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. Author manuscript; available in PMC: 2014 Oct 2.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2012 May 24;32(7):1707–1716. doi: 10.1161/ATVBAHA.112.251546

Angiopoietin-1 and Vascular Endothelial Growth Factor Regulation of Leukocyte Adhesion to Endothelial Cells: Role of Nuclear Receptor-77

Hodan Ismail 1, Mahroo Mofarrahi 1, Raquel Echavarria 1, Sharon Harel 1, Eric Verdin 1, Hyung W Lim 1, Zheng-Gen Jin 1, Jianxin Sun 1, Huiyan Zeng 1, Sabah NA Hussain 1
PMCID: PMC4183139  NIHMSID: NIHMS417042  PMID: 22628435

Abstract

Objective

Vascular endothelial growth factor (VEGF) promotes leukocyte adhesion to endothelial cells (ECs). Angiopoietin-1 (Ang-1) inhibits this response. Nuclear receptor-77 (Nur77) is a proangiogenic nuclear receptor. In the present study, we assessed the influence of Ang-1 and VEGF on Nur77 expression in ECs, and evaluated its role in Ang-1/VEGF-mediated leukocyte adhesion.

Methods and Results

Expression of Nur77 was evaluated with real-time polymerase chain reaction and immunoblotting. Adhesion of leukocytes to ECs was monitored with inverted microscopy. Nur77 expression or activity was inhibited using adenoviruses expressing dominant-negative form of Nur77, retroviruses expressing Nur77 in the antisense direction, and small interfering RNA oligos. Both Ang-1 and VEGF induce Nur77 expression, by >5- and 30-fold, respectively. When combined, Ang-1 potentiates VEGF-induced Nur77 expression. Ang-1 induces Nur77 through the phosphoinositide 3-kinase and extracellular signal-regulated protein kinase 1/2 pathways. VEGF induces Nur77 expression through the protein kinase D/histone deacetylase 7/myocyte enhancer factor 2 and extracellular signal-regulated protein kinase 1/2 pathways. VEGF induces nuclear factor-kappaB transcription factor, vascular cell adhesion molecule-1, and E-selectin expressions, and promotes leukocyte adhesion to ECs. Ang-1 inhibits these responses. This inhibitory effect of Ang-1 disappears when Nur77 expression is disrupted, restoring the inductive effects of VEGF on adhesion molecule expression, and increased leukocyte adhesion to ECs.

Conclusion

Nur77 promotes anti-inflammatory effects of Ang-1, and functions as a negative feedback inhibitor of VEGF-induced EC activation.

Keywords: angiopoietin-1, endothelial cells, inflammation, leukocyte adhesion, nuclear receptor-77, vascular endothelial growth factor

Angiopoietin-1 (Ang-1), a major ligand of Tie-2 receptors, promotes vascular development in embryos, and enhances endothelial cell (EC) integrity and survival in adults.1 Vascular endothelial growth factor (VEGF) promotes leukocyte adhesion, and induces adhesion molecules expressed on the surface of ECs, including E-selectin and vascular cell adhesion molecule-1 (VCAM1). These adhesion molecules are major regulators of leukocyte adhesion to activated ECs.2 In contrast to the proinflammatory effects that are exerted by VEGF, Ang-1 inhibits leukocyte adhesion, the expressions of adhesion molecules, and basal and VEGF-induced vascular leakage.3–5 The mechanisms through which Ang-1 exerts these suppressor effects on VEGF-induced inflammation remain unclear.

Recent studies have indicated that the proangiogenic effects of VEGF are mediated by a specific signaling pathway known as the protein kinase D (PKD)/histone deacetylase 7 (HDAC7)/myocyte enhancer factor 2 (MEF2) pathway, which involves PKD1 and PKD2, HDAC7 (a transcriptional suppressor), and MEF2 transcription factor.6,7 VEGF-induced activation of PKD1 and PKD2 results in phosphorylation of Ser178, Ser344, and Ser479 of HDAC7, which is associated with HDAC7 nuclear export and the removal of its suppressor effect on MEF2-mediated transcription.8 The influence of Ang-1 on PKD/HDAC7/MEF2 pathway in ECs is unknown.

Nuclear receptor-77 (Nur77) is a member of the nuclear receptor subfamily 4 group A member 1 subfamily of ligand-independent nuclear receptors.9 Its expression is rapidly and transiently upregulated in response to cytokines and growth factors.10,11 Nur77 regulation by Ang-1 has not been studied; however, in ECs exposed to VEGF, Nur77 is one of the most robustly upregulated genes.12 VEGF-mediated upregulation of Nur77 is mediated through activation of the PKD/HDAC7/MEF2 pathway,6,7 but whether or not Ang-1 also regulates Nur77 expression through this pathway is unclear. The first objective of the present study, therefore, is to evaluate the degrees to which Ang-1 and VEGF regulate Nur77 expression, including the nature of their interaction, and to identify the precise mechanisms through which this regulation occurs.

The functional significance of Nur77 in relation to inflammation remains unclear. In nonvascular cells, Nur77 interacts with p65 (RelA) subunits of nuclear factor-kappaB (NF-κB),13 and interferes with p65 binding to low-affinity NF-κB binding elements in promoters of proinflammatory cytokines.14 In ECs, the only evidence that Nur77 negatively regulates pro-inflammatory responses has recently been provided by You et al,15 who have described significant upregulation of Nur77 in cells exposed to tumor necrosis factor-α (TNF-α), significant inhibition by Nur77 of TNF-α–induced adhesion molecule expression, and significant inhibition of leukocyte adhesion by selective Nur77-induced inhibition of NF-κB activation.

Based on these observations, we hypothesize that Nur77 may play an important role in ECs as a feedback inhibitor, suppressing VEGF-mediated NF-κB activation and thereby limiting VEGF-induced adhesion molecule expressions and leukocyte adhesion. We also hypothesize that the suppressor effects of Ang-1 on VEGF-induced inflammation are mediated, in part, through Nur77. The second objective of the study, therefore, is to identify the importance of Nur77 to VEGF- and Ang-1–mediated activation of ECs.

Methods

Detailed methods are available in the online-only Data Supplement.

Cell Cultures

Each experimental set of cells was exposed to vehicle, Ang-1, VEGF, Ang-1 and VEGF in combination, for varying durations. One set was also exposed to Ang-2 or Ang-2 and VEGF in combination. Before experimentation, cells recovered for 48 hours after infection with viruses, or transfected with small interfering RNA (siRNA) oligos. Human umbilical vein ECs (HUVECs) were maintained in culture, as described.16 HUVECs stably expressing empty vector or a dominant-negative form of c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK; HUVECs stably expressing empty vector-JNK- alanine-proline-phenylalanine mutant) were maintained in culture, as described.17 Human leukemic leukocyte lymphoma cells (U937) were maintained in Gibco Roswell Park Memorial Institute Medium 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum.

siRNA Oligos

HUVECs were transfected with scrambled siRNA oligos (nonsilencing) or Nur77 smart pool siRNA oligos using DharmaFECT 4 reagent, according to the manufacturer’s instructions (Dharmacon Inc, Lafayette, CO). Nur77 knockdown was verified with real-time polymerase chain reaction and immunoblotting (online-only Data Supplement).

Adenoviruses and Retroviruses

Adenoviruses expressing green fluorescent protein (Ad-GFP, control), wild-type Nur77, and a dominant-negative form of Nur77 lacking the transactivation domain (Ad-dn Nur77) were generated, as described.15 Expression of Nur77 protein in response to infection with these viruses was verified with immunoblotting (online-only Data Supplement). Adenoviruses expressing wild-type PKD1, a dominant-negative form of PKD1, GFP-HDAC7, and a GFP-HDAC7 mutant in which Ser178, Ser344, and Ser479 were mutated to alanine residues (adenoviruses expressing GFP-HDAC7 S/A) were generated, as described.7 Retroviruses expressing LacZ (control), Nur77 in the sense direction, Nur77 in the antisense direction (Nur77 antisense), Nur77 lacking the ligand-binding domain (Nur77 Δligand-binding domain), transactivation domain (Nur77 Δtransactivation domain), and DNA-binding domain (Nur77 ΔDNA-binding domain) were generated, as described.18

mRNA Measurements

mRNA samples were extracted using a GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich, Oakville, ON). Total RNA was reverse transcribed using a Superscript II Reverse Transcriptase Kit and random primers (Invitrogen). The mRNA expression levels were measured using real-time polymerase chain reaction (Applied Biosystems, Foster City, CA) and specific primers (online-only Data Supplement). Results were analyzed using both the comparative threshold cycle method and absolute copy numbers normalized per 1000 copies of β-actin.

Immunoblotting

Subcellular fractionation of cell lysates into cytosolic and nuclear fractions was performed using hypotonic lysis buffer and a Dounce Tissue Grinder (Wheaton Industries Inc, Millville, NJ; online-only Data Supplement). Fractions and total cell lysates were separated using SDS-PAGE, transferred to polyvinylidene fluoride membranes, then probed with primary antibodies. Proteins were detected with horseradish peroxidase-conjugated secondary antibodies and electrochemiluminescence reagents.

HDAC7 Localization

HUVECs were infected with adenoviruses expressing GFP-HDAC7 or GFP-HDAC7 S/A adenoviruses, and exposed to angiogenesis factors, as above. Cells were washed with PBS, fixed, then visualized using fluorescence microscopy.

Luciferase Reporter Assays

HUVECs were infected with adenoviruses expressing NF-κB- Luciferase (Luc) adenoviruses, and exposed to vehicle, Ang-1, or VEGF for 6 hours. NF-κB–mediated transcription induction was evaluated by measuring luminescence (Promega Inc, Madison, WI).

Electrophoretic Mobility Shift Assay

Electrophoretic mobility shift assays for NF-κB were performed with a Gelshift NF-κB/Rel kit (Panomics, Fremont, CA) using double-stranded DNA probes for human NF-κB (online-only Data Supplement) and 20 μg of nuclear extracts, according to the manufacturer’s instructions.

Leukocyte Adhesion Assays

U937 cells were labeled with 3,3′-dioctadecylindocarbocyaniniodide dye (Biotium Inc, Hayward, CA) for 30 minutes. Cells (5×10)5 were washed, then added to 24-well culture plates containing confluent HUVECs transfected with siRNA oligos, or infected with adeno- or retroviruses. After 1 hour of incubation on a rocking platform, cells were washed 3×. Fixed and adhering leukocytes were counted in 10 fields per well using an inverted fluorescence microscope (Olympus 1×70, Olympus America Inc, Melville, NY).

Statistical Analysis

Data were expressed as means±SE. Statistical significance was determined by 1-way ANOVA. Differences were considered statistically significant at P<0.05.

Results

Ang-1 and VEGF Regulation of Nur77 Expression

Ang-1 exposure triggered dose-dependent and transient increases of Nur77 mRNA expression levels (Figure 1A and 1B). VEGF exposure induced dose- and time-dependent increases (Figure 1C and 1D). Relative inductions of VEGF-induced Nur77 expressions were higher than those observed with Ang-1 (Figure 1E). Upregulation of Nur77 expressions by Ang-1 was also observed in EC-RF24 and human microvascular endothelial cells (online-only Data Supplement). When Ang-1 and VEGF were combined, synergistic induction of Nur77 expression occurred (Figure 1F). Ang-2 exposure did not induce Nur77 expression, and had no influence on VEGF-induced Nur77 expression (Figure 1F).

Figure 1.

Figure 1

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A and B, Effect of dose on nuclear receptor-77 (Nur77) mRNA expression in human umbilical vein endothelial cells (HUVECs) exposed to angiopoietin-1 (Ang-1; 100–500 ng/mL) for 1 hour (A) or Ang-1 (500 ng/mL) for different periods (B). C and D, Effect of dose and exposure time on Nur77 mRNA expression in HUVECs exposed to vascular endothelial growth factor (VEGF). All data (A–D) are expressed as means±SEM. n=6. *P<0.05, compared with control. E, Representative immunoblottings of Nur77 protein expression in HUVECs exposed to vehicle (control), Ang-1 (500 ng/mL), or VEGF (40 ng/mL) for 1 hour. F, Effect of treatment on Nur77 mRNA expression in HUVECs exposed to vehicle (control), Ang-1 (500 ng/mL), VEGF (40 ng/mL), Ang-1+VEGF, Ang-2 (500 ng/mL), or Ang-2+VEGF for 1 hour. n=8. *P<0.05 compared with control. #P<0.05 compared with VEGF.

Ang-1– and VEGF-Induced Phosphorylation and Mobilization of HDAC7

Ang-1 induced HDAC7 phosphorylation on Ser178, Ser344, and Ser479 (Figure 2A; online-only Data Supplement). A similar effect was observed in response to VEGF (online-only Data Supplement). However, the relative rise in HDAC7 phosphorylation in response to VEGF was relatively stronger (≈9-fold) than that elicited by Ang-1 (≈6-fold; online-only Data Supplement). In control cells, HDAC7 was exclusively in the nucleus; Ang-1 exposure induced only a mild increase of HDAC7 mobilization to the cytosol, whereas VEGF exposure triggered robust mobilization (Figure 2B; online-only Data Supplement). Gradual relocalization of HDAC7 to the nucleus occurred within 24 hours. When Ang-1 and VEGF were combined, a synergistic increase in HDAC7 mobilization was observed within 1 hour of exposure (Figure 2B). In combination, they were incapable of inducing mobilization when the Ser178, Ser344, and Ser479 residues of HDAC7 were mutated to alanine (HDAC7 S/A; online-only Data Supplement).

Figure 2.

Figure 2

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A, Histone deacetylase 7 (HDAC7) phosphorylation on Ser178, Ser344, and Ser479 in human umbilical vein endothelial cells (HUVECs) exposed to vehicle (control) or angiopoietin-1 (Ang-1; 500 ng/mL) for varying durations. B, Total number of cells with cytosolic HDAC7 in response to vehicle (control), Ang-1 (500 ng/mL), vascular endothelial growth factor (VEGF; 40 ng/mL), or Ang-1+VEGF exposure for 1 or 4 hours. n=8. *P<0.05 compared with control. #P<0.05 compared with VEGF. C, Nuclear receptor-77 (Nur77) mRNA expression in HUVECs infected with adenoviruses expressing HDAC7 (Ad-HDAC7) or Ad-HDAC7 S/A viruses and then exposed to vehicle (control), Ang-1, or VEGF. n=6. *P<0.05 compared with control (adenoviruses expressing green fluorescent protein [Ad-GFP]-infected) cells. #P<0.05 compared with Ad-GFP-infected cells stimulated with VEGF. D, Nur77 mRNA expression in HUVECs infected with adenoviruses expressing wild-type protein kinase D1 (Ad-PKD1) or adenoviruses expressing dominant-negative form of PKD1 (Ad-dn PKD1) viruses and then exposed to vehicle, Ang-1, or VEGF. n=6. *P<0.05 compared with vehicle exposure of Ad-GFP–infected cells. #P<0.05 compared with Ad-GFP–infected cells stimulated with VEGF. E, Effect of pretreatment with PBS, BIRB 796 (p38 inhibitor), wortmannin (WM; phosphoinositide 3 [PI3] kinase inhibitor), PD18435 (ERK1/2 inhibitor), or SP600125 (c-Jun N-terminal kinase/stress-activated protein kinase [JNK/SAPK] inhibitor) on control, Ang-1– and VEGF-induced Nur77 expression. n=6. *P<0.05 compared with own control group. #P<0.05 compared with control condition in PBS-pretreated cells. F, Effect of pathway inhibition on Nur77 mRNA expression in HUVECs stably expressing empty vector (MSCV) or a dominant-negative form of JNK (MSCV-JNK-aggregation-promoting factor [APF]) exposed to vehicle or Ang-1 for 1 hour. n=5. *P<0.05 compared with its own control. #P<0.05 compared with MSCV cells exposed to Ang-1.

Ang-1– and VEGF-Induced Phosphorylation of PKD1 and PKD2

Ang-1 exposure induced significant and progressive increases in PKD1 (Ser744/748) and PKD2 (Ser876) phosphorylation (online-only Data Supplement). As reported previously,19 VEGF exposure also triggered robust PKD1 and PKD2 phosphorylation (online-only Data Supplement). Although both Ang-1 and VEGF induced significant PKD1 phosphorylation, the relative rise in PKD1 phosphorylation in response to VEGF is relatively stronger (≈29-fold) compared with that elicited by Ang-1 (≈5-fold; online-only Data Supplement).

Signaling Pathways Regulating Nur77 Expression

Ang-1–induced Nur77 mRNA expressions were not significantly different in cells infected with Ad-GFP (control), adenoviruses expressing HDAC7, and HDAC7 S/A viruses (Figure 2C). In contrast, VEGF-induced Nur77 expressions were strongly attenuated in cells infected with adenoviruses expressing HDAC7 S/A viruses (Figure 2C), and partially attenuated in cells infected with adenoviruses expressing dominant-negative form of PKD1 viruses (Figure 2D). Ang-1–induced Nur77 expressions were not significantly different in cells infected with adenoviruses expressing wild-type PKD1 and adenoviruses expressing dominant-negative form of PKD1 viruses (Figure 2D). To evaluate the roles of phosphoinositide 3 (PI3) kinase, extracellular signal-regulated protein kinase 1/2 (ERK1/2), p38, and JNK/SAPK pathways in Ang-1– and VEGF-induced upregulation of Nur77, we used selective pharmacological inhibitors of these pathways. The inductive effect of Ang-1 exposure on Nur77 expressions disappeared under conditions of selective inhibition of the ERK1/2 and PI3 kinase pathways, whereas inhibition of the JNK/SAPK pathway augmented both basal and Ang-1–induced Nur77 expressions (Figure 2E). Ang-1 substantially induced Nur77 expressions in cells expressing a dominant-negative form of JNK1 (Figure 2F), and a dominant-negative form of the c-Jun subunit of activator protein 1 transcription factor (online-only Data Supplement). In the case of VEGF, only ERK1/2 inhibition completely eliminated the inductive effect of VEGF exposure on Nur77 expression, whereas inhibition of p38 or JNK/SAPK pathways had no significant effect on this response (Figure 2E). Inhibition of the PI3 kinase pathway slightly attenuated but did not completely inhibit VEGF-induced Nur77 upregulation (Figure 2E).

Ang-1 and VEGF Regulation of NF-κB Activity

Within 2 hours of VEGF exposure in HUVECs, nuclear translocation of the p50 and p65 subunits of NF-κB increased significantly (Figure 3A). Ang-1 had no effect on translocation of these 2 subunits or p65 phosphorylation on Ser536, whereas VEGF exposure induced significant phosphorylation of this residue (Figure 3B; online-only Data Supplement). VEGF-induced p65 phosphorylation was attenuated when in combination with Ang-1 (Figure 3B). Ang-1 had no effect on basal NF-κB DNA-binding activity (results not shown), whereas VEGF exposure induced significant increases in NF-κB DNA-binding activity in cells infected with Ad-GFP and Ad-dn Nur77 viruses, but not adenoviruses expressing Nur77 viruses (Figure 3C). Thus, VEGF stimulates NF-κB DNA-binding activity, and Nur77 exerts a negative effect on binding. Effects on NF-κB–dependent transcription were confirmed using a luciferase promoter construct driven by 4 tandem copies of the NF-κB consensus sequence (Figure 3D). VEGF, but not Ang-1, significantly induced NF-κB–Luc promoter activity in cells infected with Ad-GFP viruses. Ang-1 inhibited VEGF-induced NF-κB–Luc activity in these cells (Figure 3D). NF-κB–Luc promoter activity was strongly attenuated in cells infected with adenoviruses expressing Nur77 viruses and augmented in cells infected with Ad-dn Nur77 viruses. The inhibitory effect of Ang-1 on VEGF-induced NF-κB–Luc activity was eliminated in cells infected with Ad-dn Nur77 (Figure 3D). Thus, VEGF significantly enhanced NF-κB–driven promoter activity, whereas Nur77 exerted an inhibitory influence. Removal of the inhibitory effect resulted in further augmentation of VEGF-induced activity and eliminated the depressor effect of Ang-1 on this activity.

Figure 3.

Figure 3

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A, Mobilization of p50 and p65 subunits of nuclear factor-kappaB (NF-κB) in the cytosolic (C) and nuclear (N) fractions in response to 1-, 2-, and 3-hour exposure to vascular endothelial growth factor (VEGF). Histone H3 was used as a nuclear marker. B, Representative immunoblottings of phosphorylated (Ser536) and total p65 subunit of NF-κB in human umbilical vein endothelial cells (HUVECs) exposed to angiopoietin-1 (Ang-1), VEGF, or Ang-1+VEGF. C, Representative electrophoretic mobility shift assay (EMSA) of NF-κB DNA-binding activity and optical densities (ODs) of NF-κB binding in HUVECs infected with adenoviruses expressing green fluorescent protein (Ad-GFP), adenoviruses expressing nuclear receptor-77 (Ad-Nur77), or a dominant-negative form of Nur77 (Ad-dn Nur77) viruses, and then exposed to vehicle (control, -) or VEGF (+) for 2 hours. Data are expressed as means±SEM. n=4. *P<0.05 compared with control. D, Effect of treatment on NF-κB activity in HUVECs infected 48 hours earlier with Ad-NF-κB-large unstained cells (Luc) viruses plus Ad-GFP, Ad-Nur77, or Ad-dn Nur77 viruses, and then exposed to vehicle (control), Ang-1, VEGF, or Ang-1+VEGF. Data are expressed as means±SEM. n=6. *P<0.05 compared with its own control. #P<0.05 compared with Ad-GFP–infected cells stimulated with VEGF. ΔP<0.05 compared with Ad-GFP–infected cells exposed to Ang-1+VEGF.

Regulation of IkappaBα Expression by Nur77

Overexpression of Nur77 with adenoviruses expressing Nur77 viruses significantly upregulated IkappaBα (IκBα) mRNA and protein expression levels (Figure 4A–4C). Likewise, IκBα protein expression levels were significantly induced in cells infected with retroviruses expressing Nur77 in the sense direction and those expressing Nur77 lacking the ligand-binding domain, compared with those infected with retroviruses expressing LacZ (control; Figure 4D; online-only Data Supplement). Iκβα protein expression levels remained unchanged in cells infected with retroviruses expressing a Nur77 lacking transactivation or DNA-binding domains (Figure 4D; online-only Data Supplement).

Figure 4.

Figure 4

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A, Effect of multiplicity of infection (MOI) on IkappaBα (IκBα) mRNA expression in human umbilical vein endothelial cells (HUVECs) untreated (control) or infected with increasing MOI of adenoviruses expressing nuclear receptor-77 (Ad-Nur77) or a dominant-negative form of Nur77 (Ad-dn Nur77) viruses. n=6. *P<0.05 compared with control. B and C, Representative IκBα blottings and means±SEM of IκBα optical densities (ODs) in HUVECs infected with Ad-Nur77 and Ad-dn Nur77 viruses at different MOI. *P<0.05 compared with control. D, IκBα protein expression in HUVECs infected with retroviruses expressing LacZ, Nur77 S, Nur77Δligand-binding domain (ΔLBD), Nur77Δtransactivation domain (ΔTAD), and Nur77ΔDNA-binding domain (ΔDBD).

Regulation of Adhesion Molecules by Ang-1 and VEGF

Ang-1 significantly inhibited both basal and VEGF-induced E-selectin and VCAM1 mRNA and protein expression levels (Figure 5A; online-only Data Supplement). In contrast, VEGF significantly induced E-selectin and VCAM1 expression levels (Figure 5A; online-only Data Supplement) but had no influence on intercellular adhesion molecule-1 levels (results not shown). In cells infected with LacZ-expressing viruses (control), Ang-1 attenuated E-selectin and VCAM1 mRNA expression levels, whereas VEGF significantly induced their expressions (Figure 5B). Suppression of Nur77 using Nur77 antisense–expressing retroviruses eliminated the inhibitory effects of Ang-1 on E-selectin and VCAM1 and significantly enhanced VEGF effects (Figure 5B). This demonstrates that Ang-1 inhibits their expressions through induction of Nur77, and that induction of Nur77 inhibits the upregulatory effects of VEGF on these adhesion molecules. This conclusion was confirmed in cells overexpressing Nur77, where the upregulatory effect of VEGF on E-selectin and VCAM1 mRNA and protein expression levels was eliminated (Figure 5C and 5D).

Figure 5.

Figure 5

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A, E-selectin and vascular cell adhesion molecule-1 (VCAM1) protein expression in human umbilical vein endothelial cells (HUVECs) exposed to vehicle (control), angiopoietin-1 (Ang-1), vascular endothelial growth factor (VEGF), or Ang-1+VEGF. B, E-selectin and VCAM1 mRNA levels in HUVECs infected with retroviruses expressing LacZ or nuclear receptor-77 antisense (Nur77 AS), and exposed to vehicle (control), Ang-1, or VEGF for 4 hours. n=6. *P<0.05 compared with control. #P<0.05 compared with cells infected with LacZ viruses and exposed to VEGF. C and D, E-selectin and VCAM1 mRNA and protein expression in HUVECs infected adenoviruses expressing green fluorescent protein (Ad-GFP), adenoviruses expressing Nur77 (Ad-Nur77), or dominant-negative form of Nur77 (Ad-dn Nur77) viruses, and exposed to vehicle (-) VEGF (+) for 4 hours. n=6. *P<0.05 compared with control. #P<0.05 compared with Ad-GFP–infected cells exposed to VEGF.

Ang-1 and VEGF Regulation of Leukocyte Adhesion to ECs

Ang-1 significantly reduced both basal and VEGF-induced leukocyte (U937) adhesion to HUVECs, whereas VEGF significantly enhanced adhesion (online-only Data Supplement). Ang-1 inhibition of basal and VEGF-induced leukocyte adhesion was observed in cells infected with LacZ-expressing viruses (Figure 6A). In cells infected with Nur77 antisense–expressing viruses, Ang-1 augmented basal leukocyte adhesion and was unable to inhibit VEGF-induced leukocyte adhesion, whereas VEGF-induced adhesion was even greater in these cells compared with cells infected with LacZ-expressing viruses (control; Figure 6A). The importance of Nur77 to the regulation of leukocyte adhesion was also assessed using siRNA oligos to inhibit Nur77 expression. In HUVECs transfected with scrambled siRNA oligos, VEGF exposure induced leukocyte adhesion whereas Ang-1 exposure inhibited it (Figure 6B). Combining Ang-1 and VEGF resulted in significant attenuation of VEGF-induced leukocyte adhesion (Figure 6B). In cells transfected with Nur77 siRNA, the inhibitory effect of Ang-1 on baseline and VEGF-induced leukocyte adhesion disappeared, whereas VEGF-induced leukocyte adhesion was further augmented in these cells (Figure 6B). Adenoviruses overexpressing GFP or a dominant-negative form of Nur77 were used to assess the influence of Nur77 on Ang-1– and VEGF-induced leukocyte adhesion to ECs (Figure 6C). In cells infected with Ad-GFP viruses, Ang-1 significantly inhibited whereas VEGF exposure significantly enhanced leukocyte adhesion. In cells infected with Ad-dn Nur77 viruses, the Ang-1 exposure actually triggered a significant increase in leukocyte adhesion whereas the stimulatory effect of VEGF was further exaggerated. Collectively, these results indicate that Nur77 played an important role in counteracting the positive regulatory effect of VEGF on leukocyte adhesion to ECs and in supporting the negative regulatory effect of Ang-1.

Figure 6.

Figure 6

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A, Number of adhered U937 cells in human umbilical vein endothelial cells (HUVECs) infected with retroviruses expressing LacZ or nuclear receptor-77 antisense (Nur77 AS) exposed to vehicle, angiopoietin-1 (Ang-1), vascular endothelial growth factor (VEGF), or Ang-1+VEGF for 8 hours, and then incubated with labeled U937 cells for 1 hour. n=6. *P<0.05 compared with control. #P<0.05 compared with cells infected with LacZ-expressing viruses and exposed to Ang-1, VEGF, or Ang-1+VEGF. B, Number of adhered U937 cells to HUVECs transfected with scrambled small interfering RNA (siRNA) oligos or Nur77 siRNA oligos, and exposed to vehicle, Ang-1, VEGF, or Ang-1+VEGF. n=6. *P<0.05 compared with control. #P<0.05 compared with scrambled siRNA-transfected cells exposed to VEGF or Ang-1+VEGF. C, Number of adhered U937 cells to HUVECs infected with adenoviruses expressing green fluorescent protein (Ad-GFP) or dominant-negative form of Nur77 (Ad-dn Nur77) viruses, and exposed to vehicle, Ang-1, or VEGF. n=6. *P<0.05 compared with control. #P<0.05 compared with Ad-GFP–infected cells exposed to VEGF. #P<0.05 compared with cells infected with Ad-GFP viruses and exposed to Ang-1 or VEGF. D, Schematic representation of Ang-1 and VEGF regulation of leukocyte adhesion to endothelial cells. ERK1/2 indicates extracellular signal-regulated protein kinase 1/2; HDAC7, histone deacetylase 7; JNK, c-Jun N-terminal kinase; MEF2, myocyte enhancer factor 2; NF-κB, nuclear factor-kappaB; PI3K, phosphoinositide 3-kinase; PKD1/2, protein kinase D 1/2; SAPK, stress-activated protein kinase; VEGFR2, VEGF receptor 2.

Discussion

The main findings of the present study are as follows: (1) Ang-1 regulates Nur77 expression in ECs through the PI3 kinase and ERK1/2 pathways; (2) Ang-1 potentiates VEGF-induced Nur77 expression, HDAC7 phosphorylation, and nuclear export; (3) VEGF induces NF-κB activation, adhesion molecule expressions, and leukocyte adhesion to ECs, and Ang-1 suppresses these responses; (4) Nur77 suppression potentiates the stimulatory effects of VEGF on NF-κB activation, adhesion molecule expressions, and leukocyte adhesion to ECs, while eliminating the inhibitory effects of Ang-1 on these responses; and (5) overexpression of Nur77 upregulates IκBα expression and suppresses VEGF-induced activation of NF-κB, adhesion molecule expressions, and leukocyte adhesion to ECs.

Regulation of Nur77 Expression

It has been established that Nur77 is one of the most highly upregulated genes triggered by VEGF-induced nuclear export of HDAC7.6,7 The present study confirms that the PKD/HDAC7/MEF2 and ERK1/2 pathways are important in VEGF-induced upregulation of Nur77 in ECs, because overexpression of mutant forms of HDAC7 and PKD1 and ERK1/2 inhibitor attenuates VEGF-induced Nur77 upregulation (Figure 2). The importance of the ERK1/2 pathway and the lack of a significant role for the PI3 kinase/protein kinase B (AKT) pathway in the stimulatory effect of VEGF on Nur77 in the present are in agreement with previous reports.12

Our study demonstrates for the first time that Ang-1, but not Ang-2, induces significant upregulation of Nur77 expression in ECs and that Ang-1 stimulates the phosphorylation of PKD1, PKD2, and HDAC7, enhances the nuclear export of HDAC7, and potentiates the effect of VEGF on HDAC7 mobilization to the cytosol (Figure 2). Despite activation of the PKD/HDAC7/MEF2 pathway by Ang-1, this pathway does not appear to be important in Ang-1–induced Nur77 upregulation but appears to be critical for VEGF-induced Nur77 upregulation (Figure 2). We attribute this difference in the importance of the PKD/HDAC7/MEF2 pathway to the fact that relative activation by VEGF of the PKD/HDAC7/MEF2 pathway as assessed by PKD1 and HDAC7 phosphorylation and HDAC7 mobilization is significantly stronger than that elicited by Ang-1 (Figure 2; online-only Data Supplement). We propose that Ang-1 activation of the PKD/HDAC7/MEF2 pathway may play an important role in other biological effects of Ang-1, those not involved with Nur77 regulation of EC activation. Such effects include enhanced EC survival and migration. This speculation is based on the fact that HDAC7 is vitally important to embryonic vascular development20 and plays a critical role in VEGF-induced promotion of EC proliferation, survival, and migration.6,7 Whether or not HDAC7 plays a similar role in Ang-1–induced angiogenesis remains to be investigated. We should point out that we could not exclude a role for PKD2 in Ang-1–induced Nur77 expression because the dominant-negative form of PKD influences PKD1- but not PKD2-mediated responses (Figure 2D).

The Ang-1/Tie-2 receptor axis exerts its biological effects on ECs through several signaling pathways, including ERK1/2, p38 mitogen-activated protein kinase, and JNK/SAPK members of the mitogen-activated protein kinases, and the PI3 kinase/AKT pathway.16,21 We found that Ang-1 induces Nur77 through the ERK1/2 and PI3 kinase pathways and that the p38 mitogen-activated protein kinase pathway has no effect on Nur77 expression (Figure 2). There are several possible mechanisms through which activation of the ERK1/2 pathway by Ang-1 and VEGF results in upregulation of Nur77 expression in ECs. One such pathway is activation of mitogen- and stress-activated protein kinases 1 and 2. In non-ECs, TNF-α and EGF trigger ERK1/2-dependent phosphorylation of mitogen- and stress-activated protein kinases 1 and 2 and activation of the cAMP response element-binding transcription factor which then binds to 2 activator protein 1–like elements adjacent to the transcription start site in the Nur77 promoter.22 An alternative pathway through which ERK1/2 regulates Nur77 expression is through JunD phosphorylation and binding of JunD to the activator protein 1–like elements of Nur77 promoter. This pathway has been implicated in the upregulation of Nur77 expression triggered by prostaglandin and nerve growth factor.23,24 Whether cAMP response element-binding and JunD transcription factors play important roles in ERK1/2-dependent upregulation of Nur77 in HUVECs exposed to Ang-1 or VEGF remains to be investigated.

The PI3 kinase/AKT pathway is a major mediator of the prosurvival and antiapoptotic effects of Ang-1 and VEGF on ECs.21,25,26 The present study revealed that PI3 kinase/AKT pathway plays an important role in Ang-1–induced Nur77 upregulation but is not a major mediator of Nur77 expression in cells exposed to VEGF (Figure 2). The reasons behind this difference are unclear primarily because little is known about how the PI3 kinase/AKT pathway regulates Nur77 in ECs. We speculate that the importance of this pathway in Nur77 regulation by Ang-1 may be related to ERK1/2 activation. In ECs exposed to Ang-1, inhibition of PI3 kinase/AKT pathway attenuated ERK1/2 activation suggesting that the ERK1/2 is downstream from the PI3 kinase/AKT pathway in Tie-2 receptor signaling.16 This is apparently not the case in VEGF receptors signaling in ECs.27 Further studies are needed to elucidate the exact molecular mechanisms through which the PI3 kinase/AKT pathway regulates Nur77 in ECs.

The present study also shows that the JNK/SAPK mitogen-activated protein kinase pathway exerts a strong inhibitory effect on Nur77 expression both at the basal level and in response to Ang-1, but it does not play a major role in VEGF-induced Nur77 expression (Figure 2). This inhibitory effect was confirmed in cells overexpressing a dominant-negative form of JNK1 and those overexpressing a dominant-negative c-Jun (Figure 2; online-only Data Supplement). The inhibitory effect of the JNK/SAPK pathway on Nur77 expression has not previously been described in ECs. What is known is that the JNK/SAPK pathway inhibits Nur77 transactivation activation.28,29 JNK proteins directly phosphorylate Nur77 on Ser95, resulting in ubiquitination and degradation of Nur77 and strong attenuation of its transactivation activity. Moreover, c-Jun directly interacts with Nur77 to inhibit its transactivation.30 The results of the present study bolster these observations and clearly demonstrate that the JNK/SAPK pathway also inhibits transcription of Nur77, as indicated by enhanced Nur77 expression, both at the basal level and after exposure to Ang-1, in response to JNK/SAPK pathway inhibition.

It should be emphasized that the present study does not rule out the possibility that other pathways such as the PKD/HDAC5/MEF2, calcineurin/NF of activated T cells, (Ca2+)I, and the protein kinase C pathways may also be involved in Ang-1– and VEGF-induced upregulation of Nur77 in ECs.12,31

Nur77 and Anti-Inflammatory Effects of Ang-1

There is increasing evidence that Nur77 regulates inflammatory processes in the vascular system. Indeed, Nur77 is expressed in macrophages in advanced human atherosclerosis, and macrophage Nur77 expression is upregulated by lipopolysaccharide and TNF-α.32 In isolated macrophages, overexpression of Nur77 inhibits expression and production of several proinflammatory cytokines and chemokines.32 Similarly, Nur77 inhibits the formation of vascular lesions in ligated carotid arteries of transgenic mice.33

Induction of EC adhesion molecules is an essential step early in the leukocyte adhesion cascade.34 VEGF promotes leukocyte adhesion to ECs as a result of NF-κB–mediated upregulation of the adhesion molecules, intercellular adhesion molecule-1, VCAM1, and E-selectin.35–37 Upregulation of NF-κB activation, VCAM1 and E-selectin expressions, and leukocyte adhesion to ECs by VEGF is confirmed by our study. Intercellular adhesion molecule-1 upregulation appears to be variable, depending on the type of EC that is under investigation.38 In the present study, we uncovered a novel role for endogenous Nur77 in VEGF signaling, namely, as a feedback inhibitor designed to limit the intensity and duration of VEGF-induced NF-κB activation, VCAM1 and E-selectin upregulation, and leukocyte adhesion to ECs. This conclusion is drawn from results from both gain-of-function and loss-of-function experiments (Figures 5 and 6).

Several reports have indicated that administration of Ang-1 recombinant protein or Ang-1 expressing adenoviruses to control mice or mice with lipopolysaccharide-induced sepsis results in significant increase in survival, attenuation of vascular leakages, and strong inhibition of adhesion molecule expression and leukocyte infiltration into the lungs.4,39,40 In cultured ECs, Ang-1 reduces basal and VEGF-induced vascular leakage, and strongly attenuates VEGF-induced adhesion by reducing intercellular adhesion molecule-1, VCAM1, and E-selectin expressions,3,4,41 although 1 published report42 has described significant increases in P-selectin translocation and neutrophil adhesion to ECs in response to Ang-1. We found that Ang-1 also attenuates expressions of basal levels of VCAM1 and E-selectin in ECs, and significantly reduces basal leukocyte adhesion to ECs. Moreover, Ang-1 antagonizes VEGF-induced upregulation of adhesion molecule expressions and leukocyte adhesion to ECs (Figures 5 and 6). We believe that these effects are largely mediated through inhibition of NF-κB activation (Figure 3). The mechanisms through which Ang-1 modulates NF-κB activity remain under investigation. In the present study, we evaluated the role of Nur77 in relation to anti-inflammatory effects of Ang-1 by using a loss-of-function (siRNA oligos or antisense transcripts) approach that revealed that Ang-1 does not inhibit VCAM1 and E-selectin expressions and leukocyte adhesion to ECs when Nur77 expression is downregulated (Figures 5 and 6). Based on these findings, we conclude that Nur77 plays an important role in mediating the anti-inflammatory effects of Ang-1 on ECs.

Mechanisms of Nur77-Mediated Inhibition of NF-κB in ECs

Previous studies have identified Nur77 as a potent inhibitor of NF-κB.43 The mechanisms through which Nur77 regulates NF-κB activity remain under investigation. You et al15 have recently reported that Nur77 inhibits TNF-α–induced NF-κB activation and p65 mobilization to the nucleus in ECs. They identified IκBα as an important transcriptional target of Nur77 in that it specifically binds to a Nur77-binding element located at the –445 and –452 base pairs of human IκBα promoter. In the present study, using both adenoviruses and retroviruses expressing wild-type Nur77, we have confirmed that Nur77 upregulates IκBα expression in ECs (Figure 4). Interestingly, mutation of either the transactivation or the DNA-binding domains results in the inability of Nur77 to induce IκBα expression, suggesting that these structural domains of Nur77 are essential for the stimulatory effect of Nur77 on IκBα transcription. These results further emphasize the importance of the DNA-binding and transactivation domains of Nur77, which have been shown to be critical for the proangiogeneic of this protein in VEGF-stimulated ECs.18

It should be emphasized that our study does not preclude the possibility that Nur77 may influence NF-κB activity through mechanisms other than upregulation of IκBα expression. One possible mechanism through which Nur77 may influence NF-κB activation independently of IκBα expression is through direct interaction between Nur77 and the p65 subunit of NF-κB, which has been reported in R2C cells, although the functional importance of this interaction in regulating NF-κB activation remains unclear.13 Another report has described interactions between Nur77 and NF-κB subunits at the level of DNA-binding sites as being an important mechanistic locus through which Nur77 suppresses NF-κB activation.14

In addition, our study does not exclude the possibility that Ang-1 may influence NF-κB activation and adhesion molecules through Nur77-independent pathways. Indeed, Hughes et al44 reported that Tie-2 receptors directly interact with a potent regulator of inflammatory gene expression, namely, A20 binding inhibitor of NF-κB activation-2. This interaction mediates the inhibitory effects of Ang-1 on protein kinase C–induced NF-κB activation. Whether Ang-1 inhibits VEGF- or TNF-induced leukocyte adhesion to ECs through A20 binding inhibitor of NF-κB activation-2 remains unclear.

In summary, we report here that in ECs Nur77 is significantly induced by Ang-1 and VEGF through the PI3 kinase/AKT, ERK1/2, and PKD/HDAC7/MEF2 pathways. The present study also demonstrates that the proinflammatory effects of VEGF on ECs are exaggerated when Nur77 expression is inhibited. Conversely, VEGF-induced EC activation is strongly attenuated when Nur77 expression is upregulated by adenoviruses. We also found that Nur77 mediates the suppressive effects of Ang-1 on NF-κB activation, adhesion molecule expression, and leukocyte adhesion to ECs. These results suggest that Nur77 functions as a negative feedback inhibitor of NF-κB and adhesion molecule expression in ECs.

Supplementary Material

supplemental materials

Acknowledgments

Sources of Funding

This work was supported by grants from the Canadian Institute of Health Research (S.N.A. Hussain) and National Institutes of Health grants 7R01HL103869 (to J. Sun) and 5R01HL080611 (to Z-G.J.).

Footnotes

Disclosures

None.

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Supplementary Materials

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