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

小鼠表皮黑素细胞的发育过程受多种毛色基因的调控。在这些基因中，agglutinase（A）和pink-eyed dilution（p）基因是调控小鼠表皮黑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、增殖活性和表皮黑色素细胞的分化与黑色素细胞的分化相比，（C57 BL/10 JHir-a/a）及其同源系无血清原代培养小鼠（C57 BL/10 JHir-A/A）。黑素细胞和黑素细胞的增殖活性以及对分化刺激因子的反应性无显著差异。然而，培养的黑素细胞具有大量的III期黑素体比培养的黑色黑素细胞的电镜观察。相反，agglomerate黑素细胞具有少量的阶段IV黑素体相比，黑色黑素细胞。黑素体成熟 关于我们 可以抑制黑素细胞中从III期到IV期的ES。此外，还测定了生后不同日龄的黑、白小鼠表皮中真黑素和褐黑素的含量。生后3.5和5.5天A/A表皮中的褐黑素含量远高于a/a表皮。RT-PCR分析表明，agglutinin mRNA在真皮中表达，而不是在表皮中。这些结果表明，A基因的产物（agglutinin蛋白）是在真皮中产生的，然后该蛋白渗透到表皮中，诱导表皮中的褐黑素合成，与黑色黑素细胞相比，粉红色眼睛的成黑素细胞/黑素细胞在原代培养中的增殖受到明显抑制。用内皮素（ET）-1、ET-2和ET-3刺激培养的红眼稀释成黑素细胞增殖。这些结果表明，p基因通过影响依赖于p53功能的调节机制，影响小鼠表皮黑素细胞的增殖活性。与黑素细胞相比，原代培养的粉眼稀释黑素细胞的分化也受到明显抑制。为了了解红眼稀释（p）基因对表皮黑素细胞分化的作用机制，从同源红眼稀释小鼠（C57 BL/10 JHir-p/p）的原代培养开始，将酪氨酸酶的底物L-酪氨酸（tyr）添加到无血清培养基中，并检查其效果。在用L-tyr培养的粉红色眼睛的稀释黑素细胞中，所有阶段（I、II、III和IV）的黑素体的数量显著增加，尽管对照黑素细胞具有有限数量的阶段I、II和III黑素体。用2 mM L-tyr培养的p/p细胞中真黑素的含量增加2倍。p/p黑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）的含量明显高于P/P黑素细胞。而p/p黑素细胞培养液中的褐黑素及其前体5-S-半胱氨酰多巴（5-S-CD）含量与P/P黑素细胞相比无明显增加。这些结果表明，原代培养的p/p黑素细胞被过量的L-Tyr诱导合成黑素，但难以在黑素体中积累。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