title: ADAM protease inhibitors reduce melanogenesis by regulating PMEL17 processing in human melanocytes.
abstract: BACKGROUND
  ADAMs (a disintegrin and metalloprotease) are a family of proteases involved in ectodomain shedding that play a role in various biological processes such as cell adhesion and migration. ADAM10 and ADAM17 are suggested to be involved in pigmentary disorders.
  
  
  OBJECTIVE
  We examined the effect of ADAM protease inhibitors on the modulation of melanogenesis in normal human epidermal melanocytes (NHEM).
  
  
  METHODS
  NHEMs and B16F10 treated with ADAM protease inhibitors were analyzed. AlamarBlue cell proliferation assay, melanin content assay, tyrosinase activity assay, Western blotting analysis, electron microscopic analysis, and RNA interference were employed.
  
  
  RESULTS
  In NHEMs, melanin content was reduced by treatment with ADAM protease inhibitors. The inhibitors did not change the protein expression of tyrosinase, TRP-1, and MITF. In B16F10 cells, treatment of the cells with ADAM protease inhibitor diminished the α-MSH-induced increase in melanin content. Electron microscopy showed that the number of fibrillar and mature melanosomes was significantly reduced and that the vacuolar compartments were filled with dense unstructured aggregates after treatment with ADAM protease inhibitors. We therefore focused on the processing of PMEL17, a melanosomal glycoprotein that forms a fibrillar matrix on which melanin gets deposited. Proteolytic processing of PMEL17 is required to form functional fibrils during melanogenesis. Recently, γ-secretase and β-site amyloid precursor protein-cleaving enzyme 2 (BACE2) were found to cleave PMEL17. We found that ADAM protease inhibitors exerted effects on the processing of C-terminal and N-terminal fragments of PMEL17. Using BACE2 siRNA and γ-secretase inhibitor, we showed that ADAM protease inhibitor affected PMEL17 processing in a γ-secretase and BACE2-independent mechanism.
  
  
  CONCLUSIONS
  Several proteases, including ADAM proteases, can contribute to the formation of fibrils and their assembly into sheets in melanosomes.
authors.affiliation: Department of Dermatology, Yamagata University, Faculty of Medicine, 2-2-2 Iida-Nishi, Yamagata 990-9585, Japan. Electronic address: kawaguch@med.id.yamagata-u.ac.jp.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Kawaguchi
authors.affiliation: Department of Dermatology, Yamagata University, Faculty of Medicine, 2-2-2 Iida-Nishi, Yamagata 990-9585, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Hozumi
authors.affiliation: Department of Dermatology, Yamagata University, Faculty of Medicine, 2-2-2 Iida-Nishi, Yamagata 990-9585, Japan.
authors.forename: Tamio
authors.initials: T
authors.lastname: Suzuki
mesh_terms: D051722:ADAM Proteins / Q000037:antagonists & inhibitors*; D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D004151:Dipeptides / Q000494:pharmacology; D006801:Humans; D007370:Interferon Type I / Q000378:metabolism; D008543:Melanins / Q000096:biosynthesis*; D008544:Melanocytes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D008546:Melanoma, Experimental / Q000378:metabolism* / Q000648:ultrastructure; D020460:Melanosomes / Q000187:drug effects / Q000648:ultrastructure; D051739:Microphthalmia-Associated Transcription Factor / Q000378:metabolism; D008854:Microscopy, Electron; D014442:Monophenol Monooxygenase / Q000378:metabolism; D011257:Pregnancy Proteins / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D000521:alpha-MSH / Q000378:metabolism; D058951:gp100 Melanoma Antigen / Q000378:metabolism*
chemical_list: D004151:Dipeptides; D007370:Interferon Type I; C497234:MITF protein, human; D008543:Melanins; D051739:Microphthalmia-Associated Transcription Factor; C419410:N-(N-(3,5-difluorophenacetyl)alanyl)phenylglycine tert-butyl ester; C548637:PMEL protein, human; D011257:Pregnancy Proteins; D011480:Protease Inhibitors; D034741:RNA, Small Interfering; D058951:gp100 Melanoma Antigen; C020947:interferon tau; D000521:alpha-MSH; D014442:Monophenol Monooxygenase; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; C509284:BACE2 protein, human; D051722:ADAM Proteins
keywords: ADAM protease; Melanocyte; Melanogenesis; Melanosome; PMEL17