Role of cytoglobin in the physiology of cellular redox disturbance in the liver

Previous data from our laboratory generated by a former BBSRC-CASE funded PhD studentship in collaboration with DRs Mark Graham and Martyn Foster at AstraZeneca has demonstrated a role for the novel globin cytoglobin in lung physiology. A detailed immunohistochemical (IHC) analysis revealed that cytoglobin expression is up-regulated in regions of the lung that have become anoxic leading us to hypothesize that cytoglobin functions by acting as a 'buffer' between normal and hypoxic tissue and that its function in these regions is either to sequester molecular oxygen to prevent normal tissue becoming hypoxic or to metabolise ROS generated during ischaemic reperfusion. In support of this hypothesis, work by our and other laboratories demonstrate that in in vitro cell culture models over expression of cytoglobin affords protection from agents that induce both oxidative stress and hypoxia. In the current proposal we wish to expand upon these observations and investigate the fundamental biological role of cytoglobin in another important organ, the liver. We will map in detail the expression of cytoglobin expression in normal liver and use an in vivo rodent model system to investigate the adaptive physiological function of cytoglobin under conditions where normal liver homeostasis is oxidatively perturbed. A study of the adaptive changes of cytoglobin expression/location will be informative regarding the normal cellular function of cytoglobin. Specific regions of the liver will be targeted using: diquat (global), CCl4 (centrilobular) and allyl alcohol (periportal) respectively. Animals will be sacrificed and analysis of liver tissue by IHC, western blotting, laser capture micro-dissection and qPCR used to investigate and map the level and profile of distribution of cytoglobin expression. Time and dose-dependency will be investigated and cytoglobin expression related to other indices of adaptive changes of liver function (e.g, fibrosis, necrosis, apoptosis, 8-oxo dG formation). We will use this data to 'map' in detail the pattern of cytoglobin expression to areas of different liver physiology to test our hypothesis that cytoglobin induction occurs at the interface between normal and hypoxic tissue. These in vivo experiments will complemented by in vitro mechanistic studies aimed at identifying the function and regulation of cytoglobin at the cellular and molecular level. We will use primary rat hepatocytes as well as the rat liver cell line MHC1 to investigate if these hepatotoxic agents induce cytoglobin expression in vitro and whether over-expression or knock down by transfection or RNAi respectively modulates sensitivity of cells to the effects of these compounds and hypoxia. Parameters related to oxidative stress will also be assessed (e.g. cellular oxidative stress by FACS analysis with DCFDA and MitoSOX, glutathione and lipid peroxidation). We will also use reporter/promoter deletion analysis and EMSA-gel shift assays to identify key regions of the cytoglobin promoter sequence and transcription factors important for regulation of induction cytoglobin gene expression. Identification of these will further our understanding of the key signaling pathways involved in cytoglobin induction and contribute to an enhanced understanding of the fundamental biology of this new globin. In addition, there is evidence that cytoglobin through interaction with protein partners may be involved directly in cell signaling but this has not been investigated in rat hepatocytes. We will use yeast two hybrid and mass spectrometry approaches to identify novel protein partners of cytoglobin in hepatocytes.