The NR4A Orphan Nuclear Receptor NOR1 Is Induced by Platelet-derived Growth Factor and Mediates Vascular Smooth Muscle Cell Proliferation

Abstract

Members of the nuclear hormone receptor superfamily function as key transcriptional regulators of inflammation and proliferation in cardiovascular diseases. In addition to the ligand-dependent peroxisome proliferator-activated receptors and liver X receptors, this family of transcription factors includes a large number of orphan receptors, and their role in vascular diseases remains to be investigated. The neuron-derived orphan receptor-1 (NOR1) belongs to the ligand-independent NR4A subfamily, which has been implicated in cell proliferation, differentiation, and apoptosis. In this study, we demonstrate NOR1 expression in vascular smooth muscle cells (SMC) of human atherosclerotic lesions. In response to mitogenic stimulation with platelet-derived growth factor (PDGF), SMC rapidly express NOR1 through an ERK-MAPK-dependent signaling pathway. 5-Deletion analysis, site-directed mutagenesis, and transactivation experiments demonstrate that PDGF-induced NOR1 expression is mediated through a cAMPresponse element-binding protein (CREB)-dependent transactivation of the NOR1 promoter. Consequently, short interfering RNAmediated depletion of CREB abolished PDGF-induced NOR1 expression in SMC. Furthermore, PDGF induced Ser-133 phosphorylation of CREB and subsequent binding to the CRE sites of the endogenous NOR1 promoter. Functional analysis demonstrated that PDGF induces NOR1 transactivation of its consensus NGFI-B-response elements (NBRE) in SMC. We finally demonstrate that SMC isolated from NOR1-deficient mice exhibit decreased cell proliferation and characterize cyclin D1 and D2 as NOR1 target genes in SMC. These experiments indicate that PDGF-induced NOR1 transcription in SMC is mediated through CREB-dependent transactivation of the NOR1 promoter and further demonstrate that NOR1 functions as a key transcriptional regulator of SMC proliferation.Atherosclerosis, the subsequent development of occlusive vascular diseases, and the failure of treatment approaches such as postangioplasty restenosis involve several interrelated processes (1, 2). In addition to endothelial dysfunction and inflammation, proliferation of smooth muscle cells (SMC) 3 is considered to play a pivotal role in the pathogenesis of atherosclerosis and the failure of interventional approaches used to treat related occlusive vascular complications (2-4). With the evolving understanding of the mechanisms contributing to the development of vascular diseases, members of the nuclear hormone receptor superfamily of transcription factors have emerged as key transcriptional regulators of inflammation and cell proliferation (5, 6). Based on this evidence, elucidation of the molecular pathways utilized by nuclear receptors to regulate programs of gene expression is expected to facilitate the development of novel pharmacological approaches for the treatment of cardiovascular diseases. Nuclear receptors of the peroxisome proliferator-activated receptor (PPAR) and liver X receptor (LXR) subfamilies are expressed in SMC and inhibit their proliferation in response ...

NR4A orphan nuclear receptor family members, NR4A2 and NR4A3, regulate neutrophil number and survival

“…33,34 A role for NR4A2 and not NR4A3 in mCMP may reflect divergent roles for NR4A members in mice, or perhaps that NR4A3 can compensate for the absence of NR4A2. Consistent with this, distinct roles are seen in other models where NR4A3 has been shown to control the proliferation of human hepatocytes and vascular smooth muscle cells, 35,36 and NR4A2 overexpression in human synoviocytes promotes proliferation and survival. 37 Although we show that NR4A2/3 may play a role in neutrophil proliferation and homeostasis, we were unable to demonstrate a role for NR4A2/3 in the resolution of inflammation in zebrafish tail injury models in vivo.…”

Transcriptional Regulation of S Phase Kinase-associated Protein 2 by NR4A Orphan Nuclear Receptor NOR1 in Vascular Smooth Muscle Cells

“…Cell Culture-Primary murine aortic VSMC were isolated from wild-type Nor1 ϩ/ϩ and littermate Nor1 Ϫ/Ϫ mice as described recently (19). Murine VSMC passage three to eight were subjected to G 0 /G 1 phase synchronization by serum-deprivation in 0.4% FBS (Invitrogen) for at least 36 h followed by stimulation with 10 or 20% FBS as indicated.…”

“…The NBRE site in the Skp2 promoter located between Ϫ1443 and Ϫ1436 bp from the transcription initiation site (23) was mutated from CAGAAAGGACAAAGT to CAGAAAAAACAAAGT using the QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA). The CMV expression vectors overexpressing GFP or NOR1 have been described previously (19,20). The NBRE(8ϫ)-luciferase reporter construct was kindly provided by Dr. Ohkura (National Cancer Center Research Institute, Tokyo, Japan) (19).…”

“…The CMV expression vectors overexpressing GFP or NOR1 have been described previously (19,20). The NBRE(8ϫ)-luciferase reporter construct was kindly provided by Dr. Ohkura (National Cancer Center Research Institute, Tokyo, Japan) (19). Murine wild-type VSMC were cultured in six-well plates and transfected with the indicated Skp2 reporter construct in DMEM supplemented with 5% FBS using 5 l of Lipofectamine 2000 (Invitrogen) and 1.6 g of total transfected plasmid DNA per well.…”

Orphan Nuclear Receptor Nurr1 as a Potential Novel Marker for Progression in Human Prostate Cancer

“…Specific ligands for the NR4A family of transcription factors have not been identified, classifying them as orphan nuclear receptors (Bonta et al, 2006). Furthermore, gene expression of NR4A members is induced by various growth factors and cytokines in a variety of cell types (Pei et al, 2005;Nomiyama et al, 2006).…”