title: Inhibition of transforming growth factor β1/Smad3 signaling decreases hypoxia-inducible factor-1α protein stability by inducing prolyl hydroxylase 2 expression in human periodontal ligament cells.
abstract: BACKGROUND
  Hypoxia-inducible factor-1α (HIF-1α), the α subunit of the heterodimeric transcription factor HIF-1, maintains oxygen homeostasis by regulating gene expression. Under normoxic conditions, HIF-1α expression is maintained at low steady-state levels by the critical oxygen sensor prolyl hydroxylase 2 (PHD2). Transforming growth factor β1 (TGF-β1) activates Smad3 signaling and contributes to HIF-1α stabilization under normoxic conditions. In chronic periodontitis, HIF-1α is expressed highly in gingival fibroblasts and upregulates inflammatory factor transcription, which promotes periodontal inflammation. Here, the authors investigated the effect of TGF-β1/Smad3 signaling and its blockade by the specific inhibitor of Smad3 (SIS3) on HIF-1α expression and stability in human periodontal ligament cells.
  
  
  METHODS
  The authors investigated the effect of TGF-β1 on HIF-1α protein stability using cycloheximide. Furthermore, they analyzed HIF-1α expression, PHD2 expression, and Smad3 phosphorylation following TGF-β1 stimulation in the presence or absence of SIS3.
  
  
  RESULTS
  The half-life of HIF-1α was prolonged in TGF-β1-treated cells. TGF-β1 treatment induced HIF-1α gene expression and enhanced HIF-1α protein stability while decreasing PHD2 expression and activating Smad3 phosphorylation. Notably, HIF-1α protein expression was not detectable prior to TGF-β1 stimulation. Furthermore, SIS3 treatment abrogated Smad3 phosphorylation, impaired TGF-β1-induced HIF-1α gene expression and protein stability, and stimulated TGF-β1-mediated PHD2 inhibition.
  
  
  CONCLUSIONS
  These results demonstrate that HIF-1α transcription and protein synthesis are controlled by TGF-β1/Smad3 signaling, whereas HIF-1α protein stability is controlled by PHD2, which is regulated by TGF-β1/Smad3 signaling.
authors.affiliation: Department of Orthodontics and Dentofacial Orthopedics, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima City, Tokushima, Japan. teppei@dent.tokushima-u.ac.jp
authors.forename: Teppei
authors.initials: T
authors.lastname: Watanabe
authors.forename: Akihiro
authors.initials: A
authors.lastname: Yasue
authors.forename: Eiji
authors.initials: E
authors.lastname: Tanaka
mesh_terms: D000293:Adolescent; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D055113:Chronic Periodontitis / Q000378:metabolism / Q000473:pathology; D003513:Cycloheximide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors* / Q000187:drug effects; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases / Q000187:drug effects*; D007546:Isoquinolines / Q000494:pharmacology; D008297:Male; D010513:Periodontal Ligament / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D051900:Smad3 Protein / Q000037:antagonists & inhibitors* / Q000187:drug effects; D013997:Time Factors; D053773:Transforming Growth Factor beta1 / Q000037:antagonists & inhibitors* / Q000494:pharmacology
chemical_list: C508504:6,7-dimethyl-2-(2E)-3-(1-methyl-2-phenyl-1H-pyrrolo(2,3-b)pyridin-3-yl-prop-2-enoyl)-1,2,3,4-tetrahydroisoquinoline hydrochloride; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007546:Isoquinolines; D011500:Protein Synthesis Inhibitors; D011725:Pyridines; D011758:Pyrroles; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D053773:Transforming Growth Factor beta1; D003513:Cycloheximide; C490190:EGLN1 protein, human; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases