Role of sphingosine 1-phosphate and lysophosphatidic acid in fibrosis

This review highlights an emerging role for sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) in many different types of fibrosis. Indeed, both LPA and S1P are involved in the multi-process pathogenesis of fibrosis, being implicated in promoting the well-established process of differentiation of fibroblasts to myofibroblasts and the more controversial epithelial–mesenchymal transition and homing of fibrocytes to fibrotic lesions. Therefore, targeting the production of these bioactive lysolipids or blocking their sites/mechanisms of action has therapeutic potential. Indeed, LPA receptor 1 (LPA₁) selective antagonists are currently being developed for the treatment of fibrosis of the lung as well as a neutralising anti-S1P antibody that is currently in Phase 1 clinical trials for treatment of age related macular degeneration. Thus, LPA- and S1P-directed therapeutics may not be too far from the clinic. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Regulation of autotaxin expression and secretion by lysophosphatidate and sphingosine 1-phosphate

Autotaxin (ATX) is a secreted enzyme, which produces extracellular lysophosphatidate (LPA) from lysophosphatidylcholine (LPC). LPA activates six G protein-coupled receptors and this is essential for vasculogenesis during embryonic development. ATX is also involved in wound healing and inflammation, and in tumor growth, metastasis, and chemo-resistance. It is, therefore, important to understand how ATX is regulated. It was proposed that ATX activity is inhibited by its product LPA, or a related lipid called sphingosine 1-phosphate (S1P). We now show that this apparent inhibition is ineffective at the high concentrations of LPC that occur in vivo. Instead, feedback regulation by LPA and S1P is mediated by inhibition of ATX expression resulting from phosphatidylinositol-3-kinase activation. Inhibiting ATX activity in mice with ONO-8430506 severely decreased plasma LPA concentrations and increased ATX mRNA in adipose tissue, which is a major site of ATX production. Consequently, the amount of inhibitor-bound ATX protein in the plasma increased. We, therefore, demonstrate the concept that accumulation of LPA in the circulation decreases ATX production. However, this feedback regulation can be overcome by the inflammatory cytokines, TNF-α or interleukin 1β. This enables high LPA and ATX levels to coexist in inflammatory conditions. The results are discussed in terms of ATX regulation in wound healing and cancer.     

Modulation of cell interactions with extracellular matrix by lysophosphatidic acid and sphingosine 1-phosphate

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (SPP) are lipid mediators released upon platelet activation. The concentration of LPA in serum is estimated at 1-10 &mgr;M whereas the concentration in plasma is considerably less [1]. The SPP concentration in serum is 0.5 &mgr;M, approximately two-fold higher than the plasma concentration [1]. The lipids are present during tissue injury and promote cellular processes involved in wound repair [2]. LPA and SPP have multiple effects on cells, many of which are pertinent to wound healing and require that the cells interact in some fashion with components of the extracellular matrix. This review focuses on modulation of cell adhesion, cell migration, collagen gel contraction, and fibronectin matrix assembly by LPA and SPP.     

Modulation of cell interactions with extracellular matrix by lysophosphatidic acid and sphingosine 1-phosphate

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (SPP) are lipid mediators released upon platelet activation. The concentration of LPA in serum is estimated at 1-10 microM whereas the concentration in plasma is considerably less. The SPP concentration in serum is 0.5 microM, approximately two-fold higher than the plasma concentration. The lipids are present during tissue injury and promote cellular processes involved in wound repair. LPA and SPP have multiple effects on cells, many of which are pertinent to wound healing and require that the cells interact in some fashion with components of the extracellular matrix. This review focuses on modulation of cell adhesion, cell migration, collagen gel contraction, and fibronectin matrix assembly by LPA and SPP.     

Differential effects of sphingosine 1-phosphate and lysophosphatidic acid on endothelial cells

This review discusses multiple effects of sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) on endothelial cells and proposes that S1P and LPA are important regulators of the vascular system. Two physiologic sources of S1P and LPA are platelets and lipoproteins. S1P is an inducer of angiogenesis in vivo whereas LPA is not. S1P and LPA act through endothelial cell surface Edg receptors. S1P stimulates endothelial cell migration, but inhibits migration of most nonendothelial cells. Edg1 and Edg3 receptors, working through G(i), play an important role in regulation of S1P-stimulated endothelial cell migration. LPA effects on endothelial cells are more restricted than the effects of S1P on endothelial cells. LPA stimulates migration of certain endothelial cells on certain extracellular matrix proteins. However, LPA acts like S1P in its effects on the endothelial cell cytoskeleton, proliferation, cell-cell adhesion molecule expression, and vascular permeability. LPA receptors on endothelial cells are likely Edg2 and Edg4. Future studies should better delineate the roles of Edg receptors and downstream pathways on effects of extracellular S1P and LPA and the contributions of intracellularly generated S1P and nitric oxide (NO).     

Ca2+ signaling induced by sphingosine 1-phosphate and lysophosphatidic acid in mouse B cells

Lysophospholipids (LPLs) such as lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are chemotactic for lymphocytes, and increases of in cytosolic [Ca²⁺] signal the regulation of lymphocyte activation and migration. Here, the authors investigated the effects of LPA and S1P on [Ca²⁺]_c in mouse B cell lines (WEHI-231 and Bal-17) and primary B cells isolated from mouse spleen and bone marrow, and focused on the modulation of store-operated Ca²⁺ entry (SOCE) by LPLs. In Bal-17 (a mature B cell line) both LPA and S1P induced a transient [Ca²⁺]_c increase via a phospholipase C pathway. In addition, pretreatment with LPLs was found to augment thapsigargin-induced SOCE in Bal-17 cells. However, in WEHI-231 (an immature B cell line) LPLs had no significant effect on [Ca²⁺]_c or SOCE. Furthermore, in freshly isolated splenic B cells (SBCs) and bone marrow B cells (BMBCs), LPLs induced only a small increase in [Ca²⁺]_c. Interestingly, however, pretreatment with LPLs markedly increased SOCE in primary B cells, and this augmentation was more prominent in BMBCs than SBCs. The unidirectional influx of Ca²⁺ was measured using Ba²⁺ as a surrogate ion. Similarly, Ba²⁺ influx was also found to be markedly increased by LPLs in SBCs and BMBCs. Summarizing, LPLs were found to strongly augment SOCE-mediated Ca²⁺-signaling in mouse B cells. However, unlike the mature Bal-17 cell line, PLC-dependent Ca²⁺ release was insignificant in primary B cells and inWEHI-231.     

Multiple mechanisms linked to platelet activation result in lysophosphatidic acid and sphingosine 1-phosphate generation in blood

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (Sph1P) production was examined in vitro under conditions that simulated blood clotting. Several approaches were utilized to elucidate the metabolic pathways. 1) Platelet phospholipids were labeled using [32P]orthophosphate, and the production of [32P]Sph1P and LPA was examined. Thrombin stimulation of platelets resulted in rapid secretion of Sph1P stored within the platelet. In contrast, LPA was neither stored within nor secreted from platelets. Nonetheless, extracellular levels of LPA gradually increased following stimulation. 2) Stable-isotope dilution mass spectrometry was used to quantify the molecular species of LPA generated from platelets in vitro. Only 10% of the LPA generated following thrombin stimulation was associated with platelets, the remaining 90% was contained within the extracellular medium. The acyl composition of LPA produced by platelets differed depending on the presence or absence of plasma in the incubation. 3) The fate of exogenously added fluorescent phospholipid analogs was determined. Incubation of [(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl-(NBD)-labeled phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine with the supernatant fractions from thrombin-stimulated platelets yielded no LPA production. However, these lipids were converted to the corresponding lysolipids by released PLA1 and PLA2 activities. When incubated with plasma or serum the NBD-labeled lysophospholipids were readily converted to LPA. Inhibitors of lysophospholipase D and the biological activity of LPA were detected in plasma. These results suggest that the bulk of LPA produced through platelet activation results from the sequential cleavage of phospholipids to lysophospholipids by released phospholipases A1 and A2 and then to LPA by plasma lysophospholipase D.     

Metabolism of sphingosine 1-phosphate and lysophosphatidic acid: a genome wide analysis of gene expression in Drosophila

Lipids, in addition to being structural components of cell membranes, can act as signaling molecules. Bioactive lipids, such as sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA), may act intracellularly as second messengers or be secreted and act as intercellular signaling molecules. Such molecules can affect a variety of cellular processes including apoptosis, proliferation, differentiation and motility. To investigate possible sources of bioactive lipids during development we have searched the Drosophila genome for homologs of genes involved in mammalian S1P and LPA metabolism. Here we report the developmental expression of 31 such genes by in situ hybridization to Drosophila embryos. Most show expression in specific tissues, with expression in the gut and nervous system being recurring patterns.     

Metabolism of sphingosine 1-phosphate and lysophosphatidic acid: a genome wide analysis of gene expression in Drosophila

Lipids, in addition to being structural components of cell membranes, can act as signaling molecules. Bioactive lipids, such as sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA), may act intracellularly as second messengers or be secreted and act as intercellular signaling molecules. Such molecules can affect a variety of cellular processes including apoptosis, proliferation, differentiation and motility. To investigate possible sources of bioactive lipids during development we have searched the Drosophila genome for homologs of genes involved in mammalian S1P and LPA metabolism. Here we report the developmental expression of 31 such genes by in situ hybridization to Drosophila embryos. Most show expression in specific tissues, with expression in the gut and nervous system being recurring patterns.     

Cellular signalling by sphingosine kinase and sphingosine 1-phosphate

Sphingosine kinases, through the formation of the bioactive phospholipid sphingosine 1-phosphate, have been implicated in a diverse range of cellular processes, including cell proliferation, apoptosis, calcium homeostasis, angiogenesis and vascular maturation. The last few years have seen a number of significant advances in understanding of the mechanisms of action, activation, cellular localisation and biological roles of these enzymes. Here we review the current understanding of the regulation of and cellular signalling by sphingosine kinase and sphingosine 1-phosphate and discuss recent findings implicating sphingosine kinase as a potential therapeutic target for the control of cancer, inflammation and a number of other diseases. We suggest that, since the activation and subcellular localization of these enzymes appear to play critical roles in their biological functions, targeting these processes may provide more specific therapeutic options than direct catalytic inhibitors.     

Biological roles of lysophosphatidic acid signaling through its production by autotaxin

Lysophosphatidic acid (LPA) exhibits a wide variety of biological functions as a bio-active lysophospholipid through G-protein-coupled receptors specific to LPA. Currently at least six LPA receptors are identified, named LPA₁ to LPA₆, while the existence of other LPA receptors has been suggested. From studies on knockout mice and hereditary diseases of these LPA receptors, it is now clear that LPA is involved in various biological processes including brain development and embryo implantation, as well as patho-physiological conditions including neuropathic pain and pulmonary and renal fibrosis. Unlike sphingosine 1-phosphate, a structurally similar bio-active lysophospholipid to LPA and produced intracellularly, LPA is produced by multiple extracellular degradative routes. A plasma enzyme called autotaxin (ATX) is responsible for the most of LPA production in our bodies. ATX converts lysophospholipids such as lysophosphatidylcholine to LPA by its lysophospholipase D activity. Recent studies on ATX have revealed new aspects of LPA. In this review, we highlight recent advances in our understanding of LPA functions and several aspects of ATX, including its activity, expression, structure, biochemical properties, the mechanism by which it stimulates cell motility and its pahto-physiological function through LPA production.     

Synthesis of fluorescence-labeled sphingosine and sphingosine 1-phosphate; effective tools for sphingosine and sphingosine 1-phosphate behavior

A fluorescence-labeled sphingosine and sphingosine 1-phosphate have been successfully synthesized from the oxazolidinone methyl ester derived from glycidol via monoalkylation and the stereoselective reduction of the resulting ketone. The labeled sphingosine was converted into its phosphate by treatment with sphingosine kinase 1 (SPHK1) from mouse, and in platelets, and it was incorporated into the Chinese Hamster Ovarian (CHO) cells. In addition, MAPK was activated by NBD-Sph-1-P through Edg-1, Sph-1-P receptor.     

Inhibition of chemotactic motility and trans-endothelial migration of human neutrophils by sphingosine 1-phosphate

In previous studies, we reported that sphingosine 1-phosphate (Sph-1-P) inhibits the chemotactic motility of some cancer cell lines such as mouse melanoma cells, as well as human smooth muscle cells, at a very low concentration, as demonstrated by a transwell migration assay method (Proc. Natl. Acad. Sci. USA 89, 9698, 1992; J. Cell Biol. 130, 193, 1995). In this study, we investigated the effect of Sph-1-P on the chemotactic motility and invasiveness of human neutrophils, utilizing three different assay systems: (a) a transwell migration assay where IL-8 or fLMP was added as a chemotactic factor, (b) a phagokinetic assay with gold colloids, and (c) a trans-endothelial migration assay with human umbilical vein endothelial cells (HUVECs) plated on collagen layers. We found that among various sphingosine derivatives, Sph-1-P specifically inhibited the IL-8- or fLMP-induced chemotactic migration of neutrophils at concentrations below 1 μM. Phagokinetic activity of neutrophils was also suppressed by Sph-1-P, but more moderately than by the PKC inhibitory sphingosine analog, trimethylsphingosine. Finally, Sph-1-P inhibited trans-endothelial migration and invasiveness of neutrophils into HUVEC-covered collagen layers, whereas no effect on their adhesion to HUVECs was observed. These observations strongly suggest that Sph-1-P can act as a specific and effective motility regulator of human neutrophils, raising the possibility of future applications of Sph-1-P, or its analogs, as anti-inflammatory agents regulating invasive migration of neutrophils through endothelial layers at injured vascular sites.     

Identification of lysophospholipid receptors in human platelets: the relation of two agonists, lysophosphatidic acid and sphingosine 1-phosphate

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (Sph-1-P) are known as structurally related bio-active lipids activating platelets through their respective receptors. Although the receptors for LPA and Sph-1-P have been recently identified in various cells, the identification and characterization of ones in platelets have been reported only preliminarily. In this report, we first investigated the distinct modes of LPA and Sph-1-P actions in platelet activation and found that LPA functioned as a much stronger agonist than Sph-1-P, and high concentrations of Sph-1-P specifically desensitized LPA-induced intracellular Ca(2+) mobilization. In order to identify the responsible receptors underlying these observations, we analyzed the LPA and Sph-1-P receptors which might be expressed in human platelets, by RT-PCR. We found for the first time that Edg2, 4, 6 and 7 mRNA are expressed in human platelets.     

New insights into the role of sphingosine 1-phosphate and lysophosphatidic acid in the regulation of skeletal muscle cell biology

Lysophospholipids are bioactive molecules that are implicated in the control of fundamental biological processes such as proliferation, differentiation, survival and motility in different cell types. Here we review the role of sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) in the regulation of skeletal muscle biology. Indeed, a wealth of experimental data indicate that these molecules are crucial players in the skeletal muscle regeneration process, acting by controllers of activation, proliferation and differentiation not only of muscle-resident satellite cells but also of mesenchymal progenitors that originate outside the skeletal muscle. Moreover, S1P and LPA are clearly involved in the regulation of skeletal muscle metabolism, muscle adaptation to different physiological needs and resistance to muscle fatigue. Notably, studies accomplished so far, have highlighted the complexity of S1P and LPA signaling in skeletal muscle cells that appears to be further complicated by their close dependence on functional cross-talks with growth factors, hormones and cytokines. Our increasing understanding of bioactive lipid signaling can individuate novel molecular targets aimed at enhancing skeletal muscle regeneration and reducing the fibrotic process that impairs full functional recovery of the tissue during aging, after a trauma or skeletal muscle diseases. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Secretion and role of autotaxin and lysophosphatidic acid in adipose tissue

In obesity, adipocyte hypertrophy is often associated with recrutement of new fat cells (adipogenesis) under the control of circulating and local regulatory factors. Among the different lipids released in the extracellular compartment of adipocytes, our group found the presence of lysophosphatidic acid (LPA). LPA is a bioactive phospholipid able to regulate several cell responses via the activation of specific G-protein coupled membrane receptors. Our group found that LPA increases preadipocyte proliferation and inhibits adipogenesis via the activation of LPA1 receptor subtype. Extracellular LPA-synthesis is catalyzed by a lysophospholipase D secreted by adipocytes: autotaxin (ATX). Adipocyte ATX expression strongly increases with adipogenesis as well as in individuals exhibiting type 2 diabetes associated with massive obesity. A possible contribution of ATX and LPA as paracrine regulators of adipogenesis and obesity associated diabetes is proposed.     

Athero- and thrombogenic actions of lysophosphatidic acid and sphingosine-1-phosphate

Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are potent bioactive phospholipids with specific and multiple effects on blood cells and cells of the vessel wall. Released by activated platelets, LPA and S1P mediate physiological wound healing processes such as vascular repair. Evidence is accumulating that these lipid mediators can, however, under certain conditions become athero- and thrombogenic molecules that might aggravate cardiovascular disease. For example, LPA present in minimally modified LDL and within the intima of atherosclerotic lesions may play a role in the early phase of atherosclerosis by inducing barrier dysfunction and increased monocyte adhesion of the endothelium, as well as in the late phase by triggering platelet activation and intra-arterial thrombus formation upon rupture of the atherosclerotic plaque. Moreover, LPA and S1P, by stimulating the proliferation of fibroblasts and by enhancing the survival of inflammatory cells are likely to play a central role in the excessive fibroproliferative and inflammatory response to vascular injury that characterizes the progression of atherosclerosis. Furthermore, LPA can cause the phenotypic dedifferentiation of medial vascular smooth muscle cells, and S1P is able to stimulate the migration and proliferation of intimal vascular smooth muscle cells; both processes ultimately lead to the formation of the neointima. Most importantly, as LPA and S1P bind to and activate multiple G-protein receptors, it emerges that the beneficial or harmful action of LPA and S1P are critically dependent on the expression profile of their receptor subtypes and their coupling to different signal transduction pathways in the target cells. By targeting specific subtypes of LPA and S1P receptors in selective cells of the vascular wall and blood, new strategies for the prevention and therapy of cardiovascular diseases can be envisioned.     

Adipose-specific disruption of autotaxin enhances nutritional fattening and reduces plasma lysophosphatidic acid

Autotaxin (ATX) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA). ATX is secreted by adipose tissue and its expression is enhanced in obese/insulin-resistant individuals. Here, we analyzed the specific contribution of adipose-ATX to fat expansion associated with nutritional obesity and its consequences on plasma LPA levels. We established ATX(F/F)/aP2-Cre (FATX-KO) transgenic mice carrying a null ATX allele specifically in adipose tissue. FATX-KO mice and their control littermates were fed either a normal or a high-fat diet (HFD) (45% fat) for 13 weeks. FATX-KO mice showed a strong decrease (up to 90%) in ATX expression in white and brown adipose tissue, but not in other ATX-expressing organs. This was associated with a 38% reduction in plasma LPA levels. When fed an HFD, FATX-KO mice showed a higher fat mass and a higher adipocyte size than control mice although food intake was unchanged. This was associated with increased expression of peroxisome proliferator-activated receptor (PPAR)γ2 and of PPAR-sensitive genes (aP2, adiponectin, leptin, glut-1) in subcutaneous white adipose tissue, as well as in an increased tolerance to glucose. These results show that adipose-ATX is a negative regulator of fat mass expansion in response to an HFD and contributes to plasma LPA levels.     

Lysophosphatidic acid and sphingosine 1-phosphate stimulate endothelial cell wound healing

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate(S1P) are potent lipid growth factors with similar abilities tostimulate cytoskeleton-based cellular functions. Their effects aremediated by a subfamily of G protein-coupled receptors (GPCRs) encoded by endothelial differentiation genes (edgs). Wehypothesize that large quantities of LPA and S1P generated by activatedplatelets may influence endothelial cell functions. Using an in vitrowound healing assay, we observed that LPA and S1P stimulated closure ofwounded monolayers of human umbilical vein endothelial cells and adultbovine aortic endothelial cells, which express LPA receptor Edg2, andS1P receptors Edg1 and Edg3. The two major components of wound healing,cell migration and proliferation, were stimulated individually by bothlipids. LPA and S1P also stimulated intracellular Ca²⁺mobilization and mitogen-activated protein kinase (MAPK)phosphorylation. Pertussis toxin partially blocked the effects of bothlipids on endothelial cell migration, MAPK phosphorylation, andCa²⁺ mobilization, implicatingG_i/_o-coupled Edg receptor signaling inendothelial cells. LPA and S1P did not cross-desensitize each other inCa²⁺ responses, suggesting involvement of distinctreceptors. Thus LPA and S1P affect endothelial cell functions throughsignaling pathways activated by distinct GPCRs and may contribute tothe healing of wounded vasculatures.