Angiotensin II Type 2 Receptor Gene Transfer Downregulates Angiotensin II Type 1a Receptor in Vascular Smooth Muscle Cells
Two distinct subtypes of angiotensin (Ang) II receptors, type 1 (AT1) and type 2 (AT2), have been identified. Vascular smooth muscle cells (VSMCs) usually express AT1 receptor. To elucidate the direct effects of the AT2 receptor on the AT1 receptor in VSMCs, we transfected AT2 receptor gene into cultured rat VSMCs. Overexpression of AT2 receptor significantly decreased expression of AT1a receptor at both the mRNA and protein levels in the presence and absence of Ang II in VSMCs. Overexpression of AT2 receptor increased expression of bradykinin and inducible NO in the presence and absence of Ang II in VSMCs. Bradykinin B2 receptor antagonist HOE–140 and NO synthase inhibitor Nω-nitro-l-arginine methyl ester (L-NAME) inhibited the decreases in AT1a receptor expression by the overexpression of AT2 receptor in VSMCs. l-Arginine augmented the decrease in AT1a receptor expression. Overexpression of AT2 receptor suppressed basal DNA synthesis and proliferation of VSMCs and abolished response of DNA synthesis to Ang II in VSMCs. Our results demonstrate that overexpression of the AT2 receptor downregulates AT1a receptor expression in rat VSMCs in a ligand-independent manner that is mediated by the bradykinin/NO pathway. Downregulation of AT1a receptor is a novel mechanism by which the AT2 receptor regulates growth and metabolism of VSMCs.
Angiotensin (Ang) II plays an important role in regulation of cardiovascular hemodynamics and growth. At least 2 distinct subtypes of Ang II receptors have been identified on the basis of their differential pharmacological and biochemical properties, and they are designated type 1 (AT1) receptor and type 2 (AT2) receptor.1,2⇓ Both the AT1 and AT2 receptors belong to the family of seven-transmembrane G protein–coupled receptors.3 To date, most of the known effects of Ang II in adult tissues have been attributed to the AT1 receptor. Less is known about the AT2 receptor.
AT2 receptor mRNA is expressed at very low levels in the aorta during early embryonic development, but it is expressed at high levels during later stages of development and in the neonate.4 After birth, AT2 receptor levels decline rapidly.5 The significance of the increased expression of AT2 receptor in many pathological conditions remains unclear. It has been reported that AT2 receptor has antigrowth, antihypertrophic, and proapototic effects.4 Thus, the function and signaling of the AT1 and AT2 receptors are quite different, and these receptors have opposing actions in terms of cell growth and blood pressure regulation.4 AT1 receptor blockades are used clinically as antihypertensive therapies. It has been reported that during chronic AT1 receptor blockade, the actions of Ang II, which increases in concentration, may be mediated by the AT2 receptor, thereby contributing to the cardioprotective effects.4,6,7⇓⇓
Vascular smooth muscle cells (VSMCs) usually express only AT1 receptor.4 In the present study, we investigated the effect of AT2 receptor gene transfer on expression of the AT1a receptor and growth of rat VSMCs.
Our investigation conformed to the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996).
A 2.6-kb genomic DNA fragment containing the entire coding region of rat AT2 receptor was cloned into the mammalian expression vector pcDNA3 (Invitrogen Japan KK). pcDNA3 alone was used as control.
Cultured VSMCs were grown from explants of aortic media of Wistar-Kyoto/Izumo rats (SHR Corp, Funabashi, Japan) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% calf serum (GIBCO Life Technologies Inc), 100 U/mL penicillin, and 100 mg/mL streptomycin. The hill-and-valley pattern was observed as typical of cultured smooth muscle cells when cells reached confluence. The purity of the VSMCs was further confirmed by immunofluorescence with an anti–α-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 phosphate-buffered saline (PBS) and incubated in 75-cm2 tissue culture flasks at a density of 105 cells/mL. Experiments were performed 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
Quiescence cells were transfected with the AT2 receptor gene expression vector by the lipofectin (GIBCO) method, according to manufacturer’s instructions. Briefly, 2 μg of plasmid DNA was diluted into 100 μL of serum-free DMEM as solution A, and 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. Solution A and solution B 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 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 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 mL of 75% ethanol and centrifuged at 12 000g for 8 minutes at 4°C, dried, and dissolved in 10 μL of 10 mmol/L Tris-HCl (pH 8.0) and 1 mmol/L EDTA buffer. 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 pH 8.3, 50 mmol/L KCl, and 2.5 mmol/L MgCl2) at room temperature for 45 minutes. Then 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.8 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-NTP, 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 AT1a receptor to yield a 198-basepair (bp) product. Sense primer (5′-GTTCAACCTCCAGCAATCCTT-3′) and antisense primer (5′-CCTCCCTAACTCCCAAATCC-3′) were used and specific for PCR amplification of AT1b receptor to yield a 195-bp product. Sense primer (5′-CTTCAGCCTGCATTTAAAGG-3′) and antisense primer (5′-CTGAGCTTCCCACACGCACT-3′) were used for PCR amplification of AT2 receptor to yield a 306-bp product. Sense primer (5′-CGACGACCCATTCGAACGTCT-3′) and antisense primer (5′-GCTATTGGAGCATGGAATTACCG-3′) were used for PCR amplification of 18S ribosomal RNA 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 and as 30 cycles of 94°C for 1 minute, 55°C for 1 minute, and 72°C for 2 minutes for the AT2 receptor. PCR using primers for 18S ribosomal RNA was incubated 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 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.9 Serial 10-fold dilutions of cDNA (100, 10, and 1 ng) were amplified; the PCR products were visible after a fewer number of 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 pH 8.0, 150 mmol/L NaCl, 0.02% sodium azide, 100 μg/mL phenylmethylsulfonyl fluoride, 1 μg/mL aprotinin, and 1% Triton X-100). Samples were dissolved in 20 μL of sample buffer, boiled, and subjected to 10% polyacrylamide gel electrophoresis. The proteins were 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), rabbit polyclonal antibody specific for bradykinin (Biogenesis Ltd), or rabbit polyclonal antibody specific for inducible NO synthase (Biotrend Chemikalien GmbH) diluted in 200 vol TBST solution (10 mmol/L Tris-HCl pH 8.0, 150 mmol/L NaCl, and 0.05% Tween 20) containing 15% Block Ace at room temperature for 3 hours. After blots were washed with TBST twice for 10 minutes, the membranes were incubated with goat anti-mouse IgG (BioRad Laboratories) diluted in 3000 vol in TBST containing 15% Block Ace at room temperature for 1 hour, washed with TBST for 10 minutes for 3 times, then visualized with ECL method.10 Membranes were reprobed with mouse monoclonal antibody specific for α-tubulin (Sigma) as an internal control. Antiserums to AT1 receptor and AT2 receptor showed no reactivity with other G protein–coupled receptors. Antiserums to bradykinin showed 57% cross-reactivity with kallidin. Antiserums to human inducible NO synthase (iNOS) showed 50% cross-reactivity with rat iNOS and no cross-reactivity with neuronal and endothelial NO synthase.
Determination of DNA Synthesis
[3H]Thymidine incorporation into newly synthesized DNA was determined as described previously.11 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 a washing with 1 mL of water, 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.
Determination of Cell Numbers
Transfected and control VSMCs were maintained in DMEM containing 5% calf serum in 24-well culture dishes at a density of 5×104 cells/cm2. Cells were detached from plates with 0.05% trypsin at 24, 48, and 72 hours after inoculation, and cell numbers were counted in a Coulter Counter (Coulter Electronics Ltd).
Results are given as the mean±SEM. The significance of differences between mean values was evaluated by Student’s t test for unpaired data and by 2-way ANOVA, followed by Duncan’s multiple range test.
Expression of AT2, AT1a, and AT1b Receptors in VSMCs After Transfection of the AT2 Receptor Gene
Expression of AT2 receptor mRNA in VSMCs is shown in Figure 1. Basal expression of AT2 receptor was low in VSMCs. Levels of AT2 receptor mRNA increased from 2 hours after transfection and was sustained until 72 hours (Figure 1a). pcDNA3 alone did not affect expression of AT2 receptor mRNA (Figure 1b).
Expression of AT1a and AT1b receptor mRNAs after transfection of the AT2 receptor gene in the absence or presence of 0.1 μmol/L Ang II is shown in Figure 2. Transfection of AT2 receptor gene significantly (P<0.01) decreased abundance of AT1a receptor mRNA in VSMCs in the presence and absence of Ang II. pcDNA3 alone did not affect expression of AT1a receptor mRNA in VSMCs. Transfection of AT2 receptor gene did not affect abundance of AT1b receptor mRNA in VSMCs in the presence and absence of Ang II.
Levels of AT2 receptor protein were increased significantly (P<0.01) in VSMCs after transfection of the AT2 receptor gene. Levels of AT1a receptor protein were decreased significantly (P<0.05) in VSMCs after transfection of the AT2 receptor gene in the presence and absence of Ang II (Figure 3). These findings indicate that overexpression of AT2 receptor decreases expression of AT1a receptor at mRNA and protein levels in the absence of Ang II as a ligand.
Expression of Bradykinin and iNOS Proteins After Transfection of the AT2 Receptor Gene
Expression of bradykinin and iNOS proteins after transfection of the AT2 receptor gene in the absence and presence of Ang II are shown in Figure 3. Ang II (0.1 μmol/L) significantly (P<0.05) increased amounts of bradykinin and iNOS proteins in VSMCs. Furthermore, levels of bradykinin and iNOS proteins were increased significantly (P<0.01) after transfection of the AT2 receptor gene in the presence and absence of Ang II.
Effect of Bradykinin B2 Receptor Antagonist, NO Synthase Inhibitor, or l-Arginine on Expression of AT1a Receptor in VSMCs After Transfection of the AT2 Receptor Gene
To assess contributions of bradykinin and NO on the decreases in expression of AT1a receptor after transfection of the AT2 receptor gene, we elucidated effect of a bradykinin B2 receptor antagonist, HOE–140, or a NO synthase inhibitor, Nω-nitro-l-arginine methyl ester (L-NAME), on the of AT1a receptor suppression. A delivery agent lipofection alone had no effect on amounts of AT1a receptor protein. HOE–140 (10 nmol/L) blocked the decreases in AT1a receptor preen levels after transfection of the AT2 receptor gene (Figure 4A). L-NAME (10 mmol/L) blocked the decreases in AT1a receptor mRNA levels after transfection of the AT2 receptor gene (Figure 4B). l-Arginine (150 mmol/L) alone did not affect AT1a receptor mRNA levels in VSMCs. However, the same dose of l-arginine augmented significantly (P<0.05) the decreases in AT1a receptor mRNA levels after the transfection of AT2 receptor gene in VSMCs (Figure 4C).
Growth of VSMCs After Transfection of the AT2 Receptor Gene
Increasing doses (0.01 to 1 μmol/L) of Ang II increased DNA synthesis in VSMCs in a dose-dependent manner. Basal DNA synthesis was decreased significantly (P<0.05), and the response of DNA synthesis to Ang II was abolished after transfection of the AT2 receptor gene (Figure 5A).
Proliferation of VSMCs after transfection of the AT2 receptor gene in the presence of 5% calf serum is shown in Figure 5B. Transfection of the AT2 receptor gene inhibited significantly (P<0.05) cell proliferation.
The present study demonstrates that overexpression of the AT2 receptor gene downregulates AT1a receptor and inhibits proliferation of rat VSMCs. An increasing body of evidence indicates that the AT2 receptor exerts antigrowth, antihypertrophic, and proapototic effects4 that may counteract the growth stimulation mediated by AT1 receptor. Several recent studies have demonstrated that there is negative crosstalk between AT1 receptor and AT2 receptor signaling.12,13⇓ It was reported that AT1 receptor expression is significantly higher in AT2 receptor knockout mice than in control animals.12 Horiuchi et al13 observed that transfection of the AT2 gene in rat VSMCs inhibited AT1 receptor–mediated tyrosine phosphorylation of signal transducers and activators of transcription (STAT). AbdAlla et al14 demonstrated that the AT2 receptor binds directly to the AT1 receptor by heterodimerization and inhibits AT1 receptor function, which is independent of AT2 receptor activation and signaling. These studies, however, did not demonstrate the downregulation of AT1a receptor by transfection of the AT2 receptor gene. We showed that AT1a receptor expression was downregulated by overexpression the AT2 receptor in rat VSMCs and that AT1a receptor levels were decreased in the absence and presence of Ang II, indicating that overexpression of the AT2 receptor downregulates AT1a receptor in a ligand-independent manner. It is possible that AT2 receptor alone seems to act as one of intracellular signalings. Miura et al15 also observed that overexpression of the AT2 receptor induces apoptosis of fibroblasts in the absence of Ang II, which is in the transfection efficiency-dependent manner, suggesting that overexpression of AT2 receptor induces apoptosis in a ligand-independent manner. Thus, the downregulation of AT1a receptor by the AT2 receptor gene transfer in these experiments may be results by the state of overexpression of AT2 receptor in VSMCs. AT2 receptor is expressed at a high level during later stages of development, in neonates, and in the neointima of artery after angioplasty.5 AT2 receptor may thus have effects to regulate AT1a receptor in cells and tissues. However, the AT1a receptor downregulation was not reported in neointima after the vascular injury in which expression of AT2 receptor is increased.13 It is possible that the AT1a receptor downregulation can be seen only by the overexpression of AT2 receptor.
Expression of AT1a receptor is regulated by various vasoactive substances, growth factors, and glucocorticoids.16 Circulating Ang II does not affect AT1 receptor expression.17 However, local Ang II has been reported to have a regulatory effect on its receptors. Ang II decreases AT1 receptor expression in kidney, vascular and bladder smooth muscle cells,18 and in rat adrenal grand.19 There are tissue- and species-dependent differences in regulation of AT1 receptor gene expression.19 AT1a receptor is mainly expressed in heart, kidney, and VSMCs, whereas AT1b receptor is mainly expressed in adrenal gland.20–22⇓⇓
AT2 receptor has been reported to mediate renal productions of bradykinin and NO.23,24⇓ Tsutsumi et al25 reported that overexpression of AT2 receptor in aortic smooth muscle cells in AT2 receptor transgenic mice blocks the amiloride-sensitive Na+–H+ exchange and promotes intracellular acidification that stimulates the production of bradykinin and NO in a paracrine manner to promote vasodilation. In addition, AT1 receptor expression has been known to be transcriptionally suppressed by NO.26 It is therefore possible that increased production of NO by overexpression of AT2 receptor could suppress AT1a receptor expression by the inhibition of its transcription.
To evaluate the mechanisms underlying the downregulation of AT1a receptor by overexpression of the AT2 receptor, we investigated the influence of AT2 receptor overexpression on levels of bradykinin and iNOS and found that overexpression of the AT2 receptor significantly increased both bradykinin and iNOS protein levels. In addition, the bradykinin B2 receptor antagonist HOE 140 and the NO synthase inhibitor L-NAME ameliorated the decrease of AT1a receptor expression, and l-arginine as substrate for NO synthesis augmented the down regulation of AT1a receptor by overexpression of the AT2 receptor in VSMCs. These findings indicate that the downregulation of AT1a receptor is mediated through the bradykinin/NO pathway induced by overexpression of the AT2 receptor in VSMCs.
The overexpression of the AT2 receptor suppressed the basal DNA synthesis and the proliferation of VSMCs in the present experiment. The growth inhibitory effect of the AT2 receptor has been reported to be mediated by the activation of protein tyrosine phosphatase which inhibits activation of MAP kinases.6,27⇓ The AT2 receptor also mediates apoptosis of cells through dephosphorylation of Bcl-2 by activation of MAP kinase phosphatase-1.28 Thus, suppression of basal DNA synthesis and proliferation of VSMCs by overexpression of the AT2 receptor is consistent with the growth inhibitory effects of AT2 receptor described originally. In addition, transfection of the AT2 receptor gene abolished the response of DNA synthesis to Ang II in VSMCs. This phenomenon is considered to be a result by the downregulation of AT1a receptor by overexpression of the AT2 receptor in VSMCs. Thus, the growth inhibition by the AT2 receptor may not only be associated with suppression of MAP kinases and the proapototic effects, but also with the downregulation of AT1a receptor in VSMCs.
We demonstrated that overexpression of AT2 receptor gene downregulates AT1a receptor in rat VSMCs in a ligand-independent manner, which is mediated by the bradykinin/NO pathway. This downregulation of AT1a receptor is novel mechanisms by which AT2 receptor regulates growth and metabolism of VSMCs in cardiovascular developments and diseases.
This work was supported by a grant from the Ministry of Education, High-Tech Research Center, Japan.
- Received December 12, 2001.
- Revision received January 11, 2002.
- Accepted March 12, 2002.
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