MMP-3-mediated cleavage of OPN is involved in copper oxide nanoparticle-induced activation of fibroblasts

Figures

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  Fig. 1. Cytotoxicity of Nano-CuO on BEAS-2B cells, U937-derived macrophages (U937*), and MRC-5 fibroblasts. BEAS-2B (a), U937* macrophages (b), and MRC-5 fibroblasts (c) were seeded into 96-well plates and treated with 0.5, 1, 2, 5, and 10 µg/mL of Nano-CuO for 24 h (a and b) and 48 h (c), respectively. Cells without treatment were used as control. The cytotoxicity was determined by alamarBlue assay. Data represent mean ± SE (n = 6). * Significant difference as compared to the control group, p < 0.05

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  Fig. 2. Increased expression and activity of MMP-3 in the conditioned media of BEAS-2B cells and U937* macrophages exposed to Nano-CuO. BEAS-2B cells (a and c) and U937* macrophages (b and d) were exposed to 0.5 and 1 µg/mL of Nano-CuO for 12 h. After treatment, the conditioned media were collected. Cells without treatment were used as control. MMP-3 protein level in the conditioned media was detected by Western blot, while its activity was detected by β-casein zymography. Equal protein loading was verified by Coomassie Brilliant Blue staining. The activity of MMP-3 was also determined in the conditioned media from co-culture system (e). a, b, and e were the results of a single experiment, while c and d were normalized band densitometry readings averaged from three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05

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  Fig. 3. Increased expression of OPN in BEAS-2B cells and U937* macrophages exposed to Nano-CuO. BEAS-2B cells, U937* macrophages, or co-culture of BEAS-2B and U937* macrophages at a ratio of 1:1 or 9:1 (BEAS-2B:U937*) were exposed to 0.5 and 1 µg/mL of Nano-CuO for 12 h. After treatment, the cells and conditioned media were collected. Cells without treatment were used as control. Full-length OPN (66 kDa) in the cells and cleaved OPN (40 kDa N-terminal fragment) in the conditioned media were detected by Western blot. Equal protein loading was verified by β-actin expression (a and b) or Coomassie Brilliant Blue staining (c and d). a and c were the results of a single experiment. b and d were results of three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05

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  Fig. 4. The role of MMP-3 on Nano-CuO-induced production of cleaved OPN in BEAS-2B cells and U937* macrophages. BEAS-2B cells (a and b) and U937* macrophages (c and d) were transfected with 30 nM of MMP-3 siRNA or Negative Control No. 2 siRNA. After transfection, the cells were exposed to 1 µg/mL of Nano-CuO for 12 h. The conditioned media were collected to detect the level of cleaved OPN (40 kDa N-terminal fragment) by Western blot. Equal protein loading was verified by Coomassie Brilliant Blue staining. a and c were the results of a single experiment. b and d were normalized band densitometry readings averaged from three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05; # Significant difference as compared to the Nano-CuO-treated group transfected with Negative Control No. 2 siRNA, p < 0.05

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  Fig. 5. Direct Nano-CuO exposure had no effects on activation of MRC-5 fibroblasts. MRC-5 cells were treated with 0.5 and 1 µg/mL of Nano-CuO for 48 h (a and b) or with 1 µg/mL of Nano-CuO for 12, 24, 48, and 72 h (c and d). Cells without treatment were used as control. Proteins were isolated from the cells to detect the expression of α-SMA, Col1A1, and fibronectin. a and c were the results of a single Western blot experiment. b and d were the average expression levels of α-SMA, Col1A1, and fibronectin normalized to β-actin from three independent experiments. Data represent mean ± SE (n = 3)

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  Fig. 6. Activation of MRC-5 fibroblasts cultured in the conditioned media from Nano-CuO-exposed BEAS-2B or U937* cells. a is the experimental protocol. BEAS-2B or U937* cells were treated with 1 µg/mL of Nano-CuO for 12 h and the conditioned media (CM) were collected to culture MRC-5 fibroblasts. After 0, 12, 24, 48, and 72 h culture, the MRC-5 fibroblasts were collected for protein isolation and Western blot. b and d were the results of a single Western blot experiment. c and e were the average expression levels of α-SMA, Col1A1, and fibronectin normalized to β-actin from three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05. Graphics in a were created with BioRender (https://BioRender.com)

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  Fig. 7. Conditioned media from Nano-CuO-exposed BEAS-2B or U937* cells enhanced migration of unexposed MRC-5 fibroblasts. BEAS-2B or U937* cells were treated with 0 or 1 µg/mL of Nano-CuO for 12 h and the conditioned media (CM) were collected to culture MRC-5 fibroblasts for 48 h. Migration of MRC-5 cells was detected by wound healing assay. a and b were representative images. c and d were the average closure area from three independent experiments. Scale bars in a and b represent 200 µm. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05

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  Fig. 8. Cultured in the conditioned media from Nano-CuO-exposed co-culture of BEAS-2B and U937* cells caused activation of unexposed MRC-5 fibroblasts. a is the experimental protocol of the co-culture experiments. Co-culture of BEAS-2B and U937* cells with ratio of 1:1 or 9:1 (BEAS-2B:U937*) was treated with 1 µg/mL of Nano-CuO for 12 h and the conditioned media (CM) were collected. MRC-5 fibroblasts were then cultured in the conditioned media for 0, 12, 24, 48, and 72 h. Proteins from MRC-5 cells were isolated to detect the expression of α-SMA, Col1A1, and fibronectin. b and d were the results of a single Western blot experiment. c and e were the average expression levels of α-SMA, Col1A1, and fibronectin normalized to β-actin from three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05. Graphics in a were created with BioRender (https://BioRender.com)

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  Fig. 9. Indirect exposure to Nano-CuO caused activation of MRC-5 fibroblasts in a triple co-culture system. a is the experimental protocol for the triple co-culture experiments. BEAS-2B cells and U937* cells were seeded at the ratio of 1:1 (b and c) or 9:1 (d and e) (BEAS-2B:U937*) in the inserts of Transwell® units and MRC-5 cells were seeded in the lower chambers of the units. After establishment of the triple co-culture system, BEAS-2B and U937* cells were exposed to 0, 0.5 and 1 µg/mL of Nano-CuO for 12 h, then the inserts were taken out. MRC-5 cells in the lower chamber were incubated for another 36 h and proteins were isolated to detect the expression of α-SMA, Col1A1, and fibronectin. b and d were the results of a single Western blot experiment. c and e were the average expression levels of α-SMA, Col1A1, and fibronectin normalized to β-actin from three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05. Graphics in a were created with BioRender (https://BioRender.com)

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  Fig. 10. The role of MMP-3 on Nano-CuO-induced activation of MRC-5 fibroblasts in the triple co-culture system. BEAS-2B and U937* cells were seeded at the ratio of 1:1 (a and b) or 9:1 (c and d) (BEAS-2B:U937*) in the inserts of Transwell® units and transfected with 30 nM of MMP-3 siRNA or Negative Control No. 2 siRNA. After transfection, the cells were exposed to 1 µg/mL of Nano-CuO for 12 h. After exposure, the inserts were taken out and the MRC-5 fibroblasts seeded in the lower chamber were incubated for another 36 h. The proteins isolated from MRC-5 fibroblasts were used to detect the expression levels of α-SMA, Col1A1, and fibronectin by Western blot. a and c were the results of a single Western blot experiment. b and d were the average expression level of α-SMA, Col1A1, and fibronectin normalized to β-actin from three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05; # Significant difference as compared to the Nano-CuO-treated group transfected with Negative Control No. 2 siRNA, p < 0.05

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  Fig. 11. MMP-3 siRNA transfection attenuated Nano-CuO-induced migration of MRC-5 fibroblasts in the triple co-culture system. BEAS-2B and U937* cells were seeded at a ratio of 1:1 (a and c) or 9:1 (b and d) (BEAS-2B:U937*) in the inserts of Transwell® units and transfected with 30 nM of MMP-3 siRNA or Negative Control No. 2 siRNA. After transfection, the cells were exposed to 1 µg/mL of Nano-CuO for 12 h. After exposure, the inserts were taken out and the MRC-5 fibroblasts seeded in the lower chamber were incubated for another 36 h. Migration of MRC-5 cells was detected by wound healing assay. Cells without any treatments were used as the control. a and b were representative images. c and d were the average closure area from three independent experiments. Scale bars in a and b represent 200 µm. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05; # Significant difference as compared to the Nano-CuO-treated group with control siRNA transfection, p < 0.05

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  Fig. 12. The role of MMP-3-cleaved OPN in Nano-CuO-induced activation of MRC-5 fibroblasts in the triple co-culture system. BEAS-2B and U937* cells were seeded at the ratio of 1:1 (a and b) or 9:1 (c and d) (BEAS-2B:U937*) in the inserts, and MRC-5 fibroblasts were seeded in the lower chambers of Transwell® units. MRC-5 fibroblasts were pretreated with 200 µg/mL of GRGDSP peptides, then BEAS-2B and U937* cells were exposed to 1 µg/mL of Nano-CuO for 12 h. After exposure, the inserts were taken out and the MRC-5 fibroblasts were incubated for another 36 h. The proteins from MRC-5 fibroblasts were isolated to determine the activation of MRC-5 fibroblasts by Western blot. a and c were the results of a single Western blot experiment. b and d were the average expression levels of α-SMA, Col1A1, and fibronectin normalized to β-actin from three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05; # Significant difference as compared to the Nano-CuO-treated alone group, p < 0.05