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Effect of CCL2 on BV2 microglial cell migration: Involvement of probable signaling pathways
Cytokine 81 (2016) 39–49
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Effect of CCL2 on BV2 microglial cell migration: Involvement of probable signaling pathways Shambhunath Bose, Sunyoung Kim, Yeonsoo Oh, Md. Moniruzzaman, Gyeongjun Lee, Jungsook Cho ⇑ College of Pharmacy, Dongguk University-Seoul, Goyang, Gyeonggi-do 10326, Republic of Korea
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Article history: Received 8 July 2015 Received in revised form 3 February 2016 Accepted 4 February 2016
Keywords: CCL2 CCR2 BV2 cells Migration Intracellular signaling pathways
a b s t r a c t Microglia, the resident macrophages of the central nervous system, play a vital role in the regulation of innate immune function and neuronal homeostasis of the brain. Currently, much interest is being generated regarding the investigation of the microglial migration that results in their accumulation at focal sites of injury. Chemokines including CCL2 are known to cause the potential induction of migration of microglial cells, although the underlying mechanisms are not well understood. In the present study, using murine neonatal BV2 microglial cells as a model, we investigate the impact of CCL2 on the migration of microglial cells and address the probable molecular events within the cellular signaling cascades mediating CCL2-induced cell migration. Our results demonstrate concentration- and time-dependent induction of BV2 cell migration by CCL2 and reveal complex mechanisms involving the activation of MEK, ERK1/2, and Akt, and their cross-talk. In addition, we demonstrate that the MEK/ERK pathway activated by CCL2 treatment mediate p90RSK activation in BV2 cells. Moreover, our findings indicate that Akt, ERK1/2, and p90RSK are the downstream effectors of PI3K in the CCL2-induced signaling. Finally, phosphorylation of the transcription factors c-jun and ATF-1 is found to be a further downstream signaling cascade in the CCL2-mediated action. Our results suggest that CCL2-induced activation of c-jun and ATF-1 is likely to be linked to the MEK/ERK and PI3K signaling pathways, respectively. Taken together, these findings contribute to a better understanding of CCL2-induced microglial migration and the probable signaling pathways involved. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Microglia, the resident cells of the central nervous system, are originally derived from the monocyte/macrophage lineage. They play a key role in the innate immune regulation and neuronal homeostasis of the brain [1], and act as a primary defense mechanism against invading pathogens [2]. In a pathological condition, microglia are triggered and activated through a cascade of events. The entire process involves proliferation, migration to the site of injury, elevated expression of immunomodulators in the cells, and finally transformation into the phagocytes that are dedicated to removing the damaged cells and debris [3] through the process of inflammation. Chemokine molecules are known to play a vital role in the regulation of the movement of microglia and their recruitment to sites of inflammation [4,5]. As such, microglial migration involves cell polarization and the generation of lamellipodia at the leading edge, known as ruffling. This event triggers the redistribution of specific ⇑ Corresponding author. E-mail address: [email protected] (J. Cho). http://dx.doi.org/10.1016/j.cyto.2016.02.001 1043-4666/Ó 2016 Elsevier Ltd. All rights reserved.
molecules, such as integrins, and the polymerization and depolymerization of filamentous actin, resulting in the deformation of shape. In association, a number of proteases are released in order to sustain the dynamic process of cell attachment and detachment [6], eventually leading to elongation of the lamellipodia and filopodia that are required for cell migration [7]. Among the chemokines, CCL2, also termed monocyte chemotactic protein (MCP)-1, has been found to be one of the most potent microglial chemoattractants. CCL2 is produced by different kinds of cells, either constitutively or following induction by various factors including oxidative stress, cytokines, or growth factors [8]. CCL2 binds to the CC-chemokine receptor 2 (CCR2), a member of the G protein-coupled receptors, expressed on microglial cell membranes, triggering the assembly of these cells at pathological sites [9]. The CCL2–CCR2 interaction has been demonstrated to induce changes in the actin polymerization and subsequent reorganization of the actin cytoskeleton, the generation of focal adhesions, and the extension of pseudopodia, which collectively are responsible for the migration of activated microglia [4,10,11]. A number of studies have been performed in order to better understand the molecular mechanism(s) underlying the
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chemokine-induced migratory activity of microglial cells. It has been demonstrated that mitogen-activated protein kinases (MAPKs) play vital roles in cell migration [12]. Accumulating genetic and pharmacological evidence strongly supports the involvement of phosphoinositide 3-kinase (PI3K) as a major biochemical signal for chemotaxis in response to a number of chemokines [13]. Further, it has been revealed that multiple independent signal transduction pathways are induced in monocytes by CCL2, and the activation of all these pathways is essential in order to produce a maximal chemoattractant response [14]. However, to the best of our knowledge, no studies have yet been conducted in order to understand the probable molecular mechanism(s) underlying the CCL2-induced migration of microglial cells. In the present study, we investigate the impact of CCL2 on the migration of microglial cells using BV2 cells as a model, which are originally derived from raf/myc-immortalized murine neonatal microglia and are the most commonly used substitute for primary microglia [15]. Furthermore, to explore the probable molecular mechanism(s) underlying the CCL2-induced microglial migration, we study the signal transduction pathways linked to CCR2 activation, with an emphasis on the MAPK and PI3K/Akt pathways, the major signaling cascades utilized by CCL2 in a variety of cell types involved in the immune system [16,17]. 2. Materials and methods 2.1. Chemicals and antibodies Dulbecco’s Modified Eagle’s Medium (DMEM) and 0.05% trypsin–EDTA were purchased from Thermo Scientific (Rockford, IL, USA). Anti-b-actin antibody (monoclonal), and AS605240 were acquired from Sigma–Aldrich (St. Louis, MO, USA). RS504393 was obtained from Tocris Cookson Ltd. (Bristol, UK). PI3Ka inhibitor VIII was purchased from Calbiochem (San Diego, CA, USA). Antiphospho-MEK1/2 (Ser217/221), anti-MEK1/2, anti-phosphoERK1/2 (Thr202/Tyr204), anti-ERK1/2, anti-phospho-p90RSK (Ser380), anti-RSK1/RSK2/RSK3, anti-phospho-c-jun (Ser63), antic-jun, anti-phospho-CREB (Ser133), and horseradish peroxidase (HRP)-linked anti-rabbit immunoglobulin G (IgG) antibodies, LY294002, U0126, and wortmannin were acquired from Cell Signaling Technology (Danvers, MA, USA). Anti-CCR2 antibody and its blocking peptide were procured from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Additional anti-CCR2 antibodies were purchased from GeneTex, Inc. (Irvine, CA, USA) and Novus Biologicals (Littleton, CO, USA). HRP-linked anti-mouse IgG antibody was the product of Merck (Darmstadt, Germany). Recombinant rat CCL2 was purchased from PeproTech Inc. (Rocky Hill, NJ, USA).
specific protein kinases or a specific antagonist of CCR2 for 1 h prior to exposure to CCL2. Following the desired treatments, the cells were washed three times with ice-cold PBS, pH 7.4, and then lysed in lysis buffer [10 mM Tris–HCl, pH 7.4; 150 mM NaCl; 2 mM EDTA; 4.5 mM sodium pyrophosphate; 10 mM bglycerophosphate; 1 mM NaF; 1 mM Na3VO4; 1% (v/v) Triton X100; 0.5% (v/v) NP-40; and one tablet of protease inhibitor cocktail (Roche Diagnostic GmbH, Mannheim, Germany)] for 30 min on ice. Lysates were subjected to centrifugation at 14,000 rpm for 30 min at 4 °C to remove the insoluble material, and the collected supernatants were stored at 80 °C until use. The protein concentrations of the supernatants were measured using a BioRad DC protein assay kit (BioRad, Hercules, CA, USA). The samples were denatured at 100 °C in Laemmli sample buffer (BioRad) containing 5% bmercaptoethanol. Equal amounts of protein (30 lg) were resolved by SDS-PAGE using 8% or 10% gels, and electrophoretically transferred to nitrocellulose membrane (GE Healthcare UK Ltd., Buckinghamshire, UK) for 90 min at 100 V [18]. The membranes were blocked with Tris–buffered saline (TBS) containing 0.1% Tween 20 (TBST) and 5% non-fat dried milk (BD Falcon, Sparks, MD, USA) for 90 min to prevent non-specific binding. Following this, the membranes were washed three times with TBST at room temperature, and then incubated overnight at 4 °C with the specific primary antibodies in 5% bovine serum albumin (USB, Canton, OH, USA). After thorough washing with TBST, the membranes were incubated for 90 min with the appropriate HRP-conjugated antiIgG secondary antibodies, and the immunoreactive bands were detected by a BioRad ChemiDoc XRS imaging system (BioRad) using Super Signal West Pico ECL reagent (Thermo Fisher Scientific, San Jose, CA, USA). For the blocking peptide experiment, anti-CCR2 antibody (Santa Cruz) was incubated overnight at 4 °C with its blocking peptide (Santa Cruz) before incubations with nitrocellulose membrane. 2.4. Assessment of cell migration
BV2 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% antibiotic–antimycotic agent at 37 °C in an incubator under a humidified atmosphere of air containing 5% CO2. The cells were then plated in either 35-mm culture dishes (2 106 cells/dish) or 24-well culture plates (5 105 cells/well) for Western blotting analysis and cell migration assays, respectively.
The migration of cells was investigated by an in vitro scratch wound assay as described previously [19,20] with some modifications. After culturing for 24 h in 24-well plates, confluent monolayers of cells were scratched using a 200 ll pipette tip to produce a wound. Cultures were gently washed with DMEM to remove loosely attached cells and then treated with vehicle (control) or CCL2 as indicated in the Figures. A Nikon (Tokyo, Japan) Eclipse TS100 inverted microscope, a Digital sight DS-U2 camera (Nikon), and NIS-Elements F 3.0 image software (Nikon) were used to capture images of the wounded edges. The images were acquired with a 4X objective. For other experiments where the roles of CCR2 and different protein kinases in CCL2-induced cell migration were evaluated, the cells were treated with specific pharmacological inhibitors of the respective protein kinases or a selective antagonist of CCR2 for 1 h prior to the wound induction, and after scratching they were treated with 30 nM CCL2 in the presence of these agents. The control group did not receive any treatment except the corresponding vehicles. The images were acquired at 0 and 12 h of CCL2 treatment. The difference between the width of the wound at time 0 and at the time point being analyzed was measured. This value reflects the relative distance migrated by the cells during a given time interval. The migration distance of the treated groups was expressed as a percentage of the respective control.
2.3. Western blotting
2.5. Statistical analyses
The cells were serum-starved overnight and then exposed to various concentrations of CCL2 for different time periods in serum-free medium as indicated in the Figures. In some experiments, the cells were treated with pharmacological inhibitors of
Data are presented as the mean ± SEM from at least three independent experiments. The Statistical Package for Social Science (SPSS) software program (version 11.5; SPSS, Chicago, IL, USA) was employed for the analyses of the data. A one way ANOVA,
2.2. Cell culture
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keeping with the previous studies, showing the two bands of CCR2 isoforms produced by alternate mRNA splicing [21–23]: CCR2A (45 kDa) and CCR2B (42 kDa). 3.2. Time-and concentration-dependent effects of CCL2 on cell migration
Fig. 1. Expression of CCR2 in BV2 cells. CCR2 protein expression was evaluated in BV2 cells using Western blot analyses. Immunoblotting was performed as described in Section 2 using anti-CCR2 antibody either unblocked (left panel) or blocked by prior incubation with its blocking peptide overnight at 4 °C (right panel).
followed by Bonferroni’s post hoc analysis, was performed to determine the statistical significance. Differences were considered significant at P < 0.05. 3. Results 3.1. Expression of CCR2 in BV2 cells Before investigating the effects of CCL2 on cell migration, we verified the expression of CCR2 in BV2 cells by Western blotting. In this study, using the anti-CCR2 antibody from Santa Cruz, we observed two prominent protein bands corresponding to 42 and 45 kDa, which were not detected in the membrane preincubated with the blocking peptide (Fig. 1). Similar profiles were also observed in the blots using anti-CCR2 antibodies from different commercial sources (data not shown). These findings are in
Treatment with CCL2 at the concentration of 30 nM caused increase in microglial motility at 6 and 12 h in a time-dependent manner, accounting for 303% and 391% cell migration, respectively, compared with the control (Fig. 2A and B). Although a marked wound healing was observed in the cells treated with CCL2 for 24 h (270% migration vs control), a substantial proliferation of the cells was also observed at this time point as reflected in the corresponding control. This event is likely to interfere with the assessment of cell migration, and because of this reason we opted a 12 h CCL2 treatment schedule for further experimental use, where a minimal intervention of the cell proliferation was noticed. In addition, we observed a concentration-dependent effect of CCL2 on the wound healing when the cells were exposed to 10, 30 and 60 nM CCL2 for 12 h (Fig. 2C and D). 3.3. Evaluation of probable roles of vital cell signaling cascades in the CCL2-induced cell migration 3.3.1. MEK/ERK and p90RSK pathways To explore the probable molecular mechanism(s) underlying CCL2-induced migration of BV2 cells, we first evaluated the impact of this chemokine on the vital cell signaling pathways. Our results
Fig. 2. Time- and concentration-dependent effects of CCL2 on the migration of BV2 cells. Microscopic images demonstrate migration of cells induced by either 30 nM CCL2 at different time points (A) or different concentrations of CCL2 at 12 h (C). The distance migrated by the cells at a particular time point or concentration of CCL2 treatment was quantitated as described in Section 2 and expressed as percentage of corresponding control (B and D, respectively). Each data point represents the mean ± SEM from at least three measurements. ⁄ P < 0.05, ⁄⁄ P < 0.01, and ⁄⁄⁄ P < 0.001 compared with corresponding control, # P < 0.05 vs 10 nM CCL2.
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Fig. 3. Time- and concentration-dependent effects of CCL2 on the phosphorylation of MEK1/2 (A and D), ERK1/2 (B and E), and p90RSK (C and F) in BV2 cells. Cells were treated with 30 nM CCL2 for the indicated periods of time or exposed to various concentrations of CCL2 for 30 min and Western blotting analyses were performed as described in Section 2. Representative blots from at least three individual experiments for each protein are shown (upper panel). The density of the bands in each blot was quantified by densitometric analysis and normalized to the amount of non-phospho forms of MEK1/2, ERK1/2, or p90RSK, wherever applicable (lower panel). Each data point represents the mean ± SEM from at least three measurements. ⁄ P < 0.05, ⁄⁄ P < 0.01, and ⁄⁄⁄ P < 0.001 vs corresponding control (0 h CCL2 treatment or without CCL2 treatment, wherever applicable); # P < 0.01 compared with 10 nM CCL2.
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Fig. 4. Effects of a CCR2 antagonist RS504393 and different protein kinase inhibitors on the CCL2-induced migration of BV2 cells. Microscopic images of cell migration induced by 12 h treatment with 30 nM CCL2 in presence or absence of RS504393 and the protein kinase inhibitors are shown (A); the distance migrated by the cells in the treated groups was quantitated as described in Section 2 and expressed as percentage of control (B). # P < 0.001 compared with control, ⁄⁄⁄ P < 0.001 compared with CCL2 alone-treated group. RS, RS504393; U, U0126; W, wortmannin; LY, LY294002; PI3K-alpha I, PI3K a inhibitor VIII; AS, AS605240.
demonstrated that treatment of cells with CCL2 at 30 nM concentration caused activation of the MEK/ERK cascade and p90RSK, effectively both at 1 and 30 min of exposures (Fig. 3A–C). Moreover, we also examined concentration-dependent effects of CCL2 on these signaling pathways by exposing the cells to 10, 30 and 60 nM CCL2 for 30 min (Fig. 3D–F). The overall results
demonstrated that the levels of phosphorylation of the above mentioned proteins in the 30 or 60 nM CCL2-tretated cells were more pronounced compared to that in cells treated with 10 nM concentration of this chemokine. Our further studies revealed that microglial migration in response to 12 h exposure to 30 nM CCL2 was potently blocked
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Fig. 5. Effect of RS504393 on the phosphorylation of MEK1/2 (A), and impact of RS504393 or U0126 on the phosphorylation of ERK1/2 (B and C, respectively), and p90RSK (D) in CCL2-induced BV2 cells. Following the treatments with RS504393 or U0126 for 1 h, cells were co-exposed to 30 nM CCL2 for 1 or 30 min and Western blotting analyses were performed as described in Section 2. Representative blots from at least three individual experiments for each protein are shown (upper panel). The density of the bands in each blot was quantified as described in the legend of Fig. 3 (lower panel). Each data point represents the mean ± SEM from at least three measurements. @ P < 0.05, and # P < 0.001 vs corresponding control; ⁄⁄⁄ P < 0.001 vs CCL2 alone-treated group.
upon co-treatment of cells with either CCR2-selective antagonist, RS504393, or MEK inhibitor, U0126 (Fig. 4A, upper panel; Fig. 4B). In parallel, the phosphorylation of MEK1/2, ERK1/2, and p90RSK in the cells exposed to 30 nM CCL2 for 1 or 30 min was completely inhibited upon co-treatment with RS504393 (Fig. 5A, B, and D), confirming CCR2-mediated activation of these kinases. Under similar experimental conditions, the activation of ERK1/2 and p90RSK was also significantly inhibited when the cells were pretreated with U0126 (Fig. 5C and D).
3.3.2. PI3K/Akt pathway and its cross-talking with MEK/ERK/p90RSK pathway Our further investigations on the time-dependent effects of CCL2 showed that an exposure of cells to this chemokine at 30 nM caused activation of Akt, effectively both at 1 and 30 min of exposure (Fig. 6A), Furthermore, a concentration-dependent effect of CCL2 was also observed on the activation of Akt signaling when the cells were exposed to 10, 30 and 60 nM concentrations of this chemokine for 30 min (Fig. 6B). The highest activation of Akt
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Fig. 6. Time- and concentration-dependent effects of CCL2 on the phosphorylation of Akt (A and B, respectively) and the impact of RS504393 and U0126 on the CCL2-induced phosphorylation of Akt (C) in BV2 cells. Cells were treated with 30 nM CCL2 for the indicated periods of time (A); or exposed to various concentrations of CCL2 for 30 min (B); or treated with 30 nM CCL2 for 1 or 30 min following an exposure to RS504393 or U0126 for 1 h (C). Western blotting analyses were performed as described in Section 2. Representative blots from at least three individual experiments are shown (upper panel). The density of the bands in each blot was quantified and normalized to the amount of non-phospho form of Akt (lower panel). Each data point represents the mean ± SEM from at least three measurements. ⁄ P < 0.05, ⁄⁄ P < 0.01, and ⁄⁄⁄ P < 0.001 vs control (0 h CCL2 treatment or without CCL2 treatment, A and B, respectively); @ P < 0.05, and # P < 0.001 vs corresponding control (C); ⁄ P < 0.05, ⁄⁄ P < 0.01, and ⁄⁄⁄ P < 0.001 vs CCL2 alonetreated group (C).
was noticed at 60 nM CCL2 (164% vs control, P < 0.01), while the effect of this chemokine on Akt phosphorylation was found to be statistically insignificant at 10 nM concentration. The activation of Akt at both 1 and 30 min exposures to 30 nM CCL2 was completely inhibited upon pretreatment of cells with RS504393 (Fig. 6C), indicating that the activation of Akt is mediated by CCL2–CCR2 interaction. Interestingly, the CCL2-induced
Akt phosphorylation at both time points was also completely prevented in cells pretreated with U0126 (Fig. 6C), suggesting the involvement of MEK in the CCL2-induced Akt phosphorylation. For further understanding the complex nature of the CCL2induced Akt phosphorylation and its impact on CCL2-induced cell migration, if any, we extended our study to explore the probable molecular mechanism(s) underlying CCL2-induced PI3K/Akt
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Fig. 7. Effects of different PI3K inhibitors on the CCL2-induced phosphorylation of Akt (A), MEK1/2 (B), ERK1/2 (C), and p90RSK (D) in BV2 cells. Cells pretreated with wortmannin, LY294002, PI3Ka inhibitor VIII, or AS605240 were exposed to 30 nM CCL2 for 30 min and Western blot analyses were performed as described in Section 2. Representative blots from at least three individual experiments for each protein are shown (upper panel). The density of the bands in each blot was quantified as described in the legends of Figs. 3 and 6 (lower panel). Each data point represents the mean ± SEM from at least three measurements. # P < 0.001 vs control; ⁄⁄⁄ P < 0.001 vs CCL2 alonetreated group.
activation and subsequent cross-talk with the MEK pathway. For this study, we used several PI3K inhibitors with different specificities for its isoforms: wortmannin and LY294002, commonly used broad-spectrum PI3K inhibitors; PI3Ka inhibitor VIII, a selective inhibitor of class I PI3K isoform p110a; and AS-605240, a selective inhibitor of class I PI3K isoform p110c. Our results demonstrated that CCL2-induced cell migration was completely blocked by all of the above PI3K inhibitors (Fig. 4A, lower panel; Fig. 4B). In parallel, we found that CCL2-induced phosphorylation of Akt was markedly inhibited in cells pretreated with any of these inhibitors,
although the degree of inhibition was found to be less in the case of PI3Ka inhibitor VIII compared with the other inhibitors (Fig. 7A). Interestingly, CCL2-induced phosphorylation of MEK1/2, ERK1/2, and p90RSK was also effectively inhibited in the cells pretreated with all of the above-mentioned PI3K inhibitors (Fig. 7B–D). 3.3.3. c-Jun and CREB/ATF-1 pathways To further understand the downstream signaling cascades of CCL2 action in BV2 cells, we examined the impact of this chemokine on the activation of c-jun and CREB/ATF-1. Using an
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(Fig. 8A and B). In addition, our time-point study revealed that an exposure of the cells to 30 nM CCL2 for 1 or 30 min caused a significant increase in the phosphorylation of both c-jun and ATF-1 (Fig. 9A and B). Our further investigations demonstrated that unlike the upstream signaling proteins as mentioned above, the phosphorylation of c-jun induced by 1 or 30 min exposure to 30 nM CCL2 was not suppressed by RS504393 (data not shown). However, CCL2-induced activation of c-jun at both time points was significantly attenuated by U0126 (Fig. 9A). In contrast, CCL2-induced phosphorylation of ATF-1 was significantly inhibited by RS504393 at both time points, but not significantly changed by U0126 (Fig. 9B). Moreover, the CCL2-induced phosphorylation of c-jun was found not to be inhibited in cells pretreated with all of the PI3K inhibitors used in this study (Fig. 9C). Even an increase in this phosphorylation was noticed in cells pretreated with LY294002, the exact reason for which is yet to be identified. In contrast, the CCL2-induced phosphorylation of ATF-1 was significantly inhibited in the cells pretreated with all types of PI3K inhibitors (Fig. 9D), almost in a similar fashion to that noticed for Akt, MEK1/2, ERK1/2, and p90RSK.
4. Discussion
Fig. 8. Concentration-dependent effects of CCL2 on the phosphorylation of c-jun (A) and ATF-1 (B) in BV2 cells. Cells were treated with various concentrations of CCL2 for 30 min and Western blot analyses were performed as described in Section 2. Representative blots from at least three individual experiments for each protein are shown (upper panel). The density of the bands in each blot was quantified and normalized to the amount of non-phospho form of c-jun or b-actin, wherever applicable (lower panel). Each data point represents the mean ± SEM from at least three measurements. ⁄ P < 0.05, ⁄⁄ P < 0.01 vs control.
anti-phospho-CREB antibody (Ser133), no band was detected at the position corresponding to phospho-CREB in lysates prepared from either control or CCL2-treated cells. However, the antibody confirmed a band corresponding to the phosphorylated ATF-1 as per specification of the antibody manufacturer (Cell Signaling). Our overall results showed concentration-dependent effects of CCL2 on the phosphorylation of both c-jun and ATF-1 when the cells were exposed to 10, 30 and 60 nM CCL2 for 30 min
Microglia are known to actively participate in the brain’s inflammatory response to injury or infection by migrating to pathological locations [2,3]. Chemokines including CCL2 have been found to be the potential inducers of migration of microglial cells and their recruitment to sites of injury [4,5]. Upon binding to its major receptor CCR2 on microglial cell membranes [9], CCL2 triggers the accumulation of these cells at the pathological sites [8,10,24]. In this study, we first examined the expression of CCR2 in BV2 cells and found two prominent bands at 42 and 45 kDa, corresponding to the CCR2 isoforms [21–23]. Our findings also revealed induction of cell migration by CCL2 in concentrationand time-dependent manners. However, no significant difference in cell motility was noticed between 30 and 60 nM concentrations, probably due to the saturation of CCR2 as found in previous reports [9,10]. Moreover, CCL2-induced migration of BV2 cells was strongly inhibited upon co-treatment with RS504393, a highly potent spiropiperidine CCR2-selective competitive antagonist with 50% inhibitory concentration (IC50) of 89 nM [25]. Thus our findings support the involvement of CCL2–CCR2 axis in CCL2-induced migration of microglial cells. Our investigations on the time- and concentration-dependent effects of CCL2 revealed CCL2-mediated activation of the MEK/ ERK pathway and p90RSK, in agreement with previous reports [16,18,26,27]. Further studies demonstrated that CCL2-induced microglial migration and phosphorylation of ERK1/2 and p90RSK were potently inhibited upon co-treatment of cells with U0126, a highly selective inhibitor of both MEK1 and MEK2. Taken together and based on previous findings [12,28], our results suggest that both CCL2–CCR2 axis and MEK/ERK/p90RSK signaling pathway are critical for the CCL2-induced microglial migration. Akt, a serine/threonine kinase activated by PI3K, is involved in the chemotaxis, migration, and invasion of various cell types [29,30]. Our results demonstrated that an exposure of BV2 cells to CCL2 induced the phosphorylation of Akt in concentrationand time-dependent manners, in keeping with a previous report [31]. Notably, CCL2-triggered activation of Akt was markedly inhibited in the cells when pretreated with RS504393, suggesting that this event may be mediated through the CCL2–CCR2 interaction. Furthermore, CCL2-induced Akt phosphorylation was also significantly suppressed upon pretreatment of cells with U0126, in agreement with earlier studies [32,33]. Collectively, our findings
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Fig. 9. Effect of U0126 on the phosphorylation of c-jun (A); impact of RS504393 or U0126 on the phosphorylation of ATF-1 (B); and effects of different PI3K inhibitors on the phosphorylation of c-jun or ATF-1 in CCL2-induced BV2 cells (C and D, respectively). Cells pretreated with RS504393, U0126, wortmannin, LY294002, PI3Ka inhibitor VIII, or AS605240 were exposed to 30 nM CCL2 for 1 or 30 min and Western blot analyses were performed as described in Section 2. Representative blots from at least three individual experiments for each protein are shown (upper panel). The density of the bands in each blot was quantified as described in the legend of Fig. 8 (lower panel). Each data point represents the mean ± SEM from at least three measurements. @ P < 0.05, & P < 0.01, and # P < 0.001vs corresponding control; ⁄ P < 0.05, ⁄⁄ P < 0.01, and ⁄⁄⁄ P < 0.001 vs CCL2 alone-treated group.
imply that there may be a cross-talk between CCL2-induced MAPK and PI3K/Akt pathways in microglial cells. To further understand the complex nature of CCL2-induced Akt activation and to find which PI3K isoform is particularly involved in this event and in CCL2-triggered cell migration, we used several PI3K inhibitors with different specificities. The results revealed that both Akt phosphorylation and cell migration were significantly inhibited in the CCL2-treated cells when they were
pre-treated with any of these inhibitors. Such findings suggest that the CCL2-mediated signal activates a broad range of PI3K isoforms which are probably involved in cell migration, as reported previously [13,34]. Additionally, we found that CCL2-induced phosphorylation of MEK1/2, ERK1/2, and p90RSK was also significantly inhibited in the cells when pretreated with all of the abovementioned PI3K inhibitors, in agreement with previous studies [13]. Taken together, our results suggest that a broad range of
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PI3K isoforms may play a vital role in the cross-talk between the PI3K/Akt and MAPK pathways in CCL2-induced signaling. To further elucidate the downstream signaling cascades of CCL2 action, we studied the impact of this chemokine on c-jun and CREB/ATF-1. These two important transcription factors which regulate a number of cellular functions and processes including migration [12] are known to be involved in the downstream signaling of CCL2 in a variety of cell types [18,26,35]. Consistent with this, we observed that treatment of cells with CCL2 triggered the phosphorylation of both c-jun and ATF-1 in concentration- and time-dependent manners. We also found that CCL2-induced phosphorylation of ATF-1, but not c-jun, was significantly inhibited by RS504393. While CCL2-triggered phosphorylation of c-jun, but not ATF-1, was significantly attenuated by U0126. Our further studies revealed that CCL2-induced phosphorylation of ATF-1, but not c-jun, was significantly inhibited in the cells pretreated with all types of PI3K inhibitors. Collectively, these results suggest that CCL2-induced stimulation of c-jun is not directly linked to CCR2 activation and PI3K/Akt pathway, but rather is being influenced by MEK/ERK signaling cascades. While, CCL2-triggered phosphorylation of ATF-1 is the effector of PI3K/Akt signaling mediated through the CCL2-CCR2 interaction and does not involve the MEK/ ERK pathway. In conclusion, the signaling mechanism(s) underlying CCL2induced migration of BV2 microglial cells appears to be complex and is associated with the activation of MEK1/2, ERK1/2, p90RSK, and Akt and their cross-talk. In addition, our results demonstrate activation of the transcription factors c-jun and ATF-1 in response to CCL2 treatment, which are likely to be linked to the MEK/ERK and PI3K signaling pathway, respectively. Collectively, our findings contribute to a better understanding of the CCL2-induced microglial migration and the probable signaling pathways involved. Further studies will be required to elucidate the exact mechanisms of cross-talking between MAPK and PI3K/Akt pathways and their downstream signaling cascades involved in the molecular events of CCL2-induced cell migration. Conflict of interests Authors declare that they don’t have any competing interests. Acknowledgment This work was supported by the Dongguk University Research Fund of 2015. References [1] S. Sugama, Stress-induced microglial activation may facilitate the progression of neurodegenerative disorders, Med. Hypotheses 73 (2009) 1031–1034. [2] G.W. Kreutzberg, Microglia: a sensor for pathological events in the CNS, Trends Neurosci. 19 (1996) 312–318. [3] X.G. Luo, S.D. Chen, The changing phenotype of microglia from homeostasis to disease, Trans. Neurodegener. 1 (2012) 9. [4] A.K. Cross, M.N. Woodroofe, Chemokines induce migration and changes in actin polymerization in adult rat brain microglia and a human fetal microglial cell line in vitro, J. Neurosci. Res. 55 (1999) 17–23. [5] E. Ambrosini, F. Aloisi, Chemokines and glial cells: a complex network in the central nervous system, Neurochem. Res. 29 (2004) 1017–1038. [6] B.A. Premack, T.J. Schall, Chemokine receptors: gateways to inflammation and infection, Nat. Med. 2 (1996) 1174–1178. [7] D.A. Lauffenburger, A.F. Horwitz, Cell migration: a physically integrated molecular process, Cell 84 (1996) 359–369. [8] S.L. Deshmane, S. Kremlev, S. Amini, B.E. Sawaya, Monocyte chemoattractant protein-1 (MCP-1): an overview, J. Interferon Cytokine Res. 29 (2009) 313–326. [9] X.S. Jiang, Y.Q. Ni, T.J. Liu, M. Zhang, H. Ren, R. Jiang, X. Huang, G.Z. Xu, CCR2 overexpression promotes the efficient recruitment of retinal microglia in vitro, Mol. Vis. 18 (2012) 2982–2992.
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Cytokine journal homepage: www.journals.elsevier.com/cytokine
Effect of CCL2 on BV2 microglial cell migration: Involvement of probable signaling pathways Shambhunath Bose, Sunyoung Kim, Yeonsoo Oh, Md. Moniruzzaman, Gyeongjun Lee, Jungsook Cho ⇑ College of Pharmacy, Dongguk University-Seoul, Goyang, Gyeonggi-do 10326, Republic of Korea
a r t i c l e
i n f o
Article history: Received 8 July 2015 Received in revised form 3 February 2016 Accepted 4 February 2016
Keywords: CCL2 CCR2 BV2 cells Migration Intracellular signaling pathways
a b s t r a c t Microglia, the resident macrophages of the central nervous system, play a vital role in the regulation of innate immune function and neuronal homeostasis of the brain. Currently, much interest is being generated regarding the investigation of the microglial migration that results in their accumulation at focal sites of injury. Chemokines including CCL2 are known to cause the potential induction of migration of microglial cells, although the underlying mechanisms are not well understood. In the present study, using murine neonatal BV2 microglial cells as a model, we investigate the impact of CCL2 on the migration of microglial cells and address the probable molecular events within the cellular signaling cascades mediating CCL2-induced cell migration. Our results demonstrate concentration- and time-dependent induction of BV2 cell migration by CCL2 and reveal complex mechanisms involving the activation of MEK, ERK1/2, and Akt, and their cross-talk. In addition, we demonstrate that the MEK/ERK pathway activated by CCL2 treatment mediate p90RSK activation in BV2 cells. Moreover, our findings indicate that Akt, ERK1/2, and p90RSK are the downstream effectors of PI3K in the CCL2-induced signaling. Finally, phosphorylation of the transcription factors c-jun and ATF-1 is found to be a further downstream signaling cascade in the CCL2-mediated action. Our results suggest that CCL2-induced activation of c-jun and ATF-1 is likely to be linked to the MEK/ERK and PI3K signaling pathways, respectively. Taken together, these findings contribute to a better understanding of CCL2-induced microglial migration and the probable signaling pathways involved. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Microglia, the resident cells of the central nervous system, are originally derived from the monocyte/macrophage lineage. They play a key role in the innate immune regulation and neuronal homeostasis of the brain [1], and act as a primary defense mechanism against invading pathogens [2]. In a pathological condition, microglia are triggered and activated through a cascade of events. The entire process involves proliferation, migration to the site of injury, elevated expression of immunomodulators in the cells, and finally transformation into the phagocytes that are dedicated to removing the damaged cells and debris [3] through the process of inflammation. Chemokine molecules are known to play a vital role in the regulation of the movement of microglia and their recruitment to sites of inflammation [4,5]. As such, microglial migration involves cell polarization and the generation of lamellipodia at the leading edge, known as ruffling. This event triggers the redistribution of specific ⇑ Corresponding author. E-mail address: [email protected] (J. Cho). http://dx.doi.org/10.1016/j.cyto.2016.02.001 1043-4666/Ó 2016 Elsevier Ltd. All rights reserved.
molecules, such as integrins, and the polymerization and depolymerization of filamentous actin, resulting in the deformation of shape. In association, a number of proteases are released in order to sustain the dynamic process of cell attachment and detachment [6], eventually leading to elongation of the lamellipodia and filopodia that are required for cell migration [7]. Among the chemokines, CCL2, also termed monocyte chemotactic protein (MCP)-1, has been found to be one of the most potent microglial chemoattractants. CCL2 is produced by different kinds of cells, either constitutively or following induction by various factors including oxidative stress, cytokines, or growth factors [8]. CCL2 binds to the CC-chemokine receptor 2 (CCR2), a member of the G protein-coupled receptors, expressed on microglial cell membranes, triggering the assembly of these cells at pathological sites [9]. The CCL2–CCR2 interaction has been demonstrated to induce changes in the actin polymerization and subsequent reorganization of the actin cytoskeleton, the generation of focal adhesions, and the extension of pseudopodia, which collectively are responsible for the migration of activated microglia [4,10,11]. A number of studies have been performed in order to better understand the molecular mechanism(s) underlying the
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chemokine-induced migratory activity of microglial cells. It has been demonstrated that mitogen-activated protein kinases (MAPKs) play vital roles in cell migration [12]. Accumulating genetic and pharmacological evidence strongly supports the involvement of phosphoinositide 3-kinase (PI3K) as a major biochemical signal for chemotaxis in response to a number of chemokines [13]. Further, it has been revealed that multiple independent signal transduction pathways are induced in monocytes by CCL2, and the activation of all these pathways is essential in order to produce a maximal chemoattractant response [14]. However, to the best of our knowledge, no studies have yet been conducted in order to understand the probable molecular mechanism(s) underlying the CCL2-induced migration of microglial cells. In the present study, we investigate the impact of CCL2 on the migration of microglial cells using BV2 cells as a model, which are originally derived from raf/myc-immortalized murine neonatal microglia and are the most commonly used substitute for primary microglia [15]. Furthermore, to explore the probable molecular mechanism(s) underlying the CCL2-induced microglial migration, we study the signal transduction pathways linked to CCR2 activation, with an emphasis on the MAPK and PI3K/Akt pathways, the major signaling cascades utilized by CCL2 in a variety of cell types involved in the immune system [16,17]. 2. Materials and methods 2.1. Chemicals and antibodies Dulbecco’s Modified Eagle’s Medium (DMEM) and 0.05% trypsin–EDTA were purchased from Thermo Scientific (Rockford, IL, USA). Anti-b-actin antibody (monoclonal), and AS605240 were acquired from Sigma–Aldrich (St. Louis, MO, USA). RS504393 was obtained from Tocris Cookson Ltd. (Bristol, UK). PI3Ka inhibitor VIII was purchased from Calbiochem (San Diego, CA, USA). Antiphospho-MEK1/2 (Ser217/221), anti-MEK1/2, anti-phosphoERK1/2 (Thr202/Tyr204), anti-ERK1/2, anti-phospho-p90RSK (Ser380), anti-RSK1/RSK2/RSK3, anti-phospho-c-jun (Ser63), antic-jun, anti-phospho-CREB (Ser133), and horseradish peroxidase (HRP)-linked anti-rabbit immunoglobulin G (IgG) antibodies, LY294002, U0126, and wortmannin were acquired from Cell Signaling Technology (Danvers, MA, USA). Anti-CCR2 antibody and its blocking peptide were procured from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Additional anti-CCR2 antibodies were purchased from GeneTex, Inc. (Irvine, CA, USA) and Novus Biologicals (Littleton, CO, USA). HRP-linked anti-mouse IgG antibody was the product of Merck (Darmstadt, Germany). Recombinant rat CCL2 was purchased from PeproTech Inc. (Rocky Hill, NJ, USA).
specific protein kinases or a specific antagonist of CCR2 for 1 h prior to exposure to CCL2. Following the desired treatments, the cells were washed three times with ice-cold PBS, pH 7.4, and then lysed in lysis buffer [10 mM Tris–HCl, pH 7.4; 150 mM NaCl; 2 mM EDTA; 4.5 mM sodium pyrophosphate; 10 mM bglycerophosphate; 1 mM NaF; 1 mM Na3VO4; 1% (v/v) Triton X100; 0.5% (v/v) NP-40; and one tablet of protease inhibitor cocktail (Roche Diagnostic GmbH, Mannheim, Germany)] for 30 min on ice. Lysates were subjected to centrifugation at 14,000 rpm for 30 min at 4 °C to remove the insoluble material, and the collected supernatants were stored at 80 °C until use. The protein concentrations of the supernatants were measured using a BioRad DC protein assay kit (BioRad, Hercules, CA, USA). The samples were denatured at 100 °C in Laemmli sample buffer (BioRad) containing 5% bmercaptoethanol. Equal amounts of protein (30 lg) were resolved by SDS-PAGE using 8% or 10% gels, and electrophoretically transferred to nitrocellulose membrane (GE Healthcare UK Ltd., Buckinghamshire, UK) for 90 min at 100 V [18]. The membranes were blocked with Tris–buffered saline (TBS) containing 0.1% Tween 20 (TBST) and 5% non-fat dried milk (BD Falcon, Sparks, MD, USA) for 90 min to prevent non-specific binding. Following this, the membranes were washed three times with TBST at room temperature, and then incubated overnight at 4 °C with the specific primary antibodies in 5% bovine serum albumin (USB, Canton, OH, USA). After thorough washing with TBST, the membranes were incubated for 90 min with the appropriate HRP-conjugated antiIgG secondary antibodies, and the immunoreactive bands were detected by a BioRad ChemiDoc XRS imaging system (BioRad) using Super Signal West Pico ECL reagent (Thermo Fisher Scientific, San Jose, CA, USA). For the blocking peptide experiment, anti-CCR2 antibody (Santa Cruz) was incubated overnight at 4 °C with its blocking peptide (Santa Cruz) before incubations with nitrocellulose membrane. 2.4. Assessment of cell migration
BV2 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% antibiotic–antimycotic agent at 37 °C in an incubator under a humidified atmosphere of air containing 5% CO2. The cells were then plated in either 35-mm culture dishes (2 106 cells/dish) or 24-well culture plates (5 105 cells/well) for Western blotting analysis and cell migration assays, respectively.
The migration of cells was investigated by an in vitro scratch wound assay as described previously [19,20] with some modifications. After culturing for 24 h in 24-well plates, confluent monolayers of cells were scratched using a 200 ll pipette tip to produce a wound. Cultures were gently washed with DMEM to remove loosely attached cells and then treated with vehicle (control) or CCL2 as indicated in the Figures. A Nikon (Tokyo, Japan) Eclipse TS100 inverted microscope, a Digital sight DS-U2 camera (Nikon), and NIS-Elements F 3.0 image software (Nikon) were used to capture images of the wounded edges. The images were acquired with a 4X objective. For other experiments where the roles of CCR2 and different protein kinases in CCL2-induced cell migration were evaluated, the cells were treated with specific pharmacological inhibitors of the respective protein kinases or a selective antagonist of CCR2 for 1 h prior to the wound induction, and after scratching they were treated with 30 nM CCL2 in the presence of these agents. The control group did not receive any treatment except the corresponding vehicles. The images were acquired at 0 and 12 h of CCL2 treatment. The difference between the width of the wound at time 0 and at the time point being analyzed was measured. This value reflects the relative distance migrated by the cells during a given time interval. The migration distance of the treated groups was expressed as a percentage of the respective control.
2.3. Western blotting
2.5. Statistical analyses
The cells were serum-starved overnight and then exposed to various concentrations of CCL2 for different time periods in serum-free medium as indicated in the Figures. In some experiments, the cells were treated with pharmacological inhibitors of
Data are presented as the mean ± SEM from at least three independent experiments. The Statistical Package for Social Science (SPSS) software program (version 11.5; SPSS, Chicago, IL, USA) was employed for the analyses of the data. A one way ANOVA,
2.2. Cell culture
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keeping with the previous studies, showing the two bands of CCR2 isoforms produced by alternate mRNA splicing [21–23]: CCR2A (45 kDa) and CCR2B (42 kDa). 3.2. Time-and concentration-dependent effects of CCL2 on cell migration
Fig. 1. Expression of CCR2 in BV2 cells. CCR2 protein expression was evaluated in BV2 cells using Western blot analyses. Immunoblotting was performed as described in Section 2 using anti-CCR2 antibody either unblocked (left panel) or blocked by prior incubation with its blocking peptide overnight at 4 °C (right panel).
followed by Bonferroni’s post hoc analysis, was performed to determine the statistical significance. Differences were considered significant at P < 0.05. 3. Results 3.1. Expression of CCR2 in BV2 cells Before investigating the effects of CCL2 on cell migration, we verified the expression of CCR2 in BV2 cells by Western blotting. In this study, using the anti-CCR2 antibody from Santa Cruz, we observed two prominent protein bands corresponding to 42 and 45 kDa, which were not detected in the membrane preincubated with the blocking peptide (Fig. 1). Similar profiles were also observed in the blots using anti-CCR2 antibodies from different commercial sources (data not shown). These findings are in
Treatment with CCL2 at the concentration of 30 nM caused increase in microglial motility at 6 and 12 h in a time-dependent manner, accounting for 303% and 391% cell migration, respectively, compared with the control (Fig. 2A and B). Although a marked wound healing was observed in the cells treated with CCL2 for 24 h (270% migration vs control), a substantial proliferation of the cells was also observed at this time point as reflected in the corresponding control. This event is likely to interfere with the assessment of cell migration, and because of this reason we opted a 12 h CCL2 treatment schedule for further experimental use, where a minimal intervention of the cell proliferation was noticed. In addition, we observed a concentration-dependent effect of CCL2 on the wound healing when the cells were exposed to 10, 30 and 60 nM CCL2 for 12 h (Fig. 2C and D). 3.3. Evaluation of probable roles of vital cell signaling cascades in the CCL2-induced cell migration 3.3.1. MEK/ERK and p90RSK pathways To explore the probable molecular mechanism(s) underlying CCL2-induced migration of BV2 cells, we first evaluated the impact of this chemokine on the vital cell signaling pathways. Our results
Fig. 2. Time- and concentration-dependent effects of CCL2 on the migration of BV2 cells. Microscopic images demonstrate migration of cells induced by either 30 nM CCL2 at different time points (A) or different concentrations of CCL2 at 12 h (C). The distance migrated by the cells at a particular time point or concentration of CCL2 treatment was quantitated as described in Section 2 and expressed as percentage of corresponding control (B and D, respectively). Each data point represents the mean ± SEM from at least three measurements. ⁄ P < 0.05, ⁄⁄ P < 0.01, and ⁄⁄⁄ P < 0.001 compared with corresponding control, # P < 0.05 vs 10 nM CCL2.
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Fig. 3. Time- and concentration-dependent effects of CCL2 on the phosphorylation of MEK1/2 (A and D), ERK1/2 (B and E), and p90RSK (C and F) in BV2 cells. Cells were treated with 30 nM CCL2 for the indicated periods of time or exposed to various concentrations of CCL2 for 30 min and Western blotting analyses were performed as described in Section 2. Representative blots from at least three individual experiments for each protein are shown (upper panel). The density of the bands in each blot was quantified by densitometric analysis and normalized to the amount of non-phospho forms of MEK1/2, ERK1/2, or p90RSK, wherever applicable (lower panel). Each data point represents the mean ± SEM from at least three measurements. ⁄ P < 0.05, ⁄⁄ P < 0.01, and ⁄⁄⁄ P < 0.001 vs corresponding control (0 h CCL2 treatment or without CCL2 treatment, wherever applicable); # P < 0.01 compared with 10 nM CCL2.
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Fig. 4. Effects of a CCR2 antagonist RS504393 and different protein kinase inhibitors on the CCL2-induced migration of BV2 cells. Microscopic images of cell migration induced by 12 h treatment with 30 nM CCL2 in presence or absence of RS504393 and the protein kinase inhibitors are shown (A); the distance migrated by the cells in the treated groups was quantitated as described in Section 2 and expressed as percentage of control (B). # P < 0.001 compared with control, ⁄⁄⁄ P < 0.001 compared with CCL2 alone-treated group. RS, RS504393; U, U0126; W, wortmannin; LY, LY294002; PI3K-alpha I, PI3K a inhibitor VIII; AS, AS605240.
demonstrated that treatment of cells with CCL2 at 30 nM concentration caused activation of the MEK/ERK cascade and p90RSK, effectively both at 1 and 30 min of exposures (Fig. 3A–C). Moreover, we also examined concentration-dependent effects of CCL2 on these signaling pathways by exposing the cells to 10, 30 and 60 nM CCL2 for 30 min (Fig. 3D–F). The overall results
demonstrated that the levels of phosphorylation of the above mentioned proteins in the 30 or 60 nM CCL2-tretated cells were more pronounced compared to that in cells treated with 10 nM concentration of this chemokine. Our further studies revealed that microglial migration in response to 12 h exposure to 30 nM CCL2 was potently blocked
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Fig. 5. Effect of RS504393 on the phosphorylation of MEK1/2 (A), and impact of RS504393 or U0126 on the phosphorylation of ERK1/2 (B and C, respectively), and p90RSK (D) in CCL2-induced BV2 cells. Following the treatments with RS504393 or U0126 for 1 h, cells were co-exposed to 30 nM CCL2 for 1 or 30 min and Western blotting analyses were performed as described in Section 2. Representative blots from at least three individual experiments for each protein are shown (upper panel). The density of the bands in each blot was quantified as described in the legend of Fig. 3 (lower panel). Each data point represents the mean ± SEM from at least three measurements. @ P < 0.05, and # P < 0.001 vs corresponding control; ⁄⁄⁄ P < 0.001 vs CCL2 alone-treated group.
upon co-treatment of cells with either CCR2-selective antagonist, RS504393, or MEK inhibitor, U0126 (Fig. 4A, upper panel; Fig. 4B). In parallel, the phosphorylation of MEK1/2, ERK1/2, and p90RSK in the cells exposed to 30 nM CCL2 for 1 or 30 min was completely inhibited upon co-treatment with RS504393 (Fig. 5A, B, and D), confirming CCR2-mediated activation of these kinases. Under similar experimental conditions, the activation of ERK1/2 and p90RSK was also significantly inhibited when the cells were pretreated with U0126 (Fig. 5C and D).
3.3.2. PI3K/Akt pathway and its cross-talking with MEK/ERK/p90RSK pathway Our further investigations on the time-dependent effects of CCL2 showed that an exposure of cells to this chemokine at 30 nM caused activation of Akt, effectively both at 1 and 30 min of exposure (Fig. 6A), Furthermore, a concentration-dependent effect of CCL2 was also observed on the activation of Akt signaling when the cells were exposed to 10, 30 and 60 nM concentrations of this chemokine for 30 min (Fig. 6B). The highest activation of Akt
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Fig. 6. Time- and concentration-dependent effects of CCL2 on the phosphorylation of Akt (A and B, respectively) and the impact of RS504393 and U0126 on the CCL2-induced phosphorylation of Akt (C) in BV2 cells. Cells were treated with 30 nM CCL2 for the indicated periods of time (A); or exposed to various concentrations of CCL2 for 30 min (B); or treated with 30 nM CCL2 for 1 or 30 min following an exposure to RS504393 or U0126 for 1 h (C). Western blotting analyses were performed as described in Section 2. Representative blots from at least three individual experiments are shown (upper panel). The density of the bands in each blot was quantified and normalized to the amount of non-phospho form of Akt (lower panel). Each data point represents the mean ± SEM from at least three measurements. ⁄ P < 0.05, ⁄⁄ P < 0.01, and ⁄⁄⁄ P < 0.001 vs control (0 h CCL2 treatment or without CCL2 treatment, A and B, respectively); @ P < 0.05, and # P < 0.001 vs corresponding control (C); ⁄ P < 0.05, ⁄⁄ P < 0.01, and ⁄⁄⁄ P < 0.001 vs CCL2 alonetreated group (C).
was noticed at 60 nM CCL2 (164% vs control, P < 0.01), while the effect of this chemokine on Akt phosphorylation was found to be statistically insignificant at 10 nM concentration. The activation of Akt at both 1 and 30 min exposures to 30 nM CCL2 was completely inhibited upon pretreatment of cells with RS504393 (Fig. 6C), indicating that the activation of Akt is mediated by CCL2–CCR2 interaction. Interestingly, the CCL2-induced
Akt phosphorylation at both time points was also completely prevented in cells pretreated with U0126 (Fig. 6C), suggesting the involvement of MEK in the CCL2-induced Akt phosphorylation. For further understanding the complex nature of the CCL2induced Akt phosphorylation and its impact on CCL2-induced cell migration, if any, we extended our study to explore the probable molecular mechanism(s) underlying CCL2-induced PI3K/Akt
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Fig. 7. Effects of different PI3K inhibitors on the CCL2-induced phosphorylation of Akt (A), MEK1/2 (B), ERK1/2 (C), and p90RSK (D) in BV2 cells. Cells pretreated with wortmannin, LY294002, PI3Ka inhibitor VIII, or AS605240 were exposed to 30 nM CCL2 for 30 min and Western blot analyses were performed as described in Section 2. Representative blots from at least three individual experiments for each protein are shown (upper panel). The density of the bands in each blot was quantified as described in the legends of Figs. 3 and 6 (lower panel). Each data point represents the mean ± SEM from at least three measurements. # P < 0.001 vs control; ⁄⁄⁄ P < 0.001 vs CCL2 alonetreated group.
activation and subsequent cross-talk with the MEK pathway. For this study, we used several PI3K inhibitors with different specificities for its isoforms: wortmannin and LY294002, commonly used broad-spectrum PI3K inhibitors; PI3Ka inhibitor VIII, a selective inhibitor of class I PI3K isoform p110a; and AS-605240, a selective inhibitor of class I PI3K isoform p110c. Our results demonstrated that CCL2-induced cell migration was completely blocked by all of the above PI3K inhibitors (Fig. 4A, lower panel; Fig. 4B). In parallel, we found that CCL2-induced phosphorylation of Akt was markedly inhibited in cells pretreated with any of these inhibitors,
although the degree of inhibition was found to be less in the case of PI3Ka inhibitor VIII compared with the other inhibitors (Fig. 7A). Interestingly, CCL2-induced phosphorylation of MEK1/2, ERK1/2, and p90RSK was also effectively inhibited in the cells pretreated with all of the above-mentioned PI3K inhibitors (Fig. 7B–D). 3.3.3. c-Jun and CREB/ATF-1 pathways To further understand the downstream signaling cascades of CCL2 action in BV2 cells, we examined the impact of this chemokine on the activation of c-jun and CREB/ATF-1. Using an
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(Fig. 8A and B). In addition, our time-point study revealed that an exposure of the cells to 30 nM CCL2 for 1 or 30 min caused a significant increase in the phosphorylation of both c-jun and ATF-1 (Fig. 9A and B). Our further investigations demonstrated that unlike the upstream signaling proteins as mentioned above, the phosphorylation of c-jun induced by 1 or 30 min exposure to 30 nM CCL2 was not suppressed by RS504393 (data not shown). However, CCL2-induced activation of c-jun at both time points was significantly attenuated by U0126 (Fig. 9A). In contrast, CCL2-induced phosphorylation of ATF-1 was significantly inhibited by RS504393 at both time points, but not significantly changed by U0126 (Fig. 9B). Moreover, the CCL2-induced phosphorylation of c-jun was found not to be inhibited in cells pretreated with all of the PI3K inhibitors used in this study (Fig. 9C). Even an increase in this phosphorylation was noticed in cells pretreated with LY294002, the exact reason for which is yet to be identified. In contrast, the CCL2-induced phosphorylation of ATF-1 was significantly inhibited in the cells pretreated with all types of PI3K inhibitors (Fig. 9D), almost in a similar fashion to that noticed for Akt, MEK1/2, ERK1/2, and p90RSK.
4. Discussion
Fig. 8. Concentration-dependent effects of CCL2 on the phosphorylation of c-jun (A) and ATF-1 (B) in BV2 cells. Cells were treated with various concentrations of CCL2 for 30 min and Western blot analyses were performed as described in Section 2. Representative blots from at least three individual experiments for each protein are shown (upper panel). The density of the bands in each blot was quantified and normalized to the amount of non-phospho form of c-jun or b-actin, wherever applicable (lower panel). Each data point represents the mean ± SEM from at least three measurements. ⁄ P < 0.05, ⁄⁄ P < 0.01 vs control.
anti-phospho-CREB antibody (Ser133), no band was detected at the position corresponding to phospho-CREB in lysates prepared from either control or CCL2-treated cells. However, the antibody confirmed a band corresponding to the phosphorylated ATF-1 as per specification of the antibody manufacturer (Cell Signaling). Our overall results showed concentration-dependent effects of CCL2 on the phosphorylation of both c-jun and ATF-1 when the cells were exposed to 10, 30 and 60 nM CCL2 for 30 min
Microglia are known to actively participate in the brain’s inflammatory response to injury or infection by migrating to pathological locations [2,3]. Chemokines including CCL2 have been found to be the potential inducers of migration of microglial cells and their recruitment to sites of injury [4,5]. Upon binding to its major receptor CCR2 on microglial cell membranes [9], CCL2 triggers the accumulation of these cells at the pathological sites [8,10,24]. In this study, we first examined the expression of CCR2 in BV2 cells and found two prominent bands at 42 and 45 kDa, corresponding to the CCR2 isoforms [21–23]. Our findings also revealed induction of cell migration by CCL2 in concentrationand time-dependent manners. However, no significant difference in cell motility was noticed between 30 and 60 nM concentrations, probably due to the saturation of CCR2 as found in previous reports [9,10]. Moreover, CCL2-induced migration of BV2 cells was strongly inhibited upon co-treatment with RS504393, a highly potent spiropiperidine CCR2-selective competitive antagonist with 50% inhibitory concentration (IC50) of 89 nM [25]. Thus our findings support the involvement of CCL2–CCR2 axis in CCL2-induced migration of microglial cells. Our investigations on the time- and concentration-dependent effects of CCL2 revealed CCL2-mediated activation of the MEK/ ERK pathway and p90RSK, in agreement with previous reports [16,18,26,27]. Further studies demonstrated that CCL2-induced microglial migration and phosphorylation of ERK1/2 and p90RSK were potently inhibited upon co-treatment of cells with U0126, a highly selective inhibitor of both MEK1 and MEK2. Taken together and based on previous findings [12,28], our results suggest that both CCL2–CCR2 axis and MEK/ERK/p90RSK signaling pathway are critical for the CCL2-induced microglial migration. Akt, a serine/threonine kinase activated by PI3K, is involved in the chemotaxis, migration, and invasion of various cell types [29,30]. Our results demonstrated that an exposure of BV2 cells to CCL2 induced the phosphorylation of Akt in concentrationand time-dependent manners, in keeping with a previous report [31]. Notably, CCL2-triggered activation of Akt was markedly inhibited in the cells when pretreated with RS504393, suggesting that this event may be mediated through the CCL2–CCR2 interaction. Furthermore, CCL2-induced Akt phosphorylation was also significantly suppressed upon pretreatment of cells with U0126, in agreement with earlier studies [32,33]. Collectively, our findings
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Fig. 9. Effect of U0126 on the phosphorylation of c-jun (A); impact of RS504393 or U0126 on the phosphorylation of ATF-1 (B); and effects of different PI3K inhibitors on the phosphorylation of c-jun or ATF-1 in CCL2-induced BV2 cells (C and D, respectively). Cells pretreated with RS504393, U0126, wortmannin, LY294002, PI3Ka inhibitor VIII, or AS605240 were exposed to 30 nM CCL2 for 1 or 30 min and Western blot analyses were performed as described in Section 2. Representative blots from at least three individual experiments for each protein are shown (upper panel). The density of the bands in each blot was quantified as described in the legend of Fig. 8 (lower panel). Each data point represents the mean ± SEM from at least three measurements. @ P < 0.05, & P < 0.01, and # P < 0.001vs corresponding control; ⁄ P < 0.05, ⁄⁄ P < 0.01, and ⁄⁄⁄ P < 0.001 vs CCL2 alone-treated group.
imply that there may be a cross-talk between CCL2-induced MAPK and PI3K/Akt pathways in microglial cells. To further understand the complex nature of CCL2-induced Akt activation and to find which PI3K isoform is particularly involved in this event and in CCL2-triggered cell migration, we used several PI3K inhibitors with different specificities. The results revealed that both Akt phosphorylation and cell migration were significantly inhibited in the CCL2-treated cells when they were
pre-treated with any of these inhibitors. Such findings suggest that the CCL2-mediated signal activates a broad range of PI3K isoforms which are probably involved in cell migration, as reported previously [13,34]. Additionally, we found that CCL2-induced phosphorylation of MEK1/2, ERK1/2, and p90RSK was also significantly inhibited in the cells when pretreated with all of the abovementioned PI3K inhibitors, in agreement with previous studies [13]. Taken together, our results suggest that a broad range of
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PI3K isoforms may play a vital role in the cross-talk between the PI3K/Akt and MAPK pathways in CCL2-induced signaling. To further elucidate the downstream signaling cascades of CCL2 action, we studied the impact of this chemokine on c-jun and CREB/ATF-1. These two important transcription factors which regulate a number of cellular functions and processes including migration [12] are known to be involved in the downstream signaling of CCL2 in a variety of cell types [18,26,35]. Consistent with this, we observed that treatment of cells with CCL2 triggered the phosphorylation of both c-jun and ATF-1 in concentration- and time-dependent manners. We also found that CCL2-induced phosphorylation of ATF-1, but not c-jun, was significantly inhibited by RS504393. While CCL2-triggered phosphorylation of c-jun, but not ATF-1, was significantly attenuated by U0126. Our further studies revealed that CCL2-induced phosphorylation of ATF-1, but not c-jun, was significantly inhibited in the cells pretreated with all types of PI3K inhibitors. Collectively, these results suggest that CCL2-induced stimulation of c-jun is not directly linked to CCR2 activation and PI3K/Akt pathway, but rather is being influenced by MEK/ERK signaling cascades. While, CCL2-triggered phosphorylation of ATF-1 is the effector of PI3K/Akt signaling mediated through the CCL2-CCR2 interaction and does not involve the MEK/ ERK pathway. In conclusion, the signaling mechanism(s) underlying CCL2induced migration of BV2 microglial cells appears to be complex and is associated with the activation of MEK1/2, ERK1/2, p90RSK, and Akt and their cross-talk. In addition, our results demonstrate activation of the transcription factors c-jun and ATF-1 in response to CCL2 treatment, which are likely to be linked to the MEK/ERK and PI3K signaling pathway, respectively. Collectively, our findings contribute to a better understanding of the CCL2-induced microglial migration and the probable signaling pathways involved. Further studies will be required to elucidate the exact mechanisms of cross-talking between MAPK and PI3K/Akt pathways and their downstream signaling cascades involved in the molecular events of CCL2-induced cell migration. Conflict of interests Authors declare that they don’t have any competing interests. Acknowledgment This work was supported by the Dongguk University Research Fund of 2015. References [1] S. Sugama, Stress-induced microglial activation may facilitate the progression of neurodegenerative disorders, Med. Hypotheses 73 (2009) 1031–1034. [2] G.W. Kreutzberg, Microglia: a sensor for pathological events in the CNS, Trends Neurosci. 19 (1996) 312–318. [3] X.G. Luo, S.D. Chen, The changing phenotype of microglia from homeostasis to disease, Trans. Neurodegener. 1 (2012) 9. [4] A.K. Cross, M.N. Woodroofe, Chemokines induce migration and changes in actin polymerization in adult rat brain microglia and a human fetal microglial cell line in vitro, J. Neurosci. Res. 55 (1999) 17–23. [5] E. Ambrosini, F. Aloisi, Chemokines and glial cells: a complex network in the central nervous system, Neurochem. Res. 29 (2004) 1017–1038. [6] B.A. Premack, T.J. Schall, Chemokine receptors: gateways to inflammation and infection, Nat. Med. 2 (1996) 1174–1178. [7] D.A. Lauffenburger, A.F. Horwitz, Cell migration: a physically integrated molecular process, Cell 84 (1996) 359–369. [8] S.L. Deshmane, S. Kremlev, S. Amini, B.E. Sawaya, Monocyte chemoattractant protein-1 (MCP-1): an overview, J. Interferon Cytokine Res. 29 (2009) 313–326. [9] X.S. Jiang, Y.Q. Ni, T.J. Liu, M. Zhang, H. Ren, R. Jiang, X. Huang, G.Z. Xu, CCR2 overexpression promotes the efficient recruitment of retinal microglia in vitro, Mol. Vis. 18 (2012) 2982–2992.
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