Hydrogen peroxide (H202) is a reactive oxygen species (ROS), which possesses the relative stability, neutrality, and small size required for a freely diffusible signaling molecule. ROS are generated in vascular cells by the mitochondria and by the enzyme NADPH oxidase, and have been shown to play an important role in a wide variety of human diseases, especially vascular diseases. Low concentrations of H202 (<10 microM) are mitogenic when applied to human cells in culture, and these low concentrations stimulate endothelial migration and tube-formation in an in vitro model of angiogenesis. In contrast, higher concentrations (50 microM to 1mM) of H202 cause growth arrest, apoptosis, and/or necrosis. Although several signal transduction pathways have been reported to be activated by the application of these higher concentrations of H202, the molecular mechanisms by which low concentrations (< 10 microM) of H202 function in mammalian cells are poorly understood. Recently, using a functional proteomics approach it has been determined that low concentrations of H202 stimulate the transient phosphorylation of heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2, and also stimulate the post-translational modification of pyruvate kinase M2 with alteration of its enzymatic activity. The overall goal of this proposal is to identify signaling pathways for sensing low physiologically relevant concentrations of H202 in human endothelial cells. To this end, the biochemical response of human endothelial cells to low concentrations of H202 will be explored in three focused specific aims: (1) Determine how low concentrations of H202 stimulate the transient phosphorylation of hnRNP C1/C2 in human endothelial cells and elucidate the functional consequences of this modification. (2) Identify the H202-stimulated post-translational modification on pyruvate kinase, determine the mechanism by which this modification occurs, and further characterize its functional consequences. (3) Identify and characterize additional protein modifications stimulated by low concentrations of H202, and use this information to establish signaling pathways for sensing H202 in human endothelial cells.
