Novel Regulatory Effect of Angiotensin II Type 1 Receptor-Interacting Molecule on Vascular Smooth Muscle Cells
We have recently cloned a novel molecule that interacts with the angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP). In this study, we tested the hypothesis that ATRAP modulates angiotensin II-induced responses in vascular smooth muscle cells. The results of immunoprecipitation and bioluminescence resonance energy transfer assay demonstrated a direct interaction between ATRAP and AT1R at baseline and showed that angiotensin II enhanced the interaction of these proteins >2-fold. The results of immunofluorescence analysis also demonstrated that >65% of ATRAP constitutively colocalized with an endosome marker. Although only 36% of ATRAP colocalized with AT1R at baseline, angiotensin II enhanced the colocalization of these molecules and made 92% of ATRAP colocalize with AT1R on a quantitative fluorescence analysis. Overexpression of ATRAP by adenoviral transfer decreased the cell surface AT1R number from 4.33 to 2.13 fmol/106 cells at baseline and from 3.04 to 1.26 fmol/106 cells even after removal of angiotensin II. ATRAP also suppressed angiotensin II-mediated increases in c-fos gene transcription and transforming growth factor-β production. Furthermore, this suppression was accompanied by inhibition of angiotensin II-induced activation of 5-bromodeoxyuridine incorporation. Finally, ATRAP knockdown by small-interference RNA activated angiotensin II-induced c-fos gene expression, which was effectively inhibited by valsartan, an AT1R-specific antagonist. These results indicate that ATRAP promotes internalization of AT1R and attenuates the angiotensin II-mediated c-fos-transforming growth factor-β pathway and proliferative response in vascular smooth muscle cells, suggesting a novel strategy to inhibit vascular fibrosis and remodeling through a novel and specific blockade of AT1R signaling.
- angiotensin receptors
- cell signaling
- growth factors and cytokines
- receptor internalization
- renin-angiotensin system
The angiotensin II type 1 receptor (AT1R) is a member of the G protein-coupled receptor superfamily and activates G proteins through the third intracellular loop and the intracellular carboxyl-terminal (C-terminal) tail of the receptor.1,2 The C-terminal cytoplasmic end of AT1R is involved in the control of AT1R internalization independent of G protein coupling, and it plays an important role in linking receptor-mediated signal transduction to the specific biological response to angiotensin II (Ang II), such as cardiovascular fibrosis and remodeling.3,4 Using a yeast 2-hybrid screening system, we recently cloned a novel AT1R-associated protein (ATRAP) that specifically interacts with the C-terminal cytoplasmic domain of AT1R.5–7 We showed that ATRAP is expressed in a variety of tissues and suppresses Ang II-mediated hypertrophic responses in cardiac myocytes.8,9 ARAP1 is another protein that was found recently to interact with the C-terminal domain of AT1R.10 Characterization of ARAP1 has revealed that ARAP1 binds and promotes recycling of AT1R to the plasma membrane, indicating its role in the receptor-recycling pathway. In this study, we examined the function of ATRAP in Ang II-induced fibrotic and proliferative responses of rat vascular smooth muscle cells (VSMCs).
Ang II was purchased from Sigma. The AT1R-specific antagonist valsartan and the Ang II type 2 receptor-specific antagonist PD123319 were supplied by Novartis Pharma and Parke Davis, respectively.
Cell Culture of Rat VSMCs
VSMCs were aseptically isolated from thoracic aortic explants of 5-week-old Sprague-Dawley rats as described previously.11 The experimental protocol was approved by the animal studies committee of Yokohama City University.
Production of Rabbit Anti-ATRAP Antibody
Originally, a 14-aa synthetic peptide corresponding with amino acids 148 to 161 of the C-terminal tail of the mouse ATRAP was used for production of the polyclonal anti-ATRAP antibody, as described previously.8,9 This amino acid sequence is completely conserved between the mouse and rat (GenBank Accession No. AF102548 and BC082019, respectively).
Coimmunoprecipitation and Western Blot Analysis
Coimmunoprecipitation analysis was performed essentially as described previously.8 Briefly, the NH2-terminal hemagglutinin epitope-tagged ATRAP (HA-ATRAP) was cotransfected with a FLAG-tagged AT1R (FLAG-AT1R) into VSMCs. The cells were then incubated with or without 100 nmol/L of Ang II for 60 minutes, and crude membrane fractions were prepared. Immunoprecipitation and immunoblotting were performed using anti-FLAG M1 monoclonal (Sigma) and anti-HA polyclonal (BETHYL Laboratories, Inc) antibodies.
Bioluminescence Resonance Energy Transfer Assay
Full-length ATRAP and the AT1R were cloned in frame into the expression vectors pRluc and pGFP2 (Biosignal, Perkin Elmer-Cetus), and bioluminescence resonance energy transfer (BRET) assay was performed as described previously.7
VSMCs cotransfected with HA-ATRAP and FLAG-AT1R were treated with 100 nmol/L of Ang II for 60 minutes and then incubated in fresh medium for 60 minutes to remove Ang II. The cells were then fixed and permeabilized with 2% paraformaldehyde and 0.1% Triton X-100, as described previously.8 Membrane and intracellular fluorescence were measured using Image J (National Institutes of Health) for a quantitative analysis, as described.12 Blind selection and analysis of the cells were performed to avoid any bias during the evaluation of the internalization data.
Cell-Surface AT1R-Binding Assay
VSMCs infected with adenoviral (Ad.)LacZ or Ad.HA-ATRAP were treated with 100 nmol/L of Ang II for 60 minutes and then incubated in fresh medium for 60 minutes to remove Ang II. AT1R binding was measured as described previously.8 The radioactivity of the lysate was measured with a gamma counter. AT1R binding was calculated as the difference between the total count and the count from samples incubated with valsartan.
5-Bromodeoxyuridine Incorporation Assay
VSMCs infected with Ad.LacZ or Ad.HA-ATRAP were incubated with or without Ang II (100 nmol/L). The activity of DNA synthesis was evaluated using the 5-bromodeoxyuridine (BrdUrd) labeling and detection kit (Boehringer Mannheim). The incorporation of BrdUrd in place of thymidine into the DNA of proliferating cells was detected by a subsequent enzyme reaction and quantified spectrophotometrically at 405 nm.
Real-Time Quantitative RT-PCR Analysis of Transforming Growth Factor-β
VSMCs infected with Ad.LacZ or Ad.HA-ATRAP were incubated with or without Ang II (100 nmol/L) for 36 hours. Real-time quantitative RT-PCR was performed by incubating the reverse transcription product with the TaqMan Universal PCR Master Mix and designed TaqMan probe (Applied Biosystems). RNA quantity was expressed relative to the 18S endogenous control.
ELISA of TGF-β
ELISA was performed to examine the effect of ATRAP on Ang II-mediated TGF-β secretion from VSMCs. VSMCs infected with Ad.LacZ or Ad.HA-ATRAP were incubated with or without Ang II (100 nmol/L) for 48 hours. Total TGF-β released into the medium was determined with an ELISA system (Promega).
The RNA interference experiment was performed essentially as described previously.13 Briefly, to knock down the endogenous ATRAP expression, a small interference RNA (siRNA) with 25 nucleotides (nucleotides positions corresponding with +158 to +182 of rat ATRAP cDNA, GenBank Accession No. BC082019) was synthesized with Invitrogen Technology (StealthTM RNAi). The siRNA duplex (20 nmol/L) was introduced into VSMCs with Lipofectamine 2000 (Invitrogen).
Transcriptional c-fos Promoter Assay
For a transcriptional fos promoter assay, the annealed double-stranded siRNA and the c-fos luciferase reporter gene (p2FTL, 1 μg) were cotransfected into VSMCs using Lipofectamine 2000 with normalization by a dual reporter assay system as described.8,13
The quantitative data are expressed as the mean±SE. For the statistical analysis of differences among groups, unpaired Student’s t test or ANOVA followed by Scheffe’s F test were used. A P<0.05 was considered statistically significant.
ATRAP Specifically Interacts With AT1R in Intact VSMCs
The results of RT-PCR, Western blot, immunofluorescence, and immunohistochemical analyses showed that ATRAP was endogenously expressed in rat VSMCs in culture and in situ in the aortic wall (Figure S1, available online at http://hyper.ahajournals.org). To examine the binding of ATRAP to AT1R in VSMCs, we performed coimmunoprecipitation analysis using VSMCs cotransfected with HA-ATRAP and the FLAG-AT1R. Immunoprecipitation of transfected ATRAP and AT1R showed that ATRAP was associated with AT1R at baseline and that Ang II (100 nmol/L) stimulation for 60 minutes promoted the association of ATRAP and AT1R by 2.1- and 2.3-fold (Figure 1A). To further examine the binding of ATRAP to AT1R in intact VSMCs, we performed BRET analysis. After transfection of chimeric forms of ATRAP fused to green fluorescent protein (GFP) at the C-terminal end and the AT1R fused to luciferase at the C-terminal cytoplasmic end, we were able to detect a significant BRET signal at baseline, and Ang II stimulation resulted in a 2.5-fold increase in the BRET signal (Figure 1B).
The association of ATRAP with AT1R in intact VSMCs was also confirmed by immunofluorescence colocalization analysis. First, further analysis to determine the exact identity of intracellular compartments showed that ATRAP was significantly colocalized with GFP-rhoB, the vesicle marker of the endocytic pathway (Figure 2A). A quantitative analysis showed that 67% of the total ATRAP was colocalized with endosomes at baseline and that the intracellular localization of ATRAP and rhoB, as well as the colocalization of these proteins, did not change on stimulation with 100 nmol/L of Ang II for 60 minutes, indicating that ATRAP is localized constitutively in the endocytic pathway (Figure 2B). Next, immunofluorescence analysis of transfected ATRAP and AT1R showed that ATRAP was colocalized to some degree with AT1R at baseline and that Ang II (100 nmol/L) stimulation for 60 minutes increased intracellular AT1R staining and the colocalization of ATRAP and AT1R (Figure 2C). On quantitative fluorescence analysis, although ≈36% of total ATRAP and 27% of total AT1R were colocalized with each other at baseline, Ang II treatment decreased the cell surface/total fluorescence ratio for AT1R from 0.63 to 0.20 (P<0.05) without the Ang II-induced change of the ratio for ATRAP (from 0.30 to 0.24; P value not significant) and enhanced the fluorescence ratio derived from the colocalization of these proteins (92% of total ATRAP, P<0.05, and 94% for AT1R, P<0.05, respectively; Figure 2D).
Adenoviral Transfer of Recombinant ATRAP Decreases AT1R Expression on the Cell Surface in VSMCs
To examine the possible effect of ATRAP on the internalization and recycling of AT1R, VSMCs infected with Ad.LacZ or Ad.HA-ATRAP were exposed to 100 nmol/L of Ang II for 60 minutes, incubated in fresh medium for 60 minutes to remove Ang II, and subjected to immunofluorescence analysis (Figure 3A). VSMCs infected with Ad.HA-ATRAP showed an enhanced AT1R endocytosis, because the cell surface/intracellular fluorescence ratio decreased from 1.03 to 0.41 (P<0.05) under basal conditions (Figure 3B). Furthermore, the recycling of AT1R after removal of Ang II from the medium was significantly suppressed in VSMCs infected with Ad.HA-ATRAP, as the cell surface/intracellular fluorescence ratio for AT1R decreased from 1.03 to 0.41 (P<0.05; Figure 3B).
To determine the changes in AT1R number on the plasma membrane, cell surface AT1R binding assay in VSMCs was performed (Figure 3C). The cell surface AT1R density at baseline was decreased in VSMCs infected with Ad.HA-ATRAP (baseline: 2.13 versus 4.33 fmol/106 cells; P<0.05). Furthermore, overexpression of ATRAP significantly inhibited the recovery of cell surface AT1R density by the removal of Ang II (washout: 1.26 versus 3.04 fmol/106 cells; P<0.05).
ATRAP Specifically Inhibits Ang II-Induced BrdUrd Incorporation and TGF-β Production in VSMCs
We next examined the effects of ATRAP on the downstream effectors of the AT1R-signaling pathway in VSMCs by performing adenoviral transfer of recombinant ATRAP. Although treatment with Ang II significantly augmented BrdUrd incorporation into VSMCs infected with Ad.LacZ by 1.4-fold at 48 hours, this Ang II-mediated activation of BrdUrd incorporation was completely suppressed by overexpression of HA-ATRAP (Figure 4A).
Ang II treatment of VSMCs infected with Ad.LacZ increased the expression of TGF-β mRNA by 1.5-fold (Figure 4B) and secretion of TGF-β protein into the medium by 3.7-fold (Figure 4C), whereas VSMCs infected with Ad.HA-ATRAP exhibited a complete inhibition of the Ang II-induced enhancement of TGF-β mRNA expression and TGF-β secretion into the medium.
ATRAP Specifically Inhibits Ang II-Induced Transcriptional Activity of c-fos in VSMCs
Because c-fos and the AP1 family of transcription factors are involved in the activation of TGF-β gene expression,14 we next determined whether ATRAP has a functional effect on the Ang II-c-fos pathway. Although treatment of VSMCs with Ang II induced the c-fos reporter gene 3.8-fold, overexpression of ATRAP, as well as pretreatment with the AT1R-specific antagonist valsartan, but not the Ang II type 2 receptor-specific antagonist PD123319, inhibited Ang II-mediated activation of c-fos reporter gene expression (Figure 4D). A combination of valsartan and PD123319 did not affect the inhibitory effect of valsartan, indicating that Ang II type 2 receptor was not involved in the Ang II-mediated regulation of c-fos gene. These results indicate that ATRAP is involved in the c-fos pathway activated by Ang II and is a negative regulator in the AT1R-c-fos-TGF-β signaling pathway.
We next examined the effect of RNA interference ATRAP knockdown in the regulation of c-fos promoter activity. Transfection of ATRAP-siRNA significantly decreased ATRAP protein levels to <10% of those of untreated cells, whereas the control siRNA had no effect on the protein levels of ATRAP (Figure 4E). Transfection of ATRAP siRNA increased the basal level of c-fos promoter activity by 1.6-fold, and knockdown of ATRAP sensitized the effect of Ang II on promoter activity, increasing it by 2.1-fold (Figure 4F). Furthermore, whereas pretreatment with valsartan did not affect the basal c-fos transcription in VSMCs transfected with control siRNA, the activation of basal and Ang II-induced c-fos gene transcription by the endogenous knockdown of ATRAP with ATRAP siRNA was suppressed by valsartan. Thus, endogenous ATRAP is actively involved in the suppression of AT1R-mediated TGF-β activation by inhibiting c-fos signaling.
In this study, the results of immunoprecipitation assay and BRET analysis indicate that ATRAP was able to interact with the AT1R under baseline conditions, but Ang II further enhanced their interaction in the form of a complex in VSMCs (Figure 1). The C-terminal tail of AT1R is clearly involved in the control of endosomal internalization/trafficking and intracellular recycling of the receptor to the plasma membrane.3 Thus, as a specific interacting molecule with the C-terminal tail of AT1R, ATRAP may be involved in some of the events responsible for the endosomal internalization/trafficking and/or the recycling of the receptor.
Our previous study showed that ATRAP contains 3 hydrophobic domains at the amino-terminal end of the protein and a hydrophilic cytoplasmic C-terminal tail, indicating that ATRAP is a transmembrane protein.7 The results of immunofluorescence staining showed that ATRAP was mainly located in the endosome, one of the membrane structures in the cytoplasm and at the cell periphery to a lesser degree (<30% of total ATRAP immunofluorescence) under baseline conditions and that Ang II stimulation did not affect the intracellular localization of ATRAP. With respect to the intracellular colocalization of ATRAP and AT1R, a quantitative analysis of immunofluorescence staining showed that <30% of the total AT1R was colocalized with ATRAP under baseline conditions. However, Ang II stimulation decreased cell-surface AT1R, increased the internalization of AT1R, and made >90% of total AT1R colocalize with ATRAP in the cytoplasmic endosome (Figure 2).
Thus, the results of immunoprecipitation assay, BRET analysis, and immunofluorescence staining indicate that ATRAP is able to interact with AT1R even without Ang II stimulation and that Ang II stimulation significantly facilitated the interaction of these proteins. Our previous real-time trafficking analysis of ATRAP vesicles also showed a constitutive translocation of ATRAP from intracellular vesicle compartments to the periphery of the cell, which was not affected by the treatment with Ang II.7 Taken together, these results suggest that ATRAP is actually able to bind to the AT1R under baseline conditions but that ATRAP interacts mainly with the AT1R that is internalized from the cell surface into the endocytic vesicles on Ang II stimulation to keep the receptor internalized even after the removal of Ang II. Furthermore, the quantitative analysis of immunofluorescence staining indicated that almost all of the internalized AT1Rs were associated with ATRAP, suggesting that internalization is a protective mechanism that does not mediate AT1R signaling. Nevertheless, determining whether ATRAP participates in a specific step of the endosomal internalization/trafficking or the recycling of AT1R to the plasma membrane will require further investigation.
The results of gain-of-function analysis using adenoviral gene transfer showed that overexpression of ATRAP did not affect c-fos gene transcription, TGF-β expression, or BrdUrd incorporation in the absence of Ang II, whereas ATRAP increased the internalized AT1R constitutively under basal conditions. On the other hand, the results of loss-of-function analysis using silencing of the ATRAP gene by ATRAP siRNA slightly but statistically significantly increased basal c-fos gene transcription in the absence of Ang II. Thus, there seems to be a discrepancy between the ATRAP-induced promoting effect on AT1R internalization and the ATRAP-mediated inhibitory action on AT1R signaling. Under basal conditions, it is likely that several networks of vasoactive receptor-mediated pathways, in addition to the AT1R pathway, also regulate downstream signaling in VSMCs.15 Thus, a decrease in basal cell surface AT1R expression by overexpression of ATRAP may be dispensable for the maintenance of downstream signaling basal activity. This possibility would be supported by the lack of an inhibitory effect of valsartan on the basal c-fos transcription in VSMCs transfected with control siRNA (Figure 4). On the other hand, several recent studies showed that a recombinant AT1R expressed at a high density exhibited basal constitutive activity for downstream signaling independent of Ang II.16,17 Therefore, it is possible that an increase in cell-surface AT1R expression by a silencing of the ATRAP gene may stimulate the basal c-fos gene transcription mediated by enhanced constitutive activity of AT1R. This hypothesis is supported by our observation that valsartan suppressed ATRAP siRNA-mediated activation of basal c-fos gene transcription (Figure 4).
In summary, we demonstrate the following new findings in rat VSMCs: (1) whereas ATRAP constitutively enhanced the internalization of AT1R, Ang II stimulation significantly promoted the interaction between ATRAP and AT1R, as revealed by immunoprecipitation and BRET assay, and >90% of ATRAP and AT1R were colocalized with each other in a series of quantitative fluorescence analyses; (2) overexpression of ATRAP by adenoviral gene transfer blocked the Ang II-mediated c-fos-TGF-β pathway activation; and (3) knockdown of endogenous ATRAP by siRNA transfection increased basal constitutive AT1R activity, as well as Ang II-induced activation of AT1R, both of which were efficiently inhibited by an AT1R-specific antagonist.
The results in this study suggest that the activation of ATRAP could be useful in suppressing vascular fibrosis and remodeling. A recent study that reported neointimal formation in the injured femoral artery to be attenuated in transgenic mice ubiquitously overexpressing the ATRAP gene also supports this hypothesis.18 It seems likely that the inhibitory mechanism of ATRAP on AT1R signaling is distinct from that of AT1R antagonists. Clinical application of ATRAP, such as the use of putative activating ligands, may enable a more efficient inhibition of the AT1R pathway in cardiovascular tissues in combination with AT1R antagonists in the near future.
Sources of Funding
This study was supported by grants from the 21st Century Centers of Excellence Program of the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Japan Society for the Promotion of Science. This study was also partially supported by the grant of Strategic Research Project of Yokohama City University. M.L-I. was supported by a Scientist Development Grant (0435427T) from the American Heart Association and an RO1 HL083154 grant from the National Institutes of Health.
- Received June 10, 2007.
- Revision received July 8, 2007.
- Accepted August 22, 2007.
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