title: Plasminogen activator inhibitor-1 promotes the proliferation and inhibits the apoptosis of pulmonary fibroblasts by Ca(2+) signaling.
abstract: OBJECTIVE
  Our previous investigation demonstrated that plasminogen activator inhibitor-1 (PAI-1) siRNA ameliorated bleomycin (BLM)-induced rat lung fibrosis. The present study was undertaken to explore the effect and the mechanism of PAI-1 siRNA and plasmid pcDNA on the proliferation and apoptosis of cultured fibroblasts from BLM-induced fibrotic lung tissues.
  
  
  METHODS
  The fibroblasts from BLM-induced fibrotic lung tissue were isolated and transfected using PAI-1 siRNA and plasmid pcDNA-PAI-1. The techniques of real time RT-PCR and/or western blot were used to determine the expression of PAI-1, α-smooth muscle actin (α-SMA) (real time RT-PCR only), collagen type-1 and type-3 (real time RT-PCR only), and the levels of caspase-3, ERK and AKT signal molecules. The proliferation of fibroblasts was measured by cell cycle with flow cytometry. The intracellular concentration of Ca(2+) was examined by confocal laser microscopy.
  
  
  RESULTS
  PAI-1 siRNA downregulated the PAI-1 mRNA expression by 70%±7% at 24h and protein expression by 73.5%±10% and 42%±3% at 48h and 72h compared to Non-specific siRNA group. Flow cytometry showed that the fibroblasts at the G(2)M+S phase were significantly reduced by 20.56±1.03% after transfecting PAI-1 siRNA and were significantly increased by 43.8±1.21% after transfecting plasmid pcDNA-PAI-1. The mRNA expressions of α-SMA, collagen type-1and type-3 were downregulated after transfecting the PAI-1 siRNA, while upregulated after the transfection of pcDNA-PAI-1. PAI-1 siRNA increased the level of caspase-3, inhibited the expressions of p-ERK and p-AKT protein molecules, while the pcDNA-PAI-1 transfection showed a reversal effect on these expressions. Intracellular Ca(2+) concentration was decreased after transfecting PAI-1 siRNA, whereas increased after transfecting pcDNA-PAI-1.
  
  
  CONCLUSIONS
  PAI-1 promotes the proliferation, transforming into myofibroblasts, collagen synthesis, and inhibits apoptosis of pulmonary fibroblasts by activating Ca(2+), ERK and AKT signaling pathway. Decreasing PAI-1 expression is an available strategy in inhibiting the progression of pulmonary fibrosis.
authors.affiliation: Department of Pathophysiology, Hebei Medical University, 361 Zhongshan East Road, Shijiazhuang 050017, PR China.
authors.forename: Yan-Ping
authors.initials: YP
authors.lastname: Zhang
authors.forename: Wei-Li
authors.initials: WL
authors.lastname: Wang
authors.forename: Jian
authors.initials: J
authors.lastname: Liu
authors.forename: Wen-Bin
authors.initials: WB
authors.lastname: Li
authors.forename: Lin-Lin
authors.initials: LL
authors.lastname: Bai
authors.forename: Ya-Dong
authors.initials: YD
authors.lastname: Yuan
authors.forename: Shu-Xia
authors.initials: SX
authors.lastname: Song
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D001761:Bleomycin; D015153:Blotting, Western; D020013:Calcium Signaling; D053148:Caspase 3 / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D024061:Collagen Type III / Q000235:genetics / Q000378:metabolism; D004195:Disease Models, Animal; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D005434:Flow Cytometry; D008168:Lung / Q000378:metabolism* / Q000473:pathology; D008297:Male; D018613:Microscopy, Confocal; D058628:Myofibroblasts / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011658:Pulmonary Fibrosis / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D034622:RNA Interference; D051381:Rats; D017208:Rats, Wistar; D060888:Real-Time Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection
chemical_list: D000199:Actins; D001761:Bleomycin; D053148:Caspase 3; D024042:Collagen Type I; D024061:Collagen Type III; D048049:Extracellular Signal-Regulated MAP Kinases; D017395:Plasminogen Activator Inhibitor 1; D051057:Proto-Oncogene Proteins c-akt; C498862:Acta2 protein, rat; C505410:Casp3 protein, rat