Identification of the human sphingolipid C4-hydroxylase, hDES2, and its up-regulation during keratinocyte differentiation

AbstractThe C4-hydroxylation of dihydrosphingosine or dihydroceramide is a key reaction in the biosynthesis of phytosphingolipids, both in yeasts and in mammalian cells. Mouse DES2 (mDES2) was recently cloned and shown to work as a Δ4-desaturase/C4-hydroxylase, when expressed in yeast cells. Here, we cloned a human homologue of mDES2, hDES2, by homology search utilizing a BLAST program. When expressed in HEK 293 cells, hDES2 exhibited hydroxylase activity for dihydroceramide. Northern blot analyses of hDES2 revealed high expression in skin, intestines, and kidney, sites reportedly possessing high levels of phytosphingolipids. Furthermore, up-regulation of hDES2 mRNA expression and subsequent phytoceramide production were observed during vitamin C/serum-induced differentiation of human keratinocytes. These results suggest that the newly cloned hDES2 plays an essential role in phytosphingolipid synthesis in human skin and other phytosphingolipid-containing tissues

Identification of lysophospholipid receptors in human platelets: the relation of two agonists, lysophosphatidic acid and sphingosine 1-phosphate

AbstractLysophosphatidic acid (LPA) and sphingosine 1-phosphate (Sph-1-P) are known as structurally related bio-active lipids activating platelets through their respective receptors. Although the receptors for LPA and Sph-1-P have been recently identified in various cells, the identification and characterization of ones in platelets have been reported only preliminarily. In this report, we first investigated the distinct modes of LPA and Sph-1-P actions in platelet activation and found that LPA functioned as a much stronger agonist than Sph-1-P, and high concentrations of Sph-1-P specifically desensitized LPA-induced intracellular Ca2+ mobilization. In order to identify the responsible receptors underlying these observations, we analyzed the LPA and Sph-1-P receptors which might be expressed in human platelets, by RT-PCR. We found for the first time that Edg2, 4, 6 and 7 mRNA are expressed in human platelets

Signaling mechanisms for positive and negative regulation of cell motility by sphingosine-1-phosphate receptors

Sphingosine-1-phosphate (S1P) exerts positive and negative effects on cell migration apparently in a cell-type-dependent manner. Our data suggest that the bimodal actions of S1P on cell migration is due to receptor subtype-specific positive and negative regulation of Rho family GTPase, Rac; S1P1 and S1P3 mediate Rac stimulation and chemotaxis whereas S1P2 mediates Rac inhibition and chemorepulsion. The stimulatory effects of S1P 1 and S1P3 on Rac and, subsequently on migration, are mediated by Gi. The inhibitory effect of SlP2 acts on G12/13 and Rho. S1P exerts inhibitory effects on some tumor cell migration and invasion via S1P2. S1P2 also mediates the inhibition of hematogenous metastasis. In contrast, exogenously expressed S1P1 has the reverse effect, it stimulates invasion and metastasis. S1P also exerts a similar bimodal action on vascular endothelial cells and, thereby, angiogenesis. The examples suggest that control of S1P receptor activity using a receptor subtype-specific agonist and antagonist may have beneficial effects on disorders, including cancer, and vascular diseases. © Springer-Verlag Tokyo 2006. All rights reserved.[Book Chapter] Y. Hirabayashi, Y. Igarashi, A.H. Merrill, Jr. (eds.), Sphingolipid biology, Springer-Verlag, c200

Phytoceramide and sphingoid bases derived from brewer's yeast Saccharomyces pastorianus activate peroxisome proliferator-activated receptors

<p>Abstract</p> <p>Background</p> <p>Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that regulate lipid and glucose metabolism. PPARα is highly expressed in the liver and controls genes involved in lipid catabolism. We previously reported that synthetic sphingolipid analogs, part of which contains shorter-length fatty acid chains than natural sphingolipids, stimulated the transcriptional activities of PPARs. Sphingosine and dihydrosphingosine (DHS) are abundant sphingoid bases, and ceramide and dihydroceramide are major ceramide species in mammals. In contrast, phytosphingosine (PHS) and DHS are the main sphingoid bases in fungi. PHS and phytoceramide exist in particular tissues such as the epidermis in mammals, and involvement of ceramide species in PPARβ activation in cultured keratinocytes has been reported. The purpose of the present study is to investigate whether natural sphingolipids with C18 fatty acid and yeast-derived sphingoid bases activate PPARs as PPAR agonists.</p> <p>Method</p> <p>Lipids of brewer's yeast contain PHS- and DHS-based sphingolipids. To obtain the sphingoid bases, lipids were extracted from brewer's yeast and acid-hydrolyzed. The sphingoid base fraction was purified and quantified. To assess the effects of sphingolipids on PPAR activation, luciferase reporter assay was carried out. NIH/3T3 and human hepatoma (HepG2) cells were transfected with expression vectors for PPARs and retinoid × receptors, and PPAR responsive element reporter vector. When indicated, the PPAR/Gal4 chimera system was performed to enhance the credibility of experiments. Sphingolipids were added to the cells and the dual luciferase reporter assay was performed to determine the transcriptional activity of PPARs.</p> <p>Results</p> <p>We observed that phytoceramide increased the transcriptional activities of PPARs significantly, whereas ceramide and dihydroceramide did not change PPAR activities. Phytoceramide also increased transactivation of PPAR/Gal4 chimera receptors. Yeast-derived sphingoid base fraction, which contained PHS and DHS, or authentic PHS or DHS increased PPAR-dependent transcription. Additionally, phytoceramide stimulated PPARα activity in HepG2 hepatocytes, suggesting that phytoceramide activates genes regulated by PPARα.</p> <p>Conclusions</p> <p>Phytoceramide and yeast-derived sphingoid bases activate PPARs, whereas ceramide and dihydroceramide do not change the PPAR activity. The present findings suggest that phytoceramide acts as a PPAR ligand that would regulate PPAR-targeted genes.</p

Malabaricone C as natural Sphingomyelin Synthase Inhibitor against diet-induced obesity and its lipid metabolism in mice

The interaction between natural occurring inhibitors and targeted membrane proteins could be an alternative medicinal strategy for the treatment of metabolic syndrome, notably, obesity. In this study, we identified malabaricones A–C and E (1–4) isolated from the fruits of Myristica cinnamomea King as natural inhibitors for sphingomyelin synthase (SMS), a membrane protein responsible for sphingolipid biosynthesis. Having the most promising inhibition, oral administration of compound 3 exhibited multiple efficacies in reducing weight gain, improving glucose tolerance, and reducing hepatic steatosis in high fat diet-induced obesity mice models. Liver lipid analysis revealed a crucial link between the SMS activities of compound 3 and its lipid metabolism in vitro and in vivo. The nontoxic nature of compound 3 makes it a suitable candidate in search of drugs which can be employed in the treatment and prevention of obesity

Circadian Disruption Accelerates Tumor Growth and Angio/Stromagenesis through a Wnt Signaling Pathway

Epidemiologic studies show a high incidence of cancer in shift workers, suggesting a possible relationship between circadian rhythms and tumorigenesis. However, the precise molecular mechanism played by circadian rhythms in tumor progression is not known. To identify the possible mechanisms underlying tumor progression related to circadian rhythms, we set up nude mouse xenograft models. HeLa cells were injected in nude mice and nude mice were moved to two different cases, one case is exposed to a 24-hour light cycle (L/L), the other is a more “normal” 12-hour light/dark cycle (L/D). We found a significant increase in tumor volume in the L/L group compared with the L/D group. In addition, tumor microvessels and stroma were strongly increased in L/L mice. Although there was a hypervascularization in L/L tumors, there was no associated increase in the production of vascular endothelial cell growth factor (VEGF). DNA microarray analysis showed enhanced expression of WNT10A, and our subsequent study revealed that WNT10A stimulates the growth of both microvascular endothelial cells and fibroblasts in tumors from light-stressed mice, along with marked increases in angio/stromagenesis. Only the tumor stroma stained positive for WNT10A and WNT10A is also highly expressed in keloid dermal fibroblasts but not in normal dermal fibroblasts indicated that WNT10A may be a novel angio/stromagenic growth factor. These findings suggest that circadian disruption induces the progression of malignant tumors via a Wnt signaling pathway

Intracellular localization and tissue-specific distribution of human and yeast DHHC cysteine-rich domain-containing proteins

Increasing evidence indicates that DHHC cysteine-rich domain-containing proteins (DHHC proteins) are protein acyltransferases. Although multiple DHHC proteins are found in eukaryotes, characterization has been examined for only a few. Here, we have cloned all the yeast and human DHHC genes and investigated their intracellular localization and tissue-specific expression. Most DHHC proteins are localized in the ER and/or Golgi, with a few localized in the plasma membrane and one in the yeast vacuole. Human DHHC mRNAs also differ in their tissue-specific expression. These results may provide clues to aid in discovering the specific function(s) of each DHHC protein

Exosomes as Carriers of Alzheimer's Amyloid-ß

The intracerebral level of the aggregation-prone peptide, amyloid-ß (Aß), is constantly maintained by multiple clearance mechanisms, including several degradation enzymes, and brain efflux. Disruption of the clearance machinery and the resultant Aß accumulation gives rise to neurotoxic assemblies, leading to the pathogenesis of Alzheimer's disease (AD). In addition to the classic mechanisms of Aß clearance, the protein may be processed by secreted vesicles, although this possibility has not been extensively investigated. We showed that neuronal exosomes, a subtype of extracellular nanovesicles, enwrap, or trap Aß and transport it into microglia for degradation. Here, we review Aß sequestration and elimination by exosomes, and discuss how this clearance machinery might contribute to AD pathogenesis and how it might be exploited for effective AD therapy