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Phosphorylation of Smad2/3 at Specific Linker Threonine Indicates Slow-Cycling Intestinal Stem-Like Cells Before Reentry to Cell Cycle

Digestive Diseases and Sciences Aims and scope Submit manuscript

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.

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Acknowledgments

This study was supported by the Grant-in-Aid for Young Scientists (B) (22790676) from the Japan Society for the Promotion of Science, by the Health and Labour Sciences Research Grant for Research on Measures for Intractable Diseases from the Ministry of Health, Labour and Welfare of Japan, and by the Grant-in-Aid from CREST Japan Science and Technology Agency, by the Grant-aided Program for the Strategic Research Foundation for Private Universities, by the NEXT-Supported Program for the Strategic Research Foundation at Private Universities from the Ministry of Education, Culture, Sports, Science and Technology in Japan.

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Correspondence to Toshiro Fukui.

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Kishimoto, M., Fukui, T., Suzuki, R. et al. Phosphorylation of Smad2/3 at Specific Linker Threonine Indicates Slow-Cycling Intestinal Stem-Like Cells Before Reentry to Cell Cycle. Dig Dis Sci 60, 362–374 (2015). https://doi.org/10.1007/s10620-014-3348-3

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  • DOI: https://doi.org/10.1007/s10620-014-3348-3

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