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Originally published online as doi:10.1189/jlb.1106672 on March 16, 2007

Published online before print March 16, 2007
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(Journal of Leukocyte Biology. 2007;81:1568-1576.)
© 2007 by Society for Leukocyte Biology

Catalase potentiates retinoic acid-induced THP-1 monocyte differentiation into macrophage through inhibition of peroxisome proliferator-activated receptor {gamma}

Qiurong Ding, Ting Jin, Zhenzhen Wang and Yan Chen1

Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai, China

1 Correspondence: Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences, 294 Tai Yuan Rd., Shanghai 200031, China. E-mail: ychen3{at}sibs.ac.cn


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ABSTRACT
 
Macrophage differentiation plays a pivotal role in cardiovascular diseases and many other physiological processes. However, the role of reaction oxygen species in macrophage differentiation has not been elucidated. Here, we report functional characterization of catalase, an enzyme that degrades hydrogen peroxide (H2O2), in THP-1 monocyte differentiation. Treatment of THP-1 cells with catalase was able to synergize with all-trans retinoic acid (ATRA) to enhance macrophage differentiation, demonstrated by changes of cell adherence, cell cycle arrest, nitroblue tetrazolium reduction, and expression of differentiation markers including CD68, CD11b, and matrix metalloproteinase 9 (MMP9). ATRA could stimulate retinoic acid (RA) receptor-mediated transcription, but this was not affected by catalase. However, ATRA and catalase were capable of reducing transcriptional activity mediated by peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}). Consistently, PPAR{gamma} antagonists enhanced, and PPAR{gamma} agonists inhibited MMP9 expression stimulated by ATRA and catalase in THP-1 cells. Therefore, these data indicate that catalase is able to potentiate ATRA-induced macrophage differentiation by inhibition of PPAR{gamma} activity, underscoring an important interplay between H2O2, RA, and PPAR{gamma} in macrophages.

Key Words: PPAR • ROS • ATRA • hydrogen peroxide


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INTRODUCTION
 
Reaction oxygen species (ROS), a major cause of oxidative stress, have been recognized to be important in host defense and pathogenesis of a variety of human diseases including cardiovascular diseases, cancers, and Type 2 diabetes [1 2 3 4 ]. ROS are broadly defined as a group of highly reactive molecules, which are generated in the redox reaction in the cells. The representative ROS are superoxide anion (O2•–), hydroxyl radical (•OH), hydrogen peroxide (H2O2), and thiyl radicals [5 ]. ROS are mainly generated during electron transfer in the mitochondrial respiratory chain by reducing molecular oxygen to form O2•–, which is converted by superoxide dismutase into H2O2 that is able to produce a highly reactive •OH in the presence of reduced metals and is removed by catalase and glutathione peroxidase. In addition, it has been well characterized that ROS, generated by NADPH oxidase (Nox) following macrophage activation, are important for host defense response against bacterial and viral infection [6 7 8 9 ]. However, the potential function of ROS in the process of monocyte-to-macrophage differentiation remains obscure. Limited reports have demonstrated potential functions of ROS on macrophage differentiation. Removal of H2O2 by catalase was demonstrated to induce differentiation of immature myeloid cells (ImC) from tumor-bearing mice into macrophages [10 ]. A large array of antioxidants such as curcumin, ebselen, and silibinin had a cooperative effect with 1,25-dihydroxyvitamin D3 in the differentiation of leukemia HL60 cells [11 ]. These findings indicate that the ROS are actively involved in the regulation of the macrophage differentiation process. However, the molecular mechanism underlying the regulatory role of ROS in macrophage differentiation has not been elucidated.

All-trans retinoic acid (ATRA) is a potent metabolite of vitamin A. ATRA has been well documented as a growth and differentiation factor in many tissues and cells, and proved to be an effective treatment to many diseases including cancers. ATRA is now widely used as a therapy for the treatment of acute promyelocytic leukemia, as it is able to induce cell cycle arrest, cell differentiation, and apoptosis [12 13 14 15 16 ]. ATRA was also found to induce macrophage differentiation [17 , 18 ]. In THP-1 cells, ATRA could induce cell cycle arrest at the G1 phase through regulation of cell cycle-related gene expression [17 ]. ATRA treatment increased the expression of the macrophage differentiation marker CD11b in THP-1 cells and enhanced macrophage phagocytosis [17 , 18 ]. However, how ATRA induces macrophage differentiation at the molecular level has been elusive.

Many nuclear factors are implicated in macrophage differentiation, such as the peroxisome proliferator-activated receptor (PPAR) family. PPARs are ligand-dependent nuclear transcription factors, which play a critical role in the regulation of lipid and glucose metabolism, cell growth, differentiation, and homeostasis [19 20 21 22 23 ]. PPAR{gamma}, one member of the PPAR family, has been shown to modulate many facets of macrophage functions including regulation of cell differentiation, proinflammatory activities, and uptake of oxidized low-density lipoprotein into the cells [24 25 26 27 28 29 ]. PPAR{gamma} was found to be a negative regulator of macrophage activation, manifested by inhibition of genes, which are up-regulated during macrophage differentiation and activation, such as matrix metalloproteinase 9 (MMP9) and NO synthase [30 ]. PPAR{gamma} agonists were also found to inhibit production of inflammatory cytokines in monocytes [31 ]. In agreement with the negative regulation of PPAR{gamma} on macrophage differentiation, deletion of PPAR{gamma} in the mouse appeared to favor macrophage differentiation [32 ].

Although ROS, ATRA, and PPAR{gamma} are shown to be important in macrophage differentiation, the functional interaction among them has not been investigated. We used THP-1 monocytic leukemia cells as a model system to dissect the functions of these factors in macrophage differentiation [33 ]. As reported here, we found that catalase, a specific scavenger of H2O2, is able to cooperate with ATRA to induce THP-1 differentiation with functional involvement of PPAR{gamma}. These findings, therefore, revealed an intriguing interplay among H2O2, retinoic acid (RA), and PPAR{gamma} in macrophage functioning.


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MATERIALS AND METHODS
 
Reagents
Bisphernol A diglycidyl ether (BADGE), ATRA, catalase, N-acetylcystein (NAC), NBT, PMA, DEAE-dextran, and dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Propidium iodide (PI) was purchased from Invitrogen (Carlsbad, CA, USA). GW9662, 15-deoxy-{Delta}12,14-PG J2 (15d-PGJ2), and rosiglitazone were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA).

Plasmids
Human PPAR{gamma} cDNA (1434 bp) was cloned by PCR with reverse-transcribed cDNA from HepG2 cells as a template with oligonucleotide primers 5'-CCGCAGAAATGACCATGGTTGACA-3' and 5'-AAGGGAAATGTTGGCAGTGGCTCA-3'. The PCR fragment was cloned into pcDNA3 vector (Invitrogen) and verified by DNA sequencing. The PPAR-responsive luciferase reporter was generated by cloning the PPAR response element sequence 5'-TGAACTAGGGCAAAGTTGA-3' [34 ] in front of the E1b TATA box (5'-TATATAAT-3') from adenovirus type 5 [35 ], followed by cloning into pGL3-basic vector (Promega, Madison, WI, USA).

Cell culture
THP-1 human monocytic leukemia cells were cultured in RPMI 1640, supplemented with 10% FBS, 100 µg/ml penicillin, and 100 µg/ml streptomycin (all from Gibco-BRL, Gaithersburg, MD, USA). Cells were incubated at 37°C in humidified air with 5% CO2. Cells were seeded at a density of ~5 x 105/ml before experimental procedures.

Cell cycle analysis
For analysis of cell cycle profiles, ~2 x 106/ml cells were incubated with ATRA and/or catalase for 48 h. The cells were then pelleted by centrifugation at 300 g and 4°C for 5 min and washed with PBS twice. Cell pellets were resuspended in 300 µl ice-cold PBS and fixed by adding 700 µl absolute ethanol at –20°C. Cells were stored at –20°C after fixation until ready for analysis. For PI staining, fixed cells were centrifuged and washed with PBS as above, followed by resuspending with PBS and RNase A treatment. PI was added at a final concentration of 50 µg/ml. Cells were incubated at room temperature for 30 min before analysis using FACSAriaTM (Becton Dickinson, Franklin Lakes, NJ, USA)

RT-PCR
Total RNA was prepared from ~2 x 106 THP-1 cells after treatments for 48 h. The RNA was reverse-transcribed with oligo(dT) primer using a SuperScript first-strand synthesis kit (Invitrogen) to generate the first-strand cDNA, followed by PCR to detect the expression of CD68, CD11b, MMP9, and GAPDH. The sequences of the PCR primers are as follows: 5'-AGGACGGCAATGCTGATG-3' and 5'-AGGGCGAGGACCATAGAGG-3' for human MMP9; 5'-CATAGCCAGCGGATAGCA-3' and 5'-GCAACTGTAGTTTCAGGGTC-3' for human CD11b; 5'-AGGCTGGCTGTGCTTTTC-3' and 5'-CTTCCCTGGACCTTGGTT-3' for human CD68; 5'-GCACTCTTCCAGCCTTTCCTG-3' and 5'-GGAGTACTTGCGCTCAGGAGGAGC-3' for human GAPDH. The amplification conditions were as follows: one cycle of denaturing at 94°C for 5 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min. This was followed by 21–31 cycles of PCR. The reaction products were separated on 1.5% agarose gel and stained with ethidium bromide.

Real-time RT-PCR
The cDNA was generated as described above. Primers used in real-time RT-PCR were designed by Primer Express software. The sequences of primers are as follows: 5'-TTGACAGCGACAAGAAGTG-3' and 5'-CTGAGGAATGATCTAAGCC-3' for MMP9; 5'-GGGAGAAGGGAGGGAGAGAA-3' and 5'-GGAAAACCCCGTCAAAGATT-3' for CD68; 5'-AGATTGTGTTTTGAGGTTTC-3' and 5'-TGTGTATGTGTGGTGTGTGT-3' for CD11b; 5'-GATCATTGCTCCTCCTGAGC-3' and 5'-ACTCCTGCTTGCTGATCCAC-3' for actin. For relative quantitation, the reactions were performed in mixtures containing 1.5 µl 10x Taq reaction buffer, 1.125 µl deoxy-NTP mixture (2.5 mM each), 0.9 µl MgCl2 stock (25 mM; Tiangen Biotech, Beijing, China), 0.75 µl EvaGreen (Biotium, Inc., Hayward, CA, USA), 0.09 µl fluorescein calibration dye stock (Bio-Rad, Hercules, CA, USA), 1 µl DNA (10 ng/µl), and 0.6 µl each primer (3 µmol) in a total volume of 15 µl. The PCR amplification and detection were carried out in an iCycler (Bio-Rad), each with 30 s at 94°C, 30 s at 56–60°C, and 1 min at 72°C for 40 cycles after the initial denaturing step for 5 min at 94°C. To exclude the presence of nonspecific products, a routine melting curve analysis was performed after finishing amplification. This was done by high-resolution data collection during an incremental temperature increase from 60°C to 95°C. All real-time PCR procedures were performed three times. The threshold cycle (CT) value was calculated using the comparative CT method. CT for each gene was determined using thermocycler software, and the average of three independent experiments was calculated. The copy number of the target genes was normalized to actin as an endogenous reference. Fold change of controls was set at 1, and normalized fold change of genes after different treatments compared with control samples was calculated.

NBT assay
For NBT assay, cells were seeded in 24-well plates (Gibco-BRL) at a density of ~5 x 105 cells/ml and incubated with ATRA and/or catalase for 48 h. After that, cells were centrifuged and resuspended in 200 µl RPMI-1640 medium containing 1 mg/ml NBT and 4 µg/ml PMA and then incubated at 37°C for 1 h. Cells were then pelleted, and the pellets were resolved in 100 µl DMSO. Absorbance at 570 nm was detected with SpectraMAX 190 (Molecular Devices Corp, Sunnyvale, CA, USA).

Dual luciferase assay
THP-1 cells were transfected using DEAE-dextran reagent. Briefly, after PBS wash, ~4 x 106 cells were collected and resuspended in 25 mM Tris-HCl at pH 7.4, 137 mM NaCl, 5 mM KCl, 0.6 mM Na2HPO4, 0.7 mM CaCl2, and 0.5 mM MgCl2 buffer. A total 4.5 µg plasmids and 15 ul DEAE-dextran/double-distilled H2O (150 µg/ml) was added together to the cells. After incubation at 37°C for 20 min, cells were washed with PBS and resuspended in RPMI-1640 medium with 10% FBS. A renilla luciferase vector phRL-SV40 (Promega) was cotransfected to monitor transfection efficiency. At 48 h after transfection, the cells were centrifuged and resuspended in lysis buffer (PBS with 0.1% Triton X-100 and 1 mM PMSF). The lysate (10 µL) was used in the dual-luciferase assay using a method described previously [36 ]. The samples were counted for 10 s with a luminometer (Berthold, Bad Wildbad, Germany). The luciferase activity was normalized to the renilla luciferase activity, and triplicate transfections were carried out for each experimental group.

H2O2 measurement
To measure the intracellular H2O2 level, THP-1 cells were incubated with 0.1 µM ATRA or 1000 units/ml catalase for 24 h. Approximately 2 x 106 cells were used for each experimental group. The cells were washed once with PBS and incubated with 20 µM DCFH-DA in RPMI-1640 medium at 37°C for 1 h. The cells were then washed three times with PBS to remove excess DCFH-DA dye and were kept on ice. The DCFH-DA density of the samples was analyzed in FACSAriaTM (Becton Dickinson) with excitation at 488 nm and emission at 525 nm.

Statistic analysis
All statistic analyses were performed with Student’s t test.


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RESULTS
 
Potentiation of ATRA-induced THP-1 differentiation by catalase
As the first step to study the potential role of H2O2 in macrophage differentiation, we analyzed the effect of catalase on ATRA-induced THP-1 adherence, a functional marker of macrophage differentiation [33 ]. When THP-1 cells were incubated without ATRA or with catalase alone at 1000 units/ml, few cells adhered to the cell culture plates (data not shown). However, treatment of the cells with low concentration of ATRA (0.1 µM) was able to moderately induce THP-1 adherence (Fig. 1A ). It is interesting that addition of catalase at 1000 units/ml appeared to increase THP-1 adherence profoundly in the presence of ATRA (Fig. 1A) . Therefore, these data provided the first piece of evidence that catalase may potentiate ATRA-induced THP-1 differentiation.


Figure 1
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  		Figure 1. Catalase enhances ATRA-induced THP-1 adherence and cell cycle arrest. (A) Effect of catalase on cell adherence. THP-1 cells were incubated with ATRA (0.1 µM) in the absence or presence of catalase (1000 units/ml) for 48 h. Suspended cells were washed off by PBS, and the images were taken using phase-contrast microscope. The same experiment was performed three times with identical results. (B) Effect of catalase on cell cycle arrest. The cell cycle profiles of THP-1 cells after treatment with ATRA (0.1 µM) and/or catalase (1000 units/ml) for 48 h were determined by FACS. Summary of cell cycle analysis is shown in the lower right panel in which the percentage of cells in each cell cycle phase is shown. G0/G1, cells in G0/G1 phase; S, S phage; G2/M, cells in G2/M phase. The same experiment was performed three times with identical results.

ATRA has been shown to induce cell cycle arrest during THP-1 differentiation [17 ]. Therefore, we analyzed the effect of catalase on cell cycle progression in THP-1 cells. As shown in Figure 1B , treatment of THP-1 cells with 0.1 µM ATRA was able to increase the cell population of G0/G1 from 55% to 70% with a corresponding decrease of cells at S, G2, and M phases. When ATRA and catalase were added to the cell culture, the proportion of G0/G1 cells was elevated further to ~90%. These data suggest that the catalase was able to enhance ATRA-mediated cell cycle arrest in THP-1 cells.

We next analyzed NBT reduction, a commonly used assay to analyze macrophage differentiation [37 , 38 ]. Previously, ATRA has been shown to increase NBT reduction in THP-1 cells [18 ]. To address whether catalase could affect NBT reduction, THP-1 cells were treated with different concentrations of catalase, with or without cotreatment of ATRA. As shown in Figure 2 , ATRA treatment itself was able to increase NBT reduction significantly. Treatment of THP-1 cells with 500 units/ml and 1000 units/ml catalase alone had no significant effect on NBT reduction. However, when the concentration of catalase reached 2000 units/ml, it was able to increase NBT reduction, indicating that high concentration of catalase by itself was sufficient to induce the differentiation of THP-1 cells. In agreement with the results of cell adherence and cell cycle arrest, catalase was capable of enhancing NBT reduction further in the presence of ATRA. These data, therefore, substantiated the cooperative effect between catalase and ATRA in macrophage differentiation.


Figure 2
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  		Figure 2. Catalase synergizes with ATRA to increase NBT reduction. THP-1 cells were incubated with ATRA (0.1 µM), with or without different concentrations of catalase as indicated for 48 h. The cells were used in the NBT reduction assay, which was measured by absorbance at 570 nm. The data were shown as mean ± SD from three independent experiments. Individual Student’s t test was performed between the experimental groups (*, P<0.05, for comparison between treated group and untreated control; #, P<0.05, and ##, P<0.01, for comparison between catalase- and ATRA-treated groups and ATRA treatment alone).

Macrophage differentiation is associated with increased expression of several genes, which are critical for the functionality of macrophage. CD68 and CD11b are classical markers of macrophage differentiation [39 , 40 ]. MMP9, a matrix metalloproteinase, is another macrophage differentiation marker important for an inflammatory response of macrophages [41 , 42 ]. To analyze the effect of catalase and ATRA on these markers, THP-1 cells were treated with different concentrations of catalase, with or without ATRA. Total RNA was isolated from these cells and used in RT-PCR to determine the RNA expression levels of CD68, CD11b, and MMP9. Catalase alone at 1000 units/ml had no effect on CD68 expression after 24 or 48 h of treatment (data not shown). However, catalase at this concentration had an obvious synergistic effect with ATRA to induce CD68 expression (Fig. 3A ). In addition, catalase by itself at the concentration range of 250 to 1000 units/ml could increase the expression of MMP9 slightly, but such effect was enhanced greatly when the cells were cotreated with ATRA (Fig. 3B) . Catalase alone at 250–1000 units/ml appeared not able to affect CD11b expression (Fig. 3B) . However, catalase at this concentration range could increase ATRA-stimulated CD11b expression. We also used quantitative RT-PCR to confirm the regulation of cell differentiation markers by ATRA and catalase. By real-time PCR, we found that ATRA could increase the mRNA levels of CD68, MMP9, and CD11b significantly (Fig. 3C) . Furthermore, catalase had a synergistic effect with ATRA on the induction of CD68 and MMP9 by quantitative PCR (Fig. 3C) . Taken together, these data suggest that catalase was able to synergize with ATRA to induce expression of genes specific for macrophage differentiation.


Figure 3
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  		Figure 3. Effect of catalase on the expression of macrophage differentiation markers. (A) THP-1 cells were incubated with ATRA (0.1 µM) and catalase (CAT; at 1000 units/ml) for 24 or 48 h. Total RNA was isolated for RT-PCR to determine the RNA levels of CD68 and GAPDH (as a control for the relative amount of RNA). The ratio of the relative density of CD68 bands compared with that of GAPDH was shown. (B) THP-1 cells were treated with ATRA (0.1 µM), with or without different concentrations of catalase, as indicated for 48 h. RT-PCR was used to determine the expression of MMP9, CD11b, and GAPDH. The ratios of the PCR band densities of MMP9 and CD11b in comparison with that of GAPDH were shown. (C) Analysis of cell differentiation markers by quantitative RT-PCR. THP-1 cells were treated with ATRA (0.1 µM) and catalase (1000 units/ml) for 48 h. Real-time RT-PCR was used to determine the expression of MMP9, CD11b, CD68, and actin. Fold change of each marker compared with actin was shown as mean ± SD, and the fold change of the control group was set to 1. *, P < 0.05, and **, P < 0.01, in comparison with the untreated group.

Reduction of H2O2 by catalase and ATRA in THP-1 cells
Three major types of ROS are generated by the respiratory chain of mitochondria: O2•–, ·OH, and H2O2. Catalase is a specific scavenger of H2O2. To confirm that catalase treatment was indeed able to remove H2O2 from THP-1 cells, a H2O2-specific fluorescence dye DCFH-DA was loaded to THP-1 cells to measure the cellular H2O2 level. As expected, catalase was able to remove H2O2 effectively (Fig. 4 ). It is surprising that ATRA could also decrease H2O2 generated by THP-1 cells, indicating that RA itself may regulate H2O2 production or remove H2O2 from the cells.


Figure 4
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  		Figure 4. Removal of H2O2 by ATRA and catalase. THP-1 cells were incubated with ATRA (0.1 µM) or catalase (1000 units/ml) for 24 h. The cells were then incubated with 20 µM DCFH-DA for 1 h to stain H2O2, followed by washing with PBS and FACS assay with excitation at 488 nm and emission at 525 nm. The cells without treatment of DCFH-DA were used as a blank control. The fluorescence data for each experimental group are superimposed to facilitate comparison.

Catalase had no stimulatory effect on RA receptor (RAR)-mediated gene transcription
ATRA exerts its cellular functions through activation of RARs, which belong to the nuclear hormone receptor superfamily of ligand-dependent transcription factors. A hallmark characteristic of these proteins is a highly conserved DNA-binding domain, which consists of two zinc fingers responsible for sequence-specific DNA binding to their cognate hormone response elements and sensitive to the oxidative status of a cell. In response to ATRA, RAR forms a heterodimer with the retinoid X receptor (RXR) and binds to a RA response element (RARE) to modulate downstream gene expression [43 ]. As catalase was able to enhance ATRA-induced THP-1 differentiation, we hypothesized that catalase may achieve this function by facilitating RAR-mediated gene transcription. To test this hypothesis, we analyzed the effect of catalase on a luciferase reporter, which contains three repeats of RARE [44 ]. The luciferase reporter was transiently transfected into THP-1 cells, followed by treatment with catalase and ATRA. As expected, treatment of the cells with ATRA was able to stimulate the luciferase activity significantly (Fig. 5 ). However, catalase did not have a synergistic effect with ATRA (Fig. 5) . Furthermore, catalase by itself could decrease RARmediated transcription. These data, therefore, indicate that the cooperative effect between catalase and ATRA in THP-1 differentiation is not likely a result of stimulation of RAR-mediated gene transcription by catalase.


Figure 5
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  		Figure 5. Effects of ATRA and catalase on RAR-mediated transcription. THP-1 cells were transiently transfected with a luciferase reporter plasmid, which contains three repeats of RARE. A renilla luciferase vector was used to monitor the transfection efficiency. The cells were treated with ATRA (0.1 µM) and catalase (1000 units/ml) at 8 h after transfection. The whole cell lysate was used in dual-luciferase assay 48 h after transfection. The fold change of luciferase activity is shown as mean ± SD. Student’s t test was performed with the data; *, P < 0.05, and **, P < 0.01, in comparison with the untreated group.

Catalase cooperates with ATRA to inhibit PPAR{gamma}-mediated transcriptional response
PPARs belong to another transcription factor family, which plays important roles in cell differentiation and other cellular processes. Several reports have indicated that PPAR{gamma} may play a negative role in modulating macrophage differentiation [28 , 30 , 31 ]. To investigate whether catalase could act on macrophage differentiation through modulation of PPAR{gamma} activity, we performed a luciferase assay using a reporter that contains a PPAR-responsive element [34 ]. When a PPAR{gamma} expression plasmid was cotransfected into THP-1 cells, the luciferase activity was increased to more than threefold (Fig. 6A ). It is interesting that ATRA treatment was able to inhibit a PPAR{gamma}-stimulated transcriptional response, indicating that RA negatively regulates the activity of PPAR{gamma}. To our surprise, catalase treatment could reduce the PPAR{gamma}-stimulated transcriptional response profoundly to a level even lower than the baseline (Fig. 6A) . Treatment of the cells with ATRA and catalase could also abrogate PPAR{gamma}-stimulated gene transcription completely. As PPAR{gamma} negatively regulates macrophage differentiation, our observation indicates that catalase may potentiate ATRA-induced THP-1 differentiation through inhibition of PPAR{gamma} activity.


Figure 6
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  		Figure 6. Involvement of PPAR{gamma} in catalase-mediated macrophage differentiation. (A) Effects of ATRA and catalase on PPAR{gamma}-mediated transcription. THP-1 cells were transiently transfected with a plasmid that contains the PPAR response element upstream of the firefly luciferase reporter. A PPAR{gamma} expression plasmid was also cotransfected as indicated. A SV40-driven renilla luciferase vector was used to monitor the transfection efficiency. The cells were treated with ATRA (0.1 µM) and catalase (1000 units/ml) at 8 h after transfection. The whole cell lysate was used in dual-luciferase assay at 48 h after transfection. The fold change of luciferase activity is shown in mean ± SD. **, P< 0.01, between treated groups with PPAR{gamma}-transfected but untreated group. (B) Enhancement of THP-1 differentiation by PPAR{gamma} antagonist. THP-1 cells were incubated with ATRA (0.1 µM), catalase (250 units/ml), and BADGE (10 µM) as indicated for 48 h. The cells in suspension were washed off with PBS, and the images were taken using phase-contrast microscope. Note that BADGE markedly enhanced ATRA and catalase-induced cell adherence. (C) Analysis of MMP9 expression by quantitative RT-PCR. THP-1 cells were incubated with ATRA (0.1 µM), catalase (1000 units/ml), PPAR{gamma} antagonists BADGE (10 µM) and GW9662 (GW; 1 µM), PPAR{gamma} agonist rosiglitazone (Rosi; at 10 µM), and 15d-PGJ2 (1.5 µM) as indicated. DMSO was added in controls with a final concentration less than 0.1%. The cells were treated for 48 h followed by real-time RT-PCR. Fold changes of MMP9 level compared with actin were shown as mean ± SD, and the control group was set to 1. *, P < 0.05, and **, P< 0.01, between treated groups and the ATRA plus catalase group.

If catalase enhances ATRA-induced THP-1 differentiation via inhibition of PPAR{gamma} activity, treatment of THP-1 cells with PPAR{gamma} antagonists would further increase macrophage differentiation induced by ATRA and catalase. To analyze this hypothesis, we treated THP-1 cells with the PPAR{gamma} antagonist BADGE. As shown in Figure 6B , BADGE was indeed able to enhance ATRA-induced THP-1 differentiation, as judged by the magnitude of cell adherence. The increase of THP-1 adherence by BADGE with ATRA appeared to be similar to the cooperative effect of ATRA with catalase at 250 units/ml. Furthermore, BADGE could markedly increase the number of adhered cells treated with ATRA and catalase. We also analyzed the effect of another PPAR{gamma} antagonist GW9662 and found that it could enhance THP-1 adherence induced by ATRA and catalase treatment (data not shown). To provide further evidence about the regulation of THP-1 differentiation by the PPAR{gamma} pathway, we analyzed the effect of PPAR{gamma} antagonists and agonists on MMP9 expression induced by ATRA and catalase. Real-time RT-PCR was used to determine the mRNA level of MMP9. As shown in Figure 6C , PPAR{gamma} antagonists BADGE and GW9662 were able to enhance MMP9 expression further upon ATRA and catalase treatment. Consistently, PPAR{gamma} agonists rosiglitazone and 15d-PGJ2 could decrease ATRA/catalase-stimulated MMP9 expression. Taken together, these data provided additional evidence that catalase is able to potentiate ATRA-induced macrophage differentiation, at least partly by inhibition of PPAR{gamma} activity.


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DISCUSSION
 
In this study, we demonstrated for the first time that catalase cooperated with ATRA to induce differentiation of human THP-1 monocyte into macrophage. Such potentiation effect of catalase on macrophage differentiation was confirmed by changes of cell adherence, cell cycle arrest, NBT reduction, and expression of differentiation markers, although the effects of catalase and ATRA on these differentiation indicators had a certain degree of concentration-dependent discrepancy as a result of the difference in experimental systems. We found that catalase and ATRA were able to reduce H2O2 generated from THP-1 cells. We investigated the possible molecular mechanism underlying the cooperative effect of catalase with ATRA. Catalase treatment could not enhance the stimulation of ATRA on RAR-mediated gene transcription. However, catalase and ATRA could inhibit a PPAR{gamma}-mediated transcriptional response. Correspondingly, PPAR{gamma} antagonists were able to enhance catalase and ATRA-induced THP-1 differentiation. PPAR{gamma} agonists could antagonize MMP9 expression induced by ATRA and catalase. Therefore, our data reveal an intriguing interplay among catalase, RA, and PPAR{gamma} in macrophage differentiation, as depicted in a hypothetical model (Fig. 7 ). In this model, ATRA stimulates a RAR-mediated transcriptional response, which in turn inhibits PPAR{gamma}-mediated transcription. Meanwhile, ATRA may also positively modulate macrophage differentiation independent of PPAR{gamma} inhibition (Y. Chen, unpublished observation). Conversely, catalase is able to inhibit the activity of PPAR{gamma}. The synergistically reduced PPAR{gamma} activity by ATRA and catalase would relieve the inhibitory effect of PPAR{gamma} on macrophage differentiation, thus explaining the cooperative effect of catalase and ATRA on macrophage.


Figure 7
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  		Figure 7. A proposed model to illustrate the functional interplay among catalase, ATRA, and PPAR{gamma} in macrophage differentiation. ATRA binds to RXR-RAR heterodimer to trigger transcription of downstream genes, leading to macrophage differentiation. PPAR{gamma} activity is negatively regulated by an ATRA signaling pathway. Catalase also negatively modulates PPAR{gamma} activity by removal of H2O2. The inhibition of PPAR{gamma} activity by ATRA and catalase relieves the inhibitory action of PPAR{gamma} on macrophage differentiation. ATRA may also regulate macrophage differentiation independent of PPAR{gamma} inhibition.

ROS play an important function in a large array of cell signaling pathways. H2O2 is more stable than O2•– and •OH and has been found to be more active in cell metabolism and signaling than other forms of ROS [45 ]. The major source of H2O2 in most cells is mitochondria, in which O2•– generated in a respiratory chain is converted to H2O2 by superoxide dismutase. In addition, H2O2 could be produced by Nox, especially in neutrophils. The biological function of H2O2 has been revealed in many studies. H2O2 could function as a signaling molecule, which is able to modulate the activity of protein tyrosine kinases by inhibition of tyrosine phosphatase or stimulation of tyrosine kinase [45 ]. Using a transgenic mouse model, it is found that overexpression of catalase in mitochondria could increase the lifespan of the animals significantly, indicating that H2O2-mediated oxidative stress is implicated in aging [46 ]. Our study suggests that H2O2 is actively involved in macrophage differentiation through functional interaction with RA and PPAR{gamma}. This is consistent with the finding that catalase was able to induce differentiation of ImC into macrophage in tumor-bearing mice [10 ]. In agreement with our finding, antioxidants have been shown to have a cooperative effect with 1,25-dihydroxyvitamin D3 in the differentiation of leukemia HL60 cells [11 ]. It is noteworthy that the functional role of ROS in macrophage differentiation might be dependent on the type of ROS and the nature of differentiation inducers. For example, the differentiation of bone marrow monocyte-macrophage lineage cells into osteoclasts was inhibited by treatment of NAC to remove •OH or diphenyleneiodonium (DPI) to inhibit Nox [47 ]. Meanwhile, DPI and NAC could abrogate PMA-mediated cell cycle arrest in THP-1 monocytes [48 ]. It is believed that the PMA-stimulated ROS production is implicated in induction of p21WAF1/Cip1 expression in these cells [48 ]. Therefore, it is worthwhile to investigate how individual ROS are involved in macrophage differentiation induced by different factors or signaling pathways in the future.

RA and PPAR{gamma} are well-studied regulators in cell metabolism and many other cellular functions. ATRA and PPAR{gamma} have been shown to play critical roles in macrophage differentiation, which is important in host defense and development of many human disorders such as cardiovascular diseases. ATRA is an effective drug for the treatment of acute promyelocytic leukemia [12 13 14 15 16 ]. Meanwhile, ATRA is also an active inducer of macrophage differentiation [17 , 18 ]. In THP-1 cells, ATRA was able to induce cell cycle arrest through regulation of several cell cycle-related genes such as cyclin E, retinoblastoma protein, and p27Kip1 [17 ]. Conversely, PPAR{gamma} has been shown to be a potent, negative regulator of macrophage differentiation and activation [28 , 30 , 31 ]. This is consistent with the finding that deletion of PPAR{gamma} in mouse is accompanied by an increment of macrophage differentiation [32 ]. However, it was previously unknown how ROS are implicated in the modulatory effects of RA and PPAR{gamma} on macrophage differentiation. Our studies provided an initial evidence that H2O2 is able to functionally collaborate with ATRA and PPAR{gamma} to affect macrophage differentiation. It is important that our data suggest that there exists a close cross-talk among ROS, ATRA, and PPAR{gamma} during macrophage activation (Fig. 7) . The stimulatory effect of ATRA on macrophage differentiation is, at least partly, mediated by inhibition of PPAR{gamma} activity, which is modulated by the intracellular level of H2O2. However, it is noteworthy that many important questions need to be addressed in the future to elucidate in detail the functional interplay among these factors, such as how RA signaling pathway and H2O2-mediated signaling modulate the activity of PPAR{gamma}. Nevertheless, our study provided a framework to start understanding the functional interaction among ROS, RA signaling, and PPAR{gamma} activity in macrophages, which are important in the pathogenesis of many human diseases.


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ACKNOWLEDGEMENTS
 
This work was supported by research grants from Chinese Academy of Sciences (One Hundred Talents program), National Natural Science Foundation of China (30470870 and 30588002), and Science and Technology Commission of Shanghai Municipality (05DJ14009 and 04dz14007) to Y.C. Q.D. designed and performed most experiments, T.J. and Z.W. performed parts of the experiment, and Y. C. overlooked the project and wrote the paper. We thank Dr. Spanjaard from Boston University School of Medicine for providing the RAR-responsive luciferase report plasmid.

Received November 12, 2006; revised January 30, 2007; accepted February 12, 2007.


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