Lactosylceramide interacts with and activates cytosolic phospholipase A2α

Abstract

Lactosylceramide (LacCer) is a member of the glycosphingolipid family and is known to be a bioactive lipid in various cell physiological processes. However, the direct targets of LacCer and cellular events mediated by LacCer are largely unknown. In this study, we examined the effect of LacCer on the release of arachidonic acid (AA) and the activity of cytosolic phospholipase A2α (cPLA2α). In CHO-W11A cells, treatment with 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), an inhibitor of glucosylceramide synthase, reduced the glycosphingolipid level, and the release of AA induced by A23187 or platelet-activating factor was inhibited. The addition of LacCer reversed the PPMP effect on the stimulus-induced AA release. Exogenous LacCer stimulated the release of AA, which was decreased by treatment with an inhibitor of cPLA2α or silencing of the enzyme. Treatment of CHO-W11A cells with LacCer induced the translocation of full-length cPLA2α and its C2 domain from the cytosol to the Golgi apparatus. LacCer also induced the translocation of the D43N mutant of cPLA2α. Treatment of L929 cells with TNF-α induced LacCer generation and mediated the translocation of cPLA2α and AA release, which was attenuated by treatment with PPMP. In vitro studies were then conducted to test whether LacCer interacts directly with cPLA2α. Phosphatidylcholine vesicles containing LacCer increased cPLA2α activity. LacCer bound to cPLA2α and its C2 domain in a Ca(2+)-independent manner. Thus, we propose that LacCer is a direct activator of cPLA2α.

Keywords: Arachidonic acid; Glycosphingolipid; Phospholipase A; Sphingolipid; Tumor Necrosis Factor (TNF).

FIGURE 1.

LacCer induces cPLA₂α-dependent AA release from cells. A, CHO-W11A cells were cultured for 48 h in culture medium supplemented with vehicle or 1 μm PPMP. After the cells were washed, glycosphingolipids were extracted and spotted onto a TLC plate. B, CHO-W11A cells were cultured for 30 h in culture medium supplemented with or without 1 μm PPMP, 10 μm GlcCer, and/or 10 μm LacCer. The cells were then labeled by incubation for 18 h in 0.1% BSA- and [³H]AA-containing medium supplemented with the same reagents. The labeled cells were washed and stimulated with 1 μm A23187 or 100 nm PAF for 30 min at 37 °C. ^a, p < 0.05, significantly different from the control without PPMP. C, [³H]AA-labeled cells were stimulated with the indicated concentrations of LacCer for 30 min at 37 °C. D, [³H]AA-labeled cells were stimulated with vehicle or 30 μm LacCer for the indicated time periods at 37 °C. ○, vehicle; ●, LacCer. E, the labeled cells were pretreated with vehicle (Control) or 2 μm pyrrophenone for 30 min prior to stimulation with vehicle (Vehi) or 30 μm LacCer for 30 min at 37 °C. ^a, p < 0.05, significantly different from the control without LacCer; ^b, p < 0.05, significantly different from the control without pyrrophenone. F, [³H]AA-labeled L929 and L929-cPLA₂α-shRNA cells were pretreated with 2 μm pyrrophenone (Pyrro) for 30 min and then stimulated with 30 μm LacCer for 30 min at 37 °C. The expression levels of cPLA₂α in both cells are shown. ^a, p < 0.05, significantly different from the control without LacCer. The data shown are the means ± S.E. for three experiments.

FIGURE 2.

LacCer induces cPLA₂α translocation in cells. A, CHO-W11A cells transiently transfected with expression vectors for GFP-cPLA₂α and GFP were treated with vehicle (Control) or 30 μm LacCer for 1 h. B, CHO-W11A cells transiently transfected with expression vectors for GFP-cPLA₂α and red fluorescent protein (RFP)-Golgi were treated with 30 μm LacCer. C, DsRed-cPLA₂α-expressing CHO-W11A cells were treated with 100 nm BODIPY-C5-LacCer for 1 h. D, GFP-C2-expressing CHO-W11A cells were treated with 30 μm LacCer for 1 h. In A–D, data are representative of three independent experiments.

FIGURE 3.

LacCer activates cPLA₂α without intracellular Ca²⁺ mobilization. A, CHO-W11A cells transiently transfected with expression vector for GFP-cPLA₂α were treated with vehicle (Control) or 30 μm LacCer for 1 h in the presence of 2 mm EGTA. B, GFP-D43N-cPLA₂α-expressing CHO-W11A cells were treated with 30 μm LacCer or 5 μm A23187 for 1 h. C, CHO-W11A cells labeled with Oregon Green 488 were washed and then treated with 30 μm LacCer for 10 or 60 min or with 5 μm A23187 for 5 min. In A–C, data are representative of three independent experiments. D, [³H]AA-labeled CHO-W11A cells were stimulated with vehicle (Vehi) or 30 μm LacCer for 30 min at 37 °C in the presence or absence of 2 mm EGTA. ^a, p < 0.05, significantly different from the control without LacCer.

FIGURE 4.

cPLA₂α binds to LacCer in a calcium-independent manner. A, the binding of cPLA₂α to LacCer was examined using the lipid-protein overlay assay as described under “Experimental Procedures.” The indicated amounts of LacCer were spotted onto a Hybond C membrane. The membrane was exposed to purified GST-cPLA₂α overnight at 4 °C. B, the lipid-protein overlay assay was repeated with 100 nmol of lipids and GST-cPLA₂α. PC, 1-palmitoyl-2-oleoylphosphatidylcholine; PE, 1-palmitoyl-2-oleoylphosphatidylethanolamine; PI, 1-palmitoyl-2-oleoylphosphatidylinositol; S1P, sphingosine 1-phosphate. C, the lipid-protein overlay assay was repeated with 100 nmol of LacCer, 20 nmol of C1P, and GST-cPLA₂α with or without 2 mm EGTA. D, the membrane spotted with 100 nmol of LacCer was exposed to the GST-tagged C2 domain of cPLA₂α overnight at 4 °C. E, the large multilamellar vesicle binding assay was performed as described under “Experimental Procedures.” In A–E, data are representative of three independent experiments.

FIGURE 5.

LacCer activates cPLA₂α in vitro. PLA₂ activity in the cytosolic fraction from HEK293T cells expressing human cPLA₂α was measured as described under “Experimental Procedures.” PLA₂ activities toward PAPC and 95:5 PAPC/LacCer-mixed vesicles were measured in reaction buffer with or without 2 μm pyrrophenone (Pyrro) or 2 mm EGTA. The data shown are the means ± S.E. for three experiments. ^a, p < 0.05, significantly different from the control without LacCer.

FIGURE 6.

TNF-α induces cPLA₂α-dependent AA release via LacCer formation in L929 cells. A, L929 cells labeled with [¹⁴C]serine for 18 h were stimulated with or without 10 nm TNF-α for 2 h. After the cells were washed, lipids were extracted and separated by TLC as described under “Experimental Procedures.” B, [³H]AA-labeled L929 cells were stimulated with 10 nm TNF-α for the indicated time periods at 37 °C. ○, none; ●, TNF-α. C, L929 cells were pretreated with vehicle or 3 μm PPMP for 30 min, and then the cells and L929-cPLA₂α-shRNA cells were stimulated with 10 nm TNF-α for 6 h at 37 °C. D, down-regulation of GlcCer synthase were performed as described under “Experimental Procedures.” L929 cells transfected with or without GlcCer synthase siRNA were stimulated with 10 nm TNF-α for 6 h at 37 °C. The expression levels of GlcCer synthase (GCS) in cells are shown. E, L929 cells were pretreated with vehicle or 3 μm PPMP for 30 min and then stimulated with 10 nm TNF-α for 2 h at 37 °C. The cytosolic and membrane fractions were prepared and subjected to immunoblot analysis. Upper panels, immunoblotting with antibodies against cPLA₂α. Lower panel, the histogram represents the expression levels of cPLA₂α in membrane fractions quantified using ImageJ. The data shown are the means ± S.E. for three experiments. ^a, p < 0.05, significantly different from the control without TNF-α; ^b, p < 0.05, significantly different from the control without PPMP or cPLA₂α knockdown. F, L929 cells transiently transfected with expression vector for GFP-cPLA₂α were treated with or without TNF-α for 2 h. PPMP were pretreated for 30 min before stimulation with TNF-α.