The role of Jam-C trafficking in endothelial vascular function

Lead Research Organisation: Queen Mary University of London
Department Name: William Harvey Research Institute

Abstract

The body's response to a pathogen or tissue injury is essential for survival. White blood cells present in the circulatory system are targeted to the site of injury by signals present on the endothelial cells that line the blood vessels near to the site of injury or infection. This results in rolling of the leukocytes on the endothelium followed by subsequent firm adhesion and crawling before the leukocyte finds a suitable point to pass through the endothelium into the infected tissue. This multi-step cascade is tightly regulated and a number of different protein receptors on both the leukocyte and the endothelium govern each step.
A family of proteins present on the endothelial cells (the junctional adhesion molecules or JAMs) have been shown to have important roles in the final step of this inflammatory cascade, the passage of leukocytes through the endothelial cell layer and into the infected tissue. Jam-C, a member of this family, is necessary for this transmigration and has been demonstrated to maintain the directionality of leukocyte movement as well as helping to control the permeability of the endothelial layer. Inhibition of Jam-C function results in leukocytes passing hesitantly through the endothelial cell monolayer and/or returning to the blood flow. Importantly Jam-C expression and function has been associated with multiple inflammatory disease states including, amongst others, arthritis and heart disease.
Jam-C is primarily localised to the junctions between endothelial cells where it binds to Jam-C or Jam-B molecules present on neighbouring cells thus helping to create a tight junction. This junction restricts the passage of some molecules and proteins through the endothelial cell layer. When a leukocyte passes through the endothelium the Jam-C present on the endothelial cell is thought to switch to bind a receptor present on the leukocyte surface. Recent research has demonstrated that Jam-C is also present in intracellular vesicular pools and that the amount of Jam-C present at the surface, the cellular junctions and in intracellular vesicles varies following an inflammatory stimulus and this is also true of endothelial cells grown in culture. This indicates that internal pools of Jam-C might be dynamically regulated depending on the stimulus that the cells are exposed to. Internalisation or redistribution of Jam-C might be a means of regulating known Jam-C functions: either by preventing the receptor binding and signalling or by concentrating it in specific pools where it might be used i.e. around transmigrating leukocytes.
We propose to develop approaches to monitor the internal trafficking of Jam-C and the cellular machinery required to do this. Often this internalisation and intracellular routing of a surface receptor depends upon amino acid motifs present in the intracellular portion of the receptor. These motifs bind to cellular machinery and determine if the protein is internalised and also what happens subsequently to the now intracellular protein. The receptor might be degraded in the lysosome or the receptor might be recycled back to the cell surface by a number of different routes.
Once we have a better mechanistic idea of how Jam-C gets internalised and traffics around the cell we intend to use this information to find novel ways to inhibit this process by knocking out key pieces of machinery or by changing the motifs themselves. This approach should allow us to define the importance of intracellular Jam-C traffic on the known functions of Jam-C. What role does it play in leukocyte migration or in increasing cell permeability? Any new information could translate into new methods for controlling these processes. Such information could eventually be important in controlling inappropriate leukocyte transmigration in chronic inflammatory situations, or in boosting the rate of leukocyte influx in situations where a patient is immune compromised.

Technical Summary

The endothelium plays a crucial role in responding to an infection or tissue injury: In the recruitment of leukocytes to the site, and in tissue repair and angiogenesis. Adhesion receptors have a fundamental role in endothelial function governing many of these processes. Recent research suggests that rather than being present at fixed levels on the plasma membrane, some adhesion receptors exist in dynamically trafficked pools that can redistribute reflecting endothelial status.
Of particular interest is the junctional adhesion molecule-C (Jam-C), a type-1 integral membrane protein of the Ig superfamily that has been shown to mediate numerous endothelial cell functions. Most notably Jam-C mediates aspects of leukocyte transendothelial cell migration, angiogenesis and vascular permeability. Importantly Jam-C expression and function has been associated with multiple inflammatory disease states such as arthritis, peritonitis, acute pancreatitis, ischemia reperfusion injury, pulmonary inflammation and atherosclerosis. Its receptor function is mediated by homophilic interactions as well as interactions with Jam-B on endothelial cells and Mac-1 on leukocytes. Jam-C is present at the cell surface and enriched at cellular junctions but there is also good evidence that it is present in intracellular pools and that the amount present in vesicles varies with the inflammatory status as well as the presence of permeability or pro-angiogenic factors. In particular, Ischemia reperfusion injury causes marked intracellular redistribution of Jam-C, the function and mechanism of this redistribution is as yet unclear.
We aim to develop biochemical, live cell imaging and EM assays to investigate intracellular Jam-C traffic. Using these assays we will determine the motifs in Jam-C and molecular machinery required. We will then use this information to determine the functional significance of Jam-C trafficking on models of leukocyte transmigration, permeability and angiogenesis.

Planned Impact

Impact Summary
The research will define novel trafficking pathways that will provide a means to regulate the known functions of the junctional adhesion receptor, Jam-C. Not only will this provide the ground work for future in vivo work it will highlight molecules that could potentially be novel drug targets for the control of inflammation.

The beneficiaries are:

1. The General public-The receptor Jam-C has been implicated in a number of inflammatory disease states including rheumatoid arthritis, peritonitis, pulmonary inflammation, atherosclerosis and ischaemia reperfusion injury. Novel means of targeting and controlling receptor function by perturbing its trafficking will potentially provide drug targets for the control of inflammation. This is a long-term goal, but the potential for impact will be maximised when the Centre's expertise in the imaging of the vasculature in vivo is taken into account. This means promising leads can be very quickly translated into a more physiologically relevant context.

2. Private sector-Any promising targets for new drugs will be of potential interest to the public sector. But such research has the potential to stimulate investment from companies and University spin-offs (in the UK and overseas). We will ensure that any likely targets are patented using the QMUL technology transfer office (Queen Mary innovation). QMI has already overseen the development of a number of spin out companies including Activiomics and PhosphonicS.

3. The UK government-Inflammatory disorders and heart disease presents a major burden to the health service. Any new drug targets found could be potentially important in terms of public health but also from an economic perspective.

4. The UK economy (transferable skills)- The PDRA hired for this position along with any students that are carrying out research in the laboratory will get extensive training in molecular cell biology, high end imaging techniques and electron microscopy. Such transferable skills would be very important both in the public sector in terms of academic research but also in carrying out science in the private sector.
 
Description British heart foundation project grant
Amount £197 (GBP)
Funding ID PG/15/72/31732 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2016 
End 03/2019
 
Description New approaches to limit angiogenic responses by controlling endothelial junction
Amount £233,773 (GBP)
Funding ID MGU0534 
Organisation Barts Charity 
Sector Charity/Non Profit
Country United Kingdom
Start 12/2020 
End 12/2023
 
Description WHRI academy post doctoral fellowship
Amount € 90,020 (EUR)
Organisation Marie Sklodowska-Curie Actions 
Sector Charity/Non Profit
Country Global
Start 03/2016 
End 09/2018
 
Title Intracellular biotinylation approach for analysis of co-trafficked proteins 
Description This research method builds on published research which uses proteins tagged with ascorbate peroxidase to indentify intracellular interacting proteins. We have modified this approach using Horse Radish Peroxidase containing protein sequences. These target in a similar way to endogenous proteins and can be used to label nearby proteins at the cell surface. By also including ascorbate you can block cell surface labelling and more specifically label nearby protein in vesicles. The labeling process is very rapid (1 minute) and therefore can provide a snapshot of a molecules transport. 
Type Of Material Biological samples 
Provided To Others? No  
Impact There is no notable impact yet. 
 
Description Endothelial cell collaboration 
Organisation University College London
Department MRC Laboratory for Molecular Cell Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution I have an ongoing collaboration with Prof Daniel Cutler to study trafficking in endothelial cells. We provide expertise and novel JAM-C constructs and techniques.
Collaborator Contribution Prof Daniel Cutler is an expert in endothelial cell function. He will provide expertise and advice as well as access to spinning disk and super resolution facilities.
Impact We have already used both spinning disk and super resolution microscopy facilities to characterise Jam-C function and we hope to extend such work to encompass all of our mutant proteins.
Start Year 2013
 
Description JAM-C collaboration 
Organisation National Center for Scientific Research (Centre National de la Recherche Scientifique CNRS)
Department Centre National de la Recherche Scientifique Marseille
Country France 
Sector Academic/University 
PI Contribution We designed an approach to analyse the intracellular trafficking routes and molecular machinery used by Jam-C. We are collaborating with Dr Aurrand Lions as he is an expert in JAM-C function in immunity.
Collaborator Contribution Dr Michel Aurrand-Lions provided a number of key constructs and expertise in the experiments we designed to determine the routes of JAM-C trafficking.
Impact With the help pf Dr Aurrand-Lions we have made over 40 different tagged constructs for further characterisation.
Start Year 2013
 
Description Mass Spectrometry collaboration 
Organisation Queen Mary University of London
Department Barts Cancer Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution We are developing a novel technique for biotin labelling co-trafficked proteins in endothelial cells. We are further delineating the pathways and machinery involved by using a series of JAM-C mutant constructs.
Collaborator Contribution Dr Pedro Cutillas is an expert at quantitative mass spectrometry and has also developed a number of novel algorithms for analysing mass spectrometry data. We are using his equipment and expertise to characterise the proteins that are co-trafficked with JAM-C during leukocyte transmigration
Impact We have already carried out mass spectrometry analysis of JAM-C interactors at the cell surface and we are going to extend this to look at mutant proteins.
Start Year 2015
 
Description Novel Biotinylation reagents for live cell HRP trafficking assay 
Organisation Queen Mary University of London
Department School of Biological and Chemical Science QMUL
Country United Kingdom 
Sector Academic/University 
PI Contribution We have set up a novel trafficking assay to monitor the proteins surrounding Jam-C. Jam-C is tagged with Horse Radish Peroxidase and we can identify nearby proteins either at the cell surface or intracellularly by feeding cells with biotin phenol and adding Hydrogen peroxide to get a snap shot of nearby proteins with a time resolution of 1 min. These proteins can then be characterised by mass spectrometry, IF or western blot. By including a membrane impermeant inhibitor we can remove the cell surface pool and highlight only intracellular proteins that are being trafficked. This approach relies on the biotin phenol drug crossing two membranes the plasma and the vesicular membrane. We have evidence to suggest that by changing the permeability of the biotin phenol reagent we can improve/modify the assay.
Collaborator Contribution Dr Nathalie Lebrasseur is designing and making novel biotin phenol like compounds for us to test in our assay.
Impact We have tested two chemicals so far. One improved labelling of intracellular pools but reduced labelling of surface pools. This is likely to be useful for identifying minor components of vesicles with greater sensitivity (such as SNAREs etc).
Start Year 2016
 
Description electron microscopy collaboration 
Organisation University College London
Department MRC Laboratory for Molecular Cell Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution We have made wild type and mutant versions of horse radish peroxidase tagged JAM-C constructs which we have transfected into primary endothelial cells. Using these constructs we will delineate the trafficking pathways of JAM-C at the ultrastructural level.
Collaborator Contribution The electron microscopy department headed by Dr Jemima Burden and Dr Ian White are providing their expertise in designing electron microscopy experiments as well as carrying out sectioning and electron microscopical analysis.
Impact We have characterised the WT JAM-C receptor and are currently working our way through the 8 different mutants we have made so far.
Start Year 2013
 
Description novel biotinlyation reagents 
Organisation Queen Mary University of London
Department School of Biological and Chemical Science QMUL
Country United Kingdom 
Sector Academic/University 
PI Contribution We are designing novel chemical reagents for intracellular modification of nearby proteins as they trafffick.
Collaborator Contribution Dr Rooney is designing and developing a number of chemicals that will be activated by peroxide radicals resulting in covalent binding to the amino acids of nearby proteins. Thus allowing them to be purified and characterised.
Impact Currently we have 5 chemicals that will be tested in the near future for their efficacy in biotinylation of nearby proteins.
Start Year 2014
 
Description Barts Science Fair 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact My post doc Katja Kostlenik helped prepare a stand to help children understand the process of inflammation. She then talked the children through the information and played games to illustrate some of the key points. This led to more discussion about the process that was led by the children. Feedback from the schools involved and the organisers suggested that the fair was well received.
Year(s) Of Engagement Activity 2016
 
Description GCSE student work experience 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact A GCSE student shadowed myself or one of my postdocs as we carried out our normal research.
Year(s) Of Engagement Activity 2017
 
Description Meeting with local politicians 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Policymakers/politicians
Results and Impact A tour of the centre and its facilities was taken by members of the British Heart foundation board and local politicians this served to highlight research and how it is relevant for heart disease and stroke. It also highlighted how our research might lead to the identification of new therapeutic targets.
Year(s) Of Engagement Activity 2016
 
Description Organisation of pint of science 
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 My post doc Chris organised an evening of talks to publicise research and to inform members of the public on its impact and importance.
Year(s) Of Engagement Activity 2018
URL https://pintofscience.co.uk/
 
Description William Harvey research institute work experience programme 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact A level students took part in a week of activities to showcase the science going at our institute. This happens biannually and the students apply to learn more about careers as well as medical research. I gave a talk summarising the labs research and the approaches we use to study haemostasis and inflammation as well as the implications for patient health. As part of this students shadowed members of my lab as they carried out their day to day experiments. My post docs explained what they were doing and the students were encouraged to ask questions about what it is like to be a scientist as well as more technical questions about the research we do.
Year(s) Of Engagement Activity 2017,2018,2019,2022
URL http://www.qmul.ac.uk/whri/patient--public-engagement/whri-work-experience-programme-/