Fig. 1.

High glucose (HG) induces activation of activator protein 1 (AP-1) in part via TRAF3 interacting protein 2 (TRAF3IP2) and JNK in human aortic endothelial cells (HAECs). A: HG (25 mM d-glucose) induces time-dependent TRAF3IP2 expression. At 70% confluency, the complete medium on HAECs was replaced with endothelial basal medium-2 (without supplements) for 2 h, and cells were then incubated with HG for the indicated time periods and analyzed for TRAF3IP2 protein expression by immunoblot analysis. B: HG (15 and 25 mM), but not mannitol, induces TRAF3IP2 expression. HAECs treated as in A with HG or mannitol for 30 min were analyzed for TRAF3IP2 by immunoblot analysis. C: HG (25 mM) induces time-dependent JNK activation. HAECs treated as in A with HG were analyzed for JNK activation using activation-specific antibodies. Total JNK served as a control. D: HG (25 mM) induces JNK activation via TRAF3IP2. At 50% confluency, HAECs were infected with lentiviral TRAF3IP2 or control enhanced green fluorescent protein (eGFP) shRNA (multiplicity of infection 0.5 for 48 h). Cells were then treated with HG for 60 min. Total and activated JNK were analyzed as in C. Knockdown of TRAF3IP2 was confirmed by immunoblot analysis and is shown on the right. ASK1 served as an off-target control. E: HG (25 mM) induced AP-1 activation via TRAF3IP2 and JNK. HAECs infected with TRAF3IP2 or JNK1 shRNA or pretreated with the JNK inhibitor SP600125 (20 µM for 30 min) were incubated with HG for 60 min and analyzed for AP-1 activation by immunoblot analysis using antibodies that specifically detect phosphorylated c-Jun at Ser⁷³. Knockdown of JNK1 is shown in the top right. Bar graphs in A–E represent densitometric analyses from 3 independent experiments. *P ≤ 0.05 vs. control (i.e., open bar); †P ≤ 0.05 vs. HG (n = 3).