cPLA2 mRNA Expression Research Articles

Hypoxia-Induced Inflammation in In Vitro Model of Human Blood-Brain Barrier: Modulatory Effects of the Olfactory Ensheathing Cell-Conditioned Medium.

Hypoxia compromises the integrity of the blood-brain barrier (BBB) and increases its permeability, thereby inducing inflammation. Olfactory ensheathing cells (OECs) garnered considerable interest due to their neuroregenerative and anti-inflammatory properties. Here, we aimed to investigate the potential modulatory effects of OEC-conditioned medium (OEC-CM) on the response of human brain microvascular endothelial cells (HBMECs), constituting the BBB, when exposed to hypoxia. HBMECs were utilized to establish the in vitro BBB model. OECs were isolated from mouse olfactory bulbs, and OEC-CM was collected after 48h of culture. The effect of OEC-CM treatment on the HBMEC viability was evaluated under both normoxic and hypoxic conditions at 6h, 24h, and 30h. Western blot and immunostaining techniques were employed to assess NF-κB/phospho-NF-κB expression. HIF-1α, VEGF-A, and cPLA2 mRNA expression levels were quantified using digital PCR. ELISA assays were performed to measure PGE2, VEGF-A, IL-8 secretion, and cPLA2 specific activity. The in vitro formation of HBMEC capillary-like structures was examined using a three-dimensional matrix system. OEC-CM attenuated pro-inflammatory responses and mitigated the HIF-1α/VEGFA signaling pathway activation in HBMECs under hypoxic condition. Hypoxia-induced damage of the BBB can be mitigated by novel therapeutic strategies harnessing OEC potential.

Dietary chitosan affects metabolism of arachidonic acid in weaned piglets

The effects of chitosan on immune function via arachidonic acid (AA) pathway in weaned piglets were investigated. A total of 180 piglets (Duroc × Yorkshire × Landrace) were randomly assigned to 5 dietary treatments and fed a basal diet supplemented with 0 (control), 100, 500, 1000, and 2000 mg chitosan/kg feed, respectively. Results showed that serum AA, prostaglandin E2 (PGE2), and leukotriene B4 (LTB4) contents in piglets were increased in a linear or quadratic dose-dependent manner with increasing chitosan on day 28 (P < 0.05). Chitosan increased serum cytosolic-phospholipase A2 (cPLA2) activity in a linear or quadratic dose-dependent manner on day 14 or 28, and improved 5-lipoxygenase (5-LOX) activity in a linear manner and cyclooxygenase-2 (COX-2) activity quadratically on day 28 (P < 0.05). Moreover, chitosan elevated gene expression of cPLA2 mRNA quadratically in the small intestine on days 14 and 28, increased the COX-2 mRNA expression in the duodenum or jejunum in a linear or quadratic manner on day 28, and improved the 5-LOX mRNA expression quadratically in the small intestine (P < 0.05). These results implied that the metabolism of AA was regulated by chitosan in a dose-dependent relationship, which may be one reason why chitosan affected immune function via AA pathway in weaned piglets.

Clinacanthus nutans Extracts Modulate Epigenetic Link to Cytosolic Phospholipase A2 Expression in SH-SY5Y Cells and Primary Cortical Neurons.

Clinacanthus nutans Lindau (C. nutans), commonly known as Sabah Snake Grass in southeast Asia, is widely used in folk medicine due to its analgesic, antiviral, and anti-inflammatory properties. Our recent study provided evidence for the regulation of cytosolic phospholipase A2 (cPLA2) mRNA expression by epigenetic factors (Tan et al. in Mol Neurobiol. doi: 10.1007/s12035-015-9314-z , 2015). This enzyme catalyzes the release of arachidonic acid from glycerophospholipids, and formation of pro-inflammatory eicosanoids or toxic lipid peroxidation products such as 4-hydroxynonenal. In this study, we examined the effects of C. nutans ethanol leaf extracts on epigenetic regulation of cPLA2 mRNA expression in SH-SY5Y human neuroblastoma cells and mouse primary cortical neurons. C. nutans modulated induction of cPLA2 expression in SH-SY5Y cells by histone deacetylase (HDAC) inhibitors, MS-275, MC-1568, and TSA. C. nutans extracts also inhibited histone acetylase (HAT) activity. Levels of cPLA2 mRNA expression were increased in primary cortical neurons subjected to 0.5-h oxygen-glucose deprivation injury (OGD). This increase was significantly inhibited by C. nutans treatment. Treatment of primary neurons with the HDAC inhibitor MS-275 augmented OGD-induced cPLA2 mRNA expression, and this increase was modulated by C. nutans extracts. OGD-stimulated increase in cPLA2 mRNA expression was also reduced by a Tip60 HAT inhibitor, NU9056. In view of a key role of cPLA2 in the production of pro-inflammatory eicosanoids and free radical damage, and the fact that epigenetic effects on genes are often long-lasting, results suggest a role for C. nutans and phytochemicals to inhibit the production of arachidonic acid-derived pro-inflammatory eicosanoids and chronic inflammation, through epigenetic regulation of cPLA2 expression.

Epigenetic Regulation of Cytosolic Phospholipase A2 in SH-SY5Y Human Neuroblastoma Cells.

Group IVA cytosolic phospholipase A2 (cPLA2 or PLA2G4A) is a key enzyme that contributes to inflammation via the generation of arachidonic acid and eicosanoids. While much is known about regulation of cPLA2 by posttranslational modification such as phosphorylation, little is known about its epigenetic regulation. In this study, treatment with histone deacetylase (HDAC) inhibitors, trichostatin A (TSA), valproic acid, tubacin and the class I HDAC inhibitor, MS-275, were found to increase cPLA2α messenger RNA (mRNA) expression in SH-SY5Y human neuroblastoma cells. Co-treatment of the histone acetyltransferase (HAT) inhibitor, anacardic acid, modulated upregulation of cPLA2α induced by TSA. Specific involvement of class I HDACs and HAT in cPLA2α regulation was further shown, and a Tip60-specific HAT inhibitor, NU9056, modulated the upregulation of cPLA2α induced by MS-275. In addition, co-treatment of with histone methyltransferase (HMT) inhibitor, 5'-deoxy-5'-methylthioadenosine (MTA) suppressed TSA-induced cPLA2α upregulation. The above changes in cPLA2 mRNA expression were reflected at the protein level by Western blots and immunocytochemistry. Chromatin immunoprecipitation (ChIP) showed TSA increased binding of trimethylated H3K4 to the proximal promoter region of the cPLA2α gene. Cell injury after TSA treatment as indicated by lactate dehydrogenase (LDH) release was modulated by anacardic acid, and a role of cPLA2 in mediating TSA-induced injury shown, after co-incubation with the cPLA2 selective inhibitor, arachidonoyl trifluoromethyl ketone (AACOCF3). Together, results indicate epigenetic regulation of cPLA2 and the potential of such regulation for treatment of chronic inflammation.

275 EXPRESSION OF THE ACYL-COA SYNTHETASE 4 IN THE PERI-IMPLANTATION MOUSE UTERUS

Acyl-CoA synthetase 4 (ACS4) is an arachidonate-preferring enzyme abundant in steroidogenic tissues and postulated to modulate eicosanoid production. Arachidonate that is esterified predominantly in phospholipids is a precursor of eicosanoids. After its release by the action of cytosolic phospholipase A2 (cPLA2), arachidonate can be converted to prostaglandins, thromboxanes, and leukotrenes via the cyclo-oxygenase (COX1 and COX2) and lioxygenase pathways, respectively, depending on the cell type. It is reported that eicosanoids have influence on inflammation, vascularization, and parturition. Then again, free arachidonate released from the plasma membrane is reesterified into phospholipids to prevent constant synthesis of potent eicosanoids. In the rodent, vasoactive prostaglandins are implicated in the implantation process. To further clarify ACS4 gene expression during pregnancy, we examined developmental expression in the peri-implantation uterus of the normal mouse and regulation of ACS4 by steroid hormone in ovariectomized mice treated with estradiol-17β (E2) and/or progesterone (P4). Adult female mice (BDF1, 6 weeks old) were mated with fertile males of the same strain. The morning on which a vaginal plug was found was designated Day 0.5 of pregnancy. Mice were sacrificed between 09:00 and 10:00 h on Days 2.5 to 6.5 of pregnancy. To induce and maintain delayed implantation, mice were ovariectomized on the morning (09:00 to 10:00 h) of Day 3.5 of pregnancy and received a daily injection of P4 from Days 4.5 to 6.5. To terminate the delay and induce implantation, the P4-primed delayed mice were given an injection of E2 on Day 6.5. COX1, COX2, cPLA2, and ACS4 mRNA were analyzed by Northern blot analysis and real-time PCR. The expression of COX1 mRNA did not show much variation on the various days in the normal pregnancy and ovariectomized mice. The levels of COX2 mRNA were high on Day 4.5 of pregnancy and dramatically decreased on Day 5.5 of pregnancy. In the ovariectomized mouse uterus, COX2 mRNA levels declined rapidly at Day 4.5 of pregnancy. The expression of cPLA2 mRNA was not altered in the normal pregnancy. However, the expression was increased in the ovariectomized mouse as compared with that in the normal pregnancy mice. An ACS4 transcript was already present in the uterus at low level on Day 3.5 until the initiation of attachment reaction after which the expression was up-regulated. In the ovariectomized mouse uterus, ACS4 mRNA was increased at Day 4.5 of pregnancy as compared with the normal pregnancy mice. To determine whether ovarian steroids influenced the induction of ACS4 gene, ovariectomized mice were treated with (E2) and/or (P4). Treatment with P4 maintained the expression of ACS4 mRNA in the ovariectomized mouse uterus. In contrast, combined treatment with P4 and E2 modestly decreased the levels of ACS4 mRNA as compared with Day 7.5 of normal pregnancy. Overall, these results suggest that the ACS4 gene is regulated in the implantation process and influenced by ovarian steroids.

BK‐induced cytosolic phospholipase A2 expression via sequential PKC‐δ, p42/p44 MAPK, and NF‐κB activation in rat brain astrocytes

Bradykinin (BK), an inflammatory mediator, has been shown to induce cytosolic phospholipase A2 (cPLA2) expression implicating in inflammatory responses in various cell types. However, the detailed mechanisms underlying BK-induced cPLA2 expression in astrocytes remain unclear. RT-PCR and Western blotting analysis showed that BK induced the expression of cPLA2 mRNA and protein, which was inhibited by Hoe140, suggesting the involvement of B2 BK receptors, confirmed by immunofluorescence staining using anti-B2 BK receptor antibody. BK-induced cPLA2 expression and phosphorylation of p42/p44 MAPK was attenuated by PD98059, indicating the involvement of MEK1/2-p42/p44 MAPK in these responses. BK-induced cPLA2 expression might be due to the translocation of NF-kappaB into nucleus which was inhibited by Hoe140, helenalin, and PD98059, implying the involvement of NF-kappaB. Moreover, BK-induced cPLA2 expression was attenuated by rottlerin, suggesting that PKC-delta might be involved in these responses. This hypothesis was supported by the transfection with a dominant negative plasmid of PKC-delta significantly attenuated BK-induced response. In addition, BK-stimulated translocation of PKC-delta from cytosol to membrane fraction was inhibited by rottlerin but not by PD98059, indicating that PKC-delta might be an upstream component of p42/p44 MAPK. Accordingly, BK-induced phosphorylation of p42/p44 MAPK was attenuated by rottlerin but not by helenalin. These results suggest that in RBA-1 cells, BK-induced cPLA2 expression was sequentially mediated through activation of PKC-delta, p42/p44 MAPK, and NF-kappaB. Understanding the regulation of cPLA2 expression induced by BK in astrocytes might provide a new therapeutic strategy of brain injury and inflammatory diseases.

Leishmania donovani-induced macrophages cyclooxygenase-2 and prostaglandin E2 synthesis.

Prostaglandin E2 (PGE2) secretion during Leishmania infection has been reported. However, the signalling mechanisms mediating this response are not well understood. Since cyclooxygenase-2 (COX-2) and cytosolic phospholipase A2 (cPLA2) are involved in PGE2 synthesis in response to various stimuli, the implication of these enzymes was evaluated in Leishmania-infected phorbol myristate acetate-differentiated U937 human monocytic cell line. Time-course experiments showed that PGE2 synthesis increased significantly in parallel with COX-2 expression when cells were incubated in the presence of Leishmania donovani promastigotes or lipopolysaccharides (LPS). Increase in cPLA2 mRNA expression was only detected when cells were stimulated with LPS. Indomethacin, genistein, and H7, which are antagonists of COX-2, protein tyrosine kinase (PTK) and protein kinase C (PKC), respectively, inhibited PGE2 production induced by L. donovani and LPS. However, only H7 inhibited COX-2 mRNA synthesis, and there was a significant correlation between PGE2 inhibition and reduced COX-2 expression. Collectively, our results indicate that infection of U937 by L. donovani leads to the generation of PGE2 in part through a PKC-dependent signalling pathway involving COX-2 expression. They further reveal that PTK-dependent events are necessary for Leishmania-induced PGE2 generation, but not for COX-2 expression. A better understanding of the mechanisms by which Leishmania can induce PGE2 production could provide insight into the pathophysiology of leishmaniasis and may help to improve therapeutic approaches.

Regulation of synoviocyte phospholipase A2 and cyclooxygenase 2 by macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with known actions in macrophage and T cell activation. MIF also has the unique capacity to reverse the inhibitory effects of glucocorticoids on these cells. We have recently demonstrated MIF expression in human rheumatoid arthritis (RA) synovium and cultured fibroblast-like synoviocytes (FLS), as well as the ability of FLS-derived MIF to induce monocyte release of tumor necrosis factor alpha. We investigated the effects of MIF on aspects of RA FLS activation, including the induction of phospholipase A2 (PLA2) and cyclooxygenase (COX). PLA2 activity was measured by 3H-arachidonic acid released from treated FLS supernatants. COX activity was measured by prostaglandin E2 enzyme-linked immunosorbent assay. Cytosolic PLA2 (cPLA2) and COX-2 messenger RNA (mRNA) were determined using semiquantitative reverse transcriptase-polymerase chain reaction. Constitutive PLA2 activity was detected in RA FLS. Recombinant human MIF up-regulated PLA2 activity (P < 0.01) and cPLA2 mRNA expression, but had no effect on secretory PLA2. Recombinant human MIF up-regulated COX activity (P < 0.05) and COX-2 mRNA, but had no observable effect on COX-1. Interleukin-1beta (IL-1beta) significantly up-regulated PLA2 activity (P < 0.005) and cPLA2 mRNA expression while anti-MIF monoclonal antibody (mAb) significantly inhibited this IL-1beta-induced PLA2 activity (P < 0.02). Anti-MIF mAb significantly reduced IL-1beta-induced COX activity (P < 0.05) and COX-2 mRNA expression. MIF exerts a proinflammatory effect on key aspects of RA FLS activation. That anti-MIF mAb inhibited IL-1beta up-regulation of FLS indicates an additional cofactor role for MIF in IL-1beta-induced FLS activation. These data suggest that MIF antagonism has important therapeutic potential in RA.

Induction of Cytosolic Phospholipase A2 mRNA Expression by Interleukin-1β and Tumor Necrosis Factor α in Human Gingival Fibroblasts

The involvement of phospholipase A2 (PLA2) enzymes, the 85 kDa cytosolic PLA2 (cPLA2) and the 14 kDa secretory PLA2 (sPLA2), on PGE2 production in human gingival fibroblasts was investigated. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the inflammatory mediators interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) induce the mRNA expression of cPLA2 in gingival fibroblasts. In addition, treatment of the cells with calcium ionophore, A23187, or the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), also induced cPLA2 mRNA expression, accompanied by enhanced PGE2 production. The anti-inflammatory steroid dexamethasone (DEX) reduced basal cPLA2 mRNA expression as well as blocked the induction of cPLA2 mRNA by IL-1β and TNFα in gingival fibroblasts. In contrast to cPLA2, the expression of sPLA2 mRNA was not detected either in untreated or in treated gingival fibroblasts. The study demonstrates that cPLA2 mRNA expression is upregulated by IL-1β and TNFα in human gingival fibroblasts suggesting an important role for the enzyme cPLA2 in the cytokine-induced PGE2 production. Furthermore, the enzyme cPLA2, rather than sPLA2, may be involved in the pathogenesis of periodontal disease by mediating PGE2 production in gingival fibroblasts.

A Role of Protein Kinase C in the Regulation of Cytosolic Phospholipase A2 in Bradykinin-Induced PGI2 Synthesis by Human Vascular Endothelial Cells

The purpose of this study was to elucidate the mechanism by which bradykinin (BK) enhances prostacyclin (PGI2) production in human umbilical vein endothelial cells (HUVEC). BK-induced enhancement of PGI2 synthesis was observed in a dose- and time-dependent manner, and it also increased [Ca2+]i followed by enhancement of cytosolic phospholipase A2 (cPLA2) activity. The PKC inhibitors GF109203X and H7 attenuated the BK-induced increase in [Ca2+]i and inhibited the BK-induced PGI2 synthesis. Phorbol 12-myristate 13-acetate increased cPLA2 activity and PGI2 synthesis but failed to alter [Ca2+]i. BK increased cPLA2 mRNA eightfold by 15 min, and this increase was inhibited by pretreatment with the PKC inhibitors. In response to cycloheximide pretreatment, cPLA2 mRNA was superinduced. These results suggest that BK stimulates PGI2 synthesis in HUVEC by activation of cPLA2 by dual mechanisms: an elevation of [Ca2+]i and a PKC-dependent pathway. Moreover, changes in calcium kinetics and expression of cPLA2 mRNA may underlie the BK-induced PGI2 enhancement in these cells.

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.

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.

Interleukin-4 inhibits prostaglandin G/H synthase-2 and cytosolic phospholipase A2 induction in neonatal mouse parietal bone cultures.

We have shown previously that prostaglandin (PG) production in 7-day-old neonatal mouse calvarial cultures is regulated largely by changes in prostaglandin G/H synthase-2 (PGHS-2) expression and to a lesser extent by changes in arachidonic acid (AA) release. In this study, we examined the effects of interleukin-4 (IL-4), and its interactions with other cytokines and with parathyroid hormone (PTH), on mRNA levels of PGHS-2, PGHS-1, and cytosolic phospholipase A2 (cPLA2) and on medium protaglandin E2 (PGE2) levels in calvarial cultures. IL-1 and tumor necrosis factor-alpha (TNF-alpha), both at 1-100 ng/ml, and PTH at 0.1-10 nM increased PGHS-2 and cPLA2 mRNA and medium PGE2 levels dose-dependently after 4 h of treatment. IL-6 and IL-11 at 1-100 ng/ml did not affect mRNA or PGE2 levels. IL-4 at 1-100 ng/ml decreased PGHS-2 and cPLA2 mRNA and PGE2 levels in control as well as IL-1, TNF-alpha, and PTH-stimulated cultures. The inhibition of PGHS-2 and cPLA2 mRNA expression by IL-4 (10 ng/ml) was present at 1 h, reached a maximum at 4 h, and persisted for 24 h. The effects were maintained in the presence of cycloheximide. IL-4 also decreased PGHS-2 protein levels in control and IL-1-stimulated cultures. PGHS-1 mRNA levels were not stimulated by any of the factors studied nor inhibited by IL-4. IL-4 partially inhibited control and PTH-stimulated 45Ca release from prelabeled mouse calvariae at 4 days. However, neither the inhibition of resorption by IL-4 nor the stimulation by IL-1 and PTH were altered by indomethacin (1 microM). We conclude that (1) IL-1, TNF-alpha, and PTH, but not IL-6 nor IL-11, can increase the expression of PGHS-2, cPLA2, and PGE2 production in cultured mouse calvariae; (2) IL-4 inhibits PGE2 production in both control and stimulated calvarial cultures by inhibiting PGHS-2 and cPLA2; and (3) IL-4 has an inhibitory effect on bone resorption which is independent of PG production.

Endothelin-1 stimulates cytosolic phospholipase A2 activity and gene expression in rat glomerular mesangial cells

Endothelin-1 (ET-1) stimulates vascular smooth muscle and mesangial cells to release prostaglandin E2 (PGE2), which can attenuate the vasoconstrictor and mitogenic effects of this peptide. Phospholipase A2 (PLA2)-mediated release of arachidonic acid from the sn-2 position of membrane phospholipids is thought to be one of the rate-limiting steps in prostaglandin (PG) synthesis. We evaluated the role of ET-1 to regulate gene expression, protein synthesis and enzymatic activity of cytosolic PLA2 (cPLA2), an intracellular form of the PLA2 enzyme family, in cultured rat mesangial cells using both acute and chronic incubation protocols. Acute ET-1-induced stimulation of cPLA2 activity was maximal after 10 minutes (181.1 +/- 6.84% of control), persisted for 40 minutes and did not require new protein synthesis. Heparin, a potent inhibitor of intracellular Ca2+ increase as well as mitogen-activated protein (MAP) kinase activation and cell proliferation, did not affect the rapid cPLA2 stimulation by ET-1. Chronic incubation of glomerular mesangial cells with ET-1 (1 to 24 hr) led to time- and dose-dependent increases in cPLA2 mRNA expression which was maximal after six hours, persisted up to 24 hours and which was accompanied by both cPLA2 protein formation, as assessed by Western analysis, as well as by stimulation of enzymatic activity. Inhibition of protein synthesis by cycloheximide increased basal cPLA2 mRNA accumulation in quiescent mesangial cells, and the combination of ET-1 and cycloheximide resulted in a greater induction of cPLA2 gene expression when compared to ET-1 alone. Actinomycin D treatment blocked the effect of ET-1 on cPLA2 mRNA accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin 1 alpha causes rapid activation of cytosolic phospholipase A2 by phosphorylation in rat mesangial cells.

We have shown previously that interleukin 1 (IL-1) stimulates eicosanoid production in glomerular mesangial cells (MC) by de novo synthesis of a 14-kD, group II phospholipase A2 (PLA2). IL-1-stimulated prostaglandin E2 synthesis precedes expression of this enzyme, suggesting that another PLA2 isoform must be more rapidly activated. In the presence but not absence of calcium inophore, [3H]arachidonate release is increased significantly as early as 5 min after addition of IL-1, and IL-1 concurrently stimulates a Ca(2+)-dependent phospholipase activity, which was characterized as the cytosolic form of PLA2 (cPLA2). IL-1 does not alter either cPLA2 mRNA expression or mass in serum-stimulated MC, suggesting that cPLA2 activity is increased by a posttranslational modification. IL-1 treatment for 30 min doubles 32P incorporation into immunoprecipitable cPLA2 protein, concordant with the increase in enzyme activity. Immunoblot analysis of extracts derived from IL-1-treated (30 min) cells demonstrates a decreased mobility of cPLA2, and treatment of MC lysates with acid phosphatase significantly reduces cytokine-activated cPLA2 activity, further indicating that IL-1 stimulates phosphorylation of the enzyme. IL-1 treatment (24 h) of serum-deprived MC doubled cPLA2 mRNA, protein, and activity. In summary, IL-1 increases cPLA2 activity in a biphasic, time-dependent manner both by posttranslational modification and de novo synthesis. We consider cPLA2 activation a key step in IL-1-stimulated synthesis of pro-inflammatory, lipid mediators, and an integral event in the phenotypic responses induced in target cells by this cytokine.