Homeostatic Roles of Connexin43 in Response to DNA Damage

描述(申请人提供)：DNA损伤可以显著改变细胞的功能和活力，因此，未修复的DNA损伤的积累被认为是衰老过程中器官功能恶化的主要影响因素。然而，细胞也以DNA修复以外的许多其他方式对DNA损伤做出反应，以维持其功能和生存能力。尤其是参与细胞间通讯的基因，如连接蛋白，已被确定为细胞对多种应激源做出反应的关键组成部分。尽管如此，遗传毒性应激后细胞间通讯被改变的分子机制还没有被描述。这个F30提案的总体目标是了解角膜内皮细胞对DNA损伤的反应，这些细胞的泵和屏障功能对于角膜透明是必不可少的，并且在体内表现出与年龄相关的DNA损伤的退化和积累。利用CE细胞的体外模型，我们最近观察到DNA损伤诱导剂暴露后缝隙连接蛋白Cx43(Cx43)的显著变化。为了解释这些变化背后的细胞机制(S)，我们认为DNA损伤和通过酪蛋白激酶-1增量(CK1？)进行的细胞通讯之间存在潜在的联系，CK1？既被DNA损伤激活，也是一种必不可少的Cx43激酶。我们的总体假设是，细胞对DNA损伤的保护性反应包括稳定缝隙连接的细胞间通讯，这种通讯是(A)通过Cx43表达的变化和CK1？的位点特异性磷酸化来调节的，以及(B)维持生存和功能所必需的。这将通过两个特定的目的进行测试，以确定1)DNA损伤介导连接蛋白43(Cx43)修饰的机制(S)和2)DNA损伤过程中这些变化的生理后果(S)，包括对CE细胞存活和功能的影响。该项目的结果将进一步加深我们对DNA损伤的应激反应的理解，并为如何潜在地增强某些老化组织的生存能力提供见解。人体细胞、组织和器官逐渐退化的衰老过程与DNA损伤的积累有关。作为对这种压力的反应，细胞的存活或死亡取决于其应对这种损伤的能力。这项建议研究了一种潜在的应对DNA损伤的机制，涉及改变受损细胞相互沟通的方式，以维持其功能和生存能力。

DESCRIPTION (provided by applicant): DNA damage can significantly alter cell function and viability, thus, the accumulation of unrepaired DNA damage is believed to be a major influence in the deterioration of organ function in aging. However, cells also respond to DNA damage in many other ways beyond DNA repair in order to maintain their function and viability. In particular, genes involved in intercellular communication, such as connexins, have been identified as key components in cellular responses to numerous stressors. Despite this, the molecular mechanisms by which intercellular communication is modified after genotoxic stress have not been described. The overall goal of this F30 proposal is to understand the responses to DNA damage in corneal endothelial (CE) cells whose pump and barrier functions are essential for corneal transparency and which in vivo display age-related degeneration and accumulation of DNA damage. Using an in vitro model of CE cells, we have recently observed significant changes in the gap junction protein connexin-43 (Cx43) after exposure to DNA damage-inducing agents. To explain the cellular mechanism(s) behind these changes, we consider a potential link between DNA damage and cell communication via casein kinase-1 delta (CK1?), which is both activated by DNA damage and an essential Cx43 kinase. Our overall hypothesis is that a protective cellular response to DNA damage involves stabilization of gap junction intercellular communication which is (a) mediated by changes in Cx43 expression and site-specific phosphorylation by CK1?), and (b) required for maintenance of viability and function. This will be tested with two specific aims that will determine 1) the mechanism(s) by which DNA damage mediates modification of connexin-43 (Cx43) and 2) the physiological consequence(s) of these changes during DNA damage including effects of CE cell viability and function. The results of this project will further our understanding of stress responses to DNA damage and provide insights into how the viability of certain aging tissues may be potentially enhanced. The aging process in which the body's cells, tissues, and organs progressively deteriorate has been associated with the accumulation of DNA damage. In response to such stress, a cell may survive or perish depending on its ability to cope with that damage. This proposal studies a potential coping mechanism to DNA damage that involves changes in how damaged cells communicate with one another to maintain their function and viability.