476-P: Analysis of Upstream Signaling Pathway of Erk2 following Transient High-Glucose Exposure in Coronary Artery Endothelial Cells

Abstract

Using an experimental approach, we investigated the mechanisms by which brief high-glucose exposure activates extracellular signal-regulated kinase 2 (Erk2) in cultured human coronary artery endothelial cells. The cells were cultured in standard medium for 14 days (5.5 mM D-glucose). Thereafter, transient glucose elevation was achieved by adding either 183 mg/dL L-glucose or 183 mg/dL D-glucose to the culture medium for 1 h. The Erk2 activation status was measured by western blot analysis of phospho-Erk2. As part of the analysis of the upstream signaling pathway of Erk2, Rap1 and Ras activation status were examined. Furthermore, guanine nucleotide-binding protein G (i) α2 (Giα2) protein and microRNA-138 (miR-138) levels were measured. Ras and Erk2 were activated, whereas Rap1 was deactivated following addition of 183 mg/dL D-glucose to the culture medium and incubation for 1 h. Furthermore, Giα2 protein levels increased and miR-138 levels decreased. The addition of 183 mg/dL L-glucose did not affect the results. In general, our findings suggest that transient exposure to high amounts of D-glucose decreased miR-138 levels, resulting in an increase in Giα2 protein levels, Rap1 deactivation, Ras activation, and Erk2 activation. Here we report a novel mechanism that links transient high-glucose levels to Erk2 activation in cultured human coronary artery endothelial cells. Disclosure J.Okada: None. E.Yamada: None. T.Saito: None. S.Okada: None.