Regulation of NF-kB by the ZO-1/ZONAB pathway

Lead Research Organisation: University College London
Department Name: Institute of Ophthalmology

Abstract

Epithelia are continuous layers of cells that delineate our tissues and organs. The integrity of epithelia is important for our organs to function normally and to protect us from our environment. For example, breaches in epithelial layers such as the skin or in the lining of the intestine can lead to serious infections. Individual epithelial cells interact with each other via molecular complexes that mediate adhesion but also function as sensors that transmit information about the environment, such as the presence or absence of neighbouring cells, to the cell interior. These sensors are important for the regulation of cell behaviour as they tell cells when to divide and when to stop to grow. If an epithelium is damaged, for example, cells divide and migrate to repair holes until they reach their neighbours and can form new adhesive complexes. Therefore, defects in these sensory mechanisms lead to severe diseases such as cancer. Many inflammatory diseases affect the functional properties of these adhesive molecular complexes. These diseases can be caused by bacteria and viruses, as well as allergens or by our own body defence system and often directly involve components of adhesive complexes. For example, a bacterium that causes gastric cancer injects molecules into epithelial cells that stimulate dissociation of adhesive complexes. Such infections also cause a cellular inflammatory response that helps our body to deal with them. However, these cellular responses can get out of control and cause damage. Here we focus on a new molecular mechanism by which adhesive complexes in epithelial cells can regulate the cellular inflammatory response. We propose to study the molecular mechanism and to determine how it is affected in two models of human disease. One model is based on the mentioned bacterium that causes gastric cancer and the other on cells isolated from colon and prostate cancers. Understanding how adhesive complexes guide the inflammatory response will help us to design new therapies for the above-mentioned diseases.

Technical Summary

Epithelial cells adhere to each other via junctional complexes, enabling them to form cellular barriers that separate different tissues and body compartments. Tight junctions are one of these intercellular junctions. They restrict paracellular permeability and are hence required for barrier formation. Tight junctions also contribute to the regulation of epithelial proliferation, differentiation and polarisation via different types of signalling mechanisms. Dissociation of tight junctions results in epithelial barrier breakdown and is associated with many diseases including chronic inflammations and cancer. Such diseases often stimulate proinflammatory signalling by activating NF-?B. Whether tight-junction-associated signalling mechanisms directly contribute to the regulation of proinflammatory signalling is not known. In previous work, we have discovered a tight-junction-associated signalling mechanism that is based on the junctional adaptor ZO-1 and the Y-box transcription factor ZONAB. By forming a junction-associated complex, ZO-1 inhibits nuclear accumulation of ZONAB/DbpA and thereby regulates gene expression and cell proliferation. As there are many pathogens that disrupt tight junctions, we asked whether this protein plays a role in NF-?B activation. Our unpublished results based on functional and biochemical assays now suggest that the ZO-1/ZONAB pathway interacts with NF-?B pathway components. Based on these observations, we hypothesize that ZO-1 and ZONAB regulate proinflammatory signalling by interacting with cellular machinery that controls NF-?B activation, and that this mechanism is functionally important under conditions that trigger the dissociation of intercellular junctions such as microbial infections or cancer. The purpose of this application is to identify the molecular mechanisms by which the ZO-1/ZONAB pathway participates in the regulation of NF-?B activation and whether these mechanisms are of pathological importance. In aim 1, we propose an experimental strategy based on biochemical, functional and morphological assays to determine how and where ZO-1 and ZONAB interact with specific NF-?B activating pathways, and, in aim 2, we plan to use two disease models, H. pylori infection and carcinoma-derived cell lines, to test the functional relevance of the identified mechanisms for the regulation of NF-?B. An additional objective will be to identify possible strategies to exploit this regulatory mechanism for future therapeutic approaches The expected results will be important for the understanding of the role of epithelial junctions in the regulation NF-?B signalling pathways and how these molecular mechanisms contribute to pathological conditions.

Publications

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Balda MS (2009) Tight junctions and the regulation of gene expression. in Biochimica et biophysica acta

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Balda MS (2014) Tight junctions in health and disease. in Seminars in cell & developmental biology

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Balda MS (2008) Tight junctions at a glance. in Journal of cell science

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Balda MS (2016) Tight junctions as regulators of tissue remodelling. in Current opinion in cell biology

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Larre I (2010) Ouabain modulates epithelial cell tight junction. in Proceedings of the National Academy of Sciences of the United States of America

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Lima WR (2010) ZONAB promotes proliferation and represses differentiation of proximal tubule epithelial cells. in Journal of the American Society of Nephrology : JASN

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Matter K (2014) SnapShot: Epithelial tight junctions. in Cell

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Nie M (2012) Stress- and Rho-activated ZO-1-associated nucleic acid binding protein binding to p21 mRNA mediates stabilization, translation, and cell survival. in Proceedings of the National Academy of Sciences of the United States of America

 
Description Apg-2: At the crossroads of tissue regeneration and degeneration
Amount £100,000 (GBP)
Funding ID R180018A 
Organisation Moorfields Eye Charity 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2018 
End 09/2021
 
Description MarvelD3 signalling and retinal tissue stress
Amount £122,242 (GBP)
Funding ID GR001000 
Organisation Moorfields Eye Charity 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2020 
End 12/2021
 
Description PhD fellowship
Amount £100,000 (GBP)
Organisation Fight for Sight 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2016 
End 09/2019
 
Description Research Grant
Amount £84,979 (GBP)
Funding ID ST 15 07 I 
Organisation Moorfields Eye Hospital NHS Foundation Trust 
Department The Special Trustees of Moorfields Eye Hospital General Fund
Sector Charity/Non Profit
Country United Kingdom
Start 04/2016 
End 03/2017
 
Title LNT-vectors 
Description Lentirviral vectors for the manipulation of junctional signalling pathways and NFkB activation in cell lines and tissues 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact Allows easy testing of pathway activity in vivo and in vitro 
 
Title MD3 reagents 
Description Reagents for the biochemical and functional analysis of MD3, a new tight junction-associated membrane protein that regulates subcellular signalling mechanisms (cDNA and siRNAs, adenoviral vectors, antibodies) 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact A first paper describing a basic analysis of this paper is in press 
 
Title MDCK cell lines 
Description MDCK cells liens expressing inflammatory regulators to study effect interplay between inflammation and epithelial barrier properites 
Type Of Material Cell line 
Provided To Others? No  
Impact Used at talks and is being incorporated into a publication 
 
Title ZONAB controls endothelial actin cytoskeleton and genes important for angiogenesis and inflammation 
Description Endothelial cell junctions have an important role in the function of the endothelium. They provide adhesive contacts between neighbouring cells and provide communication between cells. Proteins at the endothelial junctions are capable of detecting stretch, stress and damage and providing signals to intercellular effector pathways. ZONAB is a ZO-1 associated transcription factor that shuttles from the tight junction to the nucleus in epithelia. Our unpublished results demonstrate that ZONAB contributes to the regulation of endothelial cytoskeletal arrangement, tight junction protein localisation, proliferation, senescence and inflammation. Further characterization of the molecular mechanisms involved in these ZONAB-dependent pathways may therefore highlight novel therapeutic interventions for vascular diseases, such as atherosclerosis. 
Type Of Material Model of mechanisms or symptoms - human 
Year Produced 2016 
Provided To Others? No  
Impact Endothelial cell junctions have an important role in the function of the endothelium. They provide adhesive contacts between neighbouring cells and provide communication between cells. Proteins at the endothelial junctions are capable of detecting stretch, stress and damage and providing signals to intercellular effector pathways. ZONAB is a ZO-1 associated transcription factor that shuttles from the tight junction to the nucleus in epithelia. Our unpublished results demonstrate that ZONAB contributes to the regulation of endothelial cytoskeletal arrangement, tight junction protein localisation, proliferation, senescence and inflammation. Further characterization of the molecular mechanisms involved in these ZONAB-dependent pathways may therefore highlight novel therapeutic interventions for vascular diseases, such as atherosclerosis. 
 
Title ZONAB controls endothelial actin cytoskeleton and genes important for angiogenesis and inflammation > Research Databases & Models Back to top 
Description Cell-base models for testing drugs for endothelial diseases 
Type Of Material Data analysis technique 
Year Produced 2017 
Provided To Others? No  
Impact Endothelial cell junctions have an important role in the function of the endothelium. They provide adhesive contacts between neighbouring cells and provide communication between cells. Proteins at the endothelial junctions are capable of detecting stretch, stress and damage and providing signals to intercellular effector pathways. ZONAB is a ZO-1 associated transcription factor that shuttles from the tight junction to the nucleus in epithelia. Our unpublished results demonstrate that ZONAB contributes to the regulation of endothelial cytoskeletal arrangement, tight junction protein localisation, proliferation, senescence and inflammation. Further characterization of the molecular mechanisms involved in these ZONAB-dependent pathways may therefore highlight novel therapeutic interventions for vascular diseases, such as atherosclerosis. 
 
Description Analysis of ZONAB function using knockout mice 
Organisation Agency for Science, Technology and Research (A*STAR)
Department Institute of Molecular and Cell Biology,
Country Singapore 
Sector Academic/University 
PI Contribution We are analysing alterations in the retinal caused by the deletion of the ZONAB gene.
Collaborator Contribution The partner generated mice with deletions of the ZONAB gene and provides us with issue to analyse the role of ZONAB in the retina.
Impact a manuscript is in progress that includes part of this research
Start Year 2008
 
Description The role of ZONAB in H. pylori invasion 
Organisation University College Dublin
Department School of Biomolecular and Biomedical Science UCD
Country Ireland 
Sector Academic/University 
PI Contribution We are analysing the effect on ZONAB signalling in response to H. plyori invasion using the samples prepared by the collaborator
Collaborator Contribution The partner provides fixed samples of H. pylori-infected gastric epithelial cells
Impact Results from the study are included in a manuscript in preparation that focuses on ZONAB signalling and NF-kB activation The collaborator is an expert in microbiology, which complements our experience in cell and molecular biology
Start Year 2009
 
Description ZONAB signalling in inflammation and cell survival, and apical differentiation in the retinal pigment epithelium 
Organisation University College London
Department Institute of Ophthalmology UCL
Country United Kingdom 
Sector Academic/University 
PI Contribution We are performing the analysis with primary cultures in vivo and the collaborators test our in vitro results with mouse experiments
Collaborator Contribution The collaborator performs the in vivo experiments with lentiviral vectors in mouse retinas
Impact A first publication has been published in 2010 (PMID: 21209887) Follow-up work has led to an an extension of this collaboration to apical differentiation in vivo in mouse RPE, which has been funded by the BBSRC
Start Year 2007
 
Description Distinguished Lecture UCL Medicine 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Discussion of future work
Year(s) Of Engagement Activity 2017
 
Description Participation in Charity Fund Raising Event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Type Of Presentation Workshop Facilitator
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Several hundred participants

NA
Year(s) Of Engagement Activity 2011
 
Description PhD students Berlin 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Lecture for students of a PhD programme in Germany and discussions about their own research projects
Year(s) Of Engagement Activity 2017
 
Description Visit to Charity Fundraising committee 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact First meeting; 12 people attended, including staff and volunteers of Fight for Sight Charity, who were presented with a short talk about the principle of our research followed by a interactive discussion
Second meeting: large group of participants in combination of information and fund raising event

Have since then regular contacts with charity staff
Year(s) Of Engagement Activity 2009,2012