Lipid-mediated Signaling Research Articles

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.

A Peptide-Based Fluorescent Sensor for Anionic Phospholipids.

Anionic phospholipids are key cell signal mediators. The distribution of these lipids on the cell membrane and intracellular organelle membranes guides the recruitment of signaling proteins leading to the regulation of cellular processes. Hence, fluorescent sensors that can detect anionic phospholipids within living cells can provide a handle into revealing molecular mechanisms underlying lipid-mediated signal regulation. A major challenge in the detection of anionic phospholipids is related to the presence of these phospholipids mostly in the inner leaflet of the plasma membrane and in the membranes of intracellular organelles. Hence, cell-permeable sensors would provide an advantage by enabling the rapid detection and tracking of intracellular pools of anionic phospholipids. We have developed a peptide-based, cell-permeable, water-soluble, and ratiometric fluorescent sensor that entered cells within 15 min of incubation via the endosomal machinery and showed punctate labeling in the cytoplasm. The probe could also be introduced into living cells via lipofection, which allows bypassing of endosomal uptake, to image anionic phospholipids in the cell membrane. We validated the ability of the sensor toward detection of intracellular anionic phospholipids by colocalization studies with a fluorescently tagged lipid and a protein-based anionic phospholipid sensor. Further, the sensor could image the externalization of anionic phospholipids during programmed cell death, indicating the ability of the probe toward detection of both intra- and extracellular anionic phospholipids based on the biological context.

Transcriptome reveals key microRNAs involved in fat deposition between different tail sheep breeds.

MicroRNA (miRNA) is a kind of noncoding RNA whose function involved in various biological processes in neuronal maturation and adipocyte cells, such as differentiation, proliferation, development, apoptosis, and metabolism. Herein, miRNA-Seq was used to identify miRNAs in the tail fat tissue of Hu sheep (short-fat-tailed) and Tibetan sheep (short-thin-tailed). In this study, 155 differentially expression miRNAs (DE miRNAs) were identified, including 78 up-regulated and 77 down-regulated. Among these DE miRNAs, 17 miRNAs were reported and related with lipid metabolism. MiRanda and RNAhybrid software were used to predict the target genes of DE miRNAs, obtaining the number of targeting relationships is 38553. Target genes were enriched by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). 742 terms and 302 single pathways are enriched, including lipid metabolic process, response to lipid, cellular lipid catabolic process, lipid catabolic process, cellular lipid metabolic process, inositol lipid-mediated signaling, calcium channel activity, PI3K-Akt signaling pathway, MAPK signaling pathway, ECM-receptor interaction, AMPK signaling pathway, Wnt signaling pathway and TGF-beta signaling pathway. Notably, miR-379-5p was associated with tail fat deposition of sheep. Dual-Luciferase reporter assays showed miR-379-5p and HOXC9 had targeted relationship. The result of RT-qPCR showed that the expression trend of miR-379-5p and HOXC9 was opposite. miR-379-5p was down-regulated and highly expressed in tail adipose tissue of Tibetan sheep. HOXC9 was highly expressed in adipose tissue of Hu sheep. These results could provide a meaningful theoretical basis for studying the molecular mechanisms of sheep tail adipogenesis.

Selective brain regional changes in lipid profile with human aging

Fatty acids are key components in the structural diversity of lipids and play a strategic role in the functional properties of lipids which determine the integrity of neuronal and glial cell membranes, the generation of lipid signaling mediators, and the chemical reactivity of acyl chains. The present study analyzes using gas chromatography the fatty acid profiles of 13 regions of the human central nervous system in healthy individuals ranging from 40 to 80 years old. The outcomes suggest the existence of general traits in fatty acid composition such as an average chain length of 18 carbon atoms, high monounsaturated fatty acid content, and predominance in polyunsaturated fatty acids of those of series n-6 over series n-3 which are shared by all brain regions regardless of age. Our results also show a general sustained and relatively well-preserved lipid profile throughout the adult lifespan in most studied regions (olive, upper vermis, substantia nigra, thalamus, hippocampus, putamen, caudate, occipital cortex, parietal cortex, entorhinal cortex, and frontal cortex) with minor changes that are region-dependent. In contrast, of particular relevance is the involvement of the inferior temporal cortex and cingulate cortex. It is proposed that during normal human brain aging, the lipid profile is resistant to changes with age in most human brain regions to ensure cell survival and function, but some particular regions involved in specific memory domains are greatly affected.

Tri-2-Hydroxyarachidonein Induces Cytocidal Autophagy in Pancreatic Ductal Adenocarcinoma Cancer Cell Models.

Cell proliferation in pancreatic cancer is determined by a complex network of signaling pathways. Despite the extensive understanding of these protein-mediated signaling processes, there are no significant drug discoveries that could considerably improve a patient’s survival. However, the recent understanding of lipid-mediated signaling gives a new perspective on the control of the physiological state of pancreatic cells. Lipid signaling plays a major role in the induction of cytocidal autophagy and can be exploited using synthetic lipids to induce cell death in pancreatic cancer cells. In this work, we studied the activity of a synthetic lipid, tri-2-hydroxyarachidonein (TGM4), which is a triacylglycerol mimetic that contains three acyl moieties with four double bonds each, on cellular and in vivo models of pancreatic cancer. We demonstrated that TGM4 inhibited proliferation of Mia-PaCa-2 (human pancreatic carcinoma) and PANC-1 (human pancreatic carcinoma of ductal cells) in in vitro models and in an in vivo xenograft model of Mia-PaCa-2 cells. In vitro studies demonstrated that TGM4 induced cell growth inhibition paralleled with an increased expression of PARP and CHOP proteins together with the presence of sub-G0 cell cycle events, indicating cell death. This cytocidal effect was associated with elevated ER stress or autophagy markers such as BIP, LC3B, and DHFR. In addition, TGM4 activated peroxisome proliferator-activated receptor gamma (PPAR-γ), which induced elevated levels of p-AKT and downregulation of p-c-Jun. We conclude that TGM4 induced pancreatic cell death by activation of cytocidal autophagy. This work highlights the importance of lipid signaling in cancer and the use of synthetic lipid structures as novel and potential approaches to treat pancreatic cancer and other neoplasias.

Role of the Endocannabinoid System in the Regulation of Intestinal Homeostasis.

The maintenance of intestinal homeostasis is fundamentally important to health. Intestinal barrier function and immune regulation are key determinants of intestinal homeostasis and are therefore tightly regulated by a variety of signaling mechanisms. The endocannabinoid system is a lipid mediator signaling system widely expressed in the gastrointestinal tract. Accumulating evidence suggests the endocannabinoid system is a critical nexus involved in the physiological processes that underlie the control of intestinal homeostasis. In this review we will illustrate how the endocannabinoid system is involved in regulation of intestinal permeability, fluid secretion, and immune regulation. We will also demonstrate a reciprocal regulation between the endocannabinoid system and the gut microbiome. The role of the endocannabinoid system is complex and multifaceted, responding to both internal and external factors while also serving as an effector system for the maintenance of intestinal homeostasis.

Characterization of the Glehnia littoralis Non-specific Phospholipase C Gene GlNPC3 and Its Involvement in the Salt Stress Response.

Glehnia littoralis is a medicinal halophyte that inhabits sandy beaches and has high ecological and commercial value. However, the molecular mechanism of salt adaptation in G. littoralis remains largely unknown. Here, we cloned and identified a non-specific phospholipase C gene (GlNPC3) from G. littoralis, which conferred lipid-mediated signaling during the salt stress response. The expression of GlNPC3 was induced continuously by salt treatment. Overexpression of GlNPC3 in Arabidopsis thaliana increased salt tolerance compared to wild-type (WT) plants. GlNPC3-overexpressing plants had longer roots and higher fresh and dry masses under the salt treatment. The GlNPC3 expression pattern revealed that the gene was expressed in most G. littoralis tissues, particularly in roots. The subcellular localization of GlNPC3 was mainly at the plasma membrane, and partially at the tonoplast. GlNPC3 hydrolyzed common membrane phospholipids, such as phosphotidylserine (PS), phosphoethanolamine (PE), and phosphocholine (PC). In vitro enzymatic assay showed salt-induced total non-specific phospholipase C (NPC) activation in A. thaliana GlNPC3-overexpressing plants. Plant lipid profiling showed a significant change in the membrane-lipid composition of A. thaliana GlNPC3-overexpressing plants compared to WT after the salt treatment. Furthermore, downregulation of GlNPC3 expression by virus-induced gene silencing in G. littoralis reduced the expression levels of some stress-related genes, such as SnRK2, P5SC5, TPC1, and SOS1. Together, these results indicated that GlNPC3 and GlNPC3-mediated membrane lipid change played a positive role in the response of G. littoralis to a saline environment.

Lipolysis: cellular mechanisms for lipid mobilization from fat stores.

The perception that intracellular lipolysis is a straightforward process that releases fatty acids from fat stores in adipose tissue to generate energy has experienced major revisions over the last two decades. The discovery of new lipolytic enzymes and coregulators, the demonstration that lipophagy and lysosomal lipolysis contribute to the degradation of cellular lipid stores and the characterization of numerous factors and signalling pathways that regulate lipid hydrolysis on transcriptional and post-transcriptional levels have revolutionized our understanding of lipolysis. In this review, we focus on the mechanisms that facilitate intracellular fatty-acid mobilization, drawing on canonical and noncanonical enzymatic pathways. We summarize how intracellular lipolysis affects lipid-mediated signalling, metabolic regulation and energy homeostasis in multiple organs. Finally, we examine how these processes affect pathogenesis and how lipolysis may be targeted to potentially prevent or treat various diseases.

Deciphering the Dynamics of Signaling Cascades and Virulence Factors of B. cinerea during Tomato Cell Wall Degradation.

The ascomycete Botrytis cinerea is one of the most relevant plant pathogenic fungi, affecting fruits, flowers, and greenhouse-grown crops. The infection strategy used by the fungus comprises a magnificent set of tools to penetrate and overcome plant defenses. In this context, the plant-pathogen communication through membrane receptors and signal transduction cascades is essential to trigger specific routes and the final success of the infection. In previous reports, proteomics approaches to B. cinerea signal transduction cascades changes in response to different carbon source and plant-based elicitors have been performed. Analyzing the secretome, membranome, phosphoproteome, and the phosphomembranome. Moreover, phenotypic changes in fungal biology was analyzed, specifically toxin production. To obtain the whole picture of the process and reveal the network from a system biology approach, this proteomic information has been merged with the phenotypic characterization, to be analyzed using several bioinformatics algorithms (GO, STRING, MCODE) in order to unravel key points in the signal transduction regulation crucial to overcome plant defenses, as well as new virulence/pathogenicity factors that could be used as therapeutic targets in the control of the gray mold rot disease. A total of 1721 and 663 exclusive or overexpressed proteins were identified under glucose (GLU) and deproteinized tomato cell walls (TCW), summarizing all of the protein identifications under phenotypic characterized stages. Under GO analysis, there are more biological process and molecular functions described in GLU, highlighting the increase in signaling related categories. These results agree with the high number of total identified proteins in GLU, probably indicating a more varied and active metabolism of the fungus. When analyzing only GO annotations related with signal transduction, it was revealed that there were proteins related to TOR signaling, the phosphorelay signal transduction system, and inositol lipid-mediated signaling, only under GLU conditions. On the contrary, calcium-mediated signaling GO annotation is only present between the proteins identified under TCW conditions. To establish a potential relationship between expressed proteins, cluster analyses showed 41 and 14 clusters under GLU and TCW conditions, confirming an increase in biological activity in GLU, where we identified a larger number of clusters related to transcription, translation, and cell division, between others. From these analyses, clusters related to signal transduction and clusters related to mycotoxin production were found, which correlated with the phenotypic characterization. The identification of the proteins encompassed in each condition and signal transduction cascade would provide the research community with new information about the B. cinerea infection process and potential candidates of pathogenicity/virulence factors, overcoming plant defenses, and new therapeutic targets.

Abstract 2326: Differential lipid metabolism in mammary cancer cell progression

Abstract There is an association between increased lipid accumulation and aggressive breast cancer phenotypes, and therefore poorer clinical outcomes. The mechanism by which dysregulated lipid metabolism may promote metastasis remains incompletely understood. Several potential mechanisms by which the neutral lipid storage organelles, cytoplasmic lipid droplets, may promote metastasis include utilization of fatty acids (FAs) for oxidation, protection against lipotoxcity, and production of lipid signaling mediators. We have previously demonstrated that metastatic MCF10CA1a mammary cancer cells rely on fatty acid oxidation (FAO) compared to non-metastatic MCF10A-ras cells, using etomoxir to inhibit the rate-limiting enzyme of FAO, carnitine palmitoyltransferase 1 (CPT1). Previous literature indicates that there is an increase in various lipid metabolism pathways, including FA uptake, de novo lipogenesis, and FA storage in neutral lipid. We found that there is no significant difference between palmitic acid uptake between the non-metastatic and metastatic cells. In the current study, we show that MCF10CA1a cells have significantly higher levels of carbon incorporation into palmitic and oleic FAs from 13C-acetate (25% and 20% greater), 13C-glucose (21% and 11% greater), and 13C-lactate (29% and 22% greater) respectively, compared to the MCF10A-ras cells, as detected by LC-MS/MS. In order to determine the source of FAs that contributes to the migration-dependent FAO, we assessed MCF10CA1a cell migration following inhibition of lipolysis (adipose triglyceride lipase—ATGL; ATGListatin) alone or in combination with the FAO inhibitor, etomoxir. We determined that inhibition of ATGL, the initial enzyme of triglyceride lipolysis pathway, significantly reduces MCF10CA1a cell migration (20% decrease compared to vehicle). Inhibition of FAO and lipolysis did not induce additional reduction in cell migration. We also showed that MCF10CA1a cells are more dependent on FA metabolism than the non-metastatic MCF10A-ras cells, demonstrated by MCF10CA1a cells having a greater decrease in cell viability compared to the MCF10A-ras cells given each FA metabolism inhibition (p<0.05: 22% greater decrease for FAO inhibition and 16% greater decrease for dual FAO and lipolysis inhibition, p=0.05: 17% greater decrease for FA synthesis inhibition). These data suggest that metastatic MCF10CA1a cells rely on their increased FA metabolism to survive and migrate, and specifically that the catabolism of FAs from triglyceride supports the FAO-driven migration of metastatic cancer cells. Citation Format: Chaylen Andoline, Parik Pawar, Dorothy Teegarden. Differential lipid metabolism in mammary cancer cell progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2326.

Acute Knockdown of MG29 in Mouse Muscle Cells Reveals Signaling Mechanisms Associated with Polyunsaturated Fatty Acid (PUFA) – Implications for Sarcopenia

Estimates of humans with sarcopenia (i.e., the age related loss of muscle loss and strength) ranges from 30% to over 70% depending on the age group, and specific population studied. Mitsugumin 29 (MG29), a muscle-specific protein, is a member of the synaptophysin-like protein family (SYPL2) that contain MAL and related proteins for vesicle trafficking and membrane link (MARVEL) domains. MG29 is important for the formation of the transverse-tubule (TT) system in skeletal muscles, efficient signaling between RyR1 and the store-operated Ca2+ entry (SOCE) machinery, and maintenance of lipid content of the sarcolemmal membrane. Our previous in vivo studies using a global knockout model of MG29 suggested a role of MG29 in the aging decline in muscle function. To avoid potential adaptive effects of the global absence of MG29, we also tested the siRNA-mediated acute knockdown of MG29 in mouse primary skeletal muscle cells, which led to extensive myogenic changes characterized by increased myogenic differentiation that led to abnormally thinner and longer myotubes. To examine the mechanistic basis of these changes, we measured changes in lipid signaling following knockdown of MG29. We collected both cell culture medium and cell homogenates 72 h after siRNA transfection, and applied a custom-targeted LC/MS lipidomics approach to quantify specific Lipid Signaling Mediators (LMs) in cell culture media and we used our customized skeletal muscle-specific RT-qPCR gene array to investigate specific genes showing alteration expression following acute MG29 knockdown. Compared with negative control siRNA treated cells, in MG29 siRNA treated cells, the cell culture media levels of arachidonoylethanolamine (AEA), oleoylethanolamide (OEA) increased significantly (p < 0.05) and the levels of prostaglandin D2 (PGD2), 9,10-DiHOME, 9-HOTrE, 9-HODE decreased significantly (p < 0.05). In addition, the PCR gene array revealed that in MG29 siRNA treated cells the expression of Cacna1s, RyR3, Btk and Sod3 increased 2.6 ± 0.48, 4.5 ± 0.89, 2.7 ± 0.51 and 6.5 ± 1.06 fold, respectively, while Fkbp1b and Ccl2 decreased 2.8 ± 0.38 and 7.3 ± 1.74 fold, respectively. These genes have function in calcium signaling and homeostasis and oxidative stress as well as inflammation and immune-regulation. We conclude that MG29 modulation of myogenic differentiation occurs via a complex signaling mechanism related to changes in LMs derived from polyunsaturated fatty acid (PUFA). This study further positions MG29 as an important regulator of signaling pathways associated with intracellular calcium homeostasis, oxidative stress, and inflammation, which emphasizes the role of MG29 as an orchestrator of alterations in skeletal muscle during sarcopenia.

Real Time Monitoring of Transient Lipid-Lipid Interactions on Live Cell Membranes using a DNA Probe

Cell membrane is an intriguing, dynamic and complex structure. Preferential interactions among lipids and proteins promote the lateral heterogeneity and segregation in the membrane plane. This membrane heterogeneity influences the spatial and temporal distributions of signaling molecules and receptors, which further support localized signaling. Understanding and modulating membrane heterogeneity is critical for regulating cell signaling and interactions. There are still many mysteries surrounding cell membranes, especially those pertaining to lipid-mediated membrane interactions and signaling pathways.

Membrane Deformation Modulated by Hydration and Cholesterol Unveiled by Solid-State 2H NMR Spectroscopy

Phospholipid membranes are biological liquid crystals in which intermolecular interactions at mesoscopic length scales play key roles in the emergence of membrane physical properties like structure and rigidity. Such interactions modulated by membrane hydration [1] and cholesterol [2] have been investigated using solid-state 2H NMR spectroscopy. The average structure is manifested by the lineshapes through principal values of static or residual coupling tensors due to quadrupolar interactions. The residual quadrupolar couplings (RQCs) yield segmental order parameters for the individual acyl segments (i), providing average membrane properties such as bilayer thickness, area/lipid, and spontaneous curvature [3]. We show that membrane dehydration and increased cholesterol content result in greater RQCs, indicative of membrane thickening. Elastic membrane deformations are exhibited as collective order-director fluctuations (ODF) contributing to the nuclear magnetic relaxation. At mesoscopic length scales the thermal fluctuations connect to membrane mechanical properties via the fluctuation-dissipation theorem. Model-free interpretation of the functional dependence of spin-lattice relaxation rates, R1Z, on segmental order parameters (square-law) reveals emergent material properties in the liquid-crystalline state. The R1Z relaxation dispersion and temperature dependence of square-law plots indicate a nematic-like membrane hydrocarbon core. Solid-state 2H NMR relaxation studies demonstrate that both dehydration and increased cholesterol content in the membrane lead to decreased slopes of square-law plots, indicating an increase in membrane bending rigidity, consistent with neutron spin-echo (NSE) measurements. Analogous 2H NMR studies with various membrane lipid compositions and peptide-reconstituted membranes have addressed lipid-mediated cell signaling and lipid-protein interactions. Molecular dynamics simulations provide further insights on the correspondence of the solid-state NMR and NSE results. [1] K.J. Mallikarjunaiah et al. (2019) Phys. Chem. Chem. Phys. 21, 18422. [2] S. Chakraborty et al. (2020) Proc. Natl. Acad. Sci. 117, 21896. [3] Mallikarjunaiah et al. (2011) Biophys. J. 100, 98.

Revealing mechanism of Caulis Sargentodoxae for the treatment of ulcerative colitis based on network pharmacology approach.

Objective: The traditional Chinese medicine Caulis Sargentodoxae is widely used in the treatment of ulcerative colitis (UC), but the mechanism remains unknown. The present study aims to reveal its effective components, targets and pathways through network pharmacology and bioinformatics approaches.Materials and methods: Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) was used to identify effective components. The ligand-based targets prediction was achieved through SwissTargetPrediction and TargetNet. UC-related targets were identified using Gene Expression Omnibus (GEO) data and DisGeNET. The common targets of disease and components were constructed and analyzed by PPI network. Lastly, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses are used to explain the functions of these common targets. Components-Targets-Pathways network was visualized and analyzed to further reveal the connection between the components and targets.Results: Eight active components and 102 key targets were identified to play an important role in UC. These targets were related to regulation of protein serine/threonine kinase activity, positive regulation of cell motility, response to molecule of bacterial origin, response to toxic substance, ERK1 and ERK2 cascade, peptidyl-tyrosine modification, inositol lipid-mediated signaling, cellular response to drug, regulation of inflammatory response and leukocyte migration. Moreover, HIF-1 signaling pathway and PI3K-Akt signaling pathway were the key targets involved in UC-related signaling pathways.Conclusion: The eight active components of Caulis Sargentodoxae mainly play a therapeutic role for UC through synergistic regulation of HIF-1 signaling pathway and PI3K-Akt signaling pathway.

Lipid-Orchestrated Acceleration of Epstein-Barr Virus-Induced B-Cell Lymphoma Via the Secreted Phospholipase A &lt;sub&gt;2&lt;/sub&gt;-Mediated&amp;nbsp;Modification of Exosomes

Exosomes act as intercellular communicators by transferring protein and microRNA cargoes, yet the role of exosome lipids remains unclear. Here, we show that the pro-tumorigenic action of lymphoma-derived exosomes is augmented via secreted phospholipase A2(sPLA2)-driven lipid metabolism. Hydrolysis of exosomal 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 exosomes were smaller, 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; treatment with sPLA2-modified exosomes reversed this phenotype. Further, sPLA2 expression in human large B-cell lymphomas inversely correlated with patient survival. Overall, the sPLA2-mediated exosome modification promotes tumor development.

Crosstalk between ORMDL3, serine palmitoyltransferase, and 5-lipoxygenase in the sphingolipid and eicosanoid metabolic pathways

Leukotrienes (LTs) and sphingolipids are critical lipid mediators participating in numerous cellular signal transduction events and developing various disorders, such as bronchial hyperactivity leading to asthma. Enzymatic reactions initiating production of these lipid mediators involve 5-lipoxygenase (5-LO)-mediated conversion of arachidonic acid to LTs and serine palmitoyltransferase (SPT)-mediated de novo synthesis of sphingolipids. Previous studies have shown that endoplasmic reticulum membrane protein ORM1-like protein 3 (ORMDL3) inhibits the activity of SPT and subsequent sphingolipid synthesis. However, the role of ORMDL3 in the synthesis of LTs is not known. In this study, we used peritoneal-derived mast cells isolated from ORMDL3 KO or control mice and examined their calcium mobilization, degranulation, NF-κB inhibitor-α phosphorylation, and TNF-α production. We found that peritoneal-derived mast cells with ORMDL3 KO exhibited increased responsiveness to antigen. Detailed lipid analysis showed that compared with WT cells, ORMDL3-deficient cells exhibited not only enhanced production of sphingolipids but also of LT signaling mediators LTB4, 6t-LTB4, LTC4, LTB5, and 6t-LTB5. The crosstalk between ORMDL3 and 5-LO metabolic pathways was supported by the finding that endogenous ORMDL3 and 5-LO are localized in similar endoplasmic reticulum domains in human mast cells and that ORMDL3 physically interacts with 5-LO. Further experiments showed that 5-LO also interacts with the long-chain 1 and long-chain 2 subunits of SPT. In agreement with these findings, 5-LO knockdown increased ceramide levels, and silencing of SPTLC1 decreased arachidonic acid metabolism to LTs to levels observed upon 5-LO knockdown. These results demonstrate functional crosstalk between the LT and sphingolipid metabolic pathways, leading to the production of lipid signaling mediators.

Extracellular Acidity-mediated Expression of cPLA2γ Confers Resistance in Gastric Cancer Cells.

Extracellular acidity, a characteristic of solid tumors, has been proposed to be a critical factor for aggravating tumor malignancy and conferring resistance to therapeutics. Recently, acidity has been implicated in inflammatory responses, which are mediated through active lipid metabolites in various human tissues. In the present study, we investigated whether acidity can affect lipid-mediated signaling, and found that phospholipase A2 (PLA2) activity increased at acidic pH in SNU601 and AGS gastric carcinoma cell lines. To identify the PLA2 isoform that is responsible for the acidity-induced activity, we assessed mRNA levels of cPLA2 isotypes through real-time qPCR, and protein levels through immunoblot assay in cells cultured in acidic medium. It was found that acidic pH conditions markedly elevated the PLA2γ expression. A gene interference study using specific siRNA of cPLA2γ suggested that expression of cPLA2γ in acidic culture conditions may be associated with protection of cancer cells in acidic environment, as shown by cell viability and clonogenic assays. In addition, expression of cPLA2γ appeared to confer cell resistance to anticancer drugs under acidic pH conditions. Acidity-induced cPLA2γ expression may exert protective effects by imparting resistance to the gastric cancer cells under acidic environment.

Therapeutic Targeting of PTEN in Duchenne Muscular Dystrophy

Therapeutic Targeting of PTEN in Duchenne Muscular Dystrophy

Chemical Genetics to Uncover Mechanisms Underlying Lipid-Mediated Signaling Events in Plants.

Like animals, plants use various lipids as signaling molecules to guide their growth and development. The focus of our work is on the N-acylethanolamine (NAE) group of lipid mediators, which have been shown to play important physiological roles in plants. However, mechanisms by which NAEs modulate plant function remain elusive. Chemical genetics has emerged as a potent tool to elucidate signaling pathways in plants, particularly those orchestrated by plant hormones. Like plant hormones, exogenous application of NAEs elicits distinct plant growth phenotypes that can serve as biological readouts for chemical genetic screens. For example, N-lauroylethanolamide (NAE 12:0) inhibits seedling development in the model plant Arabidopsis thaliana. Thus, a library of small synthetic chemical compounds can be rapidly screened for their ability to reverse the inhibitory effect of NAE 12:0 on seedling development. Chemicals identified through such screens could be potential agonists/antagonists of NAE receptors or signaling pathways and therefore serve as additional tools for understanding NAE function in plants. In this chapter, we describe general protocols for NAE 12:0-based chemical genetic screens in Arabidopsis. Although such screens were designed primarily for NAE 12:0, they could potentially be applied for similar work with other NAE species or plant lipid mediators.

Deletion of SREBF1, a Functional Bone-Muscle Pleiotropic Gene, Alters Bone Density and Lipid Signaling in Zebrafish.

Through a genome-wide analysis of bone mineral density (BMD) and muscle mass, identification of a signaling pattern on 17p11.2 recognized the presence of sterol regulatory element-binding factor 1 (SREBF1), a gene responsible for the regulation of lipid homeostasis. In conjunction with lipid-based metabolic functions, SREBF1 also codes for the protein, SREBP-1, a transcription factor known for its role in adipocyte differentiation. We conducted a quantitative correlational study. We established a zebrafish (ZF) SREBF1 knockout (KO) model and used a targeted customized lipidomics approach to analyze the extent of SREBF1 capabilities. For lipidomics profiling, we isolated the dorsal muscles of wild type (WT) and KO fishes, and we performed liquid chromatography-tandem mass spectrometry screening assays of these samples. In our analysis, we profiled 48 lipid mediators (LMs) derived from various essential polyunsaturated fatty acids to determine potential targets regulated by SREBF1, and we found that the levels of 11,12 epoxyeicosatrienoic acid (11,12-EET) were negatively associated with the number of SREBF1 alleles (P = 0.006 for a linear model). We also compared gene expression between KO and WT ZF by genome-wide RNA-sequencing. Significantly enriched pathways included fatty acid elongation, linoleic acid metabolism, arachidonic acid metabolism, adipocytokine signaling, and DNA replication. We discovered trends indicating that BMD in adult fish was significantly lower in the KO than in the WT population (P < 0.03). These studies reinforce the importance of lipidomics investigation by detailing how the KO of SREBF1 affects both BMD and lipid-signaling mediators, thus confirming the importance of SREBF1 for musculoskeletal homeostasis.