title: The Role of Sphingosine-1-Phosphate in Adipogenesis of Graves' Orbitopathy.
abstract: OBJECTIVE
  To investigate the action of sphingosine-1-phosphate (S1P) in adipocyte differentiation of orbital fibroblasts and determine its putative role in the pathogenesis of Graves' orbitopathy (GO).
  
  
  METHODS
  Primary preadipocyte orbital fibroblast cultures were stimulated for adipogenesis. Real-time PCR was performed to evaluate the expression of S1P receptor mRNA. To evaluate the effect of S1P and S1P receptor blockers (W146 and FTY720) on adipocyte differentiation, cultures were exposed to each receptor blocker for the first 4 days of the differentiation period. Differentiated cells were stained with Oil Red O, and the production of peroxisome proliferator activator gamma (PPARγ) and CCAAT-enhancer-binding proteins (C/EBP) α and β were determined by Western blot analysis.
  
  
  RESULTS
  Sphingosine-1-phosphate receptor 1, 2, and 3 mRNA expression levels were significantly higher in GO tissue samples than non-GO. Sphingosine-1-phosphate receptor 1 through 5 mRNA expression was significantly increased during the 10 days of adipogenesis. Sphingosine-1-phosphate treatment increased the size and number of adipocytes, and increased the expression of adipogenic transcriptional regulators. Treatment with S1P1 receptor inhibitor (W146) for 4 days after induction of adipogenesis attenuated adipocyte differentiation. Sphingosine-1-phosphate receptor blocker also decreased reactive oxygen species (ROS) production in GO orbital fibroblasts and H2O2-stimulated HO-1 production in GO orbital fibroblasts. S1P1 receptor inhibitor reduced the number of adipocytes and suppressed the accumulation of lipid droplets induced by 10 μM H2O2 or 2% cigarette smoke extract (CSE) treatment.
  
  
  CONCLUSIONS
  Sphingosine-1-phosphate could play a role in orbital adipocyte differentiation of GO. Modulation of S1P actions may provide a therapeutic target for the treatment of GO.
authors.affiliation: Department of Ophthalmology International St. Mary's Hospital, Catholic Kwandong University College of Medicine, Incheon, Korea 2Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea.
authors.forename: Sung Eun
authors.initials: SE
authors.lastname: Kim
authors.affiliation: Myung-gok Eye Research Institute at Kim's Eye Hospital, Konyang University College of Medicine, Nonsan, Korea.
authors.forename: Joon H
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea.
authors.forename: Min Kyoung
authors.initials: MK
authors.lastname: Chae
authors.affiliation: Department of Endocrinology, Institute of Endocrine Research, Yonsei University College of Medicine, Seoul, Korea.
authors.forename: Eun Jig
authors.initials: EJ
authors.lastname: Lee
authors.affiliation: Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea.
authors.forename: Jin Sook
authors.initials: JS
authors.lastname: Yoon
mesh_terms: D050156:Adipogenesis / Q000502:physiology*; D000328:Adult; D015153:Blotting, Western; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D005260:Female; D005347:Fibroblasts / Q000502:physiology; D000068876:Fingolimod Hydrochloride / Q000494:pharmacology; D049970:Graves Ophthalmopathy / Q000378:metabolism*; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D008246:Lysophospholipids / Q000502:physiology*; D008297:Male; D008875:Middle Aged; D009915:Orbit / Q000166:cytology; D047495:PPAR gamma / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D049349:Receptors, Lysosphingolipid / Q000037:antagonists & inhibitors / Q000235:genetics; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000502:physiology
chemical_list: D022782:CCAAT-Enhancer-Binding Protein-beta; D022762:CCAAT-Enhancer-Binding Proteins; C515848:CEBPA protein, human; C545012:CEBPB protein, human; D007166:Immunosuppressive Agents; D008246:Lysophospholipids; D047495:PPAR gamma; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D049349:Receptors, Lysosphingolipid; C060506:sphingosine 1-phosphate; D000068876:Fingolimod Hydrochloride; D013110:Sphingosine