Control of Trabecular Meshwork Cytoskeleton

描述（由申请人提供）：这是对no - od -09-058公告的竞争性修订，标题为“NIH宣布为竞争性修订申请提供恢复法案资金”。该资助的长期目标是确定细胞基质信号机制，可作为控制类固醇性青光眼的治疗靶点。青光眼导致视网膜神经节细胞不可逆转的丧失，全世界约有6700万人患有青光眼。它们通常与眼内压（IOP）水平升高有关，这是由于房水从小梁网（TM）流出减少所致。虽然已知有许多生理因素调节流出设施，但已成为流出设施重要调节机制的关键因素之一是TM的收缩特性。目前，还不清楚是什么分子事件调节了TM的收缩性。众所周知，细胞外基质（ECM）蛋白及其受体（整合素）的信号特性在调节细胞收缩性中起着关键作用，我们提出这些信号通路中一种或多种成分的激活可能是在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。在这一竞争性修订中，我们计划通过使用新的方法来研究整合素和g蛋白信号以及类固醇诱导的培养TM细胞肌动蛋白细胞骨架变化之间的关系，从而扩大原始资助的范围。先前的目的集中在整合素和syndecan信号通路上，以确定哪些整合素和syndecan参与了CLAN的形成，并证明这些通路的激活会影响培养前节段的流出设施。这些研究表明，23整合素和CD47 （IgG超家族成员和g蛋白偶联受体家族成员）可能是类固醇性青光眼中肌动蛋白网络组织转变为交联肌动蛋白网络（CLANs）的信号机制的一部分。为了帮助鉴定23整合素/CD47信号通路中可能被类固醇上调的成分，我们计划使用基因微阵列来鉴定23整合素和CD47信号通路中不受类固醇治疗调节的成分，并在培养的TM细胞中被23整合素和CD47激活。定量磷酸化蛋白质组学将用于确定23整合素/CD47信号通路中磷酸化状态的变化和蛋白质活性是否因类固醇处理而上调，以及是否与培养的TM细胞中23整合素/CD47激活CLAN形成相关。

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.