The neuroprotective function of 2-carba-cyclic phosphatidic acid: Implications for tenascin-C via astrocytes in traumatic brain injury.

We examined the mechanism how 2-carba-cyclic phosphatidic acid (2ccPA), a lipid mediator, regulates neuronal apoptosis in traumatic brain injury (TBI). First, we found 2ccPA suppressed neuronal apoptosis after the injury, and increased the immunoreactivity of tenascin-C (TN-C), an extracellular matrix protein by 2ccPA in the vicinity of the wound region. 2ccPA increased the mRNA expression levels of Tnc in primary cultured astrocytes, and the conditioned medium of 2ccPA-treated astrocytes suppressed the apoptosis of cortical neurons. The neuroprotective effect of TN-C was abolished by knockdown of TN-C. These results indicate that 2ccPA contributes to neuroprotection via TN-C from astrocytes in TBI.

Mechanistic models of PLC/PKC signaling implicate phosphatidic acid as a key amplifier of chemotactic gradient sensing.

Chemotaxis of fibroblasts and other mesenchymal cells is critical for embryonic development and wound healing. Fibroblast chemotaxis directed by a gradient of platelet-derived growth factor (PDGF) requires signaling through the phospholipase C (PLC)/protein kinase C (PKC) pathway. Diacylglycerol (DAG), the lipid product of PLC that activates conventional PKCs, is focally enriched at the up-gradient leading edge of fibroblasts responding to a shallow gradient of PDGF, signifying polarization. To explain the underlying mechanisms, we formulated reaction-diffusion models including as many as three putative feedback loops based on known biochemistry. These include the previously analyzed mechanism of substrate-buffering by myristoylated alanine-rich C kinase substrate (MARCKS) and two newly considered feedback loops involving the lipid, phosphatidic acid (PA). DAG kinases and phospholipase D, the enzymes that produce PA, are identified as key regulators in the models. Paradoxically, increasing DAG kinase activity can enhance the robustness of DAG/active PKC polarization with respect to chemoattractant concentration while decreasing their whole-cell levels. Finally, in simulations of wound invasion, efficient collective migration is achieved with thresholds for chemotaxis matching those of polarization in the reaction-diffusion models. This multi-scale modeling framework offers testable predictions to guide further study of signal transduction and cell behavior that affect mesenchymal chemotaxis.

Role of Phospholipase D-Derived Phosphatidic Acid in Regulated Exocytosis and Neurological Disease.

Lipids play a vital role in numerous cellular functions starting from a structural role as major constituents of membranes to acting as signaling intracellular or extracellular entities. Accordingly, it has been known for decades that lipids, especially those coming from diet, are important to maintain normal physiological functions and good health. On the other side, the exact molecular nature of these beneficial or deleterious lipids, as well as their precise mode of action, is only starting to be unraveled. This recent improvement in our knowledge is largely resulting from novel pharmacological, molecular, cellular, and genetic tools to study lipids in vitro and in vivo. Among these important lipids, phosphatidic acid plays a unique and central role in a great variety of cellular functions. This review will focus on the proposed functions of phosphatidic acid generated by phospholipase D in the last steps of regulated exocytosis with a specific emphasis on hormonal and neurotransmitter release and its potential impact on different neurological diseases.

Phospholipase D and phosphatidic acid in plant immunity.

Phospholipase D (PLD) hydrolyzes membrane phospholipids to generate phosphatidic acid (PA). Both PLD and its lipid product PA are involved in various physiological processes, including plant response to pathogens. The PLD family is comprised of multiple members in higher plants, and PLDs have been reported to play positive and/or negative roles in plant immunity, depending on the types of pathogens and specific PLDs involved. Individual PLDs have distinguishable biochemical properties, such as Ca2+ and phosphatidylinositide requirements. In addition, PLDs and PA are found to interact with various proteins in hormone and stress signaling. The different biochemical and regulatory properties of PLDs and PA shed light on the mechanisms for the functional diversity of PLDs in plant defense signaling and response.

Phosphatidic acid - a simple phospholipid with multiple faces.

Phosphatidic acid (PA) is the simplest glycerophospholipid naturally occurring in living organisms, and even though its content among other cellular lipids is minor, it is drawing more and more attention due to its multiple biological functions. PA is a precursor for other phospholipids, acts as a lipid second messenger and, due to its structural properties, is also a modulator of membrane shape. Although much is known about interaction of PA with its effectors, the molecular mechanisms remain unresolved to a large degree. Throughout many of the well-characterized PA cellular sensors, no conserved binding domain can be recognized. Moreover, not much is known about the cellular dynamics of PA and how it is distributed among subcellular compartments. Remarkably, PA can play distinct roles within each of these compartments. For example, in the nucleus it behaves as a mitogen, influencing gene expression regulation, and in the Golgi membrane it plays a role in membrane trafficking. Here, we discuss how a biophysical experimental approach enabled PA behavior to be described in the context of a lipid bilayer and to what extent various physicochemical conditions may modulate the functional properties of this lipid. Understanding these aspects would help to unravel specific mechanisms of PA-driven membrane transformations and protein recruitment and thus would lead to a clearer picture of the biological role of PA.

Phosphatidic Acid and Cardiolipin Coordinate Mitochondrial Dynamics.

Membrane organelles comprise both proteins and lipids. Remodeling of these membrane structures is controlled by interactions between specific proteins and lipids. Mitochondrial structure and function depend on regulated fusion and the division of both the outer and inner membranes. Here we discuss recent advances in the regulation of mitochondrial dynamics by two critical phospholipids, phosphatidic acid (PA) and cardiolipin (CL). These two lipids interact with the core components of mitochondrial fusion and division (Opa1, mitofusin, and Drp1) to activate and inhibit these dynamin-related GTPases. Moreover, lipid-modifying enzymes such as phospholipases and lipid phosphatases may organize local lipid composition to spatially and temporarily coordinate a balance between fusion and division to establish mitochondrial morphology.

Phosphatidic acid induces EHD3-containing membrane tubulation and is required for receptor recycling.

EHD3 is localized on the tubular structures of early endosomes, and it regulates their trafficking pathway. However, the regulatory mechanism of EHD3-containing tubular structures remains poorly understood. An in vitro liposome co-sedimentation assay revealed that EHD3 interacted with phosphatidic acid through its helical domain and this interaction induced liposomal tubulations. Additionally, inhibiting phosphatidic acid synthesis with diacylglycerol kinase inhibitor or lysophosphatidic acid acyltransferase inhibitor significantly reduced the number of EHD3-containing tubules and impaired their trafficking from early endosomes. These results suggest that EHD3 and phosphatidic acid cooperatively regulate membrane deformation and trafficking from early endosomes.

Phosphatidic acid-mediated signaling.

Phosphatidic acid (PA) is recognized as an important class of lipid messengers. The cellular PA levels are dynamic; PA is produced and metabolized by several enzymatic reactions, including different phospholipases, lipid kinases, and phosphatases. PA interacts with various proteins and the interactions may modulate enzyme catalytic activities and/or tether proteins to membranes. The PA-protein interactions are impacted by changes in cellular pH and other effectors, such as cations. PA is involved in a wide range of cellular processes, including vesicular trafficking, cytoskeletal organization, secretion, cell proliferation, and survival. Manipulations of different PA production reactions alter cellular and organismal response to a wide range of abiotic and biotic stresses. Further investigations of PA's function and mechanisms of action will advance not only the understanding of cell signaling networks but also may lead to biotechnological and pharmacological applications.

[Metabolism and biological function of cardiolipin].

Cardiolipin (CL) is a phospholipid, which is exclusively located in mitochondria, and has a unique structure that consists of 2 phosphate residues and 4 kinds of fatty acyl chains. Cardiolipin plays an important role in regulating various kinds of mitochondrial proteins such as electron transport complexes, carrier proteins and phosphate kinases, and is also essential for the organization of particular mitochondrial structures such as cristae and contact sites. Mitochondrial phospholipase D hydrolyzes CL to produce phosphatidic acid, which is required for mitochondrial fusion. Oxidative stress-induced peroxidation of CL occurs because CL is rich in polyunsaturated fatty acids, especially linoleic acid. Accumulation of CL hydroperoxide (CLOOH) triggers the initiation of apoptosis. Formation of CLOOH causes the release of proapoptotic factors such as cytochrome c from the inner mitochondrial membrane and triggers opening of the permeability transition pore. Levels of CL decrease in the heart following ischemia or disease. Apoptosis is enhanced in temperature-dependent mutant cells whose amounts of CL reduce to half when compared to that of wild type cells. Low levels of CL cause the accumulation of CLOOH and enhance sensitivity to apoptosis. Accumulation of CLOOH in mitochondria causes instability of the membrane, because swelling of mitochondria is induced by the presence of CLOOH in the membrane and is significantly enhanced in CLOOH-loaded mitochondria by the addition of inducer of swelling.

Phosphatidic acid-dependent recruitment and function of the Rac activator DOCK1 during dorsal ruffle formation.

Activation of receptor tyrosine kinases leads to the formation of two different types of plasma membrane structures: peripheral ruffles and dorsal ruffles. Although the formation of both ruffle types requires activation of the small GTPase Rac, the difference in kinetics suggests that a distinct regulatory mechanism operates for their ruffle formation. DOCK1 and DOCK5 are atypical Rac activators and are both expressed in mouse embryonic fibroblasts (MEFs). We found that although PDGF-induced Rac activation and peripheral ruffle formation were coordinately regulated by DOCK1 and DOCK5 in MEFs, DOCK1 deficiency alone impaired dorsal ruffle formation in MEFs. Unlike DOCK5, DOCK1 bound to phosphatidic acid (PA) through the C-terminal polybasic amino acid cluster and was localized to dorsal ruffles. When this interaction was blocked, PDGF-induced dorsal ruffle formation was severely impaired. In addition, we show that phospholipase D, an enzyme that catalyzes PA synthesis, is required for PDGF-induced dorsal, but not peripheral, ruffle formation. These results indicate that the phospholipase D-PA axis selectively controls dorsal ruffle formation by regulating DOCK1 localization.

Role of phosphatidic acid in plant galactolipid synthesis.

Phosphatidic acid (PA) is a precursor metabolite for phosphoglycerolipids and also for galactoglycerolipids, which are essential lipids for formation of plant membranes. PA has in addition a main regulatory role in a number of developmental processes notably in the response of the plant to environmental stresses. We review here the different pools of PA dispatched at different locations in the plant cell and how these pools are modified in different growth conditions, particularly during plastid membrane biogenesis and when the plant is exposed to phosphate deprivation. We analyze how these modifications can affect galactolipid synthesis by tuning the activity of MGD1 enzyme allowing a coupling of phospho- and galactolipid metabolisms. Some mechanisms are considered to explain how physicochemical properties of PA allow this lipid to act as a central internal sensor in plant physiology.

Modulation of oxidative stress and tau phosphorylation by the mTOR activator phosphatidic acid in SH-SY5Y cells.

The mammalian target of rapamycin complex 1 (mTORC1) pathway including p70(S6K) (the 70-kDa p70 S6 kinase) and S6, controls protein synthesis, has anti-apoptotic functions and can phosphorylate tau protein. mTORC1 is triggered by nutrients such as phosphatidic acid (PA). Previous experimental studies have shown that oxidative stress may down-regulate this pathway leading to neuronal death. Our results showed that in human neuroblastoma cells, PA exposure can reduce H(2)O(2)-induced apoptosis and can increase tau protein phosphorylation on Ser214 via p70(S6K) activation. These findings reveal that PA, via the mTOR kinase, can trigger tau phosphorylation on a site known to reduce paired helical filament (PHF) formation.

Ral isoforms are implicated in Fc gamma R-mediated phagocytosis: activation of phospholipase D by RalA.

Phagocytosis is an essential element of the immune response permitting the elimination of pathogens, cellular debris, apoptotic cells, and tumor cells. Recently, both phospholipase D (PLD) isoforms, PLD1 and PLD2, were shown to be necessary for efficient FcgammaR-mediated phagocytosis. In this study, we investigated the role of a potential PLD regulator, the Ral GTPases RalA and RalB, in murine RAW 264.7 macrophages. Both Ral isoforms are expressed in macrophages and are transiently activated following FcgammaR stimulation. When Ral expression levels were varied using Ral mutants or interference RNA, phagocytosis assays revealed that Ral isoforms have antagonistic effects; RalA is a positive modulator, whereas RalB plays a negative role. We then focused on RalA and its possible relationship with PLD. The increase in PLD activity that occurs when phagocytosis is stimulated was inhibited in cells with reduced RalA protein, but it was unaffected by reduced levels of RalB. Furthermore, in macrophages transfected with dsRed-RalA and GFP-PLD1 or GFP-PLD2, RalA colocalized with PLD1 and PLD2 at the phagocytic cup during phagosome formation. Additional results obtained from immunoprecipitation of PLD from macrophages transfected with myc-RalA and hemagglutinin-tagged PLD1 or PLD2 indicated an enhanced interaction of RalA with both PLD isoforms during phagocytic stimulation. The increase in RalA and PLD1 interaction was transient and correlated with the time course of RalA activation. These findings reveal a novel pathway involving RalA and PLD in the regulation of FcgammaR-mediated phagocytosis.

Phosphatidic acid plays a regulatory role in clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) is the main route of internalization of receptor-ligand complexes. Relatively little is known about the role of specific lipids in CME, in particular that of phosphatidic acid (PA). We examined the effect of altering cellular PA levels on CME by manipulating the activities and/or levels of either phospholipase D (PLD1 and PLD2) or diacylglycerol kinase (DGK), two enzyme classes involved in PA production. DGK inhibition resulted in a dramatic reduction of cellular PA, measured directly using an enzyme-coupled reaction, which resulted in a decreased rate of EGFR internalization measured biochemically. This corresponded to a decreased rate of clathrin-coated pit (CCP) initiation and increased lifetimes of productive CCPs, as determined by quantitative live-cell total internal reflection fluorescence microscopy. Unexpectedly, PLD inhibition caused an increase in cellular PA, suggesting that PLD activity negatively regulates PA synthesis by other more productive pathways. Consistent with opposite effects on cellular PA levels, PLD inhibition had opposite effects on EGFR internalization and CCP dynamics, compared with DGK inhibition. Importantly, the constitutive internalization of transferrin receptors was unaffected by either treatment. These findings demonstrate that PA plays a regulatory rather than obligatory role in CME and differentially regulates ligand-stimulated CME of EGFR.

Phosphatidic acid mediates activation of mTORC1 through the ERK signaling pathway.

The mammalian target of rapamycin (mTOR) assembles into two distinct multiprotein complexes known as mTORC1 and mTORC2. Of the two complexes, mTORC1 acts to integrate a variety of positive and negative signals to downstream targets that regulate cell growth. The lipid second messenger, phosphatidic acid (PA), represents one positive input to mTORC1, and it is thought to act by binding directly to mTOR, thereby enhancing the protein kinase activity of mTORC1. Support for this model includes findings that PA binds directly to mTOR and addition of PA to the medium of cells in culture results in activation of mTORC1. In contrast, the results of the present study do not support a model in which PA activates mTORC1 through direct interaction with the protein kinase but, instead, show that the lipid promotes mTORC1 signaling through activation of the ERK pathway. Moreover, rather than acting directly on mTORC1, the results suggest that exogenous PA must be metabolized to lysophosphatidic acid (LPA), which subsequently activates the LPA receptor endothelial differentiation gene (EDG-2). Finally, in contrast to previous studies, the results of the present study demonstrate that leucine does not act through phospholipase D and PA to activate mTORC1 and, instead, show that the two mediators act through parallel upstream signaling pathways to activate mTORC1. Overall, the results demonstrate that leucine and PA signal through parallel pathways to activate mTORC1 and that PA mediates its effect through the ERK pathway, rather than through direct binding to mTOR.

[Relationship between lysophosphatide acid acyltransferase beta and tumor - review].

Phosphatide acid (PA) is a kind of multifunctional bioactive phospholipid. It has been proved that PA produced by lysophosphatide acid acyltransferase (LPAATbeta) was involved in several signalling pathways in tumor cells, leading to the proliferation, apoptosis, migration, invasion, respiratory burst, expression and release of cytokine form tumor cells. The fact that expression of LPAATbeta was higher in tumor tissues than in their homologous normal tissues, and that antitumor effect of inhibitng LPAATbeta on solid tumor and hematological malignancy suggested that the targeting LPAATbeta would be a promising method of antitumor treatment. In this paper, the relevant basic and preclinical researches of LPAATbeta on antitumor treatment were summarized.

Lysophosphatidic acids, cyclic phosphatidic acids and autotaxin as promising targets in therapies of cancer and other diseases.

Lysophospholipids have long been recognized as membrane phospholipid metabolites, but only recently lysophosphatidic acids (LPA) have been demonstrated to act on specific G protein-coupled receptors. The widespread expression of LPA receptors and coupling to several classes of G proteins allow LPA-dependent regulation of numerous processes, such as vascular development, neurogenesis, wound healing, immunity, and cancerogenesis. Lysophosphatidic acids have been found to induce many of the hallmarks of cancer including cellular processes such as proliferation, survival, migration, invasion, and neovascularization. Furthermore, autotaxin (ATX), the main enzyme converting lysophosphatidylcholine into LPA was identified as a tumor cell autocrine motility factor. On the other hand, cyclic phosphatidic acids (naturally occurring analogs of LPA generated by ATX) have anti-proliferative activity and inhibit tumor cell invasion and metastasis. Research achievements of the past decade suggest implementation of preclinical and clinical evaluation of LPA and its analogs, LPA receptors, as well as autotaxin as potential therapeutic targets.

Phosphatidic acid mediates demyelination in Lpin1 mutant mice.

Lipids play crucial roles in many aspects of glial cell biology, affecting processes ranging from myelin membrane biosynthesis to axo-glial interactions. In order to study the role of lipid metabolism in myelinating glial cells, we specifically deleted in Schwann cells the Lpin1 gene, which encodes the Mg2+-dependent phosphatidate phosphatase (PAP1) enzyme necessary for normal triacylglycerol biosynthesis. The affected animals developed pronounced peripheral neuropathy characterized by myelin degradation, Schwann cell dedifferentiation and proliferation, and a reduction in nerve conduction velocity. The observed demyelination is mediated by endoneurial accumulation of the substrate of the PAP1 enzyme, phosphatidic acid (PA). In addition, we show that PA is a potent activator of the MEK-Erk pathway in Schwann cells, and that this activation is required for PA-induced demyelination. Our results therefore reveal a surprising role for PA in Schwann cell fate determination and provide evidence of a direct link between diseases affecting lipid metabolism and abnormal Schwann cell function.

Dioleoyl phosphatidic acid induces morphological changes through an endogenous LPA receptor in C6 glioma cells.

Previously, we suggested that dioleoyl phosphatidic acid (PA) and lysophosphatidic acid (LPA) increased [Ca(2+)](i) through endogenous LPA receptors coupled to pertussis toxin-sensitive G proteins in rat C6 glioma cells. In the present report, we investigated morphological changes and cytotoxicity induced by PA and LPA in C6 glioma cells. Isoproterenol treatment led to changes in the cell morphology of rat C6 glioma cells, which were reverted by the addition of PA and LPA. PA-and LPA-induced morphological reversions were inhibited by treatment with Ki16425, an LPA(1)/LPA(3) receptor antagonist. VPC32183, another LPA(1)/LPA(3) receptor antagonist with a different structure, only inhibited PA-induced morphological reversion but not LPA-induced reversion. However, the reversions were not inhibited by treatment with pertussis toxin, a specific inhibitor of G(i/o) proteins. In addition, cytotoxicity was only induced by LPA but not by PA in C6 glioma cells. Our results suggest that PA may act as a partial agonist at endogenous LPA receptors, which are sensitive to Ki16425 and coupled to PTX-insensitive G proteins, to evoke morphological changes in C6 glioma cells.