FIZZ1 Expression and Function in Pulmonary Fibrosis

项目三：肺纤维化涉及多种细胞类型之间通过复杂的介质系统进行复杂的相互作用。进行性纤维化疾病，如特发性肺纤维化（通常是间质性肺炎）基本上仍然是一种无法治愈的疾病，其结果是致命的。使用微阵列方法的最新进展有助于识别以前未被怀疑的分子，这些分子似乎在纤维化中发挥重要作用。使用这种方法的初步数据显示，在博莱霉素诱导的肺纤维化啮齿动物模型中，最近发现的一种称为FIZZ1的分子（发现于炎性区1）被诱导。这是使用10k大鼠基因芯片诱导程度最高的分子，经RT-PCR证实，其诱导程度是对照肺的30倍。FIZZ1也被称为抵抗素样分子α (relm - α)，在变应性气道疾病的炎症气道上皮中也有高表达，但其功能尚不清楚。博来霉素模型的初步证据证实，表达主要定位于气道和肺泡上皮，通过分离的II型肺细胞分析证实了这一点。然而，肺成纤维细胞似乎不表达FIZZ1。将表达FIZZ1的II型肺细胞与成纤维细胞共培养，诱导其向肌成纤维细胞分化。基于这些初步数据，本项目的中心假设是肺泡上皮细胞诱导FIZZ1表达在肺泡上皮细胞凋亡中起作用

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.