Skip Navigation

Cardiovascular Research 2006 71(3):574-585; doi:10.1016/j.cardiores.2006.05.023
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Köhl, R.
Right arrow Articles by Brüne, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Köhl, R.
Right arrow Articles by Brüne, B.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2006, European Society of Cardiology

Oxidized low-density lipoprotein depletes PKC{alpha} and attenuates reactive oxygen species formation in monocytes/macrophages

Roman Köhl, Stefan Preiß, Andreas von Knethen and Bernhard Brüne*

Institute of Biochemistry I, Faculty of Medicine, Johann Wolfgang Goethe-University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany

* Corresponding author. Tel.: +49 69 6301 7424; fax: +49 69 6301 4203. Email address: bruene{at}zbc.kgu.de

Received 8 March 2006; revised 3 May 2006; accepted 23 May 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Preexposure of macrophages to oxidized low-density lipoprotein (oxLDL) attenuates formation of reactive oxygen species (ROS) upon stimulation with phorbol 12-myristate 13-acetate (PMA) or acute exposure to oxLDL. We examined whether attenuation of the oxidative burst is attributed to down-regulation of protein kinase C {alpha} (PKC{alpha}).

Methods and results Acute exposure of a mouse macrophage cell line (RAW 264.7) to both PMA and oxLDL provoked ROS generation that was blocked by the PKC{alpha}/β1 inhibitor Go 6967. However, in RAW 264.7 macrophages preincubated with oxLDL, ROS formation in response to stimulation with oxLDL or PMA was reduced. Attenuated ROS production correlated with down-regulation of the amount of PKC{alpha} protein in a time-dependent manner and was maximal at 8 h and concentrations of 50–100 µg/ml oxLDL. PMA, a well-established PKC{alpha} activator, inhibited ROS formation as well when preincubated for 8 to 16 h. In cells stably overexpressing PKC{alpha}-EGFP, we noticed that ROS formation remained intact upon pre-exposure of these cells to oxLDL. Moreover, in these cells endogenous but not overexpressed PKC{alpha}-EGFP disappeared, further substantiating the importance of PKC{alpha} in stimulating ROS production. In addition, we noticed a concentration-dependent ability of oxLDL to halt ROS production. Whereas 10 µg/ml oxLDL was insufficient in attenuating ROS formation over an 8-h incubation period in RAW 264.7 cells, 50 µg/ml oxLDL impaired ROS generation.

Conclusion These results indicate that attenuation of the oxidative burst in oxLDL-pretreated macrophages is closely associated with down-regulation of PKC{alpha}, which is elicited in a dose- and time-dependent manner.

KEYWORDS Protein kinase C; Monocytes/macrophages; Oxygen radicals; Inflammation; Lipoproteins


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
It is being appreciated that inflammation plays a fundamental role in coronary artery disease (CAD) and other manifestations of atherosclerosis (reviewed in: [1]). In early atherosclerotic lesions, immune and vascular smooth muscle cells (VSMC) accumulate in the subendothelial space of the artery wall [2]. The slow changes in the intima most likely result from a chronic inflammatory response towards oxidized low density lipoprotein (LDL) [3] with the notion that oxLDL is a prominent component of atherosclerotic lesions [4]. A local release of proinflammatory cytokines by activated immune cells and VSMCs accelerate progression of the lesion [5,6]. Monocytes recruited by the activated endothelium differentiate into macrophages [7] and macrophages express several receptors for oxLDL, known as scavenger receptors. Uptake of oxLDL by invading macrophages but also VSMCs contributes to foam cell and fatty streak formation. Ehara et al. positively correlated levels of oxLDL with the severity of acute coronary syndromes and proposed that more severe lesions contain a significantly higher percentage of oxLDL-positive macrophages [8].

Uptake of oxidized proteins or phospholipids via scavenger receptors and the interactions of macrophages with T cells or other vascular cells through cytokines provokes macrophage activation and concomitant generation of reactive oxygen species (ROS) [9]. Superoxide anion (O2–) formation in monocytes/macrophages in response to invading pathogens acts as a first line defense. Superoxide generation, primarily by NADPH oxidase, is followed by dismutation of superoxide to H2O2 in a process known as the respiratory burst [10]. NADPH oxidase is a multiprotein complex which remains inactive in resting cells with its components being distributed between cytosolic vesicles and/or the plasma membrane [11]. Numerous studies support a major role of protein kinase C (PKC) in NADPH oxidase activation by provoking phosphorylation and assembly of NADPH oxidase components [12]. Mammalian PKC comprises a family of 12 distinct members subdivided into three subfamilies based on sequence similarities and their modes of activation. Conventional PKC isoforms such as {alpha}, βI, βII, and {gamma} are activated by phospholipids, in particular phosphatidylserine (PS), diacylglycerol (DAG) and Ca2+ whereas novel PKCs such as {delta}, {varepsilon}, {eta}, and {theta} lacking a Ca2+ binding site require PS and DAG. Atypical PKC isoforms neither respond to DAG nor to Ca2+ but still necessitate phospholipids. PKC is synthesized as an inactive non-phosphorylated precursor which resides in the detergent-insoluble fractions associated with the cytoskeleton. Activation of PKC is achieved by two coordinated mechanisms. First, phosphorylation of three distinct sites within the activation loop, the turn motif, and the hydrophobic domain is required for catalytic competence. Second, binding of DAG as well as PS (for conventional PKC) and membrane targeting promotes conformational activation of the protein [13,14]. As PKC activity is also controlled by desensitization, degradation of the enzyme plays a decisive role. Dephosphorylated PKC undergoes ubiquitination and degradation by the 26S proteasome [15,16]. However, recent findings suggest a proteasome-independent degradation pathway of PKC{alpha} as demonstrated in rat intestinal epithelial cells [17]. Whereas PKC{alpha} activation by phorbol 12-myristate 13-acetate (PMA) promotes PKC{alpha} translocalization to the plasma membrane where it becomes ubiquitinated and degraded, bryostatin 1 triggered an alternative pathway of PKC{alpha} down-regulation. Besides targeting PKC{alpha} to the plasma membrane, a portion of caveolae-associated enzyme was transported to perinuclear regions, where it underwent dephosphorylation and proteasome-independent destruction.

PMA, a DAG homologue, is well established in activating the PKC{alpha} pathway and downstream ROS production via NADPH oxidase. It was demonstrated that a decreased protein amount of PKC{alpha} in response to LPS- and IFN-{gamma}-stimulation attenuated ROS formation in monocytes/macrophages following PMA addition. This type of desensitization was reversed in cells overexpressing PKC{alpha} [18]. Short-term exposure of primary monocyte-derived macrophages and macrophage cell lines to oxLDL dose-dependently provoked ROS formation. However, there is also evidence that preincubation of monocytes/macrophages with oxLDL or major lipid components attenuated the oxidative burst [19]. In monocytes of septic patients, we previously have been correlating an attenuated oxidative burst in response to PMA with depletion of PKC{alpha}. Therefore, we hypothesized that macrophage desensitization by oxLDL-treatment is facilitated by depletion of PKC{alpha}.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Chemicals
Medium and supplements were purchased from PAA (Linz, Austria). Fetal calf serum (FCS) was from Biochrom (Berlin, Germany). Phorbol myristate acetate (PMA) was ordered from Sigma (Schnelldorf, Germany). Go 6976 was obtained from Calbiochem (Schwalbach, Germany). Human lipoprotein LDL was from Intracel (Frederick, USA). The pan-caspase specific inhibitor Z-VAD-FMK was purchased from Alexis Biochemicals (Grünberg, Germany), while anti-PKC{alpha} was purchased from BD Biosciences (Heidelberg, Germany). Protease inhibitor cocktail came from Roche (Mannheim, Germany). A protein assay kit was bought from Bio-Rad (München, Germany). Nitrocellulose membrane, ECL detection system and horseradish peroxidase (HRP)-labeled anti-mouse or anti-rabbit secondary antibodies were delivered by Amershan Biosciences (Freiburg, Germany). Lipidophor Tris All In kit was purchased from Technoclone (Wien, Austria). The CoolSNAP CCD camera was from Roper Scientific (Tucson, AZ), whereas the MetaMorph software package came from Universal Imaging (West Chester, PA). Vectashield mounting medium was ordered from Linaris (Wertheim, Germany). Hydroethidine (HE) and 3,3'-dihexyloxacarbocyanine iodide (DiOC6) were purchased from Molecular Probes (Karsruhe, Germany). All other chemicals were of the highest grade of purity and commercially available.

2.2 Cell culture
The mouse monocyte/macrophage cell line RAW 264.7 was cultured in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin and 10% fetal calf serum. Cells were kept at 377 °C in a humidified atmosphere with 5% CO2 and propagated according to recommendations given by the American Type Culture Collection. Cells were transferred three times a week, and medium was changed prior to experiments.

2.3 Oxidative modification of LDL
Native LDL (density range: 1.019–1.063 g/ml) was ordered from Intracel (Frederick, USA) and oxidized immediately after delivery. Oxidized LDL was prepared by dialyzing native LDL (nLDL) against isotonic saline (pH 7.4, 4 °C) containing 5 µM CuSO4 for 25 h at RT as described previously [20,21]. Oxidation was terminated by dialyzing oxLDL against phosphate buffered saline (PBS) containing EDTA (0.67 mM, 1 l) for 3 h. OxLDL was further dialyzed against EDTA-free PBS for 24 h at 4 °C. This procedure was repeated three times. Quantification of the oxidized lipoprotein fraction was performed by an electrophoretic separation method using Lipidophor Tris All In kit (Technoclone, Wien, Austria). Briefly, after electrophoresis of nLDL and oxLDL in 1% agarose gels, lipoproteins were visualized with a solution containing 6.3 mM phosphotungstic acid, 182 mM MgCl2, 70 mM NaCl, and 12 mM NaN3 according to the manufacturer's protocol. Protein content of oxLDL was measured, and the degree of oxidation was quantified by the increase in relative mobility on agarose gels as described [22]. The index for lipoprotein oxidation thereby was typically 2.6- to 3.0-fold increased compared with native LDL. Lipoproteins were stored at 4 °C in the dark and freshly prepared every 2 weeks. Results obtained after oxLDL treatment showed no significant differences by the use of different oxLDL stocks.

2.4 Retroviral transduction
Retroviral infection of RAW 264.7 macrophages was performed essentially as described [23,24]. Vector constructs have been described [18]. Briefly, following transient transfection of the packaging plasmids encoding the vesicular stomatitis virus glycoprotein G (pczVSV-G, [25]) and the MLV-gag-pol genes (pHIT 60, [26]) together with the retroviral vectors pLXIN-PKC{alpha}-EGFP into 293T cells, target cells were incubated for 24 h with the infectious supernatant containing 8 µg/ml Polybrene. Positive selection based on EGFP-expression was performed using the sort option of a FACSCalibur flow cytometer (BD Biosciences, Heidelberg, Germany). Transduced cell populations containing 100% positive EGFP-expressing cells were used for the experiments.

2.5 Flow cytometry of oxygen-radical production
Cells were seeded 24 h prior to stimulation with a density of 3 x 105 cells/ml of culture medium. Following preincubations with oxLDL or PMA (e.g. 8 or 16 h) cells were washed off the culture dish using culture medium and transferred to FACS tubes. Now, cells were stimulated for 1 h with oxLDL (50 µg/ml) or 1 µM PMA. After 30 min of incubations, 2 µM HE was added and incubation went on for another 30 min before FACS measurement. All incubations were carried out at 37 °C in the dark. Flow cytometry analysis was performed using a FACSCalibur flow cytometer (BD Biosciences, Heidelberg, Germany) and oxidation of HE was measured in 1 x 104 cells through a 630 nm long pass filter (FL3). To discriminate the influence of oxLDL itself on HE oxidation we performed comparative experiments in which cells were incubated with oxLDL for 1 h and washed intensively before adding HE for 30 min. In these cells, we detected similar amounts of ROS compared to cells incubated with HE in medium containing oxLDL (data not shown). To rule out any effects of solvent or native LDL, appropriate controls were performed.

2.6 Fluorescence microscopy
To follow PKC{alpha} translocation, PKC{alpha}-EGFP positive cells were seeded in 6 cm dishes. After stimulation with PMA or oxLDL cells were fixed with 4% paraformaldehyde for 20 min and intracellular localization of PKC{alpha}-EGFP was analyzed using an Axioscope fluorescent microscope (Carl Zeiss, Frankfurt, Germany). Photographs were taken with a CoolSNAP CCD camera and images were created by the MetaMorph software package (Universal Imaging).

2.7 Determination of apoptosis
Apoptosis was measured by the loss of mitochondrial membrane potential as detected by decreased emission from the dye 3,3'-dihexyloxacarbocyanine iodide (DiOC6). DiOC6 was added at a final concentration of 40 nM 30 min prior to the end of incubations and the green fluorescence of the dye was measured by flow cytometry. Results are expressed as percentages of apoptotic cells (dull green fluorescence) over all viable cells (bright green fluorescence).

2.8 Immunoblotting
PKC{alpha} and actin were quantified by Western blot analysis. Briefly, equivalent numbers of cells were washed twice with PBS, lyzed in 200 µl buffer A (PBS, 0.5% Triton X-100, 1 mM PMSF, protease inhibitor cocktail, pH 8.0), and sonicated. Following centrifugation (15.000 x g, 20 min) the protein content was determined in the supernatants and 90 µg protein was added to the same volume of 2 x SDS-PAGE sample buffer (125 mM Tris/HCl, 2% SDS, 10% glycerin, 1 mM DTT, 0.002% bromophenol blue, pH 6.9) and boiled for 5 min. Proteins were resolved on 10% SDS-polyacrylamide gels and blotted onto nitrocellulose membranes. Nonspecific binding sites on the transblots were blocked with 5% milk solution in TTBS (50 mM Tris/HCl, 140 mM NaCl, 0.05% Tween-20, pH 7.2) for 1 h. The PKC{alpha} and actin antibodies (1:1000 in 5% milk/TTBS) were added and incubated overnight at 4 °C. Nitrocellulose membranes were washed with TTBS and then incubated with goat anti-mouse or goat anti-rabbit secondary antibodies conjugated with peroxidase (1:2000 in 1% milk/TTBS) for 2 h. After washing with TTBS immunoreactive proteins were detected by enhanced chemiluminescence. For each Western blot equal amounts of transferred proteins were verified by detecting actin in parallel.

2.9 Statistics
Each experiment was performed at least three times and representative data are shown. For statistical analysis, data from the three experiments were pooled. Data are expressed as means±S.E.M.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 OxLDL provoked ROS formation
Formation of reactive oxygen species (ROS) in response to oxLDL has previously been noticed in numerous cell types. Considering that monocytes enter atherosclerotic plaques and differentiate into macrophages as a critical step during development of atherosclerosis we have chosen the murine macrophage-like cell line RAW 264.7 in establishing a system to follow ROS production. ROS formation was determined by flow cytometry of cells loaded with HE which reacts with ROS, thus producing hydroxyethidium (2-OH-E+) [27].

Activation of RAW 264.7 cells with oxLDL (50 µg/ml) evoked ROS formation (Fig. 1A). Fischer et al. already demonstrated concentration-dependent ROS formation with an optimal response at 50–100 µg/ml oxLDL [19]. As a control for ROS production, we used 1 µM PMA, to activate Ca+- and phospholipid-dependent PKC and thus NADPH oxidase.


Figure 1
Figure 1
View larger version (57K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 OxLDL provoked generation of reactive oxygen species in RAW 264.7 macrophages. (A) ROS production in response to a 1-h lasting (time not indicated) stimulation with oxLDL (50 µg/ml) or PMA (1 µM) was analyzed by flow cytometry following HE oxidation. Otherwise, cells were preincubated either with 50 µg/ml oxLDL or 100 nM PMA for 8 h (time indicated) or with the PKC inhibitor Go 6976 (10 µM, 45 min preincubated) prior to stimulation for 1 h with oxLDL (50 µg/ml) or PMA (1 µM) as indicated. (B) As controls, RAW 264.7 macrophages were incubated with native LDL (nLDL, 50 µg/ml) for 1 h (upper left panel). Alternatively, nLDL was preincubated for 8 h followed by PMA stimulation prior to analysis of HE oxidation (upper right panel). Further controls were accomplished with cells treated with 50 µg/ml oxLDL or 100 nM PMA for 8 h. For details, see Methods. Data shown are representative of at least three independent experiments.

 
However, when RAW 264.7 cells were preincubated with 50 µg/ml oxLDL for 8 h ROS formation on subsequent stimulation with oxLDL for 1 h was blocked. In analogy, preincubating cells with 100 nM PMA for 8 h blocked ROS formation upon oxLDL addition. Considering activation of PKC{alpha} upon PMA addition, we questioned the role of PKC{alpha} in oxLDL-provoked ROS formation. Go 6967, which selectively blocks PKC{alpha} and PKCβ1 isoenzymes attenuated the oxidative burst in RAW 264.7 cells stimulated with either oxLDL or PMA. As controls, cells treated with 50 µg/ml nLDL neither produce ROS nor did a preincubation with nLDL attenuate PMA-elicited ROS production (Fig. 1B). Cells incubated with either oxLDL or PMA for 8 h without secondary stimulation did not show ROS production at the end of the incubation period.

3.2 OxLDL down-regulated PKC{alpha}
Having demonstrated that activation of classical PKC isoforms might control ROS production upon stimulation with oxLDL, we analyzed PKC{alpha} protein amount by Western analysis. Treatment of RAW 264.7 cells with 50 µg/ml oxLDL time-dependently reduced PKC{alpha} protein amount with a maximal decay seen at 8 h (Fig. 2A). With longer incubation times of 12 or 16 h, PKC{alpha} reappeared and reached levels comparable to controls at 16 h. This biphasic effect of oxLDL contrasted activation and subsequent degradation of PKC{alpha} by PMA (Fig. 2B). With 100 nM PMA we observed continuous reduction of PKC{alpha} starting from 2 h and remaining low up to 16 h. Higher concentrations of PMA, e.g. 1 µM, led to a faster degradation of PKC{alpha}, starting 15 min following its activation (data not shown). To rule out an effect of nLDL on PKC{alpha} degradation, we incubated cells with 50 µg/ml nLDL for 8 and 16 h and observed no alterations in the PKC{alpha} protein amount compared to controls (Fig. 2C).


Figure 2
View larger version (41K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Time-dependent depletion of PKC{alpha} in response to oxLDL. (A and B) RAW 264.7 cells were time-dependently incubated with 50 µg/ml oxLDL or 100 nM PMA or left untreated as controls (CTL). (C) RAW 264.7 cells were incubated with 50 µg/ml native LDL (nLDL) for 8 and 16 h or treated with 50 µg/ml oxLDL (8 and 16 h) in presence or absence of the pan-caspase inhibitor Z-VAD-FMK (20 µM). Cell extracts were separated by SDS-PAGE followed by Western blotting of PKC{alpha}. Data shown are representative of at least three independent experiments.

 
Reactive oxygen species often provoke oxidative damage and accumulating evidence suggests that ROS may initiate apoptotic cell death (reviewed in [28]). To analyze apoptosis in response to oxLDL, we followed the loss of mitochondrial membrane potential as detected by decreased emission from the dye DiOC6 by flow cytometry. OxLDL time- and dose-dependently increased the rate of apoptosis. The proportion of apoptosis increased from 1.2% to 8.1% when incubating RAW 264.7 cells with 50 µg/ml oxLDL for up to 16 h and from 1.4% to 10.7% with 100 µg/ml oxLDL (Table 1).


View this table:
[in this window]
[in a new window]

  		Table 1 Apoptosis of RAW 264.7 cells in response to oxLDL

 
Most experiments concerning PKC{alpha} depletion were done at concentrations of 50 µg/ml oxLDL, delivered for 8 or 16 h. As we noticed higher amounts of PKC{alpha} protein at 16 h compared to the 8-h time point, apoptotic events can be disregarded as an explanation for PKC{alpha} degradation. Furthermore, we used the pan-caspase inhibitor Z-VAD-FMK to verify that oxLDL-mediated down-regulation of PKC{alpha} seen at 8 h as well as its reappearance after 16 h is not controlled by apoptosis, i.e. active caspases. Fig. 2B demonstrates that there are no differences in the amounts of PKC{alpha} after oxLDL treatment with caspase being blocked by Z-VAD-FMK. Determinations of apoptosis by flow cytometry again assured that cell death was below 2% in those cells treated with Z-VAD-FMK.

3.3 PKC{alpha} overexpression restored ROS formation after oxLDL pretreatment
After retroviral infection with the pLXIN-PKC{alpha}-EGFP plasmid, RAW 264.7 cells not only expressed endogenous PKC{alpha} but also an approximately 30 kDa larger PKC{alpha} form, that can be detected based on its EGFP tag by Western blot analysis (Fig. 3A, right panel). To achieve high gene transfer efficiency, positive cells were sorted giving rise to 100% PKC{alpha}-EGFP positive clones. Functionality of the PKC{alpha}-EGFP protein was analyzed by its cytosol to membrane translocation in response to 100 nM PMA as determined by fluorescence microscopy. Compared to controls (Fig. 3, row B) PKC{alpha}-EGFP completely translocated from the cytosol to the cell membranes in response to PMA within 5 min (Fig. 3C, two left columns). After a 4 to 8-h PMA treatment, PKC{alpha} protein almost disappeared. In RAW 264.7 cells expressing PKC{alpha}-EGFP PKC{alpha} could be detected below the cell surface in the cytoplasm when incubated with oxLDL for 5 min (Fig. 3D). PKC{alpha} localization at the membrane became more pronounced when extending incubations up to 4 h. Longer incubation periods of oxLDL still revealed cell membrane associated and cytosolic localized PKC{alpha} protein (Fig. 3D, right column). Furthermore, we observed cytosolic accumulation of oxLDL inside lipid droplets (white arrows) as additionally determined by oil red staining.


Figure 3
View larger version (93K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Compensation of oxLDL-mediated PKC{alpha} depletion by overexpression of PKC{alpha}-EGFP in RAW 264.7 cells. (A) PKC{alpha}-EGFP-overexpressing RAW 264.7 cells compared to wild type cells were incubated with 50 µg/ml oxLDL or 100 nM PMA for 8 h. Cell extracts were separated by SDS-PAGE followed by PKC{alpha} Western analysis. Actin served as a loading control. (B–D) PKC{alpha} localization in response to 100 nM PMA (C) or 50 µg/ml oxLDL (D) and times as indicated was analyzed by fluorescence microscopy. Panel B shows untreated control cells. Photos in the left column provide an overview, whereas the three right columns show higher magnifications. Data shown are representative of at least three independent experiments.

 
Consequently, we determined the ability of oxLDL and PMA to deplete PKC{alpha} subsequent its activation by Western analysis. Incubating parent RAW 264.7 cells vs. PKC{alpha} overexpressing mutants with PMA for 8 h resulted in loss of PKC{alpha} protein (Fig. 3A). When both cell lines were treated with oxLDL for 8 h only endogenous PKC{alpha} disappeared, while the level of exogenous EGFP-tagged PKC{alpha} remained nearly unchanged.

Using RAW 264.7-PKC{alpha}-EGFP cells, we analyzed the effect of PKC{alpha} overexpression on ROS formation following stimulation with oxLDL and PMA. In contrast to wild type cells, overexpression of PKC{alpha} restored the ability of PMA and oxLDL to generate ROS in oxLDL-pretreated cells (Fig. 4). This result correlated with our observation that oxLDL did not deplete overexpressed PKC{alpha}-EGFP.


Figure 4
View larger version (38K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Overexpression of PKC{alpha} attenuated oxLDL-mediated desensitization of RAW 264.7 macrophages. PKC{alpha}-overexpressing RAW 264.7 macrophages were incubated with PMA (1 µM) or 50 µg/ml oxLDL for 1 h or remained as controls. Otherwise, cells were preincubated with 50 µg/ml oxLDL or 100 nM PMA for 8 h, followed by a 1-h lasting stimulation with PMA (1 µM) or oxLDL (50 µg/ml). ROS production was assessed by HE oxidation using flow cytometry. Data shown are representative of at least three independent experiments.

 
Taking into account that preincubation of RAW 264.7-PKC{alpha}-EGFP cells with PMA degrades PKC{alpha} we analyzed ROS production following preincubations with PMA as well. In this case, oxLDL- and PMA-elicited ROS formation were completely blocked. These results point to the requirement of PKC{alpha} in promoting ROS formation.

3.4 Long-term incubations with oxLDL recovered ROS formation after PMA/oxLDL stimulation
Time kinetics revealed that PKC{alpha} depletion by oxLDL was transient, with protein reappearing at 12- to 16-h incubations. Therefore, it was of interest whether the protein seen at 16 h was active in facilitating ROS formation upon oxLDL addition (Fig. 5A). When preexposing RAW 264.7 macrophages for 16 h to 50 µg/ml oxLDL subsequent addition of oxLDL elicited ROS formation. In contrast, preincubations with 100 nM PMA for 16 h did not allow provoking a subsequent oxLDL response.


Figure 5
View larger version (29K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Preincubation of RAW 264.7 macrophages with oxLDL affected ROS formation. (A) RAW 264.7 cells were pretreated with oxLDL (50 µg/ml) or PMA (100 nM) for 16 h. Thereafter, cells were stimulated with PMA (1 µM) or oxLDL (50 µg/ml) for 1 h prior to analyzing HE oxidation by flow cytometry. HE oxidation of control cells was determined following a 1-h incubation with oxLDL (50 µg/ml) or PMA (1 µM). (B) RAW 264.7 cells were incubated with PMA (100 nM) or oxLDL (50 µg/ml) for 8 and 16 h. Depletion of PKC{alpha} protein in cell extracts was analyzed by Western analysis. Data shown are representative of at least three independent experiments.

 
Western analysis of PKC{alpha}, performed in parallel to ROS determination, showed differences in the kinetics of PKC{alpha} depletion upon oxLDL vs. PMA stimulation (Fig. 5B).

3.5 OxLDL concentration-dependently attenuated ROS formation
In a last set of experiments we determined the concentration-dependent effect of oxLDL on ROS generation. Therefore, RAW 264.7 cells were exposed to 10 and 50 µg/ml oxLDL for 8 and 16 h prior to stimulation with PMA (Fig. 6). OxLDL preincubated for 8 h at 10 µg/ml did not attenuate ROS formation, while 50 µg/ml did.


Figure 6
View larger version (29K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Concentration-dependent attenuation of oxLDL-mediated ROS production in RAW 264.7 macrophages. RAW 264.7 cells were preincubated with different concentrations of oxLDL for 8 and 16 h. Afterwards, ROS production was initiated for 1 h (time not indicated) with PMA (1 µM) as determined by HE oxidation and flow cytometry. Data shown are representative of at least three independent experiments.

 
Extending the exposure period to 16 h revealed a different picture. At 16 h the low dose of oxLDL, i.e. 10 µg/ml oxLDL reduced ROS formation while at 50 µg/ml and an 16-h incubation period ROS formation proceeded normally. Thus, attenuation of ROS formation by oxLDL occurred dose- and time-dependently. Doses of 10 µg/ml oxLDL were insufficient to block ROS formation at 8 h, while being effective at 16 h. 50 µg/ml oxLDL prevented ROS formation at 8 h but turned out to be ineffective at longer incubation periods of i.e. 16 h. These results demonstrate a concentration-dependent as well as time-dependent effect of oxLDL in modulating the oxidative burst.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Macrophages are specialized to fight invading pathogens by activating innate immune functions. Molecular activation signatures, which aim to eliminate invaders, also permit uptake of lipoproteins, the release of reactive oxygen species and pro-inflammatory mediator formation that collectively foster atherosclerosis. It is assumed that oxidation of LDL comprises an essential step in its conversion towards a pro-atherogenic component [29] with the notion that enzymes found in macrophages such as lipoxygenases, myeloperoxidase, inducible nitric oxide synthase and NADPH oxidases possibly contribute to LDL oxidation. Therefore, macrophages accumulating in atherosclerotic lesions are likely to amplify oxidative reactions and thus to perpetuate a vicious cycle. These considerations stimulated our interest to understand macrophage activation towards long-term exposures to oxLDL as carried out in this study.

We focused on ROS formation as a prototype macrophage activation signal, taking into account that stimulation of PKC{alpha} represents an essential transducing component. Previously, it has been shown that oxLDL as well as major lipid peroxidation products of oxLDL are naturally occurring ligands for peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) [30]. It became apparent that preincubation of monocytes/macrophages with oxLDL attenuated the oxidative burst via activation of PPAR{gamma}. In these studies, activators of PPAR{gamma} such as ciglitazone or 15-deoxy-{delta}12,14-prostaglandin J2 (15dPGJ2) but also macrophage activation by LPS/IFN-{gamma} attenuated ROS formation [19,31] with the notion that activators of PPAR{gamma} as useful tools to protect against vascular inflammation and diabetic complications [32–34]. However, precise molecular mechanisms underlying their anti-inflammatory action remain unclear. Verrier et al. demonstrated, that PPAR{gamma} agonists belonging to the thiazolidinedione class of compounds (TZDs), or 15dPGJ2 were capable of activating diacylglycerol (DAG) kinase (DGK), thereby lowering the steady state concentration of DAG which impairs PKC activation [35]. To characterize the potential role of PKC in mediating anti-inflammatory responses after oxLDL pretreatment, we addressed the ability of macrophages to elicit an oxidative burst because inhibition of PKC{alpha} reduces ROS formation [36]. PMA, an established PKC activator not only provoked NADPH oxidase activation and concomitant ROS production but also desensitized macrophages for oxLDL-evoked ROS formation. As PMA resulted in activation and subsequent degradation of PKC{alpha} [37], we concluded that down-regulation of PKC{alpha} may also account for macrophage desensitization following oxLDL pretreatment. Indeed, PKC{alpha} protein amount in total cell extract was time-dependently down-regulated by oxLDL. Along that line, not only degradation of PKC{alpha} correlated with an attenuated oxidative burst but also inhibition of PKC with Go 6976 attenuated ROS production. To underscore these findings, we established a PKC{alpha}-overexpressing cell line. In PKC{alpha}-overexpressing macrophages oxLDL-mediated down-regulation of endogenous PKC{alpha} was compensated by preserving the expression of the overexpressed isoform. Functional activity of PKC{alpha}-EGFP was assured by demonstrating cytosol to membrane translocation of PKC{alpha}. As expected, in the experimental set up using PKC{alpha} overexpression, a preexposure with oxLDL no longer attenuated ROS formation. Interestingly, it appeared that oxLDL not only differed from PMA in its potency to produce ROS, but also with regard to the time-kinetics in down-regulating PKC{alpha}. Whereas PMA treatment resulted in a continuous degradation of PKC{alpha} for at least 16 h, oxLDL-mediated PKC{alpha} destruction occurred to be biphasic. With concentrations of 50–100 µg/ml oxLDL we observed a maximal decrease of PKC{alpha} after 8 h. However, with prolonged incubation periods the protein reappeared and reached an expression comparable to controls at 16 h. More interesting, reappearance of PKC{alpha} was again active in fostering ROS production. Again, this biphasic effect seemed to be concentration-dependent as higher amounts of oxLDL prolonged the period needed to gain reexpression of PKC{alpha} (data not shown). In addition, low concentrations of oxLDL (e.g. 10 µg/ml) required longer times to attenuate ROS generation compared to higher amounts of e.g. 50 µg/ml oxLDL. We must assume that the ability of oxLDL to deplete PKC{alpha} and thereby to halt ROS formation depends on the oxLDL concentration and time of exposure. In addition, PKC{alpha} localization, phosphorylation and diverse PKC{alpha} agonists may be critical determinants in affecting enzyme desensitization [17]. One can speculate that differences between PMA- and oxLDL-attenuated ROS formation arise form different signaling pathways that affect desensitization of PKC{alpha}. It is also likely, that internalization of oxLDL vs. PMA differs. Recently, it was demonstrated that the uptake of different phorbol esters not only depend on their lipophilicity but also on growth conditions, e.g. the presence of serum proteins [38].

There are manifold receptors identified to bind oxLDL, categorized as class A scavenger receptors, class B scavenger receptors such as CD36, SR-BI and type D scavenger receptors (LOX-1) (reviewed in [39]). Specific expression pattern on distinct cell types combined with the gap of knowledge on downstream signaling events make it difficult to predict cellular responses towards oxLDL. It is reported that oxLDL increases O2– production in endothelial cells via a process involving lectin-like oxLDL receptor-1 [40]. Furthermore it was shown that oxLDL acutely, within 15 min, increases the activity of PKC{alpha} in coronary artery smooth muscle cells [41] and increases global PKC activity (over 24 h) in coronary endothelial cells by activating LOX-1 [42]. Contrasting results presented by Fleming et al. indicated decreased PKC{alpha} activity in oxLDL-treated endothelial cells in association with dephosphorylation of nitric oxide synthase (eNOS) and enhanced production of eNOS-derived O2– [43]. Although it is generally accepted that PKC activation contributes to cardiovascular dysfunction [44,45] it is unclear whether the cardiovascular impact of PKC is due to an increased gene/protein expression or merely a posttranslational modulation of its kinase activity.

In summary, we suggest a model where desensitization of macrophages by oxLDL is concentration- and time-dependently mediated by depletion of PKC{alpha}. These studies provide further details to understand attenuation of the oxidative burst in macrophages by long-term oxLDL exposure.


   Acknowledgements
 
The technical assistance of Sabine Knaus is highly appreciated. The work was supported by grants from Deutsche Forschungsgemeinschaft (BR 999).


   Notes
 
Time for primary review 27 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Hansson G.K. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med (2005) 352:1685–1695.[Free Full Text]
  2. Jonasson L., Holm J., Skalli O., Bondjers G., Hansson G.K. Regional accumulations of T cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque. Arteriosclerosis (1986) 6:131–138.[Abstract/Free Full Text]
  3. Itabe H. Oxidized low-density lipoproteins: what is understood and what remains to be clarified. Biol Pharm Bull (2003) 26:1–9.[CrossRef][ISI][Medline]
  4. Palinski W., Rosenfeld M.E., Yla-Herttuala S., Gurtner G.C., Socher S.S., Butler S.W., et al. Low density lipoprotein undergoes oxidative modification in vivo. Proc Natl Acad Sci U S A (1989) 86:1372–1376.[Abstract/Free Full Text]
  5. Frostegard J., Ulfgren A.K., Nyberg P., Hedin U., Swedenborg J., Andersson U., et al. Cytokine expression in advanced human atherosclerotic plaques: dominance of pro-inflammatory (Th1) and macrophage-stimulating cytokines. Atherosclerosis (1999) 145:33–43.[CrossRef][ISI][Medline]
  6. Raines E.W., Ferri N. Thematic review series: the immune system and atherogenesis. Cytokines affecting endothelial and smooth muscle cells in vascular disease. J Lipid Res (2005) 46:1081–1092.[Abstract/Free Full Text]
  7. Smith J.D., Trogan E., Ginsberg M., Grigaux C., Tian J., Miyata M. Decreased atherosclerosis in mice deficient in both macrophage colony-stimulating factor (op) and apolipoprotein E. Proc Natl Acad Sci U S A (1995) 92:8264–8268.[Abstract/Free Full Text]
  8. Ehara S., Ueda M., Naruko T., Haze K., Itoh A., Otsuka M., et al. Elevated levels of oxidized low density lipoprotein show a positive relationship with the severity of acute coronary syndromes. Circulation (2001) 103:1955–1960.[Abstract/Free Full Text]
  9. Nguyen-Khoa T., Massy Z.A., Witko-Sarsat V., Canteloup S., Kebede M., Lacour B., et al. Oxidized low-density lipoprotein induces macrophage respiratory burst via its protein moiety: a novel pathway in atherogenesis? Biochem Biophys Res Commun (1999) 263:804–809.[CrossRef][ISI][Medline]
  10. Iles K.E., Forman H.J. Macrophage signaling and respiratory burst. Immunol Res (2002) 26:95–105.[CrossRef][ISI][Medline]
  11. Bokoch G.M., Diebold B.A. Current molecular models for NADPH oxidase regulation by Rac GTPase. Blood (2002) 100:2692–2696.[Abstract/Free Full Text]
  12. Fontayne A., Dang P.M., Gougerot-Pocidalo M.A., El-Benna J. Phosphorylation of p47phox sites by PKC alpha, beta II, delta, and zeta: effect on binding to p22phox and on NADPH oxidase activation. Biochemistry (2002) 41:7743–7750.[CrossRef][ISI][Medline]
  13. Newton A.C. Regulation of the ABC kinases by phosphorylation: protein kinase C as a paradigm. Biochem J (2003) 370:361–371.[CrossRef][ISI][Medline]
  14. Dutil E.M., Toker A., Newton A.C. Regulation of conventional protein kinase C isozymes by phosphoinositide-dependent kinase 1 (PDK-1). Curr Biol (1998) 8:1366–1375.[CrossRef][ISI][Medline]
  15. Lee H.W., Smith L., Pettit G.R., Bingham Smith J. Dephosphorylation of activated protein kinase C contributes to downregulation by bryostatin. Am J Physiol (1996) 271:C304–C311.[ISI][Medline]
  16. Lee H.W., Smith L., Pettit G.R., Vinitsky A., Smith J.B. Ubiquitination of protein kinase C-alpha and degradation by the proteasome. J Biol Chem (1996) 271:20973–20976.[Abstract/Free Full Text]
  17. Leontieva O.V., Black J.D. Identification of two distinct pathways of protein kinase Calpha down-regulation in intestinal epithelial cells. J Biol Chem (2004) 279:5788–5801.[Abstract/Free Full Text]
  18. von Knethen A., Tautenhahn A., Link H., Lindemann D., Brune B. Activation-induced depletion of protein kinase C alpha provokes desensitization of monocytes/macrophages in sepsis. J Immunol (2005) 174:4960–4965.[Abstract/Free Full Text]
  19. Fischer B., von Knethen A., Brune B. Dualism of oxidized lipoproteins in provoking and attenuating the oxidative burst in macrophages: role of peroxisome proliferator-activated receptor-gamma. J Immunol (2002) 168:2828–2834.[Abstract/Free Full Text]
  20. Cominacini L., Garbin U., Davoli A., Micciolo R., Bosello O., Gaviraghi G., et al. A simple test for predisposition to LDL oxidation based on the fluorescence development during copper-catalyzed oxidative modification. J Lipid Res (1991) 32:349–358.[Abstract]
  21. Cominacini L., Garbin U., De Santis A., Campagnola M., Davoli A., Pasini A.F., et al. Mechanisms involved in the in vitro modification of low density lipoprotein by human umbilical vein endothelial cells and copper ions. J Lipid Mediat Cell Signal (1996) 13:19–33.[CrossRef][ISI][Medline]
  22. Galle J., Wanner C. Oxidized LDL and Lp(a). Preparation, modification, and analysis. Methods Mol Biol (1998) 108:119–130.[Medline]
  23. Denk A., Goebeler M., Schmid S., Berberich I., Ritz O., Lindemann D., et al. Activation of NF-kappa B via the Ikappa B kinase complex is both essential and sufficient for proinflammatory gene expression in primary endothelial cells. J Biol Chem (2001) 276:28451–28458.[Abstract/Free Full Text]
  24. Lindemann D., Bock M., Schweizer M., Rethwilm A. Efficient pseudotyping of murine leukemia virus particles with chimeric human foamy virus envelope proteins. J Virol (1997) 71:4815–4820.[Abstract]
  25. Pietschmann T., Heinkelein M., Heldmann M., Zentgraf H., Rethwilm A., Lindemann D. Foamy virus capsids require the cognate envelope protein for particle export. J Virol (1999) 73:2613–2621.[Abstract/Free Full Text]
  26. Soneoka Y., Cannon P.M., Ramsdale E.E., Griffiths J.C., Romano G., Kingsman S.M., et al. A transient three-plasmid expression system for the production of high titer retroviral vectors. Nucleic Acids Res (1995) 23:628–633.[Abstract/Free Full Text]
  27. Zhao H., Joseph J., Fales H.M., Sokoloski E.A., Levine R.L., Vasquez-Vivar J., et al. Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence. Proc Natl Acad Sci U S A (2005) 102:5727–5732.[Abstract/Free Full Text]
  28. Droge W. Free radicals in the physiological control of cell function. Physiol Rev (2002) 82:47–95.[Abstract/Free Full Text]
  29. Gaut J.P., Heinecke J.W. Mechanisms for oxidizing low-density lipoprotein. Insights from patterns of oxidation products in the artery wall and from mouse models of atherosclerosis. Trends Cardiovasc Med (2001) 11:103–112.[CrossRef][ISI][Medline]
  30. Nagy L., Tontonoz P., Alvarez J.G., Chen H., Evans R.M. Oxidized LDL regulates macrophage gene expression through ligand activation of PPARgamma. Cell (1998) 93:229–240.[CrossRef][ISI][Medline]
  31. Von Knethen A.A., Brune B. Delayed activation of PPARgamma by LPS and IFN-gamma attenuates the oxidative burst in macrophages. FASEB J (2001) 15:535–544.[Abstract/Free Full Text]
  32. Minamikawa J., Tanaka S., Yamauchi M., Inoue D., Koshiyama H. Potent inhibitory effect of troglitazone on carotid arterial wall thickness in type 2 diabetes. J Clin Endocrinol Metab (1998) 83:1818–1820.[Abstract/Free Full Text]
  33. Marx N., Libby P., Plutzky J. Peroxisome proliferator-activated receptors (PPARs) and their role in the vessel wall: possible mediators of cardiovascular risk? J Cardiovasc Risk (2001) 8:203–210.[CrossRef][ISI][Medline]
  34. Lehrke M., Lazar M.A. The many faces of PPARgamma. Cell (2005) 123:993–999.[CrossRef][ISI][Medline]
  35. Verrier E., Wang L., Wadham C., Albanese N., Hahn C., Gamble J.R., et al. PPARgamma agonists ameliorate endothelial cell activation via inhibition of diacylglycerol-protein kinase C signaling pathway: role of diacylglycerol kinase. Circ Res (2004) 94:1515–1522.[Abstract/Free Full Text]
  36. Larsen E.C., DiGennaro J.A., Saito N., Mehta S., Loegering D.J., Mazurkiewicz J.E., et al. Differential requirement for classic and novel PKC isoforms in respiratory burst and phagocytosis in RAW 264.7 cells. J Immunol (2000) 165:2809–2817.[Abstract/Free Full Text]
  37. Nishizuka Y. Studies and perspectives of protein kinase C. Science (1986) 233:305–312.[Abstract/Free Full Text]
  38. Braun D.C., Cao Y., Wang S., Garfield S.H., Hur G.M., Blumberg P.M. Role of phorbol ester localization in determining protein kinase C or RasGRP3 translocation: real-time analysis using fluorescent ligands and proteins. Mol Cancer Ther (2005) 4:141–150.[Abstract/Free Full Text]
  39. Horiuchi S., Sakamoto Y., Sakai M. Scavenger receptors for oxidized and glycated proteins. Amino Acids (2003) 25:283–292.[CrossRef][ISI][Medline]
  40. Cominacini L., Rigoni A., Pasini A.F., Garbin U., Davoli A., Campagnola M., et al. The binding of oxidized low density lipoprotein (ox-LDL) to ox-LDL receptor-1 reduces the intracellular concentration of nitric oxide in endothelial cells through an increased production of superoxide. J Biol Chem (2001) 276:13750–13755.[Abstract/Free Full Text]
  41. Giardina J.B., Tanner D.J., Khalil R.A. Oxidized-LDL enhances coronary vasoconstriction by increasing the activity of protein kinase C isoforms alpha and epsilon. Hypertension (2001) 37:561–568.[Abstract/Free Full Text]
  42. Li D., Liu L., Chen H., Sawamura T., Mehta J.L. LOX-1, an oxidized LDL endothelial receptor, induces CD40/CD40L signaling in human coronary artery endothelial cells. Arterioscler Thromb Vasc Biol (2003) 23:816–821.[Abstract/Free Full Text]
  43. Fleming I., Mohamed A., Galle J., Turchanowa L., Brandes R.P., Fisslthaler B., et al. Oxidized low-density lipoprotein increases superoxide production by endothelial nitric oxide synthase by inhibiting PKCalpha. Cardiovasc Res (2005) 65:897–906.[Abstract/Free Full Text]
  44. Meier M., King G.L. Protein kinase C activation and its pharmacological inhibition in vascular disease. Vasc Med (2000) 5:173–185.[Abstract/Free Full Text]
  45. Idris I., Gray S., Donnelly R. Protein kinase C activation: isozyme-specific effects on metabolism and cardiovascular complications in diabetes. Diabetologia (2001) 44:659–673.[CrossRef][ISI][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Köhl, R.
Right arrow Articles by Brüne, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Köhl, R.
Right arrow Articles by Brüne, B.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?