Airway Resistance Caused by Sphingomyelin Synthase 2 Insufficiency in Response to Cigarette Smoke.

Sphingomyelin synthase is responsible for the production of sphingomyelin (SGM), the second most abundant phospholipid in mammalian plasma, from ceramide, a major sphingolipid. Knowledge of the effects of cigarette smoke on SGM production is limited. In the present study, we examined the effect of chronic cigarette smoke on sphingomyelin synthase (SGMS) activity and evaluated how the deficiency of Sgms2, one of the two isoforms of mammalian SGMS, impacts pulmonary function. Sgms2-knockout and wild-type control mice were exposed to cigarette smoke for 6 months, and pulmonary function testing was performed. SGMS2-dependent signaling was investigated in these mice and in human monocyte-derived macrophages of nonsmokers and human bronchial epithelial (HBE) cells isolated from healthy nonsmokers and subjects with chronic obstructive pulmonary disease (COPD). Chronic cigarette smoke reduces SGMS activity and Sgms2 gene expression in mouse lungs. Sgms2-deficient mice exhibited enhanced airway and tissue resistance after chronic cigarette smoke exposure, but had similar degrees of emphysema, compared with smoke-exposed wild-type mice. Sgms2-/- mice had greater AKT phosphorylation, peribronchial collagen deposition, and protease activity in their lungs after smoke inhalation. Similarly, we identified reduced SGMS2 expression and enhanced phosphorylation of AKT and protease production in HBE cells isolated from subjects with COPD. Selective inhibition of AKT activity or overexpression of SGMS2 reduced the production of several matrix metalloproteinases in HBE cells and monocyte-derived macrophages. Our study demonstrates that smoke-regulated Sgms2 gene expression influences key COPD features in mice, including airway resistance, AKT signaling, and protease production.

PECULIARITIES OF THE STRUCTURE AND EXPRESSION OF HUMAN SPHINGOMYELIN SYNTHASE 1 GENE (SGMS1).

The article is a review of the results of the study of the structural and functional organization of the human sphingomyelin synthase 1 gene (SGMS1) in Human Molecular Genetics Department of Institute of Molecular Genetics RAS. SGMS1 gene encodes an essential enzyme which is involved in the synthesis of sphingomyelin and diacylglycerol from phosphatidylcholine and ceramide, wich determines its participation in the regulation of intracellular vesicular transport, cholesterol metabolism, cell proliferation, apoptosis and other significant processes. Our research has shown that the SGMS1 gene is located on the chromosome 10, has a size of 320 kb and contains more than 20 exons. A detailed study of the SGMS1 gene's structure allowed us to identify the variety of its transcripts. mRNA isoforms with different fragments of 5R untranslated region (5R UTR) and encoding the full length protein, as well as transcripts resulting from alternative combinations of exons and containing the coding region of the gene and 3R UTR have been discovered. We have found new transcripts among the products of SGMS1 gene ­ circular RNAs, which mostly contained sequences of multi-exon 5R UTR of the gene. They are conservative and predominantly expressed in the brain. Circular RNAs of SGMS1 gene had a large number of binding sites for a microRNA that may determine the functional significance of these molecules. The review describes the latest information about the structural and functional organization of the human gene SGMS1 as well as the features of its expression.

[Alternative promoters localised in SGMS1 gene introns take part in regulation of its expression in human tissues].

Sphingomyelin synthase 1 (SMS1) is an enzyme of vital importance which is responsible for the synthesis of sphingomyelin and diacylglycerol from phosphatidylcholine and ceramide in eukaryotic cells. Previously we have investigated in detail the structure of SGMS1 human gene and identified a lot of its transcripts. We revealed the isoforms of mRNA differing in the 5'-UTR and coding the full-length protein, and also the transcripts arising from alternative combination of the exons localized in the coding region of the gene and 3'-UTR. From the results of computer analysis it follows that the synthesis of transcripts differing in the 5'-UTR is enabled by the different promoters of SGMS1 gene. It has been found in the present work by the method of real-time PCR that the content of five alternative transcripts of this gene, differing in the 5'-UTR, is substantially dissimilar among human tissues. In all the investigated tissues those transcripts are presented most prominently whose synthesis takes place under the control of the distal promoter including exon 1. In lesser extent are presented the transcripts including 5'-end exons whose synthesis is enabled by the promoters localized in introns of this gene. The differential level of content of SGMS1 gene transcripts, differing in the 5'-UTR, indicates that the use of the alternative promoters is tissue-specific and apparently strictly regulated. The structural organization of 5'-UTR variants of SGMS1 transcripts, directed by alternative promoters, is substantially different; this can provide regulation of the gene functioning on post-transcriptional level.

In situ characterizing membrane lipid phenotype of breast cancer cells using mass spectrometry profiling.

Lipid composition in cell membrane is closely associated with cell characteristics. Here, matrix-assisted laser desorption/ionization- Fourier transform ion cyclotron resonance mass spectrometry was employed to in situ determine membrane components of human mammary epithelial cells (MCF-10 A) and six different breast cancer cell lines (i.e., BT-20, MCF-7, SK-BR-3, MDA-MB-231, MDA-MB-157, and MDA-MB-361) without any lipid extraction and separation. Partial least-square discriminant analysis indicated that changes in the levels of these membrane lipids were closely correlated with the types of breast cell lines. Elevated levels of polyunsaturated lipids in MCF-10 A cells relative to six breast cancer cells and in BT-20 cells relative to other breast cancer cell lines were detected. The Western blotting assays indicated that the expression of five lipogenesis-related enzymes (i.e., fatty acid synthase 1(FASN1), stearoyl-CoA desaturase 1 (SCD1), stearoyl-CoA desaturase 5 (SCD5), choline kinase α (CKα), and sphingomyelin synthase 1) was associated with the types of the breast cells, and that the SCD1 level in MCF-7 cells was significantly increased relative to other breast cell lines. Our findings suggest that elevated expression levels of FASN1, SCD1, SCD5, and CKα may closely correlated with enhanced levels of saturated and monounsaturated lipids in breast cancer cell lines.

Global regulator Anr represses PlcH phospholipase activity in Pseudomonas aeruginosa when oxygen is limiting.

Haemolytic phospholipase C (PlcH) is a potent virulence and colonization factor that is expressed at high levels by Pseudomonas aeruginosa within the mammalian host. The phosphorylcholine liberated from phosphatidylcholine and sphingomyelin by PlcH is further catabolized into molecules that both support growth and further induce plcH expression. We have shown previously that the catabolism of PlcH-released choline leads to increased activity of Anr, a global transcriptional regulator that promotes biofilm formation and virulence. Here, we demonstrated the presence of a negative feedback loop in which Anr repressed plcH transcription and we proposed that this regulation allowed for PlcH levels to be maintained in a way that promotes productive host-pathogen interactions. Evidence for Anr-mediated regulation of PlcH came from data showing that growth at low oxygen (1%) repressed PlcH abundance and plcH transcription in the WT, and that plcH transcription was enhanced in an Δanr mutant. The plcH promoter featured an Anr consensus sequence that was conserved across all P. aeruginosa genomes and mutation of conserved nucleotides within the Anr consensus sequence increased plcH expression under hypoxic conditions. The Anr-regulated transcription factor Dnr was not required for this effect. The loss of Anr was not sufficient to completely derepress plcH transcription as GbdR, a positive regulator of plcH, was required for expression. Overexpression of Anr was sufficient to repress plcH transcription even at 21 % oxygen. Anr repressed plcH expression and phospholipase C activity in a cell culture model for P. aeruginosa-epithelial cell interactions.

Moraxella catarrhalis expresses a cardiolipin synthase that impacts adherence to human epithelial cells.

The major phospholipid constituents of Moraxella catarrhalis membranes are phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin (CL). However, very little is known regarding the synthesis and function of these phospholipids in M. catarrhalis. In this study, we discovered that M. catarrhalis expresses a cardiolipin synthase (CLS), termed MclS, that is responsible for the synthesis of CL within the bacterium. The nucleotide sequence of mclS is highly conserved among M. catarrhalis isolates and is predicted to encode a protein with significant amino acid similarity to the recently characterized YmdC/ClsC protein of Escherichia coli. Isogenic mclS mutant strains were generated in M. catarrhalis isolates O35E, O12E, and McGHS1 and contained no observable levels of CL. Site-directed mutagenesis of a highly conserved HKD motif of MclS also resulted in a CL-deficient strain. Moraxella catarrhalis, which depends on adherence to epithelial cells for colonization of the human host, displays significantly reduced levels of adherence to HEp-2 and A549 cell lines in the mclS mutant strains compared to wild-type bacteria. The reduction in adherence appears to be attributed to the absence of CL. These findings mark the first instance in which a CLS has been related to a virulence-associated trait.

Detection of soluble co-factor dependent protein expression in vivo: application to the 4'-phosphopantetheinyl transferase PptT from Mycobacterium tuberculosis.

The need for early-on diagnostic tools to assess the folding and solubility of expressed protein constructs in vivo is of great interest when dealing with recalcitrant proteins. In this paper, we took advantage of the picomolar sensitivity of the bipartite GFP1-10/GFP11 system to investigate the solubility of the Mycobacterium tuberculosis 4'-phosphopantetheinyl transferase PptT, an enzyme essential for the viability of the tubercle bacillus. In vivo and in vitro complementation assays clearly showed the improved solubility of the full-length PptT compared to its N- and C-terminally truncated counterparts. However, initial attempts to purify the full-length enzyme overexpressed in Escherichia coli cells were hampered by aggregation issues overtime that caused the protein to precipitate within hours. The fact that the naturally occurring Coenzyme A and Mg(2+), essentials for PptT to carry out its function, could play a role in stabilizing the enzyme was confirmed using DSF experiments. In vitro activity assays were performed using the ACP substrate from the type I polyketide synthase PpsC from M. tuberculosis, a 2188 amino-acid enzyme that plays a major role in the virulence and pathogenicity of this microbial pathogen. We selected the most soluble and compact ACP fragment (2042-2188), identified by genetic selection of in-frame fragments from random library experiments, to monitor the transfer of the P-pant moiety from Coenzyme A onto a conserved serine residue of this ACP domain.

Self-enhancement of hepatitis C virus replication by promotion of specific sphingolipid biosynthesis.

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.

Sphingomyelin synthase overexpression increases cholesterol accumulation and decreases cholesterol secretion in liver cells.

BACKGROUND: Studies have shown that plasma high density lipoprotein cholesterol levels are negatively correlated with the development of atherosclerosis, whereas epidemiological studies have also shown that plasma sphingomyelin level is an independent risk factor for atherosclerosis. METHODS: To evaluate the relationship between cellular sphingomyelin level and cholesterol metabolism, we created two cell lines that overexpressed sphingomyelin synthase 1 or 2 (SMS1 or SMS2), using the Tet-off expression system. RESULTS: We found that SMS1 or SMS2 overexpression in Huh7 cells, a human hepatoma cell line, significantly increased the levels of intracellular sphingomyelin, cholesterol, and apolipoprotein A-I and decreased levels of apolipoprotein A-I and cholesterol in the cell culture medium, implying a defect in both processes. CONCLUSIONS: Our findings indicate that the manipulation of sphingomyelin synthase activity could influence the metabolism of sphingomyelin, cholesterol and apolipoprotein A-I.

Adenovirus-mediated sphingomyelin synthase 2 increases atherosclerotic lesions in ApoE KO mice.

BACKGROUND: Sphingomyelin synthase 2 (SMS2) contributes to de novo sphingomyelin (SM) biosynthesis. Its activity is related to SM levels in the plasma and the cell membrane. In this study, we investigated the possibility of a direct relationship between SMS and atherosclerosis. METHODS: The Adenovirus containing SMS2 gene was given into 10-week ApoE KO C57BL/6J mice by femoral intravenous injection. In the control group, the Adenovirus containing GFP was given. To confirm this model, we took both mRNA level examination (RT-PCR) and protein level examination (SMS activity assay). RESULT: We generated recombinant adenovirus vectors containing either human SMS2 cDNA (AdV-SMS2) or GFP cDNA (AdV-GFP). On day six after intravenous infusion of 2 × 10(11) particle numbers into ten-week-old apoE KO mice, AdV-SMS2 treatment significantly increased liver SMS2 mRNA levels and SMS activity (by 2.7-fold, 2.3-fold, p < 0.001, respectively), compared to AdV-GFP treated mice. Moreover, plasma total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), and sphingomyelin (SM) levels were significantly increased by 39% (p < 0.05), 42% (p < 0.05), 68% (p < 0.001), and 45% (p < 0.05), respectively. Plasma high-density lipoprotein cholesterol (HDL-C), phosphatidylcholine (PC), and PC/SM ratio were decreased by 42% (p < 0.05), 18% (p < 0.05), and 45% (p < 0.05), respectively. On day 30, the atherosclerotic lesions on the aortic arch of AdV-SMS2 treated mice were increased, and the lesion areas on the whole aorta and in the aortic root were significantly increased (p < 0.001). Furthermore, the collagen content in the aorta root was significantly decreased (p < 0.01). CONCLUSIONS: Our results present direct morphological evidence for the pro-atherogenic capabilities of SMS2. SMS2 could be a potential target for treating atherosclerosis.

Cardiolipin synthase-1 mRNA expression does not correlate with endogenous cardiolipin synthase enzyme activity in vitro and in vivo in mammalian lipopolysaccharide models of inflammation.

We examined if lipopolysaccharide (LPS) treatment of mice affected cardiolipin (CL) synthesis. Mice were injected i.p. with LPS, the liver harvested, and CL synthase (CLS) enzyme activity and its mRNA expression examined. Treatment of mice with LPS resulted in a 55% decrease (p < 0.01) in mRNA expression of murine CLS compared to controls, but CLS enzyme activity was unaltered. The pool size of liver CL and other phospholipids were unaltered by LPS treatment. A similar effect was observed in murine epidermal fat pad and in vitro in RAW mouse macrophages and in human HepG2 cells. LPS treatment of HepG2 cells transiently expressing a histidine-tagged human cardiolipin synthase-1 (hCLS1) reduced hCLS1 mRNA and newly synthesized CLS activity indicating that LPS inhibits production of newly synthesized hCLS1 via reduction in hCLS1 mRNA. The results clearly indicate that CLS mRNA levels cannot be correlated with CLS enzyme activity nor CL content in the LPS model of inflammation.

Genetic evidence for functional interaction of the Escherichia coli signal recognition particle receptor with acidic lipids in vivo.

The mechanism underlying the interaction of the Escherichia coli signal recognition particle receptor FtsY with the cytoplasmic membrane has been studied in detail. Recently, we proposed that FtsY requires functional interaction with inner membrane lipids at a late stage of the signal recognition particle pathway. In addition, an essential lipid-binding α-helix was identified in FtsY of various origins. Theoretical considerations and in vitro studies have suggested that it interacts with acidic lipids, but this notion is not yet fully supported by in vivo experimental evidence. Here, we present an unbiased genetic clue, obtained by serendipity, supporting the involvement of acidic lipids. Utilizing a dominant negative mutant of FtsY (termed NG), which is defective in its functional interaction with lipids, we screened for E. coli genes that suppress the negative dominant phenotype. In addition to several unrelated phenotype-suppressor genes, we identified pgsA, which encodes the enzyme phosphatidylglycerophosphate synthase (PgsA). PgsA is an integral membrane protein that catalyzes the committed step to acidic phospholipid synthesis, and we show that its overexpression increases the contents of cardiolipin and phosphatidylglycerol. Remarkably, expression of PgsA also stabilizes NG and restores its biological function. Collectively, our results strongly support the notion that FtsY functionally interacts with acidic lipids.

The Escherichia coli highly expressed entD gene complements the pfaE deficiency in a pfa gene clone responsible for the biosynthesis of long-chain n-3 polyunsaturated fatty acids.

The Escherichia coli entD gene, which encodes an Sfp-type phosphopantetheinyl transferase (PPTase) that is involved in the biosynthesis of siderophore, is available as a high-expression ASKA clone (pCA24N::entD) constructed from the E. coli K-12 strain AG1. In E. coli DH5alpha, pCA24N::entD complemented a pfaE-deficient clone that comprised pfaA, pfaB, pfaC and pfaD, which are four of the five pfa genes that are responsible for the biosynthesis of eicosapentaenoic acid derived from Shewanella pneumatophori SCRC-2738. Sfp-type PPTases are classified into the EntD and PfaE groups, based on differences between their N-terminal-domain structures. Here, we showed that all Sfp-type PPTases may have the potential to promote the biosynthesis of long-chain n-3 polyunsaturated fatty acids.

Acyl carrier protein-specific 4'-phosphopantetheinyl transferase activates 10-formyltetrahydrofolate dehydrogenase.

4'-Phosphopantetheinyl transferases (PPTs) catalyze the transfer of 4'-phosphopantetheine (4-PP) from coenzyme A to a conserved serine residue of their protein substrates. In humans, the number of pathways utilizing the 4-PP post-translational modification is limited and may only require a single broad specificity PPT for all phosphopantetheinylation reactions. Recently, we have shown that one of the enzymes of folate metabolism, 10-formyltetrahydrofolate dehydrogenase (FDH), requires a 4-PP prosthetic group for catalysis. This moiety acts as a swinging arm to couple the activities of the two catalytic domains of FDH and allows the conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. In the current study, we demonstrate that the broad specificity human PPT converts apo-FDH to holoenzyme and thus activates FDH catalysis. Silencing PPT by small interfering RNA in A549 cells prevents FDH modification, indicating the lack of alternative enzymes capable of accomplishing this transferase reaction. Interestingly, PPT-silenced cells demonstrate significantly reduced proliferation and undergo strong G(1) arrest, suggesting that the enzymatic function of PPT is essential and nonredundant. Our study identifies human PPT as the FDH-modifying enzyme and supports the hypothesis that mammals utilize a single enzyme for all phosphopantetheinylation reactions.

Cardiolipin synthesis is required to support human cholesterol biosynthesis from palmitate upon serum removal in Hela cells.

We examined whether cardiolipin (CL) synthesis was required to support cholesterol (CH) production from palmitate in Hela cells. Knockdown of human cardiolipin synthase-1 (hCLS1) in Hela cells has been shown to reduce CL synthesis. Therefore Hela cells stably expressing shRNA for hCLS1 and mock control cells were incubated for 16 h with [14C(U)]palmitate bound to albumin (1:1 molar ratio) in the absence or presence of serum. Knockdown of hCLS1 in Hela cells resulted in a reduction in [14C(U)]palmitate incorporation into CL and CH. This reduction in [14C(U)]palmitate incorporation into CH was most pronounced during incubation under serum-free conditions. The reduction in [14C(U)]palmitate incorporation into CH was not due to alterations in total uptake of [14C(U)]palmitate into cells or altered palmitate metabolism, since [14C(U)]palmitate incorporation into phosphatidylcholine, the major [14C(U)]palmitate-containing lipid, and its immediate precursor, 1,2-diacyl-sn-glycerol, were unaffected by hCLS1 knockdown. In addition, knockdown of hCLS1 did not affect CH pool size, indicating that CH catabolism was unaltered. Hydroxymethylglutaryl coenzyme A reductase enzyme activity and its mRNA expression were reduced by knockdown of hCLS1 and this was most pronounced in Hela cells cultured under serum-free conditions. These data indicate that CL synthesis is required to support human de novo CH biosynthesis under conditions of increased demand for CH.

Regulated transcription of the Saccharomyces cerevisiae phosphatidylinositol biosynthetic gene, PIS1, yields pleiotropic effects on phospholipid synthesis.

Phosphatidylinositol is an important membrane lipid in Saccharomyces cerevisiae and other eukaryotes. Phosphatidylinositol and its metabolites (phosphoinositides, inositol polyphosphates, etc.) affect many cellular processes with implications in human diseases. Phosphatidylinositol synthesis in S. cerevisiae requires the essential PIS1 gene. Recent studies reveal that PIS1 expression is regulated at the level of transcription in response to carbon source, oxygen, and zinc. However, the consequence of this regulation on phosphatidylinositol levels and functions has not been thoroughly studied. To investigate this, we created a strain with a galactose-inducible GAL1-PIS1 gene. In this strain, the amount of phosphatidylinositol correlated with PIS1 expression but did not exceed c. 25% of the total phospholipid composition. Interestingly, we found that 4% phosphatidylinositol was sufficient for cell growth. We also found that reduced PIS1 expression yielded derepression of two phospholipid biosynthetic genes (INO1 and CHO1) and the INO2 regulatory gene. Consistent with this derepression, reduced PIS1 expression also yielded an overproduction of inositol (Opi(-)) phenotype. The effect on transcription of the INO1, CHO1, and INO2 genes is consistent with the accepted model that phosphatidic acid (PA) is the signal for regulation of these genes because decreased phosphatidylinositol synthesis would affect PA levels.

[Overexpression and purification of Escherichia coli holo-acyl carrier protein and synthesis of acyl carrier protein].

OBJECTIVE: To investigate the mechanism of fatty acids, lipid A and N-acylhomoserine lactones biosynthesis of bacteria by using high quality Escherichia coli holo-ACP and varied length chain acyl-ACPs as substrates. METHODS AND RESULTS: Using PCR technique we amplified the acpP and acpS gene fragments from genomic DNA of E. coli strain MG1655. Ligating these gene fragments with plasmids pBAD24 or pET28b respectively, we obtained 3 expression plasmids of acyl carrier protein: pBAD-ACP, pET-ACP and pET-ACP-ACPS, and one expression plasmid of holo-acyl carrier protein synthase: pBAD-ACPS. Then we constructed 3 acyl carrier protein producer strains: DH5alpha/pBAD-ACP, BL21 (DE3)/pET-ACP and BL21(DE3)/pET-ACP-ACPS by transforming E. coli strains DH5alpha or BL21(DE3)with pBAD-ACP, pET-ACP or pET-ACP-ACPS, respectively. Although these 3 strains could produce more acyl carrier protein under induction than strain DK574, which was used to purify holo-acyl carrier protein in general, the yield of holo-acyl carrier protein of these strains was still lower. In order to increase the yield of holo-acyl carrier protein in these strains, we introduced pBAD-ACPS into these strains. The assay of expressions of new strains was shown the that strain DH5alpha harbored pBAD-ACP and pBAD-ACPS double plasmids produced more holo-acyl carrier protein than strain DK574, and the purity of holo-acyl carrier protein was also increased (up to 99%). Then we purified high quality holo-acyl carrier protein from the culture of the strain DH5alpha harbored pBAD-ACP and pBAD-ACPS by using UNOsphere Q anion-exchange chromatography. Utilizing holo-acyl carrier protein and long chain fatty acids as substrates and under Vibrio harveyi acyl-acyl carrier protein synthetase catalyzing, we synthesized several different acyl-acyl carrier proteins. CONCLUSION: From this study we obtained a high holo-ACP producer strain and demonstrated that co-expressing acpP with acpS, E. coli strains could produce more holo-ACP.

Regulation of lipid metabolism by sphingolipids.

Sphingolipids, together with phospholipids and cholesterol are key components of membrane lipid bilayers, contribute to specialized membrane domains called rafts and function as signaling molecules. Sphingolipids have been recognized to exert a distinct role in the post-transcriptional regulation of the sterol-regulatory element binding proteins (SREBPs), key transcription factors of lipid synthesis. Sphingolipid synthesis is an obligate activator of SREBP. Inhibition of sphingolipid synthesis decreases SREBP on a post-transcriptional level. With the exception of enzymes that synthesize sphingolipids, SREBPs regulate the transcription of key enzymes that synthesize cholesterol, phospholipids and fatty acids. This observation suggests an exclusive role for sphingolipids in the regulation of lipid metabolism. Although exact mechanisms how sphingolipids regulate lipid metabolism are currently not known, this relationship has important implications with regard to cellular lipid homeostasis, composition of lipoproteins and development of atherosclerosis.

SMS overexpression and knockdown: impact on cellular sphingomyelin and diacylglycerol metabolism, and cell apoptosis.

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 detergent-insoluble microdomains and significantly increased tumor necrosis factor-alpha-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.

Resistance to alkyl-lysophospholipid-induced apoptosis due to downregulated sphingomyelin synthase 1 expression with consequent sphingomyelin- and cholesterol-deficiency in lipid rafts.

The ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; Et-18-OCH3) induces apoptosis in S49 mouse lymphoma cells. To this end, ALP is internalized by lipid raft-dependent endocytosis and inhibits phosphatidylcholine synthesis. A variant cell-line, S49AR, which is resistant to ALP, was shown previously to be unable to internalize ALP via this lipid raft pathway. The reason for this uptake failure is not understood. In the present study, we show that S49AR cells are unable to synthesize SM (sphingomyelin) due to down-regulated SMS1 (SM synthase 1) expression. In parental S49 cells, resistance to ALP could be mimicked by small interfering RNA-induced SMS1 suppression, resulting in SM deficiency and blockage of raft-dependent internalization of ALP and induction of apoptosis. Similar results were obtained by treatment of the cells with myriocin/ISP-1, an inhibitor of general sphingolipid synthesis, or with U18666A, a cholesterol homoeostasis perturbing agent. U18666A is known to inhibit Niemann-Pick C1 protein-dependent vesicular transport of cholesterol from endosomal compartments to the trans-Golgi network and the plasma membrane. U18666A reduced cholesterol partitioning in detergent-resistant lipid rafts and inhibited SM synthesis in S49 cells, causing ALP resistance similar to that observed in S49AR cells. The results are explained by the strong physical interaction between (newly synthesized) SM and available cholesterol at the Golgi, where they facilitate lipid raft formation. We propose that ALP internalization by lipid-raft-dependent endocytosis represents the retrograde route of a constitutive SMS1- and lipid-raft-dependent membrane vesicular recycling process.