• Proceedings of the PSK Conference
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• 2000.10a
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• pp.206.1-206.1
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• 2000

• Proceedings of the PSK Conference
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• 2002.04a
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• pp.99.1-99.1
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• 2002

• Molecular & Cellular Toxicology
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• v.3 no.4
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• pp.273-281
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• 2007

Ceramide is involved in cell death as a lipid mediator of stress responses. In this study, we developed an improved method of ceramide quantification based on added synthetic ceramide and thin layer chromatography (TLC) separation, and applied to biological samples. Lipids were extracted from samples spiked with N-oleoyl-D-erythro-sphingosine ($C_{17}$ ceramide) as an internal standard. Ceramide was resolved by TLC, complexed with fatty-acidfree bovine serum albumin (BSA), and deacylated by ceramidase (CDase). The released sphingosine was derivatized with o-phthalaldehyde (OPA) and measured by high performance liquid chromatography (HPLC). The limit of detection for ceramide was about 1-2 pmol and the lower limit of quantification was 5 pmol. Ceramide recovery was approximately 86-93%. Ceramide concentrations were determined in biological samples including cultured cells, mouse tissues, and mouse and human plasma. TLC separation of ceramide provides HPLC chromatogram with a clean background without any interfering peaks and the enhanced solubility of ceramide by BSAceramide complex leads to the increased deacylation of ceramide. The use of an internal standard for the determination of ceramide concentration in these samples provides an accurate and reproducible analytical method, and this method can be applicable to diverse biological samples.

• BMB Reports
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• v.42 no.10
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• pp.685-690
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• 2009

Angiogenesis is essential for tumor growth and vascular endothelial cell growth factor (VEGF) plays a key role in this process. Conversely, sphingosine 1-phosphate (S1P) is a biologically active sphingolipid known to play a key role in cancer progression by regulating endothelial cell proliferation and migration. In this study, the authors found that S1P increases the level of VEGF mRNA in human umbilical vein endothelial cells (HUVECs) and immortalized HUVECs (iHUVECs). Additionally, S1P was found to increase VEGF promoter activity in MS-1 mouse pancreatic islet endothelial cells. Furthermore, a pharmacological inhibitory study revealed that $G_{\alpha i/o}$-mediated phospholipase C, Akt, Erk, and p38 MAPK signaling are involved in this S1P-induced expression of VEGF. A component of AP1 transcription factor is important for S1P-induced VEGF expression. Taken together, these findings suggest that S1P enhances endothelial cell proliferation and migrat ion by upregulating the expression of VEGF mRNA.

• Molecules and Cells
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• v.19 no.1
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• pp.39-45
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• 2005

RPK118 is a sphingosine kinase-1-binding protein that has been implicated in sphingosine 1 phosphate-mediated signaling. It contains a PX (phox homology) domain and two pseudo-kinase domains, and co-localizes with sphingosine kinase-1 on early endosomes. In this study we identified a novel RPK118-binding protein, PRDX3 (peroxiredoxin-3), by yeast two-hybrid screening. The interaction between these proteins was confirmed by pull-down assays and co-immunoprecipitation experiments. Deletion studies showed that RPK118 interacted with PRDX3 through its pseudokinase domains, and with early endosomes through its PX domain. Double immunofluorescence experiments demonstrated that PRDX3 co-localized with RPK118 on early endosomes in COS7 cells. PRDX3 is a member of the antioxidant family of proteins synthesized in the cytoplasm and functioning in mitochondria. Our findings indicate that RPK118 is a PRDX3-binding protein that may be involved in transporting PRDX3 from the cytoplasm to its mitochondrial site of function or to other membrane structures via endosome trafficking.

• BMB Reports
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• v.52 no.3
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• pp.220-225
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• 2019

We have identified a mechanism to diminish the proliferative capacity of cells during cell expansion using human adipose-derived stromal cells (hAD-SCs) as a model of replicative senescence. hAD-SCs of high-passage numbers exhibited a reduced proliferative capacity with accelerated cellular senescence. Levels of key bioactive sphingolipids were significantly increased in these senescent hAD-SCs. Notably, the transcription of sphingosine kinase 1 (SPHK1) was down-regulated in hAD-SCs at high-passage numbers. SPHK1 knockdown as well as inhibition of its enzymatic activity impeded the proliferation of hAD-SCs, with concomitant induction of cellular senescence and accumulation of sphingolipids, as seen in high-passage cells. SPHK1 knockdown-accelerated cellular senescence was attenuated by co-treatment with sphingosine-1-phosphate and an inhibitor of ceramide synthesis, fumonisin $B_1$, but not by treatment with either one alone. Together, these results suggest that transcriptional down-regulation of SPHK1 is a critical inducer of altered sphingolipid profiles and enhances replicative senescence during multiple rounds of cell division.

• 대한약리학회:학술대회논문집
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• 2006.10a
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• pp.105.1-105.1
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• 2006

• Biomolecules & Therapeutics
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• v.28 no.6
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• pp.537-541
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• 2020

Sphingosine-1-phosphate (S1P) and its receptors have been implicated in atopic dermatitis. S1P₂ was found to function as a proallergic receptor, while its antagonist JTE-013 was found to suppress allergic asthma in mice. Topical application of JTE-013 has not been investigated in an in vivo model of atopic dermatitis. Therefore, the therapeutic potential of JTE-013 topical application was evaluated by the use of a 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis mouse model. DNCB-induced inflammation and mast cell accumulation in skin tissues were significantly suppressed by topical JTE-013 treatment in BALB/c mice. DNCB-induced increase of lymph nodes sizes and elevated inflammatory cytokines (IL-4, IL-13, IL-17, and IFN-γ) in lymph nodes were also significantly reduced by the JTE-013 treatment. Elevated serum levels of IgE were significantly suppressed by the topical treatment of JTE-013. In summary, the topical treatment of JTE-013 S1P₂ antagonist suppressed DNCB-induced atopic dermatitis symptoms and immune responses. These results suggested JTE-013 as a potential therapeutic agent for atopic dermatitis.

• Biomolecules & Therapeutics
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• v.29 no.1
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• pp.52-57
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• 2021

Fumonisin B1 (FB1) structurally resembles sphingolipids and interferes with their metabolism leading to sphingolipid dysregulation. We questioned if FB1 could exacerbate liver or kidney toxicities in glutathione peroxidase 1 (Gpx1) and catalase (Cat) knockout mice. While higher serum levels of thiobarbituric acid reactive substances (TBARS) and sphinganine (Sa) were measured in Gpx1/Cat knockout mice (Gpx1/Cat KO) than wild type mice after 5 days of FB1 treatment, serum levels of alanine aminotransferase (ALT), sphingosine-1 phosphate (So-1-P), and sphinganine-1 phosphate (Sa-1-P) were found to be relatively low. Although Sa was highly elevated in Gpx1/Cat KO mice and wild mice, lower levels of So and Sa were found in both the kidney and liver tissues of Gpx/Cat KO mice than wild type mice after FB1 treatment. Paradoxically, FB1-induced cellular apoptosis and necrosis were hastened under oxidative stress in Gpx1/Cat KO mice.

• Journal of Life Science
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• v.21 no.2
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• pp.183-193
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• 2011

Migration and differentiation of mesenchymal stem cells are crucial for tissue regeneration in response to injury. Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates a variety of biological processes, including proliferation, survival, differentiation and motility. In the present study, we determined the role of S1P in migration and differentiation of human bone marrow-derived mesenchymal stem cells (BMSCs). S1P stimulated migration of BMSCs in a dose- and time-dependent manner, and pre-incubation of the cells with pertussis toxin completely abrogated S1P-induced migration, suggesting involvement of Gi-coupled receptors in S1P-induced cell migration. S1P elicited elevation of intracellular concentration of $Ca^{2+}$ ($[Ca^{2+}]_i$) and pretreatment with VPC23019, an antagonist of $S1P_1/S1P_3$, blocked S1P-induced migration and increase of $[Ca^{2+}]_i$. Small interfering RNA-mediated knockdown of endogenous $S1P_1$ attenuated S1P-induced migration of BMSCs. Furthermore, S1P treatment induced expression of $\alpha$-smooth muscle actin ($\alpha$-SMA), a smooth muscle marker, and pretreatment with VPC23019 abrogated S1P-induced $\alpha$-SMA expression. S1P induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), and pretreatment of cells with SB202190, an inhibitor of p38 MAPK, or adenoviral overexpression of a dominant-negative mutant of the p38 MAPK blocked S1P-induced cell migration and $\alpha$-SMA expression. Taken together, these results suggest that S1P stimulates migration and smooth muscle differentiation of BMSCs through an $S1P_1$-p38 MAPK-dependent mechanism.