title: Phosphorylation of Smad2/3 at the specific linker threonine residue indicates slow-cycling esophageal stem-like cells before re-entry to the cell cycle.
abstract: The stem cell compartment in the esophageal epithelium is possibly located in the basal layer. We have identified significant expression of Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), in the epithelial cells of murine stomach and intestine, and have suggested that these cells are epithelial stem cells. In this study, we explore whether pSmad2/3L-Thr could serve as a biomarker for esophageal stem cells. We examined esophageal tissues from normal C57BL/6 mice and those with esophagitis. Double immunofluorescent staining of pSmad2/3L-Thr with Ki67, CDK4, p63, or CK14 was performed. After immunofluorescent staining, we stained the same sections with hematoxylin-eosin and observed these cells under a light microscope. We used the 5-bromo-2-deoxyuridine (BrdU) labeling assay to examine label retention of pSmad2/3L-Thr immunostaining-positive cells. We collected specimens 5, 10, 15 and 20 days after repeated BrdU administrations and observed double immunofluorescent staining of pSmad2/3L-Thr with BrdU. In the esophagus, pSmad2/3L-Thr immunostaining-positive cells were detected in the basal layer. These cells were detected between Ki67 immunostaining-positive cells, but they were not co-localized with Ki67. pSmad2/3L-Thr immunostaining-positive cells showed co-localization with CDK4, p63, and CK14. Under a light microscope, pSmad2/3L-Thr immunostaining-positive cells indicated undifferentiated morphological features. Until 20 days follow-up period, pSmad2/3L-Thr immunostaining-positive cells were co-localized with BrdU. pSmad2/3L-Thr immunostaining-positive cells significantly increased in the regeneration phase of esophagitis mucosae, as compared with control mice (esophagitis vs.
  
  
  METHODS
  6.889 ± 0.676/cm vs. 4.293 ± 0.659/cm; P < 0.001). We have identified significant expression of pSmad2/3L-Thr in the specific epithelial cells of murine esophagi. We suggest that these cells are slow-cycling epithelial stem-like cells before re-entry to the cell cycle.
authors.affiliation: The Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Takahashi
authors.affiliation: The Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Fukui
authors.affiliation: The Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Kishimoto
authors.affiliation: The Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan.
authors.forename: R
authors.initials: R
authors.lastname: Suzuki
authors.affiliation: The Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Mitsuyama
authors.affiliation: The Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Sumimoto
authors.affiliation: The Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Okazaki
authors.affiliation: The Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Sakao
authors.affiliation: The Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Sakaguchi
authors.affiliation: The Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Yoshida
authors.affiliation: The Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Uchida
authors.affiliation: The Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Nishio
authors.affiliation: The Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Matsuzaki
authors.affiliation: The Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Okazaki
mesh_terms: D000818:Animals; D002453:Cell Cycle; D059447:Cell Cycle Checkpoints; D018797:Cell Cycle Proteins / Q000032:analysis*; D051358:Cyclin-Dependent Kinase 4 / Q000032:analysis; D004847:Epithelial Cells / Q000737:chemistry; D000071041:Esophageal Mucosa / Q000166:cytology / Q000473:pathology; D004941:Esophagitis / Q000378:metabolism / Q000473:pathology; D004947:Esophagus / Q000166:cytology* / Q000473:pathology; D019394:Ki-67 Antigen / Q000032:analysis; D051379:Mice; D008810:Mice, Inbred C57BL; D010750:Phosphoproteins / Q000032:analysis; D010766:Phosphorylation; D051899:Smad2 Protein / Q000032:analysis*; D051900:Smad3 Protein / Q000032:analysis*; D013194:Staining and Labeling; D013234:Stem Cells / Q000737:chemistry* / Q000166:cytology; D013912:Threonine; D015534:Trans-Activators / Q000032:analysis
chemical_list: D018797:Cell Cycle Proteins; D019394:Ki-67 Antigen; C498355:Mki67 protein, mouse; D010750:Phosphoproteins; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D015534:Trans-Activators; C486419:Trp63 protein, mouse; D013912:Threonine; C495902:Cdk4 protein, mouse; D051358:Cyclin-Dependent Kinase 4
keywords: Smad proteins; cell cycle; phosphorylation; stem cells; threonine