Acrylamide Induces Senescence in Macrophages through a Process

Article pubs.acs.org/crt

Acrylamide Induces Senescence in Macrophages through a Process Involving ATF3, ROS, p38/JNK, and a Telomerase-Independent Pathway Kyung-Ho Kim,† Bongkyun Park,† Dong-Kwon Rhee, and Suhkneung Pyo* School of Pharmacy, Sungkyunkwan University, Suwon, Kyunggi-do, 440-746, Republic of Korea S Supporting Information *

ABSTRACT: Senescence, which is irreversible cell cycle arrest, is induced by various types of DNA damage, including genotoxic stress. Senescent cells show dysregulation of tumor suppressor genes and other regulators of cellular proliferation. Activating transcription factor 3 (ATF3) plays a pleiotropic role in biological processes through genotoxic stress. In this study, we examined the effects of acrylamide (ACR), a genotoxic carcinogen, on cellular senescence and the molecular mechanisms of ATF3 function in macrophages. Treatment of macrophages with ACR at low concentrations (97% macrophages. Assessment of Cell Proliferation. Cell proliferation was measured by a quantitative colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide (MTT). RAW 264.7 cells and peritoneal macrophages were seeded at 1 × 105 cells/well in 96well plates and treated with ACR for the indicated time. Cells were washed once with D-PBS, and 200 μL of MTT solution (25 μg/mL in media) was added and incubated for 4 h at 37 °C and 5% CO2. Blue formazan crystals, formed by the reduction of MTT, were dissolved in 150 μL of DMSO. The amount of formazan was determined by absorbance at 540 nm using a Molecular Devices microplate reader (Eugene, OR, USA) Senescence-Associated β-Gal Staining and Quantification. The senescence associated-β-galactosidase (SA-β-gal) assay was 72

DOI: 10.1021/tx500341z Chem. Res. Toxicol. 2015, 28, 71−86

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Figure 1. Induction of senescence-like growth arrest in ACR-treated macrophages. (A) RAW 264.7 and peritoneal macrophages were incubated with various concentrations of ACR for 24 h. Cell proliferation was assessed by MTT assay. The level of cell proliferation is presented as the percentage of untreated control cells. Data shown are the mean ± SEM of at least three independent experiments. *, P < 0.05 versus untreated control. (B) RAW 264.7 cells were incubated with ACR for 1, 3, and 5 days. Proliferation curve was determined by MTT assay. Results are presented as the mean ± SEM of three independent experiments. *, P < 0.05, significantly different from untreated cells (control). (C) Effect of ACR on cell cycle distribution in RAW 264.7 cells. Cells were incubated in the absence or presence of 0.5 mM ACR for 1, 3, and 5 days. Cells were stained with propidium iodide 74


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in the presence of RNase A and Triton X-100 and analyzed by FACS. DNA content was obtained from three independent experiments and plotted. * and $, P < 0.05, significantly different from ACR treated 0 day cells. (D, E) Induction of SA-β-gal activity in ACR-treated macrophages. RAW 264.7 cells and peritoneal macrophages were incubated with 0.5 mM ACR for 1, 3, and 5 days. (D) After SA-β-gal staining, cells were photographed at 40× magnification. Arrows denote β-gal positive cells. (E) SA-β-gal activity was quantified by FACS at the desired intervals. The results illustrated are from a single experiment and are representative of three separate experiments. Values are the mean ± SEM of three independent experiments. *, P < 0.05, significantly different from control cultures. (F) Expression of telomerase subunit and telomerase activity in ACR-treated macrophages. RAW 264.7 cells were incubated with ACR for 1, 3, and 5 days. (a) Level of mTERT mRNA expression was determined by RT-PCR analysis. (b, c) TRAP assay of crude nuclear extracts in ACR-treated RAW 264.7 cells at the indicated time points. Cells were harvested, and crude nuclear extracts were prepared and analyzed for telomerase activity by (b) polyacrylamide gel electrophoresis coupled with silver staining and (c) telomerase ELISA: +, positive control (TSR8 DNA template); −, negative control (lysis buffer). Representative data from three independent experiments are shown and quantitated. Immunofluorescence. Macrophages, grown on 22 mm diameter glass coverslips, were pretreated with NAC (10 mM) for 2 h, followed by addition of ACR (10 ng/mL) for 12 h. Cells were washed with PBS and fixed with 3.7% formaldehyde in PBS for 15 min at room temperature and then washed again with PBS. Ice-cold methanol was added, and cells were incubated at −20 °C for 10 min and washed with PBS. Cells were permeabilized with 1% BSA/0.2% Triton X-100/PBS for 1 h. Cells were washed with PBS and incubated with antibody against ATF3 overnight at 4 °C. After PBS washing, cells were incubated for 1 h with anti-rabbit TRITC in 1% BSA/0.05% Triton X100/PBS. Cells were washed twice with permeabilization buffer, incubated for 5 min in a Hoechst 33342-containing PBS solution, and washed with PBS. Coverslips were mounted to glass slides using ProLong Gold antifade agent and photographed with a confocal microscope (LSM 510 META; Carl Zeiss). Statistical Analysis. Results are reported as the mean ± SEM. One-way analysis of variance was used to determine significance among groups, after which a modified t-test with Bonferroni correction was used for comparison between individual groups. Significant values are represented by an asterisk: *, P < 0.05.

enhanced chemoluminescence kit (Amersham). In all immunoblotting experiments, blots were reprobed with anti-β-actin antibody as a control for protein loading. RNA Isolation, Reverse Transcription, and Quantitative PCR Analysis. Total RNA, 1 μg, was mixed with 1 μL of dNTPs and 1 μL of oligo dT for 5 min at 65 °C and incubated on ice for at least 1 min. RNA was reverse transcribed for 60 min at 42 °C using reverse transcriptase (Fermentas, Glen Burnie, MD, USA). PCR amplication of mTERT was performed using the primers listed below. PCR was performed in 50 μL reaction containing 2 μL of cDNA, 1 μL of dNTPs (10 mM), 5 μL of 10× PCR buffer (10 mM Tris-HCl, pH 9, 50 mM KCl and 0.1% Triton X-100, 15 mM MgCl2), 2 μL of senseand antisense-specific oligonucleotide primers (10 μM), and 1.25 units of EF-Taq DNA polymerase (Solgent Co., Korea). GAPDH was detected using primers 5′-CCATGGAGAAGGCTGGGG-3′ and 5′CCAAGTTGTCATGGATGACC-3′. mTERT was detected using primers 5′-CCATGGAGAAGGCTGGGG-3′ and 5′-CCAAGTTGTCATGGATGACC-3′. The PCR reaction was as follows: 94 °C for 5 min followed by 35 cycles of 30 s at 94 °C, 45 s at 52−62 °C, and 72 °C for 1 min. After a final extension at 72 °C for 10 min, PCR products were separated on a 1.2% agarose gel, and amplification products were visualized with gel red (Koma Biotech, Seoul, Korea). Quanitative real-time PCR analysis was performed by Primer Express 2.0 (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions, using SYBR Green. GAPDH was detected using primers 5′-TGCATCCTGCACCACCAA-3′ and 5′-TCCACGATGCCAAAGTTGTC-3′. ATF3 was detected using primers 5′GAGCAGGCAGGAGCATCCT-3′ and 5′-CGGGATGATAAGGCTAAATCC-3′. Plasmid and shRNA Construction. To generate the ATF3 shRNA vector, oligonucleotides shATF3#1 5′-GGAGGCGGCGAGAAAGAAA-3′, shATF3#2 5′-GCAGAAAGAGTCAGAGAAA-3′, and shATF3#3 5′-GAGTGCCTGCAGAAAGAGT-3′ were designed to target nucleotides of ATF3 mRNA (GenBank accession no. NM007498.3). Oligonucleotides were annealed and cloned into pSuper vector between BglII and HindIII sites (Oligoengine, Seattle, WA, USA). Stably transfected RAW 264.7 cells expressing shRNA were selected using puromycin. To generate HA-tagged ATF3, the coding region of ATF3 was amplified by PCR and cloned into the pcDNA3 vector. ATF3 was amplified with forward primer 5′ATATGGGCCCATGATGCTTCAACATCCA-3′ and reverse primer 5′-ATATCTCGAGTTAGCTCTGCAATGTTCC-3′. ATF3 was cloned between the ApaI and XhoI sites in the pcDNA 3/HA-ATF3 vector. Stably transfected RAW 264.7 clones expressing shRNA and HA-ATF3 were selected using medium containing G418. ROS Production Assay. CMH2-DCFDA, a redox-sensitive fluorescent dye, was used to evaluate the intracellular ROS level by flow cytometry. Macrophages (7 × 105 cells/mL) were treated with various concentrations of ACR for the indicated time. Cells were stained for 15 min at 37 °C with 5 μM CMH2-DCFDA. Cells were washed with PBS and resuspended in PBS. Cells were kept on ice in the dark, and at least 10 000 cells from each sample were analyzed using a Becton Dickinson FACSCalibur. Changes in the level of intracellular ROS are expressed as a percentage of cells not treated with ACR.

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RESULTS Induction of Senescence-Like Growth Arrest in ACRTreated Macrophages. Since stress-induced senescence can be induced by DNA-damaging agents such as ACR, we evaluated whether ACR causes a concentration-dependent reduction in cell proliferation and induction of senescence in macrophages. Our results showed that low concentrations (1 mM) for 24 h (data not shown). In another experiment, nonconfluent cells exposed to low concentrations of ACR showed slow proliferation, which is most likely associated with senescence-like growth arrest. The inhibition of cell growth was persistent and observed for up to 5 days at 0.25, 0.5, or 1 mM ACR, whereas untreated control cells proliferated exponentially (Figure 1B). These results indicated that ACR treatment resulted in sustained growth arrest at optimal concentrations and times. In subsequent experiments, macrophages were treated with 0.5 mM ACR for the indicated time. Next, we examined whether ACR-induced inhibition of cell proliferation is due to cell cycle arrest. Flow cytometry-based cell cycle analysis showed that ACR treatment resulted in the accumulation of cells in G0/G1 phase along with a concomitant reduction of those in S phase, which is a general characteristic of senescent cells (Figure 1C). Our results also showed that exposure to ACR for the indicated times markedly increased the number of SA-β-gal positive cells and SA-β-gal activity compared with that of untreated control cells over the culture period (Figure 1D,E). Thus, these results indicate that 75


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Figure 2. Involvement of ATF3 in senescence induced by ACR. (A, B) RAW 264.7 cells were incubated with various concentrations of ACR for the indicated time, and total RNA and protein were isolated and subjected to quantitative real-time PCR (A) and western blot (B) using ATF3-specific primers and antibody. The GAPDH gene and β-actin protein levels were considered as internal controls. Values are the mean ± SEM of three independent experiments.*, P < 0.05, significantly different from untreated cells. (C) Young and aged macrophages were incubated with ACR for 3 days, and total protein was isolated and subjected to western blot with antibodies specific for ATF3, p53, and p21. The β-actin protein levels were evaluated as an internal control. (D) Macrophages from young (7 weeks) and aged (18−20 months) mice were treated with ACR (0.5 mM) for 3 days. After SA-β-gal staining, cells were photographed at 40× magnification. Representative data from three independent experiments are shown and quantitated (bottom). Values are the mean ± SEM of three independent experiments.

various conditions,29 we investigated the role of ATF3 in relation to senescence in ACR-treated macrophages. We first examined ATF3 mRNA and protein levels in ACR-treated macrophages. The data obtained showed that ACR increased ATF3 mRNA expression in a concentration-dependent manner (Figure 2A). The level of ATF3 protein was also increased in a time-dependent manner (Figure 2B). We next determined whether the level of ATF3 expression differed between isolated macrophages from young and aged mice. As shown in Figure 2C, a significant enhancement of ATF3 expression by ACR was observed in peritoneal macrophages from young mice (7 weeks). However, ACR did not alter ATF3 expression in macrophages from aged mice (Figure 2C). Interestingly, ATF3 was more highly expressed in freshly untreated peritoneal

treatment of macrophages with a low concentration of ACR could cause senescence-like growth arrest. The regulation of telomerase activity and telomere length are known to play a significant role in cellular senescence. Induction of senescence is closely associated with the suppression of telomerase activity. Therefore, we assessed the effect of ACR on the level of TERT mRNA expression and telomere length, which were not altered over 5 days of compound exposure. Moreover, telomerase activity was not suppressed (Figure 1F). These data suggest that ACR-induced senescence was telomerase-independent and likely proceeded through a stress-induced senescence mechanism. Involvement of ATF3 in ACR-Induced Senescence. Since the ATF3 gene is induced in response to stress under 76


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Figure 3. Involvement of ATF3 in ACR-induced senescence. (A) RAW 264.7 cells were stably transfected with vector control, vector overexpressing ATF3 (HA-ATF3), or specific knockdown of ATF3 (shATF3). (a) Total cell lysates were subjected to western blot using ATF3, HA, or β-actin antibodies. (b) ATF3 protein was visualized using an anti-ATF3 antibody and an anti-rabbit TRITC-conjugated secondary antibody. DNA in the nuclei was stained with Hoechst 33342. Coverslips were mounted, and slides were examined using a confocal fluorescence microscope. Scale bars are 50 μm. (B, C) shATF3-stable (B) or HA-ATF3 (C) RAW 264.7 cells were treated with 0.5 mM ACR for 5 days. After SA-β-gal staining, cells were photographed using 40× magnification, and the intensity of green fluorescence (SA-β-gal activity) was analyzed by FACS. Arrows denote β-gal positive cells. Representative data from three independent experiments are shown and quantitated (bottom). Values are the mean ± SEM of three independent experiments. *, P < 0.05, significantly different from untreated cells. #, P < 0.05, significantly different from ACR-treated shControl. $, P < 0.05, significantly different from ACR-treated HA control cells.

results, we examined the effect of glycidamide (GA), an ACR metabolite, on ATF3, p53, and p21 expression (Supporting Information Figure S1). Similar changes in the expression of these genes was also observed in GA-treated macrophages, indicating that ACR and its metabolite appear to promote the same effects on cellular senescence and that ACR could promote cellular senescence in vivo. The finding that levels of ATF3 mRNA and protein were significantly increased under senescence-inducing concentrations of ACR in macrophages suggested a potential role of ATF3 in ACR-induced senescence. To further examine the role of ATF3 in the aging process, we generated RAW 264.7 cells stably expressing shATF3 and used HA-tagged stable DNA to overexpress ATF3 protein. This was confirmed by western blotting and by an immunofluorescence assay, which showed that ATF3 protein expression was not detected in shATF3transfected cell (Figure 3A). Treatment with ACR resulted in a decrease in the number of SA-β-gal positive cells and in SA-βgal activity in shATF3-stable cells at 5 days (Figure 3B). Similar changes in SA-β-gal expression were also observed in ATF3−/− MEF (Supporting Information Figure S2). In contrast, overexpression of ATF3 significantly increased the number of cells positive for SA-β-gal and SA-β-gal activity in ACR-treated cells (Figure 3C). These results suggest that ATF3 plays an important role in ACR-induced senescence. ROS Is an Important Mediator of ACR-Induced Cellular Senescence in Macrophages. Numerous studies have shown that intracellular ROS production is important in

macrophages from aged mice (18−20 months) than in macrophages from young mice. Thus, these results suggest that ATF3 may be linked to senescence. Additionally, in the present study, we demonstrated that there is a difference between young and aged mice in the percentage of SA-β-gal positive cells from fresh, untreated macrophages (Figure 2D). Moreover, treatment of macrophages from young mice with ACR resulted in a significantly higher percentage of SA-β-gal positive cells when compared with that from untreated cells.30 However, although the number of SA-β-gal positive cells was slightly increased in ACR-treated macrophages from aged mice as compared with that in untreated cells, ACR did not show a statistically significant change in the rate of senescence in aged macrophages. Furthermore, we determined whether p53 and p21 expression were related to induction of ACR-induced senescence because functional p53 and its transcriptional target, p21, are essential for the senescence-like response to DNA-damaging agents. As shown in Figure 2B, the expression of ATF3, p53, and p21 was considerably increased in ACRtreated macrophages. Similar results were obtained in primary cultures of macrophages. ACR treatment resulted in an increase in the expression of ATF3, p53, and p21 in macrophages from young mice, and the expression of these genes was upregulated in untreated macrophages from aged mice (Figure 2C). Interestingly, p53 and ATF3 protein levels were not altered in ACR-treated macrophages from aged mice, whereas ACR caused an increase in p21 expression. To further validate these 78


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Figure 4. ACR-induced ROS is an essential mediator of cellular senescence in macrophages. (A) RAW 264.7 cells were treated with increasing concentrations of ACR. After incubation for 1, 3, and 5 days, cells were incubated with CM-H2DCFDA (5 μM) for 15 min 37 °C. The intensity of CM-H2DCFDA fluorescence was measured by a flow cytometer. Representative data from three independent experiments are shown and quantitated (bottom right). (B) Control or HA-ATF3 stably overexpressing RAW 264.7 cells were treated with the indicated concentrations of ACR for 5 days. Representative data from three independent experiments are shown and quantitated (bottom). Values are the mean ± SEM of three independent experiments. *, P < 0.05, significantly different from untreated cells. #, P < 0.05, significantly different from ACR-treated HA control 79


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cells. (C, D) RAW 264.7 cells were pretreated with NAC (10 mM) for 2 h, followed by incubation with ACR for 5 days. (C) After SA-β-gal staining, cells were photographed at 40× magnification. Arrows denote β-gal positive cells. Representative data from three independent experiments are shown and quantitated (bottom). Values are the mean ± SEM of three independent experiments. *, P < 0.05, significantly different from ACRtreated cells. (D) The levels of ATF3 expression were determined by western blot using an anti-ATF3 antibody. (E) Subcellular fractionation was conducted to separate the nuclear from the cytosolic fractions, which were then subjected to western blotting using an antibody specific for ATF3. αTubulin and lamin A were used as fractionation controls.

the induction of senescence.31 Previous results also indicate that ACR is able to induce oxidative stress and ROS production.32,33 To investigate whether ROS production was regulated by ACR, macrophages were treated with various concentrations of ACR for the indicated time. As shown in Figure 4A, intracellular ROS levels significantly increased in a time-dependent manner at 0.25 and 0.5 mM ACR. However, ROS production gradually decreased after 3 days at 1 mM. One possible explanation for the decreased ROS production is that a reduction in cell viability by ACR contributes to a loss of ROS production and/or that overproduction of ROS by ACR could affect cell viability. To further elucidate whether ROS production was associated with ATF3 expression, we assessed ROS production using RAW 264.7 cells overexpressing ATF3. As shown in Figure 4B, ATF3 overexpression in ACR-treated cells caused increased ROS production compared with that of cells transfected with a mock vector. In addition, cells expressing ATF3 in combination with ACR displayed a further increase in ROS generation. To clarify the importance of the increase of ROS production in the development of senescence, cells were pretreated with N-acetyl cysteine (NAC), a wellknown antioxidant, and then incubated with ACR. We found that NAC significantly reduced senescence, as judged by SA-βgal staining (Figure 4C). Furthermore, these cells showed a decrease in ATF3 protein (Figure 4D). We also examined whether ATF3 expression was dependent on ROS production because many proteins are suggested to undergo nuclear translocation in response to regulatory signals and ROS acts as a signaling molecule. As shown in Figure 4E, ATF3 localization by western blotting showed that ACR treatment induced ATF3 translocation from the cytoplasm to the nucleus in macrophages, which reflected its activation. Furthermore, NAC partially inhibited this translocation, indicating ROS dependence. These findings suggest that ROS plays an important role in ACR-mediated ATF3 upregulation. Induction of Macrophage Senescence via the p38 and JNK Pathways. The mitogen-activated protein kinase (MAPK) signaling pathway is a key regulator of cell differentiation, proliferation, senescence, survival, and apoptosis.34 Therefore, the ERK1/2, p38 MAPK, and JNK kinase pathways were examined to determine whether MAPKs were involved in ACR-induced senescence. Figure 5A shows that ACR clearly stimulated an increase in the levels of activated ERK1/2, p38 MAPK, and JNK. However, the induced MAPK activity and ATF3 expression were significantly inhibited by pretreatment with NAC for 2 h followed by incubation with ACR for 5 days (Figure 5B), suggesting that ROS production was necessary for the induction of the MAPK pathways and ATF3 expression in ACR-treated macrophages. Next, to confirm whether the MAPK pathways were involved in ATF3 expression in ACR-treated macrophages, we examined the effects of inhibitors of ERK1/2 (PD98059), p38 MAPK (SB203580), and JNK (SP600125) on ACR-induced ATF3 expression. As shown in Figure 5C, pretreatment of macro-

phages with inhibitors SB203580 and SP600125 for 2 h before ACR exposure displayed inhibitory effects on the upregulation of ATF3 by ACR. In contrast, the ACR-induced expression of ATF3 was barely altered by pretreatment with PD98059. These results indicate that p38 and JNK are important for inducing ATF3 gene expression in ACR-treated macrophages. We subsequently examined whether the activation of MAPK pathways accompanied senescence in ACR-treated macrophages. For this experiment, cells were preincubated with inhibitors of the MAPK pathways prior to ACR treatment. As shown in Figure 5D, the inhibition of p38 or JNK abrogated the ACR-induced increase in the number of SA-β-gal positive cells compared with that for cells treated with ACR alone. However, inhibition of ERK1/2 had no significant effect on SA-β-gal activity. Collectively, our data suggests that ROS production acts as an upstream mediator of MAPKs, leading to effects on p38 kinase and JNK activation with the subsequent induction of ATF3-mediated cellular senescence in ACR-treated cells. ATF3 Regulates p53 in ACR-Induced Cellular Senescence. p53 is known to be important for the senescence-like response to genotoxic stress induced by DNA damaging agents, and p53 stability requires interaction with ATF3 after genotoxic stress.35−37 To better understand the mechanism underlying ATF3-mediated regulation of cellular senescence in response to ACR, we generated a stable cell line expressing ATF3 shRNA. As shown in Figure 6B, depletion of ATF3 significantly attenuated the p53 protein level in ACR-treated cells (Figure 6A). Interestingly, expression of the p53-regulated cell cycle protein, p21, was also decreased by ATF3 knockdown, whereas the level of p16 protein was not altered (data not shown). Furthermore, we investigated whether p53 affected the expression of ATF3. The data clearly indicated that ablation of p53 did not affect ATF3 expression (Figure 6B). Additionally, treatment of the p53 knockdown cells with ACR resulted in a decrease in the number of SA-β-gal positive cells compared with that in ACR-treated shControl cells (Figure 6C). Similar results were obtained using p53-null osteosarcoma MG63 and ATF3−/− MEF cells (Supporting Information Figure S3). Overall, these findings suggest that ATF3 is a regulator of cellular senescence through interactions with p53 and that p53 is a downstream effector of ATF3 in the process of cellular senescence in ACR-treated cells.

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DISCUSSION Immune senescence is a progressive dysfunctioning of the immune system that leads to an inappropriate, inefficient, and sometimes detrimental immune response.4,38 Macrophages play an important role in both innate and adaptive immunity by several indirect and direct mechanisms. The altered function of macrophages by stress-induced aging may play a part in agerelated immunological changes. In this study, we showed, for the first time, that treatment of macrophages with ACR induces changes in senescence-associated genes and the production of ROS. In addition, we identified the involvement of ATF3 80


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Figure 5. Induction of macrophage senescence via p38 and JNK pathways. (A) RAW 264.7 cells were treated with ACR for the indicated time, and phosphorylation levels of MAPKs were detected by western blot. The results illustrated are from a single experiment and are representative of three separate experiments. Protein levels were quantified by scanning densitometry, normalized to β-actin, and expressed as arbitrary units. *, P < 0.05, significantly different from untreated cells. (B, C) RAW 264.7 cells were pretreated with the indicated concentrations of NAC, ERK inhibitor PD98059 (PD), p38 inhibitor SB203580 (SB), and JNK inhibitor SP600125 (SP) for 2 h, followed by incubation with ACR for 30 min. Levels of 82


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ATF3 expression and MAPK phosphorylation were determined by western blotting. The results illustrated are from a single experiment and are representative of three separate experiments. Protein levels were quantified by scanning densitometry, normalized to β-actin, and expressed as arbitrary units. *, P < 0.05, significantly different from untreated cells. #, P < 0.05, significantly different from ACR-treated cells. (D) RAW 264.7 cells were incubated with ACR for the indicated time in the absence or presence of PD, SB, and SP for 2 h followed by incubation with ACR for 5 days. After SA-β-gal staining, cells were photographed at 40× magnification. Arrows denote β-gal positive cells. Representative data from three independent experiments are shown and quantitated (bottom). Data shown are the mean ± SEM of at least three independent experiments. *, P < 0.05, significantly different from ACR-treated cells.

Figure 6. Regulation of p53 by ATF3 in ACR-induced cellular senescence. (A) RAW 264.7 cells were stably constructed with vector control or ATF3 shRNA and stimulated with 0.5 mM ACR. Cell lysates were subjected to western blot. The results illustrated are from a single experiment and are representative of three separate experiments. (B, C) RAW 264.7 cells were transfected with vector control or p53-specific shRNA and stimulated with 0.5 mM ACR. (B) Cell lysates were subjected to western blot. The results illustrated are from a single experiment and are representative of three separate experiments. (C) After SA-β-gal staining, cells were photographed at 40× magnification. Arrows denote β-gal positive cells. Representative data from three independent experiments are shown and quantitated (right). Data shown are the mean ± SEM of three independent experiments. *, P < 0.05, significantly different from untreated cells. #, P < 0.05, significantly different from ACR-treated shControl cells.

senescence is known to be closely associated with the suppression of telomerase activity.2 In our study, no telomerase activity was seen in normal macrophages, and no activity was induced by ACR, suggesting that ACR-induced senescence occurred independently of telomerase activity and likely proceeded through a stress-induced senescence mechanism. ATF3 has been studied for its role as a regulator of inflammatory cytokines and apoptosis in response to various stimulations.24 However, the effect of ATF3 activation on the growth and lifespan of macrophages has been poorly investigated. Here, we show that ACR increased the levels of ATF3 mRNA and protein in RAW 264.7 cells and primary cultures of macrophages. In addition, ACR-treated macrophages led to elevated levels of ATF3 in peritoneal macrophages from young mice. Macrophages from aged mice also

protein in ACR-induced, senescence-like G0/G1 phase accumulation of macrophages. Recently, a replicative senescence state was demonstrated to be rapidly triggered by diverse forms of cellular damage or stress in different cell types.39 Our results indicated that treatment of macrophages with subtoxic concentrations of ACR resulted in the induction of a senescence-like growth arrest. In addition, an increased positive staining of SA-β-gal was observed in ACR-treated cells. FACS analysis showed that SA-β-gal activity was also significantly enhanced by treatment of macrophages with ACR. These results suggested that the treatment of macrophages with a low concentration of ACR could cause senescence-like growth arrest. Telomerase activity has been detected in several normal human somatic cells, including immune cells.40,41 Additionally, induction of 83


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pathways are critical in the expression of ATF3 and ROS production in ACR-induced senescence. Cellular proteins that interact with ATF3 play a major role in both positive and negative regulation of this transcription factor. Numerous studies show that p53 is the key regulator of major mammalian cellular functions such as cellular senescence. We found that knockdown of ATF3 causes attenuated expression of p53 and p21 along with a decrease in cellular senescence. In contrast, ATF3 overexpression enhanced the level of p53 expression and the process of cellular senescence. In addition, p53 knockdown significantly suppresses ACRinduced cellular senescence, but it has no effect on ATF3 expression. On the basis of these findings, it is conceivable that ATF3 is a regulator in the control of cellular senescence through interactions with p53 and that p53 is upregulated by binding with activated ATF3 in response to ACR. It has been suggested that ATF3 can act as either a transcriptional activator or repressor, probably depending on the promoter context.22 The present data showing that ACR upregulates the expression of p53 through ATF3 activation are consistent with previous reports that an ATF3 binding motif is located in p53’s promoter region and that ATF3 interacts with the C-terminus of p53 to increase its stability in response to genotoxic stress by preventing it from undergoing MDM2-mediated ubiquitination and degradation.45 Moreover, it is plausible to speculate that ACR causes SUMO modification of ATF3, which regulates the ATF3−p53 interaction, because a previous study has demonstrated that SUMOylation of ATF3 plays a role in regulating ATF3−p53 interaction and transactivation of a p53responsive promoter.46 However, it has been recently suggested that ectopic ATF3 expression inhibits expression of p53.46 This discrepancy possibly arose from differences in the cell type used and the experimental conditions employed. In our studies, macrophages were used and treated with ACR in vitro, but another group examined the effect of cyclosporine A on ATF3 and p53 expression in human squamous cell carcinoma of the skin.47 In summary, our studies with RAW 264.7 cells and primary macrophages suggest that regulation of ATF3 expression in response to ACR is an important event for controlling cellular senescence. p53 is a downstream effector of ATF3 in the process of cellular senescence in ACR-treated macrophages. Moreover, the induction of ROS might initiate a senescence cascade in response to ACR and subsequently increase ATF3 expression through MAPK pathways. Thus, the data presented suggest that ACR, a DNA-damaging agent, might influence various diseases through a biochemical pathway that induces immune cell senescence.

had markedly increased levels of ATF3. These results indicate that elevated ATF3 could be involved in ACR-induced senescence. Interestingly, ACR did not affect ATF3 expression in macrophages from aged mice. This might be due to a progressive increase in the level of ATF3 with aging, which subsequently leads to maximum levels of ATF3 expression in aged cells. Our work further defines the role of ATF3 in this process by demonstrating that ATF3 depletion leads to a reduction in cellular senescence, as indicated by the increased number of senescence-associated SA-β-gal positive cells in ATF3-overexpressing cells. Taken together, these data suggested that ATF3 appears to be an important regulator of aging-related cellular processes in ACR-treated macrophages. The stabilization of p53 and the induction of p21 are known to be essential under cellular senescence conditions induced by various stresses including DNA damage, oncogene activation, and telomere dysfunction.42 In addition, p21 expression is upregulated by p53-dependent and -independent mechanisms.43 Our results indicated that the levels of p53 and p21 protein expression were enhanced by ACR, whereas ACR did not induce an increase in p16 protein. On the basis of these findings, p53 and p21 might be involved in ACR-induced senescence. ROS are involved in maintaining human physiological processes, including senescence and in vivo aging.44 Indeed, ROS accumulation with age induces cell senescence and apoptosis. Also, ACR is known to induce an increase in the ROS level in various cells.32,33 The data presented here demonstrate that ACR induces the production of ROS in a time-dependent manner in macrophages. Interestingly, we found that ROS production was significantly increased in cells expressing ATF3 alone and that cells expressing ATF3 in combination with ACR displayed a further increase in ROS generation. On the basis of these observations, it is reasonable to speculate that ATF3 overexpression could increase ROS production, which, in turn, further induces ATF3 gene expression, leading to cellular senescence in the ACR-treated ATF3 overexpressing cells. In addition, the ROS inhibitor NAC suppressed ATF3 expression as well as SA-β-gal activity in ACR-induced senescent macrophages. Moreover, a significant inhibitory effect of NAC on ACR-induced ATF3 nuclear translocation was observed. These findings indicate that ROS acts as a signaling molecule regulating the expression of ATF3 following treatment with ACR. Taken together, it is possible that ACR-induced ATF3 expression and senescence may be dependent on ROS production. Multiple MAPKs play an important role in the signal transduction pathways that regulate cell differentiation, proliferation, senescence, survival, and apoptosis in response to external stimuli.34 Therefore, we studied the effect of ACR on the JNK, p38 MAPK, and ERK1/2 pathways, three wellcharacterized MAPK subtypes. Our data demonstrate that ACR significantly activates phosphorylation of JNK, p38 MAPK, and ERK1/2 in macrophages. In addition, inhibitors of p38 MAPK and JNK pathways suppress ACR-induced ATF3 protein expression and SA-β-gal activity in macrophages. Inhibition of ERK1/2 had no significant effect on ACR-induced ATF3 protein expression and SA-β-gal activity. We also found that NAC treatment decreases activation of the MAPK pathway. These results suggested that ACR induces the elevation of ATF3 expression in macrophages through the activation of a MAPK signaling pathway and that the p38 MAPK and JNK

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ASSOCIATED CONTENT

S Supporting Information *

Effect of glycidamide (GA) on senescence of macrophages; involvement of ATF3 in ACR-induced senescence; regulation of p53 by ATF3 in MG63 and ATF3−/− MEF cells. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +82-31-290-7753. Fax: +82-31-290-7733. E-mail: [email protected]. 84


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†

K.-H.K. and B.P. contributed equally to this work.

Funding

This work was funded with internal resources. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS ACR, acrylamide; ATF3, activating transcription factor 3; SA-βgal, senescence-associated β-galactosidase; ROS, reactive oxygen species; SIPS, stress-induced premature senescence; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; CMH2-DCFDA, 5,6-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester; p-JNK, phospho-cJun N-terminal kinase; p-ERK, phospho-extracellular signalregulated kinase; MEFs, mouse embryonic fibroblasts; MTT, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide; Xgal, 5-bromo-4-chloro-3-indolyl β-D-galacopyranoside; TBST, Tris-buffered saline/nonfat Tween

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Acrylamide Induces Senescence in Macrophages through a Process

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