| Co-Investigator(Kenkyū-buntansha) |
HIROBE Tomohisa National Institute of Radiological Sciences, Radiation Hazards Research Group, Team Leader, 放射線障害研究グループ, チームリーダ (10111238)
WAKAMATSU Kazumasa Fujita Health University, School of Health Sciences, Assoc. Professor, 衛生学部, 助教授 (80131259)
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| Research Abstract |
The process of development of mouse epidermal melanocytes is regulated by numerous coat color genes. Of these genes, agouti (A) and pink-eyed dilution (p) genes are important ones for the regulation of the development of mouse epidermal melanocytes.
To investigate the role of the A gene in the development of epidermal melanocytes, the activity of proliferation and, differentiation of epidermal melanocytes was compared from black (C57BL/10JHir-a/a) and its congenic agouti (C57BL/10JHir-A/A) mice in serum-free primary culture. There was no significant difference between agouti and black melanocytes in the proliferative activity as well as the reactivity to differentiation-stimulating factors. However, cultured agouti melanocytes possessed numerous stage III melanosomes than cultured black melanocytes under the electron microscopic observations. On the contrary, agouti melanocytes possessed a small number of stage IV melanosomes as compared to black melanocytes. The maturation of melanosom
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es from stage III to IV in agouti melanocytes may be inhibited. Moreover, contents of eumelanin and pheomelanin in the epidermis of black and agouti mice were measured at various days after birth. The content of pheomelanin in A/A epidermis at 3.5 and 5.5 days after birth was much greater than that of a/a epidermis. RT-PCR analysis showed that agouti mRNA expression was observed in the dermis, but not in the epidermis. These results suggest that the product of A gene (agouti protein) is produced in the dermis, then the protein permiates to the epidermis, and induces the pheomelanin synthesis there.
The proliferation of pink-eyed dilution melanoblasts/melanocytes in primary culture was greatly inhibited as compared to black melanoblasts/melanocytes. The proliferation of pink-eyed dilution melanoblasts in culture was stimulated by endothelin (ET)-1, ET-2, and ET-3. These results suggest that p gene exerts its influence on the proliferative activities of mouse epidermal melanoblasts by affecting the regulatory mechanisms dependent on the function of ETs, The differentiation of pink-eyed dilution melanocytes in primary culture was also greatly inhibited as compared to black melanocytes. To understand the mechanism of the action of the pink-eyed dilution (p) gene on the differentiation of epidermal melanocytes, L-tyrosine (tyr), the substrate of tyrosinase, was supplemented to a serum-free culture medium from initiation of primary culture of congenic pink-eyed dilution mice (C57BL/10JHir-p/p), and its effects were examined. In pink-eyed dilution melanocytes cultured with L-tyr, the numbers of melanosomes in all stages (I, II, III, and IV) were dramatically increased, though control melanocytes possessed the limited numbers of stage I, II, and III melanosomes. The content of eumelanin in p/p cells cultured with 2 mM L-tyr was increased 2-fold. Contents of eumelanin and its precursor, 5,6-dihydroxyindole-2-carboxylic acid (DHICA) of cultured media in p/p melanocytes were much more greatly increased than in P/P melanocytes. However, contents of pheomelanin and its precursor, 5-S-cysteinyldopa (5-S-CD) of cultured media in p/p melanocytes were not increased as compared with P/P melanocytes. These results suggest that p/p melanocytes in the primary culture are induced to synthesize melanins by excess L-Tyr, but difficult to accumulate them in melanosomes. Less
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