Skip Navigation


Cardiovascular Research Advance Access first published online on September 19, 2007
This version [Corrected Proof] published online on October 19, 2007
Cardiovascular Research, doi:10.1093/cvr/cvm016
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
77/1/169    most recent
cvm016v2
cvm016v1
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Li, J.
Right arrow Articles by Li, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, J.
Right arrow Articles by Li, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

FXR-mediated regulation of eNOS expression in vascular endothelial cells

Jiang Li1,{dagger}, Annette Wilson2,{dagger}, Ramalinga Kuruba1, Qiuhong Zhang1, Xiang Gao1, Fengtian He1, Li-Ming Zhang3, 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 Anesthesiology, 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

Time for primary review: 21 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 that is highly expressed in liver, kidney, adrenals, and intestine. FXR was previously proposed to play an important role in the pathogenesis of cardiovascular diseases via regulating the metabolism and transport of cholesterol. We have recently shown that FXR is also expressed in rat pulmonary vascular endothelial cells (EC) and that activation of FXR leads to inhibition of endothelin-1 expression. In the present study, we examine whether activation of FXR also affects the expression of endothelial nitric oxide synthase (eNOS) in rat, bovine, and sheep vascular EC.

Methods and results: Treatment of vascular EC with a FXR ligand resulted in upregulation of expression of eNOS mRNA and protein and an increased production of nitrite/nitrate. FXR appears to induce eNOS expression at a transcriptional level because (1) upregulation of eNOS mRNA expression was abolished by the treatment of a transcription inhibitor, actinomycin D; and (2) eNOS promoter activity was significantly increased by pharmacological or genetic activation of FXR. Functional analysis of rat eNOS promoter identified an imperfect inverted repeat DNA motif, IR2 (–628AGCTCAgtGGACCT-641), as a likely FXR-responsive element that is involved in eNOS regulation.

Conclusion: These results support the notion that vascular FXR may serve as a novel molecular target for manipulating the expression of eNOS for the treatment of vascular diseases.

KEYWORDS Endothelial cells; Endothelial nitric oxide synthase; Farnesoid X receptor; Gene regulation

Received April 20, 2007; revised August 28, 2007; accepted September 3, 2007


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Nitric oxide (NO) is a critical effector molecule in the cardiovascular system and endothelial nitric oxide synthase (eNOS or NOSIII) derived NO plays an important role in vascular homeostasis affecting vasomotor tone as well as vascular smooth muscle proliferation, platelet aggregation, expression of adhesion molecules, inhibition of lipid oxidation, and regulation of apoptosis.1,2 The eNOS activity is regulated at both transcriptional and posttranscriptional levels by numerous physiological and pathophysiological stimuli such as shear stress, hypoxia, and inflammatory mediators.2–4 Many studies have shown that eNOS expression and activity are also modulated by a number of nuclear receptors such as estrogen receptor (ER),5–7 peroxisome proliferator-activated receptors,8–10 and retinoic acid receptor.11

The farnesoid X receptor (FXR) belongs to the superfamily of ligand-activated transcriptional factors. FXR has the typical nuclear receptor structure and can be activated by structurally different ligands, including several primary and secondary bile acid (BA) species conjugated to either taurine or glycine.12–15 The most potent natural activator of human FXR appears to be chenodeoxycholic acid (CDCA). In addition to BAs, a number of synthetic FXR agonists have been developed, among which GW4064 is one of the most potent FXR agonists.16,17 FXR has previously been shown to be largely expressed in liver, intestine, and kidney. Since BAs are potentially toxic to cells at high concentrations, many FXR-mediated transcriptional regulatory events could be seen as defence mechanisms against potential BA toxicity: by acting as FXR ligands, BAs can induce expression of hepatocellular bile acid efflux mechanisms, downregulate BA uptake mechanisms, repress the crucial enzymes required for de novo synthesis of BAs, and modulate other mechanisms leading to reduced BA toxicity.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, as they affect lipid metabolism in the liver and gastrointestinal tract and lower circulating triglycerides and cholesterol.19,20 More recently, FXR has also been shown to modulate adiposity and peripheral insulin sensitivity and improve hyperglycemia and hyperlipidemia in diabetic mice, suggesting that FXR agonists are promising therapeutic agents for treatment of diabetes mellitus.20,21 Expression of FXR has been reported in other ‘non-classical’ tissues as well. Bishop-Bailey et al.22 reported that FXR was expressed in the smooth muscle cells (SMC) of normal and atherosclerotic blood vessels (aorta and coronary artery). FXR was also detected immunohistochemically on the lumen of some large vascular tissue sections, suggesting an endothelial expression of FXR. Treatment of SMC with a range of FXR ligands led to apoptosis in a manner that correlates with the ability of the ligands to activate FXR.22 We have recently shown that FXR is also expressed in human and rat pulmonary endothelial cells (EC).23 We have further shown that activation of FXR resulted in inhibition of endothelin-1 (ET-1) in rat pulmonary EC.23 In this study, we showed, for the first time, that activation of FXR also led to an increased expression of eNOS in vascular EC. These results strongly support the notion that vascular FXR may serve as a novel molecular target for manipulating the expression of vasoactive mediators for the treatment of vascular diseases.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
2.1 Materials
CDCA was purchased from Sigma (St. Louis, MO, USA). GW4064 was synthesized following a published protocol.16 All products for cell culture were purchased from Invitrogen (Carlsbad, CA, USA). pCMX, pCMX-FXR, and pCMV-ßgal were described previously.24 pCMX-vpFXR (a gift from Drs Ennique Saez and Ronald Evans at the Salk Institute) was generated by fusing the VP16 activation domain from the herpes simplex virus to the N-terminus of the FXR cDNA. pCMX-D/N FXR, a plasmid expressing dominant negative rat FXR, was constructed following a published protocol.25

2.2 Plasmid construction
Rat eNOS promoter containing fragment (3012 bp) was amplified by PCR with the oligonucleotides 5'-ctggcccacactcttcaagt-3' (forward primer) and 5'-cgatagagttgcccagataagc-3' (reverse primer) using rat genomic DNA as template. Rat eNOS promoter containing fragment (803 bp) was similarly amplified by PCR with the oligonucleotides 5'-gcaggtcagtggacctagcc-3' (forward primer) and 5'- cgatagagttgcccagataagc-3' (reverse primer). Mutated eNOS promoter containing fragment (803 bp) was similarly amplified by PCR with the oligonucleotides 5'-agtgcagtagatctagccaccagggca-3' (forward primer) and 5'-cgatagagttgcccagataagc-3' (reverse primer). The fragment was cloned into pGL3-basic vector (Promega), and the resulting plasmid was named as pGL3-eNOS.

2.3 Cells and culture
Rat pulmonary microvasculature endothelial cells (RPMVEC) were purchased from VEC Technologies, Inc. (NY, USA) and cultured in MCDB-131 complete medium. Bovine aortic endothelial cells (BAEC) and sheep pulmonary artery endothelial cells (SPAEC) were prepared from the respective artery obtained from a local slaughterhouse following a published protocol.26 Both cells were cultured in 90% Opti-MEM, 10% fetal bovine serum supplemented with antibiotics.

2.4 Quantitative real-time RT–PCR assay of eNOS
Total RNA was extracted from cells with TRIzol reagent (Invitrogen) and the first-strand cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen). Pre-designed Assays-on-Demand TaqMan probes and primer pairs for rat eNOS were obtained from Applied Biosystems Incorporated (ABI). Each amplification mixture (25 µL) contains 25 ng of cDNA, 1.25 µL of primers and FAM-labelled fluorogenic probe, and 12.5 µL of Universal PCR Master mix. Amplification was performed using the following conditions: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. Transcript abundance, normalized to ß-glucuronidase expression, is expressed as a fold increase over a calibrator sample.

Real-time PCR analysis of bovine eNOS was performed using SYBR Green-based assays with the ABI 7300 Real-Time PCR System.27

2.5 Western blot analysis for eNOS
Protein extraction and western blot analysis were performed as described.28 Rabbit anti-eNOS antibody (C-20) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). HRP-labelled goat anti-rabbit IgG and the ECL chemiluminescence kit were purchased from Amersham Biosciences (Piscataway, NJ, USA).

2.6 Nitric oxide synthase activity
The conversion of [3H]L-arginine to [3H]L-citrulline was used to assess eNOS activity in cultured EC using the NOS Activity Assay Kit (Cayman Chemicals, Inc., Ann Arbor, MI, USA). Briefly, BAEC grown in 100 mm culture dishes were treated with GW4064 vehicle (DMSO), GW4064 (5 µM), E2 (10 µM), or untreated (control) for 24 h. Cells were then suspended by brief treatment with trypsin–EDTA and washed with PBS. To prepare total cell lysates, BAEC were suspended in cold lysis buffer at 4°C for 1 h and centrifuged at 10 000 g for 20 min. Protein concentrations were determined using the BCA Protein Assay (Pierce, Rockford, IL, USA). To analyse eNOS activity, cell lysates (150 µg) were incubated with 1 mM NADPH, 0.6 mM CaCl2, 100 nM calmodulin, [3H]L-arginine (58 Ci/mmol, 1 µCi/µL), 1 µM flavin adenine dinucleotide, 1 µM flavin adenine mononucleotide, and 3 µM BH4 for 15 min at 37°C. In all experiments, replicate incubations were performed in the presence of the NOS inhibitor 1 mM L-NNA. Incubations were terminated by adding 0.4 mL of ice-cold stop buffer. [3H]L-arginine was separated from [3H]L-citrulline by passing the entire reaction mixture over 0.1 mL of equilibrated Dowex AG 50 WX-8 cation exchange resin (Bio-Rad Laboratories, Hercules, CA, USA). The effluent containing [3H]L-citrulline was collected and quantified by liquid scintillation counting. The activities are expressed as pmol citrulline per mg protein per min.

2.7 Nitrite/nitrate assays
EC grown to an 80–90% confluence in 6-well plates were exposed to various treatments. NO release was measured in culture medium using a nitrate/nitrite colorimetric assay kit (Cayman Chemical Co., MI, USA). Briefly, samples were incubated with nitrate reductase and NADPH cofactor for 3 h to convert nitrate into nitrite. The total amount of NO was then determined using the Griess reagent. A standard curve was determined using known concentrations of nitrate assayed at 550 nm using a microplate reader. The NO concentrations in samples were calculated using the standard curve. The results were expressed as µM nitrate/nitrite released by the cells.

2.8 Transfection assays
Normal monkey kidney fibroblast cells (CV-1 line) were grown to 60–70% confluence in 48-well plates. Cells were transiently transfected using Lipofectamine2000 (Invitrogen) with pGL3-eNOS in the presence or absence of pCMX-vpFXR or pCMX-FXR. pCMX was added to ensure identical amounts of DNA in each well. Transfection efficiency was monitored by co-transfection of pCMV-ßgal plasmid. Cell extracts were prepared after transfection, and the luciferase and ß-galactosidase assays were performed as described29 and luciferase activity was normalized against ß-galactosidase activity. Transfection experiments were done at least three times in triplicate. Data were represented as fold induction over reporter gene alone.

2.9 Electrophoretic mobility shift assay
FXR and retinoid X receptor (RXR) proteins were generated in vitro by coupled transcription/translation (TNT system, Promega) with pCMX-FXR and pCMX-RXR plasmids. The DNA probe eNOS IR2 (5'-CGAGCAGGTCAgtGGACCTAGCC-3') was derived from a region in the rat eNOS 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 (10 mM Hepes, pH 7.9, 10 mM EGTA, 10 mM EDTA, 0.25 mM DTT, and 10% glycerol) containing 2 µg polydI: dC (Sigma) and 6–20 x 103 cpm of DNA probe at room temperature for 20 min. For supershift assays, 0.2 µg IgG of rabbit anti-FXR was added and the samples were incubated for another 10 min. The binding mixture was then applied onto a 5% polyacrylamide gel (0.5 x TrisBorateEDTA buffer) for electrophoresis. The gels were dried and exposed at –80°C for autoradiography.

Similarly, electrophoretic mobility shift assay (EMSA) was performed with nuclear extracts generated from BAEC that were treated with GW4064.

2.10 Statistical analysis
All data are expressed as means ± SEM unless otherwise stated. Comparisons between two groups were made with unpaired Student's t-test. Comparisons between three or more groups were made with ANOVA followed by Tukey-Kramer 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 Treatment with FXR agonists led to increased NO production in vascular EC
We have previously shown that activation of FXR led to inhibition of ET-1 expression in rat pulmonary EC.23 In this study, we examined whether activation of FXR affects the NO production in EC. Figure 1A shows that treatment of CDCA led to increased amounts of nitrite/nitrate in the supernatants of cultured RPMVEC. The effect of CDCA on NO production in EC appears to be a general phenomenon as CDCA treatment similarly increased the amounts of nitrite/nitrate in BAEC and SPAEC (Figure 1B and C). A similar effect was also observed for GW4064 (Figure 1A–C). GW4064 is a synthetic ligand that is highly specific for FXR and is often used as a ‘chemical tool’ to show that BA target genes are regulated in an FXR-specific manner.16 Thus, results from Figure 1 suggest that activation of FXR plays a role in CDCA- or GW4064-mediated increased NO production in EC.


Figure 1
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1 CDCA or GW4064 treatment led to an increased NO production in vascular EC as determined by nitrite/nitrate assay. RPMVEC (A), BAEC (B) or SPAEC (C) in 6-well plates were cultured in serum-free medium overnight prior to treatment with CDCA, GW4064 or vehicle (DMSO). Estrogen (E2) was also included as a positive control. Nitric oxide production was determined 24 h later by total nitrite assay and values are normalized to vehicle control. n = 3. *P < 0.05 (vs. DMSO). The basal levels of nitrite/nitrate in EC range from 0.92 to 1.98 µM.

 
It has been reported that bile acids such as CDCA also trigger the production of reactive oxygen species (ROS) in EC at high concentrations (e.g. 100 µM CDCA).30 However, at the concentrations used in Figure 1, CDCA or GW4064 induced minimal or non-detectable levels of ROS production in BAEC (see Supplementary material online, Figure S1).

3.2 Treatment with FXR agonists led to increased NOS activity in vascular EC
To further confirm that activation of FXR led to increased production of NO, we examined the effect of GW4064 on NOS activity in BAEC. As shown in Figure 2, there was about two-fold increase in NOS activity 24 h following treatment of BAEC with GW4064. Estrogen, included as a positive control, also caused increased NOS activity in BAEC.


Figure 2
View larger version (11K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2 Treatment with GW4064 led to increases in NOS activity in vascular EC. BAEC were similarly treated with GW4064 or estrogen as described in the legend to Figure 1. Twenty-four hours later, cells were washed with PBS and lysed with lysis buffer. The NOS activity in the cell lysates was determined via examining the efficiency in conversion of [3H]L-arginine to [3H]L-citrulline. *P<0.05 (vs. Control); **P<0.01 (vs. Control).

 
3.3 Treatment with FXR agonists resulted in increased levels of eNOS protein expression in vascular EC
In a preliminary study, we showed that treatment of EC with CDCA or GW4064 did not result in direct activation of eNOS as determined by western blot analysis of phosphorylated eNOS (data not shown). We then examined whether increased production of NO was due to an increased eNOS protein expression following treatment with CDCA or GW4064. Figure 3 showed that treatment of RPMVEC with either CDCA (A) or GW4064 (B) led to an increase in the level of eNOS protein expression in a dose-dependent manner. CDCA or GW4064 treatment similarly increased the eNOS protein levels in BAEC (Figure 3C and D). GW4064-mediated increases in eNOS protein expression appeared to occur as early as 8 h following the treatment and reached the peak at 16 h (see Supplementary material online, Figure S2).


Figure 3
View larger version (33K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3 Effect of CDCA or GW4064 treatment on eNOS expression in vascular EC. RPMVEC (A and B) or BAEC (C and D) in 6-well plates were cultured in serum-free medium overnight prior to treatment with CDCA, GW4064, E2, or vehicle (DMSO). eNOS protein levels in EC were examined by western analysis at 24 h following the treatment. Shown in each panel are the representative western blots and the densitometric analysis of Western films from three independent experiments. *P < 0.05 (vs. DMSO).

 
3.4 Treatment with FXR agonists resulted in increased eNOS mRNA expression in vascular EC
To determine the basis for CDCA- or GW4064-induced upregulation of eNOS protein expression, eNOS mRNA abundance in RPMVEC was determined by quantitative real-time RT–PCR. As shown in Figure 4A, treatment with CDCA or GW4064 resulted in an upregulation of eNOS mRNA in a dose-dependent manner. GW4064 treatment similarly led to increased expression of eNOS mRNA in BAEC (Figure 4B). The GW4064-mediated induction of eNOS mRNA expression appeared to occur as early as 2 h following the treatment and reached the peak at 16 h (Figure 4C).


Figure 4
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4 Effect of CDCA or GW4064 treatment on eNOS mRNA expression in vascular EC. RPMVEC (A) or BAEC (B) was treated with CDCA or GW4064 as described in the legend to Figure 1. Twenty-four hours later, the total RNA was extracted and eNOS mRNA expression was analysed by quantitative real-time RT–PCR. In a separate experiments, BAEC were treated with GW4064 (5 µM) and expression of eNOS mRNA was examined at different times following GW4064 treatment (C). n = 4. *P < 0.05 vs. DMSO in (A) and (B) and the eNOS mRNA level at 0 h time point in (C).

 
3.5 FXR enhances transcriptional activation of the rat eNOS gene promoter
To examine whether CDCA- or GW4064-mediated increases in the levels of eNOS mRNA are due to an enhancement in gene transcription or an increased mRNA stability, eNOS mRNA abundance was similarly examined in RPMVEC that were treated with a transcription inhibitor, actinomycin D, prior to CDCA or GW4064 treatment. As shown in Figure 5, pretreatment with actinomycin D substantially inhibited CDCA- or GW4064-mediated upregulation of eNOS mRNA, suggesting that CDCA or GW4064 enhanced eNOS mRNA expression at the transcriptional level.


Figure 5
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 5 Treatment with actinomycin D abolished CDCA- or GW4064-mediated upregulation of eNOS mRNA expression. RPMVEC were treated with actinomycin D (5 µg/mL) for 30 min prior to CDCA or GW4064 treatment. Twenty-four hours later, eNOS mRNA expression in EC was similarly examined by real-time RT–PCR as described in the legend to Figure 4. n = 3. *P < 0.05 vs. actinomycin D treatment.

 
To further examine whether eNOS promoter is a transcriptional target of FXR, we constructed a luciferase reporter expression plasmid (pGL3-eNOS) that is driven by a –3012 bp rat eNOS promoter. CV-1 cells were transfected with pGL3-eNOS in the absence or presence of a FXR expression vector. CV-1 cells instead of EC were chosen in this study due to a low efficiency of transfection with EC (data not shown). As shown in Figure 6A, GW4064 alone showed no effect on eNOS promoter activity. This might be due to the fact that CV-1 cells lack the expression of endogenous FXR. Indeed, GW4064 significantly enhanced the eNOS promoter activity in CV-1 cells that were co-transfected with a FXR expression plasmid. CDCA, although effective, was less efficient than GW4064 in activating the eNOS promoter. Nonetheless, the increases in promoter activity were substantially abolished when CV-1 cells were co-transfected with a dominant-negative FXR (D/N FXR) expression plasmid, suggesting that GW4064 or CDCA enhances eNOS promoter activity largely via activation of FXR. To further elucidate a role of FXR in regulating eNOS promoter activity, we then co-transfected pGL3-eNOS with an expression plasmid encoding a constitutively activated FXR, vpFXR.23 vpFXR was generated by fusing the VP16 activation domain to the N terminus of FXR cDNA. Figure 6A shows that co-expression of vpFXR in CV-1 cells significantly enhanced the eNOS promoter activity, clearly demonstrating that a genetic activation of FXR enhances eNOS promoter activity. Similar results were observed in transfection with a luciferase reporter driven by a shorter rat eNOS promoter (~803 bp) (Figure 6B).


Figure 6
View larger version (34K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 6 FXR enhances the transcriptional activity of the eNOS gene promoter. A luciferase reporter driven by a rat eNOS promoter of 3012 bp (A) or 803 bp (B) was used to study the eNOS promoter activity. CV-1 cells were transiently transfected with pGL3-eNOS 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 presence of GW4064 or vehicle DMSO for 24 h. Luciferase assay was then performed. Data shown in the panels represent mean (SD) from triplicate assays. In some studies, cells were co-transfected with pCMX-FXR and pCMX-FXR-DN at a weight ratio of 1:2. Luciferase assay was then similarly performed. To examine the effect of genetic activation of FXR on eNOS promoter activity, CV-1 cells were transfected with pGL3-eNOS in the presence or absence of pCMX-vpFXR. Luciferase assays were then examined as described above.

 
To search for FXR-responsive elements (FXREs) that may mediate eNOS induction by CDCA and GW4064, the 803 bp eNOS promoter sequence was subjected to in silico analysis with a Web-based algorithm (NUBIScan).31 One such potential FXRE was identified and its sequence and location are shown in Figure 7A. There is a hexameric core sequence AGGTCA between –628 and –633. Two base pairs downstream from this half-site is an imperfect inverted repeat sequence GGACCT (–636 to –641). This forms an imperfect inverted repeat motif IR2 (–628AGGTCAgtGGACCT-641). To determine whether this IR2 sequence is the binding site for the FXR, a heterologous tk-luciferase reporter gene that contains three copies of the eNOS/IR2 element was generated and tested for FXR transactivation in CV-1 cells. As shown in Figure 7B, CDCA or GW4064, alone or together with co-transfection with a FXR expression plasmid, similarly activated the tk-luciferase reporter gene as they did on the reporter gene driven by a –803 bp eNOS promoter (Figure 6B).


Figure 7
View larger version (50K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 7 Analysis of putative FXR-responsive elements (FXREs) in rat eNOS promoter. (A) Identification of a putative FXRE in rat eNOS promoter via an in silico analysis with a Web-based algorithm (NUBIScan). (B) IR2 can enhance promoter activity by FXR activation. (C) Mutation of IR2 on the eNOS promoter eliminates activation by FXR. (D) EMSA analysis of the binding of FXR/RXR to IR2 in rat ENOS promoter. Double-stranded oligonucleotides (–628/–641) were end-labelled with [{gamma}-32P]-ATP using T4 polynucleotide kinase. The labelled probe was 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. In some studies, the samples were pre-incubated with anti-FXR antibody prior to gel electrophoresis. (E) EMSA analysis of the binding of nuclear proteins of GW4064-treated BAEC to eNOS IR2.

 
Figure 7C shows that the FXR-mediated activation of eNOS promoter (–803 bp) was substantially diminished when the eNOS/IR2 site was mutated, further suggesting a role of IR2 site in FXR-mediated transactivation of rat eNOS promoter.

To determine whether FXR directly binds to this element, EMSA assays were performed with a 23 bp oligonucleotide that contains the eNOS/IR2. A typical 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 eNOS/IR2 oligonucleotide with in vitro translated FXR/RXR yielded a DNA/protein band of expected mobility (Figure 7D). This binding was specific, as it was inhibited by addition of excess unlabelled (cold) eNOS/IR2 or IR1/FXRE (Figure 7D). Addition of antibody against FXR to the reaction mixture resulted in further retarded migration of the radiolabelled band (Figure 7D). This supershifting confirms the identity of the protein that interacts with the DNA as being FXR.

We also examined DNA-binding activity to eNOS/IR2 in nuclear extracts generated from GW4064-treated BAEC (Figure 7E). Incubation of the eNOS/IR2 probe with GW4064/BAEC nuclear extracts produced a shifted band. This complex represented sequence-specific interactions between eNOS/IR2 and nuclear factors, since its formation was specifically reduced by unlabelled eNOS/IR2 or IR1/FXRE probe but not by mutated eNOS/IR2 competitors. The above results strongly suggest that eNOS/IR2 is likely to mediate the transactivation of eNOS promoter by FXR.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Supplementary material
 Funding
 References
 
We have demonstrated in this study that treatment of vascular EC with a FXR ligand such as CDCA or GW4064 resulted in upregulation of expression of eNOS mRNA and protein and an increased NO production. FXR appears to regulate eNOS expression at transcriptional level because (1) ligands-mediated increase in the expression of eNOS mRNA was substantially abolished by actinomycin D; and (2) eNOS promoter activity was significantly increased via pharmacological or genetic activation of FXR. Furthermore we have identified an IR2 as a novel FXRE that appears to be involved in CDCA or GW4064-mediated upregulation of eNOS in vascular EC. It has been shown that FXR/RXR can bind to and activate a variety of elements including IR1 elements with changes in the core half-site sequence, spacing nucleotide, and flanking nucleotides although FXR/RXR binds to the consensus IR1 sequence with the highest affinity.32 For example, IR0, ER8, and IR8 have been shown to be capable of conferring responsiveness on several FXR-target genes.33–35 It remains to be examined whether the IR2-mediated FXR transactivation of eNOS promoter is species-specific.

Other than at transcriptional level, eNOS activity can also be regulated at posttranscriptional levels via phosphorylation, S-nitrosylation, and interaction of eNOS with other proteins such as caveolins.36–38 Particularly, phosphorylation of eNOS at Ser 1177 and/or Ser 635 has been shown to be an important non-genomic mechanism by which estrogen and ER mediate activation of eNOS.36 Our preliminary study showed that there was no obvious phosphorylation of eNOS following treatment of EC with either CDCA or GW4064 (J.L., data not shown), suggesting an unlikely role of this mechanism in bile acids/FXR-mediated increase of eNOS activity in vascular EC.

It is interesting to note that although CDCA was comparable to GW4064 in inducing eNOS expression, it was less efficient in activating the eNOS promoter. It remains to be determined whether CDCA also mediates the upregulation of eNOS via a mechanism(s) that is independent of FXR activation. A recent study has shown that the G-protein-coupled BA receptor TGR5 is expressed in liver sinusoidal EC and activation of this receptor by BAs resulted in the upregulation of eNOS expression in addition to its activation.39 More studies are needed in the future to determine if functional TGR5 is also expressed in pulmonary or aortic EC and involved in the eNOS regulation by BAs.

The physiological or pathophysiological significance of this study is not clearly understood at present. However, it is noteworthy that haemodynamic changes, such as arterial hypotension and reduced pressor effects of vasoconstrictors are seen in certain patients with advanced liver disease in which plasma levels of BAs are significantly increased.40,41 The mechanisms for the haemodynamic changes observed in these patients remain unknown; however, data from both clinical and experimental studies have suggested that increased production of endothelial NO plays an important role.40 Considering the effect of bile acids/FXR in regulating eNOS expression in vascular EC, a role of bile acids/FXR in the altered haemodynamics cannot be ruled out, which warrants further studies.

In summary, we have shown for the first time that activation of FXR in vascular EC led to upregulation of eNOS expression and increased NO production. These data, together with our previous work demonstrating FXR-mediated downregulation of ET-1,23 strongly suggest the notion that FXR ligands-based treatment may also benefit from their direct effects on vasculature in addition to their effects in lowering blood levels of triglyceride and cholesterol through activation of FXR in liver and intestine. 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. 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.30 More studies are required to investigate the potential of FXR/FXR ligands-based novel therapy in the treatment of cardiovascular 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.


   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 HL68688 (to S.L.), R37 HL65695, P50 GM53789, and PO1 HL70807 (to B.R.P.), and by American Heart Association Grant 0555456U (to S.L.).

Conflict of interest: none declared.


   Notes
 
{dagger} These authors contributed equally to this work. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 

  1. Steuhr DJ. Structure-function aspects in the nitric oxide synthases. Annu Rev Pharmacol Toxicol (1997) 37:339–359.[CrossRef][Web of Science][Medline]
  2. Dudzinski DM, Igarashi J, Greif D, Michel T. The regulation and pharmacology of endothelial nitric oxide synthase. Annu Rev Pharmacol Toxicol (2006) 46:235–276.[CrossRef][Web of Science][Medline]
  3. Searles CD. Transcriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression. Am J Physiol Cell Physiol (2006) 291:C803–C816.[Abstract/Free Full Text]
  4. Tai SC, Robb GB, Marsden PA. Endothelial nitric oxide synthase: a new paradigm for gene regulation in the injured blood vessel. Arterioscler Thromb Vasc Biol (2004) 24:405–412.[Abstract/Free Full Text]
  5. MacRitchie AN, Jun SS, Chen Z, German Z, Yuhanna IS, Sherman TS, et al. Estrogen upregulates endothelial nitric oxide synthase gene expression in fetal pulmonary artery endothelium. Circ Res (1997) 81:355–362.[Abstract/Free Full Text]
  6. Fleming I, Busse R. Signal transduction of eNOS activation. Cardiovasc Res (1999) 43:532–541.[Abstract/Free Full Text]
  7. Lu Q, Pallas DC, Surks HK, Baur WE, Mendelsohn ME, Karas RH. Striatin assembles a membrane signaling complex necessary for rapid, nongenomic activation of endothelial NO synthase by estrogen receptor alpha. Proc Natl Acad Sci USA (2004) 101:17126–17131.[Abstract/Free Full Text]
  8. Calnek DS, Mazzella L, Roser S, Roman J, Hart CM. Peroxisome proliferator-activated receptor gamma ligands increase release of nitric oxide from endothelial cells. Arterioscler Thromb Vasc Biol (2003) 23:52–57.[Abstract/Free Full Text]
  9. Cho DH, Choi YJ, Jo SA, Jo I. Nitric oxide production and regulation of endothelial nitric-oxide synthase phosphorylation by prolonged treatment with troglitazone: evidence for involvement of peroxisome proliferator-activated receptor (PPAR) gamma-dependent and PPARgamma-independent signaling pathways. J Biol Chem (2004) 279:2499–2506.[Abstract/Free Full Text]
  10. Polikandriotis JA, Mazzella LJ, Rupnow HL, Hart CM. Peroxisome proliferator-activated receptor gamma ligands stimulate endothelial nitric oxide production through distinct peroxisome proliferator-activated receptor gamma-dependent mechanisms. Arterioscler Thromb Vasc Biol (2005) 25:1810–1816.[Abstract/Free Full Text]
  11. Uruno A, Sugawara A, Kanatsuka H, Kagechika H, Saito A, Sato K, et al. Upregulation of nitric oxide production in vascular endothelial cells by all-trans retinoic acid through the phosphoinositide 3-kinase/Akt pathway. Circulation (2005) 112:727–736.[Abstract/Free Full Text]
  12. Makishima M, Okamoto AY, Repa JJ, Tu H, Learned RM, Luk A, et al. Identification of a nuclear receptor for bile acids. Science (1999) 284:1362–1365.[Abstract/Free Full Text]
  13. Wang H, Chen J, Hollister K, Sowers LC, Forman BM. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR. Mol Cell (1999) 3:543–553.[Web of Science][Medline]
  14. Parks DJ, Blanchard SG, Bledsoe RK, Chandra G, Consler TG, Kliewer SA, et al. Bile acids: natural ligands for an orphan nuclear receptor. Science (1999) 284:1365–1368.[Abstract/Free Full Text]
  15. Chiang JY. Bile acid regulation of gene expression: roles of nuclear hormone receptors. Endocr Rev (2002) 23:443–463.[Abstract/Free Full Text]
  16. Maloney PR, Parks DJ, Haffner CD, Fivush AM, Chandra G, Plunket KD, et al. Identification of a chemical tool for the orphan nuclear receptor FXR. J Med Chem (2000) 43:2971–2974.[CrossRef][Web of Science][Medline]
  17. Pellicciari R, Costantino G, Camaioni E, Sadeghpour BM, Entrena A, Willson TM, et al. Bile acid derivatives as ligands of the farnesoid X receptor. Synthesis, evaluation, and structure-activity relationship of a series of body and side chain modified analogues of chenodeoxycholic acid. J Med Chem (2004) 47:4559–4569.[CrossRef][Web of Science][Medline]
  18. Eloranta JJ, Kullak-Ublick GA. Coordinate transcriptional regulation of bile acid homeostasis and drug metabolism. Arch Biochem Biophys (2005) 433:397–412.[CrossRef][Web of Science][Medline]
  19. Watanabe M, Houten SM, Wang L, Moschetta A, Mangelsdorf DJ, Heyman RA, et al. Bile acids lower triglyceride levels via a pathway involving FXR, SHP, and SREBP-1c. J Clin Invest (2004) 113:1408–1418.[CrossRef][Web of Science][Medline]
  20. Zhang Y, Lee FY, Barrera G, Lee H, Vales C, Gonzalez FJ, et al. Activation of the nuclear receptor FXR improves hyperglycemia and hyperlipidemia in diabetic mice. Proc Natl Acad Sci USA (2006) 103:1006–1011.[Abstract/Free Full Text]
  21. Cariou B, van Harmelen K, Duran-Sandoval D, van Dijk TH, Grefhorst A, Abdelkarim M, et al. The farnesoid X receptor modulates adiposity and peripheral insulin sensitivity in mice. J Biol Chem (2006) 281:11039–11049.[Abstract/Free Full Text]
  22. Bishop-Bailey D, Walsh DT, Warner TD. Expression and activation of the farnesoid X receptor in the vasculature. Proc Natl Acad Sci USA (2004) 101:3668–3673.[Abstract/Free Full Text]
  23. He F, Li J, Mu Y, Kuruba R, Ma Z, Wilson A, et al. Downregulation of endothelin-1 by farnesoid X receptor in vascular endothelial cells. Circ Res (2006) 98:192–199.[Abstract/Free Full Text]
  24. Xie W, Radominska-Pandya A, Shi Y, Simon CM, Nelson MC, Ong ES, et al. An essential role for nuclear receptors SXR/PXR in detoxification of cholestatic bile acids. Proc Natl Acad Sci USA (2001) 98:3375–3380.[Abstract/Free Full Text]
  25. Kocarek TA, Shenoy SD, Mercer-Haines NA, Runge-Morris M. Use of dominant negative nuclear receptors to study xenobiotic-inducible gene expression in primary cultured hepatocytes. J Pharmacol Toxicol Methods (2002) 47:177–187.[CrossRef][Web of Science][Medline]
  26. Shiki Y, Meyrick BO, Brigham KL, Burr IM. Endotoxin increases superoxide dismutase in cultured bovine pulmonary endothelial cells. Am J Physiol (1987) 252:C436–C440.[Web of Science][Medline]
  27. Gong H, Guo P, Zhai Y, Zhou J, Uppal H, Jarzynka MJ, et al. Estrogen deprivation and inhibition of breast cancer growth in vivo through activation of the orphan nuclear receptor liver X receptor. Mol Endocrinol (2007) 21:1781–1790.[Abstract/Free Full Text]
  28. Li J, Ma Z, Tang ZL, Stevens T, Pitt B, Li S. CpG DNA-mediated immune response in pulmonary endothelial cells. Am J Physiol Lung Cell Mol Physiol (2004) 287:L552–L558.[Abstract/Free Full Text]
  29. Saini SP, Sonoda J, Xu L, Toma D, Uppal H, Mu Y, et al. A novel constitutive androstane receptor-mediated and CYP3A-independent pathway of bile acid detoxification. Mol Pharmacol (2004) 65:292–300.[Abstract/Free Full Text]
  30. Qin P, Tang X, Elloso MM, Harnish DC. Bile acids induce adhesion molecule expression in endothelial cells through activation of reactive oxygen species, NF-kappaB, and p38. Am J Physiol Heart Circ Physiol (2006) 291:H741–H747.[Abstract/Free Full Text]
  31. Podvinec M, Kaufmann MR, Handschin C, Meyer UA. NUBIScan, an in silico approach for prediction of nuclear receptor response elements. Mol Endocrinol (2002) 16:1269–1279.[Abstract/Free Full Text]
  32. Laffitte BA, Kast HR, Nguyen CM, Zavacki AM, Moore DD, Edwards PA. Identification of the DNA binding specificity and potential target genes for the farnesoid X-activated receptor. J Biol Chem (2000) 275:10638–10647.[Abstract/Free Full Text]
  33. Song CS, Echchgadda I, Baek BS, Ahn SC, Oh T, Roy AK, et al. Dehydroepiandrosterone sulfotransferase gene induction by bile acid activated farnesoid X receptor. J Biol Chem (2001) 276:42549–42556.[Abstract/Free Full Text]
  34. Kast HR, Goodwin B, Tarr PT, Jones SA, Anisfeld AM, Stoltz CM, et al. Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and constitutive androstane receptor. J Biol Chem (2002) 277:2908–2915.[Abstract/Free Full Text]
  35. Prieur X, Coste H, Rodriguez JC. The human apolipoprotein AV gene is regulated by peroxisome proliferator-activated receptor-alpha and contains a novel farnesoid X-activated receptor response element. J Biol Chem (2003) 278:25468–25480.[Abstract/Free Full Text]
  36. Kleinert H, Wallerath T, Euchenhofer C, Ihrig-Biedert I, Li H, Förstermann U. Estrogens increase transcription of the human endothelial NO synthase gene: analysis of the transcription factors involved. Hypertension (1998) 31:582–588.[Abstract/Free Full Text]
  37. Bauer PM, Fulton D, Boo YC, Sorescu GP, Kemp BE, Jo H, et al. Compensatory phosphorylation and protein-protein interactions revealed by loss of function and gain of function mutants of multiple serine phosphorylation sites in endothelial nitric oxide synthase. J Biol Chem (2003) 278:14841–14849.[Abstract/Free Full Text]
  38. Erwin PA, Lin AJ, Golan DE, Michel T. Receptor-regulated dynamic S-nitrosylation of endothelial nitric-oxide synthase in vascular endothelial cells. J Biol Chem (2005) 280:19888–19894.[Abstract/Free Full Text]
  39. Keitel V, Reinehr R, Gatsios P, Rupprecht C, Gorg B, Selbach O, et al. The G-protein coupled bile salt receptor TGR5 is expressed in liver sinusoidal endothelial cells. Hepatology (2007) 45:695–704.[CrossRef][Web of Science][Medline]
  40. Palma DT, Fallon MB. The hepatopulmonary syndrome. J Hepatol (2006) 45:617–625.[CrossRef][Web of Science][Medline]
  41. MacGilchrist AJ, Sumner D, Reid JL. Impaired pressor reactivity in cirrhosis: evidence for a peripheral vascular defect. Hepatology (1991) 13:689–694.[CrossRef][Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Immunol.Home page
A. Mencarelli, B. Renga, M. Migliorati, S. Cipriani, E. Distrutti, L. Santucci, and S. Fiorucci
The Bile Acid Sensor Farnesoid X Receptor Is a Modulator of Liver Immunity in a Rodent Model of Acute Hepatitis
J. Immunol., November 15, 2009; 183(10): 6657 - 6666.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
J. Li, A. Wilson, X. Gao, R. Kuruba, Y. Liu, S. Poloyac, B. Pitt, W. Xie, and S. Li
Coordinated Regulation of Dimethylarginine Dimethylaminohydrolase-1 and Cationic Amino Acid Transporter-1 by Farnesoid X Receptor in Mouse Liver and Kidney and Its Implication in the Control of Blood Levels of Asymmetric Dimethylarginine
J. Pharmacol. Exp. Ther., October 1, 2009; 331(1): 234 - 243.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
P. Lefebvre, B. Cariou, F. Lien, F. Kuipers, and B. Staels
Role of Bile Acids and Bile Acid Receptors in Metabolic Regulation
Physiol Rev, January 1, 2009; 89(1): 147 - 191.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
T. Kida, T. Murata, M. Hori, and H. Ozaki
Chronic stimulation of farnesoid X receptor impairs nitric oxide sensitivity of vascular smooth muscle
Am J Physiol Heart Circ Physiol, January 1, 2009; 296(1): H195 - H201.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
77/1/169    most recent
cvm016v2
cvm016v1
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Li, J.
Right arrow Articles by Li, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, J.
Right arrow Articles by Li, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?