Jacek Bielawski

The sphingosine kinase 1/sphingosine-1-phosphate pathway mediates COX-2 induction and PGE2 production in response to TNF-α

In this study we addressed the role of sphingolipid metabolism in the inflammatory response. In a L929 fibroblast model, tumor necrosis factor‐α (TNF) induced prostaglandin E2 (PGE2) production by 4 h and cyclooxygenase‐2 (COX‐2) induction as early as 2 h. This TNF‐induced PGE2 production was inhibited by NS398, a COX‐2 selective inhibitor. GC‐MS analysis revealed that only COX‐2‐generated prostanoids were produced in response to TNF, thus providing further evidence of COX‐2 selectivity. As sphingolipids have been implicated in mediating several actions of TNF, their role in COX‐2 induction and PGE2 production was evaluated. Sphingosine‐1‐phosphate (S1P) induced both COX‐2 and PGE2 in a dose‐responsive manner with an apparent ED50 of 100–300 nM. The related sphingolipid sphingosine also induced PGE2, though with much less efficacy. TNF induced a 3.5‐fold increase in sphingosine‐1‐phosphate levels at 10 min that rapidly returned to baseline by 40 min. Small interfering RNAs (siRNAs) directed against mouse SK1 decreased (typically by 80%) SK1 protein and inhibited TNF‐induced SK activity. Treatment of cells with RNAi to SK1 but not SK2 almost completely abolished the ability of TNF to induce COX‐2 or generate PGE2. By contrast, cells treated with RNAi to S1P lyase or S1P phosphatase enhanced COX‐2 induction leading to enhanced generation of PGE2. Treatment with SK1 RNAi also abolished the effects of exogenous sphin‐gosine and ceramide on PGE2, revealing that the action of sphingosine and ceramide are due to intracellular metabolism into S1P. Collectively, these results provide novel evidence that SK1 and S1P are necessary for TNF to induce COX‐2 and PGE2 production. Based on these findings, this study indicates that SK1 and S1P could be implicated in pathological inflammatory disorders and cancer.—Pettus, B. J., Bielawski, J., Porcelli, A. M., Reames, D. L., Johnson, K. R., Morrow, J., Chalfant, C. E., Obeid, L. M., Hannun, Y A. The sphingosine kinase 1/sphingosine‐1‐phosphate pathway mediates COX‐2 induction and PGE2 production in response to TNF‐α, FASEB J., 17, 1411–1421 (2003)

Glycosphingolipid synthesis requires FAPP2 transfer of glucosylceramide.

The molecular machinery responsible for the generation of transport carriers moving from the Golgi complex to the plasma membrane relies on a tight interplay between proteins and lipids. Among the lipid-binding proteins of this machinery, we previously identified the four-phosphate adaptor protein FAPP2, the pleckstrin homology domain of which binds phosphatidylinositol 4-phosphate and the small GTPase ARF1. FAPP2 also possesses a glycolipid-transfer-protein homology domain. Here we show that human FAPP2 is a glucosylceramide-transfer protein that has a pivotal role in the synthesis of complex glycosphingolipids, key structural and signalling components of the plasma membrane. The requirement for FAPP2 makes the whole glycosphingolipid synthetic pathway sensitive to regulation by phosphatidylinositol 4-phosphate and ARF1. Thus, by coupling the synthesis of glycosphingolipids with their export to the cell surface, FAPP2 emerges as crucial in determining the lipid identity and composition of the plasma membrane.

Central role of ceramide biosynthesis in body weight regulation, energy metabolism, and the metabolic syndrome

Although obesity is associated with multiple features of the metabolic syndrome (insulin resistance, leptin resistance, hepatic steatosis, chronic inflammation, etc.), the molecular changes that promote these conditions are not completely understood. Here, we tested the hypothesis that elevated ceramide biosynthesis contributes to the pathogenesis of obesity and the metabolic syndrome. Chronic treatment for 8 wk of genetically obese (ob/ob), and, high-fat diet-induced obese (DIO) mice with myriocin, an inhibitor of de novo ceramide synthesis, decreased circulating ceramides. Decreased ceramide was associated with reduced weight, enhanced metabolism and energy expenditure, decreased hepatic steatosis, and improved glucose hemostasis via enhancement of insulin signaling in the liver and muscle. Inhibition of de novo ceramide biosynthesis decreased adipose expression of suppressor of cytokine signaling-3 (SOCS-3) and induced adipose uncoupling protein-3 (UCP3). Moreover, ceramide directly induced SOCS-3 and inhibited UCP3 mRNA in cultured adipocytes suggesting a direct role for ceramide in regulation of metabolism and energy expenditure. Inhibition of de novo ceramide synthesis had no effect on adipose tumor necrosis factor-alpha (TNF-alpha) expression but dramatically reduced adipose plasminogen activator inhibitor-1 (PAI-1) and monocyte chemoattactant protein-1 (MCP-1). This study highlights a novel role for ceramide biosynthesis in body weight regulation, energy expenditure, and the metabolic syndrome.

Role for sphingosine kinase 1 in colon carcinogenesis

Sphingosine kinase 1 (SphKl) phosphory‐lates sphingosine to form sphingosine‐l‐phosphate (S1P) and is a critical regulator of sphingolipid‐mediated functions. Cell‐based studies suggest a tumor‐promoting function for the SphK1/S1P pathway. Also, our previous studies implicated the SphK1/S1P pathway in the induction of the arachidonic acid cascade, a major inflammatory pathway involved in colon carcinogenesis. Therefore, we investigated whether the SphK1/S1P pathway is necessary for mediating carcinogenesis in vivo. Here, we report that 89% (42/47) of human colon cancer samples stained positively for SphK1, whereas normal colon mucosa had negative or weak staining. Adenomas had higher expression of SphK1 vs. normal mucosa, and colon cancers with metastasis had higher expression of SphK1 than those without metastasis. In the azoxymethane (AOM) murine model of colon cancer, SphK1 and S1P were significantly elevated in colon cancer tissues compared to normal mucosa. Moreover, blood levels of S1P were higher in mice with colon cancers than in those without cancers. Notably, SphK1_/_ mice subjected to AOM had significantly less aberrant crypt foci (ACF) formation and significantly reduced colon cancer development. These results are the first in vivo evidence that the SphK1/S1P pathway contributes to colon carcinogenesis and that inhibition of this pathway is a potential target for chemoprevention.—Kawamori, T., Kaneshiro, T., Okumura, M., Maalouf, S., Uflacker, A., Bielawski, J., Hannun, Y. A., Obeid, L. M. Role for sphingosine kinase 1 in colon carcinogenesis. FASEB J. 23, 405‐414 (2009)

Ceramide targets autophagosomes to mitochondria and induces lethal mitophagy

Mechanisms by which autophagy promotes cell survival or death are unclear. We provide evidence that C18-pyridinium ceramide (C18-Pyr-Cer) treatment, or endogenous C18-ceramide generation by ceramide synthase 1 (CerS1) expression mediates autophagic cell death, independent of apoptosis in human cancer cells. C18-ceramide-induced lethal autophagy was regulated via microtubule-associated protein 1 light chain 3 beta lipidation (LC3B-II) and selective targeting of mitochondria by LC3B-II-containing autophagolysosomes (mitophagy) through direct interaction between ceramide and LC3B-II upon Drp1-dependent mitochondrial fission, leading to inhibition of mitochondrial function and oxygen consumption. Accordingly, expression of mutant LC3B with impaired ceramide binding, as predicted by molecular modeling, prevented CerS1-mediated mitochondrial targeting, recovering oxygen consumption. Moreover, knockdown of CerS1 abrogated sodium selenite-induced mitophagy, and stable LC3B knockdown protected against CerS1-C18-ceramide-dependent mitophagy and blocked tumor suppression in vivo. Thus, these data suggest a novel receptor function of ceramide for anchoring LC3B-II-autophagolysosomes to mitochondrial membranes, defining a key mechanism for the induction of lethal mitophagy.

Sphingosine kinase 1 is up-regulated in colon carcinogenesis

Sphingosine kinase 1 (SK1) phosphorylates sphingosine to form sphingosine 1‐phosphate (S1P), which has the ability to promote cell proliferation and survival and stimulate angiogenesis. The SK1/S1P pathway also plays a critical role in regulation of cyclooxygenase‐2 (COX‐2), a well‐established pathogenic factor in colon carcinogenesis. Therefore, we examined the expression of SK1 and COX‐2 in rat colon tumors induced by azoxymethane (AOM) and the relationship of these two proteins in normal and malignant intestinal epithelial cells. Strongly positive SK1 staining was found in 21/28 (75%) of rat colon adenocarcinomas induced by AOM, whereas no positive SK1 staining was observed in normal mucosa. The increase in SK1 and COX‐2 expression in AOM‐induced rat colon adenocarcinoma was confirmed at the level of mRNA by real‐time RT‐PCR. In addition, it was found that 1) down‐regulation of SK1 in HT‐29 human colon cancer cells by small interfering RNA (siRNA) decreases COX‐2 expression and PGE2 production; 2) overexpression of SK1 in RIE‐1 rat intestinal epithelial cells induces COX‐2 expression; and 3) S1P stimulates COX‐2 expression and PGE2 production in HT‐29 cells. These results suggest that the SK1/S1P pathway may play an important role in colon carcinogenesis, in part, by regulating COX‐2 expression and PGE2 production.

Altered Adipose and Plasma Sphingolipid Metabolism in Obesity A Potential Mechanism for Cardiovascular and Metabolic Risk

The adipose tissue has become a central focus in the pathogenesis of obesity-mediated cardiovascular and metabolic disease. Here we demonstrate that adipose sphingolipid metabolism is altered in genetically obese (ob/ob) mice. Expression of enzymes involved in ceramide generation (neutral sphingomyelinase [NSMase], acid sphingomyelinase [ASMase], and serine-palmitoyl-transferase [SPT]) and ceramide hydrolysis (ceramidase) are elevated in obese adipose tissues. Our data also suggest that hyperinsulinemia and elevated tumor necrosis factor (TNF)-α associated with obesity may contribute to the observed increase in adipose NSMase, ASMase, and SPT mRNA in this murine model of obesity. Liquid chromatography/mass spectroscopy revealed a decrease in total adipose sphingomyelin and ceramide levels but an increase in sphingosine in ob/ob mice compared with lean mice. In contrast to the adipose tissue, plasma levels of total sphingomyelin, ceramide, sphingosine, and sphingosine 1-phosphate (S1P) were elevated in ob/ob mice. In cultured adipocytes, ceramide, sphingosine, and S1P induced gene expression of plasminogen activator inhibitor-1, TNF-α, monocyte chemoattractant protein-1, interleukin-6, and keratinocyte-derived chemokine. Collectively, our results identify a novel role for sphingolipids in contributing to the prothrombotic and proinflammatory phenotype of the obese adipose tissue currently believed to play a major role in the pathogenesis of obesity-mediated cardiovascular and metabolic disease.

Alterations of Ceramide/Sphingosine 1-Phosphate Rheostat Involved in the Regulation of Resistance to Imatinib-induced Apoptosis in K562 Human Chronic Myeloid Leukemia Cells

In this study, mechanisms of resistance to imatinib-induced apoptosis in human K562 cells were examined. Continuous exposure to stepwise increasing concentrations of imatinib resulted in the selection of K562/IMA-0.2 and -1 cells, which expressed ∼2.3- and 19-fold resistance, respectively. Measurement of endogenous ceramides by high performance liquid chromatography/mass spectroscopy showed that treatment with imatinib increased the generation of ceramide, mainly C18-ceramide, which is generated by the human longevity assurance gene 1 (hLASS1), in sensitive, but not in resistant cells. Inhibition of hLASS1 by small interfering RNA partially prevented imatinib-induced cell death in sensitive cells. In reciprocal experiments, overexpression of hLASS1, and not hLASS6, in drug-resistant cells caused a marked increase in imatinib-induced C18-ceramide generation, and enhanced apoptosis. Interestingly, there were no defects in the levels of mRNA and enzyme activity levels of hLASS1 for ceramide generation in K562/IMA-1 cells. However, expression levels of sphingosine kinase-1 (SK1) and generation of sphingosine 1-phosphate (S1P) were increased significantly in K562/IMA-1 cells, channeling sphingoid bases to the sphingosine kinase pathway. The partial inhibition of SK1 expression by small interference RNA modulated S1P levels and increased sensitivity to imatinib-induced apoptosis in resistant cells. On the other hand, forced expression of SK1 in K562 cells increased the ratio between total S1P/C18-ceramide levels ∼6-fold and prevented apoptosis significantly in response to imatinib. Additional data indicated a role for SK1/S1P signaling in the up-regulation of the Bcr-Abl expression at the post-transcriptional level, which suggested a possible mechanism for resistance to imatinib-mediated apoptosis. In conclusion, these data suggest a role for endogenous C18-ceramide synthesis mainly via hLASS1 in imatinib-induced apoptosis in sensitive cells, whereas in resistant cells, alterations of the balance between the levels of ceramide and S1P by overexpression of SK1 result in resistance to imatinib-induced apoptosis.

Antiapoptotic roles of ceramide-synthase-6-generated C16-ceramide via selective regulation of the ATF6/CHOP arm of ER-stress-response pathways

Emerging results suggest that ceramides with different fatty acid chain lengths might play distinct functions in the regulation of tumor growth and therapy. Here we report that de novo‐generated C18‐ and C16‐ceramides by ceramide synthases 1 and 6 (CerS1 and CerS6) play opposing proapoptotic and prosurvival roles, respectively, in human head and neck squamous cell carcinomas (HNSCCs). Unexpectedly, knockdown of CerS6/C16‐ceramide using small interfering RNA induced endoplasmic reticulum (ER)‐stress‐mediated apoptosis. Reconstitution of C16‐ceramide generation by induced expression of wild‐type CerS6, but not its catalytically inactive mutant, protected cells from cell death induced by knockdown of CerS6. Moreover, using molecular tools coupled with analysis of sphingo‐lipid metabolism showed that generation of C16‐cer‐amide, and not dihydro‐C16‐ceramide, by induced expression of CerS6 rescued cells from ER stress and apoptosis. Mechanistically, regulation of ER‐stress‐in‐duced apoptosis by CerS6/C16‐ceramide was linked to the activation of a specific arm, ATF6/CHOP, of the unfolded protein response pathway. Notably, while expression of CerS1/C18‐ceramide inhibited HNSCC xenograft growth, CerS6/C16‐ceramide significantly protected ER stress, leading to enhanced tumor development and growth in vivo, consistent with their pro‐ and antiapoptotic roles, respectively. Thus, these data reveal an unexpected and novel prosur‐vival role of CerS6/C16‐ceramide involved in the protection against ER‐stress‐induced apoptosis and induction of HNSCC tumor growth.—Senkal, C. E., Ponnusamy, S., Bielawski, J., Hannun, Y. A., Ogret‐men, B. Antiapoptotic roles of ceramide‐synthase‐6‐generated C16‐ceramide via selective regulation of the ATF6/CHOP arm of ER‐stress‐response pathways. FASEB J. 24, 296–308 (2010). www.fasebj.org

Blood sphingolipidomics in healthy humans: impact of sample collection methodology

We used a HPLC-MS/MS methodology for determination of a basic metabolomic profile (18:1,18:0 sphingoid backbone, C14-C26 N-acyl part) of “normal” sphingolipid levels in human serum and plasma. Blood was collected from healthy males and nonpregnant females under fasting and nonfasting conditions with and without anticoagulants. Sphingolipids analyzed included sphingoid bases, sphingosine and dihydrosphingosine, their 1-phosphates (S1P and dhS1P), molecular species (Cn-) of ceramide (Cer), sphingomyelin (SM), hexosylceramide (HexCer), lactosylceramide (LacCer), and Cer 1-phosphate (Cer1P). SM, LacCer, HexCer, Cer, and Cer1P constituted 87.7, 5.8, 3.4, 2.8, and 0.15% of total sphingolipids, respectively. The abundant circulating SM was C16-SM (64.0 µM), and it increased with fasting (100 µM). The abundant LacCer was C16-LacCer (10.0 µM) and the abundant HexCer was C24-HexCer (2.5 µM). The abundant Cer, C24-Cer (4.0 µM), was not influenced by fasting; however, levels of C16-C20 Cers were decreased in response to fasting. S1P levels were higher in serum than plasma (0.68 µM vs. 0.32 µM). We also determined levels of sphingoid bases and SM species in isolated lipoprotein classes. HDL3 was the major carrier of S1P, dhS1P, and Sph, and LDL was the major carrier of Cer and dhSph. Per particle, VLDL contained the highest levels of SM, Cer, and S1P. HPLC-MS/MS should provide a tool for clinical testing of circulating bioactive sphingolipids in human blood.