Antithrombotic Effects of Nur77 and Nor1 Are Mediated Through Upregulating Thrombomodulin Expression in Endothelial Cells

Abstract

Objective Thrombomodulin (TM) is highly expressed on the lumenal surface of vascular endothelial cells (ECs) and possesses potent anticoagulant, anti-fibrinolytic, and anti-inflammatory activities in the vessel wall. However, the regulation of TM expression in ECs remains largely unknown. Approaches and Results In the present study, we characterized nuclear receptor 4A (NR4A) family as a novel regulator of TM expression in vascular ECs. We demonstrated that both NR4A receptor Nur77 and Nor1 robustly increase TM mRNA and protein levels in human vascular ECs and in mouse liver tissues after adenovirus-mediated transduction of Nur77 and Nor1 cDNAs. Moreover, Nur77 deficiency and knockdown of Nur77 and Nor1 expression markedly attenuated the basal and VEGF165-stimulated TM expression. Mechanistically, we found that Nur77 and Nor1 increase TM expression by acting through two different mechanisms. We show that Nur77 barely affects TM promoter activity, but significantly increases TM mRNA stability, while Nor1 enhances TM expression mainly through induction of Kruppel-like factor 2 and 4 in vascular ECs. Further, we demonstrate that both Nur77 and Nor1 significantly increase protein C activity and inhibit tumor necrosis factor alpha (TNF-α)-induced prothrombotic effects in human ECs. Deficiency of Nur77 increases susceptibility to arterial thrombosis, while enhanced expression of Nur77 and Nor1 protects mice from arterial thrombus formation. Conclusions Our results identified NR4A receptors as novel regulators of TM expression and function in vascular ECs and provided a proof-of-concept demonstration that targeted increasing expression of Nur77 and Nor1 in the vascular endothelium might represent a novel therapeutic approach for the treatment of thrombotic disorders.

A Nuclear Attack on Thrombosis and Inflammation

“…NR4A receptors are widely expressed in many cells beyond the endothelium, and they have diverse properties that may complicate interpretation of the underlying mechanisms and responsible cell(s). 4 For Nur77, even though it is upregulated in injured endothelial cells, smooth muscle cells and monocytes/macrophages, there is overwhelming in vitro and in vivo evidence that supports Yang et al's 3 findings that Nur77 is vasculoprotective. By directly upregulating KLF-2, activating target genes by binding to their promoter(s), and enhancing expression of the NF-κB inhibitor IκBα 18 Nur77 induces an anti-inflammatory endothelial cell phenotype, 19 dampens vascular smooth muscle cell proliferation and migration, and suppresses leukocyte activation and infiltration.…”

“…Yang et al 3 have taken a different approach to augment endothelial thrombomodulin and limit disease, particularly focusing on thrombosis. Going nuclear, they examined the role of 2 transcription factors, Nur77 and Nor1, members of the family of nuclear orphan NR4A receptors.…”

“…These are constitutively active, early response genes that encode transcription factors that have multiple effects, but are implicated in modulating vascular function. 4 Yang et al 3 showed that gene delivery of either Nur77 or Nor1, upregulated thrombomodulin on the surface of HUVECs (human umbilical vein endothelial cells), but interestingly, via distinctly different mechanisms. Nor1 caused an increase in thrombomodulin gene transcription by enhancing expression of Kruppel-like factors (KLF)-2 and KLF-4.…”

“…15 KLF-2 is also vasculoprotective by inducing expression of endothelial nitric oxide synthase, and suppressing PAI-1, von Willebrand factor, and tissue factor, 16,17 the latter also observed with Nor1 and Nur77. 3 Clearly, Nor1 and Nur77 are not the only nuclear receptors that upregulate thrombomodulin, able to switch the vasculature to an anti-inflammatory/antithrombotic phenotype. However, Yang et al 3 are the first to convincingly…”

“…3 Clearly, Nor1 and Nur77 are not the only nuclear receptors that upregulate thrombomodulin, able to switch the vasculature to an anti-inflammatory/antithrombotic phenotype. However, Yang et al 3 are the first to convincingly…”