The Interplay of Lipid Signaling in Musculoskeletal Cross Talk: Implications for Health and Disease.

The intricate interplay between the muscle and bone tissues is a fundamental aspect of musculoskeletal physiology. Over the past decades, emerging research has highlighted the pivotal role of lipid signaling in mediating communication between these tissues. This chapter delves into the multifaceted mechanisms through which lipids, particularly phospholipids, sphingolipids, and eicosanoids, participate in orchestrating cellular responses and metabolic pathways in both muscle and bone. Additionally, we examine the clinical implications of disrupted lipid signaling in musculoskeletal disorders, offering insights into potential therapeutic avenues. This chapter aims to shed light on the complex lipid-driven interactions between the muscle and bone tissues, paving the way for a deeper understanding of musculoskeletal health and disease.

Nanoparticles and Their Applications in Lipid Signaling.

This chapter provides an overview of the diverse range of applications associated with nanoparticles. The application of nanoparticles in the medical field has garnered considerable attention due to their unique properties and versatile compositions. They have shown promise in the treatment of cancer, fungal and viral infections, and pain management. These systems provide numerous benefits, such as increased drug stability, improved bioavailability, and targeted delivery to specific tissues or cells. The objective of this chapter is to provide a brief analysis of the differences between nanoparticles and lipid particles, focusing particularly on the importance of nanoparticle size and composition in their interactions with lipids. Additionally, the applications of nanoparticles in lipid signaling will be discussed, considering the vital roles lipids play in cellular signaling pathways. Nanoparticles have shown immense potential in the regulation and control of medical pathways. In this case, we will focus on the manufacture of liposomes, a type of nanoparticle composed of lipids. The reason behind the extensive investigation into liposomes as drug delivery vehicles is their remarkable biocompatibility and adaptability. This section will provide insights into the methods and techniques employed for liposome formulation.

Lipids and Lipid-Mediated Signaling in Plant-Pathogen Interactions.

Plant lipids are essential cell constituents with many structural, storage, signaling, and defensive functions. During plant-pathogen interactions, lipids play parts in both the preexisting passive defense mechanisms and the pathogen-induced immune responses at the local and systemic levels. They interact with various components of the plant immune network and can modulate plant defense both positively and negatively. Under biotic stress, lipid signaling is mostly associated with oxygenated natural products derived from unsaturated fatty acids, known as oxylipins; among these, jasmonic acid has been of great interest as a specific mediator of plant defense against necrotrophic pathogens. Although numerous studies have documented the contribution of oxylipins and other lipid-derived species in plant immunity, their specific roles in plant-pathogen interactions and their involvement in the signaling network require further elucidation. This review presents the most relevant and recent studies on lipids and lipid-derived signaling molecules involved in plant-pathogen interactions, with the aim of providing a deeper insight into the mechanisms underpinning lipid-mediated regulation of the plant immune system.

Market Needs and Methodologies Associated with Patient Lipidomic Diagnoses and Analyses.

This chapter conducts an in-depth exploration of the impact of musculoskeletal (MSK) disorders and injuries, with a specific emphasis on their consequences within the older population demographic. It underscores the escalating demand for innovative interventions in MSK tissue engineering. The chapter also highlights the fundamental role played by lipid signaling mediators (LSMs) in tissue regeneration, with relevance to bone and muscle recovery. Remarkably, Prostaglandin E2 (PGE2) emerges as a central orchestrator in these regenerative processes. Furthermore, the chapter investigates the complex interplay between bone and muscle tissues, explaining the important influence exerted by LSMs on their growth and differentiation. The targeted modulation of LSM pathways holds substantial promise as a beneficial way for addressing muscle disorders. In addition to these conceptual understandings, the chapter provides a comprehensive overview of methodologies employed in the identification of LSMs, with a specific focus on the Liquid Chromatography-Mass Spectrometry (LC-MS). Furthermore, it introduces a detailed LC MS/MS-based protocol tailored for the detection of PGE2, serving as an invaluable resource for researchers immersed in this dynamic field of study.

Measurement of Intracellular Ca2+ for Studying GPCR-Mediated Lipid Signaling.

Intracellular Ca2+ can be conveniently monitored by sensitive Ca2+ fluorescent dyes in live cells. The Gαq involved lipid signaling pathways and, thus, can be studied by intracellular Ca2+ imaging. Here we describe the protocols to measure intracellular Ca2+ for studying PEG2-EP1 activity in esophageal smooth muscle cells. The ratiometric Fura-2 imaging provides quantitative data, and the Fluo-4 confocal microscopic imaging has high-spatial resolution.

Quantifying ABCA1/apoA-1 Signaling Pathways with AFM Imaging and Lipidomic Analysis.

The role of lipid metabolic pathways in the pathophysiology of primary open-angle glaucoma (POAG) has been thoroughly elucidated, with pathways involved in lipid-related disorders such as hypercholesterolemia and hyperlipoprotein accumulation being of particular interest. The ABCA1/apoA-1 transduction pathway moderates reverse cholesterol transport (RCT), facilitating the transport of free cholesterol (FC) and phospholipids (PL) and preventing intracellular lipid aggregates in retinal ganglion cells (RGCs) due to excess FCs and PLs. A deficiency of ABCA1 transporters, and thus, dysregulation of the ABCA1/apoA-1 transduction pathway, may potentiate cellular lipid accumulation, which affects the structural and mechanical features of the cholesterol-rich RGC membranes. Atomic force microscopy (AFM) is a cutting-edge imaging technique suitable for imaging topographical surfaces of a biological specimen and determining its mechanical properties and structural features. The versatility and precision of this technique may prove beneficial in understanding the effects of ABCA1/apoA-1 pathway downregulation and decreased cholesterol efflux in RGCs and their membranes. In this protocol, ABCA1-/- RGC mouse models are prepared over the course of 3 days and are then compared with non-knockout ABCA1 RGC mouse models through AFM imaging of topographical surfaces to examine the difference in membrane dynamics of knockout vs. non-knockout models. Intracellular and extracellular levels of lipids are quantified through high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS).

Phospholipid Signaling in Crop Plants: A Field to Explore.

In plant models such as Arabidopsis thaliana, phosphatidic acid (PA), a key molecule of lipid signaling, was shown not only to be involved in stress responses, but also in plant development and nutrition. In this article, we highlight lipid signaling existing in crop species. Based on open access databases, we update the list of sequences encoding phospholipases D, phosphoinositide-dependent phospholipases C, and diacylglycerol-kinases, enzymes that lead to the production of PA. We show that structural features of these enzymes from model plants are conserved in equivalent proteins from selected crop species. We then present an in-depth discussion of the structural characteristics of these proteins before focusing on PA binding proteins. For the purpose of this article, we consider RESPIRATORY BURST OXIDASE HOMOLOGUEs (RBOHs), the most documented PA target proteins. Finally, we present pioneering experiments that show, by different approaches such as monitoring of gene expression, use of pharmacological agents, ectopic over-expression of genes, and the creation of silenced mutants, that lipid signaling plays major roles in crop species. Finally, we present major open questions that require attention since we have only a perception of the peak of the iceberg when it comes to the exciting field of phospholipid signaling in plants.

PPARG is dispensable for bovine embryo development up to tubular stages.

Following blastocyst hatching, ungulate embryos undergo a prolonged preimplantation period termed conceptus elongation. Conceptus elongation constitutes a highly susceptible period for embryonic loss and the embryonic requirements during this process are largely unknown, but multiple lipid compounds have been identified in the fluid nourishing the elongating conceptuses. Peroxisome proliferator-activated receptors (PPARs) mediate the signaling actions of prostaglandins and other lipids and, between them, PPARG has been pointed out to play a relevant role on conceptus elongation by a functional study that depleted PPARG in both uterus and conceptus. The objective of this study has been to determine if embryonic PPARG is required for bovine embryo development. To that aim, we have generated bovine PPARG KO embryos in vitro by two independent gene ablation strategies and assess their developmental ability. In vitro development to Day (D) 8 blastocyst was unaffected by PPARG ablation, as total, inner cell mass and trophectoderm cell numbers were similar between WT and KO D8 embryos. In vitro post-hatching development to D12 was also comparable between different genotypes, as embryo diameter, epiblast cell number, and embryonic disc formation and hypoblast migration rates were unaffected by the ablation. The development to tubular stages equivalent to E14 was assessed in vivo, following a heterologous embryo transfer experiment, observing that the development of extra-embryonic membranes and of the embryonic disc was not altered by PPARG ablation. In conclusion, PPARG ablation did not impaired bovine embryo development up to tubular stages.

Mammalian START-like phosphatidylinositol transfer proteins - Physiological perspectives and roles in cancer biology.

PtdIns and its phosphorylated derivatives, the phosphoinositides, are the biochemical components of a major pathway of intracellular signaling in all eukaryotic cells. These lipids are few in terms of cohort of unique positional isomers, and are quantitatively minor species of the bulk cellular lipidome. Nevertheless, phosphoinositides regulate an impressively diverse set of biological processes. It is from that perspective that perturbations in phosphoinositide-dependent signaling pathways are increasingly being recognized as causal foundations of many human diseases - including cancer. Although phosphatidylinositol transfer proteins (PITPs) are not enzymes, these proteins are physiologically significant regulators of phosphoinositide signaling. As such, PITPs are conserved throughout the eukaryotic kingdom. Their biological importance notwithstanding, PITPs remain understudied. Herein, we review current information regarding PITP biology primarily focusing on how derangements in PITP function disrupt key signaling/developmental pathways and are associated with a growing list of pathologies in mammals.

Genetic interaction between TTG2 and AtPLC1 reveals a role for phosphoinositide signaling in a co-regulated suite of Arabidopsis epidermal pathways.

The TTG2 transcription factor of Arabidopsis regulates a set of epidermal traits, including the differentiation of leaf trichomes, flavonoid pigment production in cells of the inner testa (or seed coat) layer and mucilage production in specialized cells of the outer testa layer. Despite the fact that TTG2 has been known for over twenty years as an important regulator of multiple developmental pathways, little has been discovered about the downstream mechanisms by which TTG2 co-regulates these epidermal features. In this study, we present evidence of phosphoinositide lipid signaling as a mechanism for the regulation of TTG2-dependent epidermal pathways. Overexpression of the AtPLC1 gene rescues the trichome and seed coat phenotypes of the ttg2-1 mutant plant. Moreover, in the case of seed coat color rescue, AtPLC1 overexpression restored expression of the TTG2 flavonoid pathway target genes, TT12 and TT13/AHA10. Consistent with these observations, a dominant AtPLC1 T-DNA insertion allele (plc1-1D) promotes trichome development in both wild-type and ttg2-3 plants. Also, AtPLC1 promoter:GUS analysis shows expression in trichomes and this expression appears dependent on TTG2. Taken together, the discovery of a genetic interaction between TTG2 and AtPLC1 suggests a role for phosphoinositide signaling in the regulation of trichome development, flavonoid pigment biosynthesis and the differentiation of mucilage-producing cells of the seed coat. This finding provides new avenues for future research at the intersection of the TTG2-dependent developmental pathways and the numerous molecular and cellular phenomena influenced by phospholipid signaling.

The Development of LAT1 Efflux Agonists as Mechanistic Probes of Cellular Amino Acid Stress.

Amino acid restriction induces cellular stress and cells often respond via the induction of autophagy. Autophagy or 'self-eating' enables the recycling of proteins and provides the essential amino acids needed for cell survival. Of the naturally occurring amino acids, methionine restriction has pleiotropic effects on cells because methionine also contributes to the intracellular methyl pools required for epigenetic controls as well as polyamine biosynthesis. In this report, we describe the chemical synthesis of four diastereomers of a methionine depletion agent and demonstrate how controlled methionine efflux from cells significantly reduces intracellular methionine, S-adenosylmethionine (SAM), S-adenosyl homocysteine (SAH), and polyamine levels. We also demonstrate that human pancreatic cancer cells respond via a lipid signaling pathway to induce autophagy. The methionine depletion agent causes the large amino acid transporter 1 (LAT1) to preferentially work in reverse and export the cell's methionine (and leucine) stores. The four diastereomers of the lead methionine/leucine depletion agent were synthesized and evaluated for their ability to (a) efflux 3H-leucine from cells, (b) dock to LAT1 in silico, (c) modulate intracellular SAM, SAH, and phosphatidylethanolamine (PE) pools, and (d) induce the formation of the autophagy-associated LC3-II marker. The ability to modulate the intracellular concentration of methionine regardless of exogenous methionine supply provides new molecular tools to better understand cancer response pathways. This information can then be used to design improved therapeutics that target downstream methionine-dependent processes like polyamines.

Lipid phosphorylation by a diacylglycerol kinase suppresses ABA biosynthesis to regulate plant stress responses.

Lipid phosphorylation by diacylglycerol kinase (DGK) that produces phosphatidic acid (PA) plays important roles in various biological processes, including stress responses, but the underlying mechanisms remain elusive. Here, we show that DGK5 and its lipid product PA suppress ABA biosynthesis by interacting with ABA-DEFICIENT 2 (ABA2), a key ABA biosynthesis enzyme, to negatively modulate plant response to abiotic stress tested in Arabidopsis thaliana. Loss of DGK5 function rendered plants less damaged, whereas overexpression (OE) of DGK5 enhanced plant damage to water and salt stress. The dgk5 mutant plants exhibited decreased total cellular and nuclear levels of PA with increased levels of diacylglycerol, whereas DGK5-OE plants displayed the opposite effect. Interestingly, we found that both DGK5 and PA bind to the ABA-synthesizing enzyme ABA2 and suppress its enzymatic activity. Consistently, the dgk5 mutant plants exhibited increased levels of ABA, while DGK5-OE plants showed reduced ABA levels. In addition, we showed that both DGK5 and ABA2 are detected in and outside the nuclei, and loss of DGK5 function decreased the nuclear association of ABA2. We found that both DGK5 activity and PA promote nuclear association of ABA2. Taken together, these results indicate that both DGK5 and PA interact with ABA2 to inhibit its enzymatic activity and promote its nuclear sequestration, thereby suppressing ABA production in response to abiotic stress. Our study reveals a sophisticated mechanism by which DGK5 and PA regulate plant stress responses.

The wide world of non-mammalian phospholipase D enzymes.

Phospholipase D (PLD) hydrolyses phosphatidylcholine (PtdCho) to produce free choline and the critically important lipid signaling molecule phosphatidic acid (PtdOH). Since the initial discovery of PLD activities in plants and bacteria, PLDs have been identified in a diverse range of organisms spanning the taxa. While widespread interest in these proteins grew following the discovery of mammalian isoforms, research into the PLDs of non-mammalian organisms has revealed a fascinating array of functions ranging from roles in microbial pathogenesis, to the stress responses of plants and the developmental patterning of flies. Furthermore, studies in non-mammalian model systems have aided our understanding of the entire PLD superfamily, with translational relevance to human biology and health. Increasingly, the promise for utilization of non-mammalian PLDs in biotechnology is also being recognized, with widespread potential applications ranging from roles in lipid synthesis, to their exploitation for agricultural and pharmaceutical applications.

Arabidopsis thaliana phosphoinositide-specific phospholipase C 2 is required for Botrytis cinerea proliferation.

Phospholipase C (PLC) plays a key role in lipid signaling during plant development and stress responses. PLC activation is one of the earliest responses during pathogen perception. Arabidopsis thaliana contains seven PLC encoding genes (AtPLC1 to AtPLC7) and two pseudogenes (AtPLC8 and AtPLC9), being AtPLC2 the most abundant isoform with constitutive expression in all plant organs. PLC has been linked to plant defense signaling, in particular to the production of reactive oxygen species (ROS). Previously, we demonstrated that AtPLC2 is involved in ROS production via the NADPH oxidase isoforms RBOHD activation during stomata plant immunity. Here we studied the role of AtPLC2 on plant resistance against the necrotrophic fungus Botrytis cinerea, a broad host-range and serious agricultural pathogen. We show that the AtPLC2-silenced (amiR PLC2) or null mutant (plc2-1) plants developed smaller B. cinerea lesions. Moreover, plc2-1 showed less ROS production and an intensified SA-dependent signaling upon infection, indicating that B. cinerea uses AtPLC2-triggered responses for a successful proliferation. Therefore, AtPLC2 is a susceptibility (S) gene that facilitates B. cinerea infection and proliferation.

The role of extended synaptotagmin at membrane contact sites in cancer research.

Membrane contact sites (MCSs) are adjacent locations between the membranes of two different organelles and play important roles in various physiological processes, including cellular calcium and lipid signaling. In cancer research, MCSs have been proposed to regulate tumor metabolism and fate, contributing to tumor progression, and this function could be exploited for tumor therapy. However, there is little evidence on how MCSs are involved in cancer progression. In this review, we use extended synaptotagmins (E-Syts) as an entry point to describe how MCSs affect cancer progression and may be used as new diagnostic biomarkers. We then introduced the role of E-Syt and its related pathways in calcium and lipid signaling, aiming to explain how MCSs affect tumor proliferation, progression, metastasis, apoptosis, drug resistance, and treatment through calcium and lipid signaling. Generally, this review will facilitate the understanding of the complex contact biology of cancer cells.

Dynamics of Docosahexaenoic Acid Utilization by Mouse Peritoneal Macrophages.

In this work, the incorporation of docosahexaenoic acid (DHA) in mouse resident peritoneal macrophages and its redistribution within the various phospholipid classes were investigated. Choline glycerophospholipids (PC) behaved as the major initial acceptors of DHA. Prolonged incubation with the fatty acid resulted in the transfer of DHA from PC to ethanolamine glycerophospholipids (PE), reflecting phospholipid remodeling. This process resulted in the cells containing similar amounts of DHA in PC and PE in the resting state. Mass spectrometry-based lipidomic analyses of phospholipid molecular species indicated a marked abundance of DHA in ether phospholipids. Stimulation of the macrophages with yeast-derived zymosan resulted in significant decreases in the levels of all DHA-containing PC and PI species; however, no PE or PS molecular species were found to decrease. In contrast, the levels of an unusual DHA-containing species, namely PI(20:4/22:6), which was barely present in resting cells, were found to markedly increase under zymosan stimulation. The levels of this phospholipid also significantly increased when the calcium-ionophore A23187 or platelet-activating factor were used instead of zymosan to stimulate the macrophages. The study of the route involved in the synthesis of PI(20:4/22:6) suggested that this species is produced through deacylation/reacylation reactions. These results define the increases in PI(20:4/22:6) as a novel lipid metabolic marker of mouse macrophage activation, and provide novel information to understand the regulation of phospholipid fatty acid turnover in activated macrophages.

Circadian clock factors regulate the first condensation reaction of fatty acid synthesis in Arabidopsis.

The circadian clock regulates temporal metabolic activities, but how it affects lipid metabolism is poorly understood. Here, we show that the central clock regulators LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) regulate the initial step of fatty acid (FA) biosynthesis in Arabidopsis. Triacylglycerol (TAG) accumulation in seeds was increased in LHY-overexpressing (LHY-OE) and decreased in lhycca1 plants. Metabolic tracking of lipids in developing seeds indicated that LHY enhanced FA synthesis. Transcript analysis revealed that the expression of genes involved in FA synthesis, including the one encoding ß-ketoacyl-ACP synthase III (KASIII), was oppositely changed in developing seeds of LHY/CCA1-OEs and lhycca1. Chromatin immunoprecipitation, electrophoretic mobility shift, and transactivation assays indicated that LHY bound and activated the promoter of KASIII. Furthermore, phosphatidic acid, a metabolic precursor to TAG, inhibited LHY binding to KASIII promoter elements. Our data show a regulatory mechanism for plant lipid biosynthesis by the molecular clock.

A live-cell ergosterol reporter for visualization of the effects of fluconazole on the human fungal pathogen Candida albicans.

IMPORTANCE: Ergosterol is a critical membrane lipid in fungi. In Candida albicans, this essential plasma membrane amphipathic lipid is important for interactions with host cells, in particular, host immune responses. Here, we use a live-cell reporter for specifically visualizing ergosterol and show that apical enrichment of this sterol is not critical for budding and filamentous growth in this human fungal pathogen. Our results highlight that this live-cell reporter is likely to be a useful tool in the analyses of azole resistance and tolerance mechanisms, including alterations in drug targets and upregulation of efflux activities.

Sphingolipids: From structural components to signaling hubs.

In late November 2019, Prof. Lina M. Obeid passed away from cancer, a disease she spent her life researching and studying its intricate molecular underpinnings. Along with her husband, Prof. Yusuf A. Hannun, Obeid laid down the foundations of sphingolipid biochemistry and oversaw its remarkable evolution over the years. Lipids are a class of macromolecules that are primarily associated with cellular architecture. In fact, lipids constitute the perimeter of the cell in such a way that without them, there cannot be cells. Hence, much of the early research on lipids identified the function of this class of biological molecules as merely structural. Nevertheless, unlike proteins, carbohydrates, and nucleic acids, lipids are elaborately diverse as they are not made up of monomers in polymeric forms. This diversity in structure is clearly mirrored by functional pleiotropy. In this chapter, we focus on a major subset of lipids, sphingolipids, and explore their historic rise from merely inert structural components of plasma membranes to lively and necessary signaling molecules that transmit various signals and control many cellular processes. We will emphasize the works of Lina Obeid since she was an integral pillar of the sphingolipid research world.

Regulation of phosphoinositide metabolism in Apicomplexan parasites.

Phosphoinositides are a biologically essential class of phospholipids that contribute to organelle membrane identity, modulate membrane trafficking pathways, and are central components of major signal transduction pathways that operate on the cytosolic face of intracellular membranes in eukaryotes. Apicomplexans (such as Toxoplasma gondii and Plasmodium spp.) are obligate intracellular parasites that are important causative agents of disease in animals and humans. Recent advances in molecular and cell biology of Apicomplexan parasites reveal important roles for phosphoinositide signaling in key aspects of parasitosis. These include invasion of host cells, intracellular survival and replication, egress from host cells, and extracellular motility. As Apicomplexans have adapted to the organization of essential signaling pathways to accommodate their complex parasitic lifestyle, these organisms offer experimentally tractable systems for studying the evolution, conservation, and repurposing of phosphoinositide signaling. In this review, we describe the regulatory mechanisms that control the spatial and temporal regulation of phosphoinositides in the Apicomplexan parasites Plasmodium and T. gondii. We further discuss the similarities and differences presented by Apicomplexan phosphoinositide signaling relative to how these pathways are regulated in other eukaryotic organisms.