Oxidative stress triggers Ca-dependent lysosome trafficking and activation of acid sphingomyelinase

Abstract

Recent studies demonstrate that rapid translocation of the acid sphingomyelinase (ASM), a lysosomal hydrolase, to the outer leaflet of the cell membrane and concomitant release of ceramide constitute a common cellular signaling cascade to various stimuli including CD95 ligation, UV-irradiation, bacterial and viral infections. Reactive oxygen species (ROS) were shown to play a crucial role in regulating this signaling cascade at least for some bacterial infections and UV-irradiation. However, the precise role of ROS for regulation of ASM is unknown. Here, by confocal microscopy and flow cytometry analysis, we demonstrate that hydrogen peroxide (H(2)O(2)), a primary form of ROS in mammalian cells, induces very rapid translocation of ASM and formation of ceramide-enriched membrane platforms in the plasma membrane of Jurkat T cells. In parallel, H(2)O(2) triggers lysosome trafficking and fusion with the plasma membrane, i.e. lysosome exocytosis, as detected by exposure of a lysosome-associated protein, LAMP1. Depletion of intracellular Ca(2+) by cell permeable EGTA-AM inhibits H(2)O(2)-induced lysosome exocytosis, ASM translocation and formation of ceramide-enriched platforms. Pharmacological inhibition or genetic deficiency of ASM did not affect H(2)O(2)-induced lysosome exocytosis. These results indicate that ROS-induced membrane translocation of ASM is mediated by exocytosis of lysosomes, which is dependent on intracellular Ca(2+) release.

Fig. 1. Oxidative stress induces ASM externalization and formation of ceramide-enriched membrane platforms

(A) Oxidative stress by H₂O₂ (1 mM) induces a translocation of ASM onto the cell surface. Surface ASM was detected by flow cytometry analysis of cells incubated with Cy3-labeled goat anti-ASM antibodies. Shown is a representative flow cytometry analysis from four independent experiments. (B) H₂O₂ (1 mM)-induced ASM translocation correlates with rapid clustering of ceramide in the membrane and formation of ceramide-enriched membrane platforms. Representative confocal fluorescence microscopy and light images from three independent experiments are shown. (C) Displayed are the summarized data showing the mean fluorescence intensity for Cy3-anti-ASM staining from four independent experiments. (D) The quantitative analysis of the data from four independent experiments, which each included analysis of 200 cells/time point, shows the formation of ceramide-enriched membrane platforms in a large proportion of the cells. Displayed is the percentage of cells with a ceramide-enriched membrane platform at indicated time points. Panel C and D show the mean ± S.D. Significant differences between treated and non-treated controls were determined by t-test and are indicated by *** (P<0.001, t-test) or * (P<0.05, t-test), respectively.

Fig. 2. Oxidative stress induces ASM translocation and activation of ASM onto membrane lipid rafts

(A) Western blot analysis of ASM and Actin in Jurkat cells stimulated with or without H₂O₂ (10 min, 1 mM). The experiments were repeated three times with similar results. (B) Confocal microscopy of Jurkat cells revealed a translocation of ASM onto the surface of the cell membrane and a co-localization of ASM with ceramide-enriched membrane platforms. Shown is a representative results from three independent experiments. (C) ASM activity in DRM fractions is increased upon H₂O₂ stimulation. Low density membrane fractions were collected and designated as DRMs (Light). High density membrane fractions were collected in parallel and designated as none-DRM fraction (Heavy). The DRM maker protein flotillin-1 was detected by Western blot analysis. Shown is the mean ± S.D. of three independent experiments. Significant differences between treated and non-treated controls were determined by t-test and are indicated by ***, respectively (P<0.001, t-test).

Fig. 3. H₂O₂induces lysosome excytosis

The translocation of LAMP1 onto the cell surface served as marker for lysosome fusion with the plasma membrane and was detected by flow cytometry analysis using PE-conjugated goat anti-LAMP1. (A) Shown is a representative flow cytometry analysis from four independent experiments. (B) Summarized data showing the mean fluorescence intensity for PE-anti-LAMP1 staining (n=4). Shown are the mean ± SD. Significant differences between treated and non-treated controls were determined by t-test and are indicated by *** (P<0.001, t-test) or * (P<0.05, t-test), respectively.

Fig. 4. H₂O₂-induced lysosomal excytosis and ASM externalization are Ca²⁺-dependent

(A, B) Jurkat cells were stimulate with or without H₂O₂ (1 mM, 10 min) in the absence (DMSO) or presence of EGTA-AM (100 μM) in a Ca²⁺-free PBS buffer. H₂O₂-induced ASM externalization and lysosomal excytosis were detected by flow cytometry analysis using Cy3-conjugated anti-ASM (A) and PE-conjugated goat anti-LAMP1 (B). Shown is a representative flow cytometry analysis from four independent experiments. (C) and (D) summarized data of four independent studies and show the mean fluorescence intensity for Cy3-anti-ASM or PE-anti-LAMP1 staining. (E) Attenuation of H₂O₂-induced ceramide-enriched membrane platforms by EGTA-AM. Displayed is the percentages of cells with a ceramide-enriched membrane platform in the cells treated with 1 mM H₂O₂ for 10 min (n = 4). Panel C–E show the mean ± S.D. Significant differences were determined by t-test (***, H₂O₂ vs. Control, P<0.001; §, EGTA+H₂O₂ vs. H₂O₂, P<0.001).

Fig. 5. Effect of ASM inhibition or deficiency on H₂O₂-induced lysosomal exocytosis

(A) Shown is a representative flow cytometry analysis diagram using PE-conjugated goat anti-LAMP1 antibodies in Jurkat cells with or without ASM inhibitor amitriptyline (10 μM) (n = 4). (B) Spleen T cells were isolated from wild-type and Asm deficient (Smpd1^−/−) mice, stimulated without (green) or with H₂O₂ (red) and stained with PE-conjugated goat anti-LAMP1 antibodies. Shown are representative flow cytometry analyses from 3 independent experiments. Summarized data from 3 independent studies show the mean fluorescence intensity for PE-anti-LAMP1 staining in Jurkat cells (C) or spleen T cells (D). Displayed is the mean ± S.D. Significant differences between treated and non-treated controls were determined by t-test and are indicated by *** (P<0.001, t-test).