title: Oxidation levels differentially impact melanocytes: low versus high concentration of hydrogen peroxide promotes melanin synthesis and melanosome transfer.
abstract: BACKGROUND
  UVB light can generate potentially harmful hydrogen peroxide (H(2)O(2)) in vivo, but it can also promote the beneficial proliferation and migration of melanocytes. The successful use of UVB monotherapy for treatment of vitiligo suggests that H(2)O(2) may have a biphasic effect on melanin synthesis and melanosome transfer.
  
  
  OBJECTIVE
  To study the beneficial role of H(2)O(2) on melanogenesis and melanosome transport in living melanocytes and keratinocytes.
  
  
  METHODS
  A co-culture system model was constructed using the primary human melanocytes and keratinocytes. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to determine cell proliferation, NaOH was used to determine the melanin content, and real-time PCR was used to determine tyrosinase expression. Western blot was used to determine Rab-27A and protease-activated receptor 2 (PAR-2) expression.
  
  
  RESULTS
  This study demonstrated that tyrosinase was activated by low concentrations of H(2)O(2) (≤0.3 mM); however, this activity was downregulated by high concentrations of H(2)O(2) (>0.3 mM). Activation of high levels of melanin synthesis was induced when cells were treated with low concentrations of H(2)O(2) (0.3 mM). Further observation using an in vitro co-culture system of fluorescein (carboxyfluorescein diacetate succinimidyl ester, CFDA-SE)-labeled melanocytes and keratinocytes indicated that melanosome transfer occurred in normal human epidermal melanocytes. Fluorescence microscopy revealed increased melanosome transfer into keratinocytes treated with 0.3 mM H(2)O(2) in the co-culture compared to the control. Examination of melanosomes in the keratinocytes by flow cytometry confirmed these results. Furthermore, treatment with H(2)O(2) (0.3 mM) upregulated the expression of Rab-27A and PAR-2, significant proteins involved in melanosome transfer, according to Western blot.
  
  
  CONCLUSIONS
  These results confirmed that low concentration levels of H(2)O(2) play a major role in the regulation of human pigmentation by increasing melanin synthesis and melanosome transfer.
authors.affiliation: Department of Dermatology, Huashan Hospital, FuDan University, Shanghai, China.
authors.forename: Luyan
authors.initials: L
authors.lastname: Tang
authors.forename: Jian
authors.initials: J
authors.lastname: Li
authors.forename: Xiao
authors.initials: X
authors.lastname: Lin
authors.forename: Wenyu
authors.initials: W
authors.lastname: Wu
authors.forename: Kefei
authors.initials: K
authors.lastname: Kang
authors.forename: Wenwen
authors.initials: W
authors.lastname: Fu
mesh_terms: D049109:Cell Proliferation; D002478:Cells, Cultured; D018920:Coculture Techniques; D015536:Down-Regulation; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D015603:Keratinocytes / Q000378:metabolism; D008543:Melanins / Q000096:biosynthesis*; D020460:Melanosomes / Q000378:metabolism*; D014442:Monophenol Monooxygenase / Q000378:metabolism; D044464:Receptor, PAR-2 / Q000096:biosynthesis; D020691:rab GTP-Binding Proteins / Q000096:biosynthesis; D000075745:rab27 GTP-Binding Proteins
chemical_list: D008543:Melanins; D044464:Receptor, PAR-2; D000075745:rab27 GTP-Binding Proteins; D006861:Hydrogen Peroxide; D014442:Monophenol Monooxygenase; C066694:RAB27A protein, human; D020691:rab GTP-Binding Proteins