doi: 10.1161/hq1101.098550
© 2001 American Heart Association, Inc.
Vascular Biology |
Induction of Nuclear Orphan Receptor NGFI-B Gene and Apoptosis in Rat Vascular Smooth Muscle Cells Treated With Pyrrolidinedithiocarbamate
From the Department of Geriatric Medicine (T.W., M.Y., M.E., M.H., K.N., Y.-Q.L., Y. Ohike, K.I., N.S., S.K., J.A., Y. Ouchi), Graduate School of Medicine, University of Tokyo; the Department of Geriatric Medicine (M.A., K.T.), Kyorin University School of Medicine; and the Department of Advanced Medicine (T.N., N.Y.), Institute of Medical Science, University of Tokyo, Tokyo, Japan.
Correspondence to Yasuyoshi Ouchi, MD, PhD, Department of Geriatric Medicine, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail youchi-tky{at}umin.ac.jp
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Abstract |
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Abstract—— NGFI-B is one of the orphan nuclear receptors, and its gene is implicated in the apoptosis of T cells. The aim of this study was to investigate the expression and the role of NGFI-B in vascular smooth muscle cells (VSMCs). Pyrrolidinedithiocarbamate (PDTC) is a modulator of an oxidative state and is reported to induce apoptosis only when the density of VSMCs is low. Under low VSMC density (10 000 cells/cm2), addition of PDTC (0.1 to 10 µmol/L) caused apoptosis of VSMCs, which was confirmed by Hoechst 33258 staining under fluorescence microscopy. At low VSMC density, expression of NGFI-B mRNA was induced 1 hour after the addition of PDTC, peaking at 6 hours, and persisted for up to 12 hours. The protein level of NGFI-B was increased 4 hours after PDTC addition and persisted for up to 12 hours. Under low VSMC density, PDTC-induced expression of NGFI-B mRNA was correlated with the magnitude of apoptosis, which was quantified by enzyme immunoassay for histone-associated DNA fragments. In contrast, when the density of VSMCs was high (50 000 cells/cm2), PDTC did not induce apoptosis, and the expression of NGFI-B was only transient. This transient expression pattern was also seen when VSMCs were treated with phorbol ester, calcium ionophore, hydrogen peroxide, or angiotensin II, even at low cell density. We next investigated whether the NGFI-B gene may act as a transcription factor under treatment with PDTC by measuring the promoter activity of luciferase reporter plasmids that contained typical NGFI-B–responsive elements. The PDTC-induced transcriptional activity of NGFI-B was 2-fold higher at low cell density than at high cell density. These data demonstrate that NGFI-B can be induced in VSMCs and suggest that NGFI-B may play a role in PDTC-induced VSMC apoptosis.
Key Words: vascular smooth muscle cells • apoptosis • nuclear receptor • cell density
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Introduction |
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Transcription factors play a central role in the regulation of their target genes in response to extracellular stimuli. In 1988, Milbrandt1 isolated a nerve growth factor (NGF)-induced gene from a rat pheochromocytoma cell line, PC12, and named it NGFI-B, as a second inducible gene by NGF. Nur77 (a mouse homologue to NGFI-B) and NAK1 (a human homologue) were also isolated from mouse fibroblasts2 and from the human fetal muscle cDNA library,3 respectively. The NGFI-B/Nur77/NAK1 gene has a transcriptional domain in the N-terminus, a DNA-binding domain with a zinc finger motif, and a ligand-binding domain in the C-terminus. Therefore, the gene is thought to be a member of the steroid receptor superfamily and is still an orphan receptor whose ligand remains unknown. NGFI-B can be induced immediately by several stimuli in various tissues, such as brain, thymus, adrenal gland, and heart.4–7 Although little is known about its function, it has been recently reported that NGFI-B/Nur77 is required for the apoptosis of T-cell hybridoma.8,9
In vascular smooth muscle cells (VSMCs), many transcription factors have been studied for the understanding of signaling pathways in pathological or pathophysiological conditions.10,11 However, to our knowledge, there is only 1 report that showed NGFI-B mRNA expression in VSMCs of rat coronary artery by using in situ hybridization histochemistry,12 although the functional role of NGFI-B in VSMCs is still unknown.
Apoptosis of VSMCs has been detected in human vasculature13,14 and is thought to have divergent roles in clinical settings; it may be effective for reducing restenosis after coronary angioplasty14 but, in contrast, may be harmful because it might be responsible for unstable plaques in atherosclerosis.15 VSMCs are unique in terms of response to the redox state; VSMCs can grow more rapidly under oxidative conditions,16 and antioxidants, including pyrrolidinedithiocarbamate (PDTC), can induce apoptosis of VSMCs.17 Recently, it has been shown that PDTC-induced apoptosis in VSMCs takes place at low cell density (LCD) but not at high cell density (HCD).10
In the present study, to test whether NGFI-B can be expressed in VSMCs and whether it plays a role in VSMC apoptosis, we used the divergent effects of PDTC on VSMC apoptosis, depending on the cell density. We treated rat VSMCs with PDTC under various conditions and found that the NGFI-B gene was strongly and persistently induced by PDTC when VSMCs were apoptotic at LCD.
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Methods |
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Chemicals
PDTC and sodium nitroprusside were purchased from Sigma-Aldrich Japan; phorbol 12-myristate 13-acetate (PMA), hydrogen peroxide (H2O2), and A23187 were from Wako; and human angiotensin II was from Peptide, Inc. Simvastatin was kindly provided by Banyu Pharmaceutical Co, Ltd, Tokyo, Japan.
Cell Culture
Rat VSMCs were harvested from the aortas of male Sprague-Dawley rats by enzymatic dissociation according to the modified method of Chamley et al.18 Cells were cultured in DMEM (Nikken Bio Medical Laboratory) supplemented with 10% FBS (Intergen Co), 25 mmol/L HEPES (pH 7.4), penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37°C in a humidified atmosphere of 95% air and 5% CO2. VSMCs from passages 6 to 9 were used in the experiments.11
Morphological Study of Apoptosis
VSMCs were plated on culture chamber slides (Becton Dickinson Laboratory) at 2 different cell densities: LCD (10 000/cm2) and HCD (50 000/cm2). After incubation for 24 hours, the cells were treated with PDTC at 1 µmol/L for 24 hours. Cells were washed once with PBS and fixed with PBS containing 1% glutaraldehyde for 30 minutes at room temperature. The cells were washed again with PBS, incubated with PBS containing 10 µmol/L Hoechst 33258 (Funakoshi Co) for 15 minutes in the dark, and examined under fluorescence microscopy. The numbers of apoptotic cells and intact cells were counted in 6 high-power fields for each culture chamber slide by an observer blinded to the treatment group.
Analysis of VSMCs by Flow Cytometry for Apoptosis
For identification of VSMC apoptosis, the binding of annexin V–FITC (Pharmingen) and the uptake of propidium iodide (Sigma) were measured by flow cytometry (EPICS XL flow cytometer, Beckman Courter Co) and Expo32 analysis software. Five thousand cells harvested 12 hours after treatment by PDTC or PBS were acquired, and apoptotic cells were quantified as the percentage of the cells that were FITC positive and propidium iodide negative.
Northern Blot Analysis
VSMCs were plated at 2 different cell densities (LCD and HCD) on 10-cm-diameter dishes 24 hours before the experiments. Total RNA was extracted from VSMCs by guanidinium isothiocyanate extraction and centrifugation through cesium chloride, as described previously.19 The RNA was fractionated on 1.3% formaldehyde-agarose gel and transferred to nylon filters (Hybond-N, Amersham Life Science Inc). The filters were hybridized at 68°C for 2 hours with a random-primed, 32P-labeled, mouse NGFI-B cDNA probe in QuikHyb solution (Stratagene) and autoradiographed. The product digested by EcoRI from mouse N10 plasmid kindly provided by Dr A. Winoto (Department of Molecular and Cell Biology, University of California, Berkeley) was used as the NGFI-B probe.20 An oligonucleotide probe complementary to 18S rRNA was used to confirm the equal loading of RNA.19 The filters were autoradiographed, and the density of the bands was scanned with NIH Image software (version 1.61, National Institutes of Health).
Western Blot Analysis
VSMCs were plated at LCD and HCD on 10-cm-diameter dishes and, after 24 hours of incubation, were treated with 1 µmol/L PDTC. Cells were washed quickly with PBS twice and lysed in RIPA buffer containing 50 mmol/L Tris/HCl, pH 7.5, 150 mmol/L NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail (Complete, Mini, Boehringer-Mannheim). The samples were separated on 12% SDS-PAGE, electroblotted onto nitrocellulose membranes, and immunoblotted with the anti–NGFI-B monoclonal antibody Mab2E1, kindly provided by Dr T. Fahrner (Department of Pathology, Washington University Medical School, St. Louis, Mo).21 Antibody was detected by horseradish peroxidase–linked secondary antibody by using an enhanced chemiluminescence system (Amersham Life Sciences Inc).
MTT Assay for Cell Viability
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed with nonradioactive cell proliferation assay (CellTiter 96, Promega), according to the manufacturer’s protocol with a little modification. Cells were plated on 24-well plates at LCD and treated with PDTC after 24 hours of incubation. After 24 hours, 150 µmol/L dye solution was added into wells and incubated for 4 hours, and then 1 mL stop solution was added. A volume of 200 µL was transferred into 96-well plates, and an absorbance of 570 nm was measured by using a microplate reader.
Enzyme Immunoassay for Histone-Associated DNA Fragments
For quantitative determination of apoptosis, we measured histone-associated DNA fragments by using a photometric enzyme immunoassay (cell death detection ELISA, Boehringer-Mannheim) according to the manufacturer’s instructions. Cells were plated on 6-well plates at LCD and, after 24 hours of incubation, were treated with PDTC for 24 hours. Floating cells were first collected from the culture medium. The attached cells were harvested with trypsin, and the cell suspension was pelleted by centrifugation. Floating and attached cells were lysed (200 µL), added to the streptavidin-coated microtiter plate, and incubated with a mixture of anti-histone biotin and anti-DNA peroxidase for 2 hours. The plate was washed 3 times, and 2,2'-azino-di-3-ethylbenzthiazoline sulfonate was added for color development. Absorbance was measured at 405 nm, and the reading represented the amount of DNA fragments in VSMCs.
Transfection and Luciferase Assays
We used NGFI-B binding response element (NBRE)2-Luc, which contains an enhancer of 2 copies of NBRE21 and a firefly luciferase reporter vector,11 as a reporter plasmid. VSMCs at LCD or HCD were transfected with 1.6 µg NBRE-Luc plasmid via FuGENE6 (Roche) according to the manufacturer’s instructions.22 To correct for variability in transfection efficiency, we cotransfected 0.4 µg of pRL-SV40 control plasmid, containing the potent simian virus 40 promoter and enhancer driving the Renilla luciferase gene (Promega).11 We measured 2 kinds of luciferase activity by a dual-luciferase reporter assay system (Promega) according to the manufacturer’s protocol, and the ratio of firefly luciferase activity to that of Renilla luciferase in each sample was used as a measure of normalized luciferase activity.11 The transcription activity in PDTC-treated VSMCs for 8 hours was compared with that in untreated cells at either LCD or HCD.
Construction of Adenovirus Vector Carrying the NGFI-B Gene and Transfer Into VSMCs
A replication-deficient adenovirus vector carrying the CMV-IE enhancer, chicken ß-actin promoter, and the coding region of NGFI-B (AxCANGFI-B) was constructed by use of adenovirus expression vector kit (Takara Shuzo Co), and the titer was determined as described.23 VSMCs were seeded at 5000 cells/cm2 onto 60-mm dishes. Twenty-four hours later, they were exposed to 600 µL DMEM containing 5% FBS and either AxCANGFI-B or a replication-deficient recombinant adenovirus carrying the Escherichia coli ß-galactosidase gene (AxCALacZ) at a different multiplicity of infection for 24 hours, followed by treatment with PDTC or PBS for 12 hours. The harvested cells were subjected to apoptosis analysis.
Statistical Analysis
The dose-response effect of PDTC on cell viability in VSMCs and the luciferase activity in PDTC-treated VSMCs were analyzed by using 1-way ANOVA. If a statistically significant effect was found, the Newman-Keuls test was performed to isolate the difference between groups. Simple correlations of NGFI-B mRNA levels with cell viability and with DNA fragmentation were analyzed. A value of P<0.05 was considered statistically significant. All data in the text and figures are expressed as mean±SD.
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Nuclear Morphology of VSMCs Treated With PDTC at LCD and HCD
VSMCs were treated with PDTC, either at LCD or at HCD, for 24 hours. At LCD, PDTC caused apoptotic changes, including condensation and shrinkage of nuclei, which were observed with Hoechst 33258 staining under fluorescence microscopy (Figure 1B). In contrast, at HCD, PDTC did not induce apoptosis of VSMCs (Figure 1D) as previously reported.10 Treatment with PBS did not cause apoptotic changes at LCD or HCD (Figure 1A and 1C). The percentage of apoptotic VSMCs was calculated by counting the cells with apoptotic changes stained with Hoechst 33258 and by analyzing the cells stained with annexin V–FITC but without propidium iodide by using flow cytometry. As shown in Table 1, the rate of apoptosis was significantly increased in PDTC-treated cells compared with untreated cells at LCD. . .
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Different Expression Patterns of NGFI-B Induced by PDTC at LCD and HCD
The time courses of NGFI-B expression induced by 1 µmol/L PDTC were analyzed by Northern blot and Western blot analyses (Figure 2). NGFI-B mRNA was expressed at a very weak level in VSMCs before PDTC treatment. At LCD, PDTC induced the expression of NGFI-B mRNA at 1 hour, which was peaking at 6 hours, and persisted for >12 hours (Figure 2A and 2B), and the protein level of NGFI-B was increased 4 hours after the addition of PDTC and persisted for >12 hours, as shown in Figure 2C. In contrast to LCD, PDTC-induced NGFI-B mRNA expression was transient only at HCD, peaking at 2 hours, and returned to the baseline level at 4 hours after the addition of PDTC (Figure 2A and 2B). Remarkable induction of NGFI-B protein by PDTC was not observed at HCD (Figure 2C). .
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Relation Between Apoptosis of VSMCs and Expression of NGFI-B
The dose-response effect of PDTC on the expression of NGFI-B mRNA was examined at 6 hours after the addition at LCD. There was an abrupt increase of NGFI-B mRNA at 0.03 to 0.1 µmol/L PDTC, and no further increase was observed at higher doses of PDTC (Figure 3A). .
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To examine the dose-response effect of PDTC on cell survival, MTT assay was performed at LCD. As shown in Figure 3B, PDTC (0.1 to 10 µmol/L) decreased the viability of VSMCs; however, an obvious dose-response effect could not be found. The dose-response effect of PDTC on the induction of apoptosis was also evaluated by an enzyme immunoassay for histone-associated DNA fragments. PDTC (0.1 to 10 µmol/L) increased the amount of histone-associated DNA fragments (Figure 3C). The change of DNA fragments was the mirror image of the result of cell viability assay. These data suggest that there might be a threshold level of PDTC (between 0.1 and 1 µmol/L) for the induction of apoptosis in VSMCs.
Figure 3D shows a strong correlation between the expression of NGFI-B mRNA and the cell viability. Also, Figure 3E shows a strong correlation between the expression of NGFI-B and the magnitude of apoptosis quantified by DNA fragmentation, indicating that the expression of NGFI-B gene is closely associated with the PDTC-induced apoptosis in VSMCs.
Expression of NGFI-B mRNA Induced by Other Stimuli in VSMCs
We tested whether the induction of NGFI-B gene took place in VSMCs undergoing apoptosis induced by other agents, such as 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors24 and sodium nitroprusside.25 Interestingly, they did not cause either persistent or transient induction of NGFI-B mRNA at LCD (Figure 4A), although they induced apoptosis of VSMCs (data not shown). Therefore, it is suggested that the induction of NGFI-B mRNA is not necessarily related to the apoptotic process itself. .
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Previous reports showed the induction of NGFI-B gene by several stimuli, such as PMA and calcium ionophore A23187 in T-cell hybridoma8 and angiotensin II in adrenal cortical cells.26 Therefore, we tested whether these stimuli would also induce the NGFI-B gene in VSMCs. Northern blot analysis revealed that 3 kinds of agents induced transient expression of NGFI-B mRNA in VSMC at LCD (Figure 4B). Although VSMCs at LCD were used, apoptotic changes were not observed with the use of these stimuli (data not shown). We also found that H2O2, one of the reactive oxygen species, also transiently induced the expression of NGFI-B in VSMCs. An expression pattern of NGFI-B mRNA similar to that of PDTC-treated VSMCs at HCD (Figure 2A and 2B) suggested that NGFI-B might function as an early-response gene under these kinds of stimuli.
Transcriptional Activation of NGFI-B Responsive Element by PDTC
We next investigated whether NGFI-B may act as a transcription factor under treatment with PDTC by measuring the promoter activity of the luciferase reporter plasmid that contained typical NGFI-B–responsive elements.21 As shown in Figure 5, at LCD, PDTC increased luciferase activity in VSMCs by 
8-fold. At HCD, PDTC also increased luciferase activity by 2.7-fold, although the increment of luciferase activity by PDTC stimulation was 50% lower at HCD than at LCD. .
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Effect of Overexpression of NGFI-B on VSMC Apoptosis
Production of the NGFI-B protein by the cells infected with adenovirus carrying NGFI-B was confirmed by Western blot analysis (data not shown). The transfer of the NGFI-B gene into VSMCs at a multiplicity of infection of 30 or 300 did not affect VSMC apoptosis regardless of cell density. Moreover, as shown in Table 2, the overexpression of NGFI-B in VSMCs also did not modify VSMC apoptosis induced by PDTC. .
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Discussion |
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In the present study, using Northern and Western blotting, we showed that the NGFI-B gene was induced in VSMCs by diverse stimuli and that the expression of NGFI-B was associated with apoptosis of VSMCs induced by PDTC.
Recently, it has been reported that NGFI-B is required for apoptosis in a lung cancer cell line and a prostate cancer cell line,27,28 as well as in T-cell hybridoma.8,9 In the present study, sustained induction of the NGFI-B gene was observed when VSMCs were apoptotic with PDTC at LCD; however, PDTC-induced NGFI-B gene expression was transient only under nonapoptotic conditions at HCD. Interestingly, our results are quite similar to the previous report by Woronicz et al,9 who reported that Nur77 (a mouse homologue of NGFI-B) mRNA expression in the apoptosis of T-cell hybridoma induced by anti-CD3 antibody was prolonged for 14 hours, whereas it was diminished 3 hours after the addition of PMA, which did not cause apoptosis of T-cell hybridoma. The induction of NGFI-B mRNA was also prolonged when cells were apoptotic in a lung cancer cell line27 and a prostate cancer cell line induced by calcium ionophore.28
Although it was shown that sustained induction of the NGFI-B gene was associated with PDTC-induced apoptosis of VSMCs at LCD, this finding was not common to the apoptosis of VSMCs. Although simvastatin, N-acetylcysteine, and sodium nitroprusside induced the apoptosis of VSMCs in our preliminary experiments, several researchers17,24,25 were unable to induce the expression of NGFI-B mRNA in VSMCs (Figure 4A and data not shown). PDTC and N-acetylcysteine are antioxidative agents and can induce the apoptosis of VSMCs by inhibiting nuclear factor-
B activity, which is important for VSMC survival.10 However, it is not known why only PDTC is able to induce the NGFI-B gene in VSMCs. One possibility is that PDTC may work as a pro-oxidant as well as an antioxidant under different conditions.29,30 NGFI-B also has the character of an immediate-early gene, which can be induced within 1 hour after the stimulation and decreased to a basal level 4 hours later. The gene is thought to be a stress-response gene in some conditions, including seizure,4 acute or chronic immobilization,31 or stretching of left ventricular muscle.32 As Liu et al8 reported in T cells, we have confirmed that acute chemical stimuli, PMA, and calcium ionophore A23187 could induce the NGFI-B gene in VSMCs. We also found that angiotensin II as well as hydrogen peroxide, inducers of oxidative stress, could induce the NGFI-B gene in VSMCs. However, the functional roles of transient expression of NGFI-B in VSMCs remain unknown because only few NGFI-B–response genes are identified in other cell types.33,34 Moreover, only one report showed the expression of NGFI-B in the vascular wall in vivo.12 In our preliminary study, the mRNA of NGFI-B was expressed at a very low level in the intact rat carotid artery and was induced 1 day after balloon injury, lasting for 2 weeks (authors’ unpublished data, 2001). The in vivo relevance of the present study needs to be addressed in a future study.
Because we have revealed that nuclear receptor NGFI-B was induced by PDTC in VSMCs at LCD and HCD, we examined whether the induction of NGFI-B was associated with transcriptional activity of this gene by using the luciferase assay of the reporter plasmids, which contain a classic NBRE.21 Although there is no definite evidence that this response element is working in PDTC-treated VSMCs at LCD as well as at HCD, higher transcriptional activity at LCD may be related to the profound induction of apoptosis in VSMCs. Our preliminary data showed that the protein expression of apoptosis-related genes, including Bcl-2, Bax, and Bcl-x, was not changed regardless of cell density and the addition of PDTC and was not altered by the adenovirus-mediated overexpression of NGFI-B in VSMCs (data not shown). The pathway involved in NGFI-B–related VSMC apoptosis might be independent of the Bcl family. Causal relation between NGFI-B expression and VSMC apoptosis is unclear. Overexpression of NGFI-B did not induce VSMC apoptosis without the addition of PDTC at LCD (Table 2) or HCD, indicating that the expression of NGFI-B alone is not sufficient to trigger VSMC apoptosis. Also, overexpression of NGFI-B did not enhance PDTC-induced apoptosis. This might be attributable to such a strong induction of NGFI-B by PDTC that the additional expression of NGFI-B by gene transfer did not amplify VSMC apoptosis. The inhibition experiment of NGFI-B by using antisense or overexpression of dominant-negative mutant is needed to address this issue. Taken together, we suppose that NGFI-B may play a part in PDTC-induced VSMC apoptosis, probably by regulating the transcription of some genes other than the Bcl family.
What is the physiological meaning of PDTC-induced VSMC apoptosis depending on cell density? As Erl et al10 stated in a study involving PDTC-induced VSMC apoptosis, a high frequency of apoptosis may be recognized in high cell turnover and tissue remodeling in growing cell populations, and these situations may be mimicked in an LCD condition. On the contrary, an HCD condition may reflect a more stable situation in normal adult tissue. Although NF-B is constitutively activated in VSMCs,35 an LCD condition has lower NF-B activity than does an HCD condition.10 It is postulated that PDTC, an inhibitor of NF-B, decreases the activity of NF-B, resulting in VSMC apoptosis only at LCD. However, the effect of PDTC on VSMCs in vivo has not been reported so far and remains to be determined. An important finding of the present study is that NGFI-B can be induced as an early-response gene or as an apoptosis-related gene by the same reagent, PDTC, depending on cell density only. Further study will be necessary to elucidate the function of the NGFI-B gene under these 2 conditions.
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Acknowledgments |
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This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (12670652), by a Grant-in-Aid from Takeda Medical Research Foundation, and by Funds for Comprehensive Research on Aging and Health from the Japan Foundation for Aging and Health. We thank Dr Astar Winoto for the gift of N10 plasmid and Dr Tim Fahrner for the gift of Mab 2E1 anti–NGFI-B antibody and NBRE-Luc plasmid. We also thank Harumi Hata for excellent technical assistance in the luciferase reporter assay.
Received October 16, 2000; accepted August 23, 2001.
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References |
|---|
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|---|
1. Milbrandt J. Nerve growth factor induces a gene homologous to the glucocorticoid receptor gene. Neuron. 1988; 1: 183–188.[Medline] [Order article via Infotrieve]
2.
Hazel TG, Nathans D, Lau LF. A gene inducible by serum growth factors encodes a member of the steroid and thyroid hormone receptor superfamily. Proc Natl Acad Sci U S A. 1988; 85: 8444–8448.
3.
Nakai A, Kartha S, Sakurai A, Toback FG, DeGroot LJ. A human early response gene homologous to murine nur77 and rat NGFI-B, and related to the nuclear receptor superfamily. Mol Endocrinol. 1990; 4: 1438–1443.
4.
Watson MA, Milbrandt J. The NGFI-B gene, a transcriptionally inducible member of the steroid receptor gene superfamily: genomic structure and expression in rat brain after seizure induction. Mol Cell Biol. 1989; 9: 4213–4219.
5.
Davis IJ, Lau LF. Endocrine and neurogenic regulation of the orphan nuclear receptors Nur77 and Nurr-1 in the adrenal glands. Mol Cell Biol. 1994; 14: 3469–3483.
6. Bandoh S, Tsukada T, Maruyama K, Ohkura N, Yamaguchi K. Differential expression of NGFI-B and RNR-1 genes in various tissues and developing brain of the rat: comparative study by quantitative reverse transcription-polymerase chain reaction. J Neuroendocrinol. 1997; 9: 3–8.[Medline] [Order article via Infotrieve]
7.
Rockman HA, Ross RS, Harris AN, Knowlton KU, Steinhelper ME, Field LJ, Ross JJr, Chien KR. Segregation of atrial-specific and inducible expression of an atrial natriuretic factor transgene in an in vivo murine model of cardiac hypertrophy. Proc Natl Acad Sci U S A. 1991; 88: 8277–8281.
8. Liu ZG, Smith SW, McLaughlin KA, Schwartz LM, Osborne BA. Apoptotic signals delivered through the T-cell receptor of a T-cell hybrid require the immediate-early gene nur77. Nature. 1994; 367: 281–284.[Medline] [Order article via Infotrieve]
9. Woronicz JD, Calnan B, Ngo V, Winoto A. Requirement for the orphan steroid receptor Nur77 in apoptosis of T-cell hybridomas. Nature. 1994; 367: 277–281.[Medline] [Order article via Infotrieve]
10.
Erl W, Hansson GK, de Martin R, Draude G, Weber KS, Weber C. Nuclear factor-kappa B regulates induction of apoptosis and inhibitor of apoptosis protein-1 expression in vascular smooth muscle cells. Circ Res. 1999; 84: 668–677.
11. Iijima K, Yoshizumi M, Ako J, Eto M, Kim S, Hashimoto M, Sugimoto N, Liang YQ, Sudoh N, Toba K, et al. Expression of peroxisome proliferator-activated receptor gamma (PPARgamma) in rat aortic smooth muscle cells. Biochem Biophys Res Commun. 1998; 247: 353–356.[Medline] [Order article via Infotrieve]
12. Ueyama T, Yoshida K, Senba E. Emotional stress induces immediate-early gene expression in rat heart via activation of alpha- and beta-adrenoceptors. Am J Physiol. 1999; 277: H1553–H1561.[Medline] [Order article via Infotrieve]
13.
Henderson EL, Geng YJ, Sukhova GK, Whittemore AD, Knox J, Libby P. Death of smooth muscle cells and expression of mediators of apoptosis by T lymphocytes in human abdominal aortic aneurysms. Circulation. 1999; 99: 96–104.
14.
Isner JM, Kearney M, Bortman S, Passeri J. Apoptosis in human atherosclerosis and restenosis. Circulation. 1995; 91: 2703–2711.
15. Bennett MR, Evan GI, Schwartz SM. Apoptosis of human vascular smooth muscle cells derived from normal vessels and coronary atherosclerotic plaques. J Clin Invest. 1995; 95: 2266–2274.[Medline] [Order article via Infotrieve]
16.
Rao GN, Berk BC. Active oxygen species stimulate vascular smooth muscle cell growth and proto-oncogene expression. Circ Res. 1992; 70: 593–599.
17.
Tsai JC, Jain M, Hsieh CM, Lee WS, Yoshizumi M, Patterson C, Perrella MA, Cooke C, Wang H, Haber E, et al. Induction of apoptosis by pyrrolidinedithiocarbamate and N-acetylcysteine in vascular smooth muscle cells. J Biol Chem. 1996; 271: 3667–3670.
18. Chamley JH, Groschel-Stewart U, Campbell GR, Burnstock G. Distinction between smooth muscle, fibroblasts and endothelial cells in culture by the use of fluoresceinated antibodies against smooth muscle actin. Cell Tissue Res. 1977; 177: 445–457.[Medline] [Order article via Infotrieve]
19.
Iijima K, Yoshizumi M, Hashimoto M, Kim S, Eto M, Ako J, Liang YQ, Sudoh N, Hosoda K, Nakahara K, et al. Red wine polyphenols inhibit proliferation of vascular smooth muscle cells and downregulate expression of cyclin A gene. Circulation. 2000; 101: 805–811.
20. Ryseck RP, Macdonald BH, Mattei MG, Ruppert S, Bravo R. Structure, mapping and expression of a growth factor inducible gene encoding a putative nuclear hormonal binding receptor. EMBO J. 1989; 8: 3327–3335.[Medline] [Order article via Infotrieve]
21.
Paulsen RE, Weaver CA, Fahrner TJ, Milbrandt J. Domains regulating transcriptional activity of the inducible orphan receptor NGFI-B. J Biol Chem. 1992; 267: 16491–16496.
22. Uyttersprot N, Costagliola S, Miot F. A new tool for efficient transfection of dog and human thyrocytes in primary culture. Mol Cell Endocrinol. 1998; 142: 35–39.[Medline] [Order article via Infotrieve]
23. Nakaoka T, Gonda K, Ogita T, Otawara Y, Okabe F, Kira Y, Harii K, Miyazono K, Takuwa Y, Fujita T. Inhibition of rat vascular smooth muscle proliferation in vitro and in vivo by bone morphogenetic protein-2. J Clin Invest. 1997; 100: 2824–2832.[Medline] [Order article via Infotrieve]
24.
Guijarro C, Blanco CL, Ortego M, Alonso C, Ortiz A, Plaza JJ, Diaz C, Hernandez G, Edigo J. 3-Hydroxy-3-methylglutaryl coenzyme A reductase and isoprenylation inhibitors induce apoptosis of vascular smooth muscle cells in culture. Circ Res. 1998; 83: 490–500.
25.
Fukuo K, Hata S, Suhara T, Nakahashi T, Shinto Y, Tsujimoto Y, Morimoto S, Ogihara T. Nitric oxide induces upregulation of Fas and apoptosis in vascular smooth muscle. Hypertension. 1996; 27: 823–826.
26. Enyeart JJ, Boyd RT, Enyeart JA. ACTH and AII differentially stimulate steroid hormone orphan receptor mRNAs in adrenal cortical cells. Mol Cell Endocrinol. 1996; 124: 97–110.[Medline] [Order article via Infotrieve]
27.
Li Y, Lin B, Agadir A, Liu R, Dawson MI, Reed JC, Fontana JA, Bost F, Hobbs PD, Zheng Y, et al. Molecular determinants of AHPN (CD437)-induced growth arrest and apoptosis in human lung cancer cell lines. Mol Cell Biol. 1998; 18: 4719–4731.
28.
Uemura H, Chang C. Antisense TR3 orphan receptor can increase prostate cancer cell viability with etoposide treatment. Endocrinology. 1998; 139: 2329–2334.
29. Chung KC, Park JH, Kim CH, Lee HW, Sato N, Uchiyama Y, Ahn YS. Novel biphasic effect of pyrrolidine dithiocarbamate on neuronal cell viability is mediated by the differential regulation of intracellular zinc and copper ion levels, NF-kappaB, and MAP kinases. J Neurosci Res. 2000; 59: 117–125.[Medline] [Order article via Infotrieve]
30.
Nobel CI, Kimland M, Lind B, Orrenius S, Slater AF. Dithiocarbamates induce apoptosis in thymocytes by raising the intracellular level of redox-active copper. J Biol Chem. 1995; 270: 26202–26208.
31. Senba E, Ueyama T. Stress-induced expression of immediate early genes in the brain and peripheral organs of the rat. Neurosci Res. 1997; 29: 183–207.[Medline] [Order article via Infotrieve]
32. Umemoto S, Kawai Y, Senba E. Differential regulation of IEGs in the rat PVH in single and repeated stress models. Neuroreport. 1994; 6: 201–204.[Medline] [Order article via Infotrieve]
33. Buono P, Conciliis LD, Izzo P, Salvatore F. The transcription of the human fructose-bisphosphate aldolase C gene is activated by nerve-growth-factor-induced B factor in human neuroblastoma cells. Biochem J. 1997; 323: 245–250.[Medline] [Order article via Infotrieve]
34.
Wilson TE, Mouw AR, Weaver CA, Milbrandt J, Parker KL. The orphan nuclear receptor NGFI-B regulates expression of the gene encoding steroid 21-hydroxylase. Mol Cell Biol. 1993; 13: 861–868.
35.
Lawrence R, Chang LJ, Siebenlist U, Bressler P, Sonenshein GE. Vascular smooth muscle cells express a constitutive NF-B-like activity. J Biol Chem. 1994; 269: 28913–28918.
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