Pathophysiology of PPARgamma in the lung

描述(申请人提供)：过氧化体增殖物激活受体γ(PPAR())是包括肺在内的各种组织系统中的抗炎分子。它在肺中的功能作用还不是很清楚。这项工作的长期目标将确定PPAR(肺发病机制的分子途径)。提出的研究的中心假设是PPAR是通过基质金属蛋白酶-12(MMP12)的分子控制来调节肺部炎症的介质，这种控制来自于溶酶体中由溶酶体酸性脂肪酶(LAL)产生的脂肪酸配体。我们先前的研究支持了这一点，即阻断溶酶体酸性脂肪酶缺陷小鼠(LAL-/-)的配体合成会导致肺部炎症、肺气肿、不想要的上皮细胞生长和基因表达异常。用PPAR(配体9-HODE和西格列酮)治疗可显著减轻LAL/肺炎症和异常基因表达。在体外瞬时转染实验中，配体和PPAR(负性调节基质金属蛋白酶-12启动子活性)。PPAR(蛋白主要存在于肺内的肺泡巨噬细胞、Clara细胞和肺泡II型上皮细胞中。因此，中心假设将在我们的两个新的转基因模型中得到验证，在这两个模型中，内源性PPAR(通过其优势负式(dnPPAR())在多西环素诱导的转基因小鼠系统中的肺泡II型上皮细胞或支气管肺泡巨噬细胞中的过度表达而失活。对一个转基因模型的初步评估显示，在CCSP启动子的控制下，dnPPAR(在2个月内过表达)显示炎症细胞进入肺和肺气肿。此外，在dnPPAR(转基因小鼠)中，MMP12的表达水平增加了55倍。DnPPAR(过度表达)的长期致病作用尚未确定。具体目标1：确定dnPPAR(在呼吸道上皮细胞中)的发病机制；特异性目标2：确定dnPPAR(在巨噬细胞中)的发病机制；特异性目标3：确定MMP12在肺上皮细胞中调控的分子机制。综上所述，这些研究将极大地提高我们对PPAR(PPAR)在肺部，特别是在肺部炎症和组织重塑中的病理生理功能的认识，并阐明MMP12基因调控的分子机制，该分子调控介导LAL-/-小鼠肺的表型。这些研究的结果将为设计抗击肺部炎症和肺气肿的新策略提供证据。项目简介：本研究提案的目的是了解过氧化体增殖物激活受体γ(PPAR())在肺中作为抗炎效应因子的关键作用。我们正在使用最先进的技术来创建转基因小鼠，这些转基因小鼠在肺细胞(肺泡II型上皮细胞或细支气管肺泡巨噬细胞)中具有细胞特异性和时间调控的显性负型PPAR(dnPPAR())的表达，并表征其肺部炎症、重塑和肺气肿表型。此外，拟议的研究还将阐明PPAR失活的分子链接物，以及通过对基质金属蛋白酶-12(MMP12)基因的转录调控来重塑和肺气肿的表型。总之，这些研究将大大增强我们对PPAR(在肺中的病理生理功能，特别是在肺部炎症和组织重塑中的病理生理功能)的了解。这些研究将为设计策略和发现抗肺气肿和慢性阻塞性肺病的药物提供新的方法。

DESCRIPTION (provided by applicant): Peroxisome proliferator-activated receptor gamma (PPAR() is an anti-inflammatory molecule in various tissue systems including the lung. Its functional role in the lung is not well understood. The long-term goal of this work will determine PPAR( molecular pathways in lung pathogenesis. The central hypothesis for the proposed studies is that PPAR( is a mediator of pulmonary inflammation via the molecular control of matrix metalloproteinase-12 (MMP-12) and that this control derives from fatty acid ligands, generated in the lysosomes by lysosomal acid lipase (LAL). This has been supported by our previous study that blockage of its ligand synthesis in lysosomal acid lipase deficient mice (lal-/-) caused pulmonary inflammation, emphysema, unwanted epithelial cell growth and aberrant gene expression. Treatment with PPAR( ligands 9-HODE and Ciglitazone significantly attenuated lal-/- pulmonary inflammation and aberrant gene expression. The ligands and PPAR( negatively regulate MMP-12 promoter activity in in vitro transient transfection assays. The PPAR( protein is primarily detected in broncho-alveolar macrophages, Clara cells and alveolar type II epithelial cells in the lung. Thus, the central hypothesis will be tested in our two new transgenic models, in which endogenous PPAR( is inactivated by over-expression of its dominant negative form (dnPPAR() in alveolar type II epithelial cells or bronchoalveolar macrophages in doxycycline-inducible transgenic mouse system. Preliminary assessment of a transgenic model, in which dnPPAR( was over- expressed for 2 months under the control of the CCSP promoter, showed inflammatory cell influx into the lung and emphysema. In addition, the expression level of MMP12 increased 55-fold in the dnPPAR( transgenic mice. The long-term pathogenic effect of dnPPAR( over-expression has not been determined. Three specific aims are designed to test the central hypothesis: Specific Aim 1: Determine pathogenesis of dnPPAR( in respiratory epithelial cells; Specific Aim 2: Determine pathogenesis of dnPPAR( in macrophages; Specific Aim 3: Determine molecular mechanism of MMP12 regulation in lung epithelial cells. Together, these studies will significantly enhance our knowledge for understanding the pathophysiological function of PPAR( in the lung, especially in pulmonary inflammation and tissue remodeling, and elucidate the molecular mechanism of gene regulation in MMP12 that mediates the phenotype in the lung of lal-/- mice. The outcomes of these studies will provide evidence to design new strategies to combat pulmonary inflammation and emphysema. PROJECT NARRATIVE: The objectives of this research proposal are to understand the critical role of peroxisome proliferator- activated receptor gamma (PPAR() as anti-inflammatory effector in the lung. We are using the state-of-art techniques to create transgenic mice that have cell specific and temporal regulated expression of dominant negative form of PPAR( (dnPPAR() in pulmonary cells (alveolar type II epithelial cells or bronchio-alveolar macrophages) and characterizing their pulmonary inflammation, remodeling, and emphysema phenotype. In addition, the proposed studies will elucidate the molecular linker of inactivation of PPAR( and the remodeling and emphysema phenotype through transcriptional regulation of matrix metallproteinase-12 (MMP-12) gene. Together, these studies will significantly enhance our knowledge for understanding the pathophysiological function of PPAR( in the lung, especially in pulmonary inflammation and tissue remodeling. These studies will provide new approaches to design strategies and discover drugs to combat emphysema and COPD.