Heterologous Expression and Immunogenic Potential of the Most Abundant Phospholipase A2 from Coral Snake Micrurus dumerilii to Develop Antivenoms.

Micrurus dumerilii is a coral snake of clinic interest in Colombia. Its venom is mainly composed of phospholipases A2 being MdumPLA2 the most abundant protein. Nevertheless, Micrurus species produce a low quantity of venom, which makes it difficult to produce anticoral antivenoms. Therefore, in this work, we present the recombinant expression of MdumPLA2 to evaluate its biological activities and its immunogenic potential to produce antivenoms. For this, a genetic construct rMdumPLA2 was cloned into the pET28a vector and expressed heterologously in bacteria. His-rMdumPLA2 was extracted from inclusion bodies, refolded in vitro, and isolated using affinity and RP-HPLC chromatography. His-rMdumPLA2 was shown to have phospholipase A2 activity, a weak anticoagulant effect, and induced myonecrosis and edema. The anti-His-rMdumPLA2 antibodies produced in rabbits recognized native PLA2, the complete venom of M. dumerilii, and a phospholipase from another species of the Micrurus genus. Antibodies neutralized 100% of the in vitro phospholipase activity of the recombinant toxin and a moderate percentage of the myotoxic activity of M. dumerilii venom in mice. These results indicate that His-rMdumPLA2 could be used as an immunogen to improve anticoral antivenoms development. This work is the first report of an M. dumerilii functional recombinant PLA2.

Protein composition and biochemical characterization of venom from Sonoran Coral Snakes (Micruroides euryxanthus).

The elapid genus, Micruroides, is considered the sister clade of all New World coral snakes (Genus Micrurus), is monotypic, and is represented by Sonoran Coral Snakes, Micruroides euryxanthus. Coral snakes of the genus Micrurus have been reported to have venoms that are predominantly composed of phospholipases A2 (PLA2) or three finger toxins (3FTx), but the venoms of the genus Micruroides are almost completely unstudied. Here, we present the first description of the venom of M. euryxanthus including identification of some proteins as well as transcriptomic, and biological activity assays. The most abundant components within M. euryxanthus venom are 3FTxs (62.3%) and there was relatively low proportion of PLA2s (14.2%). The venom phenotype supports the hypothesis that the common ancestor of Micrurus and Micruroides had a 3FTx-dominated venom. Within the venom, there were two nearly identical α-neurotoxins (α-Ntx), one of which was designated Eurytoxin, that account for approximately 60% of the venom's lethality to mice. Eurytoxin was cloned, expressed in a soluble and active form, and used to produce rabbit hyperimmune serum. This allowed the analysis of its immunochemical properties, showing them to be different from the recombinant αNTx D.H., present in the venoms of some species of Micrurus. Finally, we observed that the commercial antivenom produced in Mexico for coral snake envenomation is unable to neutralize the lethality from M. euryxanthus venom. This work allowed the classification of Micruroides venom into the 3FTx-predominant group and identified the main components responsible for toxicity to mice.

What about COVID-19 and arachidonic acid pathway?

BACKGROUND AND OBJECTIVE: COVID-19 is a highly contagious viral disease. In this study, we tried to define and discuss all the findings on the potential association between arachidonic acid (AA) pathway and COVID-19 pathophysiology. METHODS: A literature search across PubMed, Scopus, Embase, and Cochrane database was conducted. A total of 25 studies were identified. RESULTS: The data elucidated that COX-2 and prostaglandins (PGs), particularly PGE2, have pro-inflammatory action in COVID-19 pathophysiology. Arachidonic acid can act as endogenous antiviral compound. A deficiency in AA can make humans more susceptible to COVID-19. Targeting these pro-inflammatory mediators may help in decreasing the mortality and morbidity rate in COVID-19 patients. CONCLUSIONS: PGE2 levels and other PGs levels should be measured in patients with COVID-19. Lowering the PGE2 levels through inhibition of human microsomal prostaglandin E synthase-1 (mPGES-1) can enhance the host immune response against COVID-19. In addition, the hybrid compounds, such as COX-2 inhibitors/TP antagonists, can be an innovative treatment to control the overall balance between AA mediators in patients with COVID-19.

Mucosal expression of defensin-5, soluble phospholipase A2 and lysozyme in the intestine in a rat model of acute liver failure and its relationship to intestinal bacterial translocation.

INTRODUCTION: To study the expression of defensin-5 (RD-5), soluble phospholipase A2 (sPLA2) and lysozyme in the intestine in a rat model of acute liver failure and its relationship with intestinal bacterial translocation (BT). PATIENTS AND METHODS: Sprague-Dawley (SD) rats were divided into two groups. The experimental group was divided into five subgroups according to the lapsing time after the model was established, which were designated accordingly as 8h, 16h, 24h, 48h, and 72h groups. Acute liver failure (ALF) model was induced by intraperitoneal injection of 10% d-galactosamine. The homogenates of mesenteric lymph nodes (MLNs), liver and spleen from each group were cultured in agar to determine the bacterial outgrowth. The mRNA expression of RD-5, sPLA2, lysozyme and the protein expression of sPLA2, lysozyme were determined. RESULTS: No bacteria grew in the organ cultures from the control group while experimental groups had positive cultures. Expression of the RD-5 and sPLA2 mRNA in the experimental groups gradually increased at early time points and peaked 16h after induction of ALF, then progressively decreased. The mRNA expression of lysozyme in the experimental group peaked at 8h after ALF induction, then progressively decreased. Similar results were obtained with Western blot and immunohistochemical staining. DISCUSSION: The immune barrier function of the ileal mucosa in the rat model of acute liver failure was compromised as demonstrated by the decreased expression of RD-5, sPLA2 and lysozyme in Paneth cells along with increased intestinal bacterial translocation.

Venoms of Iranian Scorpions (Arachnida, Scorpiones) and Their Potential for Drug Discovery.

Scorpions, a characteristic group of arthropods, are among the earliest diverging arachnids, dating back almost 440 million years. One of the many interesting aspects of scorpions is that they have venom arsenals for capturing prey and defending against predators, which may play a critical role in their evolutionary success. Unfortunately, however, scorpion envenomation represents a serious health problem in several countries, including Iran. Iran is acknowledged as an area with a high richness of scorpion species and families. The diversity of the scorpion fauna in Iran is the subject of this review, in which we report a total of 78 species and subspecies in 19 genera and four families. We also list some of the toxins or genes studied from five species, including Androctonus crassicauda, Hottentotta zagrosensis, Mesobuthus phillipsi, Odontobuthus doriae, and Hemiscorpius lepturus, in the Buthidae and Hemiscorpiidae families. Lastly, we review the diverse functions of typical toxins from the Iranian scorpion species, including their medical applications.

cDNA cloning, heterologous expression, protein folding and immunogenic properties of a phospholipase A2 from Bothrops ammodytoides venom.

A mRNA transcript that codes for a phospholipase (PLA2) was isolated from a single venom gland of the Bothrops ammodytoides viper. The PLA2 transcript was cloned onto a pCR®2.1-TOPO vector and subsequently expressed heterologously in the E. coli strain M15, using the pQE30 vector. The recombinant phospholipase was named rBamPLA2_1, and is composed of an N-terminal fusion protein of 16 residues, along with 122 residues from the mature protein that includes 14 cysteines that form 7 disulfide bonds. Following bacterial expression, rBamPLA2_1 was obtained from inclusion bodies and extracted using a chaotropic agent. rBamPLA2_1 had an experimental molecular mass of 15,692.5 Da that concurred with its theoretical molecular mass. rBamPLA2_1 was refolded in in vitro conditions and after refolding, three main protein fractions with similar molecular masses, were identified. Although, the three fractions were considered to represent different oxidized cystine isoforms, their secondary structures were comparable. All three recombinant isoforms were active on egg-yolk phospholipid and recognized similar cell membrane phospholipids to be native PLA2s, isolated from B. ammodytoides venom. A mixture of the three rBamPLA2_1 cystine isoforms was used to immunize a horse in order to produce serum antibodies (anti-rBamPLA2_1), which partially inhibited the indirect hemolytic activity of B. ammodytoides venom. Although, anti-rBamPLA2_1 antibodies were not able to recognize crotoxin, a PLA2 from the venom of a related but different viper genus, Crotalus durissus terrificus, they recognized PLA2s in other venoms from regional species of Bothrops.

Suppression of IL-17F, but not of IL-17A, provides protection against colitis by inducing Treg cells through modification of the intestinal microbiota.

The cytokines IL-17A and IL-17F have 50% amino-acid identity and bind the same receptor; however, their functional differences have remained obscure. Here we found that Il17f-/- mice resisted chemically induced colitis, but Il17a-/- mice did not, and that Il17f-/- CD45RBhiCD4+ T cells induced milder colitis in lymphocyte-deficient Rag2-/- mice, accompanied by an increase in intestinal regulatory T cells (Treg cells). Clostridium cluster XIVa in colonic microbiota capable of inducing Treg cells was increased in both Il17f-/- mice and mice given transfer Il17f-/- T cells, due to decreased expression of a group of antimicrobial proteins. There was substantial production of IL-17F, but not of IL-17A, not only by naive T cells but also by various colon-resident cells under physiological conditions. Furthermore, antibody to IL-17F suppressed the development of colitis, but antibody to IL-17A did not. These observations suggest that IL-17F is an effective target for the treatment of colitis.

Bothrops jararaca accessory venom gland is an ancillary source of toxins to the snake.

In Viperidae snakes, it has been attributed to the main venom gland, a component of the venom gland apparatus, the function of synthesizing all venom toxins and storing them inside a basal-central lumen. However, the role of the accessory gland is still unknown. Here, we analyzed the proteome and the transcriptome of the accessory gland during venom production and secretion cycle. We showed that the accessory gland expresses and synthesizes toxins that are similar to those produced by the main venom gland such as C-type lectin/C-type lectin-like proteins, metalloproteinase, phospholipase A2, cysteine rich secretory protein, nerve growth factor, vascular endothelial growth factor, serine proteinase, and l-amino acid oxidase. Our data have shown that toxin synthesis in the accessory gland is asynchronous when compared to the same process in the venom gland. Moreover, this gland also expresses inhibitors of venom phospholipases A2 and metalloproteinases. Transcriptome analysis showed that the transcripts that correspond to toxins in the accessory gland have a good correlation to the main venom gland transcripts. Therefore, it is proposed that the accessory gland is an ancillary source of toxins to the snake, and provides inhibitors that could control venom toxicity (and integrity) during storage. SIGNIFICANCE: In this study, we propose that the accessory venom gland acts as an important ancillary source of toxins to the snake, in lieu of a depleted main venom gland, and provides inhibiting agents that control venom toxicity (and integrity) during its storage.

Eicosapentaenoic and docosahexaenoic acids have different effects on peripheral phospholipase A2 gene expressions in acute depressed patients.

INTRODUCTION: Omega-3 polyunsaturated fatty acids (PUFAs) have been proven critical in the development and management of major depressive disorder (MDD) by a number of epidemiological, clinical and preclinical studies, but the molecular mechanisms underlying this therapeutic action are yet to be understood. Although eicosapentaenoic acid (EPA) seems to be the active component of omega-3 PUFAs' antidepressant effects, the biological research about the difference of specific genetic regulations between EPA and docosahexaenoic acid (DHA), the two main components of omega-3 PUFAs, is still lacking in human subjects. METHODS: We conducted a 12-week randomized-controlled trial comparing the effects of EPA and DHA on gene expressions of phospholipase A2 (cPLA2) and cyclooxygenase-2 (COX2), serotonin transporter (5HTT), and Tryptophan hydroxylase 2 (TPH-2) in 27 MDD patients. In addition, the erythrocyte PUFA compositions and the candidate gene expressions were also compared between these 27 MDD patients and 22 healthy controls. RESULTS: EPA was associated with a significant decrease in HAM-D scores (CI: -13 to -21, p<0.001) and significant increases in erythrocyte levels of EPA (CI: +1.0% to +2.9%, p=0.001) and DHA (CI: +2.9% to +5.6%, p=0.007). DHA treatment was associated with a significant decrease in HAM-D scores (CI: -6 to -14, p<0.001) and a significant increase in DHA levels (CI: +0.2% to +2.3%, p=0.047), but not of EPA levels. The cPLA2 gene expression levels were significantly increased in patients received EPA (1.9 folds, p=0.038), but not DHA (1.08 folds, p=0.92). There was a tendency for both EPA and DHA groups to decrease COX-2 gene expressions. The gene expressions of COX-2, cPLA2, TPH-2 and 5-HTT did not differ between MDD cases and healthy controls. CONCLUSIONS: EPA differentiates from DHA in clinical antidepressant efficacy and in upregulating cPLA2 gene regulations, which supports the clinical observation showing the superiority of EPA's antidepressant effects. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02615405.

Recombinant-phospholipase A2 production and architecture of inclusion bodies are affected by pH in Escherichia coli.

Aggregation of recombinant proteins into inclusion bodies (IBs) is the major drawback of heterologous expression in Escherichia coli. Here, we evaluated the effects of a pH shift after expression induction on recombinant phospholipase A2 production and its aggregation in IBs in E. coli Origami™, as compared to cultures with pH maintained at 7.5 or uncontrolled pH. Cultures shifted from 7.5 to pH 6.5 or 8.5 produced ∼15-25% less biomass as compared with those kept at 7.5 or without pH control. The cultures shifted to pH 8.5 showed a ∼50% higher yield of acetate per biomass, and the rPLA2 yield was improved 2.4-fold. Purified IBs formed at pH 8.5 containing ∼50% of rPLA2, were more susceptible to proteinase-K cleavage and bound less thioflavin-T, indicating lower amyloid content, with the concomitant enrichment of α-helical and random-coil secondary structures, as demonstrated by FTIR. Moreover, only one IB per cell was formed at pH 8.5; instead, more than two were observed under the other culture pH conditions. Nevertheless, under uncontrolled pH conditions, ∼300nm larger IBs were observed. Our work presents evidence of the usefulness of recombinant protein expression cultivated at pH 8.5 allowing the reduction of amyloid content in IBs.

Binge alcohol alters PNPLA3 levels in liver through epigenetic mechanism involving histone H3 acetylation.

The human PNPLA3 (patatin-like phospholipase domain-containing 3) gene codes for a protein which is highly expressed in adipose tissue and liver, and is implicated in lipid homeostasis. While PNPLA3 protein contains regions homologous to functional lipolytic proteins, the regulation of its tissue expression is reflective of lipogenic genes. A naturally occurring genetic variant of PNPLA3 in humans has been linked to increased susceptibility to alcoholic liver disease. We have examined the modulatory effect of alcohol on PNPLA3 protein and mRNA expression as well as the association of its gene promoter with acetylated histone H3K9 by chromatin immunoprecipitation (ChIP) assay in rat hepatocytes in vitro, and in vivo in mouse and rat models of acute binge, chronic, and chronic followed by acute binge ethanol administration. Protein expression of PNPLA3 was significantly increased by alcohol in all three models used. PNPLA3 mRNA also increased, albeit to a varying degree. ChIP assay using H3AcK9 antibody showed increased association with the promoter of PNPLA3 in hepatocytes and in mouse liver. This was less evident in rat livers in vivo except under chronic treatment. It is concluded for the first time that histone acetylation plays a role in the modulation of PNPLA3 levels in the liver exposed to binge ethanol both in vitro and in vivo.

Effects of adenosine stimulation on the mRNA expression of CLCNKB in the basolateral medullary thick ascending limb of the rat kidney.

Adenosine is a molecule produced by several organs within the body, including the kidneys, where it acts as an autoregulatory factor. It mediates ion transport in several nephron segments, including the proximal tubule and the thick ascending limb (TAL). Ion transport is dictated in part by anionic chloride channels, which regulate crucial kidney functions, including the reabsorption of Na+ and Cl­, urine concentration, and establishing and maintaining the corticomedullary osmotic gradient. The present study investigated the effects of adenosine on the mRNA expression of chloride voltage­gated channel Kb (CLCNKB), a candidate gene involved in hypertension, which encodes for the ClC­Kb channel. Medullary thick ascending limb (mTAL) tubules were isolated from the rat kidney, and primary cultures of mTAL cells from the mTAL tubules were established. The cells were treated with adenosine and the mRNA expression of CLCNKB was detected by reverse transcription­quantitative polymerase chain reaction. The cells were also treated with pathways inhibitors (H8 and AACOCF3), and the protein expression of cyclic adenosine 3',5'­monophosphate (cAMP)­protein kinase A (PKA) and phospholipase A2 (PLA2) by were analyzed by western blotting. The findings indicated that adenosine increased the mRNA expression of CLCNKB in primary cultures of medullary TAL cells, and this stimulatory effect was regulated by the cAMP­PKA and PLA2­arachidonic acid (AA) pathways. The present study showed that adenosine affected the mRNA expression of CLCNKB, initially through the cAMP­PKA pathway and then the PLA2­AA pathway.

Phospholipase A2-dependent release of inflammatory cytokines by superantigen-stimulated nasal polyps of patients with chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) is an inflammatory/allergic disease with unclear pathophysiology, but it has been linked to an imbalance in the production of eicosanoids, which are metabolites of arachidonic acid, and results from phospholipids hydrolysis by phospholipase A2 (PLA2). As of yet, the role of PLA2 in CRS has hardly been studied, except for a report that group II PLA2 expression is elevated in interleukin (IL) 1ß or tumor necrosis factor α-stimulated CRS nasal tissues with and without polyps. The PLA2 families include extracellular (secretory) and intracellular isoforms, which are involved in the regulation of inflammatory processes in different ways. Here we comprehensively investigated the expression of PLA2s, particularly those reported to be involved in respiratory disorders, in superantigen (SAE)-stimulated nasal polyps from patients with CRS with polyps, and determined their role in inflammatory cytokine production by inhibition of PLA2 expression. METHODS: The release of IL-5, IL-13, IL-17, and interferon γ by nasal polyps dispersed cells (NPDC) was determined concomitantly with PLA2 messenger RNA expression, under SAE stimulation, with or without dexamethasone, as a regulator of PLA2 expression. RESULTS: Stimulation of NPDCs by SAE-induced cytokine secretion with enhanced expression of several secretory PLA2 and Ca(2+)-independent PLA2, while suppressing cytosolic PLA2 expression. All these were reverted to the level of unstimulated NPDCs on treatment with dexamethasone. CONCLUSION: This study further supports the key role of secretory PLA2 in the pathophysiology of respiratory disorders and presents secretory PLA2 inhibition as a therapeutic strategy for the treatment of CRS and airway pathologies in general.

Secretory expression of a phospholipase A2 from Lactobacillus casei DSM20011 in Kluyveromyces lactis.

The pla2 gene encoding a phospholipase A2 (EC 3.1.1.4) of Lactobacillus casei DSM20011 was cloned and expressed in the yeast Kluyveromyces lactis GG799 successfully for the first time. The structural pla2 gene fused in frame with the K. lactis secretion signal α-mating factor was integrated into the LAC4 locus and expressed under the control of the LAC4 promoter. sPLA2 activity was detected in the culture supernatant during shake flask culture of K. lactis/pKLAC1-pla2. In comparison with the control strain K. lactis/pKLAC1, SDS-PAGE analysis revealed a 17-kDa recombinant protein band in K. lactis/pKLAC1-pla2, which was consistent with the predicted molecular weight of the mature protein. Real-time quantitative PCR analysis indicated that the copy number of the integrated pla2 gene ranged from 2 to 6 and positively correlated with sPLA2 activity. When the inducer galactose was used as the carbon source, the sPLA2 activity in the culture supernatant of the recombinant that harbored six pla2 gene copies reached 1.96 ± 0.15 U/mL. The influence of the culture composition and conditions on the recombinant sPLA2 activity in shake flask culture were also studied. When the recombinant was cultured at 30°C in a YPD medium culture volume of 70 mL in a 250-mL shake flask with an initial pH of 7.0, the sPLA2 activity reached 2.16 ± 0.18 U/mL.

Cross-talk between p(38)MAPK and G iα in regulating cPLA 2 activity by ET-1 in pulmonary smooth muscle cells.

Endothelin-1 (ET-1) is known as the most potent vasoconstrictor yet described. Infusion of ET-1 into isolated rabbit lung has been shown to cause pulmonary vasoconstriction with the involvement of arachidonic acid metabolites. Given the potency of arachidonic acid metabolites, the activity of phospholipase A2 must be tightly regulated. Herein, we determined the mechanisms by which ET-1 stimulates cPLA2 activity during ET-1 stimulation of bovine pulmonary artery smooth muscle cells. We demonstrated that (i) treatment of bovine pulmonary artery smooth muscle cells with ET-1 stimulates cPLA2 activity in the cell membrane; (ii) ET-1 caused increase in O 2 (·-) production occurs via NADPH oxidase-dependent mechanism; (iii) ET-1-stimulated NADPH oxidase activity is markedly prevented upon pretreatment with PKC-ζ inhibitor, indicating that PKC-ζ plays a prominent role in this scenario; (iv) ET-1-induced NADPH oxidase-derived O 2 (·-) stimulates an aprotinin sensitive protease activity due to prominent increase in [Ca(2+)]i; (v) the aprotinin sensitive protease plays a pivotal role in activating PKC-α, which in turn phosphorylates p(38)MAPK and subsequently Giα leading to the activation of cPLA2. Taken together, we suggest that cross-talk between p(38)MAPK and Giα with the involvement of PKC-ζ, NADPH oxidase-derived O 2 (·-) , [Ca(2+)]i, aprotinin-sensitive protease and PKC-α play a pivotal role for full activation of cPLA2 during ET-1 stimulation of pulmonary artery smooth muscle cells.

Diapocynin, a dimer of the NADPH oxidase inhibitor apocynin, reduces ROS production and prevents force loss in eccentrically contracting dystrophic muscle.

Elevation of intracellular Ca2+, excessive ROS production and increased phospholipase A2 activity contribute to the pathology in dystrophin-deficient muscle. Moreover, Ca2+, ROS and phospholipase A2, in particular iPLA2, are thought to potentiate each other in positive feedback loops. NADPH oxidases (NOX) have been considered as a major source of ROS in muscle and have been reported to be overexpressed in muscles of mdx mice. We report here on our investigations regarding the effect of diapocynin, a dimer of the commonly used NOX inhibitor apocynin, on the activity of iPLA2, Ca2+ handling and ROS generation in dystrophic myotubes. We also examined the effects of diapocynin on force production and recovery ability of isolated EDL muscles exposed to eccentric contractions in vitro, a damaging procedure to which dystrophic muscle is extremely sensitive. In dystrophic myotubes, diapocynin inhibited ROS production, abolished iPLA2 activity and reduced Ca2+ influx through stretch-activated and store-operated channels, two major pathways responsible for excessive Ca2+ entry in dystrophic muscle. Diapocynin also prevented force loss induced by eccentric contractions of mdx muscle close to the value of wild-type muscle and reduced membrane damage as seen by Procion orange dye uptake. These findings support the central role played by NOX-ROS in the pathogenic cascade leading to muscular dystrophy and suggest diapocynin as an effective NOX inhibitor that might be helpful for future therapeutic approaches.

Corticotropin-releasing hormone receptors mediate apoptosis via cytosolic calcium-dependent phospholipase A2 and migration in prostate cancer cell RM-1.

Peripheral corticotropin-releasing hormone receptors (CRHRs) are G protein-coupled receptors that play different roles depending on tissue types. Previously, we discovered the mechanism of CRHR-mediated apoptosis of mouse prostate cancer cell line (RM-1) to be a change of Bcl-2:Bax ratio, and CRH was found to inhibit transforming growth factor ß migration of breast cancer cells via CRHRs. In the present study, we investigated cytosolic calcium-dependent phospholipase A2 (cPLA2) bridging CRHR activations and Bcl-2:Bax ratio and the effect of CRHR activation on cell migration. Silencing of cPLA2 attenuated a CRHR1 agonist, CRH-induced apoptosis, and the decrease of the Bcl-2:Bax ratio, whereas silencing of cPLA2 aggravated CRHR2 agonist, Urocortin 2 (Ucn2)-inhibited apoptosis, and the increase of the Bcl-2:Bax ratio. CRH in a time- and concentration-dependent manner increased cPLA2 expression mainly through interleukin 1ß (IL1ß) upregulation. Ucn2 decreased cPLA2 expression through neither tumor necrosis factor α nor IL1ß. CRH-suppressed decay of cPLA2 mRNA and Ucn2 merely suppressed its production. Overexpression of CRHR1 or CRHR2 in HEK293 cells correspondingly upregulated or downregulated cPLA2 expression after CRH or Ucn2 stimulation respectively. In addition, both CRH and Ucn2 induced migration of RM-1 cells. Our observation not only established a relationship between CRHRs and cell migration but also for the first time, to our knowledge, demonstrated that cPLA2 participates in CRHR1-induced apoptosis and CRHR2-inhibited apoptosis.

TNF-α induces cytosolic phospholipase A2 expression via Jak2/PDGFR-dependent Elk-1/p300 activation in human lung epithelial cells.

Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in mediating agonist-induced arachidonic acid release for prostaglandin (PG) synthesis during inflammation triggered by tumor necrosis factor-α (TNF-α). However, the mechanisms underlying TNF-α-induced cPLA2 expression in human lung epithelial cells (HPAEpiCs) were not completely understood. Here, we demonstrated that TNF-α induced cPLA2 mRNA and protein expression, promoter activity, and PGE2 secretion in HPAEpiCs. These responses induced by TNF-α were inhibited by pretreatment with the inhibitor of Jak2 (AG490), platelet-derived growth factor receptor (PDGFR) (AG1296), phosphoinositide 3 kinase (PI3K) (LY294002), or MEK1/2 (PD98059) and transfection with siRNA of Jak2, PDGFR, Akt, or p42. We showed that TNF-α markedly stimulated Jak2, PDGFR, Akt, and p42/p44 MAPK phosphorylation, which were attenuated by their respective inhibitors. Moreover, TNF-α stimulated Akt activation via a Jak2/PDGFR pathway in HPAEpiCs. In addition, TNF-α-induced p42/p44 MAPK phosphorylation was reduced by AG1296 or LY294002. On the other hand, TNF-α could induce Akt and p42/p44 MAPK translocation from the cytosol into the nucleus, which was inhibited by AG490, AG1296, or LY294002. Finally, we showed that TNF-α stimulated Elk-1 phosphorylation, which was reduced by LY294002 or PD98059. We also observed that TNF-α time dependently induced p300/Elk-1 and p300/Akt complex formation in HPAEpiCs, which was reduced by AG490, AG1296, or LY294002. The activity of cPLA2 protein upregulated by TNF-α was reflected on the PGE2 release, which was reduced by AG490, AG1296, LY294002, or PD98059. Taken together, these results demonstrated that TNF-α-induced cPLA2 expression and PGE2 release were mediated through a Jak2/PDGFR/PI3K/Akt/p42/p44 MAPK/Elk-1 pathway in HPAEpiCs.

Phospholipase A2, oxidative stress, and neurodegeneration in binge ethanol-treated organotypic slice cultures of developing rat brain.

BACKGROUND: Brain neurodamage from chronic binge ethanol (EtOH) exposure is linked to neuroinflammation and associated oxidative stress. Using rat organotypic hippocampal-entorhinal cortical (HEC) slice cultures of developing brain age, we reported that binge EtOH promotes release of a neuroinflammatory instigator, arachidonic acid (AA), concomitant with neurodegeneration, and that mepacrine, a global inhibitor of phospholipase A2 (PLA2) enzymes mobilizing AA from phospholipids, is neuroprotective. Here, we sought with binge EtOH-treated HEC cultures to establish that PLA2 activity is responsible in part for significant oxidative stress and to ascertain the PLA2 families responsible for AA release and neurodegeneration. METHODS: HEC slices, prepared from 1-week-old rats and cultured 2 to 2.5 weeks, were exposed to 100 mM EtOH over 6 successive days, with 4 daytime "withdrawals" (no EtOH). Brain 3-nitrotyrosinated (3-NT)- and 4-hydroxy-2-nonenal (4-HNE)-adducted proteins, oxidative stress footprints, were immunoassayed on days 3 through 6, and mepacrine's effect was determined on day 6. The effects of specific PLA2 inhibitors on neurodegeneration (propidium iodide staining) and AA release (ELISA levels in media) in the cultures were then determined. Also, the effect of JZL184, an inhibitor of monoacylglycerol lipase (MAGL) which is reported to mobilize AA from endocannabinoids during neuroinflammatory insults, was examined. RESULTS: 3-NT- and 4-HNE-adducted proteins were significantly increased by the binge EtOH exposure, consistent with oxidative stress, and mepacrine prevented the increases. The PLA2 inhibitor results implicated secretory PLA2 (group II sPLA2) and to some extent Ca(2+) -independent cytosolic PLA2 (group VI iPLA2) in binge EtOH-induced neurotoxicity and in AA release, but surprisingly, Ca(2+) -dependent cytosolic PLA2 (group IV cPLA2) did not appear important. Furthermore, unlike PLA2 inhibition, MAGL inhibition failed to prevent the neurodegeneration. CONCLUSIONS: In these developing HEC slice cultures, pro-oxidative signaling via sPLA2 and iPLA2, but not necessarily cPLA2 or MAGL, is involved in EtOH neurotoxicity. This study provides further insights into neuroinflammatory phospholipase signaling and oxidative stress underlying binge EtOH-induced neurodegeneration in developing (adolescent age) brain in vitro.

Role of phospholipases A2 in diabetic retinopathy: in vitro and in vivo studies.

Diabetic retinopathy is one of the leading causes of blindness and the most common complication of diabetes with no cure available. We investigated the role of phospholipases A2 (PLA2) in diabetic retinopathy using an in vitro blood-retinal barrier model (BRB) and an in vivo streptozotocin (STZ)-induced diabetic model. Mono- and co-cultures of endothelial cells (EC) and pericytes (PC), treated with high or fluctuating concentrations of glucose, to mimic the diabetic condition, were used. PLA2 activity, VEGF and PGE2 levels and cell proliferation were measured, with or without PLA2 inhibition. Diabetes was induced in rats by STZ injection and PLA2 activity along with VEGF, TNFα and ICAM-1 levels were measured in retina. High or fluctuating glucose induced BRB breakdown, and increased PLA2 activity, PGE2 and VEGF in EC/PC co-cultures; inhibition of PLA2 in mono- or co-cultures treated with high or fluctuating glucose dampened PGE2 and VEGF production down to the levels of controls. High or fluctuating glucose increased EC number and reduced PC number in co-cultures; these effects were reversed after transfecting EC with small interfering RNA targeted to PLA2. PLA2 and COX-2 protein expressions were significantly increased in microvessels from retina of diabetic rats. Diabetic rats had also high retinal levels of VEGF, ICAM-1 and TNFα that were reduced by treatment with a cPLA2 inhibitor. In conclusion, the present findings indicate that PLA2 upregulation represents an early step in glucose-induced alteration of BRB, possibly upstream of VEGF; thus, PLA2 may be an interesting target in managing diabetic retinopathy.