Figure 2
Figure 2. NFAT, SRF, and CREB bind to and transactivate the Egr-3 promoter in primary human endothelial cells. (A) Egr-3 (−2544/+80)-luc was transiently transfected into HUVECs, HDMVECs, human skin fibroblasts (H.Fibroblast), or HEK-293 cells. Cells were treated in the absence or presence of 50 ng/mL VEGF for 4 hours and assayed for reporter gene activity. The results show the mean ± SD of luciferase light units in VEGF-treated cells relative to untreated cells, obtained in triplicate from 3 independent experiments. *P < .001 compared with untreated cells. (B) 5′-deletion analysis of Egr-3 promoter activity in control versus VEGF-treated HUVECs. Successive deletions of the 5′-flanking region of Egr-3 were coupled to luciferase in pGL3, and the resulting constructs were transiently transfected into HUVECs. Cells were treated in the absence or presence of 50 ng/mL VEGF for 4 hours and assayed for reporter gene activity. The results show the mean ± SD of luciferase light units in VEGF-treated cells relative to untreated cells obtained in triplicate from 3 independent experiments. *P < .01 compared with Egr-3 (−2544/+80)-luc. (C) Analysis of mutant Egr-3 promoter activity in control versus VEGF-treated HUVECs. HUVECs were transiently transfected with either wild-type Egr-3 (−515/+80)-luc or Egr-3 (−515/+80)-luc containing point mutations of NFAT, SRF, and/or CRE motifs; treated in the absence or presence of 50 ng/mL VEGF for 4 hours; and assayed for reporter gene activity. The results show the mean ± SD of luciferase light units in VEGF-treated cells relative to untreated cells obtained in triplicate from 3 independent experiments. *P < .01 compared with Egr-3 (−515/+80)-luc. (D) ChIP assays of HUVECs treated in the absence or presence of 50 ng/mL VEGF for 30 minutes. Formalin-fixed chromatin was immunoprecipitated with monoclonal mouse antibodies to NFATc1 or NFATc2 or with mouse control immunoglobulin (IgG; left). Alternatively, formalin-fixed chromatin was immunoprecipitated with rabbit polyclonal antibodies against SRF or phospho-CREB or with rabbit control IgG (right). Precipitated chromatin was PCR amplified (30-35 cycles) and subjected to agarose gel electrophoresis (top). Binding was quantified by the use of real-time PCR (bottom). The results show the mean ± SE of binding level relative to control (without VEGF) obtained from at least 3 independent experiments. *P < .01 compared with control. (E) Immunofluorescent studies of NFAT, SRF, and phospho-CREB (pCREB) in control and VEGF-treated HUVECs. Serum-starved cells were incubated in the presence or absence of 50 ng/mL VEGF for the times indicated. The cells were then fixed and incubated with antibodies against NFATc1, NFATc2, SRF, or phospho-CREB, followed by Alexa 488–conjugated second antibody (green). The nuclei were stained with DAPI (blue). Merged images are shown in the bottom row. White bar indicates 20 μm.

NFAT, SRF, and CREB bind to and transactivate the Egr-3 promoter in primary human endothelial cells. (A) Egr-3 (−2544/+80)-luc was transiently transfected into HUVECs, HDMVECs, human skin fibroblasts (H.Fibroblast), or HEK-293 cells. Cells were treated in the absence or presence of 50 ng/mL VEGF for 4 hours and assayed for reporter gene activity. The results show the mean ± SD of luciferase light units in VEGF-treated cells relative to untreated cells, obtained in triplicate from 3 independent experiments. *P < .001 compared with untreated cells. (B) 5′-deletion analysis of Egr-3 promoter activity in control versus VEGF-treated HUVECs. Successive deletions of the 5′-flanking region of Egr-3 were coupled to luciferase in pGL3, and the resulting constructs were transiently transfected into HUVECs. Cells were treated in the absence or presence of 50 ng/mL VEGF for 4 hours and assayed for reporter gene activity. The results show the mean ± SD of luciferase light units in VEGF-treated cells relative to untreated cells obtained in triplicate from 3 independent experiments. *P < .01 compared with Egr-3 (−2544/+80)-luc. (C) Analysis of mutant Egr-3 promoter activity in control versus VEGF-treated HUVECs. HUVECs were transiently transfected with either wild-type Egr-3 (−515/+80)-luc or Egr-3 (−515/+80)-luc containing point mutations of NFAT, SRF, and/or CRE motifs; treated in the absence or presence of 50 ng/mL VEGF for 4 hours; and assayed for reporter gene activity. The results show the mean ± SD of luciferase light units in VEGF-treated cells relative to untreated cells obtained in triplicate from 3 independent experiments. *P < .01 compared with Egr-3 (−515/+80)-luc. (D) ChIP assays of HUVECs treated in the absence or presence of 50 ng/mL VEGF for 30 minutes. Formalin-fixed chromatin was immunoprecipitated with monoclonal mouse antibodies to NFATc1 or NFATc2 or with mouse control immunoglobulin (IgG; left). Alternatively, formalin-fixed chromatin was immunoprecipitated with rabbit polyclonal antibodies against SRF or phospho-CREB or with rabbit control IgG (right). Precipitated chromatin was PCR amplified (30-35 cycles) and subjected to agarose gel electrophoresis (top). Binding was quantified by the use of real-time PCR (bottom). The results show the mean ± SE of binding level relative to control (without VEGF) obtained from at least 3 independent experiments. *P < .01 compared with control. (E) Immunofluorescent studies of NFAT, SRF, and phospho-CREB (pCREB) in control and VEGF-treated HUVECs. Serum-starved cells were incubated in the presence or absence of 50 ng/mL VEGF for the times indicated. The cells were then fixed and incubated with antibodies against NFATc1, NFATc2, SRF, or phospho-CREB, followed by Alexa 488–conjugated second antibody (green). The nuclei were stained with DAPI (blue). Merged images are shown in the bottom row. White bar indicates 20 μm.

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