Destabilization of AT1 Receptor mRNA by Calreticulin
AT1 receptor activation leads to vasoconstriction, blood pressure increase, free radical release, and cell growth. AT1 receptor regulation contributes to the adaptation of the renin-angiotensin system to long-term stimulation and serves as explanation for the involvement of the AT1 receptor in the pathogenesis of cardiovascular disease. The molecular mechanisms involved in AT1 receptor regulation are poorly understood. Here, we report that angiotensin II accelerates AT1 receptor mRNA decay in vascular smooth muscle cells. A cognate mRNA region within the 3′ untranslated region at bases 2175 to 2195 governs the inducible decay of the AT1 receptor mRNA. Sequential protein purifications led to the discovery of a novel mRNA binding protein, calreticulin, which mediates destabilization of the AT1 receptor mRNA. Angiotensin II–caused phosphorylation of calreticulin enables binding of calreticulin to the AT1 receptor mRNA at bases 2175 to 2195 and propagates calreticulin-induced acceleration of AT1 receptor mRNA decay. Thus, a novel mRNA binding protein, calreticulin, is discovered, which causes AT1 receptor mRNA degradation via binding to a distinct mRNA region in the 3′ untranslated region. These findings display a novel mechanism of posttranscriptional mRNA processing.
The renin-angiotensin system (RAS) is central for the physiological regulation of blood pressure and fluid homeostasis.1,2⇓ Importantly, the RAS has been strongly implemented in the pathogenesis of hypertension and atherosclerosis.3 This assumption is predominantly based on numerous interventional studies demonstrating that pharmacological blockade of the RAS leads to diminished vascular damage, to an improved endothelial function, and ultimately to reduced mortality rates in patients suffering of hypertension, coronary heart disease, or heart failure.4–6⇓⇓ Most biological effects of the RAS such as vasoconstriction, neurohumoral activation, cell growth, and free radical release are mediated through the AT1 receptor.1,2⇓ The expression of the AT1 receptor is altered by angiotensin II, growth factors, estrogen, and lipoproteins.7–10⇓⇓⇓ Dysregulated expression of AT1 receptors may profoundly participate in the development of vascular damage.11 AT1 receptor regulation takes place at the posttranscriptional level via agonist-induced (de)stabilization of the AT1 receptor mRNA.8,9⇓ As previously shown, a family of mRNA binding proteins binds the AT1 receptor mRNA at bases 2175 to 2195.12 We further investigated the role of the 3′ untranslated region of the AT1 receptor mRNA in the degradation processing and sought to identify putative proteins involved in these pathways.
Materials and Methods
Vascular smooth muscle cells (VSMCs) were isolated from rat thoracic aorta by enzymatic dispersion and cultured over several passages. Experiments were performed with cells from passage 5 to 15.
mRNA Isolation, Northern Analysis, and Polymerase Chain Reaction
Cells were lysed with 1 mL RNA-clean (AGS), scraped, and processed according to the manufacturer’s protocol in order to obtain total cellular RNA. Northern blots were prehybridized for 2 hours at 42°C and then hybridized for 15 hours at 42°C with a random-primed, [32P]-dCTP–labeled, rat AT1 receptor cDNA probe. The rat AT1 receptor cDNA probe was a 479-bp fragment generated from an AT1 receptor cDNA template by the polymerase chain reaction (PCR) using the same primer pair as mentioned in the PCR section.
Isolated total RNA (2 μg) and the mutAT1 mRNA (10 pg) were mixed and reverse transcribed using random primers. The single-stranded cDNA was amplified by PCR using Taq DNA-polymerase (Boehringer). Twenty eight cycles were performed under the following conditions: 30 seconds, 94°C; 55°C, 45 seconds; and 72°C, 45 seconds. The sequence for AT1 receptor sense and antisense primers were 5′-ACCCTCTACAGCATCATCTTTGTGGTGGGA-3′ and 5′-GGGAGCGTCGAATTCCGAGACTCATAATGA-3′, respectively.
The same samples were used for GAPDH cDNA amplification to confirm that equal amounts of RNA were reverse transcribed. The primers employed were 5′-ACCACAGTCCATGCCATCAC-3′and 5′-TCCACCACCCTGTTGCTGTA-3′. PCR amplification gave 478 bp and 452 bp of fragments originated from the AT1 receptor mRNA and GAPDH mRNA, respectively.
UV mRNA Protein Crosslink Assays
Polysomal protein (10 μg) was mixed on ice with 4 to 10 pmol of [32P] UTP-labeled RNA transcripts (4×105–106 cpm) and UV mRNA protein crosslink experiments were performed as described earlier.12
Cell Transfections and Calreticulin Constructs
For electroporation, VSMCs grown at a confluent monolayer were removed from the culture dish by addition of trypsin and pelleted. The pellet was resuspended in 200 μL of OptiMEM I (Gibco BRL Life Technologies). Samples (106 cells) were incubated with 20 μg of the respective DNA (pcDNA3 vector, calreticulin full length cDNA) in precooled cuvettes (Promega) for 30 minutes on ice. Electroporation was performed for 16 ms at 0.3 kV and 500 μF. Cells were plated on the appropriate culture dishes or microtiter plates. Full-length calreticulin was subcloned into the eucaryotic expression vector pcDNA3 (Invitrogen). A calreticulin antisense oligonucleotide 5′-AGGTTGCCAAGCAGGAGCGGCACCGAAAGGAGCATG-3′ was subcloned in pcDNA3 with the help of HindIII and BamHI restriction sites.
In Vitro Decay Assays
In vitro decay assays were performed as described earlier.12 The included AT1 receptor mRNA fragment was detected by real-time PCR.
Anionic Exchange Chromatography
Polysomal protein extracts were used for an initial purification step on a standard chromatography system using a Source DEAE Sepharose Fast Flow column (Pharmacia Biotech) equilibrated in a buffer containing 10 mmol/L Tris-HCl (pH 7.4) and 0.5 mmol/L EDTA. Proteins were eluted in a NaCl-gradient from 100 mmol/L up to 1 mol/L. After desalting, protein fractions with RNA-binding activity were submitted to a second anionic exchange chromatography using a FPLC chromatography system with a Q-Sepharose HP column (Pharmacia Biotech). Elution gradient was between 500 and 700 mmol/L NaCl. Binding proteins were excised out of SDS-PAGE gels and identified via MALDI mass spectroscopy.
Immunoprecipitation and Western Blotting
Stimulated VSMCs were washed and lysed. After centrifugation, supernatants were incubated with an anti-calreticulin antibody (Novus Biologicals). Protein-A Sepharose (20 mg) was added and incubated for 1 hour. After centrifugation, pellets were washed and samples were boiled, electrophoresed, and blotted to nitrocellulose membranes. Immunoblotting/detection was performed with an anti-phosphotyrosine antibody, clone 4G10 (Upstate Biotechnology), an anti-mouse peroxidase conjugate antibody (Sigma, Taufkirchen, Germany), and the enhanced chemiluminescence (ECL) kit (Pharmacia Biotech).
In Vitro Phosphorylation of Calreticulin
In vitro phosphorylation of calreticulin was performed according to the manufacturer’s protocol using the following: β-insulin receptor kinase/Src kinase/JNK kinase kit (all obtained from Stratagene).
For quantitative PCR, a reverse transcription was performed (primer for AT1 receptor 5′-GAGGTAAACATACATTGCC-3′, GAPDH 5′-TGTTATGGGGTCTGGGATGGA-3′) After 1:1000 dilution, the PCR was performed according to the manufacturer’s instructions using the Sybr-Green Mastermix Kit (Applied Biosystems) and the Abi Prism 7700 Sequence Detector. PCR primer: AT1 receptor 5′-TTCAGCCAGTGTTTTAGA-3′ (sense) and 5′-GAGGTAAACATACATTGCC-3′ (antisense); GAPDH 5′-CCTGGACCACCCAGCCCAGCA-3′ (sense) and TGTTAT-GGGGTCTGGGATGGA-3′ (antisense).
Data are presented as mean±standard error of mean (SEM) obtained in at least 3 separate experiments. Statistical analysis was performed using the ANOVA test. A value of P<0.05 indicates statistical significance.
AT1 Receptor Decay
It is well established that angiotensin II downregulates AT1 receptor mRNA expression. The putative angiotensin II–elicited acceleration of AT1 receptor mRNA was tested in an in vitro decay assay. VSMCs were incubated with vehicle or 100 nmol/L angiotensin II for 2 hours, polyribosomes were isolated, and the decay of the AT1 receptor mRNA was monitored in an in vitro system. Figure 1 shows that angiotensin II causes an accelerated AT1 receptor mRNA decay within the polysomal compartment. Thus, angiotensin II causes destabilization of AT1 receptor mRNA in VSMCs.
Binding of Polysomal Proteins to the AT1 Receptor mRNA
Polysomal proteins and their binding to the 3′ untranslated region are involved in the induced AT1 receptor mRNA destabilization. UV mRNA protein crosslink assays were used to analyze protein-mRNA interactions in the AT1 receptor mRNA. Polysomal proteins isolated from VSMCs were incubated with a radioactive AT1 receptor mRNA riboprobe of the region 1864 to 2213 in the presence of unlabeled, mutated AT1 receptor mRNA transcripts. Figure 2A shows a representative autoradiography, which reveals that several polysomal proteins bind the 3′ untranslated region of the AT1 receptor mRNA. This binding is selective and is inhibited by addition of unlabeled AT1 receptor mRNA transcripts bases 1864 to 2213 and 2131 to 2213 but not by fragment bases 2131 to 2187 and 2131 to 2170, suggesting binding of the identified proteins at the very 3′ part of the AT1 receptor mRNA. Previous studies with mutated competitors showed that the cognate binding sequence is located at bases 2175 to 2195. Control experiments using multiple AT1 receptor mRNA competitors and various radiolabeled AT1 receptor mRNA probes (5′ untranslated region, open reading frame) demonstrated that mRNA binding proteins exclusively bind within the delineated region bases 2175 to 2195.12 This region comprises a AUUUUA hexamer and is considerably AU-rich (5′-AAAGUAAUUUUAUUGUAAUGU-3′).
Isolation and Characterization of Calreticulin as AT1 Receptor mRNA Binding Protein
Further clarification of the described regulative pathways is dependent on the identification of participating binding proteins. Polysomal proteins were isolated from VSMCs and subjected to sequential anionic exchange chromatography. Isolated fractions were monitored by UV crosslink assays (Figure 3A). The left panel of Figure 3A shows the initial purification step on a standard chromatography system using a Source DEAE-Sepharose Fast Flow column. Protein fractions with RNA-binding activity (lanes 7 and 8) were submitted to a second anionic exchange chromatography using a FPLC chromatography system (right panel). A purified protein (right panel, lanes 8 and 9) was extracted. Protein sequence was defined by MALDI analysis and revealed identification of calreticulin. Figure 3B shows amino acid sequences of fragments characterized during fingerprinting. Homology alignments identified calreticulin as the purified protein.
Control experiments showed that recombinant calreticulin binds indeed to the 3′ untranslated region of the AT1 receptor mRNA. However, this requires phosphorylation of calreticulin. Recombinant calreticulin was phosphorylated with Src kinase, JNK kinase, β-insulin receptor kinase (β-IR kinase), or autophosphorylated. Binding of calreticulin to the AT1 receptor riboprobe bases 1864 to 2213 was monitored by UV crosslink assay. Figure 3C reveals that phosphorylation with Src as well as JNK kinase enables interaction of calreticulin with the AT1 receptor mRNA.
In order to assess whether this different binding of calreticulin was based on differential protein phosphorylation, the latter was monitored. Figure 3D shows that JNK kinase led to a profound phosphorylation of calreticulin. Src kinase also reproducibly phosphorylated calreticulin, but not as abundant as JNK. β-IR kinase and autophosphorylation, which did not result in binding of calreticulin to the AT1 receptor mRNA, showed no detectable phosphorylation of calreticulin. Thus, phosphorylation of calreticulin is required for interaction with the AT1 receptor mRNA; however, the extend of phosphorylation seems not to influence the strength of protein-mRNA cross-talk.
Calreticulin binds the AT1 receptor mRNA in the region 2175 to 2195, as confirmed by UV crosslink assays including various competitors (Figure 3E). Calreticulin did not bind to the GAPDH mRNA, eNOS mRNA, or to an AT1 receptor mRNA lacking bases 2175 to 2195 (data not shown).
Phosphorylation of Calreticulin by Angiotensin II
As angiotensin II accelerates AT1 receptor mRNA decay and as calreticulin-AT1 receptor mRNA interaction requires phosphorylation, the ability of angiotensin II to phosphorylate calreticulin was investigated. VSMCs were stimulated with 100 nmol/L angiotensin II, calreticulin was immunoprecipitated from cell homogenates and phosphorylation was monitored with an anti-phosphotyrosine–antibody and Western blotting (Figure 4), revealing that angiotensin II led to a time-dependent phosphorylation of calreticulin.
These experimental findings identify calreticulin as an mRNA binding protein that interacts with the 3′ untranslated region of the AT1 receptor mRNA at bases 2175 to 2195, provided that phosphorylation was initiated. The latter is inducible on stimulation with angiotensin II.
Calreticulin Accelerates AT1 Receptor mRNA Decay
The functional relevance of the interaction of calreticulin with the AT1 receptor mRNA was assessed in transfection experiments. Overexpression of calreticulin in VSMCs downregulated basal AT1 receptor expression (Figure 5A).
To assess whether the AT1 receptor mRNA is destabilized by calreticulin, recombinant, phosphorylated calreticulin was added to in vitro decay assays. AT1 receptor mRNA concentrations were assessed by real-time PCR. Figure 5B demonstrates that calreticulin led to a significant destabilization of the AT1 receptor mRNA.
In addition, it was tested whether angiotensin II–induced AT1 receptor mRNA downregulation, which is based on AT1 receptor mRNA destabilization, is inhibited by transfection with antisense-calreticulin cDNA. Therefore, cells were transfected with either an insertless pcDNA3 vector or an antisense calreticulin construct and incubated with vehicle or 1 μmol/L angiotensin II for 4 hours. Effective overexpression of calreticulin or inhibition of calreticulin protein expression was monitored by Western blots (insert at the top of Figure 5C). AT1 receptor and GAPDH mRNA were quantified by real-time PCR. Figure 5C demonstrates that antisense calreticulin effectively blocks the angiotensin II–caused AT1 receptor mRNA decrease, suggesting that interaction of calreticulin with the 3′ untranslated region of the AT1 receptor mRNA causes destabilization of the AT1 receptor mRNA.
The presented data display a cognate mRNA sequence involved in posttranscriptional regulation of the AT1 receptor, and the identification of a novel mRNA binding protein is reported. Calreticulin binds, if phosphorylated, to the cognate sequence bases 2175 to 2195 of the AT1 receptor mRNA and leads to destabilization of the AT1 receptor mRNA. Angiotensin II stimulation, which causes destabilization of AT1 receptor mRNA, causes phosphorylation of calreticulin.
Cognate sequences within the 3′ untranslated region, such as the pentamer AUUUA, and nucleotide sequences, such as UUAUUUA(U/A)(U/A) and UUAUUUAUU, regulate mRNA stability by interaction with cytosolic and nuclear-associated factors.13–20⇓⇓⇓⇓⇓⇓⇓ Several genes have been explored with regard to mRNA binding protein properties and the role of the mRNA consensus sequences in the homeostasis of mRNA turnover, including immediate early genes (eg, c-fos, c-myc), cytokines (eg, colony stimulating factor, tumor necrosis factor α), growth factors (eg, vascular endothelial growth factor), and the inducible and endothelial isoforms of nitric oxide synthase.21–29⇓⇓⇓⇓⇓⇓⇓⇓ Regulation of endothelial NO synthase expression is connected to a 51-kDa binding protein interacting with a 43 base sequence in the 3′ untranslated region of the eNOS mRNA.30 Malbon and colleagues have thoroughly characterized interactions of the β2-adrenergic receptor mRNA with corresponding binding proteins.17,25,26⇓⇓ Their work revealed that mRNA processing is arranged through binding of various proteins in the 3′ untranslated region of the β2-adrenergic receptor mRNA, including a 35-kDa protein. The latter is induced on stimulation with β-adrenergic agonists and is a prerequisite for agonist-induced destabilization of the β2-adrenergic receptor mRNA acting through cognate sequences identified in the 3′ untranslated region composed of a 20 nucleotide (A+U)-rich element that compromises an AUUUUA hexamer rather than the commonly identified AUUUA pentamer.17 The β2-adrenergic receptor mRNA displays also binding to AUF-1, which probably resembles the best known mRNA binding protein.31 This 35 kDa binding protein that was cloned and functionally characterized by Brewer participates also in β2-adrenergic receptor mRNA and seems to be involved in the posttranscriptional processing of various other genes.14
Several other mRNA binding proteins ranging in size from approximately 100 to 20 kDa have been described, although only a few of them have been characterized on the level of nucleotide or amino acid sequence.14,15,27,32⇓⇓⇓
The characterization of the novel mRNA binding protein calreticulin further elucidates the detailed molecular pathways of AT1 receptor mRNA turnover. So far, calreticulin has been ascribed 3 different functions. First, calreticulin is involved in intracellular calcium homeostasis due to calcium binding sites in the protein and its ability to regulate other calcium-handling proteins.33 Second, calreticulin acts as chaperone-like molecule involved in folding and oligomerization of glycoproteins.34 Third, calreticulin has been described as a receptor for nuclear export.35 The data presented herein associate calreticulin with another biological property: based on its binding to distinct nucleotides in the 3′ untranslated region of the AT1 receptor mRNA, calreticulin is engaged in the inducible decay of the AT1 receptor mRNA. This result is in concert with the finding that calreticulin acts as mRNA binding protein in rubella virus.36 Obviously, phosphorylation causes a either a conformational change of calreticulin or activates a preformed binding site of calreticulin, which enables the interaction with the mRNA. It cannot be excluded that calreticulin binds other protein factors before or while interacting with the target mRNA. Furthermore, it is not clear if calreticulin itself activates RNases, which realize the actual AT1 receptor mRNA decay. Alternatively, calreticulin may induce a change in the tertiary structure of an mRNA, leading to a more or less pronounced interference with nucleases.
Binding of mRNA binding proteins on their corresponding mRNA is profoundly influenced by secondary and tertiary structures of the RNA. Hairpins or stem loops formed by the RNA region of interest may interact with neighboring proteins. In the case of the AT1 receptor, computer modeling showed that the identified AT1 receptor mRNA binding motif bases 2175 to 2195 forms such a stem loop. That holds true for the entire AT1 receptor mRNA and also for the isolated 20-base transcript used in our study as competitor and decoy. In concert with our finding that such a mutated mRNA binds no longer to polysomal proteins, deletion of this motif abolished the stem loop; therefore, suggesting the importance of secondary structure for protein-mRNA interaction.12
The presented findings could represent general mechanisms involved in mRNA processing and could be also applicable to other genes. However, calreticulin binds neither to the GAPDH nor to the eNOS mRNA.
AT1 receptor regulation is of broad interest because numerous studies have shown that AT1 receptor activation is closely involved in the pathogenesis of hypertension, atherosclerosis, and heart failure.1–6⇓⇓⇓⇓⇓ The identification of the involved binding protein and the cognate mRNA sequence enables investigations that need to test if dysregulated AT1 receptor expression induced by inherited variations in calreticulin or the corresponding 3′ untranslated AT1 receptor mRNA is involved in the development of cardiovascular diseases.
Our findings reveal novel molecular mechanisms involved in posttranscriptional regulation of the AT1 receptor mRNA, prompting further characterization of interactions between binding proteins and the AT1 receptor mRNA, and thus, enabling the structural identification of engaged binding factors. The latter is a prerequisite for the better understanding of the complex cellular mechanisms of cytosolic mRNA turnover. In addition, the described mechanisms for AT1 receptor regulation may have relevant implications for the pathogenesis of atherosclerosis and hypertension because pathological abnormalities of AT1 receptor regulation may drive both development and progression of these diseases.
This work was supported by the Deutsche Forschungsgemeinschaft.
↵*Both authors contributed equally to this study.
Original received June 25, 2001; revision received November 8, 2001; accepted November 9, 2001.
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