Orphan nuclear receptor TR3/Nur77 improves wound healing by upregulating the expression of integrin β4

Abstract

Tissue repair/wound healing, in which angiogenesis plays an important role, is a critical step in many diseases including chronic wound, myocardial infarction, stroke, cancer, and inflammation. Recently, we were the first to report that orphan nuclear receptor TR3/Nur77 is a critical mediator of angiogenesis and its associated microvessel permeability. Tumor growth and angiogenesis induced by VEGF-A, histamine, and serotonin are almost completely inhibited in Nur77 knockout mice. However, it is not known whether TR3/Nur77 plays any roles in wound healing. In these studies, skin wound-healing assay was performed in 3 types of genetically modified mice having various Nur77 activities. We found that ectopic induction of Nur77 in endothelial cells of mice is sufficient to improve skin wound healing. Although skin wound healing in Nur77 knockout mice is comparable to the wild-type control mice, the process is significantly delayed in the EC-Nur77-DN mice, in which a dominant negative Nur77 mutant is inducibly and specifically expressed in mouse endothelial cells. By a loss-of-function assay, we elucidate a novel feed-forward signaling pathway, integrin β4 → PI3K → Akt → FAK, by which TR3 mediates HUVEC migration. Furthermore, TR3/Nur77 regulates the expression of integrin β4 by targeting its promoter activity. In conclusion, expression of TR3/Nur77 improves wound healing by targeting integrin β4. TR3/Nur77 is a potential candidate for proangiogenic therapy. The results further suggest that TR3/Nur77 is required for pathologic angiogenesis but not for developmental/physiologic angiogenesis and that Nur77 and its family members play a redundant role in normal skin wound healing.

Keywords: VEGF; angiogenesis.

Figure 1.

Induction of angiogenesis in the flank skin of the EC-Nur77-S mice. Macroscopic images of flank skin of the EC-Nur77-S mice (B, D, and F) and their respective wild-type control littermates (A, C, and E), in which tetracycline had been withdrawn from the drinking water for 6 days (A and B) or 3 months (C, D, E, and F). Four mice were used for each condition. Immunohistochemical staining of the sections obtained from the flank skin of E and F with an antibody against CD31 (G and H). Quantification of vessel number (right panel, n = 20 views, P < 0.001).

Figure 2.

Expression of Nur77 in wound tissues. A) Protein extracts from wound tissues (w) and their respective normal control tissues (c) from the same mouse were immunoblotted with antibodies against Nur77 (top panel) and β-actin to confirm protein equal loading (bottom panel). B) Quantitative measurement of intensity in A (4, 3, or 2, P < 0.001). C) RNA were isolated from wound tissues and their control tissues, and subjected to real-time RT-PCR. Data are expressed as average fold changes of animals on each day (n = 4, P < 0.001). D) Wound tissues were immunostained with antibodies against Nur77 and CD31. Data are representative of the results from 4 mice.

Figure 3.

Skin wound healing on mice with various TR3/Nur77 activities. A) Macroscopic images of skin wounds (representative of 10 mice/group). B) Average percentage of wound area related to day 0 (n = 10, P < 0.05). C) Re-epithelialization analysis (n = 4, P < 0.05). D) Dermal closure analysis (n = 4, P < 0.05). E) Immunostaining of wound tissues obtained from the transgenic mice with various TR3/Nur77 activity. F) Quantitative measurement of vessel number and vessel density from images in E (n = 20 views, P < 0.001).

Figure 4.

Regulation of integrin β4 by TR3/Nur77. A) HUVECs were transduced without (H) or with LacZ as control, TR3 full-length cDNA. RNA were isolated and subjected to real-time RT-PCR with integrin β4-specific primers (n = 2 for real-time PCR, P < 0.001). B) Cellular extracts from HUVECs that were transduced with LacZ as control, TR3 full-length cDNA, TR3 antisense DNA, or TR3 shRNA were immunoblotted with antibodies against integrin β4 (left top panel) and β-actin for protein equal loading control (left bottom panel). Quantitative measurement of integrin β4 expression (right panel). Experiments were repeated 3 times.

Figure 5.

Integrin β4 is a downstream target of TR3/Nur77 in migration but not proliferation. A) RNA isolated from HUVECs that were transduced with LacZ, as control, shITGB4-498, or shITGB4-5719 were subjected to real-time RT-PCR (n = 2, P < 0.001). B) HUVECs were transduced as indicated and subjected to proliferation assay (n = 6, P < 0.001). C) HUVECs were transduced as indicated and subjected to scratch wound migration assay (n = 10, P < 0.001). D) Cellular extracts from HUVECs that were transduced with negative control shRNA (lane 1), HA-tagged TR3 (lane 2), HA-TR3 + shITGB4-498 (lane 3), or HA-TR3 + ITGB4-5719 (lane 4) were immunoblotted with antibodies against HA to indicate the expression of TR3 (panel I), pAkt-S473 (panel II), pAkt-T308 (panel III), Akt (panel IV), PI3K (panel V), FAK (panel VI), pMAPK42/44 (panel VII), MAPK42/44 (panel VIII), and β-actin (panel IX) as protein equal loading control. Experiments were repeated 3 times.

Figure 6.

Transcriptional regulation of integrin β4 by TR3/Nur77. A) HUVECs were transduced as indicated. RNA were subjected to real-time RT-PCR with integrin β primers (n = 2 for real-time PCR, P < 0.001). B) Cellular extracts from HUVECs that were transduced as indicated were immunoblotted with antibodies against integrin β4 (left top panel) or β-actin as protein equal loading control (left bottom panel). Quantitative measurement of integrin β4 expression (right panel). C) HUVECs were infected with adenovirus expressing Lac Z as control and TR3, and transfected with integrin β4 promoter construct and internal luciferase construct. Data are presented as fold change of luciferase activity in cells expressing TR3 related to LacZ (n = 4, P < 0.001). Experiments were repeated 3 times.