ADAM17 mediates MMP9 expression in lung epithelial cells

Abstract

The purposes were to study the role of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α/nuclear factor-κB (NF-κB) signaling in matrix metalloproteinase 9 (MMP9) expression in A549 cells and to investigate the effects of lentivirus-mediated RNAi targeting of the disintegrin and metalloproteinase 17 (ADAM17) gene on LPS-induced MMP9 expression. MMP9 expression induced by LPS in A549 cells was significantly increased in a dose- and time-dependent manner (p<0.05). Pyrrolidine dithiocarbamate (PDTC) and a TNFR1 blocking peptide (TNFR1BP) significantly inhibited LPS-induced MMP9 expression in A549 cells (p<0.05). TNFR1BP significantly inhibited LPS-induced TNF-α production (p<0.05). Both PDTC and TNFR1BP significantly inhibited the phosphorylation of IκBα and expression of phosphorylation p65 protein in response to LPS (p<0.05), and the level of IκBα in the cytoplasm was significantly increased (p<0.05). Lentivirus mediated RNA interference (RNAi) significantly inhibited ADAM17 expression in A549 cells. Lentivirus-mediated RNAi targeting of ADAM17 significantly inhibited TNF-α production in the supernatants (p<0.05), whereas the level of TNF-α in the cells was increased (p<0.05). Lentiviral ADAM17 RNAi inhibited MMP9 expression, IκBα phosphorylation and the expression of phosphorylation p65 protein in response to LPS (p<0.05). PDTC significantly inhibited the expression of MMP9 and the phosphorylation of IκBα, as well as the expression of phosphorylation p65 protein in response to TNF-α (p<0.05). Lentiviral RNAi targeting of ADAM17 down-regulates LPS-induced MMP9 expression in lung epithelial cells via inhibition of TNF-α/NF-κB signaling.

Figure 1. LPS-induced expression of MMP9 mRNA in a dose- and time-dependent manner.

(A) lane 1: DNA ladder marker; lane 2–6: MMP9 mRNA expression in A549 cells stimulated with LPS at concentrations ranging from 0.1 to 100 µg/l. (B) DNA ladder marker; lane 2–6: expression of MMP9 mRNA in A549 cells stimulated for different durations (0–24 h) with LPS at a concentration of 10 µg/l.

Figure 2. LPS-induced MMP9 expression is mediated by the TNF-α/NF-κB signaling pathway in A549 cells.

(A) A549 cells were stimulated with LPS at concentrations ranging from 0.1 to 100 µg/l and TNF-α protein production in the supernatants was detected using ELISA. (B) A549 cells stimulated for different lengths of time (0–24 h) with LPS at a concentration of 10 µg/l. (C) A549 cells were pretreated for 30 min with 5 mg/l TNFR1BP and were then stimulated for 24 h with LPS at a concentration of 100 µg/l. TNF-α protein in the supernatants was detected via ELISA. (D) TNFR1BP and PDTC inhibited LPS-induced MMP9 mRNA expression. Lane 1: DNA ladder marker; lane 2: the group not stimulated by LPS; lane 3: the group stimulated for 24 h with LPS at a concentration of 100 µg/l; lane 4: A549 cells were pretreated for 30 min with 5 mg/l TNFR1BP and then stimulated for 24 h with LPS at a concentration of 100 µg/l; Lane 5: A549 cells were pretreated for 30 min with 50 mg/l PDTC and then stimulated for 24 h with LPS at a concentration of 100 µg/l. (E) TNFR1BP and PDTC inhibited LPS-induced MMP9 expression. A549 cells were pretreated for 30 min with 5 mg/l TNFR1BP and 50 mg/l PDTC and then stimulated for 24 h with LPS at a concentration of 100 µg/l. The expression of MMP9 protein was detected via western blotting. (F) The effects of TNFR1BP and PDTC on LPS-induced the phosphorylation of IκBα and p65. A549 cells were pretreated for 30 min with 5 mg/l TNFR1BP and 50 mg/l PDTC and then stimulated for 24 h with LPS at a concentration of 100 µg/l. Both the phosphorylation of IκBα and p65 was detected via western blotting. (G) The effects of TNFR1BP and PDTC on IκBα protein expression in the cytoplasm. A549 cells were pretreated for 30 min with 5 mg/l TNFR1BP and 50 mg/l PDTC and then stimulated for 24 h with LPS at a concentration of 100 µg/l. The level of IκBα in the cytoplasm was detected via western blotting.

Figure 3. Microfluorographs of A549 cells 96 h after transfection.

A: A549 cells not stimulated with LPS. B: A549 cells only stimulated for 24 h with LPS at a concentration of 100 µg/l. C: A549 cells transfected for 72 h with LV-NC-shRNA and then stimulated for 24 h with LPS at a concentration of 100 µg/l. D: A549 cells were transfected for 72 h with LV-ADAM17-shRNA and then stimulated for 24 h with LPS at a concentration of 100 µg/l.

Figure 4. Effects of lentivirus-mediated ADAM17 RNAi on the expression of MMP9 and p-p65 protein, and the phosphorylation of IκBα, in response to LPS.

(A)The silencing effects of lentivirus mediated RNA interference on ADAM17 expression in A549 cells. (B) The effects of lentiviral ADAM17 RNA interference on LPS-induced TNF-α protein production in the supernatants. A549 cells were infected for 72 h with LV-ADAM17-shRNA or LV-NC-shRNA and then stimulated for 24 h with LPS (100 µg/l). (C) The effects of lentiviral ADAM17 RNA interference on TNF-α shedding in A549 cells. The level of TNF-α in the A549 cells was detected by western blotting. (D) The effects of lentiviral ADAM17 RNA interference on LPS-induced expression of MMP9 mRNA. Lane 1: DNA ladder marker; lane 2: the group not stimulated with LPS; lane 3: A549 cells stimulated for 24 h with LPS (100 µg/l); lane 4: A549 cells were infected for 72 h with LV-NC-shRNA and then stimulated for 24 h with LPS (100 µg/l); lane 5: A549 cells were infected for 72 h with LV-ADAM17-shRNA and then stimulated for 24 h with LPS (100 µg/l). (E) The effects of lentiviral ADAM17 RNA interference on LPS-induced expression of MMP9 protein. A549 cells were infected for 72 h with LV-ADAM17-shRNA or LV-NC-shRNA and then stimulated for 24 h with LPS at a concentration of 100 µg/l. (F) The effects of lentiviral ADAM17 RNA interference on LPS-induced phosphorylation of IκBα and p65. A549 cells were infected for 72 h with LV-ADAM17-shRNA or LV-NC-shRNA and then stimulated for 24 h with LPS at a concentration of 100 µg/l. Both the phosphorylation of IκBα and p65 was detected via western blotting. (G) The effects of lentiviral ADAM17 RNA interference on IκBα protein expression in the cytoplasm. A549 cells were infected for 72 h with LV-ADAM17-shRNA or LV-NC-shRNA and then stimulated for 24 h with LPS at a concentration of 100 µg/l. The level of IκBα in the cytoplasm was detected via western blotting.

Figure 5. Effects of lentiviral ADAM17 RNAi on MMP9 expression, IκBα phosphorylation, and p-p65 protein expression induced by TNF-α.

(A) PDTC inhibited TNF-α-induced expression of MMP9 mRNA in A549 cells. Lane 1: DNA ladder marker; lane 2: cells not stimulated with TNF-α; lane 3: cells stimulated for 24 h with TNF-α at a concentration of 1 mg/l; lane 4: cells pretreated for 30 min with 50 mg/l PDTC and then stimulated for 24 h with 1 mg/l TNF-α. (B) The effects of PDTC on TNF-α-induced expression of MMP9 protein. A549 cells were pretreated for 30 min with 50 mg/l PDTC and then stimulated for 24 h with 1 mg/l TNF-α. (C) The effects of PDTC on the phosphorylation of IκBα and p65 induced by TNF-α. A549 cells were pretreated for 30 min with 50 mg/l PDTC, and then stimulated for 24 h by TNF-α at a concentration of 1 mg/l. (D) The effects of PDTC on IκBα protein expression in the cytoplasm. A549 cells were pretreated for 30 min with 50 mg/l PDTC and then stimulated for 24 h with 1 mg/l TNF-α. The level of IκBα in the cytoplasm was detected via western blotting. (E) The effects of lentiviral ADAM17 RNA interference on TNF-α-induced expression of MMP9 mRNA in A549 cells. Lane 1: DNA ladder marker; lane 2: cells not stimulated with TNF-α; lane 3: cells stimulated only with 1 mg/l TNF-α; lane 4: the cells were infected for 72 h with LV-NC-shRNA and then stimulated for 24 h with 1 mg/l TNF-α; lane 5: cells infected for 72 h with LV-ADAM17-shRNA and stimulated for 24 h with 1 mg/l TNF-α. (F) The effects of lentiviral ADAM17 RNA interference on TNF-α-induced expression of MMP9 protein. A549 cells were infected for 72 h with LV-ADAM17-shRNA or LV-NC-shRNA and then stimulated for 24 h with 1 mg/l TNF-α. (G) The effects of lentiviral ADAM17 RNA interference on induced by TNF-α-induced phosphorylation of IκBα and p65. A549 cells were infected for 72 h with LV-ADAM17-shRNA or LV-NC-shRNA and then stimulated for 24 h with TNF-α (1 mg/l). (H) The effects of lentiviral ADAM17 RNA interference on IκBα protein expression in the cytoplasm. A549 cells were infected for 72 h with LV-ADAM17-shRNA or LV-NC-shRNA and then stimulated for 24 h with 1 mg/l TNF-α. The level of IκBα in the cytoplasm was detected via western blotting.