Project three will focus on delineating the basic mechanisms that underlie the inhibitory effect of IFNgamma on the expression of insulin-like growth factor-I expression by macrophages. IGF-I is a progression-type fibroblast growth factor that has been found in elevated levels in brochoalveolar lavage fluid of patients with pulmonary fibrosis. In addition, IGF-I stimulates collagen synthesis by lung fibroblasts in vitro. Thus, understanding the mechanisms that control IGF-I expression by macrophages will significantly improve our understanding of the mechanisms underlying the development of pulmonary fibrosis. Two finding from our studies that have led to the proposed work are (i) that TNFalpha is an autocrine/paracrine acting agonist of macrophage IGF-I expression and (ii) IFN-gamma dramatically silences the expression of this gene product. It is hypothesized that the activation of IGF-I expression by TNF-alpha is mediated by the transcriptional activator AP-1 which is activated as a result of the activation of two protein kinase cascades i.e. the mitogen- activated protein kinase cascade (MAPK) and the c-Jun kinase cascade (JNK). By contrast, IFNgamma, which dramatically silences the expression of IGF-I through an effect on the level of gene transcription, is hypothesized to induce the synthesis and/or activation of a specific repressor or IGF-I gene expression. These hypotheses will be addressed by three specific aims.
Specific aim one will address the role of the MAP kinase and c-Jun kinase cascades in the activation of IGF-I expression.
Specific aim two will investigate the regions of the IGF-I gene promoter that are responsive to IFNgamma, while in specific aim three we will characterize and potentially identify the putative repressor protein that is hypothesized to mediate the effect of IFNgamma on IGF-I gene transcription. In all three specific aims, the general approach will involve the analysis of IGF-I gene transcription in a macrophage cell line using IGF-I promoter-luciferase reporter gene constructs. The identification of novel IFNgamma responsive elements in the 5-flanking region of the IGF-I gene is expected to shed new light on how IFNgamma negatively controls the expression of other fibrogenic gene products such as PDGF and TGFbeta. In addition this project will provide information on the possibility that IFNgamma suppresses gene expression through a novel repressor protein. These collective findings will have important implication for our understanding of the mechanisms underlying the development of pulmonary fibrosis.
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