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...
Sphingomyelin plays a very important role both in cell membrane formation that may well have an impact on the development of diseases like atherosclerosis and diabetes. However, the molecular mechanism that governs intracellular and plasma membrane SM levels is largely unknown. Recently, two isoforms of sphingomyelin synthase (SMS1 and SMS2), the last enzyme for SM de novo synthesis, have been cloned. We have hypothesized that SMS1 and SMS2 are the two most likely candidates responsible for the SM levels in the cells and on the plasma membrane. To test this hypothesis, cultured cells were treated with tricyclodecan-9-yl-xanthogenate (D609), an inhibitor of SMS, or with SMS1 and SMS2 siRNAs. Cells were then pulsed with [ 14 C]-L-serine (a precursor of all sphingolipids). SMS activity and [ 14 C]-SM in the cells were monitored. We found that SMS activity was significantly decreased in cells after D609 or SMS siRNA treatment, compared with controls. SMS inhibition by D609 or SMS siRNAs significantly decreased intracellular [ 14 C]-SM levels. We measured cellular lipid levels, including SM, ceramide, phosphatidylcholine, and diacylglycerol and found that SMS1 and SMS2 siRNA treatment caused a significant decrease of SM levels (20% and 11%, respectively), compared to control siRNA treatment; SMS1 but not SMS2 siRNA treatment caused a significant increase of ceramide levels (10%). There was a decreasing tendency for diacylglycerol levels after both SMS1 and SMS2 siRNA treatment, however, it was not statistical significant. As shown by lipid rafts isolation and lipid determination, SMS1 and SMS2 siRNA treatment led to a decrease of SM content in detergent-resistant lipid rafts on the cell membrane. Furthermore, SMS1 and SMS2 siRNA-treated cells had a stronger resistance than did control siRNA-treated cells to lysenin (a protein that causes cell lysis due to its affinity for plasma membrane SM). These results indicate that both SMS1 and SMS2 contribute to SM de novo synthesis and control SM levels in the cells and on the cell membrane including plasma membrane, implying an important relationship between SMS activity and cell functions.
Sphingomyelin synthase (SMS), the last enzyme in the sphingomyelin (SM) biosynthetic pathway, uses ceramide and phosphatidylcholine as substrates to produce SM and diacylglycerol (DAG). To evaluate the role of SMS in apoptosis, we generated Chinese hamster ovary cells that stably express human SMS1 or SMS2. We found that SMS1 or SMS2 overexpression results in a significant increase in cellular levels of SM (24% or 20%) and DAG (35% or 31%), respectively, compared with controls. Cells overexpressing SMS1 or SMS2 were more likely to undergo lysis mediated by lysenin (a protein that causes lysis through its affinity with SM-rich microdomains in the plasma membrane) than were controls, indicating SM enrichment of the plasma membrane. SMS1 and SMS2 overexpression also led to higher retention of DiIC16 fluorescence compared with wild-type cells, indicating an increased number of detergentinsoluble microdomains and significantly increased tumor necrosis factor-a-mediated apoptosis. To further evaluate the relationship between SMS activity and cell apoptosis, we used SMS1 and SMS2 small interfering RNA (siRNA) to knock down their mRNA in THP-1-derived macrophages. We found that SMS1 or SMS2 siRNA significantly reduces intracellular SM (by 20% or 23%), plasma membrane SM (as indicated by the rate of lysenin-mediated cell lysis), and DAG levels (24% or 20%), respectively, while significantly reducing lipopolysaccharide-mediated apoptosis compared with controls. These results indicate that SMS1 and SMS2 are key factors in the control of SM and DAG levels within the cell and thus influence apoptosis.-Ding, T., Z. Li, T. Hailemariam, S. Mukherjee, F. R. Maxfield, M-P. Wu, and X-C. Jiang. SMS overexpression and knockdown: impact on cellular sphingomyelin and diacylglycerol metabolism, and cell apoptosis. J. Lipid Res. 2008. 49: 376-385.
Background-It has been proposed that plasma sphingomyelin (SM) plays a very important role in plasma lipoprotein metabolism and atherosclerosis. Sphingomyelin synthase (SMS) is the last enzyme for SM de novo biosynthesis. Two SMS genes, SMS1 and SMS2, have been cloned and characterized. Methods and Results-To evaluate the in vivo role of SMS2 in SM metabolism, we prepared SMS2 knockout (KO) and SMS2 liver-specific transgenic (LTg) mice and studied their plasma SM and lipoprotein metabolism. On a chow diet, SMS2 KO mice showed a significant decrease in plasma SM levels (25%, PϽ0.05), but no significant changes in total cholesterol, total phospholipids, or triglyceride, compared with wild-type (WT) littermates. On a high-fat diet, SMS2 KO mice showed a decrease in plasma SM levels (28%, PϽ0.01), whereas SMS2LTg mice showed a significant increase in those levels (29%, PϽ0.05), but no significant changes in other lipids, compared with WT littermates. Atherogenic lipoproteins from SMS2LTg mice displayed a significantly stronger tendency toward aggregation after mammalian sphingomyelinase treatment, compared with controls. Moreover, SMS2 deficiency significantly increased plasma apoE levels (2.0-fold, PϽ0.001), whereas liver-specific SMS2 overexpression significantly decreased those levels (1.8-fold, PϽ0.01). Finally, SMS2 KO mouse plasma promoted cholesterol efflux from macrophages, whereas SMS2LTg mouse plasma prevented it. Conclusions-We therefore believe that regulation of liver SMS2 activity could become a promising treatment for atherosclerosis. Key Words: SMS2 knockout mice Ⅲ SMS2 liver-specific transgenic mice Ⅲ plasma sphingomyelin Ⅲ plasma lipoproteins Ⅲ plasma cholesterol S phingomyelin (SM), which is the second most abundant phospholipid in mammalian plasma, appears in all major lipoproteins, where it is part of the monolayer of polar lipids and cholesterol that surrounds a core of neutral lipids. Up to 18% of total plasma phospholipid exists as SM, 1 with the ratio of phosphatidylcholine (PC)/SM varying widely among lipoprotein subclasses. 2 Atherogenic lipoproteins such as VLDL remnants accumulating in cholesterol-fed rabbits tend to be SM-enriched. 3 The SM content of atherosclerotic lesions is higher than that of normal arterial tissue, and SM accumulation is disproportionate to that of PC. 4 However, the role of SM deposited or synthesized in atheromata still remains undefined.It has been suggested that subendothelial retention and aggregation of atherogenic lipoproteins play a very important role in atherogenesis. 5 SM-rich LDL retained in atherosclerotic lesions is acted on by an arterial wall sphingomyelinase, which appears to promote aggregation, initiating the early phase of atherosclerosis development. 4 We have found that plasma SM levels in apoE KO mice are 4-fold higher than in WT mice, 6 and this may partially explain the increased atherosclerosis found in these animals. 7 Our laboratory and others have also discovered that inhibition of SM biosynthesis significantly decreases plasma SM and ...
In mammals, adipose tissue stores energy in the form of fat. The ability to regulate fat storage is essential for the growth, development and reproduction of most animals, thus any abnormalities caused by excess fat accumulation can result in pathological conditions which are linked to several interrelated diseases, such as cardiovascular diseases, diabetes, and obesity. In recent years significant effort has been applied to understand basic mechanism of fat accumulation in mammalian system. Work in mouse has shown that the family of Krüppel-like factors (KLFs), a conserved and important class of transcription factors, regulates adipocyte differentiation in mammals. However, how fat storage is coordinated in response to positive and negative feedback signals is still poorly understood. To address mechanisms underlying fat storage we have studied two Caenorhabditis elegans KLFs and demonstrate that both worm klfs are key regulators of fat metabolism in C. elegans. These results provide the first in vivo evidence supporting essential regulatory roles for KLFs in fat metabolism in C. elegans and shed light on the human counterpart in disease-gene association. This finding allows us to pursue a more comprehensive approach to understand fat biology and provides an opportunity to learn about the cascade of events that regulate KLF activation, repression and interaction with other factors in exerting its biological function at an organismal level. In this review, we provide an overview of the most current information on the key regulatory components in fat biology, synthesize the diverse literature, pose new questions, and propose a new model organism for understanding fat biology using KLFs as the central theme.
Background Brugia malayi, like most human filarial parasite species, harbors an endosymbiotic bacterium of the genus Wolbachia. Elimination of the endosymbiont leads to sterilization of the adult female. Previous biochemical and genetic studies have established that communication with its endobacterium is essential for survival of the worm.Methodology/Principal findingsWe used electron microscopy to examine the effects of antibiotic treatment on Wolbachia cell structure. We have also used microarray and quantitative RT-PCR analyses to examine the regulation of the B. malayi transcripts altered in response to the anti-Wolbachia treatment. Microscopy of worms taken from animals treated with tetracycline for 14 and 21 days (14 d and 21 d) demonstrated substantial morphologic effects on the Wolbachia endobacterium by 14 d and complete degeneration of the endobacterial structures by 21 d. We observed upregulation of transcripts primarily encoding proteins involved in amino acid synthesis and protein translation, and downregulation of transcripts involved in cuticle biosynthesis after both 7 d and 14 d of treatment. In worms exposed to tetracycline in culture, substantial effects on endobacteria morphology were evident by day 3, and extensive death of the endobacteria was observed by day 5. In a detailed examination of the expression kinetics of selected signaling genes carried out on such cultured worms, a bimodal pattern of regulation was observed. The selected genes were upregulated during the early phase of antibiotic treatment and quickly downregulated in the following days. These same genes were upregulated once more at 6 days post-treatment.Conclusions/SignificanceUpregulation of protein translation and amino acid synthesis may indicate a generalized stress response induced in B. malayi due to a shortage of essential nutrients/factors that are otherwise supplied by Wolbachia. Downregulation of transcripts involved in cuticle biosynthesis perhaps reflects a disruption in the normal embryogenic program. This is confirmed by the expression pattern of transcripts that may be representative of the worms' response to Wolbachia in different tissues; the early peak potentially reflects the effect of bacteria death on the embryogenic program while the second peak may be a manifestation of the adult worm response to the affected bacteria within the hypodermis.
Translocations of the genes encoding the related transcription factors TFE3 and TFEB are almost exclusively associated with a rare juvenile subset of renal cell carcinoma and lead to overexpression of TFE3 or TFEB protein sequences. A better understanding of how deregulated TFE3 and TFEB contribute to the transformation process requires elucidating more of the normal cellular processes in which they participate. Here we identify TFE3 and TFEB as cell type-specific leukemia inhibitory factor-responsive activators of E-cadherin. Overexpression of TFE3 or TFEB in 3T3 cells activated endogenous and reporter E-cadherin expression. Conversely, endogenous TFE3 and/or TFEB was required for endogenous E-cadherin expression in primary mouse embryonic fibroblasts and human embryonic kidney cells. Chromatin precipitation analyses and E-cadherin promoter reporter gene assays revealed that Ecadherin induction by TFE3 or TFEB was primarily or exclusively direct and mitogen-activated protein kinasedependent in those cell types. In mouse embryonic fibroblasts, TFE3 and TFEB activation of E-cadherin was responsive to leukemia inhibitory factor. In 3T3 cells, TFE3 and TFEB expression also induced expression of Wilms' tumor-1, another E-cadherin activator. In contrast, E-cadherin expression in model mouse and canine renal epithelial cell lines was indifferent to inhibition of endogenous TFE3 and/or TFEB and was reduced by TFE3 or TFEB overexpression. These results reveal new cell type-specific activities of TFE3 and TFEB which may be affected by their mutation.Deregulated expression of the related transcription factors TFE3 and TFEB is associated with rare, juvenile forms of the malignancy renal cell carcinoma (RCC), and TFE3 mutation with alveolar soft part sarcoma (for review, see Ref. 1). The genetic lesions are translocations that lead to dramatic overexpression of TFE3 or TFEB protein sequences. Five different genetic loci have been identified as translocation partners for TFE3, which lead to the creation of a chimeric protein containing the translocation partner at the N terminus fused to the C-terminal portion of TFE3 which includes its DNA binding and multimerization domains (1). TFEB translocations result in promoter substitution and do not change the coding sequence (2, 3). Although there is evidence that TFE3 fusion partners contribute oncogenic properties to the fusion protein (4, 5), TFEB overexpression in RCC 1 and the ability of normal TFE3 to promote clonotypic growth of melanoma cells suggest that overexpression of the TFE3 protein sequence is a critical oncogenic force (2). Moreover, TFE3, and to a lesser extent, TFEB, have been implicated in several cytokine signaling pathways that control cell growth and differentiation, but the precise mechanisms by which their dysregulation contributes to renal oncogenesis are not clear.TFE3 and TFEB are closely related members of the Mi/TFE3 (MiT) transcription factor family that includes TFEC and the microphthalmia (mi) transcription factor Mitf (6). TFE3 or TFEB overexpress...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.