title: Interferon regulatory factor-1 together with reactive oxygen species promotes the acceleration of cell cycle progression by up-regulating the cyclin E and CDK2 genes during high glucose-induced proliferation of vascular smooth muscle cells.
abstract: BACKGROUND
  The high glucose-induced proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development of diabetic vascular diseases. In a previous study, we confirmed that Interferon regulatory factor-1 (Irf-1) is a positive regulator of the high glucose-induced proliferation of VSMCs. However, the mechanisms remain to be determined.
  
  
  METHODS
  The levels of cyclin/CDK expression in two cell models involving Irf-1 knockdown and overexpression were quantified to explore the relationship between Irf-1 and its downstream effectors under normal or high glucose conditions. Subsequently, cells were treated with high glucose/NAC, normal glucose/H₂O₂, high glucose/U0126 or normal glucose/H₂O₂/U0126 during an incubation period. Then proliferation, cyclin/CDK expression and cell cycle distribution assays were performed to determine whether ROS/Erk1/2 signaling pathway was involved in the Irf-1-induced regulation of VSMC growth under high glucose conditions.
  
  
  RESULTS
  We found that Irf-1 overexpression led to down-regulation of cyclin D1/CDK4 and inhibited cell cycle progression in VSMCs under normal glucose conditions. In high glucose conditions, Irf-1 overexpression led to an up-regulation of cyclin E/CDK2 and an acceleration of cell cycle progression, whereas silencing of Irf-1 suppressed the expression of both proteins and inhibited the cell cycle during the high glucose-induced proliferation of VSMCs. Treatment of VSMCs with antioxidants prevented the Irf-1 overexpression-induced proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression in high glucose conditions. In contrast, under normal glucose conditions, H₂O₂ stimulation and Irf-1 overexpression induced cell proliferation, up-regulated cyclin E/CDK2 expression and promoted cell cycle acceleration. In addition, overexpression of Irf-1 promoted the activation of Erk1/2 and when VSMCs overexpressing Irf-1 were treated with U0126, the specific Erk1/2 inhibitor abolished the proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression under high glucose or normal glucose/H₂O₂ conditions.
  
  
  CONCLUSIONS
  These results demonstrate that the downstream effectors of Irf-1 are cyclin E/CDK2 during the high glucose-induced proliferation of VSMCs, whereas they are cyclin D1/CDK4 in normal glucose conditions. The Irf-1 overexpression-induced proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression are associated with ROS/Erk1/2 signaling pathway under high glucose conditions.
authors.affiliation: Institute of Biomedical Engineering, Second Military Medical University, Shanghai, China. zhangxicyl1126@163.com.
authors.forename: Xi
authors.initials: X
authors.lastname: Zhang
authors.forename: Long
authors.initials: L
authors.lastname: Liu
authors.forename: Chao
authors.initials: C
authors.lastname: Chen
authors.forename: Ya-Li
authors.initials: YL
authors.lastname: Chi
authors.forename: Xiang-Qun
authors.initials: XQ
authors.lastname: Yang
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
authors.forename: Xiao-Tong
authors.initials: XT
authors.lastname: Li
authors.forename: Shi-Lei
authors.initials: SL
authors.lastname: Guo
authors.forename: Shao-Hu
authors.initials: SH
authors.lastname: Xiong
authors.forename: Man-Ru
authors.initials: MR
authors.lastname: Shen
authors.forename: Yu
authors.initials: Y
authors.lastname: Sun
authors.forename: Chuan-Sen
authors.initials: CS
authors.lastname: Zhang
authors.forename: Kai-Meng
authors.initials: KM
authors.lastname: Hu
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism*; D051358:Cyclin-Dependent Kinase 4 / Q000378:metabolism; D003925:Diabetic Angiopathies / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D050856:Interferon Regulatory Factor-1 / Q000235:genetics / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection; D015854:Up-Regulation
chemical_list: D000975:Antioxidants; C530106:Ccnd1 protein, rat; D019927:Cyclin E; D050856:Interferon Regulatory Factor-1; C494259:Irf1 protein, rat; D047428:Protein Kinase Inhibitors; D017382:Reactive Oxygen Species; D019938:Cyclin D1; C495892:Cdk2 protein, rat; C495903:Cdk4 protein, rat; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D048049:Extracellular Signal-Regulated MAP Kinases; D005947:Glucose