MEDIATED SECRETION OF TUMOUR ... - Wiley Online Library

Clinical and Experimental Pharmacology and Physiology (2009) 36, 940–944

doi: 10.1111/j.1440-1681.2009.05177.x

GLUCOSE-REGULATED PROTEIN 78 PROMPTS SCAVENGER RECEPTOR A-MEDIATED SECRETION OF TUMOUR NECROSIS FACTOR- BY RAW 264.7 CELLS

Blackwell GRP78 S Gao etprompts al. Publishing secretion Asia of TNF-

Song Gao,*† Xiaozheng Zhong,* Jingjing Ben,*† Xudong Zhu,* Yuan Zheng,* Yan Zhuang,* Hui Bai,* Li Jiang,* Yaoyu Chen,‡ Yong Ji* and Qi Chen*† *Atherosclerosis Research Center, †Institute of Reproductive Medicine, Nanjing Medical University, Nanjing, China and ‡ Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA

SUMMARY 1. Activation of macrophages plays an important role in atherosclerosis. In order to investigate the effect of endoplasmic reticulum (ER) stress on cytokine release from macrophages, the RAW 264.7 mouse macrophage cell line was treated with 0.2 mmol/L 6-aminonicotinamide (6-AN) for 36 h and the secretion of tumour necrosis factor (TNF)- determined. In addition, Raw 264.7 cells were incubated in the presence of 10 g/mL acetylated low-density lipoprotein (acLDL) at 37C for 8 h. 2. Secretion of TNF- from RAW 264.7 cells was stimulated by both loading of cells with acLDL and following 6-AN treatment. In addition, the expression of glucose-regulated protein (GRP) 78 was increased in 6-AN-treated cells (by 165%). 3. In separate experiments, PD98059, a specific inhibitor of the mitogen-activated protein kinase kinase (MEK) pathway, blocked acLDL- and/or 6-AN-induced TNF- secretion, whereas LY294002, which blocks the AKT signalling pathway, had no effect. On the basis of these results, we speculate that acLDL/ 6-AN-induced secretion of TNF- from RAW 264.7 cells may be regulated by activation of the MEK signalling pathway. 4. The present study suggests that the accumulation of lipids in cells and/or ER stress could lead to macrophage apoptosis as a result of the increased production of TNF-, which integrates into atherosclerosis. Key words: acetylated low-density lipoprotein, glucoseregulated protein 78, Raw 264.7 cells, scavenger receptor A, tumour necrosis factor-.

INTRODUCTION Class A scavenger receptor (SR-A) can bind and internalize modified low-density lipoproteins (LDL) in macrophages, which initiates lipid-laden foam cell formation.1–3 Foam cell formation is a key step in the generation and progression of atherosclerosis. In addition to

Correspondence: Qi Chen, Atherosclerosis Research Center, Nanjing Medical University, Nanjing 210029, China. Email: [email protected] Received 30 October 2008; revision 24 February 2009; accepted 25 February 2009. © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Asia Pty Ltd

it role in atherogenesis, SR-A also functions in host defence,4,5 antimicrobial immunity, cell adhesion,6,7 apoptotic cell clearance, antigen processing7 and cytokine production.4,8,9 Multiple ligands, including acetylated LDL (acLDL), have been found to bind to SR-A.7 Activation of macrophages can stimulate the secretion of inflammatory cytokines, such as tumour necrosis factor (TNF)-.9–11 However, the relationship between SR-A and TNF- secretion is not clear. Recently, it was found that acLDL and fucoidan upregulate urokinase-type plasminogen activator (uPA) in macrophage cells, which, in turn, activates tyrosine-containing proteins.12 To investigate the possible link between SR-A and TNF- secretion, we induced endoplasmic reticulum (ER) stress in RAW 264.7 cells by treating the cells with 6-aminonicotinamide (6-AN). Both acLDL and 6-AN stimulated TNF- secretion from RAW 264.7 cells and the effects of 6-AN may involve upregulation of glucose-regulated protein (GRP) 78 and activation of the mitogen-activated protein kinase (MAPK)/MAPK kinase (MEK)/extracellular signal-regulated kinase (ERK) signalling pathway.

METHODS Drugs and reagents Antibodies against phosphorylated (p-) MEK and MEK were obtained from Cell Signal Technology (Danvers, MA, USA). Antibodies against SR-A, GRP78 and -actin, as well as horseradish peroxidase (HRP)-conjugated antibodies, were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 6-Aminonicotinamide was obtained from Sigma (St Louis, MO, USA) and was made up into a 10 mmol/L solution in phosphate-buffered saline (PBS); the MEK protein kinase inhibitor PD98059 and the AKT protein kinase inhibitor LY294002 were also from Sigma. 3-(4,5-Dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was obtained from Amresco (Solon, OH, USA). The bicinchoninic acid (BCA) protein assay kit was obtained from Pierce (Rockford, IL, USA). Secreted TNF- was quantified using ELISA kit from Biosource (Camarillo, CA, USA).

Cells and cell culture The RAW 264.7 macrophage cell line, obtained from the American Type Culture Collection (Rockville, MD, USA), was used in all experiments as the target cell for SR-mediated stimulation. These cells readily express detectable levels of SR, as determined using an antibody aganist SR-A (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Mycoplasma-free cell cultures were maintained in complete medium, consisting of Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT, USA) supplemented with

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GRP78 prompts secretion of TNF- 2 mmol/L glutamine, 1 mmol/L pyruvate, 50 U/mL penicillin, 50 mg/mL streptomycin and 2 g/L sodium bicarbonate (Sigma), plus 10% fetal bovine serum (FBS; Life Technologies, Rockville, MD, USA) at 37C in 5% CO2. Additional aspects of cell maintenance were as described previously.13,14 Cells were plated at a density of 4  105 cells/well in 12-well plates (Costar, Bethesda, MD, USA) and cultured for 24 h. Then, 10 g/mL acLDL was added to the culture and cells were incubated for a further 0, 4, 8, 12, 18 or 24 h. The medium bathing the cells was collected at these time-points for the measurement of TNF- levels using a commercially available ELISA kit (Biosource).

Determination of TNF- levels in culture medium Briefly, the cell culture medium was collected and centrifuged at 12 000 g for 10 min at 4C. The supernatant was then diluted in 1 : 20 in fresh DMEM and added to microtitre plates precoated with anti-human TNF- monoclonal antibody. Samples were incubated for 2 h at room temperature and were then washed thoroughly with the wash buffer provided in the ELISA kit (Biosource, Camarillo, CA, USA) before the addition of an HRP-conjugated detecting antibody. Samples were incubated with chromophore 3,3,5,5tetramethylbenzidine and H2O2 for up to 15 min at room temperature before 1 mol/L H2SO4 was added to the samples as a stop solution. The concentration of TNF- in samples was measured spectrophotometrically at 450 nm on a 96-well plate ELISA reader (CliniBio 128; ASYS Hitech, Eugendorf, Austria). Samples were assayed in triplicate. The TNF- concentration was determined by extrapolation from a standard curve generated by serial dilution of the appropriate recombinant mouse cytokine.

Fig. 1 Acetylated low-density lipoprotein (acLDL) increases the secretion of tumour necrosis factor (TNF)- by RAW 264.7 cells. After cells had been cultured for 24 h, they were treated with 10 mg/mL acLDL for 0, 4, 8, 12, 18 or 24 h. The medium was collected and the concentration of TNF- was measured by ELISA. Data are the mean±SD (n = 3). *P < 0.05 compared with control.

phosphorylation of MEK, the membrane was blocked by the addition of 3% bovine serum albumin in Tris-buffered saline (TBS) and then probed with an antibody against p-MEK or MEK. Bound primary antibody was detected with an HRP-conjugated goat anti-rabbit IgG and an enhanced chemiluminescence (ECL) system.

Statistical analysis Measurement of cell toxicity 4

Cells were cultured at a density of 8  10 cells/mL for 24 h in 96-well plates (Costar) and were treated with 0–0.5 mmol/L 6-AN for 36 h. To determine MTT release from the cells, 25 L MTT stock solution (5 mg/mL, pH 7.4) was added to each well and cells were incubated for a further 4 h. After incubation of the cells with the MTT reagent, the cell supernatant (i.e. the MTT solution) was carefully decanted off and 200 L dimethylsulphoxide (DMSO) was added to each well to dissolve the formazan crystals. The optical density of each well was measured spectrophotometrically at 490 nm using an ELISA reader. The amount of colour produced is directly proportional to the number of viable cells. Measurements were performed in triplicate.

Mitogen-activated protein kinase kinase and AKT signalling pathways The RAW 264.7 cells were plated at a density of 4  105 cells/mL in 12well tissue culture plates (Corning-Costar, Corning, NY, USA) and were incubated for 24 h in complete DMEM. Cells were treated with 6-AN (0.2 mmol/L) for a further 36 h. After removal of the medium, cells were treated with 50 mol/L PD98059 or 10 mol/L LY294002 for 4 h. Control cells were incubated for 4 h with medium alone or with medium containing 0.4% DMSO (the vehicle for the inhibitors used). After 4 h incubation, acLDL (10 g/mL) was added to the cells and the cells were incubated for a further 8 h 37C for 8 h before analysis of TNF- by ELISA.15

Western blot assay For measurements of SR-A, GRP78 or phosphorylation of MEK, RAW 264.7 cells were treated with 0.2 mmol/L 6-AN for 36 h. After lysis in lysis buffer (5 mmol/L Tris-HCl, pH 7.5, 2 mmol/L EDTA, 1% Triton X-100 and 1 mmol/L phenylmethylsulphonyl fluoride), the cell protein concentration was determined using a BCA protein assay kit. Proteins (30 g/lane) were separated by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto an Immobilon PVDF transfer membrane (Millipore, Bedford, MA, USA). The membrane was blocked by the addition of 5% fat-free milk in PBS and then probed with an antibody against SR-A, GRP78 or -actin. For measurement of the

All data are expressed as the mean±SD. Data were analysed using Student’s paired t-test and statistical significance was set at P < 0.05. All experiments were conducted three times unless indicated otherwise.

RESULTS Secretion of TNF- by RAW 264.7 cells When RAW 264.7 cells were incubated with 10 mg/mL acLDL, the concentration of TNF- in the medium was significantly higher compared with control at all time-points examined. The stimulatory effect of acLDL on TNF- secretion peaked at 8 h incubation (TNF- concentration = 182.7 pg/mL). After 8 h, TNF- secretion in the presence of acLDL started to decline (Fig. 1).

Upregulation of GRP78 prompts acLDL-induced secretion of TNF- by RAW 264.7 cells In order to evaluate the effects of ER stress on TNF- secretion, 6-AN, an nicotinamide adenine dinucleotide (NAD) antagonist and a relatively weak inhibitor of poly(ADP-ribose) polymerase (PARP) activity,16 was used to induce an ER stress in RAW 264.7 cells. The toxic effects of 6-AN on RAW 264.7 cells were tested and there was no significant MTT leak from cells for concentrations  0.2 mmol/L 6-AN (Fig. 2). Thus, 0.2 mmol/L 6-AN was used in all subsequent experiments. When cells were treated with 0.2 mmol/L 6-AN for 36 h, GRP78 expression increased by 165%. Meanwhile, no visible changes in SR-A expression were observed (Fig. 3). Following 6-AN treatment, RAW 264.7 cells exhibited markedly increased TNF- secretion (217.1 pg/mL) that was greater than that seen following treatment with acLDL alone (Fig. 4). When cells were treated simultaneously with acLDL and 6-AN, TNF- secretion was even higher, reaching 420.6 pg/mL in the medium (Fig. 4).

© 2009 The Authors Journal compilation © 2009 Blackwell Publishing Asia Pty Ltd

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Fig. 2 Toxic effects of 6-aminonicotinamide (6-AN) on RAW 264.7 cells, as determined by the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Cells were treated with various concentrations of 6-AN as indicated for 36 h before cellular MTT release was determined by reading absorbance at 490 nm. Data are the mean±SD (n = 3). *P < 0.05 compared with control (0 mmol/L 6-AN). OD, optical density. Fig. 4 Effects of 6-aminonicotinamide (6-AN) on tumour necrosis factor (TNF)- secretion by RAW 264.7 cells. Cells were incubated with 10 g/mL acetylated low-density lipoprotein (acLDL) for 8 h, with 0.2 mmol/L 6-AN for 36 h or a combination of acLDL and 6-AN. Concentrations of TNF- in the media were determined. Data are the mean±SD (n = 3). *P < 0.05 compared with control; †P < 0.05 compared with the other three groups.

Fig. 3 Western blot analysis of the expression of (a) glucose-regulated protein (GRP) 78 and (b) scavenger receptor (SR)-A. Cells were treated with 0.2 mmol/L 6-aminonicotinamide (6-AN) for 36 h and cell lysates were then subjected to 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis. Raw, untreated RAW 264.7 cells.

Increased secretion of TNF- is mediated by the MEK pathway The signal pathways underlying the increased TNF- secretion induced by acLDL and 6-AN treatment were investigated by treating RAW 264.7 cells with PD98059, an inhibitor of MEK, or LY294002, an inhibitor of AKT. We found that LY294002 treatment had no effect on TNF- secretion by RAW 264.7 cells (Fig. 5). However, PD98059 treatment inhibited TNF- secretion by 65% (Fig. 5). In addition, by determining levels of p-MEK, we demonstrated that PD98059 significantly inhibited phosphorylation of MEK in the presence of both acLDL and 6-AN (Fig. 6). However, LY294002 treatment had no effect on MEK phosphorylation (Fig. 6).

DISCUSSION Atherosclerosis is an inflammatory response of macrophages and lymphocytes to invading pathogenic lipoproteins in the arterial

Fig. 5 Effects of inhibitors of the signal pathways on the production of tumour necrosis factor (TNF)- by RAW 264.7 cells. After incubation with 0.2 mmol/L 6-aminonicotinamide (6-AN) for 36 h, cells were treated with PD98059 (50 mol/L) or LY294002 (10 mol/L) for 4 h. Control cells were incubated for 4 h with medium containing 0.4% dimethylsulphoxide. Acetylated low-density lipoprotein (acLDL; 10 g/mL) was added and cells were incubated at 37C for a further 8 h before TNF- concentrations were determined by ELISA. Data are the mean±SD (n = 3). *P < 0.05 compared with acLDL + 6-AN.

wall. Following uptake of modified LDL, macrophages become lipid-loaded foam cells and initiate lipid strip formation. Activated macrophages also contribute to the inflammatory process in the arterial wall by secreting inflammatory cytokines, such as TNF-.17,18 Several groups have reported that both lipoprotein and nonlipoprotein ligands of SR-A can induce cytokine production via SR-A-associated signalling cascades.8,15 The endocytic uptake of the SR-A ligands lipoteichoic acid (LTA) and polyinosinic–polycytidilic acid (poly I:C) is essential for macrophage production of TNF-; the production of TNF- is reduced by 95% and 80% in response to LTA and poly I:C if clathrin-coated pit formation is blocked by


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Fig. 6 Western blot analysis of mitogen-activated protein kinase kinase (MEK) phosphorylation levels in RAW 264.7 cells. Cells were treated as described in Fig. 5 and cell lysates were subjected to western blot assay for phosphorylated (p-) MEK or MEK.

the primary amine and monodansylcadaverine.15 Tumour necrosis factor- is a pluripotent cytokine that was initially described as an agent capable of stimulating cachexia/wasting.19,20 In addition, TNF- acts as an immunomodulator, like interleukin (IL)-1, IL-1, IL-6, IL-10, IL-12 and granulocyte–macrophage colony stimulating factor, which are augmented in herpes simplex virus (HSV)- 1-stimulated antigen-presenting dendritic cells.21 In the present study, we demonstrated that TNF- production increased approximately sixfold in acLDL-treated RAW 264.7 cells after 8 h incubation compared with control (0 h incubation). Thus, the uptake of modified LDL may evoke macrophages not only to accumulate lipid, but also to participate in the inflammatory process in the arterial wall, both of which are key steps in atherogenesis. In the present study, we investigated factors that influence acLDLinduced TNF- secretion in 6-AN-treated cells. It has been reported that 6-AN increases cell sensitivity to alkylating agents and ionizing radiation, interferes with poly(ADP-ribose) metabolism and reduces NAD biosynthesis.22,23 In addition, 6-AN increases cellular cisplatin accumulation and DNA adduct formation.24 In the present study, 6-AN stimulated higher TNF- secretion by RAW 264.7 cells. The effects of 6-AN on TNF- production may independent of SR-A, because treatment with 6-AN did not cause any changes in SR-A expression. Combined treatment of RAW 264.7 cells with acLDL and 6-AN resulted in a synergistic increase in TNF- secretion. Furthermore, 6-AN treatment increased expression of GRP78 in RAW 264.7 cells, which is consistent with the report of Chatterjee et al.25 Thus, we propose that 6-AN mediates TNF- production via GRP78. The relationship between GRP78 and TNF- production is not yet known. As an ER resident molecular chaperone, GRP78 regulates ER function by maintaining a permeability barrier during protein translocation, directing protein folding and assembly, preventing protein aggregation, contributing to ER calcium homeostasis and sensing conditions of stress to activate the mammalian unfolded protein response.26 Glucose-regulated protein 78 is a member of the heat shock protein (HSP) 70 family; it and other HSP proteins are thought to regulate cell survival and apoptosis.27,28 Overexpression of HSP70 enhances T cell receptor/CD3- and Fas/Apo-1/CD95mediated apoptosis.29 Upregulation of HSP90 leads to an increase in TNF-- and cycloheximide-induced apoptosis in the human U-937 cell line.29,30 Moreover, HSP70 promotes TNF-mediated cell death by binding IKK and impairing nucelar factor-B survival signalling.31 These results imply that GRP78 may be pro-apoptotic via secretion of TNF-. The mechanism underlying acLDL/6-AN-induced secretion of TNF- was further investigated in the present tudy. Several lines of evidence show that TNF- production in antigenically stimulated

mast cells is a highly regulated process.32–34 We demonstrated that PD98059, a specific inhibitor of the MEK pathway, inhibits acLDL/ 6-AN-induced TNF- production. Mitogen-activated protein kinase kinase is a signal molecule downstream of Ras-guanosine triphosphate and Raf/MEK-kinase 1 and 2. Activation of MEK is required for full activation of the MAPK cascade, including ERK, c-Jun-kinase and p38 kinases, as well as the nuclear translocation of the transcription factors targeted in that system, such as ets-like protein kinase 1, c-Jun and activating transcription factor 2.15 Blockade of the AKT signal pathway by LY294002 did not inhibit acLDL/ 6-AN-induced TNF- production, suggesting little role of the AKT pathway in TNF- production. Phosphorylation of MEK was enhanced in acLDL/6-AN-treated cells and was inhibited by the addition of PD98059. This is consistent with TNF- secretion by the cells. Thus, the acLDL/6-AN-induced secretion of TNF- by RAW 264.7 cells is likely regulated by the MEK pathway. The results of the present study indicate that both acLDL and 6-AN induce TNF- secretion from RAW 264.7 macrophages. The results suggest that the accumulation of lipid in cells and/or ER stress could lead to apoptosis as a result of TNF- production. In addition, GRP78 may play a key role in this process. Recently Seimon et al.35 showed that SR-A ligands trigger apoptosis in ER-stressed macrophages by cooperating with Toll-like receptor (TLR) 4 to redirect TLR4 signalling from prosurvival to pro-apoptotic. The relationship between SR-A and apotpsis/GRP78 requires further investigation. By secreting the inflammatory cytokine TNF-, macrophages contribute directly to the inflammatory process. In addition, the inflammatory cytokines secreted can then affect other cells in the lesion (e.g. endothelial cells, smooth muscle cells).

ACKNOWLEDGEMENTS This work was supported by grants from the National Natural Science Foundation of China (no. 30730044), National Basic Research Program (973) (no. 2006CB708509 and 2006CB503801) and Changjiang Scholars and Innovative Research Team in University to QC.

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