Mouse macrophage RAW 264.7 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum, 100 U/ml streptomycin and 100 U/ml penicillin (Gibco; Thermo Fisher Scientific, Inc.) in a humidified atmosphere at 37°C and 5% CO₂. Wntpalmitoyltransferase inhibitor (IWP-2,N-(6-M ethyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) and 30 µM was used to treat the cells.

For overexpression of ATF3, ATF3 cDNA generated from reverse transcription-quantitative PCR (RT-qPCR; The primers used for the cloning were: 5′-AAAAAGCTTATGATGCTTCAACATCCAGG-3′ and 5′-TTTGAATTCTTAGCTCTGCAATGTTCCTT-3′) was subcloned into a pcDNA™3.1 plasmid (Invitrogen; Thermo Fisher Scientific, Inc.) between Hind3 and EcoR I sites to express ATF3 in abundance in E. coli DH5α cells to generate an ATF3 expression plasmid. The third generation of cells (5×10⁵) were seeded into a 24-well plate and transfected with the pcDNA-ATF3 or the empty vector negative control plasmid, psDNA3.1(−), using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol at 37°C. After incubation for 48 h, cells were harvested and ATF3 expression was determined. To knockdown ATF3 and tenascin (TNC), The third generation of cells (5×10⁵) were seeded into a 24-well plate and ATF3 siRNA, its negative control (sham), or TNC siRNA and its negative control siRNA (scramble) were transfected at 37°C using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.; 0.1 µM siRNA with 20 µl Lipofectamine). Following transfection for 48 h, the transfection medium was exchanged for normal medium and the cells were used in the subsequent experiments or harvested for ATF3 and TNC expression measurement.

Migration assays were performed using 8 µm pore size filters within 24-well transwell cell culture chambers with polycarbonate filters (Corning Life Sciences, Glendale, Arizona, USA) as previously described (16). A total of 5×10⁵ cells were transfected with pcDNA-ATF3 or the psDNA3.1(−), and subsequently seeded into the upper chamber of the transwell. Transwells were either uncoated (5 µm pore size) or coated with Matrigel™ (BD Biosciences, Franklin Lakes, NJ, USA) diluted 1:50 (8 µm pore size). As a chemoattractant, monocyte chemoattractant protein-1 (MCP-1; 100 ng/ml; R&D Systems, Inc., Minneapolis, MN, USA) was present in the lower wells. After incubation for 18 h at 37°C in 5% CO₂, cells in the lower chambers that passed through the filter were counted under a Carl Zeiss Primo Vert microscope (Carl Zeiss AG, Oberkochen, Germany).

Total protein was lysed using radioimmunoprecipitation assay (RIPA) buffer (Beyotime Institute of Biotechnology, Nantong, China). After determining the protein concentration with a Bicinchoninic acid assay kit (Beyotime Institute of Biotechnology, Haimen, China), ~30 µg of proteins were loaded onto 10% gels and subjected to SDS-PAGE, prior to transfer onto polyvinylidene difluoride membranes (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, membranes were blocked for 1 h in a blocking solution (5% skimmed milk, 0.05% Tween 20) at 37°C and probed with the following primary antibodies: Mouse anti-β-catenin (2698; 1:1,000), rabbit anti-c-myc 9402; 1:1,000) and anti-cyclin D1 (2292; 1:1,000; all from Cell Signaling Technology, Inc., Danvers, MA, USA), and mouse anti-ATF3 (sc81189; 1:500), rabbit anti-TNC (sc20932; 1:1,000), and mouse anti-β-actin (sc130300; 1:1,000; all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight. Horseradish peroxidase-conjugated rabbit anti-mouse (sc358917; 1:3,000; Santa Cruz Biotechnology) or goat anti-rabbit (RPN4301; 1:5,000; GE Healthcare Life Sciences, Chalfont, UK) secondary antibodies were added to the membranes for 1 h at room temperature. Protein bands were detected using the Enhanced Chemiluminescence substrate detection system (Amersham Biosciences Corporation, Piscataway, USA). The intensities of the resulting bands were quantified using Carestream Molecular Imaging software version 5.0.2.30 (Carestream Health, Woodbridge, CT, USA) on a Gel Logic 2000 imaging system (Kodak, Rochester, NY, USA).

Reverse transcription PCR was performed on an Applied Biosystems^® 7500 fast sequence detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Briefly, total RNA was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed using the MMLV Reverse Transcriptase kit (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol. qPCR was performed using SYBR Green reagent (Qiagen, Inc., Valencia, CA, USA). Cycling conditions were as follows: An initial predenaturation step at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 sec, annealing at 58°C for 30 sec and extension at 72°C for 20 sec. The experiment was performed three times. The relative expression levels of the target genes were calculated using the 2^−∆∆Cq method (17) and normalized to GAPDH. Forward and reverse sequences of the primers used for all target genes and GAPDH are listed in Table I.

Data are expressed as the mean ± standard deviation. Comparisons between two groups were analyzed by unpaired Student's t-test. Experiments were repeated three times. P<0.05 was considered to indicate a statistically significant difference analyzed using SPSS version 18.0 (SPSS, Inc., Chicago, IL, USA).

To examine whether ATF3 regulates macrophage migration, the pcDNA-ATF3 plasmid and ATF3 siRNA were utilized to overexpress and knockdown the ATF3 protein, respectively (Fig. 1A). Cell viability was tested using an MTT assay and the ATF3 siRNA had no effect on cellular viability (data not show).

Subsequently, migration of macrophages was evaluated in the presence of chemotaxis-inducing agent MCP-1. Results demonstrated that overexpression of ATF3 significantly promoted the migration of macrophages compared with the empty vector [pcDNA3.1(−)], whereas knockdown of ATF3 significantly reduced migration compared with the sham group and the difference between the control and sham group were not significant (Fig. 1B).

To determine the function of ATF3 in the process of macrophage polarization, markers of the M1 phenotype [MCP-1, inducible nitric oxide synthase (iNOS), cluster of differentiation (CD) 16 and TNF-α] and the M2 phenotype [CD163, mannose receptor C type 1 (Mrc-1), arginase 1 (Arg-1) and peroxisome proliferator-activated receptor γ (PPARγ)] were measured. Results revealed that the mRNA expression levels of M1-associated genes encoding MCP-1, iNOS, CD16 and TNF-α, were reduced following transfection with pcDNA-ATF3 compared with the empty vector control group, and were enhanced following transfection with ATF3 siRNA compared with the sham group (P<0.05; Fig. 2A). In addition, overexpression of ATF3 in RAW 264.7 cells enhanced the expression levels of the genes encoding CD163, Mrc-1, Arg-1 and PPARγ compared with the empty vector control group, and these levels were then reduced by knockdown of ATF3 compared with the sham group (P<0.05; Fig. 2B). These results suggested that ATF3 promotes polarization of M2 in macrophages.

β-catenin, encoded by the CTNNB1 gene, is a transcriptional co-activator and serves a role in the inflammatory response (18,19). To explore the mechanism of ATF3 regulation of macrophage migration and M2 polarization, the effect of ATF3 on Wnt/β-catenin signaling was investigated. As demonstrated in Fig. 3, overexpression of ATF3 resulted in enhanced expression levels of β-catenin and its target genes cyclin D1 and c-myc compared with cells transfected with the empty vector control. This suggested that overexpression of ATF3 induced the activation of the Wnt/β-catenin signaling pathway in macrophages.

The gene encoding TNC is a canonical Wnt target (20), and serves a role in macrophage behavior and function (21,22). Therefore, the association between ATF3 and TNC was investigated. Results revealed that overexpression of AFT3 significantly upregulated the mRNA expression levels of TNC (Fig. 4A) and the TNC protein (Fig. 4B) compared with the empty vector control, and this effect was partially inhibited by IWP-2, an inhibitor of Wnt/β-catenin signaling, which suggested that ATF3 activates TNC via the Wnt/β-catenin signaling pathway.

To further investigate the role of TNC in ATF3-mediated macrophage migration and polarization, cells were transfected with pcDNA-ATF3 and knockdown of TNC using TNC siRNA (Fig. 5A), prior to determining cell viability and the expression levels of M2-associated genes. Results revealed that transfection of pcDNA-ATF3 significantly promoted macrophage migration compared with the empty vector control, whereas transfection with TNC siRNA reduced the migration induced by pcDNA-ATF3 compared with the scrambled control group (Fig. 5B). In addition, the expression levels of genes associated with the M1 phenotype that were downregulated by pcDNA-ATF3 were enhanced by transfection with TNC siRNA (Fig. 5C), whereas M2 gene expression levels that were upregulated by pcDNA-ATF3 were inhibited by TNC siRNA (Fig. 5D).

These results suggested that ATF3 regulates macrophage migration and M2 polarization, in part, by upregulation of TNC.