Control of fibroblast function by prostaglandin E2 and plasminogen activation

描述(申请人提供)：成纤维细胞是主要的效应细胞，通过其促进存活、增殖、胶原沉积和肌成纤维细胞分化的能力，介导特发性肺纤维化(IPF)的组织重建。虽然对肺纤维化发病机制的研究主要是对成纤维细胞激活信号的研究，但有证据表明，这种疾病的特征也是相对缺乏反调节的抗纤维化信号。两个这样的抗纤维化信号是前列腺素E2(PGE2)和纤溶酶原激活物(PA)活性。在IPF患者中，其中每一种都被证明是缺乏的，并且在肺纤维化的动物模型中，每一种都被证实是致病的重要因素。PGE2是一种脂质介质，来源于脂肪酸花生四烯酸的环氧合酶代谢，该脂肪酸通过细胞表面G蛋白偶联的E前列腺素受体发挥作用。PA系统是一个蛋白水解级联反应，包括尿激酶型PA(UPA)及其相关的抑制物(纤溶酶原激活物抑制物-1)。虽然PGE2通过细胞内cAMP(CAMP)信号抑制肺成纤维细胞中所有相关促纤维化细胞表型的激活，但其下游机制尚不完全清楚。PA系统被认为可以协调纤溶、调节细胞黏附和细胞信号，但对其对成纤维细胞或其相关表型的直接影响知之甚少。最后，在包括成纤维细胞在内的任何类型的肺细胞中，没有关于PGE2和PA系统之间的串扰的信息。本项目旨在了解这两种介质调节成纤维细胞功能的机制，表征它们之间的相互作用，并确定纤维化肺损伤如何影响成纤维细胞对它们各自的反应。一般的假设是，PGE2和PA系统相互上调并相互作用，以一种有利于肺修复而不是纤维化的方式影响肺成纤维细胞的表型。这一假设将在成纤维细胞系以及从正常和纤维化小鼠和人肺分离的原代细胞中进行验证。目的1研究cAMP效应蛋白激酶A和cAMP激活的鸟苷交换蛋白以及磷酸酶PTEN在介导PGE2对成纤维细胞表型影响中的作用。目的2将确定PGE2和PA活性相互影响彼此表达的机制，而在目标3中将探讨各自在介导彼此作用中的作用。目的4将比较PGE2和PA活性对正常和损伤小鼠肺成纤维细胞表型的影响，以及组织学正常和IPF人肺成纤维细胞表型的影响。拟议的研究将为这两种介质对成纤维细胞激活的调控提供新的见解，并将为未来针对这些分子的治疗提供信息。与公共卫生相关：一种称为肺疤痕形成(肺纤维化)的严重疾病的发展受到身体产生的两种物质--前列腺素E2和尿激酶型纤溶酶原激活剂--的反对。这项建议将研究这两种物质如何作用和相互作用，以抑制关键的肺细胞类型，即成纤维细胞的疤痕反应。这些研究将加强我们对疤痕反应是如何调节的理解，并可能为这些物质是否可以用于患者治疗这种毁灭性的疾病提供洞察力。

DESCRIPTION (provided by applicant): Fibroblasts are the principal effector cells that mediate tissue remodeling in idiopathic pulmonary fibrosis (IPF) via their capacities for enhanced survival, proliferation, collagen deposition, and myofibroblast differentiation. Although research in the pathogenesis of pulmonary fibrosis has been dominated by studies investigating fibroblast activation signals, evidence indicates that this disorder is also characterized by a relative deficiency in counter-regulatory anti-fibrotic signals. Two such anti-fibrotic signals are the prostanoid prostaglandin E2 (PGE2) and plasminogen activator (PA) activity. Each of these has been shown to be deficient in patients with IPF, and deficiency of each has been established to be pathogenically important in animal models of pulmonary fibrosis. PGE2 is a lipid mediator derived from cyclooxygenase metabolism of the fatty acid arachidonic acid that acts via cell surface G protein-coupled E prostanoid receptors. The PA system is a proteolytic cascade that includes the protease urokinase-type PA (uPA) and its associated inhibitor (plasminogen activator inhibitor-1). Although PGE2 inhibits the activation of all relevant pro-fibrotic cellular phenotypes in lung fibroblasts via intracellular cyclic AMP (cAMP) signaling, the downstream mechanisms by which it does so are incompletely understood. The PA system is recognized to orchestrate fibrinolysis and to modulate cellular adhesion and cellular signaling, but little is known about its direct effects on fibroblasts or their relevant phenotypes. Finally, there is no information about cross-talk between PGE2 and the PA system in lung cells of any kind, including fibroblasts. This project seeks to understand the mechanisms by which both mediators modulate fibroblast function, to characterize the cross-talk between them, and to determine how fibrotic lung injury influences the responses of fibroblasts to each of them. The general hypothesis is that the PGE2 and PA systems up-regulate each other and interact to influence pulmonary fibroblast phenotypes in a manner which favors lung repair over fibrosis. This hypothesis will be tested in fibroblast cell lines and in primary cells isolated from normal and fibrotic murine and human lungs. Aim 1 will examine the roles of cAMP effectors protein kinase A and guanylate exchange protein activated by cAMP as well as the phosphatase PTEN in mediating PGE2 effects on fibroblast phenotypes. Aim 2 will determine the mechanisms by which PGE2 and PA activity influence the expression of each other, while the role of each in mediating the actions of the other will be explored in Aim 3. Aim 4 will compare the effects of PGE2 and PA activity on phenotypes of fibroblasts derived from normal vs. injured mouse lungs and from histologically normal vs. IPF human lungs. The proposed studies will provide novel insights into the regulation of fibroblast activation by these two mediators, and will inform future efforts to target these molecules therapeutically. PUBLIC HEALTH RELEVANCE: The development of a serious condition known as lung scarring (pulmonary fibrosis) is opposed by two substances produced by the body, prostaglandin E2 and urokinase plasminogen activator. This proposal will examine how these two substances act and interact to suppress scarring responses of the key lung cell type known as the fibroblast. These studies will enhance our understanding of how scarring responses are regulated, and may provide insight as to whether these substances could be administered to patients to treat this devastating condition.