TR3 Orphan Receptor Is Expressed in Vascular Endothelial Cells and Mediates Cell Cycle Arrest
Objective— Endothelial cells play a pivotal role in vascular homeostasis. In this study, we investigated the function of the nerve growth factor–induced protein-B (NGFI-B) subfamily of nuclear receptors comprising the TR3 orphan receptor (TR3), mitogen-induced nuclear orphan receptor (MINOR), and nuclear orphan receptor of T cells (NOT) in endothelial cells.
Methods and Results— The mRNA expression of TR3, MINOR, and NOT in atherosclerotic lesions was assessed in human vascular specimens. Each of these factors is expressed in smooth muscle cells, as described before, and in subsets of endothelial cells, implicating that they might affect endothelial cell function. Adenoviral overexpression of TR3 in cultured endothelial cells resulted in decreased [3H]thymidine incorporation, whereas a dominant-negative TR3 variant that inhibits the activity of endogenous TR3-like factors enhanced DNA synthesis. TR3 interfered with progression of the cell cycle by upregulating p27Kip1 and downregulating cyclin A, whereas expression levels of a number of other cell cycle–associated proteins remained unchanged.
Conclusions— These findings demonstrate that TR3 is a modulator of endothelial cell proliferation and arrests endothelial cells in the G1 phase of the cell cycle by influencing cell cycle protein levels. We hypothesize involvement of TR3 in the maintenance of integrity of the vascular endothelium.
Under physiologic conditions, the vascular endothelium serves as a physical barrier between the blood compartment and the vessel wall and remains in a quiescent, nonproliferative state.1 Endothelial cell (EC) proliferation is, however, induced in pathologic situations, eg, by balloon angioplasty–mediated disruption of the monolayer. The exact molecular mechanisms regulating EC proliferation under pathophysiologic conditions are poorly understood. EC cycle progression requires activation of distinct cyclin-dependent kinases (Cdk’s) through their association with regulatory subunits called cyclins during different phases of the cell cycle.2 The Cdk inhibitors p21Cip1, p27Kip1, and p15/p15INK negatively regulate this process by inhibiting cyclin/Cdk activity.3 In ECs and a variety of other cells, the Cdk inhibitor p27Kip1 induces G1 arrest of the cell cycle and inhibits growth and migration.3–5⇓⇓
The nerve growth factor–induced protein-B (NGFI-B) subfamily6 of nuclear receptors (NR4A) belongs to the steroid/thyroid hormone superfamily of transcription factors and comprises TR3 orphan receptor (TR3), mitogen-induced nuclear orphan receptor (MINOR), and nuclear orphan receptor of T cells (NOT).7 Like other members of the nuclear receptor superfamily, the NGFI-B–like factors contain a central DNA-binding domain, comprising two zinc fingers, that recognizes response elements in the promoters of specific target genes. The amino-terminal domain mediates transactivation, and the carboxy-terminal domain is involved in (hetero)dimerization and ligand binding. At present, the ligands for TR3, MINOR, and NOT are unknown, qualifying these transcription factors as orphan receptors.8 Several lines of evidence indicate that TR3 and MINOR are involved in T-cell apoptosis. Antisense oligonucleotides directed against TR3 prevent apoptosis in cultured T cells and prostate and lung carcinoma cells.9–11⇓⇓ Furthermore, it has been shown that overexpression of TR3 or MINOR in developing T cells of transgenic mice results in massive apoptosis of thymocytes and reduced levels of peripheral T cells, whereas a dominant-negative variant of TR3 inhibits T-cell apoptosis.12,13⇓ Recently, it has been shown that in response to apoptotic stimuli, TR3 can translocate from the nucleus to the mitochondria to promote cytochrome c release and apoptosis. This proapop-totic effect of TR3 was shown to be independent of its transactivation activity.14
We have recently shown that TR3 inhibits serum-stimulated proliferation of vascular smooth muscle cells (SMCs). Moreover, overexpression of TR3 specifically in arterial SMCs of transgenic mice resulted in reduced intima formation in the carotid artery ligation model.15 Downregulation of p27Kip1 is crucial for cell cycle progression, and we showed that TR3 inhibits progression of the cell cycle in SMCs through the regulation of this important Cdk inhibitor.15
In the current study, we show expression of the NGFI-B family members in ECs in human vascular tissue. We demonstrate that TR3 regulates p27Kip1 and cyclin A protein levels in cultured ECs, consistent with inhibition of DNA synthesis and arrest of the cell cycle at G1. We discuss the physiologic relevance of TR3-like factors in protection against excessive vascular EC proliferation.
Immunohistochemistry and Radioactive In Situ Hybridization
Human tissue samples were obtained with informed consent from organ donors, according to protocols approved by the Medical Ethics Committee of the Academic Medical Center, Amsterdam. The specimens were paraffin embedded, sectioned, and mounted on glass slides (Superfrost Plus, Emergo). Vascular specimens were characterized by immunohistochemistry to establish the stage of disease according to the American Heart Association classification.16 ECs in the human vascular specimens were identified by Ulex europaeus lectin (1:50 dilution), which was detected with a rabbit anti–Ulex lectin antibody conjugated with horseradish peroxidase at a 1:50 dilution (Dako).
Radioactive in situ hybridization assays were performed as described.17 The following riboprobes were synthesized for in situ hybridization: TR3, GenBank No. L13740, bp 1221 to 1905; MINOR, GenBank No. U12767, bp 1435 to 2172; and NOT, GenBank No. X75918, bp 119 to 1003. Matching sense riboprobes were assayed for each gene and were shown to give neither background nor a nonspecific signal. The sections were exposed for 4 to 8 weeks.
HUVEC Isolation and Cell Culture
Human umbilical vein ECs (HUVECs) were isolated as described18 and cultured on fibronectin-coated, tissue-culture plates (Nunc) in “growth medium” containing medium-199 (GIBCO-BRL) supplemented with 20% (vol/vol) fetal bovine serum, 2 mmol/L l-glutamine, 50 μg/mL heparin (Sigma), 12.5 μg/mL EC growth supplement (ECGS; Sigma), and 100 U/mL penicillin/streptomycin (Gibco-BRL). Cells at passage level 1 to 2 were applied and plated on fibronectin-coated, 6-wells plates (Nunc) in growth medium.
Replication-defective adenoviruses expressing cDNAs under control of the cytomegalovirus promoter were purified by CsCl gradient centrifugation, and viral titers were determined by standard plaque assays.15 Full-length TR3 cDNA (bp 1 to 2375; GenBank X97226) was inserted into the TR3 virus, whereas mock virus did not contain the cDNA sequence. The ΔTA virus lacked bp 178 to 690 from the full-length cDNA and consequently encoded a TR3 variant without the transactivation domain.19 ΔTA exhibits normal DNA binding without mediating transcriptional activation and acts as a dominant-negative inhibitor for all 3 subfamily members:TR3, MINOR, and NOT.
HUVECs were seeded in 24-well plates and reached 70% to 80% confluence after 24 hours. Cells were infected for 2 hours with mock, ΔTA, or TR3 adenovirus at 3×108 plaque-forming units per milliliter in modified growth medium containing 5 μg/mL ECGS (instead of 12.5 μg/mL) and were allowed to recover in full growth medium for 2 hours.15 Subsequently, the cells were maintained for 60 hours in modified growth medium containing 5 μg/mL ECGS. The HUVECs were stimulated with growth medium, and after 8 hours, 0.5 μCi/mL [methyl-3H]thymidine (Amersham) was added for 18 hours. Incorporated radioactivity was determined as described.15 The experiments were performed in triplicate and repeated 3 times.
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was performed with cell lysates, and proteins were transferred to nitrocellulose-Protran (Schleicher and Schuell). TR3 was detected by Western blotting with an antiserum directed against Nur77 (M-210, 1:1000), Jab1 was detected with antiserum sc-6271 (1:200; Santa Cruz Biotechnology), pro–caspase-3 protein was assayed with an antiserum from BD Biosciences (1:1500), and cleaved caspase-3 was shown with an antiserum (D175; 1:1000) from Cell Signaling. Monoclonal antibodies were used to detect Cdk1/Cdc2 (1:2500), Cdk2 (1:2500), cyclin A (1:250), cyclin B (1:1000), cyclin D3 (1:1000), proliferating cell nuclear antigen (1:5000), p21WAF1/Cip1 (1:500), p27Kip1 (1:1000; BD Biosciences), and α-tubulin (1:2500; Cedar Lane). Proteins were visualized by enhanced chemiluminescence detection (Amersham Pharmacia Biotech). We analyzed the data quantitatively by applying a commercially available system (Lumi-LightPLUS, Roche, and the Lumi-Imager, Boehringer Mannheim).
Flow Cytometric Analysis of Cell Cycle Distribution
Cells were plated in 6-well plates and infected as described earlier to assess [3H]thymidine incorporation. After recovery of the cells for 2 hours in growth medium, the cells were incubated for 60 hours in growth medium with low (5 μg/mL) ECGS. Subsequently, the HUVECs were split 1:2 and placed overnight in growth medium. Bromodeoxyuridine (BrdU) was administered from a 100× stock solution to a final concentration of 10 μmol/L. After 4 hours, the cells were harvested, fixed in 70% ethanol in phosphate-buffered saline, and stored at −20°C. Ethanol-fixed cells were centrifuged (1 minute, 2200 rpm), resuspended in 1 mL pepsin solution (0.4 mg/mL 0.1N HCl), and incubated for 30 minutes at room temperature. Subsequently, the DNA was degraded by a 30-minute incubation at 37°C in 1 mL 2N HCl. After being washed with PBTb (phosphate-buffered saline, 0.05% [vol/vol] Tween-20, and 20 mg/mL bovine serum albumin [Sigma]), the cells were resuspended in 0.1 mL rat anti-BrdU (Harlan Sera-Laboratory Ltd, diluted 1:100 in PBTb) and incubated for 30 minutes at room temperature. After being washed with PBTg (phosphate-buffered saline, 0.05% [vol/vol] Tween-20, and 1% [vol/vol] normal goat serum [Dako]), the cells were resuspended with 0.1 mL fluorescein isothiocyanate (FITC)–conjugated goat anti-rat immunoglobulin G (Jackson ImmunoResearch Laboratories Inc; diluted 1:100 in PBTg) and incubated in the dark for 30 minutes at room temperature. Propidium iodide and ethanol were added to an end concentration of 1 μg/mL and 30% (vol/vol), respectively. Samples were syringed through a 21-gauge needle to reduce cell aggregation before flow cytometry (FACScan cytometer, Becton Dickinson).
Cells were plated in 6-well plates and infected and cultured as described previously for the flow cytometric analyses. Total RNA was extracted, and reverse transcription (RT) of 1 μg total RNA was performed with 0.5 μg (dT)12 to 18 primer with the use of SuperScript II (Gibco-BRL). Real-time RT–polymerase chain reaction (PCR) was performed with the use of the FastStart DNA Master SYBR green I kit (Roche) in the LightCycler System (Roche). Primers were as follows: for TR3, (forward) 5′-GTTCTCTGGAGGTCATC-CGCAAG -3′ and (reverse) 5′-GCAGGGACCTTGAGAAGGCCA-3′; for MINOR, (forward) 5′-CCATTACAACAGGAACCTTC-3′ and (reverse) 5′-GAGATCAGTAAATCCCGGAA-3′, and for NOT, (forward) 5′-TATTCCAGGTTCCAGGCGAA-3′ and (reverse) 5′-GCTAATCGAAGGACAAACAG-3′. As a control for equal amounts of first-strand cDNA in the different samples, we determined HPRT mRNA levels, with the primers (forward) 5′-TAATTATGGACAGGACTGAACG-5′ and (reverse) 5′-CACAA-TCAAGACATTCTTTCCAG-3′.
TR3, MINOR, and NOT Are Expressed in ECs In Vivo
We have previously shown that TR3, MINOR, and NOT are expressed in intimal SMCs of human atherosclerotic lesions, whereas no expression was observed in normal medial SMCs.15 To determine whether these genes are expressed in arterial ECs, we performed radioactive in situ hybridizations with probes specific for TR3, MINOR, or NOT. We analyzed the expression of TR3-like factors in 19 different vascular specimen derived from individuals ranging in age from 17 to 65 years. The complexity of the lesions varied from apparently normal type I to type V, according to the classification of the American Heart Association.16 In apparently healthy endothelium, we observed patchy, noncontinuous expression of TR3, MINOR, and NOT (Figure 1a–1c, respectively). Furthermore, expression of TR3-like factors was present in the vasa vasorum of normal and atherosclerotic vascular tissue (Figure 1d–1f). TR3-like factors are prominently expressed in microvascular ECs as well as in some cells surrounding the capillaries, which are probably adventitial fibroblasts (indicated by arrows). As a control, in situ hybridizations were performed with sense riboprobes (Figure 1 g–i). Next we show expression of TR3, MINOR and NOT in type II lesions (Figure 2). An overview of the specimens after hematoxylin/eosin staining is shown (Figure 2 a, 2d, and 2g), and the corresponding EC-specific staining illustrates the presence of an intact endothelial layer (Figure 2b, 2e, and 2h). In situ hybridizations were performed on consecutive sections and revealed mRNA expression of TR3, MINOR, or NOT in all ECs, as well as in some underlying intimal SMCs (Figure 2 c, 2f, and 2i). These experiments revealed that ECs overlying atherosclerotic lesions express mRNA encoding each of these transcription factors.
Adenoviral Infection of HUVECs to Overexpress TR3 and the Dominant-Negative Variant of TR3 (ΔTA)
The mRNA expression studies revealed relatively high expression of TR3 in ECs in atherosclerotic lesions, which prompted us to assess the functional activity of TR3 in cultured HUVECs. We applied adenoviruses expressing either TR3 or a dominant-negative variant of TR3, denoted ΔTA.13,15⇓ This dominant-negative variant lacks the amino-terminal transactivation domain and inhibits endogenous transcriptional activity of all 3 family members, which is important because functional redundancy has been described for members of this subfamily.20
We first assayed the expression of TR3, MINOR, and NOT in HUVECs under the conditions used in our experiments. Confluent, quiescent HUVEC cultures were exposed to complete growth medium, and the expression of TR3, MINOR, and NOT was monitored by real-time RT-PCR (Figure 3A). All 3 nuclear receptors are transiently induced in serum-stimulated HUVECs, with optimal expression 2 to 4 hours after stimulation.
Because TR3 has been associated with apoptosis, HUVECs were infected with mock, ΔTA, or TR3 adenovirus, and the extent of caspase-3 activation was determined by Western blotting with an antiserum specific for cleaved caspase-3 (19 kDa). Clearly, no activation of caspase-3 was observed in the infected cells, whereas treatment with the apoptotic agent staurosporine for 2 hours resulted in generation of a substantial amount of active caspase-3 (Figure 3). Inactive pro–caspase-3 (36 kDa) was present in all cells, with enhanced expression in ΔTA-infected cells. Pro–caspase-3 was relatively low in staurosporine-treated cells, which was correlated with less total protein loaded in this lane, as illustrated by α-tubulin expression levels. Given that only the presence of cleaved caspase-3 is indicative for the induction of apoptosis, we concluded that overexpression of TR3 or ΔTA does not affect programmed cell death in ECs.
To determine whether TR3 is involved in cellular proliferation, we performed [3H]thymidine incorporation experiments. Overexpression of TR3 resulted in a 2.6-fold lower [3H]thymidine incorporation, and, consistent with these data, ΔTA-infected HUVECs showed a 1.5-fold increase in DNA synthesis (P<0.001; Figure 4A). Moreover, TR3 blocks the cell cycle in ECs in the G1 phase, as shown by fluorescence-activated cell sorting analysis of propidium iodide–stained cells (Figure 4B). The number of cells in the S phase was determined by BrdU staining followed by fluorescence-activated cell sorting analysis and was shown to be 2.1-fold lower in the TR3-infected cells compared with the mock-infected cells (13.1% and 28.1% of the counted cells, respectively). To delineate the mechanism of this cell cycle arrest, we determined the expression level of cell cycle proteins by Western blotting, followed by quantitative luminometry (Figure 5). No changes were observed in protein expression levels of Cdk1, Cdk2, cyclin B, cyclin D3, proliferating cell nuclear antigen, and p21Cip1 (Figure 5A). Expression of the Cdk inhibitor p27Kip1 was upregulated 2-fold by TR3 and downregulated 2.2-fold by ΔTA, whereas cyclin A was regulated inversely: 1.9-fold downregulation by TR3 and 2.6-fold upregulation by ΔTA (Figure 5B and 5C). It has been described that p27Kip1 is a substrate of the ubiquitin/proteasome system and that Jab1 controls the activity of p27Kip1 by facilitating its degradation.21 However, Jab1 protein levels were not affected by TR3 or ΔTA (Figure 5A).
ECs constitute a monolayer in the vessel wall facing the lumen and display a strategic function in the regulation of many (patho)physiologic processes, such as the control of blood coagulation, vasomotor tone, ischemic and reperfusion injuries, and atherosclerosis.1 In pathologic situations, such as the initiation and progression of atherosclerosis, ECs become activated and are involved in excessive extravasation of monocytes into the vessel wall.22 In response to local insults, eg, those due to transluminal angioplasty, the endothelium is disrupted, resulting in a proliferative response of these cells.
In the current study, we demonstrated that the nuclear orphan receptor TR3 and potentially also its subfamily members NOT and MINOR induced cell cycle arrest of ECs. TR3, MINOR, and NOT were shown to be expressed in ECs in the (atherosclerotic) vessel wall. The majority of the ECs overlying atherosclerotic plaques in large arteries express TR3, MINOR, and NOT mRNA. In the apparently healthy part of vessels with relatively early, eccentric lesions, we observed patchy expression of TR3-like factors in the EC layer.
TR3-like factors have been implicated in apoptosis of T cells, prostate tumor cells, and lung tumor cells.9–14⇓⇓⇓⇓⇓ We have shown that TR3 overexpression in ECs or full inhibition of the transcriptional activity of endogenous subfamily members by ΔTA in these cells does not affect the activation of caspase-3. Furthermore, we performed a multiplex ligation-dependent probe amplification, which allowed simultaneous assessment of mRNA expression levels of 24 apoptosis-related genes and showed that both TR3- and ΔTA-overexpressing HUVECs exhibited no significant differences in the expression of each of these genes (authors’ unpublished data). On the basis of these data, we concluded that TR3 does not provoke apoptosis in ECs.
Here, we demonstrate inhibition of EC proliferation in response to TR3 overexpression, which might be explained by cyclin A downregulation and enhanced expression of the Cdk inhibitor p27Kip1, resulting in cell cycle arrest at G1, as shown by fluorescence-activated cell sorting analysis.3,4⇓ Analogous to our data, Chen et al24 have shown that p27Kip1 induction and cyclin A downregulation are crucial for cell-cell contact–mediated inhibition of EC proliferation. Furthermore, Hirano et al4 have concluded that cell-cell contact–mediated growth inhibition in vascular ECs involves, to some extent, transcriptional upregulation of the p27Kip1 gene. It is thus conceivable that TR3 modulates p27Kip1 expression at the transcriptional level, even though no consensus NGBFI-B response element (NBRE)25 or NurRE26 has been identified in the region 5 kb upstream of the p27Kip1 gene (authors’ unpublished data). An alternative mechanism might be that Jab1-mediated degradation of p27Kip1 is less rapid in the presence of TR3; however, we did not find any change in Jab1 expression levels in ΔTA- or TR3-overexpressing HUVECs, implying that this is probably not the mechanism by which TR3 influences p27Kip1 protein expression levels. Tanner et al27 have demonstrated that in human atherosclerotic lesions, p27Kip1 expression appears to be correlated inversely with vascular cell proliferation, and they proposed that p27Kip1 sets the balance between proliferating and nonproliferating cells in the vessel wall. Moreover, p27Kip1 knockout mice crossed with apolipoprotein E–knockout mice exhibit profound enhancement of atherosclerosis compared with apolipoprotein E–knockout mice when fed a Western-type diet, indicating that p27Kip1 protects against diet-induced atherosclerosis.28 However, it is difficult to assess the effect of the lack of p27Kip1 expression on EC (dys)function and its relative contribution to lesion formation in those in vivo experiments.
In adventitial microvessels, we observed relatively high mRNA levels of TR3, MINOR, and NOT. These data are in good correlation with the observation of abundant and uniform expression of p27Kip1in adventitial microvessels.27 In the same study, p27Kip1 was shown to be expressed in a patchy manner in luminal ECs of large arteries, which correlates with the vascular expression pattern that we describe for TR3.
Finally, EC proliferation is crucial for angiogenesis, and consequently, antiangiogenic therapy by inducing growth arrest of ECs has been shown to be effective in the treatment of cancer.29 Accordingly, it has been shown that overexpression of p27Kip1 inhibits EC proliferation, resulting in reduced angiogenesis5 and delayed tumor progression.30 Therefore, TR3 might be considered a potential target to inhibit angiogenesis. However, in vivo experiments are necessary to provide evidence for such an effect of TR3.
In summary, we show that TR3, MINOR, and NOT are expressed in (activated) ECs and that TR3 inhibits proliferation of these cells. EC growth inhibition might be undesirable in situations where the endothelium is denuded, such as after angioplasty. At the initiation of atherosclerosis, however, the EC layer is not disrupted. In this respect, it is important to mention that TR3 has also been shown to cause cell cycle arrest in the dopaminergic NM9D cell line, which simultaneously results in induction of differentiation of these cells.31 We envision that expression of TR3 in ECs results, by an analogous mechanism, in preservation of the normal EC characteristics, thereby influencing the vessel wall toward a reduction of its susceptibility to progression of atherosclerosis.
Recently, Gruber et al32 showed similar data on TR3 (denoted as Nur77/NAK1) expression in atherosclerotic vessels and showed that TR3 is expressed in HUVECs in response to tumor necrosis factor-α, interleukin-1, and lipopolysaccharide stimulation.
This research was supported by grants from the Netherlands Heart Foundation M93.007 and M93.001 and the Bekales Foundation (Belgium). We thank Dr Robert Kleemann for suggestions made to study Jab1 expression levels.
- Received May 22, 2003.
- Accepted June 11, 2003.
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- ↵Stary HC, Chandler AB, Dinsmore RE, Fuster W, Glagov S, Insull W Jr, Rosenfeld ME, Schwartz CJ, Wagner WD, Wissler RW. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis: a report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Arterioscler Thromb Vasc Biol. 1995; 15: 1512–1531.
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- Deleted in proof.
- ↵Chen D, Walsh K, Wang J. Regulation of cdk2 activity in endothelial cells that are inhibited from growth by cell contact. Arterioscler Thromb Vasc Biol. 2000; 20: 629–635.
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