Fig. 5

DDR2 as a receptor for cC1q in HT1080 epithelial cells. A C1q collagen tail peptide (C1qA peptide) binds with DDR2. Recombinant mouse DDR2-Fc chimera protein (7479-DR), DDR1-Fc (6416-DR), or control Fc (4460-MG) (20 μg per spot) were blotted onto the nitrocellulose membrane, and then detected using biotinylated C1qA peptide (200 nM). Representative image of three independent experiments. B, C Microtiter plates were immobilized with C1qA peptide or control peptide (8 μg/mL, B), sDDR2 (2538-DR), or BSA (8 μg/mL, C). sDDR2 (2538-DR; 0, 0.1, 0.5, 2.0 and 8 μg/mL, B) or C1qA peptide (0, 0.1, 0.5, 2.0 and 8 μg/mL, C) were added to the plates. Plate-bound peptides were detected using anti-His antibody (B) or AP-conjugated streptavidin (C). The graph shows the mean ± SE of three independent experiments. ***p < 0.001. D Pull-down assay of 0, 1, and 5 μg sDDR2 and 10 μg C1qA peptide-coupled streptavidin beads. C1qA peptide-bound sDDR2 was detected by anti-DDR2 Ab (MAB25381). The input of C1qA peptide was visualized by dot blot using infrared 800-labeled streptavidin. E Wound healing effects of C1q and C1qA peptide. HT1080 cells were treated with or without 20 μg/mL C1q or 200 nM C1qA peptide for the indicated duration. The percentage of wound closure to the initial wound areas were calculated using Image J. The graph shows the mean ± SEM of four independent experiments. F sDDR2 inhibits the wound healing effect of C1q and C1qA peptides. sDDR2 (2 μg/mL) was co-incubated with C1q or C1qA peptide for 2 h, and the wound healing rate was determined as described in E. Data represent the mean ± SEM of four independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, ****p < 0.0001 (one-way ANOVA). G C1qA peptide induces DDR2-autophosphorylation. HT1080 cells were treated with 20 μg/mL of collagen-I or C1q or C1qA peptide for 24 h. DDR2 phosphorylation was determined by immunoprecipitation with anti-DDR2 Ab and western blot with anti-phosphotyrosine (4G10) Ab. The bar graph shows the mean ± SEM of three independent experiments. * p < 0.05 (one-way ANOVA). H MMP-9 mRNA expression in HT1080 cells treated as described in 5G. Data represent the mean ± SEM of three independent experiments. ** p < 0.01 (one-way ANOVA)