Phospholipase A2 Gene Expression Research Articles

Oxygenation of European seabass (Dicentrarchus labrax L.) in cages through aeration and effect on lipid metabolism.

Two groups of the same batch of European seabass were used in an industrial-scale trial in sea cages in Vorios Evoikos, Greece. For about one month, one of the two cages was oxygenated by use of compressed air injected in seawater through an AirX frame (Oxyvision A/S, Norway) at 3.5 m depth, while oxygen concentration and temperature were monitored every 30 min. The liver, gut, and pyloric ceca samples were taken from fish of both groups for measurement of gene expression of phospholipase A2 (PLA2) and hormone-sensitive lipase (HSL), and for histological analysis at the middle and end of the experiment. Real-time qPCR was performed with housekeeping genes ACTb, L17, and EF1a. The expression of PLA2 increased in pyloric caeca samples from the oxygenated cage, suggesting that aeration improved the absorption rate of dietary phospholipids (p < 0.05). Expression of HSL increased significantly in liver samples from the control cage, in comparison with the aerated cage (p < 0.05). Histological examination of sea bass samples revealed an increased fat accumulation in the hepatocytes of fish in the oxygenated cage. The results of the present study showed increased lipolysis induced by low DO levels in farmed sea bass in cages.

The Interplay among Glucocorticoid Therapy, Platelet-Activating Factor and Endocannabinoid Release Influences the Inflammatory Response to COVID-19.

COVID-19 is associated with a dysregulated immune response. Currently, several medicines are licensed for the treatment of this disease. Due to their significant role in inhibiting pro-inflammatory cytokines and lipid mediators, glucocorticoids (GCs) have attracted a great deal of attention. Similarly, the endocannabinoid (eCB) system regulates various physiological processes including the immunological response. Additionally, during inflammatory and thrombotic processes, phospholipids from cell membranes are cleaved to produce platelet-activating factor (PAF), another lipid mediator. Nonetheless, the effect of GCs on this lipid pathway during COVID-19 therapy is still unknown. This is a cross-sectional study involving COVID-19 patients (n = 200) and healthy controls (n = 35). Target tandem mass spectrometry of plasma lipid mediators demonstrated that COVID-19 severity affected eCBs and PAF synthesis. This increased synthesis of eCB was adversely linked with systemic inflammatory markers IL-6 and sTREM-1 levels and neutrophil counts. The use of GCs altered these lipid pathways by reducing PAF and increasing 2-AG production. Corroborating this, transcriptome analysis of GC-treated patients blood leukocytes showed differential modulation of monoacylglycerol lipase and phospholipase A2 gene expression. Altogether, these findings offer a breakthrough in our understanding of COVID-19 pathophysiology, indicating that GCs may promote additional protective pharmacological effects by influencing the eCB and PAF pathways involved in the disease course.

Increased placental phospholipase A2 gene expression and free F2-isoprostane levels in response to oxidative stress in preeclampsia

Vasoactive eicosanoids such as thromboxane (TX) A2 and F2-isoprostanes (F2-isoPs) were shown to be increased in the preeclamptic placenta. Only one of the 64 possible isomers of F2-isoPs derived from the oxidation of arachidonic acid was investigated in the placenta so far. F2-isoPs are released from membrane phospholipids by phospholipases A2 (PLA2) and were shown to act on the TXA2 receptor (TBXA2R). However, the PLA2 deregulated in preeclampsia (PE) remains to be determined. In this study, we analyzed the concentrations of six isomers of F2-isoPs; 8-iso-PGF2α, 8-iso-15(R)-PGF2α, 15(R)-PGF2α, iPF2α-IV, iPF2α-VI, 5-iPF2α-VI and the concentrations of the stable metabolites of TXA2, TXB2, by high performance liquid chromatography coupled with tandem mass spectrometry in placentas of PE (n = 17) and normotensive (n = 15) pregnancies according to the biopsy site: peri-insertion or periphery. In the same biopsies, relative mRNA expression of PLA2G2A, PLA2G4A, PLA2G5, PLA2G7, the PLA2 receptor (PLA2R1), the TXA2 synthase and TBXAR2 were measured by quantitative RT-PCR. We observed similar concentrations of total F2-isoP isomers between groups whereas higher concentrations (>40%) of free F2-isoP were observed for all isomers (p ≤ 0.033) in PE than normotensive controls. As expected, we also observed higher placental concentrations of TXB2 in PE (p = 0.005). Interestingly, we concomitantly found higher mRNA expression of secretory PLA2G2A (p = 0.010), PLA2G5 (p = 0.038) and TBXA2R (p = 0.023) in PE than normotensive placentas. In sum, deregulated PLA2 could potentially be implicated in freeing F2-isoP which could participate in local hypertension observed in the PE placenta through the TX pathway.

Possible role for brain prostanoid pathways in the development of angiotensin II-salt hypertension in rats.

Prostanoids generated by the cyclooxygenase (COX) pathway appear to contribute to the neurogenic hypertension (HTN) in rats. The first goal of this study was to establish the time frame during which prostanoids participate in ANG II-salt HTN. We induced HTN using ANG II (150 ng·kg(-1)·min(-1) sc) infusion for 14 days in rats on a high-salt (2% NaCl) diet. When ketoprofen pretreatment was combined with treatment during the first 7 days of ANG II infusion, development of HTN and increased neurogenic pressor activity (indexed by the depressor response to ganglion blockade) were significantly attenuated for the entire ANG II infusion period. This suggests that prostanoid generation caused by administration of ANG II and salt leads to an increase in neurogenic pressor activity and blood pressure (BP) via a mechanism that persists without the need for continuing prostanoid input. The second goal of this study was to determine whether prostanoid products specifically in the brain contribute to HTN development. Expression of prostanoid pathway genes was measured in brain regions known to affect neurogenic BP regulation. ANG II-treated rats exhibited changes in gene expression of phospholipase A2 (upregulated in organum vasculosum of the lamina terminalis, paraventricular nucleus, nucleus of the solitary tract, and middle cerebral artery) and lipocalin-type prostaglandin D synthase (upregulated in the organum vasculosum of the lamina terminalis). On the basis of our results, we propose that activation of the brain prostanoid synthesis pathway both upstream and downstream from COX at early stages plays an important role in the development of the neurogenic component of ANG II-salt HTN.

Effect of firocoxib on cyclooxygenase 2, microsomal prostaglandin E2 synthase 1, and cytosolic phospholipase A2 gene expression in equine mononuclear cells.

To validate primer sets for use in reverse transcription quantitative PCR assays to measure gene expression of cytosolic phospholipase A2 (cPLA2) and microsomal prostaglandin E2 synthase 1 (mPGES1) in equine mononuclear cells and determine the effects of firocoxib, a selective cyclooxygenase 2 (COX-2) inhibitor, on COX-2, cPLA2, and mPGES1 gene expression following incubation of mononuclear cells with lipopolysaccharide (LPS). 8 healthy adult horses. Peripheral blood mononuclear cells were isolated by density gradient centrifugation and incubated at 37°C with medium alone, firocoxib (100 ng/mL), LPS (1 ng/mL or 1 μg/mL), or combinations of firocoxib and both LPS concentrations. After 4 hours, supernatants were collected and tested for prostaglandin E2 (PGE2) concentration with an enzyme inhibition assay, and gene expression in cell lysates was measured with PCR assays. Primer pairs for cPLA2 and mPGES1 yielded single products on dissociation curve analyses, with mean assay efficiencies of 102% and 100%, respectively. Incubation with firocoxib and LPS significantly decreased PGE2 supernatant concentrations and significantly reduced COX-2 and mPGES1 gene expression, compared with values following incubation with LPS alone. Primer sets for mPGES1 and cPLA2 gene expression in equine mononuclear cells were successfully validated. Firocoxib significantly decreased LPS-induced COX-2 and mPGES1 expression, suggesting that it may be useful in the control of diseases in which expression of these genes is upregulated.

The endometrial expression of prostaglandin cascade components in lactating dairy cows fed different polyunsaturated fatty acids

Feeding n-6 polyunsaturated fatty acids (PUFA) increases the endometrial percentages of linoleic and arachidonic acids (AA), enhances the synthesis of prostaglandin F2α (PGF2α), and improves uterine health. In contrary, the n-3 PUFA, eicosapentaenoic acid, and docosahexaenoic acid may play pivotal roles by suppressing the synthesis of uterine PGF2α, a component being centrally involved in the control of the bovine estrous cycle and in early embryo survival. The objectives of the present study were to determine the effect of feeding a diet enriched in either α-linolenic acid (n-3) or linolenic acid (n-6) on the uterine expression of genes related to prostaglandin cascade and uterine release of PGF2α (measured as 13, 14-dihydro-15-keto PGF2α [PGFM]). From calving to 60 days in milk, cows (n = 24) were fed isonitrogenous, isocaloric, and isolipidic diets that differed in the ratio of n-3/n-6 PUFA. Treatments including palm oil ([PLM]; saturated FA, n = 8), soybean whole roast ([SOY]; n-6, n = 8), and linseed extruded ([LIN]; n-3, n = 8). At 30 days in milk, the ovulatory cycles of cows were synchronized using 2 injections of PGF2α with a 14-day interval. On day 15 postovulation, cows were injected with oxytocin and blood samples were collected to monitor the uterine release of PGF2α (measured as PGFM) and uterine endometrial biopsies were prepared to evaluate the expression of genes related to prostaglandin cascade (prostaglandin F synthase [PGFS], prostaglandin E synthase [PGES], prostaglandin endoperoxide synthase-2 [PGHS-2]), phospholipase A2 (PLA2), peroxisome proliferator-activated receptors [PPAR]). Results showed that uterine endometrial PPAR-δ genes were higher in cows fed LIN (3.17-fold) compared with cows fed PLM or SOY (P < 0.05). The messenger RNA (mRNA) level of PGES in the LIN group was threefold as high as those found in SOY and PLM diets (P < 0.05). The mean relative gene expression of PLA2 and PGFS was increased in animals fed the SOY diet (2.4- and 1.7-fold, respectively) compared with LIN and PLM diets (P < 0.05). The expression of mRNA for the PGHS-2, PPAR-α, and PPAR-γ was not influenced by the diet effect. Dietary inclusion of soy FAs was associated with an increase in the PGFM concentration, possibly through an increase in the expression of genes involved in prostaglandin cascade. The uterine concentration of PGFM, however, was decreased in cows fed diets containing n-3 FAs.

Levels and values of lipoprotein-associated phospholipase A2, galectin-3, RhoA/ROCK, and endothelial progenitor cells in critical limb ischemia: pharmaco-therapeutic role of cilostazol and clopidogrel combination therapy.

ObjectiveWe tested the hypothesis that clopidogrel and cilostazol combination therapy could effectively attenuate systemic inflammatory reaction, facilitate proliferation of circulating endothelial progenitor cell (EPC), and improve the clinical outcomes of critical limb ischemia (CLI) in patients unsuitable for surgical revascularization or percutaneous transluminal angioplasty (PTA).MethodsA total 55 patients (mean age, 72 years; 56% female) were consecutively enrolled. Clopidogrel and cilostazol combination therapy was administered throughout the study period.ResultsAs compared with the baseline, circulating endothelial progenitor cell level (as shown by flow cytometry) was significantly increased (p < 0.003), whereas the CLI-related ulcers and painfulness were significantly improved (all p < 0.01) by day 90 after treatment. On the other hand, after clopidogrel and cilostazol combination therapy, galectin-3 level, lipoprotein-associated phospholipase A2 gene expression, and RhoA/ROCK-related protein expression in peripheral blood mononuclear cells were significantly suppressed (all p < 0.01). Eventually, by day 90, 5 patients (9.1%) died of other etiologies, 3 (5.5%) withdrew from the study, 6 (10.9%) required amputation, and the remaining 41 had satisfactory clinical improvement with complete wound healing in 9 (16.4%) patients.ConclusionThe results of the present study highlight that clopidogrel and cilostazol combination therapy may be considered to be an alternative method for treating patients with CLI unsuitable for surgical revascularization or PTA.

Changes in group II phospholipase A2 gene expression in rat heart during sepsis

BackgroundThis study was undertaken to investigate alterations of group II phospholipase A2 (PLA2) gene expression and its underlying mechanism in rat heart during different phases of sepsis. Materials and methodsSepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups, control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. PLA2 enzyme activity, group II PLA2 protein level, messenger RNA (mRNA) abundance, transcription rate, and half-life were measured. ResultsPLA2 activity was decreased by 29% during early sepsis but it was increased by 49% during late sepsis. Group II PLA2 protein level was decreased by 27% during early sepsis but it was increased by 35.3% during late sepsis. Group II PLA2 mRNA was decreased by 21% during early sepsis but it was increased by 141% during late sepsis. The transcription rate of group II PLA2 mRNA was reduced by 25% during early sepsis but it was elevated by 67% during late sepsis. The half-life of group II PLA2 mRNA remained unaltered during early and late phases of sepsis. ConclusionsThese results demonstrate that PLA2 activity, group II PLA2 protein level, the mRNA abundance, and transcription rate were concurrently underexpressed during early sepsis, while they were overexpressed during late sepsis, with no change in the degradation of gene transcript. These data indicate that the biphasic changes in group II PLA2 gene expression are regulated transcriptionally during sepsis.

Regulation of cyclooxygenase-2 and cytosolic phospholipase A2 gene expression by lipopolysaccharide through the RNA-binding protein HuR: involvement of NADPH oxidase, reactive oxygen species and mitogen-activated protein kinases.

BACKGROUND AND PURPOSE Lipopolysaccharide (LPS)-induced expression of cyclooxygenase-2 (COX-2) and cytosolic phospholipase A(2) (cPLA(2) ) has been implicated in several respiratory diseases. HuR is known to enhance the expression of genes by binding to 3'-untranslated region (3'-UTR) of mRNA and stabilizing mRNA. However, the exact mechanisms by which HuR affects the stability of mRNA and modulates LPS-induced COX-2 and cPLA(2) expression in human tracheal smooth muscle cells (HTSMCs) are not known. EXPERIMENTAL APPROACH The expression of prostaglandin E(2) (PGE(2) ) was measured by ELISA, and pro-inflammatory proteins were determined by use of a promoter assay, PCR or Western blot analysis. Overexpression of siRNAs to knock down the target components was used to manipulate the expression of HuR. Release of reactive oxygen species (ROS) was detected by fluorescence dye. The activation of signalling components was assessed by comparing phosphorylation levels, localization of protein kinases or coimmunoprecipitation assay. KEY RESULTS LPS induced COX-2 and cPLA(2) expression via post-translational regulation of mRNA stabilization, which were attenuated by transfection with HuR siRNA in HTSMCs. In addition, LPS-stimulated NADPH oxidase activation and ROS generation were attenuated by the NADPH oxidase inhibitors diphenyleneiodonium chloride (DPI) and apocynin (APO). Generation of ROS induced phosphorylation of p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK and JNK1/2, which was attenuated by DPI and APO and the ROS scavenger N-acetylcysteine. CONCLUSIONS AND IMPLICATIONS These results suggested that in HTSMCs, LPS-induced COX-2 and cPLA(2) expression is mediated through NADPH oxidase/ROS-dependent MAPKs associated with HuR accumulation in the cytoplasm. Activated MAPKs may regulate the nucleocytoplasmic shuttling of HuR, and thus induce the cytoplasmic accumulation of HuR.

P38 MAPK regulates group IIa phospholipase A2 expression in interleukin-1beta -stimulated rat neonatal cardiomyocytes.

Group IIa phospholipase A(2) (GIIa PLA(2)) is released by some cells in response to interleukin-1beta. The purpose of this study was to determine whether interleukin-1beta would stimulate the synthesis and release of GIIa PLA(2) from cardiomyocytes, and to define the role of p38 MAPK and cytosolic PLA(2) in the regulation of this process. Whereas GIIa PLA(2) mRNA was not identified in untreated cells, exposure to interleukin-1beta resulted in the sustained expression of GIIa PLA(2) mRNA. Interleukin-1beta also stimulated a progressive increase in cellular and extracellular GIIa PLA(2) protein levels and increased extracellular PLA(2) activity 70-fold. In addition, interleukin-1beta stimulated the p38 MAPK-dependent activation of the downstream MAPK-activated protein kinase, MAPKAP-K2. Treatment with the p38 MAPK inhibitor, SB202190, decreased interleukin-1beta stimulated MAPKAP-K2 activity, GIIa PLA(2) mRNA expression, GIIa PLA(2) protein synthesis, and the release of extracellular PLA(2) activity. Infection with an adenovirus encoding a constitutively active form of MKK6, MKK6(Glu), which selectively phosphorylates p38 MAPK, induced cellular GIIa PLA(2) protein synthesis and the release of GIIa PLA(2) and increased extracellular PLA(2) activity 3-fold. In contrast, infection with an adenovirus encoding a phosphorylation-resistant MKK6, MKK6(A), did not result in GIIa PLA(2) protein synthesis or release by unstimulated cardiomyocytes. In addition, infection with an adenovirus encoding MKK6(A) abrogated GIIa PLA(2) protein synthesis and release by interleukin-1beta-stimulated cells. These results provide direct evidence that p38 MAPK activation was necessary for interleukin-1beta-induced synthesis and release of GIIa PLA(2) by cardiomyocytes.

Regulation of phospholipase A2 gene expression by tumour necrosis factor alpha in human HepG2 cells.

Regulation of phospholipase A2 gene expression by tumour necrosis factor alpha in human HepG2 cells.

Group II phospholipase A2 gene expression is transcriptionally regulated in rat liver during sepsis.

Changes in group II phospholipase A2 (PLA2) gene expression in rat liver during different phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). The results show that PLA2 activity was increased by 41 and 92% during early hyperdynamic phase (9 h after CLP) and late hypodynamic phase (18 h after CLP) of sepsis, respectively. Western blot analysis reveals that group II PLA2 protein levels were elevated by 53 and 95% during early and late sepsis, respectively. Northern blot analysis depicts that the steady-state levels of group II PLA2 mRNA were enhanced by 39 and 114% during early and late sepsis, respectively. Nuclear run-off assay shows that the transcription rates of group II PLA2 gene transcript were stimulated by 36 and 74% during early and late sepsis, respectively. The actinomycin D pulse chase study indicates that the half-life of group II PLA2 mRNA remained unaffected during early and late phases of sepsis. These results demonstrate that group II PLA2 gene transcripts were overexpressed in rat liver during the progression of sepsis and that the overexpression was a result of the enhanced synthesis of group II PLA2 mRNA. Because PLA2 plays an important role in the maintenance of cell membrane integrity, our findings may contribute to the understanding of the pathogenesis of hepatic dysfunction during the progression of sepsis.

Posttranscriptional effect of insulin-like growth factor-I on interleukin-1beta-induced type II-secreted phospholipase A2 gene expression in rabbit articular chondrocytes.

Large amounts of type II-secreted phospholipase A2 (type II sPLA2) are secreted into inflammatory synovial fluid and they are believed to induce the synthesis of lipid mediators by articular chondrocytes. Preliminary experiments showed that insulin-like growth factor-I, which counteracts cartilage degradation in arthritis, inhibits interleukin-1beta-induced type II sPLA2 gene expression in rabbit articular chondrocytes (Berenbaum, F., G. Thomas, S. Poiraudeau, G. Bereziat, M.T. Corvol, and J. Masliah. 1994. FEBS Lett. 340: 51-55). The present study showed that IL-1beta induced the sustained synthesis of prostaglandin E2 and a parallel increase in type II sPLA2 gene expression (assessed by enzymatic activity and Northern blot analysis), but no increase in cytosolic PLA2 gene expression (assessed by Northern and Western blot analysis) or cytosolic PLA2 activity in rabbit articular chondrocytes. IGF-I inhibited both IL-1beta-stimulated PGE2 synthesis and type II sPLA2 gene expression, but had no effect on cytosolic PLA2 gene expression. Nuclear run-on experiments revealed that IL-1beta stimulated the transcription rate of type II sPLA2 gene, giving rise to long-lived mRNA in cells treated with actinomycin D. IGF-I did not affect transcription rate, suggesting that it acts as a post-transcriptional step. Sucrose density gradient analysis of the translation step showed no effect of IGF-I on the entry of type II sPLA2 mRNA into the polysomal pool or on its distribution into the various polysomal complexes, suggesting that IGF-I does not act on the translation of the mRNA. Lastly, IGF-I strongly decreased the half-life of IL-1beta-induced type II sPLA2 mRNA (from 92 to 12 h), suggesting that IGF-I destabilizes mRNA. These data demonstrate that IL-1beta stimulates the transcription rate of the type II sPLA2 gene and gives rise to a very stable mRNA. In contrast, IGF-I decreases the half-life of the type II sPLA2 message.

Tumor necrosis factor-alpha induces the 85-kDa cytosolic phospholipase A2 gene expression in human bronchial epithelial cells.

Phospholipase A2 (PLA2) activity has been suggested to mediate some of the tumor necrosis factor (TNF) induced cellular responses including cytotoxicity. We evaluated the induction of both the 85-kDa cytosolic phospholipase A2 (cPLA2) and non-pancreatic group II PLA2 gene expression by TNF-alpha in a human bronchial epithelial cell line (BEAS 2B cell). TNF-alpha (20 ng/ml) induced a significantly increased release of prelabeled [3H]arachidonic acid (AA) following 4-24 h incubation. Calcium ionophore A23187 (10(-5) M) further increased the [3H]AA release from the TNF-alpha-treated cells. In vitro activity assay revealed that TNF-alpha increased the dithiothreitol (DTT)-resistant PLA2 activity which was blocked by the cPLA2 inhibitor AACOCF3. Treatment with TNF-alpha for 4-24 h increased the cPLA2 protein and mRNA levels which were blocked by the broad inhibitor of protein kinases staurosporine, the protein kinase C (PKC) inhibitor calphostin C, and to a lesser extent the calcium/calmodulin-dependent protein kinase inhibitor W-7. Reverse transcription and polymerase chain reaction amplification of the group II PLA2 mRNA showed that it is expressed in human lung but not in the bronchial epithelial cell line. TNF-alpha failed to induce the expression of group II PLA2 in the BEAS 2B cells. These results demonstrate that the cPLA2 gene expression is up-regulated by TNF-alpha and this effect may contribute to the TNF-alpha stimulated AA release in airway epithelial cells.

Phospholipase A2 gene expression and activity in histologically normal ileal mucosa and in Crohn's ileitis.

Increased activity of phospholipase A2 (PLA2) in the ileal mucosa may contribute to the inflammation in Crohn's disease. The results of this study showed that (a) three months after ileocolonic resection for Crohn's disease the neoterminal ileal mucosa showed endoscopically new inflammation and had higher PLA2 activity than at the time of the operation (n = 8); no such findings were seen in controls (n = 7), (b) histologically normal ileal mucosa (n = 3) contained mRNA for three isoforms of PLA2 (PLA2-I, PLA2-II, and cPLA2), but the amounts of PLA2-II mRNA clearly exceeded the amounts of mRNA for PLA2-I and cPLA2, (c) ileal mucosa from Crohn's patients (n = 2) contained higher values of PLA2-II mRNA than ileal mucosa from two controls, (d) ileal mucosa from Crohn's patients (n = 4) showed increased PLA2-II mRNA three months after ileocolonic resection. In conclusion, these results show that the predominating PLA2 mRNA in the human ileal mucosa is type II PLA2, and the increased synthesis of PLA2-II might be responsible for the increased PLA2 activity found in the ileal mucosa accompanying recurrent ileal inflammation in Crohn's disease.

Cytosolic phospholipase A2 gene expression in rat mesangial cells is regulated post-transcriptionally

Cytosolic phospholipase A2 (cPLA2) is thought to be the rate-limiting enzyme in the arachidonic acid/eicosanoid cascade. The ability of various agonists to increase steady-state cPLA2 mRNA levels has previously been reported. The current study delineates the contributions of transcriptional and post-transcriptional processes to the regulation of cPLA2 gene expression in response to a variety of agonists in cultured rat glomerular mesangial cells. Epidermal growth factor, platelet-derived growth factor, serum and phorbol myristate acetate all increase the half-life of cPLA2 mRNA transcripts, indicating a role for post-transcriptional modulation of gene expression. The presence of three ATTTA motifs in the 3' untranslated region (3'UTR) of the rat cPLA2 cDNA is ascertained. Heterologous expression of chimeric constructs with different 3'UTRs ligated into the 3' end of the luciferase coding region reveals that the presence of the cPLA2 3'UTR results in reduced luciferase activity compared with constructs without the cPLA2 3'UTR. Furthermore, the luciferase activity in the constructs with the cPLA2 3'UTR is increased in response to the same agonists which stabilize endogenous cPLA2 mRNA. A negligible effect of these agonists on transcriptional control of cPLA2 is evident using promoter-reporter constructs expressed in transient and stable transfectants. Taken together, these results indicate predominant post-transcriptional regulation of cPLA2 mRNA levels.

The expression of phospholipase A2 and lipocortins (annexins) I, II and V in human fetal membranes and placenta in association with labour

We have studied the expression of the cellular, type two phospholipase A2 and lipocortins (annexins) I, II and V in human amnion, chorion-decidua and placenta using northern analysis. We found no difference in the expression of phospholipase A2 or lipocortin V in tissues obtained before or after labour. Lipocortin I expression was found to decrease in amnion and placenta but increase in chorion decidua with the onset of labour, while expression of lipocortin II increased only in amnion. These results support the hypothesis that the increased phospholipase A2 activity in the fetal membranes and placenta which is associated with labour is not due to increased phospholipase A2 gene expression, but that post translational control of phospholipase A2 activity may be mediated through changes in lipocortin I expression.

Cell-specific regulation of type II phospholipase A2 expression in rat mesangial cells.

IL-1 stimulates mesangial cells to synthesize specific proteins, including a non-pancreatic (Type II) phospholipase A2 (PLA2). We have studied the regulation of PLA2 by proinflammatory mediators, implicated in the pathogenesis of glomerulonephritis, and have assessed whether the activation of second messenger systems modulates or mimics PLA2 gene expression by cytokines. IL-1 alpha and beta, TNF alpha, and LPS, but not serum, IL-2, or PDGF, potently induce PLA2 mRNA, and enzyme expression. IL-1-stimulated mesangial cells express a 1.0 kB PLA2 mRNA transcript that is induced in a dose- and time-dependent manner. IL-1-stimulated increases in steady-state PLA2 mRNA abundance result from a moderate increase in PLA2 transcription rate that is amplified by the prolonged persistence of the transcript. Forskolin and dibutyryl cAMP potentiate IL-1-induced PLA2 mRNA and enzyme expression, but have no effect in the absence of cytokine. 12-tetradecanoyl phorbol 13-acetate, sn-1, 2-dioctanoyl glycerol or 1-oleoyl-2-acetyl-sn-glycerol fail to induce PLA2 expression or to alter the effect of IL-1 when coincubated with the cytokine. In contrast, serum deprivation for 24 h specifically enhances IL-1-stimulated PLA2. Genistein potentiates PLA2 mRNA expression in cells exposed to both IL-1 and serum. The inhibitory effect of serum on IL-1-induced PLA2 mRNA abundance is reproduced by PDGF but not dexamethasone. These data demonstrate that the signaling pathways directly engaged by IL-1 to induce PLA2 expression in mesangial cells interact with several second messenger systems in a cell-specific manner. We speculate that IL-1 induces specialized changes in mesangial cell structure and function through direct activation of a transcription factor(s), that result in induction of a specific gene set.

Cytokine regulation of Group II phospholipase A2 expression in glomerular mesangial cells

Phospholipase A2 (PLA2) is believed to play an essential role in inflammatory processes by releasing arachidonic acid from membrane phospholipids for synthesis of important lipid mediators, such as prostaglandins, leukotrienes and platelet activating factor. We have used glomerular mesangial cells as a model system to study the regulation of PLA2 under inflammatory conditions. Potent pro-inflammatory cytokines, such as interleukin 1 (IL-1) and tumour necrosis factor alpha (TNF alpha), as well as agents that increase cellular cAMP levels have been found to increase Group II PLA2 gene expression in a time- and dose-dependent manner. In all cases cytokine-induced synthesis of PLA2 occurred in parallel with cytokine-stimulated prostaglandin (PG) E2 synthesis. Three important classes of compounds that potently antagonise the stimulatory effect of IL-1, TNF alpha and cAMP on Group II PLA2 expression in mesangial cells have been identified, namely, glucocorticoids, transforming growth factors (TGF) type-beta and platelet-derived growth factor (PDGF). Those agents may act sequentially to protect the kidney from damage resulting from cytokine-stimulated mediator release and the subsequent inflammatory reactions.

Interleukin-1 beta- and forskolin-induced synthesis and secretion of group II phospholipase A2 and prostaglandin E2 in rat mesangial cells is prevented by transforming growth factor-beta 2.

Interleukin-1 beta and forskolin induce prostaglandin E2 release as well as 14-kDa group II phospholipase A2 gene expression and secretion of the enzyme from rat glomerular mesangial cells. We now report that pretreatment of mesangial cells with transforming growth factor-beta 2 prior to stimulation with interleukin-1 beta or forskolin inhibits the induced release of prostaglandin E2. At the same time the secretion of group II phospholipase A2, measured both as enzyme activity with sn-2-labeled phosphatidylethanolamine as substrate and as enzyme protein in immunoblot experiments, is dose-dependently inhibited by pretreatment of the cells with transforming growth factor-beta 2. Analyses of enzyme activity and enzyme protein levels in the cells indicated that this is not due to inhibition of enzyme secretion with a concomitant increase in cellular levels of the enzyme. Rather, pretreatment of the cells with transforming growth factor-beta 2 largely prevented both the interleukin-1 beta- and the forskolin-induced synthesis of phospholipase A2. This is the first report indicating an inhibition of group II phospholipase A2 gene expression by transforming growth factor-beta 2. In line with those results, transforming growth factor-beta 2 did not induce the synthesis and secretion of group II phospholipase A2. However, under conditions where the interleukin-1 beta-induced expression of group II phospholipase A2 is fully suppressed by transforming growth factor-beta 2, the growth factor itself stimulated prostaglandin E2 synthesis by a mechanism apparently not involving group II phospholipase A2. The immunochemical identification of the inducible and secretable phospholipase A2 from rat mesangial cells as a group II enzyme was confirmed by purification and N-terminal amino acid sequence determination.