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J. Biol. Chem., Vol. 281, Issue 38, 27991-28001, September 22, 2006
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1
From the
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106 and the Department of Cell Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195
Received for publication, May 25, 2006 , and in revised form, July 13, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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We have recently examined changes in protein and mRNA expression that accompany foam cell formation and, among other effects, observed an up-regulated antioxidant response (13). A unique component of our observations was the up-regulation of members of the peroxiredoxin family, a set of ubiquitously expressed peroxidases (13). The mammalian peroxiredoxin family consists of six proteins (Prx I–VI) expressed as unique gene products, with the capability to reduce hydrogen peroxide, lipid hydroperoxides, and peroxynitrite (14–17). Prx I–IV are categorized as typical 2-Cys peroxiredoxins, distinct from the atypical 2-Cys (Prx V) and 1-Cys peroxiredoxin (Prx VI). In humans, Prx I–IV share >65% protein sequence homology, including the two conserved cysteines responsible for peroxide reduction. These 2-Cys peroxiredoxins function as homodimers, in which one conserved cysteine (Cys51 in Prx I) forms an intermolecular disulfide with the second conserved cysteine (Cys172 in Prx I) upon reduction of a substrate. The disulfide is reduced by the electron donor thioredoxin, reactivating peroxiredoxin antioxidant capability. At elevated oxidant levels, Prx I–IV can be inactivated through the overoxidation of the active site cysteine to sulfinic acid (Cys-SO2) or sulfonic acid (Cys-SO3) (18, 19). The Cys-SO2 modification is reversed by sulfiredoxin in an ATP-dependent reductive mechanism (20, 21). The significance of this reversible inactivation as a means to regulate intracellular peroxide levels has generated interest in peroxiredoxin as a mediator of H2O2-regulated signaling pathways (22–24).
The findings presented in this report focus on Prx I, a basic (pI 8.3) 22-kDa protein localized to the cytosol. Prx I is up-regulated in a variety of cell types following exposure to oxidative stress. Up-regulation of Prx I in response to H2O2 was first demonstrated in mouse peritoneal macrophages (25) and in cultured vascular smooth muscle cells following exposure to oxLDL (26). Further studies characterized the up-regulation of Prx I resulting from exposure to ionizing radiation (27, 28), hyperoxia (29), and 4-hydroxy-2-nonenal (30). Additionally, elevated levels of Prx I have been discovered in several types of cancer, including lung (31, 32), breast (33), and pancreatic cancer tissues (34).
The induction of the peroxiredoxins is generally related to a cellular antioxidant response. However, recent findings suggest alternative functionality for the peroxiredoxin family, including the ability to modulate various signaling pathways. Prx II, for example, is believed to regulate peroxide levels that would otherwise inhibit phosphatases, such as PTEN (24). PTEN converts the signaling molecule phosphatidylinositol 3,4,5-trisphosphate to phosphatidylinositol bisphosphate, inhibiting the downstream activation of Akt kinase. Thus, increased Prx II activity is able to protect and support increased PTEN activity and prevent the downstream phosphorylation of Akt targets. Additionally, Prx II can regulate H2O2 levels produced in response to tumor necrosis factor-
and thus limit the activation of the c-Jun N-terminal kinase and p38 MAPKs in response to increased peroxide levels (35). One report invoking a similar role for Prx I in intracellular signaling has also been made. In those experiments, the Schizosaccharomyces pombe 2-Cys peroxiredoxin Tpx1, the yeast homolog for mammalian Prx I, was shown to regulate the peroxide-induced activation of StyI, the yeast homolog for mammalian p38 (36). The regulation occurs through a direct association of Tpx1 with StyI, forming an intermolecular disulfide that protects StyI from oxidant-induced inactivation (36). A parallel role for mammalian Prx I in p38 regulation in macrophages would be significant, considering the downstream effects of p38 activation (37, 38).
This report characterizes the functional significance of Prx I and its induction in macrophage foam cells. The oxLDL-induced formation of foam cells from macrophages is shown to result in the up-regulation of active, rather than oxidatively inactive, Prx I. By applying methods to either induce or inhibit Prx I protein expression, our data show that Prx I up-regulation prior to foam cell formation promotes cell survival and that the protective effect of Prx I corresponds directly to its ability to reduce reactive oxygen species. Additionally, the role of Prx I as a modulator of p38 activity is established by demonstrating the dependence of p38 phosphorylation on Prx I expression. Our results are consistent with a dual role for Prx I, in which 1) induced expression can provide antioxidant functionality by reducing cytotoxic levels of ROS, and 2) the extent of Prx I expression can govern the level of p38 MAPK activation.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Lipoprotein Preparation—Fresh human plasma was obtained from the Cleveland Clinic Blood Bank, and the LDL was isolated by differential ultracentrifugation (1.019 < d < 1.063 g/ml) (39). This LDL preparation, in NaBr solution containing 0.02% EDTA, was dialyzed against 0.9% NaCl, 0.02% NaN3, 0.02% EDTA (pH 7.4) and stored in the dark at 4 °C (40). Oxidatively modified LDL was prepared as previously described (13). Oxidative modification was assayed with the trinitrobenzene sulfonate assay of free amines, which typically resulted in 40% modified amines compared with 3% modified amines when LDL was dialyzed in TBS only.
Cell Culture Conditions—J774A.1 murine macrophages were obtained from the American Type Culture Collection (ATCC TIB 67) and maintained in culture medium (Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin) at 37 °C in 5% CO2. Cells were split every 2–3 days when 
80% confluence was reached and discarded after 20 passages. siRNA Inhibition of Prx I—Approximately 5 x 105 cells were plated in 60-mm tissue culture-treated plates and left to adhere overnight at 37 °C in 5% CO2 in normal culture media. For each 60-mm plate, 13 µl of siPORT Amine Transfection Reagent (Ambion Inc., Austin, TX) was incubated with 200 µl of Opti-MEM reduced serum media for 10 min. Annealed double-stranded siRNA (20 µM stock) was added to a separate aliquot of 200 µl of Opti-MEM to give a final siRNA concentration of 30 nM. The transfection agent and the siRNA mixtures were combined and incubated for 10 min at room temperature. Media in the 60 mm culture plates was changed to 4 ml of Dulbecco's modified Eagle's media plus 10% fetal calf serum only (no antibiotics), and the siRNA-transfection agent complexes were added dropwise. Transfection conditions were maintained for 16–24 h. The Prx I siRNA sequence, obtained from the online Ambion siRNA library (ID number 68674), targeted exon 4 and was as follows: sense, GGAUUAUGGAGUCUUAAAGtt; antisense, CUUUAAGACUCCAUAAUCCtg. Scrambled and reverse siRNA sequences were designed and used as negative controls. All siRNAs were obtained in lyophilized, annealed form, resuspended in diethylpyrocarbonate double-distilled H2O to a stock concentration of 20 µM, and stored at –30 °C in 100-µl aliquots.
Cell Toxicity Assay—Approximately 2 x 104 cells/well were seeded into 24-well tissue culture-treated plates and incubated overnight at 37 °C in 5% CO2. For oxLDL-induced toxicity, cells were treated for up to 48 h with 10–100 µg/ml oxLDL. For tert-butyl hydroperoxide-induced toxicity, cells were treated with 10–200 µM tert-butyl hydroperoxide in Dulbecco's modified Eagle's medium for 2 h, followed by a 48-h incubation in normal culture media. Cell survival was assayed with the Cell-Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI) as previously described (13). Results were calculated from an average of three independent treatments, and experimental error is expressed as ±1 S.D.
Flow Cytometric Assay of Reactive Oxygen Species—ROS were assayed using a Guava EasyCyte Flow Cytometer and the ROS-detecting agent CM-H2DCFDA. Lyophilized CM-H2DCFDA (50 µg) was suspended in Me2SO to a stock concentration of 1 mM and added to prewarmed Hanks' balanced salt solution without phenol red (HBSS) to a working concentration of 10 µM. Cells were washed with HBSS, covered with the CM-H2DCFDA/HBSS solution (typically 1.5 ml/well in a 6-well plate), and incubated at 37 °C in 5% CO2 for 10 min. Cells not exposed to CM-H2DCFDA were included to assay the autofluorescent background initiated by experimental conditions. Following the CM-H2DCFDA incubation, the medium was removed, and cells were washed with HBSS and then harvested by scraping in 1 ml of HBSS, centrifuged at 1000 rpm in an Eppendorf Microfuge, and washed once with phosphate-buffered saline. Cells were then fixed in 3.7% formalin for 15 min at room temperature. The cells were pelleted by centrifugation, washed once with phosphate-buffered saline, and resuspended in 500 µl of phosphate-buffered saline for further analysis. For flow cytometric analysis, 5000 events were counted within the gate parameters for each sample. Acquisition parameters were as follows: forward gain, 2x; side scatter, 330 V; forward scatter threshold, 215 V; green channel, 552 V; red channel, 524 V; yellow channel, 533 V. Gate parameters were set as follows: X1 = 251, X2 = 2154, Y1 = 3857, and Y2 = 574. A 5000-count acquisition required 20 µl of sample and 30 s at a flow rate of 0.59 µl/s. Background fluorescence measured in cells without CM-H2DCFDA was subtracted from values obtained for the respective CM-H2DCFDA-treated samples. This background was most significant when assaying oxLDL-treated macrophages and ranged from 10 to 30% of the total measured fluorescence. The background for all other treatments was less than 10%. Results are normalized with respect to ROS measured in untreated control cells and expressed as relative n-fold change in ROS. Results were calculated from an average of three independent treatments, and experimental error is expressed as ±1 S.D.
Two-dimensional Western Blot Analysis of Oxidized Prx I—Approximately 106 cells were seeded onto 100-mm tissue culture-treated plates and incubated overnight. Cells were treated with oxLDL, tert-butyl hydroperoxide, or an equivalent volume of buffer (20 mM Tris-buffered saline, pH 7.8), incubated for a given time period, harvested by scraping in cold Dulbecco's modified Eagle's medium, and washed once in sterile phosphate-buffered saline. Cell pellets were boiled in SDS-lysis buffer (25 mM Tris, pH 7.5, 2.5 mM MgCl2, 0.5% SDS) for 5 min, cooled to room temperature, and treated with 50 µg/ml DNase I and RNase A for 15 min. The protein concentration was determined using a SDS-compatible method (Bio-Rad DC protein assay kit), and 250 µg of total protein was precipitated in acetone (80%, v/v) overnight. The dried protein precipitate was prepared and analyzed by two-dimensional SDS-PAGE as previously described (13). The two-dimensional gel was immediately incubated in Novex Tris-glycine transfer buffer plus 10% methanol for 10 min. The gel was transferred to polyvinylidene difluoride in a Bio-Rad Criterion Gel Transfer Chamber for 1 h at 100 V, blocked in 2% nonfat dry milk, and incubated overnight at 5 °C in Prx I rabbit polyclonal antibody (1:2000). Western blots were completed with a 1-h incubation in horseradish peroxidase-conjugated donkey anti-rabbit secondary antibody, followed by enhanced ECL chemiluminescent detection. The membranes were stripped and reprobed with Prx I-SO2/-SO3 rabbit polyclonal antibody (1:1000). All samples were analyzed in parallel by one-dimensional Western blot for GAPDH to confirm equal loading.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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After 24 h of oxLDL treatment, the active form of Prx I is up-regulated by oxLDL treatment (Fig. 3C) with respect to the untreated control (Ctl) macrophages (Fig. 3A). In both the t-BOOH and oxLDL treatments, the center bands representing inactive, SO2-modified Prx I were increased, consistent with the ability of both treatments to modify Prx I. These two-dimensional data, combined with the data shown in Fig. 2, clearly demonstrate that the oxLDL-induced up-regulation of Prx I produces a significant increase in the active, unmodified form of the protein.
Induction and Inhibition of Prx I Protein Expression—In order to characterize the role of Prx I in foam cell survival, methods were developed to induce and inhibit Prx I protein expression. Prx I induction was achieved by treatment with ethoxyquin, a small molecule capable of inducing the expression of phase II detoxifying proteins (42). Fig. 4 shows a series of Western blots used to determine changes in Prx I expression that occur with different combinations of ethoxyquin and oxLDL treatment. Ethoxyquin induces Prx I protein expression in a dose-dependent manner (Fig. 4A). Fig. 4B demonstrates the combined effect of ethoxyquin pretreatment for 24 h, followed by oxLDL treatment for 24 h. In macrophages treated with 25 µg/ml oxLDL, ethoxyquin pretreatment gave an increase in Prx I expression compared with oxLDL treatment alone. When treated with 50 µg/ml oxLDL, however, the extent of Prx I up-regulation is similar in macrophages with and without ethoxyquin pretreatment. Thus, ethoxyquin may enhance oxLDL-induced expression of Prx I under certain conditions, but this increase can be saturated at higher concentrations of oxLDL.
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For these cytotoxicity experiments, four groups of macrophages were tested, including a control group and three groups with altered Prx I expression; the results are shown in Fig. 6. The Ctl macrophages were transfected with a reverse sequence siRNA that had no effect on Prx I expression. Prx I expression was knocked down with a 24-h transfection with Prx I siRNA (Prx I-KD) or increased with a 24 h treatment with ethoxyquin (+Ethx). Finally, the Prx I component of the ethoxyquin effect was tested with a combination of ethoxyquin treatment and Prx I siRNA transfection (Prx I-KD, +Ethx). The four groups of cells were then exposed to increasing concentrations of t-BOOH, and cell survival was determined (Fig. 6A). Knockdown of Prx I expression in the Prx I-KD macrophages resulted in a significant decrease in cell survival following t-BOOH treatment compared with control macrophages, consistent with a sensitizing effect in the Prx I knockdown. In contrast, Prx I induction by the ethoxyquin pretreatment was protective and increased cell survival following t-BOOH exposure. Finally, the protective effect produced by the ethoxyquin treatment was partially reduced when combined with Prx I expression knockdown (the Prx I-KD, +Ethx treatment). These data demonstrate that Prx I contributes significantly to the ethoxyquin-induced cytoprotective response.
The pattern of cytotoxicity observed with oxLDL treatments showed an intriguing difference (Fig. 6B). The protective effect of the ethoxyquin treatment (+Ethx versus Ctl) was still observed with oxLDL-induced toxicity, and a component of this protection was reversed by the Prx I knockdown (Prx I-KD, +Ethx versus Ctl), consistent with the effects seen in the t-BOOH exposure. Thus, the ethoxyquin-induced up-regulation of Prx I prior to the treatment with oxLDL or the oxLDL-component model t-BOOH decreases sensitivity to oxidant-induced cytotoxicity. In contrast to t-BOOH-exposure, however, no difference was seen in cell survival for the Prx I knockdown relative to the control macrophages (Prx I-KD versus Ctl). Therefore, these data show that whereas the up-regulation of Prx I that occurs during macrophage foam cell formation can protect against selected oxidants, the nonstimulated level of Prx I in macrophages does not have measurable effects on oxLDL-induced toxicity.
As an antioxidant, the cytoprotective effect of Prx I could be associated with its ability to reduce ROS. To test this function, t-BOOH- and oxLDL-induced ROS levels were assayed using the fluorescent ROS indicator CM-H2DCFDA to determine if changes in intracellular ROS correspond to the changes in oxidant-induced toxicity seen with Prx I induction and inhibition. These data are shown in Fig. 7. The control macrophages were transfected with reverse sequence siRNA (Ctl). Treatment with either t-BOOH or oxLDL gave corresponding dose-dependent increases in the relative ROS measurements. In Prx I-KD macrophages, a significant increase in ROS production was observed with both oxidants. Conversely, Prx I induction with 50 µM ethoxyquin (+Ethx) gave a significant decrease in ROS production, and a component of the reduction was reversed when combined with Prx I knockdown (Prx I-KD, +Ethx). As a group, these data show that changes in ROS detection correspond directly to Prx I knockdown or induction, including the demonstration that Prx I contributes to the reduction in ROS observed with ethoxyquin treatment.
Prx I Regulation of p38 MAPK Activation—Prx I was tested for its ability to regulate the activation of p38 MAPK stimulated by various methods. Control and Prx I-KD macrophages were exposed to 100 µM H2O2 for up to 2 h and analyzed by Western blot to determine expression of Prx I and phosphorylated and total p38 MAPK (Fig. 8A). In control macrophages, phosphorylation of p38 MAPK increased over time as total Prx I and p38 MAPK remained constant. In Prx I-KD macrophages, phosphorylation of p38 MAPK was negligible with respect to control macrophages until 2 h after H2O2 exposure. At 2 h, phosphorylation of p38 MAPK was still significantly decreased in the Prx I-KD macrophages compared with controls. These data are the first confirmation that the mammalian Prx I expression directly regulates p38 MAPK activation as seen with the yeast homologs Tpx1 and StyI (36).
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The observed increase in p38 MAPK activation with Prx I induction raises the possibility that this activation is responsible for the increase in antioxidant activity, rather than the Prx I induction, either directly or by downstream effects. To test this possibility, the ability of Prx I to decrease ROS levels was examined under conditions where Prx I was induced but p38 MAPK activity was inhibited (Fig. 9). Macrophages were treated with 50 µM ethoxyquin to induce Prx I or left untreated for 24 h. The compound SB-203580 was used to inhibit p38 MAPK, and the samples were exposed to 200 µM t-BOOH for 2 h and assayed for ROS levels. Ethoxyquin-exposed macrophages, which express increased levels of Prx I, showed the expected decrease in ROS compared with the untreated controls, consistent with the antioxidant activity of the induced Prx I, but the p38 MAPK inhibition did not alter this effect. Therefore, the decrease in ROS levels that results from Prx I induction (Figs. 7 and 9) is not dependent on p38 MAPK activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Whereas the oxLDL-induced increase in Prx I expression is quantitatively significant, the data do not indicate whether Prx I is in its active form. Therefore, an additional step in the evaluation of expression was required to demonstrate that the redox state of Prx I was consistent with the fully functional form. The reducing function of the 2-Cys peroxiredoxins, including Prx I, is a two-step process that includes a sulfenic acid intermediate state (Cys-SOH) en route to an intermolecular disulfide across its homodimer structure. The disulfide is reduced by thioredoxin, returning functionality to peroxiredoxin. Recent attention has focused on the reversible inactivation of Prx I–IV caused by overoxidation of the active site cysteine. In a mechanism proposed by Rhee and co-workers (18, 19), the sulfenic acid intermediate can undergo further oxidation to a sulfinic (Cys-SO2H) or sulfonic acid (Cys-SO3H) that cannot be reduced by thioredoxin. This overoxidation step is a particular concern for the oxidant-induced up-regulation of peroxiredoxin, because the conditions that lead to increased peroxiredoxin expression can also oxidatively inactivate newly expressed peroxiredoxin. The higher oxidation state is not detectable in one-dimensional SDS-PAGE analyzed by Western blot, because both the active and inactive forms of peroxiredoxin run at the same position in the gel. The modification is, however, readily apparent by the acidic shift on a two-dimensional Western blot of Prx I and confirmed when the Western blot is reprobed using an antibody specific to the Cys-SO2 and Cys-SO3 species (oxPrx I antibody). It should be noted that the oxPrx I antibody identifies the oxidized species of all typical 2-Cys peroxiredoxins (Prx I–IV). Because Prx I and Prx II are comparable in molecular weight, the use of this antibody on a one-dimensional gel is not sufficient to specifically identify inactive Prx I. However, due to a significant difference in isoelectric point, Prx I and Prx II run at distinct locations on a two-dimensional SDS-polyacrylamide gel. Therefore, the conditions of this experiment are uniquely suited for identifying active versus oxidatively inactive Prx I.
Whereas past studies have applied t-BOOH (53) or H2O2 (18) as an oxidative stress, there is currently no evidence for the oxidative inactivation of Prx I in macrophage foam cells due to an oxLDL treatment. The presence of active, up-regulated Prx I was detected by the combination of two-dimensional SDS-PAGE with Western blot analysis described above. Both untreated and oxidant-treated macrophages contain detectable amounts of modified Prx I, implying that Prx I may be modified by endogenous oxidants under normal culture conditions. The increased abundance of Cys-SO2 bands detected with both the oxLDL and t-BOOH treatments shows that oxLDL is able to oxidize Prx I in a manner that is comparable with t-BOOH (54). More importantly, however, these two-dimensional experiments also show that the increase in this modification is small and confirm that the oxLDL-induced up-regulation of active Prx I produces a substantial increase in the active form.
The next stage of this investigation sought to determine the functional significance of Prx I-up-regulation by investigating the effects of Prx I induction on foam cell survival under conditions of t-BOOH- and oxLDL-induced cytotoxicity. These experiments were based on the protective effects of the peroxiredoxins, including Prx I, described in other cells treated with other types of cytotoxins, including oxidants (27, 55–57). Our data show that inducing the expression of Prx I with ethoxyquin increased cell survival for both t-BOOH- and oxLDL-induced toxicity. In each case, the increased survival was attenuated by the Prx I siRNA transfection, thus confirming a significant role of Prx I induction in the enhanced survival.
It was notable, however, that the direct knockdown of Prx I sensitized the cells to t-BOOH-induced toxicity but had no effect on the oxLDL-induced toxicity. There are at least two possible explanations for this difference. First, the sensitization to t-BOOH-induced toxicity by Prx I knockdown may be related to the relatively uniform nature of this oxidative stress. Lipid peroxidation products, modeled by t-BOOH, are prototypical substrates for peroxiredoxin. Therefore, the decrease in Prx I activity would directly diminish the metabolism of the t-BOOH and give a corresponding reduction in cell survival. Oxidatively modified LDL, however, is a heterogeneous mixture of oxidants and oxidation by-products, including lipid hydroperoxides, aldehydes, and oxysterols (58–60). Prx I may aid in the reduction of lipid peroxidation products generated by or contained in oxLDL, but other toxic components of oxLDL would remain unaffected. Second, a number of other effective antioxidants and antioxidant enzymes are present in the untreated cells, giving an element of redundancy to the antioxidant system. In this case, the decreased antioxidant activity caused by Prx I knockdown would not be a significant loss to the overall antioxidant capability of the cell and would not produce a measurable change in the cytotoxicity of oxLDL. However, the increase in Prx I expression induced prior to the oxidant exposure appears to give a significant increase to the total anti-oxidant capacity, which translates to an increase in cell survival. This positive effect of preinduced factors is a common theme in cytoprotection (i.e. the induction of cellular stress response results in increased protection during a subsequent cellular stress) (13, 61–63).
Parallel experiments examined the ability of the differentially expressed Prx I to affect intracellular ROS. Inhibiting Prx I expression resulted in increased ROS in both t-BOOH and oxLDL-treated macrophages compared with controls. Conversely, increasing Prx I expression with ethoxyquin prior to oxidant exposure decreased the ROS levels relative to control macrophages that were not pretreated with ethoxyquin. When the cells were treated with Prx I siRNA prior to ethoxyquin pretreatment, ROS levels were increased to the levels seen in the untreated controls.
A few details of the ROS detection, which influence the interpretation of these results, deserve attention. First, not all oxidants may target CM-H2DCFDA for oxidation with equal affinity. Whereas the oxidation of dichlorofluorescin analogs by H2O2 is well characterized (64–66), some data suggest that singlet oxygen does not oxidize dichlorofluorescin to the fluorescent moiety, dichlorofluorescein (67). However, whereas some oxidants may not target dichlorofluorescin analogs for oxidation, they can still produce downstream oxidants for which dichlorofluorescin is a substrate. A second important consideration involves the presence of endogenously produced ROS in addition to the exogenous oxidants introduced by t-BOOH and oxLDL (68–70). A sublethal oxLDL exposure induces NADPH-oxidase to produce endogenous H2O2 (71), which has gained attention for its role in regulating signal transduction. As a result, the ROS assayed in these experiments include those introduced exogenously by t-BOOH and oxLDL and those induced endogenously as a response to cellular stress. With these considerations in mind, there remains a direct correlation between the induction of Prx I, the generation of ROS, and the resulting cytotoxicity. These data show that the induction of Prx I prior to foam cell formation provides a protective role through its ability to reduce ROS levels generated during oxLDL uptake. These results provide a mechanistic link between the cytoprotective effects of Prx I induction and the enzymatic activity of Prx I to metabolize, and thereby decrease, the amounts of intracellular ROS. In both cases, pretreating the cells with ethoxyquin results in decreased oxidant-induced cytotoxicity and a corresponding decrease in oxidant-induced ROS. When the induction of Prx I is eliminated from the effect of ethoxyquin treatment, both toxicity and ROS levels increase.
Overall, these data describe an oxidant-protective role for Prx I in macrophage-derived foam cells when the induction of Prx I occurs prior to oxLDL exposure. Such a situation may occur if macrophages endure chronic exposure to sublethal levels of oxLDL prior to foam cell formation, thus leading to enhanced foam cell survival (69, 70). Although several roles have been attributed to Prx I, this antioxidant functionality was the key to its discovery and remains the most commonly associated role (72, 73). At the same time, however, the lack of a direct effect on oxLDL-induced toxicity in the Prx I-KD macrophages is also consistent with other activities for Prx I, beyond the toxicity-protective antioxidant effects.
Therefore, a subsequent series of experiments were carried out to look at the role of Prx I in oxidant-dependent signaling systems. Since the initial report describing the ability of Prx I to associate with and inhibit c-Abl kinase activity (74), the interest in the peroxiredoxins as signal regulators has steadily increased. Prx I has been described as a tumor suppressor due to its ability to associate with a region of the c-Myc regulatory domain and inhibit c-Myc-mediated transformation (75). However, most of the data describing signal regulation by peroxiredoxin has focused on a different cytosolic peroxiredoxin, Prx II. A role for Prx II as an endogenous ROS scavenger that prevents the oxidant-induced deactivation of PTEN phosphatase has been reported (24). By positively regulating PTEN function, Prx II prevents the downstream activation of prosurvival Akt kinase targets. Both PTEN and Prx II, by association, are described as tumor suppressors for this regulatory role. Additionally, Prx II has been shown to reduce endogenous H2O2 produced in response to tumor necrosis factor- signaling and thus limit the activation of the c-Jun N-terminal kinase and p38 pathways while stimulating extracellular signal-regulated kinase activation (35). Regulation of the p38 MAPK pathway is directly linked to proatherogenic factors. Exposing cells to oxLDL has been shown to activate the p38 MAPK pathway and lead to oxLDL-induced toxicity (37). Additionally, oxLDL-induced p38 MAPK activation has been linked to the downstream release of the proinflammatory cytokines tumor necrosis factor- and IL-1
(76).
Recently, it was shown that Tpx1, the yeast homolog of Prx I, protects StyI, the yeast homolog of p38 MAPK, from oxidative inactivation (36), but a similar role for Prx I has not been presented in mammalian cells. Further, oxidative conditions that up-regulate Prx I have not been studied to determine the effect of differential Prx I expression on p38 MAPK activation. Therefore, p38 MAPK activation was assayed to determine if the up-regulation of Prx I during macrophage foam cell formation could regulate p38 MAPK activation.
The initial use of H2O2 to induce activation of p38 MAPK was based on the previous results from yeast (36). In our experiments, the reduced expression of Prx I by siRNA knockdown coincided with a decrease in H2O2-induced p38 MAPK phosphorylation. Conversely, ethoxyquin induction of Prx I coincided with an increase in p38 MAPK activation that was nearly eliminated by siRNA knockdown of Prx I expression. Finally, the Prx I-regulated activation of p38 MAPK was studied under the conditions of macrophage foam cell formation. In these experiments, exposure to oxLDL resulted in both the dose-dependent up-regulation of Prx I and the activation of p38 MAPK. Prx I siRNA decreased the oxLDL-induced up-regulation of Prx I and led to a highly attenuated, but dose-dependent, activation of p38 MAPK. When treated with 50 µg/ml oxLDL, activation of p38 MAPK was greater in control macrophages compared with Prx I-KD macrophages. However, the decrease in Prx I expression from control to Prx I-KD macrophages was much larger than the corresponding decrease in p38 MAPK activation. This observation contrasts with the ethoxyquin-induced activation of p38 MAPK, where negligible activation was observed in Prx I-KD macrophages. These data suggest that other factors may be induced during foam cell formation that contribute to p38 MAPK activation and are not induced by ethoxyquin treatment. A potential mechanism may exist where partial p38 MAPK activation occurs when Prx I expression is limited. As a group, however, these data demonstrate that p38 MAPK activation is largely dependent on Prx I.
Activation of p38 MAPK has been linked to the downstream initiation of Prx I gene expression in macrophages (77), as well as the posttranscriptional regulation of Prx I expression in osteoblasts (78). Accordingly, a feed-forward mechanism may exist in which Prx I maintains p38 activity, resulting in a continued increase in Prx I expression and a sustained p38 MAPK response. One could speculate that the effect of this response would be directed toward the initiation of apoptosis, considering the several proapoptotic transcription factors that are targets of p38 MAPK phosphorylation. The observation that the prosurvival Akt pathway was not activated following treatment with ethoxyquin or oxLDL and that the inhibition of Prx I had no effect on Akt activation would support this scenario.
In summary, our data support a functional model in which Prx I has at least two distinct roles: as an antioxidant enzyme and as a regulator of p38 MAPK. The antioxidant functionality of Prx I was demonstrated in this work to be cytoprotective and not dependent on the p38 MAPK activation. However, the anti-oxidant activity of Prx I may not be as much prosurvival as it is anti-necrosis. One could speculate that the antioxidant role of Prx I helps to maintain macrophage foam cells in the atherosclerotic lesion by preventing an undesirable necrotic cell death. At the same time, Prx I also maintains the activation of p38 MAPK, providing the option for the downstream initiation of apoptosis. This concept overlaps with observations made for cancerous cells in tumors, where Prx I up-regulation is well documented. In both cancerous cells and foam cells, the cells have transformed to a pathological state, where an apoptotic death may be a more beneficial result. Prolonged tumor cell survival leads to tumor growth and metastasis. Similarly, prolonged foam cell survival is proatherogenic by promoting a continued inflammatory response with its downstream effects. Further study of Prx I expression and function will continue to resolve the roles of this enzyme in these downstream effects.
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FOOTNOTES |
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1 To whom correspondence should be addressed: Dept. of Cell Biology NC10, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-7170; Fax: 216-444-9404; E-mail: kinterm{at}ccf.org.
2 The abbreviations used are: oxLDL, oxidized low density lipoprotein; LDL, low density lipoprotein; Prx, peroxiredoxin(s); oxPrx, oxidized Prx; siRNA, small interfering RNA; ROS, reactive oxygen species; CM-H2DCFDA, 5- (and 6-) chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester; MAPK, mitogen-activated protein kinase; t-BOOH, tert-butyl hydroperoxide; PTEN, phosphatase and tensin homolog; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; +Ethx, 24-h treatment with 50 µM ethoxyquin; HBSS, Hanks' balanced salt solution; Ctl, control.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, Ctl, macrophages transfected with reverse sequence siRNA; 
, Prx I-KD, macrophages transfected with Prx I siRNA for 24 h; 
, +Ethx, macrophages transfected with reverse sequence siRNA and then treated with 50 µM ethoxyquin for 24 h prior to oxidant exposure; 
, Prx I-KD, +Ethx, macrophages transfected with Prx I siRNA for 24 h and then exposed to 50 µM ethoxyquin for 24 h. Results represent an average of three independent treatments. Error bars, ±1 S.D.
