Toshiro Okazaki

Mitochondrial Dysfunction and Increased Reactive Oxygen Species Impair Insulin Secretion in Sphingomyelin Synthase 1-null Mice

Sphingomyelin synthase 1 (SMS1) catalyzes the conversion of ceramide to sphingomyelin. Here, we generated and analyzed SMS1-null mice. SMS1-null mice exhibited moderate neonatal lethality, reduced body weight, and loss of fat tissues mass, suggesting that they might have metabolic abnormality. Indeed, analysis on glucose metabolism revealed that they showed severe deficiencies in insulin secretion. Isolated mutant islets exhibited severely impaired ability to release insulin, dependent on glucose stimuli. Further analysis indicated that mitochondria in mutant islet cells cannot up-regulate ATP production in response to glucose. We also observed additional mitochondrial abnormalities, such as hyperpolarized membrane potential and increased levels of reactive oxygen species (ROS) in mutant islets. Finally, when SMS1-null mice were treated with the anti-oxidant N-acetyl cysteine, we observed partial recovery of insulin secretion, indicating that ROS overproduction underlies pancreatic β-cell dysfunction in SMS1-null mice. Altogether, our data suggest that SMS1 is important for controlling ROS generation, and that SMS1 is required for normal mitochondrial function and insulin secretion in pancreatic β-cells.

Impaired TCR signaling through dysfunction of lipid rafts in sphingomyelin synthase 1 (SMS1)-knockdown T cells

During T cell activation, TCRs cluster at the center of the T cell-antigen-presenting cell interface forming the central supramolecular activation cluster. Although it has been suggested that sphingolipid- and cholesterol-rich microdomains, termed lipid rafts, form platforms for the regulation and transduction of TCR signals, an actual role for membrane sphingomyelin (SM), a key component of lipid rafts, has not been reported. After cloning a gene responsible for SM synthesis, sphingomyelin synthase (SMS) 1, we established a SM-knockdown cell line (Jurkat-SMS1/kd) by transfection of SMS1-short-interfering RNA into Jurkat T cells, which is deficient in membrane expression of SM. Upon CD3 stimulation, expression of CD69 (the earliest leukocyte activation antigen), activation-induced cell adhesion and proliferation as well as TCR clustering was severely impaired in Jurkat-SMS1/kd cells. CD3-induced tyrosine phosphorylation and association of linker for activation of T cell with ZAP-70 and Grb2 and phosphorylation of protein kinase C (PKC) were also severely impaired in Jurkat-SMS1/kd cells. Finally, translocation of TCR, ZAP-70 and PKC into lipid rafts was markedly decreased in Jurkat-SMS1/kd cells. These findings indicate that membrane SM is crucial for TCR signal transduction, leading to full T cell activation through lipid raft function.

Regulation of Cell Migration by Sphingomyelin Synthases: Sphingomyelin in Lipid Rafts Decreases Responsiveness to Signaling by the CXCL12/CXCR4 Pathway

ABSTRACT Sphingomyelin synthase (SMS) catalyzes the formation of sphingomyelin, a major component of the plasma membrane and lipid rafts. To investigate the role of SMS in cell signaling and migration induced by binding of the chemokine CXCL12 to CXCR4, we used mouse embryonic fibroblasts deficient in SMS1 and/or SMS2 and examined the effects of SMS deficiency on cell migration. SMS deficiency promoted cell migration through a CXCL12/CXCR4-dependent signaling pathway involving extracellular signal-regulated kinase (ERK) activation. In addition, SMS1/SMS2 double-knockout cells had heightened sensitivity to CXCL12, which was significantly suppressed upon transfection with the SMS1 or SMS2 gene or when they were treated with exogenous sphingomyelin but not when they were treated with the SMS substrate ceramide. Notably, SMS deficiency facilitated relocalization of CXCR4 to lipid rafts, which form platforms for the regulation and transduction of receptor-mediated signaling. Furthermore, we found that SMS deficiency potentiated CXCR4 dimerization, which is required for signal transduction. This dimerization was significantly repressed by sphingomyelin treatment. Collectively, our data indicate that SMS-derived sphingomyelin lowers responsiveness to CXCL12, thereby reducing migration induced by this chemokine. Our findings provide the first direct evidence for an involvement of SMS-generated sphingomyelin in the regulation of cell migration.

Clonality analysis of lymphoproliferative disorders in patients with Sjögren's syndrome

The aim of this study was to clarify the nature of the clonal lymphocyte infiltration in Sjögrens syndrome (SS) patients associated with lymphoproliferative disorders. We examined B cell clonality in lymphoproliferative tissues from six primary SS patients associated with lymphoproliferative disorders or lymphoma by cloning and sequencing of the gene rearrangement of the immunoglobulin heavy chain complementarity determining region 3 (IgVH–CDR3). Three patients with sequential observation showed progressional clonal expansion with the presence of the same subclone in different tissues during the course of disease. Among them, one patient developed mucosa‐associated lymphoid tissue (MALT) lymphoma in glandular parotid. The other three SS patients concomitant with malignant B cells lymphomas showed different clonal expansion of B cells between nodal sites and salivary glands. The cloanality analysis indicated that monoclonal B cell population could spread from one glandular site to another site during the course of SS, suggesting that the malignant clone may arise from the general abnormal microenvironment, not restricted to the glandular tissue, in some SS patients.

Increased Oxidative Stress Impairs Adipose Tissue Function in Sphingomyelin Synthase 1 Null Mice

Sphingomyelin synthase 1 (SMS1) catalyzes the conversion of ceramide to sphingomyelin. Here, we found that SMS1 null mice showed lipodystrophic phenotype. Mutant mice showed up-regulation of plasma triglyceride concentrations accompanied by reduction of white adipose tissue (WAT) as they aged. Lipoprotein lipase (LPL) activity was severely reduced in mutant mice. In vivo analysis indicated that fatty acid uptake in WAT but not in liver decreased in SMS1 null compared to wild-type mice. In vitro analysis using cultured cell revealed that SMS1 depletion reduced fatty acid uptake. Proteins extracted from WAT of mutant mice were severely modified by oxidative stress, and up-regulation of mRNAs related to apoptosis, redox adjustment, mitochondrial stress response and mitochondrial biogenesis was observed. ATP content of WAT was reduced in SMS1 null mice. Blue native gel analysis indicated that accumulation of mitochondrial respiratory chain complexes was reduced. These results suggest that WAT of SMS1 null mice is severely damaged by oxidative stress and barely functional. Indeed, mutant mice treated with the anti-oxidant N-acetyl cysteine (NAC) showed partial recovery of lipodystrophic phenotypes together with normalized plasma triglyceride concentrations. Altogether, our data suggest that SMS1 is crucial to control oxidative stress in order to maintain WAT function.

Inhibitory effects of dietary glucosylceramides on squamous cell carcinoma of the head and neck in NOD/SCID mice

BackgroundSphingolipids, components of cellular membranes in eukaryotic cells, have roles in the regulation of tumor growth, inflammation, angiogenesis, and immunity. We investigated the effects of dietary glucosylceramides, sphingolipids isolated from rice bran, on tumor growth of human head and neck squamous cell carcinoma.MethodsThe tumor cell line SCCKN cells isolated from well-differentiated human head and neck cancer were subcutaneously inoculated into the right flank of NOD/SCID mice, to establish an SCCKN xenograft model. Rice bran glucosylceramides (300xa0mg/kg/day) were administered orally to the mice for 14 consecutive days.ResultsDietary glucosylceramides significantly inhibited the growth of the xenograft tumor in comparison with the control group. The TUNEL stain revealed that treatment of mice with glucosylceramides increased the number of apoptotic cells in the implanted tumor tissues and that apoptosis induction was accompanied by the formation of active/cleaved caspase-3.ConclusionThese results suggest that dietary glucosylceramides possibly exert anti-tumor activity by inducing apoptosis of head and neck squamous cell carcinoma. Therefore, their potential usefulness in treatment and prevention of human head and neck squamous cell carcinoma warrants further investigation.

Prevention of fasting-mediated bone marrow atrophy by leptin administration.

Leptin is an adipokine that regulates body weight. In the current study, we demonstrate that continuous injection of leptin prevents the lymphocyte reduction observed in fasted mice, especially the immature B cell populations in the bone marrow. Although leptin administration reduced apoptotic cells in the bone marrow of fasted mice, it did not prevent glucocorticoid-mediated apoptosis in vitro. Bone marrow atrophy has also been shown in the leptin receptor-deficient db/db mice. In order to investigate the mechanisms underlying these processes, we transplanted bone marrow cells from db/db or control (+m/+m) mice into C.B-17/lcr-scid/scid mice. We found that the spleen and bone marrow B cell populations were completely reconstituted when db/db and +m/+m cells were transplanted into scid mice. Our findings suggest that direct interactions between leptin and bone marrow cells are not essential for the development of B cells in a metabologically normal environment.

Metabolizing Enzymes Such As Sphingomyelin Synthase Induce Cell Death by Increasing Ceramide Content

Ceramide, among its other roles, serves as a pro-apoptotic lipid mediator. Ceramide-induced pro-apoptotic signals include caspases, reactive oxygen species (ROS) and c-jun-N-terminal kinase (JNK), and anti-apoptotic signals such as phosphatidylinositides (PI)-3 kinase and protein kinase C are inhibited by ceramide. Sphingosine-1-phosphate (S1P) competes with ceramide-induced cell death by blocking its generation and downstream pathways. This balanced interaction between S1P and ceramide may regulate cell death and survival/growth. Regulating ceramide-metabolizing enzymes, such as sphingomyelin synthase (SMS) and glucosylceramide synthase (GCS), as well as those of ceramide-downstream pathways are crucial to control ceramide signals. It was recently shown that inhibiting SMS and GCS induced apoptotic cell death in human leukemia cell lines. In addition cells possessing less ceramide and have high levels of GCS and SMS were chemoresistant in vivo. Membranous SM generated by SMS1 gene, which we cloned, may act as not only the source for generation of ceramide but also as the platform for transmembrane receptors such as Fas antigen. Thus, overwhelming drug-or chemo-resistance in human hematopoietic malignancies by inhibiting ceramide-metabolizing enzymes and enhancing ceramide-downstream signals to intensify ceramide-induced cell death would be one approach to treating these cancers.