title: Role of ALK5/Smad2/3 and MEK1/ERK Signaling in Transforming Growth Factor Beta 1-modulated Growth, Collagen Turnover, and Differentiation of Stem Cells from Apical Papilla of Human Tooth.
abstract: BACKGROUND
  Transforming growth factor β1 (TGF-β1) plays an important role in cell proliferation, matrix formation, and odontogenesis. This study investigated the effects of TGF-β1 on stem cells from apical papilla (SCAPs) and its signaling by MEK/ERK and Smad2.
  
  
  METHODS
  SCAPs were exposed to TGF-β1 with/without pretreatment and coincubation by SB431542 (an ALK5/Smad 2/3 inhibitor) or U0126 (a MEK/ERK inhibitor). Cell growth was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay or direct counting of viable cells. Collagen content was determined by using the Sircol collagen assay (Biocolor Ltd, Newtownabbey, Northern Ireland). Cell differentiation was evaluated by measuring alkaline phosphatase (ALP) activity. Smad2 and ERK1/2 phosphorylation was analyzed by Western blotting or PathScan phospho-enzyme-linked immunosorbent assay (Cell Signaling Technology Inc, Danvers, MA).
  
  
  RESULTS
  TGF-β1 stimulated the growth and collagen content of cultured SCAPs. TGF-β1 stimulated ERK1/2 and Smad2 phosphorylation within 60 minutes of exposure. Pretreatment by U0126 and SB431542 effectively prevented the TGF-β1-induced cell growth and collagen content in SCAPs. TGF-β1 stimulated ALP activity at lower concentrations (0.1-1 ng/mL) but down-regulated ALP at higher concentrations (>5 ng/mL). U0126 prevented 0.5 ng/mL TGF-β1-induced ALP activity but showed little effect on 10 ng/mL TGF-β1-induced decline of ALP in SCAPs. Interestingly, SB431542 attenuated both the stimulatory and inhibitory effects on ALP by TGF-β1.
  
  
  CONCLUSIONS
  TGF-β1 may affect the proliferation, collagen turnover, and differentiation of SCAPs via differential activation of ALK5/Smad2 and MEK/ERK signaling. These results highlight the future use of TGF-β1 and SCAP for engineering of pulpal regeneration and apexogenesis.
authors.affiliation: Department of Dentistry, National Taiwan University Hospital and Laboratory of Dental Pharmacology, Toxicology and Material Biocompatibility, School of Dentistry, National Taiwan University Medical College, Taipei, Taiwan.
authors.forename: Hsiao-Hua
authors.initials: HH
authors.lastname: Chang
authors.affiliation: Biomedical Science Team, Chang Gung University of Science and Technology, Kwei-Shan, Taoyuan City, Taiwan; Chang Gung Memorial Hospital, Taoyuan, Taiwan.
authors.forename: Mei-Chi
authors.initials: MC
authors.lastname: Chang
authors.affiliation: Department of Dentistry, National Taiwan University Hospital and Laboratory of Dental Pharmacology, Toxicology and Material Biocompatibility, School of Dentistry, National Taiwan University Medical College, Taipei, Taiwan.
authors.forename: I-Hua
authors.initials: IH
authors.lastname: Wu
authors.affiliation: Department of Dentistry, National Taiwan University Hospital and Laboratory of Dental Pharmacology, Toxicology and Material Biocompatibility, School of Dentistry, National Taiwan University Medical College, Taipei, Taiwan.
authors.forename: Guay-Fen
authors.initials: GF
authors.lastname: Huang
authors.affiliation: Department of Dentistry, National Taiwan University Hospital and Laboratory of Dental Pharmacology, Toxicology and Material Biocompatibility, School of Dentistry, National Taiwan University Medical College, Taipei, Taiwan.
authors.forename: Wei-Ling
authors.initials: WL
authors.lastname: Huang
authors.affiliation: Department of Dentistry, National Taiwan University Hospital and Laboratory of Dental Pharmacology, Toxicology and Material Biocompatibility, School of Dentistry, National Taiwan University Medical College, Taipei, Taiwan.
authors.forename: Yin-Lin
authors.initials: YL
authors.lastname: Wang
authors.affiliation: School of Dentistry, Taipei Medical University, Taipei, Taiwan.
authors.forename: Sheng-Yang
authors.initials: SY
authors.lastname: Lee
authors.affiliation: Department of Dentistry, National Taiwan University Hospital and Laboratory of Dental Pharmacology, Toxicology and Material Biocompatibility, School of Dentistry, National Taiwan University Medical College, Taipei, Taiwan.
authors.forename: Chien-Yang
authors.initials: CY
authors.lastname: Yeh
authors.affiliation: Department of Dentistry, National Taiwan University Hospital and Laboratory of Dental Pharmacology, Toxicology and Material Biocompatibility, School of Dentistry, National Taiwan University Medical College, Taipei, Taiwan.
authors.forename: Ming-Kuang
authors.initials: MK
authors.lastname: Guo
authors.affiliation: Department of Dentistry, Chang Gung Memorial Hospital, Taipei, Taiwan.
authors.forename: Chiu-Po
authors.initials: CP
authors.lastname: Chan
authors.affiliation: Chang Gung Memorial Hospital, Taoyuan, Taiwan.
authors.forename: Hsiang-Chi
authors.initials: HC
authors.lastname: Hsien
authors.affiliation: Department of Dentistry, National Taiwan University Hospital and Laboratory of Dental Pharmacology, Toxicology and Material Biocompatibility, School of Dentistry, National Taiwan University Medical College, Taipei, Taiwan. Electronic address: jhjeng@ntu.edu.tw.
authors.forename: Jiiang-Huei
authors.initials: JH
authors.lastname: Jeng
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D001549:Benzamides / Q000494:pharmacology; D002070:Butadienes / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002648:Child; D003094:Collagen / Q000378:metabolism*; D003771:Dental Papilla / Q000187:drug effects / Q000502:physiology*; D004149:Dioxoles / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000378:metabolism; D051899:Smad2 Protein / Q000037:antagonists & inhibitors / Q000378:metabolism; D051900:Smad3 Protein / Q000037:antagonists & inhibitors / Q000378:metabolism; D013234:Stem Cells / Q000187:drug effects / Q000502:physiology*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*
chemical_list: C459179:4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D001549:Benzamides; D002070:Butadienes; D004149:Dioxoles; D004791:Enzyme Inhibitors; D009570:Nitriles; D018125:Receptors, Transforming Growth Factor beta; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; C508960:TGFB1 protein, human; D053773:Transforming Growth Factor beta1; C113580:U 0126; D003094:Collagen; D017346:Protein Serine-Threonine Kinases; D000077293:Receptor, Transforming Growth Factor-beta Type I; C000625841:TGFBR1 protein, human; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D000469:Alkaline Phosphatase
keywords: ALK5; ERK; Smad2; alkaline phosphatase; signal transduction; stem cells from apical papilla; transforming growth factor beta