Sphingomyelin (SM) synthase has been assumed to be involved in both cell death and survival by regulating pro-apoptotic mediator ceramide and pro-survival mediator diacylglycerol. However, its precise functions are ambiguous due to the lack of molecular cloning of SM synthase gene(s). We isolated WR19L/Fas-SM(؊) mouse lymphoid cells, which show a defect of SM at the plasma membrane due to the lack of SM synthase activity and resistance to cell death induced by an SM-directed cytolytic protein lysenin. WR19L/Fas-SM(؊) cells were also highly susceptible to methyl-␤-cyclodextrin (M␤CD) as compared with the WR19L/Fas-SM(؉) cells, which are capable of SM synthesis. By expression cloning method using WR19L/Fas-SM(؊) cells and M␤CD-based selection, we have succeeded in cloning of a human cDNA responsible for SM synthase activity. The cDNA encodes a peptide of 413 amino acids named SMS1 (putative molecular mass, 48.6 kDa), which contains a sterile ␣ motif domain near the N-terminal region and four predicted transmembrane domains. WR19L/Fas-SM(؊) cells expressing SMS1 cDNA (WR19L/Fas-SMS1) restored the resistance against M␤CD, the accumulation of SM at the plasma membrane, and SM synthesis by transferring phosphocholine from phosphatidylcholine to ceramide. Furthermore, WR19L/Fas-SMS1 cells, as well as WR19L/ Fas-SM(؊) cells supplemented with exogenous SM, restored cell growth ability in serum-free conditions, where the growth of WR19L/Fas-SM(؊) cells was severely inhibited. The results suggest that SMS1 is responsible for SM synthase activity in mammalian cells and plays a critical role in cell growth of mouse lymphoid cells.Diverse kinds of phospho-and glycerolipids such as diacylglycerol (DAG), 1 inositol phosphatides, and phosphatidic acid are recognized as bioactive molecules in cell growth and survival (1, 2). Sphingolipid ceramide has recently emerged as a signal mediator of cell functions including apoptosis, differentiation, and secretion (3). Various stresses such as ultraviolet, irradiation, heat shock, hypoxia, and biological factors such as tumor necrosis factor-␣, interferon-␥, and Fas antibody require ceramide generation to execute apoptosis, suggesting the implications of SM as a source of ceramide generation in the induction of cell death (4, 5). It was reported that SM dose-dependently inhibits both deoxycholate-induced apoptosis and subsequent hyper-proliferation in colon epithelial cells (6) and decreases the number of aberrant crypts of colon (7), suggesting the implications of SM in cell death and growth.SM is produced by SM synthase, which is thought to be the only enzyme to synthesize SM in mammalian cells (8). The enzyme catalyzes the reaction in which phosphocholine moiety is transferred from phosphatidylcholine (PC) to ceramide. Thus, the activation of SM synthase subsequently increases the levels of DAG and decreases ceramide at the same time (8). DAG is an important signaling molecule for cell growth through protein kinase C activation (9 -12) and acts competitively against ceramide-induced a...

Dendritic cells (DCs) show a functional plasticity in determining Th responses depending on their maturational stage or on maturational signals delivered to the DCs. Human plasmacytoid DCs (PDCs) can induce either Th1- or Th2-type immune responses upon exposure to viruses or IL-3, respectively. In this study we have investigated the Th-polarizing capacity of PDCs after short (24-h) or long (72-h) culture with stimuli and have assessed the expression and function of OX40 ligand (OX40L) in PDC-mediated Th polarization in addition to type I IFN-dependent responses. IL-3-treated PDCs expressed OX40L, but produced almost no IFN-α in response to T cell stimulation (CD40 ligand or T cell interaction), resulting in the preferential priming of Th2 cells through OX40L-dependent mechanisms. Meanwhile, PDCs were rapidly endowed by viral infection (Sendai virus) with a high potency to develop IFN-γ-producing Th cells depending on their capacity to residually produce IFN-α. Although Sendai virus-stimulated PDCs simultaneously expressed OX40L in their maturational process, the Th1-inducing effect of endogenous type I IFNs may overcome and thus conceal the OX40L-dependent Th2 responses. However, during maturation in response to Sendai virus over the longer 72-h period, the expression level of OX40L was up-regulated, whereas the residual IFN-α-producing ability was down-regulated, and consequently, the PDCs with prolonged Sendai virus stimulation induced Th2 responses to some extent. Thus, PDCs have the distinct means to dictate an appropriate response to environmental stimuli.

Engagement of the Fas receptor (CD95) initiates multiple signaling pathways that lead to apoptosis, such as the formation of death-inducing signaling complex (DISC), activation of caspase cascades, and the generation of the lipid messenger, ceramide. Sphingomyelin (SM) is a major component of lipid rafts, which are specialized structures that enhance the efficiency of membrane receptor signaling and are a main source of ceramide. However, the functions of SM in Fas-mediated apoptosis have yet to be clearly defined, as the responsible genes have not been identified. After cloning a gene responsible for SM synthesis, SMS1, we established SM synthase–defective WR19L cells transfected with the human Fas gene (WR/Fas-SM(−)), and cells that have been functionally restored by transfection with SMS1 (WR/Fas-SMS1). We show that expression of membrane SM enhances Fas-mediated apoptosis through increasing DISC formation, activation of caspases, efficient translocation of Fas into lipid rafts, and subsequent Fas clustering. Furthermore, WR/Fas-SMS1 cells, but not WR/Fas-SM(−) cells, showed a considerable increase in ceramide generation within lipid rafts upon Fas stimulation. These data suggest that a membrane SM is important for Fas clustering through aggregation of lipid rafts, leading to Fas-mediated apoptosis.

Summary Cyclosporin A (CsA) is a potent immuno‐suppressant and is approved for the treatment of various disease conditions. The molecular biological mechanism of CsA has been investigated intensively in T cells and has been shown to involve the intracellular calcineurin pathway. Recently, it was reported that CsA has capacities to affect not only T cells but also antigen‐presenting cells such as B cells and dendritic cells (DCs). DCs are a master regulator of immune responses that have an integral capacity to prime naive T cells. In the present study, we investigated the biological effects of CsA on human peripheral blood DC subsets: CD11c+ myeloid and CD11c− lymphoid subsets. CsA inhibited the up‐regulation of co‐stimulatory molecules induced with or without microbial stimuli and CD40L on both CD11c+ and CD11c− subsets. In addition, CsA negatively regulated the endocytic activity of CD11c+ DC during the immature state. CsA inhibited the interleukin‐12 (IL‐12) production, but augmented the IL‐10 production from the LPS‐stimulated CD11c+ subset, whereas CsA reduced the interferon‐α (IFN‐α) production from the CD11c− subset infected with Sendai virus (SV). Both the LPS‐stimulated CD11c+ subset and SV‐infected CD11c− subset preferentially induced the development of IFN‐γ‐producing T helper‐type 1 (Th1) cells. Pretreatment of these DC subsets with CsA inhibited the Th1 skewing. These findings suggested a DC‐mediated mechanism of immunosupression by CsA.