Journal: Respiratory Research
Article Title: Nicotine stimulates collagen type I expression in lung via α7 nicotinic acetylcholine receptors
Figure Lengend Snippet: Matrices Derived from Nicotine-treated Fibroblasts and Mice Stimulate IL-1β Expression in Monocytic Cells. a Lung fibroblasts (5x10 4 cells/12 well) were treated with nicotine (50 μg/ml) for 120 h. Fibroblasts were removed by osmotic lysis, the plates were washed thoroughly, and human monocytic U937 cells expressing the human interleukin-1β gene promoter connected to a luciferase reporter gene were overlaid atop the fibroblast-derived matrix. Afterwards, expression of the IL-1β promoter was analyzed by luciferase assay. We found that collagen-containing matrices derived from nicotine-treated fibroblasts stimulated monocytic cells to express the pro-inflammatory cytokine IL-1β. Furthermore, nicotine-treated fibroblast matrix induction of IL-1β was inhibited by anti-α2β1 integrin antibodies. b IL-1β gene transcription was not increased in U937 cells cultured on matrices derived from nicotine-treated α7 nAChR deficient primary lung fibroblast matrix over control. c Fibroblasts pretreated with MG 624, an α7 nAChR antagonist (10 μM), concurrently with nicotine inhibited IL-1β expression without affecting baseline expression. d The nicotine-treated fibroblast matrix IL-1β induction was inhibited by MEK-1 inhibitor PD98059 (50 μM), with PD98059 alone bringing IL-1β expression below baseline. e The lungs of mice exposed to nicotine (100 μg/ml in the drinking water for 8 weeks) were isolated for RNA analysis, which showed an increase in IL-1β gene transcription by RT-PCR. f Lungs from control and nicotine-treated mice were stained by immunohistochemistry for IL-1β. Increased staining was present in mice treated with nicotine. Experiments were repeated at least 3 times. Significance was assessed using p values
Article Snippet: Blots were incubated with primary polyclonal antibody against either GAPDH (Abcam) (1:5000 dilution), collagen type I (ACRIS, San Diego, CA or Abcam, Cambridge, MA) (1:1000), total and p-Smad (Rockland Immunochemicals, Gilbertsville, PA) (1:2000), total and p-ERK 1 & 2 (Cell Signaling, Beverly, MA) (1:1000), and α7 nAChR (Sigma) (1:500).
Techniques: Derivative Assay, Mouse Assay, Expressing, Lysis, Luciferase, Cell Culture, Isolation, Reverse Transcription Polymerase Chain Reaction, Staining, Immunohistochemistry