The Contribution of EDF1 to PPARγ Transcriptional Activation in VEGF-Treated Human Endothelial Cells

Abstract

Vascular endothelial growth factor (VEGF) is important for maintaining healthy endothelium, which is crucial for vascular integrity. In this paper, we show that VEGF stimulates the nuclear translocation of endothelial differentiation-related factor 1 (EDF1), a highly conserved intracellular protein implicated in molecular events that are pivotal to endothelial function. In the nucleus, EDF1 serves as a transcriptional coactivator of peroxisome proliferator-activated receptor gamma (PPARγ), which has a protective role in the vasculature. Indeed, silencing EDF1 prevents VEGF induction of PPARγ activity as detected by gene reporter assay. Accordingly, silencing EDF1 markedly inhibits the stimulatory effect of VEGF on the expression of FABP4, a PPARγ-inducible gene. As nitric oxide is a marker of endothelial function, it is noteworthy that we report a link between EDF1 silencing, decreased levels of FABP4, and nitric oxide production. We conclude that EDF1 is required for VEGF-induced activation of the transcriptional activity of PPARγ.

Keywords: Endothelial Differentiation-related factor 1; Peroxisome proliferator-activated receptor γ; endothelial cells; vascular endothelial growth factor.

Figure 1

The total amounts of endothelial differentiation-related factor 1 (EDF1) and peroxisome proliferator-activated receptor gamma (PPARγ) in cells treated with VEGF. Human umbilical vein endothelial cells (HUVEC) were treated with 50 ng/mL of vascular endothelial growth factor (VEGF) for 0, 8, 12, and 24 h. (a) Real-Time PCR was performed on RNA samples. Two different experiments in triplicate were performed; (b) cell lysates were analyzed by western blot using antibodies against EDF1, PPARγ, and actin. A representative blot is shown.

Figure 2

Subcellular localization of EDF1 in cells treated with VEGF. (a) HUVEC were treated with VEGF (50 ng/mL) for 1, 8, 12, and 24 h. Immunofluorescence was performed using anti-EDF1 immunopurified immunoglobulin G (IgGs) and rhodamine-conjugated anti-rabbit IgGs; (b) HUVEC were treated with VEGF (50 ng/mL) for 1 h. Western blot was performed on nuclear and cytosolic fractions using antibodies against EDF1. GAPDH and TBP were used as cytosolic and nuclear markers, respectively. A representative blot is shown.

Figure 3

The interaction between EDF1 and PPARγ in HUVEC treated with VEGF. HUVEC were treated with VEGF (50 ng/mL) for different times. Cell lysates were immunoprecipitated with monoclonal antibodies against PPARγ and analyzed by western blot using rabbit antibodies against EDF1 (upper panel). The filter was then probed with rabbit anti-PPARγ antibodies to verify the equal amounts of immunoprecipitated proteins (lower panel). Densitometric analysis was performed using ImageJ software. EDF1/PPARγ ratio was calculated on three blots from separate experiments ± standard deviation.

Figure 4

PPARγ transcriptional activity in HUVEC with silenced EDF1. (a) The modulation of PPARγ was evaluated in αs1 cells (αs1) (HUVEC with stably silenced EDF1) and compared to HUVEC transfected with a scrambled nonsilencing sequence (used as control) (CTR). Cell lysates were analyzed by western blot using antibodies against EDF1, PPARγ, and actin. A representative blot is shown; (b) PPARγ activity was evaluated by luciferase assay in αs1 cells and compared to the control HUVEC; (c) Real-Time PCR was performed on RNA samples from αs1 cells and relative control, treated or not with VEGF (50/ng/mL) for 24 h. Three different experiments in triplicate were performed; (d) Nitric oxide (NO) release was measured using the Griess method for nitrate quantification. The values were expressed as the mean of three different experiments in triplicate ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001.