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Loss of Notch1 TAD interferes with niche recovery
are highly expressed in the endothelium during embryon- ic development and control EC specification13 and Notch1/Dll4 in coordination with VEGF-A/VEGFR2 sig- naling regulates sprouting angiogenesis.14,15 The function of Notch signaling in the adult vasculature is less under- stood. Studies showed that Notch1 signaling in the adult endothelium regulates expression of inflammatory genes.16 Notch1 is also known to be activated by blood flow and shear stress forces, which contribute to vascular homeostasis.17 Important, unresolved questions are whether Notch activation has a role in post-injury endothelial regeneration and whether it promotes the recovery of hematopoiesis.
The intracellular domains of Notch receptors have dis- tinct roles. The RAM domain has a high affinity for binding to RBPJ, while the Ankyrin repeat (ANK) domains interact with a Mastermind-like (MAML) protein factor and recruit other co-activators. The PEST domain localized at the C- terminal facilitates Notch degradation.18 In between the ANK and PEST domains there is a transcriptional activation domain (TAD), which is capable of autonomous transcrip- tional activity and directly binds co-activators PCAF and GNC5.19,20 The TAD is a region of significant divergence among the four mammalian Notch receptors.20,21 These dif- ferences among the Notch receptor TAD may be important in the tissue-specific variability of Notch signaling.
We previously developed a transgenic knock-in model system which deleted the TAD of Notch1.22 This model system was used to study the role of Notch1 TAD func- tion during fetal development. The loss of TAD in both Notch1 alleles (ΔTAD/ΔTAD), allowed mice to develop to late gestation when they succumbed to multiple Notch- dependent cardiovascular anomalies.22-24 While definitive HSC emerged from the aorta-gonad-mesonephros region, they failed to expand in the fetal liver of ΔTAD/ΔTAD embryos. Furthermore, when transplanted into irradiated adult recipients, ΔTAD/ΔTAD HSC underperformed in primary transplants and failed to reconstitute the hematopoietic system efficiently in secondary trans- plants.22 In contrast, mice heterozygous for one allele of Notch1ΔTAD (Notch1+/ΔTAD) survive to adulthood and exhib- it no overt hematopoietic phenotype.
In the present study, we made use of hypomorphic Notch signaling in the Notch1+/ΔTAD model to address whether the Notch pathway is crucial for the recovery of the adult BM niche and regeneration of hematopoietic cells after injury. We observed that high levels of Notch signaling were dispensable for the development of the endothelial niche and high Notch activity was not required during adult BM endothelial homeostasis. In the hematopoietic system, Notch1+/ΔTAD only displayed cell- autonomous defects in the development of the T-cell lin- eage. However, following myelosuppressive injury, robust Notch signaling was critical for recovery of the BM endothelial niche and thereby the regeneration of HSC. Notch signaling was stimulated by a burst of Tie2-depen- dent activation, which induced expression of Notch1 lig- ands. Interestingly, expression of Notch1ΔTAD protein in Notch1+/ΔTAD EC decreased expression of Notch target genes and led to severe apoptosis. This phenotype could not be rescued by enhanced activation of Tie2 signaling. Our results suggest a crucial role for TAD-regulated Notch activity in mediating EC survival and promoting recovery of hematopoiesis following chemotherapeutic stress.
Methods
Animals
The following strains of mice were used in our studies under the guidelines and protocols approved by the Institutional Animal Care and Use Committees of University of Illinois at Chicago: C57BL/6J (or CD45.2), B6.SJL-PtprcaPep3b/BoyJ (or CD45.1), Notch+/ΔTAD, Notch+/-, Notch1flf, RBPJf/f, PDGFRβ-CreERT2 and VE- Cadherin CreERT2 mice. More details about the mice can be found in the Online Supplementary Methods.
5-Fluorouracil treatment and irradiation
5-Fluorouracil (5-FU, 150 mg/kg) was peritoneally injected into mice. For irradiation treatment, mice were exposed, on a rotating platform, to a lethal dose (9.0 Gy) of total body irradia- tion in a Mark I 137Ce γ-irradiator (JL Shepherd, Glendale, CA, USA) at a dose rate of 6.38 Gy/min. More details about chemotherapy and radiotherapy can be found in the Online Supplementary Methods.
Analysis of bone marrow and thymic mononuclear cells
Mononuclear cells from BM and thymus were prepared as described in the Online Supplementary Methods. The frequencies of LSK cells (Lin-Sca1+c-kit+ cells), HSC (CD150+CD48-LSK cells), common lymphoid progenitors (CLP: Lin-Sca1lowc- kitlowCD135+CD127+), thymic early T-cell precursors (ETP: Lin-c- kit+CD25-CD44+) and double-negative 3 cells (DN3 cells: Lin- CD4-CD8-CD25+CD44-) were analyzed with a flow cytometer, Fortessa LSRII, after immunostaining with appropriate antibod- ies as described in the Online Supplementary Methods. All antibod- ies used are listed in Online Supplementary Table S1.
Isolation of CD25+ thymocytes and primary bone endothelial cells
Cell suspensions from the thymus or digested bones were incubated with dynabeads labeled with CD25 antibody and CD31 antibody (Invitrogen), respectively, for 30 min at 4°C. CD25+ thymocytes and bone CD31+ cells (pBEC) were isolated and used for western blot and quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) analysis.
Bone marrow-derived endothelial cell culture
Cultured bone marrow-derived endothelial cells (cBEC) were purchased from Cell Biologicals Inc (cat#, C57-6221) and cul- tured in EC medium. cBEC were treated with a γ-secretase inhibitor (1 mM, Sigma), a Tie2 kinase inhibitor (1 mM, Abcam), Ang1 (300 ng/mL, Peprotech) or 5-FU (100 mM, Sigma). More detailed information can be found in the Online Supplementary Methods.
Western blot analysis
Expression of cleaved/active Notch1 (Val1744), Notch1, VE- Cadherin, Tie2, and phosphorylated Tie2-Y992 was measured by western blot. The procedure is detailed in the Online Supplementary Methods.
Quantitative reverse transcriptase polymerse chain reaction
Expression of various genes was measured by RT-qPCR and cal- culated using the the comparative CT method. The sequences of all primers used in the RT-qPCR assay are listed in Online Supplementary Table S2.
Chromatin immunoprecipitation assays
RBPJ binding sites at Myc NDME, Hes1, Hey1 and Dtx1 locus
haematologica | 2019; 104(11)
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