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, 101 (22), 8491-6

Neuroprotectin D1: A Docosahexaenoic Acid-Derived Docosatriene Protects Human Retinal Pigment Epithelial Cells From Oxidative Stress

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Neuroprotectin D1: A Docosahexaenoic Acid-Derived Docosatriene Protects Human Retinal Pigment Epithelial Cells From Oxidative Stress

Pranab K Mukherjee et al. Proc Natl Acad Sci U S A.

Abstract

Docosahexaenoic acid (DHA) is a lipid peroxidation target in oxidative injury to retinal pigment epithelium (RPE) and retina. Photoreceptor and synaptic membranes share the highest content of DHA of all cell membranes. This fatty acid is required for RPE functional integrity; however, it is not known whether specific mediators generated from DHA contribute to its biological significance. We used human ARPE-19 cells and demonstrated the synthesis of 10,17S-docosatriene [neuroprotectin D1 (NPD1)]. This synthesis was enhanced by the calcium ionophore A-23187, by IL-1beta, or by supplying DHA. Under these conditions, there is a time-dependent release of endogenous free DHA followed by NPD1 formation, suggesting that phospholipase A(2) releases the mediator's precursor. Added NPD1 potently counteracted H(2)O(2)/tumor necrosis factor alpha oxidative-stress-triggered apoptotic RPE DNA damage. NPD1 also up-regulated the antiapoptotic proteins Bcl-2 and Bcl-x(L) and decreased proapoptotic Bax and Bad expression. Moreover, NPD1 (50 nM) inhibited oxidative-stress-induced caspase-3 activation. NPD1 also inhibited IL-1beta-stimulated expression of cyclooxygenase 2 promoter transfected into ARPE-19 cells. Overall, NPD1 protected RPE cells from oxidative-stress-induced apoptosis, and we predict that it will similarly protect neurons. This lipid mediator therefore may indirectly contribute to photoreceptor cell survival as well. Because both RPE and photoreceptor cells die in retinal degenerations, our findings contribute to the understanding of retinal cell survival signaling and potentially to the development of new therapeutic strategies.

Figures

Fig. 1.
Fig. 1.
10,17S-docosatriene (NPD1) is synthesized in ARPE-19 cells. (A) Elucidation of the structure of NPD1 by lipidomic analysis by LC-MS/MS (–35). MS/MS spectrum for NPD1 shows a full scan of negative ion products for selected parent ion (m/z 359). (Inset) The UV spectrum. (B) Production of NPD1 by IL-1β (6 h). (C) Calcium ionophore A-23187 (10 μM) promoted the release of free DHA (blue bars) and the formation of NPD1 (red bars) as a function of incubation time. No exogenous DHA was added. As described in Materials and Methods, cells were treated with IL-1β or A-23187, then lipids were extracted and analyzed by LC-MS/MS.
Fig. 2.
Fig. 2.
NPD1 attenuates oxidative-stress-induced apoptosis. (A) BSA–DHA enhanced NPD1 synthesis and led to decreased apoptosis. After plating, cells incubated for 72 h in the presence of either BSA (3.35 μM) or BSA plus DHA (6.7 μM). Then the cells were serum-starved for 1 h and TNF-α/H2O2 was added (14 h). Cells pretreated with BSA plus DHA yielded marked attenuation of Hoechst-positive cells. (B) NPD1 (50 nM) attenuated oxidative-stress-induced Hoechst-positive staining. After plating (72 h), cells were serum-starved for 8 h, TNF-α/H2O2 was added, and the cells were further incubated for 14 h and then stained with Hoechst reagent. The 800 and 400 μM refer to H2O2 concentrations. (C) Lack of inhibition by 50 nM prostaglandin E2, LTB4, or 20-OH-LTB4 of oxidative-stress-induced or Hoechst-positive staining. (D) DHA (50 nM) added as a free acid inhibited oxidative-stress-induced Hoechst-positive staining. Arachidonic acid (50 nM) exhibited far less protection. In C and D, 800 μM H2O2 was used; other conditions were as in B. (E) Synthesis of NPD1 in cultures exposed to DHA (50 nM) as in D. The pool size of free DHA is shown by blue bars.
Fig. 3.
Fig. 3.
Protection against oxidative-stress-induced DNA fragmentation by NPD1. (A) DNA fragmentation assay using [3H]thymidine prelabeling. (B) DNA fragmentation detection by ELISA detection of mono- and oligonucleosomes. NPD1 (50 nM) was added at the same time as TNF-α/H2O2.
Fig. 4.
Fig. 4.
Expression of selected Bcl-2 family proteins in ARPE-19 cells: NPD1 up-regulated antiapoptotic proteins and down-regulated proapoptotic protein expression. Cells were grow for 72 h after plating, placed in serum-free medium for 1 h, then incubated with TNF-α/H2O2 for 6 h. Data represent four independent experiments with triplicate samples in each case.
Fig. 5.
Fig. 5.
NPD1 protects against caspase cleavage induced by TNF-α/H2O2. ARPE-19 cells were serum-starved for 1 h before a 6- to 8-h treatment with TNF-α (10 ng/ml)/H2O2 (either 400 or 800 μM as indicated) in the presence or absence of NPD1 (50 nM). (A) Caspase-3 cleavage was detected by Western blot analysis by using poly(ADP-ribose) polymerase (Santa Cruz Biotechnology) as antibody. (B) Cleavage of caspase-3 using cells stably transfected with a lentivirus construct containing the Asp-Glu-Val caspase-3 cleavage sequence. Red bars represent coincubations with 50 nM NPD1. The values are the average of triplicate samples in three independent experiments.
Fig. 6.
Fig. 6.
NPD1 inhibits IL-1β-induced COX-2 expression. ARPE-19 cells grown in six-well plates were transfected with human COX-2 (830 bp) promoter-luciferase construct as described in Materials and Methods.

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