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We are currently developing an inspection system that will provide a low-cost means of screening prior to shipment by fully visualizing latent 1SSF (single Shockley stacking fault) defects originating from basal plane dislocations (BPDs) that cannot be detected by current defect inspection systems. The system will capture not only the defects that expand into right triangles under relatively low-level forward bias, but also the defects that expand into more serious bar-shaped 1SSFs under relatively high-level forward bias, with a particular focus on capturing TED (threading edge dislocation)-converted BPD at or below the buffer layer/substrate interface. Since these defects are known to cause forward voltage degradation during device operation, so-called "burn-in" (accelerated current stress) screening operation is currently utilized in some device manufacturers to avoid the shipping of the defective devices, but it is very time-consuming process which raises a total cost of production. The system we are developing, which can significantly reduce the screening time, has the potential to replace the "burn-in" operation.

In the previous report [1], we proposed the S-EVC (Selective Expansion-Visualization-Contraction) method (Fig. 1) that effectively screens for malignant BPDs (basal plane dislocations) in the drift and buffer layers, which expand to SSFs (Shockley-type stacking faults), leading to forward voltage degradation. The method intentionally utilizes the REDG (recombination enhanced dislocation glide) mechanism by UV (ultraviolet) irradiation in wafer sorting to replace the so-called burn-in (accelerated current stress) process, which is time-consuming during mass production. In the report, triangular SSFs were examined to verify the effectiveness of the method, but they only occupy a much smaller area of the active region on the chip than bar shaped SSFs. In this study, to improve the S-EVC method to be more practical, we focused on the more serious bar shaped SSFs which have a non-negligible impact on electrical characteristics. The bar shaped SSFs are mostly expanded from TED (threading edge dislocation)-converted BPD at or below the substrate epitaxial layer interface. In PL (photoluminescence) observation by a 710 nm LPF (long-pass filter), the TED-converted BPD and the complete TED extended from the bottom of the substrate are observed as the same dark spot, but it was confirmed that both can be distinguished by the presence or absence of their SSF expansion by UV irradiation. In addition, in order to confirm the validity of the S-EVC method even on the virgin epi wafer, UV irradiation was performed on both the aluminum doped PN structured wafer and the virgin epi wafer, and the similar SSF expansion was observed. Meanwhile, the correlation between UV irradiation and forward voltage degradation was quantified using PiN diodes by comparing the glide velocity of 30°Si (g) core partials for bar shaped SSFs by UV irradiation stress with that by current stress.

We propose the new practical and effective method, called Selective E-V-C (Expansion-Visualization-Contraction) technique, to screen out the basal plane dislocations (BPDs) which might cause the forward voltage degradation of SiC devices. Since the method can be adopted at the epi wafer receiving inspection process in early stage of production line, it may replace the very time-consuming so-called "burn-in" operation currently utilized in some device manufacturers.

Neuropathic pain is an unbearable condition caused by nervous system damage. As distinct acute pain, neuropathic pain is chronic, and it severely influences quality of life. N,N-Dimethyl-d-erythro-sphingosine (DMS), a neuropathic pain inducer, is metabolited de novo from sphingosine. In a recent study, metabolomics showed an increased concentration level of DMS in the spinal cord in mice with neuropathic pain. Nerve growth factor (NGF) is one of the peripheral nervous system targeted pain factors that interact with tropomyosin receptor kinase A (trkA). On the basis of this information, we were interested in the possibility that DMS may induce neuropathic pain-like behavior through an increase of NGF activity. In this study, we showed that DMS can enhance the binding of NGF to trkA, followed by neurite outgrowth of epidermal nerve fibers and phosphorylation of trkA. In addition, a stereoisomer, N,N-dimethyl-l-erythro-sphingosine, did not any show such biological activities. The results suggest that DMS can enhance the binding of NGF to trkA and that its stereochemistry is an essential factor for exhibiting its activity.

The extracellular accumulation of aggregated amyloid-β (Aβ) in the brain leads to the early pathology of Alzheimer’s disease (AD). The administration of exogenous plant-type ceramides into AD model mice can promote the release of neuronal exosomes, a subtype of extracellular vesicles, that can mediate Aβ clearance. In vitro studies showed that the length of fatty acids in mammalian-type ceramides is crucial for promoting neuronal exosome release. Therefore, investigating the structures of plant ceramides is important for evaluating the potential in releasing exosomes to remove Aβ. In this study, we assessed plant ceramide species with D-erythro-(4E,8Z)-sphingadienine and D-erythro-(8Z)-phytosphingenine as sphingoid bases that differ from mammalian-type species. Some plant ceramides were more effective than mammalian ceramides at stimulating exosome release. In addition, using deuterium chemistry-based lipidomics, most exogenous plant ceramides were confirmed to be derived from exosomes. These results suggest that the ceramide-dependent upregulation of exosome release may promote the release of exogenous ceramides from cells, and plant ceramides with long-chain fatty acids can effectively release neuronal exosomes and prevent AD pathology.

Ceramide levels controlled by the sphingomyelin (SM) cycle have essential roles in cancer cell fate through the regulation of cell proliferation, death, metastasis, and drug resistance. Recent studies suggest that exosomes confer cancer malignancy. However, the relationship between ceramide metabolism and exosome-mediated cancer malignancy is unclear. In this study, we elucidated the role of ceramide metabolism via the SM cycle in exosomes and drug resistance in human leukemia HL-60 and adriamycin-resistant HL-60/ADR cells. HL-60/ADR cells showed significantly increased exosome production and release compared with parental chemosensitive HL-60 cells. In HL-60/ADR cells, increased SM synthase (SMS) activity reduced ceramide levels, although released exosomes exhibited a high ceramide ratio in both HL-60- and HL-60/ADR-derived exosomes. Overexpression of SMS2 but not SMS1 suppressed intracellular ceramide levels and accelerated exosome production and release in HL-60 cells. Notably, HL-60/ADR exosomes conferred cell proliferation and doxorubicin resistance properties to HL-60 cells. Finally, microRNA analysis in HL-60 and HL-60/ADR cells and exosomes showed that miR-484 elevation in HL-60/ADR cells and exosomes was associated with exosome-mediated cell proliferation. This suggests that intracellular ceramide metabolism by SMS2 regulates exosome production and release, leading to acquisition of drug resistance and enhanced cell proliferation in leukemia cells.

Exosomes are extracellular vesicles that mediate the transport of intracellular molecules, including neurodegenerative agents. Exogenously administrated ceramides have been implicated in the acceleration of exosome production by neurons; however, the molecular machinery involved in this process is unknown. Here, we found that ceramides, especially those consisting of long fatty acids, were internalized into the endocytic pathway in neuroblastoma SH-SY5Y cells to induce exosome secretion through lysosome-associated protein transmembrane 4B (LAPTM4B). Knockdown of LAPTM4B inhibited the ceramide-mediated increase in exosome release completely. Fluorescence microscopy observations indicated that exogenous ceramides promote the transport of multivesicular bodies to the plasma membranes in a LAPTM4B-dependent manner. Similarly, inhibition of acid ceramidase, which tends to induce intracellular ceramide accumulation, increased exosome production by SH-SY5Y cells in a LAPTM4B-dependent manner. Furthermore, the level of amyloid-ß protein (Aß) was decreased in neuronal cells following treatment with exogenous ceramide or inhibition of acid ceramidase, and this effect was attributed to the LAPTM4B-dependent efflux of Aß-containing exosomes. Overall, these findings reveal the novel machinery involved in exosome secretion regulated by ceramides and LAPTM4B, and may contribute to efforts to ameliorate the cellular accumulation of neurodegenerative agents such as Aß.

Simple Summary Konjac ceramide (kCer) is a unique molecular species of plant-type ceramide, and is a potential Sema3A-like ligand of Nrp1. kCer suppresses histamine-stimulated cell migration of HaCaT keratinocytes. This effect of kCer is not due to histamine-activated GPCRs, but rather to Sema3A-Nrp1 receptor binding. The present study focused on the ability of kCer to induce cell differentiation, in addition to its anti-migratory effects. We demonstrated that the effects of kCer on cell migration and cell differentiation are perpetuated by a cascade of crosstalk between pathways downstream of Nrp1 and GPCR in HaCaT cells. Abstract Histamines suppress epidermal keratinocyte differentiation. Previously, we reported that konjac ceramide (kCer) suppresses histamine-stimulated cell migration of HaCaT keratinocytes. kCer specifically binds to Nrp1 and does not interact with histamine receptors. The signaling mechanism of kCer in HaCaT cells is also controlled by an intracellular signaling cascade activated by the Sema3A-Nrp1 pathway. In the present study, we demonstrated that kCer treatment induced HaCaT keratinocyte differentiation after migration of immature cells. kCer-induced HaCaT cell differentiation was accompanied by some features of keratinocyte differentiation markers. kCer induced activating phosphorylation of p38MAPK and c-Fos, which increased the protein levels of involucrin that was the latter differentiation marker. In addition, we demonstrated that the effects of both kCer and histamines are regulated by an intracellular mechanism of Rac1 activation/RhoA inhibition downstream of the Sema3A/Nrp1 receptor and histamine/GPCR pathways. In summary, the effects of kCer on cell migration and cell differentiation are regulated by cascade crosstalk between downstream Nrp1 and histamine-GPCR pathways in HaCaT cells.

The accumulation of amyloid-β protein (Aβ) in brain is linked to the early pathogenesis of Alzheimer’s disease (AD). We previously reported that neuron-derived exosomes promote Aβ clearance in the brains of amyloid precursor protein transgenic mice and that exosome production is modulated by ceramide metabolism. Here, we demonstrate that plant ceramides derived from Amorphophallus konjac, as well as animal-derived ceramides, enhanced production of extracellular vesicles (EVs) in neuronal cultures. Oral administration of plant glucosylceramide (GlcCer) to APP overexpressing mice markedly reduced Aβ levels and plaque burdens and improved cognition in a Y-maze learning task. Moreover, there were substantial increases in the neuronal marker NCAM-1, L1CAM, and Aβ in EVs isolated from serum and brain tissues of the GlcCer-treated AD model mice. Our data showing that plant ceramides prevent Aβ accumulation by promoting EVs-dependent Aβ clearance in vitro and in vivo provide evidence for a protective role of plant ceramides in AD. Plant ceramides might thus be used as functional food materials to ameliorate AD pathology.

[Image: see text] 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.

ABO blood antigens on the human red blood cell membrane as well as different cells in various human tissues have been thoroughly studied. Anti-A and -B antibodies of IgM are present in serum/plasma, but blood group-specific glyco-antigens have not been extensively described. In this study, we performed comprehensive and quantitative serum glycomic analyses of various glycoconjugates and free oligosaccharides in all blood groups. Our comprehensive glycomic approach revealed that blood group-specific antigens in serum/plasma are predominantly present on glycosphingolipids on lipoproteins rather than glycoproteins. Expression of the ABO antigens on glycosphingolipids depends not only on blood type but also on secretor status. Blood group-specific glycans in serum/plasma were classified as type I, whereas those on RBCs had different structures including hexose and hexosamine residues. Analysis of free oligosaccharides revealed that low-molecular-weight blood group-specific glycans, commonly containing lacto-N-difucotetraose, were expressed in serum/plasma according to blood group. Furthermore, comprehensive glycomic analysis in human cerebrospinal fluid showed that many kinds of free oligosaccharides were highly expressed, and low-molecular-weight blood group-specific glycans, which existed in plasma from the same individuals, were present. Our findings provide the first evidence for low-molecular-weight blood group-specific glycans in both serum/plasma and cerebrospinal fluid.

The somatic haploidy is unstable in diplontic animals, but cellular processes determining haploid stability remain elusive. Here, we found that inhibition of mevalonate pathway by pitavastatin, a widely used cholesterol-lowering drug, drastically destabilized the haploid state in HAP1 cells. Interestingly, cholesterol supplementation did not restore haploid stability in pitavastatin-treated cells, and cholesterol inhibitor U18666A did not phenocopy haploid destabilization. These results ruled out the involvement of cholesterol in haploid stability. Besides cholesterol perturbation, pitavastatin induced endoplasmic reticulum (ER) stress, the suppression of which by a chemical chaperon significantly restored haploid stability in pitavastatin-treated cells. Our data demonstrate the involvement of the mevalonate pathway in the stability of the haploid state in human somatic cells through managing ER stress, highlighting a novel link between ploidy and ER homeostatic control.Key words: haploid, ER stress, Mevalonate pathway.

Background:Dietary sphingolipids have various biofunctions, including skin barrier improvement, anti-inflammatory and anti-carcinoma properties. Long-chain bases (LCBs), the essential backbones of sphingolipids, are responsible for these bioactivities, and they vary structurally between species. Given these findings, however, the absorption dynamics of each LCB remain unclear.Methods:In this study, five structurally different LCBs were isolated and their absorption ratios and levels of their metabolites were analyzed using a lymph-duct-cannulated rat model by liquid chromatography tandem mass spectrometry (LC/MS/MS) with a novel multistage fragmentation method.Results:The five orally administered LCBs were absorbed and detected in lymph as free LCBs and several metabolites including ceramides, glucosylceramides, and sphingomyelins. The absorption rates of LCBs were 0.10-1.17% depending on their structure. The absorption rate of 4-trans,8-cis-sphingadienine was highest (1.17%), whereas that of 4-trans,8-trans,10-trans-sphingatrienine was lowest (0.10%). The amount of 4-trans,8-cis-sphingadienine-bound sphingomyelin in lymph was particularly higher than other four LCB-bound sphingomyelins.Conclusion:Structural differences among LCBs, particularly the geometric isomerism at the C8–C9 position, significant affect the absorption rate and amounts of metabolites. This is the first report revealed that the absorption rate and metabolism of sphingolipids are dependent on their LCB structure.

Sphingomyelin synthase 2 (SMS2) regulates sphingomyelin synthesis and contributes to obesity and hepatic steatosis. Here, we investigated the effect of SMS2 deficiency on liver fibrosis in mice fed with choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) or injected with carbon tetrachloride (CCl4), respectively. SMS2 deficiency suppressed hepatic steatosis, but exacerbated fibrosis induced by CDAHFD feeding. Sphingosine 1-phosphate (S1P), which is a key lipid mediator induces fibrosis in various organs, was increased in the liver of mice fed with CDAHFD. The increase of S1P became prominent by SMS2 deficiency. Meanwhile, SMS2 deficiency had no impact on CCl4-induced liver injury, fibrosis and S1P levels. Our findings demonstrated that SMS2 deficiency suppresses steatosis but worsens fibrosis in the liver in a specific condition with CDAHFD feeding.

Starfish Asterias amurensis produces sphingoid bases d18:3, 9-methyl-d18:3 (9Me-d18:3), and d22:2, which possess unique structural features. In this study, sphingoid bases prepared from A. amurensis glucosylceramides displayed unexpected elution behaviors from a general octadecyl silyl high-performance liquid chromatography (HPLC) column. For separation and isolation, sphingoid bases were fractionated by octadecyl silyl HPLC after N-acetylation, yielding d18:3, 9Me-d18:3, and two d22:2 isomers. To compare the biological activities of individual sphingoid bases, their effects on sphingolipid production in normal human keratinocytes were evaluated. Treatment with sphingoid bases increased the content of ceramides, glucosylceramides, and sphingomyelins in keratinocytes. Moreover, ceramides, which contain saturated ultra-long-chain fatty acids (C30-34), were significantly increased by treatment with d18:3, but not with other A. amurensis sphingoid bases. The mRNA level of the early differentiation marker keratin 10 was markedly decreased and sphingolipid synthesis-related genes were slightly increased in keratinocytes exposed to A. amurensis-derived d18:3, 9Me-d18:3, and d22:2 isomers. These results suggest that A. amurensis-derived sphingoid bases induce differentiation to varying degrees, sphingolipid production depends on their chemical structures, and d18:3 is the most promising functional sphingoid base.

Previously, we proposed the following mechanism for konjac ceramide (kCer)-mediated neurite outgrowth inhibition: kCer binds to Nrp as a Sema3A agonist, resulting in Nrp1/PlexA complex formation and activation of the Sema3A signaling pathway to induce phosphorylation of CRMP2 and microtubule depolymerization. The Sema3A/Nrp1 signaling pathway is known to be also expressed in normal human keratinocytes. To determine whether kCer can function in human keratinocytes as it does in neurites, that is, if it can bind to Nrp1 in place of Sema3A, we studied the effect of kCer on HaCaT cell migration activity. Using a trans-well chamber assay, we compared the effects of Sema3A and kCer on serum-derived cell migration activity. kCer showed Sema3A-like suppression of cell migration activity and induction of cellular Cofilin phosphorylation. In addition, kCer and Sema3A inhibited histamine (His)-enhanced migration of immature HaCaT cells. We have demonstrated that kCer does not interact with histaime receptors H1R or H4R directly, but we speculate that kCer may transduce a signal downstream of the His signaling pathway., Highlights • Konjac ceramide (kCer) inhibits HaCaT cell migration by Sema3A-like action. • kCer displaces the binding of Sema3A to cell surface receptor. • kCer inhibits histamine-induced stimulation of HaCaT cell migration. • kCer does not interact with histamine receptors, H1R and H4R.

Glycolipids are now known to be rapidly converted to mediators for inflammatory reactions or to signaling molecules that control inflammatory events in the nervous system. The present study aimed to explore whether disturbed glycolipids metabolism in the nervous system is present in patients with a neuroinflammatory disorder, encephalo-myelo-radiculo-neuropathy (EMRN), because most EMRN patients have been reported to exhibit autoantibodies against neutral glycolipids. Although molecular pathogenesis of this disorder remains unknown, we tried to search the immunochemical abnormalities in this disorder. ELISA for activated peripheral C5 complement and mass spectrometry analysis of cerebrospinal fluid clearly disclosed a significant upregulation of active C5 complement, C5a levels in sera as well as a significant accumulation of species-specific ceramides but not sphingomyelin in cerebrospinal fluid from EMRN patients. Furthermore, we confirmed the occurrence of anti-neutral glycolipids antibodies in all EMRN patients. Thus, the present study might indicate the pathophysiology of this disorder is the dysregulation of glycolipids metabolism and abnormal production of autoantibodies against neutral glycolipids resulting in the abnormal complement activation, although molecular basis for these sphingolipids dysregulation and the occurrence of autoantibodies against glycolipids remains to be elucidated at present. The present study implicates a new therapeutic strategy employing anti-ceramide and/or anti-complement therapy for this disorder.

The tuber of the konjac plant is a source enriched with GlcCer (kGlcCer), and has been used as a dietary supplement to improve the dry skin and itching that are caused by a deficiency of epidermal ceramide. Previously, we showed chemoenzymatically prepared konjac ceramide has a neurite-outgrowth inhibitory effect that is very similar to that of Sema3A and is not seen with animal-type ceramides. While, it has been unclear whether kCer may act on Sema3A or TrkA signaling pathway. In the present study, we showed kCer induces phosphorylation of CRMP2 and microtubules depolymerization via Sema3A signaling pathway not TrkA. It is concluded that kCer may be a potential Sema3A-like agonist that activates Sema3A signaling pathway directly.

Konjac ceramide (kCer) can be prepared by a chemoenzymatic method as previously published (Usuki, S.; Tamura, N.; Sakai, S.; Tamura, T.; Mukai, K.; Igarashi, Y. Biochem. Biophys. Rep. 5, 160-167 (2016)). Thus prepared kCer showed an activation effect on Sema3A signaling pathway to induce phosphorylation of CRMP2 and microtubule depolymerizaion, resulting in opposing NGF-induced neurite outgrowth. In the present study, we have shown that kCer is a potential Sema3A-like ligand that has a competitive effect on Sema3A binding to a cell surface receptor Nrp1, but animal-type ceramides have no effect on Sema3A binding to Nrp1. In addition, kCer showed a direct molecular interaction with Nrp1, but animal-type ceramides, C16Cer, C18Cer, and C24Cer show no specific bindings to Nrp1. Further, kCer showed an additive effect to activate the Sema3A signaling pathway together with low-dose Sema3A but a reversed effect to inhibit this pathway when combined with high-dose Sema3A.

Here, a new technology was developed to selectively produce areas of high and low surface Young's modulus on biomedical polymer films using micropatterns. First, an elastic polymer film was adhered to a striped micropattern to fabricate a micropattern-supported film. Next, the topography and Young's modulus of the film surface were mapped using atomic force microscopy. Contrasts between the concave and convex locations of the stripe pattern were obvious in the Young's modulus map, although the topographical map of the film surface appeared almost flat. The concave and convex locations of a polymer film supported by a different micropattern also contrasted clearly. The resulting Young's modulus map showed that the Young's modulus was higher at convex locations than at concave locations. Hence, regions of high and low stiffness can be locally generated based on the shape of the micropattern supporting the film. When cells were cultured on the micropattern-supported films, NIH3T3 fibroblasts preferentially accumulated in convex regions with high Young's moduli. These findings demonstrate that this new technology can regulate regions of high and low surface Young's modulus on a cellular scaffold with high planar resolution, as well as providing a method for directing cellular patterning. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 976-985, 2018.

Konjac ceramide (kCer) is a plant-type ceramide composed of various long-chain bases and a-hydroxyl fatty acids. The presence of d4t,8t-sphingadienine is essential for semaphorin 3A (Sema3A)-like activity. Herein, we examined the three neuropilin 1 (Nrp1) domains (a1a2, b1b2, or c), and found that a1a2 binds to d4t,8t-kCer and possesses Sema3A-like activity. kCer binds to Nrp1 with a weak affinity of mM dissociation constant (Kd). We wondered whether bovine serum albumin could influence the ligand&ndash, receptor interaction that a1a2 has with a single high affinity binding site for kCer (Kd in nM range). In the present study we demonstrated the influence of bovine serum albumin. Thermal denaturation indicates that the a1a2 domain may include intrinsically disordered region (IDR)-like flexibility. A potential interaction site on the a1 module was explored by molecular docking, which revealed a possible Nrp1 activation mechanism, in which kCer binds to Site A close to the Sema3A-binding region of the a1a2 domain. The a1 module then accesses a2 as the IDR-like flexibility becomes ordered via kCer-induced protein rigidity of a1a2. This induces intramolecular interaction between a1 and a2 through a slight change in protein secondary structure.

Sphingolipids make up a family of molecules associated with an array of biological functions, including cell death and migration. Sphingolipids are often altered in cancer, though how these alterations lead to tumor formation and progression is largely unknown. Here, we analyzed non-small-cell lung cancer (NSCLC) specimens and cell lines and determined that ceramide synthase 6 (CERS6) is markedly overexpressed compared with controls. Elevated CERS6 expression was due in part to reduction of microRNA-101 (miR-101) and was associated with increased invasion and poor prognosis. CERS6 knockdown in NSCLC cells altered the ceramide profile, resulting in decreased cell migration and invasion in vitro, and decreased the frequency of RAC1-positive lamellipodia formation while CERS6 overexpression promoted it. In murine models, CERS6 knockdown in transplanted NSCLC cells attenuated lung metastasis. Furthermore, combined treatment with l-α-dimyristoylphosphatidylcholine liposome and the glucosylceramide synthase inhibitor D-PDMP induced cell death in association with ceramide accumulation and promoted cancer cell apoptosis and tumor regression in murine models. Together, these results indicate that CERS6-dependent ceramide synthesis and maintenance of ceramide in the cellular membrane are essential for lamellipodia formation and metastasis. Moreover, these results suggest that targeting this homeostasis has potential as a therapeutic strategy for CERS6-overexpressing NSCLC.

Background Dietary sphingolipids have various biofunctions, including skin barrier improvement and anti-inflammatory and anti-carcinoma properties. Long-chain bases (LCBs), the essential backbones of sphingolipids, are expected to be important for these bioactivities, and they vary structurally between species. Given these findings, however, the absorption dynamics of each LCB remain unclear. Methods In this study, five structurally different LCBs were prepared from glucosylceramides (GlcCers) with LCB 18:2(4E,8Z);2OH and LCB 18:2(4E,8E);2OH moieties derived from konjac tuber (Amorphophallus konjac), from GlcCers with an LCB 18(9Me):2(4E,8E);2OH moiety derived from Tamogi mushroom (Pleurotus cornucopiae var. citrinopileatus), and from ceramide 2-aminoethyphosphonate with LCB 18:3(4E,8E,10E);2OH moiety and LCB 18(9Me):3(4E,8E,10E);2OH moiety derived from giant scallop (Mizuhopecten yessoensis), and their absorption percentages and metabolite levels were analyzed using a lymph-duct-cannulated rat model via liquid chromatography tandem mass spectrometry (LC/MS/MS) with a multistage fragmentation method. Results The five orally administered LCBs were absorbed and detected in chyle (lipid-containing lymph) as LCBs and several metabolites including ceramides, hexosylceramides, and sphingomyelins. The absorption percentages of LCBs were 0.10–1.17%, depending on their structure. The absorption percentage of LCB 18:2(4E,8Z);2OH was the highest (1.17%), whereas that of LCB 18:3(4E,8E,10E);2OH was the lowest (0.10%). The amount of sphingomyelin with an LCB 18:2(4E,8Z);2OH moiety in chyle was particularly higher than sphingomyelins with other LCB moieties. Conclusions Structural differences among LCBs, particularly geometric isomerism at the C8–C9 position, significantly affected the absorption percentages and ratio of metabolites. This is the first report to elucidate that the absorption and metabolism of sphingolipids are dependent on their LCB structure. These results could be used to develop functional foods that are more readily absorbed.

Fluorous solid-phase extraction (FSPE) is a useful technique for efficient selective enrichment of fluorous compounds from nonfluorous molecules. Sphingolipids and their metabolites, which are ubiquitous building blocks of eukaryotic and prokaryotic cell membranes, play crucial roles, for example, as signaling molecules. However, details of the functions and metabolic mechanisms of exogenous sphingolipids have remained unknown compared with those of their endogenous analogs. To better understand these unknown roles, chemical probes with appropriate biological and physicochemical properties are needed. In this study, we designed and synthesized new fluorous sphingolipids to reveal these roles. Furthermore, we confirmed that they could be efficiently and rapidly separated from normal sphingolipids by FSPE, and that they hardly showed any cytotoxic activity, similarly to normal sphingolipids at the same dose. We also showed that these fluorinated ceramides could act as metabolic substrates for sphingomyelin synthase 2 (SMS2). This demonstrates their potential for further biological studies.

Arachidonic acid (AA) plays a pivotal role in the development of edema via its oxidized metabolites derived from cyclooxygenase (COX) and lipoxygenase (LOX), and is recently recognized as an activator of TRPV3. However, it is not clear whether AA plays some TRPV3-mediated pathological roles in the development of edema. Pharmacological and histological studies using ICRTRPV3+/+ and ICRTRPV3-/- mice indicated that higher ear edema responses to topical application of AA were observed in ICRTRPV3+/+ mice compared with ICRTRPV3-/- mice. However, there was no difference in the ear edema response to 12-O-tetradecanoylphorbol 13-acetate, skin histology, and skin barrier function between these mouse strains. Furthermore, oxidized fatty acids from the lesional site were analyzed to elucidate the TRPV3-mediated pathological roles of AA, and the results revealed that there were no differences in the level of COX or LOX metabolites derived from AA between both mouse strains. We concluded that AA plays a role in the development of TRPV3-mediated ear edema and that this result may contribute to better understanding of the pathophysiological mechanisms involved in the development of a certain type of edema.

In preclinical models of glioblastoma, antigen escape variants can lead to tumor recurrence after treatment with CAR T cells that are redirected to single tumor antigens. Given the heterogeneous expression of antigens on glioblastomas, we hypothesized that a bispecific CAR molecule would mitigate antigen escape and improve the antitumor activity of T cells. Here, we created a CAR that joins a HER2-binding scFv and an IL13Rα2-binding IL-13 mutein to make a tandem CAR exodomain (TanCAR) and a CD28.ζ endodomain. We determined that patient TanCAR T cells showed distinct binding to HER2 or IL13Rα2 and had the capability to lyse autologous glioblastoma. TanCAR T cells exhibited activation dynamics that were comparable to those of single CAR T cells upon encounter of HER2 or IL13Rα2. We observed that TanCARs engaged HER2 and IL13Rα2 simultaneously by inducing HER2-IL13Rα2 heterodimers, which promoted superadditive T cell activation when both antigens were encountered concurrently. TanCAR T cell activity was more sustained but not more exhaustible than that of T cells that coexpressed a HER2 CAR and an IL13Rα2 CAR, T cells with a unispecific CAR, or a pooled product. In a murine glioblastoma model, TanCAR T cells mitigated antigen escape, displayed enhanced antitumor efficacy, and improved animal survival. Thus, TanCAR T cells show therapeutic potential to improve glioblastoma control by coengaging HER2 and IL13Rα2 in an augmented, bivalent immune synapse that enhances T cell functionality and reduces antigen escape.

The direction of mesenchymal stem cell (MSC) differentiation is regulated by stimulation with various growth factors and cytokines. We recently established MSC lines, [transforming growth factor-β (TGF-β)-responsive SG‑2 cells, bone morphogenetic protein (BMP)-responsive SG‑3 cells, and TGF-β/BMP-non-responsive SG‑5 cells], derived from the bone marrow of green fluorescent protein-transgenic mice. In this study, to compare gene expression profiles in these MSC lines, we used DNA microarray analysis to characterize the specific gene expression profiles observed in the TGF-β-responsive SG‑2 cells. Among the genes that were highly expressed in the SG‑2 cells, we focused on vascular endothelial growth factor (VEGF) receptor 3 (VEGFR3), the gene product of FMS-like tyrosine kinase 4 (Flt4). We found that VEGF-C, a specific ligand of VEGFR3, significantly induced the cell proliferative activity, migratory ability (as shown by Transwell migration assay), as well as the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in the SG‑2 cells. Additionally, VEGF-C significantly increased the expression of prospero homeobox 1 (Prox1) and lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1), which are lymphatic endothelial cell markers, and decreased the expression of osteogenic differentiation marker genes in these cells. By contrast, TGF-β significantly increased the expression of early-phase osteogenic differentiation marker genes in the SG‑2 cells and markedly decreased the expression of lymphatic endothelial cell markers. The findings of our study strongly suggest the following: i) that VEGF-C promotes the proliferative activity and migratory ability of MSCs; and ii) VEGF-C and TGF-β reciprocally regulate MSC commitment to differentiation into lymphatic endothelial or osteoblastic phenotypes, respectively. Our findings provide new insight into the molecular mechanisms underlying the regenerative ability of MSCs.

Dietary sphingolipids such as glucosylceramide (GlcCer) are potential nutritional factors associated with prevention of metabolic syndrome. Our current understanding is that dietary GlcCer is degraded to ceramide and further metabolized to sphingoid bases in the intestine. However, ceramide is only found in trace amounts in food plants and thus is frequently taken as GlcCer in a health supplement. In the present study, we successfully prepared konjac ceramide (kCer) using endoglycoceramidase I (EGCase I). Konjac, a plant tuber, is an enriched source of GlcCer (kGlcCer), and has been commercialized as a dietary supplement to improve dry skin and itching that are caused by a deficiency of epidermal ceramide. Nerve growth factor (NGF) produced by skin cells is one of the itch factors in the stratum corneum of the skin. Semaphorin 3A (Sema 3A) has been known to inhibit NGF-induced neurite outgrowth of epidermal nerve fibers. It is well known that the itch sensation is regulated by the balance between NGF and Sema 3A. In the present study, while kGlcCer did not show an in vitro inhibitory effect on NGF-induced neurite outgrowth of PC12 cells, kCer was demonstrated to inhibit a remarkable neurite outgrowth. In addition, the effect of kCer was similar to that of Sema 3A in cell morphological changes and neurite retractions, but different from C2-Ceramide. kCer showed a Sema 3A-like action, causing CRMP2 phosphorylation, which results in a collapse of neurite growth cones. Thus, it is expected that kCer is an advanced konjac ceramide material that may have neurite outgrowth-specific action to relieve uncontrolled and serious itching, in particular, from atopic eczema., Highlights • We prepared konjac ceramide (kCer) from konjac GlcCer using endoglycoceramidase I. • kCer inhibits NGF-induced neurite outgrowth by semaphorin 3A-like action. • Konjac GlcCer does not show NGF-induced neurite outgrowth such as kCer.

The present study demonstrates unidirectional cell migration using a novel 3D microfabricated scaffold, as revealed by the uneven sorting of cells into an area of 1 mm × 1 mm. To induce unidirectional cell migration, it is important to determine the optimal arrangement of 3D edges, and thus, the anisotropic periodic structures of micropatterns are adjusted appropriately. The cells put forth protrusions directionally along the sharp edges of these micropatterns, and migrated in the protruding direction. There are three advantages to this novel system. First, the range of applications is wide, because this system effectively induces unidirectional migration as long as 3D shapes of the scaffolds are maintained. Second, this system can contribute to the field of cell biology as a novel taxis assay. Third, this system is highly applicable to the development of medical devices. In the present report, unique 3D microfabricated scaffolds that provoked unidirectional migration of NIH3T3 cells are described. The 3D scaffolds could provoke cells to accumulate in a single target location, or could provoke a dissipated cell distribution. Because the shapes are very simple, they could be applied to the surfaces of various medical devices. Their utilization as a cell separation technology is also anticipated.

Ginkgolic acid obtained as a sphingomyelin synthase inhibitor from a plant extract library inspired the concept of sphingolipid mimics. Ginkgolic acid-derived N-acyl anilines and ginkgolic acid 2-phosphate (GA2P) respectively mimic ceramide and sphingosine 1-phosphate (S1P) in structure and function. The GA2P-induced phosphorylation of ERK and internalization of S1P receptor 1 (S1P(1)) indicated potent agonist activity. Docking studies revealed that GA2P adopts a similar binding conformation to the bound ligand ML5, which is a strong antagonist of S1P(1).

Exosomes represent a subtype of extracellular nanovesicles that are generated from the luminal budding of limiting endosomal membranes and subsequent exocytosis. They encapsulate or associate with obsolete molecules to eliminate or to transfer their cargos in intercellular communication. The exosomes are also released and transported between neurons and glia in the nervous system, having a broad impact on nerve development, activation and regeneration. Accumulating evidence suggests that the exosomes are attributed to the pathogenesis of several neurodegenerative diseases such as prion disease, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, as well as aging, in which the exosomes lack the capacity for cellular self-repair and spread their enclosed pathological agents among neurons. In this article, we review the current proposed functions of exosomes in physiological and pathological processes in the nervous system.

Asterias amurensis starfish provide several bioactive species in addition to being fishery waste. Glucosyl ceramides (GlcCers) were extracted from the viscera of these starfish and were isolated by silica gel column chromatography. Degraded GlcCers generated A. amurensis sphingoid bases (ASBs) that mainly consisted of the triene-type bases d18:3 and 9-methyl-d18:3. The effect of these bases on ceramide synthesis and content were analyzed using normal human epidermal keratinocytes (NHEKs). The bases significantly enhanced the de novo ceramide synthesis and gene expression in NHEKs for proteins, such as serine-palmitoyltransferase and ceramide synthase. Total ceramide, GlcCer, and sphingomyelin contents increased dramatically upon ASB treatment. In particular, GlcCer bearing very-long-chain fatty acids (≥C28) exhibited a significant content increase. These ASB-induced enhancements on de novo ceramide synthesis were only observed in undifferentiated NHEKs. This stimulation of the de novo sphingolipid synthesis may improve skin barrier functions.

Cytokines and their intercellular signals regulate the multipotency of mesenchymal stem cells (MSCs). The present study established the MSC lines SG-2, -3, and -5 from the bone marrow of green fluorescent protein (GFP)-transgenic mice. These cell lines clearly expressed mouse MSC markers Sca-1 and CD44, and SG-2 and -5 cells retained the potential for osteogenic and adipogenic differentiation in the absence of members of the transforming growth factor (TGF)-β superfamily. By contrast, SG-3 cells only retained adipogenic differentiation potential. Analysis of cytokine and cytokine receptor expression in these SG cell lines showed that bone morphogenetic protein (BMP) receptor 1B was most highly expressed in the SG-3 cells, which underwent osteogenesis in response to BMP, while TGF-β receptor II was most highly expressed in SG-3 and -5 cells. However, it was unexpectedly noted that phosphorylation of Smad 2, a major transcription factor, was induced by TGF-β1 in SG-2 cells but not in SG-3 or -5 cells. Furthermore, TGF-β1 clearly induced the expression of Smad-interacting transcription factor CCAAT/enhancer binding protein-β in SG-2 but not in SG-3 or -5 cells. These results demonstrated the establishment of TGF-β-responsive SG-2 MSCs, BMP-responsive SG-3 MSCs and TGF-β/BMP-unresponsive SG-5 MSCs, each of which was able to be traced by GFP fluorescence after transplantation into in vivo experimental models. In conclusion, the present study suggested that these cell lines may be used to explore how the TGF-β superfamily affects the proliferation and differentiation status of MSCs in vivo.

Background/Objectives: Adipose tissue (AT) has an important role in energy homeostasis. The dysfunction of AT or the hyper-accumulation of neutral lipids leads to various metabolic diseases. Recent studies indicate that sphingo lipid metabolism associates with the development of metabolic diseases. Sphingomyelin, a major sphingolipid in mammals, requires sphingomyelin synthase (SMS) for biosynthesis. Previously, we reported that Sms2 deficiency inhibited diet-induced obesity, fatty liver, and insulin resistance in mice. However, the contribution of Sms2 to obesity and insulin resistance in AT is largely unknown. In this study, we investigated whether Sms2 deficiency in ATs affects obesity and insulin resistance.Subjects/Methods: Wild-type and Sms2 knockout (KO) mice were fed a high-fat for 12 weeks. Body and AT weights, and the food intake, were recorded. The AT status and macrophage infiltration were evaluated by histological analysis. The expression levels of genes and proteins involved in adipogenesis, inflammation, energy expenditure, and fatty acid metabolism were examined.Results: In white adipose tissue (WAT) from Sms2 KO mice, the number of small adipocytes increased but the adipocyte size decreased. In epididymal WAT, Sms2 deficiency inhibited inflammation and macrophage infiltration. Moreover, adipogenesis was moderately suppressed. In subcutaneous WAT from Sms2 KO mice, the expression of genes involved in energy expenditure and browning (Ucp1, Cidea, Tbx1) was elevated. In brown adipose tissue (BAT) from Sms2 KO mice, the lipid droplet surface area was lower than that of WT mice and the expression of genes involved in fatty acid synthesis (Fasn, Scd1) decreased.Conclusion: These results demonstrate that Sms2 deficiency leads to moderate adipogenesis and inflammatory suppression in epididymal WAT, increased energy expenditure by the browning of subcutaneous WAT, and suppression of fatty acid synthesis in BAT, suggesting that these synergetic effects in ATs from Sms2 KO mice contribute to the suppression of diet-induced obesity and insulin resistance.

GPR120 is a receptor for long chain fatty acids and is expressed in small intestinal endocrine cells, L cells and adipose tissue. Activation of GPR120 promotes the secretion of incretin GLP-1, which is known to have effects on anti-metabolic syndrome. As such, GPR120 is a potential target of pharmaceuticals for type II diabetes. In this study, we performed ligand-screening for GPR120 on glycero- and sphingo-type lipids and their derivatives using a Transforming Growth Factor α-shedding assay. We found that phytosphingosine (PHS) activates GPR120 in a manner comparable to the natural ligand α-linolenic acid (ALA) and superior to that of the synthetic ligand GW9508. The IC50 value of PHS was 33.4 μM, of ALA was 31.0 μM and of GW9508 was 41.7 μM. Additionally, PHS-induced activation of GPR120 was inhibited by the specific antagonist AH7614. Many of the natural or synthetic ligands found thus far are compounds with carboxyl groups. However, PHS does not possess a carboxyl group, suggesting that its manner of interaction with GPR120 may be significantly different from that of other ligands. Since PHS is rich in the plasma membrane of yeast, our results imply that PHS found in fermented food could have effects on anti-diabetes through activation of GPR120.

Sphingomyelin (SM) is required for cells to proliferate, but the reason is not fully understood. In order to asses this question, we employed a cell line, ZS, which lacks both SMS1 and SMS2, isolated from mouse embryonic fibroblasts in SMS1 and 2 double knockout mouse, and SMS1 or SMS2 re-expressing cells, ZS/SMS1 or ZS/SMS2, respectively. We investigated regulation of SM in activating the mammalian target of rapamycin (mTOR) signal induced by essential amino acids (EAA), using these cells. EAA-stimulated mTOR signal was more activated in ZS/SMS1 and ZS/SMS2 cells than in controls. Treatment with methyl-b-cyclodextrin dramatically inhibited the activation. Interestingly, we found that the expression of CD98, LAT-1 and ASCT-2, amino acid transporters concerned with mTOR activation, was down-regulated in ZS cells. Transporters localized in microdomains and formed a functional complex. Our results indicate that SM affect proliferation through the transport of amino acids via SM-enriched microdomains.

The intracerebral level of the aggregation-prone peptide, amyloid-ß (Aß), is constantly maintained by multiple clearance mechanisms, including several degradation enzymes, and brain efflux. Disruption of the clearance machinery and the resultant Aß accumulation gives rise to neurotoxic assemblies, leading to the pathogenesis of Alzheimer's disease (AD). In addition to the classic mechanisms of Aß clearance, the protein may be processed by secreted vesicles, although this possibility has not been extensively investigated. We showed that neuronal exosomes, a subtype of extracellular nanovesicles, enwrap, or trap Aß and transport it into microglia for degradation. Here, we review Aß sequestration and elimination by exosomes, and discuss how this clearance machinery might contribute to AD pathogenesis and how it might be exploited for effective AD therapy.

Elevated amyloid-β peptide (Aβ) in brain contributes to Alzheimer’s disease (AD) pathogenesis. We demonstrated the presence of exosome-associated Aβ in the cerebrospinal fluid (CSF) of cynomolgus monkeys and APP transgenic mice. The levels of exosome-associated Aβ notably decreased in the CSF of aging animals. We also determined that neuronal exosomes, but not glial exosomes, had abundant glycosphingolipids and could capture Aβ. Infusion of neuronal exosomes into brains of APP transgenic mice decreased Aβ and amyloid depositions, similarly to what reported previously on neuroblastoma-derived exosomes. These findings highlight the role of neuronal exosomes in Aβ clearance, and suggest that their downregulation might relate to Aβ accumulation and, ultimately, the development of AD pathology.

Elevated levels of amyloid-β peptide (Aβ) in the human brain are linked to the pathogenesis of Alzheimer disease. Recent in vitro studies have demonstrated that extracellular Aβ can bind to exosomes, which are cell-secreted nanovesicles with lipid membranes that are known to transport their cargos intercellularly. Such findings suggest that the exosomes are involved in Aβ metabolism in brain. Here, we found that neuroblastoma-derived exosomes exogenously injected into mouse brains trapped Aβ and with the associated Aβ were internalized into brain-resident phagocyte microglia. Accordingly, continuous intracerebral administration of the exosomes into amyloid-β precursor protein transgenic mice resulted in marked reductions in Aβ levels, amyloid depositions, and Aβ-mediated synaptotoxicity in the hippocampus. In addition, we determined that glycosphingolipids (GSLs), a group of membrane glycolipids, are highly abundant in the exosomes, and the enriched glycans of the GSLs are essential for Aβ binding and assembly on the exosomes both in vitro and in vivo. Our data demonstrate that intracerebrally administered exosomes can act as potent scavengers for Aβ by carrying it on the exosome surface GSLs and suggest a role of exosomes in Aβ clearance in the central nervous system. Improving Aβ clearance by exosome administration would provide a novel therapeutic intervention for Alzheimer disease.

To determine how the three-dimensional (3D) shape of scaffolds influences cell functions, 3D micropatterned scaffolds of various sizes were fabricated on a silicon substrate. The micropatterns were equilateral triangular pores with 3-20 μm long sides, and all had the same pore ratio (total pore area per unit area) and depth. The patterns only differed in terms of their 2D size. Such scaffolds have not been previously generated, and thus the effects of pattern size on cell functions have not been addressed. NIH-3T3 cells were cultured on these micropatterned scaffolds, and their morphology, proliferation rate, migration rate, and level of F-actin expression were assessed. Cells became more rounded and F-actin expression decreased as the pattern size of the scaffold decreased. Relationships were also demonstrated between pattern size and cell proliferation and migration. These results suggest that the pattern size of 3D micropatterned scaffolds affects the level of mechanical stress that cells experience, and thereby influences F-actin expression, cell morphology, cell proliferation and cell migration.

Sphingosine kinase (SPHK), which catalyzes the phosphorylation of sphingosine to generate sphingosine 1-phosphate, has two mammalian isotypes, SPHK1 and SPHK2. Both isozymes are promising anti-cancer therapeutic targets. In this report, we found that SG-12, a synthetic analogue of sphingosine that acts as a SPHK2 inhibitor, induces apoptosis via phosphorylation by SPHK2. The present results revealed the novel anti-cancer potential of a sphingosine analogue in the pathological setting where SPHK2 is upregulated.

To evaluate the precise role of sphingomyelin synthase 2 (SMS2) in sphingomyelin (SM) metabolism and their anti-inflammatory properties, we analyzed species of major SM and ceramide (Cer) (18:1, 18:0 sphingoid backbone, C14 - C26 N-acyl part) in SMS2 knockout and wild-type mouse plasma and liver using HPLC-MS. SMS2 deficiency significantly decreased very long chain SM (SM (d18:1/22:0) and SM (d18:1/24:0 or d18:0/24:1)) and increased very long chain Cer (Cer (d18:1/24:0 or d18:0/24:1) and Cer (d18:1/24:1)), but not long chain SM (SM (d18:1/16:0), SM (d18:1/18:0 or d18:0/18:1) and SM (d18:1/18:1)) in plasma. To examine the effects of SM on inflammation, we studied the role of very long chain SM in macrophage activation. Addition of SM (d18:1/24:0) strongly upregulated several macrophage activation markers, SM (d18:1/6:0) and Cer (d18:1/24:0) however, did not. It was suggested that very long chain SM but not long chain SM were decreased in SMS2-deficient mice liver and plasma. And the exogenously added very long chain SM (d18:1/24:0) could activate macrophages directly, suggesting a novel role of plasma very long chain SM in modulating macrophage activation and resulting inflammation.

Background: Olmsted syndrome (OS) is a congenital dermatosis characterized by palmoplantar keratoderma and periorificial keratotic plaque. TRPV3 (transient receptor potential vanilloid subtype 3) encodes a thermosensitive Ca2+ channel and is the causative gene of OS. However, the molecular mechanism that causes the pathological development of OS is unclear. Objective: We aimed to investigate the molecular mechanisms underlying OS pathology from the perspective of lipid metabolism. Methods: Comprehensive lipidomics and microarray analyses were conducted on tissue samples from a non-lesional skin area of OS model rats (Ht rats) and from wild type (WT) rats as the control. Results: Infiltration of leukocytes such as eosinophils and neutrophils and an increase in the fibrotic region were detected in the unaffected skin area of Ht rats compared with the WT rats. Among about 600 lipid species examined, the levels of 15-lipoxygenase (LOX) metabolites, the precursors of anti-inflammatory and pro-resolving lipid mediators, and dihydroceramides decreased by >= 16-fold in Ht rats compared with WT rats: Consistent with the decreases in the 15-LOX metabolites, expression levels of the genes that encode the 15-LOXs, Alox15 and Alox15b, were largely reduced. Conversely, increased expression levels were detected of 1136b, Cc120, Cxcl1, and Cxcl2, which encode cytokines/chemokines, and S100a8 and S100a9, which encode the Ca2+ binding proteins that are implicated in epidermal proliferation. Conclusion: The pro-inflammatory state in the unaffected skin of Ht rats caused by decreases in 15-LOX metabolites and increases in cytokines/chemokines may contribute to the pathogenesis of OS. (C) 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Asterias amurensis starfish provide several bioactive species in addition to being fishery waste. Glucosyl ceramides (GlcCers) were extracted from the viscera of these starfish and were isolated by silica gel column chromatography. Degraded GlcCers generated A. amurensis sphingoid bases (ASBs) that mainly consisted of the triene-type bases d18:3 and 9-methyl-d18:3. The effect of these bases on ceramide synthesis and content were analyzed using normal human epidermal keratinocytes (NHEKs). The bases significantly enhanced the de novo ceramide synthesis and gene expression in NHEKs for proteins, such as serine-palmitoyltransferase and ceramide synthase. Total ceramide, GlcCer, and sphingomyelin contents increased dramatically upon ASB treatment. In particular, GlcCer bearing very-long-chain fatty acids (≥C28) exhibited a significant content increase. These ASB-induced enhancements on de novo ceramide synthesis were only observed in undifferentiated NHEKs. This stimulation of the de novo sphingolipid synthesis may improve skin barrier functions.

The role of “sphingolipid rheostat” by ceramide and sphingosine 1-phosphate (S1P) in the regulation of autophagy remains unclear. In human leukemia HL-60 cells, amino acid deprivation (AA(−)) caused autophagy with an increase in acid sphingomyleinase (SMase) activity and ceramide, which serves as an autophagy inducing lipid. Knockdown of acid SMase significantly suppressed the autophagy induction. S1P treatment counteracted autophagy induction by AA(−) or C2-ceramide. AA(−) treatment promoted mammalian target of rapamycin (mTOR) dephosphorylation/inactivation, inducing autophagy. S1P treatment suppressed mTOR inactivation and autophagy induction by AA(−). S1P exerts biological actions via cell surface receptors, and S1P3 among five S1P receptors was predominantly expressed in HL-60 cells. We evaluated the involvement of S1P3 in suppressing autophagy induction. S1P treatment of CHO cells had no effects on mTOR inactivation and autophagy induction by AA(−) or C2-ceramide. Whereas S1P treatment of S1P3 overexpressing CHO cells resulted in activation of the mTOR pathway, preventing cells from undergoing autophagy induced by AA(−) or C2-ceramide. These results indicate that S1P-S1P3 plays a role in counteracting ceramide signals that mediate mTOR-controlled autophagy. In addition, we evaluated the involvement of ceramide-activated protein phosphatases (CAPPs) in ceramide-dependent inactivation of the mTOR pathway. Inhibition of CAPP by okadaic acid in AA(−)- or C2-ceramide-treated cells suppressed dephosphorylation/inactivation of mTOR, autophagy induction, and autophagy-associated cell death, indicating a novel role of ceramide-CAPPs in autophagy induction. Moreover, S1P3 engagement by S1P counteracted cell death. Taken together, these results indicated that sphingolipid rheostat in ceramide-CAPPs and S1P-S1P3 signaling modulates autophagy and its associated cell death through regulation of the mTOR pathway.

Recent studies have revealed that sphingomyelin (SM) is involved in metabolic syndrome and is a new target of an anti-metabolic syndrome drug. Deficiencies in the enzyme SM synthase 1 (SMS1) result in severe abnormalities, whereas deficiencies in SMS2 do not. SMS1 and SMS2 synthesize SM under similar conditions, so their respective activities cannot be measured separately. We report here on a sensitive, high-throughput and reliable cell-based method to separately measure each SMS activity and to screen for SMS-specific inhibitors, using HPLC and fluorescent ceramide (Cer) analogs. We isolated SMS-null cells and stably transfected them with SMS1 or SMS2. Using these cells, individual SMS activities could be measured separately. Fluorescent Cer, SM, and glucosylceramide analogs could be separated within 4 min by HPLC using an NH2 column. SMS activities of SMS1- or SMS2-expressing cells seeded in a single well of a 96-well plate could be measured using HPLC and fluorescent Cer analogs. This method clearly demonstrated that treatment of the cells with their respective siRNA or D609, an inhibitor of SMS, resulted in a significant decrease in each SMS activity. These results indicate that our newly developed method can be utilized for screening therapeutics against metabolic syndrome that target SMS2.

Sphingosine 1-phosphate (S1P) regulates lymphocyte trafficking via type-1 S1P receptor (S1P(1)) and participates in many pathological conditions. We developed a novel type S1P(1)-selective antagonist, TASP0251078, which is structurally unrelated to S1P. This competitive antagonist inhibited binding of S1P to S1P(1) resulting in reduced signaling downstream of S1P(1), including GTPγS-binding and cAMP formation. TASP0251078 also inhibited S1P-induced cellular responses such as chemotaxis and receptor-internalization. Furthermore, when administered in vivo, TASP0251078 induced lymphopenia in blood, which is different from previously reported effects of other S1P(1)-antagonists. In a mouse contact hypersensitivity model, TASP0251078 effectively suppressed ear swelling, leukocyte infiltration, and hyperplasia. These findings provide the chemical evidence that S1P(1) antagonism is responsible for lymphocyte sequestration from the blood, and suggest that the effect of S1P(1) agonists on lymphocyte sequestration results from their functional antagonism.