Analysis of the ERK5 MAPK Signalling Pathway in Endothelial Cells

Abstract

Angiogenesis is the development of new capillary vessels from pre-existing ones. It involves the proliferation, migration and differentiation of endothelial cells. It is activated by different stimuli and requires the coordination of several signalling pathways. The most important growth factor for angiogenesis is VEGF. However, other factors also play an important role. For example, H2S is a gaseous molecule that exerts its pro-angiogenic effect through activation of the pro-survival Akt signalling pathway. In vitro studies have further shown the ability of H2S to enhance endothelial cell proliferation, adhesion, migration, and tube formation. The ERK5 signalling cascade is the most recently discovered mammalian MAPK (mitogen-activated protein kinase) pathway. ERK5 knockout mice die due to impaired heart development and endothelial cell dysfunction. Furthermore, VEGF promotes ERK5 activation in primary endothelial cells and ERK5 is required for VEGF-induced tubular morphogenesis. Importantly, VEGF activates ERK5 in endothelial cells, which in turn activates the AKT pro-survival pathway to suppress apoptosis of endothelial cells. As H2S also mediates its vasoprotective effect through AKT activation, the hypothesis of this thesis was that H2S protects endothelial cells by activating the ERK5 pathway. The aim of this thesis was therefore to investigate whether H2S activates ERK5 and to develop the necessary tools to further investigate the mechanisms involved. In this thesis, an assay detecting and quantifying an electrophoretic mobility shift by western blotting has been developed to measure ERK5 activation in mammalian cells. Furthermore, ERK5 could be shown to be activated by H2S in HeLa cells. Importantly, H2S also activated ERK5 in endothelial cells, demonstrating that ERK5 signalling mediates the pro-survival response of H2S in endothelial cells. To further analyse ERK5 activation in response to various stimuli, including H2S, additional tools to determine ERK5 phosphorylation were evaluated, including mass spectrometric determination of phosphorylation sites and the characterisation of a novel phosphor-specific antibody.