FAK Signals Controlling Endothelial Cell Survival and Motility

描述(申请人提供)：整合素与细胞外基质接触，通过促进细胞黏附、控制细胞运动和调节细胞存活，在血管生物学中发挥重要作用。由于跨膜整合素受体不具有内在催化活性，整合素产生的信号必须由相关蛋白介导。粘着斑激酶(FAK)是一种与整合素结合并被整合素激活的细胞质酪氨酸激酶。在血管生成过程中，FAK还作用于VEGFR等生长因子受体的下游，促进血管内皮细胞的运动和存活。FAK功能对小鼠EC细胞生物学至关重要，因为完整或条件性的EC特异性FAK基因敲除会导致胚胎死亡表型，并伴有血管形态发生的缺陷。然而，由于FAK既是一种支架蛋白，也是一种信号转导蛋白，基因敲除研究并不能提供区分FAK作用的这些特征的机械性见解。在这里，我们将建立在FAK的一个新的角色上，它能够定位于核，并在细胞应激条件下作为P53肿瘤抑制因子的负调节因子发挥作用。我们在原代小鼠和人成纤维细胞中发现，FAK通过N端FAK FERM(Band 4.1，Ezrin，Radixin，Moesin Homology)结构域介导的核转位和增强依赖MDM2的P53泛素化，以一种不依赖于激酶的方式灭活P53。我们认为，FAK FERM核关联通过保持低水平的P53来促进细胞存活。由于我们发现FAK既定位于整合素相关的信号转导部位，又定位于内皮细胞的核，这一提议将检验FAK分别通过差异激酶非依赖性(Ferm-核)和激酶依赖性(FAK-整合素)机制控制EC存活和运动-形态发生的总体假设。AIM-1将使用慢病毒介导的FAK shRNA击倒和功能获得FAK重新表达的研究来测试核FAK是否以一种不依赖于激酶的方式促进EC存活，以及整合素相关的FAK激活是否需要促进细胞运动。AIM-2将通过对FAK转基因小鼠的分析和利用FAK-/-小鼠胚胎干细胞刺激分化为内皮细胞的体外功能获得重建研究，确定FAK信号在发育中的血管生成-血管生成中的作用。AIM-3将在成年小鼠中使用可诱导的和有条件的EC特异性FAK基因敲除或药物抑制FAK活性来确定FAK在生长因子刺激的血管生成中的作用。这三个目的也是为了区分内皮细胞中FAK表达缺失或FAK催化活性失活之间的信号差异。这些研究结果将为设计治疗心血管疾病的药物提供重要的分子见解。公共卫生相关性：在抗癌研究中，正在采用特定的靶向抗血管生成治疗策略，并将其与常规的抗增殖化疗和放射治疗抗肿瘤方案结合使用。我们的研究将为FAK在控制内皮细胞运动和存活中的作用提供重要和新的见解。这些研究结果可能为FAK的药物抑制剂作为抗肿瘤和抗血管生成疗法的未来发展提供原理证明。

DESCRIPTION (provided by applicant): Integrin contacts with extracellular matrix play fundamental roles in vascular biology by promoting cell- adhesion, controlling cell motility, and regulating cell survival. As transmembrane integrin receptors do not possess intrinsic catalytic activity, signals generated by integrins must be mediated by associated proteins. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that associates with and is activated by integrins. FAK also acts downstream of growth factor receptors such as VEGFr to promote endothelial cell (EC) motility and survival during the processes of angiogenesis. FAK function is essential for mouse EC cell biology as complete or conditional EC-specific FAK knockouts yield embryonic lethal phenotypes with defects in blood vessel morphogenesis. However, as FAK works as both a scaffolding protein and as a signaling kinase, knockout studies do not provide mechanistic insights in distinguishing these features of FAK action. Here, we will build upon a novel role for FAK in its ability to become nuclear-localized and function as a negative regulator of the p53 tumor suppressor under conditions of cellular stress. We found that FAK inactivates p53 in a kinase-independent manner through N-terminal FAK FERM (band 4.1, ezrin, radixin, moesin homology) domain-mediated nuclear translocation and enhancement of Mdm2-dependent p53 ubiquitination using primary mouse and human fibroblasts. We propose that FAK FERM nuclear-association promotes cell survival by keeping p53 levels low. As we find that FAK localizes to both integrin-associated signaling sites and to the nucleus of ECs, this proposal will test the overall hypothesis that FAK controls EC survival and motility- morphogenesis through differential kinase-independent (FERM-nuclear) and kinase-dependent (FAK-integrin) mechanisms, respectively. Aim-1 will use lentiviral-mediated FAK shRNA knockdown and gain-of-function FAK re-expression studies to test whether nuclear FAK promotes EC survival in a kinase-independent manner and whether integrin-associated FAK activation is required to promote cell motility. Aim-2 will determine the role of FAK signaling during developmental vasculogenesis-angiogenesis through the analysis of FAK knock-in mice and in vitro gain-of-function reconstitution studies using FAK-/- mouse embryonic stem cells stimulated to differentiate into ECs. Aim-3 will use an inducible and conditional EC-specific FAK knockout in adult mice or pharmacological inhibition of FAK activity to determine the role of FAK in growth factor-stimulated angiogenesis. All three aims are also designed to differentiate the signaling differences between loss of FAK expression or inactivation of FAK catalytic activity in ECs. The results of these studies will provide important molecular insights for designing therapeutic agents for treatment of cardiovascular diseases. PUBLIC HEALTH RELEVANCE: In anti-cancer research, specific targeted anti-angiogenic therapeutic strategies are being adopted and used in combination with conventional anti-proliferative chemotherapeutic and radiotheraputic anti-tumor regimens. Our studies will provide important and novel insights into FAK function in controlling endothelial cell motility and survival. The results of these studies may serve as proof-of-principal for the future development of pharmacological inhibitors to FAK as anti-tumor and anti-angiogenic therapies.