Cellular Plasmalogen Content Does Not Influence Arachidonic Acid Levels or Distribution in Macrophages: A Role for Cytosolic Phospholipase A2γ in Phospholipid Remodeling

Abstract

Availability of free arachidonic acid (AA) constitutes a rate limiting factor for cellular eicosanoid synthesis. AA distributes differentially across membrane phospholipids, which is largely due to the action of coenzyme A-independent transacylase (CoA-IT), an enzyme that moves the fatty acid primarily from diacyl phospholipid species to ether-containing species, particularly the ethanolamine plasmalogens. In this work, we examined the dependence of AA remodeling on plasmalogen content using the murine macrophage cell line RAW264.7 and its plasmalogen-deficient variants RAW.12 and RAW.108. All three strains remodeled AA between phospholipids with similar magnitude and kinetics, thus demonstrating that cellular plasmalogen content does not influence the process. Cell stimulation with yeast-derived zymosan also had no effect on AA remodeling, but incubating the cells in AA-rich media markedly slowed down the process. Further, knockdown of cytosolic-group IVC phospholipase A₂γ (cPLA₂γ) by RNA silencing significantly reduced AA remodeling, while inhibition of other major phospholipase A₂ forms such as cytosolic phospholipase A₂α, calcium-independent phospholipase A₂β, or secreted phospholipase A₂ had no effect. These results uncover new regulatory features of CoA-IT-mediated transacylation reactions in cellular AA homeostasis and suggest a hitherto unrecognized role for cPLA₂γ in maintaining membrane phospholipid composition via regulation of AA remodeling.

Keywords: arachidonic acid; eicosanoids; inflammation; monocytes/macrophages; phospholipase A2; phospholipid remodeling.

Scheme 1

Measurement of arachidonic acid (AA) movement from choline glycerophospholipids (PC) to ethanolamine phospholipids (PE).

Figure 1

Phospholipid fatty acid composition of RAW 264.7 cells and plasmalogen-deficient variants: (A) The profiles of major fatty acids in RAW 264.7 cells (open bars), RAW.12 (light brown), and RAW.108 (dark brown) were determined in the phospholipid fraction by GC-MS after converting the fatty acid glyceryl esters into fatty acid methyl esters. 16:1n-x denotes a mix of the n-9 and n-10 isomers, which elute together. (B) The phospholipid classes of RAW 264.7 cells (open bars), RAW.12 (light turquoise), and RAW.108 (dark turquoise) were separated, and their AA content was determined by GC/MS. Results are shown as means ± S.E.M. (n = 3).

Figure 2

AA-containing phospholipid species in RAW 264.7 cells and plasmalogen-deficient variants: The profile of AA-containing PC, PS (left panel), PE, PI (right panel) was determined for RAW 264.7 cells (open bars), RAW.12 cells (light purple bars), and RAW.108 cells (dark purple bars) by LC/MS. Results are shown as means ± S.E.M. (n = 3). * p < 0.05, significantly different from the corresponding species of RAW264.7 cells.

Figure 3

Phospholipid AA remodeling in RAW 264.7 cells and plasmalogen-deficient variants: (A) RAW 264.7 cells were pulse-labeled with [³H]AA, washed, and incubated without label for the indicated periods of time. Phospholipids were separated into classes by thin-layer chromatography. The radioactivity incorporated into each phospholipid class was determined by scintillation counting and is given as a percentage of the radioactivity present in phospholipids. (B) AA remodeling was analyzed for different cell types, and the remodeling time (time at which the radioactivity content of PC equals that of PE) was determined. Results are shown as means ± S.E.M. (n = 6 for panel A; n = 3 for panel B). MRPM, mouse resident peritoneal macrophages.

Figure 4

Eicosanoid production by RAW 264.7 cells and plasmalogen-deficient variants: RAW 264.7 cells (open bars), RAW.12 cells (light green bars), and RAW.108 cells (dark green bars) were stimulated with 150 µg/mL zymosan for 8 h. Afterward, extracellular media was removed and analyzed for eicosanoid levels by LC/MS. Eicosanoids produced by untreated RAW 264.7 cells (crosshatched bars) are shown for comparison. Results are shown as means ± S.E.M. (n = 3). PGE2, prostaglandin E₂; PGD2, prostaglandin D₂; PGF2a, prostaglandin F_2α; 15d-PGD2, 15-deoxy-Δ^12,14-prostaglandin D₂; dhk-PGE2, 13,14-dihydro-15-keto-prostaglandin E₂; dhk-PGD2, 13,14-dihydro-15-keto- prostaglandin D₂; 11-HETE, 11-hydroxyeicosatetraenoic acid; 12-HHT, 12-hydroxyheptadecatrienoic acid; 15-HETE, 15-hydroxyeicosatetraenoic acid.

Figure 5

Macrophage polarization through the classic pro-inflammatory (M1) and alternative (M2) pathways: RAW 264.7 cells (open bars) and their plasmalogen-defective variants RAW.12 (light red bars) and RAW.108 (dark red bars) were treated for 8 h with LPS (250 ng/mL) plus interferon-γ (500 U/mL) (A) or with interleukin-4 (20 ng/mL) plus interleukin-13 (20 ng/mL) (B) to generate classically (M1) or alternatively (M2) polarized macrophages, respectively. Afterward, the expression of the indicated markers was studied by qPCR. Data are relative to basal expression levels and normalized to cyclophilin A. A representative experiment is shown, and the data are expressed as means ± S.E.M. of three individual replicates.

Figure 6

Phospholipid AA remodeling in AA-enriched cells: (A) RAW 264.7 cells (open bars), RAW.12 cells (light grey bars), and RAW.108 cells (dark grey bars) were incubated in media containing 25 µM exogenous AA for 48 h. Afterward, cellular AA content in phospholipids was analyzed by GC/MS. The AA content of otherwise untreated RAW 264.7 cells (cross-hatched bars) is shown for comparison. (B) AA remodeling was analyzed in the AA-enriched cells, and the remodeling time (time at which the radioactivity content of PC equals that of PE) was determined. Results are shown as means ± S.E.M. (n = 3). * p < 0.05, significantly different from AA-untreated cells.

Figure 7

cPLA₂γ role in phospholipid AA remodeling: (A) PCR analysis of cPLA₂γ expression in RAW 264.7 cells. (B) Phospholipid AA remodeling was analyzed in cells treated with a scrambled siRNA (Control) or siRNA targeting cPLA₂γ (cPLA₂γ Silenced). Results are shown as means ± S.E.M. (n = 5). The asterisk in the Figure 7B right panel denotes that the remodeling time of cPLA₂γ-silenced cells is significantly different from that of control cells (* p < 0.05).

Figure 8

Stimulated AA mobilization in RAW 264.7 cells: (A) [³H]AA-labeled cells, treated with a scrambled siRNA (Control) or siRNA for cPLA₂γ (cPLA₂γ Silenced), were left untreated (dark red bars) or treated with 150 µg/mL zymosan (dark yellow bars) for 1 h. (B) The cells, labeled with [³H]AA, were preincubated with either 2 µM pyrrophenone, 10 µM GK241, 5 µM bromoenol lactone (BEL), 10 µM FKGK18, 5 µM GK436, or neither (no inhibitor) for 30 min. Afterward they were untreated (Ctrl) or treated with 150 µg/mL zymosan for 1 h. Results are shown as means ± S.E.M. (n = 3). * p < 0.05, significantly different from zymosan-stimulated cells in the absence of inhibitors.