Genetic and non-genetic regulation mechanism of melanin production in the mammal

小鼠表皮黑色素细胞的发育过程受多种毛色基因的调控。其中，agouti (A)和pink-eyed amplification (p)基因是调控小鼠表皮黑色素细胞发育的重要基因。为了研究A基因在表皮黑素细胞发育中的作用，我们在无血清原代培养中比较了黑鼠（C57BL/10JHir-a/ A）及其同源黑鼠（C57BL/10JHir-a/ A）表皮黑素细胞的增殖和分化活性。针刺与黑色黑素细胞的增殖活性及对促分化因子的反应性无显著差异。然而，电镜下观察，培养的豚鼠黑素细胞比培养的黑色黑素细胞具有更多的III期黑素体。相反，与黑色黑色素细胞相比，针刺黑素细胞具有少量的IV期黑素小体。黑素体的成熟，从III期到IV期的更多的黑素细胞可能被抑制。测定黑鼠和刺鼠出生后不同时期表皮中真黑素和泛黑素的含量。出生后3.5和5.5 d A/A表皮中黑色素含量明显高于A/A表皮。RT-PCR分析结果显示，豚鼠mRNA在真皮中有表达，而在表皮中无表达。这些结果表明，A基因的产物（刺虱蛋白）是在真皮层产生的，然后该蛋白渗透到表皮，并诱导表皮的黑色素合成。在原代培养中，粉红色眼睛稀释的黑色素母细胞/黑色素细胞的增殖比黑色的黑色素母细胞/黑色素细胞受到极大的抑制。内皮素(ET)-1、ET-2和ET-3刺激培养的粉眼稀释黑素母细胞增殖。这些结果表明，p基因通过影响依赖于ETs功能的调控机制来影响小鼠表皮黑色素细胞的增殖活性。与黑色黑色素细胞相比，原代培养中粉眼稀释黑色素细胞的分化也受到极大抑制。为了解粉红眼稀释(p)基因对表皮黑色素细胞分化的作用机制，在基因型粉红眼稀释小鼠（C57BL/10JHir-p/p）原代培养起始的无血清培养基中添加酪氨酸酶底物l -酪氨酸（tyr），考察其作用。在用L-tyr培养的粉眼稀释黑素细胞中，所有阶段（I、II、III和IV）的黑素体数量都显著增加，尽管对照黑素细胞具有有限数量的I、II和III期黑素体。2 mM L-tyr培养的p/p细胞中真黑色素含量增加2倍。p/p黑色素细胞培养液中真黑素及其前体5,6-二羟基吲哚-2-羧酸（DHICA）含量明显高于p/p黑色素细胞。与p/p黑色素细胞相比，p/p黑色素细胞培养培养基中褐黑素及其前体5- s -半胱氨酸多巴（5-S-CD）含量没有显著升高。这些结果表明，原代培养的p/p黑色素细胞在过量的L-Tyr诱导下合成黑色素，但难以在黑色素小体中积累。少

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 … More 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