Published online before print November 11, 2002, doi: 10.1161/01.HYP.0000042096.17141.B1
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(Hypertension. 2002;40:853.)
© 2002 American Heart Association, Inc.
Scientific Contributions |
Effect of AT2 Receptor on Expression of AT1 and TGF-ß Receptors in VSMCs from SHR
From the Second Department of Internal Medicine, Nihon University School of Medicine (J.-Z.S., N.F., X.-Q.J., Y.-M.L., R.S., Y.T., H.T., Y.I., K.K.), Tokyo, Japan; and Gene Experiment Center, University of Tsukuba (H.M.), Ibaraki, Japan.
Correspondence to Noboru Fukuda, MD, PhD, Second Department of Internal Medicine, Nihon University School of Medicine, Ooyaguchi-kamimachi 30-1, Itabashi-ku, Tokyo 173-8610, Japan. E-mail nhukuda{at}med.nihon-u.ac.jp
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Abstract |
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We recently reported that overexpression of the angiotensin II type 2 (AT2) receptor downregulates the AT1a receptor through the bradykinin/NO pathway in a ligand-independent manner in vascular smooth muscle cells (VSMCs). In the present study, we investigated the effect of AT2 receptor overexpression on the expression of the AT1a receptor and transforming growth factor-ß (TGF-ß) receptor subtypes in VSMCs from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Transfection of the AT2 receptor gene downregulated expression of the AT1a receptor in VSMCs from WKY, but did not affect expression of the AT1a receptor in VSMCs from SHR. Transfection of the AT2 receptor abolished DNA synthesis in response to angiotensin II in VSMCs from WKY; in VSMCs from SHR, basal DNA synthesis was suppressed, but DNA synthesis in response to Ang II was not altered. The NO substrate L-arginine augmented downregulation of the AT1a receptor in VSMCs from WKY, whereas it did not affect expression of the AT1a receptor in VSMCs from SHR. In response to AT2 receptor transfection, expression of TGF-ß type I receptor mRNA was suppressed significantly in VSMCs from WKY, whereas expression of TGF-ß type I receptor was not altered in VSMCs from SHR. These results suggest that the AT2 receptor downregulates AT1a and TGF-ß type I receptors in normal VSMCs, but not in SHR-derived VSMCs. The lack of downregulation of the AT1a receptor may contribute, in part, to the exaggerated growth of VSMCs from SHR.
Key Words: receptors, angiotensin II • muscle, smooth, vascular • rats, spontaneously hypertensive
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Introduction |
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Vascular smooth muscle cells (VSMCs) from normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) exhibit distinct growth phenotypes in culture.1 SHR-derived VSMCs show exaggerated growth compared with those from WKY.2 We recently reported that VSMCs from SHR generate angiotensin (Ang) II in homogeneous cell culture by changing to the synthetic phenotype.3,4 We also showed that endogenous Ang II increases the expression of growth factors including transforming growth factor-ßI (TGF-ß type I), which contributes to the exaggerated growth of VSMCs from SHR.5
At least 2 distinct subtypes of Ang II receptors have been identified, type 1 (AT1) and type 2 (AT2).6,7 Most of the known effects of Ang II in adult tissues are attributable to the AT1 receptor. The AT2 receptor appears to exert opposite effects in terms of cell growth and blood pressure regulation.8 The AT2 receptor is expressed at very low levels in the aorta during early embryonic development, and at high levels during the late stages of development and in neonate.9 After birth, AT2 receptor levels decline rapidly.9 Increasingly, data have shown that AT2 receptors exert antigrowth, antihypertrophic, and proapoptotic effects.8 We10 previously transfected the AT2 receptor gene into cultured rat VSMCs to investigate the direct interaction between AT2 and AT1 receptors and found that overexpression of the AT2 receptor downregulates expression of the AT1a receptor through the bradykinin/NO pathway. Ang II induces expression of TGF-ß in multiple cell lines.11 We reported previously that Ang II also regulates TGF-ß receptors to regulate VSMC growth.12 We also demonstrated the expression of distinct TGF-ß receptor subtypes13 and abnormal regulation of TGF-ß receptors in VSMC from SHR relative to those from WKY.14 These differences in TGF-ß receptors may be associated with the exaggerated growth of VSMCs from SHR.
In the current study, we investigated the effect of AT2 receptor overexpression on the expression of AT1a and TGF-ß receptors in VSMCs from SHR and WKY.
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Methods |
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This study conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication 93-23, revised 1985).
Vectors
A 2.6-kb genomic DNA fragment containing the entire coding region of the rat AT2 receptor was cloned into the mammalian expression vector pcDNA3 (Invitrogen Japan KK). pcDNA3 alone was used as a control.
Cell Culture
Cultured VSMCs were grown from explants of aortic media of 4-week-old Wistar-Kyoto/Izumo rats and SHR/Izumo (SHR Corporation, Funabashi, Japan) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% calf serum (GIBCO), 100 U/mL penicillin, and 100 µg/mL streptomycin. The hill-and-valley pattern was observed as typical of cultured smooth muscle cells when cells reached confluence. The purity of VSMCs was further confirmed by immunofluorescence with an anti–
-smooth muscle actin antibody that showed >95% positive staining of the cultured cells. VSMCs were passaged by trypsinization with 0.02% EDTA and 0.05% trypsin (GIBCO) in Ca2+- and Mg2+-free Dulbecco’s PBS and incubated in 75-cm2 tissue culture flasks at a density of 105 cells/mL. Experiments were performed by using 3 to 5 passages.
Establishment of Quiescence
Trypsinized cells were plated into 24- or 6-well culture dishes (Corning Inc) at a density of 105 cells/cm2. Cells were allowed to grow in DMEM containing 10% calf serum for 24 hours, and the culture medium was then changed to DMEM with 0.2% calf serum. Cells were then incubated in this medium for 48 to 72 hours to establish quiescence.
AT2 Receptor Gene Transfer
Quiescent cells were transfected with the AT2 receptor gene expression vector by the lipofectin (GIBCO) method according to the manufacturer’s instructions. Briefly, 2 µg of plasmid DNA was diluted into 100 µL of serum-free DMEM as solution A; 2 µL of lipofectin reagent was diluted into 100 µL of serum-free DMEM as solution B. Solution A and solution B were allowed to stand at room temperature for 45 minutes. The solutions were then combined, mixed gently, and incubated for an additional 15 minutes at room temperature. For each transfection, 0.8 mL of serum-free medium was added to each tube containing the lipofectin reagent–DNA complexes, mixed gently, and then overlayed onto the cells. Cells were incubated for 24 hours at 37°C in an incubator. pcDNA3 alone was used to eliminate the possibility of nonspecific actions of the vector.
Reverse-Transcription and Polymerase Chain Reaction Analysis
Quiescent VSMCs were washed with PBS and lysed in 800 µL of RNAzol B (Biotecx Laboratories, Inc). Each sample was mixed with 80 µL of chloroform by vortexing for 15 seconds, kept on ice for 15 minutes, and centrifuged at 12 000g for 15 minutes to extract total RNA. The colorless upper aqueous phase was mixed with an equal volume of isopropanol, allowed to stand at -20°C for 45 minutes, and centrifuged at 12 000g for 15 minutes at 4°C to precipitate the RNA. The RNA pellet was washed twice with 500 µL of 75% ethanol and centrifuged at 12 000g for 8 minutes at 4°C, dried, and dissolved in 10 µL of TE buffer (10 mmol/L Tris-HCl at pH 8.0, 1 mmol/L EDTA). After denaturing at 65°C for 15 minutes, the RNA sample was treated with 0.5 U of DNase (GIBCO) in 0.5 µL of DNase buffer (20 mmol/L Tris-HCl at pH 8.3, 50 mmol/L KCl, and 2.5 mmol/L MgCl2) at room temperature for 45 minutes. The DNase was inactivated by addition of 0.5 µL of 20 mmol/L EDTA and heating at 98°C for 10 minutes.
Reverse transcription–polymerase chain reaction (RT-PCR) was performed as described previously.15 Briefly, aliquots of RNA (1 µg/20 µL) were reverse-transcribed into single-stranded cDNA with 0.25 U/µL avian myeloblastoma virus reverse transcriptase (Life Sciences Inc) in 10 mmol/L Tris-HCl (pH 8.3), 5 mmol/L MgCl2, 50 mmol/L KCl, 1 µmol/L deoxy-NTPs, and 2.5 µmol/L random hexamers. Five microliters of the diluted cDNA product was mixed with 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 4 mmol/L MgCl2, 0.025 U/mL Taq DNA polymerase (Takara Biochemicals), and 0.2 mmol/L each of the upstream sense primer and downstream antisense primer in a total volume of 25 µL. Sense primer (5'-CGTCATCCATGACTGTAAAATTTC-3') and antisense primer (5'-GGGCATTACATTGCCAGTGTG-3') were used for PCR amplification of the AT1a receptor to yield a 198-bp product. Sense primer (5'-CTTCAGCCTGCATTTAAAGG-3') and antisense primer (5'-CTGAGCTTCCCACACGCACT-3') were used for PCR amplification of the AT2 receptor to yield a 306-bp product. Sense primer (5'-GCTCTAGATTTCTGCCACCTCTGTAC-3') and antisense primer (5'-GCGAATTCGACAGTGCGGTTATGGCA-3') were used for PCR amplification of the TGF-ß type I receptor to yield a 329-bp product. Sense primer (5'-AAGTCTTGCATG-AGCAACTGC-3') and antisense primer (5'-GACGTCA-GAGAAGATGTCC-3') were used for PCR amplification of the TGF-ß type II receptor to yield a 699-bp product. Sense primer (5'-CGACGACCCATTCGA-ACGTCT-3') and antisense primer (5'-GCTATTGGAGCAT-GGAATTACCG-3') were used for PCR amplification of the 18S ribosomal RNA, which served as an internal control, to yield a 312-bp product. After initial denaturation at 96°C for 5 minutes, PCR amplification was performed as 35 cycles of 94°C for 1 minute, 64°C for 1 minute, and 72°C for 1 minute for the AT1a receptor; 30 cycles of 94°C for 1 minute, 55°C for 1 minute, and 72°C for 2 minutes for the AT2 receptor; and 30 cycles of 96°C for 45 seconds, 58°C for 45 seconds, and 72°C for 2 minutes for the TGF-ß type I and TGF-ß type II receptors. PCR using primers for the 18S ribosomal RNA was included in each reaction as an internal control. To confirm that no genomic DNA was co-amplified by PCR, control RT-PCR experiments without reverse transcriptase were performed. In all cases, no product was amplified. PCR was performed by using a DNA Thermal Cycler (Perkin-Elmer Cetus). For semiquantitative analysis of mRNA levels, the kinetics of the PCR reaction were monitored; the number of cycles at which each PCR product became visible on the gel was compared between the different samples.16 Serial 10-fold dilutions of cDNA (100, 10, and 1 ng) were amplified; the PCR products were visible after less cycles with increasing amounts of cDNA. PCR products were separated by electrophoresis on 1.5% agarose gels, stained with ethidium bromide, and visualized by UV illumination.
Western Blot Analysis
Quiescent VSMCs at a density of 105 cells/cm2 in 6-well culture dishes were transfected with pcDNA3 containing AT2 receptor cDNA, as described above, for 24 hours in serum-free DMEM and then incubated with 0.1 µmol/L Ang II for 24 hours. Cells were washed with PBS and incubated in lysis buffer (50 mmol/L Tris-HCl at pH 8.0, 150 mmol/L NaCl, 0.02% sodium azide, 100 µg/mL phenylmethylsulfonyl fluoride, 1 µg/mL aprotinin, 1% Triton X-100). Samples were dissolved in 20 µL of sample buffer, boiled, and subjected to 10% polyacrylamide gel electrophoresis. The proteins were then transblotted to nitrocellulose membranes. After blocking with 100% Block Ace (Dainippon Pharmaceutical Co) at 4°C overnight, the membranes were incubated with mouse monoclonal antibodies specific for the AT1a and AT2 receptors (Alpha Diagnostic International) and diluted in 200 volumes of TBST solution (10 mmol/L Tris-HCl at pH 8.0, 150 mmol/L NaCl, 0.05% Tween 20) containing 15% Block Ace at room temperature for 3 hours. After the blots were washed with TBST twice for 10 minutes, the membranes were incubated with goat anti-mouse IgG (BioRad Laboratories) diluted in 3000 volumes of TBST containing 15% Block Ace at room temperature for 1 hour, washed with TBST for 10 minutes 3 times, then visualized with the ECL method.10 Membranes were reprobed with mouse monoclonal antibody specific for -tubulin (Sigma Chemical Co) as an internal control. Antisera to AT1 and AT2 receptor showed no reactivity with other G protein–coupled receptors.
Determination of DNA Synthesis
[3H]Thymidine incorporation into newly synthesized DNA was determined as described previously.17 Transfected cells were incubated with 0.01 to 1.0 µmol/L Ang II for 24 hours. The medium was then changed to DMEM containing [3H]thymidine (0.5 µCi/mL; NEN Research Products), and cells were incubated for 2 hours. Each well was then washed with 1 mL of 150 mmol/L NaCl to remove excess [3H]thymidine, and the cells were fixed in 1 mL of ethanol/acetic acid (3:1) solution for 10 minutes. After washing with 1 mL of H2O, acid-insoluble material was precipitated with 1 mL of ice-cold perchloric acid, and DNA was extracted into 1.5 mL of perchloric acid by heating at 90°C for 20 minutes. The perchloric acid containing solubilized DNA was transferred to a scintillation vial, and the radioactivity was measured with a liquid scintillation spectrometer.
Statistical Analysis
Results are given as the mean±SEM. The significance of differences between mean values was evaluated by Student t test for unpaired data and by 2-way ANOVA followed by Duncan multiple-range test.
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Results |
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Expression of AT2 and AT1a Receptor mRNAs in VSMCs From SHR and WKY After AT2 Receptor Transfection
Expression of AT2 and AT1a receptor mRNAs after AT2 receptor transfection in VSMCs from SHR and WKY is shown in Figures 1A and 1B. Basal expression of the AT2 receptor was low in VSMCs from both rat strains. Levels of AT2 receptor mRNA significantly (P<0.01) increased after transfection (Figure 1A). Transfection of the AT2 receptor significantly (P<0.01) decreased the abundance of AT1a receptor mRNA in VSMCs from WKY. Transfection of the AT2 receptor did not affect the abundance of AT1a receptor mRNA in VSMCs from SHR (Figure 1B).
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Expression of AT2 Receptor and AT1a Receptor Proteins in VSMCs From SHR and WKY After AT2 Receptor Transfection
Expression of AT2 receptor and AT1a receptor proteins after AT2 receptor transfection in the absence or presence of Ang II is shown in Figure 2. Levels of AT2 receptor protein significantly (P<0.05) increased in VSMCs from both rat strains after AT2 receptor transfection. Levels of AT1a receptor protein significantly (P<0.05) decreased in VSMCs from WKY after transfection in the presence or absence of Ang II, whereas AT2 receptor transfection did not affect levels of AT1a receptor protein in VSMCs from SHR.
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Effect of L-Arginine on Expression of AT1a Receptor mRNA in VSMCs From SHR and WKY After AT2 Receptor Transfection
L-arginine alone (150 mmol/L) did not affect AT1a receptor mRNA levels in VSMCs from WKY. However, the same dose of L-arginine significantly (P<0.05) augmented the decrease in AT1a receptor mRNA levels in VSMCs from WKY after AT2 receptor transfection (Figure 3), whereas L-arginine did not affect AT1a receptor mRNA levels with or without AT2 receptor transfection of in VSMCs from SHR.
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Expression of TGF-ß Type I and TGF-ß Type II Receptor mRNAs in VSMCs From SHR and WKY After AT2 Receptor Transfection
Expression of TGF-ß type I and TGF-ß type II receptor mRNAs in VSMCs from SHR and WKY after AT2 receptor transfection is shown in Figures 4A and 4B. Basal expression of TGF-ß type I receptor was significantly (P<0.05) lower in VSMCs from SHR compared with WKY. Basal expression of TGF-ß type II receptor was significantly (P<0.05) higher in VSMCs from SHR compared with WKY. Transfection of the AT2 receptor significantly (P<0.01) decreased abundance of TGF-ß type I receptor mRNA in VSMCs from WKY but did not affect the abundance in VSMCs from SHR (Figure 4A). Transfection of the AT2 receptor significantly (P<0.05) increased the abundance of TGF-ß type II receptor mRNA in VSMCs from SHR but did not affect the abundance of TGF-ß type II receptor mRNA in VSMCs from WKY (Figure 4B).
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DNA Synthesis in VSMCs From SHR and WKY After AT2 Receptor Transfection
Ang II (0.01 to 1 µmol/L) increased DNA synthesis in a dose-dependent manner in VSMCs from both rat strains. After transfection of the AT2 receptor, basal DNA synthesis was decreased significantly (P<0.05) in VSMCs from both rat strains. The response of DNA synthesis to Ang II was abolished in VSMCs from WKY, whereas the response of DNA synthesis to Ang II was not altered in VSMCs from SHR after transfection of the AT2 receptor gene (Figure 5).
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Discussion |
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We have demonstrated that overexpression of the AT2 receptor downregulates expression of the AT1a receptor accompanied with increased expression of bradykinin and inducible NO in rat VSMCs. Bradykinin B2 receptor antagonist and NO synthase inhibitor inhibited the downregulation of AT1a receptor by the overexpression of AT2 receptor in VSMCs, and L-arginine augmented the downregulation of AT1a receptor. These findings indicated that the overexpression of AT2 receptor downregulates AT1a receptor in VSMCs that is mediated through the bradykinin/NO pathway. In addition, this downregulation was observed in independent of the presence of Ang II, indicating that it occurs in a ligand-independent manner.
In the present study, overexpression of the AT2 receptor downregulated the AT1a receptor in VSMCs from WKY but not in cells from SHR. Overexpression of the AT2 receptor abolished DNA synthesis in response to Ang II in VSMCs from WKY; in VSMCs from SHR, basal DNA synthesis was suppressed, but DNA synthesis in response to Ang II was not altered. These phenomena are considered to be results by downregulation of the AT1a receptor in VSMCs from WKY, and by lack of downregulation of the AT1a receptor in VSMCs from SHR.
The AT2 receptor has been reported to mediate the renal production of bradykinin and NO.18,19 In addition, AT1 receptor expression has been shown to be transcriptionally suppressed by NO.20 We recently reported that downregulation of the AT1a receptor in VSMCs in response to overexpression of the AT2 receptor is abolished by the NO synthesis inhibitor NG-nitro-L-arginine methyl ester, indicating that AT1a receptor downregulation is mediated by NO in these cells. In the present experiments, the NO substrate L-arginine augmented downregulation of the AT1a receptor in VSMCs from WKY, whereas L-arginine had no effect in VSMCs from SHR, suggesting that the mechanism underlying the lack of downregulation of AT1a receptor in VSMCs from SHR may be owing to an insensitivity of the AT1a receptor to NO. It has been reported that NO donors such as sodium nitroprusside do not increase cyclic GMP in cardiomyocytes from SHR,21 and that NO donor-induced vasodilation is impaired in carotid arteries from SHR.22
VSMCs from SHR show exaggerated growth with a higher specific growth rate, abnormal contact inhibition, and accelerated entry into the S phase of the cell cycle.1,2 These phenomena are associated with the changes in VSMCs from the contractile to the synthetic phenotype.4 During phenotypic modulation, VSMCs show changes in morphology, cell function, and biochemical characteristics.23 We recently demonstrated that VSMCs from SHR, even at the prehypertensive stage, show exaggerated growth and produce Ang II and increased expression of several enzymes, adhesion molecules, and cytokines analyzed by the microarray,24 suggesting that these alterations are independent of hypertension and are potentially associated with genetic abnormalities. The insensitivity to NO and the lack of downregulation of the AT1a receptor by overexpression of the AT2 receptor gene in VSMCs from SHR may be associated with these abnormalities.
Ang II and TGF-ß play important roles in vascular remodeling in hypertension. In this process, they may interact at various levels, including that of receptor regulation.25 TGF-ß type I and TGF-ß type II receptors mediate the biological activities of TGF-ß.26,27 The TGF-ß type I receptor is a transmembrane protein kinase that associates with the type II receptor to generate diverse heteromeric serine-threonine kinase complexes with diverse signaling capacities.26,27 TGF-ß type II receptor acts upstream of the type I receptor; only the type II receptor recognizes TGF-ß as a ligand, whereas the type I receptor recognizes the ligand-bound type II receptor.28
We have reported that Ang II regulates TGF-ß receptors on VSMCs.12 We found that SHR-derived VSMCs show distinct expression of TGF-ß receptor subtypes and abnormal regulation of these subtypes by Ang II compared with cells from WKY. SHR-derived VSMCs predominantly express TGF-ß type II receptor to induce platelet-derived growth factor A-chain stimulation of VSMC growth.13 We demonstrated that Ang II increases expression of the TGF-ß type I receptor but has no effect on TGF-ß type I or TGF-ß type II receptors in VSMCs from SHR.14 These findings indicate that an increase in the expression of the TGF-ß type I receptor by Ang II may facilitate the ability of endogenous TGF-ß to counteract the stimulatory effect of Ang II on growth of VSMCs from WKY, whereas endogenous TGF-ß induced by Ang II cannot counteract the growth-promoting action of Ang II in VSMCs from SHR.14 The abnormal regulation of TGF-ß receptors by Ang II may be associated with the exaggerated growth of VSMCs from SHR.
In the present experiments, basal levels of TGF-ß type I receptor mRNA was higher in VSMCs from WKY than in SHR. This increase in TGF-ß type I receptor mRNA was significantly suppressed in VSMCs from WKY by the AT2 receptor overexpression, whereas it was not altered in VSMCs from SHR. We previously showed that Ang II increases the expression of TGF-ß type I receptor through the AT1 receptor in VSMCs from WKY.12 These findings suggest that the TGF-ß type I receptor was decreased by the downregulation of the AT1a receptor after transfection of the AT2 receptor gene, and that there exists a molecular feedback mechanism to maintain balance between growth-stimulation and growth-inhibition in normal VSMCs from WKY. Furthermore, the TGF-ß type II receptor, which mediates the growth stimulatory effect of TGF-ß, was upregulated after transfection of the AT2 receptor, suggesting that a disturbance of gene regulation in maintaining the balance between growth stimulation and inhibition in VSMCs from SHR.
Perspective
Overexpression of the AT2 receptor downregulates AT1a and TGF-ß type I receptors in normal VSMCs but not in VSMCs from SHR. The lack of the downregulation of the AT1a receptor by the AT2 receptor in VSMCs from SHR may contribute, in part, to the exaggerated growth of VSMCs from SHR.
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Acknowledgments |
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This work was supported by a grant from the Ministry of Education, High-Tech Research Center, Japan.
Received May 20, 2002; first decision June 19, 2002; accepted October 4, 2002.
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