title: Runx2/Smad3 complex negatively regulates TGF-β-induced connective tissue growth factor gene expression in vascular smooth muscle cells.
abstract: OBJECTIVE
  Connective tissue growth factor (CTGF), a direct target gene of transforming growth factor-β (TGF-β) signaling, plays an important role in the development of atherosclerosis. We previously showed that Runx2, a key transcription factor in osteoblast differentiation, regulates osteogenic conversion and dedifferentiation of vascular smooth muscle cells (VSMCs). In this study, we investigated the hypothesis that Runx2 modulates CTGF gene expression via the regulation of TGF-β signaling.
  
  
  RESULTS
  Expression of the Runx2 gene was decreased, and CTGF mRNA levels were reciprocally increased by TGF-β in a time-dependent manner in cultured human aortic smooth muscle cells (HASMCs) and C3H10T1/2 cells. Forced expression of Runx2 decreased and the reduction of Runx2 expression by small interfering RNA enhanced both basal and TGF-β-stimulated CTGF gene expression in HASMCs. Site-directed mutation analysis of the CTGF promoter indicated that transcriptional repression by Runx2 was mediated by the Smad-binding element (SBE) under basal and TGF-β-stimulated conditions. Data obtained from immunoblots of Runx2-, Smad3- or Smad4-transfected cells and chromatin immunoprecipitation analysis indicated that Runx2 interacts with Smad3 at the SBE. Immunohistochemistry revealed that the expression of Runx2 and CTGF was distinct and almost mutually exclusive in human atherosclerotic plaque.
  
  
  CONCLUSIONS
  These results for the first time demonstrate that Runx2/Smad3 complex negatively regulates endogenous and TGF-β-induced CTGF gene expression in VSMCs. Thus, the induction of Runx2 expression contributes to the phenotypic modulation of VSMCs, in which the TGF-β/Smad pathway plays a major role.
authors: Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan.
authors: Yoshiaki
authors: Y
authors: Ohyama
authors: Toru
authors: T
authors: Tanaka
authors: Takehisa
authors: T
authors: Shimizu
authors: Hiroki
authors: H
authors: Matsui
authors: Hiroko
authors: H
authors: Sato
authors: Norimichi
authors: N
authors: Koitabashi
authors: Hiroshi
authors: H
authors: Doi
authors: Tatsuya
authors: T
authors: Iso
authors: Masashi
authors: M
authors: Arai
authors: Masahiko
authors: M
authors: Kurabayashi
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D047369:Chromatin Immunoprecipitation; D055513:Connective Tissue Growth Factor / Q000235:genetics* / Q000378:metabolism; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D006801:Humans; D007124:Immunoenzyme Techniques; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008809:Mice, Inbred C3H; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D015398:Signal Transduction; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D012333:RNA, Messenger; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; D055513:Connective Tissue Growth Factor; D008156:Luciferases