title: Phosphorylation of Smad2/3 at specific linker threonine indicates slow-cycling intestinal stem-like cells before reentry to cell cycle.
abstract: BACKGROUND
  Quiescent (slow-cycling) and active (rapid-cycling) stem cells are demonstrated in small intestines. We have identified significant expression of Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), in murine stomach, and suggested these cells are epithelial stem cells.
  
  
  OBJECTIVE
  Here, we explore whether pSmad2/3L-Thr could serve as a biomarker for small intestine and colon stem cells.
  
  
  METHODS
  We examined small intestines and colons from C57BL/6 mice and colons with dextran sulfate sodium (DSS)-induced colitis. We performed double-immunofluorescent staining of pSmad2/3L-Thr with Ki67, cytokeratin 8, chromogranin A, CDK4, DCAMKL1, and Musashi-1. Small intestines and colons from Lgr5-EGFP knock-in mice were examined by pSmad2/3L-Thr immunofluorescent staining. To examine BrdU label retention of pSmad2/3L-Thr immunostaining-positive cells, we collected specimens after BrdU administration and observed double-immunofluorescent staining of pSmad2/3L-Thr with BrdU.
  
  
  RESULTS
  In small intestines and colons, pSmad2/3L-Thr immunostaining-strongly positive cells were detected around crypt bases. Immunohistochemical co-localization of pSmad2/3L-Thr with Ki67 was not observed. pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with cytokeratin 8, CDK4, and Musashi-1 and different localization from chromogranin A and DCAMKL1 immunostaining-positive cells. Under a light microscope, pSmad2/3L-Thr immunostaining-strongly positive cells were morphologically undifferentiated. In Lgr5-EGFP knock-in mice, some but not all pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with Lgr5. pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with BrdU at 5, 10, and 15 days after administration. In DSS-induced colitis, pSmad2/3L-Thr and Ki67 immunostaining-positive cells increased in the regeneration phase and decreased in the injury phase.
  
  
  CONCLUSIONS
  In murine small intestines and colons, we suggest pSmad2/3L-Thr immunostaining-strongly positive cells are epithelial stem-like cells just before reentry to the cell cycle.
authors.affiliation: Division of Gastroenterology and Hepatology, The Third Department of Internal Medicine, Kansai Medical University, 2-5-1 Shinmachi, Hirakata, Osaka, 573-1010, Japan, kishimom@hirakata.kmu.ac.jp.
authors.forename: Masanobu
authors.initials: M
authors.lastname: Kishimoto
authors.forename: Toshiro
authors.initials: T
authors.lastname: Fukui
authors.forename: Ryo
authors.initials: R
authors.lastname: Suzuki
authors.forename: Yu
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Kimi
authors.initials: K
authors.lastname: Sumimoto
authors.forename: Takashi
authors.initials: T
authors.lastname: Okazaki
authors.forename: Masayuki
authors.initials: M
authors.lastname: Sakao
authors.forename: Yutaku
authors.initials: Y
authors.lastname: Sakaguchi
authors.forename: Katsunori
authors.initials: K
authors.lastname: Yoshida
authors.forename: Kazushige
authors.initials: K
authors.lastname: Uchida
authors.forename: Akiyoshi
authors.initials: A
authors.lastname: Nishio
authors.forename: Koichi
authors.initials: K
authors.lastname: Matsuzaki
authors.forename: Kazuichi
authors.initials: K
authors.lastname: Okazaki
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D059447:Cell Cycle Checkpoints; D018797:Cell Cycle Proteins / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D003092:Colitis / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology; D003106:Colon / Q000378:metabolism* / Q000473:pathology; D016264:Dextran Sulfate; D004195:Disease Models, Animal; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D007421:Intestine, Small / Q000378:metabolism* / Q000473:pathology; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D010641:Phenotype; D010766:Phosphorylation; D043562:Receptors, G-Protein-Coupled / Q000235:genetics; D015398:Signal Transduction; D051899:Smad2 Protein / Q000378:metabolism*; D051900:Smad3 Protein / Q000378:metabolism*; D013234:Stem Cells / Q000378:metabolism*; D013912:Threonine
chemical_list: D015415:Biomarkers; D018797:Cell Cycle Proteins; D043562:Receptors, G-Protein-Coupled; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D013912:Threonine; D016264:Dextran Sulfate