title: Sildenafil inhibits calcineurin/NFATc2-mediated cyclin A expression in pulmonary artery smooth muscle cells.
abstract: OBJECTIVE
  To examine whether calcineurin/NFAT signaling pathway leads to proliferation of pulmonary artery smooth muscle cells (PASMCs) by regulating cell cycle proteins and whether the phosphodiesterase-5 (PDE5) inhibitor sildenafil affects calcineurin/NFAT-induced cell proliferation.
  
  
  METHODS
  A [(3)H]thymidine incorporation assay was used to examine DNA synthesis (cell proliferation); cyclin A and NFATc2 expressions were determined by Western blot. Cyclin-dependent kinase 2 (CDK2) activity was measured with an in vitro kinase activity assay, and calcineurin and NFAT activity were evaluated using a calcineurin assay kit and a luciferase activity assay, respectively. A chemical inhibitor or siRNA transfection was used to inhibit calcineurin/NFAT signaling pathway.
  
  
  RESULTS
  Serotonin dose-dependently stimulated cyclin A expression in PASMCs. This effect was accompanied by dose-dependent increases in CDK2 activity and the rate of DNA synthesis. At the same time, PASMCs treated with serotonin showed dose-dependent activation of calcineurin/NFAT signaling pathway. Inhibition of calcineurin activity by cyclosporine A or loss of NFATc2 protein by siRNA transfection abolished serotonin-induced cyclin A expression and consequent CDK2 activation and DNA synthesis. We further found that pretreatment of cells with sildenafil suppressed serotonin-triggered activation of calcineurin/NFATc2 signaling pathway and resultant cyclin A expression, CDK2 activation and cell proliferation, while the presence of DT-3 [a specific protein kinase G (PKG) peptide inhibitor] reversed the effects of sildenafil on PASMCs.
  
  
  CONCLUSIONS
  Our study suggests that enhanced PKG activity suppresses calcineurin/NFATc2 cascade-mediated cyclin A expression, CDK2 activation and PASMC proliferation to contribute to the overall effects of sildenafil in the treatment of pulmonary hypertension.
authors.affiliation: Respiratory Diseases Research Center, The Second Affiliated Hospital of Medical College, Xi'an Jiaotong University, Xi'an, Shaanxi, 710004, PR China. manxiangli@hootmail.com
authors.forename: Manxiang
authors.initials: M
authors.lastname: Li
authors.forename: Yuan
authors.initials: Y
authors.lastname: Liu
authors.forename: Xiuzhen
authors.initials: X
authors.lastname: Sun
authors.forename: Zongfang
authors.initials: Z
authors.lastname: Li
authors.forename: Yun
authors.initials: Y
authors.lastname: Liu
authors.forename: Ping
authors.initials: P
authors.lastname: Fang
authors.forename: Ping
authors.initials: P
authors.lastname: He
authors.forename: Hongyang
authors.initials: H
authors.lastname: Shi
authors.forename: Mei
authors.initials: M
authors.lastname: Xie
authors.forename: Xiaochuang
authors.initials: X
authors.lastname: Wang
authors.forename: Dexin
authors.initials: D
authors.lastname: Zhang
authors.forename: Yonghong
authors.initials: Y
authors.lastname: Zhang
authors.forename: Zongjuan
authors.initials: Z
authors.lastname: Ming
authors.forename: Jing
authors.initials: J
authors.lastname: Xu
authors.forename: Jiamei
authors.initials: J
authors.lastname: Lu
authors.forename: Xinming
authors.initials: X
authors.lastname: Xie
mesh_terms: D000818:Animals; D019703:Calcineurin / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D019925:Cyclin A / Q000235:genetics*; D051357:Cyclin-Dependent Kinase 2 / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism; D050778:NFATC Transcription Factors / Q000235:genetics / Q000378:metabolism*; D058986:Phosphodiesterase 5 Inhibitors / Q000494:pharmacology*; D010879:Piperazines / Q000494:pharmacology*; D011651:Pulmonary Artery / Q000166:cytology* / Q000187:drug effects; D011687:Purines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D012701:Serotonin / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D000068677:Sildenafil Citrate; D013450:Sulfones / Q000494:pharmacology*
chemical_list: D019925:Cyclin A; D050778:NFATC Transcription Factors; D058986:Phosphodiesterase 5 Inhibitors; D010879:Piperazines; D011687:Purines; D034741:RNA, Small Interfering; D013450:Sulfones; D012701:Serotonin; D000068677:Sildenafil Citrate; D051357:Cyclin-Dependent Kinase 2; D019703:Calcineurin