Nuclear translocation of urokinase/nucleolin complexes

尿激酶/核仁素复合物的核转位

• 批准号: 7319578
• 负责人: Douglas Brock Cines
• 金额: $ 22.94万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7319578
• 关键词: Address; Aneurysm; Atherosclerosis; Binding; Binding Sites; Biological; Biological Assay; Biology; Blood Vessels; C-terminal; Candidate Disease Gene; Cardiovascular Diseases; Cell Nucleus; Cell Proliferation; Cell surface; Cells; Chromatin; Complement; Complex; Conserved Sequence; Coronary; Coronary artery; DNA; DNA Binding; DNA Microarray Chip; DNA Microarray format; Data; Electrophoretic Mobility Shift Assay; Endothelial Cells; Fibroblasts; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Growth; Growth Factor; Human; Hypertension; Immigration; In Vitro; Inflammation; Knowledge; Kringles; Mammalian Cell; Mediating; Membrane; Microarray Analysis; Mus; Neoplasm Metastasis; Nuclear; Nuclear Import; Nuclear Protein; Nuclear Proteins; Nuclear Translocation; Pathogenesis; Pathologic Processes; Pathway interactions; Personal Satisfaction; Phenotype; Pilot Projects; Plasmin; Plasminogen Activator; Plasminogen Activator Inhibitor 1; Polymerase Chain Reaction; Precipitation; Process; Property; Protease Domain; Protein Microchips; Protein Overexpression; Proteins; Proteolysis; Reporter; Reporting; Reverse Transcriptase Polymerase Chain Reaction; Screening procedure; Serine Protease; Signal Transduction; Single-Stranded DNA; Site-Directed Mutagenesis; Smooth Muscle Myocytes; Surface; System; Time; Urokinase; Vascular Diseases; Vascular remodeling; Western Blotting; Wound Healing; Yeasts; angiogenesis; atherogenesis; base; bone morphogenetic protein 6; cell motility; ds-DNA; fibulin 1; immunocytochemistry; in vivo; inhibitor/antagonist; insight; interest; migration; mouse Smc1l1 protein; mouse Smc1l2 protein; novel; novel therapeutics; nucleocytoplasmic transport; nucleolin; prevent; promoter; receptor; response; restenosis; therapeutic target; trafficking; transcription factor; tumor; vascular smooth muscle cell proliferation; yeast two hybrid system

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.

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）发病机制的重要组成部分。纤溶酶原激活物（PA）/纤溶酶系统参与动脉粥样硬化的形成是通过多项研究证实的，这些研究证实了uPA、tPA及其抑制剂派-1在人类动脉粥样硬化中的过度表达。uPA及其表面受体（uPAR）参与血管平滑肌细胞增殖/迁移和基质合成，在动脉粥样硬化的进展中起关键作用。我们的研究是第一个表明，scuPA是易位到哺乳动物细胞的细胞核在体外和体内，我们已经确定了一个关键的中介在这个过程中，核仁素。我们提出在本申请中解决的问题是描述uPA如何在细胞核内发挥作用，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粘附性、增殖和迁移的改变相关联。我们利用Affyssin基因芯片技术，鉴定了至少6个与uPA以核仁依赖方式诱导的血管重塑相关的基因。在目标1中，我们建议使用RT-PCR和蛋白质印迹法验证这些微阵列在冠状动脉SMC中的发现，为下文提供生物学基础。在目标2中，我们将遵循已经从转录因子（TF）阵列获得的结果，并使用哺乳动物双杂交系统，以确定是否scuPA或核仁素直接结合到这些或其他TF或新的合作伙伴，调节核运输或转录。在目标3中，我们将探索一种替代的和新的机制，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。总之，这些研究将阐明一个新的方面的uPA生物学，并应提供一个机会，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侵袭和生长。本课题旨在研究尿激酶型纤溶酶原激活剂（uPA）对血管平滑肌细胞基因表达、增殖和迁移的影响。这些研究将揭示一种新的途径和新的目标，以中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的进展中的致病性内膜SMC迁移。