title: Rapamycin increases CCN2 expression of lung fibroblasts via phosphoinositide 3-kinase.
abstract: Excessive production of connective tissue growth factor (CTGF, CCN2) and increased motor ability of the activated fibroblast phenotype contribute to the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, molecules and signal pathways regulating CCN2 expression and migration of lung fibroblasts are still elusive. We hypothesize that rapamycin, via binding and blocking mammalian target of rapamycin (mTOR) complex (mTORC), affects CCN2 expression and migration of lung fibroblasts in vitro. Primary normal and fibrotic human lung fibroblasts were isolated from lung tissues of three patients with primary spontaneous pneumothorax and three with IPF. Cells were incubated with regular medium, or medium containing rapamycin, human recombinant transforming growth factor (TGF)-β1, or both. CCN2 and tissue inhibitor of metalloproteinase (TIMP)-1 expression in cells or supernatant was detected. Wound healing and migration assay was used to measure the migratory potential. TGF-β type I receptor (TβRI)/Smad inhibitor, SB431542 and phosphoinositide 3-kinase (PI3K) inhibitor, LY294002 were used to determine rapamycin's mechanism of action. We demonstrated that rapamycin amplified basal or TGF-β1-induced CCN2 mRNA and protein expression in normal or fibrotic fibroblasts by Smad-independent but PI3K-dependent pathway. Additionally, rapamycin also enhanced TIMP-1 expression as indicated by ELISA. However, wound healing and migrating assay showed rapamycin did not affect the mobility of fibroblasts. Collectively, this study implies a significant fibrogenic induction activity of rapamycin by activating AKT and inducing CCN2 expression in vitro and provides the possible mechanisms for the in vivo findings which previously showed no antifibrotic effect of rapamycin on lung fibrosis.
authors.affiliation: 1] Department of Respiratory and Critical Care Medicine, Beijing Key Laboratory of Respiratory and Pulmonary Circulation Disorders, Beijing Chao-Yang Hospital, Beijing Institute of Respiratory Medicine, Capital Medical University, Beijing, China [2] National Clinical Research Centre for Respiratory Medicine, Beijing Hospital, Beijing, China.
authors.forename: Xuefeng
authors.initials: X
authors.lastname: Xu
authors.affiliation: Department of Respiratory and Critical Care Medicine, Beijing Key Laboratory of Respiratory and Pulmonary Circulation Disorders, Beijing Chao-Yang Hospital, Beijing Institute of Respiratory Medicine, Capital Medical University, Beijing, China.
authors.forename: Huaping
authors.initials: H
authors.lastname: Dai
authors.affiliation: Department of Respiratory and Critical Care Medicine, Beijing Key Laboratory of Respiratory and Pulmonary Circulation Disorders, Beijing Chao-Yang Hospital, Beijing Institute of Respiratory Medicine, Capital Medical University, Beijing, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Geng
authors.affiliation: Department of Respiratory and Critical Care Medicine, Beijing Key Laboratory of Respiratory and Pulmonary Circulation Disorders, Beijing Chao-Yang Hospital, Beijing Institute of Respiratory Medicine, Capital Medical University, Beijing, China.
authors.forename: Xuan
authors.initials: X
authors.lastname: Wan
authors.affiliation: Department of Respiratory and Critical Care Medicine, Beijing Key Laboratory of Respiratory and Pulmonary Circulation Disorders, Beijing Chao-Yang Hospital, Beijing Institute of Respiratory Medicine, Capital Medical University, Beijing, China.
authors.forename: Xiaoxi
authors.initials: X
authors.lastname: Huang
authors.affiliation: Department of Respiratory and Critical Care Medicine, Beijing Key Laboratory of Respiratory and Pulmonary Circulation Disorders, Beijing Chao-Yang Hospital, Beijing Institute of Respiratory Medicine, Capital Medical University, Beijing, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Li
authors.affiliation: Department of Medicine, Cedars Sinai Medical Center, Los Angeles, CA, USA.
authors.forename: Dianhua
authors.initials: D
authors.lastname: Jiang
authors.affiliation: 1] Department of Respiratory and Critical Care Medicine, Beijing Key Laboratory of Respiratory and Pulmonary Circulation Disorders, Beijing Chao-Yang Hospital, Beijing Institute of Respiratory Medicine, Capital Medical University, Beijing, China [2] National Clinical Research Centre for Respiratory Medicine, Beijing Hospital, Beijing, China.
authors.forename: Chen
authors.initials: C
authors.lastname: Wang
mesh_terms: D001549:Benzamides / Q000494:pharmacology; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004149:Dioxoles / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D054990:Idiopathic Pulmonary Fibrosis / Q000378:metabolism; D008168:Lung / Q000166:cytology*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D020123:Sirolimus / Q000494:pharmacology*; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism
chemical_list: C459179:4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D001549:Benzamides; C526317:CCN2 protein, human; D004149:Dioxoles; D012333:RNA, Messenger; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; D055513:Connective Tissue Growth Factor; D019869:Phosphatidylinositol 3-Kinases; D020123:Sirolimus