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Cardiovascular Research 2003 60(1):187-197; doi:10.1016/S0008-6363(03)00365-1
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Copyright © 2003, European Society of Cardiology

Reactive oxygen species mediate cyclooxygenase-2 induction during monocyte to macrophage differentiation: critical role of NADPH oxidase

Silvia Stella Barbieria, Sonia Eliginia, Marta Brambillaa, Elena Tremolia,b and Susanna Collia,*

aE. Grossi Paoletti Center, Department of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy
bDepartment of Cardiac Surgery, Centro Cardiologico Fondazione Monzino I.R.C.C.S, University of Milan, Milan, Italy

*Corresponding author. Tel.: +39-02-5031-8318; fax: +39-02-5031-8250. Email address: susanna.colli{at}unimi.it

Received 25 October 2002; accepted 28 January 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The objective of this study was to explore the relationship between monocyte differentiation into macrophages and cyclooxygenase-2 (Cox-2) expression, based upon the observation that high amounts of this enzyme, colocalizing mainly with macrophages, have been found in human atherosclerotic lesions. Moreover, the hypothesis that reactive oxygen species (ROS) could be important as mediators of Cox-2 expression during monocyte differentiation was verified. Although ROS are known as modulators of gene expression profile, their involvement in monocyte differentiation has not been explored previously. Methods: Human adherent monocytes and the promonocytic cell line U937 were differentiated into macrophages by phorbol ester (PMA). Cox-2 was evaluated in terms of protein, mRNA and activity. Intracellular ROS formation was measured by the oxidant sensitive dye 2',7'-dichlorofluorescein diacetate. NADPH oxidase subunit p47phox was evaluated by Western blot analysis. Results: Functionally active Cox-2 is expressed during PMA-induced monocyte transition into macrophages and ROS driven by the NADPH oxidase play a critical role in this event. Conclusion: Monocyte differentiation into macrophages, possibly triggered by unquenched ROS, may contribute to the increased inflammatory response within atheromata.

KEYWORDS Atherosclerosis; Free radicals; Infection/inflammation; Macrophages; Prostaglandins


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Macrophages are significant cellular participants throughout the development of atherosclerosis, which presents features of an inflammatory disease [1]. Plaque macrophages originate from blood monocytes that, once infiltrated into the intimal space, undergo differentiation [2]. These cells are engaged to ingest modified lipoproteins through a variety of scavenger receptors and to recognize oxidized phospholipids as markers of oxidized moieties, all events that ultimately lead to foam cells generation [3]. Monocyte differentiation into macrophages, favoured by oxidized lipids also [4], is accompanied by global changes in the profile of gene expression. These changes, in particular, equip the macrophage to act as a more potent producer of agents relevant for stability, thrombogenicity and inflammatory status of the atherosclerotic plaque. Metalloproteinase-9, β2 integrin CD11c, tissue factor, thrombomodulin, TNF{alpha}, and phospholipase A2, are all increased or triggered during monocyte differentiation into macrophages [5–10]. Macrophages, once activated by inflammatory stimuli, synthesize and release eicosanoids that are potent modulators of inflammation. The rate-limiting step in this event is represented by the cyclooxygenases (Cox), present in two isoforms. Cox-1, which is constitutive and detected in most human tissues and the inducible Cox-2, responsible for high-level production of prostanoids in response to proinflammatory agents, tumor promoters and growth factors [11]. High amounts of Cox-2 have been found in human atherosclerotic lesions, compared with unaffected arteries [12,13]. The enzyme colocalizes mainly with macrophages of the shoulder region and lipid core periphery, suggesting that its products are involved in the inflammatory pathways leading to plaque instability and subsequent rupture, possibly through the release of metalloproteinases [14–16].

Reactive oxygen species (ROS) are ubiquitous highly diffusable and reactive molecules produced by reduction of molecular oxygen, and include species such as superoxide anion (O2–), hydrogen peroxide (H2O2) and hydroxyl radical (OH•). ROS are produced normally during the respiratory burst of phagocytes as a defense mechanism against pathogens. They regulate, however, multiple cellular functions such as growth and differentiation, proliferation, apoptosis and gene expression, acting through both transductional and transcriptional pathways [17].

The intracellular sources contributing to ROS generation in monocytes are several, including cyclooxygenases, lipoxygenases, mitochondrial respiration and NADPH oxidase [18]. This latter, which predominates in monocytes [19], is composed by various subunits that are localized both in membranes (gp91phox and p22phox) and in cytosol (p47phox and p67phox). The fifth component of the enzyme, the GTPase Rac2 migrates to the membrane in association with p47phox, which, without it, is unable to reach the membrane [20].

The present study was designed to (i) evaluate the profile of Cox-2 expression during monocyte transition into macrophages, and (ii) understand the involvement of ROS and NADPH oxidase during the differentiation process. For this purpose both human adherent monocytes and the promonocytic cell line U937, an established model for the study of monocyte differentiation, were used. Here, we provide evidence that the sustained oxidant signaling driven by NADPH oxidase represents a previously unrecognized means of regulating Cox-2 expression during monocyte differentiation. This finding highlights the possibility that unquenched ROS actively contribute to monocyte differentiation into macrophages and to the acquisition of an inflammatory status within atheromata.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Reagents
Culture media were purchased from BioWhittaker Italia (Bergamo, Italy). RPMI 1640 was completed with 2 mM L-glutamine, 100 Units/ml penicillin, 100 µg/ml streptomycin (all from Sigma, Milan, Italy) and 10% heat-inactivated fetal bovine serum (Mascia Brunelli, Milan, Italy). The following reagents were purchased from Sigma: phorbol myristate acetate (PMA), phenazine methosulfate (PMS), phenylarsine oxide (PAO), N-acetyl-cysteine (NAC), rotenone, diphenyliodonium (DPI), 2',7'-dichlorofluorescein diacetate (DCFH-DA). PD098059 and SB203580 were from Biomol (Plymouth Meeting, PA, USA). Calcium ionophore A23187 [GenBank] , 2,3-dimethoxy-1,4-napthoquinone (DMNQ), herbimycin A, HA-1077, MK-886, and human M-CSF were from Calbiochem (Inalco, Milan, Italy). Apocynin and mevalonate (as mevalonolactone) were from Aldrich (Milan, Italy). all-E-geranylgeraniol (all-trans-geranylgeraniol) was purchased from American Radiolabeled Chemicals. Fluvastatin was from Novartis (Basel, Switzerland). Toxin lethalis (Tox L) from Clostridium sordellii, was kindly donated by M.R. Popoff, Institute Pasteur, Paris, France, whereas RO 31-8220 was gifted by C.H. Hill (Roche, London, UK).

2.2. Antibodies
Antibodies against Cox isoforms were gifted by Aida Habib (U348, INSERM, Paris). Antibody against p47phox, fluorescein isothiocyanate (FITC)-conjugated anti-human CD14 antibody and isotype control antibody (mouse IgG1) were from Becton Dickinson (Milan, Italy). Peroxidase-conjugated anti-mouse IgG antibody was from Jackson ImmunoResearch Labs, West Grove, PA, USA).

2.3. Cell culture and treatment
The human monocytic cell line U937 was purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in complete RPMI 1640. Cells were kept at 37°C in 5% CO2 and harvested at log phase of growth. Monocytes were isolated from blood of healthy donors. Blood sampling was performed in accordance with the principles outlined in the Declaration of Helsinki (Cardiovascular Research 1997;35:2–3). Mononuclear cells were separated by Ficoll-Paque density gradient (Amersham Pharmacia), as described [21]. Monocytes were then isolated by selective adherence to tissue culture dishes for 90 min at 37°C and incubated with various agents in medium M-199 supplemented with 5% human AB serum. Cell population was >90% monocytes, as determined by nonspecific esterase staining. Cells were exposed to PMA for different time periods, as indicated. Inhibitors were added to monocytes and U937 1 h before exposure to PMA. ROS generators were incubated with PMA-differentiated U937 (1 day PMA withdrawal) or adherent monocytes for 4 h. Cell viability was determined by trypan blue exclusion.

2.4. Prostanoid measurement
Experiments were carried out in undifferentiated and PMA-differentiated U937. Cells were incubated with calcium ionophore A23187 [GenBank] (5 µM) in medium containing calcium and magnesium (1 mM). Incubations were carried out for 10 min, in shaking bath at 37°C. Thromboxane B2 (TXB2) and prostaglandin E2 (PGE2) were measured in cell supernatant by enzyme immunoassay (EIA, Cayman Chemicals, Ann Arbor, MI, USA).

2.5. Flow cytometry
Level of CD14 expression in U937 either undifferentiated or PMA-differentiated was assessed by flow cytometry. Cells were incubated with FITC–CD14 antibody for 30 min at room temperature. After washing, they were suspended in medium and the percentage of CD14+ cells was assessed by FACSCalibur flow cytometer (Becton Dickinson). Tracings were obtained by displaying the log fluorescence of FITC of the samples (FL2-H) generated against the background staining of cells stained with an isotype control antibody (mouse IgG1) and the secondary, FITC-conjugated, antibody. For intracellular ROS detection, cells were pre-loaded (30 min at 37°C) with 5 µM 2',7'-dichlorofluorescein diacetate (DCFH-DA). Cells were harvested and the number of cells exhibiting increased fluorescence of oxidized DCF was measured at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

2.6. Western blot analysis
Cells were lysed in sample buffer, as previously described [22]. Whole cell lysates were separated in 7% or 10% SDS–PAGE, and transblotted to nitrocellulose membrane with a semidry transfer unit (Hoefer Scientific Instruments). Membranes were incubated for 1 h with antibodies directed against Cox-1 (5 µg/ml), Cox-2 (1/10 000) and p47phox (1/250), and subsequently with donkey anti-mouse IgG conjugated with peroxidase. β-actin was used as a control of protein loading. ECL (Amersham Pharmacia Biotech) substrates were used according to the manifacturer's instructions to reveal positive bands.

2.7. RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA (100 ng) was extracted from cells with TRIzol Reagent and reverse transcribed (42°C for 60 min) by Stratascript reverse transcriptase (Stratagene). cDNA (4 µl) was then subjected to 30 cycles of PCR (denaturation at 94°C for 30 s; annealing at 55°C for 30 s; primer extension at 72°C for 60 s) in a reaction mixture (100 µl), containing 5 U platinum Taq polymerase (Invitrogen) and 200 nM sense and antisense primers for Cox-2 and GAPDH. Cox-2 primers were 5'-TTCAAATGAGATTGTGGGAAAATTGCT-3' and 5'-AGATCATCTCTGCCTGAGTATCTT-3', giving rise to a 305-bp PCR product. All reactions were performed in a Perkin-Elmer GeneAmp PCR system 2400 thermal cycler. PCR products were analysed on 2% agarose gel containing ethidium bromide (0.1 µg/ml). GAPDH mRNA was used as a control of mRNA loading.

2.8. Nuclear extracts and electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared from cell suspensions as described [23]. After supernatant collection, the protein content was determined and EMSAs were performed as follows: the synthetic single-stranded oligonucleotides (Eurogentec, Herstal, Belgium) containing the proximal and distal NF-{kappa}B sites were annealed with the complementary primers and radiolabeled with 32PdCTP (Amersham Pharmacia Biotech). The consensus sequences for NF-{kappa}B are underlined: distal NF-{kappa}B site (upstream, within –455 to 428 from the transcriptional start site) 5'-GGCGGGAGAGGGGATTCCCTGCGCCCCC-3'; proximal NF-{kappa}B site (downstream, within –232 to 205 from the transcriptional start site) 5'-CAGGAGAGTGGGGACTACCCCCTCTGCT-3'. Protein–DNA complexes were separated from free DNA probe by electrophoresis through 5% nondenaturating acrylamide gels in 0.5 x TBE (45 mM Tris base, 45 mM boric acid, 1 mM EDTA). NF-{kappa}B specific bands were confirmed by competition with a 100-fold molar excess of an unlabeled NF-{kappa}B probe.

2.9. Statistical analysis
Data are reported as means±S.D. Computer-assisted statistical analyses used the ANOVA or unpaired t-test program. Following ANOVA test, probability values were calculated using Fisher's protected least-significant difference test. A value of P<0.05 was considered significant. n = number of individual experiments.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Monocytic differentiation is accompanied by persistent Cox-2 induction
Using primary human adherent monocytes and the human promonocytic cell line U937, it is demonstrated that the exposure to low-dose PMA (3.3–40 nM) triggers progressive differentiation toward macrophages, as documented by changes in cell morphology. Differentiation of U937 resulted in the formation of adherent cultures forming protruding pseudopodia and clumps [24]. Morphological changes were accompanied by progressive increases in the relative abundance of CD14, a surface marker of differentiated macrophages (Fig. 1, panel A). Cox-2 expression, which was absent in undifferentiated U937 and in resting adherent monocytes became evident upon cell exposure to PMA [25] and went in parallel with differentiation (Fig. 1, panel B). In contrast, Cox-2 induction was not observed in monocytes differentiated with M-CSF for 96 h (not shown). The effect of PMA was concentration and time-dependent (4–72 h). U937 cells exposure to 3.3 nM PMA for 24 h resulted in Cox-2 protein expression. At 72 h Cox-2 levels were still elevated. Cox-2 was detected also in adherent monocytes exposed to PMA for 4 h (5.1±0.13-fold increment with 3.3 nM PMA and 6.9±0.12-fold increment with 40 nM PMA) and protein levels progressively increased until 24 h (Fig. 1, panel B). In contrast, Cox-1 was unaltered during monocytic differentiation (not shown). Cox-2 was functional, as documented by the augmented levels of TXB2 and PGE2 measured in cell medium (Table 1). PGE2 net increase was more pronounced, respectively, to TXB2.


Figure 1
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  		Fig. 1 (A) Phenotype of U937 cells exposed to PMA 3.3 nM for 24–48 h. Surface expression of CD14, a marker of differentiated macrophages, was assessed by flow cytometry. Plain tracing gives control cells, and dotted tracing gives PMA-treated cells. (B) Time course of Cox-2 expression induced by PMA in U937 cells and in human adherent monocytes. Cox-2 protein has been evaluated by Western blot analysis. Blots are representative of three independent experiments. Densitometric analysis is shown in the lower part of the panel. (C) Time course of Cox-2 mRNA levels detected by RT-PCR in U937 cells exposed to 3.3 and 10 nM PMA. Results are representative of three independent experiments.

 

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  		Table 1 Prostanoid synthesis in undifferentiated and PMA-differentiated U937 cells

 
The occurrence of Cox-2 during differentiation was consequent to the appearance of Cox-2 mRNA levels, already appreciable upon U937 exposure to 3.3 nM PMA after 20 h (Fig. 1, panel C). Activation of the transcription factor NF-{kappa}B, was responsible for this event, as documented by results from EMSA analysis performed on nuclear extracts of PMA-treated U937 (Fig. 2, panel A). Moreover, experiments were carried out with the protein kinase C (PKC) inhibitor RO 31-8220, with the tyrosine kinase inhibitor herbimycin and with the MAP kinase inhibitors PD098059 and SB203580 that specifically target ERK1/2 and p38 MAP kinase, respectively. Results obtained showed that activation of PKC, tyrosine kinase and of the MAP-kinase ERK1/2 was essential for Cox-2 induction during differentiation (Fig. 2, panels B and C).


Figure 2
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  		Fig. 2 (A) NF-{kappa}B activation during PMA-induced differentiation of U937 cells. NF-{kappa}B activation has been determined by EMSA analysis performed on nuclear extracts of U937 exposed to PMA. Specificity of binding was determined on cell extracts by competition, using a 100-fold excess of unlabeled probe. EMSA analysis shown in figure is representative of two independent experiments. (B, C) Involvement of protein kinases and MAP kinases in Cox-2 expression induced in U937 cells by differentiation. (B) Inhibitor of protein kinase C Ro 31-8220 and protein tyrosine kinase inhibitor herbimycin were preincubated 1 h before PMA addition. Incubations were carried out for further 48 h. (C) The same experimental protocol was adopted for the MAP-kinase inhibitors PD098059 and SB203580, which specifically inhibit ERK1/2 and p38, respectively. Blots are representative of three independent experiments.

 
3.2 Differentiation was accompanied by intracellular ROS production and by up-regulation of the NADPH-oxidase subunit p47phox
Monocyte and U937 exposure to PMA resulted in intracellular generation of ROS, monitored by oxidation of the cell-permeable DCFH-DA probe [26]. The results depicted in Fig. 3, panels A and B, indicate that PMA induced a concentration and time-dependent increase in DCF fluorescence which was detectable between 1 and 4 h. Mean fluorescence intensity increased by 0.86±0.54-fold within 2 h in U937 challenged with 3.3 nM PMA. In monocytes, the mean fold-increase with PMA 40 nM was 1.33±0.58 (Fig. 3, panel B). Intracellular ROS formation was strictly dependent upon NADPH oxidase activation, as shown by experiments carried out with the NADPH oxidase inhibitor DPI [27] (Fig. 3, panel C). Other sources of ROS known to operate in monocytic cells, as the 5-lipoxygenase and the mitochondrial electron transport pathway were not involved, as documented by the effect of rotenone, which inhibits the mitochondrial electron transport and of MK-886, a 5-lipoxygenase inhibitor (Fig. 3, panel C).


Figure 3
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  		Fig. 3 Intracellular ROS generation induced by PMA in U937 cells and in adherent monocytes. Cells were loaded with DCFH-DA for 30 min in medium without phenol red. After exposure to PMA, the number of cells exhibiting increased fluorescence of oxidized DCF was evaluated by flow cytometry. (A) Representative flow cytometry tracings of U937 in resting condition (plain line) and exposed to PMA for 2 h. (B) Histograms represent mean values±S.D. of three independent experiments carried out in U937 and adherent monocytes exposed to 3.3 and 40 nM PMA, respectively. Time 0 represents DCF fluorescence of untreated cells (100%). *P<0.05 and **P<0.01 vs. untreated condition. (C) Effect of DPI (5 µM), rotenone (50 µM) and MK 886 (1 µM) on PMA-induced intracellular ROS formation detected by flow cytometry in adherent monocytes. Results are representative of three independent experiments.

 
A band corresponding to p47phox subunit of NADPH oxidase which was undetectable in undifferentiated U937 occurred upon cell exposure to PMA for 24–48 h (Fig. 4, panel A). In adherent monocytes, p47phox was already detectable in resting condition and levels increased upon exposure to 40 nM PMA for 4 h (Fig. 4, panel B).


Figure 4
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  		Fig. 4 PMA-induced differentiation upregulates p47phox in U937 cells (A) and in human adherent monocytes (B). p47phox was detected by Western blot analysis. Densitometric analyses are shown in the lower part of the panels. Results are means±S.D. of three independent experiments.

 
Noticeably, the time course of p47phox appearance went in parallel with the appearance of Cox-2 (see Fig. 1, panel B).

3.3. Cox-2 expression is triggered by ROS generators and prevented by NADPH oxidase inhibitors
Exposure of adherent monocytes and differentiated U937 (1-day PMA withdrawal) to ROS generators DMNQ [28] and PMS resulted in Cox-2 expression, documented by increased protein and mRNA levels (Fig. 5, panels A and B). Conversely, the antioxidant NAC prevented Cox-2 induction by PMA (Fig. 5, panel C). The NADPH oxidase inhibitors DPI, PAO and apocynin, all prevented PMA-induced Cox-2 protein and mRNA levels (Fig. 5, panels D and E). In contrast, pathways leading to intracellular ROS formation other than NADPH oxidase were not involved in Cox-2 induction (Fig. 5, panel F).


Figure 5
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  		Fig. 5 Cox-2 expression is triggered by ROS generators and prevented by antioxidant NAC and by NADPH oxidase inhibitors. (A, B) Adherent monocytes and PMA-differentiated U937 cells (1-day PMA withdrawal) were exposed to PMA, DMNQ and PMS, and Cox-2 protein and mRNA levels were evaluated after 4 and 1 h, respectively. (C) NAC was added to incubation medium 1 h before PMA. Incubation was continued for additional 4 and 48 h for adherent monocyte and U937, respectively. (D, E) NADPH oxidase inhibitors were added to monocytes 1 h before PMA. Cox-2 protein and mRNA were evaluated after 4 and 1 h incubation, respectively. (F) Rotenone and MK886 were incubated with monocytes for 1 h. PMA was then added and incubations were continued for further 4 h. Cox-2 protein has been evaluated by Western blot analysis. Blots are representative of three independent experiments. Cox-2 mRNA levels were determined by RT-PCR. Results are representative of two independent experiments.

 
Changes of Cox-2 protein and mRNA levels were consequent to changes of NF-{kappa}B binding to DNA (Fig. 6). NADPH oxidase inhibitors and the antioxidant NAC prevented NF-{kappa}B binding to DNA in U937 cells exposed to PMA (Fig. 6, panel A), whereas ROS generators increased it in PMA-differentiated U937 cells (Fig. 6, panel B).


Figure 6
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  		Fig. 6 NADPH oxidase inhibitors and NAC prevented NF-{kappa}B activation in U937 exposed to PMA whereas ROS generators induced it. (A) NF-{kappa}B activation was determined in nuclear extracts of U937 incubated for 1 h with NADPH oxidase inhibitors and NAC and subsequently exposed to PMA for 20 h. (B) ROS generators were incubated for 30 min with PMA-differentiated U937 cells (1-day PMA withdrawal). EMSA analyses shown in figure are representative of two independent experiments.

 
3.4. Rac2 is required for Cox-2 induction
Activation of Rac2, a GTPase of the Rac protein family is required for full activation of NADPH oxidase [29]. U937 exposure to Tox L, which selectively inhibits GTPases of the Rac family [30], resulted in a marked attenuation of Cox-2 levels (Fig. 7, panel A). Rac2 is characterized by geranylgeranylation, which is essential for its membrane association and, in turn, for NADPH oxidase function [31]. The role of this lipid modification was explored taking advantage from the use of the HMG-CoA reductase inhibitor fluvastatin. Fluvastatin (5 µM), added to culture medium of U937 16 h before PMA, markedly prevented Cox-2 induction. The effect was fully abrogated by the addition of mevalonate (0.1 mM) or geranylgeraniol (10 µM) to culture medium (Fig. 7, panel B).


Figure 7
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  		Fig. 7 Rac2 is essential for Cox-2 induction. (A) Lethal toxin (Tox L) and (B) fluvastatin prevented Cox-2 induction in U937 cells exposed to PMA for 48 h. Tox L and fluvastatin were added 1 and 16 h, respectively, before cell exposure to PMA. Mevalonate and geranylgeraniol were coincubated with fluvastatin. Cox-2 protein has been evaluated by Western blot analysis. Blots are representative of three independent experiments.

 
The inhibitor of Rho/Rho kinase pathway HA-1077 [32] failed to influence Cox-2 expression induced by differentiation, thus excluding the involvement of other geranylgeranylated small G-proteins, in particular those belonging to the Rho family, in the observed effect (not shown). Taken together these results indicate that Rac2 activity is cardinal to the expression of Cox-2 during monocyte differentiation.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In the present study we have delineated the profile of Cox-2 expression in monocytic cells undergoing differentiation and have explored several mechanisms, which underlie this expression. In particular, the activation of a signal transduction pathway mediated by ROS, together with the role of NADPH oxidase and its subunits has been disclosed. The results herein described highlight the involvement of intracellular ROS in inducing Cox-2 during monocyte differentiation and are supported by three lines of evidence. Firstly, ROS generators per se trigger Cox-2 protein and mRNA in monocytes. Secondly, the NADPH oxidase subunit p47phox is up-regulated during monocytic differentiation and this event is causally linked to Cox-2 expression. Thirdly, NADPH oxidase inhibitors markedly prevented Cox-2 protein and mRNA triggered by differentiation.

ROS may serve as intracellular signals for activation of gene expression through the involvement of specific redox-sensitive signaling pathways and transcription factors such as MAP-kinases and NF-{kappa}B [33,34]. We show here that Cox-2 induction during differentiation is consequent to NF-{kappa}B activation, as documented by EMSA analysis performed using probes for both distal and proximal NF-{kappa}B sequences in the Cox-2 promoter. Interestingly, NF-{kappa}B activation is increased by ROS generators whereas NADPH oxidase inhibitors prevent it. A similar preventive effect has been observed when NF-{kappa}B activation was induced by interleukin-1β that acts through a pathway requiring ROS production in monocytic cells [19]. Our data indicate that besides NF-{kappa}B, ERK1/2, but not p38 activation, is critically involved in Cox-2 induction during monocytic differentiation. This finding confirms and extends the crucial role exerted by this signaling pathway during differentiation of myeloid cell lines [35].

Moreover, PMA strongly up-regulates the NADPH subunits p47phox during monocytic differentiation and this event is causally linked to Cox-2 expression. In this respect it is worth to mention that PMA stimulates NADPH oxidase assembly in phagocytes not only in the plasma membrane, but also and mostly, in the specific granule membrane [36]. Hence PMA, besides its differentiating capacity, represents a good tool to investigate the signal transduction pathways leading to sustained ROS formation in the intracellular compartment, as a consequence of NADPH oxidase activation. Important to this discussion is the previous report that NADPH oxidase activity is strictly dependent on p47phox concentration [37].

Cox-2 appearance was observed only when monocyte differentiation was induced by PMA. Other differentiating agents, such as M-CSF did not exert the same effect. The failure of CSF-1 to affect p47phox in murine bone marrow-derived macrophages [38] may explain the inability of M-CSF to induce Cox-2 in our experimental condition.

Interestingly, Cox-2 expression induced by differentiation has been recently reported to occur in monocyte-derived dendritic cells. These cells, which play a crucial role in the initiation of immune responses, are obtained in vitro by culturing monocytes for 6 days in medium supplemented with GM-CSF plus IL-4 [39].

Moreover, our results indicate that cell-specific pathways regulate Cox-2 induction by PMA. Indeed, in contrast to what we report to occur in monocytes, Cox-2 expression is only partially dependent on oxidant stress and NADPH oxidase in mesangial cells exposed to this agent [40].

The GTPase Rac2, the fifth component of the NADPH oxidase, migrates to the membrane in association with p47phox. Both p47phox and Rac2 are therefore cardinal to the functional integrity of NADPH oxidase. The key role of Rac2 is well disclosed by our results obtained with both lethal toxin and with the HMG-CoA reductase inhibitor fluvastatin. In addition, the pivotal role of geranylgeranylation has been defined by testing the effect of geranylgeraniol, which, similarly to mevalonate, completely prevents the inhibition of Cox-2 exerted by fluvastatin. The preventive effect of fluvastatin on Cox-2 induction extends to the monocyte the importance of the isoprenoid pathway for the functional integrity of NADPH oxidase, as already reported for other cell types [30,41]. The role of protein–isoprene interaction in NADPH oxidase activation has been recently addressed in a cell-free system and shown to be essential to the formation of the complex between Rac and its Rho GDP dissociation inhibitor (Rho-GDI) [42]. This complex stabilizes Rac in an active conformation and presents it to its effector, the p67phox component of the NADPH oxidase [42].

Of interest is the observation that the Rac-mediated production of ROS is operative in monocytic cells. In contrast, alternative pathways, not NADPH oxidase-dependent, predominate in other cell types, as lymphoid or epithelial cells [19].

In conclusion, NADPH oxidase-mediated ROS production represents a previously unrecognized mean of regulating Cox-2 expression during monocyte differentiation toward macrophages. This finding may help to explain the augmented expression of Cox-2 detected in atherosclerotic lesions. Cox-2 overexpression has been reported to induce p21WAF1/Cip1, a cyclin-dependent inhibitor involved in the protection against apoptosis [24] and a feature characteristic of plaque regions showing signs of DNA synthesis/repair [43]. Moreover, Cox-2 expression driven by superoxide has been reported as necessary to protect monocytes from differentiation-induced cell death [28].

Within atheromata, the oxidant/antioxidant imbalance and the presence of chemotactic and growth factors, cytokines, oxidized lipid intermediates, and the interaction with vascular matrix [44] may all cooperate to induce monocyte differentiation and persistent Cox-2 expression. Overexpression of functionally active Cox-2, in turn, contributes both to the inflammatory response and to the attenuation of the antioxidative defense of the cell [45], leading to the progression of atherosclerosis and possibly, through an increased PGE2 production, to plaque instability.


   Acknowledgements
 
This work was supported by grants from the European Community (HIFMECH Study, contract BMH4-CT96-0272, grant to ET) and from the Italian Ministry of University and Scientific Research and University of Milan (COFIN, grant 9906203775/1999 to SC and FIRST 2001, grant to SC).


   Notes
 
Time for primary review 24 days.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Ross R. Atherosclerosis: an inflammatory disease. New Engl J Med (1999) 340:115–126.[Free Full Text]
  2. Gerrity R.G. The role of the monocyte in atherogenesis: I. Transition of blood-borne monocytes into foam cells in fatty lesions. Am J Pathol (1981) 103:181–190.[Abstract]
  3. Huh H.Y, Pearce S.F, Yesner L.M, Schindler J.L, Silverstein R.L. Regulated expression of CD36 during monocyte-to-macrophage differentiation:potential role of CD36 in foam cell formation. Blood (1996) 87:2020–2028.[Abstract/Free Full Text]
  4. Perrin-Cocon L, Coutant F, Agaugué S, et al. Oxidized low-density lipoprotein promotes mature dendritic cell transition from differentiating monocyte. J Immunol (2001) 167:3785–3891.[Abstract/Free Full Text]
  5. Xie B, Laouar A, Huberman E. Autocrine regulation of macrophage differentiation and 92-kDa gelatinase production by tumor necrosis factor-{alpha} via {alpha}5β1 integrin in HL-60 cells. J Biol Chem (1998) 273:11583–11588.[Abstract/Free Full Text]
  6. Shelley C.S, Teodoridis J.M, Park H, et al. During differentiation of the monocytic cell line U937, Pur alpha mediates induction of the CD11c β2 integrin gene promoter. J Immunol (2002) 168:3887–3893.[Abstract/Free Full Text]
  7. Colli S, Lalli M, Risè P, et al. Increased thrombogenic potential of human monocyte-derived macrophages spontaneously transformed into foam cells. Thromb Haemost (1999) 81:576–581.[ISI][Medline]
  8. Lopez-Pedrera C, Dobado-Berrios P.M, Ros R, et al. Signal transduction pathways underlying the expression of tissue factor and thrombomodulin in promyelocytic cells induced to differentiate by retinoid acid and dibutyrylcAMP. Thromb Haemost (2002) 85:1031–1036.[ISI]
  9. MacKenzie S, Fernandez-Troy N, Espel E. Post-transcriptional regulation of TNF-{alpha} during in vitro differentiation of human monocytes/macrophages in primary culture. J Leukoc Biol (2002) 71:1026–1032.[Abstract/Free Full Text]
  10. Anthonsen M.W, Stengel D, Hourton D, Ninio E, Johansen B. Mildly oxidized LDL induces expression of group IIa secretory phospholipase A2 in human monocyte-derived macrophages. Arterioscler Thromb Vasc Biol (2000) 20:1276–1282.[Abstract/Free Full Text]
  11. Dubois R.N, Abramson S.B, Crofford L, et al. Cyclooxygenase in biology and disease. FASEB J (1998) 12:1063–1073.[Abstract/Free Full Text]
  12. Baker C.S, Hall R.J, Evans T.J, et al. Cyclooxygenase-2 is widely expressed in atherosclerotic lesions affecting native and transplanted human coronary arteries and colocalizes with inducible nitric oxide synthase and nitrotyrosine particularly in macrophages. Arterioscler Thromb Vasc Biol (1999) 19:646–655.[Abstract/Free Full Text]
  13. Schonbeck U, Sukhova G.K, Graber P, Coulter S, Libby P. Augmented expression of cyclooxygenase-2 in human atherosclerotic lesions. Am J Pathol (1999) 155:1281–1291.[Abstract/Free Full Text]
  14. Stemme V, Swedenborg J, Claesson H, Hansson G.K. Expression of cyclo-oxygenase-2 in human atherosclerotic carotid arteries. Eur J Endovasc Surg (2000) 20:146–152.[CrossRef]
  15. Cipollone F, Prontera C, Pini B, et al. Overexpression of functionally coupled cyclooxygenase-2 and prostaglandin E synthase in symptomatic atherosclerotic plaques as a basis of prostaglandin E2-dependent plaque instability. Circulation (2001) 104:921–927.[Abstract/Free Full Text]
  16. Zhang Y, McCluskey K, Fujii K, Wahl L.M. Differential regulation of monocyte matrix metalloproteinase and TIMP-1 production by TNF-{alpha}, granulocyte-macrophage CSF, and IL-1β through prostaglandin-dependent and -independent mechanisms. J Immunol (1998) 161:3071–3076.[Abstract/Free Full Text]
  17. Irani K. Oxidant signaling in vascular cell growth, death, and survival. Circ Res (2000) 87:179–183.[Abstract/Free Full Text]
  18. Babior B.M. NADPH oxidase: an update. Blood (1999) 93:1464–1476.[Free Full Text]
  19. Bonizzi G, Piette J, Schoonbroodt S, et al. Reactive oxygen intermediate-dependent NF-{kappa}B activation by interleukin-1β requires 5-lipoxygenase or NADPH oxidase activity. Mol Cell Biol (1999) 19:1950–1960.[Abstract/Free Full Text]
  20. Segal A.W, Abo A. The biochemical basis of the NADPH oxidase of phagocytes. Trends Biochem Sci (1993) 18:43–47.[CrossRef][ISI][Medline]
  21. Colli S, Eligini S, Lalli M, et al. Vastatins inhibit tissue factor in cultured human macrophages: A novel mechanism of protection against atherothrombosis. Arterioscler Thromb Vasc Biol (1997) 17:265–272.[Abstract/Free Full Text]
  22. Eligini S, Colli S, Basso F, Sironi L, Tremoli E. Oxidized low density lipoprotein suppresses expression of inducible cyclooxygenase in human macrophages. Arterioscler Thromb Vasc Biol (1999) 19:1719–1725.[Abstract/Free Full Text]
  23. Eligini S, Brambilla M, Banfi C, et al. Oxidized phospholipids inhibit cyclooxygenase-2 in human macrophages via nuclear factor-{kappa}B/I{kappa}B- and ERK2-dependent mechanisms. Cardiovasc Res (2002) 55:406–415.[Abstract/Free Full Text]
  24. Pennington K.N, Taylor J.A, Bren G.D, Paya C.V. I{kappa}B kinase-dependent chronic activation of NF-{kappa}B is necessary for p21WAF1/Cip1 inhibition of differentiation-induced apoptosis of monocytes. Mol Cell Biol (2000) 21:1930–1941.[CrossRef][ISI]
  25. Hoff T, DeWitt D, Kaever V, Resch K, Goppelt-Struebe M. Differentiation-associated expression of prostaglandin G/H synthase in monocytic cells. FEBS Lett (1993) 320:38–42.[CrossRef][ISI][Medline]
  26. Rothe G, Valet G. Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2',7'-dichlorofluorescein. J Leukoc Biol (1990) 47:440–448.[Abstract]
  27. Hancock J.T, White J.I, Jones O.T, Silver I.A. The use of diphenylene iodonium and its analogues to investigate the role of the NADPH oxidase in the tumoricidal activity of macrophages in vitro. Free Radic Biol Med (1991) 11:25–29.[CrossRef][ISI][Medline]
  28. von Knethen A, Callsen D, Brune B. Superoxide attenuates macrophage apoptosis by NF-{kappa}B and AP-1 activation that promotes cyclooxygenase-2 expression. J Immunol (1999) 163:2858–2866.[Abstract/Free Full Text]
  29. Bokoch G.M. Regulation of human neutrophil NADPH oxidase by the Rac GTP-binding proteins. Curr Opin Cell Biol (1994) 6:212–218.[CrossRef][ISI][Medline]
  30. Wagner A.H, Kohler T, Ruckschloss U, Just I, Hecker M. Improvement of nitric oxide-dependent vasodilatation by HMG-CoA reductase inhibitors through attenuation of endothelial superoxide anion formation. Arterioscler Thromb Vasc Biol (2000) 20:61–69.[Abstract/Free Full Text]
  31. Sinensky M. Recent advances in the study of prenylated proteins. Biochim Biophys Acta (2000) 1484:93–106.[Medline]
  32. Li X, Liu L, Tupper J.C, et al. Inhibition of protein geranylgeranylation and RhoA/RhoA kinase pathway induces apoptosis in human endothelial cells. J Biol Chem (2002) 277:15309–15316.[Abstract/Free Full Text]
  33. Guha M, Bai W, Nadler J.L, Natarajan R. Molecular mechanisms of tumor necrosis factor{alpha} gene expression in monocytic cells via hyperglycemia-induced oxidant stress-dependent and -independent pathways. J Biol Chem (2000) 275:17728–17739.[Abstract/Free Full Text]
  34. Schreck R, Albermann K, Baeuerle P.A. Nuclear factor kappa B: an oxidative stress-responsive transcription factor of eukaryotic cells (a review). Free Radic Res Commun (1992) 17:221–227.[ISI][Medline]
  35. Miranda M.B, McGuire T.F, Johnson D.E. Importance of MEK-1/-2 signaling in monocytic and granulocytic differentiation of myeloid cell lines. Leukemia (2002) 4:683–692.
  36. Lundqvist H, Follin P, Khalfan L, Dahlgren C. Phorbol myristate acetate-induced NADPH oxidase activity in human neutrophils:only half the story has been told. J Leukoc Biol (1996) 59:270–279.[Abstract]
  37. Cross A.R, Erickson R.W, Curnutte J.T. The mechanism of activation of NADPH oxidase in the cell free system: the activation process is primarily catalytic and not through the formation of a stoichiometric complex. Biochem J (1999) 341:251–257.[CrossRef][ISI][Medline]
  38. Green S.P, Hamilton J.A, Uhlinger D.J, Phillips W.A. Expression of p47-phox and p67-phox proteins in murine bone marrow-derived macrophages: enhancement by lipopolysaccharide and tumor necrosis factor {alpha} but not colony stimulating factor 1. J Leukoc Biol (1994) 55:530–535.[Abstract]
  39. Witthaker D.S, Bahjat K.S, Moldawer L.L, Clare-Salzler M.J. Autoregulation of human monocyte-derived dendritic cell maturation and IL-12 production by cyclooxygenase-2-mediated prostanoid production. J Immunol (2000) 165:4298–4304.[Abstract/Free Full Text]
  40. Feng L, Xia Y, Garcia G.E, Hwang D, Wilson C.B. Involvement of reactive oxygen intermediates in cyclooxygenase-2 expression induced by interleukin-1, tumor necrosis factor-{alpha}, and lipopolysaccharide. J Clin Invest (1995) 95:1669–1675.[ISI][Medline]
  41. Takemoto M, Node K, Nakagami H, et al. Statins as antioxidant therapy for preventing cardiac myocyte hypertrophy. J Clin Invest (2001) 108:1429–1437.[CrossRef][ISI][Medline]
  42. Di-Poi N, Fauré J, Grizot S, et al. Mechanism of NADPH oxidase activation by the Rac/Rho–GDI complex. Biochemistry (2001) 40:10014–10022.[CrossRef][ISI][Medline]
  43. Kockx M.M, Herman A.G. Apoptosis in atherosclerosis: beneficial or detrimental? Cardiovasc Res (2000) 45:736–746.[Abstract/Free Full Text]
  44. Wesley R.B.I.I, Meng X, Godin D, Galis Z.S. Extracellular matrix modulates macrophage function characteristic to atheroma: collagen type I enhances acquisition of resident macrophage traits by human peripheral blood monocytes in vitro. Arterioscler Thromb Vasc Biol (1998) 18:432–440.[Abstract/Free Full Text]
  45. Nikolic D, van Breemen R.B. DNA oxidation induced by cyclooxygenase-2. Chem Res Toxicol (2001) 14:351–354.[CrossRef][ISI][Medline]

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