Award details

Regulation of cell fate by a novel p53-inducible ligand-independent TRAIL-R2 complex

Reference	BB/T002824/1
Principal Investigator / Supervisor	Professor Daniel Longley
Co-Investigators / Co-Supervisors	Dr Simon McDade
Institution	Queen's University of Belfast
Department	Sch of Medicine, Dentistry & Biomed Sci
Funding type	Research
Value (£)	383,540
Status	Current
Type	Research Grant
Start date	01/12/2019
End date	05/11/2023
Duration	47 months

Abstract

The genetic paralogs FLIP and caspase-8 are key regulators of the extrinsic apoptotic pathway and both play crucial roles in maintaining barrier functions of the skin and intestinal epithelium. Acute loss of either protein in skin or intestine results in uncontrolled cell death (via apoptosis and/or necroptosis), loss of barrier function and mortality in adult mice. This project will explore functions of a novel ligand-independent TRAIL-R2 death inducing signalling complex ("LI-DISC") containing FLIP and caspase-8 that we recently identified, formation of which is driven by p53-dependent transcriptional up-regulation of TRAIL-R2 and FLIP. Notably, this complex also appears to contain p53 itself, where it may be cleaved to a truncated form thereby altering its functions. We hypothesize that p53-mediated induction of and regulation by this complex has evolved to control inflammation and cell death in response to acute physiological stresses such as UV-induced DNA damage, directing cells with unresolvable damage towards non-inflammatory apoptotic cell death to prevent destruction of epithelial barriers. The proposed work is designed to increase our mechanistic understanding of the basic biological roles of the TRAIL-R2 LI-DISC, with a particular focus on its roles in coordinating cellular responses to DNA damage in epithelial cells of the intestine and skin, and has been structured into 3 work-packages. In the 1st work-package, we will identify: (i) the components of the LI-DISC; (ii) its cellular location; and (iii) the cellular stresses that induce its formation. In the 2nd WP, we will focus on the function(s) of the TRAIL-R2 LI-DISC, specifically its impact on: (i) cell death; (ii) feedback to p53 signalling; and (iii) impact on NFKB and global transcription. In the 3rd work-package, we will assess the biological function of the TRAIL-R2 LI-DISC in human skin using 3-dimensional organotypic cultures of primary human keratinocytes ("skin rafts").

Summary

The maintenance of the barrier functions of the surfaces (epithelia) of the skin, intestines (gut) and other organs (for example, lung) are critical for health. In the skin, the barrier function is essential for strong, healthy, hydrated skin and protects living cells against environmental chemicals, biological irritants and entry of infectious agents. In the gut, the epithelial barrier regulates nutrient absorption as well as preventing infection by pathogenic bacteria. When barrier functions are compromised, this can lead to inflammation, infection and ultimately loss of organ function. Therefore, in response to damage caused by a range of agents, for example ultraviolet (UV) radiation in the skin and infectious agents and toxins in food, the epithelial cells of these organs must respond in a highly regulated manner to the damage in a way that does not compromise barrier function. We have identified a novel internal cellular mechanism that is activated as an early response to cellular stress, which tightly controls the death of epithelial cells. We hypothesize that this mechanism is essential for organismal health in the face of the sorts of environmental damage that epithelial surfaces encounter on a daily basis, by preventing mass epithelial cell death, which would ultimately lead to loss of critical barrier functions in the skin, gut and other organs. By studying this mechanism at a fundamental molecular level, we aim to provide insights into the maintenance of skin and intestinal health that could inform new ways of protecting these vital barriers and combating diseases in which these barriers become compromised; these include inflammatory diseases of the skin (e.g. atopic dermatitis and psoriasis) and gut (e.g. inflammatory bowel disease, IBD). These studies could also have implications for other important epithelial barriers like those in the lining of the lungs, where inflammation can result in life-threatening conditions like asthma.

Impact Summary

p53 is a critical master regulator of cell fate and therefore is vital in maintaining tissue homeostasis and preventing cancer. However, the fundamental mechanisms underlying the switch from p53-induced cell cycle arrest to cell death induction or indeed other cell fates remain poorly understood. Classically, death receptors have been characterized as cell surface proteins that regulate cell fate via interactions with their ligands (TRAIL in the case of TRAIL-R2) expressed by immune effector cells. Binding of TRAIL to TRAIL-R2 (and TRAIL-R1) causes formation of a Death-Inducing Signalling Complex (DISC) containing FLIP and caspase-8 that can trigger non-inflammatory (apoptotic) cell death. It has long been known that p53 can upregulate TRAIL-R2 expression thereby enhancing sensitivity to TRAIL via this extrinsic pathway; We have discovered a novel intrinsic TRAIL-R2 complex induced by p53 that is not dependent on binding by extrinsic TRAIL, and which we have therefore called the Ligand-Independent TRAIL-R2 DISC. Notably, unlike the extrinsic TRAIL-R2 DISC, this complex appears to directly interact with and regulate p53. We hypothesise that this unique intrinsic death receptor complex plays a fundamental role in controlling the response of epithelial cells to stresses such as DNA damage induced by UV radiation in the skin. The proposed study therefore will be of general interest to a broad spectrum of scientists and has potential impact in the areas of Knowledge, Health, Wealth, Culture and Society. Below we describe the specific impacts of this research in each of these domains. Knowledge: Because of its essential roles in maintaining tissue homeostasis and preventing disease (cancer), p53 is the most studied protein in the human proteome. By studying the novel p53-inducible and p53-binding TRAIL-R2 LI-DISC, we will provide new insights into the biology of p53 and its fundamental role in maintaining epithelial homeostasis and preventing organ dysfunction, infection and disease. Health: While focused on a basic science question (the biology of the TRAIL-R2 LI-DISC), the results from this study will provide new insights into the regulation of normal tissue homeostasis. Compromised barrier function is the cause of a number of chronic and acute diseases, including atopic dermatitis, psoriasis, irritable bowel disease and asthma. In addition, the complex/pathway under investigation is also likely to be important in cancer development and treatment; hence this study could have important implications for improving our understanding of these diseases. Wealth: While this is a basic biology focussed project, potential commercial opportunities may arise from our findings; for example, insights into how epithelial cells respond to different environmental stresses may reveal novel therapeutic approaches to protect epithelial barriers to prevent/treat disease. Culture and Society: This project will also contribute to the development of highly skilled individuals, since during the project, the PDRA (and PhD student from a leveraged studentship) will develop skills in cutting edge techniques, including next-generation sequencing (NGS) and 3D tissue culture. These are skillsets that are in demand in the academic, industrial and clinical healthcare sectors. A further societal impact will stem from the involvement of the PDRA, student and co-PIs in promoting their research at University, School and public events in Northern Ireland, inspiring the next generation of Scientists in the Province and developing interest and understanding of the importance of scientific research. Staff employed on this project will be offered the opportunity to become STEM ambassadors, volunteer at fundraising events such as "Race for life" and be regularly involved in open days and lab tours to explain the work carried out in our lab.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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