Urokinase-type plasminogen activator (uPA) modulates monocyte-to-macrophage differentiation and prevents Ox-LDL-induced macrophage apoptosis

Atherosclerosis 231 (2013) 29e38

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Urokinase-type plasminogen activator (uPA) modulates monocyteto-macrophage differentiation and prevents Ox-LDL-induced macrophage apoptosis Nicole Paland, Saar Aharoni, Bianca Fuhrman* The Lipid Research Laboratory, Technion Faculty of Medicine, Rambam Medical Center, Haifa 31096, Israel

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 April 2013 Received in revised form 1 August 2013 Accepted 19 August 2013 Available online 26 August 2013

Objective: Monocyte-to-macrophage differentiation and macrophage death play a pivotal role in atherogenesis. uPA and its receptor uPAR are expressed in atherosclerotic lesion macrophages and contribute to atherosclerosis progression. In the present study we investigated the effect and mechanisms of action of uPA on monocyte-to-macrophage differentiation and on macrophage apoptotic death. Methods and results: The number of mouse peritoneal macrophages (MPM) harvested from uPARdeficient (uPAR
/
) mice was significantly lower by 30% in comparison to control C57BL/6 mice. In vitro, uPA intensified PMA-induced THP-1 monocyte differentiation, as determined by increased expression of the macrophage marker CD36. This effect was mediated via G1 arrest, downregulation of G2/S phase and inhibition of PMA-induced cell death. uPA attenuated MonoMac6 (MM6) macrophagelike cell line apoptosis induced by oxidized LDL (Ox-LDL) and by thapsigargin (inhibitor of sarcoendoplasmic reticulum Ca2þ-ATPase), but not by staurosporine (protein kinase inhibitor), suggesting that uPA antiapoptotic activity is Ca2þ-independent, but involves a kinase activation. The antiapoptotic activity of uPA was dependent on the presence of uPAR, and it involved ERK1/2 activation-dependent downregulation of the proapoptotic protein Bim in macrophages stimulated with Ox-LDL. Conclusions: The present study demonstrates, for the first time, that uPA stimulates the differentiation of monocytes into macrophages and attenuates Ox-LDL-induced macrophage apoptotic death via ERK1/2 activation-dependent Bim downregulation. These processes may result in prolonged macrophage survival in the lesion, increased lesion cellularity, and eventually necrosis, which accelerates lesion development. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords: uPA Atherosclerosis Monocytes Macrophages Differentiation Apoptosis

1. Introduction Monocyte/macrophages play a crucial role in the formation of the atherosclerotic lesion [1]. Macrophage numbers within lesions are influenced by monocyte recruitment and their differentiation into macrophages, as well as by macrophage survival. During atherogenesis, circulating monocytes adhere to endothelial cells, invade the intima and differentiate into macrophages. After differentiation, intimal macrophages incorporate oxidized LDL (OxLDL) via the scavenger receptors, transforming into foam cells, which eventually die, forming the lesion necrosis core, a hallmark feature of atherosclerosis. Ox-LDL is also cytotoxic, causing both necrosis and apoptosis in a variety of cell types, including

* Corresponding author. Tel.: þ972 4 8295278; fax: þ972 4 8520076. E-mail address: [email protected] (B. Fuhrman). 0021-9150/$ e see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.atherosclerosis.2013.08.016

macrophages [2,3]. Macrophage apoptosis occurs throughout all stages of atherosclerosis. Recent studies suggest that macrophage death in early lesions, which appears to be accompanied by rapid phagocytic clearance of apoptotic cells, decreases macrophage burden and slows lesion progression, whereas in late lesions macrophage death causes necrotic core formation, which promotes plaque rupture. Thus the balance between macrophage survival and death throughout atherosclerosis is an important determinant of lesion development and progression [4]. Cell death can be classified as physiological death (apoptosis) or accidental death (necrosis) [5]. Mitochondria play a pivotal role in the regulation of all forms of cell death, and they react to proapoptotic or pro-necrotic stimuli by loss of membrane potential [6]. Proteins from the Bcl-2 family are the key regulators of mitochondrial integrity. They are comprised of either pro- or antiapoptotic members of the Bcl-2 family, which can be transcriptionally regulated or translocated from the cytoplasm to the

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N. Paland et al. / Atherosclerosis 231 (2013) 29e38

mitochondrial membrane to exert their specific function, while the ratio of pro- and anti-apoptotic proteins at the mitochondria determines the eventual fate of the cell [7]. Cell death signals can originate from either extrinsic pathways involving death receptors of the tumor necrosis family [8], from intrinsic mitochondriadependent pathways [9] or from intrinsic endoplasmic-reticulum (ER) dependent pathways [10]. Macrophage overload with OxLDL leads to ER stress [11] and eventually to apoptosis. Urokinase (uPA) is a serine protease enzyme of the fibrinolytic system. uPA binding to its receptor, uPAR, is implicated in plasmin generation, and plays a pivotal role in plasmin-mediated pericellular proteolysis. The uPA/uPAR system has also a nonproteolytic role that extends beyond its role in fibrinolysis [12]. uPAR is a GPI-anchored protein that lacks a transmembrane domain, and its biological functions rely on its interaction with other cell surface and transmembrane proteins, including integrins [13], vitronectin [14], EGFR [15], and PDGFR [16]. uPA is expressed in human atherosclerotic vessel wall, mainly in association with macrophages [17]. Over-expression of macrophage uPA was found to contribute to the progression and complications of atherosclerosis [18]. We have recently shown that uPA has an impact on macrophage atherogenicity, by increasing macrophage cholesterol biosynthesis [19], or by promoting oxidative stress [20]. Moreover, uPA was implicated in cell growth and apoptosis of various cell types [21e24]. However, little is known on the impact of uPA on monocyte differentiation into macrophages and on macrophage death [25,26]. In the present study we investigated the effect and mechanisms of action of uPA on monocyte-to-macrophage differentiation and on macrophage apoptotic death. 2. Material and methods 2.1. Reagents and antibodies Phorbol 12-myristate 13-acetate (PMA), staurosporine, thapsigargin, trypan blue, propidium iodide, 3,30 -Dihexyloxacarbocyanine (DiOC6) and RNaseA were purchased from Sigma (St. Louis, MO, USA). PBS, DMEM, RPMI 1640 medium, HBSS, FCS (heatinactivated at 56  C for 30 min), penicillin, streptomycin, glutamine, sodium pyruvate, nonessential amino acids and insulin were from Biological Industries (Beit Haemek, Israel). Thioglycollate was from Becton Dickinson (Ontario, Canada). The MEK1/2 inhibitor UO126 was obtained from Calbiochem. The DNA polymerase inhibitor Aphidicolin was obtained from Sigma. Mounting medium EntellanÒ was from Merck Millipore. Fluoisothiocyanate (FITC)-conjugated antibodies against CD36 were from Serotec IQ Products (Zerinkepark, The Netherlands). Polyvinylidene difluoride membranes (PVDF) were from PerkinElmer Life Sciences. Rabbit polyclonal anti-human Bim, rabbit polyclonal anti-human phospho-p44/42 ERK1/2 (Thr-202/Tyr-204) were purchased from Cell Signaling Technology. Rabbit anti-human b-actin was from Sigma (St. Louis, MO, USA). Primary bound antibodies were visualized with goat anti-rabbit IgG or rabbit anti-mouse IgG conjugated with horseradish peroxidase (Jackson ImmunoResearch Laboratories). Chemiluminescence Detection Kit HRP was obtained from Biological Industries. 2.2. Ox-LDL preparation Oxidation of LDL was carried out as previously described [27]. Shortly, LDL was separated from plasma of normal healthy volunteers by discontinuous ultracentrifugation, diluted in PBS at a concentration of 1 mg/mL protein and subsequently three times dialyzed against PBS to remove residual EDTA. Oxidization of LDL was carried out by adding 0.5 mmol/L freshly prepared CuSO4 in PBS

for 2e4 h at 37  C in a shaking water bath. The oxidation state was determined by the formation of thiobarbituric acid-reactive substances (TBARS assay) [28] and the protein concentration was determined by the method of Bradford [29]. 2.3. Cell culture and induction of THP-1 monocytes differentiation The human myeloid leukemia cell line THP-1 was maintained in RPMI 1640 supplemented with 5% FCS, 1% glutamine and penicillin/ streptomycin. MonoMac6 cells were maintained in RPMI 1640 supplemented with 10% FCS, 1% glutamine, 1% pyruvate, 9 mg/mL insulin, 1% nonessential amino acids and penicillin/streptomycin at 37  C in a humidified incubator with 5% CO2. Exponentially growing THP-1 cells at a starting density of 5  105 were incubated with 25 ng/ml PMA for the indicated times. The differentiation status was observed by light microscopy, indirect immunofluorescence and determined by measuring cell surface expression of the macrophage markers CD36 using the FACS Calibur (Becton Dickinson, Ontario, Canada). 2.4. Immunostaining THP-1 cells were induced to differentiate on coverslips with 25 ng/ml PMA in the absence or presence of 5 nmol/L uPA for three days. After fixation with 1:1 (v/v) Methanol/Acetone at
20  C for 5 min cells were blocked and permeabilized in 3% BSA and 0.05% Tween 20 for 30 min at room temperature. Primary antibody was added in blocking buffer for 1 h. To enhance the signal from the prelabeled Fluoisothiocyanate (FITC)-conjugated antibodies against CD36 a goat anti-mouseeFITC secondary antibody was added for 45 min. Before addition of the rabbit anti-human actin antibody for 1 h cells were blocked again in 3% BSA in PBS. A goat anti-rabbit-cy3 labeled antibody was used to visualize actin staining. Coverslips were mounted and observed in a confocal microscope (LSM 700 upright). 2.5. SDS-PAGE and immunoblotting Proteins were separated by SDS-PAGE and transferred to PVDF membrane, blocked with 3% BSA in Tris-buffered saline (TBS) containing 0.5% Tween 20 (TBS-T) for 1 h before incubation with primary antibodies in TBS-T for 1 h. Unbound primary antibodies were washed with TBS-T three times prior to applying secondary antibodies. Unbound secondary antibodies were washed with TBST three times. Proteins were visualized by chemiluminescence. 2.6. Induction and determination of cell death in MM6 cells 2.5  105 MonoMac6 cells were pretreated with 0.5e10 nmol/L uPA in serum free medium supplemented with 0.2% BSA for 30 min. Apoptosis was induced with 5 nmol/L thapsigargin, 1 mmol/L staurosporine or 10 or 20 mg/mL Ox-LDL for 24 h. For some experiments the MEK1/2 inhibitor UO126 (10 ng/mL) was added as indicated. Total cell death of MonoMac6 cells was determined by measuring the uptake of trypan blue. Cells were incubated with 10% trypan blue solution for 5 min at room temperature. Cells from five different fields were counted and the percentage of cells which had taken up trypan blue as an indicator of cell death to the total cell number was determined. Proliferation was measured by using the Wst-1 assay according to manufacturer’s instructions (Promega). Shortly, cells were seeded in 96 well plates in a volume of 100 mL medium. The next day they were treated with 10 or 20 mg/mL Ox-LDL in the absence of presence of 5 nmol/L uPA or left untreated. 24 h later 5 ml of the

N. Paland et al. / Atherosclerosis 231 (2013) 29e38

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Fig. 1. uPA intensifies the PMA-stimulated differentiation of THP-1 monocytes. THP-1 monocytes were treated without (Control) or with PMA (25 ng/ml) in the absence or presence of uPA (5 nmol/L) for one or three days (A,B,C). Macrophage morphology analyzed by phase contrast microphotographs after one day. Untreated control cells (a) or uPA treated cells (b). PMA treated cells (c) and PMA and uPA treated cells (d) (A). CD36 surface expression was determined by FACS after three days. Results are expressed as mean  SD of three separate experiments. The insert shows a representative histogram. *p < 0.05, with vs. without uPA (B). CD36 expression (green) and redistribution of cellular actin (red) were monitored by immunofluorescence after three days in untreated control cells, uPA treated cells, PMA treated cells and PMA and uPA treated cells (C). The cells were taken at 630 fold magnification with a confocal microscope (LSM 700 upright). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Wst-1 reagent was added and the cells were incubated at 37  C for 4 h. Adsorption at 570 nm was determined in the EL808 Ultra Microplate Reader with KC4 V3.0 analysis software (Biotek Instruments) and is directly proportional to the growth rate of the cells. For determining the subG1 DNA content cells were harvested by centrifugation. The pellet was resuspended and fixed in 80% Ethanol in Hank’s Buffered Salt Solution (HBSS) for 30 min at
20  C. After washing the cells in PBS, they were incubated with 50 mg/mL propidium iodide (PI) and 50 mg/mL RNaseA. Cells were measured in the FL-A/FL-W to gate out duplets with the FACS Calibur (Becton Dickinson) and analyzed with FCSExpress. For measuring the loss of mitochondrial membrane potential cells were harvested by centrifugation, washed in PBS, resuspended in 40 nmol/L DiOC6 in PBS and incubated for 30 min at 37  C. The mitochondrial membrane potential was measured in the FL1 channel of the FACS Calibur (Becton Dickinson). Annexin V/Propidium Iodide staining was performed with a commercially available kit according to manufacturer’s instructions (MEBCYTO Apoptosis Kit, MBL International). 2.7. Animal studies uPAR
/
mice and the corresponding C57BL/6 control mice were bred under pathogen free conditions in the animal facility of the

Faculty of Medicine (Technion, Israel Institute of Technology, Haifa, Israel). Male mice at 8e12 weeks of age were intraperitoneally injected with thioglycollate. After three days mice were sacrificed for harvesting peritoneal macrophages. 2.8. Statistics For statistical analysis and graphs, Microsoft Excel 2010 was used. Biologically independent experiments were performed in duplicates and replicated three times (n ¼ 3). For determination of the statistical significance, Student’s t test was performed. pValues < 0.05 were considered statistically significant and are indicated as follows * ¼ p-value <0.05, ** ¼ p-value <0.001. 3. Results 3.1. uPA enhances PMA-induced monocyte-to-macrophage differentiation THP-1 cells were treated with PMA (25 ng/ml) for one or three days in the absence or presence of 5 nmol/L uPA. Morphologic analysis of adherent cells by light microscopy after 1 day in culture revealed that PMA or uPA alone induced cell adherence, however no morphological changes could be detected. On the contrary, cells that were incubated for 1 day with a combination of PMA and uPA

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exhibited the characteristic macrophage morphology, namely larger cell size, increased membrane ruffles, small nucleus and large cytoplasm filled with vacuoles (Fig. 1A), suggesting that uPA potentiated PMA-induced monocyte differentiation. For further proving this observation, we next examined the expression of CD36, a cell surface protein that marks the differentiation of monocytes into macrophages. Incubation of THP-1 monocytes with uPA alone for three days had no influence on CD36 surface expression (Fig. 1B). PMA alone increased CD36 surface expression over three days in culture by 3.4 fold, and the addition of uPA intensified PMA-induced differentiation by 41% (Fig. 1B). In order to further prove these findings, we examined actin re-organization using actin immunostaining. Fig. 1C demonstrates that control and uPA treated cells had a round shape and actin was distributed evenly over the cells. Cells stimulated with PMA, or with PMA and uPA had undergone dramatic shape changes and were characterized by the appearance of actin clusters distributed throughout the cytoplasm (Fig. 1C). Immunostaining of CD36 on these cells clearly shows that uPA intensified the effect of PMA on the abundance of cellular CD36, thus further justifying our conclusion that uPA enhances PMA-stimulated THP-1 monocyte differentiation. Next, we questioned whether uPAeuPAR interaction was involved in this process. Incubation of THP-1 monocytes with PMA in the presence of uPA significantly increased CD36 surface expression in comparison to the effect of PMA alone (Fig. 2A) and similar results were observed upon incubation of the cells with PMA in the presence of ATF, which is an amino terminal fragment of uPA that lacks catalytic activity but still binds to uPAR, suggesting that binding to uPAR may be sufficient in order to potentiate PMAinduced monocyte differentiation. Alternatively, THP-1 monocytes were incubated with PMA in combination with uPA or with ATF in the presence of a mouse anti-human uPAR blocking antibody (antiCD87). Anti-CD87 alone had no effect on PMA-induced monocyte differentiation. On the contrary, addition of anti-CD87 completely abolished the stimulatory effect of both uPA and ATF on PMAinduced monocyte differentiation, estimated as CD36 surface expression (Fig. 2A). These results indicate that binding of uPA to its receptor is necessary for promoting PMA-induced monocyte differentiation. To investigate the physiological relevance of uPAeuPAR interaction to monocyte differentiation, peritoneal macrophages were harvested from uPAR-KO and C57BL/6 mice and the number of

MFI, AU

4

3.2. uPA accelerates G1 arrest of PMA-treated THP-1 cells It is well known that cell differentiation is preceded by G1 arrest. In order to get an insight into the mechanism by which uPA stimulates monocyte differentiation, we questioned whether uPA interfered with the cell cycle of the THP-1 cells. THP-1 monocytes were treated for 5 h with PMA in the absence or presence of uPA, after which we performed cell cycle staining. Aphidicolin, a DNA polymerase inhibitor which arrests cells in the G1 phase, served as a positive control. PMA increased the cells arrested in the G1 state by 15% compared to control cells. Addition of uPA on top of PMA significantly increased the cells arrested in the G1 state by 40%, in a similar manner as aphidicolin (Fig. 3A). Consequently, 13% less cells underwent mitosis when they were treated with PMA, and this was further significantly decreased by 23% or by 34% in the presence of aphidicolin or uPA, respectively (Fig. 3B). In parallel, PMA increased by 4 fold the number of monocytes undergoing apoptosis, as measured by the appearance of a subG1 peak, and this effect was further potentiated by aphidicolin, as expected [30]. On the contrary, uPA significantly inhibited PMA-induced apoptosis by 18% (Fig. 3C). Altogether, these results confirm that uPA accelerates PMA-induced monocyte differentiation, at least in part by accelerating G1 arrest and down regulating G2/S phase, and also by inhibiting PMA-induced apoptotic cell death. 3.3. uPA inhibits Ox-LDL-induced macrophage death Next, we investigated our second hypothesis that uPA inhibits macrophage cell death. To circumvent the toxic effect of PMA we used the more mature monocytic cell line, MonoMac6 (MM6) as a macrophage model system to study cell death. MonoMac6 has the ability to produce cytokines and to phagocytose. Furthermore, it expresses certain macrophage surface markers such as CD11b, CD18, CD68 [31] and it has the ability to adhere to endothelial cells [32]. First of all, we confirmed the upregulation of the cell surface

A: CD36 Expression in THP-1

B: Mouse Peritoneal Macrophages 400

- anti uPAR Ab + anti uPAR Ab

*

350

*

300

Cell number/mL

5

macrophages per mL volume was determined. The number of peritoneal macrophages harvested from uPAR-KO was lower by 30% than the peritoneal macrophages harvested from C57BL/6 mice (Fig. 2B). This could be due to acceleration of monocyte-tomacrophage differentiation, but also to the inhibition of mature macrophage clearance via apoptosis, or both.

3

2

*

250 200 150 100

1 50 0

0

Control

PMA

PMA+ uPA

PMA+ ATF

C57BL/6 mice

uPAR-KO mice

Fig. 2. Role of uPAR. THP-1 cells were treated with PMA in absence or presence of uPA (5 nmol/L), or of the amino-terminal fragment (ATF) (5 nmol/L) in the absence (black bars) or presence (gray bars) of a blocking anti-uPAR antibody. Untreated cells served as Control. CD36 surface expression was determined by FACS. Results are expressed as mean  SD of three separate experiments. *p < 0.05, with vs. without uPA or ATF. (A) The number of peritoneal macrophages per mL volume harvested three days after intraperitoneal injection of thioglycollate from seven C57BL/6 mice (left) or seven uPAR
/
mice (right). *p < 0.05, uPAR
/
mice compared to C57BL/6 mice (B).

N. Paland et al. / Atherosclerosis 231 (2013) 29e38

A. Cells in G1 state 80

*

*

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% of Cells

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33

absence or presence of increasing concentrations of uPA, and the number of dead cells was determined by measuring the loss of mitochondrial membrane potential. Ox-LDL induced a 3.7 fold increase in macrophage apoptosis (Fig. 4A). uPA inhibited Ox-LDLinduced macrophage apoptosis in a dose dependent manner. A maximal inhibition of 51% was reached by 5 nmol/L of uPA, and therefore this concentration was used in all other experiments. Next, MM6 cells were treated with 10 or 20 mg/mL of Ox-LDL for 24 h in the absence or presence of uPA, and the number of dead cells was determined by different approaches. The trypan blue exclusion assay showed that Ox-LDL remarkably increased cell death in a dose-dependent manner, whereas addition of uPA significantly decreased Ox-LDL-induced cell death by up to 25% (Fig. 4B). In order to evidence that the attenuation of Ox-LDLinduced cell death by uPA was not due to an increase in the proliferation rate, we measured cell proliferation utilizing the Wst-1 proliferation assay. uPA alone had no effect on cell proliferation. Ox-LDL (10 or 20 mg/mL) decreased the proliferation rate of MM6 cells by 25% and 70%, respectively, and this effect was completely abolished by uPA (Fig. 4C), suggesting that uPA rendered the cells alive, but did not induce proliferation. Next, we further evaluated Ox-LDL-induced cell apoptotic death by measuring the subG1-DNA content of MM6 cells treated with 10 or with 20 mg/ml of Ox-LDL in the presence or absence of uPA. Ten or 20 mg/ml of Ox-LDL increased the cell number in the subG1 state by 2.4 and by 3.9 fold, respectively, whereas uPA inhibited this effect by 19% and 32%, respectively (Fig. 4D). Alternatively, cell death was assessed as the loss of the mitochondrial membrane potential. Loss of the mitochondrial membrane potential in MM6 cells treated with 10 or 20 mg/ml of Ox-LDL increased by 2.8 and 3.9 fold, respectively, and uPA significantly attenuated this effect by 30% (Fig. 4E). To further confirm that uPA inhibited Ox-LDL-induced cell death we performed an Annexin V/PI assay (Fig. 4F). Ox-LDL decreased the number of viable cells by 53% and in parallel increased the number of apoptotic cells (Anþ/PIþ) by 143%. However, pre-treatment with uPA prior to exposure to Ox-LDL, attenuated these effects by 17% and by 25%, respectively. Taken altogether these results evidence that uPA inhibits Ox-LDL-induced cell death. 3.4. uPA attenuates ER-stress-induced cell death

10

5

0

Control

PMA

PMA + Aphidicolin

PMA + uPA

Fig. 3. uPA accelerates G1 arrest and inhibits PMA-induced cell death. THP-1 monocytes were treated with PMA (25 ng/ml) in the absence or presence of uPA (5 nmol/L) or in the absence or presence of adiphicoline for 5 h. Graphs show quantification of cell cycle phases G1 (A), G2/S (B) and subG1 (C). Untreated cells served as Control. Results are expressed as mean  SD of three separate experiments. *p < 0.05, with vs. Control; #p < 0.05 PMA þ uPA vs. PMA.

In order to find mechanistic insights into the reduction of OxLDL-induced cell death by uPA, we examined whether uPA could also inhibit cell death by other stimuli. Thus, we treated MM6 cells with thapsigargin, an inhibitor of SERCA (sarco-endoplasmic reticulum Ca2þ-ATPase) or with staurosporine, a protein kinase inhibitor, in the absence or presence of uPA. Cell death was measured by determining the loss of the mitochondrial membrane potential. Ox-LDL, thapsigargin or staurosporine increased cell death by 4, 1.9 and 2 fold, respectively. Addition of uPA significantly attenuated Ox-LDL or thapsigargin-induced cell death by 43% and 35%, respectively, but had no effect on staurosporin-induced cell death (Fig. 5), suggesting that uPA inhibits cell death that is mediated via the ER-stress pathway, and it requires the activation of a kinase for exhibiting its anti-apoptotic action. 3.5. uPA inhibits Ox-LDL-induced cell death via ERK1/2 activationdependent Bim downregulation

macrophage markers CD11b, CD18, CD68 and CD36 compared to THP-1 monocytes. All macrophage surface markers were remarkably upregulated (Supplementary Figure S1). Apoptosis of MM6 macrophages was induced by Ox-LDL, as OxLDL was shown to induce apoptosis and necrosis of macrophages [33]. MM6 cells were treated with Ox-LDL (10 mg/ml) for 24 h in the

We have shown previously that uPA activates the MAPKinase MEK1/2, the upstream kinase of ERK1/2 in macrophages [19,34]. Furthermore, activated ERK1/2 was shown to have an antiapoptotic function [35]. In order to elucidate the signaling mechanisms involved in uPA-inhibition of Ox-LDL-induced cell death, we

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Fig. 4. uPA attenuates Ox-LDL induced apoptosis. MonoMac6 cells (MM6) were treated with Ox-LDL (10 mg of protein/ml) without or with increasing concentrations of uPA for 24 h. Cell death was measured as the loss of mitochondrial membrane potential expressed as the percentage of dead cells. Results are expressed as mean  SD of three separate experiments. *p < 0.05, with vs. without uPA (A). MM6 cells were treated with Ox-LDL (10 or 20 mg/mL) in the absence (black bar) or presence of 5 nmol/L uPA (gray bars). Cell death was measured as the percentage of dead cells obtained by the trypan blue exclusion assay (B), the absorption A450 nm obtained by the Wst-1 assay representing living cells (C), the subG1 DNA-content e left panel shows a representative histogram of subG1 staining of MM6 cells left untreated (control) or treated with 10 or 20 mg/ml Ox-LDL or in the absence of uPA (upper panel) or in the presence of uPA (lower panel) (D), the loss of mitochondrial membrane potential (E) and annexin V/PI cell death assay e upper panels show representative histograms of annexin V/PI staining of MM6 cells left untreated (control), treated with uPA, or treated with 20 mg/ml Ox-LDL alone or in the presence of uPA (F). Results are expressed as mean  SD of three separate experiments. *p < 0.01, vs. Control, #p < 0.05, Ox-LDL with vs. without uPA.

treated MM6 cells with Ox-LDL and uPA in the presence of the chemical inhibitor UO126 that targets MEK1/2. Fig. 6 shows that uPA significantly inhibited Ox-LDL-induced cell death, measured as the trypan blue exclusion (Fig. 6A), proliferation assay (Fig. 6B) and loss of the mitochondrial membrane potential (Fig. 6C), and these

effects were completely abrogated (Fig. 6A and B), or significantly attenuated (Fig. 6C) by UO126. The inhibitor UO126 alone had no effect on Ox-LDL-induced cell death. Immunoblot of activated ERK1/2 (Fig. 6D) shows that both Ox-LDL alone or uPA alone activate ERK1/2, however uPA further potentiate ERK1/2 activation

N. Paland et al. / Atherosclerosis 231 (2013) 29e38

MFI, DiOC6 (% of dead cells)

80

- uPA + uPA 60

*

40

* 20

0

Control

Ox-LDL (10 μg/mL)

Thapsigargin

Staurosporine

Fig. 5. uPA attenuates ER stress-induced cell death. MM6 cells were treated with OxLDL (20 mg/ml), Thapsigargin (5 nmol/L) or Staurosporine (1 mmol/L) in the absence (black bars) or in the presence of 5 nmol/L uPA (gray bars) for 24 h. Cell death was determined by measuring the loss of mitochondrial membrane potential. Results represent the percentage of dead cells expressed as mean  SD of three separate experiments. *p < 0.05, with vs. without uPA.

35

induced by Ox-LDL. Altogether, these results evidence that the activation of ERK1/2 by uPA is crucial for the attenuation of Ox-LDLinduced cell death by uPA. ERK1/2 is an upstream regulator of the proapoptotic protein Bim, a member of the Bcl-2 protein family, which are essential regulators of apoptosis [36]. There are three isoforms of the Bim protein, BimEL, BimL and BimS, all of which promote apoptosis but differ in potency, BimEL being the most potent [37]. Thus, next we questioned whether uPA downregulates Bim expression in macrophages stimulated with Ox-LDL. Ox-LDL strongly upregulated the protein expression of all three isoforms of Bim (Fig. 6D), whereas uPA markedly attenuated this effect. However, the MEK inhibitor UO126 completely abolished the inhibitory effect of uPA on OxLDL-mediated upregulation of Bim expression. Collectively, these results demonstrate that Ox-LDL-induced macrophage apoptotic death is attenuated by uPA via ERK1/2 activation-dependent downregulation of Bim.

4. Discussion This study demonstrates for the first time that uPA enhances the PMA-induced differentiation of monocytes into macrophages by accelerating G1 arrest, down regulating G2/S phase and inhibiting PMA-induced apoptosis. Moreover, uPA inhibited Ox-LDL-induced apoptosis in mature macrophages. These major findings are

A:Trypan blue exclusion assay

B: Proliferation assay

50

- uPA + uPA (5nmol/L)

30

*

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10

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* .4

.2

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Control

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.6

Absorption at 450 nm

% of dead cells

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Ox-LDL + UO126

C: Loss of mitochondrial membrane potential

Control

Ox-LDL

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D: ERK and Bim activation

20

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- uPA + uPA

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% of dead cells

15

**

+

-

+

-

+

-

+ uPA

-

+

-

+

-

+ uPA

pERK

10

-

+

BimEL

* 5

BimL BimS

0

Control

Ox-LDL

Ox-LDL + UO126

β-Actin

Control

Ox-LDL

Control

Ox-LDL

Fig. 6. uPA inhibits Ox-LDL-induced cell death via ERK1/2 activation-dependent Bim downregulation. MM6 cells were treated with Ox-LDL (10 mg/ml) in the absence (black bars) or presence of 5 nmol/L uPA (gray bars) for 24 h. In order to inhibit ERK1/2 activation the MEK1/2 inhibitor UO126 (10 nmol/L) was added to some samples. Cell death was assessed by trypan blue exclusion (A). Proliferation was measured with the Wst-1 assay (B). The loss of mitochondrial membrane potential was measured by DiOC6 (C). Results are expressed as mean  SD of three separate experiments. *p < 0.05, **p < 0.001 with vs. without uPA. Immunoblot of MM6 cells untreated or treated with Ox-LDL in the absence or presence of uPA and in the absence (left panel) or presence of UO126 (right panel). Membranes were probed against phosphorylated ERK1/2 (upper lanes) or against the three isoforms of Bim, BimEL, BimL or BimS (middle panel), Equal loading was monitored by b-Actin (lower panel) (D).

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Fig. 7. Proposed model for the effect of uPA on monocyte differentiation and cell death. The intima of an atherogenic blood vessel is illustrated. It is surrounded by layers of endothelial cells (EC) and smooth muscle cells (SMC). Monocytes are persistently infiltrated into the intima of the vessel. There they differentiate into macrophages. uPA binds to uPAR and accelerates monocyte differentiation into macrophages. Differentiated macrophages take up oxidized LDL (Ox-LDL), leading to foam cell formation, which eventually undergo apoptotic death. Cell death of macrophage-foam cells due to overload with Ox-LDL is inhibited by uPA, leading to increased lesion cellularity.

schematically presented in Fig. 7. The antiapoptotic activity of uPA was dependent on the presence of uPAR, and it involved ERK1/2 activation-dependent downregulation of the proapoptotic protein Bim in macrophages stimulated with Ox-LDL. The importance of uPA and its receptor uPAR for migration and differentiation was shown recently in mesenchymal stem cells [38], and in monocytes [26]. In agreement, our study further confirmed the role of uPAeuPAR interaction in this process, and expanded the knowledge on the mechanisms involved. It was previously shown that an autocrine interaction of uPA with its receptor uPAR mediates an essential step in PMA-mediated myeloid cell differentiation [26]. In agreement, our study shows that increasing extracellular concentration of uPA potentiate the stimulation of cell differentiation by PMA. PMA is known to induce not only differentiation, but also apoptosis in monocytes [39]. Our data demonstrates that uPA increased macrophage viability by inhibiting PMA-induced cell death, which would account for another option to intensify differentiation. Macrophage death constitutes one of the key events in the biology of atherosclerotic lesion development. Macrophage death in the artery wall proceeds via apoptosis and necrosis, the latter being either a primary event (primary necrosis) or secondary to apoptosis (secondary necrosis). In the present study we induced macrophage death using heavily oxidized LDL (Ox-LDL), in accordance to previous studies [40]. However, opposite effects were also demonstrated [41], which probably stem from different modes of oxidation [33], as well as the degree of oxidation [42]. Our results show that uPA inhibited apoptosis of macrophages treated with OxLDL. The consequences of this effect must be considered however in relation to the stage of the atherosclerotic lesion. Macrophage apoptosis occurs throughout all stages of atherosclerosis, however the consequences of this event may be very different in early vs. late advanced atherosclerotic lesions [43]. In early lesions,

macrophage apoptosis is associated with diminished lesion cellularity and decreased lesion progression [44]. Thus, inhibition of macrophage apoptosis by uPA at early stages of atherosclerotic lesion development may favor increased progression of atherosclerosis, and may explain the pro-atherogenic role of uPA. In late lesions, however, increased apoptosis is associated with plaque necrosis. It would seem then that inhibition of apoptosis by uPA in advanced lesions would be beneficial in relation to plaque development. However, increased number of surviving macrophages in advanced lesions might promote lesion progression by other means, such as by secreting pro-atherothrombotic molecules [45]. Furthermore, viable macrophages in advanced lesion will ingest Ox-LDL, transforming to foam cells, which eventually will undergo primary necrosis, promoting plaque instability. Moreover, Ox-LDL eventually causes accumulation of oxysterols and free cholesterol in macrophages, which can induce ER stress. Prolonged ER stress leads to cell necrotic death, and primary necrosis of macrophages in advanced lesions may not only be involved in plaque formation, but also cause more inflammatory cell infiltration, thus having a strong proinflammatory and proatherogenic role [46]. Evidence from in vivo studies in apo E knockout mice further argues that apoptosis of macrophages is beneficial even in advanced lesions [47]. In light of these adverse effects, uPA-mediated inhibition of Ox-LDLinduced apoptosis may be proatherogenic. Our present study demonstrates that the proatherogenic activity of uPA was dependent on the presence of uPAR, suggesting thus a proatherogenic role for uPAR in atherosclerosis. However, until now there is no consensus on the role of the uPAR in atherosclerosis. Farris et al., recently published that uPA-mediated atherosclerosis in the LDL
/
mouse model for atherosclerosis is largely independent of uPAR [48], however this effect could be demonstrated only in transgenic mice with macrophage-specific uPA overexpression. On the contrary, in non-transgenic mice (in the

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absence of uPA overexpression) a role for uPAR in atherogenesis was apparent, as the absence of uPAR significantly reduced both the aortic root intimal lesion and aortic surface atherosclerosis [48]. These results are in accordance with our conclusion that the uPA/ uPAR system may be proatherogenic depending on the state of the atherosclerotic lesion. In summary, our results shed light on a novel anti-apoptotic effect of the uPAeuPAR system in macrophages exposed to OxLDL. These results support the proatherogenic role of uPA and may have implications for new therapeutical interventions. Sources of funding This study was supported by a grant from the Israel Science Foundation (ISF) No. 669/09, Founded by The Israel Academy of Sciences and Humanities, and by a grant from the Israeli Ministry of Health, Chief Scientist Office, No. 3-7364. Disclosures None. Appendix A. Supplementary material Supplementary material associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. atherosclerosis.2013.08.016. References [1] Moore KJ, Tabas I. Macrophages in the pathogenesis of atherosclerosis. Cell 2011;145(3):341e55. [2] Vicca S, Hennequin C, Nguyen-Khoa T, et al. Caspase-dependent apoptosis in THP-1 cells exposed to oxidized low-density lipoproteins. Biochem Biophys Res Commun 2000;273(3):948e54. [3] Wintergerst ES, Jelk J, Rahner C, Asmis R. Apoptosis induced by oxidized low density lipoprotein in human monocyte-derived macrophages involves CD36 and activation of caspase-3. Eur J Biochem 2000;267(19):6050e9. [4] Tabas I. Macrophage death and defective inflammation resolution in atherosclerosis. Nat Rev Immunol 2010;10:36e46. [5] Leist M, Jäättelä M. Four deaths and a funeral: from caspases to alternative mechanisms. Nat Rev Mol Cell Biol 2001;2:589e98. [6] Kroemer G, Dallaporta B, Resche-Rigon M. The mitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol 1998;60:619e42. [7] Youle RJ, Strasser A. The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 2008;9:47e59. [8] Ashkenazi A, Dixit VM. Death receptors: signaling and modulation. Science 1998;281:1305e8. [9] Green DR, Kroemer G. The pathophysiology of mitochondrial cell death. Science 2004;305:626e9. [10] Scull CM, Tabas I. Mechanisms of ER stress-induced apoptosis in atherosclerosis. Arterioscler Thromb Vasc Biol 2011;31:2792e7. [11] Yao S, Zong C, Zhang Y, et al. Activating transcription factor 6 mediates oxidized LDL-induced cholesterol accumulation and apoptosis in macrophages by up-regulating CHOP expression. J Atheroscler Thromb 2013;20(1): 94e107. [12] Waltz DA, Fujita RM, Yang X, et al. Nonproteolytic role for the urokinase receptor in cellular migration in vivo. Am J Respir Cell Mol Biol 2000;22:316e 22. [13] Wei Y, Lukashev M, Simon DI, et al. Regulation of integrin function by the urokinase receptor. Science 1996;273:1551e5. [14] Wei Y, Waltz DA, Rao N, Drummond RJ, Rosenberg S, Chapman HA. Identification of the urokinase receptor as an adhesion receptor for vitronectin. J Biol Chem 1994;269:32380e8. [15] Liu D, Aguirre Ghiso J, Estrada Y, Ossowski L. EGFR is a transducer of the urokinase receptor initiated signal that is required for in vivo growth of a human carcinoma. Cancer Cell 2002;1:445e57. [16] Kiyan J, Kiyan R, Haller H, Dumler I. Urokinase-induced signaling in human vascular smooth muscle cells is mediated by PDGFR-beta. EMBO J 2005;24: 1787e97. [17] Kienast J, Padro T, Steins M, et al. Relation of urokinase-type plasminogen activator expression to presence and severity of atherosclerotic lesions in human coronary arteries. Thromb Haemost 1998;79:579e86.

37

[18] Cozen AE, Moriwaki H, Kremen M, et al. Macrophage-targeted overexpression of urokinase causes accelerated atherosclerosis, coronary artery occlusions, and premature death. Circulation 2004;109:2129e35. [19] Fuhrman B, Nitzan O, Karry R, Volkova N, Dumler I, Aviram M. Urokinase plasminogen activator (uPA) stimulates cholesterol biosynthesis in macrophages through activation of SREBP-1 in a PI3-kinase and MEK-dependent manner. Atherosclerosis 2007;195:e108e16. [20] Fuhrman B, Khateeb J, Shiner M, et al. Urokinase plasminogen activator upregulates paraoxonase 2 expression in macrophages via an NADPH oxidase-dependent mechanism. Arterioscler Thromb Vasc Biol 2008;28: 1361e7. [21] Alfano D, Franco P, Vocca I, et al. The urokinase plasminogen activator and its receptor: role in cell growth and apoptosis. Thromb Haemost 2005;93:205e 11. [22] Tkachuk N, Kiyan J, Tkachuk S, et al. Urokinase induces survival or proapoptotic signals in human mesangial cells depending on the apoptotic stimulus. Biochem J 2008;415(2):265e73. [23] Prager GW, Mihaly J, Brunner PM, Koshelnick Y, Hoyer-Hansen G, Binder BR. Urokinase mediates endothelial cell survival via induction of the X-linked inhibitor of apoptosis protein. Blood 2009;113(6):1383e90. [24] Ma Z, Webb DJ, Jo M, Gonias SL. Endogenously produced urokinase-type plasminogen activator is a major determinant of the basal level of activated ERK/MAP kinase and prevents apoptosis in MDA-MB-231 breast cancer cells. J Cell Sci 2001;114(Pt 18):3387e96. [25] Luikart S, Masri M, Wahl D, et al. Urokinase is required for the formation of mactinin, an alpha-actinin fragment that promotes monocyte/macrophage maturation. Biochim Biophys Acta 2002;1591(1e3):99e107. [26] Nusrat AR, Chapman Jr HA. An autocrine role for urokinase in phorbol estermediated differentiation of myeloid cell lines. J Clin Invest 1991;87(3):1091e 7. [27] Fuhrman B, Partoush A, Volkova N, Aviram M. Ox-LDL induces monocyte-tomacrophage differentiation in vivo: possible role for the macrophage colony stimulating factor receptor (M-CSF-R). Atherosclerosis 2008;196:598e607. [28] Janero DR. Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radic Biol Med 1990;9:515e54. [29] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteinedye binding. Anal Biochem 1976;72:248e54. [30] Kuwakado K, Kubota M, Hirota H, et al. Aphidicolin potentiates apoptosis induced by arabinosyl nucleosides in human myeloid leukemia cell lines. Biochem Pharmacol 1993 Dec 3;46(11):1909e16. [31] Ziegler-Heitbrock HW, Thiel E, Futterer A, Herzog V, Wirtz A, Riethmuller G. Establishment of a human cell line (Mono Mac 6) with characteristics of mature monocytes. Int J Cancer 1988;41:456e61. [32] Erl W, Weber C, Wardemann C, Weber PC. Adhesion properties of Mono Mac 6, a monocytic cell line with characteristics of mature human monocytes. Atherosclerosis 1995;113:99e107. [33] Ermak N, Lacour B, Goirand F, Drüeke TB, Vicca S. Differential apoptotic pathways activated in response to Cu-induced or HOCl-induced LDL oxidation in U937 monocytic cell line. Biochem Biophys Res Commun 2010;393(4): 783e7. [34] Fuhrman B, Gantman A, Khateeb J, et al. Urokinase activates macrophage PON2 gene transcription via the PI3K/ROS/MEK/SREBP-2 signalling cascade mediated by the PDGFR-beta. Cardiovasc Res 2009;84:145e54. [35] Heo J-I, Oh S-J, Kho Y-J, et al. ERK mediates anti-apoptotic effect through phosphorylation and cytoplasmic localization of p21Waf1/Cip1 in response to DNA damage in normal human embryonic fibroblast (HEF) cells. Mol Biol Rep 2011;38:2785e91. [36] Ley R, Balmanno K, Hadfield K, Weston C, Cook SJ. Activation of the ERK1/2 signaling pathway promotes phosphorylation and proteasome-dependent degradation of the BH3-only protein, Bim. J Biol Chem 2003;278(21): 18811e6. [37] O’Connor L, Strasser A, O’Reilly LA, et al. Bim: a novel member of the Bcl-2 family that promotes apoptosis. EMBO J 1998;17(2):384e95. [38] Vallabhaneni KC, Tkachuk S, Kiyan Y, et al. Urokinase receptor mediates mobilization, migration, and differentiation of mesenchymal stem cells. Cardiovasc Res 2011;90(1):113e21. [39] Ohta H, Sweeney EA, Masamune A, Yatomi Y, Hakomori S, Igarashi Y. Induction of apoptosis by sphingosine in human leukemic HL-60 cells: a possible endogenous modulator of apoptotic DNA fragmentation occurring during phorbol ester-induced differentiation. Cancer Res 1995;55(3):691e7. [40] Giovannini C, Varì R, Scazzocchio B, et al. OxLDL induced p53-dependent apoptosis by activating p38MAPK and PKCd signaling pathways in J774A.1 macrophage cells. Mol Cell Biol 2011;3(5):316e8. [41] Namgaladze D, Kollas A, Brüne B. Oxidized LDL attenuates apoptosis in monocytic cells by activating ERK signaling. J Lipid Res 2008;49(1):58e65. [42] Boullier A, Li Y, Quehenberger O, et al. Minimally oxidized LDL offsets the apoptotic effects of extensively oxidized LDL and free cholesterol in macrophages. Arterioscler Thromb Vasc Biol 2006;26(5):1169e76. [43] Gautier EL, Huby T, Witztum JL, et al. Macrophage apoptosis exerts divergent effects on atherogenesis as a function of lesion stage. Circulation 2009;119(13):1795e804. [44] Liu J, Thewke DP, Su YR, Linton MF, Fazio S, Sinensky MS. Reduced macrophage apoptosis is associated with accelerated atherosclerosis in low-density

38

N. Paland et al. / Atherosclerosis 231 (2013) 29e38

lipoprotein receptor-null mice. Arterioscler Thromb Vasc Biol 2005;25(1): 174e9. [45] Kockx MM, Herman AG. Apoptosis in atherogenesis: implications for plaque destabilization. Eur Heart J 1998;19(Suppl. G):G23e8. [46] Lin J, Li H, Yang M, et al. A role of RIP3-mediated macrophage necrosis in atherosclerosis development. Cell Rep 2013;3(1):200e10.

[47] Secchiero P, Candido R, Corallini F, et al. Systemic tumor necrosis factorrelated apoptosis-inducing ligand delivery shows antiatherosclerotic activity in apolipoprotein E-null diabetic mice. Circulation 2006;114(14):1522e30. [48] Farris SD, Hu JH, Krishnan R, et al. Mechanisms of urokinase plasminogen activator (uPA)-mediated atherosclerosis: role of the uPA receptor and S100A8/A9 proteins. J Biol Chem 2011;286(25):22665e77.