The essential nature of sphingolipids in plants as revealed by the functional identification and characterization of the Arabidopsis LCB1 subunit of serine palmitoyltransferase

Serine palmitoyltransferase (SPT) catalyzes the first step of sphingolipid biosynthesis. In yeast and mammalian cells, SPT is a heterodimer that consists of LCB1 and LCB2 subunits, which together form the active site of this enzyme. We show that the predicted gene for Arabidopsis thaliana LCB1 encodes a genuine subunit of SPT that rescues the sphingolipid long-chain base auxotrophy of Saccharomyces cerevisiae SPT mutants when coexpressed with Arabidopsis LCB2. In addition, homozygous T-DNA insertion mutants for At LCB1 were not recoverable, but viability was restored by complementation with the wild-type At LCB1 gene. Furthermore, partial RNA interference (RNAi) suppression of At LCB1 expression was accompanied by a marked reduction in plant size that resulted primarily from reduced cell expansion. Sphingolipid content on a weight basis was not changed significantly in the RNAi suppression plants, suggesting that plants compensate for the downregulation of sphingolipid synthesis by reduced growth. At LCB1 RNAi suppression plants also displayed altered leaf morphology and increases in relative amounts of saturated sphingolipid long-chain bases. These results demonstrate that plant SPT is a heteromeric enzyme and that sphingolipids are essential components of plant cells and contribute to growth and development.     

Localization,topology, and function of the LCB1 subunit of serine palmitoyltransferase in mammalian cells

Serine palmitoyltransferase (SPT), the enzyme catalyzing the initial step in the biosynthesis of sphingolipids, comprises two different subunits, LCB1 and LCB2. LCB1 has a single highly hydrophobic domain near the N terminus. Chinese hamster ovary cell mutant LY-B cells are defective in SPT activity because of the lack of expression of an endogenous LCB1 subunit. Stable expression of LCB1 having an epitope tag at either the N or C terminus restored SPT activity of LY-B cells, suggesting that the epitope tag did not affect the localization or topology of LCB1. Indirect immunostaining showed that the N- and C-terminal epitopes are oriented toward the lumenal and cytosol side, respectively, at the endoplasmic reticulum. Interestingly, there was far less LCB2 in LY-B cells than in wild-type cells, and the amount of LCB2 in LY-B cells was restored to the wild-type level by transfection with LCB1 cDNA. In addition, overproduction of the LCB2 subunit required co-overproduction of the LCB1 subunit. These results indicated that the LCB1 subunit is most likely an integral protein having a single transmembrane domain with a lumenal orientation of its N terminus in the endoplasmic reticulum and that the LCB1 subunit is indispensable for the maintenance of the LCB2 subunit in mammalian cells.     

Loss-of-function mutations and inducible RNAi suppression of Arabidopsis LCB2 genes reveal the critical role of sphingolipids in gametophytic and sporophytic cell viability

Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis, and downregulation of this enzyme provides a means for exploring sphingolipid function in cells. We have previously demonstrated that Arabidopsis SPT requires LCB1 and LCB2 subunits for activity, as is the case in other eukaryotes. In this study, we show that Arabidopsis has two genes ( AtLCB2a and AtLCB2b ) that encode functional isoforms of the LCB2 subunit. No alterations in sphingolipid content or growth were observed in T-DNA mutants for either gene, but homozygous double mutants were not recoverable, suggesting that these genes are functionally redundant. Reciprocal crosses conducted with Atlcb2a and Atlcb2b mutants indicated that lethality is associated primarily with the inability to transmit the lcb2 null genotype through the haploid pollen. Consistent with this, approximately 50% of the pollen obtained from plants homozygous for a mutation in one gene and heterozygous for a mutation in the second gene arrested during transition from uni-nucleate microspore to bicellular pollen. Ultrastructural analyses revealed that these pollen grains contained aberrant endomembranes and lacked an intine layer. To examine sphingolipid function in sporophytic cells, Arabidopsis lines were generated that allowed inducible RNAi silencing of AtLCB2b in an Atlcb2a mutant background. Studies conducted with these lines demonstrated that sphingolipids are essential throughout plant development, and that lethality resulting from LCB2 silencing in seedlings could be partially rescued by supplying exogenous long-chain bases. Overall, these studies provide insights into the genetic and biochemical properties of SPT and sphingolipid function in Arabidopsis.     

The ORMs interact with transmembrane domain 1 of Lcb1 and regulate serine palmitoyltransferase oligomerization,activity and localization

Serine palmitoyltransferase (SPT), an endoplasmic reticulum-localized membrane enzymecomposed of acatalytic LCB1/LCB2 heterodimer and a small activating subunit (Tsc3 in yeast; ssSPTs in mammals), is negatively regulated by the evolutionarily conserved family of proteins known as the ORMs. In yeast, SPT, the ORMs, and the PI4P phosphatase Sac1, copurify in the “SPOTs” complex. However, neither the mechanism of ORM inhibition of SPT nor details of the interactions of the ORMs and Sac1 with SPT are known. Here we report that the first transmembrane domain (TMD1) of Lcb1 is required for ORM binding to SPT. Loss of binding is not due to altered membrane topology of Lcb1 since replacing TMD1 with a heterologous TMD restores membrane topology but not ORM binding. TMD1 deletion also eliminates ORM-dependent formation of SPT oligomers as assessed by co-immunoprecipitation assays and in vivo imaging. Expression of ORMs lacking derepressive phosphorylation sites results in constitutive SPT oligomerization, while phosphomimetic ORMs fail to induce oligomerization under any conditions. Significantly, when LCB1–RFP and LCB1ΔTMD1–GFP were coexpressed, more LCB1ΔTMD1–GFP was in the peripheral ER, suggesting ORM regulation is partially accomplished by SPT redistribution. Tsc3 deletion does not abolish ORM inhibition of SPT, indicating the ORMs do not simply prevent activation by Tsc3. Binding of Sac1 to SPT requires Tsc3, but not the ORMs, and Sac1 does not influence ORM-mediated oligomerization of SPT. Finally, yeast mutants lacking ORM regulation of SPT require the LCB-P lyase Dpl1 to maintain long-chain bases at sublethal levels.     

Purification of the serine palmitoyltransferase complex responsible for sphingoid base synthesis by using affinity peptide chromatography techniques

Serine palmitoyltransferase (SPT), a membrane-bound enzyme of the endoplasmic reticulum, catalyzes the condensation of palmitoyl coenzyme A (CoA) and L-serine to produce 3-ketodihydrosphingosine. This enzyme contains at least two different subunits, named the LCB1 and LCB2 proteins. In the present study, we expressed a FLAG- and His(6) peptide-tagged version of the hamster LCB1 protein in a Chinese hamster ovary cell mutant strain lacking the endogenous LCB1 subunit and purified SPT from the cells near to homogeneity by affinity peptide chromatography. The endogenous LCB2 protein was co-purified with the tagged LCB1 protein in purification of SPT. In various aspects, including optimum pH, acyl-CoA specificity, and sphingofungin sensitivity, the activity of purified SPT was consistent with the activity detected in lysates of wild-type Chinese hamster ovary cells. The optimum concentration of palmitoyl-CoA for 3-ketodihydrosphingosine formation by purified SPT was approximately 25 microM, and the apparent K(m) of L-serine was 0.28 mM. Competition analysis of the SPT reaction with various serine analogs showed that all of the amino, carboxyl, and hydroxyl groups of L-serine were responsible for the substrate recognition of the enzyme. SDS-polyacrylamide gel electrophoretic analysis of purified SPT, together with immunoprecipitation analysis of metabolically labeled LCB proteins, strongly suggested that the SPT enzyme consisted of the LCB1 and LCB2 proteins with a stoichiometry of 1:1.     

A water-soluble homodimeric serine palmitoyltransferase from Sphingomonas paucimobilis EY2395T strain. Purification, characterization, cloning, and overproduction

Serine palmitoyltransferase (SPT, EC ) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of l-serine and palmitoyl-coenzyme A to 3-ketodihydrosphingosine. We found that the Gram-negative obligatory aerobic bacteria Sphingomonas paucimobilis EY2395(T) have significant SPT activity and purified SPT to homogeneity. This enzyme is a water-soluble homodimeric protein unlike eukaryotic enzymes, known as heterodimers composed of tightly membrane-bound subunits, named LCB1 and LCB2. The purified SPT shows an absorption spectrum characteristic of a pyridoxal 5'-phosphate-dependent enzyme. The substrate specificity of the Sphingomonas SPT is less strict than the SPT complex from Chinese hamster ovary cells. We isolated the SPT gene encoding 420 amino acid residues (M(r) 45,041) and succeeded in overproducing the SPT protein in Escherichia coli, in which the product amounted to about 10-20% of the total protein of the cell extract. Sphingomonas SPT shows about 30% homology with the enzymes of the alpha-oxamine synthase family, and amino acid residues supposed to be involved in catalysis are conserved. The recombinant SPT was catalytically and spectrophotometrically indistinguishable from the native enzyme. This is the first successful overproduction of an active enzyme in the sphingolipid biosynthetic pathway. Sphingomonas SPT is a prototype of the eukaryotic enzyme and would be a useful model to elucidate the reaction mechanism of SPT.     

Characterization of an Arabidopsis cDNA encoding a subunit of serine palmitoyltransferase, the initial enzyme in sphingolipid biosynthesis.

Serine palmitoyltransferase (SPT; EC 2.3.1.50) catalyzes the condensation of serine with palmitoyl-CoA to form 3-ketosphinganine in the first step of de novo sphingolipid biosynthesis. In this study, we describe the cloning and functional characterization of a cDNA from Arabidopsis thaliana encoding the LCB2 subunit of SPT. The Arabidopsis LCB2 (AtLCB2) cDNA contains an open reading frame of 1,467 nucleotides, encoding 489 amino acids. The predicted polypeptide contains three transmembrane helices and a highly conserved motif involved in pyridoxal phosphate binding. Expression of this open reading frame in the Saccharomyces cerevisiae mutant strains defective in SPT activity resulted in the expression of a significant level of sphinganine, suggesting that AtLCB2 cDNA encodes SPT. Southern blot analysis and inspection of the complete Arabidopsis genome sequence database suggest that there is a second LCB2-like gene in Arabidopsis. Expression of a green fluorescent protein (GFP) fusion product in suspension-cultured tobacco BY-2 cells showed that AtLCB2 is localized to the endoplasmic reticulum. AtLCB2 cDNA may be used to study how sphingolipid synthesis is regulated in higher plants.     

De novo ceramide accumulation due to inhibition of its conversion to complex sphingolipids in apoptotic photosensitized cells

The oxidative stress induced by photodynamic therapy (PDT) with the photosensitizer phthalocyanine 4 is accompanied by increases in ceramide mass. To assess the regulation of de novo sphingolipid metabolism during PDT-induced apoptosis, Jurkat human T lymphoma and Chinese hamster ovary cells were labeled with [14C]serine, a substrate of serine palmitoyltransferase (SPT), the enzyme catalyzing the initial step in the sphingolipid biosynthesis. A substantial elevation in [14C]ceramide with a concomitant decrease in [14C]sphingomyelin was detected. The labeling of [14C]ceramide was completely abrogated by the SPT inhibitor ISP-1. In addition, ISP-1 partly suppressed PDT-induced apoptosis. Pulse-chase experiments showed that the contribution of sphingomyelin degradation to PDT-initiated increase in de novo ceramide was absent or minor. PDT had no effect on either mRNA amounts of the SPT subunits LCB1 and LCB2, LCB1 protein expression, or SPT activity in Jurkat cells. Moreover in Chinese hamster ovary cells LCB1 protein underwent substantial photodestruction, and SPT activity was profoundly inhibited after treatment. We next examined whether PDT affects conversion of ceramide to complex sphingolipids. Sphingomyelin synthase, as well as glucosylceramide synthase, was inactivated by PDT in both cell lines in a dose-dependent manner. These results are the first to show that in the absence of SPT up-regulation PDT induces accumulation of de novo ceramide by inhibiting its conversion to complex sphingolipids.     

The topology of the Lcb1p subunit of yeast serine palmitoyltransferase

The structural organization and topology of the Lcb1p subunit of yeast and mammalian serine palmitoyltransferases (SPT) were investigated. In the yeast protein, three membrane-spanning domains were identified by insertion of glycosylation and factor Xa cleavage sites at various positions. The first domain of the yeast protein, located between residues 50 and 84, was not required for the stability, membrane association, interaction with Lcb2p, or enzymatic activity. Deletion of the comparable domain of the mammalian protein SPTLC1 also had little effect on its function, demonstrating that this region is not required for membrane localization or heterodimerization with SPTLC2. The second and third membrane-spanning domains of yeast Lcb1p, located between residues 342 and 371 and residues 425 and 457, respectively, create a luminal loop of approximately 60 residues. In contrast to the first membrane-spanning domain, the second and third membrane-spanning domains were both required for Lcb1p stability. In addition, mutations in the luminal loop destabilized the SPT heterodimer indicating that this region of the protein is important for SPT structure and function. Mutations in the extreme carboxyl-terminal region of Lcb1p also disrupted heterodimer formation. Taken together, these data suggest that in contrast to other members of the alpha-oxoamine synthases that are soluble homodimers, the Lcb1p and Lcb2p subunits of the SPT heterodimer may interact in the cytosol, as well as within the membrane and/or the lumen of the endoplasmic reticulum.     

A Disease-causing Mutation in the Active Site of Serine Palmitoyltransferase Causes Catalytic Promiscuity

The autosomal dominant peripheral sensory neuropathy HSAN1 results from mutations in the LCB1 subunit of serine palmitoyltransferase (SPT). Serum from patients and transgenic mice expressing a disease-causing mutation (C133W) contain elevated levels of 1-deoxysphinganine (1-deoxySa), which presumably arise from inappropriate condensation of alanine with palmitoyl-CoA. Mutant heterodimeric SPT is catalytically inactive. However, mutant heterotrimeric SPT has ∼10–20% of wild-type activity and supports growth of yeast cells lacking endogenous SPT. In addition, long chain base profiling revealed the synthesis of significantly more 1-deoxySa in yeast and mammalian cells expressing the heterotrimeric mutant enzyme than in cells expressing wild-type enzyme. Wild-type and mutant enzymes had similar affinities for serine. Surprisingly, the enzymes also had similar affinities for alanine, indicating that the major affect of the C133W mutation is to enhance activation of alanine for condensation with the acyl-CoA substrate. In vivo synthesis of 1-deoxySa by the mutant enzyme was proportional to the ratio of alanine to serine in the growth media, suggesting that this ratio can be used to modulate the relative synthesis of sphinganine and 1-deoxySa. By expressing SPT as a single-chain fusion protein to ensure stoichiometric expression of all three subunits, we showed that GADD153, a marker for endoplasmic reticulum stress, was significantly elevated in cells expressing mutant heterotrimers. GADD153 was also elevated in cells treated with 1-deoxySa. Taken together, these data indicate that the HSAN1 mutations perturb the active site of SPT resulting in a gain of function that is responsible for the HSAN1 phenotype.     

Characterization of enzymatic synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae: mutant strains exhibiting long-chain-base auxotrophy are deficient in serine palmitoyltransferase activity.

We have begun a biochemical-genetic analysis of the synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae and found evidence for the occurrence of serine palmitoyltransferase (SPT) and 3-ketosphinganine reductase, enzymes that catalyze the initial steps of the pathway in other organisms. SPT activity was demonstrated in vitro with crude membrane preparations from S. cerevisiae as judged by the formation of radiolabeled 3-ketosphinganine from the condensation of palmitoyl-coenzyme A (CoA) with radiolabeled serine. Shorter (C12 and C14) and longer (C18) acyl-CoAs sustain significant SPT activity, a result consistent with the finding of both C18 and C20 long-chain bases in the organism. Three products of the long-chain-base synthetic pathway, 3-ketosphinganine, erythrosphinganine, and phytosphingosine, neither directly inhibited the reaction in vitro nor affected the specific activity of the enzyme when these bases were included in the culture medium of wild-type cells. Thus, no evidence for either feedback inhibition or repression of enzyme synthesis could be found with these putative effectors. Mutant strains of S. cerevisiae that require a sphingolipid long-chain base for growth fall into two genetic complementation groups, LCB1 and LCB2. Membrane preparations from both lcb1 and lcb2 mutant strains exhibited negligible SPT activity when tested in vitro. Step 2 of the long-chain-base synthetic pathway was demonstrated by the stereospecific NADPH-dependent reduction of 3-ketosphinganine to erythrosphinganine. Membranes isolated from wild-type cells and from an lcb1 mutant exhibited substantial 3-ketosphinganine reductase activity. We conclude that the Lcb- phenotype of these mutants results from a missing or defective SPT, an activity controlled by both the LCB1 and LCB2 genes. These results and earlier work from this laboratory establish that SPT plays an essential role in sphingolipid synthesis in S. cerevisiae.     

Control of subunit stoichiometry in single-chain MspA nanopores

Transmembrane protein channels enable fast and highly sensitive detection of single molecules. Nanopore sequencing of DNA was achieved using an engineered Mycobacterium smegmatis porin A (MspA) in combination with a motor enzyme. Due to its favorable channel geometry, the octameric MspA pore exhibits the highest current level compared with other pore proteins. To date, MspA is the only protein nanopore with a published record of DNA sequencing. While widely used in commercial devices, nanopore sequencing of DNA suffers from significant base-calling errors due to stochastic events of the complex DNA-motor-pore combination and the contribution of up to five nucleotides to the signal at each position. Different mutations in specific subunits of a pore protein offer an enormous potential to improve nucleotide resolution and sequencing accuracy. However, individual subunits of MspA and other oligomeric protein pores are randomly assembled in vivo and in vitro, preventing the efficient production of designed pores with different subunit mutations. In this study, we converted octameric MspA into a single-chain pore by connecting eight subunits using peptide linkers. Lipid bilayer experiments demonstrated that single-chain MspA formed membrane-spanning channels and discriminated all four nucleotides identical to MspA produced from monomers in DNA hairpin experiments. Single-chain constructs comprising three, five, six, and seven connected subunits assembled to functional channels, demonstrating a remarkable plasticity of MspA to different subunit stoichiometries. Thus, single-chain MspA constitutes a new milestone in the optimization of MspA as a biosensor for DNA sequencing and many other applications by enabling the production of pores with distinct subunit mutations and pore diameters.     

A single-chain tetradomain glycoprotein hormone analog elicits multiple hormone activities in vivo

We previously demonstrated that genetically linking one or more of the glycoprotein hormone-specific beta subunit genes to the common alpha subunit resulted in single-chain analogues that were bioactive in vitro. The ability of such large structures to bind their cognate receptors with high affinity supported the hypothesis that extensive flexibility exists between the ligand and receptor to establish a functional complex. To further characterize the extent of this conformational flexibility, we engineered a single-chain analogue that consists of sequentially linked thyroid-stimulating hormone (TSH) beta, follicle-stimulating hormone (FSH) beta, and chorionic gonadotropin (CG) beta subunits to the alpha subunit and expressed this chimera in transfected CHO (Chinese hamster ovary) cells. Because the four subunits are genetically linked and expressed as a single-chain, this analogue presumably lacks significant native structural features of the individual heterodimers. However, it exhibited FSH, CG, and TSH activities in vitro. Here, we test whether this nonnative structure would be stable in vivo and thus biologically active. Using a variety of bioassay protocols, we demonstrate that the analogue elicits multihormone activities when injected in vivo. First, treatment with the analogue caused increases in ovarian and uterine weights and resulted in elevated serum estradiol. Second, the analogue-stimulated ovarian follicle growth and pharmacologically rescued in vivo FSH deficiency similar to recombinant human FSH or equine CG (eCG) as confirmed by induction of aromatase in the ovaries of FSHbeta knockout mice. Third, in a superovulation protocol, when primed with eCG, the analogue elicited a dose-dependent ovulatory response comparable with that by native heterodimeric human CG. Finally, the analogue-stimulated thyroxin production in hypothyroid mice similar to the pituitary-derived human TSH standard. Based on these data, we conclude that a single-chain tetradomain glycoprotein hormone analogue, despite its presumed altered conformation, is stable and biologically active in vivo. Our results establish the permissiveness and conformational plasticity with which the glycoprotein hormones are recognized in vivo by their target cell receptors.     

Characterization of sphingolipid long-chain base kinase in Arabidopsis thaliana

Sphingolipid long-chain base (LCB) kinase catalyses the phosphorylation of sphingolipid LCB to form LCB 1-phosphate. Based on sequence identity to a murine sphingosine kinase (murine SPHK1a), we isolated and characterized a LCB kinase-like cDNA in Arabidopsis thaliana. The deduced amino acid sequence of the homologous cDNA shows several regions that are highly conserved in LCB kinases from mouse, yeast, human and Caenorhabditis elegans. These regions are not similar to those of other known kinase families. For a functional identification, the homologous cDNA from A. thaliana was expressed in Escherichia coli, and LCB kinase activity was measured. The recombinant AtLcbk1 protein was found to utilize ATP and sphinganine. These results indicate the first identification of a gene coding for a LCB kinase in plants.     

Exosomal sorting of the cytoplasmic domain of bovine leukemia virus TM Env protein

Exosomes are small membrane vesicles that are released into the extracellular compartment as a consequence of fusion of multivesicular endosomes with the plasma membrane. To unravel the molecular basis of protein sorting into exosomes, we have made a chimeric protein containing the cytosolic domain of the transmembrane subunit of the viral Env protein of BLV and the ectodomain of CD8 (CDTM-BLV-CD8). When expressed in K562 cells known to constitutively secrete exosomes, the chimera was found to be very efficiently targeted to the released vesicles. Very interestingly, the cytosolic domain of the Env protein contains peptide motifs potentially recognized by components of the ESCRT machinery that could be related to chimera sorting into the vesicles. Then, quantifying the chimera secretion, we investigated the site of exosome biogenesis in K562 cells using a pharmacological approach. We present different arguments indicating that CDTM-BLV-CD8-containing exosomes are likely formed from a recycling endosomal/TGN compartment.     

Phosphorylation of the LCB1 subunit of Arabidopsis serine palmitoyltransferase stimulates its activity and modulates sphingolipid biosynthesis

Sphingolipids are the structural components of membrane lipid bilayers and act as signaling molecules in many cellular processes.Serine palmitoyltransferase(SPT) is the first committed and rate-limiting enzyme in the de novo sphingolipids biosynthetic pathway.The core SPT enzyme is a heterodimer consisting of LONG-CHAIN BASE1(LCB1) and LCB2 subunits.SPT activity is inhibited by orosomucoid proteins and stimulated by small subunits of SPT(ssSPTs).However,whether LCB1 is modified and how such modi...     

Expression of the Bacterial Type III Effector DspA/E in Saccharomyces cerevisiae Down-regulates the Sphingolipid Biosynthetic Pathway Leading to Growth Arrest

Erwinia amylovora, the bacterium responsible for fire blight, relies on a type III secretion system and a single injected effector, DspA/E, to induce disease in host plants. DspA/E belongs to the widespread AvrE family of type III effectors that suppress plant defense responses and promote bacterial growth following infection. Ectopic expression of DspA/E in plant or in Saccharomyces cerevisiae is toxic, indicating that DspA/E likely targets a cellular process conserved between yeast and plant. To unravel the mode of action of DspA/E, we screened the Euroscarf S. cerevisiae library for mutants resistant to DspA/E-induced growth arrest. The most resistant mutants (Δsur4, Δfen1, Δipt1, Δskn1, Δcsg1, Δcsg2, Δorm1, and Δorm2) were impaired in the sphingolipid biosynthetic pathway. Exogenously supplied sphingolipid precursors such as the long chain bases (LCBs) phytosphingosine and dihydrosphingosine also suppressed the DspA/E-induced yeast growth defect. Expression of DspA/E in yeast down-regulated LCB biosynthesis and induced a rapid decrease in LCB levels, indicating that serine palmitoyltransferase (SPT), the first and rate-limiting enzyme of the sphingolipid biosynthetic pathway, was repressed. SPT down-regulation was mediated by dephosphorylation and activation of Orm proteins that negatively regulate SPT. A Δcdc55 mutation affecting Cdc55-PP2A protein phosphatase activity prevented Orm dephosphorylation and suppressed DspA/E-induced growth arrest.     

Endotoxin activates de novo sphingolipid biosynthesis via nuclear factor kappa B-mediated upregulation of Sptlc2

Sphingolipids are membrane components and are involved in cell proliferation, apoptosis and metabolic regulation. In this study we investigated whether de novo sphingolipid biosynthesis in macrophages is regulated by inflammatory stimuli. Lipopolysaccharide (LPS) treatment upregulated Sptlc2, a subunit of serine palmitoyltransferase (SPT), mRNA and protein in Raw264.7 and mouse peritoneal macrophages, but Sptlc1, another subunit of SPT, was not altered. SPT activation by LPS elevated cellular levels of ceramides and sphingomyelin (SM). Pharmacological inhibition of nuclear factor kappa B (NFκB) prevented LPS-induced upregulation of Sptlc2 while transfection of p65 subunit of NFκB upregulated Sptlc2 and increased cellular ceramide levels. In contrast, MAP kinases were not involved in regulation of sphingolipid biosynthesis. Analysis of Sptlc2 promoter and chromatin immunoprecipitation (ChIP) assay showed that NFκB binding sites are located in Sptlc2 promoter region. Our results demonstrate that inflammatory stimuli activate de novo sphingolipid biosynthesis via NFκB and may play a critical role in lipid metabolism in macrophages.