Regulation of Melanocyte Development and Differentiation

黑素细胞发育和分化的调节

• 批准号: 7292188
• 负责人: thomas j hornyak
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7292188
• 关键词: Regulation; Melanocyte; Development; Differentiation

During the past year, we have worked to complete research on two topics in the regulation of melanocyte development and differentiation as the lab undergoes a transition to focus upon melanoma research: (1) understanding the role of neurofibromin in melanocyte development and differentiation, and (2) examining survival of otic melanocytes in the Microphthalmia-white heterozygous mouse, a model for human Waardenburg and Tietz syndromes. Our work on developing an inducible system for gene expression in vivo in murine melanocytes, described in this section last year, is described under Project 2 this year because it is more relevant to our work on malignant melanoma than to work we are completing on melanocyte differentiation.Our progess is as follows:(1) We have worked further this year to define differences between the activity of neurofibromin on the Kit signalling pathway during melanocyte development and during melanocyte differentiation. To test in a more direct way the hypothesis that neurofibromin regulates melanogenic gene expression via its effects upon Ras signalling, we have successfully optimized a system to purify primary mouse melanocytes by flow cytometry. This approach has permitted us to compare directly the effect of neurofibromin haploinsufficiency upon melanogenic gene expression without accounting for variables introduced by the presence of other types of cells in culture. Results from these experiments show that neurofibromin haploinsufficiency, which is associated with Erk activation in cultured melanocytes, increases melanogenic gene expression in primary murine melanocytes. Consistent with this finding, pharmacologic inactivation of Erk activation by inhibition of the upstream kinase Mek decreases melanogenic gene expression in both wild-type and neurofibromin-haploinsufficient melanocytes. These results, in addition to those obtained from primary melanocytes in mixed primary cultures, support a role for Ras-dependent signaling in melanogenic gene expression. Additional results from the genetic crossing of Nf1-deficient mice with murine coat color mutations in the Kit and Mitf genes support a role for neurofibromin in the regulation of melanocyte development in vivo. (2) Our observation that the survival of otic melanocytes, as opposed to cutaneous melanocytes, is selectively compromised in Microphthalmia-white heterozygous mice prompted us to investigate the effect of skin-derived factors, such as stem cell factor (SCF)/Kit ligand, endothelin-1 (ET-1), and basic FGF that might promote otic melanocyte survival in this genetic background. A set of in vitro experiments using cochlear organ culture suggest that a combination of factors can promote survival of these otic melanocytes, providing additional insight into the determinants of melanocyte survival in cells partially deficient in the melanocyte transcription factor Mitf. Going further, organ culture experiments with isolated stria vascularis that has been isolated from neonatal mice show that the combination of ET-1 and SCF can promote survival of these Mitf-deficient melanocytes in the strial environment. These results show that environmental factors in or near the murine follicle are likely to be important for promoting the survival of Mitf-deficient melanocytes in this location. This finding may be relevant to the survival of melanocytes in patients with the congenital disorders of pigmentation Waardenburg syndrome and Tietz syndrome.

在过去的一年中，我们致力于完成黑素细胞发育和分化调控的两个主题的研究，因为实验室经历了一个过渡，专注于黑色素瘤研究：（1）了解神经纤维蛋白在黑素细胞发育和分化中的作用，以及（2）检查小眼白杂合子小鼠中耳黑素细胞的存活率，这是一种人类Waardenburg和Tietz综合征的模型。我们的工作在开发一个诱导系统，在体内基因表达在小鼠黑素细胞，在这一节中描述了去年，今年的项目2，因为它是更相关的工作，我们对恶性黑色素瘤比工作，我们正在完成的黑素细胞分化。我们的成果如下：（1）我们今年进一步工作，以确定在黑素细胞发育和黑素细胞分化过程中的Kit信号通路的神经纤维蛋白的活性之间的差异。为了以更直接的方式测试神经纤维蛋白通过其对Ras信号传导的影响调节黑素生成基因表达的假设，我们成功地优化了通过流式细胞术纯化原代小鼠黑素细胞的系统。这种方法使我们能够直接比较神经纤维蛋白单倍不足对黑素基因表达的影响，而不考虑培养物中其他类型细胞的存在所引入的变量。来自这些实验的结果显示，与培养的黑素细胞中的Erk活化相关的神经纤维蛋白单倍不足增加原代鼠黑素细胞中的黑素生成基因表达。与这一发现相一致的是，通过抑制上游激酶Mek使Erk活化的药理学失活降低了野生型和神经纤维蛋白单倍不足黑素细胞中的黑素生成基因表达。这些结果，除了那些从原代黑素细胞在混合的原代培养物中获得的，支持Ras依赖的信号转导在黑素基因表达中的作用。来自Nf 1缺陷小鼠与Kit和Mitf基因中的鼠毛色突变的遗传杂交的其他结果支持神经纤维蛋白在体内调节黑素细胞发育中的作用。(2)我们的观察，耳黑素细胞的生存，而不是皮肤黑素细胞，是选择性损害小眼白杂合子小鼠促使我们调查皮肤衍生因子，如干细胞因子（SCF）/试剂盒配体，内皮素-1（ET-1），碱性成纤维细胞生长因子，可能会促进耳黑素细胞的生存在这种遗传背景下的影响。使用耳蜗器官培养物的一组体外实验表明，多种因素的组合可以促进这些耳黑素细胞的存活，从而为部分缺乏黑素细胞转录因子Mitf的细胞中黑素细胞存活的决定因素提供了额外的见解。更进一步，用从新生小鼠分离的分离的血管纹进行的器官培养实验表明，ET-1和SCF的组合可以促进这些Mitf缺陷的黑素细胞在哺乳动物环境中的存活。这些结果表明，在小鼠卵泡或附近的环境因素可能是重要的促进生存的Mitf缺陷的黑色素细胞在这个位置。这一发现可能与先天性色素沉着Waardenburg综合征和Tietz综合征患者的黑素细胞存活有关。