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Abstract #11500 Published in IGR 6-3
Role of eIF5A in TNF-α-mediated apoptosis of lamina cribrosa cells
Taylor CA; Senchyna M; Flanagan J; Joyce EM; Cliche DO; Boone AN; Culp-Stewart S; Thompson JEInvestigative Ophthalmology and Visual Science 2004; 45: 3568-3576
PURPOSE: To determine the role of eukaryotic translation initiation factor 5A (eIF5A) in TNF-α-induced apoptosis of lamina cribrosa (LC) cells. METHODS: LC cells were isolated from optic nerve heads of eyes of two human donors. The cells were treated with TNF-α and camptothecin, a TNF synergist, and the incidence of apoptosis was scored by Hoechst staining. Expression of eIF5A protein in response to camptothecin or a combination of camptothecin and TNF-α was determined by Western blot analysis. The ability of small inhibitory (si)RNAs directed against eIF5A to protect LC cells from TNF-α-induced apoptosis was determined by Hoechst and TUNEL staining of transfected LC cells. RESULTS: TNF-α and camptothecin synergized to induce greater than two times more apoptosis in LC cells than when the cells were treated with TNF-α or camptothecin separately. Expression of eIF5A protein increased significantly after 8 hours of exposure to TNF-α and camptothecin, but not in response to camptothecin alone. siRNAs directed against eIF5A reduced apoptosis of LC cells in response to TNF-α and camptothecin by between 35% and 69%, as determined by Hoechst staining. An siRNA against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) also reduced apoptosis of LC cells by 42%. TUNEL of transfected LC cells treated with TNF-α and camptothecin revealed an 80% reduction in apoptosis with siRNA against eIF5A. CONCLUSIONS: TNF-α, in synergy with camptothecin, induces apoptosis in human LC cells. eIF5A is upregulated by LC cells in response to TNF-α, and siRNAs against eIF5A protect LC cells from apoptosis. Thus, eIF5A appears to be a novel proapoptotic protein in the TNF pathway and a possible target for treatment of glaucoma.
Dr. C.A. Taylor, Department of Biology, University of Waterloo, Waterloo, Ontario, Canada
Classification:
2.14 Optic disc (Part of: 2 Anatomical structures in glaucoma)
3.5 Molecular biology incl. SiRNA (Part of: 3 Laboratory methods)
3.12 Proteomics (Part of: 3 Laboratory methods)