FIZZ1 Expression and Function in Pulmonary Fibrosis

FIZZ1 在肺纤维化中的表达和功能

• 批准号: 7312445
• 负责人: SEM H PHAN
• 金额: $ 35.81万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7312445
• 关键词: cellular pathology; collagen; cytokine receptors; eosinophil; fibroblasts; gene expression; genetically modified animals; idiopathic pulmonary fibrosis; immunocytochemistry; in situ hybridization; inflammation; laboratory mouse; lung injury; mixed tissue /cell culture; monocyte chemoattractant protein 1; neutralizing antibody; polymerase chain reaction; protein biosynthesis; pulmonary fibrosis /granuloma; receptor binding; receptor expression; respiratory disorder diagnosis; transforming growth factors

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.

项目III：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。进行性纤维性疾病，如特发性肺纤维化(通常为间质性肺炎)，基本上仍是一种无法治疗的疾病，其后果是致命的。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，初步数据显示，在博莱霉素诱导的肺纤维化的啮齿动物模型中，最近发现的一种名为FIZZ1的分子(在炎症区1中发现)被诱导。这是使用10k大鼠基因芯片的最高诱导性分子，经RT-PCR证实是对照肺的30倍诱导性。FIZZ1，也被称为抵抗素样分子α(RELM-α)，在过敏性呼吸道疾病的炎症气道上皮细胞中也高表达，但其功能尚不清楚。博莱霉素模型中的初步证据证实了主要在呼吸道和肺泡上皮细胞中表达的定位，这一点通过对分离的II型肺泡细胞的分析得到证实。然而，肺成纤维细胞似乎不表达FIZZ1。将表达FIZZ1的II型肺泡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，该项目的中心假设是，肺泡上皮细胞诱导FIZZ1表达在 诱导肌成纤维细胞分化在肺纤维化发病机制中的作用为了验证这一假设，本文提出了四个具体目标。首先，将在博莱霉素模型中确定肺FIZZ1表达变化的动力学，并尝试使用原位杂交和免疫组织化学相结合的方法对表达进行细胞定位。细胞表达的确认将在体外进行，在分离和纯化的肺细胞。其次，为了分析FIZZ1对纤维化的生物活性，将表达FIZZ1的II型肺泡上皮细胞与不表达FIZZ1的肺成纤维细胞进行共培养研究，以了解肺泡上皮与成纤维细胞之间的串扰是否由FIZZ1介导。确认任何 将使用FIZZ1表达质粒进行活性研究，用于肺成纤维细胞的转基因研究，并用于生产重组FIZZ1。第三，将分析FIZZ1在II型细胞中的表达调控以及相关的信号通路。最后，将产生FIZZ1基因敲除小鼠，并使用siRNA方法来确认其体外活性的体内相关性。