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(Hypertension. 2000;35:297.)
© 2000 American Heart Association, Inc.

Scientific Contributions

Downregulation of Angiotensin II Type 1 Receptor by All-trans Retinoic Acid in Vascular Smooth Muscle Cells

Kotaro Takeda; Toshihiro Ichiki; Yuko Funakoshi; Kiyoko Ito; Akira Takeshita

From the Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, Higashi-ku, Fukuoka, Japan.

Correspondence to Toshihiro Ichiki, MD, Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, 812-8582, Fukuoka, Japan. E-mail ichiki{at}cardiol.med.kyushu-u.ac.jp

	Abstract

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Abstract—All-trans retinoic acid (atRA) is a biologically active metabolite of vitamin A that plays an important role in cell differentiation and proliferation. Although neointimal formation after balloon injury of rat carotid artery is inhibited by atRA, the mechanisms are not clearly understood. Because the renin-angiotensin system is one of the crucial components of atherosclerosis, we examined the effects of atRA on the expression of angiotensin II type 1 receptor (AT₁-R) in vascular smooth muscle cells. atRA (1 µmol/L) decreased the AT₁-R mRNA level by 50% after 24 hours; AT₁-R number was also reduced to the same extent after 48 hours. atRA markedly suppressed promoter activity of the AT₁-R promoter-luciferase construct, but AT₁-R mRNA stability was not affected. Cycloheximide blocked the atRA-induced decrease in AT₁-R mRNA expression, suggesting that this process requires de novo protein synthesis. Simultaneous treatment with an agonist (Ro40-6055) specific for retinoic acid receptor (RAR) and an agonist (Ro25-7836) specific for retinoid X receptor (RXR) suppressed the AT₁-R mRNA expression comparable to that with treatment with atRA, suggesting that the RAR/RXR heterodimer mediates the effect of atRA in AT₁-R downregulation. These results suggest that atRA suppressed AT₁-R mRNA transcription through new protein synthesis induced by RAR/RXR-dependent transcription. This study provides novel insight into a role of atRA as an important molecule that regulates AT₁-R gene expression and provides possible mechanisms for the suppression of neointimal formation by atRA.

Key Words: receptors, angiotensin II • muscle, smooth, vascular • genes • all-trans retinoic acid

	Introduction

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Angiotensin II (Ang II) is a major final effector molecule of the renin-angiotensin system (RAS). Ang II has various physiological effects and plays crucial roles in the pathogenesis of atherosclerosis, hypertension, and restenosis after coronary angioplasty.^{1 2} There are 2 Ang II receptor isoforms, designated type 1 receptor (AT₁-R)³ and type 2 receptor (AT₂-R).⁴ Most of the cardiovascular effects are believed to be mediated by the AT₁-R. It has been demonstrated that Ang II stimulates vascular smooth muscle cell hypertrophy,⁵ migration,⁶ extracellular matrix production,⁷ superoxide production,⁸ and expression of various growth factors, such as transforming growth factor-ß,⁹ platelet-derived growth factor,¹⁰ and fibroblast growth factor,¹¹ through the AT₁-R. The AT₁-R is increased in atherosclerotic lesion and neointima after balloon injury.¹² Treatment with an ACE inhibitor or an AT₁-R antagonist inhibits the neointimal formation after balloon injury and the progression of atherosclerosis,^{2 13 14} suggesting a crucial role of AT₁-Rs in atherogenesis.

All-trans retinoic acid (atRA), a biologically active vitamin A metabolite, plays an important role in the regulation of cell differentiation,¹⁵ proliferation,¹⁶ and apoptosis.¹⁵ Signals of atRA are mediated by specific nuclear receptors, the retinoic acid receptor (RAR) and the retinoid X receptor (RXR), which belong to steroid/thyroid hormone receptor superfamily.¹⁷ RXR can form a homodimer (RXR/RXR) and a heterodimer with RAR (RAR/RXR).¹⁷ Both receptors have 3 isotypes: , ß, and . Rat vascular smooth muscle cells (VSMCs) express both types of RAR (, ß, and ) and RXR ( and ß).¹⁸ There are 2 natural retinoid receptor ligands: atRA and 9-cis retinoic acid. One of the signaling mechanisms of atRA is an activation of gene expression. The ligand-activated retinoid receptors bind to a specific DNA sequence, retinoic acid response element, and then activate target gene transcription.¹⁷ Another signaling pathway of atRA is inhibition of the transcription factor activator protein-1 (AP-1) activity.¹⁹ The reduction in AP-1 activity is due to a competition of limiting amounts of transcriptional coactivator cAMP response element binding protein (CBP) with retinoid receptor.¹⁹

Recently, it was reported that atRA inhibited neointimal formation after balloon withdrawal injury of the rat carotid artery.²⁰ However, the mechanisms have not been clarified. Because local RAS is activated in balloon injury model of neointimal formation, it is of much interest to determine the effect of atRA on RAS activity. Here, we showed for the first time evidence that atRA downregulates the AT₁-R gene expression in VSMCs.

	Methods

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Reagents
atRA and BSA were purchased from Sigma Chemical Co, and other chemical reagents were purchased from Wako Pure Chemicals. DMEM and FBS were purchased from GIBCO BRL. [-³²P]dCTP and [¹²⁵I]Sar¹,Ile⁸-Ang II were obtained from DuPont-NEN. An RAR-selective agonist (Ro40-6055) and an RXR-selective agonist (Ro25-7386) were generous gifts from Dr Eva-Maria Gutknecht (F. Hoffmann-La Roche Ltd).²¹ The retinoids were dissolved in 100% ethanol. A CBP-expression plasmid was a gift from Dr Fukamizu (Tsukuba University, Japan).

Cell Culture
VSMCs were isolated from the thoracic aorta of Sprague-Dawley rats and maintained as described previously.²² Passages between 5 and 12 were used for the experiment.

Northern Blot Analysis
Total RNA was prepared according to the acid guanidinium-phenol-chloroform extraction method.²³ Northern blot analyses of AT₁-R and 18S rRNA were performed as described previously.²² The radioactivity of hybridized bands of AT₁-R mRNA and 18S rRNA were quantified with a MacBAS Bioimage Analyzer (Fuji Film). To analyze the mRNA stability of AT₁-Rs, actinomycin D (5 µg/mL) was added after 12 hours of stimulation with atRA (1 µmol/L). VSMCs were harvested at 3, 6, 12, and 24 hours after the addition of actinomycin D. The expression level of AT_I-R mRNA was examined with the use of Northern blot analysis.

Estimation of AT₁-R Number
The number of AT₁-R binding sites was estimated on the basis of the binding of [¹²⁵I]Sar¹,Ile⁸-Ang II. Confluent VSMCs in 24-well dishes were starved in serum-free medium for 48 hours and incubated with vehicle or 1 µmol/L atRA for 48 hours. Then, the cells were washed twice with ice-cold saline and incubated for 3 hours at 4°C with varying concentrations of [¹²⁵I]Sar¹,Ile⁸-Ang II in binding buffer containing 50 mmol/L Tris · HCl, pH 7.4, 100 mmol/L NaCl, 5 mmol/L MgCl₂, 0.2% BSA, and 0.5 mg/mL bacitracin. Cells were washed 3 times with ice-cold saline and solubilized in 0.75 mL of 0.5 N NaOH. An aliquot was subjected to radioactivity count and protein concentration determination. Nonspecific binding was determined in the presence of unlabeled Sar¹,Ile⁸-Ang II (10 µmol/L). Values for nonspecific binding were subtracted from values for total binding.

Transfection of AT₁-R Promoter-Luciferase Fusion DNA Construct to VSMCs
Deletion mutants of the promoter region of rat AT₁-R gene were described previously.²² VSMCs (4x10⁵) were prepared in a 6-cm tissue culture dish. After 48 hours, 5 µg of AT₁-R promoter-luciferase fusion DNA construct and 2 µg of LacZ gene driven by SV40 promoter-enhancer sequence were introduced to VSMCs as previously described.²² Cotransfection of a CBP expression plasmid was performed with the use of Transfast transfection reagent according to the manufacturer’s instructions (Promega). In addition to AT₁-R promoter-luciferase fusion DNA construct (980 bp, 2 µg) and LacZ gene (2 µg), vector plasmid (3 µg) or CBP-expressing plasmid (3 µg) was introduced concomitantly to VSMCs. These cells were stimulated with 1 µmol/L atRA and cultured in DMEM with 10% FBS for 48 hours. The luciferase activity was measured and normalized by ß-galactosidase activity as described previously.²²

Statistical Analysis
Statistical analyses were performed with the use of 1-way ANOVA and Fisher’s test if appropriate. Differences in dissociation constant (K_d) and AT₁ receptor maximum binding (B_max) were compared with the use of an unpaired Student’s t test. Degradation in AT₁-R mRNA was analyzed with the use of 2-way ANOVA. Data are shown as mean±SEM. P<0.05 was considered statistically significant.

	Results

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atRA Suppresses AT₁-R mRNA Expression
VSMCs were incubated with 1 µmol/L atRA, and the mRNA level of AT₁-R was determined through Northern blot analysis. Two species of AT₁-R transcript were detected, and the radioactivity of both bands was measured for statistical analysis. Figure 1A demonstrates the time-dependent downregulation of AT₁-R gene expression by atRA. After 12 hours of incubation, the expression level was significantly reduced compared with the control level. The reduction was reached a maximum level at 24 hours (Figure 1A) and continued for 48 hours (data not shown). However, AT₁-R mRNA expression was not significantly changed with the use of 0.1% ethanol vehicle alone (Figure 1B). Figure 1C shows that the incubation with varying concentrations of atRA for 24 hours resulted in a dose-dependent decrease in the AT₁-R mRNA level.

View larger version (41K): [in this window] [in a new window]   Figure 1. Suppression of AT1-R mRNA expression by atRA in VSMCs. VSMCs were incubated with atRA (1 µmol/L) (A) or 0.1% ethanol vehicle (B) for varying periods of time, as indicated. VSMCs were also incubated with atRA at concentrations varying from 10-8 to 10-6 mol/L for 24 hours (C). Total RNA was isolated, and expression of AT1-R mRNA and 18S rRNA was determined through Northern blot analysis. Left, Representative autoradiogram. Right, Radioactivity of AT1-R mRNA bands was counted with a Bioimage Analyzer and was normalized with radioactivity of rRNA. Values (mean±SEM) are expressed as a percent of control culture (100%) (n=3). *P<0.05, **P<0.01.

atRA Downregulates the AT₁-R Number in VSMCs
Figure 2 shows a saturation curve and Scatchard plot analysis of the binding of [¹²⁵I]Sar¹,Ile⁸-Ang II to vehicle- and atRA-treated (48 hours) VSMCs. Binding to the vehicle-treated VSMCs revealed K_d value of 8.41 nmol/L and a B_max value of 2.08 pmol/mg protein. However, the atRA-stimulated cells demonstrated a significantly reduced B_max value (1.08 pmol/mg protein) and a statistically unchanged K_d value (8.57 nmol/L). These data indicate that atRA reduced AT₁-R protein expression, whereas the affinity of the receptor to Ang II did not change significantly.

View larger version (20K): [in this window] [in a new window]   Figure 2. Effect of atRA on AT1-R density in VSMCs. VSMCs were incubated with atRA (1 µmol/L) for 48 hours. Binding assay using [125I]Sar1,Ile8-Ang II were performed. Saturation curve (A) and Scatchard plot analysis (B) are shown. Maximum binding (Bmax) of vehicle-treated VSMCs was 2.08 pmol/mg protein with a dissociation constant (Kd) of 8.41 nmol/L. Bmax value of atRA-treated VSMCs was 1.08 pmol/mg protein with a Kd value of 8.57 nmol/L (n=3). Bmax value was significantly reduced with atRA (P<0.05), but Kd value was not changed.

Effect of atRA on AT₁-R mRNA Stability
We next examined whether atRA affected the stability of AT₁-R mRNA. Figure 3 shows that the half-life of AT₁-R mRNA in the absence of atRA was 6 hours, as previously reported,^{22 24} whereas that of AT₁-R mRNA in the presence of atRA also was 6 hours. These data suggest that atRA does not influence the stability of AT₁-R mRNA.

View larger version (18K): [in this window] [in a new window]   Figure 3. Effect of atRA on AT1-R mRNA stability. VSMCs were pretreated with atRA (1 µmol/L) for 12 hours () or vehicle (). Then, VSMCs were incubated with actinomycin D (5 µg/mL). Total RNA was isolated at indicated time points, and expression levels of AT1-R mRNA were determined through Northern blot analysis. Expression levels of AT1-R in VSMCs before addition of actinomycin D in each group was set as 100% (n=4).

Proximal Promoter Region Is Crucial for atRA-Induced AT₁-R Downregulation
To examine the molecular mechanism that might be responsible for the atRA-induced downregulation of AT₁-R gene expression, transient transfection was performed with 5 AT₁-R promoter-luciferase DNA constructs (Figure 4A). The successive deletion of AT₁-R promoter resulted in a reduction in promoter activity except for construct 4 (Figure 4B). The AT₁-R promoter activity was markedly inhibited by 1 µmol/L atRA in all 5 constructs (Figures 4B and 4C), suggesting that the most proximal element (from -61 to +25 bp) of the AT₁-R promoter was responsible for the atRA-induced AT₁-R downregulation.

View larger version (31K): [in this window] [in a new window]   Figure 4. Structure of AT1-R promoter-luciferase fusion DNA construct and effect of atRA. A, Deletion mutants of promoter region of rat AT1-R gene. B, AT1-R promoter-luciferase construct and LacZ gene were introduced to VSMCs. Luciferase activity was normalized by ß-galactosidase activity. Relative luciferase activity of vehicle-treated VSMCs transfected with construct 1 was set as 100%. Relative luciferase activity of vehicle-treated VSMCs (filled columns) and atRA-stimulated VSMCs (open columns) are shown as mean±SEM (n=6). *P<0.01. C, Relative luciferase activity of vehicle-treated VSMCs from each construct was set as 100%. *P<0.01 vs each control.

Downregulation of AT₁-R Expression by atRA Is Not Due to Competition of CBP
Competition of CBP, a transcriptional coactivator, is a well known mechanism to exert atRA-induced gene downregulation.¹⁹ To examine whether competition of CBP is important to downregulate the AT₁-R gene transcription, a CBP-expression vector and an AT₁-R promoter-luciferase construct were cotransfected. Overexpression of CBP induced a significant increase in luciferase activity compared with control cells (Figure 5). However, the effect of atRA on AT₁-R promoter activity was preserved in CBP-overexpressing VSMCs (Figure 5). These data suggest that competition of CBP may not be involved in atRA-induced AT₁-R downregulation.

View larger version (16K): [in this window] [in a new window]   Figure 5. Effect of overexpression of CBP on atRA-induced AT1-R mRNA downregulation. AT1-R promoter-luciferase construct (Figure 4A, construct 1), LacZ gene and CBP-expressing plasmid or vector plasmid were introduced to VSMCs according to liposome method. Then, VSMCs were stimulated with atRA (1 µmol/L) or vehicle. Luciferase activity was normalized by ß-galactosidase activity. Relative luciferase activity of vehicle-treated VSMCs transfected with AT1-R promoter-luciferase construct and vector plasmid was set as 100%. Data are shown as mean±SEM (n=4). *P<0.05 vs control. **P<0.01 vs control. #P<0.01.

RAR/RXR-Dependent De Novo Protein Synthesis Is Necessary for atRA-Induced Downregulation of AT₁-R Expression
To examine whether atRA-induced downregulation of AT₁-R expression was dependent on de novo protein synthesis, we used cycloheximide (10 µg/mL). Figure 6A shows that incubation with cycloheximide alone had no significant effect on AT₁-R expression. The addition of cycloheximide inhibited the atRA-induced AT₁-R downregulation (Figure 6A). These data suggest that downregulation of AT₁-R mRNA by atRA requires de novo protein synthesis.

View larger version (43K): [in this window] [in a new window]   Figure 6. RAR/RXR-dependent de novo protein synthesis is necessary for atRA-induced downregulation of AT1-R expression. A, VSMCs were pretreated with or without cycloheximide (10 µg/mL) and then treated with atRA (1 µmol/L) or vehicle for 24 hours. B, VSMCs were also treated with either Ro40-6055 (1 µmol/L) or Ro25-7386 (1 µmol/L) or both for 24 hours. Then, AT1-R mRNA levels were determined through Northern blot analysis. Left, Representative autoradiogram. Right, Radioactivity of AT1-R mRNA bands was counted with a Bioimage Analyzer and normalized with radioactivity of 18S rRNA. Values (mean±SEM) are expressed as a percent of control culture (100%) (n=3). *P<0.05 vs control. **P<0.01 vs control.

The intracellular effects of retinoids are mediated by RAR/RXR heterodimer and RXR/RXR homodimer.¹⁷ To determine which retinoid receptor dimer is responsible for the downregulation of AT₁-Rs, we used an RAR-selective agonist (Ro40-6055) and an RXR-selective agonist (Ro25-7386). Figure 6B shows that the treatment of VSMCs with Ro40-6055 slightly decreased AT₁-R mRNA, whereas the treatment with Ro25-7386 did not show any effect on AT₁-R mRNA expression. The coadministration of Ro40-6055 and Ro25-7386 decreased AT₁-R mRNA comparable to atRA. These data suggest that RAR/RXR heterodimer, but not RXR/RXR homodimer, is responsible for the AT₁-R downregulation.

	Discussion

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In the present study, we demonstrated that atRA reduced the expression of AT₁-Rs in cultured VSMCs at both mRNA and protein levels. The suppression of AT₁-R mRNA by atRA was both time and dose dependent. Because cycloheximide, a protein synthesis inhibitor, blocked the downregulation of AT₁-Rs by atRA (Figure 6A), we speculated that protein synthesis is involved in this process. AT₁-R expression was specifically suppressed by atRA in VSMCs, because atRA did not affect rRNA expression.

The expression of AT₁-R and ACE was significantly increased in atherosclerotic lesions and injured blood vessels.^{12 25} AT₁-R density may be one of the determinants of the response to Ang II. Elevations in Ang II–induced intracellular inositol trisphosphate level^{26 27} and intracellular calcium concentration²⁸ were reported in VSMCs with an increased number of AT₁-Rs. Increased numbers of AT₁-Rs may accelerate the progression of atherosclerosis and neointimal formation after angioplasty.

Estrogen replacement therapy has been known to reduce the risk of cardiovascular diseases.²⁹ Nickenig et al³⁰ reported that estrogen downregulated AT₁-R expression and calcium response to Ang II. The protective effects of estrogen against cardiovascular diseases may be mediated, at least in part, by the AT₁-R downregulation.³⁰ Because atRA also decreased AT₁-R expression, atRA may have similar favorable effects on the cardiovascular system as estrogen replacement treatment. Miano et al²⁰ showed that atRA treatment inhibited the neointimal formation after balloon injury of the rat carotid artery. One of the possible mechanisms is direct growth inhibition of VSMCs by atRA.^{18 31} In addition, our data suggest that AT₁-R downregulation induced by atRA may be involved in this process. Downregulation of AT₁-R may attenuate Ang II–induced expression of extracellular matrices and growth factors and result in the inhibition of neointimal formation.

A decrease in AT₁-R mRNA was observed after a 12-hour exposure to atRA. This delayed response and an inhibition of atRA-induced AT₁-R downregulation by cycloheximide suggest that de novo protein synthesis is necessary for AT₁-R downregulation. However, the exact feature of the synthesized protein responsible for the downregulation of AT₁-R expression remains to be determined. There is no consensus of retinoic acid response element in AT₁-R gene promoter up to -980 bp, suggesting that retinoid receptor downregulates AT₁-R gene expression indirectly rather than directly. It has been shown that atRA-induced downregulation of gene expression requires de novo protein synthesis in other cell lines.^{32 33} In squamous cell carcinoma cell line, atRA downregulates binding protein for fibroblast growth factor via the synthesis of de novo protein that decreases the stability of the binding protein mRNA.³³ atRA-induced suppression of -fetoprotein promoter activity in hepatoma cell is due to reduced expression of hepatocyte nuclear factor that binds to proximal promoter.³⁴ In our study, gel mobility shift assay examining DNA binding protein bound to the AT₁-R proximal promoter showed no difference between nuclear extract from control and that from atRA-treated VSMCs (data not shown). Therefore, it is unlikely that atRA regulates transcriptional factor or factors bound to the proximal promoter.

One of the important signaling pathways of atRA is an inhibition of AP-1 transcription factor activity.^{19 35} Competition of CBP between AP-1 and retinoid receptor results in the reduction in AP-1 activity.¹⁹ AP-1 consensus sequence is also found in the AT₁-R promoter region at position -387 bp.³⁶ However, atRA effectively suppressed the luciferase activity of the deletion construct that does not contain AP-1 site to similar level as that of deletion construct with AP-1 site (Figures 4B and 4C), and overexpression of CBP failed to abolish the atRA-induced reduction in AT₁-R promoter activity (Figure 5). These findings suggest that competition of CBP between AP-1 and retinoid receptor may not be involved in the atRA-induced downregulation on AT₁-R mRNA expression.

Deletion of the DNA segment between -331 to -201 bp resulted in an increase in AT₁-R promoter activity. In this DNA segment, there are consensus cis-DNA elements, such as AP-1 and nuclear factor-B. Although it is not clear whether they are functional, it is possible that they are negative regulatory elements. Alternatively, there may be unknown negative element in the DNA segment.

Expression of AT₁-R is regulated posttranscriptionally by various factors. Nickenig and Murphy²⁴ reported that Ang II–induced AT₁-R downregulation in VSMCs was due to AT₁-R mRNA instability. In contrast, insulin²⁸ and LDL³⁷ significantly increased AT₁-R mRNA stability. We demonstrated that atRA caused a significant decrease in AT₁-R promoter activity without affecting the stability of mRNA. These results suggest that the effect of atRA on AT₁-R gene expression occurs at the transcriptional level rather than at the posttranscriptional level.

It was reported that Ro40-6055 and Ro25-7836 selectively bound to RAR and RXR, respectively.¹⁵ It was also shown that treatment with atRA or both RAR- and RXR-selective agonists induced RAR/RXR heterodimer formation and activated RAR/RXR heterodimer–dependent transcription.^{38 39}

We have shown here that atRA inhibited AT₁-R gene expression. AT₁-R was downregulated by atRA through de novo protein synthesis that is dependent on the RAR/RXR heterodimer. These data provide novel insight into a role of atRA as an important molecule that regulates AT₁-R gene expression and the possible mechanism that suppresses neointimal formation by atRA.

	Acknowledgments

 
This study was supported in part by Kaibara Morikazu-Science Promotion Foundation and Fukuoka and Uehara Memorial Foundation (Tokyo, Japan). We are grateful to M. Kato for his helpful discussion and to N. Iino for her excellent technical assistance.

Received September 13, 1999; first decision October 26, 1999; accepted November 10, 1999.

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