Nuclear translocation of urokinase/nucleolin complexes

描述(由申请人提供)：血管重塑是动脉粥样硬化和其他常见心血管疾病，如动脉瘤形成、再狭窄和高血压的发病机制中的重要组成部分。大量研究表明uPA、tPA及其抑制物PAI-1在人类动脉粥样硬化中过表达，证实了纤溶酶原激活物(PA)/纤溶酶系统参与动脉粥样硬化的形成。UPA及其表面受体(UPAR)参与了血管SMC的增殖/迁移和基质合成，在动脉粥样硬化的发展过程中起着关键作用。我们的研究首次表明scuPA在体外和体内都被转移到哺乳动物细胞的细胞核中，并且我们发现了这一过程中的一个关键中介--核仁素。我们打算在这一应用中解决的问题是描绘uPA在细胞核内的功能，长期目标是将核转位和基因转录与uPA介导的血管重塑和动脉粥样硬化中细胞黏附、增殖和迁移的变化联系起来。利用Affymetrix DNA微阵列，我们已经鉴定出至少6个与uPA以核仁素依赖的方式诱导的血管重构相关的基因。在目标1中，我们建议使用RT-PCR和Western blotting在冠状动脉SMC中验证这些微阵列的结果，为后续的研究提供生物学基础。在目标2中，我们将跟踪已经从转录因子(TF)阵列中获得的结果，并使用哺乳动物双杂交系统来确定scuPA或核仁是直接与这些或其他TF结合，还是与调节核运输或转录的新伙伴结合。在目标3中，我们将探索另一种新的机制，即uPA是否通过直接与DNA和/或基因启动子结合来调节转录。总之，这些研究将阐明uPA生物学的一个新方面，并将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。这一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物(UPA)如何刺激血管SMC的基因表达、增殖和迁移。这些研究将揭示一种新的途径和新的靶点，以阻断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的病理性内膜SMC迁移。

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.