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Dexamethasone down-regulates the 85 kDa phospholipase A2 in mouse macrophages and suppresses its activation.

ABSTRACT: We have studied the effects of dexamethasone (dex) (i) on the level of the arachidonate-mobilizing phospholipase A2 (PLA2-85) in macrophages, (ii) on the stimulus-induced activation of this enzyme, and (iii) on the stimulus-induced release of arachidonate. Treatment of macrophages with 10 nM dex led to progressive reduction of PLA2-85 down to approx. 35% of control levels in 20 h in the absence of stimuli. This was accompanied by a partial inhibition of calcium-ionophore-induced arachidonate release. In contrast, the ability of zymosan or phorbol ester to cause both persistent activation of PLA2-85 and arachidonate release was greatly reduced or abolished. However, the protein phosphatase inhibitor okadaic acid, previously shown to cause enhanced phosphorylation and persistent activation of PLA2-85, was still able to exert this effect on the dex-suppressed PLA2-85. This suggests that the effect of okadaic acid was exerted at, or downstream of, the dex-sensitive step(s). Treatment with dex also led to inhibition of the characteristic changes in phosphoprotein labelling induced by phorbol ester or zymosan. However, phorbol-dibutyrate-binding isoforms of protein kinase C were not severely down-regulated. Thus dex was found to down-regulate PLA2-85 and, in addition, to affect one or more component(s) in the signal chain that normally leads to its activation. However, okadaic acid retained the ability to cause activation of PLA2-85.

SUBMITTER: Gewert K

PROVIDER: S-EPMC1136676 | biostudies-other | 1995 Apr

REPOSITORIES: biostudies-other

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Acyl-chain selectivity of the 85 kDa phospholipase A2 and of the release process in intact macrophages.

Project description:The selectivity of the intracellular 85 kDa phospholipase A2 (PLA2-85) towards fatty acids closely related to arachidonic acid has been investigated, using purified PLA2-85 from J774 cells and mixed phospholipids, dually acyl-chain-labelled in the sn-2 position. In parallel experiments, we assessed the acyl-chain selectivity of the release process in intact, dually labelled, peritoneal mouse macrophages responding to either calcium ionophore or zymosan beads in the presence of indomethacin and BSA. The results obtained in the two systems were very similar, which supports previous evidence that PLA2-85 is responsible for stimulus-induced release of eicosanoid precursor in mouse macrophages. In the in vitro system, PLA2-85 was found to exhibit a moderate selectivity towards C20 acyl chains differing in double-bond structure, while the sensitivity to acyl-chain length was more pronounced. Together with previous data, these results demonstrate a striking preference for C20 over either C18 or C22 unsaturated acyl chains.

| S-EPMC1137102 | biostudies-other

Characterization of the promoter for the human 85 kDa cytosolic phospholipase A2 gene.

Project description:The 85 kDa cytosolic phospholipase A2 (cPLA2) plays a key role in the production of arachidonic acid and lysophospholipids, the precursors of eicosanoids and platelet-activating factor. Here we report the cloning of the promoter of the human cPLA2 gene. A 5.7 kb EcoRI fragment containing the most 5' region of the cPLA2 cDNA was sequenced. The transcription initiation site was identified by rapid amplification of 5'-cDNA ends (5'-RACE) and primer extension analysis. DNA sequence analysis of the 595 base pairs 5' of the transcription start site reveals a 48 base purine-pyrimidine dinucleotide repeat (CA repeat), five interferon-gamma response elements (gamma-IRE), one interferon-gamma activated sequence (GAS) and two glucocorticoid response elements (GRE). The promoter lacks a TATA box. It contains a possible CAAT box at -111 and two octamer binding motifs. The 595 base fragment located immediately upstream of the transcriptional start site exhibited functional promoter activity in transient transfection assays in a bronchial epithelial cell line (BEAS 2B cells). Deletion analysis revealed that the CA repeat may confer an inhibitory effect on the cPLA2 promoter activity. The characterization of the human cPLA2 promoter sequence will allow further studies defining the molecular events regulating the expression of the cPLA2 enzyme, especially the cytokine mediated cPLA2 gene expression.

| S-EPMC523783 | biostudies-other

Multiple C-terminal serine phosphorylation accompanies both protein kinase C-dependent and -independent activation of cytosolic 85 kDa phospholipase A2 in macrophages.

Project description:Exposure of mouse macrophages to either phorbol ester or certain bacteria was previously shown to cause increased phosphorylation of the cytosolic 85 kDa phospholipase A2 as well as a stable increase in its catalytic activity. We have now attempted to map the major phosphorylation sites on the enzyme in such cells. Phosphorylation occurred on serine residues without a detectable increase in either phosphothreonine or phosphotyrosine. After CNBr cleavage five fragments showed increased 32P labelling. Among those the most heavily labelled fragment was identified as the most C-terminal (residues 698-749), containing six serine residues. This was true whether phorbol ester or bacteria, causing protein kinase C-independent phospholipase A2 activation, was used as stimulus. The heavy phosphorylation of the most C-terminal fragment and an analysis of tryptic peptides derived from it suggested that more than one of the six serine residues became phosphorylated. Smaller increases also occurred in other CNBr-cleaved fragments from the C-terminal part of the protein, including that carrying Ser-505, a known target of the mitogen-activated protein kinase ERK-2 (extracellular-signal regulated kinase). Dexamethasone treatment (1-100 nM for 20 h), which was earlier shown to dose-dependently down-regulate the 85 kDa phospholipase A2 and its activation by phorbol ester and zymosan, was here shown also to counteract the protein kinase C-independent activation and arachidonate release elicited by bacteria. It remains to be determined whether all phosphorylation sites are equally affected under those conditions.

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| S-EPMC2958102 | biostudies-literature

The selective release of phospholipase A2 by resident mouse peritoneal macrophages.

Project description:Resident mouse peritoneal macrophages have three phospholipase activities: a phospholipase A2 active at pH 4.5, a Ca2+-dependent phospholipase A2 active at pH 8.5 and a phosphatidylinositol-specific phospholipase C activity. When macrophages are exposed to zymosan in culture, the cellular activity of pH-4.5 phospholipase A2 is diminished in a manner dependent on zymosan concentration and time of exposure, whereas the cellular activities of pH-8.5 phospholipase A2 and phospholipase C remain unchanged. The depletion of pH-4.5 phospholipase A2 activity from the cell is paralleled by a quantitative recovery of this activity in the culture medium in a manner similar to the cellular depletion and extracellular recovery of two lysosomal enzymes. This release is specifically elicited by an inflammatory substance such as zymosan, since macrophages incubated with 6 micrometer latex spheres retain pH-4.5 phospholipase A2 activity and lysosomal enzyme activities intracellularly.

| S-EPMC1163555 | biostudies-other

Group X secretory phospholipase A2 enhances TLR4 signaling in macrophages.

Project description:Secretory phospholipase A(2)s (sPLA(2)) hydrolyze glycerophospholipids to liberate lysophospholipids and free fatty acids. Although group X (GX) sPLA(2) is recognized as the most potent mammalian sPLA(2) in vitro, its precise physiological function(s) remains unclear. We recently reported that GX sPLA(2) suppresses activation of the liver X receptor in macrophages, resulting in reduced expression of liver X receptor-responsive genes including ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1), and a consequent decrease in cellular cholesterol efflux and increase in cellular cholesterol content (Shridas et al. 2010. Arterioscler. Thromb. Vasc. Biol. 30: 2014-2021). In this study, we provide evidence that GX sPLA(2) modulates macrophage inflammatory responses by altering cellular cholesterol homeostasis. Transgenic expression or exogenous addition of GX sPLA(2) resulted in a significantly higher induction of TNF-?, IL-6, and cyclooxygenase-2 in J774 macrophage-like cells in response to LPS. This effect required GX sPLA(2) catalytic activity, and was abolished in macrophages that lack either TLR4 or MyD88. The hypersensitivity to LPS in cells overexpressing GX sPLA(2) was reversed when cellular free cholesterol was normalized using cyclodextrin. Consistent with results from gain-of-function studies, peritoneal macrophages from GX sPLA(2)-deficient mice exhibited a significantly dampened response to LPS. Plasma concentrations of inflammatory cytokines were significantly lower in GX sPLA(2)-deficient mice compared with wild-type mice after LPS administration. Thus, GX sPLA(2) amplifies signaling through TLR4 by a mechanism that is dependent on its catalytic activity. Our data indicate this effect is mediated through alterations in plasma membrane free cholesterol and lipid raft content.

| S-EPMC3119755 | biostudies-literature

Phospholipase A2 regulates eicosanoid class switching during inflammasome activation.

Project description:Initiation and resolution of inflammation are considered to be tightly connected processes. Lipoxins (LX) are proresolution lipid mediators that inhibit phlogistic neutrophil recruitment and promote wound-healing macrophage recruitment in humans via potent and specific signaling through the LXA4 receptor (ALX). One model of lipoxin biosynthesis involves sequential metabolism of arachidonic acid by two cell types expressing a combined transcellular metabolon. It is currently unclear how lipoxins are efficiently formed from precursors or if they are directly generated after receptor-mediated inflammatory commitment. Here, we provide evidence for a pathway by which lipoxins are generated in macrophages as a consequence of sequential activation of toll-like receptor 4 (TLR4), a receptor for endotoxin, and P2X7, a purinergic receptor for extracellular ATP. Initial activation of TLR4 results in accumulation of the cyclooxygenase-2-derived lipoxin precursor 15-hydroxyeicosatetraenoic acid (15-HETE) in esterified form within membrane phospholipids, which can be enhanced by aspirin (ASA) treatment. Subsequent activation of P2X7 results in efficient hydrolysis of 15-HETE from membrane phospholipids by group IVA cytosolic phospholipase A2, and its conversion to bioactive lipoxins by 5-lipoxygenase. Our results demonstrate how a single immune cell can store a proresolving lipid precursor and then release it for bioactive maturation and secretion, conceptually similar to the production and inflammasome-dependent maturation of the proinflammatory IL-1 family cytokines. These findings provide evidence for receptor-specific and combinatorial control of pro- and anti-inflammatory eicosanoid biosynthesis, and potential avenues to modulate inflammatory indices without inhibiting downstream eicosanoid pathways.

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[Identification and characterization of two phospholipase A2 activities in resident mouse peritoneal macrophages].

Project description:Resident mouse peritoneal macrophages synthesize and release large amounts of prostaglandins in response to inflammatory stimuli. Release of prostaglandin E2 and 6-oxoprostaglandin F1 alpha occurs at a rate of 1 nmol/h per mg of cell protein. The mechanisms by which substrate arachidonic acid is released have yet to be established. We have therefore initiated studies to characterize those enzymes that can catalyse its release from phospholipid and may be of significance at the cellular level. We report initially the characterization of two phospholipase A2 activities in homogenates of mouse peritoneal macrophages. The first is active at pH 4.5 and is not dependent on Ca2+. The second is Ca2+-dependent and is optimally active at pH 8.5. Either phospholipase A2 activity is capable of hydrolysing [14C] arachidonic acid from [14C] arachidonic acid-labelled phospholipids in quantities sufficient to account for the amounts of prostaglandins by macrophages in culture. Phospholipid substrates are prepared from mouse LM fibroblasts in serum-free Higuchi medium containing radiolabelled phospholipid precursors. Single-labelled phospholipids bear the 14C label in the arachidonic acid moiety. Dual-labelled phospholipids bear a 14C label in the polar head group and a 3H label in the arachidonic acid moiety. Experiments with dual-labelled substrates establish that both phospholipase activities are of the A2 type as indicated by the equimolar recovery of [3H] arachidonic acid and [14C] lysophospholipid. Studies with aqueous sonicated dispersions of purified [14C] arachidonic acid-labelled phospholipid or mixed liposomal substrates formed from mixtures of cellular polar lipids reveal that the pH 4.5 activity hydrolyses phosphatidylethanolamine and phosphatidylcholine more efficiently when they are present in a mixture of other polar lipids. The pH 8.5 activity, however, hydrolyses the purified phospholipids more efficiently.

| S-EPMC1162906 | biostudies-other

Lipid Profile of Activated Macrophages and Contribution of Group V Phospholipase A2.

Project description:Macrophages activated by Interleukin (IL)-4 (M2) or LPS+ Interferon (IFN)γ (M1) perform specific functions respectively in type 2 inflammation and killing of pathogens. Group V phospholipase A2 (Pla2g5) is required for the development and functions of IL-4-activated macrophages and phagocytosis of pathogens. Pla2g5-generated bioactive lipids, including lysophospholipids (LysoPLs), fatty acids (FAs), and eicosanoids, have a role in many diseases. However, little is known about their production by differentially activated macrophages. We performed an unbiased mass-spectrometry analysis of phospholipids (PLs), LysoPLs, FAs, and eicosanoids produced by Wild Type (WT) and Pla2g5-null IL-4-activated bone marrow-derived macrophages (IL-4)BM-Macs (M2) and (LPS+IFNγ)BM-Macs (M1). Phosphatidylcholine (PC) was preferentially metabolized in (LPS+IFNγ)BM-Macs and Phosphatidylethanolamine (PE) in (IL-4)BM-Macs, with Pla2g5 contributing mostly to metabolization of selected PE molecules. While Pla2g5 produced palmitic acid (PA) in (LPS+IFNγ)BM-Macs, the absence of Pla2g5 increased myristic acid (MA) in (IL-4)BM-Macs. Among eicosanoids, Prostaglandin E2 (PGE2) and prostaglandin D2 (PGD2) were significantly reduced in (IL-4)BM-Macs and (LPS+IFNγ)BM-Macs lacking Pla2g5. Instead, the IL-4-induced increase in 20-carboxy arachidonic acid (20CooH AA) was dependent on Pla2g5, as was the production of 12-hydroxy-heptadecatrienoic acid (12-HHTrE) in (LPS+IFNγ)BM-Macs. Thus, Pla2g5 contributes to PE metabolization, PGE2 and PGD2 production independently of the type of activation, while in (IL-4)BM-Macs, Pla2g5 regulates selective lipid pathways and likely novel functions.

| S-EPMC7823364 | biostudies-literature

A cytosolic phospholipase A2-initiated lipid mediator pathway induces autophagy in macrophages.

Project description:Autophagy delivers cytoplasmic constituents to autophagosomes and is involved in innate and adaptive immunity. Cytosolic phospholipase (cPLA(2))-initiated proinflammatory lipid mediator pathways play a critical role in host defense and inflammation. The crosstalk between the two pathways remains unclear. In this study, we report that cPLA(2) and its metabolite lipid mediators induced autophagy in the RAW246.7 macrophage cell line and in primary monocytes. IFN-γ-triggered autophagy involves activation of cPLA(2). Cysteinyl leukotrienes D(4) and E(4) and PGD(2) also induced these effects. The autophagy is independent of changes in mTOR or autophagic flux. cPLA(2) and lipid mediator-induced autophagy is ATG5 dependent. These data suggest that lipid mediators play a role in the regulation of autophagy, demonstrating a connection between the two seemingly separate innate immune responses, induction of autophagy and lipid mediator generation.

| S-EPMC3208068 | biostudies-literature

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