From INSERM U-533, Faculté des Sciences, 44322 Nantes Cedex 3, France
Received for publication, December 16, 2002, and in revised form, January 9, 2003
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ABSTRACT |
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The small G protein RhoA is a convergence point
for multiple signals that regulate smooth muscle cell functions. NO
plays a major role in the structure and function of the normal adult vessel wall, mainly through modulation of gene transcription. This
study was thus performed to analyze in vitro and in
vivo the effect of NO signaling on RhoA expression in arterial
smooth muscle cells. In rat or human artery smooth muscle cells, sodium nitroprusside or 8-(2-chlorophenylthio)-cGMP induced a rise in RhoA mRNA and protein expression, which was inhibited by the
cGMP-dependent protein kinase (PKG) inhibitor
(Rp)-8-bromo- Small G proteins of the Rho family function as tightly regulated
molecular switches that govern a wide range of cell functions (1). A
large body of evidence has now been obtained regarding the important
functions of Rho proteins in the vasculature, and RhoA has been shown
to play a major role in vascular processes such as smooth muscle cell
contraction, proliferation, and differentiation; endothelial
permeability; platelet activation; and leukocyte migration (2-4). The
activity of Rho is under the direct control of a large set of other
regulatory proteins (1). In the inactive GDP-bound form, RhoA is locked
in the cytosol by guanine dissociation inhibitors. The guanine
nucleotide exchange factors catalyze the exchange of GDP for GTP to
activate RhoA (5). Activation is then turned off by GTPase-activating
proteins that hydrolyze GTP to GDP. Therefore, both the relative
expression of these proteins (in particular, that of RhoA) and the
fraction of active GTP-bound RhoA are key determinants of RhoA protein activity.
Data are now accumulating regarding the regulation of the amount of
active GTP-bound RhoA. In vascular smooth muscle cells, several
agonists of G protein-coupled receptors, including thrombin, thromboxane A2, endothelin, carbachol, angiotensin,
Modulation of RhoA expression has been less extensively described;
however, a few recent studies reported that the RhoA mRNA level or
expression of the RhoA protein is increased in arteries from
hypertensive (15), aged (16), and diabetic (17) rats and in
atherosclerotic lesions (18). The functional consequences of these
changes in RhoA expression have been suggested by other observations
showing that RhoA-dependent pathways are involved in
excessive contraction, migration, and proliferation associated with
arterial diseases such as hypertension and atherosclerosis (4).
However, to our knowledge, the mechanisms of regulation of RhoA
expression still have not been investigated.
In the normal adult vessel wall, vascular smooth muscle cells are
continuously subjected to the action of the basal release of NO from
endothelial cells, regulating arterial tone, but also smooth muscle
cell gene expression (19). Here we analyzed the effect of the
NO/cGMP/PKG signaling pathway on RhoA expression in arterial smooth
muscle cells. We show that long-term stimulation of PKG positively
controlled RhoA expression both in vitro and in
vivo through an increase in RhoA protein stability and stimulation of rhoA gene transcription. Our data suggest that the tonic
release of NO is absolutely necessary to maintain RhoA expression in
vascular smooth muscle. This mechanism might thus provide an important regulation level of RhoA functions in vascular smooth muscle cells.
Chemicals and Drugs--
Mouse monoclonal anti-RhoA antibody
(26C4) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA). The plasmid encoding PKG I Smooth Muscle Cell Culture--
Rat aortic and human internal
mammary artery smooth muscle cells were isolated by enzymatic
dissociation as previously described (20). Cells were cultured in
Dulbecco's modified Eagle's medium with 10% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Secondary cultures
were obtained by serial passages after the cells were harvested with
0.5 g/liter trypsin and 0.2 g/liter EDTA and reseeded in fresh
Dulbecco's modified Eagle's medium containing 10% fetal calf serum
and antibiotics. Only smooth muscle cells at passage 2 were used in
this study. Stimulation of the NO/PKG pathway with sodium nitroprusside
(SNP; 10 µM) or 8-pCPT-cGMP (50 µM) was
performed in the absence of serum in the culture medium.
Western Blot Analysis--
Endothelium-denuded aortas or cells
were rapidly frozen in liquid nitrogen and homogenized in lysis buffer
containing 20 mM Hepes-NaOH, 10 mM KCl, 10 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, and Complete (one tablet/50 ml; Roche
Molecular Biochemicals). Nuclei and unlysed cells were removed by low
speed centrifugation at 10,000 × g for 15 min at
4 °C. The protein concentration of the supernatant was measured and
adjusted; Laemmli sample buffer was added; and equal amounts of protein
were loaded onto each lane of SDS-12% polyacrylamide gels, which were
then electrophoresed and transferred onto nitrocellulose. The amounts
of proteins were checked by staining with Ponceau red. Before
immunoblotting, the membrane was blocked with 50 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20, and
5% nonfat milk for 1 h at room temperature and then probed with
mouse monoclonal anti-RhoA antibody for 3 h at room temperature.
After three washes, membranes were incubated for 1 h at room
temperature with horseradish peroxidase-conjugated goat anti-mouse
antibody (16 ng/ml). Signals from immunoreactive bands were detected by
ECL and quantified using QuantityOne. Equal loading was checked by
reprobing the membrane with monoclonal anti-
For PKG I expression analysis, lysates were prepared from Swiss 3T3
cells transfected with PKG I Real-time Reverse Transcription-PCR--
Total RNA was extracted
using TRIzol reagent (Invitrogen, Cergy Pontoise, France), and reverse
transcription was performed according to standard techniques.
Quantitative real-time PCR assays were carried out with
sequence-specific primer pairs on the iCycler iQ system (Bio-Rad) using
intercalation of SYBR Green as a fluorescent probe. A SYBR Green kit
(PerkinElmer Life Sciences) was used for real-time monitoring of
amplification. Results were evaluated using iCycler iQ real-time
detection system software (Bio-Rad). The expression of
glyceraldehyde-3-phosphate dehydrogenase mRNA was used to normalize
the expression of RhoA mRNA. The primers used were as follows:
glyceraldehyde-3-phosphate dehydrogenase, 5'-CCATGCCATCACTGCCACT-3'
(forward) and 5'-TGTCATCATACTTGGCAGGTTTC-3' (reverse); and RhoA,
5'-GCAGGTAGAGTTGGCTTTATGG-3' (forward) and 5'-CTTGTGTGCTCATCATT CCGA-3' (reverse).
Northern Blotting--
Total RNA (20 µg) was separated on
formaldehyde-agarose gels and blotted onto nylon membranes
(HybondTM-N+, Amersham Biosciences, Orsay,
France). Membrane filters were hybridized with
[32P]dATP-random-primed cDNA probes prepared from a
partial rhoA cDNA fragment amplified by PCR
(sense primer, 5'-GCAGGTAGAGTTGGCTTTATGG-3'; and antisense primer,
5'-CTTGTGTGCTCATCATTCCGA-3'). Hybridized filters were washed under high
stringency conditions (0.1× SSC, 70 °C) and analyzed by
autoradiography. Equal loading of RNA was confirmed by staining of the
ribosomal RNA with ethidium bromide.
Cloning and Site-directed Mutagenesis of the rhoA
Promoter--
Sequences of 913 and 118 bp upstream of the ATG codon of
the human rhoA gene were cloned into the pCR2.1 vector
(Invitrogen) by PCR using the
5'-GAAGATCTTCCAAATTAACTGGTCTTCCTGTCA-3' sense primer
(
In vitro site-directed mutagenesis of the distal CRE (dCRE),
the proximal CRE (pCRE), and the proximal SRE was carried out according
to the QuikChange site-directed mutagenesis kit instruction manual
(Stratagene, La Jolla, CA) using the following PAGE-purified primers:
p(dCRE) up, 5'-CGGATCATGAAATCACGATCAC-3'; p(dCRE) down, 5'-GTGATCGTGATTTCATGATCCG-3'; p(pCRE) up,
5'-GCCCCATGACTACCAAAGC-3'; p(pCRE) down, 5'-GCTTTGGTAGTCATGGGGC-3';
p(SRE) up, 5'-GCATGTGTTGTCCTATAAGCTACC; and p(SRE) down,
5'-GGTAGCTTATAGGACAACACATGC-3'.
Transfections and Reporter Assays--
HeLa or Swiss 3T3
fibroblasts were plated 24 h before transfection in 24-well plates
to 60-70% confluency the day of transfection. The PKG I Plasmids--
Full-length wild-type RhoA and
RhoAA188 were cloned into the pSG5 vector (Stratagene), and
full-length PKG I Anti-phospho-CREB/ATF-1 Antibody Western
Blotting--
Lysates from PKG I-expressing 3T3 fibroblasts or aortic
smooth muscle cells treated with 8-pCPT-cGMP (50 µM) for
2 h were prepared as described (21), separated by electrophoresis,
and transferred onto nitrocellulose as described above. Membranes were
blocked for 1 h at room temperature and incubated overnight at
4 °C with a 1:1000 dilution of anti-phospho-CREB/ATF-1 antibody and,
after stripping, with rabbit anti-CREB antibodies (Cell Signaling). Immunoreactive proteins were detected as described above using a 1:5000
dilution of horseradish peroxidase-conjugated anti-rabbit secondary
antibody (Cell Signaling).
Electrophoretic Mobility Shift Assays (EMSAs)--
DNA-protein
binding assays were carried out with nuclear extract from 2-h
8-pCPT-cGMP (50 µM)-treated aortic smooth muscle cells
prepared as described previously (22). Synthetic complementary oligonucleotides were 3'-biotinylated using the biotin 3'-end DNA
labeling kit (Pierce) according to the manufacturer's instructions and
annealed for 2 h at room temperature. The sequences of the oligonucleotides used are 5'-GCCCCATGGTTACCAAAGC-3' for the wild-type pCRE, 5'-GCCCCATGACTACCAAAGC-3' for the mutant pCRE, and
5'-CGGATCATGAGGTCACG ATCAC-3' for the dCRE. Binding reactions were
carried out for 20 min at room temperature in the presence of 50 ng/µl poly(dI-dC), 0.05% Nonidet P-40, 5 mM
MgCl2, 10 mM EDTA, and 2.5% glycerol in 1×
binding buffer (LightShiftTM chemiluminescent EMSA kit,
Pierce) using 20 fmol of biotin-end-labeled target DNA and 4 µg of
nuclear extract. Unlabeled target DNA (4 pmol) or 2 µl of
anti-CREB or anti-ATF-1 antibody (TransCRUZ, Santa Cruz
Biotechnology, Inc.) was added per 20 µl of binding reaction where
indicated. Assays were loaded onto native 4% polyacrylamide gels
pre-electrophoresed for 60 min in 0.5× Tris borate/EDTA and electrophoresed at 100 V before being transferred onto a positively charged nylon membrane (HybondTM-N+) in 0.5×
Tris borate/EDTA at 100 V for 30 min. Transferred DNAs were
cross-linked to the membrane at 120 mJ/cm2 and detected
using horseradish peroxidase-conjugated streptavidin (LightShiftTM chemiluminescent EMSA kit) according to the
manufacturer's instructions.
Animal Model of Chronic Inhibition of NO Synthesis--
Four
groups of male Wistar rats (250 g) were used. The control group
received untreated drinking water, and the sildenafil-treated control
group received sildenafil orally (25 mg/kg/day) for 15 days. The third
group was given 0.5 g/liter
N NO Tension Measurements in Intact Fibers--
The aorta and
pulmonary artery were collected in physiological saline solution (130 mM NaCl, 5.6 mM KCl, 1 mM
MgCl2, 2 mM CaCl2, 11 mM glucose, and 10 mM Tris, pH 7.4, with HCl),
cleaned of fat and adherent connective tissue, and cut into rings.
Smooth muscle rings were then suspended under isometric conditions and connected to a force transducer (Pioden Controls Ltd., Canterbury, UK)
in organ baths filled with Krebs-Henseleit solution (118.4 mM NaCl, 4.7 mM KCl, 2 mM
CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM
NaHCO3, and 11 mM glucose), maintained at
37 °C, and equilibrated with 95% O2 and 5%
CO2. The preparations were initially placed under a resting
tension of 1500 mg, left to equilibrate for 1 h, and washed at
20-min intervals. Tension responses were induced by stimulation with
KCl (60 mM) and the thromboxane A2 receptor agonist U46619 (1 µM). Rings were blotted dry and weighed
(in milligrams) at the end of the experiments, and amplitude of the contraction was expressed as milligrams/mg of tissue. Amplitude of the
relaxation was expressed as percentage of the maximal amplitude of the
contraction induced by phenylephrine recorded before carbachol or SNP application.
Isometric Tension Measurement in Skinned Fibers--
Small
muscle strips (~200 µm wide and 4 mm long) were isolated from the
media of the pulmonary artery and tied at each end with a single
silk thread to the tips of two needles, one of which was connected to a
force transducer (AE 801, SensoNor, Horten, Norway). After measuring
contraction evoked by high K+ solution, the strips were
incubated in normal relaxing solution (85 mM KCl, 5 mM MgCl2, 5 mM Na2ATP,
5 mM creatine phosphate, 2 mM EGTA, and 20 mM Tris maleate, brought to pH 7.1 at 25 °C with KOH)
for few minutes, followed by treatment with Statistics--
All results are expressed as the means ± S.E. of sample size n. Significance was tested by Student's
t test. Probabilities <5% (p < 0.05) were
considered significant.
PKG Activation Increases RhoA Expression--
The effect of
NO/cGMP/PKG signaling on the total levels of RhoA was examined by
Western blot analysis. Stimulation of rat aortic smooth muscle cells
with SNP (10 µM) for 0.1-24 h induced a
time-dependent increase in RhoA expression (Fig.
1A). The rise in the amount of
RhoA protein could be observed after 3 h of incubation with SNP,
and there was a 3-3.5-fold increase in RhoA by 24 h. Similar
results were obtained in human artery smooth muscle cells (Fig.
1A). The role of PKG activation in the increase in RhoA expression has been assessed using the cGMP analog 8-pCPT-cGMP and the
PKG inhibitor (Rp)-8-Br-PET-cGMP-S (Fig.
1B). Activation of PKG by 8-pCPT-cGMP (50 µM,
24 h) produced an increase in RhoA expression similar to that
obtained in the presence of SNP (10 µM, 24 h). Under
both conditions, the effect on RhoA expression was abolished in the
presence of (Rp)-8-Br-PET-cGMP-S (100 nM), whereas (Rp)-8-CPT-cAMP-S had
no effect (data not shown), suggesting that cross-activation of
cAMP-dependent protein kinase was not involved.
The involvement of PKG in cGMP-dependent regulation of RhoA
expression was further examined in Swiss 3T3 cells transfected or not
with an expression vector for PKG I PKG-mediated Increase in RhoA Expression Involves Stabilization of
the RhoA Protein--
Regulation of protein stability has emerged as
an important mechanism for controlling biological functions (25). To
address the stability of RhoA in the context of PKG stimulation, we
examined its fate in vascular smooth muscle cells in the presence and
absence of SNP using cycloheximide (10 µg/ml) to abrogate new RhoA
synthesis. At the times indicated, extracts of total protein were
prepared and analyzed by RhoA immunoblotting. As shown in Fig.
3A, when protein neosynthesis
was blocked by cycloheximide, RhoA expression gradually decreased from
0 to 10 h. In contrast, in the presence of SNP and cycloheximide,
RhoA protein expression was maintained up to 6 h and then
decreased and reached a level similar to that observed in the absence
of SNP at 10 h. This observation suggests that PKG-mediated
up-regulation of RhoA involved change in the rate of RhoA protein
degradation. The rate of small G protein degradation has been shown to
be regulated by the phosphorylation state of the protein (26), leading
to the hypothesis that PKG-mediated phosphorylation of RhoA at
Ser188 (9) could slow down the degradation of RhoA. To
assess this hypothesis, PKG-expressing Swiss 3T3 fibroblasts were
transfected with RhoA or the phosphorylation-resistant RhoA mutant
(RhoAA188). RhoA expression was then assessed in
cycloheximide-treated cells in the presence and absence of 8-pCPT-cGMP
as described above (Fig. 3B). Stimulation with 8-pCPT-cGMP
decreased RhoA degradation in RhoA-expressing cells, but not in
RhoAA188-expressing cells, suggesting that the PKG-mediated
increase in RhoA stability depended on the phosphorylation of RhoA at
Ser188.
RhoA mRNA Is Up-regulated by the
NO/cGMP/PKG Pathway--
To address the
possibility of increases in RhoA mRNA levels in response to PKG
stimulation, the abundance of RhoA mRNA was examined by
quantitative reverse transcription-PCR in arterial smooth muscle cells
incubated with or without SNP or 8-pCPT-cGMP for the indicated times.
Both agents stimulated RhoA mRNA expression, with a maximal
2.5-fold increase after 3 h of incubation (Fib. 4A).
This effect was inhibited in the presence of the PKG inhibitor (Rp)-8-Br-PET-cGMP-S. This result suggests that
the observed modulation of RhoA protein expression induced by PKG
stimulation also involved an increase in the amount of RhoA mRNA.
To determine whether the observed changes in RhoA mRNA levels were
due to stimulation of gene transcription or involved
post-transcriptional RhoA mRNA stabilization, the RhoA mRNA
half-life was determined using the RNA polymerase inhibitor
5,6-dichlorobenzimidazole in the absence and presence of SNP. The
half-life of RhoA mRNA was 3.4 ± 0.5 h under control
conditions and 3.6 ± 0.4 h (n = 3;
p > 0.05) in the presence of 10 µM SNP,
indicating that there was no significant difference in
post-transcriptional regulation of RhoA mRNA after treatment of
aortic smooth muscle cells with SNP (Fig.
4B). Therefore, PKG-induced
stimulation of RhoA mRNA expression results from PKG-mediated
stimulation of rhoA gene transcription.
PKG Induction of RhoA mRNA Expression Does Not Require de
Novo Protein Synthesis--
PKG is known to directly control gene
expression through phosphorylation of transcription factors such as
members of the CREB/ATF family and/or indirectly by controlling
expression of immediate-early genes such as c-fos, which are
themselves transcription factors (19, 27). Therefore, to determine
whether PKG-mediated increases in rhoA gene transcription
required de novo protein synthesis, the effect of SNP on
RhoA mRNA was analyzed by Northern blotting in the presence of
cycloheximide. As shown in Fig. 5,
cycloheximide alone slightly decreased RhoA transcript levels after
3 h, but did not affect SNP-induced rises in RhoA mRNA,
suggesting that PKG-dependent stimulation of
rhoA gene transcription does not require new protein
synthesis.
rhoA Promoter Activity Is Stimulated by PKG Stimulation--
To
gain additional insight into the mechanism of regulation of
rhoA gene transcription by PKG, functional analysis of the rhoA gene promoter was performed. A 913-bp fragment upstream
of the ATG codon of the rhoA human gene sequence was cloned
into pGL2-Basic (pRhoA-Luc) as described under "Experimental
Procedures." PKG-expressing Swiss 3T3 fibroblasts were transiently
transfected with this pRhoA-Luc construct and stimulated with
8-pCPT-cGMP for 6 h. Stimulation of PKG induced a 2.5-fold
increase in rhoA-driven luciferase activity. Stimulation of
Swiss 3T3 fibroblasts that do not express PKG with SNP or 8-pCPT-cGMP
had no effect on rhoA-driven luciferase activity (data not
shown). Based on the partial sequence analysis of the rhoA
promoter, diverse consensus sequences for the binding of transcription
factors were identified, including CRE (one distal and one proximal),
SF1 (steroidogenic factor-1), MEF2
(myocyte enhancer
factor-2), and SRE (Fig.
6). To analyze the role of these
sequences in the effect of PKG, diverse rhoA promoter
constructs containing mutations or deletions were generated. As shown
in Fig. 6, mutational ablation of the dCRE or the proximal SRE had a
minimal effect on PKG stimulation, indicating that these elements do
not play a significant role in PKG regulation of rhoA. In
contrast, mutational ablation of the pCRE abolished the effect of PKG.
Consistent with a major role of the pCRE, the 118-bp fragment containing the CRE and SRE was sufficient for activation of
rhoA transcription by PKG. As expected, point mutation of
the SRE did not modify this effect (data not shown), whereas it was
abolished by mutational ablation of the pCRE (Fig. 6). Neither the
nonsense ( NO/cGMP-induced Phosphorylation of ATF-1 and Its Binding
to the pCRE of the rhoA Gene Promoter--
Phosphorylation of
CREB/ATF-1 transcription factors is necessary for CREB/ATF-1-mediated
transcriptional activation via binding to CRE (28). Using a
phospho-CREB/ATF-1-specific antibody, we found that 8-pCPT-cGMP
stimulation of PKG-expressing 3T3 fibroblasts or aortic smooth muscle
cells induced phosphorylation of CREB and ATF-1, which was inhibited by
(Rp)-8-Br-PET-cGMP-S (Fig.
7A). To determine whether CREB
and/or ATF-1 binds to the CRE of the rhoA promoter, we next
performed EMSA. Nuclear proteins from 8-pCPT-cGMP-stimulated aortic
smooth muscle cells were incubated with labeled oligonucleotide probes
corresponding to the pCRE and dCRE of the rhoA promoter. The
EMSA results shown in Fig. 7B demonstrate a significant
increase in pCRE binding for nuclear extract from
8-pCPT-cGMP-stimulated cells, but no effect on dCRE binding. pCRE
binding was inhibited by an excess of unlabeled oligonucleotides or by
anti-ATF-1 antibody, but not by anti-CREB antibody. These data are
therefore compatible with ATF-1/ATF-1 homodimer binding to the pCRE and
thereby mediation of the PKG regulation of rhoA
transcription.
The NO/cGMP Pathway Regulates RhoA Expression in
Vivo--
We next assessed the existence of NO-mediated regulation of
RhoA in rat arteries in vivo. For this purpose, we used
L-NNA-treated rat aortas and pulmonary arteries. Alteration
of NO/PKG signaling was confirmed by a 53 ± 2%
(n = 4) reduction of the
NO
To establish whether these changes in RhoA expression correlate with
alteration of RhoA-dependent functions, we next analyzed contractile responses induced by KCl and U46619 in pulmonary arteries
from control and L-NNA-treated rats. The maximal amplitude of KCl-induced contraction was not significantly altered in
L-NNA-treated arteries (550 ± 41 mg/mg
versus 611 ± 32 mg/mg in controls, n = 4; p > 0.5), suggesting that
Ca2+-dependent contraction is not modified by
chronic inhibition of NO production. On the contrary, responses to
U46619 were significantly decreased in L-NNA-treated
arteries (87 ± 11 mg/mg versus 808 ± 31 mg/mg in
controls, n = 4; p < 0.001).
Contraction mediated by U46619 is essentially due to the activation of
RhoA/Rho kinase-mediated Ca2+ sensitization (3). These
results thus suggest an alteration of RhoA/Rho kinase-mediated
Ca2+ sensitization in L-NNA-treated rats, which
has been directly assessed by tension measurements in permeabilized
smooth muscle.
Ca2+-dependent contractions and
Ca2+ sensitization of contractile proteins could be
independently evoked in The data presented in this work show that the NO/cGMP/PKG pathway
positively controls RhoA expression in vascular smooth muscle cells
both in vitro and in vivo. The small G protein
RhoA is clearly identified as a convergence point for multiple signals
that regulate cell functions. However, little is known about the
regulation of RhoA protein expression and rhoA gene
transcription. Here we show that the basal release of NO is absolutely
necessary to maintain RhoA expression and RhoA-dependent
contractile functions in vascular smooth muscle. Our results provide
the first evidence that the control of RhoA expression by physiological
signaling pathways represents a new mechanism that regulates the
ability of cells to respond to chemical or physical stimuli.
Nitric oxide is a unique lipophilic, diffusible, short-lived messenger
that modulates a variety of functions, including growth, differentiation, and apoptosis in many different cell types. A large
number of NO effects are mediated through changes in gene expression
essentially through modulation of gene transcription. NO can directly
control the activity of transcription factors such NF- In this study, we have shown that the NO donor SNP or the cGMP analog
8-pCPT-cGMP positively controls RhoA expression and that this process
is strictly dependent on PKG activity. Part of this effect was mediated
by an increase in rhoA gene transcription that did not
require de novo protein synthesis. The rhoA
reporter gene containing 913 bp 5' of the transcription start site
responded to PKG stimulation in PKG-expressing cells, but not in
untransfected cells, thus confirming the role of PKG. Mutation of the
pCRE in the rhoA promoter or in the short promoter construct
( The cycloheximide experiments (Fig. 3) reveal that activation of the
NO/PKG pathway increased the stability of the RhoA protein. Although
this observation could be due to a PKG-dependent mechanism that regulates protein degradation, transfection experiments strongly suggest that this stabilization is related to PKG-mediated
phosphorylation of RhoA at Ser188. We have previously shown
that PKG phosphorylates RhoA at Ser188, causing its
translocation from membranes to the cytosol (9). Such
regulation of the cellular location of small G proteins through phosphorylation of serine residues in the C-terminal domain seems to be
shared by several different subtypes of small G proteins. cAMP-dependent protein kinase has been shown to
phosphorylate Rap1 at Ser180, Rap1B at Ser179,
and RhoA at Ser188, causing their relocalization in the
cytosol (36-38). Our present results suggest that, in addition to this
effect, phosphorylation of RhoA at Ser188 by PKG increases
the stability of the protein. There are only very limited published
data on the regulation of stability or production/degradation of the
Rho proteins. The phosphorylated form of Ras has been shown to be more
stable in cells (26), and carboxyl methylation of RhoA and Cdc42 has
been reported to increase their half-lives (39). Our results suggest
that phosphorylation of Rho proteins in the C-terminal domain could
also protect these proteins from degradation.
The question is then to understand whether the
transcriptional/post-translational regulation of RhoA by PKG occurs
under physiological basal conditions. This modulation of RhoA through
the NO/PKG pathway is of particular interest in vascular smooth muscle
cells that are normally submitted to a low and continuous basal release
of NO from endothelial cells. The decrease in RhoA mRNA and protein expression associated with the decrease in NO production in arteries from L-NNA-treated rats suggests that RhoA expression in
vascular smooth muscle is indeed controlled by the continuous NO
release from endothelial cells in vivo. This is further
supported by the observation that elevation of cGMP levels by
sildenafil treatment completely prevented the down-regulation of RhoA
induced by L-NNA treatment. Although mechanisms regulating
RhoA expression in smooth muscle cells have never been studied,
variations of RhoA levels have already been observed in several
systems. In myometrial smooth muscle cells, up-regulation of RhoA
expression at the end of pregnancy is involved in the mechanisms
underlying the enhanced uterine contractility at term (40). In
bronchial smooth muscle cells, the RhoA protein level is increased in
airway hyper-responsive rats (41), and a rise in RhoA expression has
been detected in aortic smooth muscle from spontaneous hypertensive
rats (15). Modulation of RhoA expression thus appears to be a
regulatory mechanism that controls the capability of cells to respond
to external stimuli, and our results suggest that the basal activity of
NO/PKG exerts a tonic regulation of RhoA expression in vascular smooth
muscle cells. Indeed, functional analysis clearly shows that the
decrease in RhoA expression induced by the chronic inhibition of NO
synthesis is associated with a reduction of RhoA/Rho
kinase-dependent Ca2+ sensitization, indicating
that the level of RhoA expression is a limiting factor of
RhoA-dependent functions. The NO-dependent regulation of RhoA expression therefore appears to be a crucial component of the determinant action of NO in the structure and function
of the vessel wall under normal conditions (42).
In summary, the data presented herein indicate that RhoA expression is
regulated by NO through PKG-dependent mechanisms.
Therefore, NO-mediated regulation of the RhoA signaling pathway is more
complex than one would expect, and NO could not be solely considered as a negative regulator of RhoA activation. Given the importance of both
RhoA and NO/PKG signaling pathways in the regulation of vascular smooth
muscle cell functions, further studies are now required to identify the
functional roles of the NO-mediated regulation of RhoA expression in
normal vessels and in vascular diseases.
-phenyl-1,N2-ethenoguanosine
3':5'-phosphorothioate. The NO/PKG stimulation of RhoA expression
involved both an increase in RhoA protein stability and stimulation of
rhoA gene transcription. Cloning and functional analysis of
the human rhoA promoter revealed that the effect of NO/PKG
involved phosphorylation of ATF-1 and subsequent binding to the
cAMP-response element. Chronic inhibition of NO synthesis in
N
-nitro-L-arginine-treated rats
induced a strong decrease in RhoA mRNA and protein expression in
aorta and pulmonary artery associated with inhibition of RhoA-mediated
Ca2+ sensitization. These effects were prevented by oral
administration of the cGMP phosphodiesterase inhibitor sildenafil.
These results show that NO/PKG signaling positively controls RhoA
expression and suggest that the basal release of NO is necessary to
maintain RhoA expression and RhoA-dependent functions in
vascular smooth muscle cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-adrenergic agonists, sphingolipids, and extracellular nucleotides,
stimulate RhoA activity through the activation of guanine nucleotide
exchange factors and increases in the fraction of GTP-bound RhoA. This RhoA activation is accompanied by the membrane translocation of GTP-bound RhoA (5-8). On the other hand, the NO, cGMP, and
cGMP-dependent kinase
(PKG)1 signaling pathway
exerts inhibitory action on RhoA functions in cells stimulated by these
G protein-coupled receptor agonists. We have previously demonstrated
that PKG phosphorylates RhoA at Ser188 in vitro
and that the effects of PKG activation on actin cytoskeleton are lost
in cells expressing the non-phosphorylatable RhoAA188
mutant, suggesting that inhibitory effects of PKG on RhoA-mediated contraction and actin organization are due to phosphorylation of RhoA
at Ser188 (9). This effect involves inhibition of membrane
translocation of GTP-bound RhoA. Several additional reports have now
confirmed the inhibitory effect of the NO/cGMP/PKG signaling pathway on the RhoA-dependent component of agonist-induced contraction
(10-13). Recently, it has also been shown that PKG inhibits
RhoA-mediated serum response element (SRE)-dependent
transcription (14). PKG inhibits SRE-dependent
transcription induced by serum, constitutively active
G
12 or G
13, constitutively active Rho
exchange factor p115RhoGEF, or constitutively active
RhoAL63. This inhibition is associated with a
decrease in the amount of active GTP-bound RhoA in cells stimulated
with serum or constitutively active G
12 or
G
13, but not in p115RhoGEF- or
RhoAL63-expressing cells, suggesting that PKG can act both
upstream and downstream of RhoA. The effect on steps downstream of RhoA
has been confirmed by showing that SRE-dependent
transcription mediated by a constitutively active RhoA effector (ROK,
PKN, or PRK-2) is inhibited by PKG activation (14). Therefore, the
effects of the NO/cGMP/PKG signaling pathway on membrane-bound
active GTP-bound RhoA in stimulated cells are beginning to be well documented.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and rabbit anti-PKG I antiserum were
provided by Dr. Suzanne Lohmann (Institute of Clinical Biochemistry and Pathobiochemistry, Würzburg, Germany).
(Rp)-8-Br-PET-cGMP-S and 8-pCPT-cGMP were from
Biolog Life Science Institute (Bremen, Germany). Sildenafil was
purchased from Pfizer (Sandwich, UK), and Y-27632 was a gift from the
Institut International de Recherche Servier. All other reagents were
purchased from Sigma (Saint-Quentin Fallavier, France).
- actin antibody.
and analyzed by Western blotting using
rabbit anti-PKG I antiserum (diluted 1:1000) to control PKG expression.
The immunoreactive bands were detected by ECL and quantified using QuantityOne.
913), the 5'-GAAGATCTTCATGGTTACCAAAGCATGTGTCAT-3' sense primer (
118), the 5'-GAAGATCTTCTGCTGAAACACAAAACACAGAT-3'
antisense primer (
1), and the BAC clone RPCI-11-3B7
(CHORI-BACPAC Resources/Oakland, CA) as the matrix. These
sequences were fused to the luciferase gene in the pGL2-Basic vector
(Promega) using a BglII restriction site. The entire
sequence was verified by sequencing the insert with pGL1 and pGL2
primers (Promega).
and
RhoA plasmids and the different constructions of the rhoA
promoter were transfected with jetPEI (PolyPlus Transfection, Illkirch, France) according to the manufacturer's instructions. 24 h after transfection, 8-pCPT-cGMP (50 µM) or SNP
(10 µM) was added to the medium for 6-24 h. Cells were
then harvested and treated as described above for Western blot,
Northern blot, or real-time reverse transcription-PCR analysis. For
reporter assays, the pIRES-EGFP vector was always cotransfected to
estimate the level of transfection (fluorescence measure from lysates
with Victor2) and to normalize the luciferase activity measured with the luciferase reporter reagent (Promega) in a LB96V luminometer (Berthold Technologies, Wildbad, Germany).
was cloned into pcDNA3 (Invitrogen, Groningen,
The Netherlands). RhoA or PKG I
plasmids were transiently
transfected into Swiss 3T3 fibroblasts with jetPEI according to the
manufacturer's protocol. 48 h after transfection, cells were
stimulated with 8-pCPT-cGMP (50 µM).
-nitro-L-arginine
(L-NNA) in their drinking water for 2 weeks (L-NNA-treated rats), and the fourth group received both
L-NNA (0.5 g/liter) and sildenafil (25 mg/kg/day) for 2 weeks. At completion of this time, the main pulmonary artery and aorta
were removed and dissected under binocular control, and the adventitial
and medial layers were removed. Tissues were then prepared as indicated for tension measurements, RNA extraction, or Western blot analysis. NO
synthesis inhibition was assessed by measurement of plasma NO
-escin (50-70
µM) in relaxing solution for 35 min at 25 °C as
previously described (24). The skinned muscle strip was then washed
several times with fresh relaxing solution containing 10 mM
EGTA. Calmodulin (1.5 µM) was added to the bathing
solutions throughout the experiments. Tension developed by
permeabilized muscle strips was measured in activating solutions
containing 10 mM EGTA and a specified amount of
CaCl2 to give a desired concentration of free
Ca2+ (24).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Activation of the NO/PKG signaling pathway
increases RhoA protein levels in vascular smooth muscle cells.
A, stimulation of rat aortic (left panel
and lower panel, black bar) or human
mammary artery (right panel and lower panel,
open bars) smooth muscle cells with SNP (10 µM) for 0-24 h induced an increase in RhoA expression as
analyzed by Western blotting. B, involvement of PKG
was assessed by treatment of rat aortic smooth muscle cells with the
cGMP analog 8-pCPT-cGMP (50 µM, for 24 h) and the
selective PKG inhibitor (Rp)-8-Br-PET-cGMP-S
(100 nM, pretreatment for 1 h). The RhoA protein
level, normalized to -actin expression, is expressed relative to the
control taken as 1. Data shown are the means of four independent
experiments; **, p < 0.01 versus control;
##, p < 0.01 versus SNP or 8-pCPT-cGMP
stimulation.
(Fig.
2). PKG I
overexpression induced a
2-fold rise in the basal level of RhoA expression. In addition,
stimulation with the cGMP analog 8-pCPT-cGMP (50 µM) strongly increased RhoA expression in PKG I
-expressing cells and had
no effect in untransfected cells (Fig. 2). The effect of
8-pCPT-cGMP was inhibited in the presence of
(Rp)-8-Br-PET-cGMP-S (100 nM) (data
not shown). These data thus demonstrate the role of PKG in
SNP/cGMP-mediated control of RhoA expression.
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Fig. 2.
PKG expression is required for
8-pCPT-cGMP-mediated control of RhoA expression.
Stimulation of 3T3 fibroblasts with 8-pCPT-cGMP (50 µM, 24 h) had no effect on RhoA expression
(left panel); however, an increase in RhoA expression in
response to 8-pCPT-cGMP (50 µM, 24 h) was induced by
transfection of PKG I (right panel). The induction of the
response correlated with the expression of PKG detected by Western
blotting. The RhoA protein level, normalized to -actin expression,
is expressed relative to the control in the absence of 8-pCPT-cGMP
taken as 1. Data shown are the means of three independent experiments.
**, p < 0.01 versus control; ##,
p < 0.01 versus unstimulated
PKG-transfected cells.
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Fig. 3.
RhoA phosphorylation by PKG increases RhoA
protein stability. Rat aortic smooth muscle cells (A)
or Swiss 3T3 fibroblasts (B) transfected with wild-type RhoA
(RhoA WT; B, left panel) or the
phosphorylation-resistant mutant of RhoA, RhoAA188
(RhoA ala188; B, right panel), were
preincubated with 10 µg/ml cycloheximide (CHX) for 30 min
and then stimulated for 3, 6, and 10 h with 10 µM
SNP (A) or 50 µM 8-pCPT-cGMP (B).
RhoA expression under these various experimental conditions was
assessed by Western blot analysis. RhoA protein levels detected at
various times in the presence of cycloheximide without ( ) or with
SNP (
in A) or 8-pCPT-cGMP (
in B) are
expressed relative to the control (0 h) taken as 1. Data shown are the
means of three to four independent experiments. Examination of
-actin expression showed that, after 10 h,
-actin expression
began to decrease.
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Fig. 4.
PKG activation increases RhoA mRNA
expression. A, relative expression of RhoA mRNA
assessed by real-time PCR. Stimulation of rat aortic smooth muscle
cells with SNP (10 µM) or 8-pCPT-cGMP (50 µM) induced an increase in the amount of RhoA mRNA,
which was inhibited by the PKG inhibitor
(Rp)-8-Br-PET-cGMP-S (100 nM).
Results, normalized to glyceraldehyde-3-phosphate dehydrogenase
mRNA expression, are expressed relative to the control taken as 1. Data shown are the means of four independent experiments. **,
p < 0.01 versus control at the same time;
##, p < 0.01 versus 8-pCPT-cGMP
stimulation. B, time-dependent effect of
5,6-dichlorobenzimidazole (DRB; 50 µM) alone
( ) or in combination with SNP (10 µM;
) on
steady-state RhoA mRNA levels after 0, 2, 4, and 10 h as
analyzed by virtual Northern blotting. Band intensities were normalized
to control levels at 0 h and plotted semilogarithmically as a
function of time. The corresponding ethidium bromide-stained 28 S and
18 S band intensities were used to check loading conditions. Results
displayed are representative of three separate experiments.
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Fig. 5.
Cycloheximide treatment does not abolish the
SNP effect on RhoA mRNA expression. Rat aortic smooth muscle
cells were preincubated with 10 µg/ml cycloheximide (CHX)
for 30 min and then stimulated with 10 µM SNP for 3 h. Total RNA (20 µg) was analyzed by Northern blotting using a
rhoA cDNA probe. Equal loading of RNA was checked by
staining the ribosomal RNA (18 S and 28 S) with ethidium bromide. RhoA
mRNA, normalized to ribosomal RNA, is expressed relative to the
control in the absence of SNP and CHX taken as 1. Data shown are the
means of three independent experiments.
913) rhoA promoter nor the pGL2-Basic
construction responded to 8-pCPT-cGMP stimulation.
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Fig. 6.
The pCRE is responsible for
8-pCPT-cGMP-induced activation of rhoA promoter
activity. CRE (one distal and one proximal), SF1, MEF2, and SRE
regulatory elements were found in the first 913 bp of the sequence of
the 5'-upstream region of the human rhoA gene. A smaller
construct, which is 118 bp long, contains only the pCRE and SRE. Point
mutations of these elements are indicated (×). All these
constructions, fused to the luciferase (luc) gene, were
cotransfected with PKG I (see "Experimental Procedures")
in 3T3 fibroblasts, which were incubated without (white
bars) or with (black bars) 50 µM
8-pCPT-cGMP for 6 h. Luciferase activity was measured as described
under "Experimental Procedures" and normalized by measuring the
fluorescence of EGFP expressed by the pIRES-EGFP vector, which was
always cotransfected. Data shown are the means ± S.E. of three
independent transfections (each performed in duplicate).
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Fig. 7.
NO/cGMP signaling pathway-induced
phosphorylation of ATF-1 and its binding to the pCRE of the
rhoA promoter. A, Western blot
analysis using anti-phospho-CREB/ATF-1 antibody was performed on
lysates from PKG I-expressing 3T3 fibroblasts and aortic smooth muscle
cells treated with 8-pCPT-cGMP (50 µM, 2 h) in the
absence or presence of (Rp)-8-Br-PET-cGMP-S (100 nM, pretreatment for 30 min). B, EMSAs were
performed to analyze the binding of proteins from nuclear extract of
aortic smooth muscle cells treated for 2 h with 8-pCPT-cGMP to the
wild-type pCRE (wt pCRE), the mutant pCRE (m
pCRE), or the wild-type dCRE (wt dCRE) of the
rhoA promoter as described under "Experimental
Procedures." Lane 1, labeled oligonucleotide; lane
2, labeled oligonucleotide + nuclear extract; lane 3,
labeled oligonucleotide + nuclear extract + 200-fold molar excess of
unlabeled oligonucleotides; lane 4, labeled oligonucleotide + nuclear extract + anti-CREB antibody; lane 5, labeled
oligonucleotide + nuclear extract + anti-ATF-1 antibody.
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Fig. 8.
Effect of chronic inhibition of NO synthesis
on RhoA expression and RhoA-dependent function in
vivo. RhoA mRNA (A) and protein (B)
expression was examined in aortas and pulmonary arteries of control and
L-NNA-treated rats (15 days) and analyzed by real-time PCR
(A) and Western blotting (B). L-NNA
treatment induced a decrease in RhoA mRNA and protein expression,
which was prevented by the treatment of rats with sildenafil (25 mg/kg/day) for 15 days. RhoA mRNA expression was normalized to
glyceraldehyde-3-phosphate dehydrogenase mRNA (A), and
RhoA protein expression was normalized to -actin (B).
Results are expressed relative to control rats taken as 1. Data shown
are the means of four (A) and six (B) independent
experiments. **, p < 0.01 versus control
rats. pCa-tension relationships were measured under control
conditions (
) and in the presence of 10 µM GTP
S
without (
) and with (
) 10 µM Y-27632 in
-escin-permeabilized pulmonary artery smooth muscle cells from
control rats (C), L-NNA-treated rats
(D), and L-NNA-treated rats receiving sildenafil
(E). Data shown are the means of four independent
experiments.
-escin-permeabilized smooth muscle strips.
Ca2+-dependent contractions were induced by
gradual increases in Ca2+ concentrations (submaximal
pCa (
log[Ca2+]) = 8 to maximal
pCa = 4.5), and Ca2+ sensitization was
evoked by addition of 10 µM GTP
S. The Ca2+
sensitization appeared as a leftward shift of the
pCa-tension relationship. In
-escin-permeabilized
pulmonary artery strips from control rats, GTP
S induced an increase
in the Ca2+ sensitivity of contractile proteins,
illustrated by an increase in the pCa50 from
6.05 ± 0.01 (n = 4) to 6.67 ± 0.02 (n = 4; p < 0.01) (Fig.
8C). This shift in the pCa-tension relationship was completely abolished in the presence of the Rho kinase inhibitor Y-27632 (10 µM), indicating that the GTP
S-induced
Ca2+ sensitization in the pulmonary artery was mediated by
the RhoA/Rho kinase pathway (Fig. 8C). The
pCa-tension relationship in permeabilized pulmonary artery
strips from L-NNA-treated rats was similar to that in
controls (pCa50 = 6.03 ± 0.03, n = 4; p > 0.5), but the GTP
S-induced Ca2+ sensitization was completely lost
(pCa50 = 6.10 ± 0.03, n = 4; p > 0.5), and Y-27632 had no effect (Fig.
8D). The GTP
S-induced Ca2+ sensitization was
restored in sildenafil-treated L-NNA rats
(pCa50 = 6.69 ± 0.02 in the presence of
GTP
S versus 6.07 ± 0.03 in its absence,
n = 4; p < 0.01), and the effect of
GTP
S was blocked by Y-27632 (pCa50 = 6.10 ± 0.03, n = 4) (Fig. 8E). These
results suggest that chronic inhibition of NO synthesis-induced loss of RhoA expression is associated with impairment of the RhoA/Rho kinase
signaling pathway. Decreases in both RhoA expression and RhoA/Rho
kinase-dependent functions are prevented by sildenafil.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B, AP-1, and
c-Jun by S-nitrosylation (30-32). However, a large
number of NO effects are mediated through the activation of PKG (19).
Several sequence elements have been proposed to mediate the
transcriptional effect of PKG, notably the SRE, AP-1-binding site, and
CRE (27, 33-35).
118) abolished the effect of PKG, showing that the pCRE is the
response element that mediates the PKG regulation of rhoA
transcription. Both CREB and ATF-1 were phosphorylated by PKG in smooth
muscle cells. Furthermore, EMSA experiments strongly suggest that
ATF-1/ATF-1 homodimer binding to the pCRE is responsible for the
PKG-mediated regulation of rhoA transcription.
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ACKNOWLEDGEMENT |
---|
We thank Dr. Suzanne Lohmann for the gift of
the plasmid encoding PKG I and anti-PKG I antiserum.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from INSERM, Institut International de Recherche Servier, Région Pays de Loire, and Action Cibles Thérapeutiques et Médicaments (INSERM-CNRS).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally to this work.
§ Supported by a grant from the Association pour la Recherche contre le Cancer.
¶ To whom correspondence should be addressed: Laboratoire de Physiologie Cellulaire et Moléculaire, INSERM U-533, Faculté des Sciences, 2 rue de la Houssinière, BP 92208, 44322 Nantes Cedex 3, France. Tel.: 33-2-5112-5740; Fax: 33-2-5112-5614; E-mail: pacaud@svt.univ-nantes.fr and gervaise.loirand{at}nantes.inserm.fr.
Published, JBC Papers in Press, January 9, 2003, DOI 10.1074/jbc.M212776200
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ABBREVIATIONS |
---|
The abbreviations used are:
PKG, cGMP-dependent protein kinase;
SRE, serum response element;
(Rp)-8-Br-PET-cGMP-S, (Rp)-8-bromo--phenyl-1,N2-ethenoguanosine
3':5'-phosphorothioate;
8-pCPT-cGMP, 8-(2-chlorophenylthio)guanosine
3':5'-monophosphate;
(Rp)-8-CPT-cAMP-S, (Rp)-8-(4-chlorophenylthio)adenosine
3':5'-phosphorothioate;
SNP, sodium nitroprusside;
CRE, cAMP-response
element;
dCRE, distal cAMP-response element;
pCRE, proximal
cAMP-response element;
CREB, cAMP-response element-binding protein;
EMSA, electrophoretic mobility shift assay;
L-NNA, N
-nitro-L-arginine;
GTP
S, guanosine 5'-O-(3-thiotriphosphate).
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REFERENCES |
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