Lung fibroblasts produce large amounts of inflammatory mediators and growth factors that appear to be associated with the development of IPF. The investigators have found that fibroblasts isolated from the lungs of patients with IPF (HF-IPF) produce more IL-6 than do fibroblasts isolated from normal human lungs(HF-NL). Since the IL-6 promoter is under the control of the transcription factors nuclear factor kB(NFkB) and CAAT/enhances binding protein-beta (C/EBPbeta) they measure activation of these proteins. NfkB activation was increased by IL-1beta, but not PGE2, and the response was similar in HF-IPF and HF-NL. In contrast, C/EBPbeta (a.k.a nuclear factor IL-6 [NF-IL6]) activity was increased by both PGE2 and IL-1beta. In addition, there was greater C/EBPbeta activation in HF- IPF compared to HF-NL, which correlated with IL-6 production. C/EBPbeta mRNA contains three inframe translation start sites and, therefore, three different C/EBPbeta isoforms may be produced. The two larger isoforms (p42C/EBPbeta) are transcription activators, while the smallest isoform (p20/EBP beta) lacks a transactivating domain and displays increased DNA affinity, and therefore, is a potent transcription repressor. Recent evidence suggests that C/EBPbeta may have a role in the process of fibrosis. C/EBPbeta has been shown, in liver cells, to stimulate the expression of insulin-like growth factor (IGF)- 1, hepatocyte growth factor (HGF) and collagen-alpha1 genes. Furthermore, transforming growth factor (TGF)-beta induces gene expression of procollagen I via C/EBPbeta. The investigators also have preliminary data showing that, when compared to HF-NL, HF-IPF fail to increase the p20C/EBPbeta:p42C/EBPbeta ratio when stimulated with IL-1beta and PGE2. They hypothesize that phenotypically altered fibroblasts perpetuate the inflammatory and fibrotic response because they have an exaggerated activation of C/EBPbeta, which may be due to a higher p42C/EBPbeta activity, decreased p20C/EBPbeta production, or both.
The aim i s to establish a relationship between increased C/EPbeta activity, with growth factor (IGF-1, PDGF) production, collagen synthesis and proliferation in primary lung fibroblasts. They will determine whether transfecting primary lung fibroblasts with a plasmid expression vector containing the gene encoding p42C/EBPbeta will result in increased gene expression and protein production of collagen I, and the growth factors TGF- beta, PDGF and IGF-1. In addition, they will determine whether transfecting primary lung fibroblasts with a plasmid expression vector containing the gene encoding p20C/EBPbeta will reduce the stimulated (IL-1beta and TGF-beta) protein production and gene expression of IGF-1, PDGF-1, PDGF and collagen I, as well as fibroblast proliferation. This work could provide a rationale for treatment of IPF with either the p20C/EBPbeta protein or the gene encoding this C/EBPbeta isoform.
