Analysis of the pulmonary GR interactome, and functional validation.

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.

Lung disease, frequently with a major inflammatory component, is a major human health burden, in terms of cost, and morbidity/mortality. Glucocorticoids are widely used to treat lung inflammation, but the treatment response is variable. In addition some inflammatory diseases do not respond to glucocorticoids.
We have developed novel approaches to comprehensively measure the GR post-translational modification, and protein interactome in lung tissue. This permits, for the first time, sampling of the consequences of inflammation, and drug treatment within the target tissue under physiological conditions. As a consequence of our pilot work we have already discovered an unexpected role for the protein caveolin as a GR regulator, and seek to explore the mechanisms responsible at an early stage. We have preliminary data associating changes in pulmonary inflammatory reactivity with circulating endogenous glucocorticoids, and therefore also seek to explore how these two systems interact, using in-vivo and in-vitro approaches.
We move to cell-type specific targeting of GR expression to anchor our observations to the bronchial epithelial cells, permitting further proteomics analysis in defined cell populations from whole animal challenges. We will explore the mechanisms of GR regulation, and the consequences for GR function using cell models, and molecular analysis at two levels, the GR cistrome and transcriptome. We have unrivalled expertise in computational biology required to manage these multidimensional datasets at applicant, and collaborator level, and outcomes will include both systems level analysis, and the generation of testable hypotheses. These latter will be pursued using cell culture models, before return to experimental pulmonary inflammation.
We will translate our findings to human lung disease by associating changes in emergent target gene expression, and protein in our comprehensive lung tissue archive.

The research questions posed within this proposal are of major interest to ACADEMIC GROUPINGS in Biological,Biomedical Sciences, and Clinical science. The academic community will benefit from elucidation of novel mechanisms whereby innate immunity regulates and interacts with the principal endogenous anti-inflammatory regulator, the glucocorticoid receptor, on a molecular and anatomical level. As such, research findings will impact greatly on the HEALTH CARE COMMUNITY. We will disseminate findings by publishing primary papers and reviews in high impact journals, and presenting work at national and international meetings. We anticipate that the proposed work will produce 2-4 high-quality primary research papers.

All findings will be of high interest to the GENERAL PUBLIC due to the prevalence of inflammatory lung disease, and widespread usage of glucocorticoid medications, which often engender concern in patients due to the risk of side-effects. At its most basic, the work will engage sections of the populous who wish to learn about their health and human physiology. Research findings will be delivered to the general public through public engagement activities (e.g. annual science open days at the UoM, Café Scientifique presentations), as well as through mass media. For example, several of our recent papers have been reported widely in national and international newspapers, on local radio, and on the intranet following press releases issued by the University of Manchester and BBSRC.

The proposed research is of interest to PHARMACEUTICAL COMPANIES due to direct implications for human inflammatory disease affecting the lung, and other organs. Pharmaceutical industry investment into glucocorticoid regulating compounds, and other anti-inflammatory approaches is massive, due to the frequent involvement of inflammatory mechanisms in a wide range of human diseases. In the context of "building partnerships to enhance take-up and impact, thereby contributing to the economic competitiveness of the United Kingdom", our laboratories have taken a major lead within the extensive community of researchers at the University of Manchester by developing significant interactions and links with GSK and a joint nuclear receptor Biology Programme.

Industrial interest is evidenced by the substantive contributions to this application arising from the secondment of Stuart Farrow from GSK to work on the joint programme, and the access to tool compound libraries, and bioinformatic databases of gene expression data in human tissue and disease.

The Faculty of Medical and Human Sciences at Manchester has taken a strong proactive role in developing links with major pharmaceutical companies, enhancing public communication of science, as well as identification and development of commercialisation opportunities. There are dedicated members of staff employed within the Faculty to assist in these areas. A recent success is the establishment of the Manchester Centre for Collaborative Inflammation Research, director Prof T Hussell, a collaborator. This centre draws in substantial cash investment from both GSK and AZ in inflammation research. Our programme will link to the centre, maximising commercial exploitation potential.