Functional Insights into the Sphingolipids C1P, S1P, and SPC in Human Fibroblast-like Synoviocytes by Proteomic Analysis.

The (patho)physiological function of the sphingolipids ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P), and sphingosylphosphorylcholine (SPC) in articular joints during osteoarthritis (OA) is largely unknown. Therefore, we investigated the influence of these lipids on protein expression by fibroblast-like synoviocytes (FLSs) from OA knees. Cultured human FLSs (n = 7) were treated with 1 of 3 lipid species-C1P, S1P, or SPC-IL-1ß, or with vehicle. The expression of individual proteins was determined by tandem mass tag peptide labeling followed by high-resolution electrospray ionization (ESI) mass spectrometry after liquid chromatographic separation (LC-MS/MS/MS). The mRNA levels of selected proteins were analyzed using RT-PCR. The 3sphingolipids were quantified in the SF of 18 OA patients using LC-MS/MS. A total of 4930 proteins were determined using multiplex MS, of which 136, 9, 1, and 0 were regulated both reproducibly and significantly by IL-1ß, C1P, S1P, and SPC, respectively. In the presence of IL-1ß, all 3 sphingolipids exerted ancillary effects. Only low SF levels of C1P and SPC were found. In conclusion, the 3 lipid species regulated proteins that have not been described in OA. Our results indicate that charged multivesicular body protein 1b, metal cation symporter ZIP14, glutamine-fructose-6-P transaminase, metallothionein-1F and -2A, ferritin, and prosaposin are particularly interesting proteins due to their potential to affect inflammatory, anabolic, catabolic, and apoptotic mechanisms.

Vascular Endothelial Cell-Derived Exosomal Sphingosylphosphorylcholine Attenuates Myocardial Ischemia-Reperfusion Injury through NR4A2-Mediated Mitophagy.

Cardiomyocyte survival is a critical contributing process of host adaptive responses to cardiovascular diseases (CVD). Cells of the cardiovascular endothelium have recently been reported to promote cardiomyocyte survival through exosome-loading cargos. Sphingosylphosphorylcholine (SPC), an intermediate metabolite of sphingolipids, mediates protection against myocardial infarction (MI). Nevertheless, the mechanism of SPC delivery by vascular endothelial cell (VEC)-derived exosomes (VEC-Exos) remains uncharacterized at the time of this writing. The present study utilized a mice model of ischemia/reperfusion (I/R) to demonstrate that the administration of exosomes via tail vein injection significantly diminished the severity of I/R-induced cardiac damage and prevented apoptosis of cardiomyocytes. Moreover, SPC was here identified as the primary mediator of the observed protective effects of VEC-Exos. In addition, within this investigation, in vitro experiments using cardiomyocytes showed that SPC counteracted myocardial I/R injury by activating the Parkin and nuclear receptor subfamily group A member 2/optineurin (NR4A2/OPTN) pathways, in turn resulting in increased levels of mitophagy within I/R-affected myocardium. The present study highlights the potential therapeutic effects of SPC-rich exosomes secreted by VECs on alleviating I/R-induced apoptosis in cardiomyocytes, thereby providing strong experimental evidence to support the application of SPC as a potential therapeutic target in the prevention and treatment of myocardial infarction.

Paxillin participates in the sphingosylphosphorylcholine-induced abnormal contraction of vascular smooth muscle by regulating Rho-kinase activation.

BACKGROUND: The Ca2+-independent contraction of vascular smooth muscle is a leading cause of cardiovascular and cerebrovascular spasms. In the previous study, we demonstrated the involvement of Src family protein tyrosine kinase Fyn and Rho-kinase in the sphingosylphosphorylcholine (SPC)-induced abnormal and Ca2+-independent contraction of vascular smooth muscle, but the specific mechanism has not been completely clarified. METHODS: Paxillin knockdown human coronary artery smooth muscle cells (CASMCs) and smooth muscle-specific paxillin knockout mice were generated by using paxillin shRNA and the tamoxifen-inducible Cre-LoxP system, respectively. CASMCs contraction was observed by time-lapse recording. The vessel contractility was measured by using a myography assay. Fyn, Rho-kinase, and myosin light chain activation were assessed by immunoprecipitation and western blotting. The paxillin expression and actin stress fibers were visualized by histological analysis and immunofluorescent staining. RESULTS: The SPC-induced abnormal contraction was inhibited in paxillin knockdown CASMCs and arteries of paxillin knockout mice, indicating that paxillin is involved in this abnormal contraction. Further study showed that paxillin knockdown inhibited the SPC-induced Rho-kinase activation without affecting Fyn activation. In addition, paxillin knockdown significantly inhibited the SPC-induced actin stress fiber formation and myosin light chain phosphorylation. These results suggest that paxillin, as an upstream molecule of Rho-kinase, is involved in the SPC-induced abnormal contraction of vascular smooth muscle. CONCLUSIONS: The present study demonstrated that paxillin participates in the SPC-induced abnormal vascular smooth muscle contraction by regulating Rho-kinase activation. Video Abstract.

Sphingosylphosphorylcholine inhibits plasma cell differentiation and ameliorates experimental autoimmune encephalomyelitis.

Introduction: Multiple sclerosis (MS) is a potentially disabling disease that damages the brain and spinal cord, inducing paralysis of the body. While MS has been known as a T-cell mediated disease, recent attention has been drawn to the involvement of B cells in its pathogenesis. Autoantibodies from B cells are closely related with the damage lesion of central nervous system and worse prognosis. Therefore, regulating the activity of antibody secreting cell could be related with the severity of the MS symptoms. Methods: Total mouse B cells were stimulated with LPS to induce their differentiation into plasma cells. The differentiation of plasma cells was subsequently analyzed using flow cytometry and quantitative PCR analysis. To establish an experimental autoimmune encephalomyelitis (EAE) mouse model, mice were immunized with MOG35-55/CFA emulsion. Results: In this study, we found that plasma cell differentiation was accompanied by upregulation of autotaxin, which converts sphingosylphosphorylcholine (SPC) to sphingosine 1-phosphate in response to LPS. We observed that SPC strongly blocked plasma cell differentiation from B cells and antibody production in vitro. SPC downregulated LPS-stimulated IRF4 and Blimp 1, which are required for the generation of plasma cells. SPC-induced inhibitory effects on plasma cell differentiation were specifically blocked by VPC23019 (S1PR1/3 antagonist) or TY52159 (S1PR3 antagonist), but not by W146 (S1PR1 antagonist) and JTE013 (S1PR2 antagonist), suggesting a crucial role of S1PR3 but not S1PR1/2 in the process. Administration of SPC against an EAE mouse model significantly attenuated the symptoms of disease, showing decreased demyelinated areas of the spinal cord and decreased numbers of cells infiltrated into the spinal cord. SPC markedly decreased plasma cell generation in the EAE model, and SPC-induced therapeutic effects against EAE were not observed in µMT mice. Conclusion: Collectively, we demonstrate that SPC strongly inhibits plasma cell differentiation, which is mediated by S1PR3. SPC also elicits therapeutic outcomes against EAE, an experimental model of MS, suggesting SPC as a new material to control MS.

Multiplex Quantification of Plasma Biomarkers for Patients with Gaucher Disease Type 1.

Gaucher disease (GD) is a lysosomal storage disorder caused by a deficiency of the enzyme beta-glucocerebrosidase. This leads to the accumulation of glycolipids in macrophages and ultimately results in tissue damage. Recent metabolomic studies highlighted several potential biomarkers in plasma specimens. In hopes of better understanding the distribution, importance, and clinical significance of these potential markers, a UPLC-MS/MS method was developed and validated to quantify lyso-Gb1 and six related analogs (with the following modifications on the sphingosine moiety: -C2 H4 (-28 Da), -C2 H4 +O (-12 Da), -H2 (-2 Da), -H2 +O (+14 Da), +O (+16 Da), and +H2 O (+18 Da)), sphingosylphosphorylcholine, and N-palmitoyl-O-phosphocholineserine in plasma specimens of treated and untreated patients. This 12-min UPLC-MS/MS method involves a purification step via solid-phase extraction followed by evaporation under nitrogen flow and resuspension in an organic mix compatible with HILIC chromatography. This method is currently used for research purposes and might be used for monitoring, prognostics, and follow-up. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

Sphingosylphosphorylcholine (SPC), a Causative Factor of SPC-Induced Vascular Smooth Muscle Cells Contraction, Is Taken Up via Endocytosis.

The reaction field of abnormal vascular contraction induced by sphingosylphosphorylcholine (SPC) and the action point of SPC around the plasma membranes remain unknown. However, we found in a previous study that fisetin prevents SPC-induced vascular smooth muscle cells contraction, while the mechanism remains unknown. Therefore, in this study, we aimed to address the action point of SPC around the plasma membranes and the involvement of fisetin. We focused on microdomains and evaluated their markers flotillin-1 and caveolin-1 and the localization of SPC to investigate their action point. The results showed that microdomains of vascular smooth muscle cells were not involved in SPC-induced contraction. However, we found that after SPC had been affected on the plasma membrane, cells took up SPC via endocytosis. Moreover, SPC remained in the cells and did not undergo transcytosis, and SPC-induced contracting cells produced exosomes. These phenomena were similar to those observed in fisetin-treated cells. Thus, we speculated that, although not involved in the reaction field of SPC-induced contractions, the microdomain induced the endocytosis of SPCs, and fisetin prevented the contractions by directly targeting vascular smooth muscle cells. Notably, this preventive mechanism involves the cellular uptake of SPC via endocytosis.

MORN4 protects cardiomyocytes against ischemic injury via MFN2-mediated mitochondrial dynamics and mitophagy.

The imbalance of mitochondrial fission and fusion dynamics causes ischemic cardiomyocyte apoptosis and heart injury by affecting mitophagy. Regulation of mitochondrial dynamics is an important therapeutic strategy for ischemic heart diseases. Considering the important roles of MORN motifs in heart diseases and chloroplast fission, we aimed to investigate the possible role of MORN repeat-containing protein 4 (MORN4) in the progression of myocardial infarction (MI), ischemic cardiomyocyte apoptosis, mitochondrial dynamics, and mitophagy. We found that in the MI mouse, MORN4 knockdown remarkably accelerated cardiac injury and fibrosis with deteriorating cardiac dysfunction. Sphingosylphosphorylcholine (SPC) alleviated ischemic cardiomyocyte apoptosis and heart injury through increased level of MORN4, indicating a vital function of MORN4 in heart with SPC used to clarify the molecular mechanisms underlying the functions of MORN4. Mechanistically, we found that MORN4 directly binds to MFN2 and promotes the phosphorylation of MFN2 S442 through Rho-associated protein kinase 2 (ROCK2), which mediates beneficial mitophagy induced by mitochondrial dynamics, while SPC promoted the binding of MORN4 and MFN2 and the process. Taken together, our data reveal a new perspective role of MORN4 in ischemic heart injury, and report that SPC could regulate myocardial mitochondrial homeostasis by activating the MORN4-MFN2 axis during the ischemic situation, this finding provides novel targets for improving myocardial ischemia tolerance and rescue of acute myocardial infarction.

Plasma Lysosphingolipid Biomarker Measurement by Liquid Chromatography Tandem Mass Spectrometry.

Plasma lysosphingolipids are highly elevated in patients with Gaucher, Krabbe, Fabry, and Niemann-Pick diseases and tend to accumulate to a greater extent than their respective primary sphingolipids in the plasma of affected patients. In this chapter, we describe two liquid chromatography tandem mass spectrometry (LC-MS/MS) methods to measure plasma concentrations of four lysosphingolipids species. The first method described measures glucosylsphingosine (lyso-GL1) and galactosylsphingosine (psychosine), biomarkers that accumulate in Gaucher and Krabbe diseases, respectively. The second method measures globotriaosylsphingosine (lyso-Gb3) and sphingosylphosphorylcholine (lyso-SPM), biomarkers for Fabry and Niemann-Pick diseases, respectively. Each method utilizes isotope-labeled internal standards and multipoint calibration curves to quantify the analytes of interest. Briefly, plasma samples are mixed with five volumes of LC-MS grade methanol containing internal standard, and protein is removed via centrifugation. Supernatant is dried and resuspended in initial mobile phase. Samples are separated by liquid chromatography using either a BEH amide column (lyso-GL1 + psychosine) or a C18 column (lyso-Gb3 + lyso-SPM). Protonated analytes are measured by selected reaction monitoring (SRM) in positive electrospray ionization mode. Using these methods, we have observed elevations of these lyso- species in Gaucher, Fabry, and Niemann-Pick and successfully distinguished different subtypes reflecting the disease severity.

Sphingosylphosphorylcholine ameliorates doxorubicin-induced cardiotoxicity in zebrafish and H9c2 cells by reducing excessive mitophagy and mitochondrial dysfunction.

Doxorubicin (DOX, C27H29NO11), is an anthracycline tumor chemotherapy drug, which has significant side effects on many organs including the heart. In recent years, mitochondrial dysfunction caused by DOX was identified as an important reason for cardiotoxic injury. Sphingosylphosphorylcholine (SPC) is essential for mitochondrial homeostasis in our previous report, however, its role in DOX-caused cardiomyopathy has remained elusive. Herein, DOX treated zebrafish embryos (90 µM) and adult fish (2.5 µM/g) were used to simulate DOX-induced cardiotoxic damage. Histopathological and ultrastructural observations showed that SPC (2.5 µM) significantly ameliorated DOX-induced pericardial edema, myocardial vacuolization and apoptosis. Furthermore, SPC (2.5 µM) can significantly inhibit DOX-induced apoptosis and promote cell proliferation in DOX treated H9c2 cells (1 µM), which is dependent on the restoration of mitochondrial homeostasis, including restored mitochondrial membrane potential, mitochondrial superoxide and ATP levels. We finally confirmed that SPC restored mitochondrial homeostasis through ameliorating DOX-induced excessive mitophagy. Mechanistically, SPC reduced calmodulin (CaM) levels and thus inhibiting Parkin activation and Parkin-dependent mitophagy. These results suggest that reducing the cardiotoxicity of chemotherapeutic drugs by targeting SPC may be a new solution to rescue chemotherapy injury.

Inhibitory mechanism of tangeretin, a citrus flavone on the sphingosylphosphorylcholine (SPC)-induced vascular smooth muscle contraction.

We previously discovered that the SPC/Fyn/Rho-kinase (ROK) pathway mediates the Ca2+-sensitization of coronary arterial smooth muscle (CASM) contraction leading to vasospasm, a major cause of sudden death. Lately, we have been trying to find and develop more natural edible compounds which can treat and/or prevent the SPC-induced abnormal CASM contraction, and finally the first to discover that tangeretin (5,6,7,8,4'-pentamethoxyflavone), a natural compound extracted from citrus plants, can inhibit the SPC-induced CASM contraction both in the pretreatment and posttreatment. In porcine CASM tissues, tangeretin showed remarkable inhibitory effects on the SPC-induced contraction with modest inhibitory effects on the high K+-depolarization-induced Ca2+-dependent contraction, both in pretreatment and posttreatment at the optimal concentrations; Regarding the mechanisms, tangeretin markedly abolished the SPC-induced cell contraction through inhibiting the SPC-induced activation and translocation of Fyn and ROK from the cytoplasm to the cell membrane in cultured CASM cells, resulting in the reduction of phosphorylation of myosin light chain. Taken together, these findings indicate that tangeretin, upon pre- or post-treatment, inhibits the SPC-induced CASM contraction through suppressing the Fyn/ROK signaling pathway, thereby suggesting that tangeretin can be a potential candidate for the treatment and/or prevention of vasospasm.

Sphingosylphosphorylcholine ameliorates experimental sjögren's syndrome by regulating salivary gland inflammation and hypofunction, and regulatory B cells.

Sjögren syndrome (SS) is an autoimmune disease in which immune cells infiltrate the exocrine gland. Since SS is caused by a disorder of the immune system, treatments should regulate the immune response. Sphingosylphosphorylcholine (SPC) is a sphingolipid that mediates cellular signaling. In immune cells, SPC has several immunomodulatory functions. Accordingly, this study verifies the immunomodulatory ability and therapeutic effect of SPC in SS. To understand the function of SPC in SS, we treated SPC in female NOD/ShiJcl (NOD) mice. The mice were monitored for 10 weeks, and inflammation in the salivary glands was checked. After SPC treatment, we detected the expression of regulatory B (Breg) cells in mouse splenocytes and the level of salivary secretion-related genes in human submandibular gland (HSG) cells. Salivary flow rate was maintained in the SPC-treated group compared to the vehicle-treated group, and inflammation in the salivary gland tissues was relieved by SPC. SPC treatment in mouse cells and HSG cells enhanced Breg cells and salivary secretion markers, respectively. This study revealed that SPC can be considered as a new therapeutic agent against SS.

JNK-dependent phosphorylation and nuclear translocation of EGR-1 promotes cardiomyocyte apoptosis.

Myocardial apoptosis induced by myocardial ischemia and hyperlipemia are the main causes of high mortality of cardiovascular diseases. It is not clear whether there is a common mechanism responsible for these two kinds of cardiomyocyte apoptosis. Previous studies demonstrated that early growth response protein 1 (EGR-1) has a pro-apoptotic effect on cardiomyocytes under various stress conditions. Here, we found that EGR-1 is also involved in cardiomyocyte apoptosis induced by both ischemia and high-fat, but how EGR-1 enters the nucleus and whether nuclear EGR-1 (nEGR-1) has a universal effect on cardiomyocyte apoptosis are still unknown. By analyzing the phosphorylation sites and nucleation information of EGR-1, we constructed different mutant plasmids to confirm that the nucleus location of EGR-1 requires Ser501 phosphorylation and regulated by JNK. Furthermore, the pro-apoptotic effect of nEGR-1 was further explored through genetic methods. The results showed that EGR-1 positively regulates the mRNA levels of apoptosis-related proteins (ATF2, CTCF, HAND2, ELK1), which may be the downstream targets of EGR-1 to promote the cardiomyocyte apoptosis. Our research announced the universal pro-apoptotic function of nEGR-1 and explored the mechanism of its nucleus location in cardiomyocytes, providing a new target for the "homotherapy for heteropathy" to cardiovascular diseases.

Quantitation of a plasma biomarker profile for the early detection of Gaucher disease type 1 patients.

Aim: Gaucher disease (GD) is caused by a deficiency of the lysosomal enzyme acid ß-glucocerebrosidase. Recent metabolomic studies highlighted several new metabolites increased in the plasma of GD patients. We aimed to develop and validate a UPLC-MS/MS method allowing a relative quantitation of lyso-Gb1 and lyso-Gb1 analogs -28, -12, -2, +14, +16 and +18 Da in addition to sphingosylphosphorylcholine, N-palmitoyl-O-phosphocholine to study potential correlations with clinical manifestations. Methodology & results: Following solid-phase extraction, plasma samples were evaporated and resuspended in 100 µl of resuspension solution. Three microliter is injected into the UPLC-MS/MS for analysis. Conclusion: All biomarkers studied were increased in GD patients. Significant correlations were observed between specific analogs and hematological, and visceral complications, as well as overall disease severity.

A Novel Function of Sphingosylphosphorylcholine on the Inhibitory Effects of Acetylcholinesterase Activity.

Acetylcholine (ACh), a quaternary ammonium cation, is known as one of the itch inducer in atopic dermatitis (AD), an inflammatory skin disease with intense itching. Previous research has reported accumulation of ACh in lesional site of AD patients. Generally, ACh is metabolized by cholinesterase (ChE). Therefore, one of the causes of ACh accumulation may be the suppression of ChE activity. Increased levels of the multifunctional bioactive sphingolipid sphingosylphosphorylcholine (SPC) have also been detected in AD. Since SPC possesses a quaternary ammonium cation, like ACh, it is possible that SPC affects the activity of ChE catalyzing ACh metabolization. We investigated whether SPC influences the activity of ChE by performing enzymatic analysis of ChE in the presence of SPC. We found that SPC strongly suppressed acetylcholinesterase (AChE) activity, but the suppression of butyrylcholinesterase by SPC was quite low. The Michaelis constant (Km) of AChE in the presence of SPC increased, and the maximum velocity (Vmax) decreased, indicating that SPC acts as mixed-type inhibitor for AChE. The analysis of SPC analogs clarified the importance of both the quaternary ammonium cation and the carbon chain length of SPC for the AChE inhibitory effect and showed that SPC was unique in AChE inhibition among the sphingolipids in this study. These findings indicate a novel function of SPC on AChE inhibition. Thus, the inhibition activity of SPC may be a factor in the increase of ACh in AD.

Sphingosylphosphorylcholine blocks ovariectomy-induced bone loss by suppressing Ca2+ /calmodulin-mediated osteoclast differentiation.

Osteoporosis is a disease in which bone mineral density decreases due to abnormal activity of osteoclasts, and is commonly found in post-menopausal women who have decreased levels of female hormones. Sphingosylphosphorylcholine (SPC) is an important biological lipid that can be converted to sphingosine-1-phosphate (S1P) by autotaxin. S1P is known to be involved in osteoclast activation by stimulating osteoblasts, but bone regulation by SPC is not well understood. In this study, we found that SPC strongly inhibits RANKL-induced osteoclast differentiation. SPC-induced inhibitory effects on osteoclast differentiation were not affected by several antagonists of S1P receptors or pertussis toxin, suggesting cell surface receptor independency. However, SPC inhibited RANKL-induced calcineurin activation and subsequent NFATc1 activity, leading to decrease of the expression of Trap and Ctsk. Moreover, we found that bone loss in an experimental osteoporosis mouse model was recovered by SPC injection. SPC also blocked ovariectomy-induced body weight increase and Nfatc1 gene expression in mice. We also found that SPC inhibits RANKL-induced osteoclast differentiation in human macrophages. Since currently available treatments for osteoporosis, such as administration of female hormones or hormone receptor modulators, show serious side effects, SPC has potential as a new agent for osteoporosis treatment.

Ethacrynic acid, a loop diuretic, suppresses epithelial-mesenchymal transition of A549 lung cancer cells via blocking of NDP-induced WNT signaling.

Lung cancer is one of the leading causes of death in cancer patients. Epithelial-mesenchymal transition (EMT) plays an important role in lung cancer progression. Therefore, for lung cancer treatment, it is crucial to find substances that inhibit EMT. Ethacrynic acid (ECA) is a diuretic that inhibits cellular ion flux and exerts anticancer effects. However, the effects of ECA on EMT in lung cancer remain unclear. We examined the effects of ECA on sphingosylphosphorylcholine (SPC) or TGF-ß1-induced EMT process in A549 and H1299 cells via reverse transcription polymerase chain reaction and Western blotting. We found that ECA inhibited SPC-induced EMT and SPC-induced WNT signalling in EMT. We observed that SPC induces the expression of NDP [Norrie disease protein] and WNT-2, whereas ECA suppressed their expression. SPC-induced WNT activation, EMT, migration, and invasion were suppressed by NDP small-interfering RNA (siNDP), but NDP overexpression (pNDP) enhanced these events in A549 and H1299 cells. Accordingly, NDP expression may influence lung cancer prognosis. In summary, our results revealed that ECA inhibited SPC or TGF-ß1-induced EMT in A549 and H1299 lung cancer cells by downregulating NDP expression and inhibiting WNT activation. Therefore, ECA might be a new drug candidate for lung cancer treatment.

Sphingomyelin Deacylase, the Enzyme Involved in the Pathogenesis of Atopic Dermatitis, Is Identical to the ß-Subunit of Acid Ceramidase.

A ceramide deficiency in the stratum corneum (SC) is an essential etiologic factor for the dry and barrier-disrupted skin of patients with atopic dermatitis (AD). Previously, we reported that sphingomyelin (SM) deacylase, which hydrolyzes SM and glucosylceramide at the acyl site to yield their lysoforms sphingosylphosphorylcholine (SPC) and glucosylsphingosine, respectively, instead of ceramide and/or acylceramide, is over-expressed in AD skin and results in a ceramide deficiency. Although the enzymatic properties of SM deacylase have been clarified, the enzyme itself remains unidentified. In this study, we purified and characterized SM deacylase from rat skin. The activities of SM deacylase and acid ceramidase (aCDase) were measured using SM and ceramide as substrates by tandem mass spectrometry by monitoring the production of SPC and sphingosine, respectively. Levels of SM deacylase activity from various rat organs were higher in the order of skin > lung > heart. By successive chromatography using Phenyl-5PW, Rotofor, SP-Sepharose, Superdex 200 and Shodex RP18-415, SM deacylase was purified to homogeneity with a single band of an apparent molecular mass of 43 kDa with an enrichment of > 14,000-fold. Analysis by MALDI-TOF MS/MS using a protein spot with SM deacylase activity separated by 2D-SDS-PAGE allowed its amino acid sequence to be determined and identified as the ß-subunit of aCDase, which consists of α- and ß-subunits linked by amino bonds and a single S-S bond. Western blotting of samples treated with 2-mercaptoethanol revealed that, whereas recombinant human aCDase was recognized by antibodies to the α-subunit at ~56 kDa and ~13 kDa and the ß-subunit at ~43 kDa, the purified SM deacylase was detectable only by the antibody to the ß-subunit at ~43 kDa. Breaking the S-S bond of recombinant human aCDase with dithiothreitol elicited the activity of SM deacylase with ~40 kDa upon gel chromatography. These results provide new insights into the essential role of SM deacylase expressed as an aCDase-degrading ß-subunit that evokes the ceramide deficiency in AD skin.

Identification of a Reliable Biomarker Profile for the Diagnosis of Gaucher Disease Type 1 Patients Using a Mass Spectrometry-Based Metabolomic Approach.

Gaucher disease (GD) is a rare autosomal recessive multisystemic lysosomal storage disorder presenting a marked phenotypic and genotypic variability. GD is caused by a deficiency in the glucocerebrosidase enzyme. The diagnosis of GD remains challenging because of the large clinical spectrum associated with the disease. Moreover, GD biomarkers are often not sensitive enough and can be subject to polymorphic variations. The main objective of this study was to perform a metabolomic study using an ultra-performance liquid chromatography system coupled to a time-of-flight mass spectrometer to identify novel GD biomarkers. Following the analysis of plasma samples from patients with GD, and age- and gender-matched control samples, supervised statistical analyses were used to find the best molecules to differentiate the two groups. Targeted biomarkers were structurally elucidated using accurate mass measurements and tandem mass spectrometry. This metabolomic study was successful in highlighting seven biomarkers associated with GD. Fragmentation tests revealed that these latter biomarkers were lyso-Gb1 (glucosylsphingosine) and four related analogs (with the following modifications on the sphingosine moiety: -C2H4, -H2, -H2+O, and +H2O), sphingosylphosphorylcholine, and N-palmitoyl-O-phosphocholineserine. Based on the plasma biomarker distribution, we suggest the evaluation of this GD biomarker profile, which might facilitate early diagnosis, monitoring, and follow-up of patients.

A novel sphingosylphosphorylcholine and sphingosine-1-phosphate receptor 1 antagonist, KRO-105714, for alleviating atopic dermatitis.

BACKGROUND: Atopic dermatitis (eczema) is a type of inflammation of the skin, which presents with itchy, red, swollen, and cracked skin. The high global incidence of atopic dermatitis makes it one of the major skin diseases threatening public health. Sphingosylphosphorylcholine (SPC) and sphingosine-1-phosphate (S1P) act as pro-inflammatory mediators, as an angiogenesis factor and a mitogen in skin fibroblasts, respectively, both of which are important biological responses to atopic dermatitis. The SPC level is known to be elevated in atopic dermatitis, resulting from abnormal expression of sphingomyelin (SM) deacylase, accompanied by a deficiency in ceramide. Also, S1P and its receptor, sphingosine-1-phosphate receptor 1 (S1P1) are important targets in treating atopic dermatitis. RESULTS: In this study, we found a novel antagonist of SPC and S1P1, KRO-105714, by screening 10,000 compounds. To screen the compounds, we used an SPC-induced cell proliferation assay based on a high-throughput screening (HTS) system and a human S1P1 protein-based [35S]-GTPγS binding assay. In addition, we confirmed the inhibitory effects of KRO-105714 on atopic dermatitis through related cell-based assays, including a tube formation assay, a cell migration assay, and an ELISA assay on inflammatory cytokines. Finally, we confirmed that KRO-105714 alleviates atopic dermatitis symptoms in a series of mouse models. CONCLUSIONS: Taken together, our data suggest that SPC and S1P1 antagonist KRO-105714 has the potential to alleviate atopic dermatitis.

Advances in Neurobiology and Pharmacology of GPR12.

GPR12 is a G protein-coupled orphan receptor genetically related to type 1 and type 2 cannabinoid receptors (CB1 and CB2) which are ancient proteins expressed all over the body. Both cannabinoid receptors, but especially CB1, are involved in neurodevelopment and cognitive processes such as learning, memory, brain reward, coordination, etc. GPR12 shares with CB1 that both are mainly expressed into the brain. Regrettably, very little is known about physiology of GPR12. Concerning its pharmacology, GPR12 seems to be endogenously activated by the lysophospholipids sphingosine-1-phosphate (S1P) and sphingosyl-phosphorylcholine (SPC). Exogenously, GPR12 is a target for the phytocannabinoid cannabidiol (CBD). Functionally, GPR12 seems to be related to neurogenesis and neural inflammation, but its relationship with cognitive functions remains to be characterized. Although GPR12 was initially suggested to be a cannabinoid receptor, it does not meet the five criteria proposed in 2010 by the International Union of Basic and Clinical Pharmacology (IUPHAR). In this review, we analyze all the direct available information in PubMed database about expression, function, and pharmacology of this receptor in central nervous system (CNS) trying to provide a broad overview of its current and prospective neurophysiology. Moreover, in this mini-review we highlight the need to produce more relevant data about the functions of GPR12 in CNS. Hence, this work should motivate further research in this field.