DETAILS OF INDIVIDUAL RESEARCH PROGRAMMES
Research Programme 1: The regulation and function of MAP kinase phosphatase-2
We have several achievements in this area since cloning the gene (DUSP4) for this phosphatase in the late 90's. MKP-2 plays a key role in negatively regulating the activities of the MAP kinases and is thus implicated in several different cellular functions and diseases. In our original studies we re-defined the kinase specificity of the phosphatase in normal cells and identified for the first time, a novel site that dictated nuclear localization. We also demonstrated that over-expression of MKP-2 could limit genotoxic induced cell death, indicating its potential as a target in different forms of cancer. We also identified for the first time a novel variant of human MKP-2 and delineated its function again relevant to a potential role in cancer.
My group has also generated a novel DUSP-4 KO mouse, the first time a mouse for this deletion had been developed and a first for Strathclyde. This has enabled Strathclyde researchers to play a leading role in developing the understanding of MKP-2 function. Using the mouse we showed for the first time a role of MKP-2 in the response to pathogens such as Leishmania mexicana, work published recently in the high impact open access journal, Plos Pathogens and also Leishamania major and Toxoplasma (see submitted 2012 papers). Using cells from the MKP-2 KO we have also made important novel observations in relation to icell cycle progression.
We have also developed a number of important external collaborations one with Owen Samson (Beatson, Cancer Institute) looking at DUSP-4 in breast cancer, and with Ari Elson (Weizmann Institute), assessing the role of MKP-2 in bone development. In addition, we are involved in making a double KO mouse (MKP-1 and 2), with Dr Rosario Perona (University of Madrid), again a first for Strathclyde. We have also examined the potential of utilising Adv-MKP-2 as a therapy in cardiovascular disease (see also recent BHF grant award) and are investigating the role of MKP-2 in cardiac function (with Dr Susan Currie). We are assessing the potential of over-expression MKP-2 to be linked with the development of prostate cancer in collaboration with Drs Marie Boyd and Joanne Edwards.
Research Programme 2: The Biology of Protease-activated Receptors
The enzyme thrombin is well recognised to activate a series of cell signaling events important in mediating the growth of cells of the vasculature. This is achieved though the activation of a specific receptor called protease-activated receptor-1 (PAR-1). In examining signaling responses to thrombin, relevant to this action, we also recognised the potential for cells to be activated by other proteases through different members of this receptor family. Our review on Proteinase-activated Receptors (PARs) has now become one of the top ten cited articles from Strathclyde University (see Macfarlane et al., review). Research from two original papersresulted in substantial funding from the Japanese pharmaceutical company KOWA Company LTD and enabled a program of work to be funded via the Scottish Biomedical Foundation (SBF), the Strathclyde Institute for Drugs Research (SIDR) and the department of Chemistry at the University of Edinburgh. The funding totaled approximately £3 million. This project generated a number of research papers and additional funding from research councils and charities and has lead to a sustained collaborative effort between the Ferrell, Lockhart and Plevin laboratories, numerous grants and publications (see above and below listings). This work has demonstrated my ability to maintain a programme of research excellence over the longer period.
2a) Cell signalling through activation of PARs
Many years ago, my group was the first to show activation of MAP kinase signalling through the novel ‘thrombin like' receptor protease-activated receptor-2 (PAR-2). Using the cloned hPAR-2 in a model transfection system we established that PAR-2 was linked to both SAP kinase and NFκB signalling pathways, events linked to the proposed pro-inflammatory effects of PAR-2 . We used a series of molecular approaches to identify novel intermediates that couple PAR-2 to downstream signalling events including the activation of the MAP kinases and NFκB. Most recently we have established a novel regulatory role for PAR-2 and other GPCRs in the negative modulation of JNK as a general model for the anti-inflammatory actions of PAR-2 and other GPCRs (see recent submissions). We have established systems to examine PAR dimerisation, again a first for the University of Strathclyde and using this approach have demonstrated for the first time a novel role for PAR-2 in PAR-4 directed trafficking to the plasma membrane. In the area of pharmacology we have screened a peptide mimetic library and identified a novel peptide which mediated agonist selective signalling. Collaborations with medicinal chemists in SIPBS are developing this area further with a view to finding novel PAR-2 inhibitors and we have secured small amounts of funding (SULSA £20k and West of Scotland Cancer Consortium £8k) to develop the chemistry further. This latter work also involved collaborators in Japan.
2b) Development and utilization of PAR-2 knockout mice - revealing a role in rheumatoid- and osteoarthritis
We successfully developed PAR-2 deletion mice in collaboration with the Centre of Genome Research (CGR) at the University of Edinburgh. This was the first mouse designed using expertise within the University Strathclyde and is one of only two such colonies in the world. This success allowed the development of research relating to the physiological and pathophysiological role of PAR-2. This included Type IV allergic responses in skin but more importantly chronic arthritis. Most recently, we have identified a novel role for PAR-2 in osteoarthritis via the involvement of the enzyme matriptase. The underlying immune consequences have also been examined. In further collaborative work with Professors Ferrell and Lockhart, we have characterized a number of intervention strategies to block PAR-2 actions within the arthritic joint including antibody administration, siRNA and the first PAR-2 antagonist. This work will continue to explore avenues for therapeutic intervention in humans with the development of a novel humanized antibody with the potential for clinical trials (see recent CSO funding) and novel peptide antagonists. This work and its potential is the subject of a joint ARC programme grant proposal (result pending) and an ARC project grant.
Research Programme 3: NFκB sigalling in disease and infection
My group has had a long standing interest in the regulation of nuclear factor kappa B in disease particularly of the cardiovascular system and in inflammation.
3a) Regulation of NFκB signalling in the cardiovascular system
In a number of papers we have described the essential role of the inhibitory kappa B kinases (IKK) in key NFκB-dependent events within smooth muscle cells and have elucidated IKK2 as a potential target for drug therapy. This was made possible by using adenoviral constructs encoding inhibitory components of the pathways (see previous BHF awards and references). Using these approaches and also lentivirus we are investigating the role of individual IKK isoforms in endothelial and smooth muscle cell function and the upstream regulator of non-canonical NFkB signalling, NIK.
3b) Screening natural products as NFκB inhibitor drugs for use in cancer
Historically, phytochemists in SIPBS produced a large number of compounds however, there was no proper rapid drug screening approaches to compliment this. As part of the KOWA award, my group was able to generate reporter cell lines for this purpose. Using this approach we produced one of the first papers in Strathclyde systematically pinpointing the site and mechanism of action of a natural product.
Screening of a number of natural compounds provided elucidated their mode of action as selective IKK inhibitors. This work provided the basis for a successful CRUK programme grant award.
3c) Regulation of NFκB signaling during Leishmania infection
This work is a component of a long standing collaboration with Professor James Alexander. Initially we identified that amastigoes from Leishmania Mexicana were able to degrade NFκB intermediates and this is viewed as an important observation in the field and often cited. This work was a component of The Wellcome Trust Programme grant awarded to James Alexander. We have more recently also demonstrated, using again specific KO mice, a role for TLR-4 in the effects of promastigotes on macrophage function and a potentially novel affect of these pathogens on NFκB signaling. This work significantly augments the work ongoing with the MKP-2 deletion mouse in immunology.