The role of Jam-C trafficking in endothelial vascular function

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.

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.

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.