Regulation of Lens Cell Coupling and Differentiation

描述(申请人提供)：晶状体分化或内环境平衡缺陷可导致白内障，这是人类视力受损的主要原因。因此，了解控制晶状体发育和维持晶状体透明度的机制是解决重大国际健康问题的重要组成部分。缝隙连接参与将晶状体上皮细胞和纤维细胞连接成新陈代谢和离子合胞，对晶状体的清晰度至关重要。晶状体中缝隙连接介导的细胞间偶联(GJIC)水平最高的是赤道，这是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，从外周上皮培养原代胚胎鸡晶状体细胞，这种细胞类型在体外最接近于体内上皮到纤维分化和纤维型缝隙连接形成的过程。我们已经报道，在这个系统中，成纤维细胞生长因子(重组的或来自玻璃体液的)上调所检测的纤维分化标志物的表达，并以平行但至少部分独立的过程增加GJIC。研究表明，成纤维细胞生长因子活性从完整的玻璃体扩散出去，玻璃体是晶状体生长因子的主要体内储存库。这些和其他研究导致了一个新的模型，即成纤维细胞生长因子介导的ERK蛋白激酶的持续激活如何导致被认为对晶状体透明度至关重要的GJIC的不对称性(Le和Musil 2001 J Cell Biol 154：197-216)。本申请中提出的初步结果首次证明，BMP还上调了培养的晶状体细胞中纤维标记物的表达和(与成纤维细胞生长因子协同作用)GJIC，并且玻璃体体液中含有可扩散的BMP样活性。其他研究支持这一新的假设，即上皮到纤维的分化可能受到细胞黏附分子NCAM独特的翻译后修饰(聚唾液酸化)的调节。本研究的目的是：(1)研究成纤维细胞生长因子和/或骨形态发生蛋白上调培养的鸡和大鼠晶状体细胞GJIC和纤维标志物表达的信号转导途径，并阐明玻璃体液来源的骨形态发生蛋白在体内这些过程中的作用；(2)研究成纤维细胞生长因子和/或骨形态发生蛋白如何在不增加缝隙连接通道数目的情况下上调GJIC；(3)确定NCAM多聚体在上皮向纤维分化中的作用；以及(4)利用我在缝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，晶状体缝隙连接的周转率如何从小于或等于5小时的T1/2下降到多年的稳定。这些研究将为生长因子信号和细胞-细胞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。

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.