Characterization of the Human {alpha}1{beta}1 Soluble Guanylyl Cyclase Promoter: KEY ROLE FOR NF-{kappa}B(p50) AND CCAAT-BINDING FACTORS IN REGULATING EXPRESSION OF THE NITRIC OXIDE RECEPTOR.

Characterization of the Human �1�1 Soluble Guanylyl
Cyclase Promoter
KEY ROLE FORNF-�B(p50) AND CCAAT-BINDING FACTORS IN REGULATING EXPRESSION
OF THE NITRIC OXIDE RECEPTOR*□S
Received for publication, February 14, 2008, and in revised form, May 9, 2008 Published, JBC Papers in Press,May 12, 2008, DOI 10.1074/jbc.M801223200
Martı´n L. Marro‡, Concepcio´n Peiro´§1, Catherine M. Panayiotou‡, Reshma S. Baliga‡, Sabine Meurer¶,
Harald H. H. W. Schmidt¶, and Adrian J. Hobbs‡2
From the ‡Department of Pharmacology, University College London, Medical Sciences Building, Gower Street,
London WC1E 6BT, United Kingdom, the §Departamento de Farmacologı´a y Terape´utica, Facultad de Medicina, Universidad
Auto´noma de Madrid, C/Arzobispo Morcillo 4, 28029 Madrid, Spain, and the ¶Department of Pharmacology, Monash University,
Faculty of Medicine, Melbourne, Victoria 3168, Australia
Soluble guanylyl cyclase (sGC) is the principal receptor for
NO and plays a ubiquitous role in regulating cellular function.
This is exemplified in the cardiovascular systemwhere sGCgov-
erns smooth muscle tone and growth, vascular permeability,
leukocyte flux, and platelet aggregation. As a consequence,
aberrant NO-sGC signaling has been linked to diseases includ-
ing hypertension, atherosclerosis, and stroke. Despite these key
(patho)physiological roles, little is known about the expres-
sional regulation of sGC. To address this deficit, we have char-
acterized thepromoter activity of human�1 and�1 sGCgenes in
a cell type relevant to cardiovascular (patho)physiology, primary
human aortic smooth muscle cells. Luciferase reporter con-
structs revealed that the 0.3- and 0.5-kb regions upstream of the
transcription start sites were optimal for �1 and �1 sGC pro-
moter activity, respectively. Deletion of consensus sites for
c-Myb, GAGA, NFAT, NF-�B(p50), and CCAAT-binding fac-
tor(s) (CCAAT-BF) revealed that these are the principal tran-
scription factors regulating basal sGC expression. In addition,
under pro-inflammatory conditions, the effects of the strongest
�1 and �1 sGC repressors were enhanced, and enzyme expres-
sion and activity were reduced; in particular, NF-�B(p50) is piv-
otal in regulating enzyme expressionunder such conditions.NO
itself also elicited a cGMP-independent negative feedback effect
on sGC promoter activity that is mediated, in part, via
CCAAT-BF activity. In sum, these data provide a systematic
characterization of the promoter activity of human sGC �1 and
�1 subunits and identify key transcription factors that govern
subunit expression under basal and pro-inflammatory (i.e.
atherogenic) conditions and in the presence of ligand NO.
Nitric oxide is now well established to play key regulatory
roles in numerous, disparate physiological and (patho)physio-
logical processes (1–3). Pivotal to NO-mediated modification
of cell function is activation of the hemoprotein soluble guany-
lyl cyclase (sGC)3 and consequent production of the second
messenger cGMP, which in turn activates specific cyclic
nucleotide-dependent protein kinases, ion channels, and phos-
phodiesterases (4). This fundamental role for sGC as the prin-
cipal intracellular receptor for NO is exemplified in the cardio-
vascular system, where the enzyme governs smooth muscle
tone (5) and growth (6), vascular permeability (7, 8), platelet
reactivity (9, 10), and leukocyte extravasation (11, 12).
Soluble GC functions as an obligate heterodimer composed
of� and� subunits (13).Within the last decade, the localization
and structure of the genes encoding for sGC subunits have been
elucidated in several mammalian species, nonvertebrates, and
plants (14). In mammals, two isoforms of each subunit, called
�1,2 and �1,2, have been cloned and characterized (15). How-
ever, to date only �1/�1 and �2/�1 heterodimers have been
identified at the protein level. The chromosomal localization of
sGC genes has been determined in rodents (16, 17) and humans
(18); in both cases the�1 and�1 subunits are co-localized on the
same locus, whereas �2 and �2 lie on separate chromosomes.
Previous reports have provided evidence that expressional
regulation of the enzyme is a keymeans ofmodulatingNO-sGC
signaling. For instance, different tissues possess distinct levels
of mRNA for sGC isoforms (19), intimating tissue-specific
expression of individual sGC subunits. Furthermore, splice
variants of the�1 sGC subunitmRNAhave been identified (20).
In contrast, the human �1 subunit is encoded by a single tran-
script (�3.5kb), suggesting the translation of a single protein.
Such observations suggest that the expression of the �1 subunit
is somewhat invariant, whereas alterations in the expression of
the � subunit(s) may be an important physiological control
mechanism.
Changes in the expression of sGC have also been linked with
disease states, intimating that expressional regulation of the
* This workwas supported by theWellcome Trust. The costs of publication of
this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
Author’s Choice—Final version full access.
□S The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1–S5.
1 Supported by Comunidad Auto´noma de Madrid Grant CCG07-UAM/
BIO-1595.
2 Recipient of a Wellcome Trust Senior Research Fellowship. To whom corre-
spondence should be addressed: Dept. of Pharmacology, University Col-
lageLondon,Medical SciencesBldg., Gower St., LondonWC1E6BT,UK. Tel.:
44-20-7679-7161; Fax: 44-20-7679-7298; E-mail: a.hobbs@ucl.ac.uk.
3 The abbreviations used are: sGC, soluble guanylyl cyclase; TF, transcription
factor; LPS, lipopolysaccharide; HASMC, human aortic smooth muscle cell;
DETA-NO, DETA-NONOate; EMSA, electrophoretic mobility shift assay;
NFY, nuclear factor Y; A, � fragment; B, � fragment.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 29, pp. 20027–20036, July 18, 2008
Author’s Choice © 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
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http://www.jbc.org/cgi/content/full/M801223200/DC1
Supplemental Material can be found at:

enzyme is likely to have (patho)physiological significance. For
instance, in aged and spontaneously hypertensive rats, expres-
sion of the �1 subunit is diminished and correlates with eleva-
tions in systemic blood pressure (21, 22). Moreover, salt-sensi-
tive hypertension in Dahl rats is associated with decreased and
increased expression of the �1 and �2 subunits, respectively,
suggesting that exchange of � subunits may be critical to blood
pressure regulation (16). In animalmodels of pulmonary hyper-
tension, sGC expression is reduced (23, 24), and pulmonary
artery smoothmuscle cells exposed to hypoxia lose their ability
to express sGC (25). Reduced�1 sGC expression also correlates
with intimal thickening following balloon injury (26). Exposure
of cells and/or tissues to pro-inflammatory cytokines, or NO
itself, also induces changes in sGC expression. For example,
lipopolysaccharide (LPS), interleukin 1�, andNO donors cause
reduced sGC�1mRNAexpression in pulmonary artery smooth
muscle cells (27), and in atherogenic lesions reduced sGC
expression is linked to diminished cGMP-dependent signaling
and neointimal proliferation (28). In contrast, augmented �1
subunit expression has been linked to increased nitrovasodila-
tor potency in animals with endothelial dysfunction (29, 30)
and increased vasodilator response in aortic rings from rats
after myocardial infarction (31).
These observations intimate that expressional regulation of
sGC plays a key role in the cardiovascular system, both in terms
of physiological homeostasis and pathogenesis. Despite these
reports, however, there is a paucity of information regarding
the regulation of sGC promoter activity, particularly in the
human vasculature where the enzyme performs such an impor-
tant physiological function. Previous studies have examined the
promoter activity of individual sGC subunits in different spe-
cies (32, 33), yet only a single study has focused on the human�1
gene (34). Here, a key role for a CCAAT-binding factor
(CCAAT-BF) was revealed, although this analysis was con-
ducted in a neuronal, rather than cardiovascular, cell line. Thus,
there is a key requirement to understand the factors regulating
human �1 and �1 sGC promoter activity in the human vascula-
ture to more fully appreciate the physiological roles and regu-
lation of the NO receptor and how aberrant expression may
underlie cardiovascular pathogenesis. To address this need,
herein we investigated the transcriptional regulation of human
�1 and �1 sGC genes in human aortic smooth muscle cells
(HASMCs).
MATERIALS AND METHODS
Cell Culture—HASMCs (PromoCell) were cultured in
smooth muscle cell growth medium 2 supplemented with 5%
fetal bovine serum, 0.5 ng/ml recombinant human epidermal
growth factor, 2 ng/ml recombinant human basic fibroblast
growth factor, 5 mg/ml recombinant human insulin, and 0.62
ng/ml phenol red (PromoCell). The cells were maintained at
37 °C and 5% CO2, and cells in passages 2–10 were used.
Isolation of Genomic Clones for Human �1 and �1 sGC
Subunits—Bacterial artificial chromosome clones containing
�1 or �1 sGC subunits were purchased from BacPac Resources
(Children’s Hospital Oakland Research Institute). Bacterial
artificial chromosomeDNAwas isolated according to theman-
ufacturer’s protocol. The full promoters of human �1 and �1
sGC were isolated from the bacterial artificial chromosome
clones by PCR using AccuprimeTM Taq high fidelity DNA po-
lymerase, using primers based on the 5�-flanking regions of �1
(AY034777) and �1 (AY034778) sGC genes including the first
exon. The identity of both promoter regions was verified by
direct sequencing.
Construction of Luciferase Deletion Plasmids—Different
5�-flanking regions of the �1 and �1 sGC genes upstream of the
transcription start site (�1) and extending to the noncoding
part of the first exon (�9) were cloned into NheI-XhoI restric-
tion sites of the pGL3-basic luciferase reporter vector (Promega).
Fragments were obtained by PCR as previously described for the
full promoter. Insertions in the pGL3 reporter vector expressing
firefly luciferasewere identifiedby restrictiondigest, and the integ-
rity of the constructs was confirmed by direct sequencing. Primer
sequences used to generate the full promoter, and deletion con-
structs are shown in supplemental Fig. S1.
Site-directed Mutagenesis—The A0.3- and B0.5-kb frag-
ments, respectively, were subjected to site-directed mutagene-
sis by using the QuikChange II site-directed kit (Stratagene)
and high fidelity PfuTurboDNApolymerase (Stratagene). This
kit was also used to repair errors during the construction of
luciferase deletion constructs. All of the constructs were veri-
fied by direct sequencing.
Transient Transfections—3,500 HASMCs/well and 15,000
COS-7 cells/well were seeded in 96-well clear plates 1 day
before transfection. The cells were transfected with 50 ng of
plasmid DNA/well using FuGENE 6 reagent (Roche Applied
Science) with a 3:1 ratio (�l reagent:�g DNA). pGL3 control
vector (Promega) was used as a transfection control. In each case,
the cells were co-transfected with a pRL-TK vector expressing
Renilla luciferase (Promega) at a 10:1 ratio (test vector:pRL-TK
vector). Twenty-four hours later, where indicated cells were
treated with either (a) “pro-inflammatory mixture” (100 mg/ml
LPS (Salmonella typhimurium; Sigma), 200 units/ml IFN-�, 400
units/ml interleukin-1�, and 2000 units/ml tumor necrosis fac-
tor-� (all Preprotech) in cell culturemedium) or (b) theNOdonor
DETA-NONOate (DETA-NO; 0–200�M).
Luciferase Assays—Forty-eight hours after transfection, the
cells were washed in phosphate-buffered saline and lysed for 30
min at room temperature using the passive lysis buffer (Pro-
mega). Luciferase activity was determined using the dual lucif-
erase reporter assay system (Promega) on the Wallac Victor 2
Multilabel Counter (PerkinElmer Life Sciences). Firefly lucifer-
ase activity was normalized to Renilla luciferase derived from
pRL-TK vector, and the data are expressed as relative luciferase
light units.
Electrophoretic Mobility Shift Assays (EMSA)—HASMC
plated in 6-cm tissue culture disheswere stimulated for 2 hwith
the pro-inflammatory mixture or DETA-NO (100 or 200 �M),
nuclear extracts were prepared, and samples were subjected to
EMSA, as we have previously described (35). The primers used
to generate the double-stranded probes are shown in supple-
mental Fig. S2.
Western Blot Analysis—Protein concentrations were deter-
mined by BCA protein assay (Pierce, distributed by Perbio Sci-
ences Ltd., Northumberland,UK). Equal concentrations of pro-
tein were subjected to 15% SDS-PAGE under reducing
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conditions. The proteins were transferred to a nitrocellulose
membrane (Amersham Biosciences) using the mini Trans-blot
electrophoresis transfer cell (Bio-Rad). The membranes were
stained with Ponceau S stain to confirm equal transfer and
incubated in 5%milk inwash buffer (phosphate-buffered saline,
0.1% Tween 20) overnight at 4 °C with gentle shaking. The
membranes were then incubated with primary antibody (anti-
sGC �1) or anti- glyceraldehyde-3-phosphate dehydrogenase
(Abcam, Coventry, UK) diluted 1:2000 (�1) or 1:1000 (glyceral-
dehyde-3-phosphate dehydrogenase) in 5%milk in wash buffer
for 3 h at room temperaturewith constant agitation followed by
fivewashes (5min/wash). Themembraneswere then incubated
with shaking for 1 h at room temperature with horseradish
peroxidase-conjugated goat anti-rabbit IgG (Dako, Cam-
bridgeshire, UK) diluted 1:1000 in 5% milk in wash buffer. The
membranes were washed as described above, and the proteins
were visualized using enhanced chemiluminescence (Amer-
sham Biosciences). The bands were quantified by densitometry
using AlphaImager (Alpha Innotech, San Leandro, CA).
Cyclic GMPMeasurements—HASMC (106 cells) were plated
in 10-cmdishes and 24 h later exposed to the pro-inflammatory
mixture for a further 24 h. At this time, the cells were harvested
by trypsinization and centrifuged at 200� g for 10min. The cell
pellet was resuspended in assay buffer (2.36 mg/100 ml Tris-
HCl, 760mg/100mlNaCl, 22.2mg/100mlKCl, 29.6mg/100ml
MgSO4, 16.6 mg/100 ml Na2HPO4, 0.01 mg/100 ml CaCl2, pH
7.4) and treated with 1 mM 3-isobutyl-1-methylxanthine
(Sigma). The cells were then exposed to either 1 or 10 �M
spermine-NONOate (Axxora, Nottingham, UK) for 5 min
(“basal” samples did not receive spermine-NONOate), and the
reaction was terminated by heating at 90 °C for 10 min. The
cyclic GMP content was then measured using a commercially
available assay (Amersham Biosciences).
Bioinformatics and Statistical Analysis—BLAST was used to
identify and retrieve orthologous �1 sGC promoters frommouse,
rat, and human genomes from project Ensembl at TheWellcome
Trust Sanger Institute. The �1 sGC upstream sequence was ana-
lyzed for thepresenceof repeat regionsandtransposableelements,
using Repeat Masker version open-3.1.5. Confirmation of Pol II
promoterwas conductedwithPromoter Scan II andNovelNeural
Network Promoter Prediction. Identification of CpG islands was
performed using CpGProD (CpG Island Promoter Detection)
software. In silico analysis for transcription factor-binding sites
was performed with MatInspector Suite, release 7.4 (36). The
results are expressed as the means � S.E. A Student’s t test was
used to determine differences between data groups, where p �
0.05 was considered significant.
RESULTS
Characterization of the 5�-Flanking Region of Human �1 and
�1 sGCGenes—The transcriptional start sites were determined
by pair-wise blasting of �1 and �1 sGC 5�-flanking regions with
the completemRNA sequences (NM_000856 andNM_000857,
respectively). Scanning both �1 and �1 sGC sequences with the
Novel Neural Network Algorithms for Improved Eukaryotic
Promoter Site Recognition did not reveal any TATA box in
close proximity to the transcription start site of both �1 and �1
sGC promoters. Analysis of promoter core elements (37)
showed the presence of consensus Inr (initiator) and DPE
(downstream core promoter) elements but not TFIIB recogni-
tion (BRE) or the newly discoveredmotif 10 (MTE) elements in
the �1 sGC promoter (supplemental Fig. S3). For the�1 sGC pro-
moter, only DPE elements were found (supplemental Fig. S3). GC
andCCAAT-BFboxesweredetected inbothproximal promoters.
Furthermore, using RepeatMasker, we were able to identify three
repeats in the �1 sGC promoter but only one in the �1 promoter
region (Table 1). Finally, CpGProD software did not reveal the
presence of CpG islands in either the �1 or �1 sGC promoters.
Analysis of Promoter Activity within the 5�-Flanking Region—
Transiently transfected luciferase reporter constructs contain-
ing 0.3–3.0 kb of human �1 or 0.3–3.2 kb of human �1 sGC
upstream sequence were analyzed for promoter activity in
HASMCs. The A0.3-kb fragment showed the highest level of
activity among the �1 sGC promoter constructs (Fig. 1).
Reduced activity in the remaining (longer) constructs sug-
gested the presence of repressor element(s) further upstream.
The B0.5- and B0.8-kb fragments showed the highest level of
activity among the �1 sGC promoter constructs (Fig. 1). The
�1.4-kb fragment showed a decreased promoter activity, sug-
gesting the presence of a repressor element(s) between �830
and�1392 bp. Extension of the promoter fragment up to 3.2 kb
in length did not significantly alter the transcriptional activity
of the �1.4-kb construct, suggesting the absence of net activa-
tion or repression in that region. However, the 126-bp deletion
in B0.4 kb (to generate construct B0.3-kb) caused greater than
50% decrease in activity, suggesting that a key, putative enhanc-
er(s) is located between B0.3 and B0.4-kb. In accord, the A0.3-
and B0.5-kb constructs possessed the critical elements
necessary for maximal promoter activity of the �1 and �1 sGC
promoters, respectively, and were further analyzed.
TABLE 1
The positions are according to the transcription start site (�1)
Repeat No. of elements Begin End Length Percentage of sequence
bp
Human �1 sGC promoter repeats according to RepeatMasker
a
LTR (MaLRs) 1 �3038 �2853 185 6.1
DNA element (MER1_type) 1 �2188 �2031 158 5.2
Total interspersed repeats 343 11.2
Low complexity 1 �80 �42 39 1.3
Human �1 sGC promoter repeats according to RepeatMasker
b
LINE1 1 �2429 �1671 759 22.7
Total interspersed repeats 759 22.7
a Total length, 3048 bp; GC level, 36.5%; based masked, 382 bp (12.5%).
b Total length, 3344 bp; GC level, 40.0%; based masked, 759 bp (22.7%).
Soluble Guanylyl Cyclase Promoter Regulation
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Interestingly, the A0.3-kb se-
quence exhibited only 44% homol-
ogy with the mouse and rat
sequence homologues and the
B0.5-kb fragment possessed a 57%
homology when compared with the
rat sequence. As a consequence,
there is likely to be disparate regula-
tion of sGC expression (i.e. pro-
moter activity) between species,
highlighting the importance of
characterizing the activity of the
human promoter in human cells.
Identification of Critical Tran-
scription Binding Sites—Analysis of
putative transcription factor-bind-
ing sites in the fragments containing
the maximal promoter activity for
the human�1 and�1 sGC genes was
performed usingMatInspector soft-
ware (solution parameters: core
similarity, 1.0; matrix similarity
optimized) (supplemental Figs. S4A
and 5A) and confirmed by Tran-
scription Element Search Software.
In both A0.3-kb and B0.5-kb con-
structs, putative TF-binding sites
for several transcription factors
located at the optimal regulatory
distance from the transcription
start sitewere investigated. To func-
tionally determine the importance of these TFs for �1 and �1
sGC promoter activity, the cores of each TF-binding site were
deleted individually in both the A0.3-kb and B0.5-kb basal con-
structs via site-directed mutagenesis. These deletions did not
introduce any newbinding sites as confirmed by sequence anal-
ysis with MatInspector software. Transcriptional activity of
these mutant constructs was assessed in HASMCs and COS-7
cells; TF-binding sites that had a significant effect on sGC pro-
moter activity inHASMCs are shown in supplemental Figs. S4B
(�1 sGC) and S5B (�1 sGC).
A0.3-kb fragments harboring deletions for CCAAT-BF-,
c-Myb-, GAGA-, and NFAT-binding sites showed significant
increases in promoter activity compared with A0.3-kb control
in HASMCs (Fig. 2A), suggesting that binding of these TFs to
their recognition sites has repressor effects on �1 sGC pro-
moter activity in these cells. The effect of c-Myb was abolished
in COS-7 cells, suggesting a HASMC-specific effect (Fig. 2B).
Moreover, the effects of GAGAandNFATwere also reduced in
COS-7 cells (Fig. 2B), despite still having significant repressor
activity. However, CCAAT-BF had a similar effect in COS-7
cells, suggesting that this TF may belong to the basal transcrip-
tion machinery for �1 sGC gene in many cell types.
B0.5-kb fragments harboring a deletion for an NF-�B(p50)-
binding site showed almost 2-fold increase in promoter activity
compared with B0.5kb control in HASMCs (Fig. 2C). This
effect was identical in COS-7 (Fig. 2D), suggesting that
NF-�B(p50) plays a key repressor role in the basal expression of
FIGURE 1.Promoter activity of human�1 (A) and�1 sGC (B) deletion constructs inHASMCs. The cells were
transfected with luciferase reporter constructs containing different size promoter fragments (indicated in bp
relative to the transcription start site (�1)). Promoter activity of each construct wasmeasured as firefly lucifer-
ase activity normalized to Renilla luciferase, and the results are expressed as relative luciferase light units (RU).
The results are themeans� S.E. (n� 12). The hatched bars indicate the promoter constructs with the greatest
activity. pGL3 depicts the promoter-less vector.
FIGURE 2.Analysis of putative TFs at the proximal promoter sequence of
thehumansGCgeneunderbasal conditions inHASMC (AandC) andCOS-7
(B and D) cells. A and B, effects of core deletions of c-Myb, GAGA, NFAT, and
CCAAT-BF sites on promoter activity of the A0.3-kb construct. C andD, effects of
core deletions of NF-�B(p50)-, PU1-, SP1-, and NFY-binding sites on promoter
activity of the B0.5-kb construct. NFY-1 and NFY-2 indicate the upstream and
downstream CCAAT-BF sites with NFY-1&2 representing the double deletion.
Promoter activity wasmeasured as described in Fig. 1. The results are expressed
as themeans� S.E. (n� 5). *, p� 0.05.
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�1 sGC, independent of cell type. In
contrast, a B0.5-kb fragment har-
boring deletions for both NFY-
binding sites showed a small reduc-
tion in B0.5-kb promoter activity in
HASMCs, suggesting a slight activa-
tor effect of this TF (Fig. 2C). The
activator effect of NFY was much
stronger in COS-7 (Fig. 2D) and in a
BE2 human neuroblastoma cell line
(34), suggesting that a cell-specific
effect of NFY is not so apparent in
HASMC. PU1 and SP1 TFs showed
activator and repressor effects,
respectively, in COS-7 cells but not
in HASMCs, again suggesting cell-
specific regulation of sGC promoter
activity.
Effect of Pro-inflammatory Con-
ditions on sGC Promoter Activity in
HASMCs—To examine whether
vascular inflammation modulates
sGC expression in HASMCs,
human �1 and �1 sGC promoter
activity was examined in the pres-
ence of a pro-inflammatory mixture
using the A0.3-kb (Fig. 3A) and
B0.5-kB (Fig. 3B) constructs. Pro-
inflammatory conditions signifi-
cantly reduced both the �1 and �1
sGC promoter activity at 24 h.
Moreover, Western blot analysis
revealed a clear, time-dependent
decrease in sGC �1 expression fol-
lowing exposure of the HASMC to
the pro-inflammatory mixture
(Fig. 3C) that was mirrored by a
significant reduction in both the
basal and NO-stimulated sGC
activity (Fig. 3D).
As shown in Fig. 2, CCAAT-BF
and NF-�B exerted the most pro-
nounced effect on �1 and �1 sGC
promoter activity, respectively,
among the TFs studied. To deter-
mine whether these transcription
factors bind to the target DNA
sequences on the promoter, nuclear
extracts from HASMCs were ana-
lyzed by EMSA. In terms of the �1
sGC promoter, three complexes
were detected using the oligonu-
cleotide probe containing �93 to
�66 bp region (CCAAT-BF oligo-
nucleotide) upstream of the tran-
scriptional start site (Fig. 4A). The
specificity of these complexes was
demonstrated by a reduction of the
FIGURE 3. Temporal effects of a pro-inflammatory mixture on A0.3-kb (A) and B0.5-kb (B) promoter activity in
HASMCs. Black bars, basal conditions; empty bars, pro-inflammatory conditions. Promoter activity was meas-
ured as described in the legend to Fig. 1. The results are expressed as the means� S.E. (n � 3). *, p� 0.05. C,
expression of sGC �1 protein determined by Western blot in HASMC under basal and pro-inflammatory con-
ditions (0–48hof incubationwithmixture). Thedata are representativeof at least three similar experiments.D,
basal and NO-stimulated increases in cGMP in HASMC in the absence and presence of pro-inflammatory
mixture (24 h; n 40). *, p � 0.05 versus the corresponding response in the absence of pro-inflammatory
mixture.
FIGURE 4. Electrophoretic mobility shift assay (A and B) with densitometric quantification (C and D) of HASMC
nuclear extracts under basal and pro-inflammatory conditions (2h of incubation with the mixture) was per-
formedusing labeled primers carrying theNF-�Bor CCAAT-BF binding sites or themutated version (
NF-�Bor

CCAAT-BF). For competition experiments, a 100� excess of unlabeled double-stranded oligonucleotide
(cold probe) was added to the binding reaction. A supershift at the NF-�B site was demonstrable using a p50,
but not p65, antibody (E). The results are expressed as the means� S.E. (n� 3). *, p� 0.05.
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intensity of all three bands in the presence of an excess of unla-
beled probe. When the nuclear extract derived from HASMCs
treated with the pro-inflammatory mixture was analyzed, an
increase in the intensity of twoof these complexeswas observed
(Fig. 4C), suggesting that vascular inflammation increased the
binding of CCAAT-BF to the proximal �1 sGC promoter. The
deletion of a single c (g) of the core of the CCAAT-BF site
(position�82; supplemental Fig. S4) abolished the formation of
two of the three complexes, highlighting the importance of that
single base for the binding of the CCAAT-BF to the target
promoter.
For the �1 sGC promoter, interactions within the
NF-�B(p50)-binding region were also assessed using a NF-�B
oligonucleotide encompassing�138 to�109 bp region of this
promoter (supplemental Fig. S5B). Three complexes were
formed after incubation of the NF-�B probe with HASMC
nuclear extracts. These complexes were no longer formed in
the presence of an excess of unlabeled probe, confirming spec-
ificity (Fig. 4B). Interestingly, a supershift of the complexes was
only observed when we used an anti-p50 antibody but not anti-
p65 (Fig. 4E), suggesting that the smaller NF-�B subunit (per-
haps as a p50 homodimer) is pivotal in regulating sGC �1
expression; this finding dovetails well with the observed
increase in sGC �1 promoter activity in the construct with
deleted p50 consensus site(s) and with previous reports detail-
ing a predominantly repressor activity of p50 homodimers (38,
39). When the nuclear extract derived from HASMCs treated
with the pro-inflammatory mixture was analyzed, an increase
in the intensity of the complex bands was observed (Fig. 4D),
suggesting that vascular inflammation increased the binding of
the NF-�B(p50) factor to the proximal �1 sGC promoter. The
deletion of the 4-bp core (gggg) for the NF-�B factor-binding
site disrupted the formation of two complexes and reduced the
formation of the third one, indicating that the gggg motif is
necessary for its DNA interaction.
Furthermore, an important role for
NF-�B in regulating the expres-
sion of the �1 subunit was sup-
ported by a parallel reduction in
enzyme protein expression and
activity (Fig. 3, C and D).
Effect of NO on sGC Promoter
Activity in HASMCs—To assess the
effect of the sGC ligand NO on
expression of the enzyme in
HASMCs (to establish the exist-
ence of a feedback loop in terms of
enzyme expression, which is well
established to occur biochemically
(30, 40)), human �1 and �1 sGC
promoter activity was examined in
the presence of the NO donor
DETA-NONOate (releases NO
spontaneously in aqueous solution
(41)). Pilot studies revealed that
for the �1 subunit, the promoter
activity was inhibited to the great-
est extent in the A0.3 construct,
suggesting that key NO-dependent repressors are located in
this region of the promoter. For �1 sGC, maximal inhibition
was observed in the B0.5 construct, suggesting that this area
of the promoter contains the critical NO-responsive repres-
sor element(s). Subsequent studies were therefore con-
ducted using these constructs. Fig. 5 shows that NO exerted
a concentration-dependent inhibition of both �1 and �1 sGC
promoter activity. This effect was maximal at 200 �M DETA-
NO, which results in an approximate ambient NO concen-
tration of 200 nM (42). Moreover, the inhibition of promoter
activity by NO followed a time course very similar to that
observed under pro-inflammatory conditions with maximal
effect observed at 24–48 h (Fig. 5).
To determine whether the repressor activity of NO was
cGMP-dependent or independent, further experiments were
conducted using the A0.3 and B0.5 constructs as models of
promoter function. Here, theNO-dependent inhibition of both
the�1 and �1 sGC promoter was not reversed in the presence
of the sGC blocker ODQ (5 �M) nor mimicked by the cGMP
analogue 8-Br-cGMP (300�M; Fig. 6,A andB). Thus, it appears
that the inhibitory effect of NO on sGC promoter activity and
expression ismediated directly, rather than via activation of the
enzyme (and cGMP production).
In an attempt to identify putative transcription factors that
might mediate the repressor effects of NO, A0.3, and B0.5 con-
structs with targeted mutations in specific TF-binding sites
were analyzed for their responsiveness toNO. Interestingly, the
activity of theA0.3 fragmentwith a deletedCCAAT-BF sitewas
no longer sensitive to inhibition by DETA-NO (Fig. 6C), inti-
mating that this response element is involved in mediating the
negative feedback effect of NO on sGC expression. In contrast,
deletion of the same site in the B0.5 construct did not alter
promoter activity in the presence of NO.
FIGURE 5. Concentration- (A and B) and time- (C and D) dependent effects of the NO donor DETA-NO
(0–200 �M and 0–48-h incubation) on A0.3 (A and C) and B0.5 (B and D) promoter activity in HASMCs.
Promoter activity wasmeasured as described in the legend to Fig. 1. The results are expressed as themeans�
S.E. (n� 3). *, p� 0.05 versus response in the absence of DETA-NO.
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Finally, we confirmed the importance of NO in inhibiting
sGC promoter activity via the CCAAT-BF site, by EMSA. In
accord with the luciferase assay data, DETA-NO (100 �M)
caused an increase in the intensity of the three CCAAT-BF
complexes observed that was essentially absent when using
probes with a dysfunctional CCAAT-BF site (Fig. 7). However,
despite attempting to produce a supershift in the CCAAT-BF
complexes observed with specific
antibodies to two putative CCAAT-
BFs (e.g. CBF-B, MAZ), we were
unable to observe such a change
(data not shown), suggesting that
additional CCAAT-BFs are likely
involved.
DISCUSSION
Activation of sGC is essential for
NO-mediated regulation of vascular
smooth muscle reactivity (5) and
proliferation (6), microvascular per-
meability (7, 8), and the reactivity/
adherence of platelets (9) and leuko-
cytes (8, 11). As such, under
physiological circumstances activa-
tion of sGC is fundamental to car-
diovascular homeostasis and main-
tains an important cytoprotective/
anti-thrombotic influence.Moreover,
because inappropriate sGC activity
may be responsible for several fea-
tures of cardiovascular pathologies
including blood pressure dysregula-
tion (i.e. smooth muscle reactivity),
capillary edema, and cellular
recruitment, dysfunction of sGC is
likely to have at least an equivalent
impact on (patho)physiology as
inappropriate NO production;
changes in the expressional regula-
tion (and activity) of this enzyme are
therefore likely to exert consider-
able influence on the progression of
disease. Despite this obvious impor-
tance, there exists scant informa-
tion concerning the physiological
regulation and pathological alter-
ations of sGC expression. To
address this deficit, we have charac-
terized the human �1 and �1 sGC
promoter regions in human aortic
smooth muscle cells.
Analysis of the human �1 and �1
sGC gene promoters revealed some
intriguing aspects of transcriptional
regulation of these genes and differ-
ences in the promoter consensus
core elements. Both sGC genes have
a TATA-less promoter and show
CCAAT-BF sites in direct (�1 sGC) or reverse (�1 sGC) orien-
tation �80 bp upstream from the putative transcription start
site, in agreement with the canonical position for eukaryotic
promoters (43). Classically, the following three-way combina-
tions are highly favorable in human promoter regions to facili-
tate gene transcription: BRE-Inr-MTE and/or TATA-Inr-MTE
(37); yet, neither combination was detected in either the �1 or
FIGURE 6. Effect of the sGC inhibitor ODQ (5 �M) and cGMP analogue 8Br-cGMP (300 �M) and core
deletions of CCAAT-BF and NFY on NO-mediated (DETA-NO, 200 �M; 24 h of incubation) inhibition of
A0.3 (AandC) andB0.5 (BandD) promoteractivity inHASMC.Promoter activitywasmeasuredasdescribed
in the legend toFig. 1. The results are expressedas themeans�S.E. (n�3). *,p�0.05 versus respective control.
#, p� 0.05 versus A0.3� DETA. In C and D, promoter activity in the absence of DETA is normalized to 100%.
FIGURE 7. Electrophoretic mobility shift assay (A) with densitometric quantification (B) of HASMC
nuclear extracts under basal conditions and in the presence of 100 �M DETA-NO (2-h incubation) was
performed using labeled primers carrying the CCAAT-BF site or its mutated version (�CCAAT-BF). For
competition experiments, a 100� excess of unlabeled doubled-stranded oligonucleotide (cold probe) was
added to the binding reaction. The results are expressed as the means� S.E. (n� 3). *, p� 0.05.
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�1 sGC promoter. However, because the �1 sGC promoter
exhibits two potential initiation modules, an alternative tran-
scription start site 51 bp downstream given by the Inr-DPE
module (and characteristic of TATA-less promoters) (44) can-
not be ruled out. Surprisingly, an Inr element is not present in
the human �1 sGC promoter.
Functional analysis of constructs containing different size
fragments of the �1 and �1 sGC 5�-flanking regions showed a
slightly different profile of activity between both subunit pro-
moters in HASMCs. Despite �1 sGC showing the highest pro-
moter activity in the A0.3-kb construct, suggesting repressor
elements localized further upstream, the situationwas different
for �1 sGC. Here, the B0.5-kb construct showed the highest
promoter activity, and the presence of several enhancers and
repressors functioning at a distance from this regulatory region
was detected. The fact that B0.5-kb activity was comparable
with B0.8kb activity in HASMCs but has been shown to be over
3-fold lower than B0.8 kb in BE2 cells (34), suggests a cell-
specific regulation of the crucial promoter region necessary to
achieve the maximum �1 sGC promoter activity in HASMCs.
This cell-specific effect is also highlighted by the fact that
B0.4-kb activity is almost the same as B0.5 kb and 2-fold higher
than B0.3 kb in HASMCs, intimating strong activator elements
located in the �253 to �354 region, not observed in BE2 cells
(34). Thus, A0.3-kb and B0.5-kb constructs demonstrated the
highest promoter activity in HASMCs, indicating that the reg-
ulatory promoter of sGC has all the necessary elements for
maximum promoter activity in the 300–500-bp region
upstream of the transcription start site.
We also focused on the identification of principal TFs
responsible for the transcriptional regulation of �1 and �1 sGC
in HASMCs. A multiplicity of putative transcription factor-
binding sites predicted by MatInspector software are clustered
in both A0.3-kb (�1 sGC) and B0.5-kb (�1 sGC) promoter frag-
ments, all of which have been shown to play an important role
in the transcription of awide variety of eukaryotic genes.GATA
(GATA-binding factor, typical of the cardiovascular system),
GFI1 (growth factor independence I; typical of the hematopoi-
etic system),NFY (ubiquitous), and PBX (homeodomain factor;
typical of the hematopoietic system) have consensus sequences
in both promoters, and some of these factors may have a regu-
latory role in the co-expression of both �1 and �1 sGC genes;
this is particularly pertinent for the GFI1 (both �1 and �1 sGC)
and PBX (�1 sGC) sites, which are positioned between the
CCAAT-BF site and the transcription start site and therefore
are likely to play key roles in expressional regulation of sGC in
hematopoiesis. Indeed, the �1 and �1 sGC promoter regions
contain no less than 20 consensus sequences for TFs reported
to play a role in regulating hematopoiesis, supporting a key role
for sGC in this process (34).
Functional analysis with a luciferase �1 sGC reporter con-
struct containing a c-Myb, GAGA, NFAT, or CCAAT-BF core
deletion in the A0.3-kb promoter fragment demonstrated that
the integrity of these sites is very important for transcription
repression in HASMCs. The c-Myb and to a lesser extent the
GAGA and NFAT repressor effects were not observed in
COS-7 cells, intimating a vascular smooth muscle cell-specific
effect. In fact, c-Myb plays a crucial role in arterial smooth
muscle cell proliferation (associated with G1/S cell cycle tran-
sition), as occurs in many vasculopathies (45) and NFAT is
associated with cardiac morphogenesis, vasculogenesis, and
vascular smooth muscle hypertrophy (46); the physiological
importance of NO-sGC signaling in curbing these proliferative
processes fits with the ability of such TFs to suppress sGC pro-
moter activity and thereby promote pathogenesis. However, it
is noteworthy that when two other NFAT sites located at�174
and �161 in �1 sGC were mutated, they did not significantly
alter �1 sGC promoter activity.
Functional analysis with a luciferase �1 sGC reporter con-
struct containing a NFY core deletion in the B0.5-kb promoter
fragment revealed that this site is critical for transcription acti-
vation inHASMCs, although this effect was less evident than in
COS-7 cells (this study) and BE2 cells (34). Similar analysis also
showed that the integrity of an NF-�B(p50) site is very impor-
tant for transcription repression control, in both HASMCs and
COS-7 cells, suggesting that this TF is globally important in
constitutive gene expression.
To corroborate the importance of these TFs in regulation of
sGC promoter activity, we conducted EMSAs to confirm the
interaction of CCAAT-BF and NF-�B with the sGC promoter
region. It is clear from these data that both CCAAT-BF and
NF-�B(p50) bind strongly to the �1 and �1 sGC promoter con-
structs, respectively, and that the effects of these TFs on sGC
promoter activity are specific. A CCAAT-BF site has been
found to be prevalent in the promoters of cell cycle-regulated
eukaryotic genes (47) and is known to be essential for cell cycle-
dependent activation and repression of several mammalian
genes (48–50). In addition to the NFY site reported by Sharina
et al. (34), a second upstream site was also shown to have an
important role, highlighting the role NFY plays in �1 sGC tran-
scription. Moreover, the presence of the two closely situated
NFY sites in the immediate promoter is in agreement with the
idea that expression of �1 sGC gene could be regulated during
the cell cycle (34).
Many cardiovascular diseases, particularly atherosclerosis,
are now recognized to constitute a chronic inflammation of the
blood vessel wall. Because NO-sGC-cGMP signaling exerts an
important vasoprotective, anti-atherogenic effect, elucidation
of the expressional regulation of sGC in a pro-inflammatory
environment is likely to provide further insight into the patho-
genesis of vascular disease. To mimic such a pathological sce-
nario in the vasculature, we examined the effect of pro-inflam-
matory conditions on sGC promoter activity in HASMCs.
A0.3-kb and B0.5-kb fragments were analyzed in the presence
of a pro-inflammatory mixture. Under such conditions, sGC
A0.3-kb and B0.5-kb promoter activity at 24 h was significantly
reduced, demonstrating a coordinated regulation of both �1
and �1 sGC gene expression. The fact that the strongest repres-
sors for �1 ad �1 sGC (CCAAT-BF and NF-�B, respectively)
exhibit enhanced DNA binding under pro-inflammatory con-
ditionsmight explain, at least in part, the reduction in�1 and�1
sGC promoter activity observed under this “pathogenic” envi-
ronment. Moreover, analysis of protein expression confirmed
that the decreased promoter activity in the presence of pro-
inflammatorymixture wasmirrored by reductions in both sGC
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�1 protein expression and activity, providing a key link between
NF-�B and regulation of sGC bioactivity.
We also examined the effect of NO per se to mediate a feed-
back loop and prevent sGC promoter activity/expression.
Using the NO donor DETA-NO, we demonstrated that NO
exerts a concentration-dependent reduction in �1 and �1 sGC
promoter activity. In these studies, the inhibitory effect of NO
peaked at 200 �M DETA-NO, which results in an ambient NO
concentration of �200 nM (42), more akin to those associated
with inducible NO synthase activity and pathological condi-
tions (rather than generated by constitutive NO synthase).
Moreover, the effects of NO in this respect appear to be cGMP-
independent, implying direct effects on TF activity (as has been
shown for NF-�B (51) and HIF-1� (52)). These studies also
revealed that a CCAAT-BF site is pivotal to the inhibitory
effects of NO, at least in terms of the �1 subunit, because dele-
tion of this site leads to insensitivity toNO. Because several TFs
bind this site, it is difficult to pinpoint which might be respon-
sible; despite attempts to use EMSA-based supershifts to derive
information concerning the identity of this TF, we were unable
to elucidate the mechanism further (although we did obtain
negative results for CBF-B and MAZ, suggesting that it is not
either of these factors).
The inflammation- and NO-based reduction in sGC pro-
moter activity and protein expression might be likely to repre-
sent a common negative feedback regulation of sGC expression
in cardiovascular disorders associated with high output NO
production, such as septic shock. Here, it would be appropriate
for sGC expression to be down-regulated to offset the excessive
NO levels and thereby reverse the associated, life-threatening
hypotension. These data also suggest that the reduction in sGC
mRNA levels reported in vascular smooth muscle cells in
response to LPS/interleukin-1� (27), tumor necrosis factor-�/
LPS (53), and NO donors (54, 55) is likely to be due, at least in
part, to a direct down-regulation in sGC promoter activity via
NF-�B(p50)- and CCAAT-BF-dependent pathways.
In sum, these data provide a systematic, comparative analysis
of human sGC promoter regulation in HASMCs and has iden-
tified potentially important factors regulating human sGC
expressionwithin a cell system relevant to cardiovascular phys-
iology and (patho)physiology. These observations suggest that
expressional regulation of human sGC �1 and �1 subunit
expression, both coordinated and individually, is likely to play
an important role in the (patho)physiological regulation of
enzyme activity. As such, these findings represent a signifi-
cant advance in the understanding of expressional regula-
tion of the genes encoding sGC subunits and provide insight
into potential pathogenic mechanisms that result in aberrant
NO-sGC signaling.
REFERENCES
1. Hobbs, A. J., and Ignarro, L. J. (1996) Methods Enzymol. 269, 134–148
2. Murad, F. (1994) Adv. Pharmacol. 26, 19–33
3. Davis, K. L., Martin, E., Turko, I. V., and Murad, F. (2001) Annu. Rev.
Pharmacol. Toxicol. 41, 203–236
4. Hobbs, A. J. (1997) Trends Pharmacol. Sci. 18, 484–491
5. Rapoport, R. M., and Murad, F. (1983) J. Cyclic. Nucleotide. Protein Phos-
phor. Res. 9, 281–296
6. Garg, U. C., and Hassid, A. (1989) J. Clin. Investig. 83, 1774–1777
7. Kubes, P. (1993) Am. J. Physiol. 265, H1909–H1915
8. Kurose, I., Kubes, P., Wolf, R., Anderson, D. C., Paulson, J., Miyasaka, M.,
and Granger, D. N. (1993) Circ. Res. 73, 164–171
9. Radomski, M. W., Palmer, R. M., and Moncada, S. (1987) Biochem. Bio-
phys. Res. Commun. 148, 1482–1489
10. Hobbs, A. J., and Moncada, S. (2003) Vascul. Pharmacol. 40, 149–154
11. Niu, X. F., Ibbotson, G., and Kubes, P. (1996) Circ. Res. 79, 992–999
12. Ahluwalia, A., Foster, P., Scotland, R. S., McLean, P. G., Mathur, A., Per-
retti,M.,Moncada, S., andHobbs, A. J. (2004)Proc. Natl. Acad. Sci. U. S. A.
101, 1386–1391
13. Kamisaki, Y., Saheki, S., Nakane,M., Palmieri, J. A., Kuno, T., Chang, B. Y.,
Waldman, S. A., and Murad, F. (1986) J. Biol. Chem. 261, 7236–7241
14. Andreopoulos, S., and Papapetropoulos, A. (2000) Gen. Pharmacol. 34,
147–157
15. Zabel, U., Weeger, M., La, M., and Schmidt, H. H. (1998) Biochem. J. 335,
51–57
16. Azam, M., Gupta, G., Chen, W., Wellington, S., Warburton, D., and Dan-
ziger, R. S. (1998) Hypertension 32, 149–154
17. Sharina, I. G., Krumenacker, J. S., Martin, E., and Murad, F. (2000) Proc.
Natl. Acad. Sci. U. S. A. 97, 10878–10883
18. Giuili, G., Roechel, N., Scholl, U., Mattei, M. G., and Guellaen, G. (1993)
Hum. Genet. 91, 257–260
19. Budworth, J., Meillerais, S., Charles, I., and Powell, K. (1999) Biochem.
Biophys. Res. Commun. 263, 696–701
20. Ritter, D., Taylor, J. F., Hoffmann, J. W., Carnaghi, L., Giddings, S. J.,
Zakeri, H., and Kwok, P. Y. (2000) Biochem. J. 346, 811–816
21. Ruetten, H., Zabel, U., Linz, W., and Schmidt, H. H. (1999) Circ. Res. 85,
534–541
22. Kloss, S., Bouloumie, A., and Mulsch, A. (2000) Hypertension 35, 43–47
23. Tzao, C., Nickerson, P. A., Russell, J. A., Gugino, S. F., and Steinhorn, R. H.
(2001) Pediatr. Pulmonol. 31, 97–105
24. Wedgwood, S., Steinhorn, R. H., Bunderson, M., Wilham, J., Lakshmin-
rusimha, S., Brennan, L. A., and Black, S. M. (2005) Am. J. Physiol. 289,
L660–L666
25. Hassoun, P. M., Filippov, G., Fogel, M., Donaldson, C., Kayyali, U. S.,
Shimoda, L. A., and Bloch, K. D. (2004) Am. J. Respir. Cell Mol. Biol. 30,
908–913
26. Chen, L., Daum, G., Fischer, J. W., Hawkins, S., Bochaton-Piallat, M. L.,
Gabbiani, G., and Clowes, A. W. (2000) Circ. Res. 86, 520–525
27. Papapetropoulos, A., Abou-Mohamed, G., Marczin, N., Murad, F., Cald-
well, R. W., and Catravas, J. D. (1996) Br. J. Pharmacol. 118, 1359–1366
28. Melichar, V. O., Behr-Roussel, D., Zabel, U., Uttenthal, L. O., Rodrigo, J.,
Rupin, A., Verbeuren, T. J., Kumar, H. S. A., and Schmidt, H. H. (2004)
Proc. Natl. Acad. Sci. U. S. A. 101, 16671–16676
29. Brandes, R. P., Schmitz-Winnenthal, F. H., Feletou, M., Godecke, A.,
Huang, P. L., Vanhoutte, P.M., Fleming, I., and Busse, R. (2000) Proc. Natl.
Acad. Sci. U. S. A. 97, 9747–9752
30. Sabrane, K., Gambaryan, S., Brandes, R. P., Holtwick, R., Voss, M., and
Kuhn, M. (2003) J. Biol. Chem. 278, 17963–17968
31. Bauersachs, J., Bouloumie,A., Fraccarollo,D.,Hu,K., Busse, R., andErtl, G.
(1999) Circulation 100, 292–298
32. Vazquez-Padron, R. I., Pham, S. M., Pang, M., Li, S., and Aitouche, A.
(2004) Biochem. Biophys. Res. Commun. 314, 208–214
33. Yamamoto, T., and Suzuki, N. (2002) Biochem. J. 361, 337–345
34. Sharina, I. G., Martin, E., Thomas, A., Uray, K. L., and Murad, F. (2003)
Proc. Natl. Acad. Sci. U. S. A. 100, 11523–11528
35. Connelly, L., Palacios-Callender,M., Ameixa, C.,Moncada, S., andHobbs,
A. J. (2001) J. Immunol. 166, 3873–3881
36. Quandt, K., Frech, K., Karas, H., Wingender, E., and Werner, T. (1995)
Nucleic Acids Res. 23, 4878–4884
37. Jin, V. X., Singer, G. A., gosto-Perez, F. J., Liyanarachchi, S., and Davuluri,
R. V. (2006) BMC Bioinformatics 7, 114
38. Tong, X., Yin, L., Washington, R., Rosenberg, D. W., and Giardina, C.
(2004) Mol. Cell Biochem. 265, 171–183
39. Guan,H., Hou, S., andRicciardi, R. P. (2005) J. Biol. Chem. 280, 9957–9962
40. Madhani, M., Okorie, M., Hobbs, A. J., and MacAllister, R. J. (2006) Br. J.
Pharmacol. 149, 797–801
41. Morley, D., and Keefer, L. K. (1993) J. Cardiovasc. Pharmacol. 22, S3–S9
Soluble Guanylyl Cyclase Promoter Regulation
JULY 18, 2008•VOLUME 283•NUMBER 29 JOURNAL OF BIOLOGICAL CHEMISTRY 20035
by on Septem
ber 5, 2008
w
w
w
.jbc.org
D
ow
nloaded from

42. Palacios-Callender, M., Hollis, V., Mitchison, M., Frakich, N., Unitt, D.,
and Moncada, S. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 18508–18513
43. Breathnach, R., andChambon, P. (1981)Annu. Rev. Biochem. 50, 349–383
44. Smale, S. T. (1997) Biochim. Biophys. Acta 1351, 73–88
45. You, X. M., Mungrue, I. N., Kalair, W., Afroze, T., Ravi, B., Sadi, A. M.,
Gros, R., and Husain, M. (2003) Circ. Res. 92, 314–321
46. Hill-Eubanks, D. C., Gomez, M. F., Stevenson, A. S., and Nelson, M. T.
(2003) Trends Cardiovasc. Med. 13, 56–62
47. Mantovani, R. (1999) Gene (Amst.) 239, 15–27
48. Wang, Z. M., Liu, C., and Dziarski, R. (2000) J. Biol. Chem. 275,
20260–20267
49. Hu, Q., Bhattacharya, C., and Maity, S. N. (2002) J. Biol. Chem. 277,
37191–37200
50. Salsi, V., Caretti, G., Wasner, M., Reinhard, W., Haugwitz, U., Engeland,
K., and Mantovani, R. (2003) J. Biol. Chem. 278, 6642–6650
51. Matthews, J. R., Botting, C. H., Panico, M., Morris, H. R., and Hay, R. T.
(1996) Nucleic Acids Res. 24, 2236–2242
52. Palmer, L. A., Gaston, B., and Johns, R. A. (2000) Mol. Pharmacol. 58,
1197–1203
53. Takata, M., Filippov, G., Liu, H., Ichinose, F., Janssens, S., Bloch, D. B., and
Bloch, K. D. (2001) Am. J. Physiol. 280, L272–L278
54. Filippov, G., Bloch, D. B., and Bloch, K. D. (1997) J. Clin. Investig. 100,
942–948
55. Scott, W. S., and Nakayama, D. K. (1998) J. Surg. Res. 79, 66–70
Soluble Guanylyl Cyclase Promoter Regulation
20036 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283•NUMBER 29•JULY 18, 2008
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ow
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