Expression of Inducible Nitric-oxide Synthase and Intracellular Protein Tyrosine Nitration in Vascular Smooth Muscle Cells

ROLE OF REACTIVE OXYGEN SPECIES*

Diana M. Fries {ddagger} § ¶, Evgenia Paxinou §, Marios Themistocleous §, Eric Swanberg §, Kathy K. Griendling ||, Daniela Salvemini **, Jan W. Slot {ddagger}{ddagger}, Harry F. G. Heijnen {ddagger}{ddagger} §§, Stanley L. Hazen ¶¶ and Harry Ischiropoulos § ||||

From the §Stokes Research Institute, Children's Hospital of Pennsylvania and University of Pennsylvania, Philadelphia, Pennsylvania 19140, ||Department of Medicine, Division of Cardiology, Emory University, Atlanta, Georgia 30322, **MetaPhore Pharmaceuticals, Inc., St. Louis, Missouri 63114, {ddagger}{ddagger}Department of Cell Biology, University of Utrecht, Utrecht, Netherlands, §§Department of Hematology, University Medical Center, Utrecht, Netherlands, ¶¶Departments of Cell Biology and Cardiovascular Medicine, Center for Cardiovascular Diagnostics and Prevention, Cleveland Clinic Foundation, Cleveland, Ohio 44195, and Ministry of Health, Sao Paulo, SP, Brazil

Received for publication, October 22, 2002 , and in revised form, April 7, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A significant increase in the induction of inducible nitric-oxide synthase (iNOS) protein expression and in the levels of nitrite plus nitrate was observed in rat aortic smooth muscle cells (RASMCs) stably transfected with catalase (RASMC-2C2) as compared with empty vector-transfected RASMC-V4 cells after exposure to cytokines and lipopolysaccharide. The increased expression of iNOS protein in the RASMC-2C2 cells was associated with a significant activation of nuclear transcription factor {kappa}B, one of the transcriptional regulators of iNOS expression. The induction of iNOS was also accompanied by increased protein tyrosine nitration in both cell types as revealed by immunocytochemical staining and high pressure liquid chromatography with on-line electrospray ionization tandem mass spectrometry. Nitrotyrosine formation was inhibited by 1400W, an iNOS inhibitor, by 4-(2-aminoethyl) benzenesulfonyl fluoride, an inhibitor of NADPH oxidase, and by the superoxide dismutase mimetic M40403, but not by the peroxidase inhibitor 4-aminobenzoic hydrazide. Electron microscopy using affinity-purified anti-nitrotyrosine antibodies revealed labeling at the cytosolic side of the rough endoplasmic reticulum membranes, in the nucleus, occasionally in mitochondria, and consistently within the fibrillar layer underneath the plasma membrane. Collectively, the data in this model system indicate that hydrogen peroxide, by inhibiting the activation of nuclear transcription factor {kappa}B, prevents iNOS expression, whereas superoxide contributes in a precise pattern of intracellular protein tyrosine nitration.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure of vascular smooth muscle cells to cytokines and lipopolysaccharide (LPS)1 has been shown to induce the expression of inducible nitric-oxide synthase (iNOS), which may account for the vasorelaxation and hypotension associated with inflammatory and septic conditions (15). A number of transcriptional factors including nuclear transcription factor {kappa}B (NF-{kappa}B) have been shown to mediate the expression of iNOS in vascular smooth muscle cells (24). In addition to nitric oxide, exposure of vascular smooth muscle cells to cytokines such as TNF-{alpha} or IL-1{beta} also results in the generation of reduced oxygen species such as superoxide and hydrogen peroxide (59). The production of superoxide has been attributed to the activation of a family of flavoprotein oxidases that have homology to the NADPH oxidase of leukocytes and are expressed in a variety of cells and tissues (10). Although it has not been previously explored, the concomitant production of superoxide and hydrogen peroxide may play a role in the induction of iNOS primarily by the well-recognized redox regulation of NF-{kappa}B activation (1115). Moreover, the production of nitric oxide and reactive oxygen species could also result in the intracellular formation of nitrating species, which modify proteins by tyrosine nitration.

Tyrosine nitration, the addition of a nitro (NO2) group in the ortho position of tyrosine residues, has been detected under physiological settings and in a number of pathological conditions including inflammatory and septic conditions (1618). The biological significance of modifying tyrosine residues by nitration is under investigation, with a number of potential consequences such as alterations in secondary structure, function, and proteolytic removal, suggesting that this modification could profoundly modulate cellular function (16). Published data are also strongly supportive of the utility of 3-nitrotyrosine as a potential biological marker to determine risk associations with inflammatory diseases (1620). The pathways leading to the nitration of tyrosine residues in proteins have been extensively studied in vitro (1932). Biochemical reactions that have been shown to generate nitrating species include: (i) the reaction of superoxide with nitric oxide to generate peroxynitrite, which in turn may form more potent nitrating species by reaction with either CO2 or metal (19, 2830); (ii) the oxidation of nitrite by peroxidases and H2O2 (20, 2326, 32); (iii) the acidification of nitrite; and (iv) the reaction of tyrosyl radical with nitric oxide or nitrogen dioxide (26, 32). Recent studies utilizing mice with genetic deficiencies in myeloperoxidase (3234) and eosinophil peroxidase (32) revealed a significant contribution of these two peroxidases in the extracellular nitration of proteins at sites of inflammation. However, the mechanisms accounting for the intracellular nitration of proteins in non-inflammatory cells remain largely unexplored. In the present study, the mechanisms contributing to the formation of nitrated proteins within RASMCs in response to inflammatory stimuli are examined by using a combination of molecular, analytical, and pharmacological approaches. This cell model system was selected because stimulation with cytokines such as TNF-{alpha} or IL-1{beta} results in the generation of reduced oxygen species, such as superoxide and hydrogen peroxide (79), as well as nitric oxide, nitrite, and nitrotyrosine (1, 5, 6).


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Anti-iNOS monoclonal antibody and recombinant rat IFN-{gamma} were from BD Biosciences (Palo Alto, CA). Anti-GAPDH monoclonal antibody was from Chemicon International (Temecula, CA). Dul-becco's modified Eagle's medium with high glucose, fetal bovine serum, streptomycin, penicillin, Geneticin, and trypsin-EDTA were from Invitrogen. TNF-{alpha} and recombinant human IL-1{beta} were from Roche Diagnostics. 1400W was from Alexis Biochemicals (San Diego, CA). Goat anti-mouse IgG (H+L)-horseradish peroxidase conjugate was from Bio-Rad. [32P]ATP and chemiluminescence assay (ECL kit) were from Amersham Biosciences. Cy3-conjugated goat anti-rabbit IgG was from Jackson Immuno-Research Laboratory (West Grove, PA). Alexa Fluor 488 goat anti-mouse IgG was from Molecular Probes (Eugene, OR). Diphenylene iodonium chloride was from Toronto Research Chemicals (Toronto, Ontario, Canada), and 4-(2-amino)-benzenosulfonil fluoride (AEBSF), LPS from Escherichia coli, 4-aminobenzoic hydrazide (ABAH), protease inhibitor mixture, and all other chemicals were from Sigma-Aldrich. Nuclear Extract Kit (Active Motif, Carlsbad, CA), Chroma-Spin 10 columns (BD Biosciences), NF-{kappa}B antibody, p65 (Chemicon International), and gel shift assay systems (Promega, Madison, WI) were also used.

Cell Culture and Cell Stimulation Conditions—Primary aortic smooth muscle cells were isolated from male Sprague-Dawley rat thoracic aortas by enzymatic digestion as described previously (79). The cells were transfected with pCI-neo alone (RASMC-V4) or pCI-neo/catalase plasmid (RASMC-2C2) as described previously, and transfected cells were selected with Geneticin (7). Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, 100 µM streptomycin, and 300 µM Geneticin and transferred once a week by harvesting with trypsin-EDTA and seeding into 75-cm2 flasks. For experiments, cells between passages 17 and 22 were used at 70% confluence. Cells were maintained in selection medium until they were plated into 75-mm dishes for experiments. Catalase overexpression has been shown to effectively remove intracellular peroxide in response to agonist stimulation as compared with vector-transfected cells (7, 9). Expression of functional catalase in clonally expanded RASMC-2C2, but not RASMC-V4, was confirmed by Western blot analyses and catalase activity measurements of cell homogenates.

For induction of iNOS, RASMCs (RASMC-2C2 and RASMC-V4 clones) cultured in 6-well plates were treated with a mixture of 250 units/ml TNF-{alpha}, 600 units/ml IL-1{beta}, 300 units/ml IFN-{gamma}, and 5 µg/ml LPS for 72 h in Dulbecco's medium supplemented with 5% fetal bovine serum. In some experiments, cells were pretreated with 400 µM 1400W, 100 µM AEBSF, 5 µM M40403, 1 µg/ml, diphenylene iodonium, or 100 µM ABAH for 1 h before the addition of cytokines and LPS. After stimulation, cells were washed extensively with ice-cold PBS and processed for immunological or biochemical analyses.

Detection of iNOS—Cells were lysed with 100 µl of ice-cold lysis buffer, pH 7.4, containing 20 mM HEPES, 150 mM NaCl, 1 mM EGTA, 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100, and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 10 mM sodium pyrophosphate, 50 mM sodium fluoride, 2 mM sodium orthovanadate, and 1 µM clasto-lactacystin {beta}-lactone). The cells were scraped, and the soluble proteins were centrifuged at 14,000 x g at 4 °C for 30 min. Supernatants were collected and stored at –80 °C. Extracted proteins were quantified by the Bradford assay using IgG as standard. Proteins (50 µg) were separated on 10% polyacrylamide gels using SDS-PAGE and transferred to nitrocellulose membranes at 20 V overnight. Membranes were blocked for 2 h at room temperature with PBS-containing 5% non-fat dry milk and 0.05% Tween 20 (pH 7.4). The blots were washed with PBS-0.05% Tween 20 and incubated overnight with primary mouse monoclonal anti-iNOS or GAPDH antibodies at 1:2,000 and 1:40,000 dilution, respectively, in PBS containing 1% non-fat milk and 0.052 Tween 20 (pH 7.4). After washing with PBS-0.05% Tween 20, the gels were incubated with a goat anti-mouse IgG (H+L)-horseradish peroxidase conjugate at either a 1:2,500 (for iNOS) or 1:10,000 (for GAPDH) dilution for 1 h at room temperature. The proteins were detected by enhanced chemiluminescence.

Measurement of Nitrite plus Nitrate—Cell culture medium was centrifuged at 5,000 x g for 10 min to remove cellular debris. Samples were diluted 3-fold with cold ethanol, vortexed, and left on ice for 30 min. The samples were then centrifuged at 10,000 x g for 10 min at 0 °C, and 10 µl of the supernatant was injected in a reaction chamber containing a VCl3/HCl mixture heated at 95 °C in the chemiluminescence nitric oxide detector (Nitric Oxide Analyzer; Sievers Instruments, Boulder, CO). A standard curve was generated by injections of known concentrations of NaNO3. The levels of nitrite plus nitrate in the cell culture medium were normalized to protein concentration.

Immunocytochemical Staining for iNOS and Nitrotyrosine—Cells were fixed with cold methanol for 20 min at –20 °C and with a mixture of cold acetone/methanol (1:1) for 5 min at –20 °C. Non-specific binding was blocked by incubation for 1 h with 10% bovine serum albumin and normal goat serum in PBS plus 0.3% Triton X-100 at room temperature. The cells were then incubated with an affinity-purified anti-nitrotyrosine polyclonal antibody (raised against nitrated KLH) (1:300) and anti-iNOS monoclonal antibody (1:500) overnight at 4 °C. Cells were washed three times with PBS plus 0.3% Triton X-100 and incubated with Cy3-conjugated goat anti-rabbit IgG antibody and/or Alexa Fluor 488 goat anti-mouse IgG antibody at 1:250 or 1:500, respectively, for 1 h. The anti-nitrotyrosine antibody specificity was confirmed by control experiments showing loss of antibody recognition following reduction of samples with 10 mM sodium dithionite or following competition with excess (10 mM) 3-nitrotyrosine. Fluorescent images were obtained using an Olympus 1X70 inverted microscope as described previously (35).

Assay for NF-{kappa}B Activity—Nuclei were extracted according to instructions included in the Nuclear Extract Kit (Active Motif), and protein concentration in nuclear extracts was measured. DNA probes were end-labeled with T4 polynucleotide kinase and [{gamma}-32P]ATP by standard methods described in the gel shift assay systems (Promega). The labeled probe was separated from unincorporated [{gamma}-32P]ATP chromatographically on Chroma-Spin 10 spin columns. Electrophoretic mobility shift assays were performed using the consensus binding oligonucleotides (5'-AGTTGAGGGGACTTTCCCAGGC-3' and 3'-TCAACTCCCCTGAAAGGGTCCG-5') of NF-{kappa}B. DNA-protein binding reactions were performed with 5 µg of nuclear extract and 50,000–100,000 cpm of 32P-end-labeled double-stranded DNA probe in binding buffer composed of 1 mM MgCl2, 0.5 mM dithiothreitol, 0.5 mM EDTA, 50 mM NaCl, 10 mM Tris-HCl (pH 7.5), 4% glycerol, and 50 µg/ml poly(dI-dC). All components of the binding reaction, with the exception of the labeled probe, were combined and incubated on ice for 10 min. After the labeled probe was added, the binding reaction was incubated for an additional 20 min at room temperature. Competition experiment was performed with nearly a 100-fold excess of unlabeled oligonucleotides for NF-{kappa}B. Supershift assays were performed by the addition of 1.25 µg of antibody against the p65 component of NF-{kappa}B and incubated on ice for 30 min, before the probe was added. Samples were electrophoresed on a 4% non-denaturing polyacrylamide gel in 0.5x Tris-boric acid-EDTA buffer at 75 V at 4 °C. Gels were dried and analyzed by autoradiography at –80 °C using an image-intensifier screen.

Quantification of 3-Nitrotyrosine—Control and stimulated cells were washed extensively with ice-cold PBS and harvested after application of trypsin in PBS. Cells were sonicated (three 5-s pulses at 8 W), and the content of protein 3-nitrotyrosine in lysates was analyzed by high performance liquid chromatography with on-line electrospray ionization tandem mass spectrometry using stable isotope dilution methodology and an ion trap mass spectrometer (LCQ Deca; ThermoFinigann, San Jose, CA) as described in detail previously (32). Nitrotyrosine contents of proteins are expressed as a product/precursor ratio (i.e. nitrotyrosine/tyrosine; µmol/mol).

Immunoelectron Microscopy—Unstimulated cells (control) and cells treated for 72 h with cytokines plus LPS were fixed in 2% formaldehyde, 0.2% glutaraldehyde in 0.1 M sodium phosphate (pH 7.4) for 2 h. After fixation, cells were scraped and collected, washed with 1% paraformaldehyde in PBS, and processed for immunoelectron microscopy as described previously (36, 37). Briefly, the cells were washed in PBS, dispersed in 12% gelatin at 37 °C, and centrifuged. The gelatin was allowed to harden at 4 °C, and the pellet was cut into small blocks, immersed overnight with 2.3 M sucrose, frozen in liquid nitrogen, and transferred to a cryo-ultramicrotome (Leica Microsystems, Vienna, Austria). 50–60-nm-thick cryosections were thawed and immunolabeled with 20 µg/ml affinity-purified polyclonal anti-nitrotyrosine antibody (NT432). The antibody was raised against a synthetic decapeptide (Cys-Gly-NO2Tyr-Gly-Gly-Gly-NO2Tyr-Gly) that was synthesized to include two nitrotyrosine residues. The antibody was affinity-purified using an affinity nitrotyrosine column (amino group of nitrotyrosine covalently linked to AminoLink matrix; Pierce), and the purity of the purified antibody was confirmed by SDS electrophoresis. The specificity of NT432 was determined by competition enzyme-linked immunosorbent assay. The antibody binding at 1:3,200 dilution to nitrated proteins was competed by >90% only by 100 µM 3-nitrotyrosine, and not by 10 mM tyrosine, phosphotyrosine, L-Dopa, and 3-chlorotyrosine. The antibody labeling in the sections was visualized by protein A-gold. After labeling, the sections were stained with uranyl acetate and embedded in methyl cellulose before drying.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Induction of iNOS—Immunostimulation of RASMCs with a cytokine mixture comprised of TNF-{alpha}, IL-1{beta}, IFN-{gamma}, and LPS ("Experimental Procedures") resulted in a time-dependent induction of iNOS. Data in Fig. 1A illustrate the time course of iNOS expression after stimulation. Expression of iNOS was near maximal 72 h after the addition of cytokines. There were no differences in the time course of cytokine-triggered iNOS induction of RASMCs stably transfected with either catalase (RASMC-2C2) or empty vector (RASMC-V4) (data not shown). Immunocytochemical studies showed expression of iNOS in the majority of cells and demonstrated intense staining for iNOS in 79 ± 2% of cells. All subsequent experiments were performed after 72 h of stimulation, unless otherwise stated.



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FIG. 1.
Time course of iNOS induction. Unstimulated cells (–) or cells treated with 250 units/ml TNF-{alpha}, 600 units/ml IL-1{beta}, 300 units/ml IFN-{gamma}, and 5 µg/ml LPS (+) were harvested at the times indicated, and the protein expression of iNOS was determined. Detection of GAPDH was utilized as a control for protein loading.

 

To examine the intracellular mechanisms of protein nitration in immunostimulated RASMCs, we used both molecular and pharmacological approaches. However, it is important to first document that the panel of specific inhibitors used did not impact upon iNOS expression and activity. Accordingly, levels of cytokine-induced increases in iNOS protein and nitrite plus nitrate in cell media were evaluated (Fig. 2). Induction of iNOS expression and activity in RASMCs were unaffected by pre-exposure of cells to the NADPH oxidase inhibitor AEBSF (38), the superoxide dismutase mimetic M40403 (39), or the peroxidase inhibitor ABAH (20) (Fig. 2). Inclusion of iNOS inhibitor (1400W) significantly increased iNOS protein levels (1.5-fold), consistent with previous reports of nitric oxide-induced down-regulation of iNOS expression in inflammatory cells (40, 41). Cells stably transfected with catalase demonstrated significant increased in cytokine-mediated expression of iNOS protein as compared with vector-transfected cells (1.5-fold increase; p < 0.05). Parallel decreases in iNOS protein expression and levels of nitrite and nitrate were noted (Fig. 2). Addition of 1400W inhibited nitric oxide production, whereas addition of AEBSF, M40403, and ABAH did not alter iNOS protein levels and did not interfere with nitric oxide production, as revealed by the levels of nitrite and nitrate released in media (Fig. 2C).



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FIG. 2.
Effect of inhibitors on iNOS induction. A, representative Western blot for iNOS using GAPDH to control protein loading 72 h after stimulation with cytokines plus LPS. B, densitometric quantification of iNOS levels. Lane 1, vector-transfected RASMC-V4, lane 2, catalase-overexpressing RASMC-2C2; lane 3, same as lane 2 plus iNOS inhibitor 1400W; lane 4, same as lane 2 plus peroxidase inhibitor ABAH; lane 5, same as lane 2 plus the NADPH oxidase inhibitor AEBSF; lane 6, same as lane 2 plus superoxide dismutase mimetic M40403. All values in the RASMC-2C2 cells were significantly different (*, p < 0.05) from those in RASMC-V4 cells after analysis of variance using Tukey's test for all pairwise multiple comparisons. {ddagger}, p < 0.05, the value for the 1400W-treated cells was significantly different from the rest as determined by the same method (n = 4–5 independent determinations). C, the levels of plus were determined in the cell medium 72 h after stimulation. Values represent the mean ± S.D. for n = 7–11 independent determinations. nd, none detected. *, p < 0.05 after analysis of variance using Dunnett's method for multiple comparisons to RASMC-2C2.

 

Activation of NF-{kappa}B—The potential mechanism for the increased expression of iNOS in catalase-overexpressing cells was evaluated by determining the activation NF-{kappa}B, which has been shown to be one of the principal transcriptional factors regulating iNOS expression in smooth muscle cells (24). Utilizing a commercial NF-{kappa}B probe and nuclear extracts from RASMC-2C2 and RASMC-V4 cells, electrophoretic mobility shift assay revealed that stimulation of both cell types with cytokine plus LPS induced NF-{kappa}B binding. The NF-{kappa}B DNA binding activity was significantly higher in catalase-transfected cells as compared with vector-transfected controls (Fig. 3). The NF-{kappa}B-specific band was eliminated by the addition of a 100-fold excess of unlabeled NF-{kappa}B oligonucleotide to the reaction mixture, whereas addition of an anti-p65 antibody in both cell extracts resulted in the expected decrease in specific band and produced a clear supershifted band in the protein-DNA complex (Fig. 3).



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FIG. 3.
NF-{kappa}B binding activity in RASMC-2C2 and RASMC-V4 cells. The DNA binding activity of NF-{kappa}B in nuclear extracts from RASMC-2C2 and RASMC-V4 cells was determined by electrophoretic mobility shift assay as described under "Experimental Procedures." Lanes 1–3 are RASMC-V4 cells, and lanes 4–7 are RASMC-2C2 cells. In lanes 3 and 7, an antibody against p65 was added with the nuclear extracts. In lane 6, DNA binding was eliminated by the addition of a 100-fold excess of unlabeled probe. The gel shown is representative of three separate experiments.

 

Evaluation of Tyrosine Nitration—Nitration of tyrosine residues in proteins after induction of iNOS was initially evaluated by immunocytochemical staining with anti-nitrotyrosine antibodies. Comparable antibody labeling was observed throughout the cytoplasm in catalase- and vector-transfected cells after immunostimulation (Fig. 4). Background labeling was observed in non-stimulated cells (Fig. 4). The specificity of staining for nitrotyrosine was confirmed as described under "Experimental Procedures." To obtain a more quantitative measure of protein 3-nitrotyrosine formation after agonist stimulation, levels were quantified by liquid chromatography with on-line electrospray ionization tandem mass spectrometry as described previously (32). After stimulation, a 3-fold increase in the intracellular protein levels of 3-nitrotyrosine was observed in both catalase- and vector-transfected cells. The comparable level of nitrotyrosine in control and catalase-overexpressing cells suggests that hydrogen peroxide is not necessary for nitrotyrosine formation.



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FIG. 4.
Nitrotyrosine detection. Representative images for nitrotyrosine staining (1) 72 h after stimulation (B and D) or in control non-stimulated cells (A and C). A and B are RASMC-2C2 cells, and C and D are RASMC-V4 cells. Nuclei in the cells were stained with 4',6-diamidino-2-phenylindole (2). Images were obtained at x20 magnification. E, quantification of 3-nitrotyrosine levels by LC/ESI/MS/MS. Values represent mean ± S.D. for three to four independent determinations. *, p < 0.05 after analysis of variance using Tukey's post hoc test.

 

Effects of Inhibitors—To gain further insight into the molecular mechanism of intracellular nitration after stimulation with cytokines/LPS, cells were incubated with different inhibitors, and agonist-triggered staining for nitrotyrosine was evaluated. A role for active nitric oxide synthesis in the nitration of proteins was confirmed by demonstrating that nitrotyrosine staining was ablated by the addition of the iNOS-specific inhibitor 1400W (Fig. 5). Incubation of cells with the superoxide dismutase mimetic M40403 (40) significantly reduced the staining of cells with the anti-nitrotyrosine antibodies, consistent with a role for superoxide in the intracellular nitration of proteins after immunostimulation. M40403 did not interfere with iNOS induction or nitric oxide production (Fig. 2). Separate control studies demonstrated that 5 µM M40403 did not inhibit nitration of bovine serum albumin by bolus additions of peroxynitrite or peroxidase-H2O2- systems (data not shown), excluding M40403-dependent inhibition in intracellular aromatic nitration reactions through intercepting/scavenging peroxynitrite/nitrogen dioxide-mediated nitration. These data indicate that inhibition of intracellular protein nitration by the superoxide dismutase mimetic M40403 likely arises from suppression of superoxide concentration after cell immunostimulation. Incubation of cells with the NADPH oxidase inhibitor AEBSF, which prevent the assembly of the oxidase complex without interfering with the flavoprotein that carries out the reduction of oxygen (38), also resulted in a decrease in anti-nitrotyrosine immunoreactivity. The flavin inhibitor diphenylene iodonium, which has been extensively utilized to inhibit the vascular smooth muscle NADPH oxidases (79), inhibited the production of nitrite plus nitrate, making it difficult to distinguish between its effects on NADPH oxidases and iNOS. Therefore, this inhibitor was not used to evaluate mechanisms of intracellular protein nitration. There was no change in immunoreactivity in cells treated with ABAH, a peroxidase inhibitor shown to abolish extracellular aromatic nitration reactions by stimulated neutrophils (20). Overall, these data indicate that intracellular protein nitration elicited by immunostimulation of RASMCs is, for the most part, dependent on nitric oxide and superoxide production.



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FIG. 5.
Effect of inhibitors on nitrotyrosine formation. Representative images at x20 magnification for nitrotyrosine staining from four independent determinations (left panels) merged with 4',6-diamidino-2-phenylindole staining (right panels) 72 h after stimulation. A, after pre-treatment with the peroxidase inhibitor ABAH; B, after pre-treatment with the iNOS inhibitor 1400W; C, after pre-treatment with the superoxide dismutase mimetic M40403; D, after pre-treatment with the NADPH oxidase inhibitor AEBSF.

 

Cellular Localization of Nitrated Proteins—Post-translational modification of protein by tyrosine nitration was observed in cells with both robust and modest iNOS expression (Fig. 6). Nitrated proteins were also detected in cells without apparent labeling for iNOS (indicated by the asterisks in Fig. 6), suggesting that diffusion of nitric oxide to adjacent cells without robust expression of iNOS supports the formation of nitrating species.



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FIG. 6.
Nitrotyrosine and iNOS co-localization. Representative images at x20 magnification for nitrotyrosine (red) and iNOS (green) staining from four independent determinations. Merging of the images reveals co-localization in most but not all cells. Cells without robust iNOS staining but positive for nitrotyrosine are indicated by the asterisks.

 

To examine subcellular localization of nitrotyrosine formation within RASMCs after immunostimulation, electron microscopy studies were performed. Use of gold particle-tagged anti-nitrotyrosine polyclonal antibody revealed pronounced labeling at the cytosolic side of the rough endoplasmic reticulum membranes in agonist-treated cells (Fig. 7, A and B). Labeling of the remaining cytoplasm was much less conspicuous, except for a moderate but consistent labeling of the thin fibrillar cytoskeletal network located beneath the plasma membrane (Fig. 7A). Interestingly, cytokine-stimulated labeling within the nuclei (data not shown) and mitochondria (Fig. 7B) was observed. Notably, extracellular material (Fig. 7A) and the lumen of the endoplasmic reticulum cisternae (Fig. 7B) were not labeled. In all instances, much less frequent and intense labeling at the same intracellular sites was observed in nonstimulated cells. No significant labeling was observed in sections treated with antigen-competed antibody (Fig. 7C).



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FIG. 7.
Localization of nitrated proteins by immunoelectron microscopy. Anti-nitrotyrosine labeling was observed in the cytoplasm, where it was most prominent in close proximity to the rough endoplasmic reticulum membranes (arrows in A), and in the fibrillar layer below the plasma membrane (arrowheads in A and B). The nucleus (not shown) and mitochondria (M) were labeled. No apparent labeling was associated with the rough endoplasmic reticulum contents (asterisks), extracellular material (EC), and other cell structures such as lysosomes (L). In C, the tissue was labeled with antibody in the presence of 3-nitrotyrosine, which eliminates antibody binding. Bars, 200 nm.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, a combination of genetic and pharmacological approaches was used in a well-defined cellular model system to investigate the biochemical reactions leading to iNOS induction and formation of nitrating species. The proinflammatory cytokines (TNF-{alpha}, IL-1{beta}, and IFN-{gamma}) and LPS utilized in this study have been previously shown to induce expression of iNOS in vascular smooth muscle cells (15), and the increased production of nitric oxide that accompanies the induction of iNOS is thought to contribute in the pathophysiology of sepsis and other inflammatory disorders (15). The expression of iNOS was significantly increased after inhibition of nitric oxide production (Fig. 2), a finding consistent with the observation that nitric oxide regulates expression of iNOS in inflammatory cells (40, 41). One of the remarkable findings in the present study, however, was that iNOS expression was significantly enhanced in cells transfected with catalase compared with vector-transfected controls, suggesting that intracellular H2O2 production serves as an endogenous regulator of iNOS expression. Stable transfection with catalase significantly removes H2O2 generated primarily by vascular NADPH oxidases after agonist stimulation (79). A likely molecular mechanism responsible for the enhanced expression of iNOS in catalase-overexpressing cells is the increased activation of the dimeric transcription factor NF-{kappa}B as documented by the increased DNA binding activity (Fig. 3). NF-{kappa}B is one of the transcriptional factors that mediates transcription of iNOS in rat smooth muscle cells (24), and numerous studies have implicated reactive oxygen species in the regulation of NF-{kappa}B activity under inflammatory conditions (1115). The data in this cell model system indicate that upon stimulation with cytokines and LPS, endogenous H2O2 prevents the activation of this transcription factor, consistent with observations in alveolar type II cells and other cell types (15). In turn, the H2O2-induced suppression of NF-{kappa}B activity results in the inhibition of iNOS expression. Collectively, these results suggest that a complex and interconnected regulatory cross-talk, possibly through the activation of transcription factors such as NF-{kappa}B, exists between NADPH oxidase activation and formation of H2O2 and iNOS expression under inflammatory conditions. Such pathways might impact upon the biochemical interaction of reactive oxygen and nitrogen intermediates, resulting in the formation of nitrating species that could alter the function of specific subcellular proteins through selective aromatic nitration reactions. Indeed, the induction of iNOS in rat vascular smooth muscle cells to cytokines and LPS resulted in formation of nitrating species as indicated by the presence of immunoreactive nitrated proteins and by quantification with LC/ESI/MS/MS.

Cells stably transfected with catalase demonstrated cytokine-mediated intracellular protein nitration comparable to that of the vector-transfected control cells (Fig. 4), and exogenous addition of H2O2 together with nitrite does not result in increased protein nitration (data not shown). These results indicate that H2O2 formation does not significantly contribute to intracellular nitration of targets in this cellular model system. Furthermore, inhibition of cellular peroxidase activity failed to inhibit intracellular protein nitration, consistent with the formation of nitrating species in response to immunostimulation through pathways independent of peroxidase-H2O2 pathways. Rather, the results of both genetic and pharmacological studies support intracellular formation of a peroxynitrite-like nitrating species as the mechanism involved in cytokine-triggered aromatic nitration of intracellular RASMC proteins. Nitration of intracellular proteins was inhibited by removal of superoxide formation via inhibition of vascular cell NADPH oxidases a well-recognized source of cytokine-triggered superoxide in these cells (79). Furthermore, superoxide scavenging similarly ablated intracellular nitration, as noted in studies using the specific superoxide dismutase mimetic M40403 (39). Thus, peroxynitrite, the reaction product of nitric oxide and superoxide, is the likely proximal species responsible for nitrating tyrosine residues in proteins after stimulation in this model. It is worth noting that immunocytochemical studies revealed the presence of nitrated proteins within cells with no apparent iNOS expression. Intracellular protein nitration in these instances likely results from nitric oxide produced in adjacent cells that diffuses into cells producing superoxide. Nitration in these cells might also result from peroxynitrite formed in adjacent cells that then diffuses across cellular membranes because it is estimated that the average peroxynitrite molecule can diffuse over several cell diameters before productive encounters (42).

A number of reports have revealed that protein nitration in human disease, as well as in animal and cell model systems, is both spatially associated with the sites of injury and usually confined within the injured organ or cell (1618). Electron microscopic examination of RASMCs after stimulation indicated that nitrated proteins were localized in the endoplasmic reticulum and associated with cytoskeletal elements. Nitrated proteins were also localized within mitochondria and the cell nucleus (Fig. 7). Similar distribution of nitrated proteins in the nucleus and mitochondria was also detected in vascular tissue of aging rats (43). Localization of nitrated proteins within outer mitochondrial membranes has also been observed in dendrites in rat brain (44). The localization of nitrated proteins in the nucleus observed in this model is consistent with the recent demonstration of nitration of Tyr98 in histone 4 and Tyr42 in histone 2B in tumors (45). Considering that the intracellular compartment is densely populated by proteins, the localization of nitrated proteins in specific cellular compartments implies that specific proteins are targeted for nitration. Indeed proteomic and other immunological approaches identified a limited number of specific proteins that are modified by nitration in vivo (4649, 50). The specific targeting of certain proteins appears to be independent of the flux of nitric oxide, superoxide, and the nature of the nitrating agent but is, for the most part, dependent upon the presence of highly reactive tyrosine residues in certain protein environments (51). It should also be noted, however, that the antibodies used to recognize nitrotyrosine likely recognize nitrotyrosine differentially, depending upon the sequence context in which it is presented. Thus, some component of apparent target protein specificity for nitration may merely reflect differential recognition of proteins based upon the sequence context surrounding the nitration site. In summary, the studies presented herein and studies with peroxidase-deficient mice (32, 34) reinforce previous discussions that a number of pathways apparently contribute to the nitration of tyrosine residues in proteins in vivo independently or even simultaneously (16, 52). With the combined judicious use of both pharmacological agents and molecular approaches and depending upon the inflammatory stimuli and cell type, the sources and nature of nitrating agents in vivo are now being elucidated. Although H2O2 may not contribute to the nitration of proteins in this cell model system, it appears to play a critical role possibly through the regulation of transcription factors such as NF-{kappa}B in the induction of iNOS, thus regulating the production of nitric oxide, the precursor of nitrogen source for the aromatic protein nitration. Improved understanding of the biochemical pathways leading to the regulation of nitric oxide production and the formation of reactive oxygen and nitrogen species capable of modifying proteins by aromatic residue nitration should significantly advance our understanding of the role of these intermediates in the mechanisms of pathogenesis, diagnosis, and potential treatment of disease.


    FOOTNOTES
 
* This work was supported by National Heart, Lung and Blood Institute Grants HL5800 (to K. K. G.), HL62526 (to S. L. H.), and HL54926 (to H. I.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Recipient of a postdoctoral fellowship from CNPq-Brazil. Back

|||| To whom correspondence should be addressed: Stokes Research Institute, Children's Hospital of Philadelphia, 416D Abramson Research Center, 34th St. and Civic Center Blvd., Philadelphia, PA 19104-4318. Tel.: 215-590-5320; Fax: 215-590-4267; E-mail: ischirop{at}mail.med.upenn.edu.

1 The abbreviations used are: LPS, lipopolysaccharide; iNOS, inducible nitric-oxide synthase; TNF-{alpha}, tissue necrosis factor {alpha}; IL-1{beta}, interleukin-1{beta}; IFN-{gamma}, interferon-{gamma}; NF-{kappa}B, nuclear transcription factor {kappa}B; RASMC, rat aortic smooth muscle cell; AEBSF, 4-(2-amino)-benzenosulfonil fluoride; ABAH, 4-aminobenzoic hydrazide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline. Back


    ACKNOWLEDGMENTS
 
We thank Dr. Beatrice Blanchard-Fillion and Richard Lightfoot for support and discussions and Dr. Andrew Gow for critical reading of the manuscript. We also thank Sandra Waaijenburg and Ellie van Donselaar for excellent electron microscopy work and Xiaoming Fu for expert technical assistance in quantification of nitrotyrosine levels by mass spectrometry. Mass spectrometry studies were performed at the Cleveland Clinic Foundation Mass Spectrometry Core Facility.



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 EXPERIMENTAL PROCEDURES
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 DISCUSSION
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