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Characterization of sphingoid base-1-phosphate lyase in Arabidopsis thaliana
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Abstracts / Chemistry and Physics of Lipids 160S (2009) S27–S39
expression and activity. In the present study, we investigated the effect of S1P on HIF-1A in thyroid follicular carcinoma ML-1 cells. We showed that S1P increased HIF-1A in a time-dependent fashion. The effect was inhibited by Ptx, suggesting an effect mediated via Gi/o proteins. Preliminary results suggest that S1P receptors 1 and 3 mediate the effects of S1P. S1P did not enhance the level of HIF-1A mRNA. Inhibiting VEGF receptor 2 did not affect HIF-1A levels. However, if the VEGF receptor 2 was blocked, S1P only evoked a rapid, transient increase in HIF-1A. In addition to increasing HIF-1A, S1P also transiently increased the amount of P70S6 kinase. In addition, the proliferation and migration of ML-1 cells was investigated. Our results suggest that HIF-1A and P70S6K, a kinase upstream of HIF-1A, regulate the proliferation of these cells. HIF-1A and P70S6K did not regulate the basal migration of ML-1-cells, but the S1Pinduced migration was blunted when either P70S6K or HIF-1A was blocked. Taken together, these results suggest that S1P and VEGFR2 together regulate the expression of HIF-1A. These results can be of importance and should be considered with regard to finding new anti-angiogenetic drugs. doi:10.1016/j.chemphyslip.2009.06.041 PO 37 Perturbation of bioactive sphingolipids in Sgpl1−/− mice Paul P. Van Veldhoven LIPIT, Department of Molecular Cell Biology, K.U.Leuven, Campus Gasthuisberg, Herestraat, B-3000 Leuven, Belgium Sphingosine-1-phosphate (S1P), through binding to different Edg/S1P-receptors mediates many cellular and systemic processes. Despite the presence of (a)specific S1P-phosphatases in mammals, S1P-lyase appears to be the main player in the removal of S1P, as shown by the gradual accumulation of S1P in tissues of a Sgpl1−/− mice. These mice die early in life, with immunosuppression, respiratory distress, anemia, and apoptotic and/or inflammatory processes in most tissues. It is tempting to link all these symptoms to S1P, but given the many possible metabolic interconversions of S1P into other bioactive sphingolipids, in casu sphingosine, ceramide and ceramide-1-phosphate, these lipids were analyzed in plasma or in affected tissues of this mouse model. In addition to S1P, the saturated analogue sphinganine-1phosphate was detectable in affected tissues. Sphingosine levels were also increased, 2-fold in plasma, 100-fold in some tissues. Sphinganine was elevated as well, reaching concentrations comparable to those reported in fumonisin treated animals. Ceramide levels in affected tissues increased 5–10-fold and could underly the observed apoptotic events, although the ceramide/S1P ratio is still much lower than in control tissues. Finally, ceramide-1-phosphate turned out to elevated (15–20 fold). The latter lipid could play a role in inflammatory reactions, similar to the action of spider venoms containing sphingomyelinase D, causing skin necrosis at the bite site. Hence, S1P-lyase deficiency results in an increase of four major bioactive sphingolipids, each of them contributing likely to some extent to the observed phenotype. Hence, the use of lyase inhibitors as potential immunosuppressive agents for man should be carefully evaluated. doi:10.1016/j.chemphyslip.2009.06.042
PO 38 Characterization of sphingoid base-1-phosphate lyase in Arabidopsis thaliana Mai Katoh ∗ , Mai Ishiguro, Hiroyuki Imai Department of Biology, Graduate School of Natural Science, Konan University, 8-9-1 Okamoto, Higashinada-ku, Kobe 658-8501, Japan Sphingoid long-chain bases (LCB) 1-phosphates are degradated by LCB 1-phosphate lyase to C16 fatty aldehydes and phosphoethanolamine. Recently, we have reported that the Arabidopsis At1g27980 gene product, AtDPL1, is a functional LCB-1-phosphate lyase (Plant Cell Physiol. 49: 1758–1763, 2008). Expression of green fluorescent protein fusion products in suspension-cultured Arabidopsis cells showed that AtDPL1 is located to the endoplasmic reticulum. The deduced amino acid sequences from Arabidopsis, human, mouse and S. cerevisiae DPL (SPL) genes show that the DPL (SPL) proteins commonly contain one transmembrane region and one predicted pyridoxal 5 -phosphate binding site. In mammals, the active sites of SPL and SPH-1P phosphatase are located on the cytosolic and lumenal sides of the ER, respectively. Therefore, future studies may focus on the subcellular localization and transmembrane topology of sphingosine kinase, DPL and SPH-1P phosphatase for discussing roles in LCB-1P metabolisms in plants. Sphingosine 1-phosphate (SPH-1P) and phytosphingosine-1P (PHS-1P) have been reported as lipid messenger molecules for guard cell ABA responses in plants. To assess the functional consequence of the loss of function of AtDPL1, we characterized two independent Arabidopsis mutants (dpl1-1 and dpl1-2) with T-DNA insertions in the AtDPL1 gene. The rates of fresh weight decreases of dpl1-1 and dpl1-2 mutants were significantly slower than those of the wild-type plants. This ability to limit their transpiration reflected the leaf temperature of the mutant plants higher than that of wild-type plants, suggesting that AtDPL1 plays a role in dehydration stress. doi:10.1016/j.chemphyslip.2009.06.043 PO 39 1-Deoxy-sphingolipids are important for the THP1 monocyte to macrophage differentiation Daniela Ernst ∗ , Thorsten Hornemann Sphingolipids are a class of lipid which have structural (sphingomyeline, glycosphingolipids) and signalling functions (Ceramide, SO1P). Cellular sphingolipid biosynthesis is initiated by the condensation of serine and palmitoyl-CoA to form sphinganine—a reaction catalyzed by the serine-palmitoyltransferase (SPT). Several missense mutations in SPT have been associated with the inherited sensory neuropathy HSAN1. These mutations induce a shift in the substrate specificity of SPT which enables the enzyme to metabolise also alanine and glycine as alternative substrates. This reaction forms two atypical deoxy-sphingoid bases (DSB) 1-deoxysphinganine and 1-deoxymethyl-sphinganine. Both metabolites lack the C1 hydroxyl group and can therefore neither be converted to complex sphingolipids nor degraded. Consequently they accumulate in the cell, as demonstrated in mutant overexpressing HEK293 cells as well as in lymphoblasts and plasma of HSAN1 patients. Interestingly, we found that 1-deoxy-sphingoid bases are also present in several human and non-human cell lines, indicating that also the wildtype SPT is capable to form these metabolites to certain extent. High endogenous DSB levels were detected in HEK293 cells and various macrophage cell lines (RAW, HL60) whereas other cell lines like HepG2, Hela or NIH-3T3 showed low concentrations of
Abstracts / Chemistry and Physics of Lipids 160S (2009) S27–S39
expression and activity. In the present study, we investigated the effect of S1P on HIF-1A in thyroid follicular carcinoma ML-1 cells. We showed that S1P increased HIF-1A in a time-dependent fashion. The effect was inhibited by Ptx, suggesting an effect mediated via Gi/o proteins. Preliminary results suggest that S1P receptors 1 and 3 mediate the effects of S1P. S1P did not enhance the level of HIF-1A mRNA. Inhibiting VEGF receptor 2 did not affect HIF-1A levels. However, if the VEGF receptor 2 was blocked, S1P only evoked a rapid, transient increase in HIF-1A. In addition to increasing HIF-1A, S1P also transiently increased the amount of P70S6 kinase. In addition, the proliferation and migration of ML-1 cells was investigated. Our results suggest that HIF-1A and P70S6K, a kinase upstream of HIF-1A, regulate the proliferation of these cells. HIF-1A and P70S6K did not regulate the basal migration of ML-1-cells, but the S1Pinduced migration was blunted when either P70S6K or HIF-1A was blocked. Taken together, these results suggest that S1P and VEGFR2 together regulate the expression of HIF-1A. These results can be of importance and should be considered with regard to finding new anti-angiogenetic drugs. doi:10.1016/j.chemphyslip.2009.06.041 PO 37 Perturbation of bioactive sphingolipids in Sgpl1−/− mice Paul P. Van Veldhoven LIPIT, Department of Molecular Cell Biology, K.U.Leuven, Campus Gasthuisberg, Herestraat, B-3000 Leuven, Belgium Sphingosine-1-phosphate (S1P), through binding to different Edg/S1P-receptors mediates many cellular and systemic processes. Despite the presence of (a)specific S1P-phosphatases in mammals, S1P-lyase appears to be the main player in the removal of S1P, as shown by the gradual accumulation of S1P in tissues of a Sgpl1−/− mice. These mice die early in life, with immunosuppression, respiratory distress, anemia, and apoptotic and/or inflammatory processes in most tissues. It is tempting to link all these symptoms to S1P, but given the many possible metabolic interconversions of S1P into other bioactive sphingolipids, in casu sphingosine, ceramide and ceramide-1-phosphate, these lipids were analyzed in plasma or in affected tissues of this mouse model. In addition to S1P, the saturated analogue sphinganine-1phosphate was detectable in affected tissues. Sphingosine levels were also increased, 2-fold in plasma, 100-fold in some tissues. Sphinganine was elevated as well, reaching concentrations comparable to those reported in fumonisin treated animals. Ceramide levels in affected tissues increased 5–10-fold and could underly the observed apoptotic events, although the ceramide/S1P ratio is still much lower than in control tissues. Finally, ceramide-1-phosphate turned out to elevated (15–20 fold). The latter lipid could play a role in inflammatory reactions, similar to the action of spider venoms containing sphingomyelinase D, causing skin necrosis at the bite site. Hence, S1P-lyase deficiency results in an increase of four major bioactive sphingolipids, each of them contributing likely to some extent to the observed phenotype. Hence, the use of lyase inhibitors as potential immunosuppressive agents for man should be carefully evaluated. doi:10.1016/j.chemphyslip.2009.06.042
PO 38 Characterization of sphingoid base-1-phosphate lyase in Arabidopsis thaliana Mai Katoh ∗ , Mai Ishiguro, Hiroyuki Imai Department of Biology, Graduate School of Natural Science, Konan University, 8-9-1 Okamoto, Higashinada-ku, Kobe 658-8501, Japan Sphingoid long-chain bases (LCB) 1-phosphates are degradated by LCB 1-phosphate lyase to C16 fatty aldehydes and phosphoethanolamine. Recently, we have reported that the Arabidopsis At1g27980 gene product, AtDPL1, is a functional LCB-1-phosphate lyase (Plant Cell Physiol. 49: 1758–1763, 2008). Expression of green fluorescent protein fusion products in suspension-cultured Arabidopsis cells showed that AtDPL1 is located to the endoplasmic reticulum. The deduced amino acid sequences from Arabidopsis, human, mouse and S. cerevisiae DPL (SPL) genes show that the DPL (SPL) proteins commonly contain one transmembrane region and one predicted pyridoxal 5 -phosphate binding site. In mammals, the active sites of SPL and SPH-1P phosphatase are located on the cytosolic and lumenal sides of the ER, respectively. Therefore, future studies may focus on the subcellular localization and transmembrane topology of sphingosine kinase, DPL and SPH-1P phosphatase for discussing roles in LCB-1P metabolisms in plants. Sphingosine 1-phosphate (SPH-1P) and phytosphingosine-1P (PHS-1P) have been reported as lipid messenger molecules for guard cell ABA responses in plants. To assess the functional consequence of the loss of function of AtDPL1, we characterized two independent Arabidopsis mutants (dpl1-1 and dpl1-2) with T-DNA insertions in the AtDPL1 gene. The rates of fresh weight decreases of dpl1-1 and dpl1-2 mutants were significantly slower than those of the wild-type plants. This ability to limit their transpiration reflected the leaf temperature of the mutant plants higher than that of wild-type plants, suggesting that AtDPL1 plays a role in dehydration stress. doi:10.1016/j.chemphyslip.2009.06.043 PO 39 1-Deoxy-sphingolipids are important for the THP1 monocyte to macrophage differentiation Daniela Ernst ∗ , Thorsten Hornemann Sphingolipids are a class of lipid which have structural (sphingomyeline, glycosphingolipids) and signalling functions (Ceramide, SO1P). Cellular sphingolipid biosynthesis is initiated by the condensation of serine and palmitoyl-CoA to form sphinganine—a reaction catalyzed by the serine-palmitoyltransferase (SPT). Several missense mutations in SPT have been associated with the inherited sensory neuropathy HSAN1. These mutations induce a shift in the substrate specificity of SPT which enables the enzyme to metabolise also alanine and glycine as alternative substrates. This reaction forms two atypical deoxy-sphingoid bases (DSB) 1-deoxysphinganine and 1-deoxymethyl-sphinganine. Both metabolites lack the C1 hydroxyl group and can therefore neither be converted to complex sphingolipids nor degraded. Consequently they accumulate in the cell, as demonstrated in mutant overexpressing HEK293 cells as well as in lymphoblasts and plasma of HSAN1 patients. Interestingly, we found that 1-deoxy-sphingoid bases are also present in several human and non-human cell lines, indicating that also the wildtype SPT is capable to form these metabolites to certain extent. High endogenous DSB levels were detected in HEK293 cells and various macrophage cell lines (RAW, HL60) whereas other cell lines like HepG2, Hela or NIH-3T3 showed low concentrations of