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Journal of Lipid Research, Vol. 49, 58-65, January 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Oxidized LDL attenuates apoptosis in monocytic cells by activating ERK signaling

Dmitry Namgaladze, Andreas Kollas and Bernhard Brüne¹

Faculty of Medicine, Institute of Biochemistry I, Johann Wolfgang Goethe University, 60590 Frankfurt, Germany

Published, JLR Papers in Press, September 21, 2007.

¹ To whom correspondence should be addressed. e-mail: bruene{at}zbc.kgu.de

	ABSTRACT

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Low concentrations of oxidized low density lipoprotein (OxLDL) are cytoprotective for phagocytes, although the underlying mechanisms remain unclear. We investigated signaling pathways used by OxLDL to attenuate apoptosis in monocytic cells. OxLDL at 25–50 µg/ml inhibited staurosporine-induced apoptosis in THP-1 cells and mouse peritoneal macrophages, and it was cytoprotective in human primary monocytes upon serum withdrawal. Attenuated cell demise was reversed by blocking extracellular signal-regulated kinase (ERK) signaling. Translocation of cytochrome c to the cytosol was attenuated by OxLDL, which again demanded ERK signaling. Analysis of Bcl-2 family proteins revealed phosphorylation of Bad at serine 112 as well as ERK-dependent inhibition of Mcl-1 degradation. Although the formation of reactive oxygen species (ROS) is an established signal generated by OxLDL, ROS scavengers did not interfere with cell protection by OxLDL. Thus, activation of the ERK signaling pathway by OxLDL is important to protect phagocytes from apoptosis.

Supplementary key words atherosclerosis • oxidized low density lipoprotein • reactive oxygen species • extracellular signal-regulated kinase

Abbreviations: Ac-DEVD-AMC, N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartyl (7-amino-4-methylcoumarin); BHA, 2[3]-t-butyl-4-hydroxyanisole; DCFH, 2',7'-dichlorodihydrofluorescein; DPPD, N,N'-diphenyl-4-phenylenediamine; ERK, extracellular signal-regulated kinase; OxLDL, oxidized low density lipoprotein; OxPAPC, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine; PI3K, phosphatidylinositol 3-kinase; ROS, reactive oxygen species; TBARS, thiobarbituric acid-reactive substances

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Atherosclerotic plaque progression involves dynamic changes in cellular composition. These changes comprise the migration of cells from the bloodstream into the intima, their egress into the lymph system, and the proliferation and migration of smooth muscle cells from the media. Moreover, cell death affects cell numbers in atherosclerotic plaques. Several studies have established that phagocyte cell death diminished lesion progression during early stages of atherosclerosis (1, 2), whereas an increased death of phagocytes and smooth muscle may contribute to plaque destabilization in advanced atherosclerosis (3, 4).

Modified lipoproteins are important factors in lesion development, and the oxidation of lipoproteins is a characteristic feature of the plaque (5). Oxidized low density lipoproteins (OxLDLs) affect numerous phagocyte functions, including cell survival (6). Although high concentrations of OxLDL are cytotoxic for cells of the vascular wall, cytoprotective actions of OxLDL have been reported at lower doses in phagocytes (7, 8). Recent studies suggest that cytoprotection by OxLDL depends on the degree of its oxidative modification as well as OxLDL inclusion in immune complexes (9, 10).

Mechanistically, OxLDL-evoked cytoprotection was linked to activation of the phosphatidylinositol 3-kinase (PI3K)-Akt survival pathway (7, 9–11). However, little is known regarding other potential antiapoptotic mechanisms conveyed by OxLDL. Activation of extracellular signal-regulated kinase (ERK) constitutes a major mitogenic and antiapoptotic pathway activated by a variety of extracellular agonists, such as growth factors or hormones (12). ERK targets a number of apoptotic regulators, including proteins of the Bcl-2 family (13–15), caspases (16), and inhibitor of apoptosis proteins (17). Although some data suggest that ERK inhibition reduces the viability of OxLDL-treated phagocytes (18), other studies reported ERK not to be involved (7). Therefore, the role of ERK signaling in provoking antiapoptotic actions of OxLDL remains unclear.

In this study, we explored signaling mechanisms evoked by OxLDL in protecting THP-1 monocytic cells from staurosporine-induced cell death. The antiapoptotic effect of OxLDL involved the activation of ERK, whereas PI3K/Akt activation was not involved. Inhibition of apoptosis occurred upstream of cytochrome c translocation, involved Bad phosphorylation, and impaired Mcl-1 degradation.

	MATERIALS AND METHODS

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Materials
Cell culture medium and supplements were from PAA Laboratories (Coelbe, Germany). A protein assay kit was from Bio-Rad (Munich, Germany). Protease inhibitors came from Roche Diagnostics (Mannheim, Germany). Nitrocellulose membrane, the ECL detection system, and horseradish peroxidase-labeled anti-mouse and anti-rabbit secondary antibodies were from GE Healthcare (Munich, Germany). N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartyl (7-amino-4-methylcoumarin) (Ac-DEVD-AMC), LY294002, and PD98059 were from Alexis (Lausen, Switzerland). API2 was from EMD Biosciences (Darmstadt, Germany). All other chemicals were from Sigma (Taufkirchen, Germany). Primary antibodies against phospho-Akt serine 473 (9271), total Akt (9272), phospho-ERK threonine 183/185 (9101), total ERK (9102), phospho-Bad serine 112 (9296), phospho-Bad serine 136 (9295), Bim (4582), Mcl-1 (4572), and caspase-3 (9662) were from Cell Signaling Technology (Beverly, MA). Primary antibodies against Bcl-2 (610539), Bcl-xL (610212), and cytochrome c (556433) were from BD Biosciences (Heidelberg, Germany). Dr. Herman Schaegger (Johann Wolfgang Goethe University, Frankfurt, Germany) kindly provided antibody against ATP synthase (mitochondrial complex V).

LDL isolation and treatment
Human LDL (d = 1.02–1.06 g/ml) was isolated from plasma of healthy volunteers (DRK-Blutspendedienst Baden-Württemberg-Hessen, Institut für Transfusionsmedizin und Immunhämatologie Frankfurt am Main, Frankfurt, Germany) by sequential ultracentrifugation. OxLDL was prepared by incubating LDL with 5 µM CuSO₄ at room temperature for 24 h followed by dialysis against PBS with 100 µM EDTA. Oxidation was monitored by measuring the relative electrophoretic mobility of LDL and OxLDL in agarose gels (Lipidophor; Technoclone, Vienna, Austria) as well as by spectrophotometric detection of thiobarbituric acid-reactive substances (TBARS). Our OxLDL preparations showed a relative electrophoretic mobility of 3–4 and 6–8 nmol/mg TBARS. Endotoxin content of the preparations was <1 ng/mg OxLDL (Pyrotell assay; Associates of Cape Cod, Falmouth, MA). Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) was prepared as described (19) and was kindly provided by Dr. Valery Bochkov (Medical University of Vienna, Austria).

Cell culture
THP-1 monocytic cells were maintained in RPMI 1640 medium containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated fetal calf serum. Before the experiments, the medium was changed to fetal calf serum-free medium. Human monocytes were isolated from buffy coats (DRK-Blutspendedienst Baden-Württemberg-Hessen, Institut für Transfusionsmedizin und Immunhämatologie Frankfurt am Main) using Ficoll density centrifugation (LSM 1077; PAA Laboratories) followed by magnetic separation with positive selection (CD14 MicroBeads; Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of monocytes was assessed by flow cytometry using CD14 staining. Resident mouse peritoneal macrophages were obtained from C3H/HeJ mice and cultured in RPMI 1640 medium containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated fetal calf serum. Cells were incubated overnight in the same medium without serum before treatments. This study conformed with the principles outlined in the Declaration of Helsinki.

Western blot analysis
Cells were incubated overnight in serum-free medium, treated, collected, lysed in 200 µl of buffer A (50 mM Tris, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 10 mM NaF, 1 mM Na₃VO₄, 1 mM PMSF, and protease inhibitor mixture, pH 7.5), and sonified, followed by centrifugation (15,000 g, 10 min). A total of 30–60 µg of protein was loaded onto and resolved on SDS-polyacrylamide gels. Proteins were blotted onto nitrocellulose. Membranes were blocked and incubated with primary antibodies according to the manufacturer's instructions. Membranes were washed three times for 5 min each with TTBS (10 mM Tris, 150 mM NaCl, and 0.05% Tween 20, pH 7.5). For protein detection, blots were incubated with a horseradish peroxidase-labeled secondary antibody for 1 h and washed three times for 5 min each with TTBS, followed by ECL detection.

Cell death detection
Cells were incubated overnight in serum-free medium, treated, and processed for cell death detection by flow cytometry (FACS Canto; BD Biosciences, Heidelberg, Germany) using the Annexin V-FITC apoptosis detection kit (Beckman Coulter, Krefeld, Germany) according to the manufacturer's instructions. Apoptosis of mouse peritoneal macrophages was assessed by detecting cytoplasmic histone-associated DNA fragments using the cell death ELISA kit from Roche Diagnostics, according to the manufacturer's instructions.

Caspase activity assay
Caspase-3/7 activity was quantified fluorimetrically by following the cleavage of Ac-DEVD-AMC. Cell pellets were resuspended in lysis buffer (100 mM HEPES, pH 7.5, 10% sucrose, 0.1% CHAPS, and 1 mM PMSF), sonified, and centrifuged (10,000 g, 10 min). Cytosolic protein (30 µg) was incubated with 10 µM Ac-DEVD-AMC in the presence of 10 mM DTT at 30°C. Substrate cleavage and accumulation of AMC were followed fluorimetrically with excitation at 360 nm and emission at 460 nm for 1 h.

Cytochrome c release assay
Cytochrome c translocation to the cytosol was monitored by fractionation of cell lysates according to Leist et al. (20). Cell pellets were resuspended in 100 µl of PBS. A total of 100 µl of digitonin/sucrose solution (0.16 mg/ml digitonin and 500 mM sucrose) was added for 30 s, followed by centrifugation at 10,000 g for 1 min. A total of 50 µg of cytosolic and 25 µg of mitochondrial fractions were analyzed by Western blotting for cytochrome c as well as ATP synthase versus ERK as controls for mitochondrial and cytosolic fractions, respectively.

Reactive oxygen species determination
Reactive oxygen species (ROS) generation in THP-1 cells was analyzed by flow cytometry monitoring the oxidation of 2',7'-dichlorodihydrofluorescein (DCFH). Cells were loaded with 5 µM DCFH-diacetate for 30 min, pelleted, resuspended in PBS, and treated for 1 h with OxLDL and/or antioxidants followed by flow cytometry.

Statistical analysis
Data are expressed as means ± SEM. Two treatment groups were compared by independent Student's t-tests. Results were considered statistically significant at P < 0.05.

	RESULTS

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To investigate the influence of OxLDL on apoptosis, monocytic THP-1 cells were treated with 0.2 µg/ml staurosporine for 4 h to induce the mitochondrial apoptotic signaling pathway. Cell death was followed by Annexin V/propidium iodide staining and flow cytometry. As shown in Fig. 1A , staurosporine provoked massive apoptosis and thus cells became Annexin V-positive but basically remained propidium iodide-negative, indicating that only a small fraction of cells was necrotic. Exposing cells to staurosporine in the presence of 50 µg/ml OxLDL drastically reduced the number of apoptotic cells with minor signs of necrosis. Quantification of Annexin V staining and thus apoptosis is shown in Fig. 1B. Inhibition of staurosporine-induced apoptosis was prominent already at 12.5 µg/ml OxLDL and reached maximal protection with 50 µg/ml. We noticed that at concentrations of 100 µg/ml and greater, OxLDL induced necrotic cell death (19.2 ± 2.7% Annexin V/propidium iodide double-positive cells after 4 h with 150 µg/ml OxLDL), confirming previous data on OxLDL cytotoxicity (21). Native LDL was protective as well, but 5-fold higher LDL concentrations were needed to achieve an antiapoptotic effect similar to that of OxLDL.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Oxidized low density lipoprotein (OxLDL) attenuates apoptosis in THP-1 cells. A: Cells were treated with 0.2 µg/ml staurosporine (STS) alone or in combination with 50 µg/ml OxLDL for 4 h, stained with Annexin V/propidium iodide (AV/PI), and analyzed by flow cytometry. Representative dot plot diagrams are shown. Numbers indicate the percentage of Annexin V-positive but propidium iodide-negative cells. B: Cells were treated with 0.2 µg/ml staurosporine alone or in combination with the indicated concentrations of OxLDL or LDL for 4 h. Data are presented as means ± SEM (n > 3). PE-A, phycoerythrin channel fluorescence area.

View larger version (14K): [in this window] [in a new window]   Fig. 2. OxLDL diminishes caspase activation by staurosporine. A: Cells were treated with 0.2 µg/ml staurosporine (STS) alone or in combination with the indicated concentrations of OxLDL for 4 h followed by caspase-3/7 activity determinations in cell lysates as described in Materials and Methods. B: Cells were treated with 0.2 µg/ml staurosporine alone or in combination with 25 and 50 µg/ml OxLDL for 4 or 8 h. Cell lysates were subjected to Western analysis to visualize procaspase-3 and cleaved fragments. A representative blot of at least three similar experiments is shown. Data are presented as means ± SEM.

In a first set of experiments shown in Fig. 3A , we explored the activation of ERK and Akt by OxLDL. OxLDL elicited a transient phosphorylation of both Akt and ERK. Maximal effects were seen at 2–10 min, with no changes in the expression of total Akt.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Role of extracellular signal-regulated kinase (ERK) in the antiapoptotic effects of OxLDL and oxidized phospholipids. A: Cells were treated for the indicated times with 50 µg/ml OxLDL. Cell lysates were subjected to Western analysis using antibodies as described in Materials and Methods. Representative blots of at least three similar experiments are shown. B: Cells were preincubated for 30 min with 100 nM wortmannin (Wort) or 50 µM PD98059 (PD) before incubations with 0.2 µg/ml staurosporine (STS) and/or 50 µg/ml OxLDL for 4 h. Subsequently, cells were stained with Annexin V/propidium iodide. C: Cells were preincubated for 30 min with 50 µM PD98059 before treatments with 0.2 µg/ml staurosporine and 20 µg/ml oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) for 4 h. Subsequently, cells were stained with Annexin V/propidium iodide. D: Mouse peritoneal macrophages were preincubated for 30 min with 50 µM PD98059 before treatments with 0.02 µg/ml staurosporine and 50 µg/ml OxLDL for 4 h. Subsequently, DNA fragmentation in cell lysates was analyzed by cell death ELISA. All data are presented as means ± SEM (n > 3).

To explore whether the antiapoptotic effect of OxLDL is not limited only to cell lines, we examined its effect on apoptosis in resident mouse peritoneal macrophages. Apoptosis was assessed by measuring DNA fragmentation using the cell death ELISA kit (Roche Diagnostics). As shown in Fig. 3D, staurosporine treatment of macrophages induced DNA fragmentation, which was reduced significantly in the presence 50 µg/ml OxLDL. Cytoprotection by OxLDL was reversed if cells were preincubated with PD98059. Thus, OxLDL can exert antiapoptotic effects in an ERK-dependent manner in primary macrophages. Additionally, OxLDL increased the viability of human primary monocytes cultured in serum-free medium, a condition known to induce apoptosis in monocytes. Incubations of monocytes with 50 µg/ml OxLDL for 24 h increased cell viability, as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (absorbance at 560 nm = 0.117 ± 0.007 for control and 0.224 ± 0.031 for OxLDL), and reduced caspase-3/7 activity (4,310 ± 1,120 arbitrary units for control and 960 ± 200 arbitrary units for OxLDL).

LDL oxidation generates a diversity of biologically active substances, including oxysterols, lysophospholipids, aldehydes, and oxidized fatty acids. Oxidized phospholipids such as OxPAPC are thought to be generated early in the process of LDL oxidation and represent major components of minimally modified LDL, recently shown to be cytoprotective in macrophages (9). We observed that OxPAPC reduced staurosporine-induced apoptosis of THP-1 cells and that the protection attributed to OxPAPC was partly reversed by PD98059 (Fig. 3C). In contrast, 4-hydroxynonenal or lysophosphatidylcholine were not cytoprotective under our conditions (data not shown).

Assuming that ERK plays a dominant role in conveying protection by OxLDL raised the question regarding its potential targets. Several ERK targets have been identified in regulating mitochondrial pathways of apoptosis, including proteins of the Bcl-2 family, caspases, and inhibitor of apoptosis proteins. Cytochrome c translocation from mitochondria to the cytosol constitutes a central role in the intrinsic cell death signaling cascade. Therefore, we explored whether OxLDL interfered with cell death signaling upstream or downstream of cytochrome c release. Cell fractionation followed by Western analysis of cytochrome c revealed that staurosporine induced cytochrome c release to the cytosol compared with controls, an effect antagonized by OxLDL (Fig. 4A ). The protection afforded by OxLDL with regard to cytochrome c translocation was reversed by PD98059, again suggesting the involvement of ERK signaling. At the same time, the general caspase inhibitor z-VAD-fmk did not impair the release of cytochrome c from mitochondria but blocked staurosporine-induced apoptosis (data not shown). Cytosolic fractions were controlled for equal protein loading by probing the membranes with ERK antibodies, whereas the lack of contamination with the mitochondrial fraction was confirmed using antibodies against ATP synthase (data not shown). Conclusively, OxLDL interfered with staurosporine-induced apoptosis upstream of cytochrome c release.

View larger version (11K): [in this window] [in a new window]   Fig. 4. OxLDL interferes with Mcl-1 degradation after staurosporine treatment. A: Cells were preincubated for 30 min with 50 µM PD98059 or 20 µM zVAD-fmk before treatments with 0.2 µg/ml staurosporine (STS) and/or 50 µg/ml OxLDL for 4 h. Cytochrome c release into the cytosol was followed by Western analysis. B: Cells were treated for the times indicated with 0.2 µg/ml staurosporine and 50 µg/ml OxLDL. The protein amount of Bcl-2 family members was assessed by Western analysis. C: Cells were preincubated for 30 min with 50 µM PD98059 or 20 µM zVAD-fmk before the addition of 0.2 µg/ml staurosporine and/or 50 µg/ml OxLDL for 2 h. Mcl-1 levels were analyzed by Western analysis. Representative blots of at least three similar experiments are shown.

The generation of ROS by OxLDL has been implicated in eliciting several of its biological actions, including cytotoxicity (21) and nuclear factor-B activation (22). Recently, it was suggested that ROS, particularly peroxynitrite, might be involved in OxLDL-induced ERK activation in endothelial cells (23). To determine whether ROS play a role in the inhibition of apoptosis by OxLDL, we measured DCFH oxidation to follow ROS production in OxLDL-treated THP-1 cells. Treatment of THP-1 cells with OxLDL for 1 h increased DCFH oxidation by 3-fold (Fig. 5A ). DCFH oxidation was reduced significantly in the presence of the peroxyl radical scavenger N,N'-diphenyl-4-phenylenediamine (DPPD) or the lipophylic antioxidant 2[3]-t-butyl-4-hydroxyanisole (BHA). However, neither DPPD nor BHA affected apoptosis inhibition evoked by OxLDL (Fig. 5B), ruling out the possibility that ROS are important for blocking cell death. Interestingly, the same concentrations of DPPD and BHA suppressed toxicity achieved with high OxLDL concentrations (data not shown), thus confirming previous observations showing ROS-dependent OxLDL toxicity to macrophages (21).

View larger version (8K): [in this window] [in a new window]   Fig. 5. Reactive oxygen species (ROS) do not transmit the antiapoptotic action of OxLDL. A: Cells were loaded with 5 µM 2',7'-dichlorodihydrofluorescein (DCFH)-diacetate for 30 min followed by 1 h treatments with 50 µg/ml OxLDL in the presence or absence of 0.5 µM N,N'-diphenyl-4-phenylenediamine (DPPD) or 50 µM 2[3]-t-butyl-4-hydroxyanisole (BHA), as described in Materials and Methods. DCFH oxidation was monitored by flow cytometry. B: Cells were preincubated for 15 min with 0.5 µM DPPD or 50 µM BHA, followed by a 4 h treatment with 0.2 µg/ml staurosporine (STS) and/or 50 µg/ml OxLDL. Apoptosis was determined by Annexin V/propidium iodide staining. * Q3P < 0.05 versus OxLDL (n = 4).

	DISCUSSION

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LDL modifications are considered a hallmark during the development of atherosclerosis (5). However, native LDL can also support foam cell formation (24) and activate intracellular signaling pathways (25). We noticed that both OxLDL and native LDL attenuated apoptotic cell death, although the protection by LDL was minor compared with OxLDL. This implies that oxidation confers antiapoptotic properties to LDL. The nature of the OxLDL epitopes responsible for the antiapoptotic action as well as the receptor(s) mediating this effect remain unclear. Recently, it was shown that CD36, a major receptor for OxLDL on phagocytes, transmits only part of the OxLDL signals, with the ERK pathway remaining active in CD36^–/– cells (26). Thus, other scavenger receptors, such as LOX-1, may be involved in stimulating ERK (27). Although LDL showed minor protection, the involvement of the LDL receptor can be ruled out. Treatment of LDL with proteinase K did not avert protection induced by LDL, although apoB, the component interacting with the LDL receptor, was lost during proteolytic digestion. Therefore, lipid components of LDL appear more important and lipids such as sphingolipids may contribute to protection (25). In addition, we showed that OxPAPC, an oxidized phospholipid thought to underlie the biological effects of minimally modified LDL, was cytoprotective. Our preparations resulted in LDL being less extensively oxidized compared with the mostly used copper-oxidized LDL. Therefore, oxidized phospholipids may be present in the OxLDL used by us and may be partly responsible for its cytoprotective effect.

We observed potent inhibition of staurosporine-induced phosphatidylserine exposure and caspase activation at 25–50 µg/ml OxLDL. However, at concentrations exceeding 100 µg/ml, OxLDL induced necrosis. A biphasic behavior, with protection dominating at low concentrations but a cytotoxic one at higher doses of OxLDL, is in agreement with published data (7, 8). It is possible that the suppression of apoptotic mechanisms by OxLDL contributes to necrotic cell death at higher OxLDL concentrations and might also be relevant to necrotic core formation in advanced atherosclerosis. Similar observations have been made previously in human macrophages (21), in which OxLDL elicited a caspase-independent but ROS-dependent cell death.

PI3K-Akt and ERK signaling pathways are critical for the regulation of cell survival. We noticed that in addition to antiapoptotic actions attributed to Akt in lipoprotein-induced survival (9, 10, 28), ERK can also be responsible for the cytoprotective effects of OxLDL, as shown by pharmacological inhibition experiments. Previous data and ours imply that both ERK and Akt signaling pathways may be critical for the prosurvival action of modified lipoproteins. Recently, we showed in the same THP-1 system that phospholipase A₂-modified LDL activates Akt-dependent survival signaling when cell death was induced by H₂O₂ or toxic concentrations of OxLDL (28). However, phospholipase A₂-modified LDL at concentrations inducing prolonged Akt activation had no effect on staurosporine-induced apoptosis, supporting our notion that Akt activation alone is not sufficient to block staurosporine-induced apoptosis in this system (data not shown). The involvement of ERK signaling in cell protection was recently postulated when inhibition of ERK aggravated 7-ketocholesterol-induced apoptosis (29). As 7-ketocholesterol still induced caspase activation, despite ERK being active, this setup is different from ours, which shows a more prominent impact of ERK in protection.

The release of cytochrome c from mitochondria often is considered the "point of no return" in the cell death execution program. We show that OxLDL inhibits staurosporine-induced cytochrome c release in an ERK-dependent manner. This allows us to conclude that OxLDL interferes early in the apoptotic signaling cascade, most likely involving Bcl-2 family proteins. Indeed, one of the established ERK targets, Bad, was phosphorylated at serine 112, which constitutes the site of phosphorylation by ERK. Additionally, Mcl-1 degradation after staurosporine treatment was reduced by OxLDL, again with ERK inhibition reversing this effect. Mcl-1 is critical for the survival of myeloid cells (30), and interfering with its degradation appears to be an important antiapoptotic signal. Interestingly, BimEL, another ERK target within the Bcl-2 family (13), was not affected, suggesting some specificity in the OxLDL/ERK protective system.

Intracellular ROS generated by OxLDL may participate in initiating various signaling events (31). Along that line, the activation of ERK by OxLDL in endothelial cells involved peroxynitrite-dependent Ras glutathionylation (23). Similarly, lysophosphatidylcholine, an OxLDL constituent, induced ERK activation in endothelial cells, which was dependent on mitochondrial ROS formation (32). Although we observed increased ROS generation by OxLDL that was antioxidant-sensitive, the same inhibitors failed to reverse protection by OxLDL. These results rule out ROS as participating messengers during the antiapoptotic action of OxLDL.

Together, our data provide evidence for the existence of an ERK-dependent antiapoptotic signaling pathway induced by OxLDL in phagocytes. The critical role of ERK in OxLDL-elicited protection suggests the possibility that blocking this pathway by pharmacological interference would enhance phagocyte apoptosis in the lesion, thus reducing cell numbers and disease progression.

	ACKNOWLEDGMENTS

 
The authors thank Sabine Knaus and Franz-Josef Streb for technical assistance and Dr. Valery Bochkov (Medical University of Vienna) for providing OxPAPC. This study was supported by Grant BR999 from the Deutsche Forschungsgemeinschaft.

Manuscript received February 27, 2007 and in revised form June 8, 2007 and in re-revised form September 18, 2007.

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