Molecular, immunological, and physiological evidences of a sphingosine-activated plasma membrane Ca2+-channel in Trypanosoma equiperdum.

The hemoparasite Trypanosoma equiperdum belongs to the Trypanozoon subgenus and includes several species that are pathogenic to animals and humans in tropical and subtropical areas across the world. As with all eukaryotic organisms, Ca2+ is essential for these parasites to perform cellular processes thus ensuring their survival across their life cycle. Despite the established paradigm to study proteins related to Ca2+ homeostasis as potential drug targets, so far little is known about Ca2+ entry into trypanosomes. Therefore, in the present study, the presence of a plasma membrane Ca2+-channel in T. equiperdum (TeCC), activated by sphingosine and inhibited by verapamil, is described. The TeCC was cloned and analyzed using bioinformatic resources, which confirmed the presence of several domains, motifs, and a topology similar to the Ca2+ channels found in higher eukaryotes. Biochemical and confocal microscopy assays using antibodies raised against an internal region of human L-type Ca2+ channels indicate the presence of a protein with similar predicted molar mass to the sequence analyzed, located at the plasma membrane of T. equiperdum. Physiological assays based on Fura-2 signals and Mn2+ quenching performed on whole parasites showed a unidirectional Ca2+ entry, which is activated by sphingosine and blocked by verapamil, with the distinctive feature of insensitivity to nifedipine and Bay K 8644. This suggests a second Ca2+ entry for T. equiperdum, different from the store-operated Ca2+ entry (SOCE) previously described. Moreover, the evidence presented here for the TeCC indicates molecular and pharmacological differences with their mammal counterparts, which deserve further studies to evaluate the potential of this channel as a drug target.

Secretion of Sphinganine by Drug-Induced Cancer Cells and Modified Mimetic Sphinganine (MMS) as c-Src Kinase Inhibitor.

BACKGROUND: Cancer cells exhibit selective metabolic reprogramming to promote proliferation, invasiveness, and metastasis. Sphingolipids such as sphingosine and sphinganine have been reported to modulate cell death processes in cancer cells. However, the potential of extracellular sphinganine and its mimetic compounds as inducers of cancer cell death has not been thoroughly investigated. METHODS: We obtained extracellular conditioned medium from HCT-116 cells treated with the previously reported anticancer composition, goat urine DMSO fraction (GUDF). The extracellular metabolites were purified using a novel and in-house developed vertical tube gel electrophoresis (VTGE) technique and identified through LC-HRMS. Extracellular metabolites such as sphinganine, sphingosine, C16 sphinganine, and phytosphingosine were screened for their inhibitory role against intracellular kinases using molecular docking. Molecular dynamics (MD) simulations were performed to study the inhibitory potential of a novel designed modified mimetic sphinganine (MMS) (Pubchem CID: 162625115) upon c-Src kinase. Furthermore, inhibitory potential and ADME profile of MMS was compared with luteolin, a known c-Src kinase inhibitor. RESULTS: Data showed accumulation of sphinganine and other sphingolipids such as C16 sphinganine, phytosphingosine, and ceramide (d18:1/14:0) in the extracellular compartment of GUDF-treated HCT-116 cells. Molecular docking projected c-Src kinase as an inhibitory target of sphinganine. MD simulations projected MMS with strong (-7.1 kcal/mol) and specific (MET341, ASP404) binding to the inhibitory pocket of c-Src kinase. The projected MMS showed comparable inhibitory role and acceptable ADME profile over known inhibitors. CONCLUSION: In summary, our findings highlight the significance of extracellular sphinganine and other sphingolipids, including C16 sphinganine, phytosphingosine, and ceramide (d18:1/14:0), in the context of drug-induced cell death in HCT-116 cancer cells. Furthermore, we demonstrated the importance of extracellular sphinganine and its modified mimetic sphinganine (MMS) as a potential inhibitor of c-Src kinase. These findings suggest that MMS holds promise for future applications in targeted and combinatorial anticancer therapy.

A Near-Infrared Fluorogenic Probe for Rapid, Specific, and Ultrasensitive Detection of Sphingosine in Living Cells and In Vivo.

Sphingosine (Sph) plays important roles in various complex biological processes. Abnormalities in Sph metabolism can result in various diseases, including neurodegenerative disorders. However, due to the lack of rapid and accurate detection methods, understanding sph metabolic in related diseases is limited. Herein, a series of near-infrared fluorogenic probes DMS-X (X = 2F, F, Cl, Br, and I) are designed and synthesized. The fast oxazolidinone ring formation enables the DMS-2F to detect Sph selectively and ultrasensitively, and the detection limit reaches 9.33 ± 0.41 nm. Moreover, it is demonstrated that DMS-2F exhibited a dose- and time-dependent response to Sph and can detect sph in living cells. Importantly, for the first time, the changes in Sph levels induced by Aß42 oligomers and H2 O2 are assessed through a fluorescent imaging approach, and further validated the physiological processes by which Aß42 oligomers and reactive oxygen species (ROS)-induce changes in intracellular Sph levels. Additionally, the distribution of Sph in living zebrafish is successfully mapped by in vivo imaging of a zebrafish model. This work provides a simple and efficient method for probing Sph in living cells and in vivo, which will facilitate investigation into the metabolic process of Sph and the connection between Sph and disease pathologies.

Treatment of Staphylococcus aureus infection with sphingosine in ex vivo perfused and ventilated lungs.

BACKGROUND: Ex vivo lung perfusion (EVLP) has expanded the donor pool for lung transplantation. Pulmonary Staphylococcus aureus infection, especially that caused by multidrug-resistant strains, is a severe threat to posttransplantation outcomes. Sphingosine is a lipid compound that exhibits broad-spectrum antibacterial activity. Therefore, we aimed to evaluate the effects of S aureus infection on EVLP and whether sphingosine administration during EVLP prevents infection with S aureus. METHODS: Eighteen pigs were randomly assigned to 3 groups: uninfected, infected with S aureus with NaCl treatment, or infected with sphingosine treatment. Bacterial numbers were determined before and after treatment. Sphingosine concentrations in the lung tissues were determined using biochemical assays. Lung histology, lung physiological parameters, perfusate content, lung weight, and cell death were measured to analyze the effects of infection and sphingosine administration on EVLP. RESULTS: Sphingosine administration significantly reduced the bacterial load. The concentration of sphingosine in the bronchial epithelium was elevated after sphingosine administration. S aureus infection increased pulmonary artery pressure and pulmonary vascular resistance. Lung edema, histology scores, lactate and lactate dehydrogenase levels in the perfusate, ΔPO2 in the perfusate, static lung compliance, and lung peak airway pressure did not differ among the groups. CONCLUSIONS: Infection of S aureus did not affect the lung function during EVLP but induced higher pulmonary artery pressure and pulmonary vascular resistance. Administration of sphingosine effectively eliminated S aureus without side effects in isolated, perfused, and ventilated pig lungs.

SKI-349, a Sphingosine Kinases 1/2 Inhibitor, Suppresses Cell Viability, Invasion, and AKT/mTOR Signaling Pathway, and Shows Synergistic Cytotoxic Effects with Sorafenib in Hepatocellular Carcinoma.

SKI-349 is a novel sphingosine kinases (SPHK) inhibitor with anti-tumor effects. This study aimed to assess the effect of SKI-349 on cell biological behaviors, downstream pathways, and its synergistic effect with sorafenib in hepatocellular carcinoma (HCC). HCC cell lines (Huh7 and Hep3B) were treated with SKI-349 at concentrations of 1, 2, 4, or 8 µM. Then, SPHK1/2 activity, cell viability, proliferation, apoptosis, invasion, and protein expressions of phosphorylated-protein kinase B (p-AKT), AKT, phosphorylated-mammalian target of rapamycin (p-mTOR) and mTOR were detected. Combination index values of SKI-349 (0, 1, 2, 4, or 8 µM) and sorafenib (0, 2.5, 5, 10, or 20 µM) were calculated. SKI-349 decreased the relative SPHK1 and SPHK2 activity compared with blank control in a dose-dependent manner in the Huh7 and Hep3B cell lines. Meanwhile, SKI-349 reduced cell viability, 5-ethynyl-2'-deoxyuridine (EdU) positive cells, and invasive cells, while it increased apoptotic cells compared to blank control in a dose-dependent manner in Huh7 and Hep3B cell lines. Based on the western blot assay, SKI-349 decreased the ratio of p-AKT to AKT and that of p-mTOR to mTOR compared with blank control in a dose-dependent manner in the Huh7 and Hep3B cell lines. Additionally, SKI-349 combined with sorafenib declined cell viability with concentration gradient effects compared to SKI-349 sole treatment, and they had synergistic cytotoxic effects in Huh7 and Hep3B cell lines. SKI-349 suppresses SPHK1 and SPHK2 activity, cell viability, invasion, and AKT/mTOR signaling pathway, as well as exhibits a synergistic cytotoxic effect with sorafenib in HCC.

Sphingosine-1-Phosphate Receptor 3 Induces Endothelial Barrier Loss via ADAM10-Mediated Vascular Endothelial-Cadherin Cleavage.

Mechanical ventilation (MV) is a life-supporting strategy employed in the Intensive Care Unit (ICU). However, MV-associated mechanical stress exacerbates existing lung inflammation in ICU patients, resulting in limited improvement in mortality and a condition known as Ventilator-Induced Lung Injury (VILI). Sphingosine-1-phosphate (S1P) is a circulating bioactive lipid that maintains endothelial integrity primarily through S1P receptor 1 (S1PR1). During VILI, mechanical stress upregulates endothelial S1PR3 levels. Unlike S1PR1, S1PR3 mediates endothelial barrier disruption through Rho-dependent pathways. However, the specific impact of elevated S1PR3 on lung endothelial function, apart from Rho activation, remains poorly understood. In this study, we investigated the effects of S1PR3 in endothelial pathobiology during VILI using an S1PR3 overexpression adenovirus. S1PR3 overexpression caused cytoskeleton rearrangement, formation of paracellular gaps, and a modified endothelial response towards S1P. It resulted in a shift from S1PR1-dependent barrier enhancement to S1PR3-dependent barrier disruption. Moreover, S1PR3 overexpression induced an ADAM10-dependent cleavage of Vascular Endothelial (VE)-cadherin, which hindered endothelial barrier recovery. S1PR3-induced cleavage of VE-cadherin was at least partially regulated by S1PR3-mediated NFκB activation. Additionally, we employed an S1PR3 inhibitor TY-52156 in a murine model of VILI. TY-52156 effectively attenuated VILI-induced increases in bronchoalveolar lavage cell counts and protein concentration, suppressed the release of pro-inflammatory cytokines, and inhibited lung inflammation as assessed via a histological evaluation. These findings confirm that mechanical stress associated with VILI increases S1PR3 levels, thereby altering the pulmonary endothelial response towards S1P and impairing barrier recovery. Inhibiting S1PR3 is validated as an effective therapeutic strategy for VILI.

Imidazole-based sphingosine-1-phosphate transporter Spns2 inhibitors.

Sphingosine-1-phosphate (S1P) is a chemotactic lipid that influences immune cell positioning. S1P concentration gradients are necessary for proper egress of lymphocytes from the thymus and secondary lymphoid tissues. This trafficking is interdicted by S1P receptor modulators, and it is expected that S1P transporter (Spns2) inhibitors, by reshaping S1P concentration gradients, will do the same. We previously reported SLF1081851 as a prototype Spns2 inhibitor, which provided a scaffold to investigate the importance of the oxadiazole core and the terminal amine. In this report, we disclose a structure-activity relationship study by incorporating imidazole as both a linker and surrogate for a positive charge in SLF1081851. In vitro inhibition of Spns2-dependent S1P transport in HeLa cells identified 7b as an inhibitor with an IC50 of 1.4 ± 0.3 µM. The SAR studies reported herein indicate that imidazolium can be a substitute for the terminal amine in SLF1081851 and that Spns2 inhibition is highly dependent on the lipid alkyl tail length.

Blocking sphingosine 1-phosphate receptor 1 with modulators reduces immune cells infiltration and alleviates endometriosis in mice.

RESEARCH QUESTION: Do sphingosine 1-phosphate (S1P) modulators have therapeutic effects on endometriosis in mice and, if they do, which receptor is responsible for these effects? DESIGN: A surgically induced endometriosis mouse model was established. In the pilot experiment, lesions were harvested to assess fibrosis and inflammation and determine the optimal concentration of a broad-spectrum S1P modulator, FTY720. Subsequently, FTY720 was compared with a selective S1P receptor 1 modulator, SEW2871 to evaluate their effects on endometriotic lesion growth, fibrosis, inflammation and immune cell infiltration. RESULTS: The results demonstrated that both FTY720 and SEW2871, two S1P receptor modulators, effectively inhibited the growth and fibrosis of endometriotic lesions. SEW2871 inhibited inflammation-related cytokine expression, including PTGS-2, IL-1ß, TNF-α and TGF-ß1, more effectively compared with FTY720. Lymphopaenia was mainly caused by FTY720, whereas SEW2871 had a lesser effect. Both FTY720 and SEW2871 significantly reduced CD45+ cells (P = 0.002 and P = 0.032, respectively) and F4/80+ cells (P < 0.001 and P = 0.004, respectively) infiltration into the lesions, with FTY720 exerting a strong regulatory effect on CD4+ T cells. CONCLUSIONS: This study suggests that S1P receptor 1 could be investigated as a potential novel therapeutic target for endometriosis in the future.

A Preliminary Study of Pharmacokinetics and Pharmacodynamics of Oral Fingolimod in Dogs.

BACKGROUND/AIM: Fingolimod is a sphingosine-1-phosphate receptor modulator that prevents lymphocytes egress from lymphoid organs. It has been used as a disease-modifying drug for human multiple sclerosis and has shown better therapeutic effects than other conventional therapies. Therefore, this study was performed to obtain preclinical data of fingolimod in dogs. MATERIALS AND METHODS: Nine laboratory Beagle dogs were used and randomized into three groups for pharmacokinetics (PK) and pharmacodynamics (PD). The dogs were administered once with a low-dose (0.01 mg/kg, n=3), medium-dose (0.05 mg/kg, n=3), and high-dose (0.1 mg/kg, n=3) of fingolimod, orally. Samples were collected serially at predetermined time points, and whole blood fingolimod concentrations were measured using high-performance liquid chromatography-mass spectrometry. Differential counts of leukocytes over time were determined to identify immune cells' response to fingolimod. RESULTS: Regarding PK, the concentration of fingolimod in the blood increased in a dose-dependent manner, but it was not proportional. Regarding PD, the number of lymphocytes significantly decreased compared to baseline in all dose groups (low-dose, p=0.0002; medium-dose, p<0.0001; high-dose, p=0.0012). Eosinophils were significantly reduced in low- (p=0.0006) and medium- (p=0.0006) doses, and neutrophils were also significantly reduced in medium-(p=0.0345) and high- (p=0.0016) doses. CONCLUSION: This study provides the basis for future clinical applications of fingolimod in dogs with immune-mediated diseases, such as meningoencephalitis of unknown etiology.

In vitro bioevaluation and docking study of dihydrosphingosine and ethambutol analogues against sensitive and multi-drug resistant Mycobacterium tuberculosis.

Tuberculosis remains a major public health problem and one of the top ten causes of death worldwide. The alarming increase in multidrug-resistant and extensively resistant variants (MDR, pre-XDR, and XDR) makes the disease more difficult to treat and control. New drugs that act against MDR/XDR strains are needed for programs to contain this major epidemic. The present study aimed to evaluate new compounds related to dihydro-sphingosine and ethambutol against sensitive and pre-XDR Mycobacterium strains, as well as to characterize the pharmacological activity through in vitro and in silico approaches in mmpL3 protein. Of the 48 compounds analyzed, 11 demonstrated good to moderate activity on sensitive and MDR Mycobacterium tuberculosis (Mtb), with a Minimum Inhibitory Concentration (MIC) ranging from 1.5 to 8 µM. They presented 2 to 14 times greater potency of activity when compared to ethambutol in pre-XDR strain, and demonstrated a selectivity index varying between 2.21 and 82.17. The substance 12b when combined with rifampicin, showed a synergistic effect (FICI = 0.5) on sensitive and MDR Mtb. It has also been shown to have a concentration-dependent intracellular bactericidal effect, and a time-dependent bactericidal effect in M. smegmatis and pre-XDR M. tuberculosis. The binding mode of the compounds in its cavity was identified through molecular docking and using a predicted structural model of mmpL3. Finally, we observed by transmission electron microscopy the induction of damage to the cell wall integrity of M. tuberculosis treated with the substance 12b. With these findings, we demonstrate the potential of a 2-aminoalkanol derivative to be a prototype substance and candidate for further optimization of molecular structure and anti-tubercular activity in preclinical studies.

Platelet-derived factors dysregulate placental sphingosine-1-phosphate receptor 2 in human trophoblasts.

RESEARCH QUESTION: Sphingosine-1-phosphate (S1P) is an essential and bioactive sphingolipid with various functions, which acts through five different G-protein-coupled receptors (S1PR1-5). What is the localization of S1PR1-S1PR3 in the human placenta and what is the effect of different flow rates, various oxygen concentrations and platelet-derived factors on the expression profile of S1PR in trophoblasts? DESIGN: Expression dynamics of placental S1PR1-S1PR3 were determined in human first trimester (n = 10), pre-term (n = 9) and term (n = 10) cases. Furthermore, the study investigated the expression of these receptors in different primary cell types isolated from human placenta, verified the findings with publicly available single-cell RNA-Seq data from first trimester and immunostaining of human first trimester and term placentas. The study also tested whether the placental S1PR subtypes are dysregulated in differentiated BeWo cells under different flow rates, different oxygen concentrations or in the presence of platelet-derived factors. RESULTS: Quantitative polymerase chain reaction revealed that S1PR2 is the predominant placental S1PR in the first trimester and reduces towards term (P < 0.0001). S1PR1 and S1PR3 increased from first trimester towards term (P < 0.0001). S1PR1 was localized in endothelial cells, whereas S1PR2 and S1PR3 were predominantly found in villous trophoblasts. Furthermore, S1PR2 was found to be significantly down-regulated in BeWo cells when co-incubated with platelet-derived factors (P = 0.0055). CONCLUSION: This study suggests that the placental S1PR repertoire is differentially expressed across gestation. S1PR2 expression in villous trophoblasts is negatively influenced by platelet-derived factors, which could contribute to down-regulation of placental S1PR2 over time of gestation as platelet presence and activation in the intervillous space increases from the middle of the first trimester onwards.

Sphingosine Kinases at the Intersection of Pro-Inflammatory LPS and Anti-Inflammatory Endocannabinoid Signaling in BV2 Mouse Microglia Cells.

Microglia, the resident immune cells of the central nervous system, play important roles in brain homeostasis as well as in neuroinflammation, neurodegeneration, neurovascular diseases, and traumatic brain injury. In this context, components of the endocannabinoid (eCB) system have been shown to shift microglia towards an anti-inflammatory activation state. Instead, much less is known about the functional role of the sphingosine kinase (SphK)/sphingosine-1-phosphate (S1P) system in microglia biology. In the present study, we addressed potential crosstalk of the eCB and the S1P systems in BV2 mouse microglia cells challenged with lipopolysaccharide (LPS). We show that URB597, the selective inhibitor of fatty acid amide hydrolase (FAAH)-the main degradative enzyme of the eCB anandamide-prevented LPS-induced production of tumor necrosis factor-α (TNFα) and interleukin-1ß (IL-1ß), and caused the accumulation of anandamide itself and eCB-like molecules such as oleic acid and cis-vaccenic acid ethanolamide, palmitoylethanolamide, and docosahexaenoyl ethanolamide. Furthermore, treatment with JWH133, a selective agonist of the eCB-binding cannabinoid 2 (CB2) receptor, mimicked the anti-inflammatory effects of URB597. Interestingly, LPS induced transcription of both SphK1 and SphK2, and the selective inhibitors of SphK1 (SLP7111228) and SphK2 (SLM6031434) strongly reduced LPS-induced TNFα and IL-1ß production. Thus, the two SphKs were pro-inflammatory in BV2 cells in a non-redundant manner. Most importantly, the inhibition of FAAH by URB597, as well as the activation of CB2 by JWH133, prevented LPS-stimulated transcription of SphK1 and SphK2. These results present SphK1 and SphK2 at the intersection of pro-inflammatory LPS and anti-inflammatory eCB signaling, and suggest the further development of inhibitors of FAAH or SphKs for the treatment of neuroinflammatory diseases.

Apolipoprotein M supports S1P production and conservation and mediates prolonged Akt activation via S1PR1 and S1PR3.

Sphingosine 1-phosphate (S1P) is one of the lipid mediators involved in diverse physiological functions. S1P circulates in blood and lymph bound to carrier proteins. Three S1P carrier proteins have been reported, albumin, apolipoprotein M (ApoM) and apolipoprotein A4 (ApoA4). The carrier-bound S1P exerts its functions via specific S1P receptors (S1PR1-5) on target cells. Previous studies showed several differences in physiological functions between albumin-bound S1P and ApoM-bound S1P. However, molecular mechanisms underlying the carrier-dependent differences have not been clarified. In addition, ApoA4 is a recently identified S1P carrier protein, and its functional differences from albumin and ApoM have not been addressed. Here, we compared the three carrier proteins in the processes of S1P degradation, release from S1P-producing cells and receptor activation. ApoM retained S1P more stable than albumin and ApoA4 in the cell culture medium when compared in the equimolar amounts. ApoM facilitated theS1P release from endothelial cells most efficiently. Furthermore, ApoM-bound S1P showed a tendency to induce prolonged activation of Akt via S1PR1 and S1PR3. These results suggest that the carrier-dependent functional differences of S1P are partly ascribed to the differences in the S1P stability, S1P-releasing efficiency and signaling duration.

Potentiation of Sphingolipids and TGF-ß in the human corneal stroma reveals intricate signaling pathway crosstalks.

Corneal haze brought on by fibrosis due to insult can lead to partial or complete vision loss. Currently, corneal transplantation is the gold standard for treating severe corneal fibrosis, which comes with the risk of rejection and the issue of donor tissue shortages. Sphingolipids (SPLs) are known to be associated with fibrosis in various tissues and organs, including the cornea. We previously reported that SPLs are tightly related to Transforming Growth Factor ß (TGF-ß) signaling and corneal fibrogenesis. This study aimed to elucidate the interplay of SPLs, specifically sphingosine-1-phosphate (S1P) signaling, and its' interactions with TGF-ß signaling through detailed analyses of the corresponding downstream signaling targets in the context of corneal fibrosis, in vitro. Healthy human corneal fibroblasts (HCFs) were isolated, plated on polycarbonate membranes, and stimulated with a stable Vitamin C derivative. The 3D constructs were treated with either 5 µM sphingosine-1-phosphate (S1P), 5 µM SPHK I2 (I2; inhibitor of sphingosine kinase 1, one of the two enzymes responsible for generating S1P in mammalian cells), 0.1 ng/mL TGF-ß1, or 0.1 ng/mL TGF-ß3. Cultures with control medium-only served as controls. All 3D constructs were examined for protein expression of fibrotic markers, SPLs, TGF-ßs, and relevant downstream signaling pathways. This data revealed no significant changes in any LTBP (latent TGF-ß binding proteins) expression when stimulated with S1P or I2. However, LTBP1 was significantly upregulated via stimulation of TGF-ß1 and TGF-ß3, whereas LTBP2 was significantly upregulated only with TGF-ß3 stimulation. Significant downregulation of TGF-ß receptor II (TGF-ßRII) following S1P stimulation but significant upregulation following I2 stimulation was observed. Following TGF-ß1, S1P, and I2 stimulation, phospho-SMAD2 (pSMAD2) was significantly downregulated. Furthermore, I2 stimulation led to significant downregulation of SMAD4. Adhesion/proliferation/transcription regulation targets, SRC, FAK, and pERK 1/2 were all significantly downregulated by exogenous S1P, whereas I2 only significantly downregulated FAK. Exogenous TGF-ß3 caused significant upregulation of AKT. Interestingly, both I2 and TGF-ß3 caused significant downregulation of JNK expression. Lastly, TGF-ß1 led to significant upregulation of sphingosine kinase 1 (SphK1) and sphingosine-1-phosphate receptor 3 (S1PR3), whereas TGF-ß3 caused significant upregulation of only SphK1. Together with previously published work from our group and others, S1P inhibition exhibits great potential as an efficacious anti-fibrotic modality in human corneal stromal ECM. The current findings shed further light on a very complex and rather incompletely investigated mechanism, and cement the intricate crosstalk between SPLs and TGF-ß in corneal fibrogenesis. Future studies will dictate the potential of utilizing SPLs/TGF-ß signaling modulators as novel therapeutics in corneal fibrosis.

Sphingosine 1-Phosphate Activates S1PR3 to Induce a Proinflammatory Phenotype in Human Myometrial Cells.

One of the common mechanisms responsible for obstetric complications, affecting millions of women every year, is abnormal uterine contractility. Despite the critical importance of this process for women's health, the mechanisms of uterine contraction regulation remain poorly understood. The initiation of uterine smooth muscle (myometrial) contraction is an inflammatory process, accompanied by upregulation of proinflammatory genes and cytokine release. In this study, we show that sphingolipid metabolism is activated during human labor and that sphingosine 1-phosphate (S1P), the main bioactive sphingolipid, may modify the myometrial proinflammatory phenotype. Our data in both primary and immortalized human myometrial cells show that exogenous S1P induces a proinflammatory gene signature and upregulates the expression of known inflammatory markers of parturition, such as IL8 and COX2. Using expression of IL8 as a readout for S1P activity in myometrial cells, we established that these S1P effects are mediated through the activation of S1P receptor 3 (S1PR3) and downstream activation of ERK1/2 pathways. Inhibition of S1PR3 in human myometrial cells attenuates upregulation of IL8, COX2, and JUNB both at the mRNA and protein levels. Furthermore, activation of S1PR3 with a receptor-specific agonist recapitulated the effects seen after treatment with exogenous S1P. Collectively, these results suggest a signaling pathway activated by S1P in human myometrium during parturition and propose new targets for development of novel therapeutics to alter uterine contractility during management of preterm labor or labor dystocia.

Sphingosine-1-Phosphate Receptor 4 Attenuates Neutrophilic Airway Inflammation in Experimental Asthma via Repressing Proinflammatory Macrophage Activation.

Patients with eosinophilic asthma react well to conventional treatment of asthma while individualized therapy for non-eosinophilic endotypes have yet to be developed. Dysregulated sphingosine metabolites are associated with the pathophysiology of different asthma endotypes with their receptors involved. However, whether the sphingosine-1-phosphate receptor 4 (S1PR4) contributes to disease progression of asthma remains underappreciated. In this study, we demonstrated that sphingosine metabolism was disturbed in asthma while it could not be used to distinguish between different endotypes of asthma. S1PR4, a vital receptor of bioactive sphingosine metabolites and mainly expressed in macrophages, exhibited lower expression both in patients and experimental mice with neutrophilic airway inflammation. Additionally, S1pr4 deficiency aggravated the OVA/LPS-induced pulmonary inflammation in mice along with a significant up-regulation in M1 macrophage activation. Mechanistic studies showed that S1PR4 was strongly connected to bioactive oxylipins concurrent with bounding to formyl peptide receptor 2 to influence the phosphorylation of JNK and contributed to the macrophage M1 program, which in turn secreted amounts of inflammatory cytokines associated to the inflammatory response of neutrophilic asthma. Furthermore, treating mice with S1PR4 agonist CYM50308 was characterized by less pulmonary inflammatory infiltration. Our research indicates S1PR4 a promising therapeutic target for non-eosinophilic phenotypes of asthma.

Sphingosine 1 phosphate lyase inhibition rescues cognition in diabetic mice by promoting anti-inflammatory microglia.

Sphingosine-1-phosphate (S1P) is emerging as a crucial sphingolipid modulating neuroinflammation and cognition. S1P levels in the brain have been found to be decreased in cognitive impairment. S1P lyase (S1PL) is the key enzyme in metabolizing S1P and has been implicated in neuroinflammation. This study evaluated the effect of S1PL inhibition on cognition in type 2 diabetic mice. Fingolimod (0.5 mg/kg and 1 mg/kg) rescued cognition in high-fat diet and streptozotocin-induced diabetic mice, as evident in the Y maze and passive avoidance test. We further evaluated the effect of fingolimod on the activation of microglia in the pre-frontal cortex (PFC) and hippocampus of diabetic mice. Our study revealed that fingolimod inhibited S1PL and promoted anti-inflammatory microglia in both PFC and hippocampus of diabetic mice as it increased Ym-1 and arginase-1. The levels of p53 and apoptotic proteins (Bax and caspase-3) were elevated in the PFC and hippocampus of type 2 diabetic mice which fingolimod reversed. The underlying mechanism promoting anti-inflammatory microglial phenotype was also explored in this study. TIGAR, TP53-associated glycolysis and apoptosis regulator, is known to foster anti-inflammatory microglia and was found to be downregulated in the brain of type 2 diabetic mice. S1PL inhibition decreased the levels of p53 and promoted TIGAR, thereby increasing anti-inflammatory microglial phenotype and inhibiting apoptosis in the brain of diabetic mice. Our study reveals that S1PL inhibition could be beneficial in mitigating cognitive deficits in diabetic mice.

Upregulation of sphingosine kinase 1 in response to doxorubicin generates an angiogenic response via stabilization of Snail.

Sphingosine kinase 1 (SK1) converts the pro-death lipid sphingosine to the pro-survival sphingosine-1-phosphate (S1P) and is upregulated in several cancers. DNA damaging agents, such as the chemotherapeutic doxorubicin (Dox), have been shown to degrade SK1 protein in cancer cells, a process dependent on wild-type p53. As mutations in p53 are very common across several types of cancer, we evaluated the effects of Dox on SK1 in p53 mutant cancer cells. In the p53 mutant breast cancer cell line MDA-MB-231, we show that Dox treatment significantly increases SK1 protein and S1P. Using MDA-MB-231 cells with CRISPR-mediated knockout of SK1 or the selective SK1 inhibitor PF-543, we implicated SK1 in both Dox-induced migration and in a newly uncovered proangiogenic program induced by Dox. Mechanistically, inhibition of SK1 suppressed the induction of the cytokine BMP4 and of the EMT transcription factor Snail in response to Dox. Interestingly, induction of BMP4 by SK1 increased Snail levels following Dox treatment by stabilizing Snail protein. Furthermore, we found that SK1 was required for Dox-induced p38 MAP kinase phosphorylation and that active p38 MAPK in turn upregulated BMP4 and Snail, positioning p38 downstream of SK1 and upstream of BMP4/Snail. Modulating production of S1P by inhibition of de novo sphingolipid synthesis or knockdown of the S1P-degrading enzyme S1P lyase identified S1P as the sphingolipid activator of p38 in this model. This work establishes a novel angiogenic pathway in response to a commonly utilized chemotherapeutic and highlights the potential of SK1 as a secondary drug target for patients with p53 mutant cancer.

Opposing Roles of Sphingosine 1-Phosphate Receptors 1 and 2 in Fat Deposition and Glucose Tolerance in Obese Male Mice.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that regulates fundamental cellular processes such as proliferation, migration, apoptosis, and differentiation through 5 cognate G protein-coupled receptors (S1P1-S1P5). We previously demonstrated that blockade of S1P2 signaling in S1P2-deficient mice attenuates high-fat diet-induced adipocyte hypertrophy and glucose intolerance and an S1P2-specific antagonist JTE-013 inhibits, whereas an S1P1/S1P3 dual antagonist (VPC23019) activates, adipogenic differentiation of preadipocytes. Based on those observations, this study examined whether an S1P1-specific agonist, SEW-2871, VPC23019, or their combination acts on obesity and glucose intolerance in leptin-deficient ob/ob mice. The oral administration of SEW-2871 or JTE-013 induced significant reductions in body/epididymal fat weight gains and epididymal/inguinal fat adipocyte sizes and improved glucose intolerance and adipocyte inflammation in ob/ob mice but not in their control C57BL/6J mice. Both SEW-2871 and JTE-013 decreased messenger RNA levels of tumor necrosis factor-α and CD11c, whereas they increased those of CD206 and adiponectin in the epididymal fats isolated from ob/ob mice with no changes in the levels of peroxisome proliferator activated receptor γ and its regulated genes. By contrast, VPC23019 did not cause any such alterations but counteracted with all those SEW-2871 actions in these mice. In conclusion, the S1P1 agonist SEW-2871 acted like the S1P2 antagonist JTE-013 to reduce body/epididymal fats and improve glucose tolerance in obese mice. Therefore, this study raises the possibility that endogenous S1P could promote obesity/type 2 diabetes through the S1P2, whereas exogenous S1P could act against them through the S1P1.

Targeting neuroinflammation in neuropathic pain and opioid use.

Neuropathic pain arises from injuries to the nervous system. It affects 20% of the adult US population and poses a major socioeconomic burden yet remains exceedingly difficult to treat. Current therapeutic approaches have limited efficacy and a large side effect profile that impedes their ability to treat neuropathic pain effectively. Preclinical research over the last 30 yr has established the critical role that pro-inflammatory neuro-immune cell interactions have in the development and maintenance of neuropathic pain arising from various etiologies. Pro-inflammatory neuro-immune cell interactions also underlie the development of adverse side effects of opioids and the loss of their efficacy to treat pain. Evidence from work in our lab and others in preclinical animal models have shown that signaling from the bioactive sphingolipid, sphingosine-1-phosphate (S1P), through the S1P receptor subtype 1 (S1PR1) modulates neuro-immune cell interactions. Here, we discuss how targeting S1P/S1PR1 signaling with S1PR1 antagonists already Food and Drug Administration-approved or in clinical trials for multiple sclerosis can provide a viable pharmacotherapeutic approach to reduce neuro-immune cell inflammatory signaling and potentially treat patients suffering neuropathic pain and the adverse effects of opioids.