Orphan nuclear receptor TR3/Nur77 regulates VEGF-A-induced angiogenesis through its transcriptional activity

Abstract

Vascular endothelial growth factor (VEGF)-A has essential roles in vasculogenesis and angiogenesis, but the downstream steps and mechanisms by which human VEGF-A acts are incompletely understood. We report here that human VEGF-A exerts much of its angiogenic activity by up-regulating the expression of TR3 (mouse homologue Nur77), an immediate-early response gene and orphan nuclear receptor transcription factor previously implicated in tumor cell, lymphocyte, and neuronal growth and apoptosis. Overexpression of TR3 in human umbilical vein endothelial cells (HUVECs) resulted in VEGF-A-independent proliferation, survival, and induction of several cell cycle genes, whereas expression of antisense TR3 abrogated the response to VEGF-A in these assays and also inhibited tube formation. Nur77 was highly expressed in several types of VEGF-A-dependent pathological angiogenesis in vivo. Also, using a novel endothelial cell-selective retroviral targeting system, overexpression of Nur77 DNA potently induced angiogenesis in the absence of exogenous VEGF-A, whereas Nur77 antisense strongly inhibited VEGF-A-induced angiogenesis. B16F1 melanoma growth and angiogenesis were greatly inhibited in Nur77-/- mice. Mechanistic studies with TR3/Nur77 mutants revealed that TR3/Nur77 exerted most of its effects on cultured HUVECs and its pro-angiogenic effects in vivo, through its transactivation and DNA binding domains (i.e., through transcriptional activity).

Figure 1.

VEGF-A induces TR3 expression in cultured HUVECs. (A) Quantitative real-time RT-PCR to demonstrate TR3 and control GAPDH mRNA expression after stimulation with 10 ng/ml VEGF-A¹⁶⁵ at times indicated. (B) Immunoblot of TR3 protein expression in VEGF-A¹⁶⁵–stimulated HUVECs (top). Bottom shows MAPK protein loading control. (C) TR3 mRNA expression in HUVECs stimulated with 10 ng/ml VEGF-A¹⁶⁵, VEGF-A¹²⁰, placenta growth factor, platelet-derived growth factor (PDGF), or with 25 ng/ml bFGF over time as determined by quantitative real-time RT-PCR (n = 2).

Figure 2.

TR3 and Nur77 mutants. Expression of TR3/Nur77 sense, antisense, and mutant proteins in transfected HUVECs. (A) Schematic structure of TR3 and Nur77 genes and mutants constructed to lack TAD, DBD, or LBD domains. (B) Expression of TR3 protein in control HUVECs and in HUVECs transfected with TR3-S, TR3-AS, or LacZ cDNAs. TR3-S–transfected HUVECs expressed three- to fourfold more TR3 protein than untransfected or LacZ-transfected cells. Endogenous TR3 protein expression was strongly inhibited in cells transfected with TR3-AS. Bottom shows MAPK protein loading control. (C) Expression of Flag-TR3-ΔTAD, Flag-TR3-ΔDBD, and Flag-TR3-ΔLBD in HUVECs. (D) Subcellular localization of GFP-fused TR3 mutants in HUVECs.

Figure 3.

Assays measuring functional capabilities of cultured HUVECs transfected with TR3-S, TR3-AS, and TR3 mutant cDNAs, with or without VEGF-A¹⁶⁵ stimulation. (A) [³H]thymidine incorporation (reference ; n = 4). (B) Cell survival assay (n = 4). (C) Tube formation on Matrigel.

Figure 4.

TR3 regulation of cell cycle gene expression in HUVECs. Immunoblots of cell extracts from HUVECs transfected with LacZ, TR3-S, TR3-AS, and TR3 mutant DNAs, with or without VEGF-A¹⁶⁵ stimulation for indicated times. Actin expression serves as a protein loading control.

Figure 5.

Increased expression of Nur77 in VEGF-A–induced angiogenesis. (A) Angiogenic response in nude mouse ears 5 d after intradermal injection of Ad-VEGF-A¹⁶⁴ (left ear). Ad-LacZ was injected in right ear as a negative control (reference 15). (B) Fold activation of Nur77 mRNA over time in mouse ears after intradermal injection of Ad-VEGF-A¹⁶⁴ or Ad-LacZ as determined by quantitative real-time RT-PCR. (C) Immunoblots of Nur77 protein expression in uninjected mouse ears or in ears injected with Ad-LacZ or Ad-VEGF-A¹⁶⁴ (top). Bottom shows MAPK protein loading control. (D) Immunoblots of Nur77 protein expression (top) in healing skin punch biopsy wounds and in mesenteries of nude mice bearing TA3/St and MOT ascites tumors. Bottom shows MAPK protein loading controls.

Figure 6.

Nur77 and VEGF-A expression in Matrigel assays. (A) In situ hybridization performed on Matrigels containing SK-MEL/VEGF cells and PT67 cells packaging either Nur77-S (left) or Nur77-AS–expressing retrovirus (right) cDNAs. Probes were Nur77-AS (A and C), Nur77-S (B and D), and VEGF-A¹⁶⁵-AS (E and F). (B) In situ hybridization demonstrating Nur77 expression in newly formed blood vessels in Matrigels containing SK-MEL/VEGF cells hybridized with Nur77-AS (A) or Nur77-S (B) probes. v, vessel.

Figure 7.

Angiogenic response induced by Nur77-S or Nur77-AS in Matrigel assays in vivo. (A) Macroscopic (top) and CD-31–stained microscopic (bottom) images of the angiogenic response induced 3 d after implantation of Matrigels with indicated contents of VEGF-A¹⁶⁵–secreting SKMEL/VEGF cells and PT67 cells packaging LacZ, Nur77-S, or Nur77-AS. (B) Quantitative measurement of intravascular plasma volumes (μl/g) in Matrigels containing indicated cell mixtures at 1 and 3 d after implantation as determined by accumulation of Evan's blue dye administered i.v. 5 min before killing.

Figure 8.

Growth of B16F1 melanoma is inhibited in Nur77^−/− mice. (A) Growth of B16F1 melanomas in wild-type and Nur77^−/− mice (mean + SD, n = 4). (B, a) Gross appearance of tumors at 11 d. Histology (b and c) and CD31 immunohistochemistry (d and e) of B16F1 melanomas growing in wild-type (b and d) and Nur77^−/− (c and e) mice at day 11. Tumors in wild-type mice have numerous, large mother vessels (v), whereas vessels in Nur77^−/− mice are much less numerous and considerably smaller in size. N, necrosis; bar, 100 μm.

Figure 9.

Nur77 functions downstream of VEGFR-2/KDR in Matrigel assays in vivo. (A) SU1498, a VEGFR-2/KDR kinase inhibitor, inhibited angiogenesis induced by VEGF-A¹⁶⁵–expressing SKMEL/VEGF-A cells (lane 1) but not that induced by Nur77 (lane 2). (B) Quantitative measurement of intravascular plasma volumes (μl/g) in Matrigels containing indicated cell mixtures ± SU1498 as determined by accumulation of Evan's blue dye administered i.v. 5 min before killing.

Figure 10.

The transactivation and DNA binding domains of Nur77 are required to induce angiogenesis in Matrigel assays in vivo. Quantitative measurement of intravascular plasma volumes (μl/g) in 3 d Matrigels containing indicated cell mixtures as determined by accumulation of Evan's blue dye administered i.v. 5 min before killing.