FIG. 5.

Potent transcriptional activity of ectopically overexpressed HIF-2α. (A and B) HIF-1α and -2α were overexpressed along with the HRE-luciferase reporter in 293 cells (A) and HIF-1α null MEFs (B). Then, the cells were treated with either normoxia or 0.5% O₂ hypoxia for 22 h before being harvested for the reporter gene assay. (C) HIF-1α null MEFs were transfected with HIF-1α or HIF-2α along with the pGL-SRJ reporter. (D) Endogenous HIF-2α remains inactive after treatment of MEFs with various stimulators. HIF-1α null MEFs were transfected with a HRE-luciferase reporter and treated with various intracellular signaling pathway modulators to stimulate HIF-2α activity Relative HRE reporter gene activity is shown as induction over that of untreated cells. WT, wild type; PMA, phorbol myristate acetate. (E to G) Overexpressed HIF-2α can induce the expression of some endogenous HIF target genes. HIF-1α null MEFs were transfected with either an empty, HIF-1α, or HIF-2α expression vector and treated with either normoxia or 0.5% O₂ hypoxia for 22 h before being harvested for total RNA extraction. Expression of PGK, Glut-1, and VEGF was determined as described for Fig. 2 and normalized against that of β-actin.