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Cardiovascular Research Advance Access first published online on November 13, 2007
This version [Corrected Proof] published online on January 23, 2008
Cardiovascular Research, doi:10.1093/cvr/cvm068
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2007. For permissions please email: journals.permissions@oxfordjournals.org

FXR-mediated regulation of angiotensin type 2 receptor expression in vascular smooth muscle cells

Qiuhong Zhang1,{dagger}, Fengtian He1,{dagger}, Ramalinga Kuruba1, Xiang Gao1, Annette Wilson2, Jiang Li1, Timothy R. Billiar3, Bruce R. Pitt2, Wen Xie1 and Song Li1,*

1 Center for Pharmacogenetics, Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, 639 Salk Hall, Pittsburgh, PA 15261, USA
2 Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261, USA
3 Department of Surgery, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA

* Corresponding author. Tel: +1 412 383 7976; fax: +1 412 648 1664. E-mail address: sol4{at}pitt.edu

Received 15 August 2007; revised 17 October 2007; accepted 9 November 2007

Time for primary review: 20 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 SUPPLEMENTARY MATERIAL
 Funding
 References
 
Aims: The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily and plays an important role in the pathogenesis of cardiovascular diseases via regulating the metabolism and transport of cholesterol. We and others have recently shown that FXR is also expressed in the vasculature, including endothelial cells and smooth muscle cells (SMC). However, the biological significance of FXR activation in SMC is still poorly understood. In this study, we examine the effect of FXR ligands on the angiotensin system in rat aortic SMC (RASMC), as angiotensin II (Ang II) signalling contributes to various types of vascular lesions by promoting cell growth of vascular SMC.

Methods and results: Treatment of RASMC with a FXR ligand showed no obvious effect on the expression of angiotensinogen, Ang II type 1 receptor (AT1R) or type 4 receptor (AT4R) but led to a significant increase in the expression of type 2 receptor (AT2R). FXR ligand treatment also resulted in an inhibition of Ang II-mediated extracellular signal-regulated kinase (ERK) activation and growth proliferation. Promoter reporter gene and electrophoretic mobility-shift assays suggest that FXR upregulates AT2R expression at a transcriptional level. Upregulation of AT2R appears to play a role in the FXR-mediated inhibition of ERK activation via upregulation of Rous sarcoma oncogene (Src) homology domain-containing tyrosine phosphatase 1 (SHP-1) because FXR-mediated upregulation of SHP-1 can be blocked by an AT2R antagonist and FXR-mediated ERK inactivation was significantly attenuated via treatment with either an AT2R antagonist or a SHP-1 inhibitor.

Conclusion: FXR in SMC may serve as a novel molecular target for modulating Ang II signalling in the vasculature.

KEYWORDS FXR; Angiotensin II; Angiotensin II type 2 receptor; Smooth muscle cells; Regulation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 SUPPLEMENTARY MATERIAL
 Funding
 References
 
Angiotensin II (Ang II), an octapeptide hormone, is the active component of the renin-angiotensin system (RAS). The RAS was originally regarded as a circulating system. However, many of its components are localized in tissues indicating the existence of a local tissue RAS as well.1,2

In mammalian cells, Ang II mediates its effects via at least two high-affinity plasma membrane receptors, Ang II type 1 receptor (AT1R) and Ang II type 2 receptor (AT2R). The AT1R belongs to the seven membrane-spanning G protein-coupled receptor family and typically activates phospholipase C through the heterotrimeric Gq protein.2,3 To date, AT1R has been shown to mediate most of the physiological actions of Ang II, and this subtype is predominant in the control of Ang II-induced vascular functions. The second major isoform of the Ang II receptor, AT2R, is normally expressed at high levels in foetal tissues and decreases rapidly after birth. The AT2R is also a seven transmembrane-type, G protein-coupled receptor, comprising 363 amino acids. Although the exact functional roles of AT2R are unclear, these receptors may antagonize AT1R-mediated effects by inhibiting cell growth, and by inducing apoptosis and vasodilation.2,4,5 A number of mechanisms have been proposed for AT2R signalling including (i) activation of various protein phosphatases causing protein dephosphorylation,6,7 (ii) activation of the NO/cGMP system,7,8 and (iii) stimulation of phospholipase A2 with subsequent release of arachidonic acid.6,7

RAS has profound effects on endothelial cells (EC) and smooth muscle cells (SMC). These effects are not only haemodynamic in nature, but also comprise inflammation, thrombosis, and cell proliferation through stimulation of production of cytokines and growth factors.9 Therapeutics targeted to RAS has been a mainstay for the treatment of a number of cardiovascular diseases including hypertension and heart failure.10,11 Recently nuclear receptors, particularly peroxisome proliferator-activated receptors (PPARs) have also been explored as a molecular target for modulating RAS for the treatment of cardiovascular diseases.12–15

The farnesoid X receptor (FXR) (NR1H4) is a member of the nuclear receptor superfamily that is highly expressed in liver, kidney, adrenals, and intestine.16 FXR is activated by bile acids (BAs), such as the primary BA chenodeoxycholic acid (CDCA).17 In addition to BAs, synthetic FXR agonists have also been developed. Activation of FXR causes both feedback inhibition of cholesterol 7-{alpha} hydroxylase (CYP7A1), the rate-limiting enzyme in BA biosynthesis from cholesterol, and activation of intestinal BA binding protein.18 In addition to their application in the treatment of cholestasis, FXR ligands including BAs have recently been proposed as novel therapeutics in cardiovascular diseases based on their effectiveness in lowering circulating triglycerides and cholesterol,19–21 and improving hyperglycaemia.22 These effects are largely attributed to FXR activation in the liver. Interestingly, a study by Bishop-Bailey et al.23 has shown that FXR is also expressed in the vasculature including EC and SMC. Activation of FXR results in growth inhibition of SMC.23 We have recently extended their study by showing that activation of FXR in vascular EC resulted in down-regulation of endothelin-1 (ET-1) expression via interference of AP-1/NF-{kappa}B signalling.24 We report in this study that the activation of FXR in SMC resulted in upregulation of AT2R and inhibition of angiotensin II-mediated extracellular signal-regulated kinase (ERK) activation and growth proliferation. These studies suggest that FXR in SMC may serve as a novel molecular target for modulating Ang II signalling in vasculature.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 SUPPLEMENTARY MATERIAL
 Funding
 References
 
2.1 Materials
CDCA, angiotensin II, PD123319, and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St Louis, MO, USA). The SHP-1 inhibitor, {alpha}-bromo-4-hydroxyacephenone 4-hydroxyphenacyl Br, was purchased from Calbiochem (San Diego, CA, USA). 32P and [125I]-Sar1, Ile-angiotensin II (NEX248010UC) were purchased from PerkinElmer (Waltham, MA, USA). 5'-bromodeoxyuridine (BrdU) cell proliferation kit was purchased from Millipore (Bedford, MA, USA). NE-PER nuclear and cytoplasmic extraction reagents were purchased from Pierce (Rockford, IL, USA). GW4064 was synthesized following a published protocol.25 All products for cell culture were purchased from Invitrogen (Carlsbad, CA, USA). pCMX, pCMX-FXR, and pCMV-βgal constructs were described previously.26 pCMX-FXR-DN, a plasmid expressing dominant negative rat FXR, was constructed following a published protocol.27

2.2 Cell culture
Rat aortic artery smooth muscle cells (RASMC) were isolated from the thoracic aorta of Sprague-Dawley rats by enzymatic digestion as previously described.28 All studies conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). RASMC were cultured in Dulbecco’s modified Eagle’s/F-12 medium supplemented with 10% foetal bovine serum (FBS). RASMC identity and purity of cultures were verified by immunohistochemical analysis using a monoclonal antibody for smooth muscle {alpha}-actin.

Normal African green monkey kidney fibroblast cells (CV-1 line) were obtained from American Type Culture Collection (Manassas, VA, USA) and were cultured in DMEM supplemented with 10% FBS.

2.3 Plasmid construction
Rat AT2R promoter was amplified by PCR. The fragments (–2904, –1617, –1103, and –746 bp) were cloned into PGL3-basic vector (Promega, Madison, WI, USA) and the resulting plasmid was named as pGL3-AT2R. The fragment (–744 to –521 bp) was cloned into pTK-luciferase vector (Promega) and the resulting plasmid was named as TK-224 bp (see Methods in Supplemental material online).

2.4 RT–PCR and quantitative real-time RT–PCR
The expression of mRNA for FXR, phospholipid transfer protein (PLTP), angiotensinogen (AGT), AT1R, AT2R, and angiotensin type 4 receptor (AT4R) was examined by RT–PCR. AT2R mRNA expression was also further examined by quantitative real-time RT–PCR (see Methods in Supplemental material online).

2.5 Immunofluorescent staining
For FXR and AT2R immunostaining, RASMC were seeded subconfluently on fibronectin-treated glass coverslips, and treated with a FXR ligand CDCA or GW4064 for 24 h. Cells were then treated with rabbit anti-FXR antibody (sc-13063) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit anti-AT2R antibody (ab19134) (Abcam, Cambridge, MA, USA) (1: 100 dilution) for 1 h at 4°C. The cells were further incubated for 45 min with Alexa Fluor® 488 goat anti-rabbit IgG (A11034 [GenBank] ) (Invitrogen Life Technologies, Carlsbad, CA, USA) (1: 1000 dilution). The slides were mounted with Vectashield (Vector Lab, Burlingame, CA, USA) and observed under a fluorescent microscope.

2.6 Radioligand binding assay
Cells were seeded into 12-multiwell plates and grown in 10% FBS DMEM/F-12. Quiescence was achieved by serum deprivation in reduced-serum OPTI-MEM ® I culture medium overnight. Cells were then treated with various concentrations of GW4064 for 24 h, and were washed once in ice-cold Hanks’ balanced salt solution (HBBS) supplemented with calcium and magnesium. The HBBS was aspirated off and cells were incubated at 4°C for 4 h in 0.25 mL of serum free DMEM/F-12 containing 0.2% BSA and 0.2 nM radioligand [125I]-Sar1, Ile8-angiotensin II in the absence ( for the total count) or presence of 1 µM losartan or 1 µM PD123319 (each group was done in triplicate). After incubation, the cells were washed rapidly twice with ice-cold PBS, solubilized in 0.1 N NaOH, and counted with a {gamma} counter. AT1R binding was calculated as the difference between the total count and the count from samples incubated with losartan. AT2R binding was determined by subtracting the count of samples incubated with PD123319 from the total count. The net radioactivity count was converted to molar values by use of specific activity of the ligand.

2.7 Transfection assays
Transfection with CV-1 cells was performed as described (see Methods in Supplemental material online).29

2.8 Electrophoretic mobility shift assay
FXR and RXR proteins were generated in vitro by coupled transcription/translation (TNT system, Promega) with pCMX-FXR and pCMX-RXR plasmids. The DNA probe that contains an inverted repeat spaced by two nucleotides (IR2) (5'-TTTTGGTCAgtTGCCCTGCT-3') (AT2R/IR2) was derived from a region in the rat AT2R promoter that contains a putative FXR response element (bold). It was labelled with [{gamma}-32P] ATP by using the Klenow fragment of DNA polymerase. DNA binding reactions were carried out as follows: aliquots of in vitro translation mixture were incubated in 20 µL binding buffer containing 2 µg polydI: dC (Sigma) and 6–20 x 103 CPM of DNA probe at room temperature for 20 min. The binding mixture was then applied onto a 5% polyacrylamide gel (0.5x TrisBorateEDTA buffer) for electrophoresis. The gels were dried and exposed at –80°C for autoradiography.

Similarly, EMSA was performed with nuclear extracts generated from RASMC that were treated with GW4064.

2.9 Cell proliferation assay
Cells were seeded subconfluently on 96-multiwell plates. Quiescence was achieved by serum deprivation in standard culture medium supplemented with 1% FCS for 48 h. Cells were then treated with various concentrations of CDCA or GW4064 for 24 h. Cells were further treated with Ang II (100 nM) for another 24 h. Cell viability and proliferation were measured respectively by the MTT and BrdU incorporation assays.

2.10 Western blotting
Western blot analysis of AT2R, phospho-p44/42, phospho-p38, SHP-1, MKP-1, PP2A, or β-actin was performed as described previously (see Methods in Supplemental material online).24

2.11 Statistical analysis
All data are expressed as mean ± SD unless otherwise stated. Comparisons between two groups were made with unpaired Student’s t-tests. Non-parametric comparisons between three or more groups were made with ANOVA followed by Kruskal–Wallis post hoc analysis. In all cases, P < 0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 SUPPLEMENTARY MATERIAL
 Funding
 References
 
3.1 FXR is expressed in rat aortic artery smooth muscle cells
RT–PCR analysis of RNA from RASMC clearly displayed constitutive expression of transcript for FXR although its level was lower than that in rat liver (Figure 1A). Figure 1B shows the immunostaining of SMC with FXR antibody. FXR expression was localized in both the cytoplasm and the nucleus. As expected, FXR was found to be predominantly localized in the nucleus 24 h following treatment with a FXR agonist, CDCA or GW4064. CDCA is a natural ligand for FXR. GW4064 is a synthetic ligand that is highly specific for FXR. Preliminary studies showed that FXR was also expressed in rat pulmonary artery SMC (RPASMC) and human coronary artery smooth muscle cells (HCASMC) (data not shown).


Figure 1
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  		Figure 1 Functional FXR is expressed in rat aortic smooth muscle cells (RASMC). (A) RT–PCR analysis of FXR mRNA expression in RASMC. Liver was included as a positive control. (B) Immunostaining of RASMC with anti-FXR antibody. Cells were treated with CDCA (50 µM), GW4064 (5 µM), or vehicle (DMSO) for 24 h and were then subjected to immunostaining with anti-FXR antibody. (C) CDCA- or GW4064-mediated upregulation of PLTP or FXR mRNA expression in RASMC. RASMC were treated with various concentrations of CDCA or GW4064 and the mRNA expression of PLTP or FXR was analysed by RT–PCR 24 h following the treatment. Results shown in this figure are the representative data from at least three to five experiments.

 
3.2 FXR expressed in rat aortic artery smooth muscle cells is functional and exhibits autoregulation
Following demonstration of FXR expression in RASMC, we then determined whether FXR is functional in SMC by examining the induction of expression of phospholipid transfer protein (PLTP) following treatment with CDCA or GW4064. PLTP is one of the FXR target genes that are induced following FXR activation.30 As shown in Figure 1C, treatment of RASMC with CDCA or GW4064 led to increase in the mRNA levels of PLTP in a concentration-dependent fashion, suggesting that FXR expressed in RASMC is biologically active. CDCA- or GW4064-mediated induction of PLTP expression was similarly observed in RPASMC (data not shown).

We have previously shown that treatment with a FXR ligand, CDCA, led to upregulation of the expression of FXR itself in rat pulmonary artery EC.24 A similar FXR autoregulation was observed in RASMC. As shown in Figure 1C, treatment with either CDCA or GW4064 led to upregulation of FXR mRNA expression in a dose-dependent manner.

3.3 Treatment with CDCA or GW4064 led to upregulation of AT2R in rat aortic artery smooth muscle cells
The above studies clearly demonstrated that FXR is expressed in RASMC and is functional. As an initial approach to investigate the biological function of FXR in SMC, we examined whether activation of FXR by CDCA or GW4064 would affect expression of components of RAS including angiotensinogen (AGT), AT1R, AT2R, and AT4R. A previous study has shown that activation of FXR in rat hepatocytes resulted in downregulation of AGT expression.31 However, treatment with either CDCA or GW4064 had no effect on expression of AGT in RASMC as assessed by RT–PCR (Figure 2A), suggesting that FXR-mediated regulation of AGT may be cell-type dependent. CDCA or GW4064 treatment also had no significant effect on AT1R or AT4R expression in RASMC (Figure 2A). Interestingly, treatment with CDCA or GW4064 led to a significant increase in the mRNA levels of AT2R in RASMC as revealed by both semi-quantitative RT–PCR (Figure 2A) and quantitative real-time RT–PCR (Figure 2B). The FXR ligand effect appears to be concentration dependent (Figure 2A and B). Upregulation of AT2R following CDCA or GW4064 treatment was also confirmed by western blot analysis (Figure 2C), immunostaining (Figure 2D), and radioligand binding assay (Figure 2E).


Figure 2
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  		Figure 2 CDCA- or GW4064-mediated upregulation of AT2R expression in RASMC. (A) RASMC were treated with various concentrations of CDCA or GW4064 and the mRNA expression of AGT, AT1R, AT2R, or AT4R was analysed by semi-quantitative RT–PCR 24 h following the treatment. (B) Quantitative real-time RT–PCR analysis of AT2R mRNA expression in RASMC before and after treatment with CDCA or GW4064. *P < 0.05 (vs. DMSO), n = 3. (C) Western blot analysis of AT2R protein expression in RASMC 24 h following treatment with CDCA or GW4064. (D) Immunostaining of RASMC with anti-AT2R antibody 24 h following treatment with CDCA or GW4064. (E) Radioligand binding assay of AT1R and AT2R in RASMC before and after treatment with GW4064. *P < 0.05 (vs. DMSO), n = 3. Results shown in panels A, C, and D are the representative data from at least three to four experiments.

 
3.4 Rat AT2R promoter is a likely transcriptional target of FXR
Upregulation of AT2R mRNA expression by CDCA or GW4064 suggests that activation of FXR modulates AT2R expression at the transcriptional level. We then hypothesized that activation of FXR enhances AT2R expression via exerting its stimulatory activity on AT2R promoter. To test this hypothesis, we constructed several luciferase reporter expression plasmids (pGL3-AT2R) that are driven by up to 3 kb sequences of rat AT2R promoter. To examine promoter activation, CV-1 cells were transfected with pGL3-AT2R in the absence or presence of a FXR expression vector. CV-1 cells instead of RASMC were used for transfection due to low transfection efficiency in the RASMC. As shown in Figure 3A, treatment with CDCA or GW4064 alone resulted in a slight increase in the transcriptional activity of the 1617 bp AT2R promoter. This might be due to a low level of endogenous FXR activity in CV-1 cells. Such increase in AT2R promoter activity was more pronounced when the cells were co-transfected with a FXR expression plasmid. This enhancement in the promoter activity seems to be greater with GW4064 than with CDCA. Nonetheless, the AT2R promoter activation was diminished when the cells were co-transfected with an expression plasmid encoding a dominant-negative FXR, suggesting a role of FXR in CDCA- or GW4064-mediated increase in the transcriptional activity of AT2R promoter. Similar results were obtained in transfection with reporter gene driven by AT2R promoters of 746 bp (Figure 3B), 1103 bp, and 2904 bp (see Supplementary material online, Figure S1A and B), respectively. These results suggest that the 746 bp promoter contains sequences sufficient for FXR transactivation.


Figure 3
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  		Figure 3 FXR enhances the transcriptional activity of the rat AT2R gene promoter. CV-1 cells were transiently transfected with pGL3-AT2R in the presence or absence of pCMX-FXR. Five hours later the transfection medium was replaced with complete medium and cells were incubated for 12 h. Cells were then cultured in the conditioned medium in the presence of CDCA, GW4064, or vehicle DMSO. Luciferase assay was performed 24 h following the ligand treatment. Data shown in the figure represent mean (SD) from three experiments in triplicates. In some studies, cells were cotransfected with pCMX-FXR and pCMX-FXR-DN at a weight ratio of 1:2 and luciferase assay was then similarly performed. (A) Transfection with a reporter driven by a 1617 bp AT2R promoter; (B) transfection with a reporter driven by a 746 bp AT2R promoter.

 
To search for FXR responsive elements (FXREs) that may mediate AT2R induction by CDCA and GW4064, the 746 bp AT2R promoter sequence was subjected to in silico analysis with a Web-based algorithm (NUBIScan).32 One such potential FXRE was identified, and its sequences and location are shown in Figure 4A. As an initial approach to examine a role of this putative binding site in FXR-mediated activation of AT2R promoter, a DNA fragment of 224 bp that encompasses this site was amplified and cloned into a luciferase reporter driven by a heterologous TK promoter. As shown in Figure 4B, CDCA or GW4064, alone or together with co-transfection with a FXR expression plasmid, similarly activated the 224 bp regulatory element as they did on a 1617 bp AT2R promoter (Figure 3A).


Figure 4
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  		Figure 4 Analysis of putative FXR-responsive elements (FXREs) in rat AT2R promoter. (A) Identification of a putative FXRE in rat AT2R promoter via an in silico analysis with a Web-based algorithm (NUBIScan). (B) FXR-dependent regulation of the (–744~–521 bp) DNA fragment upon the heterologous TK promoter in CV-1 cells. Transfection and luciferase assays were similarly performed as described in the legend to Figure 3C. EMSA analysis of the binding of FXR/RXR to the putative FXRE in rat AT2R promoter. Double-stranded oligonucleotides were end-labelled with [{gamma}-32P]-ATP using T4 polynucleotide kinase. The labelled probes were incubated with in vitro-translated RXR/FXR for 20 min. The reactions were analysed by electrophoresis in a non-denaturing 5% polyacrylamide gel followed by autoradiography. An oligonucleotide containing a consensus IR1/FXRE sequence was included as a positive control. The binding of nuclear proteins of GW4064-treated RASMC to ATR2/IR2 was similarly analysed. Results shown in this panel are the representative data from four experiments.

 
Figure 4C shows the result of an EMSA with a 20 bp oligonucleotide that contains the putative FXRE. A typical inverted repeat spaced by one nucleotide (IR1)/FXRE oligonucleotide was also included as a positive control because FXR is known to bind to IR1 with high specificity and affinity. Interaction of the oligonucleotide with in vitro translated FXR/retinoid X receptor (RXR) yielded a DNA/protein band of expected mobility (lane 1). This binding was specific, as it was inhibited by addition of excess unlabelled (cold) AT2R/IR2 (lane 2) or IR1/FXRE (lane 3). Introduction of mutations to the AT2R/IR2 oligonucleotide resulted in a dramatic decrease in its binding to FXR/RXR (lane 4).

We also examined DNA binding activity to AT2R/IR2 in nuclear extracts generated from GW4064-treated RASMC (Figure 4C). Incubation of the ATR2/IR2 probe with GW4064/RASMC nuclear extracts produced a shifted band (lane 1). The addition of cold ATR2/IR2 (lane 2) or IR1/FXRE (lane 3) probe or antibody against FXR protein (lane 5) substantially diminished this binding, indicating that FXR was a principal DNA-binding component of this protein-DNA complex. No obvious interaction was noticed between the nuclear extracts and a mutated ATR2/IR2 (lane 4). The above results suggest that ATR2/IR2 is likely to mediate the transactivation of ATR2 promoter by FXR.

3.5 Upregulation of AT2R resulted in an inhibition of Ang II-mediated ERK activation
To study the biological consequence of AT2R upregulation, we examined whether treatment with CDCA or GW4064 affected the Ang II-mediated activation of mitogen-activated protein kinase (MAPK) signalling and the results are shown in Figure 5. In agreement with previous studies, treatment of RASMC with Ang II led to a significant increase in the amount of phosphorylated ERK1/2 and p38 (Figure 5A). Treatment with CDCA or GW4064 significantly inhibited the Ang II-mediated ERK activation but showed little effect on p38 signalling (Figure 5A). Upregulation of AT2R appears to play a role in CDCA- or GW4064-mediated ERK inactivation because such inhibitory effect was substantially abolished by pre-treatment with PD123319, an AT2R specific antagonist (Figure 5B).


Figure 5
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  		Figure 5 Role of AT2R in CDCA- and GW4064-mediated inhibition of Ang II-stimulated ERK signalling and growth proliferation. RASMC in 6-well plates were cultured in serum-free medium for 24 h followed by treatment with various concentrations of CDCA or GW4064 for another 24 h. Cells were then treated with Ang II (100 nM) for 15 min and cell lysates were subjected to western blot analysis of the expression of p-ERK1/2 and p-p38 (A), respectively. In a separate experiment RASMC received similar treatments as described above except that cells were pre-treated with PD123319 (10 µM) for 30 min prior to Ang II treatment. Western blot analysis of ERK and p38 (B) was then performed. Results shown in panels A and B are the representative data from at least three to five experiments. (C) RASMC were grown in 96-well plates and quiescence was achieved by serum deprivation in standard culture medium supplemented with 1% FCS for 48 h. Cells were then treated with CDCA or GW4064 for 24 h, followed by culture in the absence or presence of Ang II (100 nM) for another 24 h. Cell growth was determined by a BrdU incorporation assay (n = 3), *P < 0.05 (vs. DMSO).

 
ERK signalling has been shown to play an important role in Ang II-mediated proliferation of SMC.33 Thus, we then examined whether CDCA or GW4064 treatment will similarly affect the stimulatory effect of Ang II on RASMC proliferation. Figure 5C shows the result of a BrdU incorporation assay. In agreement with previous studies, treatment with Ang II resulted in a significant increase in cell proliferation. Such stimulatory effect was significantly abolished by CDCA or GW4064 treatment. Similar results were observed with a MTT assay (see Supplementary material online, Figure S2).

3.6 Role of SHP-1 in CDCA- and GW4064-mediated inhibition of ERK signalling
It has been shown previously that the growth inhibitory effects of the AT2R are associated with the activation and/or induction of a series of phosphatases including protein tyrosine phosphatase Src homology domain-containing tyrosine phosphatase 1 (SHP-1), mitogen-activated protein kinase phosphatase-1 (MKP-1), and serine/threonine phosphatase 2A (PP2A), which results in the inactivation of AT1R- and/or growth factor-activated ERK.34 As an initial approach to elucidate a possible role of phosphatases in FXR ligands/AT2R-mediated inhibition of Ang II signalling, we examined the effect of CDCA or GW4064 on the expression of the three phosphatases. As shown in Figure 6A, treatment with CDCA or GW4064 led to an increase in the expression of SHP-1 in a dose-dependent manner. Such increase in SHP-1 expression was significantly inhibited when the cells were pre-treated with an AT2R specific antagonist (Figure 6B), suggesting a role of AT2R in CDCA- or GW4064-mediated increase in SHP-1 expression. CDCA or GW4064 showed little effect on the expression of MKP-1 or PP2A (Figure 6A).


Figure 6
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  		Figure 6 Role of SHP-1 in CDCA- and GW4064-mediated inhibition of Ang II-stimulated ERK signalling. (A) Effect of CDCA or GW4064 treatment on the expression of SHP-1, MKP-1, or PP2A in RASMC. RASMC grown in 6-well plates were treated with various concentrations of CDCA or GW4064. Twenty-four hours later cells were lysed and cell lysates were subjected to western blot analysis of SHP-1, MKP-1, and PP2A, respectively. (B) Effect of AT2R antagonist on CDCA- and GW4064-mediated upregulation of SHP-1. RASMC were pre-treated with PD123319 (10 µM) for 30 min prior to treatment with CDCA or GW4064 for another 24 h. Western blot analysis of SHP-1 was then performed. (C) Effect of SHP-1 inhibitor on CDCA- or GW4064-medaited inhibition of Ang II/ERK signalling. RASMC starved in serum-free medium for 24 h were treated with CDCA or GW4064 for 24 h before treatment with a SHP-1 inhibitor (10 µM) and/or Ang II for 15 min. Western analysis of p-ERK1/2 was then performed. Results shown in this figure are the representative data from at least three to five experiments.

 
To further establish a role of SHP-1 in CDCA- or GW4064-mediated inhibition of Ang II signalling, we then examined whether inhibition of SHP-1 could block the inhibitory effect of CDCA or GW4064 on Ang II-mediated ERK activation. As shown in Figure 6C, treatment with a SHP-1 specific inhibitor, {alpha}-bromo-4-hydroxyacephenone 4-hydroxyphenacyl Br, drastically attenuated the inhibitory effect of CDCA or GW4064 on Ang II-stimulated ERK activation. These results, together with data from Figure 5 strongly suggest that CDCA or GW4064 inhibited Ang II-mediated ERK activation via a FXR/AT2R/SHP-1 pathway.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
SUPPLEMENTARY MATERIAL
 Funding
 References
 
We have confirmed in this study the original report by Bishop-Bailey et al.23 that functional FXR is expressed in vascular SMC. We have also shown for the first time that activation of FXR led to upregulation of AT2R. Furthermore, we have identified an IR2 as a novel FXRE that appears to be involved in CDCA or GW4064-mediated upregulation of ATR2 in vascular SMC. It has been shown that FXR/RXR can bind to and activate a variety of elements including IR-1 elements with changes in the core half-site sequence, spacing nucleotide, and flanking nucleotides although FXR/RXR binds to the consensus IR-1 sequence with the highest affinity.35 For example, IR0, ER8, and IR8 have been shown to be capable of conferring responsiveness on several FXR-target genes.36–38 In a separate study, we have recently shown that IR2 is also capable of mediating eNOS upregulation in vascular EC in a FXR-dependent manner.39 Interestingly, although CDCA was comparable to GW4064 in inducing AT2R expression (Figure 2), it was much less efficient in activating the AT2R promoter (Figures 3 and 4). It remains to be determined whether CDCA also mediates the upregulation of AT2R at a posttranscriptional level. It also remains to be tested whether CDCA induces AT2R expression via other types of nuclear receptors in RASMC.

The biological consequences of FXR-mediated upregulation of AT2R in SMC are not completely understood at present. However, data from our studies clearly establish a role of FXR/AT2R in inhibiting the Ang II-mediated ERK signalling. Upregulation of SHP-1 appears to play a role in FXR/AT2R-mediated inhibition of ERK signalling. This is in agreement with previous observations that overexpression of AT2R led to expression of different types of phosphatases such as MKP-1, PP2A, and SHP-1. Interestingly, different types of phosphatases were involved in different cell types. For example, Yamada et al.40 reported the involvement of MKP-1 but not PP2A in AT2R-mediated ERK inactivation in PC12W cells, whereas Huang et al.41 showed the participation of PP2A in neurons. Our data showed that FXR-mediated upregulation of AT2R selectively led to an increased expression of SHP-1. This was similar to most of the studies with SMC in which increased AT2R activity was associated with an increased SHP-1 activity.42 These differences might be due to a variation of cell types or might reflect the complexity of the network involved in negative regulation of ERK activity. It remains to be studied whether FXR activation can enhance SHP-1 expression in an ATR2-independent manner.

Modulation of AT2R activity or the AT1R/AT2R crosstalk has received increasing attention as an approach for the treatment of cardiovascular diseases.43 For example, overexpression of AT2R receptor by gene transfer has been shown to prevent neointimal proliferation in balloon-injured rat carotid arteries.44 The increased stimulation of AT2R that occurs in the presence of AT1R blockade was believed to contribute to the benefits of Ang II receptor blockers (ARBs).45 In a recent clinical study by Savoia et al.,46 small peripheral resistance arteries from hypertensive diabetic patients receiving long-term treatment with the AT1R blocker valsartan exhibited enhanced AT2R expression. The increased expression of AT2R appears to contribute to Ang II-induced vasodilatation in the resistance arteries. In an animal model of ischemia-reperfusion, the cardioprotection of rosiglitazone, a PPAR-{gamma} agonist, was also shown to be associated with overexpression of AT2R and inactivation of ERK signalling.47

FXR ligands have recently been proposed as a novel therapy for the treatment of cardiovascular diseases. This is largely based on their effects in lowering circulating triglycerides and cholesterol,19–21 and improving hyperglycemia22 via the FXR-mediated effects in liver and intestine. We have recently shown that activation of FXR in vascular EC resulted in inhibition of endothelin-124 and upregulation of eNOS/nitric oxide.39 These results, together with the new data presented in this study, suggest that FXR-based therapy may also benefit from its direct effect on vasculature via regulating the expression of vasoactive mediators. It should be noted that activation of FXR also leads to some unfavourable biological effects such as decreases in the blood levels of high-density lipoprotein (HDL). FXR ligands such as CDCA have also been shown to induce oxidative stress in vascular EC at high concentrations although such effect appears to be FXR-independent. Furthermore, conflicting studies have been reported regarding the role of FXR in atherosclerosis.48–51 For example, Hanniman et al.49 reported that administration of a high-fat/high-cholesterol diet to male Fxr–/–Apoe–/– mice resulted in increased plasma lipids and atherosclerosis. This is in contrast to the studies in female Fxr–/–Apoe–/– mice and male Fxr–/–Ldlr–/– mice which showed decreased atherosclerosis.50,51 More studies on the molecular mechanisms that underlie the actions of FXR modulation may help reconcile the discrepancies among these studies and further establish FXR as a novel therapeutic target for the treatment of vascular diseases.


   SUPPLEMENTARY MATERIAL
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 SUPPLEMENTARY MATERIAL
Funding
 References
 
Supplementary Material is available at European Heart Journal Online.

Conflict of interest: none declared.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 SUPPLEMENTARY MATERIAL
 Funding
References
 
This work was supported by National Institutes of Health grants HL63080 and HL68688 (to S.L.), R37 HL65695, P50 GM53789 and PO1 HL70807 (to B.P.), and CA107011 (to W.X.) and an American Heart Association grant 0555456U (to S.L.).


   Notes
 
{dagger} Equal contribution to this work. Back

This is a new version of this article as there was an error in Figure 5 in the first version.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 SUPPLEMENTARY MATERIAL
 Funding
 References
 

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