Analysis of the pulmonary GR interactome, and functional validation.

Lead Research Organisation: University of Manchester
Department Name: School of Medical Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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 econoic 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.

Publications

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Carter SJ (2016) A matter of time: study of circadian clocks and their role in inflammation. in Journal of leukocyte biology

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Matthews LC (2015) Glucocorticoid receptor regulates accurate chromosome segregation and is associated with malignancy. in Proceedings of the National Academy of Sciences of the United States of America

 
Description deputy chair MRC Clinical Research Training Fellowship Panel
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
Impact I have contributed to the assessment, selection and mentoring of clinical fellows funded by MRC over the last seven years, initially as panel member then chair.
 
Description MRC project grant
Amount £675,328 (GBP)
Funding ID MR/P011853/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 03/2017 
End 02/2020
 
Description The role of the clock in allergic asthma
Amount £89,000 (GBP)
Organisation Asthma UK 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2014 
End 04/2016
 
Description clinical research fellowship
Amount £269,390 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 08/2016 
End 07/2019
 
Description programme grant
Amount £2,000,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 09/2017 
End 08/2022
 
Description research grant
Amount £844,160 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 03/2017 
End 02/2020
 
Description wellcome investigator
Amount £1,900,000 (GBP)
Funding ID n/a 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2016 
End 05/2021
 
Title rapid high fidelity capture of nuclear receptor interactome 
Description We have developed a rapid, simple and direct method of capturing the nuclear receptor protein interactome, and analysing it using a workflow which culminates in tandem mass spec. This has revealed a number of novel targets, and potential pathways which we are now testing. 
Type Of Material Biological samples 
Provided To Others? No  
Impact we are completing work on our first manuscript, which will detail, and validate the method. 
 
Description Anna Nicoleau 
Organisation University of Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution We ran a study in patients with rheumatoid arthritis
Collaborator Contribution Anna and her team analysed serum for inflammatory lipid species
Impact none yet
Start Year 2015
 
Description Development of novel glucocorticoid ligands for inflammatory lung disease. 
Organisation Karo Bio
Country Sweden 
Sector Private 
PI Contribution We developed a proposal to access the KaroBio compound library, and some specific technologies, in particular nuclear receptor crystallography, in pursuit of new ligands for the glucocorticoid receptor
Collaborator Contribution They provided cash, and contribution in kind in support of an MRC CASE award.
Impact not yet
Start Year 2014
 
Description Established new collaboration with Prof Angela Simpson at NorthWest Regional Lung Centre 
Organisation National Institute for Health Research
Department NIHR Manchester Musculoskeletal Biomedical Research Unit
Country United Kingdom 
Sector Public 
PI Contribution We are identifying new pathways operating in the inflamed lung. We are now testing to see if these operate in chronic human lung diseases.
Collaborator Contribution They are helping with ethics, patient recruitment, recovery and processing of human lung.
Impact not yet
Start Year 2014
 
Description Ueli Schibler 
Organisation University of Geneva
Department Department of Molecular Biology
Country Switzerland 
Sector Academic/University 
PI Contribution We collected serum from patients with rheumatoid arthritis. We discovered a new circadian signature.
Collaborator Contribution Our partners provided us with STAR-PROM technology to measure serum circadian factors
Impact none yet
Start Year 2015
 
Description max quant summer school 
Organisation Max Planck Society
Department Max Planck Institute of Biochemistry
Country Germany 
Sector Public 
PI Contribution Max quant summer school oxford UK 2016 Juergen Cox, writer of software and scripts, and now a collaborator. Visiting with us 10th March 2017
Collaborator Contribution this is an interactive summer school which provides hands on training and collaborative links in computational biology especially as applied to proteomics.
Impact not yet
Start Year 2016
 
Description metadherin as a major regulator of glucocortcoid action 
Organisation Princeton University
Country United States 
Sector Academic/University 
PI Contribution We discovered a role for metadherin in glucocortiocid action. Yibin Kang had the knockout mouse, and we have been working together to work out the physiological consequences of this novel interaction.
Collaborator Contribution Our partners had the mouse, and did the in-vivo studies for us.
Impact So far the outputs are training but we are writing up the paper now.
Start Year 2015
 
Description yibin kang 
Organisation Princeton University
Country United States 
Sector Academic/University 
PI Contribution access to transgenic mice and in-vivo experimentation protocols
Collaborator Contribution they provided mice and did the physiology expts for us.
Impact none yet
Start Year 2015
 
Description BBC breakfast, and new channel, and five live phone in. on 4th November 2014 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact I discussed the impact of shift work on the body clock, and the consequences for human health.

I have had numerous contacts from people engaged in shift work asking for advice.
Year(s) Of Engagement Activity 2014
 
Description academic presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Clcok club presentation.

"newly rhythmic processes emerge in active rheumatoid arthritis."
Year(s) Of Engagement Activity 2017
 
Description optimiser programme for business engagement 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact Optimizer programme: business development training 2016-2017
Year(s) Of Engagement Activity 2016
 
Description public engagement 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Reclaim Project: Youth Mentor 2017 , Lead&Activism weekends (NOV2016/ FEB2017)
Year(s) Of Engagement Activity 2016,2017