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Medizinische Klinik mit Schwerpunkt Kardiologie, Angiologie, Pneumologie, Charité, Campus Mitte, Humboldt-Universität zu Berlin, Germany
1Correspondence: Medizinische Klinik mit Schwerpunkt Kardiologie, Angiologie, Pneumologie, Humboldt-Universität Berlin, Charité, Campus Mitte, Schumannstr. 20-21, D-10117 Berlin, Germany. E-mail: verena.stangl{at}charite.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: endothelium • nitric oxide synthase • inhibitors • vasodilation • proteasome
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4
5)
.
Regulation of eNOS occurs at the transcriptional, post-transcriptional, and post-translational level. Whereas increases in intracellular calcium and phosphorylation induce rapid transient elevation of eNOS activity, allowing fast response to changing environmental conditions (6
, 7)
, sustained alterations are primarily due to changes in the expression level of eNOS protein (8)
.
The ubiquitin–proteasome system represents the major pathway for intracellular protein degradation in eukaryotic cells (9)
. The 26S proteasome consists of a proteolytic core complex, the 20S proteasome, and two 19S regulatory complexes (10
, 11)
. Before degradation, substrates are labeled by conjugation with multi-ubiquitin chains (11)
. Rapid degradation of many rate-limiting enzymes and transcription factors is catalyzed by the proteasome.
Since it is not known whether there is an interaction between eNOS and the proteasome, the aim of our study was to investigate whether eNOS expression and activity are modulated by the ubiquitin–proteasome pathway.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture and treatments
The bovine pulmonary artery endothelial cell line (CPAE cells) was purchased from the American Type Culture Collection (ATCC, 
CCL-209) and cultured in MEM supplemented with 5% FCS, 1.5 g/L sodium bicarbonate, 0.11 g/L sodium pyruvate, 100 U/mL penicillin, and 100 µg/mL streptomycin. For experiments, cells were seeded onto 6 mm diameter dishes and treated confluently 48 h after seeding. Cell viability was assessed by Trypan blue exclusion and the XTT test.
Western blot analysis
After treatment, cells were washed twice with PBS and lysed in extraction buffer containing in mmol/L: Tris/HCl 50 (pH 7.4), KCl 154, glucose 5, EDTA 0.5, PMSF 1, DTT 2, and 1% Triton X-100. Total protein (50 µg/lane) was subjected to SDS-PAGE and membranes were probed with the respective antibodies. Bands were visualized using BCIP and Nitro Blue Tetrazolium (Sigma) for eNOS, Hsp90, and caveolin-1 and by employing the ECL detection system (Amersham) for ubiquitin.
Real-time RT-PCR
After treatment, cells were lysed in Trizol reagent (Gibco Life Technologies, Karlsruhe, Germany), and 800 ng of total RNA was reversed-transcribed with random hexamers. Primer sequences were synthesized by TIB Molbiol (Berlin, Germany). Table 1
summarizes the sequences of primers used in this study. mRNA expression was standardized to the HPRT (hypoxanthine phosphoribosyl transferase) gene as a housekeeping gene, the transcription level of which was not influenced under our experimental conditions. PCR amplification was carried out in 25 µL SybrGreen PCR Master Mix (Applied Biosystems, Foster City, CA, USA) containing either 0.3 or 0.9 µmol/L primer and 1 µL of the reverse transcription reaction in a 5700 Sequence Detection System (Applied Biosystems). Thermal cycling conditions comprised an initial denaturation step at 95°C for 10 min, followed by 95°C for 15 s and 60°C for 1 min for 40 cycles. Size and purity of the amplification products were verified on a 20% PAA gel. The CT (threshold cycle) is defined as the number of cycles required for the fluorescence signal to exceed the detection threshold. Expression of the target gene relative to the housekeeping gene was calculated as the difference between the threshold values for the two genes.
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Measurement of eNOS activity
eNOS activity was assessed by the formation of L-[3H]citrulline from L-[3H]arginine after separation of the amino acids by cation exchange chromatography. Endothelial cells were washed with PBS and lysed in the same extraction buffer as for Western blot analysis. We added 50 µg of total protein to the reaction mixture containing 50 mmol/L HEPES pH 7.4, 0.1% Triton X-100, 1 mmol/L EDTA, 1.25 mmol/L CaCl2, 1 mmol/L DTT, 1 µmol/L FAD, 15 µmol/L BH4, 1 mmol/L NADPH, and 1 µCi L-[3H]arginine. Incubation was performed for 30 min at 37°C. Reactions were terminated by adding 0.5 mL of ice-cold Dowex (Na+ form). L-[3H]Citrulline was separated from L-[3H]arginine by Dowex chromatography and L-[3H]citrulline formation was quantified by liquid scintillation counting. We subtracted the values obtained from samples incubated without cell extracts. We added 1 mmol/L nitro-L-arginine methyl ester (L-NAME) in some reactions to prove the specificity of the reaction. In some samples, CaCl2 was omitted from the reaction buffer to exclude any contribution of inducible NO synthase.
Measurement of proteasome activity in cell lysates
Chymotrypsin-like activity of the proteasome was assessed in cell lysates of CPAE by using the synthetic peptide substrate Suc-Leu-Leu-Val-Tyr linked to the fluorometric reporter aminomethyl coumarin (AMC). Cells were treated with MG132 for the times indicated. Lysates were incubated for 30 min at 37°C in incubation buffer containing an ATP regenerating system (50 mM Tris-HCl, pH 8.2, 18 mM KCl, 3 mM Mg(CH3COO)2, 3 mM MgCl2, 1.1 mM DTT, 6 mM ATP, 5 mM phosphocreatine, 0.2 U phosphocreatinkinase), and 0.2 mM Suc-Leu-Leu-Val-Tyr AMC. AMC hydrolysis was quantified in a Gemini EM microplate spectrofluorometer (Molecular Devices, Sunnyvale, CA, USA) with 355 nm excitation and 460 nm emission wavelengths. Enzymatic activity was normalized to protein concentration and expressed as percent activity of control lysates.
Transcription factor studies
Nuclear extracts were prepared as described (12)
. Protein concentrations were determined by Bradford reagent. For analysis of transcription factors, the TransFactor Profiling Kit Inflammation-2 was used (BD Biosciences Clontech, Palo Alto, CA, USA). In this assay 96-well plates were coated with oligonucleotides containing the consensus binding sequences of several transcription factors. Binding of these transcription factors to their consensus sequence was detected by specific primary antibodies included in the kit and analyzed by an ELISA-based assay. Coated oligonucleotides contained the following consensus sequences: 5'-GGGGGCGGGGC-3' for Sp1, 5'-ATTCCTGTAAG-3' for STAT1, 5'-TGACTCA-3' for c-Jun, c-Fos, and JunD. A total of 20 µg of nuclear extract was used in each experiment and processed according to the protocol of the manufacturer. In brief, nuclear extracts were incubated with the oligonucleotide-coated TransFactor wells for 60 min. The wells were then washed and incubated with the respective primary antibodies for 60 min. After incubation with a horseradish peroxidase-conjugated secondary antibody, a substrate was added to produce a blue color, which can be quantitated with a standard ELISA reader. Sp1, STAT1, c-Fos, and JunD were read at an absorbance of 655 nM and c-Jun was read at 450 nM. The findings show the results of three independent experiments.
Vasorelaxation studies
Thoracic aortas from male Wistar rats were rapidly excised, cleaned of connective tissue, and cut into rings 2 to 3 mm in length for organ chamber experiments. Rings were incubated with MG132 100 nmol/L, 250 nmol/L, or solvent for 48 h in MEM at 37°C containing 50 U/mL penicillin, 50 µg/mL streptomycin, 0.1% BSA, and 1 µg/mL polymyxin B. The rings were then mounted on platinum hooks in 10 mL jacketed organ baths containing modified Krebs-Henseleit solution (composition in mmol/L: NaCl 144, KCl 5.9, CaCl2 1.6, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, and D-glucose 11.1) and diclofenac 1 µmol/L. Tension was gradually adjusted to 2 g over 1 h. The solution in the bath was kept at 37°C with a gas mixture of 5% CO2 and 95% O2. After equilibration and submaximal precontraction with phenylephrine (0.05 µmol/L), relaxation to increasing concentrations (1 nmol/L to 1 µmol/L) of the endothelium-dependent vasodilator acetylcholine was performed to obtain cumulative concentration-response curves. Selected studies were conducted in rings treated with L-NAME (1 mmol/L) before phenylephrine exposure. Maintenance of smooth muscle integrity after incubation with the proteasome inhibitor or solvent was confirmed by evaluation of endothelium-independent vasodilation to papaverine (1 nmol/L to 1 µmol/L).
Statistical analysis
All values are expressed as mean ± SEM compared with controls. Band intensities were analyzed by densitometry. Vasorelaxation is expressed as percentage of precontraction with phenylephrine. Statistical analysis was performed using ANOVA, Mann-Whitney, or Student’s t test where appropriate. A level of P < 0.05 was considered significant in all statistical tests.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). In contrast, higher concentrations of MG132 (i.e., 500 nmol/L and above) decreased eNOS protein levels. Similar results were obtained with human umbilical vein endothelial cells (HUVECs; data not shown). ALLM, a nonproteasomal calpain and cathepsin inhibitor, failed to up-regulate eNOS (Fig. 1A
). Since proteasome inhibitors are known to exert cytotoxic effects, we further investigated whether the CPAE cells were damaged by MG132 in the doses applied by us. Cell viability as assessed by XTT testing showed that doses of >250 nmol/L reduced cell viability below control levels (Fig. 1B
). Similar results were obtained by Trypan blue exclusion (data not shown). All further experiments were accordingly performed at low nontoxic MG132 concentrations of 
250 nmol/L. Time- and dose-dependent experiments revealed that the rise in eNOS protein expression began after 16 h of incubation with MG132 and continued to increase for up to 48 h with 2.4-fold expression (P<0.05) occurring at 100 nmol/L MG132 (Fig. 1C
). This up-regulation was observed after incubation of the cells with a single dose of MG132. To demonstrate that eNOS up-regulation is specific to inhibition of the proteasome, we repeated the experiments with other proteasome inhibitors: MG262, a boronic acid derivative of MG132, epoxomycin, and lactacystin. Indeed, all proteasome inhibitors in equipotent doses induced up-regulation of eNOS protein after 24 h to an extent similar to that caused by MG132 (Fig. 1D
). Although there are reports that eNOS expression is influenced by the proliferation state of the cells (13)
and that proteasome inhibition leads to cell cycle arrest (14)
, we found no difference in eNOS expression and activity in proliferating vs. confluent CPAE (data not shown).
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We demonstrated the efficacy of proteasome inhibition with very low concentrations of MG132 used in our experiments: doses from 50 to 250 nmol/L that increased eNOS expression also led to accumulation of poly-ubiquitinylated proteins. In contrast, 20 nmol/L of MG132, which did not affect eNOS expression, had no effect (Fig. 2
A). Measurement of the chymotrypsin-like activity of the proteasome in cell extracts revealed only a partial inhibition of proteasomal activity at the low concentrations used in our study (Fig. 2B
). The higher sensitivity of this method allowed detection of a slight decrease (
25%) in proteasomal activity at levels as low as 20 nmol/L of MG132.
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To investigate whether up-regulation of eNOS protein after proteasome inhibition is due simply to accumulation of eNOS after inhibition of protein degradation, we measured eNOS mRNA levels in CPAE cells. Dose-dependent up-regulation of eNOS mRNA was determined after incubation with MG132. Up-regulation began at 16 h (data not shown), reached a maximum (6.5-fold the control level) at 24 h (100 nmol/L MG132, P<0.05), and remained elevated after 48 h (Fig. 3
A, B). Similar results were obtained with HUVECs (data not shown). The 6.5-fold up-regulation of eNOS mRNA after 24 h indicates transcriptional responses. To investigate possible transcription factors involved, cells were treated with 100 nmol/L MG132 for 24 h and nuclear extracts were prepared. Treatment with MG132 induced nuclear translocation of Sp1 and, to a lesser extent, JunD, whereas c-Fos, c-Jun, and Stat1 were not affected (Fig. 3C
). Treatment of cells with cycloheximide, which blocks protein synthesis, abolished up-regulation of eNOS expression after proteasome inhibition, indicating that de novo protein synthesis is required for the increase in eNOS protein levels (Fig. 3D
). The expression of two other endothelial cell proteins associated with eNOS activity, Hsp90 and caveolin-1, was not changed after 24 h of proteasome inhibition (Fig. 3E
).
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Increased eNOS enzymatic activity and improved endothelial function by proteasome inhibition
We next posed the question of whether the increased eNOS protein content observed after proteasome inhibition has a functional consequence in terms of enhanced enzyme activity. We measured eNOS catalytic activity in protein extracts by conversion of L-[3H]arginine to L-[3H]citrulline. In parallel with the higher eNOS protein content after MG132 treatment, eNOS activity increased in a dose-dependent manner beginning at 24 h, reaching a 2.8-fold increase at 48 h (100 nmol/L MG132, P<0.05) (Fig. 4
A, B). Similar results were obtained with other proteasome inhibitors (lactacystin, MG262, epoxomycin) (data not shown).
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To further elucidate the functional importance of eNOS regulation by the proteasome, the effect of proteasome inhibition on the vascular reactivity of isolated rat aortic ring preparations was investigated in organ chamber experiments (n=6–8/group). As shown in Fig. 5
A, the endothelium-dependent relaxant responsiveness of phenylephrine precontracted rings to the endothelium-dependent vasodilator acetylcholine was significantly enhanced after MG132 pretreatment (100 and 250 nmol/L, 48 h) in a dose-dependent manner: maximum relaxation in MG132-pretreated rings (250 nmol/L) was 89.4 ± 5.7% vs. 54.6 ± 5.8% for vehicle-treated rings (P<0.05). The NO synthase inhibitor L-NAME eliminated acetylcholine-induced vasodilation (data not shown). Endothelial-independent vasorelaxation in response to papaverine remained unaffected by proteasome inhibition (Fig. 5B
).
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Long-term up-regulation of eNOS expression and activity
To assess how long a single dose of the proteasome inhibitor MG132 is able to up-regulate eNOS expression and activity, experiments were extended up to day 10. Surprisingly, we still found enhanced protein levels as well as enzyme activity after proteasome inhibition (100 nmol/L MG132) in CPAE cells after 10 days (Fig. 6
A, B). eNOS mRNA was still increased after 7 days (data not shown). Long-term eNOS up-regulation was observed even though accumulation of poly-ubiquitinylated proteins was detectable for no longer than 3 days (Fig. 6C
). However, measurement of the chymotrypsin-like activity of the proteasome revealed reduced proteasomal activity, which gradually increased over time and reached control level after 7–10 days (Fig. 6D
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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16
17)
. We observed nuclear translocation of the transcription factors Sp1 and JunD after inhibition of the proteasome. Since the eNOS promoter contains consensus binding sites for Sp1 and AP1 (18)
, our results suggest that these two transcription factors may be involved in the transcriptional up-regulation of eNOS mRNA after inhibition of the proteasome.
Up-regulation of eNOS by proteasome inhibition occurs after 16 to 24 h, with results on the order of magnitude as described for potent drugs such as statins (19)
. Considering that even a 30% change in eNOS activity can induce corresponding increases in blood flow (20)
, the rise in eNOS activity by 200–300% observed after proteasome inhibition is important. We accordingly observed a significant increase in endothelial-dependent vasorelaxation in MG132-pretreated rat vascular rings.
In the present study we performed experiments with several endothelial cell types to demonstrate that the effects of proteasome inhibitors are prevalent throughout various vascular beds and species. The similarity of our results among species and the different proteasome inhibitors indicates that eNOS up-regulation secondary to proteasome inhibition follows a more general, even ubiquitous, principle.
To avoid cytotoxicity, we determined the lowest inhibitor doses capable of enhancing eNOS expression. Surprisingly, in our endothelial cell cultures eNOS was up-regulated starting at very low inhibitor doses of 50 nmol/L MG132. Inhibition of the chymotrypsin-like activity of the proteasome and accumulation of poly-ubiquitinylated proteins occurring at these low concentrations indicate that this eNOS up-regulation was indeed attributable to inhibition of the proteasome. When incubating endothelial cells with higher doses of proteasome inhibitors (e.g., 500 nM of MG132 for 24 h), we observed dose-dependent cytotoxic effects as assessed by XTT test and Trypan blue exclusion. Accordingly, up-regulation of the eNOS protein was prevented under these toxic conditions.
We hypothesize that despite targeting a common, basic regulatory system such as the proteasome, specific beneficial effects occur in endothelial cells at low inhibitor doses. Since the expression of Hsp90 and caveolin-1, two endothelial proteins involved in regulation of eNOS activity, is not significantly modulated by MG132, we suggest that proteasome inhibition leads to specific effects on eNOS expression without necessarily affecting overall up-regulation of cellular proteins. One may speculate that proteasome inhibitors in such low doses act only by partial inhibition in a subunit-specific manner and/or by differentially blocking subtypes of the 20S proteasome. Indeed, we found only partial inhibition of the catalytic activity of the proteasome at these concentrations. The residual proteasomal activity may be sufficient to allow the cell to cope with cellular demands for protein degradation.
Slight inhibition of the chymotrypsin-like proteasomal activity was observed at 20 nmol/L MG132, a dose at which we observed no effect on eNOS expression. This result suggests that some kind of threshold is required for proteasomal inhibition. In our study, physiological effects of proteasome inhibitors are observed only >20 nmol/L of MG132.
We think it is of particular interest that a single low dose of MG132 up-regulates eNOS expression for up to 10 days. Although ubiquitinylated proteins were observed only until day 3, measurement of chymotrypsin-like activity disclosed decreased proteasomal activity, with a gradual increase over time and a return to basal levels after 7–10 days. This finding, together with the above-stated observation, reflects the higher sensitivity of the method using synthetic peptide substrates. The parallel time course between proteasome inhibition and eNOS up-regulation suggests that this long-term eNOS up-regulation is a proteasome-dependent effect. We are not aware of another substance for which similar long-term up-regulation of eNOS has been demonstrated.
In summary, we have described in this study that inhibition of the ubiquitin–proteasome system has important effects on endothelial cells, in terms of increase of eNOS expression and activity, with consequent improvement of endothelial function. Up-regulation of eNOS after proteasome inhibition occurs via de novo protein synthesis. Increase in eNOS protein is preceded by elevation of mRNA levels, indicating that transcriptional and translational responses are involved in mediating augmented eNOS abundance after proteasome inhibition.
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ACKNOWLEDGMENTS |
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Received for publication March 5, 2003. Accepted for publication October 8, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B dependent mechanism. Intensive Care Med. 27,751-756[CrossRef][Medline]
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