FTY720 alleviates coxsackievirus B3-induced myocarditis and inhibits viral replication through regulating sphingosine 1-phosphate receptors and AKT/caspase-3 pathways.

Fingolimod (FTY720) after phosphorylation, as the ligand of sphingosine 1-phosphate receptors (S1PRs), plays an important role in cell proliferation and differentiation. In this article, FTY720 in the treatment of coxsackievirus B3 (CVB3)-induced viral myocarditis was closely related to apoptosis and AKT/caspase-3 apoptotic pathways. We found that CVB3 inhibited myocardial apoptosis at the early stage with upregulating p-AKT level and downregulating activated caspase-3 level for replication of virus progeny, whereas it promoted apoptosis at a late stage with downregulating p-AKT and upregulating activated caspase-3 for releasing the newly synthesized virus to spread. Interestingly, FTY720 could reverse this trend; it promoted apoptosis at an early stage and inhibited apoptosis at the late stage in vivo and vitro, which proved the antiviral effect. We also found that S1PR1, S1PR4, and S1PR5, rather than S1PR2 and S1PR3, were regulated by FTY720 in this process. The results confirmed that FTY720 alleviates CVB3-induced myocarditis and inhibits viral replication through regulating S1PRs and AKT/caspase-3 pathways with a bidirectional regulation of apoptosis.

Sphingosine 1-Phosphate (S1P)/ S1P Receptor Signaling and Mechanotransduction: Implications for Intrinsic Tissue Repair/Regeneration.

Tissue damage, irrespective from the underlying etiology, destroys tissue structure and, eventually, function. In attempt to achieve a morpho-functional recover of the damaged tissue, reparative/regenerative processes start in those tissues endowed with regenerative potential, mainly mediated by activated resident stem cells. These cells reside in a specialized niche that includes different components, cells and surrounding extracellular matrix (ECM), which, reciprocally interacting with stem cells, direct their cell behavior. Evidence suggests that ECM stiffness represents an instructive signal for the activation of stem cells sensing it by various mechanosensors, able to transduce mechanical cues into gene/protein expression responses. The actin cytoskeleton network dynamic acts as key mechanotransducer of ECM signal. The identification of signaling pathways influencing stem cell mechanobiology may offer therapeutic perspectives in the regenerative medicine field. Sphingosine 1-phosphate (S1P)/S1P receptor (S1PR) signaling, acting as modulator of ECM, ECM-cytoskeleton linking proteins and cytoskeleton dynamics appears a promising candidate. This review focuses on the current knowledge on the contribution of S1P/S1PR signaling in the control of mechanotransduction in stem/progenitor cells. The potential contribution of S1P/S1PR signaling in the mechanobiology of skeletal muscle stem cells will be argued based on the intriguing findings on S1P/S1PR action in this mechanically dynamic tissue.

The effect of S1P receptor signaling pathway on the survival and drug resistance in multiple myeloma cells.

Multiple myeloma (MM) remains incurable by conventional chemotherapy. Sphingosine-1-phosphate (S1P) receptor-mediated signaling has been recently demonstrated to have critical roles in cell survival and drug resistance in a number of hematological malignancies. To dissect the roles of S1P receptor pathway in MM, we systematically examined cell viability and protein expression associated with cell survival and drug resistance in MM cell lines upon treatment with either pathway activator (S1P) or inhibitor (FTY720). Our results reveal that FTY720 inhibits cell proliferation by downregulating expression of target genes, while S1P has an opposite effect. Knocking down of S1P receptor S1P5R results in a reduction of cell survival-related gene expression; however, it does not have impacts on expression of drug resistance genes. These results suggest that S1P signaling plays a role in cell proliferation and drug resistance in MM, and targeting this pathway will provide a new therapeutic direction for MM management.

Systemic distribution, subcellular localization and differential expression of sphingosine-1-phosphate receptors in benign and malignant human tissues.

AIMS: Five sphingosine-1-phosphate receptors (S1PR): S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5 (S1PR1-5) have been shown to be involved in the proliferation and progression of various cancers. However, none of the S1PRs have been systemically investigated. In this study, we performed immunohistochemistry (IHC) for S1PR1-S1PR5 on different tissues, in order to simultaneously determine the systemic distribution, subcellular localization and expression level of all five S1PRs. METHODS: We constructed tissue microarrays (TMAs) from 384 formalin-fixed paraffin-embedded (FFPE) blocks containing 183 benign and 201 malignant tissues from 34 human organs/systems. Then we performed IHC for all five S1PRs simultaneously on these TMA slides. The distribution, subcellular localization and expression of each S1PR were determined for each tissue. The data in benign and malignant tissues from the same organ/tissue were then compared using the Student's t-test. In order to reconfirm the subcellular localization of each S1PR as determined by IHC, immunocytochemistry (ICC) was performed on several malignant cell lines. RESULTS: We found that all five S1PRs are widely distributed in multiple human organs/systems. All S1PRs are expressed in both the cytoplasm and nucleus, except S1PR3, whose IHC signals are only seen in the nucleus. Interestingly, the S1PRs are rarely expressed on cellular membranes. Each S1PR is unique in its organ distribution, subcellular localization and expression level in benign and malignant tissues. Among the five S1PRs, S1PR5 has the highest expression level (in either the nucleus or cytoplasm), with S1PR1, 3, 2 and 4 following in descending order. Strong nuclear expression was seen for S1PR1, S1PR3 and S1PR5, whereas S1PR2 and S1PR4 show only weak staining. Four organs/tissues (adrenal gland, liver, brain and colon) show significant differences in IHC scores for the multiple S1PRs (nuclear and/or cytoplasmic), nine (stomach, lymphoid tissues, lung, ovary, cervix, pancreas, skin, soft tissues and uterus) show differences for only one S1PR (cytoplasmic or nuclear), and twenty three organs/tissues show no significant difference in IHC scores for any S1PR (cytoplasmic or nuclear) between benign and malignant changes. CONCLUSION: This is the first study to evaluate the expression level of all S1PRs in benign and malignant tissues from multiple human organs. This study provides data regarding the systemic distribution, subcellular localization and differences in expression of all five S1PRs in benign and malignant changes for each organ/tissue.

High-Density Lipoprotein Signaling via Sphingosine-1-Phosphate Receptors Safeguards Spontaneously Hypertensive Rats against Myocardial Ischemia/Reperfusion Injury.

BACKGROUND: High-density lipoprotein (HDL) protects against ischemia/reperfusion (I/R) injury via signaling through scavenger-receptor class B type-I (SR-BI) and sphingosine-1-phosphate receptors (S1PRs). We recently reported that HDL protects the hearts of spontaneously hypertensive rats (SHRs) against I/R injury in an SR-BI-dependent manner. OBJECTIVE: In this study, we examined the role of S1PRs in HDL-induced protection against myocardial I/R injury in hypertensive rats. METHODS: Hearts from Wistar Kyoto rats (WKYs) and SHRs were subjected to I/R injury using a modified Langendorff system. The hearts were treated with or without HDL in the presence or absence of a receptor- or kinase-specific antagonist. Cardiac hemodynamics and infarct size were measured. Target proteins were analyzed by immunoblotting and ELISA, and nitrite levels were measured using Greis reagent. RESULTS: HDL protected the hearts of WKYs and SHRs against I/R injury. HDL, however, was more protective in WKYs. HDL protection in SHRs required lipid uptake via SR-BI and S1PR1 and S1PR3 but not S1PR2. The hearts from SHRs expressed significantly lower levels of S1PR3 than the hearts from WKYs. HDL differentially activated mediators of the SAFE and RISK pathways in WKYs and SHRs and resulted in nitric oxide generation. Blockage of these pathways abrogated HDL effects. CONCLUSIONS: HDL protects against myocardial I/R injury in normotensive and hypertensive rats, albeit to varying degrees. HDL protection in hearts from hypertensive rodents involved SR-BI-mediated lipid uptake coupled with signaling through S1PR1 and S1PR3. The extent of HDL-induced cardiac protection is directly proportional to S1PR3 expression levels. Mechanistically, the safeguarding effects of HDL involved activation of the SAFE and RISK pathways and the generation of nitric oxide.

Xiaoyin Jiedu Granules may alleviate psoriasis-like skin diseases in mice by regulating sphingosine 1-phosphate receptor expression and reducing Th17 cells.

Sphingosine-1-phosphate (S1P) is associated with the onset and severity of psoriasis, a chronic inflammatory skin disease linked to innate and adaptive immune responses. This study explores the therapeutic effect of Xiaoyin Jiedu Granules, a combination of traditional Chinese medicines, on psoriasis-like skin lesions in mice and the underlying mechanism. We used imiquimod (IMQ) to induce psoriasis-like dermatitis in mice; the effects of Xiaoyin Jiedu Granules on S1P receptors (S1PRs) were investigated using histology and immunohistochemistry. The effects of Xiaoyin Jiedu Granules on the proliferation, differentiation, and activation of the NF-κB pathway in keratinocytes were verified using quantitative polymerase chain reaction (qPCR) and western blotting analyses. CD4+Th17 cells were screened using flow cytometry; the effects of Xiaoyin Jiedu Granules on the differentiation of Th17 cells and the content of related inflammatory factors were also verified. S1PR1-5 was highly expressed in psoriatic lesions. Xiaoyin Jiedu Granules significantly inhibited the secretion of proliferation-related proteins (K6, K16, K17, and IL-36γ) and proinflammatory cytokines (IL-17 and IL-22), transformation of Th17 cells, and activation of the NF-κB pathway and effectively alleviated IMQ-induced psoriasis-like dermatitis. Overall, our findings indicate that Xiaoyin Jiedu Granules have anti-inflammatory activity against S1PR expression, keratinocytes, and immune cells and can therefore mitigate psoriasis. Inhibiting the expression of S1PRs may be an effective treatment strategy against psoriasis.

Role of sphingosine 1-phosphate (S1P) in sepsis-associated intestinal injury.

Sphingosine-1-phosphate (S1P) is a widespread lipid signaling molecule that binds to five sphingosine-1-phosphate receptors (S1PRs) to regulate downstream signaling pathways. Sepsis can cause intestinal injury and intestinal injury can aggravate sepsis. Thus, intestinal injury and sepsis are mutually interdependent. S1P is more abundant in intestinal tissues as compared to other tissues, exerts anti-inflammatory effects, promotes immune cell trafficking, and protects the intestinal barrier. Despite the clinical importance of S1P in inflammation, with a very well-defined mechanism in inflammatory bowel disease, their role in sepsis-induced intestinal injury has been relatively unexplored. In addition to regulating lymphocyte exit, the S1P-S1PR pathway has been implicated in the gut microbiota, intestinal epithelial cells (IECs), and immune cells in the lamina propria. This review mainly elaborates on the physiological role of S1P in sepsis, focusing on intestinal injury. We introduce the generation and metabolism of S1P, emphasize the maintenance of intestinal barrier homeostasis in sepsis, and the protective effect of S1P in the intestine. We also review the link between sepsis-induced intestinal injury and S1P-S1PRs signaling, as well as the underlying mechanisms of action. Finally, we discuss how S1PRs affect intestinal function and become targets for future drug development to improve the translational capacity of preclinical studies to the clinic.

Decoding systems immunological model of sphingolipids with IL-6/IL-17/IL-23 axes in L. major infection.

IL-6, IL-17, IL-23 and IL-1ß are the crucial cytokines controlling inflammatory and immune response during L. major infection. During cutaneous leishmaniasis, an important T helper cell type CD4+ Th17 subset plays a deterministic role in lesion formation through channelling infected macrophages and production of IL-1ß, IL-6, IL-23 and IFN-γ. Ceramide derived sphingosine precursors may assist in pro-inflammatory cytokine response. However, the role of these metabolites in inflammation with pleiotropic pro-inflammatory cytokines in L. major infection is unknown. The present study indicates IL-6/IL-17/IL-23 and SPHK1-S1P-S1PRs signaling axes with the overexpression of SATB1 aiding in disease progression. Targeting SATB1 might modulate the secretion of pro-inflammatory cytokines and abnormal immune functioning, thereby killing the intracellular parasite. Systems immunological methods assisted in a step towards identifying the key to the mystery of crucial components and serving as an approach for therapeutic intervention in L. major infection.

Sphingosine 1-phosphate and its receptors in ischemia.

Sphingosine 1-phosphate (S1P), a metabolite of sphingolipids, is mainly derived from red blood cells (RBCs), platelets and endothelial cells (ECs). It plays important roles in regulating cell survival, vascular integrity and inflammatory responses through its receptors. S1P receptors (S1PRs), including 5 subtypes (S1PR1-5), are G protein-coupled receptors and have been proved to mediate various and complex roles of S1P in atherosclerosis, myocardial infarction (MI) and ischemic stroke by regulating endothelial function and inflammatory response as well as immune cell behavior. This review emphasizes the functions of S1PRs in atherosclerosis and ischemic diseases such as MI and ischemic stroke, enabling mechanistic studies and new S1PRs targeted therapies in atherosclerosis and ischemia in the future.

Salidroside Inhibits CCl4-Induced Liver Fibrosis in Mice by Reducing Activation and Migration of HSC Induced by Liver Sinusoidal Endothelial Cell-Derived Exosomal SphK1.

Sphingosine kinase 1 (SphK1)/Sphingosine-1-phosphate (S1P)/S1PRs signaling pathway is known to involve the advancement of liver fibrosis. Exosomal SphK1 promotes hepatic stellate cells (HSC) migration. Salidroside (Sal) inhibits liver fibrosis, but its mechanism is yet to be elucidated. This study was to explore the influences of Sal on the SphK/S1P/S1PRs signaling pathway in liver fibrosis induced by carbon tetrachloride (CCl4) in vivo, and investigated the mechanism of Sal affecting the migration and activation of HSC triggered by exosomal SphK1 in vitro. Our data showed that Sal reduced the activities of alanine transaminase (ALT), aspartate aminotransferase (AST) in serum, and hydroxyproline (Hyp) content in the liver tissue. Sal subdued the expression of α-smooth muscle actin (α-SMA), fibronectin (FN) and type I collagen (Col I) of the liver. Sal also reduced mitochondria-induced hepatocyte apoptosis and to inhibit JNK activation. Furthermore, Sal remarkably eradicated the influence of SphK1, SphK2, S1P, and S1PRs triggered by CCl4, whether stimulating or hindering. Compared with serum-derived exosomes from model group mice, serum-derived exosomes from Sal group mice expressed lower SphK1 and reduced JS 1 (mouse HSC cell line) migration. In addition, Sal was also observed to subdue Col I expression, AKT activation, and LX-2 migration induced by exosomal SphK1 from SK-HEP-1 (a kind of liver sinusoidal endothelial cells (LSEC) cell line). In conclusion, Sal could effectively alleviate liver injury, hepatocyte apoptosis, and liver fibrosis in vivo, providing supports that the protective effects of Sal might be realized by suppressing JNK activation and modulating the SphK/S1P/S1PRs axis. In vitro, it was observed that Sal might alleviate LX-2 migration and activation induced by exosomal SphK1 by inhibiting the AKT activation.

Dihydrosphingosine driven enrichment of sphingolipids attenuates TGFß induced collagen synthesis in cardiac fibroblasts.

The sphingolipid de novo synthesis pathway, encompassing the sphingolipids, the enzymes and the cell membrane receptors, are being investigated for their role in diseases and as potential therapeutic targets. The intermediate sphingolipids such as dihydrosphingosine (dhSph) and sphingosine (Sph) have not been investigated due to them being thought of as precursors to other more active lipids such as ceramide (Cer) and sphingosine 1 phosphate (S1P). Here we investigated their effects in terms of collagen synthesis in primary rat neonatal cardiac fibroblasts (NCFs). Our results in NCFs showed that both dhSph and Sph did not induce collagen synthesis, whilst dhSph reduced collagen synthesis induced by transforming growth factor ß (TGFß). The mechanisms of these inhibitory effects were associated with the increased activation of the de novo synthesis pathway that led to increased dihydrosphingosine 1 phosphate (dhS1P). Subsequently, through a negative feedback mechanism that may involve substrate-enzyme receptor interactions, S1P receptor 1 expression (S1PR1) was reduced.

Corrigendum: The Role of S1P and the Related Signaling Pathway in the Development of Tissue Fibrosis.

[This corrects the article DOI: 10.3389/fphar.2018.01504.].

Non-negligible factors in studying the ApoM-S1P axis using EA.hy926 cells.

BACKGROUND: The apolipoprotein M (ApoM)-sphingosine-1-phosphate (S1P) axis was recently identified, and research into its function has received increasing attention. However, there are some factors which might influence the results of studies into the function of the ApoM-S1P axis using the EA.hy926 cells. This study investigated related factors, including coagulation factor VIII (FVIII), ApoM, S1P receptor subtypes (S1PRs), C-myc-tagged, and His-tagged proteins in EA.hy926 cells, as well as the effects of ApoM overexpression on S1PRs. METHODS: The expression of FVIII, ApoM, S1PRs, C-myc, and His-tagged proteins in EA.hy926 cells was investigated through cellular immunofluorescence. EA.hy926 cells were infected with lentiviruses carrying (OE group) or lacking (NC group) the ApoM gene sequence. A stable cell line expressing ApoM was obtained, and the expression of ApoM mRNA was detected through single tube duplex fluorescence reverse transcription quantitative polymerase chain reaction (RT-qPCR). S1PRs expression was detected by RT-qPCR and Western blotting. RESULTS: The results showed that EA.hy926 cells expressed FVIII, ApoM, C-myc-tagged, and His-tagged proteins. Moreover, they highly expressed S1PR1, slightly expressed S1PR3, weakly expressed S1PR2, and did not express S1PR4 and S1PR5. ApoM overexpression significantly increased S1PR1 mRNA and protein expression but did not affect the expression of S1PR3. EA.hy926 cells expressed FVIII, suggesting the cell line possesses endothelial cell characteristics and could be used for in vitro studies of the ApoM-S1P axis. CONCLUSIONS: EA.hy926 cell line is suitable for investigation of the ApoM-S1P axis in vitro. However, Since EA.hy926 cells expressed endogenous ApoM, C-myc and His tagged proteins, the exogenous recombinant ApoM should not be labeled with C-myc and His tags for distinguishing from endogenous ApoM. In addition, overexpression of ApoM should be considered to significantly increase the expression of S1PR1 when studying the APOM-S1P axis.

The Role of S1P and the Related Signaling Pathway in the Development of Tissue Fibrosis.

Tissue fibrosis, including pulmonary fibrosis, hepatic fibrosis, and cardiac fibrosis, is an important stage in the development of many diseases. It can lead to structural damage and dysfunction and even severe carcinogenesis or death. There is currently no effective method for the treatment of fibrosis. At present, the molecular mechanism of tissue fibrosis has not yet been fully elucidated, but many studies have demonstrated that it is involved in conveying the complex messages between fibroblasts and various cytokines. Sphingosine 1-phosphate (S1P) is a naturally bioactive sphingolipid. S1P and the related signaling pathways are important intracellular metabolic pathways involved in many life activities, including cell proliferation, differentiation, apoptosis, and cellular signal transduction. Increasing evidence suggests that S1P and its signaling pathways play an important role in the development of tissue fibrosis; however, the mechanisms of these effects have not yet been fully elucidated, and even the role of S1P and its signaling pathways are still controversial. This article focuses on the role of S1P and the related signaling pathways in the development of fibrosis of lung, liver, heart, and other tissues, with emphasis on the application of inhibitors of some of molecules in the pathway in clinical treatment of fibrosis diseases.

Sphingolipid Metabolism: A New Therapeutic Opportunity for Brain Degenerative Disorders.

Neurodegenerative diseases represent a class of fatal brain disorders for which the number of effective therapeutic options remains limited with only symptomatic treatment accessible. Multiple studies show that defects in sphingolipid pathways are shared among different brain disorders including neurodegenerative diseases and may contribute to their complex pathogenesis. In this mini review, we discuss the hypothesis that modulation of sphingolipid metabolism and their related signaling pathways may represent a potential therapeutic approach for those devastating conditions. The plausible "druggability" of sphingolipid pathways is greatly promising and represent a relevant feature that brings real advantage to the development of new therapeutic options for these conditions. Indeed, several molecules that selectively target sphingolipds are already available and many of them currently in clinical trial for human diseases. A deeper understanding of the "sphingolipid scenario" in neurodegenerative disorders would certainly enhance therapeutic perspectives for these conditions, by taking advantage from the already available molecules and by promoting the development of new ones.

FTY720 (fingolimod) regulates key target genes essential for inflammation in microglial cells as defined by high-resolution mRNA sequencing.

Although microglial cells have an essential role in the host defense of the brain, the abnormal activation of microglia can lead to devastating outcomes, such as neuroinflammation and neurodegeneration. Emerging evidence indicates that FTY720 (fingolimod), an FDA-approved drug, has beneficial effects on brain cells in the central nervous system (CNS) and, more recently, immunosuppressive activities in microglia via modulation of the sphingosine 1 phosphate (S1P) 1 receptor. However, the exact molecular aspects of FTY720 contribution in microglia remain largely unaddressed. To understand the molecular mechanisms underlying the roles of FTY720 in microglia, we performed gene expression profiling in resting, FTY720, LPS and LPS + FTY720 challenged primary microglial (PM) cells isolated from 3-day-old ICR mice, and we identified FTY720 target genes and co-regulated modules that were critical in inflammation. By examining RNA sequencing and binding motif datasets from FTY720 suppressed LPS-induced inflammatory mediators, we also identified unexpected relationships between the inducible transcription factors (TFs), motif strength, and the transcription of key inflammatory mediators. Furthermore, we showed that FTY720 controls important inflammatory genes targets by modulating STAT1 and IRF8 levels at their promoter site. Our unprecedented findings demonstrate that FTY720 could be a useful therapeutic application for neuroinflammatory diseases associated with microglia activation, as well as provide a rich resource and framework for future analyses of FTY720 effects on microglia interaction.

Sphingosine-1-phosphate receptor therapies: Advances in clinical trials for CNS-related diseases.

The family of sphingosine-1-phosphate receptors (S1PRs) are G protein-coupled and comprise of five subtypes, S1P1-S1P5. These receptors are activated by the sphingolipid ligand, S1P, which is produced from the phosphorylation of sphingosine by sphingosine kinases. The activation of S1PRs modulates a host of cellular processes such as cell proliferation, migration and survival. These receptors are targeted by the drug fingolimod, a first in class oral therapy for multiple sclerosis. Importantly, S1PRs have also been implicated, in cellular experiments, pre-clinical studies and clinical trials in a range of other neurodegenerative diseases, neurological disorders and psychiatric illnesses, where S1PR drugs are proving beneficial. Overall, studies now highlight the importance of S1PRs as targets for modulating a variety of debilitating brain-related diseases. Here, we review the role of S1PRs in these illnesses. This article is part of the Special Issue entitled 'Lipid Sensing G Protein-Coupled Receptors in the CNS'.