Mechanisms of epidermal growth factor receptor signalling in primary rat hepatocytes
In the U.K. deaths due to liver diseases, especially alcohol related diseases, have risen considerably over the last 20 years. In 2005 up to 13,000 people died from liver related diseases within the U.K., including alcohol and viral liver failure and liver cancers. Worldwide hepatitis B affects about 2 billion people, killing 500,000 to 1 million per year. An effective way to treat liver disease is often liver surgery, such as liver resection for cancers and liver transplant for failure. However, the failure of liver regeneration by hepatocyte proliferation after resection surgery leads to a high death rate, and a shortage of liver donors means most people with liver failure die without access to a transplant. Therefore, understanding hepatocyte proliferation is a key to improving survival after resection surgery and providing hepatocytes for cell therapy in place of organ donation. The mechanism of hepatocyte proliferation has been studied both in vivo and in culture by many groups. However, only limited proliferation and preservation of function of primary human and rat hepatocytes, not suitable for clinical use, has been achieved on stimulation with growth factors. This study focuses on the mechanism of epidermal growth factor (EGF) stimulation of rat hepatocyte cell cycle progression and proliferation, including the role of PI3K/Akt/mTOR and MEK/ERK signalling pathways, EGF receptor location after activation of downstream proteins such as protein kinase B (Akt) and extracellular signal-regulated kinases 1/2 (ERK1/2), and their effect on the cell cycle. Included in this study are some comparisons between the stimulation of the EGF receptor (a tyrosine kinase receptor) and the P2Y receptor (a G protein coupled receptor). The PI3K/Akt/mTOR signalling pathway appears to be necessary for the hepatocyte response to EGF, inducing progression to S phase and DNA synthesis, while the MEK/ERK pathway is important but not necessary. The P2Y2 agonist UTP, which also stimulates these two pathways, does not result in the cell entering S phase. This suggests that the activation of these two signalling pathways is not sufficient for cell cycle progression. Furthermore, infection of cells with adenovirus to express constitutively active Akt increases hepatocytes proliferation and induces cell cycle progression, which generates a window to obtain hepatocytes proliferation in culture. It has been shown in this thesis that EGF stimulation of ERK phosphorylation continues from endosomes, while the evidence suggests that UTP stimulation is restricted to signalling at the cell surface. Furthermore, endocytic EGF/EGFR alone (without stimulation from the cell surface) is sufficient to induce cell cycle progression. This endosomal signalling with EGF but not UTP may explain the absence of cell cycle progression following UTP. EGF stimulates the appearance of phospho-EGFR in the nucleus. Furthermore, nuclear EGFR has a different apparent molecule weight than the cytoplasmic receptor; this may be due to nuclear EGFR having fewer and/or different phosphates. In vivo work by others has shown that in liver regeneration following partial hepatectomy (PH) EGF and full-length activated-EGFR were showed to be present in proliferating hepatocytes. This thesis describes the mechanism of growth factor (EGF) stimulation of primary rat hepatocyte proliferation. It shows for the first time that endosomal EGF/EGFR alone is sufficient to stimulate cell cycle progression, and that EGF induces phospho-EGFR in the nucleus in cultured rat hepatocytes. This thesis also provides the possibility to obtain cultured hepatocytes proliferation including over-expression of constitutively active form of Akt and translocation to the nucleus of full-length EGFR in the phosphorylated form. These studies improve our understanding of growth factor (e.g. EGF) stimulation of hepatocyte proliferation in vitro and help to move closer to the goal of obtaining sufficient functional hepatocytes in culture for clinical use, and of drugs that will stimulate hepatocyte proliferation following resection surgery.
- PhD