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The Journal of Immunology, 2003, 171: 3225-3232.
Copyright © 2003 by The American Association of Immunologists

Hypoxia Inactivates Inducible Nitric Oxide Synthase in Mouse Macrophages by Disrupting Its Interaction with {alpha}-Actinin 4 1

Sharon Daniliuc2,*,{dagger}, Haim Bitterman2,{dagger}, Michal A. Rahat3,*, Amalia Kinarty*, Doron Rosenzweig* and Lahat Nitza*

* Immunology Research Unit and {dagger} Ischemia-Shock Research Laboratory, Carmel Medical Center, Rappaport Family Institute for Research in the Medical Sciences, and Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nitric oxide, produced in macrophages by the high output isoform inducible NO synthase (iNOS), is associated with cytotoxic effects and modulation of Th1 inflammatory/immune responses. Ischemia and reperfusion lead to generation of high NO levels that contribute to irreversible tissue damage. Ischemia and reperfusion, as well as their in vitro simulation by hypoxia and reoxygenation, induce the expression of iNOS in macrophages. However, the molecular regulation of iNOS expression and activity in hypoxia and reoxygenation has hardly been studied. We show in this study that IFN-{gamma} induced iNOS protein expression (by 50-fold from control, p < 0.01) and nitrite accumulation (71.6 ± 14 µM, p < 0.01 relative to control), and that hypoxia inhibited NO production (7.6 ± 1.7 µM, p < 0.01) without altering iNOS protein expression. Only prolonged reoxygenation restored NO production, thus ruling out the possibility that lack of oxygen, as a substrate, was the cause of hypoxia-induced iNOS inactivation. Hypoxia did not change the ratio between iNOS monomers and dimers, which are essential for iNOS activity, but the dimers were unable to produce NO, despite the exogenous addition of all cofactors and oxygen. Using immunoprecipitation, mass spectroscopy, and confocal microscopy, we demonstrated in normoxia, but not in hypoxia, an interaction between iNOS and {alpha}-actinin 4, an adapter protein that anchors enzymes to the actin cytoskeleton. Furthermore, hypoxia caused displacement of iNOS from the submembranal zones. We suggest that the intracellular localization and interactions of iNOS with the cytoskeleton are crucial for its activity, and that hypoxia inactivates iNOS by disrupting these interactions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Ischemia is caused by occlusion of blood vessels or insufficient blood flow to organs, and occurs in acute as well as chronic pathological conditions, such as brain and cardiac infarcts, trauma, atherosclerosis, autoimmune diseases, and solid tumors. Ischemia is often accompanied by secretion of inflammatory mediators (1, 2, 3, 4), transmigration of activated immune cells to afflicted tissues (5, 6), and translocation of bacteria or their toxins (7, 8), cumulatively leading to local and remote tissue injury culminating in multisystem organ failure (1, 3, 9). Tissue reperfusion is required for cell salvage, but may temporarily aggravate the ischemic damage, partly due to excessive generation of reactive nitrogen and oxygen species (6, 10, 11, 12). Hypoxia and reoxygenation (H/R) 4 are major components of ischemia and reperfusion (I/R), but are also important independent clinical entities, and are frequently used as in vitro model systems to study molecular mechanisms exerted during I/R.

The reactive nitrogen species NO, a small molecule with a short t1/2 time, is produced by the conversion of L-arginine and oxygen to L-citrulline and NO, a reaction that is catalyzed by three isoforms of NO synthase (NOS). All isoforms require cofactors (calmodulin, tetrahydrobiopterin (BH4), flavin adenine dinucleotide (FAD), flavin adenine mononucleotide (FMN), and heme) for their activity (13). Two isoforms are constitutively expressed in the brain (neuronal NOS) and in endothelial cells (endothelial NOS). They produce low levels of NO, which exert multiple physiological activities, such as neurotransmission, vasodilatation, and inhibition of platelet and leukocyte adhesion (14, 15, 16, 17, 18). A third Ca2+-independent isoform (inducible NOS (iNOS)) is induced by inflammatory cytokines and LPS in many cell types, but mostly in monocytes/macrophages. This isoform generates high amounts of NO, which are associated with macrophage cytotoxic effects, as an essential part of normal immune mechanisms of host defense against intracellular pathogens (19, 20), and in contrast, with modulatory effects on inflammatory responses (21, 22).

Production of excessive NO by macrophage iNOS has been implicated in the pathophysiology of I/R due to its role in augmenting tissue damage and cell death (14, 19, 20). However, the isolated effects of H/R on the expression and activity of iNOS have been scarcely studied. To date, no posttranslational regulation of iNOS is known, although the bioavailability of oxygen, BH4, and L-arginine may be important to its activity (23, 24). Increased levels of iNOS mRNA were found in peritoneal and spleen macrophages isolated from rats subjected to 1 h of hypoxia without further insult (25), whereas hypoxia together with IFN-{gamma} increased iNOS mRNA and protein in murine macrophages (26) with no NO secretion (26, 27). The absence of oxygen, the substrate of iNOS, was used to explain its inactivation by hypoxia, despite the high mRNA and protein expression.

In this study, we show that incubation of mouse macrophages in hypoxic conditions for 24 h inhibited iNOS activity, but not protein expression, through mechanisms that are not related to oxygen or other cofactors required for NO production. We demonstrate that hypoxia disrupted interactions of iNOS with the cytoskeletal protein {alpha}-actinin 4, leading to iNOS displacement from the submembranal regions, which may be important to normal activity. These findings suggest that cellular localization and interaction of iNOS with the cytoskeleton may be required for the ability of this enzyme to produce NO.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells

The mouse macrophage-like cell line RAW 264.7 was cultured in DMEM with 10% heat-inactivated FCS and antibiotics. To avoid possible masking of signals initiated by the exogenous stimuli, cells were incubated with DMEM without FCS for 12 h before their exposure to the experimental conditions. After serum starvation, cells were subjected to normoxia, hypoxia, or reoxygenation, with or without the addition of LPS (1 µg/ml, Escherichia coli 055:B5, Sigma-Aldrich, St. Louis, MO), IFN-{gamma} (100 U/ml; Peprotech-Cytolab, Rehovot, Israel), or their combination. In several experiments, RAW 264.7 cells were incubated with various concentrations of cytochalasin B (Calbiochem, San Diego, CA), which disrupts the cytoskeleton. In addition, rat spleen macrophages were produced, as described before (28). Briefly, the spleen was washed with PBS and pressed with a syringe plunger through a 40-gauge stainless steel screen. Red cells were lysed, and the remaining splenocytes were washed with PBS. To obtain a macrophage-enriched cell population, 107 cells/ml were plated in 60-mm cell culture dishes in RPMI 1640 medium supplemented with 20% heat-inactivated FCS and antibiotics for 90 min. Nonadherent cells were removed by washing the cells four times with PBS, and the resulting adherent cells (comprised of 75–95% macrophages as verified by staining with FITC-conjugated mAb directed against rat monocytes and macrophages ED1; Serotec, Oxford, U.K.) were then cultured in enriched medium (RPMI 1640 medium supplemented with 1% nonessential amino acids, 1% pyruvate, and antibiotics). In all experiments, cell viability was determined using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilid (XTT) kit (Biological Industries, Kibbutz Beit-haemek, Israel).

Normoxic and hypoxic conditions

For normoxic conditions, cells were incubated in a regular incubator (21% O2, 5% CO2, 74% N2). Hypoxic incubation was performed in a sealed anaerobic workstation (Concept 400; Ruskinn Technologies, Leeds, U.K./Jouan, Saint Herblain, France), in which the hypoxic environment (O2 <0.3%, 5% CO2, 95% N2) is kept constant, and so are the temperature (37°C) and humidity (90%). Samples from the culture medium were taken at the end of the exposure to hypoxia, to determine the partial pressures of O2 and CO2, as well as pH, using a blood gas analyzer ABL510 (Radiometer, Copenhagen, Denmark). Hypoxic PO2 values were 23 ± 1.4 mmHg, PCO2 values were 39.5 ± 0.6 mmHg, and pH values were 7.38 ± 0.12. Reoxygenation was preformed by removing the cells from the hypoxic chamber and immediately transferring them to the normoxic incubator.

Western blot analyses

RAW 246.7 cells or primary spleen macrophages were harvested in the presence of RIPA buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 µg/ml PMSF, 30 µg/ml aprotinin, 5 µg/ml leupeptin), protein concentrations were determined by Bradford reagent, and equal amounts of protein (10 µg) were loaded on a 7% SDS-PAGE. After electrophoretic separation, the proteins were transferred and fixed onto cellulose nitrate membranes (Schleicher & Schuell, Dassel, Germany) in transfer buffer (25 mM Tris, 180 mM glycine, 20% methanol, pH 8.3). The membranes were incubated for 1 h in blocking buffer (20% skimmed milk, 1% BSA, 0.01% Tween 20, 10 mM Tris, pH 8.0, 150 mM NaCl) at room temperature, and then probed for 1 h at room temperature with the diluted (1/1000) rabbit polyclonal anti-iNOS (Santa Cruz Biotechnology, Santa Cruz, CA), or after stripping, with the monoclonal anti-{alpha}-tubulin (Sigma-Aldrich), to ensure equal protein loading. After washing three times in 1x TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20), the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (Jackson ImmunoReasearch Laboratories, West Grove, PA), diluted 1/5000 in blocking buffer for additional 1 h at room temperature, and then washed again. The ECL system (Amersham, Arlington Heights, IL) was used for detection. The OD of the bands was quantified using Bio-Imaging system (Dinco & Renium, Jerusalem, Israel) and TINA software (Raytest, Straubenhardt, Germany).

Accumulation of nitrites

NO rapidly reacts with oxygen and water to produce stable products: nitrites and nitrates (29). Nitrite concentrations in either cell-free supernatants or fractions collected after gel filtration were determined mixing equal volumes of sample and Griess reagent (Sigma-Aldrich). The assay is based on the reaction between nitrites and the reagent containing sulfanilic acid and N-(1-naphtyl)ethylenediamine that produces a chromophoric azo-derivative molecule that absorbs light at 540 nm. The nitrite concentrations were calculated from a nitrite standard curve.

Separation of iNOS dimers and monomers

Cells were exposed to either normoxia or hypoxia in the presence of the combined stimulation (LPS + IFN-{gamma}) for 24 h, then lysed in lysis buffer (40 mM BisTris, pH 7.4, 3 mM DTT, 4 mM BH4, 0.1% Triton X-100, 10% glycerol, 100 mM NaCl) containing cofactors needed for iNOS activity and stability, and preventing it from separating to its subunits. Cells were frozen and thawed three times in liquid nitrogen. Lysates were centrifuged at 12,000 x g for 30 min at 4°C to remove debris, filtered through a 0.45-µm filter, and loaded onto a Superdex-200 gel filtration column (1.0 x 31.0 cm; Amersham Pharmacia Biotech, Piscataway, NJ) in lysis buffer without detergents. The expected elution volumes for either the dimers or monomers were determined by m.w. markers. Fractions were collected, concentrated 8-fold by Vivaspin-2 (Sartorius AG, Göttingen, Germany), resolved on 7% SDS-PAGE, and immunoblotted, as described before. The ability to produce NO was determined by the accumulation of nitrites in the fractions after incubation in normoxia for 2 h at 37°C in a buffer containing all cofactors and substrates needed (40 mM Tris, pH 7.8, 3 mM DTT, 1 mM L-arginine, 4 mM BH4, 1 mM NADPH, 4 mM FAD, 4 mM FMN). At the end of this incubation, fractions were treated with 5 U lactate dehydrogenase and 5 µmol pyruvate to remove excess NADPH, which could inhibit the Griess reaction, and then nitrite concentrations were determined in a Griess reaction.

Immunoprecipitation

Cells were cultured and exposed to either normoxia or hypoxia in the presence of the combined stimulation for 24 h. Then the cells were harvested in RIPA buffer, and protein concentrations were measured by Bradford reagent. To avoid nonspecific binding, 4 mg of protein was incubated with 2 µg of normal rabbit serum and precipitated using protein A-agarose. The remaining proteins were incubated with 4 µg of rabbit polyclonal anti-iNOS (Santa Cruz Biotechnology) and rotated overnight at 4°C with protein A-agarose. After centrifugation, the pellet was washed four times in PBS, resuspended in loading buffer, and boiled for 15 min. Samples were separated in a 7% SDS-PAGE, and the gel was stained with Coomassie blue. Protein bands were excised from the gel, and cleaved into peptides that were sequenced and identified by mass spectroscopy at the Protein Center, Faculty of Biology (Technion, Israel).

Immunofluorescence

Cells were cultured on coverslips and exposed to normoxia or hypoxia for 24 h in the presence of the combined stimulation, and were then fixated with 3.7% formaldehyde for 10 min at room temperature. To avoid nonspecific binding, the coverslips were incubated with 4% donkey normal serum for 30 min at room temperature and then washed three times with PBS. Primary Abs (rabbit polyclonal anti-iNOS or goat polyclonal anti-{alpha}-actinin; Santa Cruz Biotechnology) were incubated for 1 h at room temperature following three washes with PBS. Because we did not have access to a specific Ab directed against {alpha}-actinin 4, we used a polyclonal Ab that recognizes all four {alpha}-actinin proteins, knowing that in macrophages only the nonmuscle {alpha}-actinin 1 and 4 are expressed (30). Secondary Abs (rhodamine Red-X-conjugated donkey anti-goat IgG or Cy2-conjugated donkey anti-rabbit IgG; Jackson ImmunoResearch) were incubated in the dark for 1 h at room temperature and then washed three times with PBS. The coverslips were then mounted on a slide with fluoromount G. Immunofluorescent images were acquired by confocal microscopy, using the Bio-Rad (Hercules, CA) MRC 1000 confocal system.

Statistical analyses

All values are presented as means ± SE. The data were analyzed using repeated measures ANOVA. The Tukey-Kramer multiple comparison test was used to evaluate significance between experimental groups, and p values exceeding 0.05 were not considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Determination of the optimal experimental conditions for the expression of iNOS and the accumulation of NO

We first determined the optimal conditions for iNOS expression and NO production in macrophages. RAW 264.7 cells were incubated in normoxia, with or without the addition of both IFN-{gamma} and LPS that maximally induce iNOS expression (31), and the expression of iNOS and nitrite accumulation were measured at different time periods. Expression of iNOS was not detected in the absence of the stimuli (Fig. 1A), but peaked after 24-h incubation with IFN-{gamma} + LPS (p < 0.01). Likewise (Fig. 1B), nitrites were not produced in the absence of the stimuli, but their addition resulted in a gradual accumulation of nitrites, reaching high levels at 24 h (p < 0.01). The expression of the housekeeping protein {alpha}-tubulin was unchanged throughout the experiment, indicating equal protein loading. Cell viability was not changed throughout the experimental protocol (Fig. 1C). Hence, the following experiments were performed in normoxia or hypoxia at 24 h. To test the importance of hypoxia-induced depletion of oxygen to iNOS activity, hypoxic macrophages were reoxygenated for short (2-h) and prolonged (24-h) periods of time.



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  		FIGURE 1. Effect of IFN-{gamma} and LPS on iNOS expression and activity in RAW 264.7 cells. A total of 3 x 106 cells was plated in 60-mm dishes for 12 h in a serum-free medium, and then incubated, with or without the addition of both IFN-{gamma} (100 U/ml) and LPS (1 µg/ml), for different time periods in normoxia. At the end of the incubation, supernatants were collected, and cells were extracted for determination of iNOS by Western blot analysis. A, Densitometric analysis (n = 4) of iNOS protein levels, as measured by Western blot analysis, relative to the control (time = 0). B, Determination of nitrites by Griess reaction (n = 4). C, Determination of cell viability by XTT (n = 4). In the absence of the combined stimulation, iNOS was not induced and NO was not produced. The addition of IFN-{gamma} and LPS induced iNOS expression, which peaked at 24 h, and nitrites accumulated, reaching high levels after 24 h. Cells did not die as a result of activation with the combined stimulation, but rather proliferated after 48 h, a trend that did not reach statistical significance. *, p < 0.05; **, p < 0.01 compared with control.

 
Effects of hypoxia and reoxygenation on iNOS protein expression and accumulation of nitrites in RAW 264.7 cells

Cells were subjected to hypoxia for 24 h (H24), or to hypoxia followed by short (2-h) or prolonged (24-h) reoxygenation (H24R2, H24R24), or to normoxia for 24 or 48 h (N24, N48) in the presence or absence of IFN-{gamma}, LPS, or their combination. Hypoxia alone (Fig. 2A) significantly (9.5 ± 5.3-fold) increased iNOS expression compared with the control (N24) (p < 0.01), whereas prolonged (but not short) reoxygenation reversed this effect (2.7 ± 0.7-fold compared with control). Presence of LPS in normoxia induced iNOS expression (5.7 ± 2-fold compared with the untriggered control), whereas LPS in hypoxia further enhanced iNOS expression by 21 ± 4.4-fold (p < 0.05). Reoxygenation did not change the effects of hypoxia and LPS on iNOS expression. The most pronounced effect on iNOS expression was achieved by the addition of IFN-{gamma} or IFN-{gamma} + LPS (48.6 ± 8 and 56.4 ± 8-fold increase, compared with the control, p < 0.01) in both normoxia and H/R.



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  		FIGURE 2. Effect of hypoxia and reoxygenation on iNOS expression and NO production in RAW 264.7 cells. A total of 3 x 106 cells was plated in 60-mm dishes for 12 h in a serum-free medium. Cells were then exposed to normoxia, hypoxia, or hypoxia, followed by reoxygenation, with or without the addition of IFN-{gamma} (100 U/ml) or LPS (1 µg/ml), or their combination. At the end of incubation, supernatants were collected and cellular proteins were extracted. A, A representative gel of iNOS Western blot analysis and densitometric analysis (n = 6) of iNOS protein levels, relative to the experimental control (N24). B, Determination of nitrites as a measure of iNOS activity (n = 6). C, Determination of cellular viability by XTT (n = 6). N24-normoxia for 24 h, N48-normoxia for 48 h, H24-hypoxia for 24 h, H24R2-hypoxia for 24 h, followed by short reoxygenation for 2 h, H24R24-hypoxia for 24 h, followed by prolonged reoxygenation for 24 h. Hypoxia alone or hypoxia with LPS increased iNOS protein levels insignificantly. However, IFN-{gamma} alone or with LPS similarly elevated iNOS expression by 50- to 60-fold from control, and hypoxia or reoxygenation did not change that expression. Accumulation of nitrites was elevated in the presence of IFN-{gamma} or IFN-{gamma} + LPS at 24 and 48 h in normoxia, but hypoxia inhibited production of NO, despite the presence of high levels of the protein. Hypoxia or reoxygenation did not cause cell death. **, p < 0.01 compared with control; §, p < 0.01 compared with N24 + IFN-{gamma} or with N24 + IFN-{gamma} + LPS, respectively.

 
Fig. 2B shows that negligible concentrations of nitrites were detected during normoxia alone (3.8 ± 1.5 µM and 8.4 ± 2.1 µM, at 24 and 48 h), while LPS increased it in normoxia (30.4 ± 5.8 µM and 49 ± 15 µM at 24 and 48 h, p < 0.05). The presence of IFN-{gamma} or the combined stimulation further increased nitrite accumulation to 71.6 ± 14 µM and 98.5 ± 17 µM, respectively, at 24 h (p < 0.01), and to 130.6 ± 24 µM and 135 ± 26 µM, respectively, at 48 h (p < 0.01). In contrast, during hypoxia, IFN-{gamma} alone or in combination with LPS did not increase the production of nitrites compared with controls. Moreover, in hypoxia, the level of nitrites following stimulation with IFN-{gamma} or IFN-{gamma} + LPS was lower than in normoxia (7.6 ± 1.7 µM and 13.5 ± 2 µM, respectively, p < 0.01). Short reoxygenation did not alleviate the inhibitory effect of hypoxia, but prolonged reoxygenation in the presence of LPS, IFN-{gamma}, or both lead to increased accumulation of nitrites (58.5 ± 20 µM, 84.9 ± 21 µM, and 96.7 ± 27 µM, respectively), reaching concentrations similar to those accumulated after 24 h of normoxia.

Fig. 2C demonstrates that hypoxia, reoxygenation, or the presence of the stimuli did not affect cell viability.

Effects of hypoxia and reoxygenation on iNOS protein expression and accumulation of nitrites in primary spleen macrophages

To corroborate our findings in the macrophage cell line RAW 264.7, we used a culture of primary rat spleen macrophages. Fig. 3A shows that, similar to the RAW 264.7 cells, addition of IFN-{gamma} + LPS in normoxia induced iNOS expression after 24 and 48 h (5 ± 0.1-fold and 9.2 ± 2.3-fold relative to the control (N24), respectively, p < 0.05). Likewise, iNOS expression remained elevated in the presence of the combined stimulation after hypoxia and H/R (6.3 ± 1.2-fold and 9.3 ± 3.7-fold from control, respectively, p < 0.05). A low basal level of iNOS expression was observed in unstimulated cells, which could result from mild activation caused by macrophage isolation and adherence to plastic plates. Because elevation of iNOS was calculated as folds from the untriggered normoxic values, a more prominent enhancement was detected in the cell line (50-fold) compared with the primary cells (9-fold).



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  		FIGURE 3. Effect of hypoxia and reoxygenation on iNOS expression and NO production in primary macrophages. Primary adherent macrophages from rat spleens were plated in a 24-well plate for 12 h in a serum-free medium. Cells were then exposed to normoxia, hypoxia, or hypoxia, followed by reoxygenation, with or without the addition of IFN-{gamma} (100 U/ml), LPS (1 µg/ml), or their combination. At the end of incubation, supernatants were collected and cellular proteins were extracted. A, A representative gel of iNOS Western blot analysis and densitometric analysis (n = 4) of iNOS protein levels relative to the experimental control (N24). B, Determination of nitrites as a measure of iNOS activity (n = 6). N24-normoxia for 24 h, N48-normoxia for 48 h, H24-hypoxia for 24 h, H24R24-hypoxia for 24 h, followed by prolonged reoxygenation for 24 h. The addition of the combined stimulation induced iNOS, and neither hypoxia nor reoxygenation changed this induction significantly. Similar to the RAW 264.7 cell line, nitrites significantly accumulated in supernatants of primary macrophages in the presence of the combined stimulation in normoxia. Hypoxia markedly inhibited the accumulation of nitrites, and only prolonged reoxygenation restored them to their normoxic concentrations. * or **, p < 0.05 compared with control; ***, p < 0.001 compared with control; §, p < 0.05 compared with N24 + IFN-{gamma} + LPS.

 
Fig. 3B demonstrates that low levels of nitrites were detected following 24 and 48 h of incubation in normoxia alone (2.6 ± 0.3 µM and 5.7 ± 1.1 µM, respectively), and were significantly elevated in the presence of IFN-{gamma} + LPS (10.2 ± 0.8 µM and 11 ± 1.8 µM, respectively, p < 0.01). As in the RAW 264.7 cells, hypoxia inhibited nitrite accumulation in spleen macrophages, both without (1.3 ± 0.6 µM) and with stimulation (2.8 ± 0.8 µM, p < 0.05 relative to stimulated normoxic cells). Again, H/R in the presence of the stimuli restored nitrites to their normoxic level (12 ± 2 µM, p < 0.001 compared with the control). The difference in nitrite levels between RAW 264.7 cells and spleen macrophages could arise from difference between primary cells and a cell line, as well as from the low number of adherent primary macrophages in culture compared with that of the cell line.

Ratio of iNOS dimers/monomers in normoxia and hypoxia in RAW 264.7 cells

To find whether hypoxia inactivates iNOS by reducing the ratio between the active homodimer (260 kDa) and inactive monomeric form (130 kDa) (13, 32), cellular extracts obtained from RAW 264.7 cells following incubation in normoxia or hypoxia in the presence of the combined stimulation were fractionated by fast protein liquid chromatography gel filtration. In normoxia, the dimeric form of iNOS (fractions 10–15) remained active and produced nitrites in the presence of iNOS substrates (L-arginine and oxygen) and cofactors (FAD, FMN, BH4), whereas the monomeric form (fractions 16–20) did not produce nitrites (Fig. 4). In contrast, in hypoxia, the dimeric form did not produce nitrites, despite the presence of all substrates and cofactors. Furthermore, although hypoxia inactivated iNOS dimers, it did not change the ratio between its dimers and monomers (Table I), suggesting that another mechanism is involved and that lack of oxygen or cofactors is not the cause of dimer inactivity.



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  		FIGURE 4. Separation of iNOS dimers and monomers by gel filtration, and their ability to produce nitrites. RAW 264.7 cells were plated on 100-mm dishes for 12 h in a serum-free medium. Cells were then exposed to normoxia or hypoxia with the addition of the stimulators IFN-{gamma} (100 U/ml) and LPS (1 µg/ml). At the end of the incubation, cellular proteins were extracted and separated on a Superdex-200 gel filtration column by fast protein liquid chromatography. Fractions were collected; each fraction was concentrated 8.5-fold and analyzed by Western blotting for the presence of iNOS and for the ability to produce nitrites (n = 3). In normoxia only, dimers (fractions 10–15), but not monomers (fractions 16–19) produced nitrites, whereas in hypoxia both dimers and monomers did not produce nitrites.

 

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  		Table I. Percentage of iNOS dimers and monomers in normoxia vs hypoxiaa

 
Identification of an iNOS-associated protein that dissociates in hypoxia

To explore the possibility that hypoxia renders iNOS inactive by allowing the binding of an inhibitory protein or by dissociating an activating factor bound in normoxia, RAW 264.7 cells were incubated in normoxia or hypoxia for 24 h with the combined stimulation, and their cellular extracts were subjected to immunoprecipitation using specific iNOS Ab. Two protein bands were observed after immunoprecipitation of the normoxic cellular extract (Fig. 5), and after excision of the bands from the gel, were identified by mass spectroscopy sequencing as iNOS (at ~130 kDa) and {alpha}-actinin 4 (at ~100 kDa). In contrast, only the iNOS protein was observed in hypoxia, suggesting that hypoxia causes dissociation of {alpha}-actinin 4 from iNOS. This experiment, including identification of the proteins by mass spectroscopy, was repeated three times, yielding identical results.



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  		FIGURE 5. Immunoprecipitation of proteins that are bound to iNOS in normoxia and hypoxia. RAW 264.7 cells were plated on 100-mm dishes for 12 h in a serum-free medium. Cells were then exposed to normoxia or hypoxia with the addition of the stimulators IFN-{gamma} (100 U/ml) and LPS (1 µg/ml). At the end of the experiment, cellular proteins were extracted and cleared from nonspecific proteins by precipitation with IgG Ab from normal rabbit serum. Anti-iNOS Ab and protein A-agarose were added to the remaining protein samples and incubated overnight at 4°C. The pellet was washed, resuspended in loading buffer, boiled for 15 min, and separated on 7% SDS-PAGE. After electrophoresis, the gel was stained with Coomassie blue. Two protein bands of 130 and 100 kDa were observed in normoxia, whereas in hypoxia only one band of 130 kDa was observed. All bands were excised from the gel and sent for sequencing. The 130-kDa band was identified as iNOS, and the 100-kDa band was identified as {alpha}-actinin 4. This experiment was repeated three times with identical results.

 
Cytochalasin B decreases accumulation of nitrites

{alpha}-Actinin 4 is an adapter protein that interacts with different proteins and anchors them to the actin cytoskeleton. To test whether normal structure of the cytoskeleton is important for iNOS activity, we used cytochalasin B, which inhibits actin polymerization and disrupts the cytoskeleton. RAW 264.7 cells were incubated in normoxia with the combined stimulation for 24 h to induce iNOS expression, together with increasing concentrations of cytochalasin B, which were nontoxic as evaluated by cell viability. A gradual decrease in the accumulation of nitrites was measured with increasing amounts of cytochalasin B (Fig. 6), which became significant at the higher concentrations of cytochalasin B (p < 0.05 at 10 µM and p < 0.001 at 25 µM).



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  		FIGURE 6. Dose-dependent effect of cytochalasin B on iNOS activity. RAW 264.7 cells were plated for 12 h in a serum-free medium. Cells were then exposed to normoxia or hypoxia with the addition of both stimulators IFN-{gamma} (100 U/ml) and LPS (1 µg/ml), as well as increasing amounts of cytochalasin B (0–25 µM). Because cytochalasin B could become toxic in high concentrations, cell viability was tested by XTT and only nontoxic concentrations were used. Cytochalasin B significantly decreased accumulation of nitrites in a dose-dependent manner.

 
Colocalization of iNOS and {alpha}-actinin 4

To follow the effects of hypoxia on the interaction between iNOS and {alpha}-actinin 4 and their colocalization, RAW 264.7 cells were incubated with or without the combined stimulation in normoxia, hypoxia, reoxygenation, or normoxia with cytochalasin B, and stained for both iNOS and {alpha}-actinin. Isotype-matched control sera yielded no immunostaining. Unstimulated cells showed no iNOS-positive cells, but {alpha}-actinin was immunostained in all cells. In normoxia, with the combined stimulation, three sites of iNOS staining were observed: cytoplasmic staining, which was diffused and evenly distributed in all cells; staining in an area of the plasma membrane or immediately adjacent to it in the submembranal zone (Fig. 7B, arrow), which was observed in 27.2% ± 2.9 of the cells; and staining of a perinuclear area (Fig. 7B, solid arrowhead) in a minority (1.3% ± 0.5) of the cells. Similarly, {alpha}-actinin proteins were stained diffusely and weakly in the cytoplasm, but a stronger staining, concentrated in small aggregates, was observed in areas immediately adjacent to the plasma membrane (Fig. 7A). The merged images of iNOS and {alpha}-actinin (Fig. 7C) indicate colocalization (in yellow) in the submembranal area, but not in the cytoplasm. Hypoxia changed the intracellular distribution of iNOS, which was present in many small clumps, and disappeared from the submembranal and perinuclear area (Fig. 7E). Staining of {alpha}-actinin after hypoxia remained largely at the submembranal area, with a much more aggregated appearance (Fig. 7A). The merged images reveal that colocalization was visible in only a small number of the aggregates (Fig. 7F), suggesting that the two proteins were mostly separated by hypoxia. Incubation with cytochalasin B led to a reduction in the submembranal staining of iNOS and to its stronger cytoplasmic staining, whereas the majority of the {alpha}-actinin staining remained submembranal. The merged images of iNOS and {alpha}-actinin after incubation with cytochalasin B show that the remaining colocalization still resides mainly in the submembranal area. Exposure of the cells to hypoxia followed by 24-h reoxygenation gradually restored the normoxic distribution and colocalization of iNOS and {alpha}-actinin (Fig. 7, G–I). Taken together, these data suggest that in normoxia, iNOS and {alpha}-actinin 4 partly colocalize in the submembranal area, but hypoxia disrupts this colocalization, as it moves iNOS away from the submembranal areas. Following treatment with cytochalasin B, a proportion of iNOS remains in the submembranal areas, as indicated by the merged images, so that cytochalasin B does not completely simulate hypoxia.



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  		FIGURE 7. Colocalization of iNOS and {alpha}-actinin 4 is disrupted in hypoxia. RAW 264.7 cells were stimulated with IFN-{gamma} (100 U/ml) and LPS (1 µg/ml) for 24 h in normoxia and hypoxia and immunostained with anti-iNOS (green, B, E, H, K) and anti-{alpha}-actinin (red, A, D, G, J). Cells were exposed to normoxia (A, B, C), hypoxia (D, E, F), reoxygenation (G, H, I), or 25 µM of cytochalasin B (J, K, L). The merged {alpha}-actinin and iNOS images from A + B, D + E, G + H, and J + K are shown (C, F, I, L). Areas of colocalization are shown in yellow. Magnification was x1800 for A–C and G–I and x600 for D–I and J–L. Arrow indicates peripheral submembranal staining of iNOS, and a solid arrow indicates iNOS staining in a perinuclear area.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown in this study that hypoxia inactivates the high output macrophage iNOS without affecting its expression, and disrupts its binding to the cytoskeletal protein {alpha}-actinin 4. We suggest that these two results are linked, and that the cellular localization of iNOS is important to its enzymatic activity.

In addition, we have observed that hypoxia alone induced iNOS expression, and that addition of LPS synergistically increased its levels. However, in both normoxia and hypoxia, addition of IFN-{gamma}, with or without LPS, similarly induced iNOS expression to maximal levels. This is different from two previous studies, demonstrating synergy between hypoxia and IFN-{gamma} in a different macrophage cell line (26), or showing such synergism in RAW 264.7 cells at the mRNA level only (32). Our results were further corroborated in primary spleen macrophages.

Transcription of iNOS, which was studied extensively, is considered to be its main regulatory mechanism of iNOS (13), although several posttranscriptional (30) and translational (33) checkpoints have been suggested. Our results, however, point to posttranslational regulatory events, involving protein-protein interactions and cytoskeletal changes in iNOS regulation.

Several mechanisms could explain posttranslational inactivation of iNOS by hypoxia, including lack of oxygen as a substrate of the enzyme, instability of iNOS spatial structure, or the presence of an auxiliary protein that either activates iNOS in normoxia or inhibits its activity in hypoxia. If lack of oxygen as a substrate was the only reason for hypoxia-induced iNOS inactivation, we could expect rapid production of NO upon renewal of oxygen supply following short reoxygenation, because high levels of iNOS protein were expressed in both hypoxia and reoxygenation. As only prolonged reoxygenation (24 h) gradually restored nitrite production, absence of oxygen does not seem to be the only factor influencing iNOS inactivation in hypoxia.

The possibility that hypoxia changes the spatial structure of the enzyme, preventing it from forming dimers that are necessary for its activity (13, 34), was ruled out by demonstrating similar dimer/monomer ratios in hypoxia and normoxia, yet inability of the dimers extracted in hypoxia to produce nitrites. Because dimers in hypoxia and normoxia were extracted and tested for nitrite production in the presence of oxygen and excess cofactors, our previous conjecture that lack of oxygen did not cause iNOS inactivation is strengthened. It may be argued that iNOS is active in hypoxia, but produces peroxynitrite rather than nitrites as it reacts with superoxide. Although not directly tested, this possibility was excluded, because activity of iNOS dimers was measured in an acellular system, with excess arginine that prevents production of superoxide by iNOS itself.

The third possibility that the inhibitory effect of hypoxia on iNOS function is due to the presence of an auxiliary protein was based on two previously described proteins, kalirin (35) and NAP-110 (36), which were suggested to interact with iNOS and inhibit its activity. In the present study, we show that {alpha}-actinin 4 interacts with iNOS in normoxia, and its dissociation in hypoxia correlates to iNOS inactivity. The {alpha}-actinin 4 protein is a member of the actinin family that binds actin, the main cytoskeletal protein. This protein is expressed in macrophages, participates in vital functions such as phagocytosis (37), and is involved in the anchoring of enzymes, such as CLP-36, to the cytoskeleton by protein-protein interactions mediated by PDZ and LIM sequences (38). {alpha}-Actinin 4 has also been demonstrated to bind to Ca2+/calmodulin-dependent protein kinase II, connecting it to the transmembranal protein densin-180 (39). The other two isoforms of NOS have been shown to require specific submembranal localization for their activity, and use similar domains to interact with proteins that guide them to their intracellular compartment (13).

These cumulative data and our findings lead us to hypothesize that iNOS is normally anchored to the cytoskeleton by {alpha}-actinin 4, and that its cellular localization, which is disrupted by hypoxia, is a prerequisite for NO production. Using cytochalasin B, which prevents actin polymerization and disrupts cytoskeletal integrity, we showed reduced NO production in normoxia, thus strengthening the importance of the cytoskeleton to the activity of macrophage iNOS. Confocal imaging analysis showed that colocalization of active iNOS and {alpha}-actinin 4 in normoxia or prolonged reoxygenation was largely lost in hypoxia, parallel to the loss of activity. This submembranal localization of iNOS has also been shown in a recent study, and the perinuclear area was mapped to the Golgi apparatus (23). Additionally, in the presence of cytochalasin B, the two proteins continued, at least in part, to colocalize, correlating to the partial activity of iNOS. Thus, intracellular localization of iNOS may serve as an additional posttranslation regulatory checkpoint. This is supported by another recent study that shows that disruption of the actin cytoskeleton altered iNOS expression (40).

In summary, we have shown that in mouse macrophages, IFN-{gamma} or IFN-{gamma} and LPS highly induce iNOS, and that hypoxia inhibits iNOS activity without interfering with the expression of the protein. The observed inhibition did not result from lack of oxygen, nor was it caused by a change in the dimer/monomer ratio. Rather, hypoxia disrupted protein-protein interactions between iNOS and {alpha}-actinin 4 and relocated iNOS, suggesting that cellular localization of iNOS is of extreme importance to its activity. The mechanism by which hypoxia causes the dissociation between iNOS and {alpha}-actinin 4 is yet unknown and remains to be elucidated.


   Footnotes
 
1 This work was partially supported by the German-Israeli Foundation (GIF, Grant I-475.035/02). Back

2 S.D. and H.B. are equally contributing senior authors. Back

3 Address correspondence and reprint requests to Dr. Michal A. Rahat, Immunology Research Unit, Carmel Medical Center, 7 Michal Street, Haifa, 34362 Israel. E-mail address: rahat_miki{at}clalit.org.il Back

4 Abbreviations used in this paper: H/R, hypoxia and reoxygenation; BH4, tetrahydrobiopterin; FAD, flavin adenine dinucleotide; FMN, flavin adenine mononucleotide; iNOS, inducible NOS; I/R, ischemia and reperfusion; NOS, NO synthase; XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilid. Back

Received for publication February 7, 2003. Accepted for publication July 16, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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