Orphan nuclear receptor TR3/Nur77 differentially regulates the expression of integrins in angiogenesis

Abstract

Pathological angiogenesis is a hallmark of many diseases. Previously, we reported that orphan nuclear receptor TR3/Nur77 (human homolog, Nur77, mouse homolog) is a critical mediator of angiogenesis to regulate tumor growth and skin wound healing via down-regulating the expression of the junctional proteins and integrin β4. However, the molecular mechanism, by which TR3/Nur77 regulated angiogenesis, was still not completely understood. In this report by analyzing the integrin expression profile in endothelial cells, we found that the TR3/Nur77 expression highly increased the expression of integrins α1 and β5, decreased the expression of integrins α2 and β3, but had some or no effect on the expression of integrins αv, α3, α4, α5, α6, β1 and β7. In the angiogenic responses mediated by TR3/Nur77, integrin α1 regulated endothelial cell proliferation and adhesion, but not migration. Integrin β5 shRNA inhibited cell migration, but increased proliferation and adhesion. Integrin α2 regulated all of the endothelial cell proliferation, migration and adhesion. However, integrin β3 did not play any role in endothelial cell proliferation, migration and adhesion. TR3/Nur77 regulated the transcription of integrins α1, α2, β3 and β5, via various amino acid fragments within its transactivation domain and DNA binding domain. Furthermore, TR3/Nur77 regulated the integrin α1 promoter activity by directly interacting with a novel DNA element within the integrin α1 promoter. These studies furthered our understanding of the molecular mechanism by which TR3/Nur77 regulated angiogenesis, and supported our previous finding that TR3/Nur77 was an excellent therapeutic target for pathological angiogenesis. Therefore, targeting TR3/Nur77 inhibits several signaling pathways that are activated by various angiogenic factors.

Keywords: Angiogenesis; Integrin; Migration; Proliferation; Promoter; TR3/Nur77.

Figure 1.. TR3/Nur77 differentially regulated the expression of integrins.

A. RNA isolated from the HUVECs that were transduced Lac Z as a control, and TR3/Nur77 were analyzed by Realtime RT-PCR with specific primers as indicated (n=4, *p < 0.05, **p < 0.01). Data represent 3 independent experiments. B-D. Proteins isolated from the HUVECs that were transduced with Lac Z as a control, TR3 cDNA (TR3) or TR3 antisense DNA (TR3-AS) (B), stimulated with or without VEGF (C) or histamine (D) were analyzed by immunoblotting with the antibodies against integrins α1, α2, β3 and β5. Data represent 3 independent experiments. Gel densities were measured and plotted (n=3, *p < 0.05, **p < 0.01, NS: no significance).

Figure 2.. Effect of integrins on the angiogenic responses induced by TR3/Nur77.

The HUVECs expressing TR3/Nur77 were transduced with shGFP as a control, shITGA1–1213 and shITGA1–596 (Panel I), shGFP as a control and shITGB5 (panel II), Lac Z as a control and ITGA2 (panel III), and Lac Z as a control and ITGB3 (panel IV). Cellular extracts were subjected to immunoblotting analysis with the antibodies as indicated, and a Flag antibody indicating the equal expression of TR3/Nur77. Data represent 3 independent experiments. Gel densities were measured and plotted (A). Cells were subjected to proliferation assay (B), migration assay (C) and adhesion assay (D). Data represent 3 independent experiments (n=3, *p < 0.05, **p < 0.01, ***, p < 0.001, NS: no significance).

Figure 3.. Effect of integrins on TR3/Nur77-activated signaling molecules.

Cellular extracts from the HUVECs expressing TR3/Nur77 that were transduced with shGFP as a control, shITGA1–1213 and shITGA1–596 (A), Lac Z as a control and shITGB5 (B), Lac Z as a control and ITGA2 cDNA (C), Lac Z as a control and ITGB3(D) were subjected to immunoblotting analysis with the antibodies as indicated. Data represent 3 independent experiments. Gel densities were measured and plotted (n=3, *p < 0.05, **p < 0.01, NS: no significance).

Figure 4.. Various TR3/Nur77 domains were required for its regulation of integrins α1, α2, β3 and β5.

A. Schematic structure of TR3 and Nur77 genes and mutants constructed to lack TAD, DBD, or LBD domains. B. Cellular extracts isolated from the HUVECs that were transduced with Lac Z as a control, TR3, TR3ΔTAD, TR3ΔDBD, or TR3ΔLBD were subjected Immunoblotting analysis with the antibodies against Flag, integrins α1, α2, β3 and β5, and β-actin as a protein equal loading control. Data represent 3 independent experiments. Gel densities were measured and plotted (n=3, *p < 0.05, **p < 0.01, NS: no significance). C. Schematic structure of TR3 mutants in the transactivation domain. D and E. Cellular extracts isolated from the HUVECs that were transduced with Lac Z as a control, TR3 cDNA, mutants as indicated were subjected Immunoblotting analysis with the antibodies against Flag, integrins α1, α2, β3 and β5, and β-actin as a protein equal loading control. Data represent 3 independent experiments. Gel densities were measured and plotted (n=3, *p < 0.05, **p < 0.01, NS: no significance).

Figure 4.. Various TR3/Nur77 domains were required for its regulation of integrins α1, α2, β3 and β5.

A. Schematic structure of TR3 and Nur77 genes and mutants constructed to lack TAD, DBD, or LBD domains. B. Cellular extracts isolated from the HUVECs that were transduced with Lac Z as a control, TR3, TR3ΔTAD, TR3ΔDBD, or TR3ΔLBD were subjected Immunoblotting analysis with the antibodies against Flag, integrins α1, α2, β3 and β5, and β-actin as a protein equal loading control. Data represent 3 independent experiments. Gel densities were measured and plotted (n=3, *p < 0.05, **p < 0.01, NS: no significance). C. Schematic structure of TR3 mutants in the transactivation domain. D and E. Cellular extracts isolated from the HUVECs that were transduced with Lac Z as a control, TR3 cDNA, mutants as indicated were subjected Immunoblotting analysis with the antibodies against Flag, integrins α1, α2, β3 and β5, and β-actin as a protein equal loading control. Data represent 3 independent experiments. Gel densities were measured and plotted (n=3, *p < 0.05, **p < 0.01, NS: no significance).

Figure 5.. TR3/Nur77 regulated the transcriptions of integrins α1, α2, β3 and β5.

A. RNA isolated from the HUVECs that were transduced with Lac Z as a control, TR3, TR3ΔTAD, TR3ΔDBD, or TR3ΔLBD were subjected to Realtime RT-PCR with the specific primers of integrins α1, α2, β3 and β5. B. The HUVECs that were transduced with Lac Z as a control, and TR3/Nur77 were treated with or without actinomycin-D. RNA were isolated and subjected to Realtime RT-PCR with specific primers of integrins α1, α2, β3 and β5. C. HUVECs were transduced with promoter luciferase constructs as indicated, and then infected with Lac Z and TR3/Nur77 for the luciferase assay (n=4, *p < 0.05, **p < 0.01, NS: no significance). Data represent of 3 independent experiments.

Figure 6.. TR3/Nur77 regulated integrin α1 promoter activity by directly interacting with a novel DNA element in the integrin α1 promoter.

A. The relative luciferase activities of integrin α1 promoter mutants in the HUVECs transduced with Lac Z as a control and TR3/Nur77 (n=4, *p < 0.05, NS: no significance). B. CHIP samples as indicated were subjected to immunoblotting with a TR3/Nur77 antibody (panel I), and to PCR with primers of integrin α1 (panel II), TSHβp (panel III) and endolas3p (panel IV). Data represent of 3 independent experiments.