title: All-trans retinoic acid induces differentiation and apoptosis of murine melanocyte precursors with induction of the microphthalmia-associated transcription factor.
abstract: The effects of all-trans retinoic acid on the differentiation and proliferation of immature melanocyte precursors were studied. NCC-melb4 cells are an immortal cloned cell line established from mouse neural crest cells using a single-cell cloning method. These cells were positive for tyrosinase-related protein 1, tyrosinase-related protein 2 and KIT, but were negative for tyrosinase and had no dihydroxyphenylalanine reaction. They contained only stage I melanosomes without any melanosomes in more advanced stages. After treatment with all-trans retinoic acid, many of the cells became tyrosinase- and dihydroxyphenylalanine-reaction-positive, changed from polygonal to dendritic in shape, and had stage III to IV melanosomes. These findings indicate that treatment with all-trans retinoic acid induced the differentiation of NCC-melb4 cells. Reverse transcription polymerase chain reaction analysis revealed a marked increase in expression of microphthalmia-associated transcription factor mRNA after all-trans retinoic acid treatment, suggesting that microphthalmia-associated transcription factor may be the key molecule in this event. Enhanced expression of protein kinase Calpha following treatment with all-trans retinoic acid was also demonstrated. The proliferation of NCC-melb4 cells was inhibited by all-trans retinoic acid in a dose-dependent manner. Increased apoptosis after all-trans retinoic acid treatment was observed by electron microscopy, the TUNEL method, DNA fragmentation assay, and flow cytometry. All-trans retinoic acid upregulated caspase-3 and downregulated bcl-2. Electron microscopy showed that apoptotic cells contained melanosomes of advanced stages, suggesting that mature melanocytes may tend to undergo apoptosis after all-trans retinoic acid treatment. This study provides important clues towards understanding the roles and working mechanisms of retinoic acids in melanocyte development and melanogenesis.
authors.affiliation: Department of Dermatology, St. Marianna University School of Medicine, Kawasaki, Japan. hifu@marianna-u.ac.jp
authors.forename: Hidenori
authors.initials: H
authors.lastname: Watabe
authors.forename: Yoshinao
authors.initials: Y
authors.lastname: Soma
authors.forename: Masaru
authors.initials: M
authors.lastname: Ito
authors.forename: Yoko
authors.initials: Y
authors.lastname: Kawa
authors.forename: Masako
authors.initials: M
authors.lastname: Mizoguchi
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004790:Enzyme Induction / Q000187:drug effects; D007527:Isoenzymes / Q000378:metabolism; D008544:Melanocytes / Q000166:cytology* / Q000648:ultrastructure; D051379:Mice; D051739:Microphthalmia-Associated Transcription Factor; D008854:Microscopy, Electron; D011493:Protein Kinase C / Q000378:metabolism; D051571:Protein Kinase C-alpha; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000648:ultrastructure; D014157:Transcription Factors; D014212:Tretinoin / Q000494:pharmacology*
chemical_list: D004268:DNA-Binding Proteins; D007527:Isoenzymes; D051739:Microphthalmia-Associated Transcription Factor; C497235:Mitf protein, mouse; D019253:Proto-Oncogene Proteins c-bcl-2; D014157:Transcription Factors; D014212:Tretinoin; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases