Effect of hypoxia on transcriptional activity of the TGF-β2 promoter.

(A) Schematic structure of recombinant plasmid construct pB2-77 containing the human TGF-β2 gene promoter between −77 and +63 bp, and that is linked to a CAT reporter gene. The transcriptional start site (arrow) driving the CAT gene and selected protein-binding sites (Smad, HIF-1) are indicated. (B) HUVECs were transfected with pB2-77, and cotransfected with indicated Smad and HIF-1α expression vectors and Rous sarcoma virus (RSV)–β-gal by electroporation. Each transfection was divided into 2 plates and exposed to 20% or 1% O₂ for 36 hours. Representative results of 1 of 3 independent CAT assays are shown in the panel on the left. CAT activity was determined in whole-cell extracts, and its level, given as percent acetylation, was normalized to protein and to β-gal activity in each experiment. Results are expressed as fold increases (±SD) in CAT activity in indicated hypoxic conditions compared with that found in their normoxic counterparts for each transfection. Fold changes observed in each experimental group are compared with the hypoxia-induced fold change in HUVECs transfected with the pB2-77-CAT alone (*P < .05).