Nuclear translocation of urokinase/nucleolin complexes

描述（由申请人提供）：血管重构是动脉粥样硬化和其他常见心血管疾病（如动脉瘤形成、再狭窄和高血压）发病机制的重要组成部分。纤溶酶原激活剂(PA)/纤溶酶系统在动脉粥样硬化中的作用已通过多项研究得到证实，这些研究表明uPA、tPA及其抑制剂PAI-1在人类动脉粥样硬化中过表达。uPA及其表面受体（uPAR）参与血管SMC增殖/迁移和基质合成，在动脉粥样硬化的进展中起关键作用。我们的研究首次表明，在体外和体内，scuPA被转移到哺乳动物细胞的细胞核中，我们已经确定了这个过程中的一个关键中介，核蛋白。我们建议在本应用中解决的问题是描述uPA如何在细胞核内发挥作用，并将核易位和基因转录与uPA介导的血管重构和动脉粥样硬化中细胞粘附、增殖和迁移的改变联系起来。使用Affymetrix DNA微阵列，我们已经确定了至少6个与血管重构相关的基因，这些基因以核蛋白依赖的方式由uPA诱导。在目标1中，我们建议使用RT-PCR和western blotting来验证这些微阵列在冠状动脉SMC中的发现，为接下来的研究提供生物学基础。在目标2中，我们将遵循转录因子（TF）阵列已经获得的结果，并使用哺乳动物双杂交系统来确定scuPA或核蛋白是否直接与这些或其他TF或调节核转运或转录的新伙伴结合。在Aim 3中，我们将通过询问uPA是否通过直接结合DNA和/或基因启动子来调节转录来探索另一种新的机制。总之，这些研究将阐明uPA生物学的一个新方面，并应提供一个机会来开发特定的手段来调节uPA介导的病理过程。这些知识将有助于确定新的治疗方法，以防止血管重塑中的细胞入侵和生长。本项目旨在研究尿激酶型纤溶酶原激活剂uPA如何刺激血管SMC中的基因表达、增殖和迁移。这些研究将揭示在动脉粥样硬化和其他常见的人类血管疾病以及许多人类肿瘤进展中中断致病性内膜SMC迁移的新途径和新靶点。

DESCRIPTION (provided by applicant): Vascular remodeling is an essential component in the pathogenesis of atherosclerosis and other prevalent cardiovascular disorders, such as aneurysm formation, restenosis, and hypertension. The involvement of plasminogen activator (PA)/plasmin system in atherogenesis is well established through multiple studies that demonstrate overexpression of uPA, tPA and their inhibitor PAI-1 in human athrosclerosis. uPA and its surface receptor (uPAR) are implicated in vascular SMC proliferation/migration and matrix synthesis, which is pivotal in the progression of atherosclerosis. Our studies are the first to indicate that scuPA is translocated to the nucleus of mammalian cells in vitro and in vivo and we have identified one critical intermediary in the process, nucleolin. The issue we propose to address in this application is to delineate how uPA functions within the nucleus with the long-term goal of relating nuclear translocation and gene transcription to uPA-mediated alterations in cell adhesivity, proliferation and migration in vascular remodeling and atherosclerosis. Using Affymetrix DNA microarray, we have identified at least 6 genes relevant to vascular remodeling that are induced by uPA in nucleolin-dependent manner. In Aim 1, we propose to validate these microarray findings in coronary arterial SMC using RT-PCR and western blotting to provide a biological underpinning for what follows. In Aim 2 we will follow results already obtained from transcription factor (TF) arrays and use a mammalian two-hybrid system to determine whether scuPA or nucleolin bind directly to these or other TFs or to novel partners that regulate nuclear transport or transcription. In Aim 3, we will explore an alternative and novel mechanism by asking whether uPA regulates transcription by binding directly to DNA and/or gene promoters. Together these studies will elucidate a new facet of uPA biology and should provide an opportunity to develop specific means to regulate uPA- mediated pathologic processes. This knowledge will help to identify novel therapeutic approaches to prevent cell invasion and growth in vascular remodeling. This project intends to investigate how urokinase-type plasminogen activator uPA) stimulates gene expression, proliferation and migration in vascular SMC. These studies will reveal a novel pathway and novel targets to interrupt pathogenic intimal SMC migration in atherosclerosis and other common human vascular disorders as well as in progression of many human tumors.