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Cardiovascular Research 2006 69(2):359-369; doi:10.1016/j.cardiores.2005.10.011
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Copyright © 2005, European Society of Cardiology

Arg–Gly–Asp (RGD)-containing peptides increase soluble guanylate cyclase in contractile cells

María L. Díez-Marquésa, María P. Ruiz-Torresa,c, Mercedes Grieraa, Susana López-Ongilc, Marta Sauraa, Diego Rodríguez-Puyolb,c,d and Manuel Rodríguez-Puyola,*

aDepartment of Physiology, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain
bNephrology Section, Hospital Príncipe de Asturias, Alcalá de Henares, Madrid, Spain
cResearch Unit, Hospital Príncipe de Asturias, Alcalá de Henares, Madrid, Spain
dDepartment of Medicine, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain

* Corresponding author. Departamento de Fisiología, Facultad de Medicina, Universidad de Alcalá, Alcalá de Henares. 28871-Madrid, Spain. Tel.: +34 918854519; fax: +34 8854590. Email address: manuel.rodriguez{at}uah.es

Received 10 March 2005; revised 21 October 2005; accepted 24 October 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objectives: Alterations in NO/cGMP signaling have been associated with vascular dysfunction. Here, we tested whether peptides containing arginine–glycine–aspartic acid (RGD) motifs, commonly found on the binding sites of extracellular matrix to integrins, could increase the expression and function of soluble guanylate cyclase (sGC) in human mesangial cell (HMC), and human aortic smooth muscle (HASMC) cells.

Methods and results: Arginine–glycine–aspartic acid–serine (RGDS) promoted an up-regulation in the sGC β1 subunit steady-state level, both in HMC and HASMC, in a time- and dose-dependent manner. The cellular effects of RGDS-stimulation of sGC expression was an enhanced cellular response to sodium nitroprusside, resulting in elevated cGMP levels and vasodilator-stimulated phosphoprotein (VASP) phosphorylation in both kinds of cells, and an increased NO relaxing effect on cells precontracted with H2O2 or Angiotensin II. Moreover, RGDS was able to restore the sGC levels that had been previously decreased by long term exposure to NO donors. RGDS effects on sGC regulation were due to the specific interaction with {alpha}5β1 integrin. To investigate the intracellular mechanisms activated after RGDS cell treatment, pharmacological kinase inhibitors were used. The effect of RGDS on sGC protein content was completely abolished by treating the cells with c-Jun N-terminal kinase (JNK) inhibitors. In addition, c-fos and c-jun were found in the cell nuclei after RGDS treatment, suggesting that the RGDS effect could be mediated by the AP-1 transcription factor.

Conclusion: Results provide evidence of a mechanism able to increase the sGC protein content linked to increased activity in contractile cells, not only in basal conditions, but also after the down-regulation of the receptor by its own substrate. Elucidation of this novel mechanism provides a rationale for future pharmacotherapy in certain vascular diseases.

KEYWORDS Soluble guanylate cyclase; Nitric oxide; Extracellular matrix; Hypertension; Smooth muscle; Vasoconstriction/dilation

See Editorial by C.S. Packer (pages 302–303) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Cardiovascular disorders are characterized by impaired vasodilatory responses after acetylcholine administration [1–3]. This abnormal response has been usually termed endothelial dysfunction, and is considered to be one of the main pathogenic mechanisms responsible for the abnormal hemodynamic status of patients with hypertension, diabetes or atherosclerosis [4–6]. Acetylcholine induces the synthesis of nitric oxide (NO) by endothelial cells [7], and a decreased synthesis of NO or an increased inactivation of this molecule has been proposed as the main cause of endothelial dysfunction [8–10]. However, alternative mechanisms may also be proposed. NO induces cell relaxation by interacting with its intracellular receptor, soluble guanylate cyclase (sGC), which leads to an increased intracellular cyclic guanosine monophosphate (cGMP) concentration [11]. Hence a reduction in sGC content or an abnormal response of the enzyme after its interaction with NO could also be involved in the previously mentioned vascular dysfunction. Some studies have shown a decreased sGC content in the vascular walls of animals with experimental hypertension or atherosclerosis [12,13,15]. The sGC deficiency seems to be a more generalized phenomenon, since an attenuated glomerular cGMP production and renal vasodilation in streptozotocin-induced diabetic rats has also been demonstrated [16].

The mechanisms responsible for the sGC down-regulation in pathophysiological conditions have not been extensively explored. The best known situation is the decreased sGC content in the presence of its own agonist. The continuous exposure of cells to NO induces an increased degradation of the enzyme [17]. Moreover, in lead-induced hypertension, it seems that reactive oxygen species could be involved in the sGC down-regulation detected [12], as antioxidants prevent the decreased enzyme content elicited by lead treatment in isolated rat aortic segments [18].

Recently, our group has proposed that endothelial dysfunction could be the consequence of the accumulation of abnormal extracellular matrix (ECM) proteins in vascular walls. In fact, cultured human umbilical vein endothelial cells exhibited a decreased NO synthase expression when cultured in collagen type I, when compared with the same cells grown on collagen type IV. This effect depended on integrin activation, and integrin-linked kinase seemed to play a significant role [19]. Increasing evidence supports a role for integrins in the phenotypic modulation of cells, through specific intracellular kinases that transmit signals from the extracellular compartment to the nucleus [20].

In the present study, we propose that the accumulation of abnormal ECM proteins in the vascular walls or glomerular structures might also modulate the sGC content in contractile cells. For that purpose, we tested the effect of an arginine–glycine–asparagine (RGD)-containing polypeptide on the sGC content of cultured human mesangial (HMC) and human aortic smooth muscle cells (HASMC). RGD motifs are present in various ECM proteins, and it is widely recognized that some integrins interact with ECM proteins through these sequences [21]. Thus, cell incubation with soluble RGD-containing polypeptides could mimic the interaction of contractile cells with their surrounding extracellular matrix.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Materials
Arg–Gly–Asp–Ser (RGDS), Arg–Gly–Glu–Ser (RGES), fibronectin, leupeptin, pepstatin A, aprotinin, phenylmethylsulfonyl fluoride (PMSF), 1H-(1,2,4)Oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), collagenase 1A and IV, ammonium persulphate, sodium nitroprusside (SNP), Angiotensin II, herbimycin, genistein, salmon sperm DNA, Triton X-100, formaldehyde, guanidinium thiocyanate, formamide and anti-β-tubulin antibody were purchased from Sigma Chemical (St. Louis, MO, USA). PD98059, SP600125, SB203580, and anti-sGC-β1and {alpha}1 antibodies were from Calbiochem (La Jolla, CA, USA). Anti-{alpha}1, anti-{alpha}v, anti {alpha}5, anti-β1 and anti-c-fos antibodies were from Chemicon (Temecula, CA, USA). Anti c-jun antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-vasodilator-stimulated phosphoprotein (Anti-P-VASP) and anti-VASP were from BD transduction laboratories (Franklin Lakes, NJ, USA). Acrylamide, bisacrylamide and Coomassie blue R-250 were purchased from MERCK (Darmstadt, Germany). RPMI 1640, DMEM/Ham's F-12 medium, fetal calf serum, trypsin-EDTA (0,02%), L-glutamine and penicillin-streptomycin were purchased from Biowhittaker (Walkersville, MD, USA). Culture plates were from Nunc (Kamstrup, Denmark). X-OMAT films were from Kodak (Rochester, NY, USA). Low and high molecular weight standards, the random prime labeling system (Rediprime II), the ECL chemiluminescence system, deoxy-[32P] cytidine triphosphate and the cGMP assay kit were from Amersham-Pharmacia Biotech (Buckinghamshire, UK). Electrophoresis equipment was from Bio-Rad (Richmond, CA,USA). The polyvinylidene difludide membrane was from Perkin Elmer (Boston, MA, USA).The bicinchoninic acid (BCA) assay kit was from Pierce (Rockford, IL, USA). All the reagents employed were of the highest commercially available grade.

2.2 Cell culture
HASMC were a gift of Drs. Peiró and Sánchez-Ferrer. They were cultured at Hospital Universitario de Getafe [22]. In our laboratory, they were maintained in DMEM with 10% fetal calf serum, at 37 °C, in a humidified atmosphere of 5% CO2. Confluent cultures were serially passaged by trypsinization (trypsin-EDTA). Cells between the fifth and tenth passages were used.

HMC were cultured according to previously described procedures [23]. Briefly, portions of macroscopically normal, cortical tissue were obtained from human kidneys immediately after nephrectomy for renal cell carcinoma. Isolated glomeruli were treated with collagenase, plated in plastic culture dishes, and maintained in RPMI 1640, supplemented with 10% FBS, in a 5% CO2 atmosphere. The identity of the cells was confirmed by morphological and functional criteria, as previously described [23]. All procedures were performed conform with the Declaration of Helsinki.

2.3 Protein extraction and Western blot analysis
Following treatment, cells were washed in PBS and solubilized (10 mmol/L Tris–HCl pH 7,4, 1 mmol/L EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 500 nmol/L sodium orthovanadate, 50 nmol/L NaF, 1 mmol/L pepstatin/leupeptin/aprotinin, 1 mmol/L PMSF) for 40 min at 4 °C. Nuclear protein extracts used to assess the c-jun and c-fos nuclear translocation were prepared by hypotonic lysis (15 min of incubation at 4 °C) in 10% Nonidet NP-40, 10 mmol/L Hepes, pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L DTT, 50 mmol/L NaF, 1 mmol/L sodium orthovanadate, 1 mmol/L PMSF, 10 mmol/L aprotinin, 1 µg/ml pepstatin. The nuclear proteins were separated from cytosolic proteins by centrifugation at 4000 x g. Nuclei (pellet) were solubilized by slow rotation for 45 min in hypertonic buffer, 20 mmol/L Hepes, pH 7.9, 1 mmol/L EDTA, 1 mmol/L EGTA, 0.4 mmol/L NaCl, 1 mmol/L DTT, 50 mmol/L NaF, 1 mmol/L sodium orthovanadate, 1 mmol/L PMSF, 10 mmol/L aprotinin at 4 °C. The soluble nuclear extracts were collected (12,000 x g), aliquoted, and stored at –80 °C. The protein concentration was determined spectrophotometrically by the BCA assay.

To perform immunoblots, total cell extracts (20 µg/lane) were size-fractionated by SDS-PAGE on 7.5% polyacrylamide gels and transferred in 20% methanol to polyvinylidene difluoride membranes. The presence of sGC (β1,{alpha}1) was detected by using rabbit anti-sGC antibodies and the peroxidase conjugated goat anti-rabbit Ig. The nuclear and cytosolic content of c-fos and c-jun were evaluated by using a mouse anti-c-fos antibody and a rabbit anti-c-jun antibody, respectively, followed by detection with the appropriate peroxidase-conjugated secondary antibodies. Results were developed with the ECL system and autoradiography. The films were then scanned and analyzed using appropriate software. The blots were stripped and reblotted against β-tubulin antibody to guarantee that an equal amount of protein was loaded in each case. In some experiments, anti-phospho-vasodilator-stimulated phosphoprotein (P-VASP) and anti-VASP antibodies were used.

2.4 RNA extraction and analysis of mRNA expression by Northern blots
Cells were homogenized using guanidinium isothiocyanate, and total RNA was isolated by repeated phenol-chloroform extractions and isopropanol precipitation as described [24]. Total RNA (10 µg per lane) was denatured and electrophoresed through a 1% agarose gel containing 0.66 mol/L formaldehyde. RNA was transferred to a nitrocellulose membrane and UV-cross-linked. The membranes were hybridized at 60 °C in hybridization solution (10% dextran sulphate, 1% SDS, 1 mol/L NaCl, 0,1 mg/ml denatured salmon sperm DNA) with an {alpha}-32[P]-dCTP (106 cpm/ml) radiolabeled sGC DNA probe. A 408 bp cDNA fragment of sGC β1 was obtained by RT-PCR using the following primers [25] (forward, base position 350, 5'-CGT GTC CTG GGC TCT AA-3' and reverse, base position 774, 5'-ACC ACT AGG TCC CGG TG-3'). The nucleotide sequence was determined to confirm that no errors were introduced by PCR amplification. Densitometric analysis of the exposed films was performed with a scanner and analyzed using appropriate software (NIH Image 1.55 from the National Institutes of Health, Bethesda, MD, USA).

2.5 Measurement of cGMP synthesized by sGC
Control and RGDS-treated cells were washed with buffer A (Tris 20 mmol/L, NaCl 130 mmol/L, KCl 5 mmol/L, sodium acetate 10 mmol/L, glucose 5 mmol/L, pH 7.45). Cells were then preincubated with the same buffer containing 2.5 mmol/L Ca2+ and IBMX 10–4 mol/L. The reactions were started by the addition of a NO donor (SNP 10–6 mol/L) for 15 min. The medium was aspirated and 1 ml of ice-cold ethanol was added to the plates and maintained at 4 °C for 30 min. Cell extracts were centrifuged for 20 min at 2000 x g and the supernatant was evaporated to dryness. cGMP levels were determined with a commercial [125I]-cGMP radioimmunoassay kit. Protein concentration was determined by the BCA assay. In some experiments, cells were preincubated for 10 min with 1 µM ODQ, sGC inhibitor.

2.6 Determination of changes in planar cell surface area (PCSA)
Cells were plated in 20-mm plates, and studies were performed before they reached confluence. Cells were incubated under different experimental conditions (see Figure legends) in Tris–glucose buffer (20 mmol/L Tris–HCl, 130 mmol/L NaCl, 5 mmol/L KCl, 10 mmol/L sodium acetate, and 5 mmol/L glucose, pH 7.45) containing 2.5 mmol/L CaCl2 and observed under phase contrast with an inverted PFX model TMS-F photomicroscope (magnification, 150 x; Nikon, Tokyo, Japan). Photographs of cells were taken at the indicated time periods. Every cell with a sharp margin suitable for the planimetric analysis was considered, and 6 to 12 cells were analyzed per photograph. PCSA was determined by computer-aided planimetric techniques. Measurements were performed by two different researchers in a blind fashion. The intraobserver and interobserver variations were 2 and 5%, respectively.

2.7 Statistical analysis
Each experiment was repeated at least three times. The data are expressed as the mean ± S.E. Non-parametric statistics were used for comparisons (Friedman's and Wilcoxon's test). A p<0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 RGDS increases sGC levels stimulating cellular responses to nitric oxide
The effect of RGDS on sGC β1 protein content in both HMC and HASMC is shown in Fig. 1. RGDS induced a significant increase in the sGC β1 subunit level, which was time- (Fig. 1A) and dose-dependent (Fig. 1B). The minimal RGDS concentration that elicited a significant change in sGC was 25 µmol/L, and the effect was detected after 2 h of incubation. RGES, a tetrapeptide similar to RGDS in which glutamic substitutes aspartic acid, did not modify the sGC β1 protein content (Fig. 1C). A slight increase in the sGC {alpha}1 subunit content was observed after cell treatment with RGDS in both cell types, but the differences with respect to control cells were not statistically significant (Fig. 2). Changes in the sGC β1 protein content were paralleled by modifications in its mRNA expression. Thus, RGDS produced an increase in the sGC β1 subunit mRNA expression, with a similar dose-dependent pattern to that observed for the protein content (Fig. 3).


Figure 1
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  		Fig. 1 Panels A and B: RGDS increases the sGC β1 subunit content in HMC and HASMC in a time- and dose-dependent manner. HMC (open bars) and HASMC (hatched bars) were treated with 50 µmol/L RGDS for different periods of time (panel A) or with different RGDS concentrations for 6 h (panel B). The sGC β1 protein content was evaluated by immunoblotting. A representative blot is shown in each case. Bar graphs represent the densitometric analysis of the bands (ratio between the densitometric signal of sGC and β-tubulin) of five independent experiments. The results are expressed as a percentage of control (cells without RGDS treatment) and are the mean ± SE. *p<0.05 vs. C (control cells). Panel C: The observed effect was specific of the RGD sequence. HMC and HASMC were treated with 50 µmol/L RGDS or RGES for 6 h. The sGC β1 subunit protein content was evaluated by immunoblotting. A representative blot is shown (n=4).

 

Figure 2
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  		Fig. 2 RGDS does not significantly increase the sGC {alpha}1 subunit content in HASMC. HASMC were treated with 50 µmol/L RGDS for different periods of time (panel A) or with different RGDS concentrations for 6 h (panel B). The sGC {alpha}1 protein content was evaluated by immunoblotting. A representative blot is shown in each case. Bar graphs represent the densitometric analysis of the bands of five independent experiments. The results are expressed as a percentage of control and are the mean ± SE.

 

Figure 3
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  		Fig. 3 RGDS stimulates the mRNA expression of sGC β1 subunit content in HMC and HASMC in a dose-dependent manner. HMC (open bars) and HASMC (hatched bars) were treated with different RGDS concentrations for 6 h, and sGC β1 subunit mRNA expression was evaluated by northern blotting. A representative blot is shown in each case. Bar graphs represent the densitometric analysis of the bands (ratio between sCG and 18S) of four independent experiments. The results are expressed as a percentage of control and are the mean ± SE. *p<0.05 vs. control.

 
Since the overexpression of the sGC β1 subunit seems to be linked to an increased activity of the enzyme [26], we confirmed this possibility by analyzing some of the cellular effect produced by the modulation of the β1 subunit by RGDS. For this purpose, three types of experiments were performed. First, cGMP synthesis after SNP treatment, in cells either pre-treated or not with RGDS, was measured. HMC (Fig. 4A) and HASMC (Fig. 5A) incubated with RGDS showed a higher cGMP production in response to SNP than control cells. Second, phosphorylation of the PKG substrate VASP at Ser-239, an established biochemical endpoint of NO/cGMP signaling, was analyzed in cells treated with SNP. NO induced a slight increase of VASP phosphorylation in control cells that was significantly stimulated by cell preincubation with RGDS (Figs. 4B and 5BGo). Finally, the ability of low-dose SNP to prevent the contraction induced by hydrogen peroxide on HMC (Fig. 4C) or Angiotensin II on HASMC (Fig. 5C) was tested. SNP alone minimally prevented the reduction of PCSA induced by H2O2 or Angiotensin II. Similar effects were observed in cells preincubated with RGES, however the relaxing SNP effect was magnified after RGDS treatment (Figs. 4C and 5CGo). To test the specificity of the observed effects, cGMP synthesis and VASP phosphorylation were measured in HASMC pretreated with ODQ. In this condition, no changes were detected among the different experimental groups (Fig. 5A and B).


Figure 4
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  		Fig. 4 RGDS enhances the HMC response to SNP treatment. Panel A: HMC were incubated with 50 µmol/L RGDS for 6 h, and then treated with 1 µmol/L SNP during 15 min. cGMP was determined by radioimmunoanalysis. Results are the mean ± SE of four different experiments. *p<0.05 vs. control cells, **p<0.05 vs. SNP. Panel B: HMC were incubated with 50 µmol/L RGDS for 6 h, and then treated with 1 µmol/L SNP during 15 min. VASP phosphorylation was evaluated by immunoblotting. A representative blot is shown. Bar graphs represent the densitometric analysis of the bands (ratio between P-VASP and VASP) of four independent experiments. The results are expressed as a percentage of control and are the mean ± SE. *p<0.05 vs. control. Panel C: HMC were incubated for 6 h with 50 µmol/L RGDS or RGES and then treated successively with 1 µmol/L SNP (5 min) and 100 µmol/L hydrogen peroxide (H2O2, 30 min). The changes in PCSA were determined by computerized planimetric techniques. The results are expressed as a percentage of control cells (without any treatment) and are the mean ± SE of four different experiments. *p<0.05 vs. control, **p<0.05 vs. cells treated with hydrogen peroxide and cells treated with RGDS plus SNP plus hydrogen peroxide.

 

Figure 5
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  		Fig. 5 RGDS enhances the HASMC response to (SNP) treatment. Panel A: HASMC were incubated with 50 µmol/L RGDS for 6 h, and then treated with 1 µmol/L SNP during 15 min, with or without 1 µM ODQ. cGMP was determined by radioimmunoanalysis. Results are the mean ± SE of four different experiments. *p<0.05 vs. control. **p<0.05 vs. SNP. Panel B: HMC were incubated with 50 µmol/L RGDS for 6 h, and then treated with 1 µmol/L SNP during 15 min, with or without 1 µM ODQ. VASP phosphorylation was evaluated by immunoblotting. A representative blot is shown. Bar graphs represent the densitometric analysis of the bands (ratio between P-VASP and VASP) of four independent experiments. The results are expressed as a percentage of control and are the mean ± SE. *p<0.05 vs. control. **p<0.05 vs. SNP. Panel C: HASMC were incubated for 6 h with 50 µmol/L RGDS or RGES and then treated successively with 1 µmol/L SNP (5 min) and 1 µmol/L Angiotensin II (AII, 30 min). The changes in PCSA were determined by computerized planimetric techniques. The results are expressed as a percentage of control cells and are the mean ± SE of four different experiments. *p<0.05 vs. control, **p<0.05 vs. cells treated with Angiotensin II and cells treated with RGDS plus SNP plus Angiotensin II.

 
The sGC stimulation by RGDS was not only evident in basal conditions, but also following the down-regulation of the enzyme by chronic exposure to NO. In Fig. 6, it can be observed that HMC treated with 1 mmol/L SNP for 8 h showed a decreased sGC β1 content. However, when RGDS was added to these cells in the last 6 h, the enzyme content was comparable to that in the control cells. Taken together, these results indicate that RGDS peptides possess the ability to up-regulate sGC content in contractile cells increasing their responses to NO and reversing the detrimental effects of chronic exposure to NO on sGC levels.


Figure 6
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  		Fig. 6 RGDS restores the sGC β1 subunit levels previously decreased by long-term exposure to a NO donor. HMC were exposed to 1 mmol/L SNP for 8 h. Then, in the last 6 h 50 µmol/L RGDS was added. sGC content was analyzed by immunoblotting. A representative blot is shown. Bar graphs represent the densitometric analysis of the bands of four independent experiments. The results are expressed as a percentage of control cells and are the mean ± SE. *p<0.05 vs. control.

 
3.2 RGDS binds to {alpha}5β1 integrin to modulate sGC levels
To analyze the integrins involved in the RGDS-induced up-regulation of sGC, HMC and HASMC were preincubated with different integrin blocking antibodies. As shown in the panel A of Fig. 7, the blockade of the β1 subunit of integrins blunted the stimulatory effect of RGDS. The importance of {alpha}subunits is reflected in the panel B of the figure. Neither the {alpha}1 nor the {alpha}v antibodies modified the RGDS effects on sGC, whereas the {alpha}5 antibody completely blocked the RGDS-induced stimulation of sGC. To further confirm the involvement of integrins in RGDS effects, both cell types were incubated with fibronectin, which binds to integrins through its RGDS motifs. Soluble fibronectin also increased the sGC β1 content (Fig. 7C) confirming the role of integrin binding in the observed effects.


Figure 7
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  		Fig. 7 RGDS increases sGC β1 protein content through interactions with {alpha}5β1 integrin. HMC (open bars) and HASMC (hatched bars) were incubated with 30 µg/ml anti-β1 (panel A), anti-{alpha}1, anti-{alpha}v or anti-{alpha}5 (panel B) blocking antibodies for 4 h; then 50 µmol/L RGES, 50 µmol/L RGDS, or vehicle were added and maintained for an additional period of 6 h. In panel C, cells were incubated with 50 µmol/L RGDS or different doses of soluble fibronectin for 6 h. sGC content was analyzed by immunoblotting. A representative blot is shown in each case. Bar graphs represent the densitometric analysis of the bands of four independent experiments. The results are expressed as a percentage of control and are the mean ± SE. *p<0.05 vs. control.

 
3.3 c-Jun kinase (JNK) activation is involved in RGDS up-regulation of sGC levels
Various pharmacological blockers were used to gain more insight into the intracellular mechanisms activated after the interaction of RGDS with the {alpha}5β1 integrin. The tyrosine kinase inhibitors genistein (Fig. 8A) and herbimycin (Fig. 8B) partially prevented the RGDS effect on the cyclase. A similar effect was observed with PD 98059, an erk-1/2 blocker (Fig. 8C), but not with the p38 inhibitor SB 203580, that did not modify the action of RGDS (Fig. 8D). A complete blockade of the RGDS-induced sGC up-regulation was observed only after inhibiting JNK with SP600125 or curcumin (Fig. 9A). To confirm the central role of JNK as a mediator of the RGDS action, the accumulation of c-fos and c-jun in the cell nuclear extracts was measured after different periods of incubation with 50 µmol/L RGDS. As shown in Fig. 9B and C, two peaks of c-fos and c-jun nuclear accumulation were detected, at 1–5 and 60 min, respectively.


Figure 8
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  		Fig. 8 The RGDS effect on sGC β1 content is partially blocked by tyrosine kinase inhibitors and erk 1–2 inhibitor. HMC were treated with 2 µg/ml genistein (panel A), 1 µmol/L herbimycin (panel B), 50 µmol/L PD 98059 (panel C) or 100 µmol/L SB202190 (panel D) or with their respective vehicles, and 1 h later, 50 µmol/L RGDS was added and maintained for 6 h more. sGC β1 content was evaluated by immunoblotting. A representative blot is shown in each case. Bar graphs represent the densitometric analysis of the bands of four independent experiments. The results are expressed as a percentage of control and are the mean ± SE. *p<0.05 vs. control.

 

Figure 9
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  		Fig. 9 The RGDS effect on sGC β1 content is mediated by JNK activation. Panel A: HMC were treated with the JNK inhibitors, 2 µmol/L curcumin, 10 µmol/L SP600125or their respective vehicles and, 1 h later, 50 µmol/L RGDS was added and maintained an additional 6 h. sGC content was evaluated by immunoblotting. Panels B and C: HMC were treated with 50 µmol/L RGDS for different periods of time. Nuclear proteins were extracted and immunoblots for c-fos (panel B) and c-jun (panel C) were performed. A representative blot is shown in each case. Bar graphs represent the densitometric analysis of the bands of four independent experiments. The results are expressed as a percentage of control and are the mean ± SE. *p<0.05 vs. control.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Soluble GC plays a central role in the regulation of vascular function, since it acts as a NO receptor. Defective NO/cGMP signaling is associated with many forms of vascular disease. However, the regulation of downstream components of this signaling cascade has not been extensively studied. Here we describe an up-regulation of sGC by RGDS that enhances its activity and the cellular responses to NO. The importance of the enzyme down-regulation has been demonstrated in several pathological cardiovascular diseases such as hypertension and late atherosclerosis and has been associated to other pathological conditions [12–16,27]. Decreased enzyme levels in vascular walls would determine a defective relaxation of these structures.

Nitrovasodilator resistance or nitrate tolerance, which is defined as a reduced sensitivity of vascular smooth muscle to vasodilatory effects of NO donors has been attributed to desensitization of soluble guanylate cyclase, by a mechanism that involves both loss of enzymatic activity and mRNA sGC down-regulation due to a decreased stability of mRNAs encoding sGC subunits [28,17]. Considering this information, an impairment of sGC function and/or expression could be viewed as a mechanism involved in vascular dysfunction. Thus, the sGC could constitute a therapeutic target for the reversal of these alterations.

In the present experiments, we tested the possibility that the activation of integrins could induce significant changes in cellular sGC content. The experiments were performed in HASMC and in HMC. In both cell types, RGDS, a tetrapeptide containing the arginine–glycine–aspartic acid (RGD) motif, induced a significant increase of the sGC β1 subunit content, whereas only minor changes were observed in the {alpha}1 subunit. RGD was selected for the study as it is the most widely recognized peptidic motif that interacts with integrins [21]. Fibronectin, that binds integrins through RGD domains, also induced an overexpression of the sGC β1, whereas the stimulatory effect was abolished by inactive analogue (RGES).

As sGC acts as an heterodimer, β1 subunit overexpression could not result in an increased enzyme activity. To test the cellular effects of the sGC overexpression, different experiments were performed. When NO interacts with sGC, cGMP synthesis increases, PKG is activated, and certain cellular actions, such as VASP phosphorylation or cell relaxation, are triggered. After treating the cells with RGDS, the ability of SNP to induce the cGMP synthesis, VASP phosphorylation or cell relaxation significantly increased, thus supporting the hypothesis that the increased β1 subunit content in HMC and HASMC determines an increased activity of sGC. It has been shown that long-term hypercholesterolemia induces sGC overexpression that is not accompanied by an increase in the enzymatic activity [29] suggesting that hypercholesterolemia induces the expression of a dysfunctional sGC. In contrast, our results show that the changes observed in the protein content of the sGC β1 subunit are linked to an augmented activity of the enzyme in the presence of NO donors.

The {alpha}vß1 integrin seems to be the main receptor for RGD and fibronectin in different cell types. In accordance with this notion, a blocking β1 antibody prevented sGC stimulation by RGDS in our cells. However, a blocking {alpha}v antibody, that was effective in the blockade of some RGDS actions in HMC [30], did not prevent the overexpression of sGC after RGDS treatment. An alternative integrin that binds RGD is {alpha}5β1. Experiments with {alpha}5 blocking antibodies confirmed the central role of this heterodimer in the effects of RGDS on sGC. {alpha}1 antibodies were introduced as negative controls, as RGD does not interact with this integrin, and effectively, the stimulatory ability of RGDS remained unaltered in the presence of this antibody.

Integrin activation elicits a complex intracellular response mechanism, in which different tyrosine and ser-threo kinases are involved. To test the relative importance of these pathways different pharmacological antagonists were used. From our experiments, it appears clear that two different jun-kinase/AP-1 inhibitors assayed [31,32] completely abolished the RGDS effect on sGC expression. Even when this kind of pharmacological experiment must be cautiously considered due to the relative non-specificity of the compounds tested, tyrosine kinase and erk-1/2 inhibitors only partially prevented the sGC overexpression whereas the p38 inhibitor did not modify this effect. Thus, a role for some tyrosine kinases can be proposed, perhaps as a previous step to JNK activation. Moreover, RGDS induced a rapid, biphasic, and transient accumulation of c-fos and c-jun at the nuclear level. On the other hand, Sharina et al. explored the regulation of the promoter activity of the sGC β1 subunit [33], and found at least two AP-1 binding sites in the promoter region. Taken together, our results and the previously published information suggest that AP-1 may be involved in the changes observed in sGC after RGDS treatment. However, alternative intracellular pathways might also be involved. RGDS increases integrin-linked kinase activity (ILK) in HMC [30], and it has been demonstrated that ILK induces AP-1 transactivation [34]. Hence, tyrosine kinases and ILK might act simultaneously to increase the promoter activity of the sGC β subunit through AP-1.

Results of the present study provide evidence of a novel mechanism that is able to increase the sGC protein content linked to increased activity in contractile cells. This mechanism of sGC up-regulation occurs not only under basal conditions, but also after the down-regulation of the receptor by its own substrate. As a decreased sGC may be a relevant feature of vascular dysfunction, at least in some pathophysiological conditions [12–16,27], these data provide the basis for future interventions in the reversal of functional abnormalities of certain vascular diseases.


   Acknowledgements
 
This work was supported by grants from the Fondo de Investigaciones Sanitarias (FIS 01/0434), Ministerio de Ciencia y Tecnologia (SAF 2001-0395, BFI 2001-1036, SAF 2004-07-845) and Comunidad de Madrid (CAM 084/0012/2001.2) and Merck Sharp and Dohme Spain (LRU 43/2003). M.P. Ruiz-Torres is supported by Fundación Investigación del Hospital Principe de Asturias, M. Griera was supported by Fundación de la Universidad de Alcalá. M. Saura is supported by the "Ramón y Cajal" program from MCyT. We are indebted to Dr. Peiró and Dr. Sánchez-Ferrer for providing us with the HASMC and Dr. Lylian Puebla for the assistance with the English language.


   Notes
 
Time for primary review 25 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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