MICA: Epigenetic regulation of GR function in pulmonary inflammation: the role of MERM1

Respiratory diseases are a significant health burden in the UK, with an estimated 3.7 million people with chronic obstructive pulmonary disease (COPD) and 5.2 million people being treated for asthma. In 2010, respiratory diseases accounted for 14% of all deaths. Glucocorticoids (Gc) are widely used to treat human inflammatory lung diseases, but their use is limited by side-effect profile resulting from the use of supraphysiological doses, or potencies of synthetic Gc. This supraphysiological requirement is necessary to overcome the partial resistance conferred by active inflammation. Various mechanisms to explain this resistance have been proposed, but none has led to improved therapy.
We have discovered a novel chromatin remodelling enzyme, MERM1, as a strong candidate for regulating GR function in pulmonary inflammation, and now propose to exploit this advance for the benefit of patients with pulmonary inflammatory disease.
MERM1 is widely expressed, but the highest expression observed in humans was in bronchial epithelium. MERM1 is required for GR to access its binding sites in the genome, and to regulate both induced and repressed target genes, including repression of pro-inflammatory cytokines. MERM1 protein is degraded in response to pro-inflammatory cytokines, in a ubiquitinylation-dependent mechanism. This is accompanied by loss of GR function. GR activity can be completely rescued by restoring MERM1 expression. Relevance to human disease was confirmed by finding marked loss of MERM1 protein in a panel of human lung inflammatory diseases.
We will now define the role of MERM1 in pulmonary inflammation.
The mechanisms of MERM1 action, and regulation of GR activity will be pursued in human cells ex-vivo. We will use primary human bronchial epithelial cells, and primary human macrophages; to maximise translation potential. We will investigate the responses of these cells in isolation to inflammatory activation, and investigate how MERM1 responds, and how MERM1 regulates the anti-inflammatory actions of the glucocorticoid receptor. We will extend our observations beyond candidate GR targets to analyse the genome-wide actions of MERM1, which extend well beyond potentiating the GR. This will be addressed by RNA-Seq and ChIP-Seq approaches targeting MERM1, and GR.
We will define the regulation of MERM1 in induced pulmonary inflammation, the function of MERM1 in innate, allergic, and viral induced lung inflammation. To specifically determine the role of MERM1 in the bronchial epithelium we will generate floxed Merm1 mice, and cross them to a bronchial epithelial-specific cre deletor line. We have the deletor mice, and have already shown the specificity and efficiency of floxed gene excision. In this way we can target MERM1 loss to the epithelium in-vivo. We will investigate the consequences of this loss for response to innate inflammatory challenge with LPS, allergic challenge using house dust mite, and viral challenge, using the common cold inducing respiratory rhinovirus. The pulmonary inflammation induced by all three stimuli responds to exogenous, high-dose Gc therapy. Therefore we will also be able to characterise how MERM1 loss in the epithelium affects Gc action in inflammation.
Translation to human inflammatory lung disease is essential, and will be achieved by analysing MERM1 expression in pathological samples of lung tissue, and lung-derived cells (mainly airway macrophages). In addition, we have access to a cohort of well-defined severe asthma patients. These subjects have been characterised as not responding to intramuscular, potent steroid therapy, either with a change in lung function, or a change in sputum eosinophilia. We will compare MERM1 expression in induced-sputum, and peripheral blood cells from these patients compared to two control groups, one with mild asthma, and one of healthy controls.

Pulmonary inflammation is a major human health concern, for which glucocorticoids (Gc) are the major treatment. However, Gc exert variable effects, for reasons which remain unclear.
We have discovered that in inflammation MERM1, an important epigenetic regulator of GR function is degraded. This is due to cytokine induction of MERM1 ubiquitinylation, and proteasome digestion. The same cytokines impair GR function. GR function is rescued by restoration of MERM1 expression, by mutating the ubiquitinylation sites. The importance of MERM1 to human lung disease is exemplified by finding loss of MERM1 protein expression across a panel of human inflammatory lung disease histological sections.
The mechanisms underlying MERM1 action in the lung will be explored using human bronchial epithelial cells, and macrophages in-vitro. The impact of MERM1 loss on responses to inflammatory challenge and Gc treatment will be measured. The broader actions of MERM1 will be measured using RNA-Seq and ChIP-Seq protocols targeting both MERM1, and GR under conditions of induced inflammation, and Gc exposure. These datasets will be subject to detailed bioinformatics analysis to reveal other MERM1 dependent transcription factors operative in these cells under inflammatory challenge, and to define the spectrum of Gc targets that are MERM1 regulated.
We now seek to make a bronchial epithelium-targeted MERM1 deletant mouse, and compare this to littermate controls in a series of pulmonary inflammatory challenges, including allergic, and rhinoviral infection protocols. The impact of MERM1 loss in the bronchial epithelium will be determined on elaboration, and resolution of inflammation. In particular we will investigate the impact of MERM1 loss on the anti-inflammatory actions of Gc.
The gene target and pathway discovery approaches above will be tested in primary human lung tissue and cells from patients with Gc resistant asthma.

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 asthma, 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.