ANP signaling inhibits TGF-beta-induced Smad2 and Smad3 nuclear translocation and extracellular matrix expression in rat pulmonary arterial smooth muscle cells.
Atrial natriuretic peptide (ANP) and transforming growth factor (TGF)-beta play important counterregulatory roles in pulmonary vascular adaptation to chronic hypoxia. To define the molecular mechanism of this important interaction, we tested whether ANP-cGMP-protein kinase G (PKG) signaling inhibits TGF-beta1-induced extracellular matrix (ECM) expression and defined the specific site(s) at which this molecular merging of signaling pathways occurs. Rat pulmonary arterial smooth muscle cells (PASMCs) were treated with ANP (1 muM) or cGMP (1 mM) with or without pretreatment with PKG inhibitors KT-5823 (1 muM) or Rp-8-bromo-cGMP (Rp-8-Br-cGMP 50 muM), then exposed to TGF-beta1 (1 ng/ml) for 5-360 min (for pSmad nuclear translocation and protein analysis) or 24 h (for ECM mRNA expression). Nuclear translocation of pSmad2 and pSmad3 was assessed by fluorescent confocal microscopy. ANP and cGMP inhibited TGF-beta1-induced pSmad2 and pSmad3 nuclear translocation and expression of periostin, osteopontin, and plasminogen activator inhibitor-1 mRNA and protein, but not TGF-beta1-induced phosphorylation of Smad2 and Smad3. KT-5823 and Rp-8-Br-cGMP blocked ANP/cGMP-induced activation of PKG and inhibition of TGF-beta1-stimulated nuclear translocation of pSmad2 and pSmad3 in PASMCs. These results reveal for the first time a precise site at which ANP-cGMP-PKG signaling exerts its antifibrogenic effect on the profibrogenic TGF-beta1 signaling pathway: by blocking TGF-beta1-induced pSmad2 and pSmad3 nuclear translocation and ECM expression in PASMCs. Blocking nuclear translocation and subsequent binding of pSmad2 and pSmad3 to TGF-beta-Smad response elements in ECM genes may be responsible for the inhibitory effects of ANP on TGF-beta-induced expression of ECM molecules.
Li
Peng
P
Vascular Biology and Hypertension Program, Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Oparil
Suzanne
S
Novak
Lea
L
Cao
Xu
X
Shi
Weibin
W
Lucas
Jason
J
Chen
Yiu-Fai
YF
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000494:pharmacology / Q000502:physiology*; D002227:Carbazoles / Q000494:pharmacology; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000502:physiology; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D007211:Indoles / Q000494:pharmacology; D008297:Male; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism*; D053495:Osteopontin / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D011651:Pulmonary Artery / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000502:physiology; D051899:Smad2 Protein / Q000378:metabolism*; D051900:Smad3 Protein / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D002227:Carbazoles; D015815:Cell Adhesion Molecules; D016326:Extracellular Matrix Proteins; D007211:Indoles; D017395:Plasminogen Activator Inhibitor 1; C512836:Postn protein, rat; D047428:Protein Kinase Inhibitors; D051899:Smad2 Protein; C497784:Smad2 protein, rat; D051900:Smad3 Protein; C497787:Smad3 protein, rat; D016212:Transforming Growth Factor beta; D053495:Osteopontin; C073601:KT 5823; C016276:8-bromocyclic GMP; D009320:Atrial Natriuretic Factor; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP