Esterified eicosanoids: Generation, characterization and function

Eicosanoids are oxidation products of C20 polyunsaturated fatty acids (e.g. arachidonic acid) that include prostaglandins, thromboxanes, leukotrienes and hydroperoxy fatty acids. They have important biological roles in vivo, including regulation of renal, cardiovascular and gastrointestinal function. Historically, eicosanoids were thought to mediate their signaling actions exclusively as free acids, however evidence is now emerging that they may also be generated attached to other functional groups including phospholipids and glycerol, and that these more complex forms are pathophysiological signaling mediators in their own right. Early studies showed that exogenously added eicosanoids could become esterified into membrane phospholipids of cells, while more recently, it was uncovered that esterified eicosanoids are formed endogenously. This review summarizes our current knowledge of this area, starting with the early discoveries documenting what is known about eicosanoid generation and their esterification, and moving on to discuss the discovery that esterified eicosanoids are generated endogenously by a number of different cell types. Recent research that is highlighting new structures and functions of these important lipid mediators will be presented. This article is part of a Special Issue entitled: Oxidized phospholipids-their properties and interactions with proteins.     

Membrane lipids as signaling molecules

PURPOSE OF REVIEW: Membrane lipids play important roles in signaling reactions. They are involved in most if not all cellular signaling cascades and in a wide variety of tissue and cell types. The purpose of this review is to highlight major pathways of signaling originating in membrane lipids. Details of lipid metabolism, and its relation to protein function, will thus advance understanding of the role of lipids in health and disease. RECENT FINDINGS: Major classes of lipids including glycerophospholipids, their metabolites (eicosanoids, endocannabinoids), and sphingolipids have recently generated interest in the field of signal transduction. These lipids are tightly regulated and have an impact on various physiological functions. Importantly, aberrant lipid metabolism often leads to onset of pathology, and thus the precise balance of signaling lipids and their effectors can serve as biomarkers. SUMMARY: Membrane lipids form precursors for second messengers and functional assembly matrices on membrane domains during cellular stimulation. Many of these modifications are rapid reactions at lipid headgroups. Metabolism of the fatty acyl portion of membrane lipids leads to the generation of a bewildering complexity of lipid mediators with extended effects in space and time.     

Diabetes and apoptosis: liver

The liver is a central regulator of glucose homeostasis and stores or releases glucose according to metabolic demands. In insulin resistant states or diabetes the dysregulation of hepatic glucose release contributes significantly to the pathophysiology of these conditions. Acute or chronic liver disease can aggravate insulin resistance and the physiological effects of insulin on hepatocytes are disturbed. Insulin resistance has also been recognized as an independent risk factor for the development of liver injury. In the healthy liver tissue homeostasis is achieved through cell turnover by apoptosis and dysregulation of the physiological process resulting in too much or too little cell death can have potentially devastating effects on liver tissue. The delineation of the signaling pathways that mediate apoptosis changed the paradigms of understanding of many liver diseases. These signaling events include cell surface based receptor-ligand systems and intracellular signaling pathways that are regulated through kinases on multiple levels. The dissection of these signaling pathways has shown that the regulators of apoptosis signaling events in hepatocytes can also modulate insulin signaling pathways and that mediators of insulin resistance in turn influence liver cell apoptosis. This review will summarize the potential crosstalk between apoptosis and insulin resistance signaling events and discuss the involved mediators.     

High-throughput lipidomic analysis of fatty acid derived eicosanoids and N-acylethanolamines

Fatty acid-derived eicosanoids and N-acylethanolamines (NAE) are important bioactive lipid mediators involved in numerous biological processes including cell signaling and disease progression. To facilitate research on these lipid mediators, we have developed a targeted high-throughput mass spectrometric based methodology to monitor and quantitate both eicosanoids and NAEs, and can be analyzed separately or together in series. Each methodology utilizes scheduled multiple reaction monitoring (sMRM) pairs in conjunction with a 25 min reverse-phase HPLC separation. The eicosanoid methodology monitors 141 unique metabolites and quantitative amounts can be determined for over 100 of these metabolites against standards. The analysis covers eicosanoids generated from cycloxygenase, lipoxygenase, cytochrome P450 enzymes, and those generated from non-enzymatic pathways. The NAE analysis monitors 36 metabolites and quantitative amounts can be determined for 33 of these metabolites against standards. The NAE method contains metabolites derived from saturated fatty acids, unsaturated fatty acids, and eicosanoids. The lower limit of detection for eicosanoids ranges from 0.1pg to 1pg, while NAEs ranges from 0.1pg to 1000pg. The rationale and design of the methodology is discussed.     

Arachidonic-acid-derived eicosanoids: roles in biology and immunopathology

Arachidonic acid (AA)-derived eicosanoids belong to a complex family of lipid mediators that regulate a wide variety of physiological responses and pathological processes. They are produced by various cell types through distinct enzymatic pathways and act on target cells via specific G-protein-coupled receptors. Although originally recognized for their capacity to elicit biological responses such as vascular homeostasis, protection of the gastric mucosa and platelet aggregation, eicosanoids are now understood to regulate immunopathological processes ranging from inflammatory responses to chronic tissue remodelling, cancer, asthma, rheumatoid arthritis and autoimmune disorders. Here, we review the major properties of eicosanoids and their expanding roles in biology and medicine.     

Electrophilic eicosanoids: Signaling and targets

Electrophilic eicosanoids are reactive mediators that arise by non-enzymatic transformations of arachidonic acid or of its products and display varied biological actions. Various electrophilic eicosanoids have shown anti-proliferative and anti-inflammatory effects, which have elicited a great interest in their study as potential therapeutic agents. A key feature of these compounds is their ability to covalently modify proteins, thus altering their structure and function. The modification of several components of the NF-κB pathway contributes to the anti-inflammatory effects of electrophilic eicosanoids, whereas addition to redox-sensitive proteins plays a key role in the antioxidant response. However, electrophilic eicosanoids may also have a dark side, and accumulating evidence points towards their involvement in neurotoxicity and/or neurodegeneration. The ability of some electrophilic eicosanoids to induce protein oligomerization or aggregation through various mechanisms may contribute to these effects. Biochemical and proteomic studies have led to the identification of numerous protein targets for modification by electrophilic eicosanoids, the number of which continues to expand, revealing novel potential functions for these compounds and providing a basis for their pleiotropic effects. The ample number of targets identified, together with the non-enzymatic nature of the modification argue against the potential specificity or regulation of electrophilic eicosanoid action. However, protein modification displays selectivity depending on structural features of the proteins and of the electrophilic compounds as well as on context factors such as cell type and GSH availability. Understanding the factors which control the extent and selectivity of protein modification by electrophilic eicosanoids is therefore essential to elucidate their pathophysiological roles and therapeutic potential in specific settings.     

Integrin-mediated signal transduction pathways.

Integrins serve as adhesion receptors for extracellular matrix proteins and also transduce biochemical signals into the cell. They regulate a variety of cellular functions, including spreading, migration, proliferation and apoptosis. Many signaling pathways downstream of integrins have been identified and characterized and are discussed here. In particular, integrins regulate many protein tyrosine kinases and phosphatases, such as FAK and Src, to coordinate many of the cell processes mentioned above. The regulation of MAP kinases by integrins is important for cell growth or other functions, and the putative roles of Ras and FAK in these pathways are discussed. Phosphatidylinositol lipids and their modifying enzymes, particularly PI 3-kinase, are strongly implicated as mediators of integrin-regulated cytoskeletal changes and cell migration. Similarly, actin cytoskeleton regulation by the Rho family of GTPases is coordinated with integrin signaling to regulate cell spreading and migration, although the exact relationship between these pathways is not clear. Finally, intracellular pH and calcium fluxes by integrins are suggested to affect a variety of cellular proteins and functions.     

Differential Regulation of NOTCH2 and NOTCH3 Contribute to Their Unique Functions in Vascular Smooth Muscle Cells

Notch signaling is a key regulator of vascular smooth muscle cell (VSMC) phenotypes, including differentiation, proliferation, and cell survival. However, the exact contribution of the individual Notch receptors has not been thoroughly delineated. In this study, we identify unique roles for NOTCH2 and NOTCH3 in regulating proliferation and cell survival in cultured VSMCs. Our results indicate that NOTCH2 inhibits PDGF-B-dependent proliferation and its expression is decreased by PDGF-B. In contrast, NOTCH3 promotes proliferation and receptor expression is increased by PDGF-B. Additionally, data show that NOTCH3, but not NOTCH2 protects VSMCs from apoptosis and apoptosis mediators degrade NOTCH3 protein. We identified three pro-survival genes specifically regulated by NOTCH3 in cultured VSMCs and in mouse aortas. This regulation is mediated through MAP kinase signaling, which we demonstrate can be activated by NOTCH3, but not NOTCH2. Overall, this study highlights discrete roles for NOTCH2 and NOTCH3 in VSMCs and connects these roles to specific upstream regulators that control their expression.     

FGF-2 inhibits TNF-α mediated apoptosis through upregulation of Bcl2-A1 and Bcl-xL in ATDC5 cells

FGF-2 is involved in cell survival, proliferation, apoptosis, and angiogenesis in a wide variety of cells. FRGRs, PI3K and MAP kinases are well known mediators of FGF signaling. Despite its known roles during many developmental processes, including osteogenesis, there are few known targets of FGF-2. In the present study, we identified Bcl2-A1 and Bcl-xL as two prominent targets involved in promoting cell survival. Pretreatment of ATDC5 cells with FGF-2 increased cell survival, while siRNAs specific for Bcl2-A1 and Bcl-xL compromised the anti- apoptotic effect of FGF-2, sensitized the cells to apoptosis triggered by TNF-α. Chemical inhibition of FGFR, NFkB, and PI3K activity by PD173074, pyrrolidine dithiocarbamate, and LY294002 respectively abrogated the FGF-2-mediated induction of Bcl2-A1 and Bcl-xL expression. Taken together, our data demonstrate that a subset of Bcl2 family proteins are the targets of FGF-2 signaling that promotes the survival of ATDC5 cells.     

The Roles of ROS and Caspases in TRAIL-Induced Apoptosis and Necroptosis in Human Pancreatic Cancer Cells

Death signaling provided by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) can induce death in cancer cells with little cytotoxicity to normal cells; this cell death has been thought to involve caspase-dependent apoptosis. Reactive oxygen species (ROS) are also mediators that induce cell death, but their roles in TRAIL-induced apoptosis have not been elucidated fully. In the current study, we investigated ROS and caspases in human pancreatic cancer cells undergoing two different types of TRAIL-induced cell death, apoptosis and necroptosis. TRAIL treatment increased ROS in two TRAIL-sensitive pancreatic cancer cell lines, MiaPaCa-2 and BxPC-3, but ROS were involved in TRAIL-induced apoptosis only in MiaPaCa-2 cells. Unexpectedly, inhibition of ROS by either N-acetyl-L-cysteine (NAC), a peroxide inhibitor, or Tempol, a superoxide inhibitor, increased the annexin V-/propidium iodide (PI)⁺ early necrotic population in TRAIL-treated cells. Additionally, both necrostatin-1, an inhibitor of receptor-interacting protein kinase 1 (RIP1), and siRNA-mediated knockdown of RIP3 decreased the annexin V^-/PI⁺ early necrotic population after TRAIL treatment. Furthermore, an increase in early apoptosis was induced in TRAIL-treated cancer cells under inhibition of either caspase-2 or -9. Caspase-2 worked upstream of caspase-9, and no crosstalk was observed between ROS and caspase-2/-9 in TRAIL-treated cells. Together, these results indicate that ROS contribute to TRAIL-induced apoptosis in MiaPaCa-2 cells, and that ROS play an inhibitory role in TRAIL-induced necroptosis of MiaPaCa-2 and BxPC-3 cells, with caspase-2 and -9 playing regulatory roles in this process.     

A house divided: ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death

Programmed cell death is an important physiological response to many forms of cellular stress. The signaling cascades that result in programmed cell death are as elaborate as those that promote cell survival, and it is clear that coordination of both protein- and lipid-mediated signals is crucial for proper cell execution. Sphingolipids are a large class of lipids whose diverse members share the common feature of a long-chain sphingoid base, e.g., sphingosine. Many sphingolipids have been shown to play essential roles in both death signaling and survival. Ceramide, an N-acylsphingosine, has been implicated in cell death following a myriad of cellular stresses. Sphingosine itself can induce cell death but via pathways both similar and dissimilar to those of ceramide. Sphingosine-1-phosphate, on the other hand, is an anti-apoptotic molecule that mediates a host of cellular effects antagonistic to those of its pro-apoptotic sphingolipid siblings. Extraordinarily, these lipid mediators are metabolically juxtaposed, suggesting that the regulation of their metabolism is of the utmost importance in determining cell fate. In this review, we briefly examine the role of ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death and highlight the potential roles that these lipids play in the pathway to apoptosis.     

Ultraviolet-radiation induced skin inflammation: dissecting the role of bioactive lipids

Acute exposure of human skin to the ultraviolet radiation (UVR) in sunlight results in the sunburn response. This is mediated in part by pro-inflammatory eicosanoids and other bioactive lipids, which are in turn produced via mechanisms including UVR-induction of oxidative stress, cell signalling and gene expression. Sunburn is a self-limiting inflammation offering a convenient and accessible system for the study of human cutaneous lipid metabolism. Recent lipidomic applications have revealed that a wider diversity of eicosanoids may be involved in the sunburn response than previously appreciated. This article reviews the effects of UVR on cutaneous lipids and examines the contribution of bioactive lipid mediators in the development of sunburn. Since human skin is an active site of polyunsaturated fatty acid (PUFA) metabolism, and these macronutrients can influence the production of eicosanoids/bioactive lipids, as well as modulate cell signalling, gene expression and oxidative stress, the application of PUFA as potential photoprotective agents is also considered.     

Physiological effects of oxidized phospholipids and their cellular signaling mechanisms in inflammation

Oxidized phospholipids, such as the products of the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine by nonenzymatic radical attack, are known to be formed in a number of inflammatory diseases. Interest in the bioactivity and signaling functions of these compounds has increased enormously, with many studies using cultured immortalized and primary cells, tissues, and animals to understand their roles in disease pathology. Initially, oxidized phospholipids were viewed largely as culprits, in line with observations that they have proinflammatory effects, enhancing inflammatory cytokine production, cell adhesion and migration, proliferation, apoptosis, and necrosis, especially in vascular endothelial cells, macrophages, and smooth muscle cells. However, evidence has emerged that these compounds also have protective effects in some situations and cell types; a notable example is their ability to interfere with signaling by certain Toll-like receptors (TLRs) induced by microbial products that normally leads to inflammation. They also have protective effects via the stimulation of small GTPases and induce up-regulation of antioxidant enzymes and cytoskeletal rearrangements that improve endothelial barrier function. Oxidized phospholipids interact with several cellular receptors, including scavenger receptors, platelet-activating factor receptors, peroxisome proliferator-activated receptors, and TLRs. The various and sometimes contradictory effects that have been observed for oxidized phospholipids depend on their concentration, their specific structure, and the cell type investigated. Nevertheless, the underlying molecular mechanisms by which oxidized phospholipids exert their effects in various pathologies are similar. Although our understanding of the actions and mechanisms of these mediators has advanced substantially, many questions do remain about their precise interactions with components of cell signaling pathways.     

The relationship between the fatty acid composition of immune cells and their function

The immune system, including its inflammatory components, is fundamental to host defence against pathogenic invaders. It is a complex system involving interactions amongst many different cell types dispersed throughout the body. Central to its actions are phagocytosis of bacteria, processing of antigens derived from intracellular and extracellular pathogens, activation of T cells with clonal expansion (proliferation) and production of cytokines that elicit effector cell functions such as antibody production and killing cell activity. Inappropriate immunologic activity, including inflammation, is a characteristic of many common human disorders. Eicosanoids produced from arachidonic acid have roles in inflammation and regulation of T and B lymphocyte functions. Eicosapentaenoic acid (EPA) also gives rise to eicosanoids and these may have differing properties from those of arachidonic acid-derived eicosanoids. EPA and docosahexaenoic acid (DHA) give rise to newly discovered resolvins which are anti-inflammatory and inflammation resolving. Human immune cells are typically rich in arachidonic acid, but arachidonic acid, EPA and DHA contents can be altered through oral administration of EPA and DHA. This results in a changed pattern of production of eicosanoids and probably also of resolvins, although the latter are not well examined in the human context. Changing the fatty acid composition of immune cells also affects phagocytosis, T cell signaling and antigen presentation capability. These effects appear to mediated at the membrane level suggesting important roles of fatty acids in membrane order, lipid raft structure and function, and membrane trafficking. Thus, the fatty acid composition of human immune cells influences their function and the cell membrane contents of arachidonic acid, EPA and DHA are important. Fatty acids influence immune cell function through a variety of complex mechanisms and these mechanisms are now beginning to be unraveled.     

The membrane and lipids as integral participants in signal transduction: lipid signal transduction for the non-lipid biochemist

Reviews of signal transduction have often focused on the cascades of protein kinases and protein phosphatases and their cytoplasmic substrates that become activated in response to extracellular signals. Lipids, lipid kinases, and lipid phosphatases have not received the same amount of attention as proteins in studies of signal transduction. However, lipids serve a variety of roles in signal transduction. They act as ligands that activate signal transduction pathways as well as mediators of signaling pathways, and lipids are the substrates of lipid kinases and lipid phosphatases. Cell membranes are the source of the lipids involved in signal transduction, but membranes also constitute lipid barriers that must be traversed by signal transduction pathways. The purpose of this review is to explore the magnitude and diversity of the roles of the cell membrane and lipids in signal transduction and to highlight the interrelatedness of families of lipid mediators in signal transduction.     

Thy-1, a versatile modulator of signaling affecting cellular adhesion, proliferation, survival, and cytokine/growth factor responses

Thy-1 is a 25-37 kDa glycosylphosphatidylinositol (GPI)-anchored protein involved in T cell activation, neurite outgrowth, apoptosis, tumor suppression, wound healing, and fibrosis. To mediate these diverse effects, Thy-1 participates in multiple signaling cascades. In this review, we discuss Thy-1 signaling primarily in non-immunologic cell types, including neurons, mesangial cells, ovarian cancer cells, nasopharyngeal carcinoma cells, endothelial cells, and fibroblasts. We review the current literature regarding Thy-1 signaling via integrins, protein tyrosine kinases, and cytokines and growth factors; and the roles of these signaling pathways in cellular adhesion, apoptosis, cell proliferation, and cell adhesion and migration. We also discuss the role of Thy-1 localization to lipid rafts, and of the GPI anchor in Thy-1 signaling. Ongoing research on the mechanisms of Thy-1 signaling will add to our understanding of the diverse physiologic and pathologic processes in which Thy-1 plays a role.     

A house divided: Ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death

Programmed cell death is an important physiological response to many forms of cellular stress. The signaling cascades that result in programmed cell death are as elaborate as those that promote cell survival, and it is clear that coordination of both protein- and lipid-mediated signals is crucial for proper cell execution. Sphingolipids are a large class of lipids whose diverse members share the common feature of a long-chain sphingoid base, e.g., sphingosine. Many sphingolipids have been shown to play essential roles in both death signaling and survival. Ceramide, an N-acylsphingosine, has been implicated in cell death following a myriad of cellular stresses. Sphingosine itself can induce cell death but via pathways both similar and dissimilar to those of ceramide. Sphingosine-1-phosphate, on the other hand, is an anti-apoptotic molecule that mediates a host of cellular effects antagonistic to those of its pro-apoptotic sphingolipid siblings. Extraordinarily, these lipid mediators are metabolically juxtaposed, suggesting that the regulation of their metabolism is of the utmost importance in determining cell fate. In this review, we briefly examine the role of ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death and highlight the potential roles that these lipids play in the pathway to apoptosis.     

Mouse Mammary Tumor Virus Suppresses Apoptosis of Mammary Epithelial Cells through ITAM-Mediated Signaling

Many receptors in hematopoietic cells use a common signaling pathway that relies on a highly conserved immunoreceptor tyrosine-based activation motif (ITAM), which signals through Src family tyrosine kinases. ITAM-bearing proteins are also found in many oncogenic viruses, including the mouse mammary tumor virus (MMTV) envelope (Env). We previously showed that MMTV Env expression transformed normal mammary epithelial cells and that Src kinases were important mediators in this transformation. To study how ITAM signaling affects mammary cell transformation, we utilized mammary cell lines expressing two different ITAM-containing proteins, one encoding a MMTV provirus and the other a B cell receptor fusion protein. ITAM-expressing cells were resistant to both serum starvation- and chemotherapeutic drug-induced apoptosis, whereas cells transduced with these molecules bearing ITAM mutations were indistinguishable from untransduced cells in their sensitivity to these treatments. We also found that Src kinase was activated in the MMTV-expressing cells and that MMTV-induced apoptosis resistance was completely restored by the Src inhibitor PP2. In vivo, MMTV infection delayed involution-induced apoptosis in the mouse mammary gland. Our results show that MMTV suppresses apoptosis through ITAM-mediated Src tyrosine kinase signaling. These studies could lead to the development of effective treatment of nonhematopoietic cell cancers in which ITAM-mediated signaling plays a role.