Intercellular Communication in the Eye Lens

项目摘要 晶状体是一种无血管器官，严重依赖于运输系统的网络来输送营养和其他 粗大的晶状体纤维和排泄物的基本成分。分化的核纤维细胞永远不会翻转 氧化应激是老年性白内障的主要原因。连接蛋白(CX)-形成缝隙连接通道 对晶状体的代谢动态平衡起着至关重要的作用。除了缝隙连接外，连接蛋白还形成 半通道，允许分子在细胞和细胞外环境之间的运输。缝隙连接蛋白 在核纤维中被截断，这种裂解随着年龄和氧化应激而增加。然而，几乎没有什么是 已知由全长和截断形成的半通道的功能重要性和调节 晶状体纤维中的连接蛋白。我们的初步研究表明，皮质晶状体纤维中的半管被激活。 通过机械负荷，介导葡萄糖和谷胱甘肽的摄取，并表现出自我保护作用 氧化损伤。因此，我们假设(1)由CX50/Cx46形成的半通道被激活 通过机械刺激皮质晶状体纤维和介导营养/抗氧化剂的吸收，这些营养物质/抗氧化剂 通过缝隙连接传递给内皮质和核纤维，以维持细胞的动态平衡和活性； 由全长和截短CX50/Cx46形成的功能性半通道显示出自我保护作用 氧化损伤。目的是了解连接蛋白半通道在晶状体纤维中的独特新作用。 细胞在正常生理和病理条件下(如氧化应激)。在这项建议中，首先，我们 将确定机械负荷激活的连接蛋白半通道是否是主要的运输途径 促进营养物质和抗氧化剂进入皮质晶状体纤维，以及整合素在调节中的作用 半条腿。其次，我们将测试皮质纤维中的半脑管吸收的营养物质/抗氧化剂是否 通过缝隙连接传递到内部皮质和核纤维，以满足细胞的代谢需求 动态平衡，保护内纤维细胞。第三，我们将确定由两个全长的 截短的CX50/Cx46提供了一种抵抗氧化损伤的自我保护机制。其中一个主要的 创新之处在于，这项提议旨在揭示连接蛋白半突形成的一个新角色 全长和截短连接蛋白在促进晶状体纤维代谢功能和保护纤维细胞中发挥作用 防止氧化损伤。我们将使用已建立的晶状体原代培养和逆转录病毒在晶状体中的表达 一种新发展的显性阴性体外方法，以及基因敲除小鼠模型。这是我们的 希望阐明晶状体纤维中连接蛋白通道的机制作用将提供更好的 了解晶状体在正常和病理条件下的一般内稳态过程。这个 我们的研究结果将是重要的，因为这些发现将使新的和有益的 对新的治疗策略和确定治疗晶状体疾病的药物靶点的贡献，如 老年性白内障。

Project Summary Lens, an avascular organ, relies heavily on a network of transporting systems to deliver nutrients and other essential components to bulky lens fibers and excrete wastes. Differentiated nuclear fiber cells never turn over and oxidative stress is a major cause of age related cataracts. Connexin (Cx)-forming gap junction channels play an essential role for the metabolic homeostasis of the lens. Besides gap junctions, connexins form hemichannels, permitting transport of molecules between the cell and its extracellular environment. Connexins are truncated in nuclear fibers and this cleavage is increased with aging and oxidative stress. However, little is known regarding the functional importance and regulation of hemichannels formed by full-length and truncated connexins in lens fibers. Our preliminary studies indicate that hemichannels in cortical lens fibers are activated by mechanical loading, mediate uptake of glucose and glutathione, and exhibit self-protective roles againist oxidative damages. As such, we hypothesize that (1) The hemichannels formed by Cx50/Cx46 are activated by mechanical stimulation in cortical lens fibers and mediate uptake of nutrients/antioxidants, which are delivered to inner cortical and nuclear fibers via gap junctions to maintain cell homeostasis and viability; (2) Functional hemichannels formed by both full length and truncated Cx50/Cx46 exhibit self-protection against oxidative damages. The goal is to understand distinctive, new roles of connexin hemichannels in lens fiber cells under normal physiological and pathological (e.g. oxidative stress) conditions. In this proposal, first, we will determine if connexin hemichannels activated by mechanical loading serve as a major transport pathway facilitating the uptake of nutrients and antioxidants into cortical lens fibers, and the role of integrins in regulating hemichannels. Second, we will test if nutrients/antioxidants uptaken by hemichannels in cortical fibers are delivered through gap junctions to inner cortical and nuclear fibers to meet metabolic needs of cell homeostasis and protect inner fiber cells. Third, we will determine if hemichannels formed by both full-length and truncated Cx50/Cx46 offer a self-protective mechanism against oxidative insult. One of the major innovative aspects is that this proposal aims to uncover a novel role that connexin hemichannels formed by full-length and truncated connexins play in facilitating metabolic function of lens fibers and protecting fiber cells against oxidative damages. We will use established lens primary cultures and retroviral expression in lens in situ, a newly developed dominant negative ex vivo approach, and knockout mouse models. It is our expectation that elucidation of mechanistic roles of connexin channels in lens fibers will provide a better understanding of the general homeostatic process of lens under normal and pathological conditions. The outcomes of our research will be significant because the discoveries should make novel and beneficial contributions to new therapeutic strategies and identify drug targets for the treatment of lens disorders such as age-related cataracts.