From the Institut für Kardiovaskuläre
Physiologie, Johann Wolfgang Goethe-Universität, Theodor
Stern-Kai 7, Frankfurt/Main D60590, Germany and the
§ Department of Biochemistry, University of Connecticut
Medical School, Farmington, Connecticut 06030
Received for publication, June 28, 2002, and in revised form, November 4, 2002
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ABSTRACT |
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We investigated the molecular mechanism of
cyclic GMP-induced down-regulation of soluble guanylyl cyclase
expression in rat aorta. 3-(5'-Hydroxymethyl-2'-furyl)-1-benzyl
indazole (YC-1), an allosteric activator of this enzyme, decreased the
expression of soluble guanylyl cyclase The hemoprotein soluble guanylyl cyclase
(sGC)1 is the
predominant intracellular nitric oxide (NO) receptor in vascular
smooth muscle cells (1). The active enzyme exists as an obligate
heterodimer, the most abundant isoform consisting of an
In addition to an acute activation of sGC, the output of the NO-cGMP
pathway can also be controlled at the level of sGC expression. Thus, a
reduced vasodilator response to exogenous NO consistent with a
down-regulation of sGC has been observed in aortic tissue of aged
spontaneously hypertensive rats (4). On the other hand, an
up-regulation of sGC expression was found in aortic tissue from
nitroglycerin-tolerant rats (5) and from rats suffering from chronic
heart failure (6), despite diminished vasodilator responses to NO. This
apparently discrepant finding indicates that altered sGC expression
does not necessarily translate into predictable changes in
cGMP-dependent functional responses, but that other
mechanisms, such as altered NO bio-availability, may overrun the
influence of altered sGC expression.
These findings exemplify the need for understanding the molecular
mechanisms accounting for regulation of sGC expression. There is
evidence that expression of sGC is controlled by second messenger
cyclic nucleotides via a post-transcriptional mechanism: in various
cells cyclic AMP-eliciting agonists decrease the expression of sGC
mRNA and protein (7, 8) by a destabilization of the sGC mRNA.
This effect is mimicked by activation of the cGMP signaling pathway,
e.g. application of NO donors, stimulation of particulate guanyl cyclase by atrial natriuretic factor, and stimulation of cGMP-dependent protein kinase by the stable cGMP-analogue
8-chlorophenylthio-cGMP (9).
The objective of the present investigation was to characterize the
mechanism accounting for sGC The polyclonal chicken antibody directed against the
Rat Aortic Tissue--
Male normotensive Wistar Kyoto rats (300 g) were obtained from Möllegaard (Skensved, Denmark) and were
kept according to institutional guidelines, in compliance with German
laws. The thoracic aorta was isolated from anesthetized rats (200 mg/kg ketamine (ExalgonTM), 100 mg/kg xylazine
(RompunTM), cleaned from fat and connective tissue, and cut
into rings of equal length (3 mm). The endothelium was removed by
gentle forcing and rolling a glass rod through the lumen. The aortic rings were kept in culture dishes (six well) in MEM under a
CO2-enriched atmosphere (4.5% CO2) at
37 °C. The rings were exposed to YC-1, NS2028, and/or actinomycin D
for different periods of time and were then snap-frozen in liquid
nitrogen and stored at Isolation of Total RNA from Rat Aorta and RT-PCR--
Frozen
tissue was ground in liquid nitrogen with porcelain mortar and a
pestle. Total RNA was extracted by the modified guanidine isothiocyanate method of Chomczynski and Sacchi (11). The reverse transcriptase-polymerase chain reaction (RT-PCR) for the
sGC Poly(A)+ RNA (mRNA) Isolation from Rat
Lung--
Poly(A)+ mRNA was purified from total RNA by
means of the Messagemaker kit (Invitrogen). Total RNA (2 mg; 0.55 mg/ml) was denatured for 5 min at 65 °C. The salt concentration was
adjusted to 0.5 M NaCl. Subsequently the RNA was incubated
with the oligo(dT) cellulose suspension and heated for 10 min at
37 °C. After filtration the suspension was washed with 20 mM Tris/HCl, pH 7.5, 0.5 M NaCl and then with
20 mM Tris/HCl, pH 7.5, 0.1 M NaCl. The
mRNA was eluted with RNase-free water.
Northern Blots--
The poly(A)+ RNA sample was
denatured for 15 min at 65 °C in 0.5× MOPS buffer containing 25%
formamide, 1.1% formaldehyde, 1% Ficoll 400, 0.02% bromphenol blue
(sodium salt). The mRNA was fractionated in a 1.2%
agarose-formaldehyde gel and blotted overnight onto nylon membrane
(pore size: 0.45 µm, Biodyne B, Pall) in 10× saline-sodium citrate
(SSC) buffer solution (1.5 M NaCl, 150 mM sodium citrate, pH 7.5). Membranes were washed in 2× SSC, and the
mRNA was fixed by UV-cross-linking. Subsequently the membranes were
baked at 80 °C for 2 h and then pre-hybridized for 2 h in 50% formamide, 0.8 M NaCl, 0.1% Sarkosyl, 0.1% Ficoll
400, 0.1% polyvinyl pyrrolidone, 0.1% bovine serum albumin, 0.2%
SDS, and 250 µg/ml sheared salmon sperm DNA at 42 °C.
Hybridization occurred at 42 °C overnight in 10% dextran
sulfate with biotinylated-DNA probes (5 ng/cm2) specific
for elongation factor II and sGC Preparation of sGC RNA-Protein Binding Reactions and Supershift
Assays--
Electrophoretic mobility shift assays (EMSA) were carried
out by a modification of the method of Wang (12). The
oligoribonucleotide (3UTRSK2, 50-200 ng) was incubated with 40-100
µg of native nuclear extract (prepared according to Ref. 13) from
endothelium-denuded rat aorta, and a 10× reaction buffer (15 mM Hepes, pH 7.9, 600 mM KCl, 10 mM
dithiothreitol, 50% glycerol, 30 mM MgCl2, 2 units/µl RNase inhibitors (40 units/µl, RNaseOUT, Invitrogen), 200 ng/ml total RNA) for 30 min at room temperature. Complexes were
resolved by native 2% TAE-agarose gel electrophoresis (40 mM Tris pH 8.5, 0.1% acetic acid, 2 mM EDTA)
for 2 h and blotted onto a nylon membrane (Biodyne B, Pall)
overnight in 10× SSC. Blocking and detection of biotin-labeled bands
was performed as described for Northern blots. For supershifts, 4-15
µg of the monoclonal HuR-antibody was incubated with the native
nuclear extract for 1 h on ice before the specific riboprobe was
added; all subsequent steps were performed as described for native gels.
Immunodetection of the sGC Design of HuR-specific siRNA--
An HuR-specific siRNA
(HuR-siRNA) was designed by selecting a target region from base
position 163 to 183 relative to the start codon, which fulfilled the
specific sequence requirements as follows: AA(N19)dTdT (N
is any nucleotide); 21-nt sense and 21-nt antisense strand; ~50% G/C
content; and a symmetric 2-desoxythymidine 3' overhang. Sense and
antisense oligonucleotides were synthesized by Xeragon Oligonucleotides
(Xeragon-Qiagen). The lyophilized siRNA was dissolved in sterile
annealing buffer (100 mM potassium acetate, 30 mM Hepes-KOH, 2 mM magnesium acetate, pH 7.4)
to obtain a 20 µM solution. Then oligonucleotides were
heated to 90 °C for 1 min followed by 1 h at 37 °C. In
addition, we also used a HuR-siRNA targeting base position 564-584
relative to the start codon (kindly provided by S. Sengupta) for cell
transfections. Lyophilized or dissolved siRNAs were stored at
Transfection of Cultured Vascular Smooth Muscle
Cells--
Cultured smooth muscle cells from rat aortas were grown in
minimal essential medium (MEM, Invitrogen) containing 10% fetal calf
serum and 1% penicillin and streptomycin at 37 °C and 5% CO2. SMCs were trypsinized, mixed with fresh MEM (plus
antibiotics) without fetal calf serum, and seeded onto six-well plates
(2 ml per well). 24 h later the cells had reached 80-90%
confluence and were transfected with siRNA (final concentration, 100 nM) using the TransMessenger transfection reagent (TMTR;
Qiagen). Therefore, 6 µg of siRNA was diluted with 12 µl of
Enhancer R (ratio: micrograms of RNA to micrograms of Enhancer R, 1:2)
and 168 µl of Buffer EC-R (Qiagen) and mixed by vortexing for 10 s. After incubation (5 min, room temperature) 24 µl of TMTR (ratio: micrograms of RNA to microliters of TMTR, 1:4) was added to the RNA-Enhancer R mixture. During this incubation (10 min, 15-25 °C)
SMCs were washed with 2 ml (per well) of sterile Dulbecco's phosphate-buffered saline (Invitrogen). Then the RNA-transfection mixture was diluted in 1.78 ml of MEM (plus antibiotics) and added dropwise to the cells (2 ml of transfection complex per well). The
cells were cultured for 2 days at 37 °C (5% CO2).
Thereafter the HuR and sGC mRNA and protein expression was assayed
by RT-PCR and Western blot experiments.
Influence of YC-1 on the Expression of sGC YC-1 Decreases the sGC
The time course of YC-1-induced sGC YC-1-induced sGC Identification of HuR as a sGC Activation of sGC by YC-1 Decreases HuR-ARE-binding
Activity--
To clarify whether activation of sGC decreases HuR-like
binding activity, endothelium-denuded rat aortic segments were kept for
12 h under organ culture conditions (see "Materials and
Methods"), either in the absence or presence of YC-1 (100 µM), and NS2028 (100 µM), or the solvent
control (0.2% Me2SO). Nuclear protein extracts were
prepared from the vascular tissue, and the expression of HuR-like ARE
binding activity was assessed by RNA-EMSA using the 3UTRSK2 probe. In
the presence of protein (80 µg) from control aortas a similar
bandshift as shown in Fig. 5 was observed (Fig. 6, lanes 2 and 3).
The protein extract from Me2SO-treated aorta induced a
quite similar shift (Fig. 6, lanes 4 and 5). In
contrast, with protein from YC-1-exposed aorta the shifted band
markedly decreased (Fig. 6, lanes 6 and 7). This
effect of YC-1 was prevented by concomitant exposure of the aorta to
the sGC inhibitor NS2028 (Fig. 6, lanes 8 and 9).
Addition of the monoclonal HuR antibody induced a strong supershift,
which under these chromatographic conditions unfortunately superimposed
with the shift (Fig. 6, lane 10). These findings indicate
that YC-1 either induces a reduction of the HuR affinity for the 3'-UTR
of GC Activation of sGC in Rat Aorta by YC-1 Decreases Expression of
HuR--
By Western blot analysis we assessed whether YC-1 affected
HuR expression. Protein from rat aorta incubated with YC-1 and NS2028
as shown in Fig. 6 was loaded for SDS-PAGE analysis, blotted, and
immunoprobed for HuR. The protein from the control and
Me2SO-treated aortas showed a marked HuR-positive band at
34 kDa (Fig. 7A, lanes 1-4 from left), similar to the nuclear extract from
HeLa cells applied as a positive control (+contr.,
lane 9). This band was significantly reduced
(p > 0.05, ANOVA) in YC-1-treated aortas (lanes
5 and 6, and densitometric analysis shown below in Fig. 7B). The sGC inhibitor NS2028 prevented the down-regulation
of HuR expression by YC-1 (lanes 7 and 8).
HuR Knockdown by RNA Interference Decreases Expression of
sGC The heterodimeric hemoprotein and NO receptor sGC is a key
component of the NO/cGMP signal transduction pathway in vascular smooth
muscle and other tissues. In addition to an acute regulation by
positive (NO) or negative (superoxide radical) input signals (1) the
activity of this pathway can also be controlled at the level of sGC
expression (4). Previous studies have shown a feedback inhibition of
sGC expression by its product cGMP (9, 17, 18). This finding was
related to an accelerated decay of the sGC We set out to reveal the mechanism accounting for the down-regulation
of the sGC The 3'-UTR of the rat sGC To confirm the hypothesis that a decrease in HuR expression induces a
decrease in sGC expression, we used the RNA knockdown (RNA
interference) technique (16). RNA interference is a gene-silencing mechanism that uses double-stranded RNA as a signal to trigger the
degradation of the targeted mRNA. 48 h after transfection of
cultured rat aortic smooth muscle cells with a 21-mer double-stranded RNA, homologous to base position 163-183 relative to the start codon
of the HuR message (HuR siRNA), we observed a strong decrease in HuR as
well as in sGC The signaling cascade accounting for cGMP-dependent
down-regulation of HuR could not be revealed in this study. Because
concomitant application of Act D during exposure of the rat aorta to
YC-1 prevented the down-regulation of HuR expression and binding
activity, it is likely that cGMP induces the transcriptional activation of (unknown) factors that decrease HuR expression. Our preliminary data
indicate that a cGMP-activated protein kinase and the transcription factor AP-1 are involved in down-regulation of HuR by sGC
activators,2 but an in-depth
study is required. AP-1 sites are present in the mouse HuR promoter
region (24). Interestingly, CREB sites were also found in this promoter
region (24). Agents that increase intracellular cyclic AMP decrease sGC
subunit mRNA levels and cellular cGMP formation in response to
NO-donor compounds (7, 8). We observed that cyclic AMP-eliciting
agonists decrease expression of HuR in rat aortic smooth muscle cells
as well,2 suggesting that HuR also mediates the
down-regulation of sGC in response to increased cAMP levels. It appears
that HuR can integrate cyclic nucleotide second messenger signaling and
translate changes in cAMP and cGMP levels in altered gene expression.
This underscores the increasing importance of mRNA stability
regulation for gene expression (25), as compared with transcriptional
regulation. In addition to sGC, other components of the NO/cGMP pathway
are also regulated by altered mRNA stability. In human mesangial
cells, which exhibit a smooth muscle cell-like phenotype, the
expression of the cytokine-inducible NO synthase II is also
down-regulated by NO and cGMP. Part of this negative modulation is
caused by decreased mRNA stability (26). The 3'-UTR of NO synthase
II also bears AREs, and HuR was shown to stabilize the NOS III mRNA by binding to several of these AREs. The expression of HuR in cytokine-exposed DLD1 cells (human intestinal epithelium) decreased concomitantly with enhanced NOS III-derived NO formation (27). Furthermore, an increase or decrease in HuR expression brought about by
stable transfection with HuR-sense or -antisense vectors increased or
decreased NO synthase II expression. Collectively, these examples and
our present findings emphasize that several major components of the
NO/cGMP pathway are controlled at a post-transcriptional level by HuR
in a negative feedback manner. Future studies will be needed to reveal
the relative importance of HuR-regulated mRNA stability
versus transcriptional processes for
NO/cGMP-dependent gene expression.
1 subunit
mRNA and protein. This effect was blocked by the enzyme
inhibitor
4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b-1,4)oxazin-1-one (NS2028) and by actinomycin D. Guanylyl cyclase
1
mRNA-degrading activity was increased in protein extracts from
YC-1-exposed aorta and was attenuated by pretreatment with actinomycin
D and NS2028. Gelshift and supershift analyses using an
adenylate-uridylate-rich ribonucleotide from the 3'-untranslated region
of the
1 mRNA and a monoclonal antibody directed
against the mRNA-stabilizing protein HuR revealed HuR mRNA
binding activity in aortic extracts, which was absent in extracts from
YC-1-stimulated aortas. YC-1 decreased the expression of HuR, and this
decrease was prevented by NS2028. Similarly, down-regulation of HuR by
RNA interference in cultured rat aortic smooth muscle cells decreased
1 mRNA and protein expression. We conclude that HuR
protects the guanylyl cyclase
1 mRNA by binding to
the 3'-untranslated region. Activation of guanylyl cyclase decreases
HuR expression, inducing a rapid degradation of guanylyl cyclase
1 mRNA and lowering
1 subunit expression as a negative feedback response.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 (76-81.5 kDa) and a
1 (70 kDa) subunit
(2). sGC mediates NO signaling via formation of guanosine 3':5'-cyclic
monophosphate (cGMP), which induces, for instance, vascular smooth
muscle relaxation by activating cGMP-dependent protein
kinase, prevention of contractile agonist-elicited intracellular free
Ca2+ mobilization, and dephosphorylation of
myosin light chain kinase (3).
1 mRNA destabilization
induced by increased cGMP formation in isolated rat aorta. We observed that the elav family protein HuR (10) stabilizes the
sGC
1 mRNA by binding to AU-rich elements (ARE) in
its 3'-untranslated region (UTR), and that an increase in intracellular
cGMP strongly decreases HuR expression and sGC
1 mRNA
binding activity, leading to accelerated mRNA degradation.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 and
1 subunits of the rat lung sGC was
produced by BioGenes GmbH (Berlin, Germany), which also provided the
rabbit anti-chicken antibody. For some experiments an
sGC
1-specific peptide antibody was obtained from Dr.
Stasch, Bayer AG, Leverkusen. The oligonucleotides for RT-PCR, in
vitro transcription, and gelshift analysis were synthesized by
BioSpring GmbH and MWG-Biotech.
4H-8-Bromo-1,2,4-oxadiazolo(3,4-d)benz(b-1,4)oxazin-1-one (NS2028) was from Neurosearch (Copenhagen, Denmark).
3-(5'-Hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) was a kind gift
from Aventis Pharma (Strasbourg, France). The HuR-specific siRNA
oligonucleotides were from Xeragon-Qiagen (Germantown, MD).
70 °C.
1 mRNA (product size 826 bp), and elongation
factor II (225 bp) was performed exactly as described previously
(4).
1 mRNA. Blots were then washed twice at 65 °C in 5× SSC, 0.5% SDS, washed for
30 min at 50 °C in 0.1× SSC, 1% SDS, and washed for 1 min with
TBS-Tween 20 (0.05% Tween 20, 150 mM NaCl, 100 mM Tris/HCl, pH 7.5). Afterward membranes were blocked for
1 h at 65 °C in TBS-Tween 20 containing 3% bovine serum
albumin and incubated with a streptavidin-alkaline phosphatase
conjugate (7 µl/100 cm2; 1:1000 in TBS-Tween 20) for 10 min at room temperature. The blots were washed twice in
TBS-Tween 20 and then in 100 mM NaCl, 50 mM
MgCl2, 100 mM Tris/HCl, pH 9.5. Immunoreactive
mRNA bands were visualized by chemiluminescence and exposure to
x-ray film.
1 Transcripts by in Vitro
Transcription--
Total RNA from rat lungs was used as a
template for RT-PCR amplification of the 3'-UTR of sGC
1
cDNA regions. A Pfu DNA polymerase (Pyrococcus
furiosus DSM3638, 92 kDa, Promega) with 3'
5' exonuclease (proofreading) activity was used for the PCR reaction. The 5'-sense primer contained the T7 promoter sequence
5'-CCAAGCTTCTAATACGACTCACTATAGGGAGA-3' (T7). For
generating the 3UTRSK2 (424 bp) template, sense primer
(5'-T7CCAGCTACATCTTTGTGCC-3') and antisense
primer (5'-ACTGTCCTCTACAGTAGGC-3'), corresponding to positions
3049-3067 and 3423-3441 of the sGC
1 cDNA, were
used (GenBankTM accession number U60835).
Subsequently, PCR fragments encompassing the 3'-UTR of
sGC
1 mRNA were synthesized bearing a T7
sequence at the 5'-end. Biotinylation of transcripts was performed with the North2SouthTM Biotin in vitro transcription
kit from Pierce (Rockford, IL).
1 Subunit--
Total
protein was precipitated (1.5 ml of 100% isopropanol) from the
phenol-ethanol supernatant (TRIzol method) of the RNA extraction, and
the precipitate was dissolved in 1% SDS. The protein (40 µg per
lane) was fractionated on Laemmli gels and electroblotted onto
nitrocellulose filters (Protrans; Schleicher & Schuell). The blots were
blocked at 4 °C overnight and then incubated for 2 h at room
temperature with either a polyclonal chicken antibody (IgY) or a rabbit
antibody (IgG) directed against the
1 subunits of sGC
(1:100 dilution in blocking buffer). The blots were washed and then
developed either with a peroxidase A-conjugated anti-chicken IgY (IgG,
rabbit, 1:5,000 in blocking buffer) or anti-rabbit IgG (goat,
1:10,000). Immunoreactive peptides were visualized by chemiluminescence and exposure to x-ray film. The autoradiographs were analyzed by
scanning densitometry. Equal protein loading and blotting was verified
by
-actin immunostaining.
20 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 mRNA
and Protein in Rat Aorta--
To assess the effect of increased cGMP
formation on the sGC
1 subunit expression in rat aorta,
freshly isolated endothelium-denuded aortic rings from Wistar Kyoto
rats were kept under organ culture conditions, either in the
absence (control ± 0.2% Me2SO) or presence of
the sGC activator molecule YC-1 (10 µM), and the specific
sGC inhibitor NS2028 (10 µM). After 24 h the
vascular tissue was snap-frozen and homogenized in liquid nitrogen,
then further processed for sGC
1 subunit mRNA and
protein expression by RT-PCR (Fig.
1A) and Western blot (Fig.
1B), respectively. According to densitometric analysis of
the RT-PCR product (Fig. 1A) and the immunoreactive protein
(
1 = 82 kDa, Fig. 1B) the abundance of
sGC
1 subunit mRNA and protein was markedly lower in
YC-1-exposed aorta compared with controls
(
1-mRNA = 93% lower,
1-protein = 56% lower versus controls;
bar graphs; Fig. 1, A and B), whereas
the levels of elongation factor II mRNA (Fig. 1A) and
-actin protein (Fig. 1B) were not affected by YC-1. In
the presence of NS2028 the ability of YC-1 to decrease sGC subunit gene
expression was almost completely blocked (Fig. 1, A and
B). These findings show that long lasting activation of sGC
in the rat aorta decreases sGC
1 subunit expression at
the mRNA and protein level.
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Fig. 1.
Influence of YC-1 on
sGC 1 subunit expression in rat
aorta. A, RT-PCR analysis of mRNA isolated from rat
aorta kept for 24 h in organ culture (control), in the presence of
0.2% Me2SO, 10 µM YC-1, or YC-1 and 10 µM NS2028. The upper fluorographs show
ethidium bromide-stained agarose gels containing RT-PCR products of the
sGC
1 mRNA and elongation factor (ef) II
mRNA amplified from 2 µg of total RNA. The bar graph
below shows a densitometric analysis of the sGC
1
mRNA intensity, normalized by ef II mRNA intensity. Summarized
data (mean value ± S.E.) from four rats. *, significantly
different from control (contr.), Me2SO
(DMSO), and YC1/NS2028 (p < 0.05, ANOVA).
B, Western blot. The protein from the same aorta as used for
RT-PCR analysis was separated by SDS-PAGE, and the sGC
1
subunit (82 kDa) was identified by a polyclonal antibody raised in
rabbit. The blot was then stripped for
-actin (47 kDa) to verify
equal loading and blotting efficiency. The bar graph below
shows a densitometric evaluation of the sGC
1-specific
band, normalized by
-actin-staining. Summarized data (mean
value ± S.E.) from four rats. *, significantly different from
control (contr.), Me2SO (DMSO), and
YC1/NS2028 (p < 0.05, ANOVA).
1 mRNA Expression in Rat
Aorta by a Mechanism Requiring Transcription--
To assess whether
YC-1 affects the stability of sGC
1 mRNA, we
investigated the time course of sGC
1 mRNA expression
after inhibition of cell transcription by actinomycin D (Act D). The aortic rings were held in organ culture for 3, 6, or 9 h, in the presence of YC-1 (10 µM), or Act D (10 µM),
or both. The tissue level of sGC
1 mRNA was estimated
by semi-quantitative RT-PCR. As illustrated by the fluorographs in Fig.
2A and the densitometric analysis shown below, the level of sGC
1 mRNA did not
decrease in Act D-exposed aortic rings for up to 9 h,
indicating that the half-life of this mRNA exceeds 9 h when
sGC is not activated. In contrast, in the presence of YC-1 and the
absence of Act D, the sGC
1 mRNA levels decreased
with a half-life of about 6 h. Preincubation (45 min) of the
aortic rings with Act D prevented the YC-1-induced decrease of
sGC
1 mRNA abundance (Fig. 2A). The mRNA levels of elongation factor II remained stable for up to 9 h and were not affected by Act D and YC-1 (Fig. 2A).
These results suggest that YC-1 decreases the stability of
sGC
1 mRNA by a mechanism requiring transcriptional
activation of an unknown factor.
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Fig. 2.
Effect of YC-1 on the
sGC 1 mRNA and protein
expression in rat aorta in the absence and presence of actinomycin
D. Rat aortic rings were exposed in organ culture to 10 µM YC-1 and/or 10 µM actinomycin D for 3, 6, and 9 h, frozen, and processed for RT-PCR and Western blot
analysis of sGC
1. A, the upper
fluorographs show ethidium bromide-stained agarose gels containing
RT-PCR products of the sGC
1 mRNA amplified from 2 µg of total RNA as well as of elongation factor (ef) II
mRNA. The sGC
1 RT-PCR product intensities were
normalized for ef II intensities. Summarized data (mean value ± S.D.) from three rats. *, significantly different from
YC1/act.D (9 h) and act.D (9 h)
(p < 0.05, ANOVA). B, Western blot. The
protein from the same aorta as used for RT-PCR analysis was separated
by SDS-PAGE, and the sGC
1 subunit (82 kDa) was detected
by a polyclonal antibody raised in chicken (IgY). The blot was stripped
and probed for
-actin. The bar graph below shows a
densitometric evaluation of the sGC
1-specific band,
normalized by
-actin staining. Summarized data (mean value ± S.D.) from three rats. *, significantly different from
YC1/act.D (9 h) and act.D (9 h)
(p < 0.05, ANOVA).
1 mRNA decay was
mirrored by a quite similar time course of sGC
1 protein
expression, as assessed by Western blots analysis (Fig. 2B).
In contrast, the expression of
-actin was constant for the same
period of time (Fig. 2B).
1 Poly(A)+
RNA-destabilizing Activity in the Native Protein Extract from Rat
Aorta--
To further corroborate our finding of a YC-1-induced
destabilization/accelerated degradation of sGC
1 mRNA
in the rat aorta, we assessed the effect of a protein extract from
YC-1-exposed rat aorta on the rate of sGC
1 mRNA
degradation. Therefore, total native protein was isolated from a part
of the aortic rings used in the previous experiments (Figs. 1 and 2),
and 20 µg of protein was incubated at 37 °C with 1 µg of
enriched poly(A)+ RNA isolated from rat lung (see
"Materials and Methods"). After different periods of time (10-50
min (Fig. 3A) or 15-45 min
(Fig. 3B)) an aliquot of the incubation mixture was probed
for sGC
1 and elongation factor II mRNA by Northern
blotting (see "Materials and Methods"). In the absence of aortic
protein (control) the sGC
1 mRNA was stable for up to
50 min under these assay conditions (Fig. 3A). In the
presence of protein from aortas exposed to 0.2% Me2SO
(solvent control), a moderate time-dependent decrease in sGC
1 mRNA abundance was observed (Fig. 3,
A and B, "DMSO" lanes). The rate of sGC
1 mRNA decay was considerably
accelerated by protein isolated from YC-1-exposed aortas (Fig. 3,
A and B, "YC-1" lanes). In contrast, elongation factor II mRNA was quite stable even in the
presence of protein from YC-1-exposed aorta (Fig. 3, A and B, lower autoradiographs), indicating that YC-1
specifically induced factors that led to accelerated decay of
sGC
1 mRNA. The formation of these factors was
apparently prevented by a preincubation of the aortas with Act D (Fig.
3A) or NS2028 (Fig. 3B), because under these
conditions the aortic protein extract exhibited markedly less
sGC
1 mRNA degrading activity.
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Fig. 3.
Effect of native protein from YC-1-, NS2028-,
and Act D-treated rat aorta on the
sGC 1 mRNA stability.
Representative Northern blots of sGC
1 subunit (5.5 kb)
and ef II mRNA. Native protein (20 µg) isolated from the same
aortas as used in the experiments shown in Figs. 1 and 2 was incubated
at 37 °C with 1 µg of poly(A)+ RNA isolated from rat
lung, and the amount of sGC
1 and ef II mRNA
remaining after different periods of time (A, 10-50 min;
B, 15-45 min) was assessed by Northern blotting. Two
different experiments yielded qualitatively similar results.
1 mRNA-binding
Protein in Rat Aorta--
The 3'-UTR of the rat sGC
1
mRNA bears several AUUUA motifs (AU-rich elements, AREs) (Fig.
4A), which target the mRNA
for rapid degradation by specific endonucleases (14) and enable regulation of the mRNA stability by trans-acting factors (15). One
specific protective factor is the RNA-binding protein HuR (34-38 kDa)
(10). To investigate whether HuR can interact with 3'-UTR of
sGC
1 mRNA we synthesized a biotin-labeled
oligoribonucleotide comprising bases 3049-3441 of the
sGC
1 mRNA (3UTRSK2, 424 bp, Fig. 4) containing
several AREs. This probe was incubated with native protein extracted
from rat aorta. RNA-protein complex formation was assessed by
electrophoretic mobility shift assays (EMSAs). The free probe migrated
in two bands at the bottom (front) (Fig. 5, lane 1), very likely
representing monomeric and oligomeric forms. In the presence of aortic
protein the probe was retarded (shifted upwards), indicating
interaction with a protein present in the extract. The extent of this
shift was increased with increasing amount of protein added (Fig. 5,
lanes 2 and 3). Addition of an 100-fold excess of
an unlabeled synthetic ARE, [AUUUA]4, to the RNA-protein
mixture prior to electrophoresis prevented the probe shift, indicating
competition between the synthetic ARE and the truncated 3'-UTR of
sGC
1 mRNA (Fig. 5, lanes 4 and
5). When the aortic protein was preincubated (45 min,
4 °C) with a monoclonal HuR antibody, the RNA-protein band was
further retarded (supershifted), demonstrating that HuR forms a complex
with the ARE-containing sequence of sGC
1 mRNA (Fig.
5, lanes 6 and 7).
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Fig. 4.
AU-rich elements (AREs) present in the 3'-UTR
of rat sGC 1 mRNA. The
oligonucleotides (black arrows) for the PCR synthesis of
3UTRSK2 (424 bp) are marked by a gray background. Sequence
data taken from GenBankTM accession number U60835.
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Fig. 5.
HuR present in rat aortic nuclear extract
binds to the 3'-UTR of sGC 1
mRNA. RNA electrophoretic mobility shift analysis (RNA-EMSA)
representative of two experiments. A biotin-labeled RNA probe (3UTRSK2,
424 bp, 250 ng) was incubated for 30 min with nuclear protein (40 and
80 µg) extracted from freshly isolated endothelium-denuded rat aorta
(see "Materials and Methods") then loaded and electrophoresed on a
6% TBE-acrylamide gel. The protein-RNA complexes were electroblotted
onto a nylon membrane and visualized by chemiluminescence, as described
under "Materials and Methods." Lanes (from
left to right): 1, free probe;
2 and 3, probe-shift induced by 40 and 80 µg of
rat aortic protein; 4 and 5, specific shift
blocked by unlabeled competitor [AUUUA]4 probe (25 µg);
6 and 7, supershift induced by a monoclonal HuR
antibody (HuR-AB; 7.5 and 15 µg); and 8, negative control
with aortic protein and HuR antibody.
1 mRNA or down-regulates HuR expression.
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Fig. 6.
HuR binding activity in rat aorta is
decreased by sGC activation. RNA-EMSA performed with protein from
two rat aortas. Rings of endothelium-denuded rat aorta were kept for
12 h in organ culture without additions (control), with solvent
control (0.2% Me2SO), with YC-1 (100 µM), or
with YC-1 and NS2028 (both 100 µM) (see "Materials and
Methods") then frozen and homogenized. Biotin-labeled
sGC 1 RNA from the 3'-UTR (3UTRSK2; 250 ng) was incubated
for 30 min with aortic protein (80 µg), and the EMSA was performed as
in Fig. 6, except that an 8% TBE-AA gel was used. Lanes
(from left to right): 1, free probe;
2 and 3, protein from control aortas;
4 and 5, protein from solvent (0.2%
Me2SO)-treated aortas; 6 and 7,
protein from YC-1-exposed aortas; 8 and 9,
protein from YC1/NS2028-exposed aortas; 10, supershift
induced by addition of 5 µg of HuR antibody to aortic protein from
control aortas. Representative data from three rats.
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Fig. 7.
Activation of sGC in rat aorta decreases
expression of HuR. A, representative Western blot with
aortas from two rats. Endothelium-denuded rat aortic segments were
incubated for 12 h as described in Fig. 6. Total native protein
(40 µg) was probed for HuR using a monoclonal antibody.
Lanes (from left to right):
1 and 2, control aortas; 3 and
4, solvent (0.2% Me2SO)-treated aortas;
5 and 6, YC-1-exposed aortas; 7 and
8, YC1/NS2028-exposed aortas; 9, positive control
(HeLa nuclear extract); 10, marker proteins. Equal protein
loading was verified by immunostaining for -actin (47 kDa) shown
below. B, densitometric analysis of the HuR-specific bands,
normalized by
-actin staining. Summarized data (mean value ± S.D.) from three rats. *, significantly different from control
(contr.), Me2SO (DMSO), and
YC1/NS2028 (p < 0.05, ANOVA).
1--
RNA interference allows targeted genes to be
easily and efficiently "switched off," using short stretches of
double-stranded RNA that contain the same sequence as mRNA
transcribed from the target gene (16). We used this approach to assess
whether specific knockdown of HuR in cultured rat aortic smooth muscle
cells (RASMC) affects expression of sGC. Incubation of RASMC for
24 h with two different HuR siRNA oligonucleotides decreased HuR
expression at the protein (Fig.
8A, "siRNA")
and mRNA level (Fig. 8B, "siRNA"). In the
same cell extracts the expression of sGC
1 protein and mRNA was decreased as well (Fig. 8, A and B),
compared with controls. Expression of actin protein (Fig.
8A) and elongation factor II mRNA (Fig. 8B)
was not affected by HuR siRNA. This finding clearly shows that specific
knockdown of HuR decreases sGC
1 expression in
vascular smooth muscle cells.
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Fig. 8.
HuR RNA silencing by siRNA transfection
decreases sGC 1 expression.
RASMC (16th to 18th passage) were transfected with two different
double-stranded HuR-specific siRNAs (final concentration: 100 nM; second lane from left: siRNA from
S. Sengupta; fourth lane: siRNA as described under
"Materials and Methods") and cultured for 48 h at 37 °C. As
a control RASMCs were incubated without siRNA (C) or with
the transfection reagent only (TR). A, Western
blot. Total native protein (10 µg) was probed for HuR (34 kDa) and
sGC
1 subunit using specific antibodies. Equal protein
loading was verified by immunostaining for smooth muscle actin (47 kDa). B, RT-PCR analysis of mRNA isolated from RASMC.
The graph shows ethidium bromide-stained agarose gels
containing RT-PCR products of the HuR, sGC
1, and
elongation factor II (ef II) mRNA amplified from 2 µg
of total RNA. Representative data out of two experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 and
1 mRNA (17).
1 subunit expression in response to sGC
activation. Our rationale for studying sGC
1 was that in
preliminary studies we found that this subunit was less expressed in
rat vascular tissues than the
1 subunit and, therefore,
formation of the NO-sensitive
1
1
holoenzyme would be limited by the
1 subunit. A
NO-independent activator of sGC, YC-1 (19), was chosen here to avoid
possible interference by cGMP-independent effects of NO on gene
expression (20, 21).
1 mRNA bears several
AUUUA-motifs (AU-rich elements, AREs) that are targeted by trans-acting
factors for regulation of mRNA stability (15, 22). One of these
factors is the ubiquitous 34-kDa protein HuR, which binds to AREs with high affinity and selectivity (10), thereby protecting the respective mRNA from accelerated decay (12). By Western blot analysis we were
able to show for the first time that HuR is constitutively expressed in
the rat aorta. We provide evidence by in vitro mRNA degradation assay and RNA-EMSA that HuR protects the rat
sGC
1 mRNA by binding to ARE present in the 3'-UTR.
Furthermore, we could demonstrate that prolonged (12 h) sGC activation
by YC-1 decreases the expression of HuR protein and HuR binding
activity for sGC
1 mRNA. Consequently, the expression
of the sGC
1 subunit was decreased at the mRNA and
protein level. All these effects could be blocked by an inhibitor of
YC-1-stimulated sGC activity, NS2028 (23), indicating that they were
caused by an increased sGC activity/cGMP formation. In this regard, sGC
is not just another HuR-regulated gene but also a regulator of HuR
expression, linking increased cGMP levels to depression of HuR activity
and lower sGC expression. Although we did not investigate whether
lowering of resting cGMP levels will increase HuR expression, our
findings suggest the existence of a negative feedback loop formed by
sGC and HuR.
1 expression at the mRNA and protein level (Fig. 8). This experiment proves that down-regulation of sGC
mRNA is a consequence of decreased HuR expression.
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ACKNOWLEDGEMENTS |
---|
We thank H.-P. Stasch (Bayer AG, Leverkusen)
for the sGC1-specific rabbit antibody, S. Sengupta
(Dept. of Biochemistry, University of Connecticut Medical School,
Farmington, CT) for providing the second HuR-siRNA, and H. Kleinert
(Dept. of Pharmacology, University of Mainz) for valuable suggestions.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the Deutsche Forschungsgemeinschaft, project C10 of the Collaborative Research Center SFB 553 (to A. M.).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.
¶ To whom correspondence should be addressed. Tel.: 49-69-6301-7423; Fax: 49-69-6301-6050; E-mail: muelsch@em.uni-frankfurt.de.
Published, JBC Papers in Press, November 18, 2002, DOI 10.1074/jbc.M206453200
2 S. Kloess and A. Mülsch, unpublished.
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ABBREVIATIONS |
---|
The abbreviations used are:
sGC, soluble
guanylyl cyclase, GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2;
YC-1, 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole;
NS2028, 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b-1,4)oxazin-1-one;
AU, adenylate-uridylate;
HuR, "Human R" embryonic lethal abnormal
visual (ELAV)-like RNA-binding protein;
NO, nitric oxide;
ARE, AU-rich
element;
UTR, untranslated region;
RT, reverse transcriptase;
MEM, modified Eagle's medium;
MOPS, 3-morpholinopropanesulfonic acid;
TBS, Tris-buffered saline;
3UTRSK2, truncated 3'-UTR of sGC 1
mRNA;
EMSA, electrophoretic mobility shift assay;
Act D, actinomycin D;
RASMC, rat aortic smooth muscle cells;
ANOVA, analysis
of variance;
nt, nucleotide(s);
siRNA, short interfering
RNA.
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