title: Autophagy is involved in the initiation and progression of Graves' orbitopathy.
abstract: BACKGROUND
  Differentiation of orbital fibroblasts into mature adipocytes and subsequent accumulation of adipose tissue has been shown in the progression of Graves' orbitopathy (GO). Autophagy is involved in adipogenesis, but little is known about the role of autophagy in the initiation and progression of GO. The aim of this study is to investigate the role of autophagy in the pathogenesis of GO.
  
  
  METHODS
  Orbital adipose/connective tissue explants from patients with GO and from normal subjects, as well as isolated orbital fibroblasts, were analyzed. Adipogenesis was induced using differentiating medium with or without hydrogen peroxide, and autophagy was manipulated using bafilomycin A1 and Atg5-targeted short hairpin RNA (shRNA). Autophagosomes were identified by electron microscopy. Expression of autophagy-related genes and adipogenesis-related transcription factors were analyzed by real time reverse transcription-polymerase chain reaction and/or Western blot analysis. Lipid droplet accumulation was examined by Oil Red O staining.
  
  
  RESULTS
  Autophagic vacuoles were more abundant in GO cells than in non-GO cells (p<0.05). Expression of autophagy-related genes was significantly higher in GO tissues and cells than in their non-GO counterparts, respectively. Interleukin-1β increased LC3-II, p62, and Atg7 protein in GO cells. Autophagosome accumulation was shown at day 4 of adipogenesis and decreased by day 10, along with lipid droplet formation. Expression of LC3 and p62 proteins increased within 48 hours of differentiation and diminished gradually from day 4 to 10. Bafilomycin A1 treatment and Atg5 knockdown by shRNA inhibited lipid droplet accumulation and suppressed expression of adipogenic markers.
  
  
  CONCLUSIONS
  Autophagy was increased in GO tissue and cells compared to non-GO tissue and cells, suggesting that autophagy plays a role in GO pathogenesis. Autophagy manipulation may be a therapeutic target for GO.
authors: 1 Department of Ophthalmology, Institute of Vision Research, Yonsei University College of Medicine , Seoul, Korea.
authors: Jin Sook
authors: JS
authors: Yoon
authors: Hyun Jung
authors: HJ
authors: Lee
authors: Min Kyung
authors: MK
authors: Chae
authors: Eun Jig
authors: EJ
authors: Lee
mesh_terms: D017667:Adipocytes / Q000378:metabolism / Q000473:pathology*; D050156:Adipogenesis; D001343:Autophagy / Q000187:drug effects; D000071187:Autophagy-Related Protein 5; D016022:Case-Control Studies; D054338:Cell Transdifferentiation; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D005786:Gene Expression Regulation; D049970:Graves Ophthalmopathy / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D053583:Interleukin-1beta / Q000494:pharmacology; D018942:Macrolides / Q000494:pharmacology; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D013997:Time Factors; D046509:Tissue Culture Techniques; D014162:Transfection
chemical_list: C502778:ATG5 protein, human; D000071187:Autophagy-Related Protein 5; D053583:Interleukin-1beta; D018942:Macrolides; D008869:Microtubule-Associated Proteins; C040929:bafilomycin A1