Placenta growth factor-induced early growth response 1 (Egr-1) regulates hypoxia-inducible factor-1alpha (HIF-1alpha) in endothelial cells

Abstract

Leukotrienes, the lipid inflammatory products derived from arachidonic acid, are involved in the pathogenesis of respiratory and cardiovascular diseases and reactive airway disease in sickle cell disease. Placenta growth factor (PlGF), elaborated from erythroid cells, increased the mRNA expression of 5-lipoxygenase and 5-lipoxygenase-activating protein (FLAP) in human pulmonary microvascular endothelial cells. PlGF-induced both promoter activity and mRNA expression of hypoxia-inducible factor-1alpha (HIF-1alpha), which was abrogated by early growth response-1 (EGR-1) small interfering RNA. PlGF showed a temporal reciprocal relationship in the mRNA levels of EGR-1 and NAB2, the latter a repressor of Egr-1. Moreover, Nab2, but not mutant Nab2, significantly reduced promoter activity and mRNA expression of HIF-1alpha and also reduced expression of the HIF-1alpha target gene FLAP. Furthermore, overexpression of Egr-1 led to increased promoter activities for both HIF-1alpha and FLAP in the absence of PlGF. Additionally, the Egr-1-mediated induction of HIF-1alpha and FLAP promoters was reduced to basal levels by EGR-1 small interfering RNA. The binding of Egr-1 to HIF-1alpha promoter was corroborated by electrophoretic mobility shift assay and chromatin immunoprecipitation assay, which showed increased Egr-1 binding to the HIF-1alpha promoter in response to PlGF stimulation. These studies provide a novel mechanism for PlGF-mediated regulation of HIF-1alpha via Egr-1, which results in increased FLAP expression. This study provides a new therapeutic target, namely Egr-1, for attenuation of elevated leukotriene levels in patients with sickle cell disease and other inflammatory diseases.

FIGURE 1.

Role of Egr-1 and HIF-1α in PlGF-induced 5-LO and FLAP mRNA expression in HPMVEC. A, RPA analysis of FLAP, 5-LO, and GAPDH in total RNA isolated from untreated and PlGF (250 ng/ml)-treated HPMVEC at the indicated times. B, qRT-PCR analysis of HIF-1α and FLAP mRNA in PlGF-treated HPMVEC at the indicated times. C, qRT-PCR analysis of EGR-1, NAB2, HIF-1α, FLAP, 5-LO, and VCAM-1 mRNA in VEGF-treated (250 ng/ml) HPMVEC at the indicated times. D, HPMVEC were pretreated for 30 min with pharmacological inhibitors of HIF-1α, ascorbate (25 μm), PI3K, LY294002 (10 μm), NADPH oxidase, and diphenyleneiodonium (DPI) (10 μm) followed by PlGF treatment for 6 h. Total RNA was isolated and subjected to RPA for the expression of the indicated genes. E, HPMVEC were transfected with indicated siRNA or scrambled siRNA constructs followed by PlGF treatment for 6 h. Total RNA was subjected to RPA analysis of HIF-1α, FLAP, 5-LO, and GAPDH. Data are representative of three independent experiments.

FIGURE 2.

PlGF-induced Egr-1 transcription activity is repressed by Nab2. A, qRT-PCR analysis of EGR-1 and NAB2 mRNAs in total RNA isolated from untreated and PlGF-treated HPMVEC at the indicated times. B, EMSA for Egr-1 binding to its consensus DNA binding sequence in HPMVEC nuclear extracts (10 μg). Where indicated, 50-fold excess of unbiotinylated probe (3rd lane) or Egr-1 antibody (2 μg, 4th lane) was added. Protein-DNA complexes were visualized by autoradiography. * indicates the band of interest. C, cytosolic extracts from t-HBEC treated with PlGF at the indicated times (1–8 h) were subjected to Western blotting to detect Egr-1, HIF-1α, FLAP, 5-LO, and β-actin proteins using appropriate antibodies. D, HPMVEC were cotransfected with either control reporter plasmid (pCtrl-Luc) or pEgr-1Luc (containing four repeats of Egr-1-binding sites) or pEgr-1-Luc along with Nab2 WT expression plasmid or pEgr-1-Luc along with Nab2 mutant (Nab2:ΔNCD2) plasmid and β-galactosidase plasmid prior to PlGF treatment for 4 h. Each construct was used at 0.5 μg for transfection. Luciferase and β-galactosidase activities were measured in cell lysates as described under “Experimental Procedures.” The data represent the means ± S.E. of three independent experiments. Where indicated, the vertical lines show repositioned lanes from a single gel. ***, p < 0.001; ns, nonsignificant.

FIGURE 3.

PlGF promotes Egr-1 binding to HIF-1α promoter in vitro (EMSA) and in vivo (ChIP). A, schematic of HIF-1α promoter (−863/+5 bp) indicating the presence of different transcription factor-binding sites, including Egr-1, AP-1, and NF-κB. B, HPMVEC nuclear extracts (10 μg) were incubated either with a biotinylated WT or with mutant oligonucleotide probe corresponding to the proximal Egr-1-binding site (ERE) (−74/−68 bp) in the HIF-1α promoter. Where indicated, a 50-fold excess of unlabeled WT probe (3rd lane) or Egr-1 antibody (2 μg, 4th lane) was added. * denotes supershifted band. C, HPMVEC were pretreated with indicated pharmacological inhibitors for 30 min prior to PlGF treatment for 2 h. The chromatin samples were immunoprecipitated with either Egr-1 antibody (1st and bottom panels) or control rabbit IgG (3rd panel). ChIP products were amplified using primers either flanking the Egr-1-binding sites in HIF-1α promoter or corresponding to the FLAP promoter region (−310/+9 bp, bottom panel) as indicated in Table 1. The 2nd panel shows amplification of input DNA prior to immunoprecipitation. Data are representative of two independent experiments. D, HPMVEC transfected with p9HIF1-Luc construct (containing nine repeats of HRE) along with β-galactosidase plasmid were cotransfected with either Nab2 WT or mutant (Nab2:ΔNCD2) expression plasmid prior to 6 h of PlGF stimulation. Each construct was used at 0.5 μg for transfection. Luciferase and β-galactosidase activities were estimated as mentioned under “Experimental Procedures.” The data represent the means ± S.E. of three independent experiments. The repositioned lanes from a single gel are indicated as vertical lines. ***, p < 0.001; ns, nonsignificant.

FIGURE 4.

Role of Egr-1 and Nab2 in PlGF-induced HIF-1α promoter activity. HPMVEC transfected with the HIF-1α promoter construct phHIF1A (−863/+5 bp)-Luc along with β-galactosidase plasmid were either cotransfected with indicated siRNA (A) or with expression plasmids (B) prior to 6 h of PlGF stimulation. Each construct was used at 0.5 μg for transfection. Estimation of luciferase and β-galactosidase activities was carried out as described under “Experimental Procedures.” The data represent the means ± S.E. of three independent experiments. ***, p < 0.001; ns, nonsignificant.

FIGURE 5.

Egr-1 regulates FLAP mRNA expression through HIF-1α. A, HPMVEC transfected with −371FLAP-Luc promoter construct along with β-galactosidase plasmid were either cotransfected with the indicated expression plasmid or with siRNA construct prior to PlGF exposure for 6 h. Each construct was used at 0.5 μg for transfection. Luciferase and β-galactosidase activities were estimated as described under “Experimental Procedures.” ***, p < 0.001; ns, nonsignificant. B, qRT-PCR analysis of EGR-1, NAB2, HIF-1α, and FLAP in untreated and PlGF-treated HPMVEC. Where indicated, HPMVEC were transfected with either NAB WT or Nab2 mutant construct. C, HPMVEC were cotransfected with either WT (−371FLAP-Luc) or mutant FLAP promoter constructs (HRE-M1 or HRE-M2 or HRE-M1+2 or NF-κB) and β-galactosidase plasmid, followed by PlGF treatment for 6 h. The luciferase activity was normalized with that of the promoter-less pGL3 basic vector. The data represent means ± S.E. of three independent experiments. ***, p < 0.001; **, p < 0.01; ns, nonsignificant. D, HPMVEC were pretreated with either ascorbate or LY294002 or diphenyleneiodonium (DPI) prior to PlGF stimulation for 4 h. The chromatin samples were subjected to immunoprecipitation with either HIF-1α antibody (1 μg, upper panel) or control rabbit IgG (1 μg, lower panel). Purified DNA was PCR-amplified with the primers (listed in Table 1) corresponding to the region containing both HRE sites (−310/+9 bp) in the FLAP promoter. The input DNA panel represents the amplification of samples before immunoprecipitation. Data are representative of three independent experiments.

FIGURE 6.

Schematics of the PlGF-mediated regulation of gene expression in endothelial cells. PlGF stimulation of endothelial cells leads to an early increase in Egr-1 protein, which regulates the transcription of 5-LO and HIF-1α gene by binding to their respective promoters. HIF-1α protein translocates to the nucleus, binds with its partner HIF-1β, and stimulates transcription of FLAP after binding to HREs. Subsequently, the increased expression of 5-LO and FLAP leads to elevated levels of LT, which may promote the adhesion and migration of leukocytes from blood to the alveolar space of lungs contributing to organ injury.