Signaling and Regulatory Functions of Bioactive Sphingolipids as Therapeutic Targets in Multiple Sclerosis

Spingolipids (SLs) are an important component of central nervous system (CNS) myelin sheaths and affect the viability of brain cells (oligodendrocytes, neurons and astrocytes) that is determined by signaling mediated by bioactive sphingoids (lyso-SLs). Recent studies indicate that two lipids, ceramide and sphingosine 1-phosphate (S1P), are particularly involved in many human diseases including the autoimmune inflammatory demyelination of multiple sclerosis (MS). In this review we: (1) Discuss possible sources of ceramide in CNS; (2) Summarize the features of the metabolism of S1P and its downstream signaling through G-protein-coupled receptors; (3) Link perturbations in bioactive SLs metabolism to MS neurodegeneration and (4) Compile ceramide and S1P relationships to this process. In addition, we described recent preclinical and clinical trials of therapies targeting S1P signaling, including 2-amino-2-propane-1,3-diol hydrochloride (FTY720, fingolimod) as well as proposed intervention to specify critical SL levels that tilt balances of apoptotic/active ceramide versus anti-apoptotic/inactive dihydroceramide that may offer a novel and important therapeutic approach to MS.     

Role of sphingosine kinase localization in sphingolipid signaling

The sphingosine kinases, SK1 and SK2, produce the potent signaling lipid sphingosine-1-phosphate (S1P). These enzymes have garnered increasing interest for their roles in tumorigenesis, inflammation, vascular diseases, and immunity, as well as other functions. The sphingosine kinases are considered signaling enzymes by producing S1P, and their activity is acutely regulated by a variety of agonists. However, these enzymes are also key players in the control of sphingolipid metabolism. A variety of sphingolipids, such as sphingosine and the ceramides, are potent signaling molecules in their own right. The role of sphingosine kinases in regulating sphingolipid metabolism is potentially a critical aspect of their signaling function. A central aspect of signaling lipids is that their hydrophobic nature constrains them to membranes. Most enzymes of sphingolipid metabolism, including the enzymes that degrade S1P, are membrane enzymes. Therefore the localization of the sphingosine kinases and S1P is likely to be important in S1P signaling. Sphingosine kinase localization affects sphingolipid signaling in several ways. Translocation of SK1 to the plasma membrane promotes extracellular secretion of S1P. SK1 and SK2 localization to specific sites appears to direct S1P to intracellular protein effectors. SK localization also determines the access of these enzymes to their substrates. This may be an important mechanism for the regulation of ceramide biosynthesis by diverting dihydrosphingosine, a precursor in the ceramide biosynthetic pathway, from the de novo production of ceramide.     

Androgen and estrogen receptors in placental physiology and dysfunction

Background

The placenta is recognized as an endocrine organ, largely due to its secretions of steroid hormones, including progesterone, androgens, and estrogens. Steroid hormones play an essential role in the progression of pregnancy, fetal development, and growth. Furthermore, steroids are necessary for establishment and maintenance of a normal pregnancy, preparing the endometrium for implantation, stimulating endometrial secretions, and regulating uterine blood flow, however the exact mechanism of sex steroid signaling through their receptors in placental function is unknown.

Objective

In this review, we will provide an overview of the current knowledge on sex steroid receptors in normal placental development, as well as evidence of abnormal signaling associated with placental dysfunction.

Methods

A systematic literature search was performed using the NCBI PubMed search engine, including the following key works: estrogen receptor, androgen receptor, placenta, placental development, cytotrophoblast, and differentiation.

Results

Of the over 700 articles that were returned, 125 studies focused on estrogen and androgen receptors in human placenta development and function during normal and abnormal pregnancy, as well as in rodents and ruminants placentae.

Conclusion

Receptors for both estrogens and androgens have been localized within the mammalian placenta, but surprisingly little is known about their signaling in trophoblast cell differentiation and function. An emerging picture is developing in which estrogen receptors possibly play role in cytotrophoblast proliferation and extravillous trophoblast invasion, whereas androgen receptors are involved in syncytiotrophoblast differentiation and function.

     

Down-regulation of stearoyl-CoA desaturase-1 increases susceptibility to palmitic-acid-induced lipotoxicity in human trophoblast cells

In early pregnancy, adequate dietary factors are important for the growth of human trophoblast cells, followed by placental development. Although stearoyl-CoA desaturase 1 (SCD1) is expected to relieve palmitic acid (PA)-induced lipotoxicity by regulating diacylglycerol and ceramide, its function is unclear in human trophoblast cells. The aim was to investigate inhibitory effects of SCD1 activity on PA-induced trophoblast cell death. PA induces cell death and inhibits the invasion of human trophoblast cells (HTR8/SVneo). In addition, we demonstrate that SCD1 has a protective role against PA in human trophoblast cells by regulating AKT-mediated signaling pathway and mitochondrial membrane potential. The knockdown of SCD1 enhances the proapoptotic activity of PA in HTR8/SVneo cells. Lastly, we investigated microRNA expression predicted to target SCD1 and diacylglycerol O-acyltransferase 1 (DGAT1) by PA. Collectively, the results suggest potential roles of SCD1 and DGAT1 in alleviating the toxicity of PA and maintaining lipid homeostasis for normal placentation.     

Calcium signaling in placenta

The placenta sustains the developing fetus throughout gestation and its major functions include nutrition, gas and waste exchange via a variety of passive or active mechanisms. Up to 30 g of calcium (Ca(2+)) actively crosses the trophoblast layer during human pregnancy. The Ca(2+) ion not only plays an important role for skeletal development but is also an essential second messenger. This review is intended to highlight the implications of Ca(2+) signaling during reproduction and specifically placentation. Initially, a Ca(2+) wave induces fertilization of the oocyte. The intracellular Ca(2+) concentration is key for the blastocyst implantation, proper placental development and function. Current knowledge of many proteins involved in placental Ca(2+) regulation and their function in pathologic conditions is largely limited. Recent studies, however, point to alterations in Ca(2+) homeostasis in placental pathologies such as pre-eclampsia (PE) and intrauterine growth restriction (IUGR). A broader understanding of the role of Ca(2+) signaling during human reproduction may offer insight into impaired pregnancy outcomes.     

Lipid phosphate phosphatases regulate signal transduction through glycerolipids and sphingolipids

Lipid phosphate esters including lysophosphatidate (LPA), phosphatidate (PA), sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) are bioactive in mammalian cells and serve as mediators of signal transduction. LPA and S1P are present in biological fluids and activate cells through stimulation of their respective G-protein-coupled receptors, LPA(1-3) and S1P(1-5). LPA stimulates fibroblast division and is important in wound repair. It is also active in maintaining the growth of ovarian cancers. S1P stimulates chemotaxis, proliferation and differentiation of vascular endothelial and smooth muscle cells and is an important participant in the angiogenic response and neovessel maturation. PA and C1P are believed to act primarily inside the cell where they facilitate vesicle transport. The lipid phosphates are substrates for a family of lipid phosphate phosphatases (LPPs) that dramatically alter the signaling balance between the phosphate esters and their dephosphorylated products. In the case of PA, S1P and C1P, the products are diacylglycerol (DAG), sphingosine and ceramide, respectively. These latter lipids are also bioactive and, thus, the LPPs change signals that the cell receives. The LPPs are integral membrane proteins that act both inside and outside the cell. The "ecto-activity" of the LPPs regulates the circulating and locally effective concentrations of LPA and S1P. Conversely, the internal activity controls the relative accumulation of PA or C1P in response to stimulation by various agonists thereby affecting cell signaling downstream of EDG and other receptors. This article will review the various LPPs and discuss how these enzymes could regulate signal transduction by lipid mediators.     

The role of sphingosine-1-phosphate in skeletal muscle: Physiology,mechanisms, and clinical perspectives

Sphingolipids were discovered more than a century ago and were simply considered as a class of cell membrane lipids for a long time. However, after the discovery of several intracellular functions and their role in the control of many physiological and pathophysiological conditions, these molecules have gained much attention. For instance, the sphingosine-1-phosphate (S1P) is a circulating bioactive sphingolipid capable of triggering strong intracellular reactions through the family of S1P receptors (S1PRs) spread in several cell types and tissues. Recently, the role of S1P in the control of skeletal muscle metabolism, atrophy, regeneration, and metabolic disorders has been widely investigated. In this review, we summarized the knowledge of S1P and its effects in skeletal muscle metabolism, highlighting the role of S1P/S1PRs axis in skeletal muscle regeneration, fatigue, ceramide accumulation, and insulin resistance. Finally, we discussed the physical exercise role in S1P/S1PRs signaling in skeletal muscle cells, and how this nonpharmacological strategy may be prospective for future investigations due to its ability to increase S1P levels.     

S1P metabolism in cancer and other pathological conditions

Nearly two decades ago, the sphingolipid metabolite sphingosine 1-phosphate was discovered to function as a lipid mediator and regulator of cell proliferation. Since that time, sphingosine 1-phosphate has been shown to mediate a diverse array of fundamental biological processes including cell proliferation, migration, invasion, angiogenesis, vascular maturation and lymphocyte trafficking. Sphingosine 1-phosphate acts primarily via signaling through five ubiquitously expressed G protein-coupled receptors. Intracellular sphingosine 1-phosphate molecules are transported extracellularly and gain access to cognate receptors for autocrine and paracrine signaling and for signaling at distant sites reached through blood and lymphatic circulation systems. Intracellular pools of sphingosine 1-phosphate available for signaling are tightly regulated primarily by three enzymes: sphinosine kinase, S1P lyase and S1P phosphatase. Alterations in sphingosine 1-phosphate as well as the enzymes involved in its synthesis and catabolism have been observed in many types of malignancy. These enzymes are being evaluated for their role in mediating cancer formation and progression, as well as their potential to serve as targets of anti-cancer therapeutics. In this review, the impact of sphingosine 1-phosphate, its cognate receptors, and the enzymes of sphingosine 1-phosphate metabolism on cell survival, apoptosis, autophagy, cellular transformation, invasion, angiogenesis and hypoxia in relation to cancer biology and treatment are discussed.     

Role of sphingosine-1-phosphate phosphatase 1 in epidermal growth factor-induced chemotaxis

Sphingosine-1-phosphate (S1P) is the ligand for a family of specific G protein-coupled receptors that regulate a wide variety of cellular functions, including cytoskeletal rearrangements and cell motility. Because of the pivotal role of S1P, its levels are low and tightly regulated in a spatial-temporal manner through its synthesis catalyzed by sphingosine kinases and degradation by an S1P lyase and specific S1P phosphatases (SPP). Surprisingly, down-regulation of SPP-1 enhanced migration toward epidermal growth factor (EGF); conversely, overexpression of SPP-1, which is localized in the endoplasmic reticulum, attenuated migration toward EGF. To determine whether the inhibitory effect on EGF-induced migration was because of decreased S1P or increased ceramide as a consequence of acylation of increased sphingosine by ceramide synthase, we used fumonisin B1, a specific inhibitor of ceramide synthase. Although fumonisin B1 blocked ceramide production and increased sphingosine, it did not reverse the negative effect of SPP-1 expression on EGF- or S1P-induced chemotaxis. EGF activated the epidermal growth factor receptor to the same extent in SPP-1-expressing cells, yet ERK1/2 activation was impaired. In agreement, PD98059, an inhibitor of the ERK-activating enzyme MEK, decreased EGF-stimulated migration. We next examined the possibility that intracellularly generated S1P might be involved in activating a G protein-coupled S1P receptor important for EGF-directed migration. Treatment with pertussis toxin to inactivate Galpha(i) suppressed EGF-induced migration. Moreover, expression of regulator of G protein signaling 3, which inhibits S1P receptor signaling and completely prevented ERK1/2 activation mediated by S1P receptors, not only reduced migration toward S1P but also markedly reduced migration toward EGF. Collectively, these results suggest that metabolism of S1P by SPP-1 is important for EGF-directed cell migration.     

Epidermal growth factor inhibits ceramide-induced apoptosis and lowers ceramide levels in primary placental trophoblasts

The activation of sphingomyelinase and the subsequent generation of ceramide are emerging as important components of signaling pathways leading to apoptosis. The combination of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) induces apoptosis of primary placental trophoblasts in vitro. This apoptosis is inhibited completely by cotreatment with epidermal growth factor (EGF). We therefore examined the role of sphingomyelinase and ceramide in trophoblast apoptosis and how this may be influenced by EGF. Exogenous C₁₆-ceramide (20 μM) and acid sphingomyelinase induced trophoblast apoptosis, an effect abrogated completely by cotreatment with 10 ng/ml EGF. Neutral sphingomyelinase also increased ceramide levels but did not induce apoptosis. Treatment with EGF alone decreased cellular ceramide levels. This decrease could be blocked by cotreatment with the acid ceramidase inhibitor N-oleoylethanolamine (OE). OE alone increased ceramide levels and induced apoptosis that could not be blocked by cotreatment with EGF. In contrast, the alkaline ceramidase inhibitor D-MAPP, although it also increased ceramide levels, did not induce apoptosis nor did it affect TNF-α/IFN-α-induced cell death. These results implicate sphingolipids as important mediators in trophoblast apoptosis and suggest that the antiapoptotic properties of EGF can in part be explained by its control of ceramide concentrations in trophoblasts. J. Cell. Physiol. 180:263–270, 1999. © 1999 Wiley-Liss, Inc.     

Circulating cord blood HDL-S1P complex preserves the integrity of the feto-placental vasculature

Perinatal and long-term offspring morbidities are strongly dependent on the preservation of placental vascular homeostasis during pregnancy. In adults, the HDL-apoM-S1P complex protects the endothelium and maintains vascular integrity. However, the metabolism and biology of cord blood-derived HDLs (referred to as neonatal HDL, nHDL) strikingly differ from those in adults. Here, we investigate the role of neonatal HDLs in the regulation of placental vascular function. We show that nHDL is a major carrier of sphingosine-1-phosphate (S1P), which is anchored to the particle through apoM (r^s = 0.90, p < 0.0001) in the fetal circulation. Furthermore, this complex interacts with S1P receptors on the feto-placental endothelium and activates specifically extracellular signal-regulated protein kinases 1 and 2 (ERK) and phospholipase C (PLC) downstream signaling, promotes endothelial cell proliferation and calcium flux. Notably, the nHDL-S1P complex triggers actin filaments reorganization, leading to an enhancement of placental endothelial barrier function. Additionally, nHDL induces vasorelaxation of isolated placental chorionic arteries. Taken together, these results suggest that circulating nHDL exerts vasoprotective effects on the feto-placental endothelial barrier mainly via S1P signaling.     

Control of human trophoblast function

The trophoblast, i.e. the peripheral part of the human conceptus, exerts a crucial role in implantation and placentation. Both processes properly occur as a consequence of an intimate dialogue between fetal and maternal tissues, fulfilled by membrane ligands and receptors, as well as by hormone and local factor release. During blastocyst implantation, generation of distinct trophoblast cell types begins, namely the villous and the extravillous trophoblast, the former of which is devoted to fetal-maternal exchanges and the latter binds the placental body to the uterine wall. Physiological placentation is characterized by the invasion of the uterine spiral arteries by extravillous trophoblast cells arising from anchoring villi. Due to this invasion, the arterial structure is replaced by amorphous fibrinoid material and endovascular trophoblastic cells. This transformation establishes a low-resistance, high-capacity perfusion system from the radial arteries to the intervillous space, in which the villous tree is embedded. The physiology of pregnancy depends upon the orderly progress of structural and functional changes of villous and extravillous trophoblast, whereas a derangement of such processes can lead to different types of complications of varying degrees of gravity, including possible pregnancy loss and maternal life-threatening diseases. In this review we describe the mechanisms which regulate trophoblast differentiation, proliferation, migration and invasiveness, and the alterations in these mechanisms which lead to pathological conditions. Furthermore, based on the growing evidence that proper inflammatory changes and oxidative balance are needed for successful gestation, we explain the mechanisms by which agents able to influence such processes may be useful in the prevention and treatment of pregnancy disorders.     

Roles of sphingosine 1-phosphate on tumorigenesis

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid with a variety of biological activities.It is generated from the conversion of ceramide to sphingosine by ceramidase and the subsequent conversion of sphingosine to S1P,which is catalyzed by sphingosine kinases.Through increasing its intracellular levels by sphingolipid metabolism and binding to its cell surface receptors,S1P regulates several physiological and pathological processes,including cell proliferation,migration,angiogenesis and autophagy.These processes are responsible for tumor growth,metastasis and invasion and promote tumor survival.Since ceramide and S1P have distinct functions in regulating in cell fate decision,the balance between the ceramide/sphingosine/S1P rheostat becomes a potent therapeutic target for cancer cells.Herein,we summarize our current understanding of S1P signaling on tumorigenesis and its potential as a target for cancer therapy.     

Sphingosine-1-phosphate and lipid phosphohydrolases

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that acts as both an extracellular ligand for the endothelial differentiation gene-1 (EDG-1) G-protein coupled receptor (GPCR) family and as an intracellular messenger. Cellular levels of S1P are low and tightly regulated in a spatial-temporal manner not only by sphingosine kinase (SPHK) but also by degradation catalyzed by S1P lyase, specific S1P phosphohydrolases, and by general lipid phosphate phosphohydrolases (LPPs). LPPs are characterized as magnesium-independent, insensitive to inhibition by N-ethylmaleimide (NEM) and possessing broad substrate specificity with a variety of phosphorylated lipids, including S1P, phosphatidic acid (PA), and lysophosphatidic acid (LPA). LPPs contain three highly conserved domains that define a phosphohydrolase superfamily. Recently, several specific S1P phosphohydrolases have been identified in yeast and mammalian cells. Phylogenetic and biochemical analyses indicate that these enzymes constitute a new subset of the LPP family. As further evidence, S1P phosphohydrolases exhibit high specificity for phosphorylated sphingoid bases. Enforced expression of S1P phosphohydrolase alters the cellular levels of sphingolipid metabolites in yeast and mammalian cells, increasing sphingosine and ceramide, bioactive sphingolipids that often have opposing biological actions to S1P. By regulating the cellular ratio between ceramide/sphingosine and S1P, S1P phosphohydrolase is poised to be a critical factor in cell survival/cell death decisions. Indeed, expression of S1P phosphohydrolase in mammalian cells increases apoptosis, whereas deletion of S1P phosphohydrolases in yeast correlates with resistance to heat stress. In this review, we discuss the role of phosphohydrolases in the metabolism of S1P and how turnover of S1P can regulate sphingolipid metabolites signaling.     

Adipokines, an adipose tissue and placental product with biological functions during pregnancy

Latter half of pregnancy is characterized by a "physiological diabetogenic state" since changes in insulin-sensitivity have been well documented. These changes ensure continuous supply of nutrients to the growing fetus. In the last years the role of adipocyte-derived signaling molecules, collectively known as adipokines has been object of different in vitro and in vivo studies. Of interest, adipokines and/or their receptors are expressed in the placental tissue which, therefore, can contribute to development of maternal insulin-resistance and, as a consequence, fetal growth. Leptin, adiponectin, and resistin represent the most well studied adipokines and, with the exception of adiponectin, their serum and placental levels increase as pregnancy progresses. High levels of adipokines have also been detected in umbilical plasma hence suggesting a possible role on fetal development and metabolism; however, it remains still unclear if such adipokines can directly stimulate fetal tissues development acting as growth factors. In addition to their well known metabolic effects, we also reported studies describing the role of adipokines in promoting proliferation and invasiveness of trophoblast cells and affecting local angiogenic processes. These observations strongly suggest that adipokines, by alternatively interfering with placental development, may affect pregnancy outcome and fetal growth. However, further studies are needed to better understand the local regulation of their expression. ? 2012 International Union of Biochemistry and Molecular Biology, Inc.     

Expression and regulation of calcitonin gene-related Peptide receptor in rat placentas

Calcitonin gene-related peptide (CGRP), one of the most potent vasodilators known, exerts its biological action by interacting with its receptors. Recent reports suggest the existence of two types of CGRP receptors, CGRP-A and CGRP-B. The current study was designed to examine whether CGRP-B receptors are present in the rat placenta, and if they are, whether they are modulated by gestational age and by sex-steroid hormones. Placentas were obtained from timed pregnant Sprague-Dawley rats that were killed on Days 17-21 and 22 before and during labor (n = 6 for each gestational age). In addition, placentas were also obtained from pregnant rats injected with progesterone (P(4); 4 mg per rat per day s.c. on Days 20-22), antiprogesterone RU-486 (10 mg/rat s.c. on Day 17), 17beta-estradiol (5 micro g/rat s.c. on Day 17), and antiestrogen ICI 182780 (0.3 micro g/rat s.c. on Day 17). Results showed that first, immunoflourescent staining of rat placentas using monoclonal anti-CGRP-B receptor antibody revealed the presence of CGRP-B receptors in the labyrinthine layer of the placenta, specifically to the trophoblast and blood vessel endothelium and underlying smooth muscle cells. The intensity of staining was lower in placentas obtained during labor. Second, a single band of 66 kDa, reactive to CGRP-B receptor antibody, was obtained in Western blotting of the rat placenta; third, densitometric analysis of protein bands showed that CGRP-B receptors were increased from Day 17 to Day 22, with maximal levels obtained on Day 22 before labor, which was 10 times higher than that of Day 17 (P < 0.01); fourth, expression of CGRP-B receptors in rat placenta decreased during labor (8% vs. 100% on Day 22 before labor, P < 0.01); fifth, P(4) given during Days 20-22 attenuated the fall in placental CGRP-B receptors at term labor; sixth, RU-486 given on Day 17 of gestation significantly decreased expression of placental CGRP-B receptors (18% vs. 100% in controls at 6 h, P < 0.01); seventh, a significant decrease in CGRP-B receptor expression was noted 48 h after estrogen administration; and eighth, ICI 182780 treatment on Day 17 increased placental CGRP-B receptors (152% vs. 100% in control at 48 h, P < 0.01). These results indicate that CGRP-B receptors are present in rat placenta and that receptor levels are higher with gestational age and lower at term labor. Progesterone stimulated and estrogen inhibited placental CGRP-B receptor expression. Thus, elevations in placental CGRP-B receptors in late pregnancy could play a role in increasing blood flow through the fetoplacental unit associated with rapid fetal growth during late gestation.