Localization of VEGF 5′-flanking sequences that mediate transcriptional response to hypoxia or SNAP.
VEGF sequences were cloned to the promoterless pGL2 basic vector (A) or 5′ to an HSV-TK promoter-luciferase transcription unit of pT81luc0 (B, C). The locations of restriction sites are shown relative to the transcription start site. Relative luc activity is defined as the mean ratio of luciferase/β-galactosidase activity ± SEM. Fold induction, by hypoxia or SNAP, represents the ratio of relative luc activity in cells at 1% O2 or 0.5 mmol/L SNAP in 0.1% DMSO to those at 21% O2 or 0.1% DMSO, respectively. (A) n = 8 (hypoxia) or n = 11 (SNAP); *P < 0.05 versus phVEGF1 and <0.01 versus phVEGF3 in hypoxia, #P < 0.01 versus phVEGF1 and < 0.05 versus phVEGF3 in SNAP. (B) n = 8; *P < 0.01 versus pHRB in hypoxia, **P < 0.05 versus pHRE in hypoxia, #P < 0.01 versus pHRB in SNAP, and ##P < 0.05 versus pHREL in SNAP. (C) Nucleotides of transcription factor binding sites (HIF-1 binding site, HIF-1 ancillary sequence, AP-1) are underlined (-975 to -968, -962 to -956, and -937 to -931, respectively), and substituted bases are shown in lowercase letters. n = 6; *P < 0.01 versus pHRE and < 0.05 versus pHREm3 in hypoxia, **P < 0.05 versus pHRE in hypoxia, #P < 0.01 versus pHRE and < 0.05 versus pHREm3 in SNAP, and ##P < 0.05 versus pHRE in SNAP.
