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J. Biol. Chem., Vol. 279, Issue 27, 28681-28688, July 2, 2004

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Induction of Heme Oxygenase-1 Inhibits NAD(P)H Oxidase Activity by Down-regulating Cytochrome b⁵⁵⁸ Expression via the Reduction of Heme Availability^*

Camille Taillé, Jamel El-Benna, Sophie Lanone, My-Chan Dang, Eric Ogier-Denis, Michel Aubier, and Jorge Boczkowski¶

From the INSERM, Unité 408 and Unité 479, Institut Fédératif de Recherche 02, Faculté de Médecine Xavier Bichat, 75018 Paris, France

Received for publication, September 26, 2003 , and in revised form, March 3, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

 
Heme-oxygenase-1 (HO-1), the rate-limiting enzyme of heme degradation, has powerful anti-oxidant properties related to the production of the reactive oxygen species scavenger bilirubin. However, some data suggest that HO-1 could also inhibit the cellular production of reactive oxygen species. Therefore, we investigated whether the anti-oxidant properties of HO-1 could be mediated by modulation of the activity and/or expression of the heme-containing NAD(P)H oxidase, the main source of the superoxide anion () in phagocytic cells. Increasing HO-1 expression in RAW 264.7 macrophages effectively decreased NAD(P)H oxidase activity and expression of gp91^phox, its heme-containing catalytic component, because of deficient protein maturation and increased degradation. Loading cells with heme reversed the decrease in production and gp91^phox expression induced by HO-1 overexpression. Similar results were obtained in vivo in rat alveolar macrophages after pharmacological modulation of HO-1 expression or activity. These results show that a decrease in heme content due to HO-1 activation limits heme availability for maturation of the gp91^phox subunit and assembly of the functional NAD(P)H oxidase. This study provides a new mechanism to explain HO-1 anti-oxidant properties.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

 
In phagocytes the NAD(P)H oxidase is the major source of the superoxide anion that represents, with hydrogen peroxide, a critical part of their armory of bactericidal mechanisms (1). The whole oxidase is made of an assembly of gp91^phox and p22^phox (which heterodimerize to form the cytochrome b₅₅₈) and p67^phox, p47^phox, and Rac1 or Rac2 subunits. Gp91^phox, the catalytic moiety of the oxidase, is a plasma membrane-associated flavohemoprotein, containing one flavin-adenine dinucleotide and two hemes, that catalyzes the NAD(P)H-dependent reduction of oxygen to form superoxide (2). Non-phagocytic cells such as vascular (3) and bronchial (4) smooth muscle cells, endothelial cells (5), fibroblasts (6), thyroid cells (7), and prostate epithelial cells (8) also express gp91^phox or the recently described homolog termed NOX (9). In these cells the superoxide anion drives the expression of numerous oxidant sensitive genes and is therefore involved in redox signaling as a second intracellular messenger that controls important functions such as cell proliferation, apoptosis, inflammation, cell recruitment, and matrix turnover. Thus, it appears that understanding the mechanisms of regulation of superoxide production by the oxidase could have important physiological, pathophysiological, and, probably, clinical implications in different conditions and disease states.

Heme oxygenase-1 (HO-1)¹ is the rate-limiting enzyme of heme degradation and is therefore involved in the control of cellular heme content. HO-1 has also shown increasing number of anti-oxidant properties, which confer to the enzyme overexpression protective effects in various models of oxidative injury. Until now, this anti-oxidant effect has been mainly attributed to production of bilirubin (10–12), one of the end products of heme catabolism with reactive oxygen species scavenging properties (13, 14), or to ferritin induction after release of the central iron from heme (15). Recently, we have shown that HO-1 overexpression decreases superoxide production in airway smooth muscle (12, 16), which is known to express the heme-containing gp91^phox (4), suggesting that HO-1 could affect NAD(P)H oxidase activity. This could be related to the heme content regulatory properties of HO-1. However, no data is available in the current literature concerning the effect of HO-1 on NAD(P)H oxidase activity and protein expression. Therefore, we investigated in vitro in murine RAW 264.7 macrophages and in vivo in rat alveolar macrophages whether anti-oxidant properties of HO-1 could be mediated by the modulation of NAD(P)H oxidase activity and subunits expression.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

 
Reagents—RAW 264.7 macrophages were purchased from the American Type Culture Collection (Manassas, VA). CoPP, heme, and SnPP were from Porphyrin Products (Frontier Scientific, London, UK). Cells were transfected with the Superfect® transfection reagent (Qiagen SA, Courtaboeuf, France). The polyclonal anti-HO-1 antibody was from StressGen (Tebu, Le-Perray-en-Yvelines, France), the monoclonal anti--actin antibody was from Sigma, and the anti-p22^phox, anti-p47^phox, and the anti-gp91^phox antibodies were from UBI. Secondary fluorescein isothiocyanate-coupled antibodies were from Molecular Probes (Eugene, OR). The proteasome inhibitors lactacystin and MG-132 were from Calbiochem. Culture media, supplements, and fetal calf serum were from Invitrogen. Plastic tissue culture plates were supplied by Costar Corp. (Cambridge, MA). Reagents for Western blot were from Bi-Rad. Other reagents were from Sigma.

RAW 264.7 Macrophage Culture—Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum and antibiotics (10 mg/ml streptomycin and 10,000 IU/ml penicillin G) and amphotericin B (25 µg/ml) in a humidified atmosphere of 5% CO₂/95% air at 37 °C. Cells were used when reaching 80–90% confluence.

Cellular Toxicity and Viability—Cellular toxicity and viability were assessed by two different methods, namely cell count and trypan blue exclusion and lactate dehydrogenase release in the medium.

HO-1 pcDNA Transfection—The plasmid encoding for rat HO-1 was a kind gift from Dr. Josef Dulak (Jagiellonian University, Krakow, Poland). Transfection was achieved with the Superfect® transfection reagent following the manufacturer's instructions. Briefly, cells were seeded in 6-well plates at a density of 100,000 cells/well 24 h before transfection. The proportions used were 4 µg of DNA to 20 µl of transfection reagent per well. Cells were incubated for 3 h, and then the medium was replaced with fresh medium containing 10% serum. Cytochrome c reduction assay and Western blot for HO-1 expression was performed 48 h after transfection.

HO-1, p22^phox, p47^phox, and gp91^phox Expression—HO-1, p22^phox, p47^phox, and gp91^phox protein expression was measured by Western blot. Briefly, cells were seeded in 75 cm² plates. When confluent, they were incubated with heme or the synthetic metalloporphyrins for 2 h, and then the medium was removed, cells were washed with PBS, and fresh medium was added for 24 or 48 h. The cells were scrapped in lysis buffer, sonicated for 10 min, and frozen at -80 °C until being used for Western blot analysis. Western blot was performed as described previously (17, 18). The concentration of the primary antibodies was 1:1000. Detection was performed by a chemiluminescence substrate. Using the same blots, the expression of the housekeeping protein -actin was evaluated using a monoclonal anti--actin antibody. Optical densities were measured with a Perfect Image 2.01 image analysis system (Iconix, Courtaboeuf, France). Results were expressed as the ratio of the expression of HO-1, p22^phox, p47^phox, or gp91^phox to that of -actin.

Immunofluorescence Analysis by Confocal Microscopy—Cells were cultured in four-well slides (SonicSeal®, Nalgene Nunc International, Rochester, NY) at a density of 20,000 cells/well. Cells were treated with 20 µM CoPP for 2 h, washed with PBS, and then fresh medium was added. Twenty-four hours later the medium was removed, and the cells were washed twice with cold PBS, fixed in methanol for 10 min at room temperature, and then washed again before being incubated with anti-HO-1 and anti-gp91^phox antibody (1:1000 and 1:500 dilution, respectively) for 1 h at room temperature. Secondary antibodies were used at 1:400 dilution for 30 min at room temperature. Cells were observed with a ZEISS LSM510 META confocal microscope using a X63/1.4 objective.

Reverse Transcription PCR Amplification—Total RNA of RAW 264.7 cells were prepared with TRIzol reagent. A polymerase chain reaction was performed using the high fidelity Taq Platinium PCR Supermix (Invitrogen) and the following couples of primers: gp91^phox,5'-GCA CAC CGC CAT CCA CAC AA-3' and 5'-CCC CTC CGT CCA GTC TCC AA-3'; p22^phox, 5'-TTC CTG TTG TCG GTG CCT GC-3' and 5'-TTC TTT CGG ACC TCT GCG GG-3'; and p47^phox, 5'-GTG GAG AAG AGC GAG AGC GG-3' and 5'-GGT GGA TGC TCT GTG CGT TG-3'. DNA probes were amplified by an initial cycle at 94 °C for 3 min followed by 10 cycles of 94 °C for 30 s, 58 °C for 45 s, 72 °C for 1 min, and ending with a 5-min extension at 72 °C. PCR products were separated on 1% agarose gels.

Extracellular Superoxide Anion Release/Cytochrome c Reduction—Ferricytochrome c reduction was measured as described previously (18). Briefly, cells were cultured in 6-well plates. Upon reaching confluence they were treated with CoPP (20 µM), heme (1, 10, and 100 µM), or SnPP (20 µM) as described for Western blot analysis. Twenty-four hours after treatment the medium was removed, and the cells were washed twice with PBS. The medium was replaced with 1 mg/ml ferricytochrome c in Hanks' balanced salt solution without phenol red. Each condition was made with and without 300 units/ml superoxide dismutase. Cells were stimulated with 0.5 mg/ml opsonized zymosan for 15 min. The buffer was removed, and absorbance at 550 nm was read immediately. Superoxide anion production was calculated from the differences in the absorbances between samples with and without superoxide dismutase using an extinction coefficient of 21.1 mM^-1 cm^-1 for reduced ferricytochrome c. Results are expressed as nanomoles of superoxide anion produced per well during a 15-min period.

Assay for Heme Content Measurement—For intracellular heme content assessment, cells were cultured in 24-well plates. After 2 h of treatment with heme or porphyrins the cells were washed twice with PBS, and then fresh medium was added until an assay was performed 24 or 48 h later. Heme content was determined according to the method of Motterlini et al. (19). Briefly, cells were washed with PBS and solubilized by adding 1 ml of concentrated formic acid. The heme concentration of the formic acid solution was determined spectrophotometrically at 398 nm (extinction coefficient = 1.56 10⁵ M^-1 cm^-1). Heme content was expressed as pmol/ml.

Luminol-amplified Chemiluminescence Assay in Rat Alveolar Macrophages—Male Sprague-Dawley rats (250 g) were purchased from Iffa Credo (France). They were treated with CoPP (15 mg/kg intraperitoneally), SnPP (30 mg/kg intraperitoneally), or vehicle. Forty-eight hours after injection the rats were anesthetized, and the trachea was cannulated with a 22-gauge catheter through a tracheotomy. For bronchoalveolar lavage,15 ml of cold sterile 0.9% sodium chloride was injected and reaspirated five times. The bronchoalveolar lavage was centrifuged at 300 x g for 5 min at 4 °C. Then the supernatant was removed, and the pellet was resuspended in 1 ml of Hanks' balanced salt solution.

For the chemiluminescence assay, 200,000 cells for each condition were incubated in 200 µl of Hanks' balanced salt solution in the presence of 10 µM luminol and 5 units of horseradish peroxidase and then stimulated with opsonized zymosan (1 mg/ml) or PMA (0.5 µg/ml). Chemiluminescence was measured during a 30-min period in a Berthold chemiluminometer. The areas under the curve were compared. Results are expressed as 10⁴ cpm.

At the end of the experiment, cells (10⁶) were centrifuged and resuspended in lysis buffer. They were frozen at -80 °C before being used for Western blot determination of HO-1 and gp91^phox expression.

Statistical Analysis—The values are given as the means ± S.E. The data were analyzed by one-way analysis of variance, and the differences between the means were analyzed with Fischer's protected least significant difference multiple comparison test or non-parametric analysis when appropriate. The significance for all statistics was accepted at p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

 
Cellular Toxicity and Viability—No toxicity was observed with metalloporphyrins and heme at the doses used in the present study. Twenty-four hours after treatment with metalloporphyrins or heme, the percentage of increase in lactate dehydrogenase concentration in culture media ranged from 2.01 ± 0.91 to 7.12 ± 2.91 of the values for untreated cells (not significant). The percentage of positive cells with blue trypan exclusion was 2.20 ± 1.02 for untreated cells and ranged from 2.61 ± 0.91 to 5.75 ± 0.98 in cells treated with metalloporphyrins or heme (not significant as compared with untreated cells).

HO-1 Induction Decreased Cellular Heme Content—We first checked with Western blot and confocal microscopy to determine whether the incubation of RAW 264.7 with 20 µM CoPP for 2 h induced a significant dose-dependent and time-dependent increase in HO-1 protein expression (Fig. 1, A and B). In addition (and as expected), the transfection with rat HO-1 cDNA induced a significant increase in HO-1 expression (Fig. 1C).

View larger version (25K): [in this window] [in a new window]   FIG. 1. HO-1 expression in RAW 264.7 macrophages. A, Western blot analysis of HO-1 expression after treatment with CoPP as a function of concentration (left panel) and a function of time (right panel). B, typical aspect of HO-1 expression in confocal microscopy in non-treated (left panel) or in cells 24 h after treatment with CoPP 20 µM (right panel). C, Western blot analysis of HO-1 expression 48 h after transfection with a plasmid containing HO-1 cDNA. Western blot and immunofluorescence were performed as described under "Experimental Procedures." Each experiment was repeated three times.

View larger version (27K): [in this window] [in a new window]   FIG. 2. Superoxide production in RAW 264.7 macrophages. Superoxide production by RAW 264.7 after 15 min of stimulation with 0.5 mg/ml opsonized zymosan (Zop) was assessed by a cytochrome c reduction assay. Cells were treated as described under "Experimental Procedures" 24 h before the assay with CoPP (20 µM), SnPP (20 µM), heme (1, 10, and 100 µM), or both or transfected with rat HO-1 cDNA or control cDNA 48 h before the assay. Each bar represents mean ± S.E. (n = 5–10). *, different from unstimulated cells, p < 0.05; #, different from zymosan-stimulated cells, p < 0.05; &, different from CoPP-treated zymosan-stimulated cells, p < 0.05.

HO-1 Induction Impaired gp91^phox and p22^phox Expression through a Heme-dependent Mechanism—HO-1 induction by CoPP induced a dose-dependent decrease in gp91^phox expression (Fig. 3A) that was reversed by the HO inhibitor SnPP (Fig. 3B)or by heme loading (Fig. 3C). As DeLeo et al. (20) have described that heme depletion increases gp91^phox degradation by proteasome, we checked whether two different proteasome inhibitors, lactacystin or MG-132 (21), could reverse the effect of CoPP. Treatment of cells by lactacystin or MG-132 (10 µM, 30 min before CoPP) effectively inhibited the CoPP-induced decrease in gp91^phox expression (Fig. 4), suggesting that heme restriction accelerated gp91^phox degradation by the proteasome.

View larger version (37K): [in this window] [in a new window]   FIG. 3. Effect of HO-1 induction on gp91phox expression. Typical Western blot aspect of gp91phox and -actin expression after treatment with increasing concentrations of CoPP (5–20 µM) (A), treatment with the HO inhibitor SnPP (20 µM) (B), or treatment with increasing concentrations of Heme (1–100 µM) (C). Each bar represents the ratio of the optical density of gp91phox to -actin (mean ± S.E., n = 3). *, different from untreated cells, p < 0.05; &, different from CoPP-treated cells, p < 0.05.

View larger version (44K): [in this window] [in a new window]   FIG. 4. Role of proteasome degradation in the decrease of gp91phox expression induced by HO-1. Typical Western blot aspect of gp91phox and -actin expression 24 h after treatment with CoPP + Me2SO (20 µM) (A) with and without pre-treatment with the proteasome inhibitors lactacystin and MG-132 (10 µM in Me2SO, respectively). Each bar represents the ratio of optical density of gp91phox to -actin (mean ± S.E., n = 3). &: different from cells incubated without any proteasome inhibitor and treated with CoPP, p < 0.05.

View larger version (33K): [in this window] [in a new window]   FIG. 5. Typical confocal microscopy aspect of gp91phox expression (green) and colocalization with HO-1 (red) in non-treated cells (A and C, respectively) or 24 h after treatment with CoPP (20 µM) (B and D). The experiment was repeated four times as described under "Experimental Procedures." Bars, 10 µm.

View larger version (29K): [in this window] [in a new window]   FIG. 6. Role of HO-1 induction on p22phox and p47phox expression. Typical Western blot aspect of p22phox, p47phox, and -actin expression 24 or 48 h after treatment with CoPP (20 µM) or with CoPP + heme (100 µM). Each bar represents the ratio of optical density of gp91phox to -actin (mean ± S.E., n = 3). *, different from untreated cells, p < 0.05

View larger version (30K): [in this window] [in a new window]   FIG. 7. Effect of HO-1 induction on gp91phox, p22phox, or p47phox mRNA expression. Cells were treated with different concentrations of CoPP for 24 h or with 20 µM CoPP for different times. Total RNA was extracted as described under "Experimental Procedures" and analyzed by reverse transcription PCR for gp91phox, p22phox, p47phox, and actin expression

View larger version (31K): [in this window] [in a new window]   FIG. 8. Role of HO-1 induction on gp 91phox expression and superoxide production in rat bronchoalveolar lavage macrophages. A, Western blot analysis of HO-1, gp91phox, and -actin expression in rat alveolar macrophages after a 48-h treatment with 15 mg/kg CoPP, 30 mg/kg SnPP, or vehicle (n = 3 for each group). Each bar represents the ratio of optical density of gp91phox to -actin (mean ± S.E., n = 3). *, different from vehicle-treated animals, p < 0.05. B, kinetic analysis of superoxide release by rat alveolar macrophages before (black symbols) and after (white symbols) stimulation with PMA, measured by chemiluminescence during a 30-min period. Each curve represents analysis of one animal and is from 3–5 different experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

Heme oxygenase is the rate-limiting enzyme in heme break-down and an important regulator of cellular heme availability. Heme degradation is critical for cell homeostasis because of its potential pro-oxidant effects when it is in the free form (23). Heme availability is also critical for the activity of heme-dependent enzymes such as HO-1 itself, which needs heme as its substrate (24), or for the activity of heme-containing enzymes. The expression and activity of cyclooxygenase-2 (25, 26) and nitric oxide synthase (27, 28), two enzymes requiring heme in their catalytic site, are decreased after HO-1-induced heme depletion. Similarly, rats treated with CoPP exhibited a severe depletion in cellular cytochrome P450 (29). We show in the present study that HO-1 can also modulate NAD(P)H oxidase activity through a similar mechanism. Indeed, HO-1 induction by CoPP in vitro in RAW 264.7 macrophages resulted in a decrease in both gp91^phox subunit protein expression and the whole NAD(P)H oxidase activity. These effects were associated in vitro with a decrease in heme content and were dose-dependently reversed by heme loading, suggesting that HO-1 controlled gp91^phox protein expression and activity via control of the cellular heme pool.

The heme requirement for the phagocyte NAD(P)H oxidase complex assembly and activity has been well demonstrated (20). The gp91^phox subunit contains two heme molecules that presumably mediate the transfer of electrons from NAD(P)H to oxygen (30). It has been demonstrated that heme is posttranslationally integrated to a p65 precursor of gp91^phox without influencing apoprotein synthesis (31, 32). Heme incorporation into gp91^phox is required for ensuring protein stability and the formation of an heterodimer with p22^phox, the consequent translocation to the cell membrane, and the assembly for forming the flavocytochrome b₅₅₈, the functional component of the oxidase (20). When heme synthesis is blocked, the formation of the heterodimer between gp91^phox and p22^phox is inhibited, and unassembled p65 and p22^phox monomers are degraded by the cytosolic proteasome (20). Our results show that HO, by modulating the cellular heme pool, controls the different steps of gp91^phox maturation; HO-1 induction did not modify gp91^phox mRNA expression but resulted in gp91^phox protein degradation by the proteasome and an impaired translocation to the membrane. Furthermore, the CoPP-induced decrease in NAD(P)H oxidase activity is independent from the stimulus used (PMA or zymozan), suggesting that HO-1 does not affect the activation pathways of NAD(P)H oxidase. HO-1 induction also decreased p22^phox protein expression, but this decrease happened later than the decrease of gp91^phox (at 48 h versus 24 h, respectively) without changes at the mRNA level. This finding is probably a consequence of a reduced heterodimerization of p22^phox with heme-depleted gp91^phox (31, 32). Finally, protein and mRNA expression of p47^phox, a cytosolic component of the NAD(P)H oxidase complex independent from heme metabolism, was not modified by HO-1 induction, stressing the heme dependence of the HO effect on NAD(P)H oxidase.

It is noteworthy that, despite the fact that the powerful and long lasting induction of HO-1 expression and activity by CoPP is well characterized (33), uncertainties about the specificity of synthetic metalloporphyrins still remain (34). Indeed, CoPP has been shown to inhibit the rate-limiting enzyme of heme synthesis -aminolevulinic acid synthase (29). Therefore, we can hypothesize that the decrease in heme content induced by CoPP resulted from both a direct inhibition of heme synthesis and an increase in heme degradation due to HO-1 induction. Furthermore, CoPP may have inhibited NAD(P)H oxidase by competing with heme to gp91^phox binding. Although we cannot exclude these possibilities, we are confident about the participation of HO-1 in the present results because of the following observations. 1) Overexpressing HO-1 by cell transfection induced an inhibition of the oxidase activity similar to that of CoPP. 2) No toxicity was observed with the concentrations of metalloporphyrins and heme used in the study. 3) Pharmacological inhibition of HO with SnPP induced the opposite effect and was able to reverse CoPP effects.

The lack of heme availability is probably not the only mechanism through which HO-1 can interact with NAD(P)H oxidase, as was strongly suggested by the total inhibition of superoxide production by bronchoalveolar lavage macrophages, contrasting with a significant but much lower decrease in protein expression. We have previously demonstrated in airway smooth muscle that HO-1-released or exogenously added bilirubin could decrease superoxide anion production (12, 16). This effect could be related to the oxidant-scavenging properties of bilirubin (14) or to the inhibition of NAD(P)H oxidase activation by this molecule (35). Carbon monoxide, another HO product, has been also suggested to inhibit NAD(P)H oxidase activity because of its high affinity for heme moiety (36). Thus, it appears that HO can inhibit NAD(P)H oxidase through different mechanisms.

In view of the present data, one can wonder whether NAD(P)H oxidase inhibition could take part in the anti-oxidant effect of HO and its beneficial consequences that have been observed in different models of HO overexpression, especially in cardiovascular diseases. Indeed, NAD(P)H oxidase-generated superoxide anion takes part in the control of expression of redox-sensitive genes and is strongly involved in atherosclerosis, hypertension, and vascular remodeling (22, 37). Interestingly, HO has shown a protective role in similar animal models of cardiovascular diseases by decreasing vascular tone and limiting smooth muscle proliferation and the expression of endothelial adhesion molecules (38–41). Similarly, some authors have described a microsatellite polymorphism in the HO-1 promoter (a small number of GT repeats in the promoter region responsible for a higher HO activity) in humans that is associated with a reduced susceptibility to coronary disease (42) or restenosis after angioplasty (43). In these studies, the anti-oxidant role of HO has been advocated to explain its protective effect, but an interaction with the oxidase function has never been postulated. Results from the present experiments in rats show that such a mechanism can be observed in vivo, because a single injection of CoPP strongly inhibits superoxide production. Further studies would be required to elucidate this point in pathological models.

	CONCLUSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

 
This study provides evidence for an interaction between HO-1 activity and NAD(P)H oxidase expression through regulation of cellular heme content. This interaction could explain the anti-oxidant properties of the HO system and the beneficial effects observed in different conditions such as cardiovascular diseases in which involvement of the oxidase is well described. Moreover, given the wide distribution of gp91^phox or its heme-containing homolog (NOX) (9), we can imagine that HO-1 induction could regulate the different biological functions mediated by superoxide anion, providing a new hypothesis to explain the wide protective effects of HO such those observed in sepsis, transplantation, or ischemia-reperfusion.

	FOOTNOTES

 
^* 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.

^¶ To whom correspondence should be addressed: INSERM, U408, Faculté X. Bichat, BP416, 75870 Paris Cedex 18, France. Tel.: 33-1-44856251; Fax: 33-1-42263330; E-mail: jbb2{at}bichat.inserm.fr.

¹ The abbreviations used are: HO-1, heme oxygenase-1; CoPP, cobalt (III) protoporphyrin IX chloride; SnPP, tin (IV) protoporphyrin IX dichloride; MG-132, carbobenzoxy-L-leucyl-L-leucyl-L-leucinal; PMA, phorbol myristate acetate; PBS, phosphate-buffered saline.

	ACKNOWLEDGMENTS

 
We thank Cécile Pouzet (Service de Microscopie Electronique, Institut Federatif de Recherche 02, Faculte Xavier Bichat) and Marcelle Bens (INSERM, U478) for help in confocal microscopy analysis.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

 

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