Regulation of Lens Cell Coupling and Differentiation

晶状体细胞耦合和分化的调节

• 批准号: 6860984
• 负责人: LINDA S MUSIL
• 金额: $ 30.08万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6860984
• 关键词: SDS polyacrylamide gel electrophoresis; biological signal transduction; bone morphogenetic proteins; cell cell interaction; cell differentiation; cell growth regulation; chickens; fibroblast growth factor; gap junctions; growth factor; immunoprecipitation; laboratory rat; lens; membrane channels; mitogen activated protein kinase; neural cell adhesion molecules; organ culture; protein metabolism; western blottings

DESCRIPTION (provided by applicant): Defects in lens differentiation or homeostasis can result in cataract, the major cause of visual impairment in humans. An understanding of the mechanisms that control lens development and maintain lens transparency is therefore an essential part of addressing a major international health issue. Gap junctions participate in joining lens epithelial and fiber cells into a metabolic and ionic syncytium essential for lens clarity. The highest level of gap junction-mediated intercellular coupling (GJIC) in the lens is at the equator, the region where epithelial cells differentiate into secondary fibers. The applicant has previously developed a serum-free system to culture primary embryonic chick lens cells from the peripheral epithelium, the cell type that in vitro most closely recapitulates the in vivo processes of epithelial-to-fiber differentiation and fiber-type gap junction formation. We have reported that in this system, FGF (either recombinant or from vitreous humor) upregulates the expression of the fiber differentiation markers examined and increases GJIC in parallel but at least partially independent processes. FGF activity was shown to diffuse out of intact vitreous bodies, the major in vivo reservoir of growth factors for the lens. These and additional studies led to a novel model of how FGF-mediated sustained activation of the ERK MAP kinase contributes to the asymmetry of GJIC believed to be essential for lens clarity (Le and Musil 2001 J Cell Biol 154:197-216). Preliminary results presented in this application provide the first evidence that BMP also upregulates fiber marker expression and (synergistically with FGF) GJIC in cultured lens cells, and that vitreous humor contains a diffusible BMP-like activity. Other studies support the novel hypothesis that epithelial-to-fiber differentiation may be modulated by a unique postranslational modification (polysialylation) of the cell adhesion molecule NCAM. The goals of the proposed studies are: (1) to investigate the signal transduction pathways by which FGF and/or BMP upregulate GJIC and fiber marker expression in cultured chick and rat lens cells, and elucidate the role of vitreous humor-derived BMP in these processes in vivo; (2) to study how FGF and/or BMP upregulate GJIC without increasing gap junction channel number; (3) to determine the role of NCAM polysialylation in epithelial-to-fiber differentiation; and (4) to utilize my expertise in gap junction formation and degradation to investigate how the turnover of lens gap junctions is decreased from a t1/2 less than or equal too 5 h to being stable for years upon differentiation of epithelial cells to mature fibers. These studies will provide new insights into how growth factor signaling and cell-cell interactions contribute to the unique structure and function of the lens.

描述（由申请人提供）：晶状体分化或体内平衡缺陷可导致白内障，这是人类视力障碍的主要原因。因此，了解控制晶状体发育和保持晶状体透明度的机制是解决重大国际卫生问题的重要组成部分。间隙连接参与将晶状体上皮细胞和纤维细胞连接成晶状体清晰度所必需的代谢和离子合胞体。晶状体中间隙连接介导的细胞间偶联（GJIC）的最高水平是在赤道，上皮细胞分化为次级纤维的区域。申请人之前已经开发了一种无血清系统来培养来自外周上皮的原代胚鸡晶状体细胞，这种细胞类型在体外最接近地概括了体内上皮向纤维分化和纤维型间隙连接形成的过程。我们已经报道，在这个系统中，FGF（无论是重组的还是来自玻璃体的）上调所检测的纤维分化标志物的表达，并在平行但至少部分独立的过程中增加GJIC。完整的玻璃体是晶状体生长因子在体内的主要储存库，FGF的活性可以从玻璃体中扩散出去。这些和其他的研究导致了一个新的模型，关于fgf介导的ERK MAP激酶的持续激活如何促进GJIC的不对称性，而GJIC被认为是晶状体清晰度所必需的（Le and Musil 2001 J Cell Biol 154:197-216）。本应用的初步结果提供了第一个证据，证明BMP也上调培养晶状体细胞中纤维标记物的表达和（与FGF协同）GJIC，并且玻璃体房颤含有弥漫性BMP样活性。其他研究支持了一种新的假设，即上皮细胞向纤维的分化可能是由细胞粘附分子NCAM的独特翻译后修饰（多唾液化）调节的。本研究的目的是：(1)研究FGF和/或BMP在鸡和大鼠晶状体细胞中上调GJIC和纤维标记物表达的信号转导途径，并阐明玻璃体体液来源的BMP在这些过程中的作用；(2)研究FGF和/或BMP如何在不增加间隙连接通道数量的情况下上调GJIC；(3)确定NCAM多唾液化在上皮细胞向纤维分化中的作用；(4)利用我在间隙连接形成和降解方面的专业知识，研究在上皮细胞分化为成熟纤维后，晶状体间隙连接的周转如何从少于或等于5小时的t1/2减少到稳定数年。这些研究将为生长因子信号和细胞间相互作用如何影响晶状体的独特结构和功能提供新的见解。