Modulation of the activity of cytosolic phospholipase A2alpha (cPLA2alpha) by cellular sphingolipids and inhibition of cPLA2alpha by sphingomyelin

Abstract

We examined the effect of the cellular sphingolipid level on the release of arachidonic acid (AA) and activity of cytosolic phospholipase A2alpha (cPLA2alpha) using two Chinese hamster ovary (CHO)-K1-derived mutants deficient in sphingolipid synthesis: LY-B cells defective in the LCB1 subunit of serine palmitoyltransferase for de novo synthesis of sphingolipid species, and LY-A cells defective in the ceramide transfer protein CERT for SM synthesis. When LY-B and LY-A cells were cultured in Nutridoma medium and the sphingolipid level was reduced, the release of AA stimulated by the Ca(2+) ionophore A23187 increased 2-fold and 1.7-fold, respectively, compared with that from control cells. The enhancement in LY-B cells was decreased by adding sphingosine and treatment with the cPLA2alpha inhibitor. When CHO cells were treated with an acid sphingomyelinase inhibitor to increase the cellular SM level, the release of AA induced by A23187 or PAF was decreased. In vitro studies were then conducted to test whether SM interacts directly with cPLA2alpha. Phosphatidylcholine vesicles containing SM reduced cPLA2alpha activity. Furthermore, SM disturbed the binding of cPLA2alpha to glycerophospholipids. These results suggest that SM at the biomembrane plays important roles in regulating the cPLA2alpha-dependent release of AA by inhibiting the binding of cPLA2alpha to glycerophospholipids.

Fig. 1.

Enhancement of cPLA2α-dependent AA release in sphingolipid-deficient cells. Cells were cultured in Nutridoma medium at 37°C for 30 h. They were then incubated for 18 h in Ham's F-12 medium containing 0.1% BSA. A: After the cells were washed, lipids were extracted and separated by TLC. B: Cells were cultured in Normal medium or Nutridoma medium at 37°C for 30 h. They were then labeled by incubation for 18 h in Ham's F-12 medium containing [³H]AA and 0.1% BSA. The labeled cells were stimulated with 1 μM A23187 for 30 min at 37°C. C: Cells were prepared as above. [³H]AA was further added with 0.1% BSA for 18 h. The labeled cells were pretreated for 30 min with or without 2 μM pyrrophenone. The cells were washed and then stimulated with vehicle, 1 μM A23187, and 2 μM pyrrophenone for 30 min at 37°C. The data shown are the mean ± SEM. for three experiments. *p < 0.05, significantly different from the values in LY-B/cLCB1 cells.

Fig. 2.

Effect of sphingolipid-deficiency on the protein levels, translocation and phosphorylation of cPLA2α in LY-B cells. Cells were cultured in Nutridoma medium at 37°C for 30 h, then incubated in Ham's F-12 medium containing 0.1% BSA for 18 h. A: The protein levels of cPLA2α in cell lysates were determined using anti-cPLA2α antibody. Upper panels, immunoblotting with antibodies against cPLA2α and β-tubulin. The histograms represent ratio of cPLA2α to β-tubulin as assessed with pooled densitometric data (mean ± SD) from three independent experiments. Data were normalized to ratio of cPLA2α to β-tubulin of LY-B cells. B: Cells transiently transfected with an expression vector for GFP-cPLA2α were stimulated with 1 μM A23187 for 2 min. C: Cells were prepared as above. The cells were stimulated with 1 μM A23187 for 20 min at 37°C and were subjected to immunoblot analysis. Upper panels, immunoblotting with antibodies against phospho-ERK1/2 (p-ERK1/2) and ERK1/2. The histograms represent ratio of p-ERK1/2 to total ERK1/2 as assessed with pooled densitometric data (mean ± SD) from three independent experiments. Data were normalized to ratio of p-ERK1/2 to total ERK1/2 of vehicle-treated LY-B cells. D: Cells were prepared as above. [³H]AA was further added with 0.1% BSA for 18 h. The labeled cells were incubated for 30 min with or without 20 μM U0126 and stimulated with 1 μM A23187 for 30 min at 37°C. The data shown are the mean ± SEM for three experiments. *p < 0.05, significantly different from the values in the absence of A23187. In A–C, data are representative of three independent experiments.

Fig. 3.

Enhancement of AA release is reversed by adding sphingosine in LY-B cells. Cells were cultured in Nutridoma medium with or without 1 μM D-erythro-sphingosine at 37°C for 30 h, followed by Ham's F-12 medium containing 0.1% BSA with or without 1 μM D-erythro-sphingosine for 18 h. A: After the cells were washed, lipids were extracted and separated by TLC. B: Cells were prepared as above. [³H]AA was further added for 18 h. After the cells were washed, the amount of AA released from the cells stimulated with 1 μM A23187 was measured. The data shown are the mean ± SEM for three experiments. *p < 0.05, significantly different from the values in LY-B/cLCB1 cells.

Fig. 4.

Enhancement of AA release in LY-A cells. Cells were cultured in Nutridoma medium at 37°C for 30 h, then incubated in Ham's F-12 medium containing 0.1% BSA for 18 h. A: After the cells were washed, lipids were extracted and separated by TLC. B: Cells were prepared as above. [³H]AA was further added with 0.1% BSA for 18 h. The labeled cells were washed and stimulated with 1 μM A23187 for 30 min at 37°C. The data shown are the mean ± SEM for three experiments. *p < 0.05, significantly different from the values in LY-A/hCERT cells.

Fig. 5.

Effect of SM accumulation on AA release in CHO-W11A cells. Cells were cultured for 30 h in Normal medium with or without acid SMase inhibitors, 30 μM desipramine, 10 μM imipramine, or 30 μM amitriptiline. A: The cells were further incubated for 18 h in Normal medium with or without acid SMase inhibitors. After the cells were washed, lipids were extracted and separated by TLC. B: The amounts of SM shown in A were quantified using software for densitometric analyses. C: Cells were prepared as above. They were then labeled through incubation for 18 h in Normal medium containing [³H]AA supplemented with or without acid SMase inhibitors. The labeled cells were washed and stimulated with 1 μM A23187 or 100 nM PAF for 30 min at 37°C. The data shown are the mean ± SEM for three experiments. *p < 0.05, significantly different from the values in the absence of inhibitors. D: The amount of SM increased by the acid SMase inhibitor and the release of AA induced by A23187 or PAF are shown on the x axis and y axis, respectively. Data on the amount of SM are presented as percentages of the control value. Data from three independent experiments were used for the analysis.

Fig. 6.

SM reduces the activity of cPLA2α in vitro. PLA2 activity in the cytosolic fraction from HEK293T cells expressing human cPLA2α was measured as described in Materials and Methods. A: Liposomes containing 2 μM of labeled PAPC and phospholipids, SM, PS or PE respectively, at a molar ratio of 1:1 were prepared by sonication. B: The liposomes containing 2 μM of labeled PAPC and the 2 μM SM vesicles were generated separately by sonication. Then, sources of enzyme were added and incubated for 30 min at 37°C. The data are the mean ± SEM for three experiments.

Fig. 7.

SM disturbs the binding of cPLA2α to glycerophospholipids. The binding of cPLA2α to various glycerophospholipids was examined using the lipid-protein overlay assay as described in Materials and Methods. A: The binding of cPLA2α to glycerophospholipids. Several lipids, 100 nmol PC, 100 nmol PS, 100 nmol PE, 5 nmol PIP2, and 100 nmol SM, were spotted onto a Hybond C membrane. B: The effect of SM on the binding of cPLA2α to PS or PIP2. The mixed lipids in the solvent (chloroform / methanol = 1:1) were spotted onto a Hybond C membrane. The membrane was exposed to enzyme sources overnight at 4°C. Three independent experiments gave similar results.