FIGURE 7.

SP-1 transcription factor binding is required for hypoxia-induced VEGF expression in NSCLC. SP-1 reporter activity and Sp-1 transcription factor binding to the native VEGF promoter increased with hypoxia. Hypoxia increased VEGF secretion and mRNA accumulation. A and B, four NSCLC cell lines were cultured for 24 h under normoxic (open bars) or hypoxic conditions (closed bars) (1% O₂), and the culture supernatants were analyzed by VEGF ELISA (A) with total VEGF secretion induced by hypoxia in each cell line (B). C, after 4 h of hypoxia (closed bars) or control (open bars) cell RNA were extracted for RT-QPCR analysis of VEGF mRNA accumulation. Mithramycin blocks VEGF mRNA accumulation in response to hypoxia. D, NSCLC cell lines were treated with mithramycin A (1 × 10⁻⁶ m) for 30 min prior to 4 h of control conditions (open bars, untreated; hatched bars, treated) or 4 h of hypoxia (filled bars, untreated; cross-hatched bars, treated). Mutation of SP-1-binding sites in the VEGF minimal (135) promoter luciferase-reporter eliminates the hypoxic response in all four NSCLC cell lines. E–H, NSCLC cell lines A549 (E), H460 (F), H1299 (G), and MORP (H) transfected with wild-type (VEGF-135wt), SP-1 site mutant (VEGF-SP-1m), and EGR site mutant (VEGF-EGRm) VEGF-135-luciferase reporter constructs (Fig. 2E) with normoxia (open bars) and hypoxia (closed bars) for 20 h. Hypoxia increases SP-1 reporter-luciferase activity in NSCLC cell lines. I–L, all four NSCLC cell lines, A549 (I), H460 (J), H1299 (K), and MORP (L), were transfected with the control plasmid with mutant SP-1-binding sites (191) and the wild-type SP-1 reporter construct (194) followed by normoxia (open bars) or hypoxia (closed bars) for 20 h. Hypoxia-induced SP-1 binding to the native VEGF promoter is greater in NSCLC expressing higher SP-1 levels. A549 (low Sp-1 expression) and H1299 (high SP-1 expression) were exposed to normoxia or hypoxia for 4 h. M, ChIP was performed for transcription factor SP-1 (filled bars) from chromatin samples from NSCLC. IP and input DNAs were subjected to QPCR for the VEGF promoter between −85 and +50 bp relative to the transcription start site of the VEGF promoter. Control ChIP were performed with equivalent rabbit IgG for all chromatin samples (open bars). All of the measurements represent the means ± S.E. of three independent experiments. RLU, relative light units.