Unique lipid composition maintained by extracellular blockade leads to prooncogenicity.

Lipid-mediated inflammation is involved in the development and malignancy of cancer. We previously demonstrated the existence of a novel oncogenic mechanism utilizing membrane lipids of extracellular vesicles in Epstein-Barr virus (EBV)-positive lymphomas and found that the lipid composition of lymphoma cells is skewed toward ω-3 fatty acids, which are anti-inflammatory lipids, suggesting an alteration in systemic lipid composition. The results showed that arachidonic acid (AA), an inflammatory lipid, was significantly reduced in the infected cells but detected at high levels in the sera of EBV-positive patients lead to the finding of the blockade of extracellular AA influx by downregulating FATP2, a long-chain fatty acid transporter that mainly transports AA in EBV-infected lymphoma cells. Low AA levels in tumor cells induced by downregulation of FATP2 expression confer resistance to ferroptosis and support tumor growth. TCGA data analysis and xenograft models have demonstrated that the axis plays a critical role in several types of cancers, especially poor prognostic cancers, such as glioblastoma and melanoma. Overall, our in vitro, in vivo, in silico, and clinical data suggest that several cancers exert oncogenic activity by maintaining their special lipid composition via extracellular blockade.

Hepatic phosphatidylcholine catabolism driven by PNPLA7 and PNPLA8 supplies endogenous choline to replenish the methionine cycle with methyl groups.

Choline supplies methyl groups for regeneration of methionine and the methyl donor S-adenosylmethionine in the liver. Here, we report that the catabolism of membrane phosphatidylcholine (PC) into water-soluble glycerophosphocholine (GPC) by the phospholipase/lysophospholipase PNPLA8-PNPLA7 axis enables endogenous choline stored in hepatic PC to be utilized in methyl metabolism. PNPLA7-deficient mice show marked decreases in hepatic GPC, choline, and several metabolites related to the methionine cycle, accompanied by various signs of methionine insufficiency, including growth retardation, hypoglycemia, hypolipidemia, increased energy consumption, reduced adiposity, increased fibroblast growth factor 21 (FGF21), and an altered histone/DNA methylation landscape. Moreover, PNPLA8-deficient mice recapitulate most of these phenotypes. In contrast to wild-type mice fed a methionine/choline-deficient diet, both knockout strains display decreased hepatic triglyceride, likely via reductions of lipogenesis and GPC-derived glycerol flux. Collectively, our findings highlight the biological importance of phospholipid catabolism driven by PNPLA8/PNPLA7 in methyl group flux and triglyceride synthesis in the liver.

Macrophage SREBP1 regulates skeletal muscle regeneration.

Macrophages are essential for the proper inflammatory and reparative processes that lead to regeneration of skeletal muscle after injury. Recent studies have demonstrated close links between the function of activated macrophages and their cellular metabolism. Sterol regulatory element-binding protein 1 (SREBP1) is a key regulator of lipid metabolism and has been shown to affect the activated states of macrophages. However, its role in tissue repair and regeneration is poorly understood. Here we show that systemic deletion of Srebf1, encoding SREBP1, or macrophage-specific deletion of Srebf1a, encoding SREBP1a, delays resolution of inflammation and impairs skeletal muscle regeneration after injury. Srebf1 deficiency impairs mitochondrial function in macrophages and suppresses the accumulation of macrophages at sites of muscle injury. Lipidomic analyses showed the reduction of major phospholipid species in Srebf1 -/- muscle myeloid cells. Moreover, diet supplementation with eicosapentaenoic acid restored the accumulation of macrophages and their mitochondrial gene expression and improved muscle regeneration. Collectively, our results demonstrate that SREBP1 in macrophages is essential for repair and regeneration of skeletal muscle after injury and suggest that SREBP1-mediated fatty acid metabolism and phospholipid remodeling are critical for proper macrophage function in tissue repair.

Old but New: Group IIA Phospholipase A2 as a Modulator of Gut Microbiota.

Among the phospholipase A2 (PLA2) superfamily, the secreted PLA2 (sPLA2) family contains 11 mammalian isoforms that exhibit unique tissue or cellular distributions and enzymatic properties. Current studies using sPLA2-deficient or -overexpressed mouse strains, along with mass spectrometric lipidomics to determine sPLA2-driven lipid pathways, have revealed the diverse pathophysiological roles of sPLA2s in various biological events. In general, individual sPLA2s exert their specific functions within tissue microenvironments, where they are intrinsically expressed through hydrolysis of extracellular phospholipids. Recent studies have uncovered a new aspect of group IIA sPLA2 (sPLA2-IIA), a prototypic sPLA2 with the oldest research history among the mammalian PLA2s, as a modulator of the gut microbiota. In the intestine, Paneth cell-derived sPLA2-IIA acts as an antimicrobial protein to shape the gut microbiota, thereby secondarily affecting inflammation, allergy, and cancer in proximal and distal tissues. Knockout of intestinal sPLA2-IIA in BALB/c mice leads to alterations in skin cancer, psoriasis, and anaphylaxis, while overexpression of sPLA2-IIA in Pla2g2a-null C57BL/6 mice induces systemic inflammation and exacerbates arthritis. These phenotypes are associated with notable changes in gut microbiota and fecal metabolites, are variable in different animal facilities, and are abrogated after antibiotic treatment, co-housing, or fecal transfer. These studies open a new mechanistic action of this old sPLA2 and add the sPLA2 family to the growing list of endogenous factors capable of affecting the microbe-host interaction and thereby systemic homeostasis and diseases.

Secreted phospholipase A2 modifies extracellular vesicles and accelerates B cell lymphoma.

Extracellular vesicles (EVs) including exosomes act as intercellular communicators by transferring protein and microRNA cargoes, yet the role of EV lipids remains unclear. Here, we show that the pro-tumorigenic action of lymphoma-derived EVs is augmented via secreted phospholipase A2 (sPLA2)-driven lipid metabolism. Hydrolysis of EV phospholipids by group X sPLA2, which was induced in macrophages of Epstein-Barr virus (EBV) lymphoma, increased the production of fatty acids, lysophospholipids, and their metabolites. sPLA2-treated EVs were smaller and self-aggregated, showed better uptake, and increased cytokine expression and lipid mediator signaling in tumor-associated macrophages. Pharmacological inhibition of endogenous sPLA2 suppressed lymphoma growth in EBV-infected humanized mice, while treatment with sPLA2-modified EVs reversed this phenotype. Furthermore, sPLA2 expression in human large B cell lymphomas inversely correlated with patient survival. Overall, the sPLA2-mediated EV modification promotes tumor development, highlighting a non-canonical mechanistic action of EVs as an extracellular hydrolytic platform of sPLA2.

Group IIA secreted phospholipase A2 controls skin carcinogenesis and psoriasis by shaping the gut microbiota.

Besides promoting inflammation by mobilizing lipid mediators, group IIA secreted phospholipase A2 (sPLA2-IIA) prevents bacterial infection by degrading bacterial membranes. Here, we show that, despite the restricted intestinal expression of sPLA2-IIA in BALB/c mice, its genetic deletion leads to amelioration of cancer and exacerbation of psoriasis in distal skin. Intestinal expression of sPLA2-IIA is reduced after treatment with antibiotics or under germ-free conditions, suggesting its upregulation by gut microbiota. Metagenome, transcriptome, and metabolome analyses have revealed that sPLA2-IIA deficiency alters the gut microbiota, accompanied by notable changes in the intestinal expression of genes related to immunity and metabolism, as well as in the levels of various blood metabolites and fecal bacterial lipids, suggesting that sPLA2-IIA contributes to shaping of the gut microbiota. The skin phenotypes in Pla2g2a-/- mice are lost (a) when they are cohoused with littermate WT mice, resulting in the mixing of the microbiota between the genotypes, or (b) when they are housed in a more stringent pathogen-free facility, where Pla2g2a expression in WT mice is low and the gut microbial compositions in both genotypes are nearly identical. Thus, our results highlight a potentially new aspect of sPLA2-IIA as a modulator of gut microbiota, perturbation of which affects distal skin responses.

Autophagy Is Required for Maturation of Surfactant-Containing Lamellar Bodies in the Lung and Swim Bladder.

Autophagy is an intracellular degradation system, but its physiological functions in vertebrates are not yet fully understood. Here, we show that autophagy is required for inflation of air-filled organs: zebrafish swim bladder and mouse lung. In wild-type zebrafish swim bladder and mouse lung type II pulmonary epithelial cells, autophagosomes are formed and frequently fuse with lamellar bodies. The lamellar body is a lysosome-related organelle that stores a phospholipid-containing surfactant complex that lines the air-liquid interface and reduces surface tension. We find that autophagy is critical for maturation of the lamellar body. Accordingly, atg-deficient zebrafish fail to maintain their position in the water, and type-II-pneumocyte-specific Fip200-deficient mice show neonatal lethality with respiratory failure. Autophagy suppression does not affect synthesis of the surfactant phospholipid, suggesting that autophagy supplies lipids and membranes to lamellar bodies. These results demonstrate an evolutionarily conserved role of autophagy in lamellar body maturation.

Secreted Phospholipase PLA2G2D Contributes to Metabolic Health by Mobilizing ω3 Polyunsaturated Fatty Acids in WAT.

Polyunsaturated fatty acids (PUFAs) confer health benefits by preventing inflammation and obesity and by increasing thermogenesis in brown and beige adipocytes. As well as being supplied exogenously as nutrients, PUFAs are largely stored in membrane glycerophospholipids and released by phospholipase A2s (PLA2s). However, the molecular identity of the PLA2 subtype(s) that supplies endogenous PUFAs for metabolic homeostasis remains unclear. Here we show that PLA2G2D, a secreted PLA2 isoform, is constitutively expressed in M2-type macrophages in white adipose tissue (WAT) and shows a reciprocal correlation with obesity. Studies using global and macrophage-specific Pla2g2d-deficient mice reveal that PLA2G2D increases energy expenditure and thermogenesis by facilitating adipocyte browning, thereby ameliorating diet-induced obesity, insulin resistance, and WAT inflammation. Mechanistically, PLA2G2D constitutively supplies a pool of PUFAs, ω3 in particular, in WAT. Thus, our present findings underscore the contribution of the macrophage-driven PLA2G2D-ω3 PUFA axis to metabolic health.

Activation of inflammation and resolution pathways of lipid mediators in synovial fluid from patients with severe rheumatoid arthritis compared with severe osteoarthritis.

BACKGROUND: The upregulation of the cyclooxygenase and lipoxygenase pathways of arachidonic acid is thought to be involved in the development of rheumatoid arthritis. Recently, the presence of specialized pro-resolving lipid mediators in synovial tissues from patients with osteoarthritis has been reported. OBJECTIVE: To clarify the quantitative and qualitative changes in lipid mediators in the synovium of severe rheumatoid arthritis patients, we compared the profiles of lipid mediators in synovial fluid obtained from patients with severe rheumatoid arthritis and from those with severe osteoarthritis. METHODS: We enrolled 18 patients with rheumatoid arthritis and 26 patients with osteoarthritis. All the patients had undergone total knee replacement surgery. Synovial fluid samples had been obtained during the surgery. Lipid profiling in the synovial fluid from these patients was performed using liquid chromatography-tandem mass spectrometry/mass spectrometry. RESULTS: Among the 150 oxidized fatty acids examined so far, 119 were substantially detected in synovial fluid from the patients. Not only the concentrations of pro-inflammatory lipid mediators such as prostaglandins and leukotrienes, but also those of specialized pro-resolving lipid mediators such as lipoxins, resolvins, and protectin D1 were significantly higher in synovial fluid obtained from rheumatoid arthritis patients than from synovial fluid obtained from osteoarthritis patients. CONCLUSION: The activation of both inflammation and resolution pathways of lipid mediators might be a fatty acid signature in the synovial fluid of patients with severe rheumatoid arthritis. Inflammatory, anti-inflammatory and pro-resolving mediators in synovial fluid could be good biomarkers for differentiating between severe rheumatoid arthritis and severe osteoarthritis.

Red-kerneled rice proanthocyanidin inhibits arachidonate 5-lipoxygenase and decreases psoriasis-like skin inflammation.

5-lipoxygenase is a key enzyme in the synthesis of leukotrienes from arachidonic acid. The produced leukotrienes are involved in inflammatory diseases including psoriasis, asthma, and atherosclerosis. A suitable 5-lipoxygenase inhibitor might be useful for preventing and improving the symptoms of leukotriene-related inflammatory diseases. Here, we investigate the mechanism underlying the anti-inflammatory effect of a proanthocyanidin found in red-kerneled rice. Red-kerneled rice proanthocyanidin exhibited potent mixed noncompetitive inhibition of human and rat 5-lipoxygenases, with an IC50 values of 15.1 µM against human enzyme, and 7.0 µM against rat enzyme, respectively. This compound decreased leukotriene B4 production in rat basophilic leukemia-2H3 cells. In imiquimod-induced psoriasis-like mouse skin, topical application of the proanthocyanidin suppressed hyperplasia, decreased inflammatory cell infiltration, and down-regulated expression of the psoriasis-associated genes Il17a, Il22, S100a9, and Krt1. Lipid metabolome analysis by electrospray ionization mass spectrometry showed that red-kerneled rice proanthocyanidin treatment of psoriasis-like mouse skin dose-dependently decreased the production of leukotriene B4 but no other arachidonate metabolites. Red-kerneled rice proanthocyanidin inhibits 5-lipoxygenase, resulting in a decrease in leukotriene B4 production and psoriasis-like mouse skin inflammation. These results suggest that this proanthocyanidin may be therapeutically effective for treating leukotriene-related diseases.

Preventive effect of Dioscorea japonica on squamous cell carcinoma of mouse skin involving down-regulation of prostaglandin E2 synthetic pathway.

Hyperproduced prostaglandin E2 by cyclooxygenase-2 and microsomal prostaglandin E synthase-1 evokes several pathophysiological responses such as inflammation and carcinogenesis. Our recent study demonstrated that Dioscorea japonica extract suppressed the expression of cyclooxygenase-2 and microsomal prostaglandin E synthase-1 and induced apoptosis in lung carcinoma A549 cells. In the present study, we investigated the effects of Dioscorea japonica on squamous cell carcinoma of mouse skin. Dioscorea japonica feeding and Dioscorea japonica extract topical application suppressed the expression of cyclooxygenase-2, microsomal prostaglandin E synthase-1, interleukin-1ß and interleukin-6 and inhibited tumor formation, hyperplasia and inflammatory cell infiltration. Immunohistochemical analyses showed the immunoreactivities of cyclooxygenase-2 and microsomal prostaglandin E synthase-1 in tumor keratinocytes and stronger immunoreactivities of cyclooxygenase-2 and hematopoietic prostaglandin D synthase in epidermal dendritic cells (Langerhans cells). Treatment with Dioscorea japonica decreased the immunoreactivity of cyclooxygenase-2 and microsomal prostaglandin E synthase-1. These results indicate that Dioscorea japonica may have inhibitory effects on inflammation and carcinogenesis via suppression of the prostaglandin E2 synthetic pathway.

Group III phospholipase A2 promotes colitis and colorectal cancer.

Lipid mediators play pivotal roles in colorectal cancer and colitis, but only a limited member of the phospholipase A2 (PLA2) subtypes, which lie upstream of various lipid mediators, have been implicated in the positive or negative regulation of these diseases. Clinical and biochemical evidence suggests that secreted PLA2 group III (sPLA2-III) is associated with colorectal cancer, although its precise role remains obscure. Here we have found that sPLA2-III-null (Pla2g3 -/-) mice are highly resistant to colon carcinogenesis. Furthermore, Pla2g3 -/- mice are less susceptible to dextran sulfate-induced colitis, implying that the amelioration of colonic inflammation by sPLA2-III ablation may underlie the protective effect against colon cancer. Lipidomics analysis of the colon revealed significant reduction of pro-inflammatory/pro-tumorigenic lysophosholipids as well as unusual steady-state elevation of colon-protective fatty acids and their oxygenated metabolites in Pla2g3 -/- mice. Overall, our results establish a role of sPLA2-III in the promotion of colorectal inflammation and cancer, expand our understanding of the divergent roles of multiple PLA2 enzymes in the gastrointestinal tract, and point to sPLA2-III as a novel druggable target for colorectal diseases.

Dual Roles of Group IID Phospholipase A2 in Inflammation and Cancer.

Phospholipase A2 enzymes have long been implicated in the promotion of inflammation by mobilizing pro-inflammatory lipid mediators, yet recent evidence suggests that they also contribute to anti-inflammatory or pro-resolving programs. Group IID-secreted phospholipase A2 (sPLA2-IID) is abundantly expressed in dendritic cells in lymphoid tissues and resolves the Th1 immune response by controlling the steady-state levels of anti-inflammatory lipids such as docosahexaenoic acid and its metabolites. Here, we show that psoriasis and contact dermatitis were exacerbated in Pla2g2d-null mice, whereas they were ameliorated in Pla2g2d-overexpressing transgenic mice, relative to littermate wild-type mice. These phenotypes were associated with concomitant alterations in the tissue levels of ω3 polyunsaturated fatty acid (PUFA) metabolites, which had the capacity to reduce the expression of pro-inflammatory and Th1/Th17-type cytokines in dendritic cells or lymph node cells. In the context of cancer, however, Pla2g2d deficiency resulted in marked attenuation of skin carcinogenesis, likely because of the augmented anti-tumor immunity. Altogether, these results underscore a general role of sPLA2-IID as an immunosuppressive sPLA2 that allows the microenvironmental lipid balance toward an anti-inflammatory state, exerting beneficial or detrimental impact depending upon distinct pathophysiological contexts in inflammation and cancer.

Expression and Function of Group IIE Phospholipase A2 in Mouse Skin.

Recent studies using knock-out mice for various secreted phospholipase A2 (sPLA2) isoforms have revealed their non-redundant roles in diverse biological events. In the skin, group IIF sPLA2 (sPLA2-IIF), an "epidermal sPLA2" expressed in the suprabasal keratinocytes, plays a fundamental role in epidermal-hyperplasic diseases such as psoriasis and skin cancer. In this study, we found that group IIE sPLA2 (sPLA2-IIE) was expressed abundantly in hair follicles and to a lesser extent in basal epidermal keratinocytes in mouse skin. Mice lacking sPLA2-IIE exhibited skin abnormalities distinct from those in mice lacking sPLA2-IIF, with perturbation of hair follicle ultrastructure, modest changes in the steady-state expression of a subset of skin genes, and no changes in the features of psoriasis or contact dermatitis. Lipidomics analysis revealed that sPLA2-IIE and -IIF were coupled with distinct lipid pathways in the skin. Overall, two skin sPLA2s, hair follicular sPLA2-IIE and epidermal sPLA2-IIF, play non-redundant roles in distinct compartments of mouse skin, underscoring the functional diversity of multiple sPLA2s in the coordinated regulation of skin homeostasis and diseases.

The role of group IIF-secreted phospholipase A2 in epidermal homeostasis and hyperplasia.

Epidermal lipids are important for skin homeostasis. However, the entire picture of the roles of lipids, particularly nonceramide lipid species, in epidermal biology still remains obscure. Here, we report that PLA2G2F, a functionally orphan-secreted phospholipase A2 expressed in the suprabasal epidermis, regulates skin homeostasis and hyperplasic disorders. Pla2g2f(-/-) mice had a fragile stratum corneum and were strikingly protected from psoriasis, contact dermatitis, and skin cancer. Conversely, Pla2g2f-overexpressing transgenic mice displayed psoriasis-like epidermal hyperplasia. Primary keratinocytes from Pla2g2f(-) (/-) mice showed defective differentiation and activation. PLA2G2F was induced by calcium or IL-22 in keratinocytes and preferentially hydrolyzed ethanolamine plasmalogen-bearing docosahexaenoic acid secreted from keratinocytes to give rise to unique bioactive lipids (i.e., protectin D1 and 9S-hydroxyoctadecadienoic acid) that were distinct from canonical arachidonate metabolites (prostaglandins and leukotrienes). Ethanolamine lysoplasmalogen, a PLA2G2F-derived marker product, rescued defective activation of Pla2g2f(-/-) keratinocytes both in vitro and in vivo. Our results highlight PLA2G2F as a previously unrecognized regulator of skin pathophysiology and point to this enzyme as a novel drug target for epidermal-hyperplasic diseases.

The adipocyte-inducible secreted phospholipases PLA2G5 and PLA2G2E play distinct roles in obesity.

Metabolic disorders, including obesity and insulin resistance, have their basis in dysregulated lipid metabolism and low-grade inflammation. In a microarray search of unique lipase-related genes whose expressions are associated with obesity, we found that two secreted phospholipase A2s (sPLA2s), PLA2G5 and PLA2G2E, were robustly induced in adipocytes of obese mice. Analyses of Pla2g5(-/-) and Pla2g2e(-/-) mice revealed distinct roles of these sPLA2s in diet-induced obesity. PLA2G5 hydrolyzed phosphatidylcholine in fat-overladen low-density lipoprotein to release unsaturated fatty acids, which prevented palmitate-induced M1 macrophage polarization. As such, PLA2G5 tipped the immune balance toward an M2 state, thereby counteracting adipose tissue inflammation, insulin resistance, hyperlipidemia, and obesity. PLA2G2E altered minor lipoprotein phospholipids, phosphatidylserine and phosphatidylethanolamine, and moderately facilitated lipid accumulation in adipose tissue and liver. Collectively, the identification of "metabolic sPLA2s" adds this gene family to a growing list of lipolytic enzymes that act as metabolic coordinators.

Lymphoid tissue phospholipase A2 group IID resolves contact hypersensitivity by driving antiinflammatory lipid mediators.

Resolution of inflammation is an active process that is mediated in part by antiinflammatory lipid mediators. Although phospholipase A2 (PLA2) enzymes have been implicated in the promotion of inflammation through mobilizing lipid mediators, the molecular entity of PLA2 subtypes acting upstream of antiinflammatory lipid mediators remains unknown. Herein, we show that secreted PLA2 group IID (PLA2G2D) is preferentially expressed in CD11c(+) dendritic cells (DCs) and macrophages and displays a pro-resolving function. In hapten-induced contact dermatitis, resolution, not propagation, of inflammation was compromised in skin and LNs of PLA2G2D-deficient mice (Pla2g2d(-/-)), in which the immune balance was shifted toward a proinflammatory state over an antiinflammatory state. Bone marrow-derived DCs from Pla2g2d(-/-) mice were hyperactivated and elicited skin inflammation after intravenous transfer into mice. Lipidomics analysis revealed that PLA2G2D in the LNs contributed to mobilization of a pool of polyunsaturated fatty acids that could serve as precursors for antiinflammatory/pro-resolving lipid mediators such as resolvin D1 and 15-deoxy-Δ(12,14)-prostaglandin J2, which reduced Th1 cytokine production and surface MHC class II expression in LN cells or DCs. Altogether, our results highlight PLA2G2D as a "resolving sPLA2" that ameliorates inflammation through mobilizing pro-resolving lipid mediators and points to a potential use of this enzyme for treatment of inflammatory disorders.

Evaluation of a new rehabilitation program for postoperative patients with breast cancer.

The purpose of the present study was to evaluate the efficacy of a newly designed rehabilitation program for postoperative patients with breast cancer. This was achieved through examinations to clarify the range of motion (ROM) of the shoulder joint, postoperative pain and recovery of activities of daily living (ADL). The ROM of the shoulder joint was evaluated with respect to five items and determinations were carried out five times. Patients were orally questioned four times regarding their pain after surgery, movement-associated chest pain, pain at night and operative wound pain. Evaluation of the ADL after discharge was conducted using a three-level rating method at postoperative week 4 and week 12. A total of 72 patients were studied, comprising 39 who underwent pectoral muscle-conserving mastectomy and 33 who underwent breast-conserving surgery. The arm movement of forward raising showed the greatest decline, followed by lateral raising of the arm. Postoperative time-course changes in internal/external arm rotation, as well as backward arm raising were slight. Comparison of the operative procedures revealed differences only for lateral arm raising at postoperative week 4, while there were no differences in any of the shoulder joint movements. Lowering of the ROM of the shoulder joint was more marked at an earlier time (week 1 to week 2) after surgery, but it was not statistically significant. Pain at night and operative wound pain were prolonged over the period, and reported in postoperative week 1 to week 12 by 3-15% of patients. All ADL items became almost normal in approximately 90% of patients at postoperative week 4. The efficacy of the present rehabilitation program early after surgery was demonstrated in terms of recovery of ADL as well as the ROM of the arm on the affected side.