Mechanism of PAI-1 Polarization to Myoendothelial Junctions

描述(申请人提供)：在代谢综合征的病程中，纤溶酶原激活物抑制物-1(PAI-1)显著增加；但PAI-1在疾病病程中所起的病理作用尚不清楚。我们最近发现PAI-1在肌内皮细胞连接(MEJ)的调节中起关键作用，MEJ是连接阻力血管系统中内皮细胞和平滑肌的关键异细胞结构，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，我们发现PAI-1在MEJ中积聚，并增加了这些结构的总数，表明PAI-1可能在改变异质细胞通讯方面发挥作用。为了了解PAI-1如何改变MEJ的形成，我们使用肿瘤坏死因子-α来模拟代谢综合征的影响，并显示PAI-1mRNA在内皮、MEJ和平滑肌中显著增加。然而，在应用肿瘤坏死因子-α后，仅在MEJ处PAI-1蛋白显著增加。这表明PAI-1可能在MEJ被局部翻译。最近，蛇毒结合蛋白1(SERBP1)被描述为一种新的RNA结合蛋白(RBP)，它特异性地与PAI-1mRNA结合，促进翻译过程中mRNA的稳定。然而，RBP需要锚定到细胞骨架上，以保持在细胞的特定区域(例如，MEJ)。为此，我们在新的蛋白烟酰胺磷酸核糖转移酶(NAMPT)中发现了一个微管结合域，在代谢综合征过程中与PAI-1同时增加，我们发现它定位于MEJ。因此，我们假设SERBP1和NAMPT共同作用于PAI-1RBP复合体，将PAI-1mRNA定位到MEJ，从而快速而特异地传播PAI-1蛋白。为了验证这一假说，我们提出了两个具体目标：1)PAI-1mRNA在MEJ局部翻译；2)NAMPT调节SERBP1在MEJ的定位。这项提案将集中在PAI-1如何能够极化到MEJ，使用Flash/ReAsH等技术来检测新生PAI-1蛋白质的产生，以及RBP蛋白质如何使用针对NAMPT微管结合结构域的多肽被锚定在细胞骨架上。这些实验的总和将首次为代谢综合征中PAI-1增加的影响提供一个病理机制。 公共卫生相关性：代谢综合征在超过50%的美国人口中普遍存在，如果不加以治疗，可能会导致严重的心血管疾病，如高血压和II型糖尿病。然而，目前还没有已知的治疗方法。众所周知，随着代谢综合征的进展，蛋白纤溶酶原激活物抑制物-1(PAI-1)在循环中总是以较高的浓度存在，最近我们实验室的工作表明，PAI-1可以在血管壁的一个非常特殊的位置--肌内皮连接处发现。我们的资助建议剖析PAI-1定位于肌内皮细胞连接的机制，以期了解PAI-1如何促进或加重血管系统中的代谢综合征。这项提议的成功完成可能会导致代谢综合征治疗的新的治疗目标。

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.