Selective Silencing of Angiotensin Receptor Subtype 1a (AT1aR) by RNA Interference
Angiotensin II exerts its physiological effects by activating multiple subtypes of its receptor such as AT1a-, AT1b-, and AT2-receptors. Because of a high degree of similarity among these G-protein–coupled receptors, it has been difficult to assign diverse physiological actions of angiotensin II through these receptor subtypes. We have developed small interfering RNAs to selectively inhibit the expression of the AT1a receptor (AT1aR) subtype. A dsRNA, AT1 47, was found to be highly selective and efficient in reducing the levels of AT1aR subtype. Transfection of AT1aR-expressing CHO cells with dsRNA AT1 47 resulted in an 80% decrease in the AT1aR expression. In contrast, dsRNA AT1 47 showed no significant effects on both AT1bR and AT2R subtypes. Thus, AT1 47 provides us with a powerful tool to selectively silence this subtype of receptor to investigate its role in cardiovascular physiology.
Angiotensin II (Ang II) exerts profound physiological effects on the cardiovascular system by regulating such diverse functions as increases in blood pressure (BP), extracellular fluid volume, hormone secretion, vascular and cardiac remodeling, stimulation of sympathetic nerve activity, and damping of baroreflexes.1,2 Most, if not all, of these effects are mediated by activation of a single angiotensin receptor subtype (AT1R). Since the discovery of 2 subtypes of AT1R, AT1aR, and AT1bR in rodents, it has been proposed that this diversity of Ang II actions resides in these 2 subtypes.3 Because AT1aR and AT1bR R share 94% sequence similarity,4 it has been difficult to develop subtype specific antagonists to link AT1aR and AT1bR to distinct cardiovascular effects of Ang II. Thus, investigators have relied on the use of genetic targeting of these receptors in mouse models to link them with various phenotypes.5–8 For example, AT1aR knockout studies have indicated the role of this receptor in BP regulation, sodium handling, and central dipsogenic responses.9 However, knockout studies indicate that AT1bR can partially replace the BP regulatory functions of AT1aR.10 These observations indicate that although knockout technology enables us to delineate an overall physiological perspective, it is limited because of the possible expression of compensatory mechanisms during development. Therefore, new and more selective alternatives need to be developed in which the expression of AT1aR and AT1bR could be attenuated in adult animals after development.
We have taken advantage of recent advances in RNA interference (RNAi) technology to determine whether it can be used to selectively silence the expression of these receptor subtypes.11 Thus, our objective in this study was to determine whether we could identify a double stranded RNA (dsRNA) sequence that is highly effective and selective for the AT1aR subtype.
Materials and Methods
Complete coding sequence of Rattus rattus AT1aR (Gen Bank accession number X62295) was used for the selection of target sequences. The sequence was used in the Target Finder and Design Tool provided by Ambion Inc (Austin, Tex). For screening, 3 target sequences spaced throughout the gene were chosen from the list of candidate siRNAs. Selected sequences did not show near-exact matches to any other known sequence on a BLAST search, confirming their sequence specificity to the AT1aR. Synthetic duplexed–deprotected siRNAs were purchased from Dharmacon Research, Inc (Lafayette, Col). Three 21-bp dsRNA sequences were designated as AT1 9 (5′-AAACAGCTTGGTGGTGGTGATTGT′-3) directed to nucleotides 135 to 156, AT1 39 (5′-AACAACTGCCTGAACCCTCTG-3′) directed to nucleotides 880 to 901, and AT1 47 (5′-AAAGGCCAAGTCCCACTCAAG-3′) directed to nucleotides 966 to 987. In addition, a scrambled sequence with no near-exact match to any known sequence, Scr (5′-AATGTACTCACTACGAGTGCG-3′) was designed as control for AT1 9, AT1 39, and AT1 47 dsRNAs.
Cell Culture and Transfection
Chinese hamster ovary cells (CHO) stably expressing either rat AT1aR, AT1bR, or AT2R were maintained at 37°C in Ham F12 medium supplemented with 10% fetal bovine serum. Cells were seeded in 12-well plates at a cell density of 1×105 cells/well, 24 hours before their use in transfection. Oligofectamine-mediated (Invitrogen Corporation) transfection of the dsRNA was performed after manufacturer’s instructions. Briefly, 2 μL of oligofectamine was mixed with 8 μL/well of OptiMEM (Invitrogen Corporation); similarly, the dsRNA was diluted a final volume of 90 μL/well in OptiMEM. Both solutions were incubated at room temperature for 10 minutes, mixed, and further incubated for 10 to 15 minutes. Cells were washed with phosphate-buffered saline (PBS) and 400 μL/well of serum-free Ham F12 media added. After transfection complexes were formed, the transfection mixture was added to the cells and incubation continued for 4 hours at 37°C. This was followed by addition of 250 μL/well of Ham F12 to 30%. Cells were maintained for 3 days and used for measurements of Ang II binding activity.
Measurement of [125I]-Sar1-Ile8 Ang II Binding to AT1R and AT2R
Cell cultures were rinsed with PBS (pH 7.2) and incubated with the Binding Buffer (PBS; pH 7.2; 0.16% heat-inactivated bovine serum albumin) containing 10 nmol/L [125I]-Sar1-Ile8 Ang II for 1 hour at room temperature in the absence and presence of 1 μmol/L of either losartan (AT1R-specific antagonist) or PD-123319 (AT2R-specific antagonist). Unbound radioligand was removed by rinsing the cells 3 times with ice-cold PBS pH 7.2. Cells were removed from the dish by incubation with NaOH–SDS (0.1 mol/L NaOH and 10% SDS solution) for 2 hours at room temperature. Bound radioactivity was measured in a DP5500 gamma counter (Beckman Coulter, Fullerton, Calif). Specific binding was calculated by subtracting the [125I]-Sar1-Ile8 Ang II bound in the presence of either losartan or PD123319 from that bound in its absence. Scatchard analysis was performed from saturation binding experiments with increasing Ang II concentration for the calculation of Kd and Bmax values.
Semi-Quantitative Reverse Transcription Polymerase Chain Reaction
Total RNA was isolated using 4-PCR RNA isolation kit (Ambion, Austin, Tex) according to the manufacturer’s instructions. Polymerase chain reaction (PCR) specific primers for AT1R and AT2R sequence were obtained from GenoMechanix LLC (Gainesville, Fla). Sequences for these primers were as follows: AT1R: sense, 5′-TACGCTATGCAGATGGTGATGGG-3′; antisense, 5′-CTGGCTGATGGCTGGCTTGG-3′; AT2R: sense, 5′-GCACCAATGAGTCCGCATTTA-3′; antisense, 5′-CAGAAAAGGGTAGATAACCGATTGG-3′. We used as internal control the amplification of 18 rRNA using the primer set provided by on the TaqMan Ribosomal RNA Control Reagents kit (Applied Biosystems, Foster City, Calif). Reverse transcription reactions were performed using 2 μg of total RNA in a volume of 100 μL. The PCR conditions were: 95°C for 5 minutes after of 30 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute, with a final extension step of 72°C for 10 minutes. The resulting PCR products were subjected to a 2% agarose gel electrophoresis. Bands representing PCR products for the AT1 and AT2R were quantitated and data normalized with PCR bands for 18S rRNA.
Cells were transfected with either dsRNA AT1 47 or a dsRNA Scr, as described. Two days after transfection, Ang II-stimulated [45Ca2+] uptake was tested as described elsewhere.12 Briefly, cells were washed 3 times with Tyrodes solution (137 mmol/L NaCl, 2.68 mmol/L KCl, 1.36 mmol/L CaCl2, 0.46 mmol/L MgCl2, 5.6 mmol/L glucose, 12 mmol/L NaHCO3, 0.36 mmol/L NaH2PO4, pH 7.4) and equilibrated in this solution for 1 hour at 37°C. Cells were incubated for 20 seconds in Tyrode solution containing 490 mCi/mmol [45Ca2+] in the presence or absence of 0.1 μmol/L Ang II. Immediately after incubation, the assay medium was removed and the cells were quickly rinsed twice with 1 mL of ice-cold Wash solution (100 mmol/L MgCl2, 10 mmol/L HEPES, 10 mmol/L CdCl2, 10 mmol/L MnCl2) and then rinsed rapidly with 10 mL of ice-cold 100 mmol/L MgCl2 containing 10 mmol/L HEPES. Cells were solubilized in 1 mL of 0.1 mol/L NaOH and [45Ca2+] was quantified in a LS6500 liquid scintillation counter (Beckman Coulter, Fullerton, Calif).
Data are shown as mean±SEM. Statistical differences between 2 means were determined by unpaired Student t test.
In the first series of experiments, our objective was to identify target sequences that would trigger a highly efficient and specific silencing of the AT1R. For this, we selected 3 dsRNA sequences targeting the 5′ middle and 3′ regions of AT1aR coding sequence.
Of these dsRNAs, 2 (AT1 9 and AT1 47) significantly reduced specific Ang II binding in CHO cells that overexpress AT1R (Figure 1A). These dsRNAs were without effect on Ang II binding in the CHO cells that overexpress AT2R (Figure 1A). Whereas AT1 47 was able to decrease binding of Ang II to cells expressing AT1R by 95%, AT1 9 caused a decrease of only 60% (Figure 1A). Scrambled dsRNA failed to influence the binding of Ang II. In contrast, the AT1 39 sequence was found to be nonselective, decreasing the binding of Ang II in both AT1R- and AT2R-expressing cells (Figure 1A). These data indicate that AT1 47 sequence is the most effective and selective for AT1R silencing. Next, we determined the AT1R subtype specificity of dsRNA AT1 47. For this, both AT1a and AT1b overexpressing cells were transfected with either AT1 47 or Scr dsRNA. Figure 1B shows that AT1 47 was able to completely silence AT1aR subtype with only modest effect on the AT1bR subtype. Transfection with Scr dsRNA did not influence binding of either receptor (data not shown). This indicated that AT1 47 was predominantly selective for the AT1aR subtype.
Next, we characterized the effect of AT1 47 on AT1a R. dsRNA AT1 47 caused a dose-dependent decrease in the specific binding of [125I]-Sar1-Ile8 Ang II to AT1aR-expressing CHO cells, with a maximal inhibition of 90% observed at 50 nmol/L dsRNA (Figure 2A). The IC50 was calculated to be 1.2 nmol/L. No effect was seen on AT2 R-expressing CHO cells with these concentrations of dsRNA. Incubation with 100 nmol/L dsRNA AT1 47 caused a time-dependent silencing of AT1aR expression (Figure 2B). An 80% decrease in binding was observed within 1 day, followed by a 97% decrease at 2 days after transfection. This level of decrease was maintained for an additional 24 hours. The receptor levels returned to control levels 7 days after transfection.
Saturation analyses of [125I]-Sar1-Ile8 Ang II binding were performed in Scr and AT1 47-transfected AT1aR CHO cells (Figure 3). Scatchard analyses revealed a 20-fold decrease in the Bmax for AT1aR in the AT1 47-treated cells when compared with Scr controls (0.46±0.01 versus 8.80±0.46 pmol/mg protein). In contrast, the Kd values for both treatments remained comparable (0.9±0.09 versus 2.0±0.30 nmol/L). The observed decrease in the number of AT1aR was also reflected in a significant decrease in AT1aR mRNA levels (Figure 4A). An 80% decrease in the mRNA was observed 48 hours after transfection (Figure 4B).
Finally, we determined the effect of AT1 47 on Ca2+ uptake. To find out if decreases in AT1aR-specific binding was associated to a reduction in functional AT1aR, we determined the effects of dsRNA transfection on Ang II-stimulated calcium uptake. Ang II (100 nmol/L) caused a 54% increase in [45Ca2+] uptake in CHO cells expressing AT1aR. Transfection of these cells with dsRNA Scr did not influence this stimulation and it was comparable to that of nontransfected cells. In contrast, the Ang II-induced increase in [45Ca2+] uptake was completely abolished in by dsRNA AT1 47 transfected cells (Figure 5).
The most significant finding of our study is that it establishes, for the first time to our knowledge, that RNAi technology can be used to silence the AT1aR subtype. This provides us with a powerful tool for elucidating the respective roles of AT1aR and AT1bR subtypes in the diverse and profound physiological effects of Ang II in the cardiovascular system in normal animals. The observed reduction in [125I]-Sar1-Ile8 Ang II binding, AT1aR mRNA levels, and Ang II-stimulated [45Ca2+] uptake demonstrates that at least 1 of the 3 dsRNA targeted for the AT1aR subtype is selective and efficient in silencing the AT1aR subtype gene. The dsRNA AT1 47 is more efficient in reducing AT1aR numbers compared with its sister subtype, the AT1bR, despite the fact that these subtypes share 96% similarity. The dsRNA AT1 47 is highly efficient and produces AT1aR silencing that lasts for ≈3 days. Until now, the popular technology to reduce angiotensin receptors has been the use of antisense oligonucleotides.13–18 RNAi technology not only is highly selective for the subtypes of AT1aR and AT1bR but also is more effective than antisense oligonucleotides. For example, a previous study19 demonstrated a reduction of AT1R by 57% to 73% with 1 μmol/L concentration of AT1R-specific antisense oligonucleotides. In contrast, in the present study, a concentration of 100 nmol/L of dsRNA was found to silence AT1R by 95% in 48 hours.
The efficacy of the dsRNA 47 to silence AT1R in a physiologically relevant cell was confirmed with the use of astroglia cells in primary cultures. Our observations indicated an 80% decrease in AT1 binding elicited by this dsRNA in these cultures, confirming the validity of the silencing data obtained for the CHO cells.
In summary, our observations demonstrate that RNAi technology can be successfully used to silence 2 closely related genes for Ang II receptors. This provides us with a powerful tool to selectively silence 1 or more subtypes of AT1R and to study their role in cardiovascular physiology and pathophysiology in a way that was not previously possible.
Delineating the roles of the AT1R subtypes in various cardiovascular functions has been difficult in the past because of lack of selective antagonists or antisense targeting. This study uses RNAi technology and successfully demonstrates the effectiveness of a dsRNA that selectively targets AT1aR subtype. Thus, our study provides a well-needed tool to dissect AT1aR functions in vivo.
- Received July 22, 2004.
- Revision received August 19, 2004.
- Accepted October 31, 2004.
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