Cytosolic Phospholipase A2 Expression Research Articles

Granulocyte-Macrophage Colony-Stimulating Factor Upregulates Reduced 5-Lipoxygenase Metabolism in Peripheral Blood Monocytes and Neutrophils in Acquired Immunodeficiency Syndrome

Leukotrienes (LT) are mediators derived from the 5-lipoxygenase (5-LO) pathway, which play a role in host defense, and are synthesized by both monocytes (peripheral blood monocyte [PBM]) and neutrophils (PMN). Because 5-LO metabolism is reduced in alveolar macrophages and PMN from acquired immunodeficiency syndrome (AIDS) subjects, we investigated the synthesis of LT by PBM and PMN from these subjects. There was a reduction (74.2% ± 8.8% of control) in LT synthesis in PBM from human immunodeficiency virus (HIV)-infected compared with normal subjects. Expression of 5-LO (51.2% ± 8.8% of control), and 5-LO activating protein (FLAP) (48.5% ± 8.0% of control) was reduced in parallel. We hypothesized that this reduction in LT synthetic capacity in PBM and PMN was due to reduced cytokine production by CD4 T cells, such as granulocyte-macrophage colony-stimulating factor (GM-CSF). We treated 10 AIDS subjects with GM-CSF for 5 days. PBM 5-LO metabolism ex vivo was selectively increased after GM-CSF therapy and was associated with increased 5-LO and FLAP expression. PMN leukotriene B4(LTB4) synthesis was also augmented and associated with increased 5-LO, FLAP, and cytosolic phospholipase A2 expression. In conclusion, as previously demonstrated for PMN, PBM from AIDS subjects also demonstrate reduced 5-LO metabolism. GM-CSF therapy reversed this defect in both PBM and PMN. In view of the role of LT in antimicrobial function, cytokine administration in AIDS may play a role as adjunct therapy for infections.

Pregnancy induces expression of cPLA2 in ovine uterine artery but not systemic artery endothelium.

To determine whether pregnancy increases cytosolic phospholipase A2 (cPLA2) expression in uterine artery (UA) endothelial cells and vascular smooth-muscle (VSM) cells, and whether ovarian steroids mediate this effect. Uterine arteries and omental arteries (systemic control) were isolated from pregnant (120-130 days' gestation) ewes and from nonpregnant ewes synchronized to the corresponding phases of the ovarian cycle (follicular or luteal) or ovariectomized. In addition, ovariectomized ewes were treated with vehicle, estradiol-17 beta (E2 beta), progesterone (P4), or combined E2 beta/P4 for 10 days, and UAs were collected. Arteries from all studies were separated into mechanically isolated endothelial and VSM fractions. Proteins were then solubilized and separated on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western immunoblotting using an affinity-purified mouse monoclonal antibody for cPLA2. Both UA endothelium and VSM fractions were seen to express cPLA2, detected as a single band with a molecular mass similar to that observed in myometrium (about 100 kD). Distribution of cPLA2, when expressed per microgram of protein, was observed as 40% in UA endothelium compared with 60% in VSM fractions. Uterine artery endothelial cPLA2 expression was specifically increased 1.9-fold in pregnancy (P < .05), whereas there was no significant change from VSM. Furthermore, in ovariectomized sheep versus intact luteal or follicular phase animals, there was no significant change in cPLA2 expression in endothelium or VSM. Administration of E2 beta, P4, or their combination in ovariectomized sheep also failed to reproduce the pregnancy-induced increase in cPLA2 expression in UA endothelium. Omental artery endothelial and VSM cPLA2 expression was observed at similar magnitudes as UA expression, but levels were consistently unchanged by pregnancy, the ovarian cycle, or ovariectomy. cPLA2 was expressed throughout the endothelium and VSM of both uterine and omental arteries, but only in UA endothelium was pregnancy associated with elevated cPLA2 expression. The lack of change in cPLA2 expression with the ovarian cycle or in ovariectomized animals, even after prolonged treatment with E2 beta, P4, or E2 beta/P4 combined suggests this pregnancy-induced increase in cPLA2 may not be estrogen and/or progesterone dependent.

Regulation of Cytosolic Phospholipase A2 Expression by Cytokines in Human Amnion Cells

The metabolism of arachidonic acid results in the production of prostaglandins (PGs), which are involved in the initiation of labour at term and preterm. The fetal membranes are a source of pro-inflammatory cytokines which promote increased PG biosynthesis via increased release of arachidonic acid and its conversion to biologically active metabolites such as PGE2and PGF2α. In the amnion, the liberation of arachidonic acid from membrane glycerophospholipid stores can be catalysed by cytosolic phospholipase A2(cPLA2). In amnion-derived WISH cells, the addition of tumour-necrosis factor alpha (TNF-α) (50ng/ml) provoked a time-dependent increase in the expression of the cPLA2mRNA which was greatest at 8 and 16h post-treatment (3.62±0.52 and 3.15±0.45-fold of control, n=3). The increase in cPLA2mRNA expression by TNF-α was unaffected by the prior addition of interleukin-4 (IL-4) (10ng/ml), a known inhibitor of prostaglandin endoperoxide H synthase (PGHS)-2 mRNA and protein expression in WISH cells. TNF-α also increased the level of immunoreactive cPLA2protein in a time-dependent manner with the highest levels evident after 8 and 16h. As with the mRNA, cPLA2protein levels were unaffected by pre-incubation with IL-4. The inclusion of the cPLA2-specific inhibitor arachidonyl trifluoromethyl ketone (AACOCF3) resulted in a concentration-dependent inhibition of PGE2biosynthesis in WISH cells treated with TNF-α (>95 per cent at 2μm). We conclude that TNF-α increases the abundance of the cPLA2mRNA and protein in amnion epithelial cells, an effect which plays an important role in amnion PG biosynthesis in the presence of intrauterine infection.

Upregulation of prostacyclin synthesis-related gene expression by shear stress in vascular endothelial cells.

Prostacyclin (prostaglandin I2, PGI2) has a variety of functions, including inhibition of smooth muscle cell proliferation, vasodilation, and antiplatelet aggregation. PGI2 production in endothelial cells has been reported to increase biphasically after shear loading, but the underlying mechanism is not well understood. To clarify the mechanism for the second phase of PGI2 upregulation, we examined the gene expression of the enzymes involved in PGI2 production in human umbilical vein endothelial cells (HUVECs) after shear-stress (24 dyne/cm2) loading. The production of 6-keto-PGF1alpha, a stable metabolite of PGI2, increased time-dependently under shear stress. The arachidonic acid liberation from membrane phospholipids in HUVECs after 12 hours of shear loading was increased significantly compared with the static condition. No change was observed for cytosolic phospholipase A2 expression, as detected by reverse transcription-polymerase chain reaction and Western blotting. Cyclooxygenase (COX)-1 mRNA increased after 1 hour of shear loading, and the increase lasted for 12 hours, the longest time tested, whereas COX-2 mRNA increased after 1 hour of shear loading and peaked at 6 hours. An increase of COX-1 expression was detected at 12 hours of shear loading by Western blotting. No expression of COX-2 was detected in the static control, but induced expression was observed at 6 hours after shear loading. PGI2 synthase was also found to be upregulated. These results suggest that the elevated PGI2 production by shear stress is mediated by increased arachidonic acid release and a combination of increased expression of COXs and PGI2 synthase.

Interleukin-1alpha and tumor necrosis factor alpha synergistically stimulate prostaglandin E2-dependent production of interleukin-11 in rheumatoid synovial fibroblasts.

Interleukin-11 (IL-11), an IL-6-type cytokine, is thought to be involved in bone resorption via osteoclast differentiation. Here, we characterized the combined effect of IL-1alpha and tumor necrosis factor alpha (TNFalpha), major cytokines in the rheumatoid synovium, on the production of IL-11 by cultured rheumatoid synovial fibroblasts (RSFs). The amounts of IL-11, IL-6, and prostaglandin E2 (PGE2) were measured by enzyme-linked immunosorbent assay. IL-11 messenger RNA (mRNA) levels were determined by Northern blotting. Protein expression of cytosolic phospholipase A2 (cPLA2), cyclooxygenase 2 (COX-2), and protein kinase C (PKC) isoforms were determined by Western blotting. IL-1alpha and TNFalpha synergistically stimulated RSFs to produce IL-11 at both the mRNA and protein levels. This synergistic effect was completely inhibited by indomethacin. The inhibition was prevented by PGE2, indicating that the synergistic effect of IL-1alpha and TNFalpha was PGE2-mediated. The cooperative effects of these 2 cytokines were also observed in the production of PGE2 and the expression of 2 regulatory enzymes in PGE2 production, cPLA2 and COX-2. The synergistic induction of IL-11 by IL-1alpha and TNFalpha was completely inhibited by a potent inhibitor of all isoforms of PKC, GF109203X. In contrast, phorbol myristate acetate, which induced a down-regulation of PKC, degrading all PKC isoforms except atypical PKC, did not affect the induction of IL-11. These findings suggest that IL-1alpha and TNFalpha synergistically stimulate the production of IL-11 via their effects on PGE2 production in the rheumatoid joint, and that atypical PKC may be another target for down-regulation of IL-11, the bone resorption-associated cytokine.

Vitamin E and Atherosclerosis

Vitamin E was advocated as an effective treatment for heart disease by Dr. Even Shute of London, Ontario more than 50 years ago. His pioneering claims, which were unacceptable to the medical community at large, have been confirmed by recent findings from epidemiologic studies and clinical trials. This review integrates our current knowledge of atherogenesis with the biological functions of vitamin E. The response-to-injury hypothesis explains atherosclerosis as a chronic inflammatory response to injury of the endothelium, which leads to complex cellular and molecular interactions among cells derived from the endothelium, smooth muscle and several blood cell components. Inflammatory and other stimuli trigger an overproduction of free radicals, which promote peroxidation of lipids in LDL trapped in the subendothelial space. Products of LDL oxidation are bioactive, and they induce endothelial expression and secretion of cytokines, growth factors and several cell surface adhesion molecules. The last-mentioned are capable of recruiting circulating monocytes and T lymphocytes into the intima where monocytes are differentiated into macrophages, the precursor of foam cells. In response to the growth factors and cytokines, smooth muscle cells proliferate in the intima, resulting in the narrowing of the lumen. Oxidized LDL can also inhibit endothelial production of prostacyclin and nitric oxide, two potent autacoids that are vasodilators and inhibitors of platelet aggregation. Evidence is presented that vitamin E is protective against the development of atherosclerosis. Vitamin E enrichment has been shown to retard LDL oxidation, inhibit the proliferation of smooth muscle cells, inhibit platelet adhesion and aggregation, inhibit the expression and function of adhesion molecules, attenuate the synthesis of leukotrienes and potentiate the release of prostacyclin through up-regulating the expression of cytosolic phospholipase A2 and cyclooxygenase. Collectively, these biological functions of vitamin E may account for its protection against the development of atherosclerosis.

Cholecalciferol Induces Prostaglandin E2 Biosynthesis and Transglutaminase Activity in Human Keratinocytes

In this study, we examined the effects of cholecalciferol, a primary keratinocyte metabolite and precursor of the hydroxylated form of vitamin D3, 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], on prostaglandin E2 (PGE2) production in human keratinocytes by examining its respective effects on cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and cytosolic phospholipase A2 (cPLA2) expression, the rate-limiting enzymes regulating PGE2 biosynthesis and differentiation of keratinocytes. Cholecalciferol induced PGE2 production, whereas 1alpha,25(OH)2D3 had no effect on PGE2 production both in normal human epidermal keratinocytes and in the immortalized human keratinocyte cell line, HaCaT. In HaCaT cells, neither COX-1 mRNA nor protein was detectable without stimulation and COX-1 expression did not increase in response to cholecalciferol treatment. Although cPLA2 mRNA and protein were constitutively expressed in untreated HaCaT cells, expression levels did not increase in response to cholecalciferol treatment; however, unlike COX-1 and cPLA2 expression, COX-2 mRNA and COX-2 protein expression increased in response to cholecalciferol treatment. Calphostin C, a potent protein kinase C inhibitor, significantly reduced cholecalciferol-induced PGE2 production by inhibiting cholecalciferol-enhanced COX-2 mRNA and protein expression. These results indicate that (i) 1alpha,25(OH)2D3 does not induce PGE2 biosynthesis in keratinocytes, (ii) cholecalciferol-induced PGE2 production is primarily COX-2 dependent, and (iii) cholecalciferol enhances both COX-2 mRNA and protein expression, via a protein kinase C-dependent mechanism in human keratinocytes. Furthermore, cholecalciferol increased total cellular transglutaminase activity dose dependently, suggesting a potential role for cholecalciferol in regulating the differentiation of human keratinocytes.

Transforming growth factor beta1 regulates the expression of cyclooxygenase in cultured cortical astrocytes and neurons.

The hypothesis that transforming growth factor beta1 (TGFbeta1) regulates the synthesis of prostaglandins by CNS tissue was tested by using purified cultures of cortical astrocytes or neurons that were obtained from rat pups on postnatal day 4 or 5 or fetuses on gestational day 16, respectively. The cells were exposed to TGFbeta1 for 2 days. The synthesis of prostaglandins depends upon the production and conversion of arachidonic acid, steps that are catalyzed by phospholipase A2 (PLA2) and cyclooxygenase (COX), respectively. Prostaglandin E2 (PGE2) concentration was determined by radioimmunoassay. The expression of cytosolic PLA2 and COX (the constitutive COX1 and the inducible COX2) was assessed by using immunohistochemical and quantitative immunoblotting procedures. Astrocytes produced much more PGE2 than neurons, suggesting that glial cells are an important source of PGE2 in the CNS. TGFbeta1 increased the production of PGE2 by astrocytes and neurons in a concentration-dependent manner. Furthermore, TGFbeta1 enhanced COX activity; the inhibitor indomethacin completely blocked TGFbeta1-mediated PGE2 synthesis. Cultured astrocytes and neurons expressed the three enzymes: cytosolic PLA2, COX1, and COX2. Cytosolic PLA2 expression was unaffected by TGFbeta1 treatment. In contrast, COX expression was altered by TGFbeta1 treatment in a concentration-dependent fashion. COX1 was increased by TGFbeta1, but only in astrocytes. TGFbeta1 increased COX2 expression in astrocytes and neurons. Thus, TGFbeta1-induced increases in PGE2 concentration are regulated by COX. This study suggests that TGFbeta1 is an important regulator of immune and inflammatory processes in the CNS.

Expression and localization of cyclooxygenase isoforms and cytosolic phospholipase A2 in anti-Thy-1 glomerulonephritis.

Glomerular expression of the major rate-limiting enzymes for prostanoid synthesis, cyclooxygenase isoforms (COX-1 and COX-2) and cytosolic phospholipase A2 (cPLA2), was investigated in anti-Thy-1 nephritis in rats. Ribonuclease protection assay demonstrated minimal COX-1 mRNA expression in glomeruli of control rat kidneys and a gradual increase of expression from day 1 to day 10 after administration of monoclonal anti-rat Thy-1 antibody. On the other hand, COX-2 mRNA expression, also minimal in the normal glomeruli, was enhanced in a biphasic pattern with two peaks at 1 h and day 10. Expression of cPLA2 mRNA, which was undetectable in normal glomeruli, was induced on day 1 and increased gradually in a pattern similar to that of COX-1 mRNA expression. Immunofluorescence microscopy, using antibodies against COX isoforms, showed that both COX-1 and COX-2 were negligible or faintly detectable in the glomeruli of control rat kidneys. In contrast, the immunofluorescence for COX-1 was intensified on days 4 and 10 along the glomerular capillary walls probably in glomerular epithelial and/or endothelial cells, whereas COX-2 staining was exclusively enhanced in the glomerular epithelial cells at 1 h and day 10 during the course of anti-Thy-1 nephritis. These findings indicate that prostanoids generated through induction of COX-1, COX-2, and cPLA2 are implicated in the mediation of the mesangial cell injury model. In particular, the upregulation of COX-2 expression in glomerular epithelial cells in the selective mesangial cell injury model suggests an intercellular interaction between mesangial cells, and glomerular epithelial cells.

Compensatory prostaglandin E2 biosynthesis in cyclooxygenase 1 or 2 null cells.

Prostaglandin E2 (PGE2) production in immortalized, nontransformed cells derived from wild-type, cyclooxygenase 1-deficient (COX-1(-/-)) or cyclooxygenase 2-deficient (COX-2(-/-)) mice was examined after treatment with interleukin (IL)-1beta, tumor necrosis factor alpha, acidic fibroblast growth factor, and phorbol ester (phorbol myristate acetate). Compared with their wild-type counterparts, COX-1(-/-) or COX-2(-/-) cells exhibited substantially enhanced expression of the remaining functional COX gene. Furthermore, both basal and IL-1-induced expression of cytosolic phospholipase A2 (cPLA2), a key enzyme-regulating substrate mobilization for PGE2 biosynthesis, was also more pronounced in both COX-1(-/-) and COX-2(-/-) cells. Thus, COX-1(-/-) and COX-2(-/-) cells have the ability to coordinate the upregulation of the alternate COX isozyme as well as cPLA2 genes to overcome defects in prostaglandin biosynthetic machinery. The potential for cells to alter and thereby compensate for defects in the expression of specific genes such as COX has significant clinical implications given the central role of COX in a variety of disease processes and the widespread use of COX inhibitors as therapeutic agents.

Effects of Bradykinin on the Regulatory Mechanism of PGI2 Synthesis in Human Vascular Endothelial Cells with Reference to the Intracellular Ca2+ Kinetics and mRNA Expression of Cytosolic Phospholipase A2 (cPLA2) and Prostaglandin H2 Synthase-1 (PGHS-1)

We investigated the effects of bradykinin (BK) on the regulatory mechanism of PGI2 synthesis in endothelial cells with reference to intracellular Ca2+ kinetics, mRNA expression of cytosolic phospholipase A2 (cPLA2) and prostaglandin H2 synthase-1 (PGHS-1).PGI2 generation increased with BK stimulation in a time dependent manner for 180 minutes. In the early phase of BK stimulation, intracellular Ca2+ ([Ca2+]i) concentration was increased by the influx of extracellular Ca2+ and a release from the Ca2+ storage site. However the increase of [Ca2+] i was observed for only 2 minutes.cPLA2 mRNA expression was 87 amol/μg RNA at rest. The additon of BK produced 746amol/μg RNA at 15 minutes. After pretreatment with cycloheximide, cPLA mRNA was superinduced. PGHS-1 mRNA expression was 562 amol/μg RNA at rest. The additon ofBK produced 10608 amol/μg RNA at 180 minutes. After pretreatment with cycloheximide, PGHS-1 mRNA expression was inhibited.These results indicate that in the early phase of BK stimulation, [Ca2+]i activates cPLA2, and cPLA2 is considered to increase PGI2 generation. After 2 minutes, the increase of cPLA2 and PGHS-1 mRNA with BK stimulation seems to lead to an increase in these enzymes, but the time courses of these mRNA expressions were not the same.It is suggested that BK induced PGI2 generation depends not only on the increase of [Ca2+]i but also on the mRNA of cPLA2 and PGHS-1.

Hypercalcemia stimulates expression of intrarenal phospholipase A2 and prostaglandin H synthase-2 in rats. Role of angiotensin II AT1 receptors.

In chronic hypercalcemia, inhibition of thick ascending limb sodium chloride reabsorption is mediated by elevated intrarenal PGE2. The mechanisms and source of elevated PGE2 in hypercalcemia are not known. We determined the effect of hypercalcemia on intrarenal expression of cytosolic phospholipase A2 (cPLA2), prostaglandin H synthase-1 (PGHS-1), and prostaglandin H synthase-2 (PGHS-2), enzymes important in prostaglandin production. In rats fed dihydrotachysterol to induce hypercalcemia, Western blot analysis revealed significant upregulation of both cPLA2 and PGHS-2 in the kidney cortex and the inner and outer medulla. Immunofluorescence localized intrarenal cPLA2 and PGHS-2 to interstitial cells of the inner and outer medulla, and to macula densa and cortical thick ascending limbs in both control and hypercalcemic rats. Hypercalcemia had no effect on intrarenal expression of PGHS-1. To determine if AT1 angiotensin II receptor activation was involved in the stimulation of cPLA2 and PGHS-2 in hypercalcemia, we treated rats with the AT1 receptor antagonist, losartan. Losartan abolished the polydipsia associated with hypercalcemia, prevented the increase in cPLA2 protein in all regions of the kidney, and diminished PGHS-2 expression in the inner medulla. In addition, losartan completely prevented the increase in urinary PGE2 excretion in hypercalcemic rats. Intrarenal levels of angiotensin II were unchanged in hypercalcemia. These data indicate that hypercalcemia stimulates intrarenal cPLA2 and PGHS-2 protein expression. Our results further support a role for angiotensin II, acting on AT1 receptors, in mediating this stimulation.

Induction of Cytosolic Phospholipase A2 by Oncogenic Ras in Human Non-small Cell Lung Cancer

Mutations in Ras family members that confer oncogenic potential are frequently observed in specific human cancers. We report that human non-small cell lung cancer (NSCLC) lines that harbor oncogenic mutations in Ki-Ras (H460, A549, H2122) synthesized high levels of prostaglandin E2 (PGE2) compared with NSCLC lacking Ras mutations or non-transformed lung epithelial cells (BEAS-2B). This increased PGE2 production was mediated by constitutively high expression of 85-kDa cytosolic phospholipase A2 (cPLA2) and cyclooxygenase 2 (COX-2). The increased expression of cPLA2 protein was mediated through elevated mRNA levels and activation of the cPLA2 promoter. Induction of cPLA2 promoter activity was blocked by expression of dominant-negative forms of Ras. Inhibition of Ras by the farnesyltransferase inhibitor BZA-5B inhibited prostaglandin synthesis in H2122 cells by decreasing expression of both cPLA2 and COX-2. Finally, inhibitors of eicosanoid synthesis blocked anchorage-independent growth of NSCLC lines exhibiting Ki-Ras mutations. These results identify cPLA2 as a novel Ras-inducible regulator of eicosanoid synthesis that participates in cellular transformation.

Interleukin-1 Enhances the ATP-Evoked Release of Arachidonic Acid from Mouse Astrocytes

During neuropathological states associated with inflammation, the levels of cytokines such as interleukin-1beta (IL-1beta) are increased. Several studies have suggested that the neuronal damage observed in pathogenesis implicating IL-1beta are caused by an alteration in the neurochemical interactions between neurons and astrocytes. We report here that treating striatal astrocytes in primary culture with IL-1beta for 22-24 hr enhances the ATP-evoked release of arachidonic acid (AA) with no effect on the ATP-induced accumulation of inositol phosphates. The molecular mechanism responsible for this effect involves the expression of P2Y2 receptors (a subtype of purinoceptor activated by ATP) and cytosolic phospholipase A2 (cPLA2, an enzyme that mediates AA release). Indeed, P2Y2 antisense oligonucleotides reduce the ATP-evoked release of AA only from IL-1beta-treated astrocytes. Further, both the amount of cPLA2 (as assessed by Western blotting) and the release of AA resulting from direct activation of cPLA2 increased fourfold in cells treated with IL-1beta. We also report evidence indicating that the coupling of newly expressed P2Y2 receptors to cPLA2 is dependent on PKC activity. These results suggest that during inflammatory conditions, IL-1beta reveals a functional P2Y2 signaling pathway in astrocytes that results in a dramatic increase in the levels of free AA. This pathway may thus contribute to the neuronal loss associated with cerebral ischemia or traumatic brain injury.

A Novel Cytosolic Calcium-independent Phospholipase A2 Contains Eight Ankyrin Motifs

We report the purification, molecular cloning, and expression of a novel cytosolic calcium-independent phospholipase A2 (iPLA2) from Chinese hamster ovary cells, which lacks extended homology to other phospholipases. iPLA2 is an 85-kDa protein that exists as a multimeric complex of 270-350 kDa with a specific activity of 1 micromol/min/mg. The full-length cDNA clone encodes a 752-amino acid cytoplasmic protein with one lipase motif (GXS465XG) and eight ankyrin repeats. Expression of the cDNA in mammalian cells generates an active 85-kDa protein. Mutagenesis studies show that Ser465 and the ankyrin repeats are required for activity. We demonstrate that iPLA2 selectively hydrolyzes the sn-2 over sn-1 fatty acid by 5-fold for 1,2-dipalmitoyl phosphatidylcholine in a mixed micelle. Moreover, we found the fatty acid preference at the sn-2 position to be highly dependent upon substrate presentation. However, iPLA2 does have a marked preference for 1,2-dipalmitoyl phosphatidic acid presented in a vesicle, generating the lipid second messenger lysophosphatidic acid. Finally the enzyme is able to hydrolyze the acetyl moiety at the sn-2 position of platelet-activating factor.

Interleukin-4 Inhibits the Gene Expression and Biosynthesis of Cytosolic Phospholipase A2 in Lipopolysaccharide Stimulated U937 Macrophage Cell Line and Freshly Prepared Adherent Rheumatoid Synovial Cells

We recently reported that interleukin-4 (IL-4) inhibited prostanoid synthesis through inhibiting cyclooxygenase 2 biosynthesis. In the present study, we examined the effect of IL-4 on the expression of cytosolic phospholipase A2(cPLA2). The amounts of protein and mRNA of cPLA2were determined by western blotting and reverse transcription polymerase chain reaction (RT-PCR), respectively. Although interleukin-1α (IL-1α) and tumor necrosis factor α (TNFα) had little effect on the biosynthesis of cPLA2in phorbol myristate acetate (PMA)-differentiated U937 cells, lipopolysaccharide (LPS) increased the protein level of cPLA2in a dose-dependent manner. IL-4 inhibited the increased synthesis of cPLA2at the mRNA level. In addition, IL-4 inhibited the biosynthesis of cPLA2in untreated or LPS treated freshly prepared rheumatoid synovial cells at the mRNA level. These findings suggest that IL-4 inhibits prostanoid synthesis through inhibiting the expression of both cPLA2and cyclooxygenase 2.

Activation of the mitogen-activated protein kinase cascade is suppressed by low concentrations of dexamethasone in mast cells.

Antigen stimulation of mast cells via the IgE receptor, Fc epsilon RI, results in recruitment of the cytosolic tyrosine kinases, Lyn and Syk, and the phosphorylation of proteins. We examined the effects of the glucocorticoid dexamethasone on these events in a cultured (RBL-2H3) mast cell line. Nanomolar concentrations of dexamethasone suppressed phosphorylation of proteins that were associated with the activation of the mitogen-activated protein (MAP) kinase/phospholipase A2 pathway without inhibiting initial events. For example, tyrosine phosphorylation of the subunits of Fc epsilon RI, Lyn, or Syk or of the Ras-guanine nucleotide exchange factor, Vav, was not suppressed in cells treated with up to 1 microM dexamethasone. In contrast, phosphorylation of Raf1, MEK1, p42mapk, and cytosolic phospholipase A2, as well as the associated increase in MAP kinase activity and release of arachidonic acid, were markedly inhibited in cells treated with as little as 10 nM dexamethasone--a concentration that only partially inhibited hydrolysis of inositol phospholipids or release of secretory granules. Prolonged exposure to dexamethasone also resulted in a partial decrease in expression of MEK1, p42mapk, and cytosolic phospholipase A2, which may contribute further to the effects of dexamethasone on this pathway. Activation of the MAP kinase/phospholipase A2 pathway by the calcium-mobilizing agent thapsigargin was similarly suppressed in dexamethasone-treated cells. These findings suggested that an early step in the pathway, possibly a step immediately before the activation of Raf1, was suppressed by low concentrations of dexamethasone.

The expression of cytosolic phospholipase A2 and prostaglandin endoperoxide synthase in ovine maternal uterine and fetal tissues during late gestation and labor.

Gene expression of cytosolic phospholipase A2 (cPLA2) and prostaglandin endoperoxide synthase (PGHS) 1 and 2 was studied in ovine maternal uterine and fetal tissues during glucocorticoid induced premature labor and spontaneous labor and compared with gestational age matched controls not in labor. Northern blot analysis demonstrated that the messenger RNA (mRNA) abundance of cPLA2 and PGHS-2, but not PGHS-1, increased significantly in the endometrium during both glucocorticoid induced premature labor and spontaneous labor. Protein levels of the two enzymes measured by Western blot analyses were also elevated in the endometrium during glucocorticoid induced premature labor. cPLA2 mRNA was detected in the myometrium, but no difference was observed between ewes in labor and their gestational age matched controls not in labor. While the level of PGHS-2 mRNA was below detection in the myometrium with the methods used, the enzyme protein for PGHS-2 in the myometrium was significantly increased in both glucocorticoid induced premature labor and spontaneous labor. Using immunocytochemical methods, PGHS-2 enzyme protein was identified in the endometrial gland epithelial cells and myometrial cells. PGHS-2 mRNA was also detected in the ovine fetal cotyledons in which no differences for PGHS-2 mRNA existed between tissues collected from ewes in labor and ewes not in labor. In contrast, there were no detectable signals for cPLA2 or PGHS-2 mRNA in either amnion or chorion. We conclude that the ovine endometrium and myometrium, but not fetal membranes, are important tissue sites of prostaglandin biosynthesis involved in the process of ovine parturition.

The expression of cytosolic phospholipase A2 and prostaglandin endoperoxide synthase in ovine maternal uterine and fetal tissues during late gestation and labor

The expression of cytosolic phospholipase A2 and prostaglandin endoperoxide synthase in ovine maternal uterine and fetal tissues during late gestation and labor

Cytosolic Phospholipase A2 (PLA2), but Not Secretory PLA2, Potentiates Hydrogen Peroxide Cytotoxicity in Kidney Epithelial Cells

Phospholipase A2 (PLA2) and reactive oxygen species have been implicated both individually and synergistically in various forms of cellular injury. The form(s) of PLA2 important for cell injury and the implications of enhanced activity of the enzyme, however, have not been discerned. Previous studies reveal an increase in PLA2 activity associated with cell injury, but this association does not establish a causal relationship between the increase in activity and the injury. LLC-PK1 cell lines were created that express either the cytosolic PLA2 or a group II PLA2. The susceptibility of these cells to hydrogen peroxide toxicity was determined in order to evaluate the relative importance of these two forms of PLA2 in oxidant injury. Expression of cytosolic PLA2 in the LLC-cPLA2 cell line was associated with a 50-fold increase in PLA2 activity in the cytosolic fraction, an increase in agonist-stimulated arachidonate release, and immunodetection of the cytosolic PLA2 protein that was undetectable in control cells. Exposure to hydrogen peroxide or menadione, but not mercuric chloride, resulted in significantly greater lactate dehydrogenase release in LLC-cPLA2 cells when compared with control cells. Exogenous arachidonic acid (150 microM) did not enhance hydrogen peroxide-induced injury. The intracellular calcium chelator, 1,2-bis-(o-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid/tetra(acetoxymethyl) ester, protected the cells against injury, but the calcium ionophore, A23187, did not increase injury. Glycine conferred no protective effect against hydrogen peroxide toxicity. By contrast to these results with cytosolic PLA2-expressing cells, secretory PLA2 expression to very high levels did not increase susceptibility to hydrogen peroxide. Thus, cytosolic PLA2 may an be an important mediator of oxidant damage to renal epithelial cells.