May 2006
Volume 47, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2006
Sphingosine–1–Phosphate Is an Activator of Neuroprotectin D1 (NPD1) Synthesis and Attenuates Oxidative Stress–Induced Apoptosis in Retinal Pigment Epithelial Cells
Author Affiliations & Notes
  • V.L. Marcheselli
    Ophthalmology/Neuroscience, LSUHSC, New Orleans, LA
  • P.K. Mukherjee
    Ophthalmology/Neuroscience, LSUHSC, New Orleans, LA
  • N.G. Bazan
    Ophthalmology/Neuroscience, LSUHSC, New Orleans, LA
  • Footnotes
    Commercial Relationships  V.L. Marcheselli, None; P.K. Mukherjee, None; N.G. Bazan, None.
  • Footnotes
    Support  Supported by the American Health Assistance Foundation
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 4832. doi:
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      V.L. Marcheselli, P.K. Mukherjee, N.G. Bazan; Sphingosine–1–Phosphate Is an Activator of Neuroprotectin D1 (NPD1) Synthesis and Attenuates Oxidative Stress–Induced Apoptosis in Retinal Pigment Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4832.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Sphingosine–1 phosphate (S–1–P), a ceramide derivative, has been implicated as a mediator in cellular proliferation, phospholipase–D activation, inhibition of chemotactic mobility, and invasiveness of tumor cells. Here we tested the ability of S–1–P as a potential agonist for NPD1 synthesis and cytoprotection, using ARPE–19 cells undergoing oxidative stress triggered by serum starvation/ H2O2/TNF alpha. We also investigated the effect of S–1–P on ASK–1 and AKT kinases, pro–apoptotic and anti–apoptotic proteins respectively.

Methods: : Serum–starved ARPE–19 cells were preincubated for 2 hours with 10 µM either S–1–P or DHS–1–P (dihydrosphingosine–1–phosphate, a biologically inactive saturated analog of S–1–P). Then oxidative stress was induced by exposing the cells to TNF/H2O2 for 15 hours. Methods to assess apoptosis included Hoechst 33258 staining, ELISA detection of mono– and oligonucleosomes, and DNA fragmentation by differential sedimentation after [3H]thymidine labeling. Cell and incubation media were extracted, purified, and subjected to LC–PDA–ESI–MS/MS–based lipidomic analysis. Proteins were assessed by Western–blot analysis.

Results: : S–1–P, but not DHS–1–P, inhibited oxidative stress–induced apoptosis in human ARPE–19 cells. S–1–P alone did not cause any detectable effect on cellular integrity and function. The inhibitory effect of S–1–P on cell survival was reversible. We also found up–regulation of anti–apoptotic signaling kinase AKT, and down–regulation of pro–apoptotic kinase ASK–1, by S–1–P in oxidatively stressed cells. S–1–P was a potent activator of NPD1 (10,17S–DHA) synthesis, since endogenous concentration of this lipid messenger increased in the cells as well as in the incubation medium when cells where incubated in the presence of S–1–P.

Conclusions: : S–1–P counteracts oxidative stress–induced apoptosis, suggesting that S–1–P participates in RPE cell survival. Conversely, the lack of effect on apoptosis by DHS–1–P indicates specificity at the receptor level. The S–1–P action on cell survival is reversible. Moreover, the changes in pro– and anti–apoptotic proteins induced by S–1–P imply their participation in these actions. S–1–P enhancement of NPD1 synthesis and release suggests that the release of this lipid messenger may elicit autocrine and/or paracrine actions. Overall, the cytoprotection by S–1–P against apoptosis, and the involvement of NPD1 in this growth factor–like bioactivity of S–1–P, open central questions regarding RPE cell–survival signaling.

Keywords: cytokines/chemokines • apoptosis/cell death • retinal pigment epithelium 
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