title: Expression of cytosolic NADP(+)-dependent isocitrate dehydrogenase in melanocytes and its role as an antioxidant.
abstract: BACKGROUND
  Cytosolic NADP(+)-dependent ICDH (IDPc) has an antioxidant effect as a supplier of NADPH to the cytosol, which is needed for the production of glutathione.
  
  
  OBJECTIVE
  To evaluate the expression of IDPc in melanocytes and to elucidate its role as an antioxidant.
  
  
  METHODS
  The knock-down of IDPc expression in immortalized mouse melanocyte cell lines (melan-a) was performed using the short interfering RNA (siRNA)-targeted gene silencing method. After confirming the silencing of IDPc expression with mRNA and protein levels, viability, apoptosis and necrosis, as well as ROS production in IDPc-silenced melanocytes were monitored under conditions of oxidative stress and non-stress. Also, the ratio of oxidized glutathione to total glutathione was examined, and whether the addition of glutathione recovered cell viability, decreased by oxidant stress, was checked.
  
  
  RESULTS
  The expression of IDPc in both primary human melanocytes and melan-a cells was confirmed by Western blot and RT-PCR. The silencing of IDPc expression by transfecting IDPc siRNA in melan-a cells was observed by Western blotting and real-time RT-PCR. IDPc knock-down cells showed significantly decreased cell viability and an increased number of cells under apoptosis and necrosis. IDPc siRNA-treated melanocytes demonstrated a higher intensity of DCFDA after the addition of H(2)O(2) compared with scrambled siRNA-treated melanocytes, and a lower ratio of reduced glutathione to oxidized glutathione were observed in IDPc siRNA transfected melanocytes. In addition, the addition of glutathione recovered cell viability, which was previously decreased after incubation with H(2)O(2).
  
  
  CONCLUSIONS
  This study suggests that decreased IDPc expression renders melanocytes more vulnerable to oxidative stress, and IDPc plays an important antioxidant function in melanocytes.
authors.affiliation: Department of Dermatology & Cutaneous Biology Research Institute, Yonsei University College of Medicine, Seoul, South Korea.
authors.forename: Ji Young
authors.initials: JY
authors.lastname: Kim
authors.forename: Jae Yong
authors.initials: JY
authors.lastname: Shin
authors.forename: Miri
authors.initials: M
authors.lastname: Kim
authors.forename: Seung-Kyung
authors.initials: SK
authors.lastname: Hann
authors.forename: Sang Ho
authors.initials: SH
authors.lastname: Oh
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000378:metabolism*; D017209:Apoptosis; D015153:Blotting, Western; D002460:Cell Line; D002470:Cell Survival; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D008544:Melanocytes / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D051379:Mice; D009336:Necrosis; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection; D014820:Vitiligo / Q000201:enzymology
chemical_list: D000975:Antioxidants; D016877:Oxidants; D017382:Reactive Oxygen Species; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+); C543588:IDH1 protein, human; D005978:Glutathione