INDUCTION OF TERMINAL DIFFERENTIATION IN HUMAN MELANOCYTES

人黑素细胞末端分化的诱导

• 批准号: 6299369
• 负责人: ESTELA E MEDRANO
• 金额: $ 21.16万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-15 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6299369
• 关键词: cAMP response element binding protein; cell cycle; cell differentiation; cell growth regulation; cell senescence; cholera toxin; environmental stressor; gene induction /repression; guanine nucleotide binding protein; human tissue; melanocyte; nucleotide analog; oncoprotein p21; proliferating cell nuclear antigen; tissue /cell culture; ultraviolet radiation

Melanocytes are pigment producing cells located in the basal layer of the epidermis, the bulb of the hair sheaths, the choroid of the eye and the leptomeninges of the brain. As humans age, there is a progressive decline in melanocyte number and function in the skin, and hair and eye, while nothing is known about the melanocyte's fate in the brain. Loss of melanocytes in the ear and skin is associated with partial deafness and imbalance, and with skin cancers after the 5th decade of age respectively. Since the skin is constantly exposed to the environment, loss of epidermal melanocytes may be accentuated by environmental stress (ultraviolet radiation, heat, cold) which may result in early terminal differentiation/senescence of these cells. We were the first to develop an "in vitro" model for human adult epidermal melanocytes which allowed us to define a terminally differentiated melanocyte. We demonstrated that the potent cAMP inducer cholera toxin, induced a dramatic increase in melanin deposition in melanosomes, morphological changes and irreversible withdrawal from the cell cycle. We propose to extend these studies and characterize molecular pathways involved in melanocyte proliferation, terminal differentiation and senescence. We will analyze terminal differentiation by defining the molecular consequences of steady, high levels of cAMP and melanin content by the use of cAMP analogues and by the constitutive expression of activated Galphas proteins, kinetics of activation by phosphorylation of the cAMP responsive element binding protein CREB and the repressor CREM, and binding of CREB to the cAMP enhancer element. Using the yeast two-hybrid system, we will define novel, melanocyte specific CREB-protein interactions between CREB and CREB-repressors in proliferating and terminally differentiated melanocytes. We will determine also the contribution of the melanogenic enzymes tyrosinase, TRP-1, TRP-2 and Pmel 17 to the induction of terminal differentiation and their possible use as markers for terminal differentiation/senescence. In addition we will define similarities/differences between terminally differentiated and senescent melanocytes, by analyzing the expression and function of the cell cycle inhibitors p21/p27/p16/p15 and the proliferating cell nuclear antigen (PCNA) in both phenotypes, their modulation by ultraviolet radiation and by making stable melanocyte cell strains capable of inducible expression of sense and antisense sequences of these inhibitors. The studies proposed will be the basis for future studies aimed at understanding the role of melanocyte aging in the skin and also in other organs like the eye and the brain.

黑素细胞是位于基底层的产生色素的细胞 表皮、毛鞘球、眼睛脉络膜和 大脑的软脑膜。 随着人类年龄的增长，有一个进步 皮肤、头发和眼睛的黑素细胞数量和功能下降， 而对于大脑中黑色素细胞的命运却一无所知。 损失 耳朵和皮肤中的黑色素细胞与部分耳聋和 失衡，五岁后患皮肤癌 分别。 由于皮肤经常暴露在环境中， 环境压力可能会加剧表皮黑素细胞的损失 （紫外线、热、冷）可能导致早期终末期 这些细胞的分化/衰老。 我们是第一个开发的 人类成人表皮黑色素细胞的“体外”模型 我们来定义终末分化的黑素细胞。 我们展示了 有效的 cAMP 诱导剂霍乱毒素，诱导急剧增加 黑素体中黑色素沉积、形态变化和 不可逆转地退出细胞周期。 我们建议延长这些 研究并表征黑素细胞涉及的分子途径 增殖、终末分化和衰老。 我们将分析 通过定义分子结果来进行终末分化 使用 cAMP 稳定、高水平的 cAMP 和黑色素含量 类似物和激活的Galphas的组成型表达 蛋白质，cAMP 磷酸化激活动力学 反应元件结合蛋白CREB和阻遏物CREM，以及 CREB ​​与 cAMP 增强子元件的结合。 使用酵母双杂交 系统，我们将定义新型黑素细胞特异性 CREB ​​蛋白 CREB ​​和 CREB ​​抑制因子在增殖和增殖过程中的相互作用 终末分化的黑素细胞。 我们还将确定 黑色素生成酶酪氨酸酶、TRP-1、TRP-2 和 Pmel 的贡献 17 终末分化的诱导及其可能的用途 终末分化/衰老的标记。 此外我们还将 定义终末分化和终末分化之间的相似/差异 通过分析衰老黑素细胞的表达和功能 细胞周期抑制剂 p21/p27/p16/p15 和增殖细胞核 两种表型中的抗原（PCNA），它们受紫外线的调节 辐射并通过使稳定的黑素细胞株能够 这些有义和反义序列的诱导表达 抑制剂。 提出的研究将作为未来研究的基础 旨在了解黑色素细胞老化在皮肤中的作用，以及 其他器官，如眼睛和大脑。