Overexpression of acid ceramidase (ASAH1) protects retinal cells (ARPE19) from oxidative stress - PubMed (original) (raw)

Overexpression of acid ceramidase (ASAH1) protects retinal cells (ARPE19) from oxidative stress

Eriko Sugano et al. J Lipid Res. 2019 Jan.

Abstract

Over 11 million people in the United States alone have some form of age-related macular degeneration (AMD). Oxidative stress, cell death, and the degeneration of retinal pigment epithelial (RPE) cells contribute to AMD pathology. Recent evidence suggests that ceramide (Cer), a cellular sphingolipid mediator that acts as a second messenger to induce apoptosis, might play a role in RPE cell death. The lysosomal breakdown of Cer by acid ceramidase [_N_-acylsphingosine amidohydrolase (ASAH)1] into sphingosine (Sph) is the major source for Sph 1-phosphate production, which has an opposing role to Cer and provides cytoprotection. Here, we investigated the role of Cer in human RPE-derived ARPE19 cells under hydrogen peroxide-induced oxidative stress, and show that Cer and hexosyl-Cer levels increase in the oxidatively stressed ARPE19 cells, which can be prevented by overexpression of lysosomal ASAH1. This study demonstrates that oxidative stress generates sphingolipid death mediators in retinal cells and that induction of ASAH1 could rescue retinal cells from oxidative stress by hydrolyzing excess Cers.

Keywords: N-acylsphingosine amidohydrolase 1; age-related macular degeneration; ceramide; hexosyl-ceramide; lysosome, retinal pigment epithelium; retinal degeneration.

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Figures

Fig. 1.

Generation of ASAH1-expressed ARPE19 cells. Immunocytochemistry of ASAH1-expressed cells. ASAH1 expression vector was electroporated into ARPE19 cells, and ASAH1-Venus-expressing cells were selected after sorting to develop stable pure culture. A: Cells were stained with a lysosomal marker, LysoTracker Red. B: Venus (green) expression was observed in ASAH1 cells. C: Venus-ASAH1 expression was majorly colocalized with the lysosomal marker. D: Enlarged image of ASAH1 expression with LysoTracker Red; ASAH1 shows cytoplasmic expression and ultimately moving to the punctate lysosomes. Scale bars: 20 μm (A–C) and 10 μm (D). E: Transgene expression was confirmed by RT-PCR, and a 10-fold increase in ASAH1 was seen compared with ARPE19. F: Protein expression indicates endogenous ASAH1 expression in both transfected and untransfected ARPE19 cells. G: ASAH1 protein levels were normalized against β-actin, showing a >2-fold increase in ASAH1 expressionQ21. ###P < 0.001 between cell lines. Q31

Fig. 2.

Overexpression of Ly-ASAH1 protects against short-chain Cer-mediated cell death. A: ARPE19 cells were treated with exogenous short-chain C2-Cer, which mimics the biological activity of endogenous Cer species. Treatment with C2-Cer concentrations over 20 μM for 24 h was sufficient to induce significant reductions in cell viability. B: ARPE19 cells and ARPE19 cells transfected with Ly-ASAH1 were subjected to 10–20 μM C2-Cer concentrations for 24 h in serum-free media. Increasing C2-Cer concentration was correlated with decreasing cell viability for both ARPE19 cells and ARPE19-ASAH1 cells, but ARPE19-ASAH1 cells demonstrated resistance to Cer-mediated cell death at up to 10 μM C2-Cer. Cell viability was higher in ARPE19-ASAH1 cells relative to ARPE19 cells at all concentrations of C2-Cer. ARPE19, n = 4; ARPE19-ASAH1, n = 4; **P < 0.01 and ***P < 0.001 between treatment groups belonging to the same cell line; ###P < 0.001 between cell lines in a given treatment group.

Fig. 3.

Analysis of major sphingolipid content in untreated ARPE19 and ARPE19-ASAH1 cells. A: ARPE19 and ARPE19-ASAH1 cells were counted 24 and 48 h after culturing, with ARPE19-ASAH1 cells being significantly more numerous than ARPE19 cells at 24 and 48 h. B: Bar graphs represent mean overall sphingolipid concentrations (pmol/million cells) ± SEM. There was significantly less total Hex-Cer and SM in ARPE19-ASAH1 cells after 48 h. C: Bar diagram comparing mean ± SEM lyso-sphingolipid species (pmol/million cells). ARPE19-ASAH1 cells had greater levels of Sph and dh-Sph compared with ARPE19 cells. ARPE19, n = 4; ARPE19-ASAH1, n = 4. *P < 0.05; P < 0.01; ***P < 0.001 between cell lines.

Fig. 4.

Overexpression of Ly-ASAH1 protects cells against oxidative cell death. A: ARPE19 cells underwent cell death following a concentration greater than 800 μM H2O2. B: Bar diagrams depicting mean ± SEM. Cells stably transfected with Ly-ASAH1 (ARPE19-ASAH1) had significantly greater cell viability with an increase in oxidative stress. ARPE19, n = 4; ARPE19-ASAH1, n = 4. ***P < 0.001 between H2O2 concentrations; ###P < 0.001 between cell groups.

Fig. 5.

Analysis of major sphingolipids with oxidative stress for 3 h. A: Bar diagram of mean total SM ± SEM normalized with protein. No difference was observed between ARPE19 cells and ARPE19 cells transfected with Ly-ASAH1, nor was any difference observed between Veh-treated cells and cells under oxidative stress conditions for either group. B: Bar diagram of mean total Cer ± SEM, normalized with protein. Veh-treated cells had no significant differences, but H2O2 treatment significantly increased Cer in both ARPE19 (P < 0.01) and ARPE19-ASAH1 (P < 0.05) cells, but oxidatively stressed ARPE19-ASAH1 cells had significantly lower Cer levels relative to ARPE19 cells (P < 0.05). C: Bar diagram of mean total Lac-Cer ± SEM. No significant changes were seen in Lac-Cer levels between the ARPE19 cells and ARPE19-ASAH1 cells or following oxidative stress in either group. D: Bar diagram of mean total Hex-Cer ± SEM. ARPE19 cells with Veh treatment exhibited significantly higher levels of Hex-Cer relative to ARPE19-ASAH1 cells. Following oxidative stress, the Hex-Cer levels increased significantly among both ARPE19 and ARPE19-ASAH1 cells, while the ARPE19-ASAH1 cells maintained significantly lower Hex-Cer levels relative to the H2O2-treated ARPE19 cells. ARPE19, n = 4; ARPE19-ASAH1, n = 4. *P < 0.05, **P < 0.01, and ***P < 0.001 between H2O2 concentrations. #P < 0.05 and ###P < 0.001 between cell groups.

Fig. 6.

Quantification of bioactive sphingoid species in ARPE19 and ARPE19-ASAH1 cells. Bar diagrams depict mean ± SEM of Sph, dh-Sph, and S1P content within ARPE19 and ARPE19-ASAH1 cells. An oxidative stress-dependent effect was observed in both cell types. H2O2 treatment significantly reduced the content of Sph in both cell types. dh-Sph content within ARPE19 cells was significantly lower than dh-Sph levels in ARPE19-ASAH1 cells, but increased following oxidative stress. S1P content was significantly lower in Veh-treated ARPE19-ASAH1 cells. A significant increase in S1P was seen in an oxidative stress-dependent manner in both cell types, with ARPE19-ASAH1 cells containing relatively lower levels of S1P among both treatment groups. Significant difference between Veh and H2O2 groups: *P < 0.05, ***P < 0.001; significant difference between ARPE19 and ARPE19-ASAH1 cells: ##P < 0.01, ###P < 0.001; n = 4/group.

Fig. 7.

RT-PCR analysis of sphingolipid metabolic genes with oxidative stress. Each sample included in a condition that consisted of three independent RT-PCR experiments that were averaged and normalized against housekeeping genes. Bar graphs are represented as mean ± SEM. Change in sphingolipid genes as a result of overexpressed ASAH1 and stress condition. Significant difference between cell types depicted: #P < 0.05; n = 4. SMPD1, SM phosphodiesterase 1; SGPL1, S1P lyase 1; Cers2, Cer synthase 2_._

Fig. 8.

RT-PCR analysis of inflammatory and apoptotic genes with oxidative stress. Each sample included in a condition that consisted of three independent RT-PCR experiments that were averaged and normalized against housekeeping genes. Bar graphs are represented as fold over control mean ± SEM from n = 6 biological replications. Change in inflammatory and apoptotic markers as a result of cell type and oxidative stress. Significant difference between treatment groups: *P < 0.05, **P < 0.01. Significant difference between cell types depicted: #P < 0.05. FOSL1, FOS-like 1; BAX, BCL2-associated X protein; FAS, Fas cell surface death receptor; IL6, interleukin 6.

Fig. 9.

Schematic model of bioactive sphingolipid regulation of ARPE cell viability under oxidative stress. Oxidative stress induced by H2O2 causes an increase in the levels of Cer and Hex-Cer species in ARPE cells and leads to cell death and subsequent RD. ARPE cell survival under oxidative stress can be achieved through overexpression of ASAH1.

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Overexpression of acid ceramidase (ASAH1) protects retinal cells (ARPE19) from oxidative stress - PubMed (original) (raw)