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Cardiovascular Research 2006 72(1):184-190; doi:10.1016/j.cardiores.2006.07.014
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Copyright © 2006, European Society of Cardiology

Telmisartan downregulates angiotensin II type 1 receptor through activation of peroxisome proliferator-activated receptor {gamma}

Ikuyo Imayama, Toshihiro Ichiki*, Keita Inanaga, Hideki Ohtsubo, Kae Fukuyama, Hiroki Ono, Yasuko Hashiguchi and Kenji Sunagawa

Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, 812-8582 Fukuoka, Japan

* Corresponding author. Tel.: +81 92 642 5361; fax: +81 92 642 5374. Email address: ichiki{at}cardiol.med.kyushu-u.ac.jp

Received 2 May 2006; revised 4 July 2006; accepted 6 July 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Telmisartan, an angiotensin II type 1 receptor (AT1R) antagonist, was found to have a unique property: it is a partial agonist of peroxisome proliferator-activated receptor gamma (PPAR{gamma}). Since previous studies have demonstrated that PPAR{gamma} activators suppressed AT1R expression, we examined whether telmisartan affects AT1R expression in vascular smooth muscle cells.

Methods: Vascular smooth muscle cells were derived from the thoracic aorta of Wistar–Kyoto rat. Northern and Western blotting analysis were used to examine AT1R mRNA and protein expression, respectively. The DEAE-dextran method was used for transfection, and the promoter activity of AT1R was examined by luciferase assay.

Results: Telmisartan decreased the expression of AT1R at the mRNA and protein levels in a dose- and time-dependent manner. Decreased AT1R promoter activity with unchanged mRNA stability suggested that telmisartan suppressed AT1R gene expression at the transcriptional level. However, the expression of AT1R was not suppressed by other AT1R antagonists such as candesartan or olmesartan. Since the suppression of AT1R expression was prevented by pretreatment with GW9662, a PPAR{gamma} antagonist, PPAR{gamma} should have participated in the process. The deletion and mutation analysis of the AT1R gene promoter indicated that a GC box located in the proximal promoter region is responsible for the telmisartan-induced downregulation.

Conclusion: Our data provides a novel insight into an effect of telmisartan: telmisartan inhibits AT1R gene expression through PPAR{gamma} activation. The dual inhibition of angiotensin II function by telmisartan – AT1R blockade and downregulation – would contribute to more complete inhibition of the renin–angiotensin system.

KEYWORDS Antihypertensive; Diuretic drugs; Atherosclerosis; Gene expression; Receptors; Renin–angiotensin system


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Angiontensin (Ang) II is a main final effector molecule of the renin–angiotensin system. Physiologically, Ang II plays an important role in controlling the blood pressure and the fluid volume [1]. However, Ang II is also involved in pathological conditions such as renal insufficiency [2], cardiovascular diseases [3] and metabolic disorders [4].

The effect of Ang II are mediated by Ang II receptors and so far two isoforms, type 1 receptor (AT1R) and type 2 receptor (AT2R), have been identified [5]. AT1R mediates most of the traditional effects of Ang II such as vasoconstriction, sodium retention, aldosterone secretion, and cell proliferation [1]. In contrast, AT2R mediates vasodilation and growth inhibition that opposes to the effects of AT1R [6]. However, it was reported that AT2R was hardly detected in blood vessel of adult animal [6].

Ang I converting enzyme inhibitors and AT1R antagonists are clinically used. Many clinical trials have demonstrated that these drugs are beneficial in the treatment of heart failure, renal failure and myocardial infarction. These drugs are also useful in preventing new-onset diabetes mellitus [7] and atrial fibrillation [8]. Telmisartan (Tel), one of the clinically available AT1R antagonists, was recently reported to have a partial agonistic effect on peroxisome proliferator activated receptor gamma (PPAR{gamma}) [9,10]. PPAR{gamma} is a nuclear receptor that regulates specific gene transcription [11]. The target genes of PPAR{gamma} are involved in the regulation of lipid and glucose metabolism [12], and inflammatory responses. Moreover, several studies have demonstrated that PPAR{gamma} activators are effective in preventing atherogenesis [13,14]. Therefore, Tel is focused for its additional therapeutic values in patients with metabolic disorders.

Previously, we and another group reported that activators of PPAR{gamma} such as 15-deoxy-{delta}12,14-prostaglandin J2 and pioglitazone (Pio) decreased the expression of AT1R in vascular smooth muscle cells (VSMCs) [15,16]. We, therefore, examined whether Tel, a partial agonist of PPAR{gamma}, affects the expression of AT1R in a similar way to PPAR{gamma} agonists in VSMCs.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Materials
Tel and olmesartan (Olm) were generous gifts from Boehringer Ingelheim Co. and Sankyo Co., respectively. Candesartan (Can) and Pio were provided by Takeda Pharmaceutical Company. Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO BRL. Bovine serum albumin (BSA) and Actinomycin D (ActD) were purchased from Sigma Chemical Co. Rabbit polyclonal antibody against AT1R [17,18] and {alpha}-tubulin were from Santa Cruz Biotechnology. Mouse polyclonal antibody against pERK and rabbit polyclonal antibody against ERK were from Cell Signaling Technology, Inc. Horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG and anti-mouse IgG) were purchased from VECTOR Laboratories Inc. [{alpha}-32P] dCTP was purchased from Perkin-Elmer Life Sciences.

2.2. Cell culture
All procedures and care of the animals were approved by the Committee on Ethics of Animal Experiments, Kyushu University and this study conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). VSMCs were isolated from the thoracic aorta of Wistar–Kyoto rat by an explant method and maintained in DMEM supplemented with 10% FBS at 37 °C in a humidified atmosphere of 95% air –5% CO2. VSMCs were cultured until grown to confluence, cultured in DMEM with 0.1% BSA for additional 2 days and used in the experiment. Cells between passages 4 and 14 were used.

2.3. Northern blotting
Total RNA was prepared by acid guanidinium thiocyanate–phenol–chloroform extraction method [19]. Total RNA was electrophoresed on a 1.0% agarose, 1.0% formaldehyde gel, transferred to Hybond-N+ membrane (Amersham Biosciences) by a capillary transfer method in 10xSSC (1xSSC is 150 mmol/L of sodium and 15 mmol/L of sodium citrate) buffer overnight. The membrane was cross-linked by a UV cross-linker (Funakoshi Corporation). Prehybridization and hybridization were performed in a buffer containing 50% formamide, 5xSSC, 80 mmol/L sodium phosphate (pH 7.5), 2xDenhardt's Solution, 1% SDS, and 100 µg/L of heat-denatured salmon sperm DNA for 1 h and 16 h, respectively, at 42 °C. An ECORI fragment of the third exon of rat AT1A gene [20] and ribosomal RNA were labeled with 32P by a Random Primer DNA Labeling Kit Ver.2 (Takara Bio Inc.) and used as a probe after heat denaturation. The hybridized membrane was washed twice with 2xSSC at room temperature, followed by two washes with 2xSSC/1% SDS for 30 min at 55 °C. The membrane was then exposed to a KODAK BioMax XAR Film at –80 °C. The hybridized membrane was stripped by boiling in 0.5% SDS solution and hybridized to a 32P-labeled ribosomal RNA probe to obtain reference for the amount of applied RNA. Autoradiography was scanned and analyzed by a MacBAS Bioimage Analyzer (Fuji Photo Film Co). To analyze mRNA stability of AT1R, Actinomycin (Act) D (5 µg/mL) was added after 6 h of stimulation with Tel (10 µmol/L). In a control experiment, only ActD was added. Cells were harvested after 3, 6, 12, and 24 h of addition of ActD and expression level of AT1R mRNA was examined by Northern blot analysis.

2.4. Measurement of AT1R gene promoter activity
Five deletion mutants of AT1A gene promoter were prepared by digestion with restriction endonucleases and ligated to luciferase gene [21]. Confluent VSMCs were split by trypsin/EDTA solution and cells were prepared in a 6 cm tissue culture dish. At 80% confluence, 5 µg of AT1 promoter-luciferase fusion DNA and 2 µg of β-galactosidase gene were introduced to VSMC by the DEAE-dextran method according to the manufacturer's instruction (Promega Corporation). The cells were cultured in DMEM with 10% FBS for 18 h, washed twice with phosphate buffered saline, cultured in DMEM with 0.1% BSA for 24 h and stimulated with Tel (10 µmol/L) for 12 h. Then, the cells were lysed in 200 µL of Reporter lysis buffer (Promega Corporation). 100 µL of lysate was used for luciferase activity assay in a Lumat luminometer (LB 9501, Berthold, Germany). The β-galactosidase activity in the same sample was measured spectrophotometrically according to Sambrook et al. [22] and used to normalize the luciferase activity.

The AT1R promoter-luciferase construct with mutation in the GC-box-related sequence (wild type: TGCAGAGCAGC GACGCCCCCTAGGC mutant: TGCAGAGCAGCGA CGTTTCCTAGGC) was a generous gift from Dr. Sugawara (Tohoku University) [16].

2.5. Western blot analysis
VSMCs were lysed in a lysis buffer containing RIPA (100 mM sodium, 60 mM Na2HPO4, 100 mM NaF 10 mM EDTA, and 20 mM Tris), 1% aprotinin, 0.5% pepstatin A, 1 mmol/L PMSF, and 0.05% leupeptin. Protein concentrations were determined with the bicinchoninic acid protein assay kit (Pierce Chemical Co). Cell lysates were heated in a sample buffer (62.5 mmol/L Tris–HCl [pH 6.8], 10% glycerol, 2% SDS, 0.05% bromophenolblue, and 715 mmol/L 2-mercaptoethanol) at 95 °C for 3 min, electrophoresed on 12% SDS-polyacrylamide gel, and transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore). The blots were blocked with TBS-T (20 mmol/L Tris–HCl [pH 7.6], 137 mmol/L NaCl, 0.1% Tween 20) containing 5% skim milk at room temperature for 30 min. The AT1R protein expressions were detected by ECL chemiluminescence (Amersham Pharmacia Biotech) according to the manufacturer's instructions. The membranes were exposed to X-ray film. The membranes were stripped by incubating them in a buffer containing 62.5 mmol/L Tris–HCl, 2% SDS, and 100 mmol/L 2-mercaptoethanol at 50 °C for 30 min and reprobed with an antibody against {alpha}-tubulin by the same procedure. Phospholylated ERK and ERK (which recognizes both phosphorylated and nonphosphorylated forms) were examined by the same method.

2.6. Statistical analysis
Statistical analysis was performed with 1- or 2-way ANOVA and Fisher test, if appropriate. Statistical significance was designated as P<0.05. Values are expressed as mean±S.E.M.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Tel reduced the expression of AT1R
VMSCs were incubated with Tel (10 µmol/L) for various periods. The expression level of AT1R mRNA was gradually decreased with a peak suppression at 6 h of incubation (Fig. 1). Though we did not remove Tel from the medium, the expression of AT1R demonstrated a transient suppression. The mechanism of its recovery at 12 h of incubation is unknown. However, there are some reports that demonstrated a transient or a biphasic gene expression induced by thiazolidinediones (TZDs) [23,24], which seems to be consistent with our results. Western blot analysis revealed that Tel reduced AT1R protein level with a peak reduction at 12 h of incubation (Fig. 2A), and that Tel suppressed AT1R expression in a dose-dependent manner (Fig. 2B). As shown in Fig. 2C, preincubation with Tel at the same concentration as used in Fig. 2B almost completely inhibited the Ang II-induced ERK phosphorylation.


Figure 1
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  		Fig. 1 Telmisartan (Tel) suppressed AT1R mRNA expression in VSMCs. VSMCs were incubated with Tel (10 µmol/L) for various periods as indicated in the figure. Total RNA was isolated and expression of AT1R mRNA and 18S rRNA (rRNA) was determined by Northern blot analysis. Radioactivity of AT1R mRNA was measured with an imaging analyzer and was normalized by radioactivity of rRNA. Values (mean±S.E.M.) are expressed as a percent of control culture in the bar graph (100%) (n=5). *P<0.05 vs control (c).

 

Figure 2
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  		Fig. 2 Suppression of AT1R protein by telmisartan (Tel) in VSMCs. (A) VSMCs were incubated with Tel (10 µmol/L) for various periods as indicated in the figure. (B) VSMCs were incubated with Tel at concentrations varying from 1 to 20 µmol/L for 12 h. Expression of AT1R protein and {alpha}-tubulin was detected by Western blot analysis. The density of the specific band was scanned and quantified with an imaging analyzer. The ratio of AT1R to {alpha}-tubulin is shown in the bar graph. (C) VSMCs were incubated with Tel at various concentrations as indicated in the figure and stimulated by Ang II (100 µmol/L). Expressions of pERK and ERK protein were detected by Western blot analysis. The density of the specific band was scanned and quantified with an imaging analyzer. The ratio of pERK to ERK is shown in the bar graph. Values (mean±S.E.M.) are expressed as a percent of control (c) culture (100%) (n=5). *P<0.05 vs control. {dagger} P<0.01 vs control.

 
Following experiment used 10 µmol/L of Tel, which is the minimal dose that suppressed AT1R expression. Pio, one of TZDs and a full PPAR{gamma} agonist, inhibited AT1R expression as previously described [16]. We examined the effect of other AT1R antagonists on AT1R expression. Can (Fig. 3) and Olm (data not shown) had no effect on AT1R expression.


Figure 3
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  		Fig. 3 Suppression of AT1R protein by telmisartan (Tel) but not by candesartan (Can) in VSMCs. VSMCs were incubated with Tel (10 µmol/L, 12 h), Can (10 µmol/L, 12 h), and Pio (10 µmol/L, 6 h). Expression of AT1R protein and {alpha}-tubulin was determined by Western blot analysis. Densitometric analysis was performed as described in the legend to Fig. 2. Values (mean±S.E.M.) are expressed as a percent of control (c) culture (100%) (n=5). *P<0.05 vs control. {dagger} P<0.01 vs control.

 
3.2. Tel inhibits AT1R expression at the transcriptional level
Deletion mutants of AT1 promoter/luciferase fusion DNA were used to locate the response element responsible for Tel-induced AT1R suppression (Fig. 4A). The suppression was observed in all constructs from –980/+25-luc to –61/+25-luc, so we supposed that the response element may exist in the DNA segment between –61 bp and +25 bp. Since Sugawara et al. [16] had previously reported the crucial role of a GC-box-related sequence within the –58/–34 region of the AT1R gene promoter in PPAR{gamma}-induced AT1R suppression, we hypothesized that the same site may also be important in Tel-induced suppression. The luciferase construct with mutation in GC box (Sp1 site) failed to respond to Tel (Fig. 4A), indicating the important role of Sp1 site in Tel-induced downregulation. In addition, Tel did not affect the degradation rate of AT1R mRNA (Fig. 4B). These data suggested that Tel inhibits AT1R gene transcription and does not affect AT1R mRNA stability.


Figure 4
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  		Fig. 4 Effect of telmisartan (Tel) on AT1R gene promoter activity and AT1R mRNA stability. (A) The scheme of deletion mutants of AT1R promoter/luciferase fusion DNA construct and SP-1 mutant construct is indicated. These luciferase constructs were introduced to VSMCs with LacZ expression plasmid by the DEAE-dextran method. Then VSMCs were stimulated with Tel (10 µmol/L) for 12 h. Relative luciferase activity of unstimulated VSMCs (control) was set as 100%. Solid and open bars indicate the relative luciferase activity of unstimulated and Tel-stimulated VSMCs transfected with the same construct indicated in the left panel, respectively. Values (mean±S.E.M.) are expressed as a percent of control culture (n=6). *P<0.05 vs unstimulated cells. n.s. not siginificant. (B) VSMCs were incubated with Tel (10 µmol/L) for 6 h and then ActD (5 µg/mL) was added. In a control experiment, only ActD was added to the medium. Total RNA was isolated at the indicated time after ActD supplementation and expression levels of AT1R mRNA and rRNA were determined with method described in the legend to Fig. 1. Expression level of AT1R mRNA was normalized with that of rRNA. The normalized AT1R mRNA expression before addition of ActD in each group was set as 100% (c), (n=3). n.s. not siginificant.

 
3.3. Tel-induced AT1R downregulation is PPAR{gamma} dependent
To examine the role of PPAR{gamma} in Tel-induced AT1R suppression, we examined the effect of GW9662, a PPAR{gamma} antagonist. Although GW9662 itself did not affect AT1R expression, preincubation with GW9662 blocked AT1R suppression induced by Tel (Fig. 5).


Figure 5
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  		Fig. 5 The effect of GW9662, a PPAR {gamma} antagonist, on telmisartan (Tel)-induced AT1R downregulation. VSMCs were preincubated with GW9662 (5 µg/mL) for 30 min and incubated with or without Tel (10 µmol/L) for 12 h. Expression of AT1R protein and {alpha}-tubulin was determined by Western blot analysis. Densitometric analysis was performed as described in the legend to Fig. 2. Values (mean±S.E.M.) are expressed as a percent of control culture (100%) (n=5). *P<0.05 vs control. n.s. not significant.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
In the present study, we demonstrated that Tel, an AT1R antagonist, suppressed the AT1R expression through the PPAR{gamma}-mediated pathway. This is the first study that demonstrates the suppression of AT1R expression by AT1R antagonist in VSMCs.

The expression of AT1R was suppressed at both mRNA and protein levels. The results of the promoter assay and the mRNA stability assay suggested that the suppression occurred at the transciptional level rather than the post-transcriptional level.

The involvement of PPAR{gamma} on AT1R suppression was confirmed by experiments using GW9662. GW9662 prevented the Tel-induced suppression of ATR expression, indicating the critical role of PPAR{gamma} in this pathway. The ability to activate PPAR{gamma} is reported to be unique to Tel and irbesartan (Irb) among several AT1R antagonists [10]. In our study, though we have not examined the effect of Irb, Can (Fig. 3) and Olm (data not shown) had no effect on AT1R expression. Our results seem to be consistent with the previous report [10].

Schupp et al. reported that a subset of AT1R blockers (ARB), Tel and Irb, induced PPAR{gamma} activity and promoted PPAR{gamma}-dependent differentiation in 3T3-L1 adipocytes [9]. These ARB activated PPAR{gamma} by direct interaction with the ligand binding domain (LBD) of PPAR{gamma} [9].

The docking studies of the molecular binding model explained the difference in the ability to activate PPAR{gamma} among several ARB and full agonists of PPAR{gamma} by comparing their interaction with residues of several helixes [10]. According to this model, Tel fits in the LBD of PPAR{gamma} surrounded by helixes H3, H6, and H7. However, Tel does not interact with activation function-2 helix that is responsible for receptor activation and stabilization by full PPAR{gamma} agonists. Irb, Can, and Olm, so-called tetrazole-containing ARBs, made contact with helix H3 but not H7. The difference in the interaction with these helixes of LBD of PPAR{gamma} might contribute to their potential to activate PPAR{gamma}.

On activation by a ligand, PPAR{gamma} regulates the expression of several genes involved in lipid and carbohydrate metabolism [25,26] and inflammatory responses [27]. From the molecular insight, these effects are basically due to two different transcriptional regulatory mechanisms, one is transactivation and the other is transrepression. Transactivation is dependent on PPAR{gamma} response element (PPRE). Upon activation, PPAR{gamma} forms a heterodimer with retinoid X receptor and binds to PPRE in the promoter region of the target genes such as CD36 and glucose transporter 4. In contrast, transrepression involves interference with other transcription factors such as NF-{kappa}B and AP-1. Because there is no consensus sequence of PPRE in the AT1R gene promoter up to –980 bp, the suppression of AT1R gene transcription should have occurred through the latter mechanism.

This assumption is well substantiated by a previous study which reported that suppression of AT1R gene expression by PPAR{gamma} activation was independent of PPRE [16]. The authors demonstrated that the –58/–34 region of the AT1R gene promoter, including Sp1 binding site, was essential to the PPAR{gamma} activator-induced AT1R suppression. They concluded that activated PPAR{gamma} inhibited Sp1 function by direct protein–protein interaction. Our study with Tel also demonstrated the essential role of Sp1 binding site in the suppression of AT1R expression. Therefore, Tel may inhibit Sp1 function through the activation of PPAR{gamma} resulting in downregulation of the AT1R expression.

Our previous studies showed that downregulation of AT1R by PPAR{gamma} activator attenuated cellular response to Ang II [15]. However, Ang II induced ERK activation was almost completely blocked by Tel even at 1 µmol/L, which did not affect AT1R expression because of AT1R blocking effect (Fig. 2C). Therefore, AT1R binding effect is expected at the lower concentration of Tel and dual effect of AT1R binding and AT1R downregulation is expected at higher concentration.

TZDs, synthetic PPAR{gamma} activators, are reported to inhibit atherogenesis by regulating various gene expressions. Several studies have demonstrated the anti-atherogenic effects of PPAR{gamma} activators in both animal models and human. Rosiglitazone (Rosi), one of TZDs, was shown to have additive effects on plaque regression in the combination treatment with simvastatin in an atherosclerotic rabbit model [28]. Anti-atherogenic effect of Rosi was also reported in a diabetes–atherosclerosis mouse model [29]. AT1R antagonists are reported to suppress atherogenesis. Strawn et al. demonstrated that losartan attenuated atherogenesis in monkeys with hypercholesterolemia [30]. Based on these studies, Tel may be more efficient in suppressing atherosclerotic vascular diseases due to its properties of PPAR{gamma} activation and AT1R antagonism.

TZDs are also effective in improving insulin sensitivity and they are already utilized to treat patients with type 2 diabetes [31]. Although the precise mechanism for PPAR{gamma}-mediated insulin sensitization is not clear, TZDs reduced fasting and postprandial glucose concentration, and insulin level. Ang II also affects insulin sensitivity [32]. The inhibition of AT1R by losartan improved insulin sensitivity [33] and, in the LIFE study, losartan significantly reduced the incidence of new-onset diabetes in patients with hypertension compared with atenolol [34]. Therefore, the dual effects of Tel may synergistically improve insulin sensitivity.

The dual function of Tel, an AT1R antagonist and a partial agonist of PPAR{gamma}, may be quite useful for the treatment of patients with hypertension with complications such as diabetes and atherosclerosis. Considering the blockade of Ang II, the downregulation of AT1R through the activation of PPAR{gamma} adds further possibility of Tel. This may result in more complete inhibition of the Ang II. However, it remains to be determined whether Tel suppresses the expression of AT1R in vivo and is superior to other AT1R antagonists in terms of the inhibition of the progression of cardiovascular diseases. Further study is needed.


   Acknowledgements
 
This study was supported in part by grants from Takeda Science Foundation, Kimura Memorial Heart Foundation Research Grant for 2005, and Grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (17590742) to T.I.


   Notes
 
Time for primary review 26 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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