Phosphatidic Acid, Lysophosphatidic Acid, and Related Lipids
Phosphatidic acid or 1,2-diacyl-sn-glycero-3-phosphate is a key intermediate in the biosynthesis both of other glycerophospholipids and of triacylglycerols. It is structurally one of the simplest of the phospholipids and was long thought to be important only as a precursor of other lipids, where it is indeed a key molecule, but it is now known to have many other functions in animals, plants, and other organisms by its influence on membrane structure and dynamics, and by its interactions with various proteins. As a lipid mediator, it modulates various signalling and cellular processes, such as membrane tethering, conformational changes and enzymatic activities of specific proteins, and vesicular trafficking. Moreover, its metabolite lysophosphatidic acid is recognized as a key signalling molecule with a myriad of biological effects mediated through specific receptors.
1. Phosphatidic Acid – Occurrence and Biosynthesis
Phosphatidic acid is not an abundant lipid constituent of any living organism, seldom greater than picomolar concentrations in cells, but it is extremely important both as an intermediate in the biosynthesis of other glycerophospholipids and triacylglycerols and as a signalling molecule or a precursor of signalling molecules. Indeed, it is often over-estimated in tissues as it can arise by inadvertent enzymatic hydrolysis during inappropriate storage or extraction conditions during analysis. It is the simplest diacyl-glycerophospholipid, and the only one with a phosphomonoester as the head group. The molecule is acidic and carries a negative charge, i.e., it is an anionic lipid.
There are at least four important biosynthetic pathways for phosphatidic acid biosynthesis in different organelles under various conditions, and possibly resulting in the formation of different molecular species. The main pathway involves sequential acylation of sn‑glycerol-3-phosphate, derived from catabolism of glucose, by coenzyme A derivatives of fatty acids (acyl-CoA) as illustrated (see also the web page on biosynthesis of triacylglycerols). First, one type of acyltransferase catalyses the acylation of position sn-1 to form lysophosphatidic acid (1‑acyl-sn-glycerol-3-phosphate), and then a second specific acyltransferase catalyses the acylation of position sn‑2 to yield phosphatidic acid.
In mammals, the glycerol-3-phosphate acyltransferase that catalyses the first step exists in four isoforms, two in the mitochondrial outer membrane (designated GPAT1 and 2) and two in the endoplasmic reticulum (GPAT3 and 4); all are membrane-bound enzymes, which are believed to span the membranes. GPAT1 is highly expressed in the liver and adipose tissue, where it is responsive to changes in feeding status via the sterol regulatory element binding protein-1 (SREBP-1), a master transcriptional regulator of lipogenic enzymes. It is essential in directing fatty acyl-CoA esters towards glycerolipid synthesis as opposed to β-oxidation. GPAT3 is especially important for triacylglycerol storage in adipocytes, while GPAT4 is the main contributor to lysophosphatidic acid synthesis in liver and brown adipose tissue.
For the second step in phosphatidic acid biosynthesis, five mammalian acyl-CoA:lysophosphatidic acid acyltransferases are known of which three are in the endoplasmic reticulum (LPAAT or LPAT or AGPAT1, 2 and 3), with a further two (LPAT4 and 5) on the outer mitochondrial membrane. While LPAT1 and 2 have strict specificity for lysophosphatidic acid as acyl acceptor, other isoforms can esterify other lysophospholipids. Human LPAT1 showed higher activity with 14:0-, 16:0- and 18:2‑CoAs, while LPAT2 prefers 20:4-CoA and LPAT3 produces phosphatidic acid containing docosahexaenoic acid (22:6(n-3)); the last is especially important in retina and testes. LPAT4 and 5 have a preference for oleoyl-CoA and polyunsaturated acyl-CoAs as the acyl donor, suggesting a dual role in glycerolipid synthesis and remodelling (c.f. the Lands' cycle). The activity in the endoplasmic reticulum predominates in adipose tissue, but the mitochondrial forms are believed to be responsible for half the activity in liver. However, as there is traffic of phosphatidic acid between the mitochondria and endoplasmic reticulum for remodelling or for synthesis of other lipids, the relative contributions of the two can be difficult to assess.
In plants, the sn-glycerol-3-phosphate pathway exists both in plastids and at the endoplasmic reticulum with multiple isoforms of the two types of acyltransferase as well as differences in the acyl substrates. This is the main pathway towards structural lipids, and it is discussed in some detail in our web page on galactosyldiacylglycerols. In brief, most plant lipid biosynthesis begins with fatty acid biosynthesis in the chloroplasts. The acyltransferase ATS1 transfers 18:1 acyl groups from acyl-acyl carrier protein (acyl-ACP) to position sn-1 of glycerol 3-phosphate, before ATS2 transfers a palmitoyl group from ACP to position sn-2, producing phosphatidic acid at the inner leaflet of the chloroplast inner envelope membrane (IEM). Fatty acids intended for the endoplasmic reticulum are released from ACP in the chloroplast stroma by IEM-associated thioesterases, exported and then activated by acyl-CoA synthases of the outer envelope membrane to produce species with C18 fatty acids in both positions. Thus, those fatty acids used for phosphatidic acid biosynthesis in the endoplasmic reticulum are markedly different in composition from those in the plastids. Subsequently, phosphatidic acid in the plastids is utilized for biosynthesis of galactosyldiacylglycerols, while that in the endoplasmic reticulum is used for synthesis of triacylglycerols and phospholipids.
In bacteria, two families of enzymes are responsible for acylation of position sn-1 of glycerol-3-phosphate. One present in Escherichia coli, for example, utilizes the acyl-acyl carrier protein (acyl-ACP) products of fatty acid synthesis as acyl donors as well as acyl-CoA derived from exogenous fatty acids. In a second wider group of bacteria, including cyanobacteria, there are enzymes (PlsX and PlsY) that make use of the unique acyl donors, acyl-phosphates derived in part from acyl-ACP (see this web page), to acylate position sn-1. Acylation of position sn-2 in this instance is performed by a further family of enzymes (PlsC) that uses acyl-ACP as the acyl donor, although some bacterial species may use acyl-CoA also.
In animals, a second biosynthetic pathway utilizes dihydroxyacetone phosphate (DHAP) as the primary precursor for the peroxisomal enzyme, DHAP acyltransferase, which produces acyl-DHAP. This intermediate is converted to lysophosphatidic acid in a NADPH-dependent reaction catalysed by acyl-DHAP reductase, and this is in turn acylated to form phosphatidic acid by the same LPAT as in the previous mechanism. This pathway is of particular importance in the biosynthesis of ether lipids.
A third important route to phosphatidic acid is via hydrolysis of other phospholipids, but especially phosphatidylcholine, by the enzyme phospholipase D (or by a family or related enzymes of this kind), which utilizes water as the nucleophile to catalyse the hydrolysis of phosphodiester bonds in phospholipids. The enzyme is readily available for study in plants, where the special functions of phosphatidic acid have long been known (see below), but it is now recognized that phospholipase D is present in bacteria, yeasts and most animal cells. In the last, it exists in two main isoforms with differing specificities and cellular locations; PLD1 is found mainly in the Golgi-lysosome continuum, while PLD2 is present mainly in the plasma membrane. They are phosphoproteins, the activity of which is regulated by kinases and phosphatases and by binding to phosphatidylinositol-4,5-bisphosphate. In mitochondria, a distinctive enzyme of this type utilizes cardiolipin as substrate. Phospholipase D activity is dependent on and regulated by neurotransmitters, hormones, small monomeric GTPases, and lipids.
In addition to its function in generating phosphatidic acid mainly for signalling purposes but also for the maintenance of membrane composition, phospholipase D is involved in intracellular protein trafficking, cytoskeletal dynamics, cell migration and cell proliferation, partly through protein-protein interactions; it is considered to be important in inflammation and in cancer growth and metastasis as a downstream transcriptional target of proteins involved in the pathophysiology of these diseases. It also has an unusual activity as a guanine nucleotide exchange factor. By a transphosphatidylation reaction with ethanol, it generates phosphatidylethanol, a useful biomarker for ethanol consumption in humans.
Under some conditions, phosphatidic acid can be generated from 1,2-diacyl-sn-glycerols by the action of diacylglycerol kinases (see our web page on diacylglycerols), for example those produced from other phospholipids by the action of phospholipase C. Such enzymes appear to be ubiquitous in nature, although those in bacteria and yeast are structurally different from the mammalian enzymes. In plants, there are two classes of phospholipase C, a phosphatidylinositol-specific phospholipase C and one that hydrolyses structural lipids (phosphatidylcholine-specific or non-specific phospholipase C). Diacylglycerol kinases, of which at least ten isoforms (DGKα to DGKκ) exist with different sub-cellular locations and functions in animals, use ATP as the phosphate donor. While the epsilon isoform (DGKε) utilizes the 1-stearoyl-2-arachidonoyl species of diacyl-sn-glycerols preferentially to produce phosphatidic acid for the biosynthesis of phosphatidylinositol, other isoenzymes phosphorylate diverse diacylglycerol species. Aside from producing phosphatidic acid for phospholipid production or signalling, these enzymes may attenuate the signalling effects of diacylglycerols. For example, diacylglycerol kinases can contribute to cellular asymmetry and control the polarity of cells by regulating the gradients in diacylglycerol and phosphatidic acid concentrations.
The reverse reaction, i.e., hydrolysis by lipins (phosphatidic acid phosphohydrolases), is discussed in our web page on triacylglycerols and briefly below. These enzymes are of importance in regulating the local concentrations of phosphatidic acid and thence its biological activity.
A further possible route to phosphatidic acid production for signalling specifically is via acylation of lysophosphatidic acid, which can be produced independently for signalling purposes as discussed below. This pathway may be especially relevant in membranes, where the protein endophilin has LPAT activity and is believed to generate phosphatidic acid from lysophosphatidic acid in order to alter the curvature of the membrane bilayer.
2. Phosphatidic Acid - Role as a Lipid Precursor
In summary, phosphatidic acid generated via 1-acyl-sn-glycerol-3-phosphate is the primary precursor of other glycerolipids, although other pathways may be more important for generating this lipid for signalling functions. Whether separate pools of phosphatidic acid for specific purposes really exist is not certain, as dynamic changes of intracellular distribution occur under various cellular conditions. These are attributed to inter-organelle transfer via vesicular transport or at membrane contact sites by lipid transfer proteins. Control of its concentration in membranes, especially in the endoplasmic reticulum, is therefore of great importance, and a transcriptional repressor 'Opi1', which binds specifically to phosphatidic acid in membranes, is a key regulatory factor. However, many other phosphatidic acid-binding proteins have been identified that influence how it is used either as a biosynthetic precursor or for signalling purposes. The mechanisms for phosphatidic acid homeostasis differ among animals, plants, yeasts, and bacteria in response to the differing functional requirements in these organisms.
In addition to dietary, hormonal and tissue-specific factors in animals, the extent to which fatty acids are channelling either into triacylglycerol synthesis for storage in lipid droplets and secretion in lipoproteins or into glycerophospholipids for membrane formation depends to a large extent upon the enzymes of glycerol-3-phosphate pathway, their isoform expression, activities, and locations. On the other hand, phosphatidic acid is not only a biosynthetic precursor of other lipids but also a regulatory molecule in the transcriptional control of the genes for glycerolipid synthesis, and regulation of its concentration in cells for this purpose is essential. For example, the local concentration of phosphatidic acid in the endoplasmic reticulum is an important factor in the biogenesis of lipid droplets.
The subsequent steps in the utilization of phosphatidic acid in the biosynthesis of triacylglycerols and of the various glycerophospholipids are described in separate documents of this website. Thus, hydrolysis of phosphatidic acid by phosphatidate phosphatase enzymes (including lipins 1, 2 and 3) is the source of most other glycerolipids, e.g. sn‑1,2‑diacylglycerols (DG), which are the precursors for the biosynthesis of triacylglycerols (TAG), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) via the so-called Kennedy pathway (also of monogalactosyldiacylglycerols in plants). Via reaction with cytidine triphosphate, phosphatidic acid is the precursor of cytidine diphosphate diacylglycerol, which is the key intermediate in the synthesis of phosphatidylglycerol (PG), and thence of cardiolipin (CL), and of phosphatidylinositol (PI), and in prokaryotes and yeast but not animals, phosphatidylserine (PS). Depending on the organism and other factors, phosphatidylserine can be a precursor for phosphatidylethanolamine, while the latter can give rise to phosphatidylcholine by way of mono- and dimethyl-phosphatidylethanolamine intermediates. The cytidine diphosphate diacylglycerol synthase is another enzyme that consumes phosphatidic acid and is important for modulating the concentration of phosphatidic acid in cells and for regulating processes mediated by this lipid.
While the fatty acid composition of phosphatidic acid can resemble that of the eventual products, the latter are generally much altered by re-modelling after synthesis via deacylation-reacylation reactions (the Lands' cycle - see the web page on phosphatidylcholine).
3. Phosphatidic Acid - Biological Functions in Animals
In addition to its role as an intermediate in lipid biosynthesis, phosphatidic acid and especially that generated by the action of phospholipase D and by diacylglycerol kinases may have signalling functions as a second messenger, although it is not certain whether all the activities suggested by studies in vitro operate in vivo. Nonetheless, phosphatidic acid has been implicated in many aspects of animal cell biochemistry and physiology.
Some of the observed effects may be explained simply by the physical properties of phosphatidic acid, which has a propensity to form a hexagonal II phase, especially in the presence of calcium ions. Thus, hydrolysis of phosphatidylcholine, a cylindrical non-fusogenic lipid, converts it into cone-shaped phosphatidic acid, which promotes negative membrane curvature and fusion of membranes. It differs from other anionic phospholipids in that its small anionic phosphomonoester head group lies very close to the hydrophobic interior of the lipid bilayer. In model systems, phosphatidic acid can bring about membrane fusion, probably because of its ability to form non-bilayer phases. For example, phosphatidic acid biosynthesis is believed to favour intraluminal budding of endosomal membranes with the formation of exosomes, and in many cell types, vesicle trafficking, secretion, and endocytosis may require phosphatidic acid derived from the action of phospholipase D.
Also of relevance in this context is its overall negative charge, and it is not always clear whether some of the observed biological effects are specific to phosphatidic acid or simply to negatively charged phospholipids in general. In contrast to phosphoinositide-interacting proteins, which have defined structural folds, the binding motifs of effector proteins with phosphatidic acid are not highly conserved. However, it has been demonstrated that the positively charged lysine and arginine residues of proteins can bind with some specificity to phosphatidic acid through hydrogen bonding with the phosphate group, thus distinguishing it from other phospholipids. An ‘electrostatic-hydrogen bond switch model’ has been proposed in which the head group of phosphatidic acid forms a hydrogen bond to an amino acid residue, leading to de-protonation of the head group, increasing its negative charge from -1 to -2, and thus enabling stronger interactions with basic residues and tight docking with the membrane interacting protein. In this way, phosphatidic acid can tether certain proteins to membranes, and it can simultaneously induce conformational changes, hinder ligand binding, and/or oligomerize proteins to alter their catalytic activity, stability, and interactions with other molecules. It functions as a cellular pH sensor in effect in that binding to proteins is dependent on intracellular pH and the protonation state of its phosphate headgroup.
One key target of the lipid is mTOR, a serine/threonine protein kinase with a signalling cascade that regulates cell growth, proliferation, motility, and survival, together with protein synthesis and transcription, by integrating both nutrient and growth factor signals. This forms two distinct complexes of accessory proteins that regulate downstream targets. Of these, mTORC1 interacts directly with phosphatidic acid, and this interaction allosterically activates the enzyme complex to regulate protein synthesis, mitochondrial metabolism, and the transcription of enzymes involved in lipid synthesis. In contrast, phosphatidic acid appears to inhibit mTORC2 activity, for example in relation to insulin signalling.
Phosphatidic acid is believed to regulate membrane trafficking events, and it is involved in activation of the enzyme NADPH oxidase, which operates as part of the defence mechanism against infection and tissue damage during inflammation. By binding to targeted proteins, including protein kinases, protein phosphatases and G-proteins, it may increase or inhibit their activities. Effects on gene transcription have been observed that are linked to inhibition of peroxisome proliferator-activated receptor (PPAR) activity. In yeast, phosphatidic acid in the endoplasmic reticulum binds directly to a specific transcriptional repressor to keep it inactive outside the nucleus; when the lipid precursor inositol is added, this phosphatidic acid is rapidly depleted, releasing the transcriptional factor so that it can be translocated to the nucleus where it is able to repress target genes. The overall effect is a mechanism to control phospholipid synthesis.
In addition, phosphatidic acid regulates many aspects of phosphoinositide function. For example, the murine phosphatidylinositol 4‑phosphate 5-kinase, the main enzyme generating the lipid second messenger phosphatidylinositol 4,5-bisphosphate, does not appear to function unless phosphatidic acid is bound to it; this lipid, generated by the action of phospholipase D, recruits the kinase to the membrane and induces a conformational change that regulates its activity. It may have a role in promoting phospholipase A2 activity, a key enzyme in eicosanoid production from phosphoinositide precursors.
In relation to signalling activities, it should be noted that phosphatidic acid can be metabolized to sn-1,2-diacylglycerols or to lysophosphatidic acid (see next section), both of which have distinctive signalling functions in their own right. Conversely, both of these lipids can in effect be de-activated by conversion back to phosphatidic acid.
Phospholipase D isoforms and phosphatidic acid have been implicated in a variety of pathologies, including neurodegenerative diseases, blood disorders, late-onset Alzheimer's disease and cancer, leading to attempts to develop specific inhibitors of the enzyme for therapeutic purposes. Similarly, the expression of LPAT isoforms can enhance the proliferation and chemoresistance of some cancer cells. Diacylglycerol kinase alpha (DGKα) is highly expressed in several refractory cancer cells, where it attenuates apoptosis and promotes proliferation. In addition, DGKα is highly abundant in T cells and induces a nonresponsive state, which enables advanced cancers to escape immune action. Inhibition of this enzyme also is seen as a promising treatment strategy.
4. Phosphatidic Acid - Biological Functions in Plants
Phosphatidic acid is present at higher levels in roots of plants in comparison to leaves and is believed to have a function in root architecture. Similarly, its concentration is elevated in flowers and reproductive tissues, but the significance of this is not known. In addition to its role as one of the central molecules in lipid biosynthesis, it facilitates the transport of lipids across plant membranes, and it is also the key plant lipid second messenger, which is rapidly and transiently generated in response to many different biotic and abiotic stresses. In contrast to animal metabolism, the diacylglycerol signalling pathway is believed to be relatively insignificant in plants. One further difference from animal metabolism is that diacylglycerol pyrophosphate can be synthesised from phosphatidic acid in plants (see below).
The main source of phosphatidic acid for signalling purposes is the action of phospholipase D (PLD) on membrane phospholipids, such as phosphatidylcholine and phosphatidylethanolamine. Plants contain numerous related enzymes of this type, 12 in Arabidopsis and 17 in rice, in comparison with two in humans and one in yeast, and individual iso-enzymes can be characteristic of certain tissues and may elicit specific responses. In the former, the isoforms are grouped into six classes, based on the genic architecture, sequence similarities, domain structures, and biochemical properties. These depend mainly on their lipid-binding domains, with some homologous to the human and yeast enzymes and with most containing a characteristic ‘C2’ (calcium- and lipid-binding) domain. The most widespread of these is PLDα, which does not require binding to phosphatidylinositol 4,5-bisphosphate, in contrast to other PLD isoforms and the mammalian enzyme, but millimolar levels of Ca2+ are necessary. The composition of the molecular species formed depends on the nature of the stimulus and that of the phospholipase D isoform. Studies with fluorescent biosensors suggest that phosphatidic acid accumulates in the subapical region of the cytosolic leaflet of the plasma membrane.
Many phosphatidic acid-binding proteins have been identified that play a role in plant signal transduction, although no characteristic recognition motif has yet been identified. It is involved in many different cell responses induced by hormones, stress, and developmental processes, and it often acts in concert with phosphatidylinositol 4,5-bisphosphate by binding to specific proteins rather than acting via a receptor. As in mammalian cells, targets for such signalling include protein kinases and phosphatases in addition to proteins involved in membrane trafficking and the organization of the cytoskeleton. It can both activate or inhibit enzymes. If the target protein is soluble, binding to phosphatidic acid can cause the protein to be sequestered into a membrane with effects upon downstream targets.
For example, phosphatidic acid is required to bind and allosterically activate the monogalactosyldiacylglycerol synthase (MGDG1), located in the inner envelope membrane of the chloroplast, and it may be a regulator of the biosynthesis of thylakoid membranes. Phospholipase D activity and the phosphatidic acid produced have long been recognized as of importance during seed germination and leaf senescence, and they have an essential role in the response to stress damage and pathogen attack, both in higher plants and in green algae. A high content of phosphatidic acid induced by phospholipase D action during wounding or senescence brings about a loss of the membrane bilayer phase, because of the conical shape of this negatively charged phospholipid in comparison to the cylindrical shape of structural phospholipids. This change in ionization properties has crucial effects upon lipid-protein interactions, "the electrostatic-hydrogen bond switch model" described above. By promoting negative curvature at the plasma membrane and binding to clathrin proteins, it is believed to facilitate the process of endocytosis. Similar phenomena may explain why phosphatidic acid is important in the response to other forms of stress, including osmotic stress (salinity or drought), cold and oxidation.
Although much remains to be learned of the mechanism by which it exerts its effects, phosphatidic acid is believed to promote the response to the plant hormone abscisic acid, and it may interact with salicylic acid to mediate defence responses. It is involved in promoting the growth of pollen-tubes and root hairs, decreasing peroxide-induced cell death, and mediating the signalling processes that lead to responses to ethylene and again to the hormone abscisic acid. Thus, in the 'model' plant Arabidopsis, phosphatidic acid interacts with a protein phosphatase to signal the closure of stomata promoted by abscisic acid; it interacts also with a further enzyme to mediate the inhibition of stomatal opening effected by abscisic acid. Together these reactions constitute a signalling pathway that regulates water loss from plants.
It is noteworthy that phosphatidic acid production can be initiated by opposing stress factors, such as cold and heat, as well as by hormones that are considered to be antagonistic, such as abscisic acid and salicylic acid. There is evidence that phosphatidic acid molecules synthesised by the two main pathways differ in composition and cellular distributions and so may produce different responses. Certainly, during low temperature stress, phosphatidic acid is generated by the action of diacylglycerol kinase. It also seems likely that these differing activities are controlled by the cellular environment where the lipid is produced and by the availability of target proteins or other molecules with which it can act synergistically. In addition, phosphatidic acid is of importance for the interactions of plants with pathogens, nitrogen-fixing bacteria, and arbuscular mycorrhizal fungi. Genes encoding enzymes involved in phosphatidic acid metabolism have been manipulated to explore their potential application for crop improvements, based on effects on plant growth, development, and stress responses.
As in animals, phosphatidic acid is catabolized and its signalling functions are terminated by lipid phosphate phosphatases and phosphatidic acid hydrolases, and by acyl-hydrolases and lipoxygenases with the production of fatty acids and other small molecules, which are subsequently absorbed and recycled.
5. Lysophosphatidic Acid
Lysophosphatidic acid (LPA) or 1-acyl-sn-glycerol-3-phosphate differs structurally from phosphatidic acid in having only one mole of fatty acid per mole of lipid, and as such, it is one of the simplest possible glycerophospholipids. It exists in the form of many different molecular species, i.e., esterified to 16:0 to 22:6 fatty acids, and there is preliminary evidence that saturated and polyunsaturated species may differ in their biological properties in some circumstances. As the sn-1-acylated form is more stable thermodynamically, facile isomerization ensures that this tends to predominate. As it lacks one fatty acid in comparison to phosphatidic acid, it is a much more hydrophilic molecule, while the additional hydroxyl group strengthens hydrogen bonding within membranes, properties that may be important for its function in cells.
Although lysophosphatidic acid is present at very low levels only in animal tissues, it is extremely important biologically, influencing many biochemical processes. It is a biosynthetic precursor of phosphatidic acid, but there is particular interest in its role as a lipid mediator with growth factor-like activities. For example, it is rapidly produced and released from activated platelets to influence target cells.
Biosynthesis: In the circulation, the most important source of lysophosphatidic acid is the activity of an enzyme with lysophospholipase D-like activity and known as ‘autotaxin’ on lysophosphatidylcholine (200 μM in plasma) to yield LPA in an albumin-bound form mainly, although it is also relatively soluble in aqueous media because of its polarity and small size. This lipid is more abundant in serum (1 to 5 μM) than in plasma (100 nM), because of the release of its main precursor, lysophosphatidylcholine, from activated platelets during coagulation. Autotaxin is a member of the nucleotide pyrophosphatase-phosphodiesterase family and is also present in cerebrospinal and seminal fluids and many other tissues, including cancer cell lines from which it was first isolated and characterized. Indeed, the name derives from the finding that it promoted chemotaxis on melanoma cells in an autocrine fashion. It binds to target cells via integrin and heparan sulfate proteoglycans, and this may assist the delivery of lysophosphatidic acid to its receptors. Genetic deletion of the enzyme in mice results in aberrant vascular and neuronal development and soon leads to death of the embryos. However, overexpression of autotaxin causes physical defects also and is eventually lethal to embryos.
While autotaxin is the primary source of extracellular lysophosphatidic acid, it is now established that it is produced intracellularly by a wide variety of cell types by various mechanisms often with phosphatidic acid, derived from other phospholipids by the action of phospholipase D, as the primary precursor. For example, hydrolysis of phosphatidic acid by a phospholipase A2 (PLA2) is the main mechanism in platelets, but other cellular enzymes involved include a phosphatidic acid-selective phospholipase A1 (PLA1) producing sn‑2‑acyl-lysophosphatidic acid, a monoacylglycerol kinase (utilizing monoacylglycerols produced by the action of lipid phosphate phosphatases) and glycerol-3-phosphate acyltransferase (the first step in phosphatidic acid biosynthesis). Upon stimulation by pro-inflammatory cytokines, secretory PLA2-IIA (sPLA2-IIA) can induce the release of LPA from phosphatidic acid exposed on the surface of extracellular vesicles derived from platelets and Ca2+-loaded erythrocytes.
General function: Although lysophospholipids are relatively small molecules, they carry a high content of information through the nature of the phosphate head group, the positional distribution of the fatty acids on the glycerol moiety, the presence of ether or ester linkages to the glycerol backbone, and the chain-length and degree and position of unsaturation of the fatty acyl chains. Lysophosphatidic acid acts upon nearly all cell types, often as a proliferative and pro-survival signal, inducing cellular invasion, migration and differentiation, while stimulating smooth muscle and fibroblast contraction, cytoskeletal rearrangement, secretion of cytokines/chemokines, and numerous other effects. Many of these activities are displayed also by the 1-O-alkyl- and alkenyl-ether forms, which can be derived from platelet-activating factor. On the other hand, it is possible that much of the lysophosphatidic acid produced intracellularly is used for synthesis of other phospholipids rather than for signalling purposes.
Receptors: The informational content of the lysophosphatidic acid molecule leads to selectivity in the functional relationship with cell receptors. As most mammalian cells express receptors for lysophosphatidic acid, this lipid may initiate signalling in the cells in which it is produced, as well as affecting neighbouring cells. Characterization of cloned lysophosphatidic acid receptors in combination with strategies of molecular genetics has allowed determination of both signalling and biological effects that are dependent on receptor mechanisms. At least six G protein-coupled receptors that are specific for lysophosphatidic acid have now been identified in vertebrates, and designated LPAR1 to LPAR6, each found in particular organs and coupled to at least one or more of the four heterotrimeric Gα. Of these receptors, LPAR1 is virtually ubiquitous in tissues. These vary appreciably amino acid sequences but are classified into two subgroups, the EDG (LPAR1-3) and P2Y (LPAR4-6) families, with differing tissue distributions. Most cell types express these receptors in different combinations. There is also some interaction with transient receptor potential cation channel V1 (TRPV1), peroxisome proliferator-activated receptor gamma (PPARγ), and other proteins. Plasma lysophosphatidic acid binds to its receptors while it is bound to albumin.
Experimental activation of the LPAR receptors has shown that a range of downstream signalling cascades are mediated by lysophosphatidic acid signalling. These include activation of adenylyl cyclase, cAMP production, intracellular Ca2+ and K+ production (by activating ion channels), protein kinases, phospholipase C, phosphatidylinositol 3-kinase, small GTPases (Ras, Rho, Rac), release of arachidonic acid, and much more. In this way, lysophosphatidic acid regulates cell survival, proliferation, cytoskeleton re-arrangement, motility, cytokine secretion, cell differentiation, and many other vital cellular processes. Sometimes, lysophosphatidic acid appears to function in contradictory ways, and there is evidence that it is involved in cell survival in some circumstances and in programmed cell death in others, for example.
Signalling by lysophosphatidic acid has regulatory functions in the mammalian reproductive system, both male and female, facilitating oocyte maturation and spermatogenesis through the action of the receptors LPAR1 to LPAR3. During early gestation, it regulates vascular remodelling at the maternal-foetal interface. There is also evidence that the lipid is involved in brain development, through its activity in neural progenitor cells, neurons, and glia, and in vascular remodelling. In the central nervous system, these receptors are thought to play a central role in both triggering and maintaining neuropathic pain by mechanisms that may involve demyelination of damaged nerves.
Lysophosphatidic acid has been found in saliva in significant amounts, and it has been suggested that it is involved in wound healing in the upper digestive organs such as the mouth, pharynx, and oesophagus. When applied topically to skin wounds, it has similar effects probably by stimulating proliferation of new cells to seal the wound. Receptor LPAR6 together with the phospholipase A1 is required for the development of hair follicles, and this receptor is also involved in the regulation of endothelial blood-brain barrier function. The proliferation and survival of stem cells and their progenitors is regulated by lysophosphatidic acid signalling, while acting via LPAR1 in bone cells, lysophosphatidic acid is important for bone mineralization and repair.
Cancer: There is great interest in the activity of lysophosphatidic acid in various disease states and cancer especially, as increased expression of autotaxin and subsequent increased levels of lysophosphatidic acid have been reported in several primary tumours. For example, a finding that lysophosphatidic acid is markedly elevated in the plasma and peritoneal fluid (ascites) of ovarian cancer patients compared to healthy controls may be especially significant. Also, elevated plasma levels of this lipid were found in patients in the first stage of ovarian cancer, suggesting that it may represent a useful marker for the early detection of the disease. It is believed that the secretory form of phospholipase A2 acts preferentially on lipids from damaged membranes or microvesicles, such as those produced by malignant cells, and this eventually results in increased levels of lysophosphatidic acid. This has been shown to stimulate the expression of genes for many different enzymes that lead to the proliferation of ovarian and other cancer cells and may induce cell migration via receptors LPAR1 to LPAR3 and possibly LPAR6, while LPAR4 and LPAR5 have opposing effects. Autotaxin and LPARs have been implicated in resistance to chemotherapy and radiation treatment during cancer therapy.
As lysophosphatidic acid has growth-factor-like activities that induce cell proliferation and migration in many cell types, changes in cellular shape, and increases of endothelial permeability, it is perhaps not surprising that it is relevant to tumour biology. Treatment of various cancer cell types with lysophosphatidic acid promotes the expression and release of interleukin 8 (IL-8), which is a potent angiogenic factor, and thus it has a critical role in the growth and spread of cancers by enhancing the availability of nutrients and oxygen. There is evidence that signalling by lysophosphatidic acid is causally linked to hyperactive lipogenesis in cancer. For example, it activates the sterol regulatory element-binding protein (SREBP), together with the fatty acid synthase and AMP-activated protein kinase–ACC lipogenic cascades that lead to elevated synthesis of lipids de novo. Increased autotaxin expression has been demonstrated in many different cancer cell lines, and the expression of many of the surface receptors for lysophosphatidic acid in cancer cells is aberrant. Cancer cells must evade the immune system during metastasis, and lysophosphatidic acid facilitates this process by inhibiting the activation of T cells. Therefore, lysophosphatidic acid metabolism is a target of the pharmaceutical industry in the search for new drugs for cancer therapy, aided by a knowledge of the crystal structures of three of the receptors. At least one inhibitor of autotaxin is under evaluation in a phase III clinical trial.
Inflammation: Signalling by lysophosphatidic acid has been implicated in many aspects of chronic inflammation, which it promotes by affecting the endothelium in several ways, for example by stimulating endothelial cell migration, the secretion of chemokines-cytokines, and regulating the integrity of the endothelial barrier. Problems with lysophosphatidic acid signalling together with changes in autotaxin expression are believed to be factors in such metabolic and inflammatory disorders as obesity, insulin resistance, non-alcoholic fatty liver disease, rheumatoid arthritis, multiple sclerosis, and cardiovascular disease. Further, there is evidence it contributes to neurological disorders, such as Alzheimer's disease and neuropathic pain, and to asthma, fibrosis, and bone malfunction. Drugs that interact with the lysophosphatidic acid receptors are reported to be effective in attenuating symptoms of several diseases in animal models, and three have passed phase I and II clinical trials for idiopathic pulmonary fibrosis and systemic sclerosis in human patients. Drugs that target autotaxin production and catabolism of lysophosphatidic acid are also in development, and the steroidal anti-inflammatory agent, dexamethasone, appears to be especially useful.
Under certain conditions, lysophosphatidic acid can become athero- and thrombogenic and might aggravate cardiovascular disease. As oxidized low-density lipoproteins promote the production of lysophosphatidic acid, its content in atherosclerotic plaques is high, suggesting that it might serve as a biomarker for cardiovascular disease. Indeed, lysophosphatidic acid promotes pro-inflammatory events that lead to the development of atheroma as well encouraging progression of the disease. By mediating platelet aggregation, it could lead to arterial thrombus formation.
Related lipids: The sphingolipid analogue, sphingosine-1-phosphate, shows a similar range of activities to lysophosphatidic acid and the two lipids are often discussed together in the same contexts, although they may sometimes have opposing effects. Acute leukemia cells produce methyl-lysophosphatidic acids (the polar head-group is methylated). As these act as antigens to which a specific group of human T cells react strongly, it is possible that they might be a target for the immunotherapy of hematological malignancies. Other lysophospholipids are known to have distinctive biological functions (see separate web pages).
Catabolism: Deactivation of lysophosphatidic acid is accomplished by dephosphorylation to produce monoacylglycerols by a family of three lipid phosphate phosphatases (LPP1, 2 and 3), which also de-phosphorylate sphingosine-1-phosphate, phosphatidic acid, and ceramide 1-phosphate in a non-specific manner. These are integral membrane proteins with the active site in the plasma membrane facing the extracellular environment, enabling them to access and hydrolyse extracellular lysophosphatidic acid and other phospholipids. Mice with a constitutive LPP3 deficiency are not viable, but this is not true for LPP1 and LPP2 knockout mice. Lysophosphatidic acid can be converted back to phosphatidic acid, and in effect deactivated, by a membrane-bound O-acyltransferase (MBOAT2), which is specific for lysophosphatidic acid (and lysophosphatidylethanolamine) with a preference for oleoyl-CoA as substrate.
6. Cyclic Phosphatidic Acid
Cyclic phosphatidic acid (sometimes termed ‘cyclic lysophosphatidic acid or cPA’) was isolated originally from a slime mould, but it has since been detected in a wide range of organisms including humans, especially in the brain but also bound to albumin in serum (at a concentration of 10-7M, or a tenth that of lysophosphatidic acid), and it is most abundant in tissues subject to injury). It has a cyclic phosphate at the sn-2 and sn-3 positions of the glycerol carbons, and this structure is necessary for its biological activity. In human serum, the main molecular species contains palmitic acid, though 18:0, 18:1, and 18:2 are also present. An unusual plasmenylcyclic phosphatidic acid has been isolated from the intestinal bacterium Bifidobacterium longum subs. infantis.
Studies of the biosynthesis of cyclic phosphatidic acid in fetal bovine serum suggest that it is the product of the human enzyme autotaxin, the serum lysophospholipase D that produces lysophosphatidic acid (see above), or by the action of phospholipase D2 after appropriate stimulation. These enzymes appear to produce cyclic phosphatidic acid in serum by an intramolecular transphosphatidylation reaction (this also occurs by the action of a phospholipase D in the venom of the brown spider on lysophosphatidylcholine). However, it can be formed artefactually by the addition of strong acid to serum.
While cyclic phosphatidic acid may have some similar signalling functions to lysophosphatidic acid per se in that it binds to some of the same receptors, it also has some quite distinct activities in animal tissues. For example, cyclic phosphatidic acid is known to be a specific inhibitor of DNA polymerase alpha. It has an appreciable effect on the inhibition of cancer cell invasion and metastasis, a finding that is currently attracting great pharmacological interest; derivatives of cPA, in which the sn-2 or sn-3 oxygen of the glycerol backbone is replaced by a methylene group ('2- and 3-carba-cPA') are stable analogues that are being tested for this purpose. In addition, it inhibits the platelet aggregation induced by lysophosphatidic acid, possibly by inhibiting autotaxin. In the central nervous system, it can enhance cell survival and neurite extension in neurons, while exerting neuroprotective effects against apoptosis. It has beneficial effects in an animal model of multiple sclerosis, it attenuates neuropathic pain, and it relieves the symptoms of osteoarthritis. In skin, it improves hydration by stimulating the synthesis of hyaluronic acid, a major component of the extracellular matrix. Cyclic phosphatidic acid is a high-affinity and specific ligand for the nuclear receptor PPARγ, which is involved in the regulation of adipogenesis, glucose homoeostasis, and processes related to type 2 diabetes.
7. Pyrophosphatidic Acid
Pyrophosphatidic acid or sn-1,2-diacylglycero-3-pyrophosphate is an unusual and little-known phospholipid that was first identified as a minor component in yeasts, and is also known to be present in mushrooms and higher plants as a product of the enzyme phosphatidic acid kinase, which is present in all plant tissues but especially the plasma membrane.
It is rapidly metabolized back to phosphatidic acid by a specific phosphatase and thence to diacylglycerols, and it may have a function in the phospholipase D signalling cascade in plants, perhaps by attenuating the effects of phosphatidic acid. Pyrophosphatidic acid is barely detectable in non-stimulated plant cells, but its concentration increases very rapidly in response to stress situations, including osmotic stress and attack by pathogens. Such findings add to the belief that it is an important signalling molecule in plants under stress, especially in relation to abscisic acid responses. In yeasts, it may have a role in the regulation of the synthesis and metabolism of phospholipids, especially phosphatidylserine.
Phosphatidic acid and related lipids are not the easiest to analyse. On adsorption chromatography, retention times tend to be variable and may be dependent to some extent on the nature of the cations associated with the acidic lipids, but two-dimensional TLC can give good results. Phosphatidic acid, bis(monoacyl)glycerophosphate and pyrophosphatidic acid are never easy to distinguish, but modern liquid chromatography-mass spectrometric methods appear to be the answer.
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