DEFICIENT CYCLOOXYGENASE EXPRESSION IN IPF FIBROBLASTS

特发性肺纤维化（IPF）是一种慢性且往往致命的疾病 特征在于成纤维细胞（F）和F- 衍生的基质蛋白。据信， F表型的改变有利于纤维化， IPF和其他纤维化疾病的发病机制。其中最有效的 已知下调F增殖和胶原合成的物质 前列腺素E2（PGE 2）是一种由F 自己PGE 2合成的初始和限速步骤是 由PG H合酶或环氧合酶（考克斯）催化。我们有 最近报道，与正常肺F（F-nl）相比，肺F IPF（F-IPF）患者的基础PGE 2 合成.此外，F-IPF表现出不能上调其表达。 PGE 2合成能力-响应各种炎症 刺激。这是由于它们表达mRNA的能力缺陷， 对应于考克斯的诱导型同种型，考克斯-2的蛋白质。额外 初步数据表明，这些细胞中的考克斯-2缺陷是由于 可逆抑制基因表达的异常激酶途径。 项目2的一般假设是，PGE 2合成中的这种缺陷 容量是纤维形成和纤维化的重要决定因素。 表征F-IPF的表型改变，例如 增殖率和胶原合成。这一假设将是 在从肺活检标本中获得的F原代培养物中检查 来自未经治疗的IPF患者。总体目标是阐明 细胞表型的后果，预后效用，以及 PGE 2合成和考克斯-2诱导缺陷的分子基础。的 具体目标如下。 （一）研究与 考克斯-2诱导因子/PGE 2合成缺陷和表型改变 在促进纤维化的F-IPF中。这将通过研究 从具有不同程度纤维化的肺区域分离的细胞，和 通过免疫组化分析这些组织中考克斯-2的表达 在原地。2)研究不同的体外培养条件对F表型的影响 操作导致PGE 2水平的改变，包括 加入外源性PGE 2，抑制内源性PGE 2合成， 转染考克斯-2，并加入激酶抑制剂， 考克斯-2诱导。3)确定是否a）F的PGE 2合成能力- IPF或B）支气管肺泡灌洗液中的PGE 2水平具有预后 在IPF临床病程中的效用或与IPF临床病程相关。4)确定 考克斯-2诱导缺陷的分子机制 在F-IPF中。特别是，分析导致考克斯-2的级联事件 诱导（涉及酪氨酸激酶，丝裂原活化蛋白激酶， 和转录因子），以鉴定 异常激酶途径发挥其抑制作用。通过 理解F-IPF中考克斯-2诱导缺陷的性质， 以及考克斯-2表达和PGE 2合成在调节F 功能，我们希望获得的见解，这可能会导致新的 IPF和其他破坏性纤维化疾病的治疗方法 肺的。

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.