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L. Shao et al.
(Figure 6E), was significantly increased in pBEC from both BM (Online Supplementary Figure S8E) and digested bone (Figure 6D, E). Notch ligand upregulation corresponded with the upregulation of Tie2, Notch1 and the Tie2 signaling target Socs3 (Figure 6D, E, Online Supplementary Figure S8E).
Our initial expression timeline (Figure 6A) indicated that Notch1 expression is upregulated prior to Tie2 upregula- tion. To determine whether the induction of Tie2 and Notch ligands after chemotherapy depended on active Notch signaling, we employed the previously described Notch1f/fVE-cadherin-CreERT2+ model system and analyzed freshly harvested pBEC from WT (Cre-) and Notch1f/fVE- cadherin-CreERT2+ (Cre+) mice after tamoxifen and 5-FU treatment (Figure 6F). Our data indicate that 5-FU treat- ment significantly increased expression of Tie2, Dll4 and Jag1 in pBEC in WT and Notch1f/fVE-cadherin-CreERT2+ mice. However, loss of endothelial Notch1 expression did not significantly affect upregulation of Tie2 after 5-FU treat- ment (Figure 6G, left panel). Furthermore, loss of Notch signaling did not significantly affect the expression of Notch ligands Dll4 and Jag1 (Figure 6G), upregulation of which coincided with the increase in Tie2 expression and activity (Figure 6A, B). Taken together, we show that Tie2 signaling is independent of Notch1 signaling during BM endothelial regeneration and recovery.
Notch1 functions downstream of Tie2 signaling during the recovery of bone marrow endothelium
Previous studies have shown that hematopoietic pro- genitors and mesenchymal cells are responsible for the secretion of Ang1 in the BM.10 To test this, hematopoietic progenitor (Lin-Sca-1+) cells and bone osteoblasts (CD51+) were sorted from the BM 5 days after 5-FU treatment. Expression of Ang1 was markedly increased in 5-FU-treat- ed hematopoietic progenitors (up to 150-fold) and osteoblasts (up to 30-fold) when compared to the same untreated cell populations (Figure 7A). To further analyze any possible crosstalk between the Notch and Tie-2 sig- naling pathways, we supplemented cBEC culture media with Ang1, which caused an increase in the levels of p- Tie2 and cleaved Notch1 (Figure 7B). Ang1 stimulation also induced the expression of Notch ligands (Dll4 and Jag1) and its targets, such as Hes1, Hey1 and Myc (Figure 7C). To determine whether Tie2 stimulation by Ang1 was essential for Notch ligand upregulation, we treated cBEC with a Tie2 inhibitor. Our results show that increased expression of Dll4 and Jag1 by Ang1 stimulation was com- pletely blocked by pre-treatment with the Tie2 kinase inhibitor (Figure 7D). Our findings support a role for the Tie2 signaling pathway in the induction of Notch signal- ing by enhancing expression of Notch ligands.
To determine whether Notch signaling also influenced Tie2 expression, we inhibited Notch activity by addition of a γ secretase inhibitor for 48 h followed by a 3 h washout. Notch targets, inhibited by γ secretase inhibitors, are rapidly upregulated during washout. We observed that this was the case for Hes1 in cBEC, but not for Tie2 and Ang2 (Figure 7E). Interestingly, Myc expression in cBEC was not entirely dependent on Notch activity; however, Myc levels markedly increased after the washout of the γ secretase inhibitor, indicating that in EC Myc is responsive to higher levels of Notch signaling (Figure 7E). These data showed that, in BM EC, Tie2 activation enhanced downstream Notch signaling while Notch activ- ity had no effect on Tie2 signaling.
To test whether Notch functioned downstream of Tie2 activation, ICNΔTAD-cBEC cells were treated with 5-FU for 24 h and then the medium was supplemented with Ang1 (Figure 7F, left). Ang1 stimulated moderate growth of resting cBEC but had no effect on the ICNΔTAD-trans- duced cBEC. After 5-FU treatment, Ang1 significantly increased growth of control cBEC but did not rescue ICNΔTAD-cBEC (Figure 7F, right). Tie2 activation by Ang1 had the expected result of increasing Socs3 expres- sion.41 However, Tie-2 activation had no effect on the Notch targets Hes1 and Hey1 in ICNΔTAD-expressing cells (Figure 7G). These results show that whereas Tie2 activation accelerates the recovery of the BM EC niche after chemotherapy by promoting Notch ligand expres- sion, ultimately, the resolution of niche recovery depends on activation of robust Notch signaling.
Discussion
The Notch1 receptor is crucial for the emergence of the first definitive HSC from the embryonic hemangioblast42 and expansion of fetal liver HSC.22 The role of Notch sig- naling in adult BM HSC is not well understood and mired in controversy.43-46 It has been suggested that Notch signal- ing is essential for HSC self-renewal, specifically with regard to Jagged1 activation of Notch1 signaling in HSC.46 Even so, pan-inhibition of Notch signaling in HSC by dele- tion of the key transcriptional complex member and DNA-binding factor RBPJ, showed no adverse effects in maintenance and expansion of the stem and progenitor pool.47 A separate recent study showed depletion of HSC numbers after complete ablation of Notch signaling in the endothelium, thus suggesting that the Notch1 receptor participates in the development of the EC niche.48 An even more recent article indicated a possible alternative to Notch1 BM signaling by suggesting that Jagged2 triggers Notch2 signaling in HSC after myelosuppression.49
Regardless of its proposed involvement during hematopoietic and endothelial development, the specific role of Notch signaling, its mechanistic interplay with other known regenerative pathways and its temporal acti- vation during and following myelosuppression are not known. In the present study, we investigated Notch sig- naling in adult hematopoietic and endothelial tissues by deleting the TAD from a single allele of Notch1. This muta- tion has the capacity to interfere with the activity of the WT Notch1 transcriptional complex and created a hypo- morphic signaling environment. Our Notch1+/ΔTAD heterozy- gous model system matured to adulthood and under homeostatic conditions exhibited no adverse effects in the bone and vasculature. We show that this heterozygous hypomorphic model system is useful for determining the requirement for robust Notch signaling activation in adult tissues. Using the Notch1+/ΔTAD mice we determined that basal Notch1 signaling was sufficient for development of the endothelial hematopoietic niche; however, robust acti- vation of Notch1 was required for the regeneration of the hematopoietic system.
We observed increased Notch1 receptor expression and cleavage in BM EC following treatment with the chemotherapeutic agent 5-FU. The WT BM microenviron- ment began to recover at ~9 days after exposure to 5-FU. However, the BM niche was persistently disrupted in Notch1+/ΔTAD mice as evidenced by the presence of few CD31+
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haematologica | 2019; 104(11)