Sphingomyelin Synthase Research Articles

Uncovering Ecdysozoa-specific Sphingomyelin Synthase by Phylogenetic Analysis of Metazoan Sequences.

Sphingomyelin (SM) is a membrane phospholipid that is widely distributed in Metazoa; it is the major constituent of myelin sheaths in vertebrates. In mammals, two genes (SMS1 and SMS2) are responsible for its synthesis. No SM-producing genes have been clearly identified in insects and crustaceans (Ecdysozoa) despite the presence of a myelin sheath-like structure in shrimps. Since the rapid transmission of electrical signals requires the use of an insulating material in the nerve, it is possible that the convergent evolution of enzymes to synthesize the insulating compounds for the nervous system also occurred in animals other than vertebrates. Our exhaustive phylogenetic search for metazoan SM synthase identified an Ecdysozoa-specific SM synthase candidate, SMSe, which is absent in Drosophila and Lophotrochozoa. All Ecdysozoa lack the homolog of myelin basic- and proteolipid proteins present in mammals. We propose an evolutionary path of SM synthase and discuss the origin of the myelin structure in Metazoa.

Malabaricone C as Natural Sphingomyelin Synthase Inhibitor against Diet-Induced Obesity and Its Lipid Metabolism in Mice.

The interaction between natural occurring inhibitors and targeted membrane proteins could be an alternative medicinal strategy for the treatment of metabolic syndrome, notably, obesity. In this study, we identified malabaricones A-C and E (1-4) isolated from the fruits of Myristica cinnamomea King as natural inhibitors for sphingomyelin synthase (SMS), a membrane protein responsible for sphingolipid biosynthesis. Having the most promising inhibition, oral administration of compound 3 exhibited multiple efficacies in reducing weight gain, improving glucose tolerance, and reducing hepatic steatosis in high fat diet-induced obesity mice models. Liver lipid analysis revealed a crucial link between the SMS activities of compound 3 and its lipid metabolism in vitro and in vivo. The nontoxic nature of compound 3 makes it a suitable candidate in search of drugs which can be employed in the treatment and prevention of obesity.

Abstract 3603: Sphingomyelin synthase inhibition in sarcomas results in ceramide accumulation and apoptosis

Abstract Sphingolipid metabolism is critical to cellular homeostasis especially as cells undergo mitosis. It is, therefore, unsurprising that the sphingolipid pathway is upregulated in a number of a cancers. Sphingoid bases such as sphingosine and ceramide are rapidly synthesized to provide the building blocks of other sphingolipids. Inhibition of downstream enzymes that convert sphingosine and ceramide into sphingolipids can lead to an increase of ceramide, which results in cellular toxicity and initiation of apoptosis. Using the marine natural product Jaspine B and chemically synthesized analogues, we investigated the potency of inhibiting sphingomyelin synthase in sarcoma cell lines. Sphingomyelin synthase is a downstream enzyme that converts ceramide into sphingomyelin by adding the polar head group phosphorylcholine to ceramide. Jaspine B and its analogues exhibited submicromolar potency against several human sarcoma cell lines. It was determined that the inhibition of sphingomyelin synthase resulted in an increase in ceramide and sphingosine levels and that sphingomyelinase enzymatic activity increased. While the mitochondria mass remained the same, the membrane potential of the mitochondria was significantly affected by the presence of the inhibitors. In conclusion, inhibitors of sphingolipid metabolism are viable therapeutic options with high untapped potential to treat cancer. Citation Format: Jared J. Barrott, Trevor Meldrum, Farjana Afrin, Srinath Pashikanti. Sphingomyelin synthase inhibition in sarcomas results in ceramide accumulation and apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3603.

Sphingomyelin synthase 1 enhances BCR signaling to promote lupus-like autoimmune response.

BackgroundSphingomyelin synthase 1 (SMS1) has been reported to participate in hepatitis and atherosclerosis. However, its role in autoimmune response is not clear. This study investigates the possible involvement of SMS1 in B-cell activation and lupus-like autoimmunity.MethodsSMS1 knockout lupus-like animal model and SLE patient samples were utilized. B-cell activation and associated signal transduction were detected by flow cytometry, confocal analysis and western blotting. The SMS1 expression in B cells was measured by real-time qPCR.FindingsSMS1 deficiency suppressed B-cell activation in culture, which was restored by exogenous SM supplementation. The BCR-mediated early signal transduction including the colocalization of BCR with F-actin or pY/pBtk, and the phosphorylation of intracellular Fyn and Syk were impaired in SMS1 knockout B cells. Furthermore, SMS1 knockout mice showed reduced production and deposition of autoantibodies, accompanied by less severe kidney pathological changes after pristane induction. SMS1 deficiency also displayed lower autoantibody titers and 24 h urine protein excretion in bm12-induced lupus, which were associated with reduced B-cell activation. Adoptively transferred wide-type B cells partially recovered B-cell activation and autoantibody production in SMS1 deficient bm12-induced lupus mice. Moreover, the SMS1 mRNA level in B cells of SLE patients was increased and positively correlated with the serum anti-dsDNA level, IgG and globulin titers.InterpretationThese data suggest that SMS1 is involved in lupus-like autoimmunity via regulating BCR signal transduction and B cell activation. (Word count for the abstract: 230).

Sphingomyelin Synthase 2 Promotes Endothelial Dysfunction by Inducing Endoplasmic Reticulum Stress.

Endothelial dysfunction (ED) is an important contributor to atherosclerotic cardiovascular disease. Our previous study demonstrated that sphingomyelin synthase 2 (SMS2) promotes ED. Moreover, endoplasmic reticulum (ER) stress can lead to ED. However, whether there is a correlation between SMS2 and ER stress is unclear. To examine their correlation and determine the detailed mechanism of this process, we constructed a human umbilical vein endothelial cell (HUVEC) model with SMS2 overexpression. These cells were treated with 4-PBA or simvastatin and with LiCl and salinomycin alone. The results showed that SMS2 can promote the phosphorylation of lipoprotein receptor-related protein 6 (LRP6) and activate the Wnt/β-catenin pathway and that activation or inhibition of the Wnt/β-catenin pathway can induce or block ER stress, respectively. However, inhibition of ER stress by 4-PBA can decrease ER stress and ED. Furthermore, when the biosynthesis of cholesterol is inhibited by simvastatin, the reduction in intracellular cholesterol coincides with a decrease in ER stress and ED. Collectively, our results demonstrate that SMS2 can activate the Wnt/β-catenin pathway and promote intracellular cholesterol accumulation, both of which can contribute to the induction of ER stress and finally lead to ED.

Design and Synthesis of the Diazirine-based Clickable Photo-affinity Probe Targeting Sphingomyelin Synthase 2

Background:SMS family plays a very important role in sphingolipids metabolism and is involved in the membrane mobility and signaling transduction.Methods:SMS2 subtype was related to a variety of diseases and could be regarded as a promising potential drug target. However, the uncertainty of the binding sites and the molecular mechanism of action limited the development of SMS2 inhibitors. Herein, we discovered a photo-affinity probe PAL-1 targeting SMS2.Results:The enzyme inhibitory activity and the photo-affinity labeling experiments showed that PAL-1 could be mono-labeled on SMS2.Conclusion:In summary, starting from the N-arylbenzamides core structure and the minimalist terminal alkyne-containing diazirine photo-crosslinker, we designed and synthesized a photoaffinity probe PAL-1 targeting SMS2. The enzymatic inhibitory activity study showed that PAL-1 exhibited superior selectivities for SMS2 with an IC50 of 0.37 µM over SMS1.

Sphingomyelin Synthase 1 (SMS1) Downregulation Is Associated With Sphingolipid Reprogramming and a Worse Prognosis in Melanoma.

Sphingolipid (SL) metabolism alterations have been frequently reported in cancer including in melanoma, a bad-prognosis skin cancer. In normal cells, de novo synthesized ceramide is mainly converted to sphingomyelin (SM), the most abundant SL, by sphingomyelin synthase 1 (SMS1) and, albeit to a lesser extent, SMS2, encoded by the SGMS1 and SGMS2 genes, respectively. Alternatively, ceramide can be converted to glucosylceramide (GlcCer) by the GlcCer synthase (GCS), encoded by the UGCG gene. Herein, we provide evidence for the first time that SMS1 is frequently downregulated in various solid cancers, more particularly in melanoma. Accordingly, various human melanoma cells displayed a SL metabolism signature associated with (i) a robust and a low expression of UGCG and SGMS1/2, respectively, (ii) higher in situ enzyme activity of GCS than SMS, and (iii) higher intracellular levels of GlcCer than SM. SMS1 was expressed at low levels in most of the human melanoma biopsies. In addition, several mutations and increased CpG island methylation in the SGMS1 gene were identified that likely affect SMS1 expression. Finally, low SMS1 expression was associated with a worse prognosis in metastatic melanoma patients. Collectively, our study indicates that SMS1 downregulation in melanoma enhances GlcCer synthesis, triggering an imbalance in the SM/GlcCer homeostasis, which likely contributes to melanoma progression. Evaluating SMS1 expression level in tumor samples might serve as a biomarker to predict clinical outcome in advanced melanoma patients.

Osteoporosis and skeletal dysplasia caused by pathogenic variants in SGMS2.

Mechanisms leading to osteoporosis are incompletely understood. Genetic disorders with skeletal fragility provide insight into metabolic pathways contributing to bone strength. We evaluated 6 families with rare skeletal phenotypes and osteoporosis by next-generation sequencing. In all the families, we identified a heterozygous variant in SGMS2, a gene prominently expressed in cortical bone and encoding the plasma membrane–resident sphingomyelin synthase SMS2. Four unrelated families shared the same nonsense variant, c.148C>T (p.Arg50*), whereas the other families had a missense variant, c.185T>G (p.Ile62Ser) or c.191T>G (p.Met64Arg). Subjects with p.Arg50* presented with childhood-onset osteoporosis with or without cranial sclerosis. Patients with p.Ile62Ser or p.Met64Arg had a more severe presentation, with neonatal fractures, severe short stature, and spondylometaphyseal dysplasia. Several subjects had experienced peripheral facial nerve palsy or other neurological manifestations. Bone biopsies showed markedly altered bone material characteristics, including defective bone mineralization. Osteoclast formation and function in vitro was normal. While the p.Arg50* mutation yielded a catalytically inactive enzyme, p.Ile62Ser and p.Met64Arg each enhanced the rate of de novo sphingomyelin production by blocking export of a functional enzyme from the endoplasmic reticulum. SGMS2 pathogenic variants underlie a spectrum of skeletal conditions, ranging from isolated osteoporosis to complex skeletal dysplasia, suggesting a critical role for plasma membrane–bound sphingomyelin metabolism in skeletal homeostasis.

Sphingomyelin synthase inhibition in sarcomas results in ceramide accumulation and apoptosis

Sphingolipid metabolism is critical to cellular homeostasis especially as cells undergo mitosis. It is, therefore, unsurprising that the sphingolipid pathway is upregulated in a number of a cancers. Sphingoid bases such as sphingosine and ceramide are rapidly synthesized to provide the building blocks of other sphingolipids. Inhibition of downstream enzymes that convert sphingosine and ceramide into sphingolipids can lead to an increase of ceramide, which results in cellular toxicity and initiation of apoptosis. Using the marine natural product Jaspine B and chemically synthesized analogues, we investigated the potency of inhibiting sphingomyelin synthase in sarcoma cell lines. Sphingomyelin synthase is a downstream enzyme that converts ceramide into sphingomyelin by adding the polar head group phosphorylcholine to ceramide. Jaspine B and its analogues exhibited submicromolar potency against several human sarcoma cell lines. It was determined that the inhibition of sphingomyelin synthase resulted in an increase in ceramide and sphingosine levels and that sphingomyelinase enzymatic activity increased. While the mitochondria mass remained the same, the membrane potential of the mitochondria was significantly affected by the presence of the inhibitors. In conclusion, inhibitors of sphingolipid metabolism are viable therapeutic options with high untapped potential.Support or Funding InformationIdaho State University Start UpThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Multiple actions of doxorubicin on the sphingolipid network revealed by flux analysis

Sphingolipids (SLs) have been implicated in numerous important cellular biologies; however, their study has been hindered by the complexities of SL metabolism. Furthermore, enzymes of SL metabolism represent a dynamic and interconnected network in which one metabolite can be transformed into other bioactive SLs through further metabolism, resulting in diverse cellular responses. Here we explore the effects of both lethal and sublethal doses of doxorubicin (Dox) in MCF-7 cells. The two concentrations of Dox resulted in the regulation of SLs, including accumulations in sphingosine, sphingosine-1-phosphate, dihydroceramide, and ceramide, as well as reduced levels of hexosylceramide. To further define the effects of Dox on SLs, metabolic flux experiments utilizing a d17 dihydrosphingosine probe were conducted. Results indicated the regulation of ceramidases and sphingomyelin synthase components specifically in response to the cytostatic dose. The results also unexpectedly demonstrated dose-dependent inhibition of dihydroceramide desaturase and glucosylceramide synthase in response to Dox. Taken together, this study uncovers novel targets in the SL network for the action of Dox, and the results reveal the significant complexity of SL response to even a single agent. This approach helps to define the role of specific SL enzymes, their metabolic products, and the resulting biologies in response to chemotherapeutics and other stimuli.

077 Comparison of the Actomyosin ATPase Inhibitor Calponin and the Sphingosine-1-phosphate Cell Differentiation Pathway Regulatory Enzymes Sphingosine Kinase 1 and Sphingomyelin Synthase in Vaginal Wall Smooth Muscle of Women with and without Pelvic Organ Pro

077 Comparison of the Actomyosin ATPase Inhibitor Calponin and the Sphingosine-1-phosphate Cell Differentiation Pathway Regulatory Enzymes Sphingosine Kinase 1 and Sphingomyelin Synthase in Vaginal Wall Smooth Muscle of Women with and without Pelvic Organ Pro

Involvement of exosomes in dopaminergic neurodegeneration by microglial activation in midbrain slice cultures

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive degeneration of dopamine neurons in the substantia nigra. Microglial activation is frequently observed in the brains of patients with PD and animal models. Interferon-γ (IFN-γ)/lipopolysaccharide (LPS) treatment triggers microglial activation and the reduction of dopamine neurons in midbrain slice cultures. We have previously reported that nitric oxide (NO) is mainly involved in this dopaminergic degeneration. However, this degeneration was not completely suppressed by the inhibition of NO synthesis, suggesting that factors other than NO also contribute to dopaminergic neurodegeneration. Exosomes are extracellular vesicles with diameters of 40–200 nm that contain various proteins and micro RNAs and are regarded as a novel factor that mediates cell-to-cell interactions. Previous studies have demonstrated that exosome release is enhanced by microglial stimulation and that microglia-derived exosomes increases neuronal apoptosis. In the present study, we investigated whether exosomes are involved in dopaminergic neurodegeneration triggered by microglial activation in midbrain slice cultures. IFN-γ/LPS treatment to the midbrain slice cultures activated microglia, increased exosomal release, and decreased dopamine neurons. GW4869, an inhibitor of a neutral sphingomyelinase 2, decreased exosomal release and significantly prevented dopaminergic neurodegeneration by IFN-γ/LPS without affecting NO production. In contrast, D609, an inhibitor of sphingomyelin synthase and NO synthase, did not affect dopaminergic neurodegeneration, although it strongly inhibited NO production. The protective effect mediated by inhibition of NO synthase would be counteracted by enhanced exosomal release caused by D609 treatment. In addition, dopaminergic neurodegeneration is triggered by the treatment of exosomes isolated from culture media of IFN-γ/LPS-treated slices. These results suggest that exosomes are involved in dopaminergic neurodegeneration by microglial activation.

Sphingomyelin Synthase 1 Enhances BCR Signaling to Promote Lupus-Like Autoimmune Response

Sphingomyelin synthase 1 (SMS1) has been reported to participate in hepatitis and athereosclerosis. However, its role in autoimmune response is not clear. In this study, we investigate the possible involvement of SMS1 in B-cell activation and lupus-like autoimmunity. SMS1 knockout lupus-like animal model and SLE patient samples were utilized. We found that SMS1 deficiency suppressed B-cell activation in culture, which was restored by exogenous SM supplementation. Accordingly, the BCR-mediated early signal transduction including the colocalization of BCR with F-actin or pY/pBtk, and the phosphorylation of intracellular Fyn and Syk were impaired in SMS1 knockout B cells. Furthermore, SMS1 knockout mice showed reduced production and deposition of autoantibodies, accompanied by less severe kidney pathological changes after pristane induction. SMS1 deficiency also displayed lower autoantibody titers and 24h urine protein excretion in bm12-induced lupus, which were associated with reduced B-cell activation. Adoptively transferred wide-type B cells partially recovered B-cell activation and autoantibody production in SMS1 deficient bm12-induced lupus mice. Moreover, the SMS1 mRNA level in B cells of SLE patients was increased and positively correlated with the serum IgG production and globulin titers. These data suggest that SMS1 is involved in lupus-like autoimmunity via regulating BCR signal transduction and B cell activation. Funding Statement: This work was supported by grants from National Natural Science Foundation of China (no. 31270965 and 81771754 to Lingli Dong) and Integrated Innovative Team for Major Human Diseases Program of Tongji Medical College, HUST to Lingli Dong and Xiangping Yang. Declaration of Interests: The authors declare that there is no conflict of interest in this study. Ethics Approval Statement: This study was approved by the ethic committee of Tongji Hospital. Informed consents were obtained from all participants.

Sphingomyelin synthase 2 promotes an aggressive breast cancer phenotype by disrupting the homoeostasis of ceramide and sphingomyelin

Breast cancer is the most common type of carcinoma in women worldwide, but the mechanisms underlying tumour development and progression remain unclear. Sphingomyelin synthase 2 (SGMS2) is a crucial regulator involved in ceramide (Cer) and sphingomyelin (SM) homoeostasis that is mostly studied for its role in lipid metabolism. Our primary study indicated that high SGMS2 expression is associated with breast cancer metastasis. Gain- and loss-of-function assays in vitro and in vivo revealed that SGMS2 promotes cancer cell proliferation by suppressing apoptosis through a Cer-associated pathway and promotes cancer cell invasiveness by enhancing epithelial-to-mesenchymal transition (EMT) initiation through the TGF-β/Smad signalling pathway. Further study determined that SGMS2 activated the TGF-β/Smad signalling pathway primarily by increasing TGF-β1 secretion, which was likely associated with aberrant expression of SM. Thus, our findings indicate that SGMS2-mediated activation of the TGF-β/Smad signalling pathway is important in breast cancer progression, which provides new insight into the mechanisms underlying breast cancer metastasis and suggests a possible anticancer therapy for breast cancer.

Optical manipulation of sphingolipid biosynthesis using photoswitchable ceramides.

Ceramides are central intermediates of sphingolipid metabolism that also function as potent messengers in stress signaling and apoptosis. Progress in understanding how ceramides execute their biological roles is hampered by a lack of methods to manipulate their cellular levels and metabolic fate with appropriate spatiotemporal precision. Here, we report on clickable, azobenzene-containing ceramides, caCers, as photoswitchable metabolic substrates to exert optical control over sphingolipid production in cells. Combining atomic force microscopy on model bilayers with metabolic tracing studies in cells, we demonstrate that light-induced alterations in the lateral packing of caCers lead to marked differences in their metabolic conversion by sphingomyelin synthase and glucosylceramide synthase. These changes in metabolic rates are instant and reversible over several cycles of photoswitching. Our findings disclose new opportunities to probe the causal roles of ceramides and their metabolic derivatives in a wide array of sphingolipid-dependent cellular processes with the spatiotemporal precision of light.

Cardioprotective Regimen of Adaptation to Chronic Hypoxia Diversely Alters Myocardial Gene Expression in SHR and SHR-mtBN Conplastic Rat Strains.

Adaptation to continuous normobaric hypoxia (CNH) protects the heart against acute ischemia/reperfusion injury. Recently, we have demonstrated the infarct size-limiting effect of CNH also in hearts of spontaneously hypertensive rats (SHR) and in conplastic SHR-mtBN strain characterized by the selective replacement of the mitochondrial genome of SHR with that of more ischemia-resistant Brown Norway rats. Importantly, cardioprotective effect of CNH was more pronounced in SHR-mtBN than in SHR. Thus, here we aimed to identify candidate genes which may contribute to this difference between the strains. Rats were adapted to CNH (FiO2 0.1) for 3 weeks or kept at room air as normoxic controls. Screening of 45 transcripts was performed in left ventricles using Biomark Chip. Significant differences between the groups were analyzed by univariate analysis (ANOVA) and the genes contributing to the differences between the strains unmasked by CNH were identified by multivariate analyses (PCA, SOM). ANOVA with Bonferroni correction revealed that transcripts differently affected by CNH in SHR and SHR-mtBN belong predominantly to lipid metabolism and antioxidant defense. PCA divided four experimental groups into two main clusters corresponding to chronically hypoxic and normoxic groups, and differences between the strains were more pronounced after CNH. Subsequently, the following 14 candidate transcripts were selected by PCA, and confirmed by SOM analyses, that can contribute to the strain differences in cardioprotective phenotype afforded by CNH: Alkaline ceramidase 2 (Acer2), Fatty acid translocase (Cd36), Aconitase 1 (Aco1), Peroxisome proliferator activated receptor gamma (Pparg), Hemoxygenase 2 (Hmox2), Phospholipase A2 group IIA (Ppla2g2a), Dynamin-related protein (Drp), Protein kinase C epsilon (Pkce), Hexokinase 2 (Hk2), Sphingomyelin synthase 2 (Sgms2), Caspase 3 (Casp3), Mitofussin 1 (Mfn1), Phospholipase A2 group V (Pla2g5), and Catalase (Cat). Our data suggest that the stronger cardioprotective phenotype of conplastic SHR-mtBN strain afforded by CNH is associated with either preventing the drop or increasing the expression of transcripts related to energy metabolism, antioxidant response and mitochondrial dynamics.

Minerval (2-hydroxyoleic acid) causes cancer cell selective toxicity by uncoupling oxidative phosphorylation and compromising bioenergetic compensation capacity.

This work tests bioenergetic and cell-biological implications of the synthetic fatty acid Minerval (2-hydroxyoleic acid), previously demonstrated to act by activation of sphingomyelin synthase in the plasma membrane (PM) and lowering of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) and their carcinogenic signaling. We show here that Minerval also acts, selectively in cancer cell lines, as an ATP depleting uncoupler of mitochondrial oxidative phosphorylation (OxPhos). As a function of its exposure time, Minerval compromised the capacity of glioblastoma U87-MG cells to compensate for aberrant respiration by up-modulation of glycolysis. This effect was not exposure time-dependent in the lung carcinoma A549 cell line, which was more sensitive to Minerval. Compared with OxPhos inhibitors FCCP (uncoupler), rotenone (electron transfer inhibitor), and oligomycin (F1F0-ATPase inhibitor), Minerval action was similar only to that of FCCP. This similarity was manifested by mitochondrial membrane potential (MMP) depolarization, facilitation of oxygen consumption rate (OCR), restriction of mitochondrial and cellular reactive oxygen species (ROS) generation and mitochondrial fragmentation. Additionally, compared with other OxPhos inhibitors, Minerval uniquely induced ER stress in cancer cell lines. These new modes of action for Minerval, capitalizing on the high fatty acid requirements of cancer cells, can potentially enhance its cancer-selective toxicity and improve its therapeutic capacity.

Plasma membrane sphingomyelin modulates thymocyte development by inhibiting TCR-induced apoptosis.

Sphingomyelin (SM) in combination with cholesterol forms specialized membrane lipid microdomains in which specific receptors and signaling molecules are localized or recruited to mediate intracellular signaling. SM-microdomain levels in mouse thymus were low in the early CD4+CD8+ double-positive (DP) stage prior to thymic selection and increased >10-fold during late selection. T-cell receptor (TCR) signal strength is a key factor determining whether DP thymocytes undergo positive or negative selection. We examined the role of SM-microdomains in thymocyte development and related TCR signaling, using SM synthase 1 (SMS1)-deficient (SMS1-/-) mice which display low SM expression in all thymocyte populations. SMS1 deficiency caused reduced cell numbers after late DP stages in TCR transgenic models. TCR-dependent apoptosis induced by anti-CD3 treatment was enhanced in SMS1-/- DP thymocytes both in vivo and in vitro. SMS1-/- DP thymocytes, relative to controls, showed increased phosphorylation of TCR-proximal kinase ZAP-70 and increased expression of Bim and Nur77 proteins involved in negative selection following TCR stimulation. Addition of SM to cultured normal DP thymocytes led to greatly increased surface expression of SM-microdomains, with associated reduction of TCR signaling and TCR-induced apoptosis. Our findings indicate that SM-microdomains are increased in late DP stages, function as negative regulators of TCR signaling and modulate the efficiency of TCR-proximal signaling to promote thymic selection events leading to subsequent developmental stages.

Discovery, synthesis and anti-atherosclerotic activities of a novel selective sphingomyelin synthase 2 inhibitor

The sphingomyelin synthase 2 (SMS2) is a potential target for pharmacological intervention in atherosclerosis. However, so far, few selective SMS2 inhibitors and their pharmacological activities were reported. In this study, a class of 2-benzyloxybenzamides were discovered as novel SMS2 inhibitors through scaffold hopping and structural optimization. Among them, Ly93 as one of the most potent inhibitors exhibited IC50 values of 91 nM and 133.9 μM against purified SMS2 and SMS1 respectively. The selectivity ratio of Ly93 was more than 1400-fold for purified SMS2 over SMS1. The in vitro studies indicated that Ly93 not only dose-dependently diminished apoB secretion from Huh7 cells, but also significantly reduced the SMS activity and increased cholesterol efflux from macrophages. Meanwhile, Ly93 inhibited the secretion of LPS-mediated pro-inflammatory cytokine and chemokine in macrophages. The pharmacokinetic profiles of Ly93 performed on C57BL/6J mice demonstrated that Ly93 was orally efficacious. As a potent selective SMS2 inhibitor, Ly93 significantly decreased the plasma SM levels of C57BL/6J mice. Furthermore, Ly93 was capable of dose-dependently attenuating the atherosclerotic lesions in the root and the entire aorta as well as macrophage content in lesions, in apolipoprotein E gene knockout mice treated with Ly93. In conclusion, we discovered a novel selective SMS2 inhibitor Ly93 and demonstrated its anti-atherosclerotic activities in vivo. The preliminary molecular mechanism-of-action studies revealed its function in lipid homeostasis and inflammation process, which indicated that the selective inhibition of SMS2 would be a promising treatment for atherosclerosis.

Sphingomyelin synthase 1 regulates the epithelial‑to‑mesenchymal transition mediated by the TGF‑β/Smad pathway in MDA‑MB‑231 cells.

Breast cancer is the most common cancer in women and a leading cause of cancer-associated mortalities in the world. Epithelial-to-mesenchymal transition (EMT) serves an important role in the process of metastasis and invasive ability in cancer cells, and transforming growth factor β1 (TGF-β1) have been investigated for promoting EMT. However, in the present study, the role of the sphingomyelin synthase 1 (SMS1) in TGF-β1-induced EMT development was investigated. Firstly, bioinformatics analysis demonstrated that the overexpression of SMS1 negatively regulated the TGFβ receptor I (TβRI) level of expression. Subsequently, the expression of SMS1 was decreased, whereas, SMS2 had no significant difference when MDA-MB-231 cells were treated by TGF-β1 for 72 h. Furthermore, the present study constructed an overexpression cells model of SMS1 and these cells were treated by TGF-β1. These results demonstrated that overexpression of SMS1 inhibited TGF-β1-induced EMT and the migration and invasion of MDA-MB-231 cells, increasing the expression of E-cadherin while decreasing the expression of vimentin. Furthermore, the present study further confirmed that SMS1 overexpression could decrease TβRI expression levels and blocked smad family member 2 phosphorylation. Overall, the present results suggested that SMS1 could inhibit EMT and the migration and invasion of MDA-MB-231 cells via TGF-β/Smad signaling pathway.