Analysis of the pulmonary GR interactome, and functional validation.

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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.