sPLA2 IIA Research Articles

Phospholipase A2 enzymes differently impact PUFA release and oxylipin formation ex vivo in rat hearts.

Phospholipase A2 (PLA2) enzymes cleave cell membrane phospholipids and release polyunsaturated fatty acids (PUFA), which can be converted into oxylipins. However, little is known about PLA2 preference for PUFA, and even less is known about how this further impacts oxylipin formation. Therefore, we investigated the role of different PLA2 groups in PUFA release and oxylipin formation in rat hearts. Sprague-Dawley rat heart homogenates were incubated without or with varespladib (VAR), methyl arachidonyl fluorophosphonate (MAFP) or EDTA. Free PUFA and oxylipins were determined by HPLC-MS/MS, and isoform expressions by RT-qPCR. Inhibition of sPLA2 IIA and/or V by VAR reduced the release of ARA and DHA, but only DHA oxylipins were inhibited. MAFP reduced the release of ARA, DHA, ALA, and EPA, and the formation of ARA, LA, DGLA, DHA, ALA, and EPA oxylipins. Interestingly, cyclooxygenase and 12-lipoxygenase oxylipins were not inhibited. mRNA expression levels of sPLA2 and iPLA2 isoforms were highest whereas levels of cPLA2 were low, consistent with activity. In conclusion, sPLA2 enzymes lead to the formation of DHA oxylipins, while iPLA2 is likely responsible for the formation of most other oxylipins in healthy rat hearts. Oxylipin formation cannot be implied from PUFA release, thus, both should be evaluated in PLA2 activity studies.

Reduced production of isoprostanes by peri-pancreatic adipose tissue from Zucker fa/fa rats as a new mechanism for β-cell compensation in insulin resistance and obesity

During early stages of type 2 diabetes, named prediabetes, pancreatic β-cells compensate for insulin resistance through increased insulin secretion in order to maintain normoglycemia. Obesity leads to the development of ectopic fat deposits, among which peri-pancreatic white adipose tissue (pWAT) can communicate with β-cells through soluble mediators. Thus we investigated whether pWAT produced oxygenated lipids, namely isoprostanes and neuroprostanes and whether they can influence β-cell function in obesity. In the Zucker fa/fa rat model, pWAT and epididymal white adipose tissue (eWAT) displayed different inflammatory profiles. In obese rats, pWAT, but not eWAT, released less amounts of 5-F2t-isoprostanes, 15-F2t-isoprostanes, 4-F4t-neuroprostanes and 10-F4t-neuroprostane compared to lean animals. These differences could be explained by a greater induction of antioxidant defenses enzymes such as SOD-1, SOD-2, and catalase in pWAT of obese animals compared to eWAT. In addition, sPLA2 IIA, involved in the release of isoprostanoids from cellular membranes, was decreased in pWAT of obese animals, but not in eWAT, and may also account for the reduced release of oxidized lipids by this tissue. At a functional level, 15-F2t-isoprostane epimers, but not 5-F2t-isoprostanes, were able to decrease glucose-induced insulin secretion in pancreatic islets from Wistar rats. This effect appeared to be mediated through activation of the thromboxane A2 receptor and reduction of cAMP signaling in pancreatic islets. In conclusion, through the removal of an inhibitory tone exerted by isoprostanes, we have shown, for the first time, a new mechanism allowing β-cells to compensate for insulin resistance in obesity that is linked to a biocommunication between adipose tissue and β-cells.

Moderate blood alcohol and brain neurovulnerability: Selective depletion of calcium-independent phospholipase A2, omega-3 docosahexaenoic acid, and its synaptamide derivative as a potential harbinger of deficits in anti-inflammatory reserve.

Repetitive, highly elevated blood alcohol (ethanol) concentrations (BACs) of 350 to 450mg/dl over several days cause brain neurodegeneration and coincident neuroinflammation in adult rats localized in the hippocampus (HC), temporal cortex (especially the entorhinal cortex; ECX), and olfactory bulb (OB). The profuse neuroinflammation involves microgliosis, increased proinflammatory cytokines, and elevations of Ca+2 -dependent phospholipase A2 (cPLA2) and secretory PLA2 (sPLA2), which both mobilize proinflammatory ω-6 arachidonic acid (ARA). In contrast, Ca+2 -independent PLA2 (iPLA2) and anti-inflammatory ω-3 docosahexaenoic acid (DHA), a polyunsaturated fatty acid regulated primarily by iPLA2, are diminished. Furthermore, supplemented DHA exerts neuroprotection. Given uncertainties about the possible effects of lower circulating BACs that are common occurring during short- term binges, we examined how moderate BACs affected the above inflammatory events, and the impact of supplemented DHA. Young adult male rats sustaining upper-moderate BACs (~150mg/dl) from once-daily alcohol intubations were sacrificed with appropriate controls after 1week. The HC, ECX and OB were quantitatively examined using immunoblotting, neurodegeneration staining, and lipidomics assays. Whereas neurodegeneration, increases in cPLA2 IVA, sPLA2 IIA, and ARA, and microglial activation were not detected, the HC and ECX regions demonstrated significantly reduced iPLA2 levels. Levels of DHA and synaptamide, its anti-inflammatory N-docosahexaenoylethanolamide derivative, also were lower in HC, and DHA supplementation prevented the iPLA2 decrements in HC. Additionally, adult mice maintaining upper-moderate BACs from limited alcohol binges had reduced midbrain iPLA2 levels. The apparently selective depletion by moderate BACs of the metabolically linked anti-inflammatory triad of hippocampal iPLA2, DHA, and synaptamide, and of iPLA2 in the ECX, potentially indicates an unappreciated deficit in brain anti-inflammatory reserve that may be a harbinger of regional neurovulnerability.

The Influence of Phospholipase A 2 (PLA 2 ) Enzymes on Fatty Acid Release and Oxylipin Formation in Rat Hearts

Polyunsaturated fatty acids (PUFA) released by PLA2 are converted into diverse bioactive lipid mediators called oxylipins. However, little is known about the preference of PLA2 isoforms for PUFA release, and even less is known about how this further impacts the formation of oxylipins. Further, oxylipin levels in tissues do not always mimic the levels of their precursor PUFA, so conclusions about oxylipins cannot be drawn from PUFA content. Therefore, we aimed to determine the impact of PLA2 isoforms on PUFA release and oxylipin formation. We hypothesized that different PLA2 isoforms would impact PUFA release and oxylipin formation differently, and that oxylipin formation would not necessarily reflect changes seen in PUFA. Rat heart homogenates were incubated at 37C for 20 min with or without inhibitors, as the increase of both free PUFA and oxylipins was linear at 20 min incubation. Free PUFA and oxylipins were analyzed by HPLC-MS/MS. Two inhibitors were used: Varespladib (VAR) targeting secreted (sPLA2) isoforms IIA, V and X, and methyl arachidonyl fluorophosphonate (MAFP) targeting cytosolic (cPLA2) and calcium-independent (iPLA2) isoforms. Arachidonic Acid (ARA) release was similarly inhibited by both VAR and MAFP, however, while 15 out of 29 ARA oxylipins were inhibited with MAFP, only 3 out of 29 were inhibited with VAR. Docosahexaenoic acid (DHA) release was more strongly inhibited by VAR than by MAFP, which corresponded with 7 out of 12 DHA oxylipins being more strongly inhibited by VAR than by MAFP. Both alpha-linolenic acid (ALA) and eicosapentaenoic acid (EPA) were only inhibited by MAFP, which also inhibited 3 out of 5 ALA and 1 out of 3 EPA oxylipins. Further, 9 out of 10 LA oxylipins were inhibited by MAFP while none were affected by VAR. In conclusion, in the rat heart ARA is released by sPLA2 IIA, V, and/or X as well as by cPLA2 and/or iPLA2, DHA is released more by sPLA2 IIA, V, and/or X than by cPLA2 and/or iPLA2, and EPA and ALA are released primarily by cPLA2 and/or iPLA2. Effects on oxylipin formation largely reflected those on precursor PUFA release, with an exception, as inhibition of sPLA2 IIA, V and X by VAR mostly did not affect ARA oxylipin formation even though it inhibited ARA release. Therefore, careful evaluation of the effects on the oxylipin profile is needed when considering PLA2 inhibitors for potential treatments of diseases.

Secretory Phospholipase A2 IIa Mediates Expression of Growth Factor Receptors in Esophageal Adenocarcinoma.

Receptor tyrosine kinases of the epidermal growth factor receptor (EGFR) family such as human epidermal receptor-2 (HER2) are involved in the development and progression of esophageal adenocarcinoma (EAC). Prior studies have demonstrated that group IIa secretory phospholipase A2 (sPLA2 IIa) can function as a ligand for the EGFR family of receptors and lead to an increase in receptor signaling. We hypothesized that sPLA2 IIa inhibition downregulates the expression of EGFR and HER-2 in EAC and through this mechanism decreases proliferation in EAC. Normal human esophageal epithelium, Barrett's esophagus (BE), and EAC tissue samples were assayed for baseline expression of EGFR, HER-2, and sPLA2 IIa. sPLA2 IIa was attenuated via inhibitor or lentiviral knockdown in esophageal cell lines, and cells were assayed for EGFR and HER2 expression as well as proliferation. FLO1 EAC cells were injected into the flank of nude mice. After randomization, mice received daily group IIA sPLA2 inhibitor or a control solution, and tumor volume was measured with calipers. sPLA2 IIa, EGFR, and HER2 expression increased across the spectrum of normal esophageal epithelium to EAC. sPLA2 IIa inhibition and knockdown decreased the expression of HER-2 and EGFR and proliferation. Mice treated with sPLA2 IIa inhibitor had smaller tumors than controls. sPLA2 IIa inhibition decreases EGFR and HER2 expression and lowers proliferation of human EAC. The discovery of sPLA2 IIa inhibition's ability to attenuate growth factor receptor signaling underscores the exciting potential of sPLA2 IIa inhibitors as therapeutics in the treatment of EAC.

Inhibition of secretory phospholipase A2 IIa attenuates prostaglandin E2-induced invasiveness in lung adenocarcinoma.

Secretory phospholipase A2 IIa (sPLA2 IIa) catalyzes the production of multiple inflammatory mediators that influence the development of lung and other cancers. The most potent of these carcinogenic mediators is prostaglandin E2 (PGE2). We hypothesize that sPLA2 IIa inhibition modulates the production of PGE2, and sPLA2 IIa inhibition exerts its antineoplastic effects via downregulation of PGE2 production. We aim to evaluate these relationships via analysis of PGE2-mediated growth regulation pathways. A549 and H1650 lung adenocarcinoma cells were assayed for PGE2 production in the presence of sPLA2 IIa inhibitor. A549 and H1650 cells were treated with PGE2 and immune blotting was performed to assess ICAM-1 expression and STAT-3 activity. PGE2-induced ICAM-1 expression was measured via immunofluorescence. A549 and H1650 cells were treated with PGE2 in the presence of STAT3 inhibitor and assayed for ICAM-1 expression. A549 cells were treated with PGE2 in the presence ICAM-1 blocking antibody and assayed for invasion. PGE2 stimulation significantly increased the invasiveness and proliferation of lung adenocarcinoma (invasion p < 0.05, proliferation p < 0.05 A549 cells, p < 0.005 H1650 cells). sPLA2 IIa inhibition reduced PGE2 secretion (p < 0.05). PGE2 stimulation significantly upregulated the expression of cell adhesion molecule ICAM-1 and the phosphorylation of anti-apoptotic transcription factor STAT3 (p < 0.05). STAT3 inhibition attenuated ICAM-1 expression demonstrating the dependence of ICAM-1 on the STAT3 pathway (p < 0.05). ICAM-1 blockade attenuated the pro-invasive effects of PGE2 (p < 0.05). sPLA2 IIa inhibition attenuates the potent effects of PGE2-induced invasiveness. This is mediated by decreasing pro-inflammatory and invasion-promoting ICAM-1via the STAT-3 pathway. These data further describe how sPLA2 IIa inhibition mechanistically exerts its anticancer effects and support its use as an antineoplastic agent.

PARP Inhibition Prevents Ethanol-Induced Neuroinflammatory Signaling and Neurodegeneration in Rat Adult-Age Brain Slice Cultures.

Using rat adult-age hippocampal-entorhinal cortical (HEC) slice cultures, we examined the role of poly [ADP-ribose] polymerase (PARP) in binge ethanol's brain inflammatory and neurodegenerative mechanisms. Activated by DNA strand breaks, PARP (principally PARP1 in the brain) promotes DNA repair via poly [ADP-ribose] (PAR) products, but PARP overactivation triggers regulated neuronal necrosis (e.g., parthanatos). Previously, we found that brain PARP1 levels were upregulated by neurotoxic ethanol binges in adult rats and HEC slices, and PARP inhibitor PJ34 abrogated slice neurodegeneration. Binged HEC slices also exhibited increased Ca+2-dependent phospholipase A2 (PLA2) isoenzymes (cPLA2 IVA and sPLA2 IIA) that mobilize proinflammatory ω6 arachidonic acid (ARA). We now find in 4-day-binged HEC slice cultures (100 mM ethanol) that PARP1 elevations after two overnight binges precede PAR, cPLA2, and sPLA2 enhancements by 1 day and high-mobility group box-1 (HMGB1), an ethanol-responsive alarmin that augments proinflammatory cytokines via toll-like receptor-4 (TLR4), by 2 days. After verifying that PJ34 effectively blocks PARP activity (↑PAR), we demonstrated that, like PJ34, three other PARP inhibitors-olaparib, veliparib, and 4-aminobenzamide-provided neuroprotection from ethanol. Importantly, PJ34 and olaparib also prevented ethanol's amplification of the PLA2 isoenzymes, and two PLA2 inhibitors were neuroprotective-thus coupling PARP to PLA2, with PLA2 activity promoting neurodegeneration. Also, PJ34 and olaparib blocked ethanol-induced HMGB1 elevations, linking brain PARP induction to TLR4 activation. The results provide evidence in adult brains that induction of PARP1 may mediate dual neuroinflammatory pathways (PLA2→phospholipid→ARA and HMGB1→TLR4→proinflammatory cytokines) that are complicit in binge ethanol-induced neurodegeneration.

AMPK Signaling Involvement for the Repression of the IL-1β-Induced Group IIA Secretory Phospholipase A2 Expression in VSMCs.

Secretory Phospholipase A2 of type IIA (sPLA2 IIA) plays a crucial role in the production of lipid mediators by amplifying the neointimal inflammatory context of the vascular smooth muscle cells (VSMCs), especially during atherogenesis. Phenformin, a biguanide family member, by its anti-inflammatory properties presents potential for promoting beneficial effects upon vascular cells, however its impact upon the IL-1β-induced sPLA2 gene expression has not been deeply investigated so far. The present study was designed to determine the relationship between phenformin coupling AMP-activated protein kinase (AMPK) function and the molecular mechanism by which the sPLA2 IIA expression was modulated in VSMCs. Here we find that 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleotide (AICAR) treatment strongly repressed IL-1β-induced sPLA2 expression at least at the transcriptional level. Our study reveals that phenformin elicited a dose-dependent inhibition of the sPLA2 IIA expression and transient overexpression experiments of constitutively active AMPK demonstrate clearly that AMPK signaling is involved in the transcriptional inhibition of sPLA2-IIA gene expression. Furthermore, although the expression of the transcriptional repressor B-cell lymphoma-6 protein (BCL-6) was markedly enhanced by phenformin and AICAR, the repression of sPLA2 gene occurs through a mechanism independent of BCL-6 DNA binding site. In addition we show that activation of AMPK limits IL-1β-induced NF-κB pathway activation. Our results indicate that BCL-6, once activated by AMPK, functions as a competitor of the IL-1β induced NF-κB transcription complex. Our findings provide insights on a new anti-inflammatory pathway linking phenformin, AMPK and molecular control of sPLA2 IIA gene expression in VSMCs.

Diverse activity of human secretory phospholipases A2 on the migration of human vascular smooth muscle cells.

Investigation of the diversity of human secretory phospholipases A2 (sPLA2) on the migration of human vascular smooth muscle cells (VSMC). We investigated the impact of sPLA2 IIA, V, and X and of oleic acid, linoleic acid and lysophosphatidylcholine on the migration of human VSMC. Recombinant human sPLA2's and Boyden's chamber method were applied. sPLA2, IIA but not V or X enhanced migration of VSMC in a dose/time dependent manner. Oleic and linoleic acids, and lysophosphatidylcholine markedly enhanced migration. These results imply that sPLA2 IIA, which is known to be present in the arterial wall in the vicinity of VSMC, as well as products of lipid hydrolysis induced by sPLA2, enhance the migration of VSMC, and thus may contribute to atherogenic process.

Association between serum level of secretory type II phospholipase A2 and atherosclerosis in elderly patients with metabolic syndrome

Objective To investigate the association between serum level of secretory type Ⅱ phospholipase A2 (sPLA2 Ⅱa) and carotid intima-media thickness (CIMT) in elderly patients with metabolic syndrome (MS). Methods A total of 124 consecutive MS patients aged over 65 years were enrolled and another 90 elderly non-MS subjects were served as controls. The serum concentration of sPLA2Ⅱa was measured by enzyme-linked immunosorbent assay. Blood pressure, blood lipids and blood glucose levels etc. were also measured. The carotid intima-media thickness was examined by color Doppler ultrasonography. Results Serum level of sPLA2 Ⅱa was significantly higher in elderly MS patients than in control group 〔(4.41±1.55)μg/L vs.(2.39±0.97)μg/L, P<0.05〕. Compared with the control group, elderly MS patients showed CIMT was significantly increased 〔(1.17±0.24) mm vs.(0.89±0.24)mm, P<0.05〕. Serum level of sPLA2 Ⅱa were higher in elderly obesity patients than in normal BMI subjects 〔(4.76±0.99)μg/L vs.(2.84±0.54)μg/L, P<0.05〕. Multiple regression analysis indicated that sPLA2 Ⅱa level was an independent risk factor for CIMT in elderly MS patients after adjusting for the effects of BMI, IL-6 and high sensitive C-reactive protein (hs CRP) on CIMT(r=0.38, P<0.01). Conclusions Serum level of sPLA2 Ⅱa is increased in elderly MS patients and is independently correlated with CIMT, which has a predictive value for atherosclerosis. sPLA2 Ⅱa may be involved in the process of vascular endothelial injury. Key words: Metabolic syndrome X; Atherosclerosis; Phospholipase A2, secretory; C-reactive protein

THU0551 Secretory Phospholipases as Mediators of Inflammatory and Proatheromatous Changes in Rheumatic Diseases

BackgroundInflammatory vascular changes and enhanced atheromatosis are well known in rheumatic diseases. Mitogenesis of vascular smooth muscle cells (VSMC) in above conditions is of prime importance. Secretory phospholipase A2 (sPLA2),...

Filaggrin Deficiency Leads to Impaired Lipid Profile and Altered Acidification Pathways in a 3D Skin Construct

Mutations in the filaggrin (FLG) gene are strongly associated with common dermatological disorders such as atopic dermatitis. However, the exact underlying pathomechanism is still ambiguous. Here, we investigated the impact of FLG on skin lipid composition, organization, and skin acidification using a FLG knockdown (FLG-) skin construct. Initially, sodium/hydrogen antiporter (NHE-1) activity was sufficient to maintain the acidic pH (5.5) of the reconstructed skin. At day 7, the FLG degradation products urocanic (UCA) and pyrrolidone-5-carboxylic acid (PCA) were significantly decreased in FLG- constructs, but the skin surface pH was still physiological owing to an upregulation of NHE-1. At day 14, secretory phospholipase A2 (sPLA2) IIA, which converts phospholipids to fatty acids, was significantly more activated in FLG- than in FLG+. Although NHE-1 and sPLA2 were able to compensate the FLG deficiency, maintain the skin surface pH, and ensured ceramide processing (no differences detected), an accumulation of free fatty acids (2-fold increase) led to less ordered intercellular lipid lamellae and higher permeability of the FLG- constructs. The interplay of the UCA/PCA and the sPLA2/NHE-1 acidification pathways of the skin and the impact of FLG insufficiency on skin lipid composition and organization in reconstructed skin are described.

Association of Biomarkers of Lipid Modification with Functional and Morphological Indices of Coronary Stenosis Severity in Stable Coronary Artery Disease

Biomarkers of blood lipid modification and oxidative stress have been associated with increased cardiovascular morbidity. We sought to determine whether these biomarkers were related to functional indices of stenosis severity among patients with stable coronary artery disease. We studied 197 consecutive patients with stable coronary artery disease due to single vessel disease. Fractional flow reserve (FFR) ≤ 0.80 was assessed as index of a functionally significant lesion. Serum levels of secretory phospholipase A2 (sPLA2) activity, secretory phospholipase A2 type IIA (sPLA2-IIA), myeloperoxydase (MPO), lipoprotein-associated phospholipase A2 (Lp-PLA2), and oxidized low-density lipoprotein (OxLDL) were assessed using commercially available assays. Patients with FFR > 0.8 had higher sPLA2 activity, sPLA2 IIA, and OxLDL levels than patients with FFR ≤ 0.8 (21.25 [16.03-27.28] vs 25.85 [20.58-34.63] U/mL, p < 0.001, 2.0 [1.5-3.4] vs 2.6 [2.0-3.4] ng/mL, p < 0.01; and 53.0 [36.0-71.0] vs 64.5 [50-89.25], p < 0.001 respectively). Patients with FFR > 0.80 had similar Lp-PLA2 and MPO levels versus those with FFR ≤ 0.8. sPLA2 activity, sPLA2 IIA significantly increased area under the curve over baseline characteristics to predict FFR ≤ 0.8 (0.67 to 0.77 (95 % confidence interval [CI]: 0.69-0.85) p < 0.01 and 0.67 to 0.77 (95 % CI: 0.69-0.84) p < 0.01, respectively). Serum sPLA2 activity as well as sPLA2-IIA level is related to functional characteristics of coronary stenoses in patients with stable coronary artery disease.

Knockdown of secretory phospholipase A2 IIa reduces lung cancer growth in vitro and in vivo

Group IIa secretory phospholipase A2 (sPLA2 IIa) plays a role in the malignant potential of several epithelial cancers. Nuclear factor kappa B (NF-κB) regulates cancer cell growth and is modulated by phospholipase activity in many cancer cells. We hypothesized that knockdown of sPLA2 in lung cancer cells would reduce cell proliferation and NF-κB activity in vitro and attenuate tumor growth in vivo. Two human non-small cell lung cancer cell lines (A549 and H358) were transduced with short hairpin RNA targeting sPLA2 group IIa. Quantitative reverse transcriptase-polymerase chain reaction and immunoblotting confirmed knockdown of sPLA2 IIa messenger RNA and protein, respectively. Cell proliferation was evaluated by the 5-bromo-2'-deoxyuridine DNA labeling assay. NF-κB phosphorylation was assayed by western blot. 1 × 10(6) of A549 or A549 sPLA2 knockdown cells were injected into the left flanks of nude mice (aged 6 to 8 weeks). Tumors were followed for 23 days, then removed and stained with hematoxylin and eosin, stained with Ki-67, and analyzed for sPLA2 IIa messenger RNA expression. sPLA2 knockdown reduced NF-κB phosphorylation and tumor growth in vivo. A549 wild-type tumors grew twice as fast as knockdown tumors. Ki-67 staining was more prominent throughout the wild-type tumors compared with knockdown tumors. Explanted knockdown tumors maintained lower sPLA2 levels compared with wild-type, confirmed by reverse transcriptase-polymerase chain reaction. Knockdown of sPLA2 IIa suppresses lung cancer growth in part by attenuating NF-κB activity. These findings justify further investigation into the cellular mechanisms of sPLA2 in lung cancer and its potential role as a therapeutic target.

Circulating phospholipase-A2 activity in obstructive sleep apnea and recurrent tonsillitis

ObjectivePhospholipase A2 (PLA2) plays a major part in growth regulation, differentiation and inflammation. It has been proposed as an evaluating marker for infection and inflammation. The aim of this study was to investigate activity of serum type II secretory PLA2 (sPLA2 IIa) in obstructive sleep apnea (OSA) and recurrent infective tonsillitis (RT) in children. MethodsActivity of serum sPLA2 IIa was determined in children who underwent tonsillectomy, including OSA in 126 cases and RT in 60. Serum enzyme activities were measured using the standard assay with Diheptanoyl Thio-Phosphatidylcholin as substrate. ResultsThe sPLA2 IIa activity of serum was significantly higher in RT than in OSA (P<0.01). Serum sPLA2 IIa activity in the RT patients was positively correlated with BMI (r=0.26; P=0.02), which was not apparent in OSA (r=0.14; P=0.09). ConclusionThis study suggests that serum sPLA2 IIa activity may be considered as a supportive diagnostic marker in suspected or clinically unclear cases of RT children.

Differential roles of phospholipases A 2 in neuronal death and neurogenesis: Implications for Alzheimer disease

The involvement of phospholipase A 2 (PLA 2) in Alzheimer disease (AD) was first investigated nearly 15 years ago. Over the years, several PLA 2 isoforms have been detected in brain tissue: calcium-dependent secreted PLA 2 or sPLA 2 (IIA, IIC, IIE, V, X, and XII), calcium-dependent cytosolic PLA 2 or cPLA 2 (IVA, IVB, and IVC), and calcium-independent PLA 2 or iPLA 2 (VIA and VIB). Additionally, numerous in vivo and in vitro studies have suggested the role of different brain PLA 2 in both physiological and pathological events. This review aimed to summarize the findings in the literature relating the different brain PLA 2 isoforms with alterations found in AD, such as neuronal cell death and impaired neurogenesis process. The review showed that sPLA 2-IIA, sPLA 2-V and cPLA 2-IVA are involved in neuronal death, whereas sPLA 2-III and sPLA 2-X are related to the process of neurogenesis, and that the cPLA 2 and iPLA 2 groups can be involved in both neuronal death and neurogenesis. In AD, there are reports of reduced activity of the cPLA 2 and iPLA 2 groups and increased expression of sPLA 2-IIA and cPLA 2-IVA. The findings suggest that the inhibition of cPLA 2 and iPLA 2 isoforms (yet to be determined) might contribute to impaired neurogenesis, whereas stimulation of sPLA 2-IIA and cPLA 2-IVA might contribute to neurodegeneration in AD.

Impaired phospholipases A 2 production by stimulated macrophages from patients with acute respiratory distress syndrome

The aim of this study was to investigate whether early phase of acute respiratory distress syndrome (ARDS) is associated with changes in immune response, either systemic or localized to the lung. ARDS and control mechanically ventilated patients, as well as healthy volunteers were studied. Alveolar macrophages (AM Φ) and blood monocytes (BM) were treated ex vivo with lipopolysaccharide (LPS), interferon-γ (IFNγ), and surfactant. Phospholipase A 2 (PLA 2) activity and TLR4 expression were evaluated as markers of cell response. AM Φ from ARDS patients did not respond upon treatment with either LPS or IFN-γ by inducing PLA 2 production. On the contrary, upon stimulation, in control patients the intracellular PLA 2, (mainly cPLA 2) levels were increased, but secretion of PLA 2 (mainly sPLA 2-IIA) was observed only after treatment with LPS. Surfactant suppressed PLA 2 production in cells from both groups of patients. Increased relative changes of total PLA 2 activity and an upregulation of TLR4 expression upon stimulation was observed in BM from primary ARDS, control patients and healthy volunteers. In BM from secondary ARDS patients, however, no PLA 2 induction was observed, with a concomitant down-regulation of TLR4 expression. Cytosolic PLA 2, its activated form, p-cPLA 2, and sPLA 2-IIA were the predominant PLA 2 types within the cells, while extracellularly only sPLA 2-IIA was identified. These results support the concept of down-regulated innate immunity in early ARDS that is compartmentalized in primary and systemic in secondary ARDS. PLA 2 isoforms could serve as markers of the immunity status in ARDS. Finally, our data highlight the role of surfactant in controlling inflammation.

Expression profile of multiple secretory phospholipase A 2 isoforms in the rat CNS: Enriched expression of sPLA 2-IIA in brainstem and spinal cord

Phospholipases A 2 (PLA 2) are enzymes which cleave the sn-2 ester bond in membrane phospholipids to release free fatty acids and lysophospholipids. The present study aimed to elucidate the expression profile of multiple secretory phospholipase A 2 (sPLA 2) isoforms in the normal rat CNS with focus on sPLA 2-IIA in the brainstem and spinal cord. Quantitative RT-PCR analysis showed that sPLA 2-IB expression was low throughout the CNS, sPLA 2-IIA expression was high in the brainstem and spinal cord, sPLA 2-IIC expression was high in the cerebral neocortex, hippocampus and thalamus/hypothalamus, sPLA 2-V expression was high in the olfactory bulb and cerebellum, and sPLA 2-X was expressed at very low levels in the normal CNS. Of the isoforms, sPLA 2-IIA mRNA expression was highest in the brainstem and spinal cord suggesting that this could be the most relevant isoform in the ascending pain pathway. Western blot analysis showed high level of sPLA 2-IIA expression in the brainstem and cervical, thoracic and lumbar spinal segments but low level of expression in other parts of the brain. sPLA 2-IIA was localized by immunohistochemistry to the spinal trigeminal and facial motor nuclei and dorsal- and ventral-horns of the spinal cord. The enzyme was found on the endoplasmic reticulum of neuronal cell bodies and small diameter dendrites or dendritic spines at electron microscopy. The expression of sPLA 2-IIA in the dorsal horn and spinal trigeminal nucleus is consistent with previous results which showed an important role of CNS sPLA 2 in nociceptive transmission.

Anthrax lethal toxin down-regulates type-IIA secreted phospholipase A 2 expression through MAPK/NF-κB inactivation

Bacillus anthracis, the etiological agent of anthrax, produces lethal toxin (LT) that displays a metallo-proteolytic activity toward the N-terminus of the MAPK-kinases. We have previously shown that secreted type-IIA phospholipase A 2 (sPLA 2-IIA) exhibits potent anthracidal activity. In vitro expression of sPLA 2-IIA in guinea pig alveolar macrophages (AMs), the major source of this enzyme in lung tissues, is inhibited by LT. Here, we examined the mechanisms involved in sPLA 2-IIA inhibition by LT. We first showed that chemical inhibitors of p38 and ERK MAPKs reduced sPLA 2-IIA expression in AMs indicating that these kinases play a role in sPLA 2-IIA expression. LT inhibited IL-1β-induced p38 phosphorylation as well as sPLA 2-IIA promoter activity in CHO cells. Inhibition of sPLA 2-IIA promoter activity was mimicked by co-transfection with dominant negative construct of p38 (DN-p38) and reversed by the active form of p38-MAPK (AC-p38). Both LT and DN-p38 decreased IL-1β-induced NF-κB luciferase activity. This contrasted with the effect of AC-p38, which enhanced this activity. However, neither LT nor specific p-38 inhibitor interfered with LPS-induced IκBα degradation or NF-κB nuclear translocation in AMs. Subcutaneous administration of LT to guinea pig before LPS challenge reduced sPLA 2-IIA levels in broncho-alveolar lavages and ears. We conclude that sPLA 2-IIA expression is induced via a sequential MAPK-NF-κB activation and that LT inhibits this expression likely by interfering with the transactivation of NF-κB in the nucleus. This inhibition, which is operating both in vitro and in vivo, may represent a mechanism by which B. anthracis subvert host defense.

Differential contributions of protein kinase C isoforms in the regulation of group IIA secreted phospholipase A 2 expression in cytokine-stimulated rat fibroblasts

Protein kinase C (PKC) is a family of serine/threonine kinases involved in various signal transduction pathways. We investigated the roles of PKC in the regulation of group IIA secreted phospholipase A 2 (sPLA 2-IIA) expression in cytokine-stimulated rat fibroblastic 3Y1 cells. Here we show that the induction of sPLA 2-IIA by proinflammatory cytokines was under the control of both classical cPKCα and atypical aPKCλ/ι pathways by using PKC inhibitors, a PKC activator, and PKC knockdowns. Treatment of 3Y1 cells with PKC selective inhibitors having broad specificity, such as chelerythrine chloride and GF109203X, blocked IL-1β/TNFα-dependent induction of sPLA 2-IIA protein in a dose-dependent manner. Treatment with the PKC activator phorbol 12-myristate 13-acetate (PMA), which activates cPKC and novel nPKC isoforms, markedly attenuated the cytokine-dependent induction of sPLA 2-IIA expression. In comparison, 24-h pretreatment with PMA, which down-regulates these PKC isoforms, markedly enhanced sPLA 2-IIA expression. Results with short hairpin RNA (shRNA)-mediated knockdown of PKC isoforms revealed that the cytokine-induced sPLA 2-IIA expression was markedly enhanced in cPKCα knockdown cells compared to those in replicate control cells. In contrast, knockdown of the aPKCλ/ι isoform reduced the cytokine-induced expression of sPLA 2-IIA. These results suggest that the aPKCλ/ι pathway is required for the induction of sPLA 2-IIA expression and that the cPKCα pathway acts as a negative regulator of sPLA 2-IIA expression in cytokine-stimulated rat fibroblasts.