title: Glycogen synthase kinase-3 (GSK-3) regulates TGF-β₁-induced differentiation of pulmonary fibroblasts.
abstract: BACKGROUND
  Chronic lung diseases such as asthma, COPD and pulmonary fibrosis are characterized by abnormal extracellular matrix (ECM) turnover. TGF-β is a key mediator stimulating ECM production by recruiting and activating lung fibroblasts and initiating their differentiation process into more active myofibroblasts. Glycogen synthase kinase-3 (GSK-3) regulates various intracellular signalling pathways; its role in TGF-β₁-induced myofibroblast differentiation is currently largely unknown.
  
  
  OBJECTIVE
  To determine the contribution of GSK-3 signalling in TGF-β₁-induced myofibroblast differentiation.
  
  
  METHODS
  We used MRC5 human lung fibroblasts and primary pulmonary fibroblasts of individuals with and without COPD. Protein and mRNA expression were determined by immunoblotting and RT-PCR analysis respectively.
  
  
  RESULTS
  Stimulation of MRC5 and primary human lung fibroblasts with TGF-β₁ resulted in time- and dose-dependent increases of α-sm-actin and fibronectin expression, indicative of myofibroblast differentiation. Pharmacological inhibition of GSK-3 by SB216763 dose-dependently attenuated TGF-β₁-induced expression of these myofibroblasts markers. Moreover, silencing of GSK-3 by siRNA or pharmacological inhibition by CT/CHIR99021 fully inhibited the TGF-β₁-induced expression of α-sm-actin and fibronectin. The effect of GSK-3 inhibition on α-sm-actin expression was similar in fibroblasts from individuals with and without COPD. Neither smad, NF-κB nor ERK1/2 were involved in the inhibitory actions of GSK-3 inhibition by SB126763 on myofibroblast differentiation. Rather, SB216763 increased the phosphorylation of CREB, which in its phosphorylated form acts as a functional antagonist of TGF-β/smad signalling.
  
  
  CONCLUSIONS
  We demonstrate that GSK-3 signalling regulates TGF-β₁-induced myofibroblast differentiation by regulating CREB phosphorylation. GSK-3 may constitute a useful target for treatment of chronic lung diseases.
authors.affiliation: Department of Molecular Pharmacology, GRIAC Research Institute, University of Groningen, Groningen, The Netherlands. h.a.baarsma@rug.nl
authors.forename: Hoeke A
authors.initials: HA
authors.lastname: Baarsma
authors.forename: Lilian H J M
authors.initials: LH
authors.lastname: Engelbertink
authors.forename: Lonneke J
authors.initials: LJ
authors.lastname: van Hees
authors.forename: Mark H
authors.initials: MH
authors.lastname: Menzen
authors.forename: Herman
authors.initials: H
authors.lastname: Meurs
authors.forename: Wim
authors.initials: W
authors.lastname: Timens
authors.forename: Dirkje S
authors.initials: DS
authors.lastname: Postma
authors.forename: Huib A M
authors.initials: HA
authors.lastname: Kerstjens
authors.forename: Reinoud
authors.initials: R
authors.lastname: Gosens
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000819:agonists* / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D005786:Gene Expression Regulation / Q000187:drug effects; D020868:Gene Silencing; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008168:Lung / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D058628:Myofibroblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D029424:Pulmonary Disease, Chronic Obstructive / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D053773:Transforming Growth Factor beta1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D015415:Biomarkers; C115163:CREB1 protein, human; D017362:Cyclic AMP Response Element-Binding Protein; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D011994:Recombinant Proteins; C508960:TGFB1 protein, human; D053773:Transforming Growth Factor beta1; D038362:Glycogen Synthase Kinase 3