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众多毛色基因的调控。为探讨A基因在小鼠表皮黑素细胞发育中的作用，比较了无血清原代培养条件下黑鼠(C57BL/10JHir-a/a)及其同源基因刺鼠(C57BL/10JHir-A/A)表皮黑素细胞的增殖和分化活性。刺鼠黑素细胞与黑色黑素细胞的增殖活性及对分化刺激因子的反应性无明显差异。但在电子显微镜下，培养的刺鼠黑素细胞比培养的黑色黑素细胞具有更多的III期黑素小体。相反，与黑色黑素细胞相比，刺鼠黑素细胞具有少量的IV期黑素小体。黑素体…的成熟刺鼠黑素细胞从III期到IV期更多的ES可能被抑制。此外，还测定了出生后不同天数黑鼠和雄鼠表皮中真黑素和褐黑素的含量。生后3.5天和5.5天，A/A表皮中的褐黑素含量明显高于A/A表皮。RT-PCR分析表明，刺参mRNA在真皮中有表达，而在表皮中没有表达。这些结果表明，A基因的产物(刺鼠蛋白)在真皮中产生，然后该蛋白进入表皮，并在那里诱导黑素的合成。原代培养的粉眼稀释型黑素母细胞/黑素细胞的增殖比黑色黑素母细胞/黑素细胞显著抑制。内皮素(ET)-1、ET-2、ET-3刺激培养的粉红眼稀释型黑素母细胞增殖。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素母细胞的增殖活性，与黑素细胞相比，粉眼稀释型黑素细胞在原代培养中的分化也受到极大的抑制。为了了解粉眼稀释(P)基因对表皮黑素细胞分化的作用机制，从同源粉眼稀释小鼠(C57BL/10JHir-p/p)的原代培养开始，将酪氨酸酶底物L酪氨酸(Tyr)添加到无血清培养液中，并检测其作用效果。经L-酪氨酸处理的粉眼稀释型黑素细胞，I、II、III、IV期黑素小体数量显著增加，而对照组黑素细胞I、II、III期黑素小体数量较少。经2 mM L-酪氨酸处理后，p/p细胞真黑素含量增加2倍。P/P黑素细胞培养上清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。但与P/P黑素细胞相比，P/P黑素细胞培养上清液中的褐黑素及其前体5-S-半胱氨酸多巴(5-S-CD)的含量并未增加。这些结果表明，过多的L-酪氨酸可诱导原代培养的p/p黑素细胞合成黑素，但难以在黑素小体中积累。较少

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