FIZZ1 Stimulation of Myofibroblast Differentiation

Abstract

Pulmonary fibrosis is not an uncommon end result of a multitude of lung diseases and lung injury. Many of these result in progressive fibrosis that terminates in end-stage pulmonary disease and death from respiratory failure. Fibrosis associated with the idiopathic interstitial pneumonias is commonly an untreatable disease with significant mortality. Because the natural history of many of these diseases is unknown, animal model studies have been undertaken to fill in the gaps and to seek out clues for important pathogenic mechanisms. One of the models that have been extensively studied used bleomycin (BLM) to induce lung injury, inflammation, and fibrosis. BLM is an anti-neoplastic antibiotic, and is used clinically in treatment of various squamous cell carcinomas.¹ BLM-induced lung fibrosis is a well-characterized animal model in which a progressive fibrotic process is developed after initial lung inflammation. It is characterized by increased proliferation of fibroblasts, increased expression of cytokines such as transforming growth factor (TGF)-β1, the de novo appearance of myofibroblasts with their distinct α-smooth muscle actin (α-SMA)-expressing phenotype, as well as the deposition of extracellular matrix.^2–4 Accumulated evidence revealed that expression of a number of genes was elevated after BLM administration including fibrogenic cytokines such as TGF-β1 and interleukin (IL)-5, which reaches peak expression on approximately day 7 after BLM injection.^5–7 Increases in extracellular matrix expression, such as elastin, collagen I, collagen III, and α-SMA are also well established.^4,8–10 These diverse processes likely involve complex patterns of gene expression that remain to be fully elucidated. Identification of these genes and their biological activities should provide important clues to their roles in pulmonary fibrosis, and thus provide novel insights into pathogenesis and potential therapeutic approaches.

Previous studies of gene expression have provided some clues as to the potential role of certain genes in pathogenesis of fibrosis, but elucidated little in the way of uncovering the whole spectrum of possible genes that may be involved because of the limitation of standard analysis of one or a few genes. The development of oligonucleotide array or cDNA microarray technology allows the global analysis of gene expression and provides the opportunity to explore simultaneously complex patterns of gene expression in an animal model, wherein evolution from initial injury to fibrosis can be sequentially studied. Recent studies using this approach have yielded some interesting data on patterns of gene expression, in one of which a previously unsuspected role for matrilysin in BLM-induced pulmonary fibrosis is uncovered. Analysis of known genes confirmed previous evidence of early up-regulation of genes associated with inflammation followed subsequently with up-regulation of those associated with fibrosis.^11–13 A limitation is that only genes included in the microarray can be detected, nevertheless the approach has the potential of uncovering novel (or previously not suspected) genes that may play important roles in the pathogenesis of pulmonary fibrosis.

To further examine global changes in gene expression and help identify additional, potentially novel genes that may be associated with pulmonary fibrosis, we used a 10,000 element (10 K) rat cDNA microarray to analyze the gene expression profiles in a rat model of BLM-induced pulmonary fibrosis. This approach enables screening for possible novel expressed genes and further investigation of their function. The results show more than 600 genes whose expression was significantly altered in BLM-treated lungs, some of which have been previously reported and known to be relevant to the pathogenesis of fibrosis. However, an unexpected finding was the dramatic induction of a novel molecule, previously identified as FIZZ1 (found in inflammatory zone; also known as RELM-α or resistin-like molecule-α) in BLM-induced lung injury. Initial characterization suggests expression to localize primarily to alveolar and airway epithelium, but was not expressed by fibroblasts. Studies using co-cultures of type II alveolar epithelial cells (AECs) and fibroblasts, as well as studies using a FIZZ1-expressing plasmid or rat glutathione S-transferase (GST)-FIZZ1 fusion protein indicated that FIZZ1 could promote myofibroblast differentiation that is independent of endogenous TGF-β1 activity. These findings suggest a novel activity of FIZZ1 that is consistent with a profibrotic role not dissimilar to that for TGF-β, at least with respect to de novo emergence of the myofibroblast phenotype.

FIZZ1 and TGF-β1 mRNA abundance (expressed as 2^{, and shown as means ± SE of triplicate samples) was measured by real-time PCR in AECs and fibroblasts isolated from day 7 saline or BLM-treated animals.}

To rule out a role for TGF-β1, BLM-AECs were co-cultured with fibroblasts in the presence of neutralizing anti-TGF-β1 antibody or nonimmune IgG, and the effects on α-SMA (Western blotting) and collagen type I (ELISA) expression examined. Results were expressed as a percentage of untreated fibroblasts (without AEC co-culture) and the means ± SE (triplicate samples) are shown. The antibody failed to affect the stimulatory effects of co-culture with BLM-AECs.

Acknowledgments

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