The Emerging Role of LPA as an Oncometabolite.

Lysophosphatidic acid (LPA) is a phospholipid that displays potent signalling activities that are regulated in both an autocrine and paracrine manner. It can be found both extra- and intracellularly, where it interacts with different receptors to activate signalling pathways that regulate a plethora of cellular processes, including mitosis, proliferation and migration. LPA metabolism is complex, and its biosynthesis and catabolism are under tight control to ensure proper LPA levels in the body. In cancer patient specimens, LPA levels are frequently higher compared to those of healthy individuals and often correlate with poor responses and more aggressive disease. Accordingly, LPA, through promoting cancer cell migration and invasion, enhances the metastasis and dissemination of tumour cells. In this review, we summarise the role of LPA in the regulation of critical aspects of tumour biology and further discuss the available pre-clinical and clinical evidence regarding the feasibility and efficacy of targeting LPA metabolism for effective anticancer therapy.

Sphingosine-1-Phosphate Shapes Healthy Monocytes into An Immunosuppressive Phenotype.

BACKGROUND/AIMS: The physiological phenotype of individuals can influence and shape real-life phenomena in that it can contribute to the development of specific characteristics that can affect the immune response to specific stimuli. In this study we aimed to understand whether the sphingosine/sphingosine-1-phoshate (S1P) axis can modulate the immunotype of circulating cells. METHODS: To pursue this goal, we performed bioinformatic analyses of public datasets. RESULTS: The transcriptomic profile of healthy subjects of GSE192829 dataset identified two clusters with different transcriptional repertoire. Cluster 1 expressed higher levels of enzymes for S1P formation than cluster 0 which was characterized by enzymes that lead to ceramide formation, which represent the opposite metabolic direction. Inference analysis showed that cluster 1 was higher populated by monocytes, CD4+ T and B cells than cluster 0. Of particular interest was the phenotype of the monocytes in cluster 1 which showed an immunosuppressive nature compared to those in cluster 0. The role of S1P signature in healthy PBMCs was confirmed with other dataset analyses, supporting that circulating monocytes positive to the ceramidase, unlike the negative ones, had an immunosuppressive phenotype characterized by hub immunosuppressive markers (i.e. TYROBP, FCER1G, SYK, SIRPA, CSF1R, AIF1, FCGR2A, CLEC7A, LYN, PLCG2, LILRs, HCK, GAB2). This hub genes well discriminated the immunotype of healthy subjects. CONCLUSION: In conclusion this study highlights that S1P-associated hub markers can be useful to discriminate subjects with pronounced immunosuppression.

How do sphingosine-1-phosphate affect immune cells to resolve inflammation?

Inflammation is an important immune response of the body. It is a physiological process of self-repair and defense against pathogens taken up by biological tissues when stimulated by damage factors such as trauma and infection. Inflammation is the main cause of high morbidity and mortality in most diseases and is the physiological basis of the disease. Targeted therapeutic strategies can achieve efficient toxicity clearance at the inflammatory site, reduce complications, and reduce mortality. Sphingosine-1-phosphate (S1P), a lipid signaling molecule, is involved in immune cell transport by binding to S1P receptors (S1PRs). It plays a key role in innate and adaptive immune responses and is closely related to inflammation. In homeostasis, lymphocytes follow an S1P concentration gradient from the tissues into circulation. One widely accepted mechanism is that during the inflammatory immune response, the S1P gradient is altered, and lymphocytes are blocked from entering the circulation and are, therefore, unable to reach the inflammatory site. However, the full mechanism of its involvement in inflammation is not fully understood. This review focuses on bacterial and viral infections, autoimmune diseases, and immunological aspects of the Sphks/S1P/S1PRs signaling pathway, highlighting their role in promoting intradial-adaptive immune interactions. How S1P signaling is regulated in inflammation and how S1P shapes immune responses through immune cells are explained in detail. We teased apart the immune cell composition of S1P signaling and the critical role of S1P pathway modulators in the host inflammatory immune system. By understanding the role of S1P in the pathogenesis of inflammatory diseases, we linked the genomic studies of S1P-targeted drugs in inflammatory diseases to provide a basis for targeted drug development.

Linking the Autotaxin-LPA Axis to Medicinal Cannabis and the Endocannabinoid System.

Over the past few decades, many current uses for cannabinoids have been described, ranging from controlling epilepsy to neuropathic pain and anxiety treatment. Medicines containing cannabinoids have been approved by both the FDA and the EMA for the control of specific diseases for which there are few alternatives. However, the molecular-level mechanism of action of cannabinoids is still poorly understood. Recently, cannabinoids have been shown to interact with autotaxin (ATX), a secreted lysophospholipase D enzyme responsible for catalyzing lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), a pleiotropic growth factor that interacts with LPA receptors. In addition, a high-resolution structure of ATX in complex with THC has recently been published, accompanied by biochemical studies investigating this interaction. Due to their LPA-like structure, endocannabinoids have been shown to interact with ATX in a less potent manner. This finding opens new areas of research regarding cannabinoids and endocannabinoids, as it could establish the effect of these compounds at the molecular level, particularly in relation to inflammation, which cannot be explained by the interaction with CB1 and CB2 receptors alone. Further research is needed to elucidate the mechanism behind the interaction between cannabinoids and endocannabinoids in humans and to fully explore the therapeutic potential of such approaches.

Identification of two novel chemical classes of Autotaxin (ATX) inhibitors using Enalos Asclepios KNIME nodes.

Autotaxin is a secreted lysophospholipase D which is a member of the ectonucleotide pyrophosphatase/phosphodiesterase family converting extracellular lysophosphatidylcholine and other non-choline lysophospholipids, such as lysophosphatidylethanolamine and lysophosphatidylserine, to the lipid mediator lysophosphatidic acid. Autotaxin is implicated in various fibroproliferative diseases including interstitial lung diseases, such as idiopathic pulmonary fibrosis and hepatic fibrosis, as well as in cancer. In this study, we present an effort of identifying ATX inhibitors that bind to allosteric ATX binding sites using the Enalos Asclepios KNIME Node. All the available PDB crystal structures of ATX were collected, prepared, and aligned. Visual examination of these structures led to the identification of four crystal structures of human ATX co-crystallized with four known inhibitors. These inhibitors bind to five binding sites with five different binding modes. These five binding sites were thereafter used to virtually screen a compound library of 14,000 compounds to identify molecules that bind to allosteric sites. Based on the binding mode and interactions, the docking score, and the frequency that a compound comes up as a top-ranked among the five binding sites, 24 compounds were selected for in vitro testing. Finally, two compounds emerged with inhibitory activity against ATX in the low micromolar range, while their mode of inhibition and binding pattern were also studied. The two derivatives identified herein can serve as "hits" towards developing novel classes of ATX allosteric inhibitors.

Rewiring Endothelial Sphingolipid Metabolism to Favor S1P Over Ceramide Protects From Coronary Atherosclerosis.

BACKGROUND: Growing evidence correlated changes in bioactive sphingolipids, particularly S1P (sphingosine-1-phosphate) and ceramides, with coronary artery diseases. Furthermore, specific plasma ceramide species can predict major cardiovascular events. Dysfunction of the endothelium lining lesion-prone areas plays a pivotal role in atherosclerosis. Yet, how sphingolipid metabolism and signaling change and contribute to endothelial dysfunction and atherosclerosis remain poorly understood. METHODS: We used an established model of coronary atherosclerosis in mice, combined with sphingolipidomics, RNA-sequencing, flow cytometry, and immunostaining to investigate the contribution of sphingolipid metabolism and signaling to endothelial cell (EC) activation and dysfunction. RESULTS: We demonstrated that hemodynamic stress induced an early metabolic rewiring towards endothelial sphingolipid de novo biosynthesis, favoring S1P signaling over ceramides as a protective response. This finding is a paradigm shift from the current belief that ceramide accrual contributes to endothelial dysfunction. The enzyme SPT (serine palmitoyltransferase) commences de novo biosynthesis of sphingolipids and is inhibited by NOGO-B (reticulon-4B), an ER membrane protein. Here, we showed that NOGO-B is upregulated by hemodynamic stress in myocardial EC of ApoE-/- mice and is expressed in the endothelium lining coronary lesions in mice and humans. We demonstrated that mice lacking NOGO-B specifically in EC (Nogo-A/BECKOApoE-/-) were resistant to coronary atherosclerosis development and progression, and mortality. Fibrous cap thickness was significantly increased in Nogo-A/BECKOApoE-/- mice and correlated with reduced necrotic core and macrophage infiltration. Mechanistically, the deletion of NOGO-B in EC sustained the rewiring of sphingolipid metabolism towards S1P, imparting an atheroprotective endothelial transcriptional signature. CONCLUSIONS: These data demonstrated that hemodynamic stress induced a protective rewiring of sphingolipid metabolism, favoring S1P over ceramide. NOGO-B deletion sustained the rewiring of sphingolipid metabolism toward S1P protecting EC from activation under hemodynamic stress and refraining coronary atherosclerosis. These findings also set forth the foundation for sphingolipid-based therapeutics to limit atheroprogression.

Bis(Monoacylglycero)Phosphate Promotes Membrane Fusion Facilitated by the SARS-CoV-2 Fusion Domain.

Membrane fusion is a critical component of the viral lifecycle. For SARS-CoV-2, fusion is facilitated by the spike glycoprotein and can take place via either the plasma membrane or the endocytic pathway. The fusion domain (FD), which is found within the spike glycoprotein, is primarily responsible for the initiation of fusion as it embeds itself within the target cell's membrane. A preference for SARS-CoV-2 to fuse at low pH akin to the environment of the endocytic pathway has already been established; however, the impact of the target cell's lipid composition on the FD has yet to be explored. Here, we have shown that the SARS-CoV-2 FD preferentially initiates fusion at the late endosomal membrane over the plasma membrane, on the basis of lipid composition alone. A positive, fusogenic relationship with anionic lipids from the plasma membrane (POPS: 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine) and endosomal membrane (BMP: bis(monoacylglycero)phosphate) was established, with a large preference demonstrated for the latter. When comparing the binding affinity and secondary structure of the FD in the presence of different anionic lipids, little deviation was evident while the charge was maintained. However, it was discovered that BMP had a subtle, negative impact on lipid packing in comparison to that of POPS. Furthermore, an inverse relationship between lipid packing and the fusogenecity of the SARS-CoV-2 FD was witnessed. In conclusion, the SARS-CoV-2 FD preferentially initiates fusion at a membrane resembling that of the late endosomal compartment, predominately due to the presence of BMP and its impact on lipid packing.

Global Proteomics Analysis of Lysophosphatidic Acid Signaling in PC-3 Human Prostate Cancer Cells: Role of CCN1.

Cysteine-rich angiogenic factor 61 (CCN1/Cyr61) is a matricellular protein that is induced and secreted in response to growth factors. Our previous work showed that 18:1-lysophosphatidic acid (LPA), which activates the G protein-coupled receptor LPAR1, induces CCN1 between 2-4 h in PC-3 human prostate cancer cells in a manner than enhances cell-substrate adhesion. While the time course of induction suggests that CCN1 contributes to intermediate events in LPA action, the roles of CCN1 in LPA-mediated signal transduction have not been fully elucidated. This study utilized a comprehensive global proteomics approach to identify proteins up- or down-regulated in response to treatment of PC-3 cells with LPA for three hours, during the time of peak CCN1 levels. In addition, the effects of siRNA-mediated CCN1 knockdown on LPA responses were analyzed. The results show that, in addition to CCN1, LPA increased the levels of multiple proteins. Proteins up-regulated by LPA included metastasis-associated in colon cancer protein 1 (MACC1) and thrombospondin-1 (TSP1/THBS1); both MACC1 and TSP1 regulated cancer cell adhesion and motility. LPA down-regulated thioredoxin interacting protein (TXNIP). CCN1 knockdown suppressed the LPA-induced up-regulation of 30 proteins; these included MACC1 and TSP1, as confirmed by immunoblotting. Gene ontology and STRING analyses revealed multiple pathways impacted by LPA and CCN1. These results indicate that CCN1 contributes to LPA signaling cascades that occur during the intermediate phase after the initial stimulus. The study provides a rationale for the development of interventions to disrupt the LPA-CCN1 axis.

Plasma S1P Orchestrates the Reverse Transendothelial Migration of Aortic Intimal Myeloid Cells in Mice.

BACKGROUND: Myeloid cells (MCs) reside in the aortic intima at regions predisposed to atherosclerosis. Systemic inflammation triggers reverse transendothelial migration (RTM) of intimal MCs into the arterial blood, which orchestrates a protective immune response that clears intracellular pathogens from the arterial intima. Molecular pathways that regulate RTM remain poorly understood. S1P (sphingosine-1-phosphate) is a lipid mediator that regulates immune cell trafficking by signaling via 5 G-protein-coupled receptors (S1PRs [S1P receptors]). We investigated the role of S1P in the RTM of aortic intimal MCs. METHODS: Intravenous injection of lipopolysaccharide was used to model a systemic inflammatory stimulus that triggers RTM. CD11c+ intimal MCs in the lesser curvature of the ascending aortic arch were enumerated by en face confocal microscopy. Local gene expression was evaluated by transcriptomic analysis of microdissected intimal cells. RESULTS: In wild-type C57BL/6 mice, lipopolysaccharide induced intimal cell expression of S1pr1, S1pr3, and Sphk1 (a kinase responsible for S1P production). Pharmacological modulation of multiple S1PRs blocked lipopolysaccharide-induced RTM and modulation of S1PR1 and S1PR3 reduced RTM in an additive manner. Cre-mediated deletion of S1pr1 in MCs blocked lipopolysaccharide-induced RTM, confirming a role for myeloid-specific S1PR1 signaling. Global or hematopoietic deficiency of Sphk1 reduced plasma S1P levels, the abundance of CD11c+ MCs in the aortic intima, and blunted lipopolysaccharide-induced RTM. In contrast, plasma S1P levels, the abundance of intimal MCs, and lipopolysaccharide-induced RTM were rescued in Sphk1-/- mice transplanted with Sphk1+/+ or mixed Sphk1+/+ and Sphk1-/- bone marrow. Stimulation with lipopolysaccharide increased endothelial permeability and intimal MC exposure to circulating factors such as S1P. CONCLUSIONS: Functional and expression studies support a novel role for S1P signaling in the regulation of lipopolysaccharide-induced RTM and the homeostatic maintenance of aortic intimal MCs. Our data provide insight into how circulating plasma mediators help orchestrate intimal MC dynamics.

Designer high-density lipoprotein particles enhance endothelial barrier function and suppress inflammation.

High-density lipoprotein (HDL) nanoparticles promote endothelial cell (EC) function and suppress inflammation, but their utility in treating EC dysfunction has not been fully explored. Here, we describe a fusion protein named ApoA1-ApoM (A1M) consisting of apolipoprotein A1 (ApoA1), the principal structural protein of HDL that forms lipid nanoparticles, and ApoM, a chaperone for the bioactive lipid sphingosine 1-phosphate (S1P). A1M forms HDL-like particles, binds to S1P, and is signaling competent. Molecular dynamics simulations showed that the S1P-bound ApoM moiety in A1M efficiently activated EC surface receptors. Treatment of human umbilical vein ECs with A1M-S1P stimulated barrier function either alone or cooperatively with other barrier-enhancing molecules, including the stable prostacyclin analog iloprost, and suppressed cytokine-induced inflammation. A1M-S1P injection into mice during sterile inflammation suppressed neutrophil influx and inflammatory mediator secretion. Moreover, systemic A1M administration led to a sustained increase in circulating HDL-bound S1P and suppressed inflammation in a murine model of LPS-induced endotoxemia. We propose that A1M administration may enhance vascular endothelial barrier function, suppress cytokine storm, and promote resilience of the vascular endothelium.

GT-11 impairs insulin signaling through modulation of sphingolipid metabolism in C2C12 myotubes.

AIMS: Sphingolipids are involved in the regulation of insulin signaling, which is linked to the development of insulin resistance, leading to diabetes mellitus. We aimed to study whether modulation of sphingolipid levels by GT-11 may regulate insulin signaling in C2C12 myotubes. MAIN METHODS: We investigated the effects of sphingolipid metabolism on Akt phosphorylation and glucose uptake using C2C12 myotubes. Either GT-11, an inhibitor of dihydroceramide desaturase 1 and S1P lyase, or siRNA targeting Sgpl1, the gene encoding the enzyme, was employed to determine the effect of sphingolipid metabolism modulation on insulin signaling. Western blotting and glucose uptake assays were used to evaluate the effect of treatments on insulin signaling. Sphingolipid metabolites were analyzed by high performance liquid chromatography (HPLC). KEY FINDINGS: Treatment with GT-11 resulted in decreased Akt phosphorylation and reduced glucose uptake. Silencing the Sgpl1 gene, which encodes S1P lyase, mimicked these findings, suggesting the potential for regulating insulin signaling through S1P lyase modulation. GT-11 modulated sphingolipid metabolism, inducing the accumulation of sphingolipids. Using PF-543 and ARN14974 to inhibit sphingosine kinases and acid ceramidase, respectively, we identified a significant interplay between sphingosine, S1P lyase, and insulin signaling. Treatment with either exogenous sphingosine or palmitic acid inhibited Akt phosphorylation, and reduced S1P lyase activity. SIGNIFICANCE: Our findings highlight the importance of close relationship between sphingolipid metabolism and insulin signaling in C2C12 myotubes, pointing to its potential therapeutic relevance for diabetes mellitus.

Structural basis for lysophosphatidylserine recognition by GPR34.

GPR34 is a recently identified G-protein coupled receptor, which has an immunomodulatory role and recognizes lysophosphatidylserine (LysoPS) as a putative ligand. Here, we report cryo-electron microscopy structures of human GPR34-Gi complex bound with one of two ligands bound: either the LysoPS analogue S3E-LysoPS, or M1, a derivative of S3E-LysoPS in which oleic acid is substituted with a metabolically stable aromatic fatty acid surrogate. The ligand-binding pocket is laterally open toward the membrane, allowing lateral entry of lipidic agonists into the cavity. The amine and carboxylate groups of the serine moiety are recognized by the charged residue cluster. The acyl chain of S3E-LysoPS is bent and fits into the L-shaped hydrophobic pocket in TM4-5 gap, and the aromatic fatty acid surrogate of M1 fits more appropriately. Molecular dynamics simulations further account for the LysoPS-regioselectivity of GPR34. Thus, using a series of structural and physiological experiments, we provide evidence that chemically unstable 2-acyl LysoPS is the physiological ligand for GPR34. Overall, we anticipate the present structures will pave the way for development of novel anticancer drugs that specifically target GPR34.

Lysophosphatidic Acid-Mediated Inflammation at the Heart of Heart Failure.

PURPOSE OF REVIEW: The primary aim of this review is to provide an in-depth examination of the role bioactive lipids-namely lysophosphatidic acid (LPA) and ceramides-play in inflammation-mediated cardiac remodeling during heart failure. With the global prevalence of heart failure on the rise, it is critical to understand the underlying molecular mechanisms contributing to its pathogenesis. Traditional studies have emphasized factors such as oxidative stress and neurohormonal activation, but emerging research has shed light on bioactive lipids as central mediators in heart failure pathology. By elucidating these intricacies, this review aims to: Bridge the gap between basic research and clinical practice by highlighting clinically relevant pathways contributing to the pathogenesis and prognosis of heart failure. Provide a foundation for the development of targeted therapies that could mitigate the effects of LPA and ceramides on heart failure. Serve as a comprehensive resource for clinicians and researchers interested in the molecular biology of heart failure, aiding in better diagnostic and therapeutic decisions. RECENT FINDINGS: Recent findings have shed light on the central role of bioactive lipids, specifically lysophosphatidic acid (LPA) and ceramides, in heart failure pathology. Traditional studies have emphasized factors such as hypoxia-mediated cardiomyocyte loss and neurohormonal activation in the development of heart failure. Emerging research has elucidated the intricacies of bioactive lipid-mediated inflammation in cardiac remodeling and the development of heart failure. Studies have shown that LPA and ceramides contribute to the pathogenesis of heart failure by promoting inflammation, fibrosis, and apoptosis in cardiac cells. Additionally, recent studies have identified potential targeted therapies that could mitigate the effects of bioactive lipids on heart failure, including LPA receptor antagonists and ceramide synthase inhibitors. These recent findings provide a promising avenue for the development of targeted therapies that could improve the diagnosis and treatment of heart failure. In this review, we highlight the pivotal role of inflammation induced by bioactive lipid signaling and its influence on the pathogenesis of heart failure. By critically assessing the existing literature, we provide a comprehensive resource for clinicians and researchers interested in the molecular mechanisms of heart failure. Our review aims to bridge the gap between basic research and clinical practice by providing actionable insights and a foundation for the development of targeted therapies that could mitigate the effects of bioactive lipids on heart failure. We hope that this review will aid in better diagnostic and therapeutic decisions, further advancing our collective understanding and management of heart failure.

Altered ovarian tissue level of lysophosphatidic acid and mRNA expressions of its metabolic enzymes and receptors in rats received gonadotropin-hyperstimulation.

Lysophosphatidic acid (LPA), a well-studied member of the lysophospholipid family, is known to exert an important bio-effect on oocyte maturation and ovulation in mammals. We attempted to determine how follicle maturation in the rat ovary affects the levels of LPA and its precursor lysophospholipids, as well as mRNA levels of LPA-producing and -degrading enzymes and LPA receptors in rats that received gonadotropin-hyper-stimulation. Tissue levels of lysophospholipids were quantified by LC-MS/MS, and relative mRNA expression levels of LPA-producing and -degrading enzymes, and LPA receptors were measured by RT-PCR. Tissue levels of n-6 polyunsaturated LPAs and LPCs were higher in the ovaries of rats after receiving human chorionic gonadotropin, unlike the distinct profiles of n-3 polyunsaturated LPAs, which had lower levels, and LPCs which had higher levels, after the gonadotropin treatment. The effects of different levels of other polyunsaturated lysophospholipids were variable: decreased levels of lysophosphatidylglycerol, and unaltered levels of lysophosphatidylethanolamine, lysophosphatidylinositol, and lysophosphatidylserine. The results indicate that expression of mRNA levels of autotaxin and acylglycerol kinase were reduced and expression of lipid phosphate phosphatase 3 was elevated, whereas expressions of two membrane phosphatidic acid phosphatases (A1α and A1ß) and lipid phosphate phosphatase 1 were essentially unaltered in rat ovary at several stages after ovary hyperstimulation. After the gonadotropin treatment, the expression levels of all LPA receptors except LPA3 were decreased at various times. These results are discussed with respect to the physiological processes of the ovarian environment and development in rats.

Sphingosine 1-phosphate signaling axis mediates neuropeptide S-induced invasive phenotype of endometriotic cells.

Endometriosis is a chronic gynecological syndrome characterized by endometrial cell invasion of the extra-uterine milieu, pelvic pain and infertility. Treatment relies on either symptomatic drugs or hormonal therapies, even though the mechanism involved in the onset of endometriosis is yet to be elucidated. The signaling of sphingolipid sphingosine 1-phosphate (S1P) is profoundly dysregulated in endometriosis. Indeed, sphingosine kinase (SK)1, one of the two isoenzymes responsible for S1P biosynthesis, and S1P1, S1P3 and S1P5, three of its five specific receptors, are more highly expressed in endometriotic lesions compared to healthy endometrium. Recently, missense coding variants of the gene encoding the receptor 1 for neuropeptide S (NPS) have been robustly associated with endometriosis in humans. This study aimed to characterize the biological effect of NPS in endometriotic epithelial cells and the possible involvement of the S1P signaling axis in its action. NPS was found to potently induce cell invasion and actin cytoskeletal remodeling. Of note, the NPS-induced invasive phenotype was dependent on SK1 and SK2 as well as on S1P1 and S1P3, given that the biological action of the neuropeptide was fully prevented when one of the two biosynthetic enzymes or one of the two selective receptors was inhibited or silenced. Furthermore, the RhoA/Rho kinase pathway, downstream to S1P receptor signaling, was found to be critically implicated in invasion and cytoskeletal remodeling elicited by NPS. These findings provide new information to the understanding of the molecular mechanisms implicated in endometriosis pathogenesis, establishing the rationale for non-hormonal therapeutic targets for its treatment.

Targeting the SPHK1/S1P/S1PR2 axis ameliorates GH-secreted pituitary adenoma progression.

BACKGROUND: Growth hormone-secreted pituitary adenoma (GHPA) is a prominent subtype of pituitary adenoma (PA) associated with progressive somatic disfigurement, various complications, and elevated mortality rates. Existing treatment options have limited efficacy, highlighting the urgent need for novel pharmacological interventions. Previous studies have revealed that sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P)/S1P receptors (S1PRs) signalling have critical roles in the tumour microenvironment, but their role in GHPA remains unclear. METHODS: We performed integrative analyses including bioinformatics analyses, functional studies, and clinical validation to investigate the pathological roles of SPHK1/S1P and evaluated the effectiveness of the S1P receptor 2 (S1PR2) inhibitor JTE-013 in GHPA treatment. RESULTS: SPHK1/S1P signalling is abnormally expressed in patients with GHPA. Knockdown of SPHK1 suppresses S1P-mediated cell proliferation in GH3 Cells. Mechanistically, S1P inhibits apoptosis and autophagy while promoting the secretion of Growth Hormone (GH) by binding to the S1P receptor subtype 2 (S1PR2) in GH3 cells. Moreover, the function of S1PR2 in GH3 cells is mediated by the downstream Akt-Creb pathway. We then identify the S1PR2 as a novel target for therapeutic intervention in GHPA. Systemic administration of the potent and selective S1PR2 antagonist, JTE-013, significantly reduces both tumour size and GH secretion. Importantly, we identify preoperative serum S1P levels as a biomarker predicting poor prognosis in GHPA patients at follow-up. CONCLUSION: Our study shows that blocking SPHK1/S1P/S1PR2 axis can ameliorate the progression of GHPA, providing evidence of a promising therapeutic target for GHPA.

The role of vitamin D through SphK1/S1P in the regulation of MS progression.

Sphingosine-1-phosphate (S1P) is biologically active lipid, leading to neuroinflammation and macrophage invasion in central nervous system, plays an important role in the development of multiple sclerosis (MS) model in experimental allergic encephalomyelitis (EAE) rats. Vitamin D is observed to be a key factor in regulating cell S1P levels. We detected vitamin D can alleviate the symptoms of EAE rats, but the exact mechanism is unclear. In PC12 cells, vitamin D can reverse S1P-induced cell death, but the signaling pathway unclear. This study was aimed to investigate S1P regulation mechanism or signaling pathway mediated by vitamin D in EAE and PC12 model. In our experiments, S1P and Sphingosine kinase type 1 (SphK1) mRNA and protein expression in EAE rats group, control group, vitamin D feeding group were detected by HPLC, ELISA, RT-PCR and western blot. PC12 cell death was detected by Propidium (PI) staining. VDR plasmid overexpression and RNA interference, immunofluorescence, real-time cell analysis, protein immunoblotting was used to detect SphK1 transcriptional regulation, cell-substrate attachment quality, the signaling pathway of cell apoptosis and inflammation related gene expression (Bax/Bcl-2, Casepase-3, Il-6, TGF-ß, TNF-α). Our study showed vitamin D can reverse the elevation of S1P level in EAE rats, reduce the severity and shorten the course of EAE. 1,25-(OH) 2D3 coupled with vitamin D receptor (VDR) inhibited SphK1 transcription. 1,25-(OH)2D3 significantly reduced PC12 cell death rate induced by S1P, in addition improved the cell substrate attachment quality. 1,25-(OH) 2D3 can block S1P-induced p-ERK activation and PI3K /Akt signaling pathway reduced Il-6, TGF-ß, TNF-α cytokine release and Bax/Bcl-2, Casepase-3 apoptosis protein expression. On the other hand, immunofluorescence staining showed 1,25-(OH) 2D3 can increase the expression of neuronal perinuclear protein MAP2 in PC12 cells probably protect nerve cells further. In summary, the ameliorative effect of vitamin D was derived from its ability to reduce S1P levels, provides an idea for vitamin D as a combination therapy for disease.

Density of Sphingosine-1-Phosphate Receptors Is Altered in Cortical Nerve-Terminals of Insulin-Resistant Goto-Kakizaki Rats and Diet-Induced Obese Mice.

Sphingosine-1-phosphate (S1P) is a phosphosphingolipid with pleiotropic biological functions. S1P acts as an intracellular second messenger, as well as extracellular ligand to five G-protein coupled receptors (S1PR1-5). In the brain, S1P regulates neuronal proliferation, apoptosis, synaptic activity and neuroglia activation. Moreover, S1P metabolism alterations have been reported in neurodegenerative disorders. We have previously reported that S1PRs are present in nerve terminals, exhibiting distinct sub-synaptic localization and neuromodulation actions. Since type 2 diabetes (T2D) causes synaptic dysfunction, we hypothesized that S1P signaling is modified in nerve terminals. In this study, we determined the density of S1PRs in cortical synaptosomes from insulin-resistant Goto-Kakizaki (GK) rats and Wistar controls, and from mice fed a high-fat diet (HFD) and low-fat-fed controls. Relative to their controls, GK rats showed similar cortical S1P concentration despite higher S1P levels in plasma, yet lower density of S1PR1, S1PR2 and S1PR4 in nerve-terminal-enriched membranes. HFD-fed mice exhibited increased plasma and cortical concentrations of S1P, and decreased density of S1PR1 and S1PR4. These findings point towards altered S1P signaling in synapses of insulin resistance and diet-induced obesity models, suggesting a role of S1P signaling in T2D-associated synaptic dysfunction.

Functional modulation of lysophosphatidic acid type 2 G-protein coupled receptor facilitates alveolar bone formation.

Lipid biosynthesis is recently studied its functions in a range of cellular physiology including differentiation and regeneration. However, it still remains to be elucidated in its precise function. To reveal this, we evaluated the roles of lysophosphatidic acid (LPA) signaling in alveolar bone formation using the LPA type 2 receptor (LPAR2) antagonist AMG-35 (Amgen Compound 35) using tooth loss without periodontal disease model which would be caused by trauma and usually requires a dental implant to restore masticatory function. In this study, in vitro cell culture experiments in osteoblasts and periodontal ligament fibroblasts revealed cell type-specific responses, with AMG-35 modulating osteogenic differentiation in osteoblasts in vitro. To confirm the in vivo results, we employed a mouse model of tooth loss without periodontal disease. Five to 10 days after tooth extraction, AMG-35 facilitated bone formation in the tooth root socket as measured by immunohistochemistry for differentiation markers KI67, Osteocalcin, Periostin, RUNX2, transforming growth factor beta 1 (TGF-ß1) and SMAD2/3. The increased expression and the localization of these proteins suggest that AMG-35 elicits osteoblast differentiation through TGF-ß1 and SMAD2/3 signaling. These results indicate that LPAR2/TGF-ß1/SMAD2/3 represents a new signaling pathway in alveolar bone formation and that local application of AMG-35 in traumatic tooth loss can be used to facilitate bone regeneration and healing for further clinical treatment.

Understanding the modulations of glycero-lysophospholipids in an elastase-induced murine emphysema model.

BACKGROUND: Increasing evidence indicates that bioactive lipid mediators are involved in chronic obstructive pulmonary disease (COPD) pathogenesis. Recently, glycero-lysophospholipids, such as lysophosphatidic acid (LysoPA) and lysophosphatidylserine (LysoPS), have been recognized as significant inflammation-related lipid mediators. However, their association with COPD remains unclear. METHODS: We used an elastase-induced murine emphysema model to analyze the levels of lysophospholipids and diacyl-phospholipids in the lungs. Additionally, we assessed the expression of LysoPS-related genes and published data on smokers. RESULTS: In the early phase of an elastase-induced murine emphysema model, the levels of LysoPS and its precursor (phosphatidylserine [PS]) were significantly reduced, without significant modulations in other glycero-lysophospholipids. Additionally, there was an upregulation in the expression of lysoPS receptors, specifically GPR34, observed in the lungs of a cigarette smoke-exposed mouse model and the alveolar macrophages of human smokers. Elastase stimulation induces GPR34 expression in a human macrophage cell line in vitro. CONCLUSIONS: Elastase-induced lung emphysema affects the LysoPS/PS-GPR34 axis, and cigarette smoking or elastase upregulates GPR34 expression in alveolar macrophages. This novel association may serve as a potential pharmacological target for COPD treatment.