Control of Trabecular Meshwork Cytoskeleton

描述（由申请人提供）：这是一个竞争性修订，以响应通知NOT-OD-09-058，标题为“NIH宣布恢复法案资金的可用性，用于竞争性修订申请”。该资助的长期目标是确定可用作治疗靶点的细胞基质信号传导机制，以控制类固醇诱导的青光眼。青光眼导致视网膜神经节细胞的不可逆损失，影响全球约6700万人。它们通常与眼内压（IOP）水平升高相关，这是由于小梁网（TM）的房水流出减少。尽管已知许多生理因素调节流出功能，但作为流出功能的重要调节机制出现的关键因素之一是TM的收缩特性。目前，还不清楚是什么分子事件调节TM的收缩性。细胞外基质（ECM）蛋白及其受体（整合素）的信号传导特性在调节细胞收缩性中起着关键作用，我们提出这些信号传导途径中一种或多种组分的激活可能是导致类固醇诱导的青光眼中观察到的交联肌动蛋白网络（CLANs）形成的原因。在这个竞争性的修订，我们计划利用新的方法来研究整合素和G-蛋白信号和类固醇诱导的肌动蛋白细胞骨架的培养TM细胞的变化之间的关系，以扩大原来的授权范围。先前的目的集中在整合素和多配体蛋白聚糖信号通路，以确定哪些整合素和多配体蛋白聚糖参与CLAN形成，并证明这些通路的激活影响培养的眼前节的流出设施。这些研究表明，23整合素和CD 47（IgG超家族和G蛋白偶联受体家族的成员）可能是在类固醇诱导的青光眼中将肌动蛋白网络的组织改变为交联肌动蛋白网络（CLAN）的信号传导机制的一部分。为了帮助鉴定23整合素/CD 47信号通路中可能被类固醇上调的组分，我们计划使用基因微阵列来鉴定23整合素和CD 47信号通路中不被类固醇治疗调节并被培养的TM细胞中的23整合素和CD 47激活的组分。定量磷酸蛋白质组学将用于确定类固醇处理是否上调了23整联蛋白/CD 47信号传导途径中蛋白质的磷酸化状态和活性的变化，并与培养的TM细胞中23整联蛋白/CD 47对CLAN形成的激活相关。 公共卫生相关性：青光眼是美国第二大常见的致盲原因，也是非洲裔美国人最常见的致盲原因。由于缺乏明确的细胞培养系统，试图了解这种疾病的原因受到阻碍。该项目的目标是确定类固醇诱导的青光眼（SIG）期间激活的信号通路，从而确定潜在的治疗靶点以减少SIG。

DESCRIPTION (provided by applicant): This is a competitive revision in response to notice NOT-OD-09-058 entitled "NIH announces the availability of recovery act funds for competitive revision applications" The long-term objective of this grant is to identify cell-matrix signaling mechanisms that can be used as therapeutic targets to control steroid induced glaucoma. The glaucomas, which lead to irreversible loss of retinal ganglion cells, affect approximately 67 million people worldwide. They are commonly associated with elevated levels of intraocular pressure (IOP) due to a reduction in aqueous humor outflow from the trabecular meshwork (TM). Although a number of physiological factors are known to regulate outflow facility, one of the key factors that have emerged as an important regulatory mechanism for outflow facility is the contractile properties of the TM. At the present, it is unclear what molecular events regulate contractility in the TM. It is well established that the signaling properties of extracellular matrix (ECM) proteins and their receptors (integrins) play a critical role in regulating cell contractility and we proposed that activation of one or more of the components in these signaling pathways may be responsible for the formation of cross-linked actin networks (CLANs) observed in steroid induced glaucoma. In this competitive revision we plan to expand the scope of the original grant by utilizing new methodology to examine the relationship between integrin and G-protein signaling and steroid induced changes in the actin cytoskeleton of cultured TM cells. The prior aims focused on integrin and syndecan signaling pathways in order to determine which integrins and syndecans were involved in CLAN formation and demonstrating that activation of these pathways influence outflow facility in cultured anterior segments. These studies indicated that both 23 integrins and CD47, a member of both the IgG superfamily and the G-protein- coupled receptor family, may be part of the signaling mechanism that alters the organization of the actin network into cross- linked actin networks (CLANs) in steroid induced glaucoma. To aid in the identification of components in this 23 integrin/CD47 signaling pathway that may be upregulated by steroids we plan to use gene microarrays to identify components of the 23 integrin and CD47 signaling pathways that are unregulated by steroid treatment and activated by both 23 integrin and CD47 in TM cells in culture. Quantitative phosphoproteomics will be used to determine if changes in the phosphorylation state and hence activity of proteins in the 23 integrin/CD47 signaling pathway(s) are upregulated by steroid treatment and correlate with the activation of CLAN formation by 23 integrin/CD47 in TM cells in culture. PUBLIC HEALTH RELEVANCE: Glaucoma is the second most common cause of blindness in the U.S. and the most common cause of blindness among African-Americans. Attempts to understand the cause(s) of this disease have been hampered by the lack of well defined cell culture systems. The goal of this project is to identify the signaling pathway(s) activated during steroid induced glaucoma (SIG) so we identify potential therapeutic targets to reduce SIG.