Urokinase Plasminogen Activator Upregulates Paraoxonase 2 Expression in Macrophages Via an NADPH Oxidase-Dependent Mechanism
Objective— Macrophage foam cells are characterized by increased oxidative stress. Macrophage urokinase plasminogen activator (uPA) was shown to contribute to atherosclerosis progression. We hypothesized that uPA atherogenicity is related to its ability to increase macrophage oxidative stress. Increased macrophage oxidative stress in turn was shown to enhance PON2 expression. In the present study we investigated the effect of uPA on macrophage PON2 expression in relation to cellular oxidative stress.
Methods and Results— uPA increased PON2 expression in THP-1 macrophages in a dose-dependent manner. This effect required uPA/uPAR interaction and was abolished by cell treatment with antioxidants. uPA increased macrophage oxidative stress, measured by increased lipid peroxides, reactive oxygen species formation, superoxide anion release, and cell-mediated LDL oxidation. These effects were related to uPA-mediated activation of NADPH oxidase, and could not be reproduced in mouse peritoneal macrophages (MPM) harvested from p47phox−/− mice, suggesting a causal relationship between NADPH oxidase activation and the effects of uPA on macrophage oxidative stress and PON2 expression. Finally, MPM from PON2−/− mice were more susceptible to uPA-induced cellular oxidative stress than wild-type MPM, suggesting that PON2 protects against uPA-stimulated macrophage oxidative stress.
Conclusions— Upregulation of macrophage PON2 may provide a compensatory protective mechanism against uPA-stimulation of macrophage oxidative stress during atherogenesis.
The formation of atherosclerotic lesion is characterized by accelerated oxidative stress and production of reactive oxygen species (ROS).1 We have previously shown that ROS formation induces macrophage lipid peroxidation, and subsequently, increases macrophage atherogenicity. These “oxidized macrophages” exhibit an increased capability to oxidize LDL,2 to accumulate cholesterol,3,4 and to form macrophage foam cells.
uPA is a serine protease enzyme of the fibrinolytic system, and uPA binding to its receptor, uPAR, is implicated in plasmin generation. The uPA/uPAR system has also a nonproteolytic role that extends beyond its role in fibrinolysis.5 Increased plasma levels of uPA are associated with plaque rupture.6 uPA is expressed in human atherosclerotic vessel wall, mainly in association with macrophages,7,8,9 and uPA overexpression contributes to the progression and complications of atherosclerosis.10 We have recently shown11 that uPA enhanced macrophage atherogenicity by increasing cellular cholesterol accumulation. However, as atherosclerotic lesion macrophage foam cells are characterized by increased oxidative stress, we hypothesized that uPA atherogenicity could be related to its ability to increase macrophage oxidative stress.
Paraoxonase 2 (PON2) is a member of a multigene family of the paraoxonases genes PON 1, 2, and 3.12 PON2 is expressed in arterial macrophages,13 and it was shown to possess antiatherogenic14,15 and antioxidative13,16 properties. We have previously demonstrated that in contrast to PON1 and PON3, which are inactivated under oxidative stress, PON2 expression is upregulated by cellular oxidative stress.17–20
In the present study we investigated the effect of uPA on PON2 expression and its relationship to oxidative stress in macrophages. Our results demonstrate that uPA induced a dose-dependent increase in PON2 expression. This effect could be part of a cellular compensatory response to uPA-induced NADPH oxidase-dependent increase in cellular oxidative stress. This newly identified pathway is the first description in macrophages, of oxidative stress stimulation, and initiation of oxidative stress-response, by uPA. This information may have a relevance to macrophage activation in the arterial atherosclerotic plaque, thus representing a new paradigm by which uPA may be implicated in atherogenesis, beyond its classical role in the fibrinolytic system.
Materials and Methods
For detailed Material and Methods please see online supplement Materials and Methods, Legend to Figure, at http://atvb.ahajournals.org.
Human 2-chain high molecular weight uPA activity standard, ATF (amino terminal fragment)-uPA, and fluorescein conjugated high molecular weight human urokinase were from American Diagnostica Inc. Active 2-chain high molecular weight mouse uPA, recombinantly produced in insect cells, was from Innovative Research.
Exponentially growing human myeloid leukemia cell line THP-1.
Balb/C, C57Bl/6, p47phox−/−, uPAR−/−, and PON2−/− mice were euthanized and peritoneal macrophages were harvested. The experimental protocol was approved by the Animal Care and Use Committee of the Technion, No. IL-005 to 01-2005.
uPA activity was determined by an amidolytic assay using the chromogenic substrate S-2251. For plasmin calibration curve and uPA activity results please see supplemental Figures I and II at http://atvb.ahajournals.org.
Detection of Intracellular Oxidative Stress
Intracellular oxidative stress was assayed through the oxidation of 2′, 7′-dichlorofluorescin diacetate (DCFH-DA, Sigma) using flow cytometry.21
Formation of lipid peroxides was assayed using the CHOD iodide method.22
Superoxide Anion Production
Production of superoxide anion was measured by the cytochrom C reduction assay.23
RT-PCR for Mouse PON2 and PON3, and for Human and Mouse p47phox
Total RNA extraction and RT-PCR reaction was carried out as previously described.20 Please see Supplemental Materials and Methods for details of primers and PCR protocols.
Reverse Transcriptase Quantitative PCR for Human PON2
Total RNA was extracted with Epicenter commercial kit (Tamar). cDNA was generated from 1 μg of total RNA using Thermo Scientific commercial kit (Tamar). RT products were subjected to Quantitative PCR using TaqMan Gene Expression Assays.
PON2-Associated (Lactonase) Activity
Lactonase activity was measured in cell sonicate using dihydrocumarin (DHC) as substrate.24
PON3-Associated Statinase Activity
Statinase activity was measured high-performance liquid chromatography (HPLC) in cell sonicates using lovastastin as substrate.24
Each separate experiment was performed in triplicate, and each individual experiment was replicated 3 times (n=3) to achieve statistical meaning. Statistical analyses used Student t test for comparing differences between the 2 groups, and 1-way ANOVA followed by the Student-Newman-Keuls test was used for comparing differences between multiple groups.
uPA Increases Macrophage PON2 Expression and Activity
To test whether uPA has an influence on macrophage PON2 expression, THP-1 macrophage-like cells were incubated with increasing concentrations (0 to 40 nmol/L) of uPA for 24 hours. uPA induced a dose-dependent increase in PON2 protein by up to 2.6-fold, which reached saturation already at 20 nmol/L (Figure 1A). Confocal microscopy analysis of uPA (20 nmol/L)-treated macrophages confirmed that PON2 protein abundance in cell cytoplasm, as well as in cell nucleus, markedly increased (Figure 1B through 1E). In accordance, PON2-associated lactonase activity increased by up to 3-fold in uPA-treated macrophages. This effect was uPA dose-dependent and reached saturation at 20 nmol/L (the difference between the effect of 20 and 40 nmol/L of uPA was not statistically significant; Figure 1F). Furthermore, uPA upregulated PON2 mRNA expression in a linear, dose-dependent manner (Figure 1G).
uPA Requires uPA/uPAR Interaction for Increasing Macrophage PON2 Expression
To ascertain the role of uPA enzymatic activity, and the role of its receptor, uPAR, we used alternative independent techniques.
uPA proteolytic activity, determined as plasmin generation (estimated as the increase in OD at 405 nm) in conditioned media (CM) from cells grown in presence of BSA (0.2%, control), uPA (20nmol/L), plasmin (1 μg/mL), plasminogen (1 μg/mL), or uPA with mouse antihuman uPAR blocking antibody (anti-CD87), increased in a linear manner up to 180 minutes in all CM tested (supplemental Figure III). Plasmin generation in CM from cells incubated with uPA in presence of the blocking antibody was below that of control cells, suggesting that even when uPA cannot bind to its receptor, there is still a minor quantity of plasmin in the CM. Because the calibration curve of plasmin generation using the S-2251 substrate reaches saturation after 30 minutes, as shown in supplemental Figure I, CM was then incubated with S-2251 for 30 minutes only. Supplemental Figure IV shows that uPA significantly induced plasmin generation by cells grown in serum-containing media (containing plasminogen), and to a much lower extent by cells grown in serum free-media alone, and in presence of ATF-uPA, or amiloride.
Next, we determined the role of uPA enzymatic activity, and the role of its receptor, uPAR, on uPA-mediated increase of PON2. Incubation of THP-1 cells with human uPA (20 nmol/L) induced a 2-fold increase in macrophage PON2 activity (Figure 2A) and a 2.5-fold increase in PON2 protein abundance (Figure 2B). Similar results were observed on using ATF-uPA, which lacks catalytic activity, suggesting that binding to uPAR may be sufficient to induce PON2 upregulation.
Consistent with these results, blocking the enzyme proteolytic activity by the selective inhibitor amiloride (2.5 μmol/L), did not abolish uPA-stimulation of PON2 (Figure 2). Furthermore, incubation of the cells with uPA in presence of aprotinin, a plasmin inhibitor, resulted in a substantial reduction in plasmin generation, but did not affect the stimulatory effect of uPA on PON2 expression (supplemental Table I). Taken together, these results suggest that the ability of uPA to induce PON2 upregulation is independent of its proteolytic activity.
To address the requirement of uPA interaction with uPAR, the macrophages were incubated with uPA in the presence of anti-CD87 blocking antibody. The antibody concentration used (10 μg/mL) was found in a preliminary study to maximally inhibit the binding of fluorescein isothiocyanate (FITC)-conjugated uPA to its receptor on the cells (data not shown). Incubation of macrophages with anti-CD87 alone had no effect on PON2 expression. Pretreatment of macrophages with anti-CD87 completely abolished the uPA-induced increase in PON2 activity (Figure 2A) and protein expression (Figure 2B), indicating that this process requires uPA/uPAR interaction.
To test whether receptor binding is critical, we performed a cross-species experiment, because it is well established that mouse uPA does not bind to human uPAR and human uPA does not bind to mouse uPAR.25 THP-1 cells and MPM from Balb/C mice were incubated for 18 hours with human or mouse uPA (20nmol/L), respectively. Mouse uPA did not stimulate PON2-associated lactonase activity in THP-1 cells (0.9±0.05 and 1.1±0.09 U/mg cell protein in cells incubated in absence or presence of uPA, respectively), and similarly, human uPA did not increase PON2 lactonase activity in MPM (0.22±0.04 and 0.219±0.03 U/mg cell protein in cells incubated in absence or presence of uPA, respectively).
To further consolidate uPAR functional role in PON2 upregulation by an inhibitor-independent approach, we performed additional experiments in MPM harvested from uPAR−/− mice. uPA significantly increased PON2 protein expression, lactonase activity, and mRNA expression by 1.5-, 2.0-, and 2.2-fold in MPM harvested from control C57Bl/6 mice, respectively, whereas no significant increase could be monitored in uPAR−/− mice MPM (Figure 2C, 2D, 2E). These results provide convincing evidence that uPAR is necessary for uPA upregulation of PON2.
uPA Increases Macrophage Oxidative Stress
uPA-mediated increase in macrophage PON2 lactonase activity (from 0.94±0.07 to 1.63±0.12 U/mg cell protein) was completely abolished on incubation of the cells with uPA in the presence of vitamin E (25μmol/L) or diphenyleneiodonium (DPI, 5 μmol/L; 0.93±0.08 and 0.88±0.09 U/mg cell protein, respectively), suggesting a possible role for cellular oxidative stress.
Thus, next we sought to determine whether uPA has a stimulatory effect on macrophage oxidative stress. uPA dose-dependently and significantly increased macrophage lipid peroxide content (Figure 3A) and oxidation of DCFH-DA (Figure 3B), by 3- and 2.6-fold, respectively. This effect was inhibited by pretreatment of the cells with increasing concentrations of pomegranate juice (PJ), which was previously shown to possess potent polyphenol antioxidants that protect macrophages against oxidative stress,26 as well as by DPI, an inhibitor of flavin containing oxidases, or by apocynin, which is a potent and selective inhibitor of NADPH oxidase (Figure 3C). Treatment of THP-1 cells with DPI (10μmol/L) in the presence of uPA reduced the level of oxidative stress below that of untreated cells, suggesting that the control cells are at a certain stage of activation, attributable probably to the cell culture conditions, which imposes a state of oxidative stress on the cells.27 Plasminogen activation by PJ, DPI, or apocynin, which might represent an alternative for their effects, either alone, or in the presence of uPA (20 nmol/L) was not significantly influenced (supplemental Figure IV). These results point to a possible role for superoxide anions production by NADPH oxidase. Indeed, uPA dose-dependently increased superoxide anion release from macrophages by up to 2-fold, in comparison to untreated cells (Figure 4A). Furthermore, uPA significantly increased by up to 2-fold the expression of P47phox mRNA, the cytosolic component of NADPH oxidase in macrophages (Figure 4B). Functionally, uPA increased in a dose-dependent manner the capacity of macrophages to oxidize LDL by up to 2-fold (Figure 4C).
uPA Increases Macrophage PON2 Expression and Activity Through NADPH Oxidase Activation
Next, we examined a causal relationship between the effect of uPA on NADPH oxidase and on PON2 in macrophages. For this purpose uPA was incubated with MPM harvested from p47phox−/− mice (p47−/− MPM), which lack NADPH oxidase activity because of a mutation in the cellular subunit p47phox,28 in comparison to MPM from control C57Bl/6 mice. Cellular oxidative stress, measured by DCFH oxidation (Figure 5A), and expression of p47phox mRNA (Figure 5B) were significantly increased by uPA by up to 2- and 4-fold, respectively, in control MPM, whereas no significant effect was recorded in p47−/− MPM. In parallel, uPA significantly increased PON2 activity (Figure 5C) and mRNA expression (Figure 5D) by 2.3- and 2.6-fold, respectively, in control MPM. On the contrary, uPA had no effect on PON2 expression in p47−/− MPM, thus evidencing that uPA-upregulation of PON2 is secondary to uPA-stimulation of NADPH oxidase.
Because mouse macrophages express PON3 along with PON2, we analyzed whether uPA has also an impact on PON3 expression. Statinase activity, which is specific for PON3, was not significantly affected by uPA (1217±34 and 1001±28 U/mg cell protein in cells incubated in absence or presence of uPA, respectively). Furthermore, no significant difference could be demonstrated in PON3 mRNA expression in either wt MPM, or in p47−/− MPM that were treated with uPA, in comparison to untreated cells (4.8 and 4.3 in wt MPM versus 4.8 and 4.7 in p47−/− MPM, untreated versus treated with uPA, respectively), suggesting that uPA stimulates macrophage PON2, but not PON3, expression and activity.
PON2 Protects Macrophages Against uPA-Stimulated Oxidative Stress
To test whether endogenous PON2 confer macrophages protection against uPA-stimulated oxidative stress, we used MPM from PON2 knockout mice (PON2−/− MPM), in comparison to MPM from C57Bl/6 mice (C57Bl/6 MPM). uPA induced a moderate, nonetheless a significant, increase in cellular oxidative stress in C57Bl/6 MPM (by 20%, 17%, and by 13%, as determined by DCFH oxidation, superoxide anion release, and cell capacity to oxidize LDL, respectively), and a more substantiated increase (by 2-fold, 35% and 3.3-fold higher, respectively) in PON2−/− MPM (by 43%, 23%, and 43%, respectively, Figure 6). These results suggest that endogenous PON2 protects macrophages against uPA- stimulated oxidative stress. uPA had no effect on glutathione levels (another endogenous antioxidant system) in THP-1 macrophages (19.19±1.8 and 19.24±2.1 nmol/mg cell protein in cells incubated without or with uPA, respectively).
In the present study we demonstrate for the first time that uPA increases intracellular oxidative stress in macrophages, which in turn upregulates macrophage PON2 expression. Both effects are related to uPA-induced NADPH oxidase activation. Furthermore, we show that endogenous PON2 expression confers the cells protection against oxidative stress, in accordance with previous reports,17 as macrophages from PON2-deficient mice were more susceptible to uPA-induced oxidative stress.
uPA has been suggested to play a role in the initiation and development of atherosclerosis, but the mechanisms involved remained questionable. uPA was shown to stimulate the migration and proliferation of smooth muscle cells and of macrophages, either directly through its binding to the uPA-R29,30 or indirectly through the activation of metalloproteases. Our present study extends these findings to show that uPA is atherogenic because it increases oxidative stress in macrophages. The importance of uPA as a regulator of NADPH oxidase activation and ROS production in macrophages links uPA to macrophage capacity to oxidize LDL,31 which is believed to contribute to the early atherosclerotic lesion development.32
Several approaches were taken in the present study to demonstrate the stimulatory effect of uPA on macrophage ROS production. All results led us to the conclusion that the primary species leading to the oxidative stress stimulated by uPA in macrophages was NADPH oxidase-derived superoxide anions. However, possible contribution of other cellular sources for ROS in the stimulation of macrophage oxidative stress by uPA cannot be ruled out. uPA was shown previously to prime neutrophils for superoxide anion release but could not induce NADPH oxidase activation.33 On the contrary, uPA was reported to stimulate NADPH oxidase-dependent ROS production in SMC,34 however the regulation of NADPH oxidase in phagocytic cells differ from that in nonphagocytic cells.35 Thus, we report herein for the first time on the capacity of uPA to regulate the macrophage NADPH oxidase complex. As the macrophages play a key role in foam cell formation during early atherogenesis, this information may have a relevance to macrophage activation in the arterial atherosclerotic plaque.
The results of the present study demonstrate that in response to the increased cellular oxidative stress, uPA upregulated macrophage PON2 expression.
This effect was demonstrated in THP-1 macrophage-like cell line, as well as in primary mouse peritoneal macrophages, thus reflecting a physiological importance of our results. In THP-1 cells, uPA induced a rapid saturation of PON2 protein and activity, whereas mRNA expression showed a linear relationship. This does not seem, however, to be a discrepancy, because previous reports based on a large-scale data sets showed that the correlation between mRNA and protein abundance is typically weak.36
PON3, another member of the paraoxonases gene family who exhibit lactonase activity, is also regulated by cellular oxidative status.17 However, because PON3 is not expressed in human macrophages, the uPA-induced increase in lactonase activity demonstrated in the present study cannot possibly reflect an effect on PON3. Furthermore, in mouse macrophages that express both PON2 and PON3, uPA had no effect on either PON3-associated statinase activity or on PON3 mRNA expression. These data strengthen our conclusion that the effects of uPA are only on PON2 expression.
Studies using p47phox−/− mice that lack functional NADPH oxidase complex revealed that upregulation of macrophage PON2 by uPA was caused primarily by an increase in cellular oxidative stress, which required activation of the NADPH oxidase. These results further confirm our previous findings on the regulatory effect of NADPH oxidase activation on PON2 expression.18
Information regarding the physiological functions of PON2 is very limited. An ROS reducing activity of PON2 was reported in Hela cells,15 and in human EC, SMC, and AoAF cells.16 Macrophage incubation with purified PON2 also resulted in reduced cellular oxidative stress.17 In the present study we extend these findings and report herein that endogenous PON2 confer macrophages increased resistance against uPA-induced oxidative stress. Thus, PON2 represent an additional enzyme that contributes to the vascular antioxidant defense, along with other known available mechanisms, including superoxide dismutase, catalase, and the glutathione system.37 Nevertheless, upregulation of PON2 was not sufficient to completely overcome the increase in NADPH-oxidase superoxide anion production in THP-1 cells. Future studies exploring the relation between uPA and other cellular oxidative stress response enzymes are needed.
uPA is a multifunctional molecule that serves as a proteolytic enzyme or as a signal-inducing ligand. As a result of the uPA proteolytic activity, plasmin was generated at a minimal level in the culture dishes, although the experiments were performed in serum-free media. This could be attributed to plasminogen that was probably carried along on the cell surface and was then converted to plasmin after uPA addition. However, our results demonstrated that uPA-induced increase in PON2 expression was independent of its proteolytic activity, but required binding of uPA to uPAR. This conclusion is supported by the following findings: (1) ATF-uPA, which was shown in our laboratory to lack uPA or plasmin activity, increased PON2 expression similarly to active uPA; (2) Incubation of the cells with uPA in presence of the plasmin inhibitor aprotinin, resulted in a substantial reduction in plasmin generation, but did not affect the stimulatory effect of uPA on PON2 expression; (3) Inhibition of uPA catalytic activity by the specific inhibitor amiloride, did not abolish the uPA-increase of PON2 expression; (4) Incubation of the cells with uPA in the presence of an uPAR blocking antibody, abolished the stimulatory effect of uPA, in spite of the fact that a low level of plasmin was generated; (5) The mouse uPA used in the present study, which was shown in our laboratory to be active and to completely lack plasmin activity, stimulated an increase in PON2 expression in mouse macrophages, but could not reproduce this effect in human cells, because of its inability to bind to the human uPAR; (6) uPA-mediated increase in PON2 expression could not be reproduced in uPAR−/− macrophages. Taken together, these results support our conclusion that the ability of uPA to induce PON2 upregulation is independent of its proteolytic activity and requires binding of uPA to its receptor uPAR. It might be that on binding to uPAR, uPA initiates intracellular signal transduction pathways, which are known to be activated by uPAR activation, leading to NADPH oxidase activation and subsequently PON2 upregulation.
Plasma concentration of uPA is around 20 pmol/L, and a Kd of 0.2 to 0.5 nmol/L was reported for uPA/uPAR interaction.38 However, because macrophages synthesize uPA, the local concentration of uPA found at inflammatory sites such as atherosclerotic lesions, is expected to be substantially greater than that in the circulation, thus may account for our observations at saturating uPA levels.
Our study further suggests that uPA may be acting in an autocrine fashion. Further studies will be required to determine whether upregulation of uPA synthesis by the macrophage will have a similar influence on macrophage PON2 and NADPH oxidase expression.
In conclusion, the novelty of the present study lies in the implication of uPA, beyond its role in the fibrinolytic system, in cellular processes involved in macrophage oxidative stress and foam cell formation during atherogenesis.
Sources of Funding
This study was supported by a grant from the The Israeli Ministry of Health, Chief Scientist Office, No. 5995-6.
Original received November 29, 2007; final version accepted April 13, 2008.
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