Project III: Pulmonary fibrosis involves complex interactions between multiple cell types via an intricate system of mediators. Progressive fibrotic diseases such as idiopathic pulmonary fibrosis (usual interstitial pneumonitis) remain essentially an untreatable disease with a fatal outcome. Recent progress using microarray approaches have helped to identify previously unsuspected molecules that appear to play significant roles in fibrosis. Using such an approach preliminary data revealed induction of a recently discovered molecule termed FIZZ1 (Found in Inflammatory Zone 1) in a rodent model of bleomycin-induced pulmonary fibrosis. This was the most highly induced molecule using a 10k rat gene chip, which was confirmed by RT-PCR to be >30-fold induced over control lung. FIZZ1, also known as resistin-like molecule alpha (RELM-alpha) is also highly expressed in inflamed airway epithelium in allergic airway disease, but its function remains unclear. Preliminary evidence in the bleomycin model confirms localization of expression mainly to be in airway and alveolar epithelium, which was confirmed by analysis of isolated type II pneumocytes. Lung fibroblasts however appear not to express FIZZ1. Co-culture of such FIZZ1 expressing type II pneumocytes with fibroblasts induced their differentiation to myofibroblasts. Based on these preliminary data, the central hypothesis of this project is that induction of FIZZ1 expression by alveolar epithelial cells plays a role in the pathogenesis of pulmonary fibrosis by inducing myofibroblast differentiation. To test this hypothesis four Specific Aims are proposed. First, the kinetics of alteration in lung FIZZ1 expression will be determined in the bleomycin model, and the cellular localization of expression attempted using a combination of in situ hybridization and immunohistochemistry. Confirmation of cellular expression will be undertaken in vitro in isolated and purified lung cells. Second, to analyze its biological activity vis-&-vis fibrosis, co-culture studies will be undertaken between FIZZ1 expressing type II pneumocytes and non-expressing lung fibroblasts to see if crosstalk between the alveolar epithelium and the fibroblast is mediated by FIZZ1. Confirmation of any activity will be undertaken using a FIZZ1 expressing plasmid for transfection studies using lung fibroblasts, and for the production of recombinant FIZZ1. Third, regulation of FIZZ1 expression in type II cells and associated signaling pathways will be analyzed. Finally, FIZZ1 knockout mice will be generated, and siRNA approaches used to confirm the in vivo relevance of its in vitro activity.
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