Analysis of the pulmonary GR interactome, and functional validation.

人类炎症性疾病在世界范围内造成了重大的财政和人力成本。特别是炎性肺病导致生产力损失、治疗成本、发病率和过早死亡率的主要负担。治疗的支柱是局部和全身性糖皮质激素的应用，但个体之间的反应是可变的，需要高剂量的强效合成药物，这导致脱靶效应。此外，一些患者完全没有反应，原因尚不清楚，一些疾病的特征是无反应，如慢性阻塞性肺疾病和纤维化。我们小组的新技术的发展现在允许无偏见的发现方法，靶向肺部炎症中的糖皮质激素反应。使用体内研究获得的见解将在体外进行测试和开发，最后我们将在广泛的人类肺部疾病样本中应用这些新的见解，以确定疾病和/或治疗反应相关的表达模式。这些新出现的途径和蛋白质将形成生物标志物开发的基础，但更重要的是将标志着可以通过设计新药或考虑可与糖皮质激素联合使用的额外治疗来改善使用糖皮质激素的现有方法的新方法，以获得治疗效果，并减少脱靶效应。这些新的见解也将刺激新的药物发现计划，针对新兴的途径调节糖皮质激素的行动。我们的初步方法将测量炎症在肺部的影响，修饰和糖皮质激素受体（GR）的相互作用。我们将首先研究肺部炎症，然后将这些发现带入肺细胞进行进一步表征。我们需要在肺中进行早期发现阶段，使用分离的肺细胞测试作用机制，然后在人类肺样本中测试其影响。我们已经优化了这些方法，并且已经有了令人兴奋的新见解，确定了GR相互作用蛋白，小窝蛋白，作为肺糖皮质激素作用的主要调节因子。由于许多肺部疾病在其表达中显示出一天中不同时间的变化，我们还研究了生物钟的作用，其作为代谢和炎症的关键稳态调节剂的重要性正在迅速显现。 我们现在能够遗传靶向GR删除支气管上皮细胞特异性，使用一种新的和验证的转基因小鼠品系。这将使我们能够第一次检查糖皮质激素在这种特定细胞类型中的调节作用，而且还将改进我们对特定细胞的蛋白质组学分析，通过比较完整肺和GR从支气管上皮中缺失的肺中的修饰和相互作用谱，我们对支气管上皮中糖皮质激素作用的分析进行了改进，测量炎症信号对GR功能的影响。这将使用分离的支气管上皮细胞，以减少复杂性，并作为一个易于处理的模型，用于测试假设，因为它们出现在整个肺分析。在细胞中，我们将验证新出现的蛋白质相互作用和修饰，并分析由GR驱动的所有基因调控位点，从而首次允许分析这种重要细胞类型中GR活性的全谱。此外，这项分析将使我们能够发现炎症如何影响GR功能。我们将分析在肺中获得的所有分子结果，并从中确定负责调节炎症效应的信号网络。这些将使用肺细胞进行改进，然后在人类肺部疾病样本中测试这些介质的表达，以找到疾病的新解释及其对治疗的反应。

Human inflammatory disease imposes a major financial and human cost world-wide. In particular inflammatory lung disease causes a major burden in lost productivity, treatment costs, morbidity, and premature mortality. A mainstay of therapy is the application of topical, and systemic glucocorticoids, but the response is variable between individuals, requiring high doses of powerful, synthetic drugs, which leads to off target effects. In addition, some patients fail to respond at all, for reasons which remain unclear, and some diseases are characteristically unresponsive, such as chronic obstructive pulmonary disease, and fibrosis. Development of new technology in our group now permits unbiased discovery approaches targeting the glucocorticoid response in pulmonary inflammation. Insights gained using in-vivo studies will be tested, and developed in-vitro, and finally we will apply these new insights across a broad panel of human pulmonary disease samples to identify patterns of expression with disease, and/or treatment response association. These emergent pathways, and proteins will form the basis for biomarker development, but more importantly will signal new ways in which the existing approaches using glucocorticoids can be improved, by designing new drugs, or considering additional treatments that can be used along with glucocorticoids adjunctive to gain therapeutic efficacy, and reduce off-target effects. These new insights will also stimulate new programmes of drug discovery, targeting emerging pathways regulating glucocorticoid action.Our initial approach will measure the impact of inflammation in the lung on modification, and interactions of the glucocorticoid receptor (GR). We will study lung inflammation first, and then take these findings into lung cells for further characterization. We need to undertake the early discovery phase in lung, test the mechanisms of action using isolated lung cells, before testing the implications in human lung samples. We have optimized these approaches, and already have exciting new insights identifying a GR interacting protein, caveolin, as a major regulator of the pulmonary glucocorticoid effect. As many lung diseases show a time of day variation in their expression we have also examined the role of the circadian clock, the importance of which as a key homeostatic regulator of metabolism and inflammation is rapidly emerging. We are now able to genetically target GR deletion to the bronchial epithelium specifically, using a new and validated transgenic mouse strains. This will permit us to examine both the regulatory role of glucocorticoid action in this specific cell type for the first time, but will also refine our proteomics analyses to specific cells, by comparing the spectrum of modifications and interactions in intact lungs with those where GR has been deleted from the bronchial epithelium.Having refined our analysis to glucocorticoid action in the bronchial epithelium we will apply an unbiased approach to measure the impact of inflammatory signaling on GR function. This will use isolated bronchial epithelial cells, to reduce complexity, and as a tractable model for testing hypotheses as they emerge from the whole lung analysis. In the cells we will validate emerging protein interactions, and modifications, and analyze all the sites of gene regulation that are driven by the GR, so permitting analysis of the full spectrum of GR activity in this important cell type for the first time. Moreover, this analysis will allow us to discover how inflammation impacts on GR function.We will analyse all the molecular results obtained in lung, and from this identify signaling networks responsible for regulating the inflammatory effect. These will be refined using lung cells, and then the expression of these mediators will be tested in human lung disease samples, to find new explanations for disease, and its response to treatment.