The Role of Tight and Intercellular Junctions in Liver Disease

Liver Immunbiology Laboratory

http://www.centenary.org.au/p/res/liver/rp/

Supervisors: Dr. Fiona Warner and Dr. Nick Shackel

Contact: f.warner@centenary.usyd.edu.au Tel: 95656152

Liver injury has many diverse causes including viral hepatitis, autoimmune disease and metabolic disorders. Surprisingly, all of the causes of liver disease typically result in a common progression of injury characterised by loss of hepatic architecture and cirrhosis (endstage liver disease). The loss of liver architecture is due largely to changes in cell-cell contact (junctions). Changes in cell junctions result in deteriorated barrier function as cells dissociate from their neighbours, and increased intercellular cell permeability. We are interested in study the expression and localisation of tight and adheren junctions between cells under normal and diseased conditions. This work has particular significance as a recent study has identified a cell junction protein, claudin-1 as the functional receptor for the Hepatitis C. Hence, understanding the localisation and expression of claudin-1 and its relationship to other junction proteins is crucial to understanding Hepatitis C pathophysiology.

Hypothesis: Liver injury is associated with the loss of cell junctions, which correlates with altered tight- and adheren-junction protein expression and facilitates hepatitis C viral entry.

Aim: To compare the cellular localisation and expression of junction proteins, claudin-1, occludin, zona occludens and E-cadherin in whole liver from normal and diseased rats, together with in vitro studies in HepG2 cells.

Proposal: Initially, we will establish the subcellular localisation of junction proteins (claudin-1, ZO-1, occludin, E-cadherin) in normal and diseased liver by indirect immunofluorescence and confocal microscopy. Liver sections from humans and two rat models of liver injury (bile duct ligation and carbon tetrachloride) will be used. In parallel experiments, Western blotting and quantitative real-time PCR will measure the protein and mRNA levels, respectively of each junction protein.

To complement the experiments above, we will be to determine if Hepatitis C infection is affected by the level of claudin-1 expression and/or the expression of other junction proteins, ZO-1 and occludin. Using the cell model of hepatocytes (HepG2) and bile duct epithelial cells (SK-ChA-1), this study will use siRNA to produce gene silencing of targeted proteins. This approach will determine if a direct relationship exists between hepatitis C and proteins associated with the maintenance of tight junctions.

Techniques learned: Immunofluorescence, confocal microscopy, cell culture, siRNA targeted protein silencing, ELIZA assays, quantitative real time PCR, animal handling.

Outcomes This study will establish the effect of liver injury on the localisation and expression levels of tight and adherens junction proteins in whole human and rat liver. Experiments in HepG2 cells in vitro will also provide critical information about whether alterations in tight and/or adherens junctions are directly involved in the Hepatitis C induced infection.