan
authors.initials: F
authors.lastname: Rizvi
authors.forename: Tom
authors.initials: T
authors.lastname: Heimann
authors.forename: Anja
authors.initials: A
authors.lastname: Herrnreiter
authors.forename: William J
authors.initials: WJ
authors.lastname: O'Brien
mesh_terms: D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D002518:Ceramides / Q000494:pharmacology*; D003319:Corneal Stroma / Q000166:cytology; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species / Q000378:metabolism; D051025:bcl-Associated Death Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; C494819:BAD protein, human; D002518:Ceramides; C484586:HRK protein, human; D024101:Mitochondrial Proteins; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D051025:bcl-Associated Death Protein; C109343:dihydroceramide