Monocyte adhesion constitutes a critical event for the initiation of atherosclerosis; 17
however, the molecular mechanisms that orchestrate monocyte-endothelial cell interactions are incompletely understood. In the present study, we report a previously unrecognized role for the nuclear receptor NOR1 to serve as a transcriptional regulator of adhesion molecule expression and monocyte recruitment during atherosclerosis. In endothelial cells, NOR1 is rapidly induced by inflammatory stimuli via an NF-κB-dependent trans
activation of the NOR1 promoter. Loss and gain-of-function studies establish that NOR1 positively regulates VCAM-1 and ICAM-1 expression in endothelial cells leading to increased monocyte adhesion. Consistent with the key role of NOR1 to promote monocyte adhesion to the endothelium, our studies further demonstrate that NOR1 deficiency results in decreased atherosclerosis development and macrophage recruitment in apoE-deficient mice.
Consistent with recent studies, we identified abundant NOR1 expression in endothelial cells12
as well as in cells of the subendothelial space likely representing macrophages and SMC. 14, 18-20
However, the transcriptional mechanisms governing inducible NOR1 expression in endothelial cells remain elusive. Our data provides evidence for an NF-κB-dependent induction of NOR1 transcription during endothelial cell inflammation. Inhibition of NF-κB signaling in endothelial cells repressed inducible NOR1 expression in response to inflammatory stimulation. Using site-directed mutagenesis and ChIP assays, we identified two functional NF-κB sites in the NOR1 promoter, to which p65 is recruited during inflammatory activation of endothelial cells. Earlier studies have demonstrated that NOR1 expression in endothelial cells is highly induced by mitogens through a cAMP response element binding protein (CREB)-dependent activation of the proximal region of NOR1 promoter. 16
Furthermore, hypoxia has recently been reported to induce NOR1 in endothelial cells through a mechanism involving the hypoxia-inducible factor (HIF) family of transcription factors. 21
These studies, in concert with our data characterizing NOR1 as a NF-κB target gene in endothelial cells, point to distinct transcriptional mechanisms regulating the rapid NOR1 induction in response to various environmental cues.
Compared with the well-studied early-response genes encoding proteins of the AP-1 complex, little is known about the physiological function of NOR1 and its regulated target genes, yet the high degree of conservation points to an important role in the control of gene expression. A previous study has provided initial evidence to support a functional role of NOR1 in endothelial cells by demonstrating that NOR1 regulates growth of this cell type. 16
The data presented here extends these findings and points to an unsuspected function of NOR1 to serve as a positive regulator of monocyte adhesion. In experiments using adenoviral-mediated overexpression, NOR1 induced VCAM-1 and ICAM-1 expression resulting in increased monocyte adhesion. The observation that this inducible adhesion was abolished when VCAM-1 and ICAM-1 function were blocked, suggests that the induction of both adhesion molecules by NOR1 constitutes a primary mechanism by which NOR1 induces monocyte adhesion. Conversely, the inducible expression of both adhesion molecules in response to inflammatory activation was attenuated in NOR1-deficient endothelial cells. Furthermore, consistent with these findings, TNFα-induced monocyte adhesion to HUVEC transfected with NOR1 siRNA or to the endothelium of NOR-deficient mice was altered ex vivo
, suggesting that vascular NOR1 expression is not only sufficient but also required for monocyte adhesion.
An intriguing question that arises from the observation that NOR1 induces the expression of VCAM-1 and ICAM-1 relates to the mechanisms by which NOR1 positively regulates these genes. Initial sequence analysis identified putative NBRE consensus sites in both the VCAM-1 and ICAM-1 promoters. Exemplified by the VCAM-1 promoter, our studies demonstrate that the molecular mechanisms underlying this novel function of NOR1 involve at least in part a direct trans
activation of the VCAM-1 promoter by NOR1. In response to inflammatory activation NOR1 is recruited to a canonical NBRE site in the VCAM-1 promoter. Moreover, the observation that NOR1-dependent VCAM-1 promoter trans
activation was attenuated upon mutation of this NBRE site confirms the functionality of this NBRE motif. However, the residual induction of the VCAM-1 promoter bearing a mutation of this NBRE site suggests that additional transcriptional mechanisms may regulate NOR1 expression. NOR1 has recently been shown to transactivate the inducible IκB kinase (IKKi/IKKepsilon) promoter, which phosphorylates IκBα and induces NF-κB activation. 22
Therefore, in addition to a direct trans
activation, NOR1 may function as a positive upstream regulator of NF-κB signaling and indirectly activate the VCAM-1 and ICAM-1 promoters. Alternatively, NOR1 deficiency may affect other transcriptional networks acting on these promoters, including for example the AP-1 complex. We have recently demonstrated that the combined deficiency of NOR1 and its sibling Nur77 decreases the expression of AP-1 transcription factors, 23
which may regulate adhesion molecule expression. 24
Clearly, the findings presented here provide justification for further investigating the transcriptional mechanisms by which NOR1 regulates endothelial cell gene expression and promotes monocyte adhesion.
The protein products of the VCAM-1 and ICAM-1 genes are well established to participate in atherogenesis by promoting macrophage accumulation in the arterial intima. 17
Consistent with this evidence and with the observed regulation of endothelial cell adhesion molecule expression by NOR1, we provide the first evidence that NOR1 deficiency decreases atherosclerosis in apoE-deficient mice. Considering that all three members of the NR4A receptor subfamily bind to an NBRE site, functional redundancy in certain cell types between Nur77 and NOR1 has been suggested. 25
However, NOR1-deficiency did not result in a compensatory upregulation of the siblings Nur77 and Nurr1 in endothelial cells (Supplemental Figure 1
) or in the aortae of NOR1−/−apoE−/− mice (Supplemental Figure V
). Therefore, the previously reported phenotypes in NOR1-deficient mice 10, 11, 15
in concert with the decreased atherosclerosis in NOR1−/−apoE−/− mice presented in this study, point to a function of NOR1 that is distinct and not compensated by Nur77 or Nurr1. As evidenced by the decreased accumulation of macrophages in the vascular wall of NOR1−/−apoE−/− mice, monocyte recruitment represents at least one plausible mechanism by which NOR1 acts atherogenic. However, it is possible if not likely that there are additional mechanisms by which NOR1 promotes atherosclerosis development. NOR1 induces neointimal proliferation of SMC 15
and inflammatory gene expression in macrophages 22
, which both could affect lesion development. Therefore, characterization of the cell-specific role of NOR1 in atherosclerosis will be necessary and will have to rely on tissue-specific gene targeting strategies. In conclusion, data presented here characterize the orphan nuclear receptor NOR1 as a novel positive regulator of monocyte adhesion by inducing VCAM-1 and ICAM-1 transcription. Continued investigation of the transcriptional networks regulated by NOR1 will provide new insights into how this orphan nuclear receptor participates in the development of vascular diseases.
NOVELTY AND SIGNIFICANCE
What is known?
- NR4A orphan nuclear receptors constitute a group of conserved transcription factors that function as early response genes.
- Members of the NR4A orphan nuclear receptor subfamily, including Nur77, NOR1, and Nurr1, are highly expressed in atherosclerotic lesions.
- NOR1 expression is inducible in endothelial cells.
What New Information Does This Article Contribute?
- NOR1 expression in endothelial cells is mediated via an NF-κB-dependent activation of its promoter.
- NOR1 induces monocyte adhesion through a transcriptional induction of the adhesion molecules VCAM-1 and ICAM-1 in endothelial cells
- NOR1 transactivates the VCAM-1 promoter in endothelial cells by binding to a canonical response element for NR4A receptors.
- Deficiency of NOR1 in apoE−/− mice reduces atherosclerosis formation and macrophage content of the lesion.
Monocyte adhesion and recruitment into the subendothelial space constitute critical first events for the initiation of atherosclerosis. In this study, we introduce a novel transcriptional mechanism mediating monocyte adhesion to the endothelium and subsequent atherosclerosis development. Although NR4A orphan nuclear receptors are highly conserved transcription factors expressed in atherosclerosis, surprisingly little is known about their function, and whether these receptors play a causal role in atherosclerosis has not been investigated. Expression of the NR4A receptor NOR1 was induced in endothelial cells by proatherogenic stimuli and mediated through an NF-κB-dependent transcriptional mechanism. Using loss- and gain-of-function approaches, we establish that NOR1 induces expression of the key adhesion molecules VCAM-1 and ICAM-1 resulting in increased monocyte adhesion. VCAM-1 constitutes a bona fide target gene for NOR1, which is activated by binding to a canonical response element. Consistent with this novel activity of NOR1, we demonstrate for the first time that deletion of this NR4A receptor reduces atherosclerotic lesion formation and monocyte recruitment into the arterial wall. Collectively, these experiments characterize a novel transcriptional cascade underlying atherogenesis and have important implications for our understanding of this disease.