Effect of hypoxia on transcriptional activity of the TGF-β2 promoter in HUVECs.
(A) Recombinant constructs containing the human TGF-β2 gene promoter between −778 and +63 bp. The transcriptional start site (arrow) driving the CAT gene and selected protein-binding sites (Smad, HIF-1, AP-1) are indicated. (B) Transcriptional activity of the TGF-β2 promoter in response to hypoxia. HUVECs were transfected with pB2-778 or its deletion constructs pB2-77 or pB2-40; pB2-77 was also cotransfected with indicated Smad expression vectors and RSV-β-gal by electroporation. Each transfection was divided into 2 plates and exposed to either to 20% or 1% O2 for 36 hours. CAT activity was determined in whole-cell extracts, and its level, given as percent acetylation, was normalized to protein (Bradford assay, Bio-Rad) and to β-gal activity in each experiment. Results are expressed as fold increases in hypoxic CAT activity compared with normoxic CAT activity in each transfection. The mean ± SD of 4 independent experiments is shown.
