Fig 4

The mechanism underlying the CHI3L1-activated metastasis of gastric and breast cancer cells. a Identification of IL-13Rα2 as a CHI3L1-binding membrane receptor. Glutathione-Sepharose beads coupled with GST or GST-CHI3L1 were incubated with isolated gastric and breast cancer cell membrane proteins. The proteins bound to CHI3L1 protein were analyzed by 10% SDS-PAGE with Coomassie staining and were identified by mass spectroscopy. b Interaction between CHI3L1 and IL-13Rα2. Gastric and breast cancer cells were incubated with recombinant CHI3L1 (rCHI3L1) for 3 h. Then, the cell membrane proteins were prepared for analysis of the levels of CHI3L1 and IL-13Rα2 using western blot assays (top). Additionally, the cell membrane proteins were immunoprecipitated (IP) using either anti-CHI3L1 or an isotype IgG antibody and were analyzed by western blotting (bottom). Cells not treated with rCHI3L1 were used as controls. c Co-localization of CHI3L1 and IL-13Rα2 proteins on the plasma membrane of cancer cells. MDA-MB-231 cells were incubated with rCHI3L1 for 3 h. Subsequently, the cells were stained with fluorescently labeled anti-CHI3L1 and anti-IL-13Rα2. The nuclei were counterstained with DAPI. The cells were examined using confocal microscopy. d Effect of CHI3L1-mediated IL-13Rα2 activation on triggering the activity of the MAPK and PI3K/AKT signaling pathways. Gastric and breast cancer cells with or without IL-13Rα2-siRNA transfection were incubated with rCHI3L1 protein for 3 h. Western blot analysis was conducted to evaluate the levels of total and phosphorylated ERK, JNK, Src, and AKT proteins. β-actin was used as the control. e Effects of CHI3L1-mediated IL-13Rα2 activation on AP-1 transcriptional activity in cancer cells. Cancer cells with or without IL-13Rα2-siRNA transfection were transfected with plasmids containing the binding sequence of the AP-1 transcription factor family. Then, recombinant CHI3L1 protein (rCHI3L1) was added to the cell cultures. Dual-luciferase reporter assays were conducted. The level of AP-1 transcriptional activity of the cancer cells was normalized to that of the untreated cells. f Influence of CHI3L1-IL-13Rα2 interaction on c-Fos and c-Jun protein expression in the nuclei of cancer cells. Gastric and breast cancer cells with or without IL-13Rα2-siRNA transfection were treated with rCHI3L1 at 10 nM for 3 h. Then, the levels of c-Fos and c-Jun proteins in the cancer cell nuclei were determined by western blotting. g Evaluation of MMP expression. Cancer cells with or without IL-13Rα2-siRNA transfection were incubated with rCHI3L1 at 10 nM for 3 h. Subsequently, the cells were subjected to quantitative real-time PCR to determine the levels of MMP expression. h, i Effects of ERK inhibitor (U0126) and JNK inhibitor (SP600125) on the CHI3L1-induced aggregation of c-Fos and c-Jun protein in the nuclei h and MMP gene expression i in MKN-45 cells (left) and MDA-MB-231 cells (right). Prior to the addition of recombinant CHI3L1 protein, cancer cells were treated with U0126 or/and SP600125 at 10 μM for 2 h. Then the cancer cells were incubated with rCHI3L1 protein for 3 h. Significant differences between the treatments are indicated by asterisks (*p < 0.05, **p < 0.01)