Post-transcriptional Regulation of Soluble Guanylyl Cyclase Expression in Rat Aorta*

Stephan KlößDagger , Henry Furneaux§, and Alexander MülschDagger

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 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 alpha 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 alpha 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 alpha 1 mRNA and protein expression. We conclude that HuR protects the guanylyl cyclase alpha 1 mRNA by binding to the 3'-untranslated region. Activation of guanylyl cyclase decreases HuR expression, inducing a rapid degradation of guanylyl cyclase alpha 1 mRNA and lowering alpha 1 subunit expression as a negative feedback response.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 1 (76-81.5 kDa) and a beta 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).

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 sGCalpha 1 mRNA destabilization induced by increased cGMP formation in isolated rat aorta. We observed that the elav family protein HuR (10) stabilizes the sGCalpha 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 sGCalpha 1 mRNA binding activity, leading to accelerated mRNA degradation.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The polyclonal chicken antibody directed against the alpha 1 and beta 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 sGCalpha 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).

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 -70 °C.

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 sGCalpha 1 mRNA (product size 826 bp), and elongation factor II (225 bp) was performed exactly as described previously (4).

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 alpha 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.

Preparation of sGCalpha 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 sGCalpha 1 cDNA regions. A Pfu DNA polymerase (Pyrococcus furiosus DSM3638, 92 kDa, Promega) with 3'right-arrow5' 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 sGCalpha 1 cDNA, were used (GenBankTM accession number U60835). Subsequently, PCR fragments encompassing the 3'-UTR of sGCalpha 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).

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 sGCalpha 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 alpha 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 alpha -actin immunostaining.

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 -20 °C.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Influence of YC-1 on the Expression of sGCalpha 1 mRNA and Protein in Rat Aorta-- To assess the effect of increased cGMP formation on the sGCalpha 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 sGCalpha 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 (alpha 1 = 82 kDa, Fig. 1B) the abundance of sGCalpha 1 subunit mRNA and protein was markedly lower in YC-1-exposed aorta compared with controls (alpha 1-mRNA = 93% lower, alpha 1-protein = 56% lower versus controls; bar graphs; Fig. 1, A and B), whereas the levels of elongation factor II mRNA (Fig. 1A) and alpha -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 sGCalpha 1 subunit expression at the mRNA and protein level.


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Fig. 1.   Influence of YC-1 on sGCalpha 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 sGCalpha 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 sGCalpha 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 sGCalpha 1 subunit (82 kDa) was identified by a polyclonal antibody raised in rabbit. The blot was then stripped for alpha -actin (47 kDa) to verify equal loading and blotting efficiency. The bar graph below shows a densitometric evaluation of the sGCalpha 1-specific band, normalized by alpha -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).

YC-1 Decreases the sGCalpha 1 mRNA Expression in Rat Aorta by a Mechanism Requiring Transcription-- To assess whether YC-1 affects the stability of sGCalpha 1 mRNA, we investigated the time course of sGCalpha 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 sGCalpha 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 sGCalpha 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 sGCalpha 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 sGCalpha 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 sGCalpha 1 mRNA by a mechanism requiring transcriptional activation of an unknown factor.


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Fig. 2.   Effect of YC-1 on the sGCalpha 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 sGCalpha 1. A, the upper fluorographs show ethidium bromide-stained agarose gels containing RT-PCR products of the sGCalpha 1 mRNA amplified from 2 µg of total RNA as well as of elongation factor (ef) II mRNA. The sGCalpha 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 sGCalpha 1 subunit (82 kDa) was detected by a polyclonal antibody raised in chicken (IgY). The blot was stripped and probed for alpha -actin. The bar graph below shows a densitometric evaluation of the sGCalpha 1-specific band, normalized by alpha -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).

The time course of YC-1-induced sGCalpha 1 mRNA decay was mirrored by a quite similar time course of sGCalpha 1 protein expression, as assessed by Western blots analysis (Fig. 2B). In contrast, the expression of alpha -actin was constant for the same period of time (Fig. 2B).

YC-1-induced sGCalpha 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 sGCalpha 1 mRNA in the rat aorta, we assessed the effect of a protein extract from YC-1-exposed rat aorta on the rate of sGCalpha 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 sGCalpha 1 and elongation factor II mRNA by Northern blotting (see "Materials and Methods"). In the absence of aortic protein (control) the sGCalpha 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 sGCalpha 1 mRNA abundance was observed (Fig. 3, A and B, "DMSO" lanes). The rate of sGCalpha 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 sGCalpha 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 sGCalpha 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 sGCalpha 1 mRNA stability. Representative Northern blots of sGCalpha 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 sGCalpha 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.

Identification of HuR as a sGCalpha 1 mRNA-binding Protein in Rat Aorta-- The 3'-UTR of the rat sGCalpha 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 sGCalpha 1 mRNA we synthesized a biotin-labeled oligoribonucleotide comprising bases 3049-3441 of the sGCalpha 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 sGCalpha 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 sGCalpha 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 sGCalpha 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 sGCalpha 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.

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 GCalpha 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 sGCalpha 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.

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).


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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 alpha -actin (47 kDa) shown below. B, densitometric analysis of the HuR-specific bands, normalized by alpha -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).

HuR Knockdown by RNA Interference Decreases Expression of sGCalpha 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 sGCalpha 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 sGCalpha 1 expression in vascular smooth muscle cells.


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Fig. 8.   HuR RNA silencing by siRNA transfection decreases sGCalpha 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 sGCalpha 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, sGCalpha 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

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 sGCalpha 1 and beta 1 mRNA (17).

We set out to reveal the mechanism accounting for the down-regulation of the sGCalpha 1 subunit expression in response to sGC activation. Our rationale for studying sGCalpha 1 was that in preliminary studies we found that this subunit was less expressed in rat vascular tissues than the beta 1 subunit and, therefore, formation of the NO-sensitive alpha 1beta 1 holoenzyme would be limited by the alpha 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).

The 3'-UTR of the rat sGCalpha 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 sGCalpha 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 sGCalpha 1 mRNA. Consequently, the expression of the sGCalpha 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.

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 sGCalpha 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.

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.

    ACKNOWLEDGEMENTS

We thank H.-P. Stasch (Bayer AG, Leverkusen) for the sGCalpha 1-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.

    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 alpha 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.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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