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Sphingolipids play a very important role in cell membrane formation, signal transduction, and plasma lipoprotein metabolism, all of which may well have an impact on the development of atherosclerosis. To investigate the relationship between sphingolipid metabolism and atherosclerosis, we utilized myriocin to inhibit mouse serine palmitoyl-CoA transferase (SPT), the key enzyme for sphingolipid biosynthesis. We injected 8-week-old apoE-deficient mice with myriocin (0.3 mg/ kg/every other day, intraperitoneal) for 60 days. On a chow diet, myriocin treatment caused a significant decrease (50%) in liver SPT activity (p < 0.001), significant decreases in plasma sphingomyelin, ceramide, and sphingosine-1-phosphate levels (54, 32, and 73%, respectively) (p < 0.0001), and a significant increase in plasma phosphatidylcholine levels (91%) (p < 0.0001). Plasma total cholesterol and triglyceride levels demonstrated no significant changes, but there was a significant decrease in atherosclerotic lesion area (42% in root and 36% in en face assays) (p < 0.01). On a high fat diet, myriocin treatment caused marked decreases in plasma sphingomyelin, ceramide, and sphingosine-1-phosphate levels (59, 66, and 81%, respectively) (p < 0.0001), and a marked increase in plasma phosphatidylcholine levels (100%) (p < 0.0001). Total cholesterol and triglyceride demonstrated no significant changes, but there was a significant decrease in atherosclerotic lesion area (39% in root and 37% in en face assays) (p < 0.01). These results indicate that, apart from cholesterol levels, sphingolipids have an effect on atherosclerotic development and that SPT has proatherogenic properties. Thus, inhibition of SPT activity could be an alternative treatment for atherosclerosis.Sphingolipids have many biological functions, including cell membrane formation, signal transduction, and lipid metabolism, and all of these may be related to the development of atherosclerosis. Serine palmitoyl-CoA transferase (SPT) 1 is the rate-limiting enzyme in the biosynthesis of sphingolipids (1). It has long been known that SPT plays an important role in the metabolism of sphingolipids, but its role in other lipid metabolisms and atherosclerosis has not been unequivocally determined. When SPT activity is increased in rat liver (2) and lung (3), sphingolipid formation is likewise increased. The activity of SPT is heightened in the aortas of rabbits fed a high cholesterol diet (4). Two candidate cDNAs for yeast SPT, termed LCB1 and LCB2, have been cloned (5, 6), and the translated sequences indicate that their gene products have a 21% amino acid sequence identity (6). The lack of SPT activity in a yeast strain defective in LCB1 or LCB2, together with the protein similarity data, suggest that the two genes encode subunits of SPT (6). Mouse and human LCB1 and LCB2 cDNA homologues have also been cloned (7,8). In mouse, the two mRNAs have the same tissue distribution (lung, kidney Ͼ brain Ͼ cartilage, skin Ͼ heart Ͼ liver Ͼ muscle), and the ratio of the amounts of the two transcri...

Background-NFB has long been regarded as a proatherogenic factor, mainly because of its regulation of many of the proinflammatory genes linked to atherosclerosis. Metabolism of sphingomyelin (SM) has been suggested to affect NFB activation, but the mechanism is largely unknown. SMS2 regulates SM levels in cell plasma membrane and lipid rafts and has a potential to regulate NFB activation. Methods and Results-To investigate the role of SMS2 in NFB activation we used macrophages from SMS2 knockout (KO) mice and SMS2 siRNA-treated HEK 293 cells. We found that NFB activation and its target gene expression are attenuated in macrophages from SMS2 KO mice in response to lipopolysaccharide (LPS) stimulation and in SMS2 siRNA-treated HEK 293 cells after tumor necrosis factor (TNF)-␣ simulation. In line with attenuated NFB activation, we found that SMS2 deficiency substantially diminished the abundance of toll like receptor 4 (TLR4)-MD2 complex levels on the surface of macrophages after LPS stimulation, and SMS2 siRNA treatment reduced TNF-␣-stimulated lipid raft recruitment of TNF receptor-1 (TNFR1) in HEK293 cells. SMS2 deficiency decreased the relative amounts of SM and diacylglycerol (DAG) and increased ceramide, suggesting multiple mechanisms for the decrease in NFB activation. Conclusions-SMS2 is a modulator of NFB activation, and thus it could play an important role in NFB-mediated proatherogenic process. Key Words: sphingomyelin synthase 2 Ⅲ sphingomyelin Ⅲ lipid rafts Ⅲ NFB Ⅲ atherosclerosis A therosclerosis is an inflammatory disease. The accumulation of macrophage-derived foam cells in the vessel wall is always accompanied by the production of a wide range of chemokines, cytokines, and growth factors. 1 These factors regulate the turnover and differentiation of immigrating and resident cells, eventually influencing plaque development. One of the key regulators of inflammation is NFB, 2 which has long been regarded as a proatherogenic factor, mainly because of its regulation of many of the proinflammatory genes linked to atherosclerosis. 3,4 Sphingomyelin (SM) is one of the major lipids on the plasma membrane and is enriched in lipid rafts, which are considered microdomains of plasma membrane critical for signal transduction. 5,6 Depletion of cholesterol from rafts causes a redistribution of TNF-␣ receptor 1 to nonraft plasma membrane, preventing NFB activation 7 or ligand-induced RhoA activation, 8 and such treatment also inhibits proinflammatory signals mediated by TLRs. 9 Studies also suggest that NFB activation is triggered by SM-derived ceramide. 10,11 On the contrary, it has been shown that ceramide is not necessary or even inhibits NFB activation. 12 SM biosynthesis might also affect NFB activation. SM is synthesized by sphingomyelin synthase (SMS), which transfers the phosphorylcholine moiety from phosphatidylcholine (PC) onto ceramide, producing SM and diacylglycerol (DAG). 13 Luberto et al 14 found that D609, a nonspecific SMS inhibitor, blocks TNF-␣-and phorbol ester-mediated NFB activation that was c...

Rationale: Sphingomyelin synthase (SMS)2 contributes to de novo sphingomyelin (SM) biosynthesis and plasma membrane SM levels. SMS2 deficiency in macrophages diminishes nuclear factor B and mitogen-activated protein kinase activation induced by inflammatory stimuli. Objective: The effects of SMS2 deficiency on the development of atherosclerosis are investigated. Methods and Results: We measured cholesterol efflux from macrophages of wild-type (WT) and SMS2 knockout (KO) mice. We transplanted SMS2 KO mouse bone marrow into low-density lipoprotein (LDL) receptor (LDLr) knockout mice (SMS2 ؊/؊ 3 LDLr ؊/؊ ), creating a mouse model of SMS2 deficiency in the macrophages. We found that SMS2 deficiency caused significant induction of cholesterol efflux in vitro and in vivo. Moreover, we found that SMS2 KO mice had less interleukin-6 and tumor necrosis factor ␣ in the circulation before and after endotoxin stimulation, compared with controls. More importantly, after 3 months on a western-type diet, SMS2 ؊/؊ 3 LDLr ؊/؊ mice showed decreased atherosclerotic lesions in the aortic arch, root (57%, P<0.001), and the entire aorta (42%, P<0.01), compared with WT3 LDLr ؊/؊ mice. Analysis of plaque morphology revealed that SMS2 ؊/؊ 3 LDLr ؊/؊ mice had significantly less necrotic core area (71%, P<0.001), less macrophage content (37%, P<0.01), and more collagen content (35%, P<0.05) in atherosclerotic lesions. We also found that SMS2 ؊/؊ 3 LDLr ؊/؊ mice had significantly lower free cholesterol and cholesteryl ester levels in the brachiocephalic artery than WT3 LDLr Key Words: macrophage sphingomyelin synthase deficiency Ⅲ sphingomyelin biosynthesis Ⅲ cholesterol efflux Ⅲ inflammation Ⅲ atherosclerosis F oam cell formation caused by excessive accumulation of cholesterol in the macrophages is a pathological hallmark of atherosclerosis, 1 which is also known to be an inflammatory disease. 2 The accumulation of macrophage-derived foam cells in the vessel wall is always accompanied by the production of a wide range of chemokines and cytokines that regulate the turnover and differentiation of immigrating and resident cells and subsequent plaque development. 2 Thus, promoting cholesterol efflux from cholesterol-laden macrophages, as well as diminishing their inflammatory response, can both be significant antiatherogenic approaches.The interaction between sphingomyelin (SM), cholesterol, and glycosphingolipid drives the formation of plasma membrane rafts, 3 and in some cells, caveolae. 4,5 SM is synthesized by sphingomyelin synthase (SMS), which transfers the phosphorylcholine moiety from phosphatidylcholine (PC) onto ceramide. 6 Two SMS genes, SMS1 and SMS2, have been cloned, and their subcellular localization characterized. 7,8 SMS1 is found in the trans-Golgi apparatus, whereas SMS2 is predominantly located in the plasma membranes. 7,9 Our laboratory and others have shown that SMS1 and SMS2 expression levels correlate positively with those of SM in the lipid rafts. 10 -12 Furthermore, SMS1 has been implicated in the regulation of lipi...

Objective Sphingomyelin synthase (SMS) catalyzes the conversion of ceramide to sphingomyelin (SM), and sits at the crossroads of sphingolipid biosynthesis. SMS has two isoforms: SMS1 and SMS2. Although they have the same SMS activity, they are different enzymes with distinguishable subcellular localizations and cell expression patterns. It is conceivable that these differences could yield different consequences, in terms of sphingolipid metabolism and its related atherogenesis. Methods and Results we created Sms1 gene knockout (KO) mice and found that Sms1 deficiency significantly decreased plasma, liver, and macrophage SM (59%, 45%, and 54%, respectively), but had only a marginal effect on ceramide levels. Surprisingly, we found that Sms1 deficiency dramatically increased glucosylceramide and GM3 levels in plasma, liver, and macrophages (4 to 12 fold), while Sms2 deficiency had no such effect. We evaluated total SMS activity in tissues and found that Sms1 deficiency causes 77% reduction of SMS activity, indicating SMS1 is the major SMS in macrophages. Moreover, Sms1 deficient-macrophages have significantly higher glucosylceramide synthase activity. We also found that Sms1 deficiency significantly attenuated toll-like 4 receptor-mediated NF-κB and MAP kinase activation after LPS treatment. To evaluate atherogenicity, we transplanted Sms1 KO mouse bone marrow into LDL receptor KO mice (Sms1−/−→Ldlr−/−). After 3 months on a Western diet, these animals showed a significant decrease of atherosclerotic lesions in the root and the entire aorta (35% and 44%, P<0.01, respectively), and macrophage content in lesions (51%, P<0.05), compared with WT→Ldlr−/−) mice. Conclusions Sms1 deficiency decreases SM, but dramatically increases the levels of glycosphingolipids. Atherosclerosis in Sms1−/−→Ldlr−/− mice is significantly decreased.

TFE3 and TFEB are broadly expressed transcription factors related to the transcription factor Mitf. Although they have been linked to cytokine signaling pathways in nonlymphoid cells, their function in T cells is unknown. TFE3-deficient mice are phenotypically normal, whereas TFEB deficiency causes early embryonic death. We now show that combined inactivation of TFE3 and TFEB in T cells resulted in a hyper-immunoglobulin M syndrome due to impaired expression of CD40 ligand by CD4 + T cells. Native TFE3 and TFEB bound to multiple cognate sites in the promoter of the gene encoding CD40 ligand (Cd40lg), and maximum Cd40lg promoter activity and gene expression required TFE3 or TFEB. Thus, TFE3 and TFEB are direct, physiological and mutually redundant activators of Cd40lg expression in activated CD4 + T cells critical for T cell-dependent antibody responses.Transcription factors TFE3 and TFEB are the most closely related members of a functionally interactive DNA-binding family known as Mitf-TFE (MiT) that includes the microphthalmiaassociated transcription factor Mitf and TFEC 1 . MiT proteins bind to μE3 sites, a subset of Eboxes that match a general CANNTG consensus sequence 2 , with those binding to TFE3 in vitro first identified and characterized in immunoglobulin heavy-chain and T cell receptor (TCR) enhancers 3-5 . DNA binding is mediated by nearly identical basic regions and requires homo-or heterodimer formation mediated by conserved helix-loop-helix and leucine zipper domains 5-7 . Such interactions are restricted in the MiT family.MiT proteins share similar structures and are often expressed together, yet genetic studies have demonstrated both overlapping and nonoverlapping functions for MiT proteins in different cell

Serine palmitoyltransferase (SPT) is the first and rate-limiting enzyme of the de novo biosynthetic pathway of sphingomyelin (SM). Both SPT and SM have been implicated in the pathogenesis of atherosclerosis, the development of which is driven by macrophages; however, the role of SPT in macrophage-mediated atherogenesis is unknown. To address this issue, we have analyzed macrophage inflammatory responses and reverse cholesterol transport, 2 key mediators of atherogenesis, in SPT subunit 2-haploinsufficient (Sptlc2 +/-) macrophages. We found that Sptlc2 +/-macrophages have significantly lower SM levels in plasma membrane and lipid rafts. This reduction not only impaired inflammatory responses triggered by TLR4 and its downstream NF-κB and MAPK pathways, but also enhanced reverse cholesterol transport mediated by ABC transporters. LDL receptor-deficient (Ldlr -/-) mice transplanted with Sptlc2 +/-bone marrow cells exhibited significantly fewer atherosclerotic lesions after high-fat and high-cholesterol diet feeding. Additionally, Ldlr -/-mice with myeloid cell-specific Sptlc2 haploinsufficiency exhibited significantly less atherosclerosis than controls. These findings suggest that SPT could be a novel therapeutic target in atherosclerosis.

Serine palmitoyltransferase (SPT) is the rate-limiting enzyme for sphingolipid biosynthesis. SPT has two major subunits, SPTLC1 and SPTLC2. We previously found that liver Sptlc2 deficiency in early life impairs the development of adherens junctions. Here, we investigated the role of Sptlc2 deficiency in intestine. We treated Sptlc2-Flox/villin-Cre-ERT2 mice with tamoxifen (days 1, 2, and 3) to ablate Sptlc2 specifically in the intestine. At day 6 after tamoxifen treatment, Sptlc2-deficient mice had significantly decreased body weight with concurrent diarrhea and rectal bleeding. The number of goblet cells was reduced in both large and small intestine of Sptlc2-deficient mice compared with controls. Sptlc2 deficiency suppressed the level of mucin2 in the colon and increased circulating lipopolysaccharides, suggesting that SPT activity has a housekeeping function in the intestine. All Sptlc2-deficient mice died 7–10 days after tamoxifen treatment. Notably, supplementation with antibiotics and dexamethasone reduced lethality by 70%. We also found that colon specimens from patients with inflammatory bowel diseases had significantly reduced Sptlc2 expression, SPTLC2 staining, and goblet cell numbers. SPT activity is crucial for intestinal cell survival and barrier function.