Investigating NF-kappaB p50 phosphorylation and the regulation of transcription

Inflammation is the complex biological response of the body's immune system to harmful stimuli, such as microbes, tissue damage or irritants. It is a protective attempt to remove the injurious stimuli as well as initiate the healing process for the affected tissue. In the absence of inflammation, wounds and infections would never heal and progressive destruction of the affected tissue would eventually lead to serious illness or death. However, inflammation which runs unchecked can also lead to a host of diseases including inflammatory bowel disease, rheumatoid arthritis, autoimmunity, sepsis and atherosclerosis. Recent research has also revealed a role for unregulated or dysregulated inflammatory processes in cancer and neurodegeneration. It is for this reason that inflammation is normally tightly regulated by the body. Thus, a thorough knowledge of the immune system and immune cells is of fundamental biological importance, and has the potential to improve our understanding and treatment of a broad spectrum of human diseases.

The proposed research will focus on a family of proteins collectively known as Nuclear Factor kappaB (NF-kB). NF-kB is a critical regulator of gene expression and plays key roles in the immune response to infection and inflammation. The triggers of such responses may include microbes or their associated products as well as signals from neighbouring cells or tissues. These stimuli induce NF-kB to increase the expression of genes that encode for pro-inflammatory factors such as cytokines and chemokines which orchestrate the movement and activation of immune cells to sites of infection or injury. While these processes are essential for the clearance of infection and the repair of damaged tissue they pose serious threats to healthy tissue and can cause severe damage in areas of inflammation. For this reason the expression of these pro-inflammatory mediators by NF-kB is normally tightly controlled. However in certain diseases, including those listed above, NF-kB is inadequately regulated and sustained expression of these potentially harmful mediators of inflammation occurs.

We are broadly interested in understanding how these complex intracellular processes are orchestrated and regulated, with a view to developing new therapies that manipulate immune cell function to control disease. In recent years it has emerged that the modification of individual NF-kB proteins by the addition of phosphate groups, a process termed phosphorylation, plays a critical role in determining the pattern of genes activated during inflammation. In this proposal we aim to comprehensively investigate the phosphorylation of NF-kB and how this modification contributes to the regulation of inflammation. The work we are proposing exploits and integrates state-of-the-art technologies to examine the importance of NF-kB phosphorylation at the molecular, cellular and genomic level. We will also incorporate computational modelling of the complex intracellular processes regulating NF-kB phosphorylation to predict potential therapeutic targets in inflammatory disease. The results of our study will provide novel and unprecedented insights into how the control of NF-kB phosphorylation contributes to the regulation of inflammation with broad physiological and pathological implications. The data generated will be relevant to our understanding of human diseases where NF-kB has been demonstrated to be important, such as inflammatory disease and cancer, and will be important in directing future therapeutic strategies aimed at inhibiting inflammation.

NF-kB is the master regulator of the immune response and is essential for the development and homeostasis of the immune system. The NF-kB subunit p50, encoded by NFKB1, is the transcription factor most highly expressed in human macrophage and is a critical factor in human inflammatory disease. Functional polymorphisms in NFKB1 are significant risk factors for inflammatory disease and cancer. Whilst the importance of NF-kB in human health and disease is indisputable, we lack fundamental information on the biochemistry of NF-kB activity. Phosphorylation of NF-kB is critically important for the transcription of target genes. The impact of specific NF-kB phosphorylation events is highly selective and directs transcription in a gene-specific fashion. However, a comprehensive analysis of p50 phosphorylation and its role in regulating transcription is notably lacking. We propose to generate a unique dataset of transcriptional events that are dependent on site-specific p50 phosphorylation, using a human macrophage model. Our approach will develop a series of gene-expression signatures predictive of functional classes of p50 phosphorylation, the activity of the kinase responsible, and determine how these influence overall transcriptional responses in macrophages. Existing transcriptomic data obtained from human macrophage or tissue from patients with inflammatory disease will be interrogated with phospho-p50 specific transcriptomic signatures. We will generate a co-expression network model of p50 target genes regulated by site-specific phosphorylation. The network will integrate public data on chromatin modifications from human monocytes, macrophages and dendritic cells. The model will provide a powerful tool for the development of novel therapeutic strategies based on the regulation of gene expression by p50 phosphorylation. This proposal will enable a highly innovative approach to tackle fundamental questions in NF-kB biology of high importance to human health and disease.

This proposal will generate a unique dataset of transcriptional profiles that will enable the development of gene expression signatures that are predictive of NF-kB modification and regulatory pathway activity. In doing so, we will provide unprecedented resolution into the biochemistry underlying NF-kB p50 transcriptional control of inflammation and macrophage activation. The scope of the project is ambitious, and will generate new paradigms for NF-kB signalling and new approaches for the therapeutic-targeting of specific NF-kB p50 mediated events. More generally, the approach and findings will have broad physiological and pathological implications for the study of gene regulation. The foundation approach, the molecular toolkit, as well as the data generated, will be of enormous utility to the wider research community. This will be enabled via a publicly accessible web-based interface, using the established 3iiiformatics platform at the University of Glasgow, which will allow researchers to visualise NF-kB signatures in our and other datasets, and facilitate requests for distribution of molecular constructs and cell lines. This provides for lasting impact for future studies where NF-kB is a major regulator of the underlying biology or pathogenesis.
The impact of the proposed research can therefore be identified as follows:

1) Academic
The data from the proposed research will be of importance to academic and private sector researchers both in the UK and internationally, and in a wide range of disciplines including, but not limited to, immunology, cancer biology, cardiovascular biology and neurobiology. The toolkit, and open dissemination of information via 3iiiformatics provides a major enabling platform for other researchers. Bioinformatics code will be made freely available to others wishing to adopt similar approaches with their own datasets. The impact of this proposal on UK based researchers will be to advance the knowledge economy. In addition, by communicating the research through our continued teaching and outreach activities we also hope to inform and educate, with impact, other beneficiaries in the University of Glasgow and local communities.

2) Private sector/biotech industry
This research addresses one of the major barriers to targeting NF-kB therapeutically, that is, that global inhibition of immunoreceptor signalling leads to blanket immunosuppression and the risk of severe toxicity. The fine-scale resolution provided by this research is necessary for any potential exploitation of NF-kB for therapeutic benefit, to circumvent the potential dangers seen with more global approaches. The data generated in this proposal will be used to predict new therapeutic targets (e.g. kinases) relevant to the treatment of inflammatory disease. The data from this proposal will be of benefit in assessing the anti-inflammatory potential of these targets with potential for attracting R&D investment and developing intellectual property in this area.

3) Economic and societal impact
NF-kB is intimately linked to the pathology of a wide range of diseases with profound social and economic impact. These include chronic inflammatory disorders such as rheumatoid arthritis, inflammatory bowel disease and autoimmunity, as well as cancer, atherosclerosis and neurodegenerative disease. This proposal focuses on a fundamental aspect of NF-kB biology, so is relevant to the future development of therapies in these areas to benefit the quality of life, health and well being of UK citizens.

4) Training of researchers at an advanced level
This proposal exploits state of the art techniques in transcription factor analysis including sophisticated gene-targeting, sequencing, kinase identification, proteomics and the development of a network based informatics based model. The multidisciplinary approach to addressing our research questions offers exceptional opportunities for the training of researchers at an advanced level.