title: Glycogen synthase kinase 3beta contributes to proliferation of arterial smooth muscle cells in pulmonary hypertension.
abstract: BACKGROUND
  Pulmonary arterial hypertension (PAH) is a rare progressive pulmonary vascular disorder associated with vascular remodeling and right heart failure. Vascular remodeling involves numerous signaling cascades governing pulmonary arterial smooth muscle cell (PASMC) proliferation, migration and differentiation. Glycogen synthase kinase 3beta (GSK3ß) is a serine/threonine kinase and can act as a downstream regulatory switch for numerous signaling pathways. Hence, we hypothesized that GSK3ß plays a crucial role in pulmonary vascular remodeling.
  
  
  METHODS
  All experiments were done with lung tissue or isolated PASMCs in a well-established monocrotaline (MCT)-induced PAH rat model. The mRNA expression of Wnt ligands (Wnt1, Wnt3a, Wnt5a), upstream Wnt signaling regulator genes (Frizzled Receptors 1, 2 and secreted Frizzled related protein sFRP-1) and canonical Wnt intracellular effectors (GSK3ß, Axin1) were assessed by real-time polymerase chain reaction and protein levels of GSK3ß, phospho-GSK3ß (ser 9) by western blotting and localization by immunohistochemistry. The role of GSK3ß in PASMCs proliferation was assessed by overexpression of wild-type GSK3ß (WT) and constitutively active GSK3ß S9A by [(3)H]-thymidine incorporation assay.
  
  
  RESULTS
  Increased levels of total and phosphorylated GSK3ß (inhibitory phosphorylation) were observed in lungs and PASMCs isolated from MCT-induced PAH rats compared to controls. Further, stimulation of MCT-PASMCs with growth factors induced GSK3ß inactivation. Most importantly, treatment with the PDGFR inhibitor, Imatinib, attenuated PDGF-BB and FCS induced GSK3ß phosphorylation. Increased expression of GSK3ß observed in lungs and PASMC isolated from MCT-induced PAH rats was confirmed to be clinically relevant as the same observation was identified in human iPAH lung explants. Overexpression of GSK3ß significantly increased MCT-PASMCs proliferation by regulating ERK phosphorylation. Constitutive activation of GSK3ß (GSK3ß S9A, 9th serine replaced to alanine) inhibited MCT-PASMCs proliferation by decreasing ERK phosphorylation.
  
  
  CONCLUSIONS
  This study supports a central role for GSK3ß in vascular remodeling processes and suggests a novel therapeutic opportunity for the treatment of PAH.
authors.affiliation: Medical Clinic II/V, University Hospital, Giessen, Germany.
authors.forename: Piotr
authors.initials: P
authors.lastname: Sklepkiewicz
authors.forename: Ralph Theo
authors.initials: RT
authors.lastname: Schermuly
authors.forename: Xia
authors.initials: X
authors.lastname: Tian
authors.forename: Hossein Ardeschir
authors.initials: HA
authors.lastname: Ghofrani
authors.forename: Norbert
authors.initials: N
authors.lastname: Weissmann
authors.forename: Daniel
authors.initials: D
authors.lastname: Sedding
authors.forename: Tarek
authors.initials: T
authors.lastname: Kashour
authors.forename: Werner
authors.initials: W
authors.lastname: Seeger
authors.forename: Friedrich
authors.initials: F
authors.lastname: Grimminger
authors.forename: Soni Savai
authors.initials: SS
authors.lastname: Pullamsetti
mesh_terms: D000818:Animals; D001158:Arteries / Q000201:enzymology / Q000473:pathology*; D001483:Base Sequence; D015153:Blotting, Western; D049109:Cell Proliferation; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017931:DNA Primers; D004195:Disease Models, Animal; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D006976:Hypertension, Pulmonary / Q000201:enzymology / Q000473:pathology*; D007150:Immunohistochemistry; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000201:enzymology / Q000473:pathology*; D016296:Mutagenesis; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D051381:Rats; D051153:Wnt Proteins / Q000378:metabolism
chemical_list: D017931:DNA Primers; D051153:Wnt Proteins; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D038362:Glycogen Synthase Kinase 3