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D. Zhu, P.G. Sreekumar, C. Spee, R. Kannan, S.J. Ryan, D.R. Hinton; Effects of Modulating Ceramide Pathway Enzymes on Human RPE and PR Cell Survival/Death . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2866.
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© ARVO (1962-2015); The Authors (2016-present)
Ceramide and some of its metabolic derivatives are important modulators of cell apoptosis and proliferation. Dysregulation or imbalance of their metabolic pathways may promote the development of retinal degenerations. We sought to modulate the expressions of the enzymes involved in the ceramide metabolic pathway, and examine their effects on the survival/death of human retinal pigmented epithelial (RPE) and photoreceptor (PR) cells.
We used RT–PCR to screen the enzymes in the ceramide metabolic pathway that are expressed in RPE (ARPE19) and PR (661W) cell lines. The ARPE19 and 661W cells were challenged with C2–ceramide at concentrations of 10 to 50 ug/ml. The pcDNA3.1 vector inserted with either neutral sphingomyelinase–2 (SMDP3) or sphingosine kinase1 (SphK1) cDNA was introduced into ARPE19 and 661W cells with Lipofectamine (Invitrogen). The enzyme cDNA–transfected or untransfected ARPE19 and 661W cells were then nutrition–depleted for 24 to 72 hours. The numbers of apoptotic or proliferating cells were measured with Tunel and BrdU assays.
SMDP2, 3 and ceramide kinase2 (CERK2) are highly expressed in human ARPE19 and 661W cell lines. Acid sphingomyelinase, acid ceramidase, and SphKs are weakly expressed in ARPE19 and 661W cells. The percentage of apoptotic cells increases with the C2–ceramide treatment in a dose–dependent manner. Over–expression of SMDP3 in the ARPE19 and 661W cells increases cell apoptosis, while over–expression of SphK1 promotes cell proliferation and protects RPE and PR cells from apoptosis.
The results indicate that RPE and PR cells may synthesize ceramide through SMDP and may phosphorylate ceramide through CERK2. In addition, over–expression of the ceramide–producing enzyme in RPE or PR cells increases cell apoptosis, while over–expression of SphK1 increases cellular S1P, promotes the proliferation of RPE and PR cells, and protects RPE and PR from ceramide–induced apoptosis. Understanding the relationship between the metabolism of sphingolipids and their effects on RPE and PR cell survival/death may help us develop effective and efficient new therapies for these blinding diseases.
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