Fig. 6.

Endothelin-1 (ET-1) induces TRAF3 interacting protein 2 (TRAF3IP2) expression. A and B: ET-1 induces TRAF3IP2 expression in a dose- and time-dependent manner. At 70% confluency, the complete medium on human aortic endothelial cells (HAECs) was replaced with endothelial basal medium-2 (without supplements) for 2 h, and cells were then treated with ET-1 at the indicated concentrations for 1 h (A) or for up to 6 h with ET-1 at 10 nM (B). TRAF3IP2 expression was analyzed by immunoblot analysis. C: ET-1 induces TRAF3IP2 mRNA expression. HAECs were treated with ET-1 (10 nM) for up to 6 h and then analyzed for TRAF3IP2 mRNA expression by quantitative RT-PCR. D: ET-1 stimulates TRAF3IP2 promoter-dependent reporter gene activation in part via CCAAT/enhancer binding protein-β (c/EBPb) and activator protein 1. HAECs were transfected with a reporter vector containing a 200-bp fragment of the 5′-flanking region of the human TRAF3IP2 gene (3 µg for 24 h) with and without mutations (n = 6). pGL3-basic served as a vector control. Cells were cotransfected with the Renilla luciferase vector (100 ng). After transfection, cells were treated with ET-1 (10 nM) for 12 h and harvested for the dual-luciferase assay. Firefly luciferase data were normalized to that of corresponding Renilla luciferase activity. Bar graphs in A and B represent densitometric analyses from 3 independent experiments. A–D: *P ≤ 0.01 vs. control (i.e., open bar); †P ≤ 0.05 vs. control or untreated HAECs transfected with the intact TRAF3IP2 promoter reporter construct (n = 3–6).