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The Journal of Immunology, 2005, 175: 5423-5429.
Copyright © 2005 by The American Association of Immunologists

Oxidative Stress Augments the Production of Matrix Metalloproteinase-1, Cyclooxygenase-2, and Prostaglandin E2 through Enhancement of NF-{kappa}B Activity in Lipopolysaccharide-Activated Human Primary Monocytes1

Yunbiao Lu and Larry M. Wahl2

Immunopathology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The excessive production of reactive oxidative species (ROS) associated with inflammation leads to a condition of oxidative stress. Cyclooxygenase-2 (COX-2), PGE2, and matrix metalloproteinases (MMPs) are important mediators during the process of inflammation. In this paper we report on studies examining how the ROS hydrogen peroxide (H2O2) affects the production of MMP-1, COX-2, and PGE2. Addition of H2O2 to LPS-activated monocytes, but not naive monocytes, caused a significant enhancement of the LPS-induced production of MMP-1, COX-2, and PGE2. The mechanism by which H2O2 increased these mediators was through enhancement of I{kappa}B{alpha} degradation, with subsequent increases in NF-{kappa}B activation and NF-{kappa}B p50 translocation to the nucleus. The effects of H2O2 on I{kappa}B{alpha} degradation, NF-{kappa}B activation, and NF-{kappa}B p50 localization to the nucleus were demonstrated through studies of coimmunoprecipitation of I{kappa}B{alpha} with p50, ELISA of NF-{kappa}B p65 activity, and Western blot analysis of the nuclear fraction extract for p50. The key role for NF-{kappa}B in this process was demonstrated by the ability of MG-132 or lactacystin (proteasome inhibitors) to block the enhanced production of MMP-1, COX-2, and PGE2. In contrast, indomethacin, which inhibited PGE2 production, partially blocked the enhanced MMP-1 production. Moreover, although PGE2 restored MMP-1 production in indomethacin-treated monocyte cultures; it failed to significantly restore MMP-1 production in proteasome inhibitor-treated cultures. Thus, in the presence of LPS and H2O2, NF-{kappa}B plays a dominate role in the regulation of MMP-1, COX-2, and PGE2 expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Reactive oxygen species (ROS)3 have been associated with the initiation or aggravation of diverse pathological states. The ability of ROS to induce biochemical alterations in macromolecules such as DNA, lipids, and proteins is believed to be the mechanism through which these molecules mediate their effects on the inflammatory process. The physiological production of ROS by leukocytes and macrophages serves as a bactericidal mechanism of host defense, but can also damage surrounding tissue (1, 2). Recent studies have shown that the production of ROS by monocytes/macrophages and endothelial cells at inflammatory sites affects the process of inflammation (3, 4, 5, 6, 7, 8). ROS, such as superoxide anion and hydrogen peroxide (H2O2), have been implicated as intracellular second messengers in an autocrine or a paracrine fashion. In chronic inflammation, such as atherosclerosis (9, 10), Chagas disease (11), arthritis (12), asthma (4), and sepsis (2), significant amounts of ROS are produced by monocytes/macrophages or other cell types at these inflammatory sites. There is substantial evidence demonstrating that many genes and signal transduction pathways are influenced by ROS and antioxidants (13).

Matrix metalloproteinases (MMPs) are believed to be responsible for the destruction of connective tissue at sites of chronic inflammation, such as arthritis, atherosclerosis, and periodontal disease. MMPs are comprised of a family of enzymes that includes interstitial collagenases, gelatinases, stromelysin, matrilysin, metalloelastase, and membrane-type MMPs (14, 15, 16). Collectively, these enzymes can degrade all the components of the extracellular matrix. Monocytes/macrophages are prominent cells at sites of chronic inflammation and have been shown to produce MMPs when activated by agents such as LPS, Con A, cytokines, and extracellular matrix components (17, 18, 19, 20, 21). Stimulation of monocytes with LPS induces a number of MMPs, including MMP-1. MMP-1 cleaves fibrillar collagens, such as types I, II, and III, and thus may have an important role in the connective tissue turnover or remodeling associated with inflammation and wound healing.

Other inflammatory mediators produced by activated monocytes include PGs, in particular PGE2, which contributes to vasodilation, pain, and fever (22). Inhibition of PG synthesis suppresses inflammation and confers analgesia (23). The synthesis of PGs is regulated by two main enzyme isoforms, cyclooxygenase-1 (COX-1) and COX-2. COX-1 is constitutively produced and detectible in most human tissues. In contrast, COX-2, normally expressed at low levels, is strongly induced by proinflammatory agents, including LPS, tumor promoters, and growth factors (24). At inflammation sites, such as human atherosclerotic lesions, high levels of COX-2 have been found compared with low levels in unaffected arteries (25, 26). The COX-2/PGE2 pathway has also been shown to be involved in the production of MMP-1 by LPS-stimulated human primary monocytes (27).

In this study we report that exposure of LPS-activated human primary monocytes to H2O2 increases I{kappa}B{alpha} degradation, leading to an augmentation of NF-{kappa}B activation, p50 localization to the nucleus, and the subsequent enhancement of COX-2, PGE2, and MMP-1. Furthermore, NF-{kappa}B is a required transcription factor in the induction of MMP-1 and COX-2 by LPS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

The following reagents were used: hydrogen peroxide (H2O2; Fisher Scientific), anti-MMP-1 (gift from Dr. H. Birkedal-Hansen, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD), anti-NF-{kappa}B p50 bound to agarose beads, anti-I{kappa}B{alpha}, anti-NF-{kappa}B p50, anti-lamin B1 (Santa Cruz Biotechnology), LPS, indomethacin (Sigma-Aldrich), lactacystin, MG-132 (Z-Leu-Leu-Leu-CHO; BIOMOL), anti-COX-1 and -2 (Cayman Chemical), nuclear extract kit and TransAM NF-{kappa}B p65 transcription factor assay kit (Active Motif), and PGE2 ELISA kit (Neogen).

Purification of human monocytes and culture conditions

Human PBMC were obtained by leukapheresis of normal volunteers at the Department of Transfusion Medicine, National Institutes of Health, and monocytes were subsequently purified by counterflow centrifugal elutriation (model J.6M; Beckman Coulter) as previously described (27). Monocytes were enriched to >90%, as determined by morphology, nonspecific esterase staining, and flow cytometry. The remaining 5–10% nonmonocyte cells were lymphocytes and did not affect any of the functions influenced by LPS treatment. Purified monocytes were cultured in DMEM (Cambrex Bioscience) supplemented with 2 mM L-glutamine (Mediatech) and 10 µg/ml gentamicin sulfate (Cambrex Bioscience) at 37°C in a humidified atmosphere containing 5% CO2. Unless otherwise stated, monocytes were adhered for 30 min before the addition of reagents. Each experiment was repeated a minimum of three times, with different donors.

Detection of MMP-1 by Western blot analysis

For determination of the protein levels of MMP-1 produced by monocytes, proteins in the 0.5 ml of supernatants collected at 48 h from cultures of 5 x 106 monocytes/ml were precipitated with 0.75 ml of cold ethanol (final concentration, 60%) at –70°C as previously described (27). The proteins from the conditioned medium were separated on a 12% Tris-glycine polyacrylamide gel and then transferred onto a nitrocellulose membrane. The nitrocellulose membranes were blocked with 5% nonfat dry milk in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20. The membranes were then incubated overnight with Abs against MMP-1. Western blots were analyzed by the addition of Alexa Fluor 680 goat anti-rabbit second Ab (Molecular Probes), and infrared fluorescence was detected and quantified using the Odyssey infrared imaging system (LI-COR).

Immunoprecipitation and Western blot assay

Monocytes were harvested at specific times after treatment with the desired reagents and were lysed in lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM {beta}-glycerolphosphate, 1 mM Na3VO4, and proteinase inhibitor mixture; Roche). After equalizing the protein to 1.5 mg/300 µl in a 1.5-ml Eppendorf tube, the appropriate Ab bound to agarose beads was added, and the samples were rotated overnight at 4°C. After washing the beads three times with lysis buffer, the samples were subjected to Western blot analysis as described above. The membranes were then stripped with elution buffer and reprobed with Abs against the nonphosphorylated protein as a measure of equal loading.

Analysis of COX-1 and COX-2 proteins

Membrane proteins for COX-1 and COX-2 determination were prepared as previously described (28). Equal amounts of cell membrane proteins (50 µg) were fractionated on a 12% Tris-glycine-polyacrylamide gel and then transferred to a nitrocellulose membrane. The levels of COX-1 and COX-2 protein were determined by Western blot analysis as described above.

PGE2 analysis

PGE2 levels were determined in the 48-h culture medium by ELISA according to the instructions of the manufacturer. Values were expressed as the mean ± SD of triplicate samples.

NF-{kappa}B p65 activation assay

Monocytes were harvested at specific times after treatment with reagents, and the nuclear extracts (2 µg) were assayed in triplicate for p65 activity with a TransAM NF-{kappa}B kit according to the instructions of the manufacturer. The OD450 was read on a Wallac Victor 1420 multilabel counter (PerkinElmer). Values were expressed as the mean ± SD.

Nuclear localization of p50

Monocytes were harvested at specific times after treatment with various reagents, and the nuclear fractions were prepared according to the instructions of the manufacturer (Santa Cruz Biotechnology). Nuclear extracts (30 µg protein/sample) were assayed by Western blot as described above. The membranes were reprobed with anti-lamin B1 (Mr, 75 kDa) as a control for the amount of nuclear protein loaded.

Cell viability assay

To determine cell viability, monocyte cultures were stained with trypan blue stain (Invitrogen Life Technologies) at specific times after treatment with reagents. Stained cells vs live cells were counted under a microscope. Each value was the average from three different donors and was expressed as the mean ± SD.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
H2O2 augments MMP-1, COX-2, and PGE2 production by LPS-stimulated human monocytes

To determine the effect of H2O2 on human primary monocytes, we first examined the MMP-1 levels in culture supernatants after treatment of monocytes with H2O2 or LPS plus H2O2 (Fig. 1A). Although H2O2 alone failed to stimulate the production of MMP-1 by unstimulated monocytes, it dramatically enhanced the production of MMP-1 by LPS-activated monocytes. The dose-dependent enhancement of MMP-1 by H2O2 was most evident at 1 mM, with a subsequent decline at 2 mM. Because we have previously shown that MMP production by monocytes is regulated at least in part by PGE2, we investigated the effect of H2O2 on the production of COX-2 and COX-1 (Fig. 1B) and PGE2 (Fig. 1C) in human primary monocytes. H2O2 alone also failed to stimulate the monocytes to produce COX-2 and PGE2, whereas, similar to MMP-1, it enhanced the production of COX-2 and PGE2 by LPS-activated monocytes. COX-1, which is constitutively expressed in human monocytes, was not changed, as shown by equal levels of protein at all experimental conditions.



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  		FIGURE 1. H2O2 enhances MMP-1, COX-2, and PGE2 production by LPS-stimulated human primary monocytes. A, Human primary monocytes in DMEM were plated in 12-well culture plates at a concentration of 5 x 106 cells/ml/well. The monocytes were cultured for 30 min to allow them to adhere, and then LPS and H2O2 were added to some of the wells as indicated. Monocytes cultured with medium only are the controls. Culture supernatants were harvested after 48 h, ethanol precipitated, and analyzed for MMP-1 by Western blot. B, Monocytes (20 x 106/4 ml DMEM) were adhered in 60-mm petri dishes for 30 min, then LPS and H2O2 were added to some of the cultures as indicated. After 16 h, the cultures were harvested, the monocyte membrane proteins were isolated, and equal amounts of protein (50 µg) from each sample were analyzed on SDS-PAGE and immunoblotted with Abs against COX-2 or COX-1. C, Monocytes in DMEM were plated in 12-well culture plates at a concentration of 5 x 106 cells/ml/well. The monocytes were cultured for 30 min to allow them to adhere, and then LPS and H2O2 were added to some of the wells as indicated. Culture supernatants were harvested after 48 h and analyzed for PGE2 by ELISA. The data represent the mean ± SD of triplicate samples. *, p > 0.05.

 
Next, we compared the kinetics of MMP-1 vs COX-2 production by monocytes stimulated with LPS or LPS plus H2O2. LPS stimulated a low level of MMP-1 at 12 h, with a significant increase at 24 and 48 h (Fig. 2A). However, the greatest increase at these time points occurred when monocytes were exposed to LPS and H2O2. In contrast to MMP-1, significant levels of LPS-induced COX-2 were detected as early as 3 h, with additional increases occurring at 6 and 12 h, unlike COX-1, which was not changed compared with control monocytes (Fig. 2B). Similar to MMP-1, the greatest increase in COX-2 occurred in the presence of LPS and H2O2. Thus, the earlier induction of COX-2 is consistent with the requirement for PGE2 preceding MMP-1 synthesis. Moreover, H2O2 increased LPS-stimulated MMP-1 and COX-2 at all time points.



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  		FIGURE 2. Kinetics of MMP-1 and COX-2 production by LPS-stimulated or LPS- plus H2O2-stimulated human primary monocytes. A, Human primary monocytes in DMEM were plated in 12-well culture plates at a concentration of 5 x 106 cells/ml/well. The monocytes were cultured for 30 min to allow them to adhere, and then LPS and H2O2 were added to some of the wells as indicated. Monocytes cultured with medium only are the controls. Culture supernatants were harvested after 12, 24, and 48 h; ethanol precipitated; and analyzed for MMP-1 by Western blot. B, Monocytes (20 x 106/4 ml DMEM) were adhered in 60-mm petri dishes for 30 min, and then LPS and H2O2 were added to some of the cultures as indicated. After 3, 6, and 16 h, the cultures were harvested, monocyte membrane proteins were isolated, and equal amounts of protein (50 µg) from each sample were analyzed on SDS-PAGE and immunoblotted with Abs against COX-2 or COX-1.

 
Enhancement of LPS-induced monocyte MMP-1 by H2O2 is suppressed by inhibitors of NF-{kappa}B and COX-2

To address the mechanism of enhancement of MMP-1 production by H2O2, we investigated whether MMP-1 is regulated by NF-{kappa}B and/or COX-1 or -2 activities. We first tested two proteasome inhibitors, MG-132 and lactacystin, that prevent the degradation of I{kappa}B by proteasomes and thus inhibit the activation of NF-{kappa}B, because I{kappa}B keeps NF-{kappa}B in an inactive form. Addition of MG-132 (Fig. 3A) or lactacystin (Fig. 3B) blocked MMP-1 induction by LPS and enhancement by H2O2. Inhibition of MMP-1 production with either NF-{kappa}B inhibitor was detected at 0.5 µM, with complete inhibition occurring at 2 µM. Addition of PGE2 to cultures treated with either proteosome inhibitor caused only a slight restoration of MMP-1 production. Comparison of the NF-{kappa}B inhibitors with indomethacin, a PG synthesis inhibitor, demonstrated that indomethacin reduced the production of MMP-1, but low levels of MMP-1 were still detectable in the medium from LPS or LPS plus H2O2 cultures (Fig. 3C). In contrast to the proteasome inhibitor-treated cultures, the inhibition of MMP-1 by indomethacin was fully restored by exogenous PGE2.



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  		FIGURE 3. Suppression of MMP-1 by inhibitors of NF-{kappa}B activity and PG synthesis is reversed by PGE2 only in indomethacin-treated cultures. Monocytes in DMEM were plated in 12-well culture plates at a concentration of 5 x 106 cells/ml/well. The monocytes were cultured for 30 min to allow them to adhere. Some cultures were incubated with MG-132 (A), lactacystin (B), or indomethacin (C) for 30 min before addition of LPS, H2O2, and PGE2. The 48-h culture supernatants were analyzed for MMP-1 by Western blot.

 
H2O2, proteasome inhibitors and indomethacin did not affect monocyte viability

Examination of monocyte viability at 24 (Fig. 4A) and 48 h (Fig. 4B) revealed that 1 mM H2O2 as well as the proteasome inhibitors and indomethacin at the indicated concentrations were not toxic to the monocytes (Fig. 4D). However, there was a decrease in control monocyte viability by 48 h.



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  		FIGURE 4. Monocyte viability was unaffected by H2O2, MG-132, lactacystin, or indomethacin. Monocytes in DMEM were plated in 12-well culture plates at a concentration of 5 x 106 cells/ml/well. The monocytes were cultured for 30 min to allow them to adhere, then inhibitors of MG132, lactacystin, and indomethacin were added for another 30 min before the addition of LPS and H2O2 to the wells as indicated. Monocytes cultured with medium only are the controls. Monocyte viability was determined by trypan blue staining after 24 h (A) and 48 h (B) of culture. The data represent the mean ± SD of triplicate donors.

 
NF-{kappa}B has a central role in H2O2-mediated increases in COX-2 and PGE2 by LPS-stimulated monocytes

NF-{kappa}B has been reported to be a transcription factor of COX-2 (29). Therefore, we examined COX-2 as well as COX-1 and PGE2 after treatment of monocytes with the proteasome inhibitors. Lactacystin or MG-132 caused an almost complete inhibition of COX-2 protein expression induced by LPS or LPS plus H2O2 at 2 µM, but did not affect COX-1 protein levels (Fig. 5, A and B). There was no effect of indomethacin on COX-2 expression (Fig. 5C), whereas it inhibited PGE2 production (Fig. 5D). When PGE2 levels were measured in similarly treated monocyte cultures, MG-132 or lactacystin also inhibited PGE2 to comparable levels as the PG synthesis inhibitor, indomethacin. These results demonstrate that NF-{kappa}B regulates COX-2 expression as well as other PGE2-independent transcription factors involved in the regulation of MMP-1, because PGE2 does not cause a significant restoration of MMP-1 in proteasome inhibitor-treated monocytes (Fig. 3).



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  		FIGURE 5. NF-{kappa}B inhibitors suppress the production of COX-2 and PGE2, but not that of COX-1. Monocytes (20 x 106) were treated with lactacystin (A), MG-132 (B), or indomethacin (C) at the indicated doses for 30 min before the addition of LPS or LPS plus H2O2. The monocyte cultures were harvested at 16 h, and the membrane proteins were analyzed for COX-2 and COX-1 by Western blot. D, Monocytes (5 x 106/ml in DMEM) were incubated with indomethacin, MG-132, or lactacystin for 30 min before the addition of LPS or LPS plus H2O2. The 48-h culture supernatants were analyzed for PGE2 by ELISA. The data represent the mean ± SD of triplicate samples. *, p < 0.05.

 
LPS mediated and H2O2 enhanced I{kappa}B{alpha} degradation and NF-{kappa}B activation and was blocked by inhibitors of NF-{kappa}B, but not by indomethacin

The above findings indicated that H2O2 regulated the increase in MMP-1 production through NF-{kappa}B. Therefore, we next examined NF-{kappa}B activity as reflected by the degradation of I{kappa}B{alpha}, p50 translocation to the nucleus, and p65 binding to its specific oligonucleotide. Cell extracts from monocytes that had been treated with H2O2, LPS, or LPS plus H2O2 were assessed for bound I{kappa}B{alpha} by NF-{kappa}B (p50) immunoprecipitation and Western analysis. H2O2 in the presence of LPS, but not alone, caused a decrease in I{kappa}B{alpha} bound to NF-{kappa}B p50 (Fig. 6A), with a reciprocal increase in the translocation of p50 to the nucleus (Fig. 6B). Maximum degradation of I{kappa}B{alpha} and nuclear localization of p50 occurred at 1 mM H2O2. MG-132 and lactacystin were then compared with indomethacin to demonstrate specificity for inhibition of NF-{kappa}B activity. MG-132 and lactacystin inhibited I{kappa}B{alpha} degradation, nuclear localization of p50, and activation of p65, whereas indomethacin did not (Fig. 6, C and D). We also examined the effect of H2O2 on NF-{kappa}B p65 activity. NF-{kappa}B p65 activity stimulated by LPS was increased by H2O2 and was inhibited by lactacystin and MG-132, but not by indomethacin (Fig. 7).



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  		FIGURE 6. I{kappa}B{alpha} degradation mediated by LPS and enhanced by H2O2 is blocked by inhibitors of NF-{kappa}B, but not by indomethacin. A, Monocytes (20 x 106 cells/experimental condition) were stimulated with LPS or LPS plus H2O2 for 2 h. Cell lysates were prepared and immunoprecipitated with anti-p50 and then analyzed by Western blot with anti-I{kappa}B{alpha} or anti-p50. B, Monocytes (20 x 106 cells/experimental condition) were stimulated with LPS or LPS plus H2O2 for 2 h. Nuclear extract fractions were prepared and analyzed by Western blot with anti-p50 or anti-lamin B1. C, After 30 min of treatment with indomethacin, MG-132, or lactacystin, monocytes were stimulated with LPS or LPS plus H2O2 for 2 h. Cell lysates were prepared and immunoprecipitated with anti-p50 and then analyzed by Western blot with anti-I{kappa}B or anti-p50. D, After 30 min of treatment with indomethacin, MG-132, or lactacystin, monocytes were stimulated with LPS or LPS plus H2O2 for 2 h. Nuclear extract fractions were prepared and analyzed by Western blot with anti-p50 or anti-lamin B1.

 


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  		FIGURE 7. NF-{kappa}B activation mediated by LPS and enhanced by H2O2 is blocked by inhibitors of NF-{kappa}B, but not by indomethacin. A, Monocytes were treated with LPS or LPS plus H2O2 for 2 h. Cell nuclear extracts were prepared, and the activation of p65 was analyzed by ELISA. The data represent the mean ± SD of triplicate samples. *, p < 0.05. B, Monocytes were treated with MG-132, lactacystin, and indomethacin for 30 min before the addition of LPS or LPS plus H2O2 for 2 h. Nuclear extracts were prepared, and the activation of p65 was analyzed by ELISA. The data represent the mean ± SD of triplicate samples. *, p < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Oxidative stress and the oxidative modification of biomolecules are involved in a number of physiological and pathophysiological processes, such as aging, atherosclerosis, inflammation, carcinogenesis, arthritis, asthma, and drug toxicity (2, 3, 4, 7, 12). ROS are ubiquitous, highly diffusible, reactive molecules produced by reduction of molecular oxygen and include species such as superoxide anion, H2O2, and hydroxyl radical (OH–). ROS are known to affect the expression of a number of genes through their effects on MAPKs, protein phosphatases, and transcription factors (13, 30, 31). It has become increasingly clear that ROS generated endogenously or exogenously, such as superoxide anion and H2O2 may act as second messengers. Our study shows that H2O2 facilitates the production of MMP-1 (Fig. 1A), COX-2 (Fig. 1B), and PGE2 (Fig. 1C) only in LPS-stimulated human primary monocytes, whereas H2O2 alone does not activate monocytes. As shown in Fig. 2, the H2O2 enhancement of LPS-induced COX-2 occurred before the appearance of MMP-1, which is consistent with the requirement for PGE2 in the signaling pathway leading to MMP-1 production.

The first signaling proteins to be recognized as oxidative stress-sensitive molecules were actually transcription factors, such as NF-{kappa}B (32, 33). Several studies have shown that ROS strongly affects the activation of NF-{kappa}B (13, 31, 34, 35). Nevertheless, NF-{kappa}B is not activated by H2O2 in various immortal human T cell lines, monocytic cell lines, and peripheral T cells (36, 37, 38, 39). In contrast, NF-{kappa}B-binding activity can be inhibited by treatment of some tissues with H2O2 (40, 41). Thus, the effects of ROS on oxidative stress signaling are cell and stimulus dependent. Our findings with monocytes demonstrate that the state of cellular activation may be critical in determining the cellular response to H2O2. This may at least partially explain the failure of H2O2-mediated activation of NF-{kappa}B in various immortal human T cell lines, monocytic cell lines, and peripheral T cells.

The activation of NF-{kappa}B is initiated through the rapid phosphorylation of two serine residues on I{kappa}B, the inhibitory subunit of NF-{kappa}B, by I{kappa}B kinase. Once phosphorylated, I{kappa}B is polyubiquitinylated and rapidly degraded by the proteasome, allowing NF-{kappa}B to translocate to the nucleus and activate transcription. Recently, novel mechanisms have been proposed for H2O2-induced activation of NF-{kappa}B, including Syk-mediated tyrosine phosphorylation of I{kappa}B{alpha} and serine phosphorylation of p65 (42), activation of I{kappa}B kinase (43), and inhibition of phosphatases (44, 45, 46). I{kappa}B-independent mechanisms of activation of NF-{kappa}B involving modulatory phosphorylation of its DNA binding subunits have also been reported. However, ROS activation of NF-{kappa}B is highly cell type dependent (47). Our study shows that H2O2 increases the degradation of I{kappa}B{alpha} (Fig. 6A), the nuclear localization of p50 (Fig. 6B), and the activation of NF-{kappa}B p65 (Fig. 7A) only in LPS-activated human primary monocytes, and proteasome inhibitors blocked this augmentation of I{kappa}B{alpha} degradation (Fig. 6C), nuclear localization of p50 (Fig. 6D), and activation of NF-{kappa}B p65 (Fig. 7B). This indicates that H2O2 modulation of NF-{kappa}B activity is largely dependent on the degradation of I{kappa}B{alpha} with subsequent activation of NF-{kappa}B, a process that requires an activation stimulus, such as LPS. It has been reported that induction of MMP-1 or COX-2 and PGE2 involves the activation of NF-{kappa}B (29, 48, 49). Our data show that the enhancement of LPS-stimulated MMP-1 production by H2O2 is largely dependent on NF-{kappa}B activation, which, in turn, activates other signal transduction components involved in the induction of MMP-1, such as COX-2 and PGE2. The H2O2-mediated increase in COX-2 through NF-{kappa}B activation results in additional generation of PGE2 that enhances MMP-1 production by a pathway involving cAMP and CREB (27). Thus, NF-{kappa}B serves as a dominant transcriptional factor in the regulation of MMP-1 production, whereas the induction of COX-2 and the subsequent generation of PGE2 are not sufficient to stimulate MMP-1 production, but require the activation of NF-{kappa}B.

COXs are the key enzymes that mediate the production of PGs from arachidonic acid. Two COX isoforms have been identified, COX-1 and COX-2. COX-1 is expressed constitutively, whereas COX-2 is induced by inflammatory agents, such as LPS, cytokines, and growth factors. The induction of COX-2 in cells is responsible for the major increase in PGE2 that is critical for many cellular functions. We have reported previously that COX-2 and PGE2 are involved in the regulation of MMP-1 and MMP-9 (27). It has been reported that oxidative stress regulates COX-2 up-regulation in neuronal cells through the MAPK pathway (50). Our study shows that H2O2 up-regulates the production of COX-2 (Fig. 1B) and PGE2 (Fig. 1C) only in monocytes activated by LPS. Furthermore, the up-regulation of COX-2 and PGE2 can be blocked by proteasome inhibitors (Fig. 5, A, B, and D), whereas there was no effect of indomethacin on COX-2 expression (Fig. 5C). These data indicate that H2O2 modulates the production of COX-2 and PGE2 mainly through the enhancement of NF-{kappa}B activity, and this only occurs when monocytes are activated by LPS. This conclusion is supported by our data (Fig. 3) showing that NF-{kappa}B inhibitors completely block the production of MMP-1, whereas there is only a slight restoration by exogenous PGE2 (Fig. 3, A and B). In contrast, indomethacin partially reduced the production of MMP-1, and exogenous PGE2 completely restored MMP-1 production (Fig. 3C). These findings demonstrate that H2O2 enhances the production of MMP-1, COX-2, and PGE2 through activation of NF-{kappa}B in human primary monocytes, a response that requires priming or activation by LPS.


   Acknowledgments
 
We thank Drs. Nancy McCartney-Francis and Nancy Vázquez for their review of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   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.

1 Address correspondence and reprint requests to Dr. Larry M. Wahl, Immunopathology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Building 30, Room 3A300, 30 Convent Drive, Bethesda, MD 20892-4352. E-mail address: lwahl{at}dir.nidcr.nih.gov Back

2 This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Dental and Craniofacial Research. Back

3 Abbreviations used in this paper: ROS, reactive oxidative species; COX, cyclooxygenase; MMP-1, matrix metalloproteinase-1. Back

Received for publication March 21, 2005. Accepted for publication August 2, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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