Differentiation of the retinal pigment epithelium: the role of the tight junction associated transcription factor ZONAB

Lead Research Organisation: University College London
Department Name: UNLISTED

Abstract

Epithelial cells line all our organs and mediate many organ-specific functions. They form barriers consisting of cellular sheets that separate compartments of different composition, such as in the eye, the blood-retinal barrier. The development and functioning of these cellular sheets requires a careful timing of cell proliferation and differentiation. After an operation, during retinal wound healing, for example, epithelial cells need to divide to fill the gaps and then re-differentiate into the organ-specific cell type. Individual epithelial cells are connected to each other via specialised intercellular junctions that mediate cell-cell adhesion, regulate transepithelial permeability, and help to control epithelial proliferation and differentiation. One of these structures, the tight junction, regulates diffusion of solutes across epithelia. We have recently discovered two tight junction regulatory system that control cell proliferation and differentiation in a cell density-dependent manner. One of these systems is based on a transcription factor, a protein that controls expressionof genes in the nucleus. The other system is based on a regulator of actin and also controls gene expression and cell shape. Here we propose to elucidate how these regulatory systems turn on and off the cellular machinery required for retinal pigment epithelial cells to stop to divide, to differentiate and to form the blood-retinal epithelial barrier.

Technical Summary

The retinal pigment epithelium (RPE) is located between the neural retina and the vascular choroid and forms the outer blood-retinal barrier. One of the key features of a differentiated RPE is the establishment of tight junctions, the cell-cell junctional structures that regulate selective diffusion of solutes along the paracellular space and that are also involved in the control of cell proliferation and gene expression. We recently identified ZONAB, a Y-box transcription factor that interacts with the tight junction protein ZO-1. ZO-1 and ZONAB functionally interact to regulate the expression of the co-growth factor receptor erbB-2 and cell cycle progression in a cell density-dependent manner. Since ZONAB is transcriptionally active in proliferating cells, our working hypothesis is that stimulation of ZONAB plays an important role in epithelial proliferation and destabilises the epithelial phenotype. This is supported by experiments with RPE cell lines, ARPE-19 and RPE-J, that revealed that overexpression of ZONAB causes dedifferentiation as manifested by disorganisation of cell-cell junctional and cytoskeletal proteins such as ZO-1, beta-catenin, actin and alpha-smooth muscle actin. These are features of an epithelial-mesenchymal transition that can be triggered by several physiological or pathological conditions, in which epithelial cells lose cell-cell and/or cell-matrix contacts. Our cell culture data are supported by an in vivo model of retinal degeneration, the Royal College of Surgeons dystrophic rat, in which the RPE-photoreceptor attachment is disrupted, and where we observed that upregulation of ZONAB and alpha-smooth muscle actin, a marker for dedifferentiating epithelial cells, coincides. Therefore, the goal of this project is to characterise the ZONAB-regulated signal transduction pathways that are important for retinal pigment epithelial differentiation.We will determine the mechanism(s) by which signalling by ZONAB and the interacting Rho exchange factor GEF-H1 regulate the expression of alpha-smooth muscle actin, modify adherens junction-associated signalling, and whether junctional adhesion and extracellular matrix proteins regulate ZONAB. To perform these studies we will use RPE cells in culture, the in vivo model of RPE degeneration of the RCS rats, and rat explants with intact RPE cells. We will also test whether the main molecules identified are affected in human eye tissue with RPE pathology. Investigating the cellular mechanisms by which tight junctions regulate epithelial differentiation is important for the understanding of tissue biogenesis, wound repair, as well as pathological conditions such as proliferative or degenerative retinal diseases, and provides the basis for the development of novel therapies for the treatment of such diseases.

Publications

10 25 50
 
Description Training and career development of researchers: Tsapara, Anna
Geographic Reach Asia 
Policy Influence Type Influenced training of practitioners or researchers
 
Description Inhibition of the Rho signalling activator GEF-H1 for the prevention of epithelial degeneration
Amount £100,000 (GBP)
Funding ID 1393/94 
Organisation Fight for Sight 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2012 
End 09/2015
 
Description MRC Project Grant (G0700793)
Amount £450,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2010 
End 04/2013
 
Description ZONAB controls endothelial actin cytoskeleton and genes important for angiogenesis and inflammation
Amount £100,000 (GBP)
Funding ID FS/16/23/32071 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2016 
End 09/2019
 
Title GEF-H1 as TGF-beta target gene and effector as well as biomarker for retina detachment 
Description We established an experimental system based on primary procine RPE (PP-RPE) cells. They form well-differentiated monolayers with well-assembled intercellular junctions and functional epithelial barriers with a transepithelial electrical resistance of 106±5Ocm2. They respond to both high glucose concentrations and TGF-ß, as both treatments result in increased paracellular permeability, indicating that PP-RPE cells can be used as an in vitro model to study diabetic signalling mechanisms (MS in preparation). The porcine genome has been sequenced; hence, we can also use functional genomics approaches (e.g., cDNA microarrays; see below). 
Type Of Material Model of mechanisms or symptoms - human 
Year Produced 2008 
Provided To Others? Yes  
Impact As these cells can be isolated from eyes obtained from the slaughterhouse, this system also supports the MRC policy to promote the 3Rs of animal welfare in research. 
 
Description Dr. Christophe E. Pierreux and Professor Pierre J. Courtoy 
Organisation de Duve Institute
Country Belgium 
Sector Academic/University 
PI Contribution We have contributed with different types of reagents to analyse ZONAB function, the discussion of the data, the writing and revision of a manuscript that it is in press in the Journal of the American Society of Nephrology.
Collaborator Contribution Contributions of tools to understand how ZONAB promotes proliferation and represses differentiation of proximal tubule epithelial cells
Impact ZONAB is regulated by cell polarization and represses apical differentiation
Start Year 2007
 
Description Functional analysis of proteins encoded by retinal disease genes and analysis of patient derived induced pluripotent stem cells 
Organisation Andalusian Center for Molecular Biology and Regenerative Medicine
Country Spain 
Sector Private 
PI Contribution Design of the project
Collaborator Contribution Provision of human induced pluripotent stem cells from patients with inherited retinal degeneration
Impact grant application
Start Year 2016
 
Description MRCK signalling in epithelial polarity and function 
Organisation Beatson Institute for Cancer Research
Country United Kingdom 
Sector Academic/University 
PI Contribution We are determining the functional importance of MRCK signalling in epithelia
Collaborator Contribution BICR provides small molecule inhibitors of MRCK
Impact A first paper has been published in 2017 describing part of this research
Start Year 2016
 
Description Professor Anna M. Randi 
Organisation Imperial College London
Department Imperial College Academic Health Science Centre
Country United Kingdom 
Sector Academic/University 
PI Contribution Generation of data, equipment and facilities, training of staff and significant intellectual input into the research project to understand how tight junctions regulate endothelial functions and angiogenesis
Collaborator Contribution Access to methods, reagents and intellectual input into the research project.
Impact Generation and discussion of data to obtain the current grant funding
Start Year 2006
 
Description Professor Caroline Hill 
Organisation Cancer Research UK
Department Cancer Research UK London Research Institute (LRI)
Country United Kingdom 
Sector Academic/University 
PI Contribution Tools and discussion for the analysis of TGF-beta signalling.
Collaborator Contribution GEF-H1 induction is Smad4-dependant and led to Rho activation and cell migration
Impact GEF-H1 induction is Smad4-dependant and led to Rho activation and cell migration
Start Year 2006
 
Description Professor Gary Owens 
Organisation University of Virginia (UVa)
Department Department of Molecular Physiology and Biological Physics
Country United States 
Sector Academic/University 
PI Contribution He has provided several alpha-smooth muscle actin promoter constructs
Collaborator Contribution GEF-H1 induction is induced by TGF-beta in Smad4-dependant and led to Rho activation and a-SMA expression. GEF-H1 activation can regulate a-SMA transcription and inhibition counteracted a-SMA expression and cell migration induced by TGF-beta.
Impact GEF-H1 induction is induced by TGF-beta in Smad4-dependant and led to Rho activation and a-SMA expression. GEF-H1 activation can regulate a-SMA transcription and inhibition counteracted a-SMA expression and cell migration induced by TGF-beta.
Start Year 2006
 
Description Professor Phil Luthert. 
Organisation University College London
Department Institute of Ophthalmology UCL
Country United Kingdom 
Sector Academic/University 
PI Contribution Analysis of GEF-H1 expression in diseased human retina.
Collaborator Contribution Institute of Ophthalmology
Impact Analysis of GEF-H1 expression in diseased human retina.
Start Year 2007
 
Description Professor Robin R. Ali 
Organisation University College London
Department Institute of Ophthalmology UCL
Country United Kingdom 
Sector Academic/University 
PI Contribution Analysis of the role of ZONAB and ZO-1 expression in vivo in mouse RPE by developing lentiviral vectors
Collaborator Contribution Analysis of the role of ZONAB and ZO-1 expression in vivo in mouse RPE by developing lentiviral vectors
Impact Analysis of the role of ZONAB and ZO-1 expression in vivo in mouse RPE by developing lentiviral vectors
Start Year 2006
 
Description Adhesion and Signalling 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Health professionals
Results and Impact 2008 Annual Meeting of the German Society of Cell Biology, Symposium on Adhesion and Signalling, Marburg, Germany

NA
Year(s) Of Engagement Activity 2008
 
Description BICEL symposium, University of Geneva, Switzerland (2010) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Tight junctions proteins and functions

Discussion of data and interchange of knowledge
Year(s) Of Engagement Activity 2011
 
Description Blood-brain barrier 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Health professionals
Results and Impact 2006 Sixth Symposium on Signal transduction in the blood-brain barrier, Berlin, Germany

NA
Year(s) Of Engagement Activity 2006
 
Description Cell contact and adhesion 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Health professionals
Results and Impact 2007 Gordon Research Conference: Cell contact and adhesion. Lucca (Barga), Italy

NA
Year(s) Of Engagement Activity 2007
 
Description Cerebral Vascular Biology 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Health professionals
Results and Impact 2006 Cerebral Vascular Biology Meeting, Muenster, Germany

NA
Year(s) Of Engagement Activity 2006
 
Description De Duve Institute, Universite catholique de Louvain. Brussels, Belgium (2009) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Towards understanding tight junction functions


Discussion of data and collabortion
Year(s) Of Engagement Activity 2009
 
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 Eye Research - an equal partner 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Vision Bridge is an organisation dedicated to informing the general public about contemporary eye research and to provide a platform to enable exchange between researchers, the general public and patients.
Year(s) Of Engagement Activity 2018,2019
URL http://visionbridge.org.uk/
 
Description Gastroenterology 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Health professionals
Results and Impact 2006 British Society for Gastroenterology, Epithelial differentiation, London, UK

NA
Year(s) Of Engagement Activity 2006
 
Description International Union of Physiological Sciences 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Health professionals
Results and Impact 2006 International Union of Physiological Sciences (IUPS) Congress, San Diego, USA

NA
Year(s) Of Engagement Activity 2006
 
Description Molecular structure and function of the tight junction 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Health professionals
Results and Impact 2008 Molecular structure and function of the tight junction - From basic mechanisms to clinical manifestations, Berlin, Germany

NA
Year(s) Of Engagement Activity 2008
 
Description Ocular Cell Biology 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Health professionals
Results and Impact 2006 First International Society for Ocular Cell Biology , Cambridge, UK

NA
Year(s) Of Engagement Activity 2006
 
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 Signal transduction in the blood-brain barriers 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Health professionals
Results and Impact 2006 Signal transduction in the blood-brain barriers, Salzburg, Austria

NA
Year(s) Of Engagement Activity 2006
 
Description Signalling and Pathogens 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Health professionals
Results and Impact 2008 DFG Meeting on Signalling and Pathogens, Freiburg, Germany

NA
Year(s) Of Engagement Activity 2008
 
Description The 10th Biennial Alex Mowat meeting, titled "Cholestasis in children", held at King's College Hospital on 12th and 13th September 2014. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact To discuss and improve understand the role of tight junctions in Cholestasis within Paediatric Liver Research
Year(s) Of Engagement Activity 2014