PLA2G4A and ACHE modulate lipid profiles via glycerophospholipid metabolism in platinum-resistant gastric cancer.

BACKGROUND: Bioactive lipids involved in the progression of various diseases. Nevertheless, there is still a lack of biomarkers and relative regulatory targets. The lipidomic analysis of the samples from platinum-resistant in gastric cancer patients is expected to help us further improve our understanding of it. METHODS: We employed LC-MS based untargeted lipidomic analysis to search for potential candidate biomarkers for platinum resistance in GC patients. Partial least squares discriminant analysis (PLS-DA) and variable importance in projection (VIP) analysis were used to identify differential lipids. The possible molecular mechanisms and targets were obtained by metabolite set enrichment analysis and potential gene network screened. Finally, verified them by immunohistochemical of a tissue microarray. RESULTS: There were 71 differential lipid metabolites identified in GC samples between the chemotherapy-sensitivity group and the chemotherapy resistance group. According to Foldchange (FC) value, VIP value, P values (FC > 2, VIP > 1.5, p < 0.05), a total of 15 potential biomarkers were obtained, including MGDG(43:11)-H, Cer(d18:1/24:0) + HCOO, PI(18:0/18:1)-H, PE(16:1/18:1)-H, PE(36:2) + H, PE(34:2p)-H, Cer(d18:1 + hO/24:0) + HCOO, Cer(d18:1/23:0) + HCOO, PC(34:2e) + H, SM(d34:0) + H, LPC(18:2) + HCOO, PI(18:1/22:5)-H, PG(18:1/18:1)-H, Cer(d18:1/24:0) + H and PC(35:2) + H. Furthermore, we obtained five potential key targets (PLA2G4A, PLA2G3, DGKA, ACHE, and CHKA), and a metabolite-reaction-enzyme-gene interaction network was built to reveal the biological process of how they could disorder the endogenous lipid profile of platinum resistance in GC patients through the glycerophospholipid metabolism pathway. Finally, we further identified PLA2G4A and ACHE as core targets of the process by correlation analysis and tissue microarray immunohistochemical verification. CONCLUSION: PLA2G4A and ACHE regulated endogenous lipid profile in the platinum resistance in GC patients through the glycerophospholipid metabolism pathway. The screening of lipid biomarkers will facilitate earlier precision medicine interventions for chemotherapy-resistant gastric cancer. The development of therapies targeting PLA2G4A and ACHE could enhance platinum chemotherapy effectiveness.

Phospholipase D1 activity is crucial for cytosolic phospholipase A2 -dependent prostaglandin E2 formation in murine osteoblastic MC3T3-E1 cells.

In bone, prostaglandin E2 (PGE2) is highly osteogenic and formed by osteoblasts, a key modulatory event in the regulation of bone cell activity. MC3T3-E1 cells are widely used as an in vitro model of osteoblast function. It is still not clear which pathways contribute to the release of AA in these cells. In this study we have focussed on the contribution of phospholipase D (PLD) enzymes to osteoblastic PGE2 formation after stimulation with endothelin-1 (ET-1). Using specific inhibitors of PLD1 and PLD2 we could show that PGE2 formation was strictly dependent on PLD1 but not PLD2 activity and cytosolic phospholipase A2 (cPLA2) was activated by triggering through PLD1. We have identified diacyl glycerol (DAG) as a possible effector molecule which may serve as a triggering signal for PKC activation and subsequent cPLA2 phosphorylation.

Skewing cPLA2α activity toward oxoeicosanoid production promotes neutrophil N2 polarization, wound healing, and the response to sepsis.

Uncontrolled inflammation is linked to poor outcomes in sepsis and wound healing, both of which proceed through distinct inflammatory and resolution phases. Eicosanoids are a class of bioactive lipids that recruit neutrophils and other innate immune cells. The interaction of ceramide 1-phosphate (C1P) with the eicosanoid biosynthetic enzyme cytosolic phospholipase A2 (cPLA2) reduces the production of a subtype of eicosanoids called oxoeicosanoids. We investigated the effect of shifting the balance in eicosanoid biosynthesis on neutrophil polarization and function. Knockin mice expressing a cPLA2 mutant lacking the C1P binding site (cPLA2αKI/KI mice) showed enhanced and sustained neutrophil infiltration into wounds and the peritoneum during the inflammatory phase of wound healing and sepsis, respectively. The mice exhibited improved wound healing and reduced susceptibility to sepsis, which was associated with an increase in anti-inflammatory N2-type neutrophils demonstrating proresolution behaviors and a decrease in proinflammatory N1-type neutrophils. The N2 polarization of cPLA2αKI/KI neutrophils resulted from increased oxoeicosanoid biosynthesis and autocrine signaling through the oxoeicosanoid receptor OXER1 and partially depended on OXER1-dependent inhibition of the pentose phosphate pathway (PPP). Thus, C1P binding to cPLA2α suppresses neutrophil N2 polarization, thereby impairing wound healing and the response to sepsis.

Synthesis, activity, metabolic stability and cell permeability of new cytosolic phospholipase A2α inhibitors with 1-indolyl-3-phenoxypropan-2-one structure.

Cytosolic phospholipase A2α (cPLA2α), the key enzyme of the arachidonic acid cascade, is considered to be an interesting target for the development of new anti-inflammatory drugs. Potent inhibitors of the enzyme include indole-5-carboxylic acids with propan-2-one residues in position 1 of the indole. Previously, it was found that central pharmacophoric elements of these compounds are their ketone and carboxylic acid groups, which unfortunately are subject to pronounced metabolism by carbonyl reductases and glucuronosyltransferases, respectively. Here we show that the metabolic stability of these inhibitors can be improved by introducing alkyl substituents in the vicinity of the ketone group or by increasing their rigidity. Furthermore, permeability tests with Caco-2 cells revealed that the indole derivatives have only low permeability, which can be attributed to their affinity to efflux transporters. Among other things, the polar ketone group in the center of the molecules seems to be a decisive factor for their reverse transport. After its removal, the permeability increased significantly. The enhancement in metabolic stability and permeability achieved by the structural variations carried out was accompanied by a more or less pronounced decrease in the inhibitory potency of the compounds against cPLA2α.

Integrative Analysis of Transcriptome and Metabolome to Illuminate the Protective Effects of Didymin against Acute Hepatic Injury.

Based on the multiomics analysis, this study is aimed at investigating the underlying mechanism of didymin against acute liver injury (ALI). The mice were administrated with didymin for 2 weeks, followed by injection with lipopolysaccharide (LPS) plus D-galactosamine (D-Gal) to induce ALI. The pathological examination revealed that didymin significantly ameliorated LPS/D-Gal-induced hepatic damage. Also, it markedly reduced proinflammatory cytokines release by inhibiting the TLR4/NF-κB pathway activation, alleviating inflammatory injury. A transcriptome analysis proved 2680 differently expressed genes (DEGs) between the model and didymin groups and suggested that the PI3K/Akt and metabolic pathways might be the most relevant targets. Meanwhile, the metabolome analysis revealed 67 differently expressed metabolites (DEMs) between the didymin and model groups that were mainly clustered into the glycerophospholipid metabolism, which was consistent with the transcriptome study. Importantly, a comprehensive analysis of both the omics indicated a strong correlation between the DEGs and DEMs, and an in-depth study demonstrated that didymin alleviated metabolic disorder and hepatocyte injury likely by inhibiting the glycerophospholipid metabolism pathway through the regulation of PLA2G4B, LPCAT3, and CEPT1 expression. In conclusion, this study demonstrates that didymin can ameliorate LPS/D-Gal-induced ALI by inhibiting the glycerophospholipid metabolism and PI3K/Akt and TLR4/NF-κB pathways.

Synthesis and pharmacokinetic properties of novel cPLA2α inhibitors with 1-(carboxyalkylpyrrolyl)-3-aryloxypropan-2-one structure.

Indole-5-carboxylic acids with 3-aryloxy-2­oxopropyl residues in position 1 have been shown to be potent inhibitors of cytosolic phospholipase A2α (cPLA2α), an enzyme involved in the formation of pro-inflammatory lipid mediators. Unfortunately, in animal experiments, only very low plasma concentrations could be achieved after peroral administration of this type of compound. Since insufficient metabolic stability was suspected as the cause, structural modifications were made to optimize this property. These included the conversion of the aromatic into an aliphatic carboxylic acid function as well as the rigidification of the lipophilic structural elements. A selected pyrrole-3-propionic acid was tested for its peroral in vivo bioavailability in mice. However, higher plasma concentrations could not be achieved also with this compound. Using the Caco2 cell permeation assay, substances investigated were found to be very good substrates for gastrointestinal efflux transporters, which explains their poor peroral absorption.

Calcium-dependent cytosolic phospholipase A2 activation is implicated in neuroinflammation and oxidative stress associated with ApoE4.

BACKGROUND: Apolipoprotein E4 (APOE4) is associated with a greater response to neuroinflammation and the risk of developing late-onset Alzheimer's disease (AD), but the mechanisms for this association are not clear. The activation of calcium-dependent cytosolic phospholipase A2 (cPLA2) is involved in inflammatory signaling and is elevated within the plaques of AD brains. The relation between APOE4 genotype and cPLA2 activity is not known. METHODS: Mouse primary astrocytes, mouse and human brain samples differing by APOE genotypes were collected for measuring cPLA2 expression, phosphorylation, and activity in relation to measures of inflammation and oxidative stress. RESULTS: Greater cPLA2 phosphorylation, cPLA2 activity and leukotriene B4 (LTB4) levels were identified in ApoE4 compared to ApoE3 in primary astrocytes, brains of ApoE-targeted replacement (ApoE-TR) mice, and in human brain homogenates from the inferior frontal cortex of persons with AD dementia carrying APOE3/4 compared to APOE3/3. Higher phosphorylated p38 MAPK but not ERK1/2 was found in ApoE4 primary astrocytes and mouse brains than that in ApoE3. Greater cPLA2 translocation to cytosol was observed in human postmortem frontal cortical synaptosomes with recombinant ApoE4 than ApoE3 ex vivo. In ApoE4 astrocytes, the greater levels of LTB4, reactive oxygen species (ROS), and inducible nitric oxide synthase (iNOS) were reduced after cPLA2 inhibition. CONCLUSIONS: Our findings implicate greater activation of cPLA2 signaling system with APOE4, which could represent a potential drug target for mitigating the increased neuroinflammation with APOE4 and AD.

Neuronal STAT3/HIF-1α/PTRF axis-mediated bioenergetic disturbance exacerbates cerebral ischemia-reperfusion injury via PLA2G4A.

Ischemic stroke is an acute and severe neurological disease with high mortality and disability rates worldwide. Polymerase I and transcript release factor (PTRF) plays a pivotal role in regulating cellular senescence, glucose intolerance, lipid metabolism, and mitochondrial bioenergetics, but its mechanism, characteristics, and functions in neuronal cells following the cerebral ischemia-reperfusion (I/R) injury remain to be determined. Methods: Transcription factor motif analysis, chromatin immunoprecipitation (ChIP), luciferase and co-Immunoprecipitation (co-IP) assays were performed to investigate the mechanisms of PTRF in neuronal cells after I/R injury. Lentiviral-sgRNA against PTRF gene was introduced to HT22 cells, and adeno-associated virus (AAV) encoding a human synapsin (hSyn) promoter-driven construct was transduced a short hairpin RNA (shRNA) against PTRF mRNA in primary neuronal cells and the cortex of the cerebral I/R mice for investigating the role of PTRF in neuronal damage and PLA2G4A change induced by the cerebral I/R injury. Results: Here, we reported that neuronal PTRF was remarkably increased in the cerebral penumbra after I/R injury, and HIF-1α and STAT3 regulated the I/R-dependent expression of PTRF via binding to its promoter in neuronal cells. Moreover, overexpression of neuronal PTRF enhanced the activity and stability of PLA2G4A by decreasing its proteasome-mediated degradation pathway. Subsequently, PTRF promoted reprogramming of lipid metabolism and altered mitochondrial bioenergetics, which could lead to oxidative damage, involving autophagy, lipid peroxidation, and ferroptosis via PLA2G4A in neuronal cells. Furthermore, inhibition of neuronal PTRF/PLA2G4A-axis markedly reduced the neurological deficits, cerebral infarct volumes, and mortality rates in the mice following cerebral I/R injury. Conclusion: Our results thus identify that the STAT3/HIF-1α/PTRF-axis in neurons, aggravating cerebral I/R injury by regulating the activity and stability of PLA2G4A, might be a novel therapeutic target for ischemic stroke.

Phosphorylation of cPLA2α at Ser505 Is Necessary for Its Translocation to PtdInsP2-Enriched Membranes.

Group IVA cytosolic phospholipase A2α (cPLA2α) is a key enzyme in physiology and pathophysiology because it constitutes a rate-limiting step in the pathway for the generation of pro- and anti-inflammatory eicosanoid lipid mediators. cPLA2α activity is tightly regulated by multiple factors, including the intracellular Ca2+ concentration, phosphorylation reactions, and cellular phosphatidylinositol (4,5) bisphosphate levels (PtdInsP2). In the present work, we demonstrate that phosphorylation of the enzyme at Ser505 is an important step for the translocation of the enzyme to PtdInsP2-enriched membranes in human cells. Constructs of eGFP-cPLA2 mutated in Ser505 to Ala (S505A) exhibit a delayed translocation in response to elevated intracellular Ca2+, and also in response to increases in intracellular PtdInsP2 levels. Conversely, translocation of a phosphorylation mimic mutant (S505E) is fully observed in response to cellular increases in PtdInsP2 levels. Collectively, these results suggest that phosphorylation of cPLA2α at Ser505 is necessary for the enzyme to translocate to internal membranes and mobilize arachidonic acid for eicosanoid synthesis.

Altered hippocampal expression and function of cytosolic phospholipase A2 (cPLA2) in temporal lobe epilepsy (TLE).

OBJECTIVES: Temporal lobe epilepsy (TLE) is the most common form of drug-resistant epilepsy. Blood-brain barrier (BBB) leakage occurs during epileptogenesis and several pieces of evidence suggest that this might contribute to the progression of epilepsy. Seizures trigger a pathway involving glutamate signalling through cytosolic phospholipase A2 (cPLA2). This pathway leads to BBB leakage and induces the expression of drug efflux transporters, leading to drug resistance. Therefore, this study aims to determine the mRNA and protein levels of cPLA2, along with its functional activity, in the hippocampus of pilocarpine model of TLE as well as in the surgically resected hippocampal samples of patients with TLE. METHODS: mRNA levels and protein levels of cPLA2 were evaluated by real-time PCR and western blot analysis respectively in animal model of TLE as well as surgically resected hippocampal tissue specimens of TLE. cPLA2 functional activity was measured spectrophotometrically. RESULTS: Significant up-regulation of cPLA2 mRNA was observed in the hippocampal samples obtained from TLE rats (p < 0.05) and-TLE patients (p < 0.01). Increased protein expression of cPLA2 was also demonstrated in the hippocampal samples of TLE rats (p < 0.01) as well as TLE patients (p < 0.01). Similarly, functional activity of cPLA2 was found to be up-regulated in the hippocampus of pilocarpine model of TLE rats (p < 0.01) as well as in the TLE patients (p < 0.01). DISCUSSION: These findings suggest that alterations in cPLA2 expression and activity level in the hippocampus could potentially be a part of dynamic changes associated with TLE.

Contribution of cyclooxygenase-1-dependent prostacyclin synthesis to bradykinin-induced dermal extravasation.

BACKGROUND: Non-allergic angioedema is a potentially life-threatening condition caused by accumulation of bradykinin and subsequent activation of bradykinin type 2 receptors (B2). Since COX activity plays a pivotal role in B2 signaling, the aim of this study was to determine which prostaglandins are the key mediators and which COX, COX-1 or COX-2, is predominantly involved. METHODS: We used Miles assays to assess the effects of inhibitors of COX, 5-lipoxygenase, epoxyeicosatrienoic acid generation, cytosolic phospholipase A2α and a variety of prostaglandin receptor antagonists on bradykinin-induced dermal extravasation in C57BL/6 and COX-1-deficient mice (COX-1-/-). In addition, the prostacyclin metabolite 6-keto-PGF1α was quantified by ELISA in subcutaneous tissue from C57BL/6 and human dermal microvascular endothelial cells. In the latter, 6-keto-PGF1α was also quantified and identified by LC-MS/MS. RESULTS: Unspecific COX inhibition by ibuprofen and diclofenac significantly reduced B2-mediated dermal extravasation in C57BL/6 but not COX-1-/-. Likewise, inhibition of cytosolic phospholipase A2α showed similar effects. Furthermore, extravasation in COX-1-/- was generally lower than in C57BL/6. Of the prostaglandin antagonists used, only the prostacyclin receptor antagonist RO1138452 showed a significant reduction of dermal extravasation. Moreover, 6-keto-PGF1α concentrations were increased after bradykinin treatment in subcutaneous tissue from C57BL/6 as well as in human dermal microvascular endothelial cells and this increase was abolished by diclofenac. CONCLUSION: Our findings suggest that COX-1-dependent prostacyclin production is critically involved in dermal extravasation after activation of B2 in small dermal blood vessels. Targeting prostacyclin production and/or signaling appears to be a suitable option for acute treatment of non-allergic angioedema.

A defective lysophosphatidic acid-autophagy axis increases miscarriage risk by restricting decidual macrophage residence.

Massive infiltrated and enriched decidual macrophages (dMφ) have been widely regarded as important regulators of maternal-fetal immune tolerance and trophoblast invasion, contributing to normal pregnancy. However, the characteristics of metabolic profile and the underlying mechanism of dMφ residence remain largely unknown. Here, we observe that dMφ display an active glycerophospholipid metabolism. The activation of ENPP2-lysophosphatidic acid (LPA) facilitates the adhesion and retention, and M2 differentiation of dMφ during normal pregnancy. Mechanistically, this process is mediated through activation of the LPA receptors (LPAR1 and PPARG/PPARγ)-DDIT4-macroautophagy/autophagy axis, and further upregulation of multiple adhesion factors (e.g., cadherins and selectins) in a CLDN7 (claudin 7)-dependent manner. Additionally, poor trophoblast invasion and placenta development, and a high ratio of embryo loss are observed in Enpp2±, lpar1-/- or PPARG-blocked pregnant mice. Patients with unexplained spontaneous abortion display insufficient autophagy and cell residence of dMφ. In therapeutic studies, supplementation with LPA or the autophagy inducer rapamycin significantly promotes dMφ autophagy and cell residence, and improves embryo resorption in Enpp2± and spontaneous abortion mouse models, which should be dependent on the activation of DDIT4-autophagy-CLDN7-adhesion molecules axis. This observation reveals that inactivation of ENPP2-LPA metabolism and insufficient autophagy of dMφ result in resident obstacle of dMφ and further increase the risk of spontaneous abortion, and provides potential therapeutic strategies to prevent spontaneous abortion.Abbreviations: ACTB: actin beta; ADGRE1/F4/80: adhesion G protein-coupled receptor E1; Atg5: autophagy related 5; ATG13: autophagy related 13; BECN1: beclin 1; CDH1/E-cadherin: cadherin 1; CDH5/VE-cadherin: cadherin 5; CFSE: carboxyfluorescein succinimidyl ester; CLDN7: claudin 7; CSF1/M-CSF: colony stimulating factor 1; CSF2/GM-CSF: colony stimulating factor 2; Ctrl: control; CXCL10/IP-10: chemokine (C-X-C) ligand 10; DDIT4: DNA damage inducible transcript 4; dMφ: decidual macrophage; DSC: decidual stromal cells; ENPP2/ATX: ectonucleotide pyrophosphatase/phosphodiesterase 2; Enpp2±: Enpp2 heterozygous knockout mouse; ENPP2i/PF-8380: ENPP2 inhibitor; EPCAM: epithelial cell adhesion molecule; ESC: endometrial stromal cells; FGF2/b-FGF: fibroblast growth factor 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GPCPD1: glycerophosphocholine phosphodiesterase 1; HE: heterozygote; HIF1A: hypoxia inducible factor 1 subunit alpha; HNF4A: hepatocyte nuclear factor 4 alpha; HO: homozygote; ICAM2: intercellular adhesion molecule 2; IL: interleukin; ITGAV/CD51: integrin subunit alpha V; ITGAM/CD11b: integrin subunit alpha M; ITGAX/CD11b: integrin subunit alpha X; ITGB3/CD61: integrin subunit beta 3; KLRB1/NK1.1: killer cell lectin like receptor B1; KRT7/cytokeratin 7: keratin 7; LPA: lysophosphatidic acid; LPAR: lysophosphatidic acid receptor; lpar1-/-: lpar1 homozygous knockout mouse; LPAR1i/AM966: LPAR1 inhibitor; LY6C: lymphocyte antigen 6 complex, locus C1; LYPLA1: lysophospholipase 1; LYPLA2: lysophospholipase 2; Lyz2: lysozyme 2; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MARVELD2: MARVEL domain containing 2; 3-MA: 3-methyladenine; MBOAT2: membrane bound O-acyltransferase domain containing 2; MGLL: monoglyceride lipase; MRC1/CD206: mannose receptor C-type 1; MTOR: mechanistic target of rapamycin kinase; NP: normal pregnancy; PDGF: platelet derived growth factor; PLA1A: phospholipase A1 member A; PLA2G4A: phospholipase A2 group IVA; PLPP1: phospholipid phosphatase 1; pMo: peripheral blood monocytes; p-MTOR: phosphorylated MTOR; PPAR: peroxisome proliferator activated receptor; PPARG/PPARγ: peroxisome proliferator activated receptor gamma; PPARGi/GW9662: PPARG inhibitor; PTPRC/CD45: protein tyrosine phosphatase receptor type, C; Rapa: rapamycin; RHEB: Ras homolog, mTORC1 binding; SA: spontaneous abortion; SELE: selectin E; SELL: selectin L; siCLDN7: CLDN7-silenced; STAT: signal transducer and activator of transcription; SQSTM1: sequestosome 1; TJP1: tight junction protein 1; VCAM1: vascular cell adhesion molecule 1; WT: wild type.

Calcium-Dependent Cytosolic Phospholipase A2α as Key Factor in Calcification of Subdermally Implanted Aortic Valve Leaflets.

Calcium-dependent cytosolic phospholipase A2α (cPLA2α) had been previously found to be overexpressed by aortic valve interstitial cells (AVICs) subjected to in vitro calcific induction. Here, cPLA2α expression was immunohistochemically assayed in porcine aortic valve leaflets (iAVLs) that had undergone accelerated calcification subsequent to 2- to 28-day-long implantation in rat subcutis. A time-dependent increase in cPLA2α-positive AVICs paralleled mineralization progression depending on dramatic cell membrane degeneration with the release of hydroxyapatite-nucleating acidic lipid material, as revealed by immunogold particles decorating organelle membranes in 2d-iAVLs, as well as membrane-derived lipid byproducts in 7d- to 28d-iAVLs. Additional positivity was detected for (i) pro-inflammatory IL-6, mostly exhibited by rat peri-implant cells surrounding 14d- and 28d-iAVLs; (ii) calcium-binding osteopontin, with time-dependent increase and no ossification occurrence; (iii) anti-calcific fetuin-A, mostly restricted to blood plasma within vessels irrorating the connective envelopes of 28d-iAVLs; (iv) early apoptosis marker annexin-V, limited to sporadic AVICs in all iAVLs. No positivity was found for either apoptosis executioner cleaved caspase-3 or autophagy marker MAP1. In conclusion, cPLA2α appears to be a factor characterizing AVL calcification concurrently with a distinct still uncoded cell death form also in an animal model, as well as a putative target for the prevention and treatment of calcific valve diseases.

ATF6α promotes prostate cancer progression by enhancing PLA2G4A-mediated arachidonic acid metabolism and protecting tumor cells against ferroptosis.

BACKGROUND: Despite the clinical success of androgen receptor (AR)-targeted therapies, prostate cancer (PCa) inevitably progresses to castration-resistant prostate cancer (CRPC). Transcription factor 6 α (ATF6α), an effector of the unfolded protein response (UPR) that modulates the cellular response to endoplasmic reticulum (ER) stress, has been linked to tumor development, metastasis, and relapse. However, the role of ATF6α in CRPC remains unclear. METHODS: The effect of ATF6α on the CRPC-like phenotype in PCa cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carb-Oxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS), 5-Bromo-2-deoxyUridine (BrdU) incorporation analysis, and cell death assay. Mechanistically, bioinformatic analysis was utilized to evaluate the potential of PLA2G4A as the target of ATF6α. Moreover, Western blot analysis, real-time polymerase chain reaction, chromatin immunoprecipitation, arachidonic acid (AA), and prostaglandin E2 (PGE2) assays were performed to identify the regulatory effect of ATF6α on PLA2G4A. RESULTS: In this study, we found that the increase of ATF6α expression in response to androgen deprivation generates PCa cells with a CRPC-like phenotype. PCa cells with high levels of ATF6α expression are resistant to ferroptosis, and genetic and pharmacological inhibition of ATF6α could, therefore, promote the ferroptotic death of tumor cells and delay PCa progression. Molecular analyses linked ATF6α regulation of ferroptosis to the PLA2G4A-mediated release of AA and the resulting increase in PGE2 production, the latter of which acts as an antiferroptotic factor. CONCLUSIONS: This study defines ATF6α as a novel antiferroptotic regulator that exacerbates PCa progression. In addition, our data establish ATF6α-PLA2G4A signaling as an important pathological pathway in PCa, and targeting this pathway may be a novel treatment strategy.

Characterization of the binding of cytosolic phospholipase A2 alpha and NOX2 NADPH oxidase in mouse macrophages.

BACKGROUND: Previous studies have demonstrated that cytosolic phospholipase A2α (cPLA2α) is required for NOX2 NADPH oxidase activation in human and mouse phagocytes. Moreover, upon stimulation, cPLA2α translocates to the plasma membranes by binding to the assembled oxidase, forming a complex between its C2 domain and the PX domain of the cytosolic oxidase factor, p47phox in human phagocytes. Intravenous administration of antisense against cPLA2α that significantly inhibited its expression in mouse peritoneal neutrophils and macrophages also inhibited superoxide production, in contrast to cPLA2α knockout mice that showed normal superoxide production. The present study aimed to determine whether there is a binding between cPLA2α-C2 domain and p47phox-PX in mouse macrophages, to further support the role of cPLA2α in oxidase regulation also in mouse phagocytes. METHODS AND RESULTS: A significant binding of mouse GST-p47phox-PX domain fusion protein and cPLA2α in stimulated mouse phagocyte membranes was demonstrated by pull-down experiments, although lower than that detected by the human p47phox-PX domain. Substituting the amino acids Phe98, Asn99, and Gly100 to Cys98, Ser99, and Thr100 in the mouse p47phox-PX domain (present in the human p47phox-PX domain) caused strong binding that was similar to that detected by the human p47phox-PX domain CONCLUSIONS: The binding between cPLA2α-C2 and p47phox-PX domains exists in mouse macrophages and is not unique to human phagocytes. The binding between the two proteins is lower in the mice, probably due to the absence of amino acids Cys98, Ser 99, and Thr100in the p47phox-PX domain that facilitate the binding to cPLA2α.

Endothelial group IVA phospholipase A2 promotes hepatic fibrosis with sinusoidal capillarization in the early stage of non-alcoholic steatohepatitis in mice.

AIM: Non-alcoholic steatohepatitis (NASH) is characterized by steatosis, inflammatory responses and fibrosis. Our previous studies provided evidence that group IVA phospholipase A2 (IVA-PLA2), a key PLA2 isozyme in the arachidonic acid cascade, is involved in the development of NASH. However, which types of cells are critical for the IVA-PLA2-dependent onset and progression of NASH is unclear. We elucidated the effects of the cell-type-specific deficiency of IVA-PLA2 in mice on the development of NASH. MAIN METHODS: Cell-type-specific IVA-PLA2-conditional knockout (cKO) mice and littermate controls were fed a choline-deficient, L-amino-acid-defined, high-fat diet with 0.1% methionine as a NASH model. The degree of hepatic fibrosis was evaluated by staining with picric acid-Sirius red, and the number of activated hepatic stellate cells was determined by immunoblotting and immunostaining for α-smooth muscle actin. Sinusoidal capillarization was analyzed by scanning electron microscopy. KEY FINDINGS: The deposition of collagen and number of activated hepatic stellate cells were markedly reduced in endothelial cell/liver sinusoidal endothelial cell (EC/LSEC)-specific IVA-PLA2 cKO mice but not in hepatocyte-, monocyte/macrophage-, or hepatic stellate cell-specific IVA-PLA2 cKO mice. In addition, EC/LSEC-specific IVA-PLA2-deficient mice showed more fenestrae than control mice fed a CDAHFD, indicating suppression of sinusoidal capillarization. SIGNIFICANCE: These results suggest that ECs/LSECs contribute to the IVA-PLA2-dependent onset and/or progression of NASH. Endothelial IVA-PLA2 is a promising factor for promoting sinusoidal capillarization and the ensuing HSC activation and fibrosis; thus IVA-PLA2 in ECs/LSECs is a potential therapeutic target for NASH.

Narciclasine inhibits phospholipase A2 and regulates phospholipid metabolism to ameliorate psoriasis-like dermatitis.

Introduction: Psoriasis is a common inflammatory skin disease recognized by the World Health Organization as "an incurable chronic, noninfectious, painful, disfiguring and disabling disease." The fact that metabolic syndrome (MetS) is the most common and important comorbidities of psoriasis suggests an important role of lipid metabolism in the pathogenesis of psoriasis. Narciclasine (Ncs) is an alkaloid isolated from the Amaryllidaceae plants. Its biological activities include antitumor, antibacterial, antiinflammatory, anti-angiogenic and promoting energy expenditure to improve dietinduced obesity. Here, we report that Ncs may be a potential candidate for psoriasis, acting at both the organismal and cellular levels. Methods: The therapeutic effect of Ncs was assessed in IMQ-induced psoriasis-like mouse model. Then, through in vitro experiments, we explored the inhibitory effect of Ncs on HaCaT cell proliferation and Th17 cell polarization; Transcriptomics and lipidomics were used to analyze the major targets of Ncs; Single-cell sequencing data was used to identify the target cells of Ncs action. Results: Ncs can inhibit keratinocyte proliferation and reduce the recruitment of immune cells in the skin by inhibiting psoriasis-associated inflammatory mediators. In addition, it showed a direct repression effect on Th17 cell polarization. Transcriptomic and lipidomic data further revealed that Ncs extensively regulated lipid metabolismrelated genes, especially the Phospholipase A2 (PLA2) family, and increased antiinflammatory lipid molecules. Combined with single-cell data analysis, we confirmed that keratinocytes are the main cells in which Ncs functions. Discussion: Taken together, our findings indicate that Ncs alleviates psoriasiform skin inflammation in mice, which is associated with inhibition of PLA2 in keratinocytes and improved phospholipid metabolism. Ncs has the potential for further development as a novel anti-psoriasis drug.

A synergy between mechanosensitive calcium- and membrane-binding mediates tension-sensing by C2-like domains.

When nuclear membranes are stretched, the peripheral membrane enzyme cytosolic phospholipase A2 (cPLA2) binds via its calcium-dependent C2 domain (cPLA2-C2) and initiates bioactive lipid signaling and tissue inflammation. More than 150 C2-like domains are encoded in vertebrate genomes. How many of them are mechanosensors and quantitative relationships between tension and membrane recruitment remain unexplored, leaving a knowledge gap in the mechanotransduction field. In this study, we imaged the mechanosensitive adsorption of cPLA2 and its C2 domain to nuclear membranes and artificial lipid bilayers, comparing it to related C2-like motifs. Stretch increased the Ca2+ sensitivity of all tested domains, promoting half-maximal binding of cPLA2 at cytoplasmic resting-Ca2+ concentrations. cPLA2-C2 bound up to 50 times tighter to stretched than to unstretched membranes. Our data suggest that a synergy of mechanosensitive Ca2+ interactions and deep, hydrophobic membrane insertion enables cPLA2-C2 to detect stretched membranes with antibody-like affinity, providing a quantitative basis for understanding mechanotransduction by C2-like domains.

Inhibition of PLA2G4E/cPLA2 promotes survival of random skin flaps by alleviating Lysosomal membrane permeabilization-Induced necroptosis.

Necrosis that appears at the ischemic distal end of random-pattern skin flaps increases the pain and economic burden of patients. Necroptosis is thought to contribute to flap necrosis. Lysosomal membrane permeabilization (LMP) plays an indispensable role in the regulation of necroptosis. Nonetheless, the mechanisms by which lysosomal membranes become leaky and the relationship between necroptosis and lysosomes are still unclear in ischemic flaps. Based on Western blotting, immunofluorescence, enzyme-linked immunosorbent assay, and liquid chromatography-mass spectrometry (LC-MS) analysis results, we found that LMP was presented in the ischemic distal portion of random-pattern skin flaps, which leads to disruption of lysosomal function and macroautophagic/autophagic flux, increased necroptosis, and aggravated necrosis of the ischemic flaps. Moreover, bioinformatics analysis of the LC-MS results enabled us to focus on the role of PLA2G4E/cPLA2 (phospholipase A2, group IVE) in LMP of the ischemic flaps. In vivo inhibition of PLA2G4E with an adeno-associated virus vector attenuated LMP and necroptosis, and promoted flap survival. In addition, microRNA-seq helped us determine that Mir504-5p was differentially expressed in ischemic flaps. A string of in vitro and in vivo tests was employed to verify the inhibitory effect of Mir504-5p on PLA2G4E, LMP and necroptosis. Finally, we concluded that the inhibition of PLA2G4E by Mir504-5p reduced LMP-induced necroptosis, thereby promoting the survival of random-pattern skin flaps.Abbreviations: AAV: adeno-associated virus; ACTA2/α;-SMA: actin alpha 2, smooth muscle, aorta; ALOX15/12/15-LOX: arachidonate 15- lipoxygenase; c-CASP8: cleaved caspase; c-CASP3: cleaved caspase 3; CTSD: cathepsin D; CTSB: cathepsin B; CTSL: cathepsin L; DMECs: primary mouse dermal microvascular endothelial cells; ELISA: enzyme-linked immunosorbent assay; F-CHP: 5-FAM-conjugated collagen hybridizing peptide; FISH: fluorescence in situ hybridization; HUVECs: human umbilical vein endothelial cells; LAMP1: lysosomal-associated membrane protein 1; LAMP2: lysosomal-associated membrane protein 2; LC-MS: liquid chromatography-mass spectrometry; LDBF: laser doppler blood flow; LMP: lysosomal membrane permeabilization; LPE: lysophosphatidylethanolamine; LPC: lysophosphatidylcholine; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MLKL: mixed lineage kinase domain-like; NDI: N-dodecylimidazole; PECAM1/CD31: platelet/endothelial cell adhesion molecule 1; PLA2G4A/cPLA2: phospholipase A2, group IVA (cytosolic, calcium-dependent); PLA2G4E/cPLA2: phospholipase A2, group IVE; qPCR: quantitative real-time polymerase chain reaction; RIPK1: receptor (TNFRSF)-interacting serine-threonine kinase 1; RIPK3: receptor-interacting serine-threonine kinase 3; RISC: RNA-induced silencing complex; ROS: reactive oxygen species; shRNA: short hairpin RNA; SQSTM1: sequestosome 1; TBHP: tert-butyl hydroperoxide; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labelling.