DEFICIENT CYCLOOXYGENASE EXPRESSION IN IPF FIBROBLASTS

特发性肺纤维化(Ipf)是一种慢性且常常致命的疾病。 以成纤维细胞(F)和F-细胞过度聚集为特征 肺中衍生的基质蛋白。据信，这一现象的出现 有利于纤维化的F表型的改变导致了 IPF和其他纤维化疾病的发病机制。最强大的力量之一 已知可下调F增殖和胶原合成的物质 前列腺素E_2(PGE_2)是由F_2合成的一种介质 他们自己。前列腺素E_2合成的初始和限速步骤是 由PG H合成酶或环氧合酶(COX)催化。我们有 最近报道，与正常肺F(F-NL)相比，肺F 来自IPF患者(F-IPF)的基础PGE2水平降低 综合。此外，F-IPF表现出无法上调其 前列腺素E_2合成能力对多种炎症的反应 刺激物。这是由于它们表达信使核糖核酸和 与COX的诱导亚型COX-2相对应的蛋白质。其他内容 初步数据表明，这些细胞中的COX-2缺陷是由于 可逆地抑制基因表达的一种异常的激酶途径。 项目2的一般假设是PGE2中的这种缺陷是合成的 能力是纤维形成的一个重要决定因素 以F-IPF为特征的表型改变，如 增殖率和胶原合成。这一假设将是 从肺活检标本中获得的F原代培养的检测 来自未经治疗的IPF患者。总体目标是阐明 对细胞表型、预后效用和 PGE2合成缺陷和COX-2诱导缺陷的分子基础。这个 具体目标如下。L)考察了两者之间的关系 COX-2诱导性/PGE2合成缺陷与表型改变 在促进纤维化的F-IPF中。这将通过研究来实现 从不同程度纤维化的肺组织分离出的细胞，以及 通过免疫组织化学分析COX-2在这些组织中的表达 在原地。2)研究不同体外培养条件对F_1表型的影响 导致PGE2水平变化的操作，包括 外源性PGE2的加入，内源性PGE2合成的抑制， 转染环氧合酶-2，并加入能揭开面纱的激酶抑制剂 COX-2诱导。3)确定a)F-PGE_2合成能力 肺泡灌洗液中的IPF或PGE2水平可预测预后 在IPF的临床病程中的效用或与之相关。4)确定 COX-2诱导性缺陷的分子机制 在F-IPF中。特别是，剖析导致COX-2的一连串事件 诱导(涉及酪氨酸激酶、丝裂原活化蛋白激酶、 和转录因子)，以确定其所在的位置(S 异常的激酶途径发挥其抑制作用。通过 对F-IPF AS中COX-2诱导缺陷本质的认识 以及COX-2表达和PGE2合成在调节F_2中的作用 功能，我们希望获得可能导致新奇的见解 IPF和其他破坏性纤维化疾病的治疗方法 对肺部的影响。

Idiopathic pulmonary fibrosis (IPF) is a chronic and often fatal disorder characterized by an excessive accumulation of fibroblasts (F) and F- derived matrix proteins in the lung. It is believed that the emergence of an altered F phenotype which favors fibrosis contributes to the pathogenesis of IPF and other fibrotic diseases. One of the most potent substances known to down-regulate F proliferation and collagen synthesis is prostaglandin E2 (PGE2), a mediator which is synthesized by F themselves. The initial and rate-limiting step in PGE2 synthesis is catalyzed by the enzyme PG H synthase, or cyclooxygenase (COX). We have recently reported that, as compared with normal lung F (F-nl), lung F from patients with IPF (F-IPF) exhibit a reduction in basal PGE2 synthesis. In addition, F-IPF manifest an inability to upregulate their PGE2 synthetic capacity in -response to a variety of inflammatory stimuli. This is due to a defect in their ability to express mRNA and protein corresponding to the inducible isoform of COX, COX-2. Additional preliminary data suggest that the COX-2 defect in these cells is due to an aberrant kinase pathway which reversibly suppresses gene expression. The general hypothesis of Project 2 is that this defect in PGE2 synthetic capacity is an important determinant of fibrogenesis and of the phenotypic alterations which characterize F-IPF, such as increases in proliferative rate and collagen synthesis. This hypothesis will be examined in primary cultures of F obtained from lung biopsy specimens from patients with untreated IPF. The overall goal is to elucidate the consequences for cellular phenotype, the prognostic utility, and the molecular basis of this defect in PGE2 synthesis and COX-2 induction. The specific aims are as follows. l) Examine the relationship between the defect in COX-2 inducibility/PGE2 synthesis and phenotypic alterations in F-IPF which promote fibrosis. This will be accomplished by studying cells isolated from regions of lung with varying degrees of fibrosis, and by immunohistochemical analysis of COX-2 expression in these same tissues in situ. 2) Study the effects on F phenotype of various in vitro manipulations which result in alterations in PGE2 levels, including the addition of exogenous PGE2, inhibition of endogenous PGE2 synthesis, transfection of COX-2, and addition of kinase inhibitors which unmask COX-2 induction. 3) Determine whether a) PGE2 synthetic capacity of F- IPF, or b) PGE2 levels in bronchoalveolar lavage fluid, has prognostic utility in, or correlates with the clinical course of, IPF. 4) Determine the molecular mechanisms responsible for the defect in COX-2 inducibility in F-IPF. In particular, dissect the cascade of events leading to COX-2 induction (involving tyrosine kinases, mitogen-activated protein kinase, and transcription factors) in F in order to identify the site(s) at which the aberrant kinase pathway exerts its suppressive actions. By understanding the nature of the defect in COX-2 induction in F-IPF as well as the role of COX-2 expression and PGE2 synthesis in regulating F function, we hope to gain insights which might result in novel therapeutic approaches for IPF and other devastating fibrotic diseases of the lungs.