Ceramide 1-phosphate is required for the translocation of group IVA cytosolic phospholipase A2 and prostaglandin synthesis

Abstract

Little is known about the regulation of eicosanoid synthesis proximal to the activation of cytosolic phospholipase A(2)alpha (cPLA(2)alpha), the initial rate-limiting step. The current view is that cPLA(2)alpha associates with intracellular/phosphatidylcholine-rich membranes strictly via hydrophobic interactions in response to an increase of intracellular calcium. In opposition to this accepted mechanism of two decades, ceramide 1-phosphate (C1P) has been shown to increase the membrane association of cPLA(2)alpha in vitro via a novel site in the cationic beta-groove of the C2 domain (Stahelin, R. V., Subramanian, P., Vora, M., Cho, W., and Chalfant, C. E. (2007) J. Biol. Chem. 282, 20467-204741). In this study we demonstrate that C1P is a proximal and required bioactive lipid for the translocation of cPLA(2)alpha to intracellular membranes in response to inflammatory agonists (e.g. calcium ionophore and ATP). Last, the absolute requirement of the C1P/cPLA(2)alpha interaction was demonstrated for the production of eicosanoids using murine embryonic fibroblasts (cPLA(2)alpha(-/-)) coupled to "rescue" studies. Therefore, this study provides a paradigm shift in how cPLA(2)alpha is activated during inflammation.

FIGURE 1.

Endogenous C1P is required for the translocation of cPLA₂α in response to calcium ionophore. A, A549 cells (0.5 × 10⁵ cells/35-mm plate) were transfected with siRNA specific to CERK using Dharmafect1 reagent as previously described (19, 21). After 36 h, the total RNA was isolated and analyzed by quantitative PCR. Data are representative of n = 4 from 2 separate occasions. B, A549 cells (0.5 × 10⁶ cells/10-cm plate) were simultaneously transfected with CERK siRNA as described in panel A. Protein extracts were produced, and the level of CERK was examined by Western blot analysis using a monoclonal antibody specific to CERK (21). β-Actin was used as a loading control. C, A549 cells (0.5 × 10⁶ cells/10-cm plate) were again transfected with CERK siRNA as described in panel A. C1P levels were then analyzed by steady state labeling with [³²P]orthophosphate as described under “Experimental Procedures.” The lipids were harvested and subjected to lipid analysis using TLC and autoradiography. The spots on the TLC plate corresponding to C1P as denoted by the standards were scraped and counted by scintillation counting. The graph represents the amount of C1P detected and is representative of n = 6 from 2 separate occasions. Statistical significance was evaluated with Student's t test (*, p < 0.001). D, using the same methodology as panel B; the cells were processed for mass spectrometry as described under “Experimental Procedures.” The graph represents the level of the different C1P subspecies in fmol of C1P/1 × 10⁶ cells. Data are representative of n = 4 from 2 separate occasions. Statistical significance was evaluated with Student's t test (*, p < 0.05). E, A549 cells (0.5 × 10⁶ cells per 10-cm plate) were transfected with control siRNA or siRNA-specific to CERK using Dharmafect reagent as described under “Experimental Procedures.” After 36 h of incubation, the cells were treated with DMSO or A₂₃₁₈₇ for 5 min, and C1P levels were then analyzed by mass spectrometry as described previously. The graph represents the % control of the level of d-erytho-C16 C1P. Data are representative of n = 6 from 3 separate occasions. Statistical significance was evaluated with Student's t test (*, p < 0.001; **, no statistical difference compared with untreated samples). F, A549 cells (0.5 × 10⁵ cells) were seeded overnight onto 22 × 22-mm coverslips in 35-mm plates. The cells were infected with GFP-cPLA₂α(WT) adenovirus. After 24 h, siRNA specific to CERK was transfected into the cells using Dharmafect1 reagent. After 36 h, slides were analyzed by confocal microscopy. The figure depicts translocation of GFP-cPLA₂α(WT) treated with CERK siRNA or control siRNA upon activation with calcium ionophore (A₂₃₁₈₇ (10 μm, 5 min) or ionomycin (10 μm, 5 min)). DIC, differential interference contrast. G, a graph representing the % of translocation from the data depicted in panel D. Data are the mean ± S.E. and are representative of at least three different experiments. Statistical significance was evaluated with Student's t test (*, p < 0.05).

FIGURE 1.

Endogenous C1P is required for the translocation of cPLA₂α in response to calcium ionophore. A, A549 cells (0.5 × 10⁵ cells/35-mm plate) were transfected with siRNA specific to CERK using Dharmafect1 reagent as previously described (19, 21). After 36 h, the total RNA was isolated and analyzed by quantitative PCR. Data are representative of n = 4 from 2 separate occasions. B, A549 cells (0.5 × 10⁶ cells/10-cm plate) were simultaneously transfected with CERK siRNA as described in panel A. Protein extracts were produced, and the level of CERK was examined by Western blot analysis using a monoclonal antibody specific to CERK (21). β-Actin was used as a loading control. C, A549 cells (0.5 × 10⁶ cells/10-cm plate) were again transfected with CERK siRNA as described in panel A. C1P levels were then analyzed by steady state labeling with [³²P]orthophosphate as described under “Experimental Procedures.” The lipids were harvested and subjected to lipid analysis using TLC and autoradiography. The spots on the TLC plate corresponding to C1P as denoted by the standards were scraped and counted by scintillation counting. The graph represents the amount of C1P detected and is representative of n = 6 from 2 separate occasions. Statistical significance was evaluated with Student's t test (*, p < 0.001). D, using the same methodology as panel B; the cells were processed for mass spectrometry as described under “Experimental Procedures.” The graph represents the level of the different C1P subspecies in fmol of C1P/1 × 10⁶ cells. Data are representative of n = 4 from 2 separate occasions. Statistical significance was evaluated with Student's t test (*, p < 0.05). E, A549 cells (0.5 × 10⁶ cells per 10-cm plate) were transfected with control siRNA or siRNA-specific to CERK using Dharmafect reagent as described under “Experimental Procedures.” After 36 h of incubation, the cells were treated with DMSO or A₂₃₁₈₇ for 5 min, and C1P levels were then analyzed by mass spectrometry as described previously. The graph represents the % control of the level of d-erytho-C16 C1P. Data are representative of n = 6 from 3 separate occasions. Statistical significance was evaluated with Student's t test (*, p < 0.001; **, no statistical difference compared with untreated samples). F, A549 cells (0.5 × 10⁵ cells) were seeded overnight onto 22 × 22-mm coverslips in 35-mm plates. The cells were infected with GFP-cPLA₂α(WT) adenovirus. After 24 h, siRNA specific to CERK was transfected into the cells using Dharmafect1 reagent. After 36 h, slides were analyzed by confocal microscopy. The figure depicts translocation of GFP-cPLA₂α(WT) treated with CERK siRNA or control siRNA upon activation with calcium ionophore (A₂₃₁₈₇ (10 μm, 5 min) or ionomycin (10 μm, 5 min)). DIC, differential interference contrast. G, a graph representing the % of translocation from the data depicted in panel D. Data are the mean ± S.E. and are representative of at least three different experiments. Statistical significance was evaluated with Student's t test (*, p < 0.05).

FIGURE 2.

The C1P/cPLA₂α interaction is required for translocation of the enzyme in response to calcium ionophore. A549 cells (0.5 × 10⁵ cells) were seeded overnight onto 22 × 22-m coverslips in 35-m plates. The cells were infected with adenovirus containing GFP-cPLA₂α(WT) (panel A), GFP-cPLA₂α(R57A/K58A/R59A) (panel B), or CFP-cPLA₂α(WT) and YFP-cPLA₂α(R57A/K58A/R59A) (panel F). After 48 h, the cells were treated with calcium ionophore (A₂₃₁₈₇ (10 μm, 5 min)) and viewed using a Leica confocal microscope. A, effect of calcium ionophore on GFP-cPLA₂α(WT). DIC, differential interference contrast. B, effect of calcium ionophore on GFP-cPLA₂α(R57A/K58A/R59A). C, a graph representing the % of translocation from the data depicted in panels A and B. Data are the mean ± S.E. and are representative of at least six different experiments. Statistical significance was evaluated with Student's t test (*, p < 0.05). D, A549 cells were plated into a 10-cm dish at the concentration of 1 × 10⁶ cells per dish in their appropriate media and incubated under standard incubator conditions overnight. The following day the cells were rested for 2 h in 2% serum-containing media and then treated with calcium ionophore (10 μm A₂₃₁₈₇ for 5 min). The cells were fractionated into cytosol and membrane fractions. Protein samples were analyzed by Western immunoblotting analysis using anti-cPLA₂α antibody (1:1000) (Santa Cruz), anti-protein-disulfide isomerase antibody (1:1000) (Stressgen) to normalize the membrane fractions, and anti β-actin (1:5000) (Sigma) to normalize the cytosolic fractions. E, mouse embryonic fibroblasts, cPLA₂α^−/− (0.2 × 10⁵ cells/35-mm plate), were infected with adenovirus containing GFP-cPLA₂α(WT) or GFP-cPLA₂α(R57A/K58A/R59A). After 48 h the cells were rested for 2 h in 2% serum-containing media and then treated with calcium ionophore (10 μm A₂₃₁₈₇ for 5 min). The cells were fractionated as detailed under “Experimental Procedures” into cytosol fraction and membrane fraction. Protein samples were analyzed by Western immunoblotting analysis using anti-cPLA₂α antibody (1:1000) (Santa Cruz), anti-protein-disulfide isomerase antibody (1:1000) (Stressgen) to normalize the membrane fractions and anti-β-actin (1:5000) (sigma) to normalize the cytosolic fractions. F, effect of calcium ionophore on co-transfected CFP-cPLA₂α(WT) and YFP-cPLA₂α(R57A/K58A/R59A). G, a graph representing the % of translocation from the data depicted in panel F. Data are the mean ± S.E. and are representative of at least six different experiments. Statistical significance was evaluated with Student's t test. (*, p < 0.05). H, cPLA₂α^−/− MEFs (0.2 × 10⁵ cells) were seeded overnight onto 22 × 22-mm coverslips in 35-mm plates. The cells were then co-infected with adenovirus containing CFP-cPLA₂α(WT) and YFP-cPLA₂α(R57A/K58A/R59A). After 48 h of incubation, the cells were treated with of calcium ionophore (A_23187, 10 μm, 5 min) and analyzed by confocal microscopy. Data are representative of at least n = 3 on 3 separate occasions.

FIGURE 2.

The C1P/cPLA₂α interaction is required for translocation of the enzyme in response to calcium ionophore. A549 cells (0.5 × 10⁵ cells) were seeded overnight onto 22 × 22-m coverslips in 35-m plates. The cells were infected with adenovirus containing GFP-cPLA₂α(WT) (panel A), GFP-cPLA₂α(R57A/K58A/R59A) (panel B), or CFP-cPLA₂α(WT) and YFP-cPLA₂α(R57A/K58A/R59A) (panel F). After 48 h, the cells were treated with calcium ionophore (A₂₃₁₈₇ (10 μm, 5 min)) and viewed using a Leica confocal microscope. A, effect of calcium ionophore on GFP-cPLA₂α(WT). DIC, differential interference contrast. B, effect of calcium ionophore on GFP-cPLA₂α(R57A/K58A/R59A). C, a graph representing the % of translocation from the data depicted in panels A and B. Data are the mean ± S.E. and are representative of at least six different experiments. Statistical significance was evaluated with Student's t test (*, p < 0.05). D, A549 cells were plated into a 10-cm dish at the concentration of 1 × 10⁶ cells per dish in their appropriate media and incubated under standard incubator conditions overnight. The following day the cells were rested for 2 h in 2% serum-containing media and then treated with calcium ionophore (10 μm A₂₃₁₈₇ for 5 min). The cells were fractionated into cytosol and membrane fractions. Protein samples were analyzed by Western immunoblotting analysis using anti-cPLA₂α antibody (1:1000) (Santa Cruz), anti-protein-disulfide isomerase antibody (1:1000) (Stressgen) to normalize the membrane fractions, and anti β-actin (1:5000) (Sigma) to normalize the cytosolic fractions. E, mouse embryonic fibroblasts, cPLA₂α^−/− (0.2 × 10⁵ cells/35-mm plate), were infected with adenovirus containing GFP-cPLA₂α(WT) or GFP-cPLA₂α(R57A/K58A/R59A). After 48 h the cells were rested for 2 h in 2% serum-containing media and then treated with calcium ionophore (10 μm A₂₃₁₈₇ for 5 min). The cells were fractionated as detailed under “Experimental Procedures” into cytosol fraction and membrane fraction. Protein samples were analyzed by Western immunoblotting analysis using anti-cPLA₂α antibody (1:1000) (Santa Cruz), anti-protein-disulfide isomerase antibody (1:1000) (Stressgen) to normalize the membrane fractions and anti-β-actin (1:5000) (sigma) to normalize the cytosolic fractions. F, effect of calcium ionophore on co-transfected CFP-cPLA₂α(WT) and YFP-cPLA₂α(R57A/K58A/R59A). G, a graph representing the % of translocation from the data depicted in panel F. Data are the mean ± S.E. and are representative of at least six different experiments. Statistical significance was evaluated with Student's t test. (*, p < 0.05). H, cPLA₂α^−/− MEFs (0.2 × 10⁵ cells) were seeded overnight onto 22 × 22-mm coverslips in 35-mm plates. The cells were then co-infected with adenovirus containing CFP-cPLA₂α(WT) and YFP-cPLA₂α(R57A/K58A/R59A). After 48 h of incubation, the cells were treated with of calcium ionophore (A_23187, 10 μm, 5 min) and analyzed by confocal microscopy. Data are representative of at least n = 3 on 3 separate occasions.

FIGURE 3.

The C1P/cPLA₂α interaction is required for translocation of the enzyme in response to ATP. cPLA₂α^−/− MEFs (0.2 × 10⁵ cells) were seeded overnight onto 22 × 22-mm coverslips in 35-mm plates. The cells were infected with adenovirus containing CFP-cPLA₂α(WT) or YFP-cPLA₂α(R57A/K58A/R59A). After 48 h incubation, the cells were treated with DMSO (A) or ATP 100 μm (B) for 4 h and then analyzed by confocal microscopy as detailed under “Experimental Procedures.” Data are representative of at least n = 3 on 3 separate occasions. DIC, differential interference contrast.

FIGURE 4.

The C1P/cPLA₂α interaction is required for production PGE₂ in response to inflammatory agonists. Mouse embryonic fibroblasts, cPLA₂α^+/+ and cPLA₂α^−/− (0.2 × 10⁵ cells/35-mm plate) were infected with adenovirus containing GFP-cPLA₂α(WT) or GFP-cPLA₂α(R57A/K58A/R59A). After 48 h, the cells were treated with Il-1β (100 ng/ml), ATP (100 μm) or TNFα (50 ng/ml), and the media were collected at 0.5, 2, 4, 6, and 8 h. Simultaneously, the cells were collected and proteins extracted. A, effect of ATP on the PGE₂ production of MEFs transfected by cPLA₂α(WT) viruses or cPLA₂α(R57A/K58A/R59A) viruses (multiplicity of infection = 1). Mut, mutant. B, Western immunoblot showing the expression of cPLA₂α(WT) and cPLA₂α(R57A/K58A/R59A) in MEFs from the depicted experiment. C, effect of Il-1β on the PGE₂ production of MEFs transfected by either cPLA₂α(WT) virus or cPLA₂α(R57A/K58A/R59A) viruses (multiplicity of infection = 1). D, Western immunoblot showing the expression of cPLA₂α(WT) and cPLA₂α(R57A/K58A/R59A) in MEFs from the panel C experiment. E, effect of TNFα on the PGE₂ production of MEFs transfected by either cPLA₂α(WT) virus or cPLA₂α(R57A/K58A/R59A) viruses (multiplicity of infection = 1). F, Western immunoblot showing the expression of cPLA₂α(WT) and cPLA₂α(R57A/K58A/R59A) in MEFs from the panel E experiment. Data are representative of at least n = 3 on 3 separate occasions. Statistical significance was evaluated with Student's t test (*, p < 0.05). G, mouse embryonic fibroblasts, cPLA₂α^−/− (0.2 × 10⁵ cells/35-mm plate) were infected with adenovirus containing GFP-cPLA₂α(WT) or GFP-cPLA₂α(R57A/K58A/R59A). After 48 h the cells were collected in buffer containing 50 mm Tris (pH 7.4), 0.1 m KCl, and 30% glycerol. The cells were sonicated 3 times for 10 s, and they were analyzed for cPLA₂α activity as detailed under “Experimental Procedures.” Data are representative of n = 4 for 2 separate occasions.

FIGURE 5.

The hypothetical mechanism of cPLA₂α activation in response to inflammatory agonists and the generation of ceramide 1-phosphate. The role of C1P in cPLA₂α activation and eicosanoid synthesis in response to inflammatory agonists begins with the activation of CERK by increases in intracellular calcium (light blue) and other possibly other unknown mechanisms (e.g. CAMKII phosphorylation) after activation of a receptor by an inflammatory agonists (e.g. ATP via the purinergic receptor). CERK utilizes ceramide (dark purple) provided by CERT (ceramide transport protein) to produce C1P membrane structures (dark green) localized near phosphoinositides (dark blue). Simultaneously, cPLA₂α is phosphorylated by CAMKII and enzymes of the mitogen-activated protein kinase pathway in the C terminus leading to a fully active enzyme. cPLA₂α is then recruited to the trans-Golgi via C1P association with the C1P interaction site (light pink) in the CaLB domain, which penetrates the membrane via calcium binding subdomains I and III (dark red). Membrane penetration of the catalytic (cat)domain follows, which is dramatically enhanced by association with PIPs via the specific PIP interaction site (yellow). AA is then produced via the action of this enzyme and utilized by cyclooxygenases or lipoxygenases (e.g. COX-2 or 5-LO) beginning the eicosanoid biosynthetic pathway.