Abstract
The extracellular N-terminus of G-protein-coupled receptors may be involved in signalling events. We examined this in the angiotensin II type 1 receptor (AT1-R) using monoclonal antibody 6313/G2, raised against a conserved sequence in the N-terminal domain, and found it evokes inhibitory and stimulatory responses. In rat aortic smooth muscle cell (RASMC) primary cultures, 6313/G2 (2.5 μg/ml) inhibited both basal and angiotensin II (Ang II; 10−7 mol/l)-stimulated [H3]thymidine incorporation. Exposure to 6313/G2 gave sustained increases in phosphorylated protein kinase Cα (PKCα) but gave a decrease in phosphorylated p44/42 extracellular signal-regulated kinases (ERK1/2) sustained from 10 min to 48 h compared with untreated control RASMC. In contrast, Ang II had no effect on PKCα, and, though it is acutely stimulatory (up to 5 min), it had no sustained effect on ERK1/2 either. Using Fura-2 and microfluorimetry, 6313/G2 added alone induced a transient increase in intracellular calcium ([Ca2+]i), with a characteristic response curve different from that of Ang II itself. The antibody was without effect on an Ang II-stimulated activator protein-1 reporter system, though it reduced unstimulated reporter activity. Such discriminatory effects on intracellular signalling suggest that the AT1-R N-terminus itself might be a target for therapeutic intervention in chronic vascular disease.
Introduction
Vascular smooth muscle cell (VSMC) growth (hyperplasia/hypertrophy) occurs in chronic vascular diseases including atherosclerosis and hypertension. Angiotensin II (Ang II), produced by the renin–angiotensin system (RAS), plays a crucial role in the VSMC proliferative activity (Berk 2001, Cheng et al. 2005). In addition to the systemic RAS, local tissue RASs may also be involved with VSMC growth (Berk 2001, Xiao et al. 2004, Kobori et al. 2007). For these reasons, the RAS is a key target for drugs combating cardiovascular disease (Weir & Dzau 1999). The actions of angiotensin-converting enzyme inhibitors and Ang II type 1 receptor (AT1-R) blockers are well studied (Bohm 2007). There is still scope for development, however, and one view is that targeting the RAS by gene therapy or antibody (Cheng et al. 2005) treatment may provide a longer term treatment for hypertension (Leckie 2005, Oro et al. 2007).
The AT1 receptor is a member of the G-protein-coupled receptor superfamily (Griendling & Alexander 1993) and has been shown to mediate most of the physiological actions of Ang II (Higuchi et al. 2007). Although the ligand-binding region of AT1-R is generally thought to be partly in the barrel formed by the seven transmembrane domains (Griendling & Alexander 1993, Correa et al. 2002, Perodin et al. 2002), and also to involve extracellular domains, including part of the N-terminal region (Hjorth et al. 1994, Santos et al. 2004, Pignatari et al. 2006, Oliveira et al. 2007) and there is now a growing body of evidence that suggests that the N-terminus undergoes activation-dependent conformational changes and influences receptor activity (Hjorth et al. 1994, Lecat et al. 2002, Motta et al. 2003, Gupta et al. 2007). However, relatively little is known about the N-terminus-mediated signalling pathways.
Through interactions with AT1-R and phospholipase C activation, Ang II triggers the inositol trisphosphate (IP3)/calcium and the protein kinase C (PKC) signalling pathways (Malarkey et al. 1996). Of the 12 distinct members of the PKC family (Mellor & Parker 1998), which all exhibit distinct properties and activation profiles (Mellor & Parker 1998), PKCα is the major Ca2+-dependent phorbol-ester-sensitive PKC in neonatal rat ventricular myocytes (Puceat et al. 1994) and after phosphorylation it has been shown to inhibit cell proliferation (Acs et al. 1997). Although other members of the PKC family are involved in mitogenic signal transduction (Toker 1998), the precise mechanism is still not clear, though one common route is by activating the mitogen-activated protein (MAP) kinases through multiple mechanisms, including increased extracellular signal-regulated kinases (ERK1/2) phosphorylation (Toker 1998, Heidkamp et al. 2001, Daniels et al. 2007).
The MAP kinase superfamily of serine/threonine protein kinases is associated with the increased expression of the early response genes c-fos, c-myc and c-jun (Lyall et al. 1992), DNA synthesis and cell growth (Morrell et al. 1999). In cultured VSMC and in intact arteries, Ang II activates ERK1/2, c-Jun N-terminal kinases (JNKs) and p38 that are the three major members of the MAP kinase family, via AT1-R (Touyz et al. 1999), and although their exact functional effects are ill-defined, regulation of cell proliferation may be an important result of ERK1/2 activation (Touyz & Schiffrin 2000). Downstream activator protein-1 (AP-1) consisting of a variety of dimers composed of members of the Fos, Jun and activating transcription factor (ATF) families of proteins (Angel & Karin 1991) convert extracellular signals into changes in the expression of specific targets which harbour AP-1-binding site(s) in their promoter or enhancer regions. Among factors regulating AP-1 are the ERK cascade that induces hyperphosphorylation of Fos and increases its activity, as well as JNKs that phosphorylate Jun and ATF within their N-terminal transactivation domains and enhance their transactivation potential (Wagner 2001). AP-1 family members regulate cell proliferation and differentiation in the context of both normal development and disease (Hess et al. 2004).
Our previous results showed that a monoclonal antibody (6313/G2) to be a conserved sequence in the extracellular domain of the AT1-R stimulates IP3 and aldosterone production, while non-competitively inhibiting Ang II-induced PKC activation (Kapas et al. 1994). The present studies were designed to explore action of this antibody on the proliferation of rat arterial smooth muscle cells (RASMCs). Understanding the relationship between the structure and functions of this receptor could lead to novel therapies in chronic vascular diseases.
Materials and Methods
Materials
Male Wistar rats, 200–300 g, were obtained from commercial suppliers. RPMI-1640 culture medium and fetal bovine serum (FBS) were from Sigma. Methyl-[3H]thymidine-5.0 Ci/mmol was obtained from Amersham Biosciences, Fura-2 from Molecular Probes (Paisley, UK), TransFast Transfection Reagent and Dual-Luciferase Reporter Assay System from Promega, the vectors pAP1(PMA)-TA-Luc from BD Biosciences Clontech and pCVM-β-gal (Smith et al. 2001) was generously given by Dr Paul Lavender (Department of Respiratory Medicine and Allergy, Kings College London, Guy's Hospital, London, UK). Anti-phospho-p44/42 ERK1/2 (Thr202/Tyr204) monoclonal antibody and anti-p44/42 ERK1/2 polyclonal antibody were obtained from Cell Signalling Technology Inc. (Hitchin, UK). Anti-phospho-PKCα (Ser657) and anti-PKCα were purchased from Upstate Ltd (Milton Keynes, UK). Anti-AT1-R monoclonal antibody (6313/G2, mouse IgG subclass 2a, hybridoma supernatant) was produced as previously described (Barker et al. 1993) and purified using ImmunoPure(L) Immunoglobulin Purification Kit from Perbio Science UK Ltd (Cramlington, UK) according to the manufacturer's protocol. The purified 6313/G2 was used for all experiments, except both purified 6313/G2 and 6313/G2 hybridoma supernatant were used in the cell proliferation and 6313/G2 specificity studies. Unless otherwise stated, all other reagents were purchased from Sigma.
Cell culture
RASMCs were isolated from rat thoracic and abdominal arteries by the media explant method and cultured over several passages according to previously described methods (Xiao et al. 2004). RASMCs were grown in RPMI-1640 culture medium supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, 4 μmol/l l-glutamine and 20% FBS. Cultures were incubated at 37 °C in a humidified atmosphere of 5% CO2 in air. Cells were passaged using a solution of 0.125% trypsin and 0.02% EDTA in PBS. Experiments were performed after three to five passages. Stimulants added as appropriate were Ang II and bFGF (basic fibroblast growth factor) and the specific AT1R inhibitor losartan was also used (generously given by Merck Sharp & Dohme Ltd).
Tritiated thymidine uptake
A suspension of RASMC (105 cells/ml) was prepared on the first day of the experiment using RPMI-1640 supplemented with 20% FBS. The cells were synchronised in the G0/G1 phase by serum deprivation for 24 h. The quiescent (serum-deprived) cells were incubated with the appropriate experimental media in the presence of 2% FBS for 48 h with four wells per group. Methyl-[3H]thymidine (0.1 mCi/ml) in 10 μl medium was added to each well, which contained 1 ml medium/well. After 24 h of the addition of radioactive thymidine 20 nM, cells were collected as described previously (Xiao et al. 2004). Cells were then dissolved in 0.5 ml of 0.1 M NaOH and a 0.3 ml aliquot was mixed with 3.5 ml scintillation fluid (Packard Instrument BV Chemical Operations, Groningen, The Netherlands) and, after standing overnight at room temperature, tritium content was assayed in a liquid scintillation counter.
Cell count
A suspension of RASMC (0.5×105 cells/ml) was prepared on the first day of the experiment using RPMI-1640 supplemented with 20% FBS. One millilitre of this cell suspension was distributed to each well of a 24-well multiwell dish. The medium was replaced 24 h after the subculture with appropriate experimental media with three wells per group. Experiments were terminated after an additional 48-h incubation. The cells were resuspended by washing them with PBS and treatment with 0.3 ml PBS containing 0.125% trypsin and 0.02% EDTA. Digestion was stopped by the addition of 0.7 ml of 20% FBS RPMI-1640 to each well. Cells were counted in a haemocytometer using light microscopy.
Immunoblotting analysis
Sample preparations
Cultured cells were treated with Ang II at 10−7 mol/l for 1, 5, 10, 20, 30 and 60 min to examine the effects of Ang II on phosphorylations of PKCα and ERK1/2. The short-term (10 min) and long-term (48 h) effects of 6313/G2 on activities of ERK1/2 and PKCα were carried out by incubating these cells with purified 6313/G2 at 2.5 μg/ml, or as negative control with mouse non-specific IgG (whole molecule, unconjugate) from Perbio Science UK Ltd. Solubilised total cell protein from cultured cells was prepared as described previously (Xiao et al. 2000), and the membrane fraction was extracted using a Compartmental Protein Extraction kit (CHEMICON Europe Ltd, Eastleigh, Hampshire, UK). Extracted protein was estimated using DC Protein Assay (Bio-Rad).
SDS-PAGE
Unless otherwise stated, 50 μg/lane protein was separated on 9% SDS-PAGE to identify the activation of ERK1/2 and 30 μg/lane on 7.4% SDS-PAGE to examine the activation of PKCα. Proteins were separated on SDS-PAGE together with Precision Protein Standard (Bio-Rad) and transferred to Hybond-ECL nitrocellulose membrane (Amersham Biosciences) overnight at 30 V at 4 °C. Membranes were blocked in 5% non-fat dry milk in Tris-buffered saline with Tween-20 (TBS-T; 20 mM Tris base, 150 mM NaCl and 0.1% Tween-20, pH 7.6) for 2 h at room temperature followed by incubation with diluted 1:1000 rabbit monoclonal anti-phospho-p44/42 ERK1/2 antibodies and/or rabbit polyclonal anti-p44/42 ERK1/2 antibodies, in TBS-T with 5% BSA, or with diluted 1:1000 rabbit polyclonal anti-phospho-PKCα (Ser657) antibody and/or mouse anti-PKCα monoclonal antibody in TBS-T at 4 °C with gentle shaking overnight. To test specificities of purified 6313/G2 and 6313/G2 hybridoma supernatant, membrane protein amounts ranging from 2.5 to 40 μg were used, and membranes with transferred proteins were incubated with unpurified 6313/G2 undiluted supernatant or purified antibody, 2.5 μg/ml, at 4 °C overnight. In another experiment, serial dilutions of 6313/G2 supernatant were used with a fixed concentration of membrane protein (30 μg per lane).
Detection of protein
Protein-bound antibody was detected by incubation of the membrane with horseradish peroxidase-labelled second antibody at room temperature for 1.5 h. Conjugated donkey anti-rabbit IgG (Amersham Biosciences) diluted 1:2000 in TBS-T was used for detection of anti-phospho-ERK1/2/anti-ERK1/2 antibody and anti-phospho-PKCα and sheep anti-mouse IgG (1:1000) was used to detect anti-PKCα and 6313/G2. Bands were detected using enhanced chemiluminescence assay reagents and quantified by Image Acquisition and Analysis software (UVP, Cambridge, UK).
Measurement of [Ca2+]i in RASMC
RASMCs cultured on ultrathin glass coverslips were loaded with 1 μM Fura-2 acetoxymethyl ester in serum-free RPMI-1640 medium for 30 min at 37 °C in a humidified atmosphere of 5% CO2 in air. The cells were then washed three times using modified Krebs–Ringer bicarbonate solution (3.6 mM K+, 1.2 mM Ca2+, 0.5 mM Mg2+, 5 mM HEPES and 20 mM HCO-) to remove excess dye.
Fura-2 microfluorimetry was carried out using a DeltaRam Fast Fluorescence Imaging Microscopy System (Photo Technology International Inc. (PTI), Twickenham, UK) using a modified inverted fluorescence microscope (Olympus, PA, USA). The coverslips containing the Fura-2-loaded cells were placed in a temperature-controlled perfusion chamber (Intracel Ltd, Royston, UK), which were set on the stage of the microscope. The cells were illuminated by alternating excitation light (340 and 380 nm), as obtained spectroscopically from a 75 watt xenon light source. Fluorescence from the cells was collected using the 40× u.v. Apochromatic oil immersion objective lens and Model IC-200 intensified CCD camera (PTI). Fluorescence (emission wavelength 510 nm) intensities at excitation wavelengths 340 nm (F340) and 380 nm (F380), and the F340/F380 ratio were continuously measured.
Transient transfection-AP1 reporter assay
pAP1(PMA)-TA-Luc is designed for monitoring the induction of the PKC signal transduction pathway, as well as related pathways such as the MAP kinase pathway, by assaying luciferase activity. pAP1(PMA)-TA-Luc contains six tandem copies of AP1 enhancer, located upstream of the sense encoding firefly luciferase (Luc). Transfections and luciferase assays were conducted using TransFast Transfection Reagent and Dual-Luciferase Reporter Assay System, according to the manufacturer's protocol. Briefly, cells were prepared as for the thymidine incorporation experiments. Cells, cultured in 24-well dishes with serum-free culture medium, were transiently transfected with 0.5 μg pAP1(PMA)-TA-Luc. As a control for transfection efficiency, samples were cotransfected with 0.25 μg pCVM-β-gal. After 3-h incubation at 37 °C, serum-free culture medium was replaced with 20% FBS culture medium. After 12 h of transfection, cells were washed with PBS and then incubated in the absence or presence of Ang II at 10−7 mol/l and/or purified 6313/G2, 2.5 μg/ml, in 2% FBS culture medium for 24 h. Luciferase assays were performed using a Luminoskan microplate luminometer and Ascent Software (ThermoLabsystems, Helsinki, Finland), and β-galactosidase was assayed using standard procedures (Smith et al. 2001).
Statistical analysis
Values are expressed as means±s.e.m. The level of significance for difference between means was evaluated by ANOVA and Student's t-test.
Results
Cell proliferation
Tritiated thymidine incorporation into RASMC was significantly increased by Ang II (10−7 mol/l) in 2% FBS medium compared with controls, but this was inhibited by purified 6313/G2 (2.5 μg/ml). 6313/G2 added alone to RASMC also significantly decreased tritiated thymidine incorporation when compared with controls (Fig. 1A). Similar results were obtained from treatment with unpurified 6313/G2 hybridoma supernatant diluted 1:10 in culture medium (Fig. 1B).
(A) The effects of 6313/G2 on [3H]thymidine incorporation into RASMC. Cultured RASMCs were incubated for 48 h alone or with Ang II 10−7 mol/l, and/or purified 6313/G2 (2.5 μg/ml) in 2% FBS RPMI-1640 medium. (B) Similar experiments as for (A) but using 6313/G2 hybridoma supernatant diluted 1:10 in culture medium. In both (A) and (B), Ang II significantly increased [3H]thymidine incorporation compared with controls, and this stimulation was inhibited by 6313/G2. In both (A) and (B), 6313/G2 alone also significantly inhibited [3H]thymidine incorporation. Values are means±s.e.m. 4 per group. Ang II, angiotensin II; Ab, purified 6313/G2; Abs, 6313/G2 hybridoma supernatant. (C) RASMCs (0.5×105/ml/well) were incubated with 20% FBS RPMI-1640 medium containing Ang II in the absence and presence of losartan (10−5 mol/l) or bFGF (50 ng/ml) with or without losartan and purified 6313/G2 (2.5 μg/ml) for 48 h. Cell numbers were obtained by counting, values are means±s.e.m. N=3 throughout. ANOVA: P<0.001; Student's t-test: **P<0.01 and *P<0.05.
Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0498
Using cell counts as measure of proliferation (Fig. 1C), Ang II again stimulated proliferation, and this was inhibited by losartan 10−5 mol/l (P<0.01). Cell number was also increased by bFGF (50 ng/ml; P<0.05), but bFGF stimulation was blocked by neither losartan nor 6313/G2.
Immunoblotting
Both purified and unpurified 6313/G2 showed linear reactivity in immunoblots of serially diluted membrane proteins (Fig. 2A and B) and there was also a linear relationship with dilutions of antibody (Fig. 2C). In all cases, 6313/G2 recognised a single discrete protein band of approximate molecular weight 60 kDa.
Immunoblotting for testing specificity of 6313/G2. Serially diluted cell membrane protein over the range 2.5–40 μg was separated on 9% SDS-PAGE. The blot was probed with (A) 6313/G2 supernatant without dilution or (B) purified 6313/G2 (2.5 μg/ml). (C) Immunoblotting of 30 μg protein/lane detected using undiluted or 4- to 16-fold dilutions of the 6313/G2 hybridoma supernatant. A single band 60 kDa was detected in (A), (B) and (C).
Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0498
Phosphorylated ERK1/2 and total ERK1/2 with molecular weights of 44 and 42 kDa and phosphorylated PKCα and total PKCα with a molecular weight of 82 kDa were detected by immunoblot analysis in RASMC incubated with or without 6313/G2 (2.5 μg/ml) or Ang II (10−7 mol/l) in 2% FBS medium for 10 min and 48 h (Fig. 3). Treatment with 6313/G2 significantly inhibited phosphorylation of ERK1/2 and increased phosphorylation of PKCα, compared with controls. As negative controls, non-specific IgG treatment gave no changes in either phosphorylated ERK1/2 or phosphorylated PKCα in short-term treatment. Conversely, immunoblotting for phosphorylated ERK1/2 and PKCα demonstrated that although Ang II induced rapid (1–5 min) activation of ERK1/2 (data not shown) it had no sustained effect on ERK1/2 phosphorylation. It also had no effects on PKCα phosphorylation nor on total ERK1/2 and PKCα protein.
(A) RASMCs were incubated alone or treated with purified 6313/G2 (2.5 μg/ml), Ang II 10−7 mol/l, and/or non-specific IgG (2.5 μg/ml) in 2% FBS RPMI-1640 medium for 10 min (short term) and 48 h (long term). Two phospho-ERK bands with the expected sizes of 44 and 42 kDa and a specific band with the expected size of 82 kDa for phospho-PKCα were detected, and both used for quantitation. (B) Quantification of phosphorylated proteins shows treatment with 6313/G2 significantly decreased ERK1/2 phosphorylation and increased PKCα phosphorylation in short- and long-term treatment, and Ang II had no such sustained effect. As negative controls, non-specific IgG had no effects on the phosphorylation of ERK1/2 and PKCα in short treatment. Data are means±s.e.m. from three experiments. Values are expressed as a percentage of control values. Student's t-test: **P<0.01 and * P<0.05 versus control culture. C, control culture; Ab, purified 6313/G2; Ang, Ang II and IgG, non-specific IgG.
Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0498
Measurement of intracellular Ca2+
Transient elevation of intracellular Ca2+ by 6313/G2 (2.5 μg/ml; Fig. 4A and C) and Ang II (10−7 mol/l; Fig. 4B and D) was detected in cultured RASMC. Figure 4A and B shows pseudocolour images of cultured RASMC measured by ratiometry of Fura-2. After 10 s of stimulation, [Ca2+]i increased in these cells. The effects of 6313/G2 and Ang II on [Ca2+]i dynamics are different. The ratio of fluorescence intensities, F340/F380 (Fig. 4C and D), shows that Ang II gave a single peak at 19 s, whereas 6313/G2 invariably showed two peaks at 12 s and a larger subsequent peak at 25 s. A non-specific IgG control gave no response (not shown).
Effect of 6313/G2 on intracellular Ca2+ elevation in cultured RASMC. After loading with 1 μM Fura-2 acetoxymethyl ester, RASMCs were stimulated with purified 6313/G2 (2.5 μg/ml) or Ang II 10−7 mol/l in the presence of extracellular Ca2+ (1.2 mM). Pseudocolour images of RASMC show changes in [Ca2+]i after the addition of (A) 6313/G2 or (B) Ang II. (C and D) After 10 s of stimulation, [Ca2+]i was increased by both treatments. The ratio of fluorescence intensities (F340/F380) showed a major peak 25 and 19 s after stimulation of (C) 6313/G2 and (D) Ang II respectively. This was preceded by a smaller peak after 12 s on 6313/G2 stimulation. Results are representative of three separate experiments giving similar results. Three cells were selected in each experiment. Ab, purified 6313/G2. Colour scale bar: fluorescence ratio represents [Ca2+]i. Arrow indicates the addition of effector.
Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0498
AP-1 reporter assay
When cultured RASMCs were transfected with vector pAP1(PMA)-TA-Luc, treatment with Ang II (10−7 mol/l) enhanced luciferase activity compared with controls (Fig. 5), but this enhancement was not inhibited by 6313/G2 (2.5 μg/ml). In contrast, 6313/G2 alone added to RASMC significantly decreased luciferase activity when compared with controls.
Effect of 6313/G2 on Ang II-induced AP-1 promoter activity. After transfection with 0.5 μg vector pAP1(PMA)-TA-Luc, RASMCs were treated with purified 6313/G2 (2.5 μg/ml), or Ang II 10−7 mol/l with or without purified 6313/G2 for 24 h. Ang II significantly increased luciferase activity, representing AP-1 reporter activity. 6313/G2 had no effect on AP-1 reporter activity stimulated by Ang II, but 6313/G2 alone decreased AP-1 reporter activity. Values are expressed as a percentage of controls. Student's t-test: *P<0.05. ANOVA: P<0.01. Ab, purified 6313/G2. Means±s.e.m., n=3.
Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0498
Discussion
Monoclonal antibody 6313/G2 recognises AT1R with high specificity (Harrison-Bernard et al. 1997, Yang et al. 1997, Cheng et al. 2005). Immunoblots from most target tissues show a single immunoreactive band, though of varying molecular weight, apparently because of differences in degrees of glycosylation. Cells that do not express the receptor are not immunoactive (Barker et al. 1993). The high specificity of 6313/G2 was confirmed by the single 60 kDa immunoreactive band detected in RASMC membrane proteins, regardless of antibody or membrane protein concentration, or indeed antibody purity, over the concentration range used (Fig. 2). The molecular weight is significantly higher than predicted by the primary structure of the receptor, but consistent with other results, and probably reflects the degree of glycosylation (Barker et al. 1993).
Many studies have examined Ang II-induced mitogenic effects in cultured RASMC and their inhibition by the AT1-R antagonist losartan (Weber et al. 1994, Xiao et al. 2000). Monoclonal antibody 6313/G2, whether used in an unpurified or purified form, was also effective in inhibiting Ang II-stimulated RASMC proliferation (Fig. 1). A number of studies, including our own (Xiao et al. 2000), have demonstrated the autocrine function of Ang II in cultured VSMC, which may explain the inhibition of 6313/G2 on basal cell proliferation in the absence of Ang II (Fig. 1A and B). We can additionally confirm that AT1 receptor inhibition by losartan, the specific AT1-R antagonist, also produces inhibition of cell proliferation in the absence of any added stimulation (data not shown).
Although it is known that AT1-R signals crosstalk with other receptors, for example, the α-adrenergic or endothelin, so that part of the actions of these ligands are mediated by AT1-R (Cook et al. 1993, Dzimiri 2002, Lemos et al. 2002, Nossaman et al. 2007, Wang et al. 2007), in these experiments, bFGF-stimulated proliferation was blocked by neither losartan nor 6313/G2 (Fig. 1C), demonstrating its independence of AT1-R signalling, again supporting the specificity of 6313/G2's action.
The intracellular signalling evoked by the AT1-R has been extensively studied in VSMC (Higuchi et al. 2007). Ang II elicits complex highly regulated cascades of intracellular signal transduction that lead to short-term (seconds or minutes) vascular effects, such as contraction, and to long-term (hours or days) effects, such as cell growth, migration, extracellular matrix deposition and inflammation (Touyz & Schiffrin 2000). Ang II-stimulated long-term effects are mediated via activation of various kinases, including MAP kinase, induction of proto-oncogene expression and stimulation of nuclear signalling cascades, ultimately resulting in cellular growth (Touyz & Schiffrin 2000). To understand whether the MAP kinases were involved in the inhibitory effects of 6313/G2, their activation was assessed by immunoblot analysis in treated RASMC. The data demonstrate that 6313/G2 caused a reduction in the level of phosphorylated ERK1/2 (Fig. 3) in RASMC after 10-min and 48-h exposure to 6313/G2, without any effect on total p44/42 ERK1/2, whereas non-specific IgG had no effect. These data may appear consistent with the inhibition by 6313/G2 of Ang II-stimulated cell proliferation. In contrast, as others have shown (Morrell et al. 1999), Ang II itself induced a transient activation of ERK1/2 at 2–5 m (data not shown) but had no effect at longer periods (Fig. 3).
The mechanisms underlying AT1-mediated MAP kinase responses are related to amplification by PKCs (Malarkey et al. 1996, Oro et al. 2007). In myocytes, PKC isoenzymes are differentially activated by various stimuli (Clerk et al. 1994, Puceat et al. 1994, Strait & Samarel 2000) and PKCs differently regulate MAP kinase cascades in an isoenzyme-specific and time-dependent manner (Heidkamp et al. 2001). Previously, we showed that 6313/G2 alters Ang II-induced PKC localisation in rat-dispersed adrenal glomerulosa cells (Kapas et al. 1994). However, in the present study, 6313/G2 rapidly induced phosphorylation of PKCα and this was sustained over 48 h (Fig. 3). This is perhaps consistent with inhibition of proliferation, as others have suggested (Acs et al. 1997). In contrast, Ang II had no such long-term effect on PKCα phosphorylation (Fig. 3). In RASMC, ERK1/2 is activated by PKCε (Malarkey et al. 1996) and by PKCζ (Berra et al. 1995), which lack the C2 domain, thus their kinase activities are Ca2+ independent (Liao et al. 1997). It is relevant that PKCα is calcium activated (Toker 1998), though its interactions with ERK1/2 (Seo et al. 2004), and indeed cell hypertrophy/proliferation remains unclear (Stewart & O'Brian 2005).
The integrated Ca2+ signal stimulated by Ang II is complex and elicited via multiple pathways, including release from calcium stores and calcium channel activation (Touyz & Schiffrin 2000). Our studies demonstrated that 6313/G2 also induced a transient Ca2+ signal response in RASMC (Fig. 4) and it is possible that this Ca2+ transient triggered the increased phosphorylation of PKCα. Interestingly, the Ca2+ signal from 6313/G2, with two peaks, differs from that evoked by Ang II (Fig. 4), suggesting different kinetics of Ca2+ release or entry.
Many of the signal events relevant to cell proliferation are mediated through activation of AP-1, by the MAP kinase family, and these have also been shown to be activated by interactions of Ang II and AT1-R (Ishida et al. 1998). It is believed that Ang II rapidly increases the DNA-binding activity of AP-1 in RASMC through activation of JNKs (Viedt et al. 2000). Our results confirmed that 6313/G2 inhibited basal activity of the AP-1 reporter, but had no effect on Ang II-stimulated activation of AP-1 (Fig. 5), perhaps indicating that this antibody exerts inhibitory effects on AP-1 activity via an alternative route. Downregulation of ERK1/2 (Fig. 3) may contribute to 6313/G2-mediated inhibition of AP-1 activity, whereas, as noted, Ang II activates AP-1.
In total, the results show that in RASMC, as in the rat adrenal (Kapas et al. 1994), 6313/G2 has unique actions on AT1 receptor-linked signalling pathways. It exerts inhibitory effects on RASMC growth perhaps through increased phosphorylation of PKCα, decreased ERK1/2 activation and inhibition of AP-1 activity, but in contrast it also produces a calcium signal. These properties are somewhat different from those described for agonist antibodies in pre-eclampsia (Thway et al. 2004), and suggest that antibodies raised against different receptor domains might evoke different activities, though such studies have not been done. Furthermore, such discriminatory effects on intracellular signalling may indicate new routes for therapy in atherosclerosis and hypertension.
Acknowledgements
We are grateful to Prof. Mitsuhiro Okamoto and Dr Hiroshi Takemori, Department of Biochemistry and Molecular Biology, (H-1), Osaka University Osaka, 565-0871, Japan, for the vector pAP1(PMA)-TA-Luc. Part of this research was funded by Innathera Inc.
References
Acs P, Wang QJ, Bogi K, Marquez AM, Lorenzo PS, Biro T, Szallasi Z, Mushinski JF & Blumberg PM 1997 Both the catalytic and regulatory domains of protein kinase C chimeras modulate the proliferative properties of NIH 3T3 cells. Journal of Biological Chemistry 272 28793–28799.
Angel P & Karin M 1991 The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochimica et Biophysica Acta 1072 129–157.
Barker S, Marchant W, Ho MM, Puddefoot JR, Hinson JP, Clark AJ & Vinson GP 1993 A monoclonal antibody to a conserved sequence in the extracellular domain recognizes the angiotensin II AT1 receptor in mammalian target tissues. Journal of Molecular Endocrinology 11 241–245.
Berk BC 2001 Vascular smooth muscle growth: autocrine growth mechanisms. Physiological Reviews 81 999–1030.
Berra E, Diaz-Meco MT, Lozano J, Frutos S, Municio MM, Sanchez P, Sanz L & Moscat J 1995 Evidence for a role of MEK and MAPK during signal transduction by protein kinase C zeta. EMBO Journal 14 6157–6163.
Bohm M 2007 Angiotensin receptor blockers versus angiotensin-converting enzyme inhibitors: where do we stand now? American Journal of Cardiology 100 38J–44J.
Cheng ZJ, Vapaatalo H & Mervaala E 2005 Angiotensin II and vascular inflammation. Medical Science Monitor 11 RA194–RA205.
Clerk A, Bogoyevitch MA, Anderson MB & Sugden PH 1994 Differential activation of protein kinase C isoforms by endothelin-1 and phenylephrine and subsequent stimulation of p42 and p44 mitogen-activated protein kinases in ventricular myocytes cultured from neonatal rat hearts. Journal of Biological Chemistry 269 32848–32857.
Cook MD, Phillips MI, Cook VI, Kimura B & Wilcox CS 1993 Angiotensin II receptor subtypes on adrenal adenoma in primary hyperaldosteronism. Journal of the American Society of Nephrology 4 111–116.
Correa SA, Franca LP, Costa-Neto CM, Oliveira L, Paiva AC & Shimuta SI 2002 Relevant role of Leu265 in helix VI of the angiotensin AT1 receptor in agonist binding and activity. Canadian Journal of Physiology and Pharmacology 80 426–430.
Daniels D, Yee DK & Fluharty SJ 2007 Angiotensin II receptor signalling. Experimental Physiology 92 523–527.
Dzimiri N 2002 Receptor crosstalk. Implications for cardiovascular function, disease and therapy. European Journal of Biochemistry 269 4713–4730.
Griendling KK & Alexander RW 1993 The angiotensin (AT1) receptor. Seminars in Nephrology 13 558–566.
Gupta A, Decaillot FM, Gomes I, Tkalych O, Heimann AS, Ferro ES & Devi LA 2007 Conformation state-sensitive antibodies to G-protein-coupled receptors. Journal of Biological Chemistry 282 5116–5124.
Harrison-Bernard LM, Navar LG, Ho MM, Vinson GP & el-Dahr SS 1997 Immunohistochemical localization of ANG II AT1 receptor in adult rat kidney using a monoclonal antibody. American Journal of Physiology 273 F170–F177.
Heidkamp MC, Bayer AL, Martin JL & Samarel AM 2001 Differential activation of mitogen-activated protein kinase cascades and apoptosis by protein kinase C epsilon and delta in neonatal rat ventricular myocytes. Circulation Research 89 882–890.
Hess J, Angel P & Schorpp-Kistner M 2004 AP-1 subunits: quarrel and harmony among siblings. Journal of Cell Science 117 5965–5973.
Higuchi S, Ohtsu H, Suzuki H, Shirai H, Frank GD & Eguchi S 2007 Angiotensin II signal transduction through the AT1 receptor: novel insights into mechanisms and pathophysiology. Clinical Science 112 417–428.
Hjorth SA, Schambye HT, Greenlee WJ & Schwartz TW 1994 Identification of peptide binding residues in the extracellular domains of the AT1 receptor. Journal of Biological Chemistry 269 30953–30959.
Ishida M, Ishida T, Thomas SM & Berk BC 1998 Activation of extracellular signal-regulated kinases (ERK1/2) by angiotensin II is dependent on c-Src in vascular smooth muscle cells. Circulation Research 82 7–12.
Kapas S, Hinson JP, Puddefoot JR, Ho MM & Vinson GP 1994 Internalization of the type I angiotensin II receptor (AT1) is required for protein kinase C activation but not for inositol trisphosphate release in the angiotensin II stimulated rat adrenal zona glomerulosa cell. Biochemical and Biophysical Research Communications 204 1292–1298.
Kobori H, Nangaku M, Navar LG & Nishiyama A 2007 The intrarenal renin-angiotensin system: from physiology to the pathobiology of hypertension and kidney disease. Pharmacological Reviews 59 251–287.
Lecat S, Bucher B, Mely Y & Galzi JL 2002 Mutations in the extracellular amino-terminal domain of the NK2 neurokinin receptor abolish cAMP signaling but preserve intracellular calcium responses. Journal of Biological Chemistry 277 42034–42048.
Leckie BJ 2005 Targeting the renin–angiotensin system: what's new? Current Medicinal Chemistry – Cardiovascular and Hematological Agents 3 23–32.
Lemos VS, Cortes SF, Silva DM, Campagnole-Santos MJ & Santos RA 2002 Angiotensin-(1-7) is involved in the endothelium-dependent modulation of phenylephrine-induced contraction in the aorta of mRen-2 transgenic rats. British Journal of Pharmacology 135 1743–1748.
Liao DF, Monia B, Dean N & Berk BC 1997 Protein kinase C-zeta mediates angiotensin II activation of ERK1/2 in vascular smooth muscle cells. Journal of Biological Chemistry 272 6146–6150.
Lyall F, Dornan ES, McQueen J, Boswell F & Kelly M 1992 Angiotensin II increases proto-oncogene expression and phosphoinositide turnover in vascular smooth muscle cells via the angiotensin II AT1 receptor. Journal of Hypertension 10 1463–1469.
Malarkey K, McLees A, Paul A, Gould GW & Plevin R 1996 The role of protein kinase C in activation and termination of mitogen-activated protein kinase activity in angiotensin II-stimulated rat aortic smooth-muscle cells. Cellular Signalling 8 123–129.
Mellor H & Parker PJ 1998 The extended protein kinase C superfamily. Biochemical Journal 332 281–292.
Morrell NW, Upton PD, Kotecha S, Huntley A, Yacoub MH, Polak JM & Wharton J 1999 Angiotensin II activates MAPK and stimulates growth of human pulmonary artery smooth muscle via AT1 receptors. American Journal of Physiology 277 L440–L448.
Motta SC, Poletti EF, Souza SE, Correa SA, Jubilut GN, Paiva AC, Shimuta SI & Nakaie CR 2003 Tachyphylactic properties of angiotensin II analogs with bulky and hydrophobic substituents at the N-terminus. Journal of Peptide Research 62 227–232.
Nossaman BD, Baber SR, Nazim MM, Detrolio JD & Kadowitz PJ 2007 Differential effects of losartan and candesartan on vasoconstrictor responses in the rat. Canadian Journal of Physiology and Pharmacology 85 360–371.
Oliveira L, Costa-Neto CM, Nakaie CR, Schreier S, Shimuta SI & Paiva AC 2007 The angiotensin II AT1 receptor structure–activity correlations in the light of rhodopsin structure. Physiological Reviews 87 565–592.
Oro C, Qian H & Thomas WG 2007 Type 1 angiotensin receptor pharmacology: signaling beyond G proteins. Pharmacology and Therapeutics 113 210–226.
Perodin J, Deraet M, Auger-Messier M, Boucard AA, Rihakova L, Beaulieu ME, Lavigne P, Parent JL, Guillemette G & Leduc R et al. 2002 Residues 293 and 294 are ligand contact points of the human angiotensin type 1 receptor. Biochemistry 41 14348–14356.
Pignatari GC, Rozenfeld R, Ferro ES, Oliveira L, Paiva AC & Devi LA 2006 A role for transmembrane domains V and VI in ligand binding and maturation of the angiotensin II AT1 receptor. Biological Chemistry 387 269–276.
Puceat M, Hilal-Dandan R, Strulovici B, Brunton LL & Brown JH 1994 Differential regulation of protein kinase C isoforms in isolated neonatal and adult rat cardiomyocytes. Journal of Biological Chemistry 269 16938–16944.
Santos EL, Pesquero JB, Oliveira L, Paiva AC & Costa-Neto CM 2004 Mutagenesis of the AT1 receptor reveals different binding modes of angiotensin II and [Sar1]-angiotensin II. Regulatory Peptides 119 183–188.
Seo HR, Kwan YW, Cho CK, Bae S, Lee SJ, Soh JW, Chung HY & Lee YS 2004 PKCalpha induces differentiation through ERK1/2 phosphorylation in mouse keratinocytes. Experimental and Molecular Medicine 36 292–299.
Smith PJ, Cousins DJ, Jee YK, Staynov DZ, Lee TH & Lavender P 2001 Suppression of granulocyte-macrophage colony-stimulating factor expression by glucocorticoids involves inhibition of enhancer function by the glucocorticoid receptor binding to composite NF-AT/activator protein-1 elements. Journal of Immunology 167 2502–2510.
Stewart JR & O'Brian CA 2005 Protein kinase C-α mediates epidermal growth factor receptor transactivation in human prostate cancer cells. Molecular Cancer Therapeutics 4 726–732.
Strait JB & Samarel AM 2000 Isoenzyme-specific protein kinase C and c-Jun N-terminal kinase activation by electrically stimulated contraction of neonatal rat ventricular myocytes. Journal of Molecular and Cellular Cardiology 32 1553–1566.
Thway TM, Shlykov SG, Day MC, Sanborn BM, Gilstrap LC III, Xia Y & Kellems RE 2004 Antibodies from preeclamptic patients stimulate increased intracellular Ca2+ mobilization through angiotensin receptor activation. Circulation 110 1612–1619.
Toker A 1998 Signaling through protein kinase C. Frontiers in Bioscience 3 D1134–D1147.
Touyz RM & Schiffrin EL 2000 Signal transduction mechanisms mediating the physiological and pathophysiological actions of angiotensin II in vascular smooth muscle cells. Pharmacological Reviews 52 639–672.
Touyz RM, El Mabrouk M, He G, Wu XH & Schiffrin EL 1999 Mitogen-activated protein/extracellular signal-regulated kinase inhibition attenuates angiotensin II-mediated signaling and contraction in spontaneously hypertensive rat vascular smooth muscle cells. Circulation Research 84 505–515.
Viedt C, Soto U, Krieger-Brauer HI, Fei J, Elsing C, Kubler W & Kreuzer J 2000 Differential activation of mitogen-activated protein kinases in smooth muscle cells by angiotensin II: involvement of p22phox and reactive oxygen species. Arteriosclerosis, Thrombosis, and Vascular Biology 20 940–948.
Wagner EF 2001 AP-1 – introductory remarks. Oncogene 20 2334–2335.
Wang MH, Fok A, Huang MH & Wong NL 2007 Interaction between endothelin and angiotensin II in the up-regulation of vasopressin messenger RNA in the inner medullary collecting duct of the rat. Metabolism 56 1372–1376.
Weber H, Webb ML, Serafino R, Taylor DS, Moreland S, Norman J & Molloy CJ 1994 Endothelin-1 and angiotensin-II stimulate delayed mitogenesis in cultured rat aortic smooth muscle cells: evidence for common signaling mechanisms. Molecular Endocrinology 8 148–158.
Weir MR & Dzau VJ 1999 The renin–angiotensin–aldosterone system: a specific target for hypertension management. American Journal of Hypertension 12 205S–213S.
Xiao F, Puddefoot JR & Vinson GP 2000 The expression of renin and the formation of angiotensin II in bovine aortic endothelial cells. Journal of Endocrinology 164 207–214.
Xiao F, Puddefoot JR, Barker S & Vinson GP 2004 Mechanism for aldosterone potentiation of angiotensin II-stimulated rat arterial smooth muscle cell proliferation. Hypertension 44 340–345.
Yang H, Lu D, Vinson GP & Raizada MK 1997 Involvement of MAP kinase in angiotensin II-induced phosphorylation and intracellular targeting of neuronal AT1 receptors. Journal of Neuroscience 17 1660–1669.
