Absolute requirement for STAT3 function in small-intestine crypt stem cell survival (original) (raw)

Cell Death & Differentiation volume 18, pages 1934–1943 (2011)Cite this article

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Abstract

The transcription factor signal transducer and activator of transcription 3 (STAT3) is frequently activated in human cancers. Interestingly, STAT3 also maintains the pluripotency and self-renewal of murine embryonic stem cells, and several tissue stem cell types. To investigate whether STAT3 also maintains the small-intestine crypt stem cell, we conditionally inactivated a Floxed Stat3 allele (Stat3 fl) in murine small-intestine crypt stem cells. Following Cre recombinase expression, apoptosis increased in Stat3 fl/− experimental crypts relative to Stat3 wt/− controls before declining. Control Stat3 wt/− mice carrying a Flox-STOP LacZ reporter transgene stably expressed LacZ after Cre induction. In contrast, Stat3 fl/− intestine LacZ expression initially increased modestly, before declining to background levels. Quantitative PCRs revealed a similar transient in recombined Stat3 fl allele levels. Long-term bromodeoxyuridine labelling directly demonstrated that functional STAT3 is required for +4 to +6 region label-retaining small-intestine stem cell survival. Rapid clearance of recombined Stat3 fl/− cells involves apoptosis potentially induced by elevated c-Myc in non-recombined cells and involves elevated p53 expression and caspase 3 activation. Intriguingly, Stat3 fl/− intestine recombination triggered dramatically upregulated polycomb transcriptional repressor Bmi1 – potentially accelerating recombined crypt repopulation. In summary, STAT3 activity is absolutely required for small-intestine crypt stem cell survival at both the +4 to +6 label-retaining and crypt base columnar cell locations.

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Abbreviations

AH-Cre:

Cyp1A1 promoter-Cre recombinase

BrdU:

bromodeoxyuridine

CBC:

crypt base columnar cell

Ct:

threshold cycle

DAPI:

4′,6-diamidino-2-phenylindole

Fl:

floxed

H+E:

haematoxylin plus eosin

HRP:

horseradish peroxidase

mES:

murine embryonic stem cell

NGS:

normal goat serum

PcG:

polycomb group

Q-PCR:

quantitative polymerase chain reaction

SH2:

src homology 2

STAT:

signal transducer and activator of transcription

TBS:

Tris-buffered saline

WT:

wild type

–:

null

References

  1. Schindler C, Darnell JE . Transcriptional responses to polypeptide ligands – the JAK-STAT pathway. Ann Rev Biochem 1995; 64: 621–651.
    Article CAS Google Scholar
  2. Zhong Z, Wen ZL, Darnell JE . STAT3 – a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6. Science 1994; 264: 95–98.
    Article CAS Google Scholar
  3. Yu H, Jove R . The stats of cancer – new molecular targets come of age. Nat Rev Cancer 2004; 4: 97–105.
    Article CAS Google Scholar
  4. Haura EB, Turkson J, Jove R . Mechanisms of disease: insights into the emerging role of signal transducers and activators of transcription in cancer. Nat Clin Pract Oncol 2005; 2: 315–324.
    Article CAS Google Scholar
  5. Niwa H, Burdon T, Chambers I, Smith A . Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev 1998; 12: 2048–2060.
    Article CAS Google Scholar
  6. Dyce PW, Zhu H, Craig J, Li JL . Stem cells with multilineage potential derived from porcine skin. Biochem Biophys Res Comm 2004; 316: 651–658.
    Article CAS Google Scholar
  7. Oh IH, Eaves CJ . Overexpression of a dominant negative form of STAT3 selectively impairs hematopoietic stem cell activity. Oncogene 2002; 21: 4778–4787.
    Article CAS Google Scholar
  8. Barker N, van de Wetering M, Clevers H . The intestinal stem cell. Genes Dev 2008; 22: 1856–1864.
    Article CAS Google Scholar
  9. Potten CS, Booth C, Pritchard DM . The intestinal epithelial stem cell: the mucosal governor. Int J Exp Path 1997; 78: 219–243.
    Article CAS Google Scholar
  10. Cheng H, Leblond CP . Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine V. Unitarian theory of the origin of the four epithelial cell types. Am J Anat 1974; 141: 537–561.
    Article CAS Google Scholar
  11. Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 2007; 449: 1003–1007.
    Article CAS Google Scholar
  12. Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 2009; 459: 262–265.
    Article CAS Google Scholar
  13. Ireland H, Kemp R, Houghton C, Howard L, Clarke AR, Sansom OJ et al. Inducible Cre-mediated control of gene expression in the murine gastrointestinal tract: effect of loss of beta-catenin. Gastroenterology 2004; 126: 1236–1246.
    Article CAS Google Scholar
  14. Takeda K, Kaisho T, Yoshida N, Takeda J, Kishimoto T, Akira S . Stat3 activation is responsible for IL-6-dependent T cell proliferation through preventing apoptosis: generation and characterization of T cell-specific Stat3-deficient mice. J Immunol 1998; 161: 4652–4660.
    CAS PubMed Google Scholar
  15. Ireland H, Houghton C, Howard L, Winton DJ . Cellular inheritance of a Cre-activated reporter gene to determine Paneth cell longevity in the murine small intestine. Dev Dynam 2005; 233: 1332–1336.
    Article CAS Google Scholar
  16. Takeda K, Noguchi K, Shi W, Tanaka T, Matsumoto M, Yoshida N et al. Targeted disruption of the mouse Stat3 gene leads to early embryonic lethality. Proc Natl Acad Sci USA 1997; 94: 3801–3804.
    Article CAS Google Scholar
  17. Bowman T, Broome MA, Sinibaldi D, Wharton W, Pledger WJ, Sedivy JM et al. Stat3-mediated Myc expression is required for Src transformation and PDGF-induced mitogenesis. Proc Natl Acad Sci USA 2001; 98: 7319–7324.
    Article CAS Google Scholar
  18. Moreno E, Basler K . dMyc Transforms cells into super-competitors. Cell 2004; 117: 117–129.
    Article CAS Google Scholar
  19. Sangiorgi E, Capecchi MR . Bmi1 is expressed in vivo in intestinal stem cells. Nat Genet 2008; 40: 915–920.
    Article CAS Google Scholar
  20. Ho L, Jothi R, Ronan JL, Cui K, Zhao K, Crabtree GR . An embryonic stem cell chromatin remodeling complex, esBAF, is an essential component of the core pluripotency transcriptional network. Proc Natl Acad Sci USA 2009; 106: 5187–5191.
    Article CAS Google Scholar
  21. Ura H, Usuda M, Kinoshita K, Sun C, Mori K, Akagi T et al. STAT3 and Oct-3/4 control histone modification through induction of Eed in embryonic stem cells. J Biol Chem 2008; 283: 9713–9723.
    Article CAS Google Scholar
  22. Lessard J, Schumacher A, Thorsteinsdottir U, van Lohuizen M, Magnuson T, Sauvageau G . Functional antagonism of the Polycomb-group genes eed and Bmi1 in hemopoietic cell proliferation. Genes Dev 1999; 13: 2691–2703.
    Article CAS Google Scholar

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Acknowledgements

This work was supported by a Cancer Research UK programme grant, we also gratefully thank M Bishop for providing mouse genotyping services, A Hayes for performing confocal fluorescence immunohistochemistry, C Oliver for preliminary data and thank L Parry for providing two cycles of Cre induction small-intestine slides.

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Authors and Affiliations

  1. Division of Pathophysiology and Repair, School of Biosciences, Cardiff University, Cardiff, UK
    J R Matthews & A R Clarke
  2. CRUK Beatson Institute for Cancer Research, Bearsden, Glasgow, UK
    O J Sansom

Authors

  1. J R Matthews
  2. O J Sansom
  3. A R Clarke

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Correspondence toA R Clarke.

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The authors declare no conflict of interest.

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Matthews, J., Sansom, O. & Clarke, A. Absolute requirement for STAT3 function in small-intestine crypt stem cell survival.Cell Death Differ 18, 1934–1943 (2011). https://doi.org/10.1038/cdd.2011.77

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