Yong-Moon Lee

Vascular Endothelium As a Contributor of Plasma Sphingosine 1-Phosphate

Sphingosine 1-phosphate (S1P), an abundant lipid mediator in plasma, regulates vascular and immune cells by activating S1P receptors. In this report, we investigated the mechanisms by which high plasma S1P levels are maintained in mice. We found that plasma S1P turns over rapidly with a half-life of ≈15 minutes, suggesting the existence of a high-capacity biosynthetic source(s). Transplantation of bone marrow from wild-type to Sphk1−/−Sphk2+/− mice restored plasma S1P levels, suggesting that hematopoietic cells are capable of secreting S1P into plasma. However, plasma S1P levels were not appreciably altered in mice that were thrombocytopenic, anemic, or leukopenic. Surprisingly, reconstitution of Sphk1−/−Sphk2+/− bone marrow cells into wild-type hosts failed to reduce plasma S1P, suggesting the existence of an additional, nonhematopoietic source for plasma S1P. Adenoviral expression of Sphk1 in the liver of Sphk1−/− mice restored plasma S1P levels. In vitro, vascular endothelial cells, but not hepatocytes, secreted S1P in a constitutive manner. Interestingly, laminar shear stress downregulated the expression of S1P lyase (Sgpl) and S1P phosphatase-1 (Sgpp1) while concomitantly stimulating S1P release from endothelial cells in vitro. Modulation of expression of endothelial S1P lyase with small interfering RNA and adenoviral expression altered S1P secretion, suggesting an important role played by this enzyme. These data suggest that the vascular endothelium, in addition to the hematopoietic system, is a major contributor of plasma S1P.

Intracellular Role for Sphingosine Kinase 1 in Intestinal Adenoma Cell Proliferation

ABSTRACT Sphingosine kinase (Sphk) enzymes are important in intracellular sphingolipid metabolism as well as in the biosynthesis of sphingosine 1-phosphate (S1P), an extracellular lipid mediator. Here, we show that Sphk1 is expressed and is required for small intestinal tumor cell proliferation in ApcMin/+ mice. Adenoma size but not incidence was dramatically reduced in ApcMin/+Sphk−/− mice. Concomitantly, epithelial cell proliferation in the polyps was significantly attenuated, suggesting that Sphk1 regulates adenoma progression. Although the S1P receptors (S1P1R, S1P2R, and S1P3R) are expressed, polyp incidence or size was unaltered in ApcMin/+S1p2r−/−, ApcMin/+S1p3r−/−, and ApcMin/+S1p1r+/− bigenic mice. These data suggest that extracellular S1P signaling via its receptors is not involved in adenoma cell proliferation. Interestingly, tissue sphingosine content was elevated in the adenomas of ApcMin/+Sphk1−/− mice, whereas S1P levels were not significantly altered. Concomitantly, epithelial cell proliferation and the expression of the G1/S cell cycle regulator CDK4 and c-myc were diminished in the polyps of ApcMin/+Sphk1−/− mice. In rat intestinal epithelial (RIE) cells in vitro, Sphk1 overexpression enhanced cell cycle traverse at the G1/S boundary. In addition, RIE cells treated with sphingosine but not C6-ceramide exhibited reduced cell proliferation, reduced retinoblastoma protein phosphorylation, and cyclin-dependent kinase 4 (Cdk4) expression. Our findings suggest that Sphk1 plays a critical role in intestinal tumor cell proliferation and that inhibitors of Sphk1 may be useful in the control of intestinal cancer.

Extracellular export of sphingosine kinase-1a contributes to the vascular S1P gradient

Sphingosine 1-phosphate (S1P), produced by Sphks (sphingosine kinases), is a multifunctional lipid mediator that regulates immune cell trafficking and vascular development. Mammals maintain a large concentration gradient of S1P between vascular and extravascular compartments. Mechanisms by which S1P is released from cells and concentrated in the plasma are poorly understood. We recently demonstrated [Ancellin, Colmont, Su, Li, Mittereder, Chae, Stefansson, Liau and Hla (2002) J. Biol. Chem. 277, 6667-6675] that Sphk1 activity is constitutively secreted by vascular endothelial cells. In the present study, we show that among the five Sphk isoforms expressed in endothelial cells, the Sphk-1a isoform is selectively secreted in HEK-293 cells (human embryonic kidney cells) and human umbilical-vein endothelial cells. In sharp contrast, Sphk2 is not secreted. The exported Sphk-1a isoform is enzymatically active and produced sufficient S1P to induce S1P receptor internalization. Wild-type mouse plasma contains significant Sphk activity (179 pmol x min(-1) x g(-1)). In contrast, Sphk1-/- mouse plasma has undetectable Sphk activity and approx. 65% reduction in S1P levels. Moreover, human plasma contains enzymatically active Sphk1 (46 pmol x min(-1) x g(-1)). These results suggest that export of Sphk-1a occurs under physiological conditions and may contribute to the establishment of the vascular S1P gradient.

Sulforaphane attenuates obesity by inhibiting adipogenesis and activating the AMPK pathway in obese mice

Obesity is associated with metabolic disorders. Sulforaphane, an isothiocyanate, inhibits adipogenesis and the occurrence of cardiovascular disease. In this study, we investigated whether sulforaphane could prevent high-fat diet (HFD)-induced obesity in C57BL/6N mice. Mice were fed a normal diet (ND), HFD or HFD plus 0.1% sulforaphane (SFN) for 6 weeks. Food efficiency ratios and body weight were lower in HFD-SFN-fed mice than in HFD-fed mice. SFN attenuated HFD-induced visceral adiposity, adipocyte hypertrophy and fat accumulation in the liver. Serum total cholesterol and leptin, and liver triglyceride levels were lower in HFD-SFN-fed mice than in HFD-fed mice. SFN decreased the expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα) and leptin in the adipose tissue of HFD-SFN mice and increased adiponectin expression. Phosphorylation of AMP-activated protein kinase α (AMPKα) and acetyl-CoA carboxylase in the adipose tissue of HFD-SFN-fed mice was elevated, and HMG-CoA reductase expression was decreased compared with HFD-fed mice. Thus, these results suggest that SFN may induce antiobesity activity by inhibiting adipogenesis through down-regulation of PPARγ and C/EBPα and by suppressing lipogenesis through activation of the AMPK pathway.

Products by the sphingosine kinase/sphingosine 1‐phosphate (S1P) lyase pathway but not S1P stimulate mitogenesis

Sphingosine 1‐phosphate (S1P) functions as a ligand for the S1P/EDG family receptors. For years, intracellular signaling roles for S1P have also been suggested, especially in cell proliferation. Now, we have generated several mouse F9 embryonic carcinoma cell lines varying in expression of the S1P‐degrading enzyme, S1P lyase (SPL) and/or sphingosine kinase (SPHK1). All these cell lines accumulated S1P compared to the wild‐type F9 cells, but the amounts varied. We investigated the ability of these cells to proliferate under low serum conditions, as measured by a thymidine uptake assay. Although F9 cells over‐expressing SPHK1 did exhibit enhanced DNA synthesis, other S1P‐accumulating cells (SPL‐null cells and SPL‐null cells over‐expressing SPHK1) did not. The overproduction of both SPL and SPHK1 resulted in the most striking mitogenic effect. Moreover, nM concentrations of sphingosine (or dihydrosphingosine) stimulated DNA synthesis in an SPL‐dependent manner. These results indicate that products by the SPL pathway, not S1P itself, function in mitogenesis.

Sulforaphane Inhibits Mitotic Clonal Expansion During Adipogenesis Through Cell Cycle Arrest

Obesity is a risk factor for numerous metabolic disorders such as type 2 diabetes, hypertension, and coronary heart disease. Adipocyte differentiation is triggered by adipocyte hyperplasia, which leads to obesity. In this study, the inhibitory effect of sulforaphane, an isothiocyanate, on adipogenesis in 3T3–L1 cells was investigated. Sulforaphane decreased the accumulation of lipid droplets stained with Oil Red O and inhibited the elevation of triglycerides in the adipocytes (half‐maximal inhibitory concentration = 7.3 µmol/l). The expression of peroxisome proliferator‐activated receptor γ (PPARγ) and CCAAT/enhancer‐binding protein α (C/EBPα), major transcription factors for adipocyte differentiation, was significantly reduced by sulforaphane. The major effects of sulforaphane on the inhibition of adipocyte differentiation occurred during the early stage of adipogenesis. Thus, the expression of C/EBPβ, an early‐stage biomarker of adipogenesis, decreased in a concentration‐dependent manner when the adipocytes were exposed to sulforaphane (0, 5, 10, and 20 µmol/l). The proliferation of adipocytes treated with 20 µmol/l sulforaphane for 24 and 48 h was also suppressed. These results indicate that sulforaphane may specifically affect mitotic clonal expansion to inhibit adipocyte differentiation. Sulforaphane arrested the cell cycle at the G0/G1 phase, increased p27 expression, and decreased retinoblastoma (Rb) phosphorylation. Additionally, sulforaphane modestly decreased the phosphorylation of ERK1/2 and Akt. Our results indicate that the inhibition of early‐stage adipocyte differentiation by sulforaphane may be associated with cell cycle arrest at the G0/G1 phase through upregulation of p27 expression.

Downregulation of cell-free miR-198 as a diagnostic biomarker for lung adenocarcinoma-associated malignant pleural effusion.

Circulating cell‐free microRNAs (miRNAs) are potential cancer biomarkers. The aim of this study was to identify miRNAs that are differentially expressed between benign pleural effusion (BPE) and lung adenocarcinoma‐associated malignant pleural effusion (LA‐MPE). The expression level of cell‐free miRNA was investigated in 107 patients with pleural effusion. Microarrays were used to screen 160 miRNAs in a discovery set comprising 20 effusion samples (ten BPEs and ten LA‐MPEs). Real‐time quantitative reverse transcription polymerase chain reaction (qRT‐PCR) was used to validate the profiling results obtained for the discovery set and those obtained for a validation set comprising 42 BPEs and 45 LA‐MPEs. The area under the receiver operating characteristic curve (AUC) was used to evaluate the diagnostic performance of the identified miRNAs and other common tumor markers, such as carcinoembryonic antigen (CEA) and cytokeratin fragment (CYFRA) 21‐1. Microarray profiling showed that miR‐198 was significantly downregulated in LA‐MPE compared with BPE (p = 0.002). The miRNA microarray analysis results were confirmed by qRT‐PCR (p < 0.001) using the validation set. The AUCs for miR‐198, CEA and CYFRA 21‐1 in the validation set were 0.887, 0.898 and 0.836, respectively. The diagnostic performance of miR‐198 was comparable with that of CEA, but better than that of CYFRA 21‐1. The AUC for all three markers combined was 0.926 (95% confidence interval, 0.843–0.973) with a sensitivity of 89.2% and a specificity of 85.0%. The present study suggests that cell‐free miR‐198 from patients with pleural effusion might have diagnostic potential for differentiating LA‐MPE from BPE.

Cardiomyocyte Specific Deficiency of Serine Palmitoyltransferase Subunit 2 Reduces Ceramide but Leads to Cardiac Dysfunction

Background: The importance of de novo ceramide biosynthesis in maintaining cardiac function is unknown. Results: Deletion of serine palmitoyltransferase subunit Sptlc2 reduced cardiac ceramide and caused cardiac dysfunction associated with activation of ER stress. Conclusion: Reduced ceramide content by Sptlc2 deficiency does not protect against lipid toxicity associated with increased saturated acyl CoAs. Significance: Development of disease by lipotoxicity is caused by a number of changes in lipidome. The role of serine palmitoyltransferase (SPT) and de novo ceramide biosynthesis in cardiac ceramide and sphingomyelin metabolism is unclear. To determine whether the de novo synthetic pathways, rather than ceramide uptake from circulating lipoproteins, is important for heart ceramide levels, we created cardiomyocyte-specific deficiency of Sptlc2, a subunit of SPT. Heart-specific Sptlc2-deficient (hSptlc2 KO) mice had a >35% reduction in ceramide, which was limited to C18:0 and very long chain ceramides. Sphingomyelinase expression, and levels of sphingomyelin and diacylglycerol were unchanged. But surprisingly phospholipids and acyl CoAs contained increased saturated long chain fatty acids. hSptlc2 KO mice had decreased fractional shortening and thinning of the cardiac wall. While the genes regulating glucose and fatty acid metabolism were not changed, expression of cardiac failure markers and the genes involved in the formation of extracellular matrices were up-regulated in hSptlc2 KO hearts. In addition, ER-stress markers were up-regulated leading to increased apoptosis. These results suggest that Sptlc2-mediated de novo ceramide synthesis is an essential source of C18:0 and very long chain, but not of shorter chain, ceramides in the heart. Changes in heart lipids other than ceramide levels lead to cardiac toxicity.

Hydrolytic Pathway Protects against Ceramide-Induced Apoptosis in Keratinocytes Exposed to UVB

Although ceramides (Cers) are key constituents of the epidermal permeability barrier, they also function as apoptogenic signals for UVB irradiation-induced apoptosis in epidermal keratinocytes. As epidermis is continuously exposed to UV irradiation, we hypothesized that Cer hydrolysis protects keratinocytes from UVB-induced apoptosis by attenuating Cer levels. Both low-dose UVB (L-UVB) (< 35 mJ cm(-2)) and high-dose UVB (H-UVB) (> or = 45 mJ cm(-2)) irradiation inhibited DNA synthesis in cultured human keratinocytes, but apoptosis occurred only after H-UVB. Whereas Cer production increased after both L- and H-UVB, it normalized only in L-UVB-exposed keratinocytes, but remained elevated after H-UVB. Both acidic ceramidase (aCDase) and neutral ceramidase (nCDase) activities declined after L- and H-UVB, but returned to normal only in L-UVB cells, with decreased CDase activities or mRNA or protein levels being sustained in H-UVB cells. Inhibition of CDase using either a CDase inhibitor, N-oleoylethanolamine, or small interfering RNA (siRNA) (either to a- and/or n-CDase(s)) sensitized keratinocytes to L-UVB-induced apoptosis in parallel with further Cer accumulation. Blockade of sphingosine kinase 1 (SPHK1) (but not SPHK2) by siRNA also increased apoptosis in L-UVB keratinocytes, revealing that conversion of sphingosine to sphingosine-1-phosphate (S1P) further protects keratinocytes from UVB-induced cell death. Thus, Cer → sphingosine → S1Pmetabolic conversion protects against UVB-induced, Cer-mediated apoptosis in keratinocytes, but excessive UVB overwhelms this mechanism, thereby leading to keratinocyte apoptosis.

Green tomato extract attenuates high-fat-diet-induced obesity through activation of the AMPK pathway in C57BL/6 mice

Obesity is a risk factor for numerous metabolic disorders. Recently, natural compounds that may be beneficial for improving obesity have received increasing attention. In this study, we investigated whether red and green tomato extracts attenuate high-fat-diet-induced obesity in C57BL/6 mice. The mice were maintained on a normal diet (ND) or high-fat diet (HFD) for 4 weeks and then fed ND, HFD, HFD plus 2% red tomato extract (RTE) or HFD plus 2% green tomato extract (GTE) for 13 weeks. The weekly food intakes among the groups were not significantly different. Body weight of mice fed HFD plus GTE was significantly decreased to the level of mice fed ND, but the body weight was only slightly reduced in mice fed HFD plus RTE. Epididymal adipose tissue and liver weights were significantly decreased in mice fed HFD plus GTE compared to those in HFD. Serum total cholesterol and low-density lipoprotein cholesterol levels in mice fed GTE were modestly reduced, and liver total cholesterol level was strongly decreased in HFD plus GTE-fed mice compared to that in HFD-fed mice. Adenosine-monophosphate-activated protein kinase (AMPK) and acetyl-CoA carboxylase phosphorylation in liver from HFD plus GTE-fed mice was significantly elevated, and HMG-CoA reductase expression was also significantly decreased. GTE strongly decreased the expression of peroxisome proliferator-activated receptor gamma, CCAAT/enhancer-binding protein alpha and perilipin in the adipose tissue of mice fed HFD plus GTE. Our results indicate that the antiobesity effects of GTE may be associated with activation of the AMPK pathway.