Function of IKKbeta in the progression and regression of liver fibrosis

Liver cirrhosis is the end stage of a process known as fibrosis in which the normal healthy tissues of the liver are over several years progressively replaced with non-functional scar tissue. This process leaves the liver in a state of structural and functional disorder for which the only option is replacement by transplantation to prevent death. This disease process can be caused by a wide variety of injuries including those inflicted by viruses (hepatitis C and B), alcohol, metabolic agents and autoimmune attack. Recent work in the applicants laboratory as well as in other laboratories across the world has given new hope to the treatment of liver fibrosis by demonstrating that it is a process that can be reversed. Reversibility of fibrosis is associated with the removal from the liver of a specialised cell (hepatic myofibroblast) that is responsible for production of scar tissue, however removal of these cells is very inefficient in livers that are undergoing persistent injury. In pioneering studies the applicants have shown that drugs which target a survival factor called NF-kB will promote the removal of hepatic myofibroblasts from the liver without affecting other liver cells required for healthy function. The aim of the project for which grant income is requested is to learn more about one of the key molecules that control NF-kB. This molecule known as IKKbeta is already a target for emerging anti-inflammatory drugs which may eventually offer an alternative to transplantation if we can find evidence that it plays an important role in fibrosis. The experiments that are proposed are designed to discover if loss of IKKbeta and NF-kB activities in hepatic myofibroblasts will result in reduced severity of fibrosis and enhanced rates of recovery from liver disease. The results will be used to inform colleagues in hepatology clinics as to the likely benefits of the use of IKKbeta inhibitors in the treatment of liver disease patients. The results of the work will also be presented to the general public via press releases (organised through the University of Southampton and the Medical Research Council Press Offices and Websites) as well as in the form of public lectures and publication of data in high quality scientific journals.

Liver fibrosis involves progressive replacement of liver tissue with scar tissue in response to chronic injury (alcohol, HCV, fat etc) and leads to the end-stage disease of cirrhosis. In the past 5 years, clinical and experimental studies have revealed a new paradigm, that fibrosis can regress as well as progress. However, spontaneous regression is only achieved in a small number of patients that can be effectively treated for the underlying cause of liver disease and even in these cases recovery is slow and incomplete. Studies pioneered by the applicants shows that stimulation of apoptosis of scar producing hepatic myofibroblasts (HM) accelerates the rate at which experimental fibrosis is spontaneously resolved. These studies identified NF-kB and its regulatory IkappaKinases (IKK) complex as critical controllers of HM apoptosis. Inhibition of NF-kB/IKK activity is associated with elevated rates of HM apoptosis and rapid recovery from fibrosis. The overarching aim of the current proposal is to determine the role played by the IKKbeta component of the IKK complex and to develop a highly specific inhibitor of IKKbeta action in HM. The role of IKKbeta in both the progressive and regressive phases of fibrosis will be investigated using a mouse in which the IKKbeta gene can be deleted at will and selectively in fibroblasts and myofibroblasts. Mice in which the IKKbeta allele is flanked by loxP sites will be crossed with mice in which Cre is under the transcriptional control of the alpha2 (I) collagen enhancer and post-translational control by Tamoxifen providing regulated and cell-specific knockout of IKKbeta expression. Importantly, this system will enable the fibrogenic role of IKKbeta to be studied independently from its pro-inflammatory function in the injured liver. The mice will be used in models of fibrogenesis (CCl4 injury) and recovery from fibrosis (following chronic injury with CCl4). Histological (Sirius Red for collagen I/III and alphaSMA for numbers of HM) and biochemical (qRT-PCR for fibrogenic markers, hydroxyproline and MMP activity assays). We anticipate that loss of IKKbeta will be associated with prevention of fibrosis and improved rates of recovery from fibrosis. Work in the second part of the proposal will address the way in which IKKbeta regulates NF-kB which from previous work and pilot data appears to operate via phosphorylation of the p65 subunit of NF-kB. In addition a specific peptide inhibitor of IKKbeta-mediated p65 phosphorylation will be tested for its ability to inhibit NF-kB, promote apoptosis and stimulate recovery from fibrosis.