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，随着实验室经历着重点关注黑色素瘤研究的过渡，我们一直致力于对两个主题进行调节中的两个主题的研究：（1）了解神经纤维蛋白在黑素细胞发育和分化中的作用，（2）研究在微生物中散发性绿色蛋白质的生存的生物蛋白质，并研究了生物刺激性的老鼠盐味， Tietz综合征。我们在本节中描述的在本节中描述的鼠类黑素细胞中基因表达的诱导型系统的工作已在今年的项目2下进行了描述，因为它与我们在恶性黑色素瘤方面的工作更相关，而不是在黑素细胞上完成的工作。黑素细胞发育和黑色素细胞分化。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色素基因表达的假设，我们成功地优化了一个系统，以通过流式细胞仪纯化原代小鼠黑色素细胞。这种方法使我们能够直接比较神经纤维蛋白单倍症对黑色素生成基因表达的影响，而不会考虑培养中其他类型的细胞引入的变量。这些实验的结果表明，与培养的黑素细胞中ERK活化有关的神经纤维蛋白单倍氨基弥倍不足会增加原代鼠黑素细胞中黑色素生成基因的表达。与这一发现一致，通过抑制上游激酶MEK对ERK激活的药理失活，可降低野生型和神经纤维纤维蛋白 - 荷兰蛋白富含黑色素细胞的黑色素生成基因表达。除了从混合原代培养物中的原代黑素细胞获得的结果外，这些结果还支持RAS依赖性信号在黑色素生成基因表达中的作用。 NF1缺陷型小鼠在试剂盒和MITF基因中使用鼠涂层颜色突变的遗传交叉的进一步结果支持神经纤维蛋白在体内的黑素细胞发育中的作用。 （2）我们观察到，在微观粒细胞 - 白色杂合小鼠中选择性地妥协了眼球黑素细胞的存活，而不是皮肤黑色素细胞，促使我们研究了皮肤衍生因素的效果遗传背景。一组使用人工耳蜗培养的体外实验表明，多种因素可以促进这些耳黑细胞的存活，从而提供了对黑素细胞转录因子MITF部分缺陷的细胞中黑素细胞存活决定因素的更多见解。进一步的是，已经从新生儿小鼠中分离出的分离的血管中的器官培养实验表明，ET-1和SCF的组合可以促进这些缺陷型黑素细胞在曲线环境中的生存。这些结果表明，鼠卵泡中或附近的环境因素对于在该位置促进MITF缺陷型黑素细胞的存活可能很重要。这一发现可能与色素沉着先天性疾病Waardenburg综合征和Tietz综合征患者的黑素细胞生存有关。