Phosphorylation of Smad2/3 at specific linker threonine indicates slow-cycling intestinal stem-like cells before reentry to cell cycle

Dig Dis Sci. 2015 Feb;60(2):362-74. doi: 10.1007/s10620-014-3348-3. Epub 2014 Sep 4.

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.

Aim: 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.

Conclusion: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biomarkers / metabolism
  • Cell Cycle Checkpoints*
  • Cell Cycle Proteins / metabolism
  • Cell Differentiation
  • Cell Proliferation*
  • Colitis / chemically induced
  • Colitis / metabolism*
  • Colitis / pathology
  • Colon / metabolism*
  • Colon / pathology
  • Dextran Sulfate
  • Disease Models, Animal
  • Green Fluorescent Proteins / biosynthesis
  • Green Fluorescent Proteins / genetics
  • Intestine, Small / metabolism*
  • Intestine, Small / pathology
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Phenotype
  • Phosphorylation
  • Receptors, G-Protein-Coupled / genetics
  • Signal Transduction
  • Smad2 Protein / metabolism*
  • Smad3 Protein / metabolism*
  • Stem Cells / metabolism*
  • Threonine

Substances

  • Biomarkers
  • Cell Cycle Proteins
  • Receptors, G-Protein-Coupled
  • Smad2 Protein
  • Smad2 protein, mouse
  • Smad3 Protein
  • Smad3 protein, mouse
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Threonine
  • Dextran Sulfate