title: Cigarette smoke extract promotes human pulmonary artery smooth muscle cells proliferation through protein kinase C alpha-dependent induction of cyclin D1.
abstract: BACKGROUND
  Exposure to cigarette smoke stimulates the proliferation of human pulmonary artery smooth muscle cells (HPASMCs) in vivo and in vitro. However, the molecular mechanism remains unclear. This study aimed at investigating the role of signaling pathways involving protein kinase C alpha (PKCα) and cyclin D1 in the cigarette smoke extract (CSE)-induced HPASMCs proliferation.
  
  
  METHODS
  Synchronized HPASMCs were treated with different concentrations of CSE. Cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell counting. Cell cycle was analyzed by flow cytometry with propidium iodide staining. Activation of PKCα was measured by detecting the expression of PKCα protein in the cytosolic and membrane fractions using Western blotting analysis. Small interfering RNA (siRNA) was used to knockdown PKCα and cyclin D1. The cyclin D1 mRNA was assessed by real-time RT-PCR. The PKCα and cyclin D1 protein levels were detected by Western blotting.
  
  
  RESULTS
  Low concentrations of CSE (1% - 10%) stimulated proliferation of HPASMCs, with its maximal effect at 5%. CSE (5%) led to PKCα activation. Inhibition of PKCα activity using Gö 6976 or siRNA-mediated knockdown of PKCα significantly attenuated CSE-induced cell proliferation and G1/S transition. Cyclin D1, one of key regulators of G1/S transition, was found to be upregulated by 5% CSE at both the mRNA and protein levels. CSE-stimulated cell proliferation and G1/S transition was abolished by cyclin D1 siRNA. Moreover, Gö 6976 or PKCα siRNA significantly suppressed CSE-induced upregulation of cyclin D1 at both the mRNA and protein levels.
  
  
  CONCLUSIONS
  PKCα-cyclin D1 pathway at least partially mediates the CSE-induced proliferation in HPASMCs.
authors.affiliation: Department of Respiratory Medicine, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China.
authors.forename: Min
authors.initials: M
authors.lastname: Xiang
authors.forename: Yong-Jian
authors.initials: YJ
authors.lastname: Xu
authors.forename: Xian-Sheng
authors.initials: XS
authors.lastname: Liu
authors.forename: Da-Xiong
authors.initials: DX
authors.lastname: Zeng
mesh_terms: D049109:Cell Proliferation; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000502:physiology*; D016193:G1 Phase; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000473:pathology*; D032389:Myocytes, Smooth Muscle / Q000473:pathology*; D051571:Protein Kinase C-alpha / Q000502:physiology*; D011651:Pulmonary Artery / Q000473:pathology*; D016196:S Phase; D015398:Signal Transduction; D012906:Smoke / Q000009:adverse effects*; D014026:Nicotiana / Q000009:adverse effects*
chemical_list: C530104:CCND1 protein, human; D012906:Smoke; D019938:Cyclin D1; D051571:Protein Kinase C-alpha