Mechanism of PAI-1 Polarization to Myoendothelial Junctions

PAI-1 极化至肌内皮连接的机制

• 批准号: 8240123
• 负责人: Brant E Isakson
• 金额: $ 23.1万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-01-15 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8240123
• 关键词: American; Area; Arteries; Binding; Binding Proteins; Biological Assay; Biological Markers; Blood Circulation; Blood Vessels; Cardiovascular Diseases; Cartoons; Cells; Co-Immunoprecipitations; Coculture Techniques; Communication; Complex; Cytoskeleton; Data; Detection; Disease; Endothelium; Fatty acid glycerol esters; Figs - dietary; Genetic Translation; Goals; Grant; Hypertension; Immunoblotting; In Vitro; Inflammatory; Label; Laboratories; Lead; Left; Link; Location; Messenger RNA; Metabolic; Metabolic syndrome; Microtubules; Modeling; Movement; Mus; Non-Insulin-Dependent Diabetes Mellitus; Oranges; Peptides; Phosphorylation; Physiological; Plasminogen Activator Inhibitor 1; Play; Population; Production; Proteins; RNA-Binding Proteins; Regulation; Resistance; Ribosomes; Role; Smooth Muscle; Stimulus; Structure; TNF gene; Techniques; Testing; Therapeutic; Time; Translating; Translations; Work; base; cell type; feeding; in vivo; mutant; new therapeutic target; nicotinamide phosphoribosyltransferase; novel; protein complex; protein transport; research study; response; tau Proteins

DESCRIPTION (provided by applicant): During the course of metabolic syndrome, there is a significant increase in plasminogen activator inhibitor-1 (PAI-1); however the pathological role that PAI-1 plays during the course of the disease is not clear. We have recently identified PAI-1 as being key to regulation of myoendothelial junctions (MEJs), which are critical heterocellular structures linking the endothelium and smooth muscle in the resistance vasculature, coordinating communication between the two cell types. In mice with metabolic syndrome, we have shown that PAI-1 accumulates in MEJs and increases the total number of these structures, indicating a possible role in altering heterocellular communication. In order to understand how PAI-1 may alter MEJ formation, we used TNF-a to mimic the effect of metabolic syndrome and demonstrate significant increases in PAI-1 mRNA throughout endothelium, MEJ, and smooth muscle. However, after application of TNF-a, there was only a significant increase in PAI-1 protein at the MEJ. This indicates that PAI-1 may be locally translated at the MEJ. Recently, the serpine binding protein 1 (SERBP1) has been described as a novel RNA binding protein (RBP) specifically for PAI-1 mRNA that promotes stabilization of the mRNA for translation. However, an RBP requires anchoring to the cytoskeleton to remain in a specific area of the cell (e.g., the MEJ). To that end, we discovered a microtubule binding domain in the novel protein nicotinamide phosphoribosyltransferase (NAMPT), demonstrated to increase concurrently with PAI-1 during the course of metabolic syndrome, which we found to be localized to the MEJ. Therefore, we hypothesize that SERBP1 and NAMPT act together as a PAI-1 RBP complex to localize PAI-1 mRNA to the MEJ for the rapid and specific dissemination of PAI-1 protein. To test this hypothesis, we have put forth two specific aims: 1) PAI-1 mRNA is locally translated at the MEJ and 2) NAMPT regulates localization of SERBP1 to the MEJ. This proposal will focus on how PAI-1 is capable of being polarized to the MEJ using such techniques as FlAsH/ReAsH to examine nascent PAI-1 protein production and how RBP proteins could be anchored to the cytoskeleton using peptides against the microtubule binding domain of NAMPT. The aggregate of the experiments will provide for the first time a pathological mechanism for the effect of increased PAI-1 seen in metabolic syndrome. PUBLIC HEALTH RELEVANCE: Metabolic syndrome is prevalent in over 50% of the American population, which if left untreated, can lead to severe cardiovascular diseases, such as hypertension and type II diabetes. However, there is currently no known therapeutic treatment. It is known that the protein plasminogen activator inhibitor- 1 (PAI-1) is always prevalent in high concentrations in the circulation as metabolic syndrome progresses and recent work by our laboratory has indicated that PAI-1 can be found in a very specialized location in the walls of blood vessels, the myoendothelial junction. Our grant proposes to dissect the mechanisms whereby PAI-1 is localized to the myoendothelial junction in hopes of understanding how PAI-1 may contribute to, or exacerbate, metabolic syndrome in the vasculature. Successful completion of this proposal could lead to novel therapeutic targets for treatment of metabolic syndrome.

描述（申请人提供）：在代谢综合征过程中，纤溶酶原激活物抑制剂-1（派-1）显著增加;然而，派-1在疾病过程中发挥的病理作用尚不清楚。我们最近已经确定派-1是调节肌内皮连接（MEJ）的关键，MEJ是连接阻力血管系统中内皮和平滑肌的关键异质细胞结构，协调两种细胞类型之间的通讯。在患有代谢综合征的小鼠中，我们已经表明派-1在MEJ中积累并增加这些结构的总数，这表明在改变异细胞通讯中可能起作用。为了了解派-1如何改变MEJ的形成，我们使用TNF-α来模拟代谢综合征的作用，并证明派-1 mRNA在整个内皮、MEJ和平滑肌中显著增加。然而，在应用TNF-α后，仅在MEJ处存在派-1蛋白的显著增加。这表明派-1可能在MEJ局部翻译。最近，丝氨酸蛋白酶结合蛋白1（SERBP 1）已被描述为特异性针对派-1 mRNA的新的RNA结合蛋白（RBP），其促进用于翻译的mRNA的稳定化。然而，RBP需要锚定到细胞骨架以保持在细胞的特定区域（例如，MEJ）。为此，我们发现了一种新的蛋白质烟酰胺磷酸核糖转移酶（NAMPT）中的微管结合域，证明在代谢综合征过程中与派-1同时增加，我们发现其定位于MEJ。因此，我们推测，SERBP 1和NAMPT一起作为派-1 RBP复合物定位PAI-1 mRNA的MEJ的PAI-1蛋白的快速和特异性的传播。为了验证这一假设，我们提出了两个具体的目标：1）派-1 mRNA在MEJ局部翻译和2）NAMPT调节SERBP 1在MEJ的定位。该提案将集中于派-1是如何能够被极化的MEJ使用FlAsH/ReAsH等技术，以检查新生派-1蛋白的生产和如何RBP蛋白可以锚定到细胞骨架使用肽对微管结合结构域的NAMPT。这些实验的总和将首次提供代谢综合征中派-1增加的作用的病理机制。 公共卫生相关性：代谢综合征在超过50%的美国人口中流行，如果不治疗，可能导致严重的心血管疾病，如高血压和II型糖尿病。然而，目前没有已知的治疗方法。众所周知，随着代谢综合征的进展，蛋白质纤溶酶原激活物抑制剂-1（派-1）总是以高浓度在循环中普遍存在，并且我们实验室最近的工作表明派-1可以在血管壁中的非常专门的位置（肌内皮连接）中发现。我们的资助计划是剖析派-1定位于肌内皮连接处的机制，希望了解派-1如何导致或加重血管系统中的代谢综合征。该提案的成功完成可能会为代谢综合征的治疗带来新的治疗靶点。