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.
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