Regulation of vascular contractility and blood pressure by the E2F2 transcription factor.

Abstract

Recent studies have identified a polymorphism in the endothelin-converting enzyme (ECE)-1b promoter (-338C/A) that is strongly associated with hypertension in women. The polymorphism is located in a consensus binding sequence for the E2F family of transcription factors. E2F proteins are crucially involved in cell-cycle regulation, but their roles in cardiovascular function are poorly understood. Here, we investigated the potential role of E2F2 in blood pressure regulation.
Tail-cuff measurements of systolic and diastolic blood pressures were significantly higher in E2F2-null (E2F2(-/-)) mice than in their wild-type littermates, and in ex vivo ring assays, aortas from the E2F2(-/-) mice exhibited significantly greater contractility in response to big endothelin-1. Big endothelin-1 is activated by ECE-1, and mRNA levels of ECE-1b, the repressive ECE-1 isoform, were significantly lower in E2F2(-/-) mice than in wild-type mice. In endothelial cells, chromatin immunoprecipitation assays confirmed that E2F2 binds the ECE-1b promoter, and promoter-reporter assays indicated that E2F2 activates ECE-1b transcription. Furthermore, loss or downregulation of E2F2 led to a decline in ECE-1b levels, to higher levels of the membranous ECE-1 isoforms (ie, ECE-1a, -1c, and -1d), and to deregulated ECE-1 activity. Finally, Sam68 coimmunoprecipitated with E2F2, occupied the ECE-1b promoter (chromatin immunoprecipitation), and repressed E2F2-mediated ECE-1b promoter activity (promoter-reporter assays).
Our results identify a cell-cycle-independent mechanism by which E2F2 regulates endothelial function, arterial contractility, and blood pressure.

Full text

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ISSN: 1524-4539
Copyright © 2009 American Heart Association. All rights reserved. Print ISSN: 0009-7322. Online
72514
Circulation is published by the American Heart Association. 7272 Greenville Avenue, Dallas, TX
DOI: 10.1161/CIRCULATIONAHA.109.859207
published online Sep 14, 2009;
Circulation
Douglas W. Losordo and Gangjian Qin
Dongxu Liu, Chantal Botros, Yao Liang Tang, Nichole Reisdorph, Raj Kishore,
Junlan Zhou, Yan Zhu, Min Cheng, Deepika Dinesh, Tina Thorne, Kian Keong Poh,

Transcription Factor
Regulation of Vascular Contractility and Blood Pressure by the E2F2
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Regulation of Vascular Contractility and Blood Pressure by
the E2F2 Transcription Factor
Junlan Zhou, MD, PhD*; Yan Zhu, PhD*; Min Cheng, MD; Deepika Dinesh, MS; Tina Thorne, MS;
Kian Keong Poh, MD; Dongxu Liu, MD, PhD; Chantal Botros, BS; Yao Liang Tang, MD, PhD;
Nichole Reisdorph, PhD; Raj Kishore, PhD; Douglas W. Losordo, MD; Gangjian Qin, MD
Background—Recent studies have identified a polymorphism in the endothelin-converting enzyme (ECE)–1b promoter
(?338C/A) that is strongly associated with hypertension in women. The polymorphism is located in a consensus binding
sequence for the E2F family of transcription factors. E2F proteins are crucially involved in cell-cycle regulation, but
their roles in cardiovascular function are poorly understood. Here, we investigated the potential role of E2F2 in blood
pressure regulation.
Methods and Results—Tail-cuff measurements of systolic and diastolic blood pressures were significantly higher in
E2F2-null (E2F2?/?) mice than in their wild-type littermates, and in ex vivo ring assays, aortas from the E2F2?/?mice
exhibited significantly greater contractility in response to big endothelin-1. Big endothelin-1 is activated by ECE-1, and
mRNA levels of ECE-1b, the repressive ECE-1 isoform, were significantly lower in E2F2?/?mice than in wild-type
mice. In endothelial cells, chromatin immunoprecipitation assays confirmed that E2F2 binds the ECE-1b promoter, and
promoter-reporter assays indicated that E2F2 activates ECE-1b transcription. Furthermore, loss or downregulation of
E2F2 led to a decline in ECE-1b levels, to higher levels of the membranous ECE-1 isoforms (ie, ECE-1a, -1c, and -1d),
and to deregulated ECE-1 activity. Finally, Sam68 coimmunoprecipitated with E2F2, occupied the ECE-1b promoter
(chromatin immunoprecipitation), and repressed E2F2-mediated ECE-1b promoter activity (promoter-reporter assays).
Conclusion—Our results identify a cell-cycle–independent mechanism by which E2F2 regulates endothelial function,
arterial contractility, and blood pressure. (Circulation. 2009;120:1213-1221.)
Key Words: blood pressure ? endothelin ? endothelium ? E2F transcription factors ? Sam68 protein, mouse
E
larger preproET-1 precursor that is cleaved into an inactive
38–amino-acid peptide, big ET-1 (BigET-1), and then further
processed by endothelin-converting enzyme-1 (ECE-1) into
the active, 21–amino-acid ET-1.2Subsequently, ET-1 acts on
the smooth muscle cells through 2 receptors of the G-protein–
coupled receptor family, ETAand ETB.3,4Ample evidence
from genetic mouse models and from the blockade of ET-1
receptors in humans has demonstrated that the ET-1/ECE-1
system plays a critical role in the regulation of blood pressure
(BP) homeostasis.5,6
ndothelin-1 (ET-1) is a potent vessel-constricting pep-
tide.1It is synthesized in endothelial cells (ECs) from a
Clinical Perspective on p 1221
ECE-1 is a membrane-bound zinc metalloendopeptidase
expressed predominantly in the vascular endothelium7and
catalyzes the rate-limiting step during biogenesis of ET-1.2
The physiological importance of the conversion of BigET-1
to mature ET-1 is demonstrated by observations in ECE-1–,
ET-1–, and ETA-knockout animals, which exhibit virtually
identical cardiac and craniofacial abnormalities during em-
bryonic development,5,8and by the ?140-fold greater vaso-
constrictive potency of ET-1.2There are 4 isoforms of
ECE-1: ECE-1a, -1b, -1c, and -1d, which are expressed via
alternative promoters from the same gene located on human
chromosome 1 (1p36)9,10(Figure IA of the online-only Data
Supplement). These isoforms exist primarily as homodimers
and localize to different subcellular regions because of their
dissimilar N-termini; ECE-1a, -1c, and -1d are located on the
plasma membrane, and ECE-1b is intracellular9,11,12(Figure
IB of the online-only Data Supplement). It has been shown
Received February 20, 2009; accepted July 17, 2009.
From the Feinberg Cardiovascular Research Institute, Northwestern University Feinberg School of Medicine, Chicago, Ill (J.Z., M.C., T.T., C.B., R.K.,
D.W.L., G.Q.); Molecular Cardiology Research Institute, Tufts University School of Medicine, Tufts Medical Center, Boston, Mass (Y.Z.);
Cardiovascular Research, Caritas St. Elizabeth’s Medical Center, Tufts University School of Medicine, Boston, Mass (D.D., K.K.P.); Center for Infection
and Immunity Research, School of Life Sciences, Hubei University, Wuhan, Hubei, P.R. China (D.L.); Department of Internal Medicine, College of
Medicine, University of Cincinnati, Cincinnati, Ohio (Y.L.T.); and Proteomics Facility, National Jewish Medical and Research Center, Denver, Colo
(N.R.).
*Drs Zhou and Zhu contributed equally to this work.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.109.859207/DC1.
Correspondence to Gangjian Qin, MD, Assistant Professor, Feinberg Cardiovascular Research Institute, Northwestern University, 303 E Chicago Ave,
Tarry 14–751, Chicago, IL 60611. E-mail g-qin@northwestern.edu
© 2009 American Heart Association, Inc.
Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.109.859207
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that ECE-1b is located intracellularly in the late endosomes/
multivesicular bodies,13and its N-terminal leucine-based
motifs are involved in this intracellular retention. Interaction
of a plasma-membrane ECE-1 isoform with ECE-1b results
in its intracellular localization and decreases its extracel-
lular activity.12,14,15Therefore, the targeting signals specific
for ECE-1b constitute a regulatory domain that modulates the
localization and activity of other ECE-1 isoforms.13
Recently, 2 independent clinical studies have revealed a
polymorphism in the 5?-regulatory region of the ECE-1b gene
(ECE1 C-338A, 338 bp upstream from the translation start
site) that is strongly correlated with increases in systolic,
diastolic, and mean BP levels in women.16,17Moreover, the
EDN1 K198N polymorphism in the coding region of the
preproET-1 gene, previously known to be associated with BP
in overweight people,18interacts with the ECE1 C-338A
variant to influence BP levels.17Interestingly, the ECE1
C-338A polymorphism is located in a consensus site for the
E2F family of transcription factors and alters its binding
affinity specifically to E2F2.17However, it is unknown
whether E2F2 plays a role in the regulation of ECE-1b gene
expression and the pathogenesis of hypertension.
The E2F family of transcription factors regulate cell
growth, differentiation, and survival.19The classically de-
scribed mechanism by which E2F family members regulate
transcription involves binding to DNA as heterodimers with
members of the DP protein family and the recruitment of
other regulatory elements.19Accumulating evidence indicates
that the 9 known E2F proteins (E2F1 through E2F8) target
both common and unique genes in the genome20,21and that
each E2F family member has diverse physiological functions
that are specific to the tissue type and biological context.22–24
The functional diversity of E2F members is exemplified by
the strikingly different phenotypes found in the correspond-
ing knockout animals.22The specific roles of E2F family
members in regulating the vasculature, however, are poorly
characterized.
Recently, we have shown that E2F1 is involved in the
regulation of angiogenesis. Genetic deletion of E2F1 en-
hanced blood flow recovery after ischemic injury in mice.24
To determine whether other E2F family members are in-
volved in angiogenesis, we performed experiments in E2F2-
null (E2F2?/?) mice. Angiogenesis was unchanged; however,
the E2F2?/?mice were hypertensive with altered vasomotion.
Here, we provide genetic evidence that E2F2 regulates BP via
specific regulation of endothelial ECE-1b expression, subcel-
lular localization, and hence vascular reactivity. Furthermore,
our studies reveal that Sam68, an RNA-binding protein and
oncogene, acts as a novel transcription cofactor of E2F2 in
the regulation of ECE-1b.
Methods
Mice
The heterozygote E2F2?/?mice were obtained from Dr Gustavo
Leone’s laboratory (Ohio State University)25and were bred, main-
tained, and operated on in the Center for Comparative Medicine of
Northwestern University following protocols approved by the Insti-
tutional Animal Care and Use Committee. All animals were geno-
typed by polymerase chain reaction (PCR) of tail DNA. Age- and
sex-matched E2F2?/?, E2F2?/?, and their wild-type (WT) littermates
were used.
BP Measurement
Arterial BP and heart rates from WT and E2F2?/?mice were
measured by the standard noninvasive tail-cuff method (CODA
System, Kent Scientific, Torrington, Conn).26Measurements were
performed during the day after 5 days of training. On each day of BP
determination, 20 measurements were obtained and averaged for
each mouse.
Aortic Ring Assay
The contractile properties of aortic arteries were analyzed by the ring
assay27as described previously and as summarized in the Methods
section of the online-only Data Supplement.
Measurement of ET-1 Peptides
Levels of ET-1 in mouse plasma and in cell culture medium were
measured with a commercially available ELISA kit (Biomedica,
Vienna, Austria) by following the manufacturer’s instructions.
Tissue RNA Isolation and Real-Time
Reverse-Transcription PCR
Tissue RNA was extracted and real-time PCR was performed as
described previously.24Primer sequences are listed in Figure II of the
online-only Data Supplement.
Plasmids and Small Interfering RNA
Human pECE-1b/C-alkaline phosphatase (AP) and pECE-1b/A-AP
plasmids were provided by Dr Benoit Funalot (INSERM, France).28
pRC/CMV-E2F1 and pRC/CMV-E2F2 plasmids were provided by
Dr Farbio Martelli (IRCSS, Italy). Myc-Sam68 plasmid was pro-
vided by Dr Chi Wai Eric So (Institute of Cancer Research, Sutton,
UK). Human and bovine E2F1 small interfering RNA (siRNA) and
E2F2 siRNA were synthesized by Dharmacon, Inc (Lafayette, Colo).
The mouse ECE-1b promoter–AP reporter plasmid was constructed
by following standard cloning techniques (see the Methods section of
the online-only Data Supplement for details).
Cell Culture, Plasmid and siRNA Transient
Transfection, and Reporter Assays
Human umbilical vein ECs (HUVECs) and bovine aortic ECs
(BAECs) were obtained from ATCC (Manassas, Va), cultured as
described,24and used within passage 5. For plasmid transfection of
BAECs, TransFast (Promega, Madison, Wis) or Arrest-In (Open
Biosystems, Huntsville, Ala) was used. For siRNA transfection of
HUVECs, Arrest-In or RNAiFect (QIAGEN, Valencia, Calif) was
used. Control ?-Gal–expressing plasmid (pCMV–?-Gal) was co-
transfected to normalize for transduction efficiency. Chemilumines-
cent reporter gene assays for the detection of AP and ?-Gal activities
were performed as described previously.24
Isolation of Mouse Primary Lung ECs
Primary ECs were isolated from mouse lung tissues by following the
published protocol29(see the Methods section of the online-only
Data Supplement for details). The cells were used before passage 5,
and the EC identity was confirmed before each experiment by
staining with FITC-conjugated anti-CD31 antibody.24
Subcellular Fractionation, Western Blotting,
and Coimmunoprecipitation
Plasma membrane and cytosolic proteins were fractionated by using
the Pinpoint Cell Surface Protein Isolation Kit (Pierce Biotechnology
Inc, Rockford, Ill) (see the Methods of the online-only Data
Supplement for details) and then analyzed by Western blotting.24
The primary antibodies used were anti-E2F2, anti-E2F1, anti-Sam68,
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anti-HA, anti–Tie-2, anti-actin (Santa Cruz Biotechnology, Inc,
Santa Cruz, Calif), anti-human ECE-1 (R&D Systems, Minneapolis,
Minn), anti-Calnexin (Stressgen, Ann Arbor, Mich), and anti-Myc
(Cell Signaling Technology, Danvers, Mass). For coimmunoprecipi-
tation, cell lysates were incubated overnight at 4°C with the
appropriate antibody, then incubated with protein A/G plus agarose
(Santa Cruz) for 1 hour at 4°C. After washing, the immunoprecipi-
tates were eluted by boiling for 5 minutes, and extracts were
analyzed by immunoblotting as described above. Band intensities
were determined densitometrically with Image J software.
Chromatin Immunoprecipitation Assay
Chromatin immunoprecipitation (ChIP) assays were performed in
HUVECs with a ChIP assay kit (Upstate, NY) (see the Methods
section of the online-only Data Supplement for details). Primer
sequences are listed in Figure II of the online-only Data Supplement.
Mass Spectrometry
Experiments followed a standard protein identification strategy30as
described in the Methods section of the online-only Data
Supplement.
Fluorescent Immunohistochemistry
Fluorescent immunohistochemical staining was performed by fol-
lowing standard techniques31(see the Methods section of the
online-only Data Supplement for details), and the slides were
examined under a confocal microscope (Zeiss LSM 510 META).
ECE-1 Enzymatic Activity in Intact Primary ECs
Conversion of the exogenous substrate BigET-1 (Alexis, Lausen,
Switzerland) into mature ET-1 was determined in primary lung ECs
after incubation with 10?7mol/L BigET-1 for 60 minutes in a
96-well plate at 37°C. The reaction was stopped by the addition of
1 mmol/L NaEDTA, and the ET-1 generated was measured in the
culture medium with an ELISA kit (Biomedica, Vienna, Austria).
The value obtained from the well without cells was considered the
ELISA cross-reactivity for BigET-1 and subtracted from all the
measurements in which BigET-1 had been added. The ET-1 level in
each well was normalized to the quantity of cells as determined with
a crystal violet staining method that we have described previously.31
Statistical Analysis
All values are expressed as mean?SEM. Comparisons between 2
means were performed with an unpaired Student t test, whereas
ANOVA with Fisher protected least significant differences and
Bonferroni-Dunn posthoc analysis were used for comparisons of ?2
means. Comparisons between concentration-response curves were
performed with ANOVA for repeated measures. Significance was
defined as P?0.05.
The authors had full access to and take full responsibility for the
integrity of the data. All authors have read and agree to the
manuscript as written.
Results
Loss of E2F2 Expression in Mice Results in
Elevated BP and Exaggerated Arterial Contraction
in Response to BigET-1
We measured BP in E2F2?/?mice and their WT littermates
and found that despite similar heart rates, the E2F2?/?mice
displayed significantly higher systolic and diastolic BPs
(Figure 1A). Using ex vivo aortic ring assays, we compared
the contractility of aortas isolated from E2F2?/?mice and
their WT littermates in response to BigET-1 and a variety of
vasoactive substances. Contraction induced by KCl
(30 mmol/L) and phenylephrine (1?10?9to 3?10?6mol/L)
and relaxation in response to acetylcholine (1?10?9to
3?10?6mol/L) were similar in the 2 strains, but aortas from
the E2F2?/?mice exhibited significantly greater contractility
Figure 1. Loss of E2F2 expression in mice
results in elevated BP and exaggerated arteri-
al contraction in response to BigET-1. A, Tail-
cuff arterial BP (left) and heart rates (right) in
E2F2?/?mice and their WT littermates at 6
months of age. Data are mean?SEM (n?12
per group; **P?0.01; *P?0.05; NS, not signifi-
cant vs WT). B and C, The contractility of en-
dothelium-intact aortas isolated from E2F2?/?
mice and their WT littermates was evaluated
via aortic ring assay. B, Representative re-
cordings of the ring assay. Arrows indicate
the time points when K?(black arrow),
BigET-1 (red arrow), or vehicle control (blue
arrow) was added. K?30 mmol/L; phenyleph-
rine 1?10?9to 3?10?6mol/L; BigET-1,
2.7?10?7to 21?10?7mol/L. Contractile
response to the addition of K?at the end of
each BigET-1 assessment was similar
between groups, indicating that the differ-
ences in response to BigET-1 were caused
by altered ECE-1 activity rather than by a
general change in contractile function. C,
Quantification of contraction in response to
BigET-1 (WT, dotted line; E2F2?/?, solid line).
The percentage of BigET-1–induced contrac-
tion was calculated with K?(30 mmol/L)-
induced contraction set to 100%. Data are
mean?SEM (n?4 per group; *P?0.05,
**P?0.01 vs WT). D, Plasma ET-1 levels in
the E2F2?/?mice and WT littermates (n?12
per group; ***P?0.001 vs WT).
Zhou et alEndothelial E2F2 Regulates Blood Pressure
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in response to serial doses of BigET-1 (2.7?10?7to 2.1?10?6
mol/L) (Figure 1B and 1C).
Because BigET-1 is converted to active ET-1 by ECE-1,
thereby increasing vessel contractility, our observations sug-
gest that the influence of E2F2 on contractility could be
generated, at least in part, through the regulation of ECE-1
activity. If so, the elevated BP in E2F2?/?mice may be
attributable to an increase in ET-1 level and activity. Plasma
ET-1 levels were significantly higher in E2F2?/?mice than in
WT mice (Figure 1D), and selective antagonism of the ETA
receptor with BQ123 dramatically reduced BP in E2F2?/?
mice to levels that did not differ significantly from those in
WT mice (Figure III of the online-only Data Supplement). In
addition, we found that E2F2?/?mice express a higher level
of ETAprotein in the lung tissues (Figure IV of the online-
only Data Supplement). Collectively, these findings indicate
that enhanced ET-1 biosynthesis and activity, resulting from
increased ECE-1 activity, contribute to the BP elevations
observed in E2F2?/?mice.
E2F2 Regulates ECE-1b Transcription
Because recent clinical studies have identified a strong associa-
tion between an E2F binding site polymorphism on the ECE-1b
promoter (C-338A) and hypertension in women,16,17our find-
ings may be relevant to human disease. Accordingly, we
evaluated whether E2F2 regulates ECE-1b mRNA expression
in vivo via real-time reverse-transcription PCR analyses. In
lung tissues, the levels of ECE-1b mRNA were 50% lower in
E2F2?/?mice than in their WT littermates (Figure 2A),
suggesting that endogenous E2F2 levels maintain basal
ECE-1b expression in WT tissue; the levels of ECE-1a, -1c,
and -1d mRNA, however, were similar between E2F2?/?
mice and WT controls (Figure V of the online-only Data
Supplement). Interestingly, ECE-1b mRNA levels were sim-
ilar in the lung tissues from E2F1?/?and WT mice (Figure VI
of the online-only Data Supplement), indicating that the
reduced ECE-1b expression observed in E2F2?/?mice was an
E2F2-specific effect.
To investigate whether E2F2-regulated ECE-1b mRNA
expression is allele specific for the ECE-1b promoter, BAECs
were cotransfected with an E2F2-expressing plasmid and a
reporter plasmid that expressed AP from either the native
(?338C) human ECE-1b promoter or the polymorphic
(?338A) promoter associated with hypertension in women
(Figure 2B and Figure IA of the online-only Data Supple-
Figure 2. E2F2 regulates ECE-1b transcription. A, ECE-1b (left) and E2F2 (right) mRNA expression in mouse lung tissue. Real-time
RT-PCR was performed with RNA freshly isolated from the lung tissues of E2F2?/?mice, E2F2?/?mice, and their WT littermates (n?4).
ECE-1b and E2F2 mRNA levels were normalized to GAPDH mRNA levels. B, Effect of E2F2 overexpression on human ECE-1b pro-
moter activity. BAECs were cotransfected with plasmids expressing E2F1 (pE2F1) or E2F2 (pE2F2) and with plasmids expressing AP
from either the native (?338C) ECE-1b promoter (pECE1b/C-AP) or the polymorphic (?338A) ECE-1b promoter (pECE1b/A-AP) that
has been linked to hypertension in women (n?3). C, Effect of E2F2 knockdown on human ECE-1b promoter activity. The native or poly-
morphic human promoter-reporter plasmids were transfected with or without bovine E2F2 siRNA (n?3). D, Effect of E2F2 overexpres-
sion on mouse ECE-1b promoter activity. A mouse ECE-1 proximal promoter (1.6 kb) –AP (pMuECE1b-AP) was cotransfected with
pE2F1 or pE2F2 (n?4). E, ChIP assay showing E2F2 occupation of the ECE-1b promoter in vivo. ChIP assays were performed in
HUVECs with E2F2 antibody, E2F1 antibody, and control IgG. The promoter region for the dihydrofolate reductase gene (DHFR), which
is regulated by both E2F1 and E2F2, was used as a positive control, and the 3? untranslated region served as a negative control. Data
are mean?SEM for A through D. AP activities were normalized to ?-Gal activity (*P?0.05; **P?0.01).
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ment); cells were harvested 24 hours after transfection for AP
activity assays. In the absence of E2F2 overexpression, the
activity of the polymorphic promoter was significantly higher
than native promoter activity (Figure 2B). Overexpression of
E2F2 significantly increased the activity of both the poly-
morphic and native ECE-1b promoters, but the enhancement
of native promoter activity was significantly greater (native,
28.7-fold; C-338A, 1.9-fold; Figure 2B). These observations
suggest that E2F2 activates ECE-1b expression, but this
activation is impaired by the C-338A polymorphism. Results
from similar experiments indicate that the human ECE-1b
promoter is not regulated by E2F1 (Figure 2B). To determine
whether endogenous E2F2 regulates ECE-1b promoter activ-
ity, we performed promoter-reporter assays in BAECs after
knocking down endogenous E2F2 expression with siRNA.
Both polymorphic and native ECE-1b promoter activity was
significantly reduced in the presence of E2F2 siRNA, but the
activity of the polymorphic promoter was reduced to a greater
extent (Figure 2C).
To correlate our observations from experiments in E2F2?/?
mice with the results from our human ECE-1b promoter-
reporter assays, we investigated whether regulation of the
mouse ECE-1b promoter is disrupted in E2F2?/?mice.
BAECs were cotransfected with a plasmid that expressed AP
from a murine ECE-1b proximal (1.6 kb) promoter, which
contains 3 putative E2F binding sites, and a plasmid express-
ing either E2F2 or E2F1. Overexpression of E2F2, but not
E2F1, significantly upregulated murine ECE-1b promoter
activity (Figure 2D), confirming that E2F2 regulates ECE-1b
expression in both mice and humans.
To determine whether E2F2 physically interacts with the
ECE-1b promoter, we performed ChIP assays with HUVECs.
Antibodies to E2F2, but not E2F1, coprecipitated with the
ECE-1b promoter, indicating that endogenous E2F2 occupies
the ECE-1b promoter region in vivo and that ECE-1b is a
direct transcriptional target of E2F2 (Figure 2E).
Sam68 Suppresses E2F2-Mediated
ECE-1b Transcription
To identify cofactors that may interact with E2F2 and
modulate ECE-1b regulation, we isolated nuclear protein
extracts from cultured HUVECs, coimmunoprecipitated the
extracts with the E2F2 antibody, resolved the E2F2-
coprecipitated proteins on a 1-dimensional SDS-PAGE gel,
and identified the proteins via mass spectrometry. One of the
proteins identified was Sam68, an RNA-binding protein.32
Both endogenous and overexpressed Sam68 protein coimmu-
noprecipitated with E2F2 (Figure 3A and 3B), confirming
that Sam68 binds E2F2 in vivo and identifying Sam68 as a
possible cofactor involved in E2F2 transactivity. We then
performed ChIP assays and confirmed that Sam68, like E2F2,
occupies the ECE-1b promoter region in vivo (Figure 3C).
To determine whether Sam68 modulates E2F2 transactiv-
ity at the ECE-1b promoter, we performed cotransfection
Figure 3. Sam68 suppresses E2F2-mediated ECE-1b transcription. A, Interaction between endogenous Sam68 and E2F2 proteins.
HUVEC lysates were immunoprecipitated (IP) with anti-E2F2 antibody, immunoblotted (IB) with anti-Sam68 antibody, and reblotted with
anti-E2F2 antibody. B, Interaction between overexpressed Sam68 and E2F2 proteins. The 293T cells were transfected with HA-tagged
E2F2 and Myc-tagged Sam68. Cell lysates were immunoprecipitated with anti-HA antibody, blotted with anti-Myc antibody, and reblot-
ted with anti-HA antibody. C, ChIP assay showing Sam68 occupation of the ECE-1b promoter in vivo in HUVECs. D, Effect of Sam68
overexpression on basal and E2F2-induced ECE-1b promoter activity. BAECs were cotransfected with pECE1b/C-AP and with plas-
mids expressing E2F2 (pE2F2), Sam68 (pSam68), or both; AP activity was assessed 24 hours after transfection and expressed as the
fold difference from the level observed in the pECE1b/C-AP–only group. Data are mean?SEM. AP activities were normalized to ?-Gal
activity (n?9 per treatment; **P?0.01; ***P?0.001).
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experiments with plasmids expressing E2F2, Sam68, and the
native human ECE-1b promoter–AP construct. Overexpres-
sion of Sam68 suppressed E2F2-induced ECE-1b promoter
activity in a dose-dependent manner, as well as basal ECE-1b
promoter activity (Figure 3D). Collectively, our data indicate
that Sam68 is a cofactor that represses E2F2-mediated
ECE-1b transcription.
Loss or Downregulation of E2F2 Expression
Increases Levels of the Plasma Membrane ECE-1
Isoforms and Reduces Levels of the Intracellular
ECE-1 Isoforms
Because E2F2 regulates ECE-1b transcription, we investi-
gated whether ECE-1 protein levels differ in tissues harvested
from E2F2?/?mice and their WT littermates. Because the
available antibodies cannot distinguish between the isoforms
of ECE-1 (ie, ECE-1a, -1b, -1c, and -1d), we performed
Western blotting with a polyclonal antibody that identifies a
C-terminal epitope present in all 4 ECE-1 isoforms. Total
ECE-1 protein levels in the lung tissues of E2F2?/?and WT
mice were similar (Figure 4A). We then isolated primary ECs
from the lung tissues of E2F2?/?and WT mice (Figure VII of
the online-only Data Supplement). To differentiate between
the plasma membrane and intracellular ECE-1 isoforms, we
labeled the surface of the lung ECs in culture with nonper-
meable Sulfo-NHS-SS-Biotin and then isolated the plasma
membrane and cytosolic protein compartments by fraction-
ation. Compared with WT lung ECs, E2F2?/?lung ECs
displayed significantly less ECE-1 immunoreactivity in the
cytosolic fraction (ie, ECE-1b immunoreactivity) and signif-
icantly greater ECE-1 immunoreactivity in the plasma mem-
brane fraction (ie, ECE-1a, -1c, and -1d immunoreactivity;
Figure 4. Loss of E2F2 increases levels of the plasma membrane ECE-1 isoforms and reduces levels of the intracellular ECE-1 iso-
forms. A, Representative Western blotting for ECE-1 (top) and quantification of relative ECE-1 levels (bottom) in the whole-cell lysates
from the lung tissues of E2F2?/?mice and their WT littermates. The levels of ECE-1 were determined densitometrically, normalized to
the levels of ?-actin, and expressed as the fold difference from the levels measured in WT lung tissues. Control assessments evaluated
ECE-1 levels in HUVECs and HeLa cell lysates, and recombinant human ECE-1 (rhECE-1) protein levels (n?6 per group; **P?0.01; NS,
not significant vs WT lung tissue; N/A for normalization to ?-actin). B, Western blotting analyses of ECE-1 protein levels at the plasma
membrane and in the cytosol of the primary lung ECs isolated from E2F2?/?and WT mice. Cells were fractionated with a Sulfo-NHS-
SS-Biotin kit. The levels of ECE-1 in the membrane and cytosol were normalized to the levels of Tie-2 and calnexin, respectively, and
expressed as the fold difference from the levels measured in WT ECs (n?5 per group; **P?0.01 vs WT). C, Confocal microscopy of
mouse lung ECs stained with an anti–ECE-1 antibody (red) and counterstained with DAPI (blue) (original magnification ?400); represen-
tative results from 4 separate experiments are shown. The anti–ECE-1 antibody recognizes an epitope shared by all 4 ECE-1 isoforms
(ECE-1a, -1b, -1c, -1d). D, ET-1 levels in the culture media of WT and E2F2?/?lung ECs after incubation with 10?7mol/L BigET-1 for
60 minutes (n?6 per group; ***P?0.001). The ET-1 levels were measured with ELISA and normalized to the quantity of cells.
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Figure 4B). These results were confirmed via confocal
microscopic analyses of cells immunofluorescently stained
for ECE-1 (Figure 4C), and experiments in HUVECs yielded
equivalent results: siRNA-mediated knockdown of E2F2
reduced cytoplasmic ECE-1 (ECE-1b) levels and increased
plasma membrane ECE-1 (ECE-1a, -1c, -1d) levels (Figure
VIII of the online-only Data Supplement). Importantly, the
increase in plasma membrane ECE-1 in the E2F2?/?lung ECs
was associated with an enhanced conversion of exogenous
BigET-1 into ET-1 (Figure 4D). These observations are
consistent with a study by Muller et al,13who showed that
ECE-1b decreases the expression and activity of ECE-1a, -1c,
and -1d. Collectively, the results presented here and by
Muller et al suggest that the enhanced contractility of E2F2?/?
aortic vessels in response to BigET-1 (Figure 1B and 1C)
may evolve indirectly through the downregulation of late
endosomal ECE-1b levels and a subsequent increase in
ECE-1a, -1c, and -1d activity.
Discussion
Hypertension is a multifactorial disease in which genetic
traits play an important role.33,34Recent studies indicate that
transcriptional regulators in both vascular ECs and smooth
muscle cells can critically affect BP control.27,35Our inves-
tigations provide the first evidence that E2F2, a classic
cell-cycle regulator, influences BP. Data from our studies
suggest that endogenous levels of E2F2 transcriptional activ-
ity in nonproliferating endothelium regulate vessel tone
through the ECE-1/ET-1 system: E2F2 acts as a transactiva-
tor of ECE-1b transcription, and Sam68 functions as a
cofactor that represses E2F2-induced ECE-1b transcription in
ECs. Thus, the physiological state could regulate BP, at least
in part, by balancing E2F2 and Sam68 activity to fine-tune
ECE-1b expression. If so, deregulated E2F2 transcriptional
activity (eg, in E2F2?/?mice or in women carrying the
C-338A ECE-1b promoter polymorphism) may contribute to
the pathogenesis of hypertension.
The ECE-1/ET-1 system has been recognized to regulate
BP through multiple mechanisms, including maintenance of
the system vessel resistance and renal functions.6,36–38In-
creasing evidence emphasizes aberrant vessel tone in the
pathogenesis of hypertension.39,40We observed a striking
abnormality of vascular reactivity in E2F2?/?mice. How-
ever, increased ECE-1/ET-1 activity has also been shown
to increase oxidative stress41and to mediate angiotensin
II–induced hypertension.42Importantly, the kidney has been
shown to be both a source of ET-1 generation and an
important target organ of this peptide, where it mediates
natriuretic and diuretic effects through the ETB receptor
subtype.43,44Therefore, we cannot rule out a possible contri-
bution of these factors to the hypertension phenotype of
E2F2?/?mice.
The (C-338A) ECE-1b promoter polymorphism has been
linked to hypertension in women but not in men.16,17This
gender difference may be partially due to the fact that the
higher levels of estrogen in women suppress ET-1 synthesis
to a greater extent, so women are more sensitive to the
increase in ECE-1 activity.45However, this effect was not
seen in a related study of human Alzheimer disease in which
ECE-1 activity also plays an important role28(see below). In
our experimental settings, the elevated BP existed in both
female and male E2F2?/?mice.
Higher levels of basal promoter activity were observed for
the polymorphic ECE-1b/A promoter than for the ECE-1b/C
promoter, and E2F2 siRNA strongly inhibited both promot-
ers, yielding comparable levels of residual activity. Never-
theless, the ECE-1b/A promoter was only marginally acti-
vated by E2F2 overexpression. This apparent discrepancy
could evolve from intrinsic competition between E2F2 and
other cofactors that bind to the ECE-1b promoter. Overex-
pression of 1 factor (eg, E2F2) could induce conformational
changes in other cofactors, and interactions between the
cofactor complex and their cognate cis elements in the
ECE-1b/A and ECE-1b/C promoters could differ. However,
this explanation has yet to be evaluated experimentally.
Funalot et al28have recently reported that the E2F2 binding
site polymorphism in the ECE-1b promoter is associated with
a lower risk of late-onset Alzheimer disease. Because the
deposition of ?-amyloid in the brain is a pathological hall-
mark of Alzheimer disease and ECE-1 activity degrades
?-amyloid,46these data corroborate our findings regarding
the regulation of ECE-1 activity by this E2F2 binding site in
the ECE-1b promoter. In addition, E2F2 may have a role in
other pathological conditions that have been linked to ECE-1
activity, including pulmonary hypertension, myocardial in-
farction, and renal failure.47Thus, the implications of our
findings presented here may extend to a broad range of
pathological conditions and normal developmental processes.
Acknowledgments
We thank Dr Gustavo Leone (Ohio State University) for providing
E2F2-knockout mice, Dr Funalot (INSERM, France) for ECE-1b/A
and ECE-1b/C promoter–AP reporter plasmids, Dr Farbio Martelli
(IRCSS, Italy) for E2F2 expression plasmid, Dr Chi Wai Eric So
(Institute of Cancer Research, Sutton, UK) for myc-Sam68 plasmid,
Dr Yves Goldberg (INSERM, Grenoble, France) for pEGFP-Sam68
plasmid, and Drs Lizhao Wu (Ohio State University), Ping Lu
(CSEMC, Boston), Hiromichi Hamada (CSEMC, Boston), T. Oui-
met (INSERM, France), and Jianing Zhang (Northwestern Univer-
sity), as well as Andrea Wecker (CSEMC, Boston) and Shirley Yang
(Northwestern University), for technical help. We thank Dr W.
Kevin Meisner for editorial assistance.
Sources of Funding
This work was supported by American Heart Association grant
0430135N (to Dr Qin) and National Institutes of Health grants
HL-077378 (to Dr M.E. Mendelsohn), HL-53354, HL-57516, HL-
80137, HL-63414, HL-77428, and HL-66957 (to Dr Losordo).
Disclosures
None.
References
1. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui
Y, Yazaki Y, Goto K, Masaki T. A novel potent vasoconstrictor peptide
produced by vascular endothelial cells. Nature. 1988;332:411–415.
2. D’Orleans-Juste P, Plante M, Honore JC, Carrier E, Labonte J. Synthesis
and degradation of endothelin-1. Can J Physiol Pharmacol. 2003;81:
503–510.
3. Arai H, Hori S, Aramori I, Ohkubo H, Nakanishi S. Cloning and
expression of a cDNA encoding an endothelin receptor. Nature. 1990;
348:730–732.
Zhou et al Endothelial E2F2 Regulates Blood Pressure
1219
by on May 11, 2011 circ.ahajournals.org Downloaded from

Page 9

4. Sakurai T, Yanagisawa M, Takuwa Y, Miyazaki H, Kimura S, Goto K,
Masaki T. Cloning of a cDNA encoding a non-isopeptide-selective
subtype of the endothelin receptor. Nature. 1990;348:732–735.
5. Kurihara Y, Kurihara H, Suzuki H, Kodama T, Maemura K, Nagai R,
Oda H, Kuwaki T, Cao WH, Kamada N. Elevated blood pressure and
craniofacial abnormalities in mice deficient in endothelin-1. Nature.
1994;368:703–710.
6. Schiffrin EL. Vascular endothelin in hypertension. Vascul Pharmacol.
2005;43:19–29.
7. Xu D, Emoto N, Giaid A, Slaughter C, Kaw S, deWit D, Yanagisawa M.
ECE-1: a membrane-bound metalloprotease that catalyzes the proteolytic
activation of big endothelin-1. Cell. 1994;78:473–485.
8. Yanagisawa H, Yanagisawa M, Kapur RP, Richardson JA, Williams SC,
Clouthier DE, de Wit D, Emoto N, Hammer RE. Dual genetic pathways
of endothelin-mediated intercellular signaling revealed by targeted dis-
ruption of endothelin converting enzyme-1 gene. Development. 1998;125:
825–836.
9. Schweizer A, Valdenaire O, Nelbock P, Deuschle U, Dumas Milne
Edwards JB, Stumpf JG, Loffler BM. Human endothelin-converting
enzyme (ECE-1): three isoforms with distinct subcellular localizations.
Biochem J. 1997;328(pt 3):871–877.
10. Valdenaire O, Lepailleur-Enouf D, Egidy G, Thouard A, Barret A,
Vranckx R, Tougard C, Michel JB. A fourth isoform of endothelin-
converting enzyme (ECE-1) is generated from an additional promoter
molecular cloning and characterization. Eur J Biochem. 1999;264:
341–349.
11. Azarani A, Boileau G, Crine P. Recombinant human endothelin-
converting enzyme ECE-1b is located in an intracellular compartment
when expressed in polarized Madin-Darby canine kidney cells. Biochem
J. 1998;333(pt 2):439–448.
12. Takahashi M, Fukuda K, Shimada K, Barnes K, Turner AJ, Ikeda M,
Koike H, Yamamoto Y, Tanzawa K. Localization of rat endothelin-
converting enzyme to vascular endothelial cells and some secretory cells.
Biochem J. 1995;311(pt 2):657–665.
13. Muller L, Barret A, Etienne E, Meidan R, Valdenaire O, Corvol P,
Tougard C. Heterodimerization of endothelin-converting enzyme-1
isoforms regulates the subcellular distribution of this metalloprotease.
J Biol Chem. 2003;278:545–555.
14. Schmidt M, Kroger B, Jacob E, Seulberger H, Subkowski T, Otter R,
Meyer T, Schmalzing G, Hillen H. Molecular characterization of human
and bovine endothelin converting enzyme (ECE-1). FEBS Lett. 1994;356:
238–243.
15. Shimada K, Takahashi M, Turner AJ, Tanzawa K. Rat endothelin-
converting enzyme-1 forms a dimer through Cys412 with a similar
catalytic mechanism and a distinct substrate binding mechanism
compared with neutral endopeptidase-24.11. Biochem J. 1996;315(pt
3):863–867.
16. Funalot B, Courbon D, Brousseau T, Poirier O, Berr C, Cambien F,
Amouyel P, Schwartz JC, Ducimetiere P. Genes encoding endothelin-
converting enzyme-1 and endothelin-1 interact to influence blood
pressure in women: the EVA study. J Hypertens. 2004;22:739–743.
17. Funke-Kaiser H, Reichenberger F, Kopke K, Herrmann SM, Pfeifer J,
Orzechowski HD, Zidek W, Paul M, Brand E. Differential binding of
transcription factor E2F-2 to the endothelin-converting enzyme-1b
promoter affects blood pressure regulation. Hum Mol Genet. 2003;12:
423–433.
18. Tiret L, Poirier O, Hallet V, McDonagh TA, Morrison C, McMurray JJ,
Dargie HJ, Arveiler D, Ruidavets JB, Luc G, Evans A, Cambien F. The
Lys198Asn polymorphism in the endothelin-1 gene is associated with
blood pressure in overweight people. Hypertension. 1999;33:1169–1174.
19. Attwooll C, Lazzerini Denchi E, Helin K. The E2F family: specific
functions and overlapping interests. EMBO J. 2004;23:4709–4716.
20. Muller H, Bracken AP, Vernell R, Moroni MC, Christians F, Grassilli E,
Prosperini E, Vigo E, Oliner JD, Helin K. E2Fs regulate the expression of
genes involved in differentiation, development, proliferation, and apopto-
sis. Genes Dev. 2001;15:267–285.
21. Wells J, Graveel CR, Bartley SM, Madore SJ, Farnham PJ. The identi-
fication of E2F1-specific target genes. Proc Natl Acad Sci U S A. 2002;
99:3890–3895.
22. DeGregori J. The genetics of the E2F family of transcription factors:
shared functions and unique roles. Biochim Biophys Acta. 2002;1602:
131–150.
23. Murga M, Fernandez-Capetillo O, Field SJ, Moreno B, Borlado LR,
Fujiwara Y, Balomenos D, Vicario A, Carrera AC, Orkin SH, Greenberg
ME, Zubiaga AM. Mutation of E2F2 in mice causes enhanced T lym-
phocyte proliferation, leading to the development of autoimmunity.
Immunity. 2001;15:959–970.
24. Qin G, Kishore R, Dolan CM, Silver M, Wecker A, Luedemann CN,
Thorne T, Hanley A, Curry C, Heyd L, Dinesh D, Kearney M, Martelli F,
Murayama T, Goukassian DA, Zhu Y, Losordo DW. Cell cycle regulator
E2F1 modulates angiogenesis via p53-dependent transcriptional control
of VEGF. Proc Natl Acad Sci U S A. 2006;103:11015–11020.
25. Wu L, Timmers C, Maiti B, Saavedra HI, Sang L, Chong GT, Nuckolls
F, Giangrande P, Wright FA, Field SJ, Greenberg ME, Orkin S, Nevins
JR, Robinson ML, Leone G. The E2F1–3 transcription factors are
essential for cellular proliferation. Nature. 2001;414:457–462.
26. Feng M, Whitesall S, Zhang Y, Beibel M, D’Alecy L, DiPetrillo K.
Validation of volume-pressure recording tail-cuff blood pressure mea-
surements. Am J Hypertens. 2008;21:1288–1291.
27. Zhu Y, Bian Z, Lu P, Karas RH, Bao L, Cox D, Hodgin J, Shaul PW,
Thoren P, Smithies O, Gustafsson JA, Mendelsohn ME. Abnormal
vascular function and hypertension in mice deficient in estrogen receptor
beta. Science. 2002;295:505–508.
28. Funalot B, Ouimet T, Claperon A, Fallet C, Delacourte A, Epelbaum J,
Subkowski T, Leonard N, Codron V, David JP, Amouyel P, Schwartz JC,
Helbecque N. Endothelin-converting enzyme-1 is expressed in human
cerebral cortex and protects against Alzheimer’s disease. Mol Psychiatry.
2004;9:1122–1128, 1059.
29. van Beijnum JR, Rousch M, Castermans K, van der Linden E, Griffioen
AW. Isolation of endothelial cells from fresh tissues. Nat Protoc. 2008;
3:1085–1091.
30. Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M. In-gel digestion
for mass spectrometric characterization of proteins and proteomes. Nat
Protoc. 2006;1:2856–2860.
31. Qin G, Ii M, Silver M, Wecker A, Bord E, Ma H, Gavin M, Goukassian
DA, Yoon YS, Papayannopoulou T, Asahara T, Kearney M, Thorne T,
Curry C, Eaton L, Heyd L, Dinesh D, Kishore R, Zhu Y, Losordo DW.
Functional disruption of alpha4 integrin mobilizes bone marrow-derived
endothelial progenitors and augments ischemic neovascularization. J Exp
Med. 2006;203:153–163.
32. Lukong KE, Richard S. Sam68, the KH domain-containing superSTAR.
Biochim Biophys Acta. 2003;1653:73–86.
33. Lifton RP, Gharavi AG, Geller DS. Molecular mechanisms of human
hypertension. Cell. 2001;104:545–556.
34. Takahashi N, Smithies O. Human genetics, animal models and computer
simulations for studying hypertension. Trends Genet. 2004;20:136–145.
35. Kleinert H, Wallerath T, Euchenhofer C, Ihrig-Biedert I, Li H, Forstermann
U. Estrogens increase transcription of the human endothelial NO synthase
gene: analysis of the transcription factors involved. Hypertension. 1998;31:
582–588.
36. Haynes WG, Ferro CJ, O’Kane KP, Somerville D, Lomax CC, Webb DJ.
Systemic endothelin receptor blockade decreases peripheral vascular
resistance and blood pressure in humans. Circulation. 1996;93:
1860–1870.
37. Krum H, Viskoper RJ, Lacourciere Y, Budde M, Charlon V. The effect
of an endothelin-receptor antagonist, bosentan, on blood pressure in
patients with essential hypertension: Bosentan Hypertension Investi-
gators. N Engl J Med. 1998;338:784–790.
38. Perez-Rivero G, Ruiz-Torres MP, Rivas-Elena JV, Jerkic M, Diez-
Marques ML, Lopez-Novoa JM, Blasco MA, Rodriguez-Puyol D. Mice
deficient in telomerase activity develop hypertension because of an
excess of endothelin production. Circulation. 2006;114:309–317.
39. Mendelsohn ME. In hypertension, the kidney is not always the heart of
the matter. J Clin Invest. 2005;115:840–844.
40. Zacchigna L, Vecchione C, Notte A, Cordenonsi M, Dupont S, Maretto
S, Cifelli G, Ferrari A, Maffei A, Fabbro C, Braghetta P, Marino G,
Selvetella G, Aretini A, Colonnese C, Bettarini U, Russo G, Soligo S,
Adorno M, Bonaldo P, Volpin D, Piccolo S, Lembo G, Bressan GM.
Emilin1 links TGF-beta maturation to blood pressure homeostasis. Cell.
2006;124:929–942.
41. Laplante MA, de Champlain J. The interrelation of the angiotensin and
endothelin systems on the modulation of NAD(P)H oxidase. Can
J Physiol Pharmacol. 2006;84:21–28.
42. Laplante MA, Wu R, Moreau P, de Champlain J. Endothelin mediates
superoxide production in angiotensin II-induced hypertension in rats.
Free Radic Biol Med. 2005;38:589–596.
43. Hoffman A, Abassi ZA, Brodsky S, Ramadan R, Winaver J. Mechanisms
of big endothelin-1-induced diuresis and natriuresis: role of ET(B)
receptors. Hypertension. 2000;35:732–739.
1220Circulation
September 29, 2009
by on May 11, 2011 circ.ahajournals.orgDownloaded from

Page 10

44. Rothermund L, Luckert S, Kossmehl P, Paul M, Kreutz R. Renal endo-
thelin ET(A)/ET(B) receptor imbalance differentiates salt-sensitive from
salt-resistant spontaneous hypertension. Hypertension. 2001;37:275–280.
45. Webb CM, Ghatei MA, McNeill JG, Collins P. 17Beta-estradiol decreases
endothelin-1 levels in the coronary circulation of postmenopausal women
with coronary artery disease. Circulation. 2000;102:1617–1622.
46. Eckman EA, Watson M, Marlow L, Sambamurti K, Eckman CB. Alz-
heimer’s disease beta-amyloid peptide is increased in mice deficient in
endothelin-converting enzyme. J Biol Chem. 2003;278:2081–2084.
47. Attina T, Camidge R, Newby DE, Webb DJ. Endothelin antagonism in
pulmonary hypertension, heart failure, and beyond. Heart. 2005;91:
825–831.
CLINICAL PERSPECTIVE
Hypertension is largely attributed to abnormal renal sodium handling; however, a growing body of evidence now suggests
that primary abnormalities in vessels can also cause aberrations in blood pressure. Very often, the source of the abnormality
resides in the endothelial cells that regulate the functional state of the entire vessel, and this knowledge has directed our
search for new diagnostic and therapeutic targets. To date, the role of transcriptional mechanisms in blood pressure
regulation is poorly characterized. In this study, we found that E2F2, a transcription factor involved in cell-cycle control,
regulates blood pressure by modulating vessel contractility. This previously unknown function of E2F2 evolves from the
unique role of the molecule in endothelial cells: suppression of endothelin-converting enzyme 1 (ECE-1). ECE-1 converts
the inactive precursor molecule big endothelin-1 into the potent vasoconstrictor endothelin-1, and genetic deletion of E2F2
in mice was associated with both exaggerated vessel contractility in response to big endothelin-1 stimulation and high
blood pressure. E2F2 suppresses ECE-1 activity indirectly by promoting transcription of ECE-1b, the negative isoform of
ECE-1, and our results indicate that Sam68 functions as a cofactor that represses E2F2-mediated ECE-1b transcription.
These findings are especially provocative because clinical studies have identified an E2F binding site polymorphism on the
ECE-1b promoter that is strongly associated with hypertension in women. Collectively, our results and observations from
other laboratories may have identified a previously unknown mechanism of blood pressure maintenance that operates
through the E2F2/Sam68–ECE-1 pathway, and deregulation of this pathway may contribute to blood pressure disorders,
including hypertension in humans.
Zhou et alEndothelial E2F2 Regulates Blood Pressure
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1
Regulation of Vascular Contractility and Blood Pressure by the
E2F2 Transcription Factor

Zhou J et al
SUPPLEMENTAL MATERIAL

Supplemental Methods

Aortic ring assay
The aortic ring assay was performed as previously described1. Briefly, segments of thoracic aorta
(3 mm) were dissected with the endothelium intact and mounted in organ baths containing 15
mL Krebs buffer; optimal resting tension was determined in baseline experiments performed
before treatment with the compounds under investigation. Vessels were submaximally
precontracted with phenylephrine (PE), and endothelial function was evaluated by vascular
relaxation in response to acetylcholine (Ach). At the end of each experiment, vessels were
contracted by the addition of K+ to verify that baseline contractile function was preserved during
the course of the study. Isometric forces were recorded with force transducers connected to a
PowerLab/800 Eight-channel 100 kHz A/D converter (AD Instruments, Mountainview, CA).

Mouse ECE-1b promoter-AP reporter plasmid
To clone murine ECE-1b promoter, genomic DNA was isolated from C57BL/6 mouse tail with
QIAGEN kit and used for high fidelity PCR amplification with primers listed in Supplemental
Figure S2. The PCR product was first cloned into TOPO TA cloning plasmid (Invitrogen) and
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2
subsequently cloned into pSEAP plasmid2 by the use of MluI and ClaI restriction enzymes. The
murine ECE-1b promoter sequence in the final reporter construct was verified by sequencing.

Chromatin immunoprecipitation (ChIP) assay
Cells were lysed and sheared by sonication, then extracts were pre-cleared with protein A
agarose/salmon sperm DNA and immunoprecipitated overnight. Precipitates were washed for 5
min with buffer and extracted twice with 1% SDS, 0.1 M NaHCO3, then the elutes were pooled
and heated at 65°C for 5 h to reverse the formaldehyde cross-linking. DNA fragments were
purified via phenol/chloroform extraction, then 2 μL of DNA was added to a standard PCR
mixture, and genes were amplified for 23 cycles; primer sequences are listed in Supplemental
Figure S2. PCR products were separated on a 2% agarose gel.

Mass spectrometry (MS)
Experiments followed a standard protein identification strategy3. Candidate proteins were
excised from the gel, reduced with dithiothreitol, and alkylated with iodoacetamide, then the
bands were digested overnight with trypsin, and the extracted peptides were speed vacuumed to
reduce volume and remove organic. Peptides were chromatographically resolved on-line with a
C18 column and a 1200-series high-performance liquid chromatography (HPLC) apparatus, then
analyzed by using a 6340 liquid-chromatography ion-trap mass spectrometer (Agilent
Technologies, Palo Alto, CA) with an HPLC-chip interface (Agilent Technologies). Raw data
was extracted and searched using the Spectrum Mill search engine (Rev A.03.03.038 SR1,
Agilent Technologies), and “peak picking” was performed with a signal-to-noise ratio of 25:1
and a maximum charge state of 4 (z<4); the program was directed to “find” a precursor charge
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3
state. Searches were performed in the International Protein Index human database (IPI Human rel.
3.28, 16-APR-2007) with the following criteria: fixed carbamidomethylation modification,
trypsin, no more than 1 missed cleavage, precursor mass tolerance ±1.7, product mass tolerance
±0.7, and maximum ambiguous precursor charge = 3. Protein identification was considered
significant if the following confidence thresholds were met: 2 or more peptides per protein,
individual peptide scores ≥10, protein score >20, and Scored Percent Intensity (SPI) ≥70%; the
SPI indicates what percentage of the total ion intensity matches the peptide’s MS/MS spectrum.
A reverse (random) database search was simultaneously performed, and the spectra were
inspected manually to validate the match of the spectrum to the predicted peptide fragmentation
pattern. Quality-control standards were run at the beginning of each day and at the end of each
set of analyses.

Isolation of mouse primary lung ECs
Primary ECs were isolated from mouse lung tissues as described in a published protocol4. Briefly,
tissues were minced and digested with collagenase and dispase, then the mixture was passed
through a 100-μm cell strainer to obtain single-cell suspensions. Red blood cells, granulocytes,
nonvital cells, and cell debris were removed via density centrifugation with Histopaque-1.083
(Sigma)5, then the ECs were immunostained with CD31-PE, FACS sorted to ≥ 95% purity, and
cultured in EBM-2 medium (Lonza, Walkersville, MD).

Subcellular fractionation
Plasma-membrane and cytosolic proteins were fractionated by using the Pinpoint Cell Surface
Protein Isolation Kit (Pierce) according to the manufacturer’s protocol. Briefly, cells were
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4
labeled with the cell-impermeable and cleavable biotinylation reagent Sulfo-NHS-SS-Biotin
(sulfosuccinimidyl-2-[biotinamido] ethyl-1, 3-dithiopropionate) for 30 min at 4°C, then lysed on
ice and sonicated. Labeled proteins were isolated with immobilized NeutrAvidin Gel and eluted
with SDS-PAGE sample buffer containing 50 mM DTT. The reducing agent (DTT) cleaves the
disulfide bond in the biotin label, and nearly 100% of the bound proteins are recovered.

Immunofluorescent staining
Primary lung ECs were briefly rinsed with buffer A (1 mM MgCl2 and 0.1 mM CaCl2 in PBS),
fixed in methanol at room temperature for 10 min, rinsed again, then permeabilized for 15 min in
Buffer B (Buffer A with 0.3% Triton X-100, 0.1% BSA); nonspecific protein binding was
blocked for 30 min with buffer C (Buffer A with 0.3% Triton X-100, 1% BSA). Cells were
incubated overnight at 4°C with goat polyclonal anti-ECE-1 antibodies (0.6 µg/ml) diluted in
Buffer C, washed 3 times for 5 min (15 min total) in buffer B, then incubated with PE-
conjugated anti-goat IgG antibodies (Jackson ImmunoResearch). One hour later, cells were
washed 3 times for 5 min (15 minutes total) in Buffer B and once for 10 min in 5 mM NaPi (pH
7.5), then mounted with a solution containing 0.1% p-phenylenediamine (Sigma-Aldrich Corp.),
70% glycerol, 150 mM NaCl, and 10 mM NaPi (pH 7.4).


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5
Supplemental Figures













Figure S1: ECE-1 promoter regions and N-terminal amino-acid sequences. (A) The four
ECE-1 isoforms (top), organization of the upstream regions of the human ECE-1 gene (middle),
and the reporter plasmid containing the ECE-1b promoter and alkaline phosphatase (AP)
(bottom); each isoform-specific promoter is identified by a dark bulb, and the E2F binding-site
polymorphism (-338C/A) is identified with inverted red triangles. P denotes promoter; d, splicing
donor; a, splicing acceptor. (B) The N-terminal amino acid sequences of the ECE-1 isoforms,
which determine their subcellular location. TMD denotes transmembrane domain.
A

B

ECE-1d ECE-1dECE-1c ECE-1cECE-1b ECE-1b
augaug aug aug augaug aug aug
ECE-1a ECE-1a
ECE-1 protein isoforms result from
alternative promotersalternative promoters
100 bp100 bp
Organization of the upstream
regions of the human ECE-1 generegions of the human ECE-1 gene
The cloned human pECE-1b/C (or A)-
AP promoter reporter constructs AP promoter reporter constructs
822 bp 822 bp
pECE-1b/C-AP; pECE-1b/A-APpECE-1b/C-AP; pECE-1b/A-AP
APAP
atgatgatgatgatgatgatgatgatgatgatg atg
PPPPPPPP
5’ 5’3’ 3’
ddddaaaadd
dd
11kb11kb
55kb55kb
C/AC/A
-338-338
C/AC/A
ECE-1 protein isoforms result from
Organization of the upstream
The cloned human pECE-1b/C (or A)-
MPLQGLGLQRNPFLQGKRGPGLTSSPPLLPPSLQVNFHSPRSGQRCWAARTQVEKR MPLQGLGLQRNPFLQGKRGPGLTSSPPLLPPSLQVNFHSPRSGQRCWAARTQVEKR
MRGVWPPPVSALLSALGMSTYKRATLDEEDLVDSLSEGDAYPNGLQVNFHSPRSGQRCWAARTQVEKRMRGVWPPPVSALLSALGMSTYKRATLDEEDLVDSLSEGDAYPNGLQVNFHSPRSGQRCWAARTQVEKR
MMSTYKRATLDEEDLVDSLSEGDAYPNGLQVNFHSPRSGQRCWAARTQVEKRMMSTYKRATLDEEDLVDSLSEGDAYPNGLQVNFHSPRSGQRCWAARTQVEKR
ECE-1a(758aa)ECE-1a(758aa)
ECE-1b(770aa)ECE-1b(770aa)
ECE-1c(754aa)ECE-1c(754aa)
ECE-1d(784aa) ECE-1d(784aa)
MRGVWPPPVSALLSALGMEALRESVLHLALQMSTYKRATLDEEDLVDSLSEGDAYPNGLQVNFHSPRSGQRCWAARTQVEKRMRGVWPPPVSALLSALGMEALRESVLHLALQMSTYKRATLDEEDLVDSLSEGDAYPNGLQVNFHSPRSGQRCWAARTQVEKR
TMDTMDTMD
TMDTMDTMD
TMDTMD TMD
TMD TMD TMD
22aa 22aa29aa29aa
52aa52aa
N-ter cytoplasmic tailN-ter cytoplasmic tail
TMD
(Transmembrane
domain) domain)
TMD
(Transmembrane
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