Abstract
Signaling mediated by activation of the transmembrane receptor Notch influences cell-fate decisions, differentiation, proliferation, and cell survival. Activated Notch reduces proliferation by altering cell-cycle kinetics and promotes differentiation in hematopoietic progenitor cells. Here, we investigated if the G1 arrest and differentiation induced by activated mNotch1 are dependent on tumor suppressor p53, a critical mediator of cellular growth arrest. Multipotent wild-type p53-expressing (p53wt) and p53-deficient (p53null) hematopoietic progenitor cell lines (FDCP-mix) carrying an inducible mNotch1 system were used to investigate the effects of proliferation and differentiation upon mNotch1 signaling. While activated Notch reduced proliferation of p53wt-cells, no change was observed in p53null-cells. Activated Notch upregulated the p53 target p21cip/waf in p53wt-cells, but not in p53null-cells. Induction of the p21cip/waf gene by activated Notch was mediated by increased binding of p53 to p53-binding sites in the p21cip/waf promoter and was independent of the canonical RBP-J binding site. Re-expression of p53wt in p53null cells restored the inhibition of proliferation by activated Notch. Thus, activated Notch inhibits proliferation of multipotent hematopoietic progenitor cells via a p53-dependent pathway. In contrast, myeloid and erythroid differentiation was similarly induced in p53wt and p53null cells. These data suggest that Notch signaling triggers two distinct pathways, a p53-dependent one leading to a block in proliferation and a p53-independent one promoting differentiation.
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Abbreviations
- p53wt:
-
wild type p53
- p53null:
-
p53-deficient
- FDCP-mix:
-
factor-dependent cell established at the Paterson Institute with mixed differentiation potential
- mNotch1:
-
murine Notch1
- NotchIC:
-
intracellular domain of Notch
- RBP-J:
-
recombination recognition sequence binding protein at the Jκ site
- Hes:
-
hairy and enhancer of split
- Hey/Herp:
-
Hes-related repressor
- mN1IC:
-
intracellular domain of murine Notch1
- NERT:
-
mN1IC fused to the hormone binding domain of the human estrogen receptor tamoxifen-sensitive mutant
- ERT:
-
estrogen receptor tamoxifen-sensitive mutant
- rneo:
-
retroviral control vector carrying the neomyin resistance gene
- rNERTneo:
-
retroviral control vector carrying the NERT and neomyin resistance gene
- rNERTneo FDCP-mix:
-
FDCP-mix cells expressing an OHT-inducible form of Notch1IC
- rneo FDCP-mix:
-
FDCP-mix cells carrying the retroviral control vector conferring neomycin resistance
- G418:
-
neomycin
- OHT:
-
4-hydroxytamoxifen
- CFSE:
-
carboxyfluorescein diacetate succinimidyl ester
- p21RBP-Jmut promoter:
-
p21 promoter compromised in RBP-J binding
- IMDM:
-
Iscove's modified Dulbecco's medium
- FCS:
-
fetal calf serum
- PE:
-
Phycoerytrin
- APC:
-
allophycocyanin
- PBS:
-
phosphate-buffered saline
- RT:
-
room temperature
- p21luc:
-
reporter plasmid carrying a luciferase gene under the control of the p21 promoter
- p21Δp53BS1luc:
-
p21 promoter lacking the first p53 binding site
- p21Δp53BS2luc:
-
p21 promoter lacking the second p53 binding site
- p21Δp53BS1+2luc:
-
p21 promoter lacking both p53 binding sites
- ChIP:
-
chromatin immunoprecipitation
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Acknowledgements
We thank Silke Horn, Cornelia Kuklik-Roos and Gabriele Warnecke for expert technical support and Elaine Spooncer and Michael Dexter for the p53null FDCP-mix cell lines. This work was supported by the Deutsche Forschungsgemeinschaft (Priority Program 1109 ‘Stem Cells’ and SFB 415 ‘Signal Transduction’ project B8 to UJ). This report represents a part of the doctoral thesis by KH and of the diploma thesis of JH.
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Henning, K., Heering, J., Schwanbeck, R. et al. Notch1 activation reduces proliferation in the multipotent hematopoietic progenitor cell line FDCP-mix through a p53-dependent pathway but Notch1 effects on myeloid and erythroid differentiation are independent of p53. Cell Death Differ 15, 398–407 (2008). https://doi.org/10.1038/sj.cdd.4402277
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DOI: https://doi.org/10.1038/sj.cdd.4402277
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