Abstract
To determine the role of eukaryotic translation initiation factor 5A (eIF5A) in TNF-alpha-induced apoptosis of lamina cribrosa (LC) cells. LC cells were isolated from optic nerve heads of eyes of two human donors. The cells were treated with TNF-alpha 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-alpha was determined by Western blot analysis. The ability of small inhibitory (si)RNAs directed against eIF5A to protect LC cells from TNF-alpha-induced apoptosis was determined by Hoechst and TUNEL staining of transfected LC cells. TNF-alpha and camptothecin synergized to induce greater than two times more apoptosis in LC cells than when the cells were treated with TNF-alpha or camptothecin separately. Expression of eIF5A protein increased significantly after 8 hours of exposure to TNF-alpha and camptothecin, but not in response to camptothecin alone. siRNAs directed against eIF5A reduced apoptosis of LC cells in response to TNF-alpha 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-alpha and camptothecin revealed an 80% reduction in apoptosis with siRNA against eIF5A. TNF-alpha, in synergy with camptothecin, induces apoptosis in human LC cells. eIF5A is upregulated by LC cells in response to TNF-alpha, 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.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have