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Superoxide targets calcineurin signaling in vascular endothelium
BBRC Biochemical and Biophysical Research Communications 334 (2005) 1061–1067 www.elsevier.com/locate/ybbrc
Superoxide targets calcineurin signaling in vascular endothelium Dmitry Namgaladze a,*, Ivanna Shcherbyna b, Joachim Kienho¨fer b, H. Werner Hofer b, Volker Ullrich b a
Faculty of Medicine, Institute of Biochemistry I, Johann Wolfgang Goethe-University, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany b Faculty of Biology, University of Konstanz, D-78457 Konstanz, Germany Received 29 June 2005 Available online 14 July 2005
Abstract Superoxide emerges as key regulatory molecule in many aspects of vascular physiology and disease, but identification of superoxide targets in the vasculature remains elusive. In this work, we investigated the possibility of inhibition of protein phosphatase calcineurin by superoxide in endothelial cells. We employed a redox cycler 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) to generate superoxide inside the cells. DMNQ caused inhibition of cellular calcineurin phosphatase activity, which was reversible upon DMNQ removal. Inhibition was suppressed by pre-incubating the cells with copper/zinc superoxide dismutase (Cu,ZnSOD). In addition, reducing cellular Cu,ZnSOD activity by diethylthiocarbamic acid treatment resulted in calcineurin inhibition and enhanced sensitivity to DMNQ. Further, we could show that DMNQ inhibits calcineurin-dependent nuclear translocation and transcriptional activation of NFAT transcription factor, and Cu,ZnSOD or superoxide scavenger Tiron reduced the inhibition. Thus, superoxide generation in endothelial cells results in inhibition of calcineurin signaling, which could have important pathophysiological implications in the vasculature. 2005 Elsevier Inc. All rights reserved. Keywords: Protein phosphatase; Superoxide; Oxidative stress; Endothelium; Redox regulation; Calcineurin; Calmodulin; NFAT; Superoxide dismutase; Vascular disease
Superoxide anion-radical is a small reactive molecule formed in vivo by univalent reduction of molecular oxygen via a variety of enzymatic systems. Increased superoxide generation is one of the major factors in the development of vascular disease, such as atherosclerosis, hypertension or diabetes [1,2]. The deleterious effect of superoxide is attributed mostly to its reaction with nitric oxide (NO). The consequence of this reaction is not only the consumption of NO and impairment of its vasorelaxating and antiatherogenic properties, but also the formation of peroxynitrite, a potent oxidant able to react with lipids, proteins, and nucleic acids [3,4]. Comparatively less attention is paid to direct reactions of superoxide with proteins. Identification of protein targets of superoxide should increase our understanding of super*
Corresponding author. Fax: +49 69 6301 4203. E-mail address: [email protected] (D. Namgaladze).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.07.003
oxide role in vascular (patho)physiology. Several protein targets of superoxide play important role in metabolism and signal transduction, indicating potential physiological importance of their interaction with superoxide. Some of the best known are Fe–S cluster proteins, particularly aconitase [5,6], which are rapidly oxidized and lose iron in response to superoxide, resulting in reduced mitochondrial function and altered iron homeostasis. Other targets include protein tyrosine phosphatases [7] and protein kinase C (PKC) [8], key players in intracellular signal transduction. However, the specificity of these enzymes for superoxide in cellular context remains unclear, since all of them also react with a variety of reactive oxygen and nitrogen species. We have found recently that superoxide potently inhibits enzymatic activity of the protein serine/threonine phosphatase calcineurin (CaN, also protein phosphatase 2B) [9]. CaN is an ubiquitous protein involved in a
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plenitude of physiological processes, including regulation of gene expression, muscle remodeling, neuronal activity, and apoptosis [10–13]. CaN also plays a critical role in vascular development, particularly in vascular patterning [14], and is involved in VEGF-induced expression of inflammatory markers [15] and angiogenesis [16]. In these cases, CaN action is mediated by activation of transcription factors of the NFAT family. CaN directly dephosphorylates multiple serine residues of NFAT, resulting in its nuclear translocation and activation of transcriptional activity [13]. Thus, CaN–NFAT signaling pathway is involved in many aspects of vascular physiology. The CaN active site contains a binuclear iron–zinc center, which is critical for catalytic activity. We have shown that in the native enzyme the iron of this center is Fe2+, and it is rapidly oxidized by superoxide to Fe3+, which is unable to support CaN catalysis [9]. In vitro CaN is much more sensitive to superoxide as compared to hydrogen peroxide or peroxynitrite, which is probably due to the formation of l-oxo complex bridging Fe3+ and Zn2+ of the binuclear center. This provides a biochemical basis for previous work by Klee and coworkers [17] who showed that copper/zinc superoxide dismutase (Cu,ZnSOD) protects CaN against inactivation. Although in vitro data indicate that CaN might be targeted by superoxide, little information exists regarding the possibility of this reaction in intact cells and its importance for cell physiology. It was proposed that CaN inhibition by superoxide might regulate phosphorylation of transcription factor CREB in hippocampal neurons [18,19]. In addition, redox sensitivity of CaN signaling was shown in T-cells [20] and neutrophils [21], but in this case exogenous hydrogen peroxide was applied. The possibility of specific regulation of CaN by intracellular superoxide in non-neuronal cells, particularly in the vascular system, therefore remains unclear. In this work, we investigated the effect of intracellular superoxide generation on CaN activity in endothelial cells. We employed the redox-cycling agent 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) to generate superoxide inside the cells. In addition, we investigated the effects of DMNQ on CaN-dependent nuclear translocation and transcriptional activity of NFAT. Finally, we tested whether modulation of intracellular Cu,ZnSOD activity could influence CaN-dependent signaling in vascular endothelium.
(50 mM Tris–HCl, pH 7.5, 0.2 mM EGTA, 50 lg/ml PMSF, and 10 lg/ml leupeptin) and lysed by three cycles of freezing–thawing in liquid N2. The suspension was centrifuged for 10 min at 18,000g and 4 C, and the supernatant was collected. After measuring the protein with Bradford reagent (Bio-Rad), its concentration was adjusted to 0.5–1 mg/ml with the lysis buffer. Twenty microliters of protein solution was added to 20 ll assay buffer containing 50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 1 mg/ml bovine serum albumin, 6 lM 32P-phosphorylated RII peptide (DLDVPIPGRFDRRVSVAAE), 1 lM okadaic acid, and 0.4 mM CaCl2, and incubated for 5–10 min at 30 C. The reaction was stopped by adding 50 ll of 20% DOWEX 50-X8 (200–400 mesh, Bio-Rad) in 10% trichloroacetic acid. The mix was centrifuged for 6 min at 18,000g, and 40 ll of supernatant was taken for Cherenkov counting. The amount of protein was adjusted so that the substrate consumption did not exceed 25%. Duplicate cpm values from the phosphatase assay were averaged, and the resulting value was adjusted by subtracting the counts in blanks measured in the presence of 5 mM EGTA. Superoxide measurement. BAEC were incubated in 6-well plates in HBSS with DMNQ for 0.5 h and labeled with 2 lM hydroethidine for another 0.5 h. After washing and trypsinization, cell suspension was subjected to analysis by flow cytometry using FACScan (Becton– Dickinson) with excitation at 488 and emission at 585 nm. Cells (10,000) of gated population were counted. SOD activity gels. BAEC incubated in 6-well plates were scraped into PBS, and subjected to native electrophoresis on polyacrylamide gels. SOD activity in these gels was assayed as described [23]. Immunofluorescence. HUVEC were grown on 8-well chamber slides (Lab Tek II, Nunc). After treatments cells were washed with PBS, fixed in 4% paraformaldehyde/PBS, and permeabilized with 0.5% Triton X100/PBS. After blocking with 5% BSA/PBS, cells were incubated for 60 min with 1:200 dilution of anti-NFAT-1 monoclonal antibody (N58820, Transduction Laboratories) followed by 1:300 dilution of anti-mouse antibody conjugated with Alexa Fluor 568 (Molecular Probes). Fluorescence was observed using 25· objective with a Leica DMIRB microscope equipped with a digital camera (Visitron System). Luciferase reporter assay. BAEC seeded on 12-well plates were transfected using Fugene 6 transfection reagent (Roche) according to the manufacturerÕs instructions. 1.5 lg pNFAT-luc reporter plasmid (provided by Dr. Gerald Crabtree, Stanford University) and 0.15 lg pRL-TK Renilla luciferase reporter plasmid (Promega) were used together with 1 lg pSOD1 plasmid in pcDNA6/Myc-His vector (provided by Dr. Thomas Kietzmann, University of Go¨ttingen, Germany) or with 1 lg control vector. After 24 h, cells were stimulated with 32 nM phorbol 12-myristate 13-acetate (PMA) and 2 lM A23187 for another 6 h. Reporter activity was measured using a Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase reporter activities were normalized against Renilla luciferase activities. Statistical analysis. Results are expressed as means ± SEM. Significance was determined by using the Student t test, and the level of statistical significance was defined as P < 0.05.
Results Intracellular superoxide generation by DMNQ
Experimental procedures Cell culture. Primary bovine aorta endothelial cells (BAEC) and human umbilical vein endothelial cells (HUVEC) were isolated as described in [22] and maintained in DMEM containing 10% FBS and penicillin/streptomycin (BAEC) or in EGM-2 medium (Clonetics) (HUVEC). Cells of passages 1–2 were used. Calcineurin activity. Calcineurin activity in cell lysates was measured as described [9]. Briefly, cells were scraped into lysis buffer
In order to investigate the effects of intracellular superoxide on CaN activity in BAEC, we used the redox-cycling quinone DMNQ. In the cell DMNQ undergoes reduction to its semiquinone and hydroquinone species, and those are in turn oxidized by molecular oxygen with concomitant superoxide generation [24]. Intracellular production of superoxide in BAEC treated with
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DMNQ was determined by flow cytometry using hydroethidine (HE), which reacts with intracellular superoxide giving rise to a fluorescent product [25]. Measurements in HE-labeled BAEC showed that HE oxidation was significantly increased after treatment with 10 lM DMNQ (Fig. 1). DMNQ-induced HE oxidation was suppressed by pre-incubating the cells for 4 h with Cu,ZnSOD, validating HE specificity for superoxide detection. These data confirm that DMNQ induces elevation of intracellular superoxide in BAEC, which can be prevented by increasing cellular Cu,ZnSOD activity. Effects of DMNQ on calcineurin activity in endothelial cells When applied to BAEC culture, DMNQ caused concentration-dependent inhibition of CaN activity in cell lysates (Fig. 2A). The magnitude of DMNQ inhibition reached levels comparable to maximal inhibitory effect of cyclosporin A and FK506, two well-known pharmacological inhibitors of CaN activity (data not shown). The range of effective DMNQ concentrations was similar to that reported previously [26]. CaN protein levels in cell lysates determined by Western blotting were not changed after DMNQ treatment (data not shown). A similar inhibitory effect on CaN activity in endothelial cells had another redox-cycling agent, menadione. In this case, 20 lM menadione reduced CaN activity to 14.9 ± 4.9% of control after 1 h incubation. The inhibition of cellular CaN activity by DMNQ was reversible. As Fig. 2B shows, following DMNQ removal CaN activity returned to the level observed before treatment already after 15 min. This indicates that redox homeostasis in BAEC is not disturbed by DMNQ, and the cell is able to restore CaN activity when oxidative insult is eliminated.
Fig. 1. DMNQ induces elevation of superoxide levels in BAEC. Cells were treated with 10 lM DMNQ for 0.5 h followed by 0.5 h incubation with 2 lM hydroethidine and analysis by flow cytometry. *Significantly different from control. **Significantly different from DMNQ (n = 4).
Fig. 2. Inhibition of CaN phosphatase activity by DMNQ in BAEC. (A) Cells were treated with DMNQ at indicated concentrations for 1 h, and phosphatase activity of cell lysates was measured using RII phosphopeptide (n = 3). (B) Cells were treated with DMNQ at 10 lM for 1 h followed by medium change and further incubation for indicated times (n = 3).
Redox-cycling agents generate not only superoxide, but also hydrogen peroxide (H2O2), which might inhibit CaN activity [20,27]. To prove the role of superoxide in CaN inhibition by DMNQ, we pre-incubated the cells with 1 mg/ml Cu,ZnSOD (5000 U/ml) for 4 h before DMNQ treatment. It was shown previously [28] that under these conditions intracellular SOD activity rises approximately 2-fold with no further increase at longer pre-incubation times. Intracellular accumulation of Cu,ZnSOD was confirmed by Western blot and NBT activity gels (data not shown). Pre-incubation with Cu,ZnSOD significantly decreased the inhibitory effect of DMNQ on CaN activity (Fig. 3). Another strategy to increase intracellular superoxide levels is to inhibit endogenous SOD activity. For this purpose, we employed diethylthiocarbamic acid (DDC), an inhibitor of Cu,ZnSOD, which acts by extracting copper out of the enzymeÕs active site. Application of DDC to BAEC resulted in inhibition of SOD activity as judged by SOD activity gels (Fig. 4A). When cells were treated with DDC, CaN activity in the cell lysate was suppressed (Fig. 4B). Furthermore, DDC
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120
*
Relative activity, %
100 80 60 40 20 0
Control
DMNQ
+SOD
Fig. 3. Cu,ZnSOD protects CaN against inhibitory action of DMNQ. BAEC were pre-incubated with 5000 U/ml Cu,ZnSOD for 4 h followed by 1 h incubation with 10 lM DMNQ, and phosphatase activity of cell lysates was measured using RII phosphopeptide. *Significantly different from DMNQ (n = 3).
ment of NFAT activation, we examined CaN-dependent NFAT-1 nuclear translocation in human umbilical vein endothelial cells (HUVEC) by immunofluorescence microscopy. The phosphatase activity test showed that the inhibitory action of DMNQ in these cells was comparable to that in BAEC (data not shown). Fig. 5A shows that in resting cells NFAT-1 is evenly distributed throughout the cell body with nuclei occasionally excluded. Stimulation with calcium ionophore caused nuclear translocation of NFAT-1 (Fig. 5B), which was fully suppressed by CsA (data not shown). Treatment with DMNQ inhibited ionophore-stimulated translocation (Fig. 5C), while pre-treatment with superoxide scavenger Tiron could restore the translocation in the presence of DMNQ (Fig. 5D). To analyze the influence of superoxide on NFAT transcriptional activity we used a luciferase reporter assay, in which the expression of luciferase was driven by IL-2 promoter region containing 3 tandem NFAT/ AP-1 sites. Stimulation of BAEC transfected with a NFAT-luc construct with a mixture of calcium ionophore and phorbol ester resulted in approximately 10fold elevation of reporter activity (data not shown). Pre-treatment of the cells with DMNQ caused dose-dependent inhibition of reporter activity (Fig. 6). Similar data were obtained when calcium ionophore alone was used to stimulate reporter expression (data not shown). When BAEC were transfected with Cu,ZnSOD-encoding plasmid inhibition of reporter activity by DMNQ was significantly reduced. The protective effect of SOD was less pronounced at 5 lM DMNQ; this probably reflects inhibition of reporter activation by H2O2 formed in substantial amounts when increasing concentrations of DMNQ are used. Taken together, the results suggest substantial involvement of superoxide in inhibition of CaN-dependent NFAT activation by DMNQ.
Discussion Fig. 4. Inhibition of Cu,ZnSOD by DDC reduces CaN activity. (A) representative SOD activity gel of BAEC treated with DDC for 1 h. (B) Cells were incubated with 5 mM DDC, 5 lM DMNQ or both substances together for 1 h, and phosphatase activity of cell lysates was measured using RII phosphopeptide (n = 3).
enhanced the inhibitory action of DMNQ. DDC alone did not affect phosphatase activity of isolated porcine CaN (data not shown), thus ruling out the possibility that its metal-chelating properties directly affect the CaN binuclear metal center. Inhibition of calcineurin-mediated signaling by DMNQ Next, we wanted to investigate whether signaling of CaN to NFAT is affected by superoxide. To investigate whether superoxide inhibition of CaN results in impair-
The major finding of this study is the ability of superoxide to inhibit CaN activity and CaN-dependent signal transduction in vascular endothelium. Although the effects of superoxide on vascular function via its reaction with NO are widely recognized less attention is paid to direct reactions of superoxide with enzymatic targets. In our previous work with isolated CaN in vitro, we found that CaN is potently inhibited by superoxide, and is much less sensitive to other ROS, including H2O2 and peroxynitrite. However, the conditions in the in vitro system are quite different from those of cellular milieu, where superoxide levels are kept low due to the presence of SOD. In addition, ascorbic acid and NO, both present in endothelium, should protect CaN against inhibitory action of superoxide [9]. Still, the results of the present study show that excess superoxide
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B
C
D
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Fig. 5. DMNQ inhibits calcium ionophore-stimulated NFAT-1 nuclear translocation in HUVEC. Cells were pre-incubated for 1 h with 5 mM Tiron followed by 1 h treatment with 5 lM DMNQ. NFAT nuclear translocation was induced by 20 min treatment with 2 lM A23187. Cells were fixed and NFAT-1 localization was analyzed by immunofluorescence microscopy using anti-NFAT-1 antibody. (A) Control cells. (B) A23187-treated cells. (C) Cells treated with DMNQ before A23187 stimulation. (D) Cells pre-treated with Tiron before DMNQ treatment.
Control SOD
Reporter activity, % of control
120 100
*
80 60
*
40 20 0
Control
DMNQ 2
DMNQ 5
Fig. 6. DMNQ inhibits PMA- and A23187-stimulated activity of NFAT-luc reporter in BAEC. Cells were transfected with Cu,ZnSOD plasmid or with control vector together with pNFAT-luc and pRL-TK plasmids for 24 h, treated with DMNQ at 2 lM or 5 lM for 1 h and stimulated subsequently with a mix of 32 nM PMA and 2 lM A23187 for 6 h. Data are expressed as normalized reporter activity. *Significantly different from respective DMNQ values in vector-transfected cells (n = 3).
generation is able to overwhelm these defence systems and cause CaN inhibition. This work was done by using DMNQ as a trigger of superoxide production. This is likely to model a situation of oxidative stress, such as ischemia/reperfusion, which is accompanied by massive generation of reactive oxygen species (ROS). Indeed, it was reported that CaN activity in hippocampus is decreased by ischemia [29], although the involvement of superoxide in this process was not investigated. Increased ROS formation in the
vasculature underlies several pathophysiological conditions, including hypertension, coronary artery disease, atherosclerosis, or diabetes. The major feature of superoxide-mediated processes is the local action of superoxide. In contrast, CaN is a highly abundant enzyme likely to be present at micromolar concentrations in the cytosol. Thus, localized fluxes of superoxide might not be able to affect total CaN activity measured in cell lysates. However, CaN is known to be present in signaling complexes, such as those assembled around AKAP proteins [30]. If such signaling complexes are located close to superoxide sources, there is a possibility of local CaN inhibition by superoxide. In this case, only localized dephosphorylation of specific substrates would be affected. The other substantial finding is the reversibility of CaN inhibition by superoxide in the cell. Generally, reversibility is a major criterion for a particular enzyme modification to be involved in the physiological regulatory mechanisms. This is especially important for the emerging field of redox signaling, where reversible oxidative modifications of proteins, like formation of cysteine sulfenic acid or cyclic sulfenyl-amide [31] are opposed to non-specific oxidative damage to the proteins, such as protein carbonyl formation or cysteine oxidation to sulfonic acid [32]. Thus, rapid recovery of CaN activity after removal of oxidative signal indicates that CaN redox regulation can participate in physiological signaling. The mechanism whereby CaN re-activation is accomplished within the cell remains to be elucidated. In vitro, CaN re-activation is achieved by the addition of ferrous iron and ascorbate [9]. For
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aconitase it could be shown that its inactivation by superoxide in the cells is also reversible [33]. In this case, iron insertion to the [3Fe–4S] cluster appears to be the critical step in the re-activation mechanism, since addition of iron chelator desferrioxamine reduced re-activation rate. In our hands, desferrioxamine had no effect on the recovery of CaN activity in BAEC (data not shown), indicating that iron probably remains associated with CaN during DMNQ treatment. The findings of our study also point to the role of Cu,ZnSOD in modulation of CaN activity. Inhibition of endogenous SOD by DDC resulted in CaN inhibition. In contrast, elevation of intracellular Cu,ZnSOD levels either by adding SOD extracellularly or by overexpressing it prevented inhibition of CaN signaling by DMNQ. This highlights the critical role of Cu,ZnSOD for cellular CaN activity first suggested by Claude Klee and co-workers [17]. Previous studies showed that Cu,ZnSOD plays an important role in maintaining vascular homeostasis [34,35]. In this work, we also show that Cu,ZnSOD is essential for maintaining CaN activity in endothelial cells. Relatively little is known about CaN targets in endothelium. Several reports showed that the CaN–NFAT pathway is important for vascular physiology, including vascular patterning [14], as well as in VEGF-stimulated angiogenesis [16] and inflammatory marker expression [15]. Our data indicate that superoxide targets the CaN-dependent nuclear translocation and transcriptional activity of NFAT in endothelial cells. Thus, CaN-dependent gene expression in endothelial cells is sensitive to redox regulation. Previously, the possibility of superoxide regulation of CaN-dependent gene regulation was postulated in neurons, where it could affect memory development [18,19]. Our data provide the first example of superoxide as specific regulatory molecule in CaN-dependent signaling outside of the neural system. Although redox sensitivity of CaN signaling in cells of the immune system was shown previously [20,21], the use of exogenous hydrogen peroxide to inhibit CaN did not allow the specification of the oxidizing molecule. In our system, we could show the importance of superoxide in modulating CaN signaling, in accordance with its proposed role as a messenger molecule in endothelium [1]. Further investigation, with growing knowledge about mechanisms and outcomes of CaN-dependent signaling in the vasculature, should shed more light on the importance of CaN.
Acknowledgments This study was supported by the grant from the Deutsche Forschungsgemeinschaft NA 429/1-1. The authors are indebted to Drs. Gerald Crabtree and Thomas Kietzmann for supplying plasmid constructs,
to Vera Lorenz, Elena Dormeneva, and Regina Holz for expert technical assistance, and to Bernhard Bru¨ne and Beverly Rothermel for critical reading of the manuscript.
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Superoxide targets calcineurin signaling in vascular endothelium Dmitry Namgaladze a,*, Ivanna Shcherbyna b, Joachim Kienho¨fer b, H. Werner Hofer b, Volker Ullrich b a
Faculty of Medicine, Institute of Biochemistry I, Johann Wolfgang Goethe-University, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany b Faculty of Biology, University of Konstanz, D-78457 Konstanz, Germany Received 29 June 2005 Available online 14 July 2005
Abstract Superoxide emerges as key regulatory molecule in many aspects of vascular physiology and disease, but identification of superoxide targets in the vasculature remains elusive. In this work, we investigated the possibility of inhibition of protein phosphatase calcineurin by superoxide in endothelial cells. We employed a redox cycler 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) to generate superoxide inside the cells. DMNQ caused inhibition of cellular calcineurin phosphatase activity, which was reversible upon DMNQ removal. Inhibition was suppressed by pre-incubating the cells with copper/zinc superoxide dismutase (Cu,ZnSOD). In addition, reducing cellular Cu,ZnSOD activity by diethylthiocarbamic acid treatment resulted in calcineurin inhibition and enhanced sensitivity to DMNQ. Further, we could show that DMNQ inhibits calcineurin-dependent nuclear translocation and transcriptional activation of NFAT transcription factor, and Cu,ZnSOD or superoxide scavenger Tiron reduced the inhibition. Thus, superoxide generation in endothelial cells results in inhibition of calcineurin signaling, which could have important pathophysiological implications in the vasculature. 2005 Elsevier Inc. All rights reserved. Keywords: Protein phosphatase; Superoxide; Oxidative stress; Endothelium; Redox regulation; Calcineurin; Calmodulin; NFAT; Superoxide dismutase; Vascular disease
Superoxide anion-radical is a small reactive molecule formed in vivo by univalent reduction of molecular oxygen via a variety of enzymatic systems. Increased superoxide generation is one of the major factors in the development of vascular disease, such as atherosclerosis, hypertension or diabetes [1,2]. The deleterious effect of superoxide is attributed mostly to its reaction with nitric oxide (NO). The consequence of this reaction is not only the consumption of NO and impairment of its vasorelaxating and antiatherogenic properties, but also the formation of peroxynitrite, a potent oxidant able to react with lipids, proteins, and nucleic acids [3,4]. Comparatively less attention is paid to direct reactions of superoxide with proteins. Identification of protein targets of superoxide should increase our understanding of super*
Corresponding author. Fax: +49 69 6301 4203. E-mail address: [email protected] (D. Namgaladze).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.07.003
oxide role in vascular (patho)physiology. Several protein targets of superoxide play important role in metabolism and signal transduction, indicating potential physiological importance of their interaction with superoxide. Some of the best known are Fe–S cluster proteins, particularly aconitase [5,6], which are rapidly oxidized and lose iron in response to superoxide, resulting in reduced mitochondrial function and altered iron homeostasis. Other targets include protein tyrosine phosphatases [7] and protein kinase C (PKC) [8], key players in intracellular signal transduction. However, the specificity of these enzymes for superoxide in cellular context remains unclear, since all of them also react with a variety of reactive oxygen and nitrogen species. We have found recently that superoxide potently inhibits enzymatic activity of the protein serine/threonine phosphatase calcineurin (CaN, also protein phosphatase 2B) [9]. CaN is an ubiquitous protein involved in a
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plenitude of physiological processes, including regulation of gene expression, muscle remodeling, neuronal activity, and apoptosis [10–13]. CaN also plays a critical role in vascular development, particularly in vascular patterning [14], and is involved in VEGF-induced expression of inflammatory markers [15] and angiogenesis [16]. In these cases, CaN action is mediated by activation of transcription factors of the NFAT family. CaN directly dephosphorylates multiple serine residues of NFAT, resulting in its nuclear translocation and activation of transcriptional activity [13]. Thus, CaN–NFAT signaling pathway is involved in many aspects of vascular physiology. The CaN active site contains a binuclear iron–zinc center, which is critical for catalytic activity. We have shown that in the native enzyme the iron of this center is Fe2+, and it is rapidly oxidized by superoxide to Fe3+, which is unable to support CaN catalysis [9]. In vitro CaN is much more sensitive to superoxide as compared to hydrogen peroxide or peroxynitrite, which is probably due to the formation of l-oxo complex bridging Fe3+ and Zn2+ of the binuclear center. This provides a biochemical basis for previous work by Klee and coworkers [17] who showed that copper/zinc superoxide dismutase (Cu,ZnSOD) protects CaN against inactivation. Although in vitro data indicate that CaN might be targeted by superoxide, little information exists regarding the possibility of this reaction in intact cells and its importance for cell physiology. It was proposed that CaN inhibition by superoxide might regulate phosphorylation of transcription factor CREB in hippocampal neurons [18,19]. In addition, redox sensitivity of CaN signaling was shown in T-cells [20] and neutrophils [21], but in this case exogenous hydrogen peroxide was applied. The possibility of specific regulation of CaN by intracellular superoxide in non-neuronal cells, particularly in the vascular system, therefore remains unclear. In this work, we investigated the effect of intracellular superoxide generation on CaN activity in endothelial cells. We employed the redox-cycling agent 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) to generate superoxide inside the cells. In addition, we investigated the effects of DMNQ on CaN-dependent nuclear translocation and transcriptional activity of NFAT. Finally, we tested whether modulation of intracellular Cu,ZnSOD activity could influence CaN-dependent signaling in vascular endothelium.
(50 mM Tris–HCl, pH 7.5, 0.2 mM EGTA, 50 lg/ml PMSF, and 10 lg/ml leupeptin) and lysed by three cycles of freezing–thawing in liquid N2. The suspension was centrifuged for 10 min at 18,000g and 4 C, and the supernatant was collected. After measuring the protein with Bradford reagent (Bio-Rad), its concentration was adjusted to 0.5–1 mg/ml with the lysis buffer. Twenty microliters of protein solution was added to 20 ll assay buffer containing 50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 1 mg/ml bovine serum albumin, 6 lM 32P-phosphorylated RII peptide (DLDVPIPGRFDRRVSVAAE), 1 lM okadaic acid, and 0.4 mM CaCl2, and incubated for 5–10 min at 30 C. The reaction was stopped by adding 50 ll of 20% DOWEX 50-X8 (200–400 mesh, Bio-Rad) in 10% trichloroacetic acid. The mix was centrifuged for 6 min at 18,000g, and 40 ll of supernatant was taken for Cherenkov counting. The amount of protein was adjusted so that the substrate consumption did not exceed 25%. Duplicate cpm values from the phosphatase assay were averaged, and the resulting value was adjusted by subtracting the counts in blanks measured in the presence of 5 mM EGTA. Superoxide measurement. BAEC were incubated in 6-well plates in HBSS with DMNQ for 0.5 h and labeled with 2 lM hydroethidine for another 0.5 h. After washing and trypsinization, cell suspension was subjected to analysis by flow cytometry using FACScan (Becton– Dickinson) with excitation at 488 and emission at 585 nm. Cells (10,000) of gated population were counted. SOD activity gels. BAEC incubated in 6-well plates were scraped into PBS, and subjected to native electrophoresis on polyacrylamide gels. SOD activity in these gels was assayed as described [23]. Immunofluorescence. HUVEC were grown on 8-well chamber slides (Lab Tek II, Nunc). After treatments cells were washed with PBS, fixed in 4% paraformaldehyde/PBS, and permeabilized with 0.5% Triton X100/PBS. After blocking with 5% BSA/PBS, cells were incubated for 60 min with 1:200 dilution of anti-NFAT-1 monoclonal antibody (N58820, Transduction Laboratories) followed by 1:300 dilution of anti-mouse antibody conjugated with Alexa Fluor 568 (Molecular Probes). Fluorescence was observed using 25· objective with a Leica DMIRB microscope equipped with a digital camera (Visitron System). Luciferase reporter assay. BAEC seeded on 12-well plates were transfected using Fugene 6 transfection reagent (Roche) according to the manufacturerÕs instructions. 1.5 lg pNFAT-luc reporter plasmid (provided by Dr. Gerald Crabtree, Stanford University) and 0.15 lg pRL-TK Renilla luciferase reporter plasmid (Promega) were used together with 1 lg pSOD1 plasmid in pcDNA6/Myc-His vector (provided by Dr. Thomas Kietzmann, University of Go¨ttingen, Germany) or with 1 lg control vector. After 24 h, cells were stimulated with 32 nM phorbol 12-myristate 13-acetate (PMA) and 2 lM A23187 for another 6 h. Reporter activity was measured using a Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase reporter activities were normalized against Renilla luciferase activities. Statistical analysis. Results are expressed as means ± SEM. Significance was determined by using the Student t test, and the level of statistical significance was defined as P < 0.05.
Results Intracellular superoxide generation by DMNQ
Experimental procedures Cell culture. Primary bovine aorta endothelial cells (BAEC) and human umbilical vein endothelial cells (HUVEC) were isolated as described in [22] and maintained in DMEM containing 10% FBS and penicillin/streptomycin (BAEC) or in EGM-2 medium (Clonetics) (HUVEC). Cells of passages 1–2 were used. Calcineurin activity. Calcineurin activity in cell lysates was measured as described [9]. Briefly, cells were scraped into lysis buffer
In order to investigate the effects of intracellular superoxide on CaN activity in BAEC, we used the redox-cycling quinone DMNQ. In the cell DMNQ undergoes reduction to its semiquinone and hydroquinone species, and those are in turn oxidized by molecular oxygen with concomitant superoxide generation [24]. Intracellular production of superoxide in BAEC treated with
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DMNQ was determined by flow cytometry using hydroethidine (HE), which reacts with intracellular superoxide giving rise to a fluorescent product [25]. Measurements in HE-labeled BAEC showed that HE oxidation was significantly increased after treatment with 10 lM DMNQ (Fig. 1). DMNQ-induced HE oxidation was suppressed by pre-incubating the cells for 4 h with Cu,ZnSOD, validating HE specificity for superoxide detection. These data confirm that DMNQ induces elevation of intracellular superoxide in BAEC, which can be prevented by increasing cellular Cu,ZnSOD activity. Effects of DMNQ on calcineurin activity in endothelial cells When applied to BAEC culture, DMNQ caused concentration-dependent inhibition of CaN activity in cell lysates (Fig. 2A). The magnitude of DMNQ inhibition reached levels comparable to maximal inhibitory effect of cyclosporin A and FK506, two well-known pharmacological inhibitors of CaN activity (data not shown). The range of effective DMNQ concentrations was similar to that reported previously [26]. CaN protein levels in cell lysates determined by Western blotting were not changed after DMNQ treatment (data not shown). A similar inhibitory effect on CaN activity in endothelial cells had another redox-cycling agent, menadione. In this case, 20 lM menadione reduced CaN activity to 14.9 ± 4.9% of control after 1 h incubation. The inhibition of cellular CaN activity by DMNQ was reversible. As Fig. 2B shows, following DMNQ removal CaN activity returned to the level observed before treatment already after 15 min. This indicates that redox homeostasis in BAEC is not disturbed by DMNQ, and the cell is able to restore CaN activity when oxidative insult is eliminated.
Fig. 1. DMNQ induces elevation of superoxide levels in BAEC. Cells were treated with 10 lM DMNQ for 0.5 h followed by 0.5 h incubation with 2 lM hydroethidine and analysis by flow cytometry. *Significantly different from control. **Significantly different from DMNQ (n = 4).
Fig. 2. Inhibition of CaN phosphatase activity by DMNQ in BAEC. (A) Cells were treated with DMNQ at indicated concentrations for 1 h, and phosphatase activity of cell lysates was measured using RII phosphopeptide (n = 3). (B) Cells were treated with DMNQ at 10 lM for 1 h followed by medium change and further incubation for indicated times (n = 3).
Redox-cycling agents generate not only superoxide, but also hydrogen peroxide (H2O2), which might inhibit CaN activity [20,27]. To prove the role of superoxide in CaN inhibition by DMNQ, we pre-incubated the cells with 1 mg/ml Cu,ZnSOD (5000 U/ml) for 4 h before DMNQ treatment. It was shown previously [28] that under these conditions intracellular SOD activity rises approximately 2-fold with no further increase at longer pre-incubation times. Intracellular accumulation of Cu,ZnSOD was confirmed by Western blot and NBT activity gels (data not shown). Pre-incubation with Cu,ZnSOD significantly decreased the inhibitory effect of DMNQ on CaN activity (Fig. 3). Another strategy to increase intracellular superoxide levels is to inhibit endogenous SOD activity. For this purpose, we employed diethylthiocarbamic acid (DDC), an inhibitor of Cu,ZnSOD, which acts by extracting copper out of the enzymeÕs active site. Application of DDC to BAEC resulted in inhibition of SOD activity as judged by SOD activity gels (Fig. 4A). When cells were treated with DDC, CaN activity in the cell lysate was suppressed (Fig. 4B). Furthermore, DDC
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120
*
Relative activity, %
100 80 60 40 20 0
Control
DMNQ
+SOD
Fig. 3. Cu,ZnSOD protects CaN against inhibitory action of DMNQ. BAEC were pre-incubated with 5000 U/ml Cu,ZnSOD for 4 h followed by 1 h incubation with 10 lM DMNQ, and phosphatase activity of cell lysates was measured using RII phosphopeptide. *Significantly different from DMNQ (n = 3).
ment of NFAT activation, we examined CaN-dependent NFAT-1 nuclear translocation in human umbilical vein endothelial cells (HUVEC) by immunofluorescence microscopy. The phosphatase activity test showed that the inhibitory action of DMNQ in these cells was comparable to that in BAEC (data not shown). Fig. 5A shows that in resting cells NFAT-1 is evenly distributed throughout the cell body with nuclei occasionally excluded. Stimulation with calcium ionophore caused nuclear translocation of NFAT-1 (Fig. 5B), which was fully suppressed by CsA (data not shown). Treatment with DMNQ inhibited ionophore-stimulated translocation (Fig. 5C), while pre-treatment with superoxide scavenger Tiron could restore the translocation in the presence of DMNQ (Fig. 5D). To analyze the influence of superoxide on NFAT transcriptional activity we used a luciferase reporter assay, in which the expression of luciferase was driven by IL-2 promoter region containing 3 tandem NFAT/ AP-1 sites. Stimulation of BAEC transfected with a NFAT-luc construct with a mixture of calcium ionophore and phorbol ester resulted in approximately 10fold elevation of reporter activity (data not shown). Pre-treatment of the cells with DMNQ caused dose-dependent inhibition of reporter activity (Fig. 6). Similar data were obtained when calcium ionophore alone was used to stimulate reporter expression (data not shown). When BAEC were transfected with Cu,ZnSOD-encoding plasmid inhibition of reporter activity by DMNQ was significantly reduced. The protective effect of SOD was less pronounced at 5 lM DMNQ; this probably reflects inhibition of reporter activation by H2O2 formed in substantial amounts when increasing concentrations of DMNQ are used. Taken together, the results suggest substantial involvement of superoxide in inhibition of CaN-dependent NFAT activation by DMNQ.
Discussion Fig. 4. Inhibition of Cu,ZnSOD by DDC reduces CaN activity. (A) representative SOD activity gel of BAEC treated with DDC for 1 h. (B) Cells were incubated with 5 mM DDC, 5 lM DMNQ or both substances together for 1 h, and phosphatase activity of cell lysates was measured using RII phosphopeptide (n = 3).
enhanced the inhibitory action of DMNQ. DDC alone did not affect phosphatase activity of isolated porcine CaN (data not shown), thus ruling out the possibility that its metal-chelating properties directly affect the CaN binuclear metal center. Inhibition of calcineurin-mediated signaling by DMNQ Next, we wanted to investigate whether signaling of CaN to NFAT is affected by superoxide. To investigate whether superoxide inhibition of CaN results in impair-
The major finding of this study is the ability of superoxide to inhibit CaN activity and CaN-dependent signal transduction in vascular endothelium. Although the effects of superoxide on vascular function via its reaction with NO are widely recognized less attention is paid to direct reactions of superoxide with enzymatic targets. In our previous work with isolated CaN in vitro, we found that CaN is potently inhibited by superoxide, and is much less sensitive to other ROS, including H2O2 and peroxynitrite. However, the conditions in the in vitro system are quite different from those of cellular milieu, where superoxide levels are kept low due to the presence of SOD. In addition, ascorbic acid and NO, both present in endothelium, should protect CaN against inhibitory action of superoxide [9]. Still, the results of the present study show that excess superoxide
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B
C
D
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Fig. 5. DMNQ inhibits calcium ionophore-stimulated NFAT-1 nuclear translocation in HUVEC. Cells were pre-incubated for 1 h with 5 mM Tiron followed by 1 h treatment with 5 lM DMNQ. NFAT nuclear translocation was induced by 20 min treatment with 2 lM A23187. Cells were fixed and NFAT-1 localization was analyzed by immunofluorescence microscopy using anti-NFAT-1 antibody. (A) Control cells. (B) A23187-treated cells. (C) Cells treated with DMNQ before A23187 stimulation. (D) Cells pre-treated with Tiron before DMNQ treatment.
Control SOD
Reporter activity, % of control
120 100
*
80 60
*
40 20 0
Control
DMNQ 2
DMNQ 5
Fig. 6. DMNQ inhibits PMA- and A23187-stimulated activity of NFAT-luc reporter in BAEC. Cells were transfected with Cu,ZnSOD plasmid or with control vector together with pNFAT-luc and pRL-TK plasmids for 24 h, treated with DMNQ at 2 lM or 5 lM for 1 h and stimulated subsequently with a mix of 32 nM PMA and 2 lM A23187 for 6 h. Data are expressed as normalized reporter activity. *Significantly different from respective DMNQ values in vector-transfected cells (n = 3).
generation is able to overwhelm these defence systems and cause CaN inhibition. This work was done by using DMNQ as a trigger of superoxide production. This is likely to model a situation of oxidative stress, such as ischemia/reperfusion, which is accompanied by massive generation of reactive oxygen species (ROS). Indeed, it was reported that CaN activity in hippocampus is decreased by ischemia [29], although the involvement of superoxide in this process was not investigated. Increased ROS formation in the
vasculature underlies several pathophysiological conditions, including hypertension, coronary artery disease, atherosclerosis, or diabetes. The major feature of superoxide-mediated processes is the local action of superoxide. In contrast, CaN is a highly abundant enzyme likely to be present at micromolar concentrations in the cytosol. Thus, localized fluxes of superoxide might not be able to affect total CaN activity measured in cell lysates. However, CaN is known to be present in signaling complexes, such as those assembled around AKAP proteins [30]. If such signaling complexes are located close to superoxide sources, there is a possibility of local CaN inhibition by superoxide. In this case, only localized dephosphorylation of specific substrates would be affected. The other substantial finding is the reversibility of CaN inhibition by superoxide in the cell. Generally, reversibility is a major criterion for a particular enzyme modification to be involved in the physiological regulatory mechanisms. This is especially important for the emerging field of redox signaling, where reversible oxidative modifications of proteins, like formation of cysteine sulfenic acid or cyclic sulfenyl-amide [31] are opposed to non-specific oxidative damage to the proteins, such as protein carbonyl formation or cysteine oxidation to sulfonic acid [32]. Thus, rapid recovery of CaN activity after removal of oxidative signal indicates that CaN redox regulation can participate in physiological signaling. The mechanism whereby CaN re-activation is accomplished within the cell remains to be elucidated. In vitro, CaN re-activation is achieved by the addition of ferrous iron and ascorbate [9]. For
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aconitase it could be shown that its inactivation by superoxide in the cells is also reversible [33]. In this case, iron insertion to the [3Fe–4S] cluster appears to be the critical step in the re-activation mechanism, since addition of iron chelator desferrioxamine reduced re-activation rate. In our hands, desferrioxamine had no effect on the recovery of CaN activity in BAEC (data not shown), indicating that iron probably remains associated with CaN during DMNQ treatment. The findings of our study also point to the role of Cu,ZnSOD in modulation of CaN activity. Inhibition of endogenous SOD by DDC resulted in CaN inhibition. In contrast, elevation of intracellular Cu,ZnSOD levels either by adding SOD extracellularly or by overexpressing it prevented inhibition of CaN signaling by DMNQ. This highlights the critical role of Cu,ZnSOD for cellular CaN activity first suggested by Claude Klee and co-workers [17]. Previous studies showed that Cu,ZnSOD plays an important role in maintaining vascular homeostasis [34,35]. In this work, we also show that Cu,ZnSOD is essential for maintaining CaN activity in endothelial cells. Relatively little is known about CaN targets in endothelium. Several reports showed that the CaN–NFAT pathway is important for vascular physiology, including vascular patterning [14], as well as in VEGF-stimulated angiogenesis [16] and inflammatory marker expression [15]. Our data indicate that superoxide targets the CaN-dependent nuclear translocation and transcriptional activity of NFAT in endothelial cells. Thus, CaN-dependent gene expression in endothelial cells is sensitive to redox regulation. Previously, the possibility of superoxide regulation of CaN-dependent gene regulation was postulated in neurons, where it could affect memory development [18,19]. Our data provide the first example of superoxide as specific regulatory molecule in CaN-dependent signaling outside of the neural system. Although redox sensitivity of CaN signaling in cells of the immune system was shown previously [20,21], the use of exogenous hydrogen peroxide to inhibit CaN did not allow the specification of the oxidizing molecule. In our system, we could show the importance of superoxide in modulating CaN signaling, in accordance with its proposed role as a messenger molecule in endothelium [1]. Further investigation, with growing knowledge about mechanisms and outcomes of CaN-dependent signaling in the vasculature, should shed more light on the importance of CaN.
Acknowledgments This study was supported by the grant from the Deutsche Forschungsgemeinschaft NA 429/1-1. The authors are indebted to Drs. Gerald Crabtree and Thomas Kietzmann for supplying plasmid constructs,
to Vera Lorenz, Elena Dormeneva, and Regina Holz for expert technical assistance, and to Bernhard Bru¨ne and Beverly Rothermel for critical reading of the manuscript.
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